Microorganisms with modified lactose permease and methods for producing lactose-containing oligosaccharides

Abstract

The object of the present invention is to provide a method for producing lactose-containing oligosaccharides more efficiently by fermentation. According to the present invention, by using a microorganism having the ability to produce a protein having lactose permease activity modified such as a specific amino acid residue being replaced with another amino acid residue, lactose-containing oligosaccharides such as 2'-fucosyllactose can be produced more efficiently than when using a microorganism having the ability to produce a wild-type protein having lactose permease activity.

Description

Technical Field

[0001] The present invention relates to a method for producing lactose-containing oligosaccharides capable of efficiently producing lactose-containing oligosaccharides. Background Art

[0002] It is reported that milk oligosaccharides (HMO) contained in human breast milk have an effect of protecting against infection by pathogenic bacteria and function as a prebiotic, and have attracted attention as an additive for infant formula milk powder due to their physiological activity (Non-Patent Document 1).

[0003] More than 130 types of HMOs are known to date, and most of them are lactose-containing oligosaccharides having a free lactose unit at the reducing terminal.

[0004] In the production method of lactose-containing oligosaccharides produced by fermentation, a method of adding relatively cheap lactose as a substrate is known (Non-Patent Document 2). In addition, it is known that lactose is taken up from outside microbial cells by lactose permease (Non-Patent Document 3).

[0005] On the other hand, the method for producing lactose-containing oligosaccharides using a microorganism having a mutant lactose permease and the effect of the mutation of the lactose permease on the productivity of lactose-containing oligosaccharides are unknown. On the other hand, from the perspective of the attention paid to lactose-containing oligosaccharides, a more efficient method for producing lactose-containing oligosaccharides is required.

[0006] Prior art literature

[0007] Non-patent literature

[0008] Non-patent literature 1: J Biotechnol (2016) 235: 61-83

[0009] Non-patent literature 2: Curr Opin Biotechnol (2019) 56: 130-137

[0010] Non-patent literature 3: J Biotechnol (2015) 210: 107-115 Summary of the invention

[0011] Problems to be solved by the invention

[0012] As described above, a more efficient method for producing lactose-containing oligosaccharides is required. An object of the present invention is to provide a more efficient method for producing lactose-containing oligosaccharides by fermentation.

[0013] Methods used to solve problems

[0014] The present invention relates to the following aspects.

[0015] 1. A microorganism having a protein consisting of an amino acid sequence comprising a substitution of the amino acid residue corresponding to position 319 of the amino acid sequence set forth in 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], wherein the microorganism has a higher ability to produce lactose-containing oligosaccharides than a parent strain,

[0016] [1] A protein consisting of the amino acid sequence shown in SEQ ID NO: 2,

[0017] [2] A mutant protein having lactose permease activity, which comprises an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 2,

[0018] [3] A homologous protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having lactose permease activity.

[0019] 2. A microorganism according to item 1 above, which has a protein consisting of an amino acid sequence comprising a substitution of the amino acid residue corresponding to position 319 of the amino acid sequence shown in 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 items [1] to [3] above.

[0020] 3. A microorganism according to 1 or 2 above, which has a protein consisting of an amino acid sequence comprising a substitution of L-glutamic acid for the amino acid residue corresponding to position 319 in the amino acid sequence shown in SEQ ID NO: 2 in the amino acid sequence of the protein described in any one of [1] to [3] above.

[0021] 4. A microorganism according to any one of 1 to 3 above, which is obtained by transforming a parent strain with a recombinant DNA, wherein the recombinant DNA contains a DNA encoding a protein composed of an amino acid sequence containing a substitution of the amino acid residue corresponding to position 319 of the amino acid sequence shown in sequence number 2 with other amino acid residues in the amino acid sequence of the protein recorded in any one of [1] to [3] above.

[0022] 5. A microorganism according to 4 above, which is obtained by integrating a recombinant DNA into a chromosome, wherein the recombinant DNA contains a DNA encoding a protein composed of an amino acid sequence containing a substitution of the amino acid residue corresponding to position 319 of the amino acid sequence shown in sequence number 2 with another amino acid residue in the amino acid sequence of the protein recorded in any one of [1] to [3] above.

[0023] 6. The microorganism according to any one of 1 to 5 above, which is obtained by transforming a parent strain with a recombinant DNA containing a DNA having the base sequence shown in SEQ ID NO: 3.

[0024] 7. The microorganism according to 6 above, which is obtained by integrating a recombinant DNA containing a DNA having the base sequence shown in SEQ ID NO: 3 into a chromosome.

[0025] 8. The microorganism according to any one of 1 to 7 above, wherein the parent strain is a microorganism having an ability to produce lactose-containing oligosaccharides.

[0026] 9. A method for producing lactose-containing oligosaccharides, comprising culturing the microorganism according to any one of 1 to 8 above in a culture medium to produce lactose-containing oligosaccharides in the culture.

[0027] 10. The production method according to 9 above, wherein the lactose-containing oligosaccharide is 2'-fucosyllactose.

[0028] Effects of the Invention

[0029] According to the present invention, a method for producing lactose-containing oligosaccharides capable of efficiently producing lactose-containing oligosaccharides can be provided. DETAILED DESCRIPTION

[0030] 1. Microorganisms of the present invention

[0031] The microorganism of the present invention is a microorganism having the protein described in (1) or (2) below and having an ability to produce lactose-containing oligosaccharides higher than that of a parent strain.

[0032] (1) A protein consisting of an amino acid sequence comprising a substitution of the amino acid residue corresponding to position 319 of the amino acid sequence of the protein described in any one of the following [1] to [3] with another amino acid residue.

[0033] [1] A protein consisting of the amino acid sequence shown in SEQ ID NO: 2

[0034] [2] A mutant protein having lactose permease activity, which comprises an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted and / or added to the amino acid sequence shown in SEQ ID NO: 2.

[0035] [3] A homologous protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having lactose permease activity.

[0036] (2) The protein described in (1) above, which comprises an amino acid sequence in which the amino acid residue corresponding to position 319 of the amino acid sequence shown in SEQ ID NO: 2 is substituted with L-glutamic acid in the amino acid sequence of the protein described in any one of (1) to (3) above.

[0037] The amino acid residue corresponding to position 319 of the amino acid sequence shown in SEQ ID NO: 2 in the amino acid sequence of the protein serving as the basis as recorded in any one of [1] to [3] above refers to the amino acid residue arranged at the same position as the amino acid residue at position 319 in the amino acid sequence of SEQ ID NO: 2 when the amino acid sequence of the protein serving as the basis is compared with the amino acid sequence of SEQ ID NO: 2.

[0038] The fact that the amino acid residue corresponding to position 319 of the amino acid sequence shown in SEQ ID NO: 2 in the amino acid sequence of the protein described in (1) or (2) above is replaced by another amino acid residue can be confirmed, for example, by comparing the amino acid sequence of the protein described in (1) or (2) above whose amino acid residue is to be confirmed with the amino acid sequence of the protein described in any one of the above [1] to [3] as a basis.

[0039] The alignment of amino acid sequences can be created, for example, using the well-known alignment program ClustalW [Nucelic Acids Research 22, 4673, (1994)]. For example, ClustalW can be used from http: / / www.ebi.ac.uk / clustalw / (European Bioinformatics Institute). When using ClustalW to create an alignment, the parameters can be used, for example, with the default values.

[0040] The other amino acid residues described in (1) above may be natural or non-natural. 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, and L-cysteine.

[0041] Examples of amino acids that can be substituted for each other are shown below. Amino acids included in the same group can be substituted for each other.

[0042] Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutyric acid, methionine, o-methylserine, tert-butylglycine, tert-butylalanine, cyclohexylalanine

[0043] Group B: Aspartic acid, Glutamic acid, Isoaspartic acid, Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid

[0044] Group C: Asparagine, Glutamine

[0045] Group D: lysine, arginine, ornithine, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid

[0046] Group E: Proline, 3-hydroxyproline, 4-hydroxyproline

[0047] Group F: serine, threonine, homoserine

[0048] Group G: Phenylalanine, Tyrosine

[0049] As the other amino acid residue described in (1) above, it is preferably one selected from the amino acid residues described in Group B (aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid), and more preferably the amino acid residue is an L-form. As the other amino acid residue described in (1) above, L-glutamic acid is further preferred.

[0050] A mutant protein refers to a protein in which amino acid residues in a base protein are artificially deleted or substituted, or amino acid residues are artificially inserted or added into the protein.

[0051] In the mutant protein, the deletion, substitution, insertion or addition of amino acids may be 1 to 20 amino acids deleted, substituted, inserted or added at any position in the same sequence.

[0052] The amino acid to be deleted, substituted, inserted or added may be natural or non-natural. Examples of natural amino acids include the natural amino acids mentioned above.

[0053] Examples of amino acids that can be substituted for each other are as described above. Amino acids included in the same group can be substituted for each other.

[0054] Homologous proteins refer to a group of proteins that are present in nature and that have an evolutionary origin from the same protein. Homologous proteins are similar in structure and function to each other. The identity of amino acid sequences and base sequences can be determined using the algorithms BLAST [Pro. Nat. Acad. Sci. USA, 90, 5873 (1993)] and FASTA [Methods Enzymol., 183, 63 (1990)] proposed by Karlin and Altschul. Based on the algorithm BLAST, programs called BLASTN and BLASTX were developed [J. Mol. Biol., 215, 403 (1990)]. When using BLASTN based on BLAST to analyze base sequences, the parameters are set to, for example, score = 100 and word length = 12. In addition, when using BLASTX based on BLAST to analyze amino acid sequences, the parameters are set to, for example, score = 50 and word length = 3. When using BLAST and Gap ped BLAST programs, the default parameters of each program are used. Specific methods of these analytical methods are publicly known.

[0055] The fact that the 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 a mutant protein or homologous protein whose activity is to be confirmed is prepared by the method described below. Then, a microorganism that does not have lactose permease activity, such as Escherichia coli W3110 strain lacking lactose permease, is transformed with the recombinant DNA. Finally, the microorganism is cultured in a medium containing lactose as a sugar source. When it is confirmed that the growth is improved compared to the parent strain, it can be confirmed that the mutant protein or homologous protein has lactose permease activity.

[0056] As a specific example of a protein in which the amino acid sequence of the protein described in (1) or (2) above contains a substitution of the amino acid residue corresponding to position 319 of the amino acid sequence shown in SEQ ID NO: 2 with another amino acid residue, a protein consisting of the amino acid sequence shown in SEQ ID NO: 32 can be cited.

[0057] The parent strain refers to the starting strain that is the target of genetic modification, transformation, etc. The starting strain that is the target of transformation by gene introduction is also called the host strain.

[0058] The parent strain of the microorganism of the present invention may be any microorganism as long as it has the ability to produce lactose-containing oligosaccharides.

[0059] Lactose-containing oligosaccharides are 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, and 3'-sialylo-3-fucosyllactose.

[0060] As the microorganism having the ability to produce lactose-containing oligosaccharides, a selected strain in which the ability to produce lactose-containing oligosaccharides has been artificially imparted or enhanced can be preferably used.

[0061] As methods for artificially imparting or enhancing the ability to produce lactose-containing oligosaccharides to a microorganism used as a parent strain, the following methods can be listed: (a) a method for enhancing the expression of at least one of the enzymes related to the biosynthetic pathway for producing lactose-containing oligosaccharides from sugars; (b) a method for increasing the copy number of at least one of the genes encoding enzymes related to the biosynthetic pathway for producing lactose-containing oligosaccharides from sugars; (c) a method for relaxing or releasing at least one of the regulatory mechanisms of the biosynthetic pathway for producing lactose-containing oligosaccharides from sugars; (d) a method for weakening or blocking at least one of the metabolic pathways that branch from the biosynthetic pathway for producing lactose-containing oligosaccharides from sugars to metabolites other than the target substance; etc. The above-mentioned known methods can be used alone or in combination.

[0062] Specific examples of the enzymes involved in the biosynthetic pathway for producing lactose-containing oligosaccharides from sugars include, for example, known enzymes such as an enzyme having α1,2-fucosyltransferase activity for producing 2'-fucosyllactose using GDP-salinose and lactose as substrates, an enzyme having α1,3-fucosyltransferase activity for producing 3-fucosyllactose using GDP-salinose and lactose as substrates, an enzyme having α2,3-sialyltransferase activity for producing 3'-sialyllactose using CMP-sialic acid and lactose as substrates, and an enzyme having α2,6-sialyltransferase activity for producing 6'-sialyllactose using CMP-sialic acid and lactose as substrates.

[0063] Specific examples of the method for imparting or enhancing the ability to produce lactose-containing oligosaccharides include known methods such as methods for enhancing the ability to produce 2'-fucosyllactose or 3-fucosyllactose by various genetic engineering techniques (Metabolic Engineering (2017) 41: 23-38).

[0064] Alternatively, a strain obtained by artificially imparting or enhancing the ability to supply lactose as a precursor to a microorganism having the ability to produce lactose-containing oligosaccharides may be used.

[0065] Methods for artificially imparting or enhancing the ability to supply lactose from sugar to a microorganism used as a parent strain include: (a) a method for relaxing or releasing at least one of the regulatory mechanisms of the biosynthetic pathway for producing lactose from sugar; (b) a method for enhancing the expression of at least one of the enzymes related to the biosynthetic pathway for producing lactose from sugar; (c) a method for increasing the copy number of at least one of the genes encoding the enzymes related to the biosynthetic pathway for producing lactose from sugar; (d) a method for relaxing or releasing at least one of the lactose decomposition mechanisms; (e) a method for weakening or blocking at least one of the metabolic pathways that branch from the biosynthetic pathway for producing lactose from sugar to metabolites other than the target substance; etc. The above-mentioned known methods can be used alone or in combination.

[0066] Specific examples of the enzyme involved in the biosynthetic pathway for producing lactose from sugars include known enzymes such as an enzyme having lactose synthase activity for producing lactose using glucose and UDP-galactose as substrates.

[0067] Specific examples of the method for imparting or enhancing the ability to supply lactose include known methods such as a method of reducing the activity of β-galactosidase involved in the decomposition of lactose or a method of inactivating the enzyme (Metabolic Engineering (2017) 41: 23-38).

[0068] The microorganism having the ability to produce lactose-containing oligosaccharides may be any microorganism, and preferably a prokaryotic organism or a yeast strain may be listed, more preferably a prokaryotic organism belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium or Pseudomonas, or a yeast strain belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwann yeast, Pichia or Candida, and most preferably: Escherichia coli BL21 codon plus, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue (all manufactured by Agilent Technologies, Ltd.); 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 (Deposit No.: NITE BP-03062), Serratia ficaria, Serratia fonticola, Serratia liquefaciens 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 ammoniaphilus 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.

[0069] The above-mentioned Escherichia coli KY3591 is deposited in the National Institute of Technology for Product Evaluation (NITE), an independent administrative institution, located at Room 122, 2-5-8, Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan (zip code 292-0818). The deposit date (commission date) is November 18, 2019, and the deposit number is NITE BP-03062.

[0070] A microorganism obtained by subsequently imparting or enhancing the ability to produce lactose-containing oligosaccharides to a microorganism having the protein of the above-mentioned (1) or (2) produced using the above-mentioned host strain, and a microorganism obtained by further imparting or enhancing the ability to supply lactose to the microorganism, and a microorganism having an ability to produce lactose-containing oligosaccharides higher than that of the parent strain are also the microorganisms of the present invention.

[0071] Examples of microorganisms having the protein described in (1) or (2) above include microorganisms obtained by transforming a parent strain with a recombinant DNA having the DNA described in any of (3) to (6) below, and microorganisms obtained by integrating the recombinant DNA into chromosomes.

[0072] (3) DNA encoding the protein described in (1) or (2) above

[0073] (4) A DNA encoding a protein consisting of an amino acid sequence comprising a substitution of the amino acid residue corresponding to position 319 of the amino acid sequence set forth in 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]

[0074] [4] DNA having the base sequence shown in SEQ ID NO: 1

[0075] [5] A 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

[0076] [6] A DNA comprising 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 of SEQ ID NO: 1 and encoding a homologous protein having lactose permease activity

[0077] (5) The DNA described in (4) above, encoding an amino acid sequence comprising a substitution of the amino acid residue corresponding to position 319 of the amino acid sequence shown in SEQ ID NO: 2 with L-glutamic acid.

[0078] (6) DNA having the base sequence shown in SEQ ID NO: 3

[0079] In the above, hybridization refers to a process of hybridizing DNA to a DNA having a specific base sequence or a part of the DNA. Therefore, the base sequence of the DNA hybridized to the DNA having a specific base sequence or a part of the DNA can be a DNA useful as a probe for Northern blot analysis or Southern blot analysis, or a DNA of a length that can be used as an oligonucleotide primer for PCR analysis.

[0080] As the DNA used as a probe, for example, a DNA of at least 100 bases, preferably 200 bases, and more preferably 500 bases, can be cited. In addition, as the DNA used as a primer, for example, a DNA of at least 10 bases, preferably 15 bases, can be cited.

[0081] DNA hybridization experimental methods are well known. For example, hybridization conditions can be determined and experiments can be performed according 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)), and various other standard textbooks.

[0082] Alternatively, DNA that hybridizes under stringent conditions can be obtained by following the instructions provided with commercially available hybridization kits. Examples of commercially available hybridization kits include the Random Primer DNA Labeling Kit (manufactured by Roche Diagnostics GmbH) that prepares probes using the random primer method and hybridizes under stringent conditions.

[0083] The above-mentioned stringent conditions refer to, for example, the following conditions: the filter to which the DNA is fixed and the probe DNA are incubated 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 denatured fish sperm DNA at 42°C overnight, and then the filter is washed in a 0.2×SSC solution at, for example, about 65°C.

[0084] The above-mentioned various conditions can also be set by adding or changing a blocking reagent for suppressing the background of hybridization experiments. The hybridization conditions can also be changed along with the addition of the above-mentioned blocking reagent to make the conditions suitable.

[0085] As the above-mentioned DNA capable of hybridizing under stringent conditions, for example, there can be listed: a DNA composed of a base sequence that has at least 95% or more, preferably 97% or more, further preferably 98% or more, and most preferably 99% or more identity with the base sequence shown in sequence number 1 when calculated based on the above-mentioned parameters using the above-mentioned BLAST, FASTA and other programs.

[0086] A microorganism obtained by transforming a parent strain with a recombinant DNA having the DNA described in any one of (3) to (6) above, and a microorganism obtained by integrating the recombinant DNA into a chromosome can be prepared by the following method.

[0087] The DNA described in any one of the above (3) to (6) can be obtained, for example, as follows: using a DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, a base sequence encoding a portion of the amino acid residue corresponding to position 319 of the amino acid sequence shown in SEQ ID NO: 2 is introduced into the base sequence by a mutation using, for example, the site-directed mutagenesis method described in Molecular Cloning, 4th edition (Cold Spring Harbor Laboratory Press (2012)) and Current Protocols in Molecular Biology (JOHNWILEY & SONS, INC.), and replaced with a base sequence encoding an arbitrary amino acid residue, thereby obtaining. Alternatively, the DNA of the present invention can also be obtained using PrimeSTAR Mutagenesis Basal Kit (manufactured by Takara Bio Inc.).

[0088] It can also be obtained by the same method as follows: using a DNA encoding a mutant protein composed of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 2 and having lactose permease activity, a mutation is introduced into the base sequence encoding the portion of the amino acid residue corresponding to position 319 in the amino acid sequence shown in SEQ ID NO: 2 when the amino acid sequence shown in SEQ ID NO: 2 and the amino acid sequence of the mutant protein are aligned using the above method, thereby obtaining it.

[0089] Alternatively, it can also be obtained as follows: using a DNA encoding a homologous protein composed of an amino acid sequence having more than 80% identity with the amino acid sequence shown in sequence number 2 and having lactose permease activity, a mutation is introduced into the base sequence encoding the portion of the amino acid residue corresponding to position 319 of the amino acid sequence shown in sequence number 2 when the amino acid sequence of the homologous protein is compared with the amino acid sequence shown in sequence number 2 using the above-mentioned method, thereby obtaining it.

[0090] A DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 can be obtained, for example, by performing Southern blot hybridization on a chromosomal DNA library of a microorganism, preferably Escherichia, and more preferably Escherichia coli W3110 strain, using a probe that can be designed based on the base sequence of the DNA encoding the protein consisting of the amino acid sequence shown in SEQ ID NO: 2, or by performing PCR [PCR Protocols, Academic Press (1990)] using a chromosomal DNA of Escherichia coli W3110 strain as a template using a primer DNA that can be designed based on the DNA encoding the protein consisting of the amino acid sequence shown in SEQ ID NO: 2.

[0091] Escherichia coli W3110 (ATCC27325) strain can be obtained from the American Type Culture Collection (ATCC).

[0092] Examples of DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 include a DNA consisting of the base sequence shown in SEQ ID NO: 1.

[0093] The DNA encoding the mutant protein described in [2] above, which is composed of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted and / or added to the amino acid sequence shown in SEQ ID NO: 2 and has lactose permease activity, can be obtained, for example, as follows: using the DNA composed of the base sequence shown in SEQ ID NO: 1 as a template, and obtaining it by subjecting it to error-prone PCR, etc.

[0094] Alternatively, a DNA encoding a mutant protein having lactose permease activity and consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted or added to the amino acid sequence shown in SEQ ID NO: 2 can be obtained by using a set of PCR primers having a base sequence designed at each 5' end so as to introduce the target mutation (deletion, substitution, insertion or addition) [Gene, 77, 51 (1989)].

[0095] In addition, the DNA can also be obtained according to the instructions attached to a commercially available local specific mutation introduction kit. As a commercially available local specific mutation introduction kit, for example, PrimeSTAR (registered trademark) Mutagenesis Basal Kit (manufactured by Takara Bio Inc.) can be cited, which can introduce a mutation (deletion, substitution, insertion or addition) at the position where the target mutation is to be introduced.

[0096] That is, first, a plasmid having a base sequence designed in a manner to introduce a target mutation (deletion, substitution, insertion or addition) is used as a template, and a pair of primers for mutation introduction with 15 bases overlapping on the 5' side is designed. At this time, the overlapping portion contains the target mutation. Then, using the primer for mutation introduction, PCR is performed using a plasmid having a base sequence into which the target mutation is to be introduced as a template. The amplified fragment thus obtained is transformed into Escherichia coli, and a plasmid having a base sequence into which the target mutation is introduced can be obtained.

[0097] A DNA encoding a homologous protein having lactose permease activity and composed of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2 as described in [3] above can be obtained, for example, by searching various gene sequence databases for base sequences having 80% or more identity, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more identity with the base sequence shown in SEQ ID NO: 2, and using a probe DNA or primer DNA designed based on the base sequence or amino acid sequence obtained by the search, and a microorganism having the DNA, the DNA can be obtained by the same method as the above-mentioned method for obtaining a DNA encoding a protein composed of the amino acid sequence shown in SEQ ID NO: 2. The identity of the base sequence and the amino acid sequence can be determined by the same method as described above.

[0098] A recombinant DNA having the DNA described in any one of (3) to (6) above refers, for example, to a recombinant DNA that is autonomously replicable in a parent strain and in which the DNA described in any one of (3) to (6) above is integrated into an expression vector containing a promoter at a position capable of transcribing the DNA described in any one of (3) to (6) above.

[0099] The recombinant DNA described in any one of (3) to (6) above that can be integrated into the chromosome of the parent strain is also a recombinant DNA having the DNA described in any one of (3) to (6) above.

[0100] When the recombinant DNA is recombinant DNA that can be integrated into chromosome, it is not necessary to contain a promoter.

[0101] When prokaryotes such as bacteria are used as host strains, the recombinant DNA capable of autonomous replication in the parent strain is preferably composed of a promoter, a ribosome binding sequence, the DNA described in any one of (3) to (6) above, and a transcription termination sequence. A gene controlling the promoter may be included.

[0102] It is preferred that the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the start codon be adjusted to an appropriate distance, for example, 6 to 18 bases.

[0103] In the recombinant DNA that can replicate autonomously in the parent strain, a transcription termination sequence is not essential for the expression of the DNA, but it is preferably arranged immediately downstream of the structural gene.

[0104] When a microorganism belonging to the genus Escherichia is used as the 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 Inc.); pGEX-4T-1, pTrc99A (all manufactured by GE Healthcare Bio-Sciences); pTrcHis, pSE280 (all manufactured by Thermo Fisher Scientific); pGEMEX-1 (manufactured by Promega Corp.); pQE-30, pQE-60, pQE80L (all manufactured by Qiagen); pET-3, pBluescriptII SK(+), pBluescriptII KS(-) (all manufactured by Agilent Technologies Inc.); Technologies, Ltd.); 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)]; 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 Application Publication No. 63-233798), etc.

[0105] As a promoter when using the above expression vector, any promoter may be used as long as it functions in cells of microorganisms belonging to the genus Escherichia, and for example, promoters derived from Escherichia coli, bacteriophages, etc., such as trp promoter, gapA promoter, lac promoter, PL promoter, PR promoter, PSE promoter, etc., can be used. In addition, artificially designed and modified promoters such as a promoter in which two trp promoters are linked in series, a tac promoter, a trc promoter, a lacT5 promoter, a lacT7 promoter, and a let I promoter can also be used.

[0106] When using coryneform bacteria 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 (all in Japanese Patent Publication No. 58-105999), pCE51, pCE52, pCE53 [all in Molecular and General Genetics, 196, 175 (1984)], and the like.

[0107] When the expression vector is used, any promoter may be used as long as it functions in cells of coryneform bacteria, and for example, the P54-6 promoter [Appl. Microbiol. Biotechnol., 53, p674-679 (2000)] can be used.

[0108] When a yeast strain is used as a parent strain, examples of expression vectors include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.

[0109] As a promoter when using the above expression vector, any promoter may be used as long as it functions in cells of a yeast strain, and examples thereof include promoters such as PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock polypeptide promoter, MFα1 promoter, and CUP1 promoter.

[0110] The microorganism obtained by transforming the parent strain with the recombinant DNA refers to a microorganism capable of producing a protein encoded by the DNA by transcribed the DNA by introducing the recombinant DNA in the form of a plasmid capable of autonomously replicating in the parent strain or integrating the recombinant DNA into the chromosome of the parent strain.

[0111] As a method for confirming that the DNA described in (3) to (6) above is transcribed and the protein encoded by the DNA is produced, for example, it can be confirmed by measuring the transcription amount of the DNA by Northern blotting or measuring the production amount of the protein by Western blotting.

[0112] The obtained DNA described in (3) to (6) above can be directly or cut with an appropriate restriction enzyme and then integrated into a vector using a conventional method. After the obtained recombinant DNA is introduced into a host cell, it can be analyzed using a commonly used base sequence analysis method, such as the dideoxy method [Proc. Nat. Acad. Sci., USA, 74, 5463 (1977)] or a base sequence analysis device such as Applied Biosystems 3500 Gene Analyzer, Applied Biosystems 3730 DNA Analyzer (both manufactured by Thermo Fisher Scientific), thereby determining the base sequence of the DNA.

[0113] Examples of host cells that can be used when determining the base 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 (all manufactured by Agilent Technologies, Ltd.), Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli W3110, Escherichia coli MP347, and Escherichia coli NM522.

[0114] Examples of the vector include pBluescriptII KS(+), pPCR-Script Amp SK(+) (both manufactured by Agilent Technologies, Ltd.); pT7Blue (manufactured by Merck Millipore); pCRII (manufactured by Thermo Fisher Scientific), pCR-TRAP (manufactured by GenHunter Corporation), and pDIRECT [Nucleic Acids Res., 18, 6069 (1990)].

[0115] 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, and examples thereof include a method using calcium ions [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], a protoplast method (Japanese Patent Application Laid-Open No. 63-248394), and an electroporation method [Nucleic Acids Res., 16, 6127 (1988)].

[0116] When the result of base sequence determination is that the obtained DNA is of partial length, the full-length DNA can be obtained by Southern blotting hybridization with a chromosomal DNA library using the partial-length DNA as a probe.

[0117] Furthermore, the target DNA can be prepared by chemical synthesis based on the determined DNA base sequence using a DNA synthesizer model 8905 manufactured by PerSeptive Biosystems, Inc. or the like.

[0118] Here, by performing base substitution on the base sequence of the DNA described in (3) to (6) above to make it into codons suitable for expression of the parent strain, the expression level of the protein encoded by the DNA can be increased. The codon usage frequency information in the host cell can be obtained from a public database.

[0119] The DNA fragment prepared as described above can be inserted downstream of the promoter of the above-mentioned appropriate expression vector to prepare the recombinant DNA possessed by the microorganism of the present invention.

[0120] An example of such a recombinant DNA is pYHA2 described in Examples.

[0121] Examples of a method for introducing a recombinant DNA in the form of a plasmid capable of autonomously replicating in a parent strain include the above-mentioned method using calcium ions, the protoplast method, the electroporation method, and the like.

[0122] As a method for integrating recombinant DNA into the chromosome of the parent strain, for example, homologous recombination can be cited. As a homologous recombination method, for example, a method using a homologous recombination plasmid can be cited, wherein the homologous recombination plasmid is prepared by connecting with a plasmid DNA having a drug resistance gene that cannot replicate autonomously in the host cell to be introduced. In addition, as a method using homologous recombination widely used in Escherichia coli, for example, a method for introducing recombinant DNA using the homologous recombination system of λ phage can be cited [Proc. Natl. Acad. Sci. USA, 97, 6641-6645 (2000)].

[0123] Furthermore, by using a selection method utilizing the fact that Escherichia coli becomes sensitive to sucrose due to the integration of Bacillus subtilis L-sucrase into the chromosome together with the recombinant DNA, or a selection method utilizing the fact that Escherichia coli becomes sensitive to streptomycin by integrating the wild-type rpsL gene into Escherichia coli having a mutant rpsL gene having streptomycin resistance [Mol. Microbiol., 55, 137 (2005), Biosci. Biotechnol. Biochem., 71, 2905 (2007)], it is possible to obtain Escherichia coli in which the target region on the chromosomal DNA of the host cell is replaced with the recombinant DNA.

[0124] The fact that the microorganism obtained by the above method is a microorganism having a higher ability to produce lactose-containing oligosaccharides than the parent strain can be confirmed by, for example, culturing the parent strain and the obtained microorganism in a culture medium and comparing the amount of lactose-containing oligosaccharides produced. As an example of such a microorganism, the KFL / pYHA2 strain described in the Examples can be cited.

[0125] 2. Method for producing the salt algae oligosaccharide of the present invention

[0126] Examples of the method for producing the sal algal oligosaccharide of the present invention include a method for producing the sal algal oligosaccharide characterized in that the microorganism described in 1 above is cultured in a culture medium and the sal algal oligosaccharide is produced in the culture.

[0127] The method for cultivating the microorganism of 1 above can be carried out according to the conventional method used when cultivating microorganisms. As a culture medium for cultivating the microorganism, as long as it contains a carbon source, nitrogen source, inorganic salts, etc. that the microorganism can assimilate and can efficiently cultivate the microorganism, any of natural culture medium and synthetic culture medium can be used.

[0128] The carbon source may be any carbon source assimilable by the microorganism, and examples thereof include sugars such as glucose, fructose, sucrose, molasses containing these, starch, or starch hydrolyzate; organic acids such as acetic acid or propionic acid; or alcohols such as glycerol, ethanol, or propanol.

[0129] As nitrogen sources, 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; as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented bacteria and their digests, etc. can be used.

[0130] Examples of the inorganic salt include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

[0131] Cultivation 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 cultivation is usually maintained at 3.0 to 9.0. The pH can be adjusted using inorganic acids or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

[0132] In addition, antibiotics such as ampicillin and tetracycline can be added to the culture medium as needed during the culture. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer can be added to the culture medium as needed. For example, when culturing a microorganism transformed with an expression vector using a lac promoter, isopropyl-β-D-thiogalactopyranoside (IPTG) and the like can be added to the culture medium, and when culturing a microorganism transformed with an expression vector using a trp promoter, indole acrylic acid and the like can be added to the culture medium.

[0133] During the above culture, precursors necessary for the production of lactose-containing oligosaccharides such as lactose and N-acetyllactosamine may be added to the medium as necessary.

[0134] By the above-mentioned culturing, lactose-containing oligosaccharides are generated and accumulated in the culture, and lactose-containing oligosaccharides are collected from the culture, thereby producing lactose-containing oligosaccharides.

[0135] The generated lactose-containing oligosaccharides can be analyzed using conventional methods such as sugar ion chromatography. Collection of lactose-containing oligosaccharides from the above-mentioned culture or the processed material of the culture can be carried out by conventional methods such as activated carbon, ion exchange resin, etc. When lactose-containing oligosaccharides accumulate in the bacterial body, for example, the lactose-containing oligosaccharides can be collected from the supernatant obtained by breaking the bacterial body by ultrasound or the like and removing the bacterial body by centrifugation using activated carbon, ion exchange resin, etc.

[0136] [Analysis example]

[0137] In the examples, 2'-fucosyllactose was analyzed and quantified by the following steps. The culture solution containing the microorganisms after culture was centrifuged to recover the supernatant. The 2'-fucosyllactose contained in the supernatant was analyzed using a sugar analyzer ICS-5000 (manufactured by ThermoFisher Scientific).

[0138] [Analysis conditions]

[0139] Column: CarboPAC PA1

[0140] Column temperature: 25°C

[0141] Mobile phase: (Mobile phase A) water

[0142] (Mobile phase B) 500mmol / L sodium hydroxide

[0143] (Mobile phase C) 300mmol / L sodium acetate

[0144] Mixing ratio of mobile phase A, mobile phase B and mobile phase C:

[0145] (0-10 minutes) Gradient from 80:20:0 to 70:20:10

[0146] (10-18 minutes)70:20:10

[0147] (18-25 minutes)80:20:0

[0148] Flow rate: 0.8 mL / min

[0149] Detector: Pulse current detector

[0150] Example

[0151] Although the details of the Examples are shown below, the present invention is not limited to these Examples.

[0152] [Example 1] Preparation of microorganisms used in the production of 2'-fucosyllactose

[0153] (1) Obtaining DNA fragments used as markers for gene deletion

[0154] PCR was performed using a DNA consisting of a base sequence shown in "Primer Set" in Table 1 as a primer set and a DNA described in "Template" in Table 1 as a template to obtain each amplified DNA fragment.

[0155] [Table 1]

[0156]

[0157] The genomic DNA of Bacillus subtilis 168 was prepared according to a conventional method. The amplified DNA fragment cat contained about 200 bp upstream to about 50 bp downstream of the cat gene on pHSG396. The amplified DNA fragment sacB contained about 300 bp upstream to about 100 bp downstream of the sacB gene on the genomic DNA of Bacillus subtilis 168.

[0158] Then, PCR was performed using cat and sacB of the amplified DNA fragments as templates and DNA consisting of the base sequences shown in SEQ ID NOs. 4 and 7 as a primer set to obtain a DNA fragment containing the cat gene and the sacB gene (hereinafter referred to as cat-sacB).

[0159] (2) Preparation of Escherichia coli that has lost β-galactosidase activity, lactose permease activity and cholanic acid production activity

[0160] Escherichia coli in which DNA encoding β-galactosidase (hereinafter referred to as lacZ gene), DNA encoding lactose permease (hereinafter referred to as lacY gene) and DNA encoding cholanic acid production-related protein (hereinafter referred to as wcaJ, wzxC, wcaK, wcaL or wcaM gene) are deficient are prepared by the following method. It should be noted 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.

[0161] PCR was performed using genomic DNA of Escherichia coli KY3591 strain prepared according to a conventional method as a template and using DNA consisting of the base sequence shown in "Primer Set" in Table 2 as a primer set to obtain each amplified DNA fragment.

[0162] [Table 2]

[0163]

[0164] lacZ upstream 1 and lacZ upstream 2 contain about 900 bp upstream from the start codon of the lacZ gene, and lacY downstream 1 and lacY downstream 2 contain about 800 bp downstream from the stop codon of the lacY gene.

[0165] PCR was performed using an equimolar mixture of lacZ upstream 1, lacY downstream 1 and cat-sacB fragments as a template and a DNA consisting of the base sequence shown in sequence numbers 9 and 11 as a primer set to obtain a DNA fragment consisting of a sequence in which the cat-sacB fragment was inserted into the sequence of the surrounding region of the lacZ and lacY genes (hereinafter referred to as lacZY::cat-sacB).

[0166] PCR was performed using an equimolar mixture of lacZ upstream 2 and lacY downstream 2 as a template and a DNA consisting of the base sequences shown in sequence numbers 9 and 11 as a primer set to obtain a DNA fragment that does not contain lacZ and lacY and consists of a sequence directly connected between lacZ upstream and lacY downstream (hereinafter referred to as ΔlacZY).

[0167] The lacZY::cat-sacB fragment was introduced into the Escherichia coli W3110 strain containing the plasmid pKD46 [Datsenko, KA, Warner, BL, Proc. Natl. Acad. Sci., USA, Vol. 97, 6640-6645 (2000)] containing the gene encoding λ recombinase by electroporation, and transformants showing chloramphenicol resistance and sucrose sensitivity were obtained (transformants in which the lacZ and lacY genes were replaced by lacZY::cat-sacB).

[0168] The ΔlacZY fragment was introduced into the transformant by electroporation, and a transformant showing chloramphenicol sensitivity and sucrose resistance was obtained (a transformant in which lacZY::cat-sacB was replaced with ΔlacZY). From these, a transformant showing ampicillin sensitivity was further obtained (a transformant in which pKD46 was lost). This transformant was named W3110ΔlacZY.

[0169] Similarly, PCR was performed using genomic DNA of Escherichia coli strain KY3591 (accession number: NITE BP-03062) as a template and a DNA consisting of the base sequence shown in "Primer set" in Table 3 as a primer set to obtain each amplified DNA fragment.

[0170] [Table 3]

[0171]

[0172] wcaJ upstream 1 and wcaJ upstream 2 contain about 900 bp upstream from the start codon of the wcaJ gene, and wcaM downstream 1 and wcaM downstream 2 contain about 800 bp downstream from the stop codon of the wcaM gene.

[0173] PCR was performed using an equimolar mixture of wcaJ upstream 1, wcaM downstream 1 and cat-sacB fragments as a template and a DNA consisting of the base sequences shown in sequence numbers 15 and 17 as a primer set, resulting in a DNA fragment consisting of a sequence in which the cat-sacB fragment was inserted into the sequence of the region surrounding the wcaJ-wzxC-wcaKLM operon (hereinafter referred to as wcaJ-wzxC-wcaKLM::cat-sacB).

[0174] PCR was performed using an equimolar mixture of wcaJ upstream 2 and wcaM downstream 2 as a template and a DNA consisting of the base sequences shown in sequence numbers 15 and 17 as a primer set, and a DNA fragment (hereinafter referred to as ΔwcaJ-wzxC-wcaKLM) was obtained, which did not contain wcaJ-wzxC-wcaKLM and consisted of the sequence directly connected between wcaJ upstream and wcaM downstream.

[0175] The wcaJ-wzxC-wcaKLM::cat-sacB fragment was introduced into the W3110ΔlacZY strain prepared above by electroporation to obtain a transformant showing chloramphenicol resistance and sucrose sensitivity (a transformant in which wcaJ-wzxC-wcaKLM was replaced with wcaJ-wzxC-wcaKLM::cat-sacB).

[0176] The ΔwcaJM fragment was introduced into the transformant by electroporation, and a transformant showing chloramphenicol sensitivity and sucrose resistance was obtained (a transformant in which wcaJ-wzxC-wcaKLM::cat-sacB was replaced with ΔwcaJ-wzxC-wcaKLM). Furthermore, a transformant showing ampicillin sensitivity was obtained (a transformant in which pKD46 was lost). This transformant was named KFL strain.

[0177] (3) Preparation of lactose permease (LacY) expression vector from Escherichia coli

[0178] PCR was performed using a DNA consisting of a base sequence shown in "Primer Set" in Table 4 as a primer set and a DNA described in "Template" in Table 4 as a template to obtain each amplified DNA fragment.

[0179] [Table 4]

[0180]

[0181] The genomic DNA of Escherichia coli W3110 strain was prepared by a conventional method. In addition, the base sequences shown in SEQ ID NOs. 21 and 22, 23 and 24 contain complementary sequences at their respective 5' ends.

[0182] PCR was performed using an equimolar mixture of lacY, HMFT and rcsA fragments as a template and a DNA consisting of the base sequences shown in SEQ ID NOs: 26 and 27 as a primer set to obtain a DNA fragment (hereinafter referred to as lacY-HMFT-rcsA) formed by connecting the three fragments.

[0183] PCR was performed using a DNA consisting of the base sequences shown in SEQ ID NOs. 28 and 29 as a primer set and 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, the base sequences shown in SEQ ID NOs. 26 and 29 and SEQ ID NOs. 27 and 28 contain complementary sequences at their respective 5' ends.

[0184] The lacY-HMFT-rcsA fragment obtained above and the vector fragment were ligated using In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.) to obtain an expression plasmid pYHA1.

[0185] (4) Preparation of microorganisms expressing mutant LacY

[0186] Plasmid pYHA2 in which the L-lysine residue at position 319 in the amino acid sequence of LacY was substituted with an L-glutamic acid residue was prepared using Prime STAR Mutagenesis Basal Kit (manufactured by Takara Bio Inc.) as a template. DNA consisting of the base sequences shown in SEQ ID NOs: 30 and 31 was used as a primer set.

[0187] The KFL strain prepared in the above (2) was transformed using the plasmid pYHA1 obtained in (3) and the above pYHA2 to obtain KFL / pYHA1 strain and KFL / pYHA2 strain.

[0188] [Example 2] Production of 2'-fucosyllactose by fermentation using a microorganism expressing mutant LacY

[0189] The KFL / pYHA1 strain and KFL / pYHA2 strain obtained in Example 1 were cultured on LB plates at 30°C for 24 hours, inoculated into a large test tube containing 5 mL of LB medium containing 100 mg / L kanamycin, and cultured with shaking at 30°C for 16 hours. Then, 0.1 mL of the obtained culture solution was inoculated 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, the pH was adjusted to 7.2 with an aqueous sodium hydroxide solution and then sterilized by high pressure) (the aqueous solution containing glucose, lactose monohydrate and magnesium sulfate heptahydrate was prepared separately and then sterilized, and then cooled and mixed)] containing 100 mg / L of kanamycin, and cultured with shaking at 30°C for 30 hours.

[0190] After the culture was completed, the culture solution was appropriately diluted and centrifuged, and 2'-fucosyllactose contained in the supernatant was analyzed by HPLC.

[0191] [Table 5]

[0192] Strain name 2'-fucosyllactose (g / L) KFL / pYHA1 0.38 KFL / pYHA2 2.58

[0193] As a result, the KFL / pYHA2 strain showed higher 2'-fucosyllactose productivity than the KFL / pYHA1 strain.

[0194] From the above, it was found that the productivity of 2'-fucosyllactose can be improved by using a microorganism having a mutant LacY, compared with a microorganism having a wild-type LacY.

[0195] The present invention is described in detail with reference to a specific mode, but various changes and modifications can be made without departing from the spirit and scope of the present invention, which is obvious to those skilled in the art. It should be noted that this application is based on the Japanese patent application (Japanese patent application 2019-226278) filed on December 16, 2019, and is cited in its entirety by reference. In addition, all references cited in this application are introduced as a whole.

[0196] Industrial Applicability

[0197] According to the present invention, there can be provided a method for producing lactose-containing oligosaccharides using a microorganism having an ability to produce a protein having modified lactose permease activity. Sequence Listing <110> Kyowa Hakko Biochemical Co., Ltd. <120> Microorganisms with modified lactose permease and methods for producing lactose-containing oligosaccharides <130> W530413 <150> JP2019-226278 <151> 2019-12-16 <160> 32 <170> PatentIn version 3.5 <210> 1 <211> 1254 <212> DNA <213> Escherichia coli <400> 1 atgtactatt taaaaaacac aaacttttgg atgttcggtt tattcttttt cttttacttt 60 tttatcatgg gagcctactt cccgtttttc ccgatttggc tacatgacat caaccatatc 120 agcaaaagtg atacgggtat tatttttgcc gctatttctc tgttctcgct attattccaa 180 ccgctgtttg gtctgctttc tgacaaactc gggctgcgca aatacctgct gtggattatt 240 accggcatgt tagtgatgtt tgcgccgttc tttattttta tcttcgggcc actgttacaa 300 tacaacattt tagtaggatc gattgttggt ggtatttatc taggcttttg ttttaacgcc 360 ggtgcgccag cagtagaggc atttattgag aaagtcagcc gtcgcagtaa tttcgaattt 420 ggtcgcgcgc ggatgtttgg ctgtgttggc tgggcgctgt gtgcctcgat tgtcggcatc 480 atgttcacca tcaataatca gtttgttttc tggctgggct ctggctgtgc actcatccctc 540 gccgttttac tctttttcgc caaaacggat gcgccctctt 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Leu His Asp Ile Asn His Ile Ser Lys Ser Asp Thr Gly Ile 35 40 45 Phe Ala Ala Ile Ser Leu Phe Ser Leu Phe Gln Pro Leu Phe Gly 50 55 60 You Ser Asp Lys You Gly You Arg Lys Tyr You Trp Ile Ile 65 70 75 80 Thr Gly Met Leu Val Met Phe Ala Pro Phe Phe Ile Phe Ile Phe Gly 85 90 95 Pro Leu Leu Gln Tyr Asn Ile Leu Val Gly Ser Ile Val Gly Gly Ile 100 105 110 Tyr Leu Gly Phe Cys Phe Asn Ala Gly Ala Pro Ala Val Glu Ala Phe 115 120 125 Ile Glu Lys Val Ser Arg Arg Ser Asn Phe Glu Phe Gly Arg Ala Arg 130 135 140 Met Phe Gly Cys Val Gly Trp Ala Leu Cys Ala Ser Ile Val Gly Ile 145 150 155 160 Met Phe Thr Ile Asn Asn Gln Phe Val Phe Trp Leu Gly Ser Gly Cys 165 170 175 Ala Leu Ile Leu Ala Val Leu Leu Phe Phe Ala Lys Thr Asp Ala Pro 180 185 190 Ser Ser Ala Thr Val Ala Asn Ala Val Gly Ala Asn His Ser Ala Phe 195 200 205 Ser Leu Lys Leu Ala Leu Glu Leu Phe Arg Gln Pro Lys Leu Trp Phe 210 215 220 Leu Ser Leu Tyr Val Ile Gly Val Ser Cys Thr Tyr Asp Val Phe Asp 225 230 235 240 Gln Gln Phe Ala Asn Phe Phe Thr Ser Phe Phe Ala Thr Gly Glu Gln 245 250 255 Gly Thr Arg Val Phe Gly Tyr Val Thr Thr Met Gly Glu Leu Leu Asn 260 265 270 Ala Ser Ile Met Phe Phe Ala Pro Leu Ile Ile Asn Arg Ile Gly Gly 275 280 285 Lys Asn Ala Leu Leu Leu Ala Gly Thr Ile Met Ser Val Arg Ile Ile 290 295 300 Gly Ser Ser Phe Ala Thr Ser Ala Leu Glu Val Val Ile Leu Lys Thr 305 310 315 320 Leu His Met Phe Glu Val Pro Phe Leu Leu Val Gly Cys Phe Lys Tyr 325 330 335 Ile Thr Ser Gln Phe Glu Val Arg Phe Ser Ala Thr Ile Tyr Leu Val 340 345 350 Cys Phe Cys Phe Phe Lys Gln Leu Ala Met Ile Phe Met Ser Val Leu 355 360 365 Ala Gly Asn Met Tyr Glu Ser Ile Gly Phe Gln Gly Ala Tyr Leu Val 370 375 380 Leu Gly Leu Val Ala Leu Gly Phe Thr Leu Ile Ser Val Phe Thr Leu 385 390 395 400 Ser Gly Pro Gly Pro Leu Ser Leu Leu Arg Arg Gln Val Asn Glu Val 405 410 415 That <210> 3 <211> 1254 <212> Ms <213> artificial sequence <220> <223> DNA synthesis <400> 3 atgtactatt taaaaaacac aaacttttgg atgttcggtt tattcttttt cttttacttt 60 tttatcatgg gagcctactt cccgtttttc ccgatttggc tacatgacat caaccatatc 120 agcaaaagtg atacgggtat tatttttgcc gctatttctc tgttctcgct attattccaa 180 ccgctgtttg gtctgctttc tgacaaactc gggctgcgca aatacctgct gtggattatt 240 accggcatgt tagtgatgtt tgcgccgttc tttattttta tcttcgggcc actgttacaa 300 tacaacattt tagtaggatc gattgttggt ggtatttatc taggcttttg ttttaacgcc 360 ggtgcgccag cagtagaggc atttattgag aaagtcagcc gtcgcagtaa tttcgaattt 420 ggtcgcgcgc ggatgtttgg ctgtgttggc tgggcgctgt gtgcctcgat tgtcggcatc 480 atgttcacca tcaataatca gtttgttttc tggctgggct ctggctgtgc actcatcctc 540 gccgttttac tctttttcgc caaaacggat gcgccctctt ctgccacggt tgccaatgcg 600 gtaggtgcca accattcggc atttagcctt aagctggcac tggaactgtt cagacagcca 660 aaactgtggt ttttgtcact gtatgttatt ggcgtttcct gcacctacga tgtttttgac 720 caacagtttg ctaatttctt tacttcgttc tttgctaccg gtgaacaggg tacgcgggta 780 tttggctacg taacgacaat gggcgaatta cttaacgcct cgattatgtt ctttgcgcca 840 ctgatcatta atcgcatcgg tgggaaaaac gccctgctgc tggctggcac tattatgtct 900 gtacgtatta ttggctcatc gttcgccacc tcagcgctgg aagtggttat tctggaaacg 960 ctgcatatgt ttgaagtacc gttcctgctg gtgggctgct ttaaatatat taccagccag 1020 tttgaagtgc gtttttcagc gacgatttat ctggtctgtt tctgcttctt taagcaactg 1080 gcgatgattt ttatgtctgt actggcgggc aatatgtatg aaagcatcgg tttccagggc 1140 gcttatctgg tgctgggtct ggtggcgctg ggcttcacct taatttccgt gttcacgctt 1200 agcggccccg gcccgctttc cctgctgcgt cgtcaggtga atgaagtcgc ttaa 1254 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 4 accaggcgtt taagggcacc 20 <210> 5 <211> 45 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 5 gactacgggc ctaaagtcga cagaataaat aaatcctggt gtccc 45 <210> 6 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 6 gtcgacttta ggcccgtagt ctgcaaat 28 <210> 7 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 7 tacggttagc catttgcctg c 21 <210> 8 <211> 44 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 8 ggtgccctta aacgcctggt agctgtttcc tgtgtgaaat tgtt 44 <210> 9 <211> 19 <212> DNA <213> Artificial sequence <220> 9 <223> Synthetic DNA <400> tcgcgcaacg cgtcagtgg 19 <210> 10 <211> 44 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 10 gcaggcaaat ggctaaccgt aatgaacatg tcgatgacag aaag 44 <210> 11 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 11 tgcgctgtgt gtcgttgggc 20 <210> 12 <211> 45 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 12 ttctgtcatc gacatgttca tagctgtttc ctgtgtgaaa ttgtt 45 <210> 13 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 13 atgaacatgt cgatgacaga aag 23 <210> 14 <211> 42 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 14 ggtgccctta aacgcctggt cgttgttcct gttatttgcc cc 42 <210> 15 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 15 aagctggtga ttaactccgg g 21 <210> 16 <211> 44 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 16 gcaggcaaat ggctaaccgt aatttgcgac cattcctgga aaaa 44 <210> 17 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 17 gcacatagcg accaacggcc 20 <210> 18 <211> 45 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 18 tttttccagg aatggtcgca aatcgttgtt cctgttattt gcccc 45 <210> 19 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 19 atttgcgacc attcctggaa aaa 23 <210> 20 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 20 atgtactatt taaaaaacac aaactttt 28 <210> twenty one <211> 44 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> twenty one atccattcta gacctcctta attaagcgac ttcattcacc tgac 44 <210> twenty two <211> 39 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> twenty two ttaaggaggt ctagaatgga ttttaagatt gtgcaagtg 39 <210> twenty three <211> 45 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> twenty three atggcatacc ctcactcaat gtcaagattt tgtctcaaat tggga 45 <210> twenty four <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> twenty four cattgagtga gggtatgcca tg 22 <210> 25 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 25 ttagcgcatg ttgacaaaaa tacc 24 <210> 26 <211> 43 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 26 gaggagaaat taaccatgta ctatttaaaa aacacaaact ttt 43 <210> 27 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 27 agggcatcgg tcgacttagc gcatgttgac aaaaatacc 39 <210> 28 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 28 gtcgaccgat gcccttgaga g 21 <210> 29 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 29 ggttaatttc tcctctttaa tatcg 25 <210> 30 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 30 cagcgtttcc agaataacca cttccag 27 <210> 31 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 31 attctggaaa cgctgcatat gtttgaa 27 <210> 32 <211> 417 <212> PRT <213> Artificial sequence <220> <223> Artificial protein <400> 32 Met Tyr Tyr Leu Lys Asn Thr Asn Phe Trp Met Phe Gly Leu Phe Phe 1 5 10 15 Phe Phe Tyr Phe Phe Ile Met Gly Ala Tyr Phe Pro Phe Phe Pro Ile 20 25 30 Trp Leu His Asp Ile Asn His Ile Ser Lys Ser Asp Thr Gly Ile Ile 35 40 45 Phe Ala Ala Ile Ser Leu Phe Ser Leu Leu Phe Gln Pro Leu Phe Gly 50 55 60 Leu Leu Ser Asp Lys Leu Gly Leu Arg Lys Tyr Leu Leu Trp Ile Ile 65 70 75 80 Thr Gly Met Leu Val Met Phe Ala Pro Phe Phe Ile Phe Ile Phe Gly 85 90 95 Pro Leu Leu Gln Tyr Asn Ile Leu Val Gly Ser Ile Val Gly Gly Ile 100 105 110 Tyr Leu Gly Phe Cys Phe Asn Ala Gly Ala Pro Ala Val Glu Ala Phe 115 120 125 Ile Glu Lys Val Ser Arg Arg Ser Asn Phe Glu Phe Gly Arg Ala Arg 130 135 140 Met Phe Gly Cys Val Gly Trp Ala Leu Cys Ala Ser Ile Val Gly Ile 145 150 155 160 Met Phe Thr Ile Asn Asn Gln Phe Val Phe Trp Leu Gly Ser Gly Cys 165 170 175 Ala Leu Ile Leu Ala Val Leu Leu Phe Phe Ala Lys Thr Asp Ala Pro 180 185 190 Ser Ser Ala Thr Val Ala Asn Ala Val Gly Ala Asn His Ser Ala Phe 195 200 205 Ser Leu Lys Leu Ala Leu Glu Leu Phe Arg Gln Pro Lys Leu Trp Phe 210 215 220 Leu Ser Leu Tyr Val Ile Gly Val Ser Cys Thr Tyr Asp Val Phe Asp 225 230 235 240 Gln Gln Phe Ala Asn Phe Phe Thr Ser Phe Phe Ala Thr Gly Glu Gln 245 250 255 Gly Thr Arg Val Phe Gly Tyr Val Thr Thr Met Gly Glu Leu Leu Asn 260 265 270 Ala Ser Ile Met Phe Phe Ala Pro Leu Ile Ile Asn Arg Ile Gly Gly 275 280 285 Lys Asn Ala Leu Leu Leu Ala Gly Thr Ile Met Ser Val Arg Ile Ile 290 295 300 Gly Ser Ser Phe Ala Thr Ser Ala Leu Glu Val Val Ile Leu Glu Thr 305 310 315 320 Leu His Met Phe Glu Val Pro Phe Leu Leu Val Gly Cys Phe Lys Tyr 325 330 335 Ile Thr Ser Gln Phe Glu Val Arg Phe Ser Ala Thr Ile Tyr Leu Val 340 345 350 Cys Phe Cys Phe Phe Lys Gln Leu Ala Met Ile Phe Met Ser Val Leu 355 360 365 Ala Gly Asn Met Tyr Glu Ser Ile Gly Phe Gln Gly Ala Tyr Leu Val 370 375 380 Leu Gly Leu Val Ala Leu Gly Phe Thr Leu Ile Ser Val Phe Thr Leu 385 390 395 400 Ser Gly Pro Gly Pro Leu Ser Leu Leu Arg Arg Gln Val Asn Glu Val 405 410 415 That

Claims

1. A microorganism having a protein consisting of an amino acid sequence comprising a substitution of the amino acid residue corresponding to position 319 of the amino acid sequence shown in SEQ ID NO: 2 with L-glutamic acid in the amino acid sequence of the protein described in [1] below, wherein the microorganism has a higher ability to produce lactose-containing oligosaccharides than a parent strain, [1] A protein consisting of the amino acid sequence shown in SEQ ID NO:

2.

2. A microorganism according to claim 1, which is obtained by transforming a parent strain with a recombinant DNA, wherein the recombinant DNA contains a DNA encoding a protein composed of an amino acid sequence containing a substitution of L-glutamic acid in the amino acid sequence of the protein described in [1] corresponding to the amino acid residue at position 319 of the amino acid sequence shown in sequence number 2.

3. A microorganism according to claim 2, which is obtained by integrating a recombinant DNA into a chromosome, wherein the recombinant DNA contains a DNA encoding a protein consisting of an amino acid sequence in which the amino acid residue corresponding to position 319 of the amino acid sequence shown in sequence number 2 is substituted by L-glutamic acid in the amino acid sequence of the protein described in [1].

4. The microorganism according to claim 1, which is obtained by transforming a parent strain with a recombinant DNA containing a DNA having the base sequence shown in SEQ ID NO:

3. 5 . The microorganism according to claim 4 , which is obtained by integrating a recombinant DNA containing a DNA having the base sequence shown in SEQ ID NO: 3 into a 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 in that: The microorganism according to any one of claims 1 to 6 is cultured in a culture medium to produce lactose-containing oligosaccharides in the culture.

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

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