Method for producing 3-fucosyl lactose and transformed Escherichia coli

Abstract

The purpose of the present invention is to provide: a protein involved in the transport of a fucose-containing sugar; and a method for producing a fucose-containing sugar efficiently using a microorganism capable of producing the protein. The present invention relates to any one protein selected from [1] a protein which comprises the amino acid sequence represented by SEQ ID NO: 2 or 4, [2] a mutant protein which comprises an amino acid sequence having a structure such that 1 to 20 amino acid residues are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 2 or 4 and has a fucose-containing sugar transporting activity, and [3] a homologous protein which comprises an amino acid sequence having a 90% or higher identity to the amino acid sequence represented by SEQ ID NO: 2 or 4 and has a fucose-containing sugar transporting activity.

Description

Technical Field

[0001] The present invention relates to a transporter involved in the excretion of fucose-containing carbohydrates and a method for producing fucose-containing carbohydrates such as 3-fucosyllactose.

Background Art

[0002] Human milk oligosaccharides (HMOs) contained in human breast milk have been reported to have health functions such as improving the intestinal environment as prebiotics, activating immunity, and developing the cognitive function of infants. Due to their physiological activity, they are expected to be used as additives in infant formula milk and as health functional materials for adults (Non-Patent Document 1).

[0003] Among the carbohydrates known as HMOs, fucose-containing carbohydrates such as 2'-fucosyllactose and 3-fucosyllactose account for about 60% of the total HMOs in terms of the molar ratio (Non-Patent Document 2), and their functionality has attracted particular attention among HMOs.

[0004] As a method for producing fucose-containing carbohydrates, a microbial fermentation method using fucose transferase is widely used. Non-Patent Document 3 discloses a method for producing 2'-fucosyllactose or 3-fucosyllactose by a fermentation method using microorganisms expressing fucose transferase derived from microorganisms such as Helicobacter pylori and Bacteroides fragilis, with lactose and GDP-fucose as substrates.

[0005] Patent Document 1 shows that the SetA protein, known as a transporter involved in sugar excretion in Escherichia coli, is also involved in the transport of fucose-containing carbohydrates such as 2'-fucosyllactose, and discloses a method for improving the productivity of fucose-containing carbohydrates by overexpressing the protein.

[0006] The SetA protein is a transport protein belonging to the SET family, which is part of the Major Facilitator Superfamily (MFS) transporters (Non-Patent Literature 4). However, no other proteins belonging to the same SET family are known to be involved in the transport of fucose-containing carbohydrates.

[0007] Similar to the SetA protein, the CDT2 protein derived from Neurospora crassa, which belongs to the SP family, has been disclosed to be involved in the transport of 2'-fucosyl lactose as a transporter responsible for the transport of oligosaccharides (Non-Patent Literature 5). [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent No. 5580408 [Non-patent literature]

[0009] [Non-Patent Document 1] Int J Pediatrics(2019)2390240:1-8 [Non-Patent Document 2] Curr Opin Biotechnol(2019)56:130-137 [Non-Patent Document 3] Metabolic Engineering(2017)41:23-38 [Non-Patent Document 4] J Biol Chem(1999)274:22977-22984 [Non-Patent Document 5] Metabolic Engineering(2019)52:232-242 [Overview of the project] [Problems that the invention aims to solve]

[0010] However, other than the SetA and CDT2 proteins, no other proteins involved in the excretion of fucose-containing carbohydrates have been reported. Therefore, further exploration of proteins involved in the transport of fucose-containing carbohydrates is needed as a means of realizing an efficient method for producing fucose-containing carbohydrates.

[0011] Therefore, the present invention aims to provide a protein involved in the transport of fucose-containing carbohydrates, and a method for efficiently producing fucose-containing carbohydrates using microorganisms capable of producing proteins. [Means for solving the problem]

[0012] The inventors of the present invention have discovered that 3-fucosyl lactose can be produced more efficiently than by conventional methods by using microorganisms capable of producing proteins that have transport activity for fucose-containing carbohydrates containing a specific amino acid sequence, thereby completing the present invention.

[0013] In other words, the present invention is as follows: 1. One of the proteins listed in [1] to [3] below. [1] A protein consisting of an amino acid sequence represented by Sequence ID No. 2 or 4. [2] A mutant protein having an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by Sequence ID No. 2 or 4, and which has fucose-containing carbohydrate transport activity. [3] A homologous protein having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by Sequence ID No. 2 or 4, and which has fucose-containing carbohydrate transport activity. 2. The protein according to paragraph 1, wherein the fucose-containing carbohydrate is an oligosaccharide. 3. DNA comprising the base sequence represented by Sequence ID No. 1 or 3 or its homologous sequence, and encoding the protein described in 1 or 2 above. 4. Recombinant DNA containing the DNA described in item 3 above. 5. A transformant obtained by transforming a host cell with the recombinant DNA described in item 4 above. 6. The transformant according to claim 5, which is a microorganism in which the activity of any one of the proteins [1] to [3] described in 1 above and the productivity of the fucose-containing carbohydrate are enhanced. 7. The transformant according to claim 6, wherein the microorganism is Escherichia coli having the ability to produce the fucose-containing carbohydrate. 8. A method for producing a fucose-containing carbohydrate, comprising culturing the transformant according to any one of claims 5 to 7 in a medium to produce the fucose-containing carbohydrate in the culture. 9. The production method according to claim 8, wherein the fucose-containing carbohydrate is an oligosaccharide. 10. The production method according to claim 9, wherein the oligosaccharide is 3-fucosyllactose.

Advantages of the Invention

[0014] The protein of the present invention has excellent transport activity for fucose-containing carbohydrates due to consisting of a specific amino acid sequence. By using a microorganism having the ability to produce the protein of the present invention, fucose-containing carbohydrates such as 3-fucosyllactose can be produced more efficiently than before.

Modes for Carrying Out the Invention

[0015] 1. The protein of the present invention The protein of the present invention is a 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 or 4. [2] A mutant protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 2 or 4, and having transport activity for fucose-containing carbohydrates. [3] A homologous protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or 4, and having transport activity for fucose-containing carbohydrates.

[0016] "Amino acid deletion" means the absence or disappearance of an amino acid residue in a sequence; "amino acid substitution" means that an amino acid residue in a sequence is replaced by another amino acid residue; "amino acid insertion" means that a new amino acid is inserted into a sequence; and "amino acid addition" means that a new amino acid residue is added to a sequence in an insertional manner.

[0017] Specific examples of "deletion, substitution, insertion, or addition of 1 to 20 amino acids" include cases where 1 to 20 amino acids are replaced with other chemically similar amino acids. For example, this could involve substituting one hydrophobic amino acid with another hydrophobic amino acid, or substituting one polar amino acid with another polar amino acid having the same charge. Such chemically similar amino acids are known in the relevant art for each amino acid.

[0018] To give specific examples, nonpolar (hydrophobic) amino acids include alanine, valine, glycine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Basic amino acids with a positive charge include arginine, histidine, and lysine. Acidic amino acids with a negative charge include aspartic acid and glutamic acid.

[0019] Amino acid sequences having 1 to 20 amino acid deletions, substitutions, insertions, or additions in the amino acid sequence of the target protein include amino acid sequences that have a certain degree of sequence identity with the amino acid sequence of the target protein. For example, amino acid sequences that have a preferably 60% or more, more preferably 65% ​​or more, 70% or more, 75% or more, 80% or more, 85% or more, even more preferably 90% or more, and particularly preferably 95% or more, identity with the amino acid sequence of the target protein are included.

[0020] The absence, substitution, insertion, or addition of 1 to 20 amino acids in the amino acid sequence represented by Sequence ID No. 2 or 4 can be confirmed, for example, by aligning the amino acid sequence of the protein in question with the amino acid sequence of the original protein.

[0021] Amino acid sequence alignments can be created, for example, using a well-known alignment program such as ClustalW [Nucelic Acids Research 22, 4673, (1994)]. ClustalW is available, for example, from http: / / www.ebi.ac.uk / clustalw / (European Bioinformatics Institute). When creating alignments using ClustalW, default values ​​can be used for parameters.

[0022] In the amino acid sequence represented by SEQ ID NO: 2 or 4, when 1 to 20 amino acids are substituted, inserted, or added, the amino acid residues to be substituted, inserted, or added may be native or unnatural. Examples of native amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and L-cysteine.

[0023] Examples of mutually substituted amino acids are shown below. Amino acids belonging to the same group are mutually substituted. Group A: Leucine, Isoleucine, Norleucine, Valine, Norvaline, Alanine, 2-Aminobutanoic acid, Methionine, o-Methylserine, t-Butylglycine, t-Butylalanine, Cyclohexylalanine Group B: Aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid Group C: Asparagine, Glutamine Group D: Lysine, Arginine, Ornithine, 2,4-Diaminobutanoic acid, 2,3-Diaminopropionic acid Group E: Proline, 3-hydroxyproline, 4-hydroxyproline Group F: Serine, Threonine, Homoserine Group G: Phenylalanine, tyrosine

[0024] The transport activity of fucose-containing carbohydrates refers to the activity of transporting fucose-containing carbohydrates from inside a cell to the outside of the cell.

[0025] As fucose-containing carbohydrates, for example, oligosaccharides containing fucose as a constituent monosaccharide are preferred, and oligosaccharides having a chain length of 3 to 6 and having a lactose terminus are more preferred. More specifically, examples include 3-fucosyllactose, 2'-fucosyllactose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lactodifucotetraose, lacto-N-difucohexaose I, and lacto-N-difucohexaose II, among which 3-fucosyllactose is preferred.

[0026] A mutant protein is a protein obtained by artificially deleting or substituting amino acid residues in an original protein, or by artificially inserting or adding amino acid residues to that protein. In a mutant protein, the deletion, substitution, insertion, or addition of amino acids may refer to the deletion, substitution, insertion, or addition of 1 to 20 amino acids at any position within the same sequence.

[0027] The amino acids that are substituted, inserted, or added may be either native or unnatural forms. Examples of native amino acids include those mentioned above. Examples of mutually substituted amino acids are as described above. Amino acids belonging to the same group are mutually substituted.

[0028] Homologous proteins are a group of proteins found in naturally occurring organisms that originate from the same protein in terms of evolution. Homologous proteins are similar in structure and function to each other.

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

[0030] The fact that the above-mentioned mutant protein or homologous protein has fucose-containing carbohydrate transport activity can be confirmed, for example, by the following method. First, recombinant DNA containing the DNA encoding the mutant protein or homologous protein whose activity is to be confirmed is produced by the method described later. Next, a transformant with higher activity of the protein than the parent strain is produced by transforming the parent strain with the recombinant DNA, and this can be confirmed by comparing the amount of fucose-containing carbohydrate produced and accumulated in the culture medium of the parent strain or the transformant. In this specification, "parent strain" refers to the original strain that is the subject of genetic modification and transformation.

[0031] 2. DNA of the present invention The DNA of the present invention is DNA that codes for the proteins described in [1] to [3] above. Specifically, the DNA of the present invention includes the following DNAs [A1] to [A3]. [A1] The base sequence represented by Sequence ID No. 1 or 3. [A2] DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by Sequence ID No. 1 or 3, and encodes a homologous protein having fucose-containing carbohydrate transport activity. [A3] DNA comprising a base sequence having at least 95%, preferably 97%, more preferably 98%, and most preferably 99% identity with the base sequence represented by Sequence ID No. 1 or 3, and encoding a homologous protein having fucose-containing carbohydrate transport activity.

[0032] In the above, hybridization refers to the process of one DNA hybridizing to another DNA having a specific base sequence or to a portion of said DNA. Therefore, the base sequence of the DNA that hybridizes to the DNA having the specific base sequence or to a portion of said DNA may be of a length that is useful as a probe for Northern or Southern blot analysis, or can be used as an oligonucleotide primer for PCR analysis.

[0033] Examples of DNA used as a probe include DNA with at least 100 bases, preferably 200 bases, and more preferably 500 bases. Examples of DNA used as a primer include DNA with at least 10 bases, preferably 15 bases.

[0034] The methods for DNA hybridization experiments are well known, and the conditions for hybridization can be determined and the experiment performed according to numerous other standard textbooks, such as Molecular Cloning, 4th Edition (Cold Spring Harbor Laboratory Press, 2012), Methods for General and Molecular Bacteriology (ASM Press, 1994), and Immunology Methods Manual (Academic Press, 1997).

[0035] Alternatively, DNA that hybridizes under stringent conditions can be obtained by following the instructions included with commercially available hybridization kits. An example of a commercially available hybridization kit is the Random Primed DNA Labeling Kit (manufactured by Roche Diagnostics), which uses the Random Prime method to prepare probes and performs hybridization under stringent conditions.

[0036] The stringent conditions described above include, for example, incubating a DNA-immobilized filter 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 denatured salmon sperm DNA, followed by washing the filter in a 0.2×SSC solution at approximately 65°C.

[0037] The various conditions described above can also be set by adding or changing blocking reagents used to suppress the background of the hybridization experiment. Adding the blocking reagents described above may involve changing the hybridization conditions to suit the desired conditions.

[0038] Examples of DNA that can hybridize under the stringent conditions described above include DNA consisting of a base sequence that, when calculated using a program such as BLAST or FASTA based on the above parameters, has at least 95%, preferably 97%, more preferably 98%, and most preferably 99% or more identity with the base sequence represented by SEQ ID NO: 1 or 3.

[0039] The DNA of the present invention can be obtained, for example, by introducing mutations into DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4, using site-directed mutagenesis methods 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 base sequence encoding another amino acid residue. Alternatively, the DNA of the present invention can also be obtained using the PrimeSTAR Mutagenesis Basal Kit (Takara Bio Inc.), etc.

[0040] DNA encoding a protein consisting of the amino acid sequence represented by Sequence ID No. 2 can be obtained, for example, by Southern hybridization of a chromosomal DNA library of a microorganism, preferably of the genus Escherichia, more preferably Escherichia coli W3110 strain, using a probe that can be designed based on the base sequence of DNA encoding a protein consisting of the amino acid sequence represented by Sequence ID No. 2, or by PCR using chromosomal DNA of Escherichia coli W3110 strain as a template using primer DNA that can be designed based on DNA encoding a protein having the amino acid sequence represented by Sequence ID No. 2 [PCR Protocols, Academic Press (1990)]. Specifically, DNA encoding a protein consisting of the amino acid sequence represented by Sequence ID No. 2 can be defined as DNA consisting of the base sequence represented by Sequence ID No. 1.

[0041] DNA encoding the amino acid sequence represented by Sequence ID No. 4 can be obtained, for example, by Southern hybridization of a chromosomal DNA library of a microorganism, preferably of the genus Escherichia, more preferably Escherichia coli W3110 strain, using a probe that can be designed based on the base sequence of DNA encoding a protein consisting of the amino acid sequence represented by Sequence ID No. 4, or by PCR using chromosomal DNA of Escherichia coli W3110 strain as a template, using primer DNA that can be designed based on DNA encoding a protein consisting of the amino acid sequence represented by Sequence ID No. 4. Specifically, DNA encoding a protein consisting of the amino acid sequence represented by Sequence ID No. 4 can be the DNA consisting of the base sequence represented by Sequence ID No. 3.

[0042] DNA encoding a mutant protein having fucose-containing carbohydrate transport activity, which includes 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 or 4 as described in [2] above, can be obtained, for example, by subjecting DNA consisting of the base sequence represented by SEQ ID NO: 1 or 3 to error-prone PCR or the like using it as a template.

[0043] Alternatively, DNA encoding a mutant protein that has fucose-containing carbohydrate transport activity can also be obtained by a partially specific mutagenesis method using PCR (Gene, 77, 51, 1989), which uses a set of PCR primers having a nucleotide sequence designed to introduce the desired mutation (deletion, substitution, insertion, or addition) at each of its 5' ends. This mutant protein contains 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 or 4 of [2] above.

[0044] Alternatively, the DNA can be obtained by following the instructions provided with a commercially available partially specific mutagenesis kit. An example of a commercially available partially specific mutagenesis kit is the PrimeSTAR® Mutagenesis Basal Kit (manufactured by Takara Bio Inc.), which can introduce mutations (deletions, substitutions, insertions, or additions) at the desired location.

[0045] Specifically, first, a plasmid containing a nucleotide sequence designed to introduce the target mutation (deletion, substitution, insertion, or addition) is used as a template to design a pair of mutation-introducing primers with a 15-nucleotide overlap at the 5' end. The overlapping portion contains the target mutation. Next, PCR is performed using these mutation-introducing primers on a plasmid containing the nucleotide sequence into which the target mutation is to be introduced. When the resulting amplified fragment is transformed into E. coli, a plasmid containing the nucleotide sequence with the target mutation introduced is obtained.

[0046] DNA encoding a homologous protein that contains an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or 4, and that has fucose-containing carbohydrate transport activity, can be obtained, for example, by the following method. Specifically, for example, DNA encoding the homologous protein can be obtained by searching various gene sequence databases for a base sequence that has preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 99% or more identity with the base sequence represented by SEQ ID NO: 1 or 3, and then using a probe DNA or primer DNA that can be designed based on the base sequence or amino acid sequence obtained by the search, and a microorganism having said DNA, in a method similar to the method for obtaining DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 2 or 4.

[0047] The identity of the base sequence and amino acid sequence can be determined by the same method as described in 1 above. The DNA of the present invention obtained by the above method can be used as is, or cut with a suitable restriction enzyme, incorporated into a vector by a conventional method, and the resulting recombinant DNA can be introduced into host cells. The base sequence of the DNA can then be determined by analyzing it using a commonly used base sequence analysis method, such as the dideoxy method (Proc. Nat. Acad. Sci., USA, 74, 5463, 1977), or by using a base sequence analyzer such as the Applied Biosystems 3500 Genetic Analyzer or the Applied Biosystems 3730 DNA Analyzer (both manufactured by Thermo Fisher Scientific).

[0048] Examples of vectors that can be used to determine the DNA base sequence according to the present invention include pBluescriptII KS(+), pPCR-Script Amp SK(+) (both manufactured by Agilent Technologies), pT7Blue (manufactured by Merck Millipore), pCRII (manufactured by Thermo Fisher Scientific), pCR-TRAP (manufactured by Gene Hunter), and pDIRECT (Nucleic Acids Res., 18, 6069, 1990).

[0049] Any host cell capable of growing after introducing the vector can be used as the host cell, for example, Escherichia coli DH5α, Escherichia coli HST08Premium, Escherichia coli HST02, Escherichia coli HST04 dam - / dcm -Examples include Escherichia coli JM109, Escherichia coli HB101, Escherichia coli CJ236, Escherichia coli BMH71-18 mutS, Escherichia coli MV1184, Escherichia coli TH2 (all manufactured by Takara Bio), Escherichia coli XL1-Blue, Escherichia coli XL2-Blue (both manufactured by Agilent Technologies), Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli W3110, Escherichia coli MP347, and Escherichia coli NM522.

[0050] Any method for introducing recombinant DNA obtained by incorporating the DNA of the present invention into host cells can be used, including methods for introducing DNA into host cells, such as the calcium ion method (Proc. Natl. Acad. Sci., USA, 69, 2110, 1972), the protoplast method (Japanese Patent Publication No. 63-248394), and the electroporation method (Nucleic Acids Res., 16, 6127, 1988).

[0051] If the DNA obtained as a result of sequencing is only a partial length, the full-length DNA can be obtained by Southern hybridization or other methods using the partial-length DNA as a probe in a chromosomal DNA library.

[0052] Furthermore, based on the determined DNA base sequence, the target DNA can also be prepared by chemical synthesis using an NTS M-series DNA synthesizer manufactured by Nippon Techno Service Co., Ltd.

[0053] 3. Recombinant DNA of the present invention The recombinant DNA of the present invention is DNA that can autonomously replicate in a host cell, and is DNA into which the DNA of the present invention is incorporated in an expression vector that contains a promoter at a position where the DNA of the present invention described in 2 above can be transcribed.

[0054] DNA that can be incorporated into chromosomes in a host cell and that contains the DNA of the present invention is also the recombinant DNA of the present invention. If the recombinant DNA is recombinant DNA that can be incorporated into chromosomes, it does not need to contain a promoter.

[0055] When using prokaryotes such as bacteria as host cells, the recombinant DNA of the present invention is preferably recombinant DNA composed of a promoter, a ribosome binding sequence, the DNA of the present invention as described in 2 above, and a transcription termination sequence. Furthermore, it may also include a gene that controls the promoter.

[0056] Here, 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. Furthermore, in the recombinant DNA of the present invention, a transcription termination sequence is not necessarily required for the expression of the DNA of the present invention, but it is preferable to place the transcription termination sequence directly below the structural gene.

[0057] When using microorganisms belonging to the genus Escherichia as host cells to which the recombinant DNA of the present invention is introduced, the expression vectors include, for example, pColdI, pSTV28, pUC118 (all manufactured by Takara Bio Inc.), pET21a, pCDF-1b, pRSF-1b (all manufactured by Merck Millipore Corporation), pMAL-c5x (manufactured by New England Biolabs Corporation), pGEX-4T-1, pTrc99A (all manufactured by GE Healthcare Biosciences Corporation), pTrcHis, pSE280 (all manufactured by Thermo Fisher Scientific Corporation), pGEMEX-1 (manufactured by Promega Corporation), pQE-30, pQE-60, pQE80L (all manufactured by Qiagen Corporation), pET-3, pBluescriptII SK(+), and pBluescriptII KS(-) (both manufactured by Agilent Technologies), pKYP10 (Japanese Patent Publication No. 58-110600), pKYP200 (Agric. Biol. Chem., 48, 669, 1984), pLSA1 (Agric. Biol. Chem., 53, 277, 1989), pGEL1 (Proc. Natl. Acad. Sci., USA, 82, 4306 (1985), pTrS30 [prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrS32 [prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)], pTK31 [APPLIED AND ENVIRONMENTAL Examples include MICROBIOLOGY, 2007, Vol.73, No.20, pp. 6378-6385, pPE167 (Appl. Environ. Microbiol. 2007, 73:6378-6385), pPAC31 (International Publication No. 1998 / 12343), pUC19 (Gene, 33, 103, 1985), and pPA1 (Japanese Patent Publication No. 63-233798).

[0058] When using the expression vector described above, any promoter that functions in the cells of microorganisms belonging to the genus Escherichia may be used. Examples include promoters derived from Escherichia coli or phages, such as the trp promoter, gapA promoter, lac promoter, PL promoter, PR promoter, and PSE promoter. Additionally, artificially designed and modified promoters such as two trp promoters in series, the tac promoter, the trc promoter, the lacT5 promoter, the lacT7 promoter, and the letI promoter are also acceptable.

[0059] When using Corynebacteria as host cells to which the recombinant DNA of the present invention is introduced, examples of expression vectors include pCG1 (Japanese Patent Publication No. 57-134500), pCG2 (Japanese Patent Publication No. 58-35197), pCG4 (Japanese Patent Publication No. 57-183799), pCG11 (Japanese Patent Publication No. 57-134500), pCG116, pCE54, pCB101 (both Japanese Patent Publication No. 58-105999), pCE51, pCE52, pCE53 (all Molecular and General Genetics, 196, 175, 1984), etc.

[0060] When using the expression vector described above, any promoter that functions in the cells of Corynebacterium can be used, but an example is the P54-6 promoter (Appl. Microbiol. Biotechnol., 53, p674-679, 2000).

[0061] When using a yeast strain as the host cell into which the recombinant DNA of the present invention is introduced, examples of expression vectors include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.

[0062] When using the aforementioned expression vector, any promoter that functions in the cells of the yeast strain may be used, but examples of promoters include the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock polypeptide promoter, MFα1 promoter, and CUP1 promoter.

[0063] The recombinant DNA of the present invention can be prepared, for example, by restricting the DNA fragment prepared by the method described in step 2 above and inserting it downstream of the promoter of a suitable expression vector.

[0064] Here, by substituting bases in the base sequence constituting the DNA of the present invention to create codons optimal for expression in host cells, the expression level of the protein encoded by the DNA can be improved. Information on codon usage frequency in host cells can be obtained through public databases.

[0065] 4. Transformed bodies of the present invention The transformant of the present invention is a transformant obtained by transforming a host cell with recombinant DNA containing the DNA of the present invention as described in item 2 above. In this specification, "host cell" refers to the original cell that is the target of transformation by gene transfer.

[0066] Specifically, examples of transformants of the present invention include microorganisms in which the activity of any one of the proteins described in [1] to [3] above and the productivity of fucose-containing carbohydrates are enhanced. Examples of microorganisms in which the activity of the protein described in any one of the proteins described in [1] to [3] above is enhanced include microorganisms obtained by transforming a parent strain with recombinant DNA having the DNA described in any one of the proteins described in [A1] to [A3] above, and which have enhanced productivity of fucose-containing carbohydrates compared to the parent strain.

[0067] Examples of microorganisms obtained by transforming a parent strain with the recombinant DNA, which have enhanced fucose-containing carbohydrate productivity compared to the parent strain, include the following microorganisms i) to iii). i) A microorganism in which recombinant DNA having any one of the DNAs described in [A1] to [A3] above is introduced into the parent strain as an autonomously replicating plasmid, or is incorporated into the chromosome of the parent strain, thereby increasing the transcription rate of the DNA or the production rate of the protein encoded by the DNA. ii) A microorganism in which the productivity of fucose-containing carbohydrates is enhanced by producing a protein having enhanced specific activity of a protein having fucose-containing carbohydrate transport activity as the mutant protein described in [2] above. iii) Microorganisms in which the productivity of fucose-containing carbohydrates is enhanced by producing a protein having enhanced specific activity of a protein having fucose-containing carbohydrate transport activity as a homologous protein of the above [3].

[0068] A method for confirming an increase in the transcription rate of the DNA described in any one of [A1] to [A3] above, or the production rate of the protein encoded by said DNA, is to measure the transcription rate of said DNA by Northern blotting or the production rate of said protein by Western blotting and compare it with that of the parent strain.

[0069] One way to confirm that the specific activity of a protein having fucose-containing carbohydrate transport activity has been enhanced is, for example, to transform a parent strain with DNA encoding a mutant protein, culture the transformed strain in a culture medium, measure the specific activity from the amount of fucose-containing carbohydrates produced and the amount of the protein accumulated in the culture, and compare it with the specific activity of a protein having fucose-containing carbohydrate transport activity and in which no mutation has been introduced, which has been measured in the same manner.

[0070] Examples of such transformants of the present invention include the FucT / pSTV_YdeA strain and the FucT / pMW118_MdfA strain, which will be described later in the examples.

[0071] The host cells into which the recombinant DNA of the present invention is introduced may be any prokaryotes, yeasts, animal cells, insect cells, plant cells, etc., but preferably prokaryotes or yeast strains, more preferably prokaryotes belonging to the genera Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, or Pseudomonas, etc., or yeast strains belonging to the genera Saccharomyces, Schizosaccharomyces, Kluiveromyces, Trichosporon, Siwaniomyces, Pitia, or Candida, etc., and most preferably Escherichia coli [for example, 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 HST08Premium, 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 MG1655, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli MP347, Escherichia coli NM522, Escherichia coli ATCC9637, Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium immariophilum [e.g., Brevibacterium immariophilum ATCC14068], Brevibacterium saccharolyticum [e.g., Brevibacterium saccharolyticum ATCC14066], Corynebacterium ammoniagenes, Corynebacterium glutamicum [Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869], Corynebacterium acetoacidophilum [Corynebacterium acetoacidophilum ATCC13870], Microbacterium ammoniaphilum [Microbacterium ammoniaphilumExamples include prokaryotes such as ATCC15354 or Pseudomonas (e.g., Pseudomonas sp.D-0110), or yeast strains such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris, or Candida utilis.

[0072] Suitable host cells include microorganisms capable of producing fucose-containing carbohydrates, and microorganisms with higher activity of proteins involved in the biosynthesis of fucose-containing carbohydrates than the parent strain are preferred. Proteins involved in the biosynthesis of fucose-containing carbohydrates include GDP-fucose, a reaction substrate for fucosetransferase, a receptor carbohydrate, and fucosetransferase, both reaction substrates for fucosetransferase.

[0073] Examples of host cells include the following 1) to 3): 1) Microorganisms used as host cells in which the ability to produce GDP-fucose, a reaction substrate for fucosetransferase, has been artificially conferred or enhanced. 2) Microorganisms used as host cells that have been artificially given or enhanced the ability to supply receptor carbohydrates, which are reaction substrates for fucose transferases. 3) Microorganisms used as host cells in which fucose transferase activity has been artificially conferred or enhanced. The following describes each host cell type.

[0074] 1) Microorganisms used as host cells in which the ability to produce GDP-fucose, a reaction substrate for fucosetransferase, has been artificially conferred or enhanced. Methods for conferring or enhancing the ability to produce GDP-fucose in microorganisms used as host cells include, for example, known methods such as various genetic manipulation techniques (Metabolic Engineering (2017) 41:23-38).

[0075] The ability to produce GDP-fucose includes the ability to produce GDP-fucose from sugars. Methods for artificially conferring or enhancing the ability to produce GDP-fucose from sugars to microorganisms used as host cells include, for example, the following methods (1a) to (1d). These methods may be used individually or in combination. (1a) A method of relaxing or releasing at least one mechanism that controls the biosynthetic pathway that produces GDP-fucose from sugars. (1b) A method for enhancing the expression of at least one enzyme involved in the biosynthetic pathway that produces GDP-fucose from sugar. (1c) A method to increase the copy number of at least one gene encoding an enzyme involved in the biosynthetic pathway that produces GDP-fucose from sugar. (1d) A method of weakening or blocking at least one metabolic pathway that branches off from a biosynthetic pathway that produces GDP-fucose from sugar to metabolites other than the target substance.

[0076] Specific examples of mechanisms that control the biosynthetic pathway that produces GDP-fucose from sugars include known mechanisms such as regulatory mechanisms by transcription factors (e.g., RcsA) involved in the control of the biosynthetic pathway.

[0077] Specific examples of enzymes involved in the biosynthetic pathway that produces GDP-fucose from sugars include well-known enzymes such as mannose-6-phosphate isomerase, phosphomannomutase, mannose-1-phosphate guanylyltransferase, GDP-mannose-4,6-dehydratase, and GDP-L-fucose synthase.

[0078] Specific examples of metabolic pathways that branch off from the biosynthetic pathway that produces GDP-fucose from sugar to metabolites other than the target substance include, for example, the metabolic pathway from GDP-fucose to cholanic acid, which are well known metabolic pathways.

[0079] 2) Microorganisms used as host cells that have been artificially given or enhanced the ability to supply receptor carbohydrates, which are reaction substrates for fucose transferases. Methods for artificially conferring or enhancing the ability to supply receptor carbohydrates to microorganisms used as host cells include, for example, (2a) to (2g) below. These methods may be used individually or in combination. (2a) A method of relaxing or releasing at least one mechanism that controls the biosynthetic pathway that produces receptor carbohydrates from sugars. (2b) A method for enhancing the expression of at least one enzyme involved in the biosynthetic pathway that generates receptor carbohydrates from sugars. (2c) A method to increase the copy number of at least one gene encoding an enzyme involved in the biosynthetic pathway that produces receptor carbohydrates from sugars. (2d) A method for mitigating or disabling at least one mechanism for the breakdown of receptor carbohydrates. (2e) A method to enhance the expression of at least one enzyme involved in the intracellular uptake of receptor carbohydrates. (2f) A method to increase the copy number of at least one gene encoding an enzyme involved in the intracellular uptake of receptor carbohydrates. (2g) A method for weakening or blocking at least one metabolic pathway that branches from a receptor carbohydrate to metabolites other than the target substance.

[0080] Examples of receptor carbohydrates include N-acetylglucosamine, N-acetyllactosamine, galactose, fucose, sialic acid, glucose, lactose, or combinations thereof, as well as sugar chains containing these as partial structures. Among these, lactose is preferred.

[0081] Specific examples of enzymes involved in the biosynthetic pathway that produces receptor carbohydrates from sugars include, for example, known enzymes such as lactose synthase enzymes that produce lactose using glucose and UDP-galactose as substrates. Specific examples of mechanisms that degrade receptor carbohydrates include, for example, known enzymes such as β-galactosidase that catalyzes the hydrolysis of lactose to produce glucose and galactose.

[0082] Specific examples of enzymes involved in the intracellular uptake of receptor carbohydrates include, for example, known enzymes such as lactose permyase, which is involved in the intracellular uptake of lactose. Specific examples of methods for conferring or enhancing the ability to supply the above-mentioned receptor carbohydrates include known methods such as reducing or inactivating the activity of β-galactosidase through genetic manipulation (Metabolic Engineering, 2017, 41:23-38).

[0083] 3) Microorganisms used as host cells in which fucose transferase activity has been artificially conferred or enhanced. Examples of methods for artificially conferring or enhancing fucose transferase activity in microorganisms used as host cells include (3a) and (3b) below. These methods may be used individually or in combination. (3a) A method for enhancing the expression of at least one fucose transferase. (3b) A method to increase the copy number of at least one gene encoding a fucose transferase.

[0084] Examples of fucose transferases include α1,2-fucosyltransferase, α1,3-fucosyltransferase, α1,4-fucosyltransferase, and α1,6-fucosyltransferase, with α1,3-fucosyltransferase being preferred among these.

[0085] Examples of α1,3-fucosyltransferases include the following proteins I) to III). I) Protein consisting of the amino acid sequence represented by Sequence ID No. 6 II) Mutant proteins having α1,3-fucosyltransferase activity, consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by Sequence ID No. 6. III) Homologous protein having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by Sequence ID No. 6, and possessing α1,3-fucosyltransferase activity.

[0086] Methods for introducing the recombinant DNA described in 3 above as a plasmid capable of autonomous replication in host cells include, for example, the calcium ion method described above, the protoplast method, the electroporation method, and methods such as the spheroplast method (Proc. Natl. Acad. Sci., USA, 81, 4889, 1984) and the lithium acetate method (J. Bacteriol., 153, 163, 1983).

[0087] One method for incorporating recombinant DNA into the chromosomes of a host cell is homologous recombination. Homologous recombination methods include, for example, the use of homologous recombination plasmids, which can be produced by linking plasmid DNA containing a drug resistance gene that cannot autonomously replicate within the host cell to be introduced. A frequently used homologous recombination method in Escherichia coli is the introduction of recombinant DNA using a lambda phage homologous recombination system (Proc. Natl. Acad. Sci. USA, 97, 6641-6645, 2000).

[0088] Furthermore, by using selection methods that utilize the fact that E. coli becomes sucrose-sensitive due to Bacillus subtilis levanseucrase incorporated into the chromosome along with recombinant DNA, or by using selection methods that utilize the fact that E. coli becomes streptomycin-sensitive by incorporating the wild-type rpsL gene into E. coli having a streptomycin-resistant mutant rpsL gene [Mol.Microbiol.,55,137(2005), Biosci.Biotechnol.Biochem.,71,2905(2007)], E. coli in which the target region on the chromosomal DNA of the host cell has been replaced with recombinant DNA can be obtained.

[0089] The fact that the transformant obtained by the above method has the DNA of the present invention as described in 2 above can be confirmed, for example, by culturing the transformant in a culture medium and comparing the amount of fucose-containing carbohydrates accumulated in the culture with that of the parent strain, in the case of a transformant obtained by transforming a host cell that has the ability to produce GDP-fucose or receptor carbohydrates from sugars.

[0090] 5. Method for producing fucose-containing carbohydrates according to the present invention A method for producing fucose-containing carbohydrates according to the present invention includes a fermentation method for producing fucose-containing carbohydrates, which involves culturing the transformant described in item 4 above in a culture medium to generate fucose-containing carbohydrates in the culture.

[0091] In the method for producing fucose-containing carbohydrates by fermentation, the transformant of the present invention is preferably a transformant that has the ability to produce GDP-fucose from sugar.

[0092] Furthermore, as the transformant of the present invention used in the method for producing fucose-containing carbohydrates by fermentation, a transformant having the ability to produce receptor carbohydrates may be used.

[0093] The method for culturing the transformants described in item 4 above can be carried out according to the usual methods used for culturing microorganisms.

[0094] As a culture medium for the transformant, either a natural medium or a synthetic medium may be used, as long as it contains a carbon source, nitrogen source, inorganic salts, etc. that the transformant can utilize, and is capable of efficiently culturing the transformant.

[0095] The carbon source can be anything that the transformant can utilize, and examples include glucose, fructose, sucrose, molasses containing these, sugars such as starch or starch hydrolysates, organic acids such as acetic acid or propionic acid, or alcohols such as glycerol, ethanol or propanol.

[0096] Examples of nitrogen sources include ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, or ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal, soybean meal hydrolysate, various fermentation microorganisms and their digests.

[0097] Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate. Receptor carbohydrates such as lactose may be added to the culture medium as precursors of fucose-containing carbohydrates, and GTP or mannose may be added as precursors of GDP-fucose.

[0098] In the method for producing fucose-containing carbohydrates by fermentation, if the transformant used does not have the ability to produce GDP-fucose, GDP-fucose may be added to the culture medium during cultivation. Alternatively, in the method for producing fucose-containing carbohydrates by fermentation, if the transformant used does not have the ability to produce GDP-fucose, instead of adding GDP-fucose to the culture medium during cultivation, GDP-fucose may be supplied to the transformant by culturing a microorganism capable of producing GDP-fucose from sugar simultaneously with the transformant of the present invention.

[0099] Furthermore, microorganisms capable of producing GTP may be cultured simultaneously to supply or enhance GTP, which is a precursor of GDP-fucose. Examples of microorganisms capable of producing GTP include known microorganisms in which the expression of enzymes involved in the GTP biosynthesis pathway has been enhanced through various genetic manipulations (Biotechnol Bioeng, Sep3:2019,2412-2417).

[0100] In a method for producing fucose-containing carbohydrates by fermentation, if the transformant used does not have the ability to produce receptor carbohydrates such as lactose, receptor carbohydrates such as lactose are added to the culture medium during cultivation.

[0101] Furthermore, in the method for producing fucose-containing carbohydrates by fermentation, if the transformant used does not have the ability to produce receptor carbohydrates such as lactose, instead of adding receptor carbohydrates such as lactose to the culture medium during cultivation, a microorganism capable of producing receptor carbohydrates such as lactose from sugar may be cultured simultaneously with the transformant of the present invention to supply the transformant of the present invention with receptor carbohydrates such as lactose.

[0102] Culturing is usually 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 medium during cultivation is usually maintained at 3.0 to 9.0. pH adjustment is performed using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

[0103] [Example of analysis] In the examples, the analysis and quantification of 3-fucosyl lactose were performed using the following procedure. The culture medium containing the microorganisms after cultivation was centrifuged, and the supernatant was collected. The 3-fucosyl lactose contained in the supernatant was analyzed using a sugar analyzer ICS-5000 (Thermo Fisher Scientific). [Analysis conditions] Column: CarboPAC PA1 Column temperature: 25℃ 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)80:20:0 (10-15 minutes) Gradient from 80:20:0 to 70:20:10 (15-17 minutes) Gradient from 70:20:10 to 0:20:80 (17~25 minutes)0:20:80 (25~35 minutes)80:20:0 Flow rate: 1.0mL / min Detector: Pulsed amperometry detector [Examples]

[0104] The following are examples of the invention, but the present invention is not limited to these examples.

[0105] [Example 1] Creation of microorganisms used in the production of 3-fucosyl lactose (1) Obtaining DNA fragments to be used as markers in the case of gene defects PCR was performed using DNA consisting of the base sequences shown in "Primer Set" in Table 1 as the primer set, and the DNA described in "Template" in Table 1 as the template, to obtain each amplified DNA fragment.

[0106] [Table 1]

[0107] The genomic DNA of 168 strains of Bacillus subtilis was prepared using standard methods. The amplified DNA fragment cat contains approximately 200 bp upstream to 50 bp downstream of the cat gene on pHSG396. The amplified DNA fragment sacB contains approximately 300 bp upstream to 100 bp downstream of the sacB gene on the genomic DNA of 168 strains of Bacillus subtilis.

[0108] Next, PCR was performed using the amplified DNA fragments cat and sacB as templates, and DNA consisting of the base sequences represented by SEQ ID NOs. 8 and 11 as primers to obtain a DNA fragment containing the cat gene and the sacB gene (hereinafter referred to as cat-sacB).

[0109] (2) Creation of Escherichia coli that has lost β-galactosidase activity, lactose permyase activity, and cholanic acid production activity. Escherichia coli lacking the DNA encoding β-galactosidase (hereinafter referred to as the lacZ gene), the DNA encoding lactose permyase (hereinafter referred to as the lacY gene), and the DNA encoding cholanic acid production-related proteins (hereinafter referred to as the wcaJ, wzxC, wcaK, wcaL, or wcaM genes) was created using the following method. Note that lacZ and lacY (hereinafter referred to as lacZY), as well as wcaJ, wzxC, wcaK, wcaL, and wcaM (hereinafter referred to as wcaJ-wzxC-wcaKLM), each form an operon on the Escherichia coli genome.

[0110] Using the genomic DNA of Escherichia coli strain W3110, prepared by conventional methods, PCR was performed using primer sets consisting of DNA sequences represented in the "primer sets" in Table 2, and each DNA fragment was amplified.

[0111] [Table 2]

[0112] lacZ upstream 1 and lacZ upstream 2 include the region of the lacZ gene from the start codon to approximately 1000 bp upstream of the start codon. lacY downstream 1 and lacY downstream 2 include the region of the lacY gene from approximately 50 bp downstream of the stop codon to approximately 1000 bp.

[0113] Using a template made by mixing lacZ upstream 1, lacY downstream 1, and cat-sacB fragments in equimolar proportions, PCR was performed using a primer set consisting of DNA with the nucleotide sequences represented by SEQ ID NOs: 13 and 15 to obtain a DNA fragment (hereinafter referred to as lacZY::cat-sacB) in which the cat-sacB fragment was inserted into the sequences surrounding the lacZ and lacY genes.

[0114] Using a template made by mixing lacZ upstream 2 and lacY downstream 2 in equimolar ratios, PCR was performed using a primer set consisting of DNA with the nucleotide sequences represented by SEQ ID NOs. 13 and 15 to obtain a DNA fragment (hereinafter referred to as ΔlacZY) that does not contain lacZ or lacY, and consists of a sequence in which lacZ upstream and lacY downstream are directly linked.

[0115] The lacZY::cat-sacB fragment was introduced by electroporation into the W3110 strain, which carries plasmid pKD46 [Datsenko, KA, Warner, BL, Proc. Natl. Acad. Sci., USA, Vol. 97, 6640-6645 (2000)] containing the gene encoding λ recombinase, to obtain transformants that were chloramphenicol resistant and sucrose sensitive (transformers in which the lacZ and lacY genes were replaced with lacZY::cat-sacB).

[0116] The ΔlacZY fragment was introduced into the transformant by electroporation to obtain a transformant that was chloramphenicol-sensitive and sucrose-resistant (a transformant in which lacZY::cat-sacB was replaced with ΔlacZY). From these, a transformant that was ampicillin-sensitive (a transformant in which pKD46 was removed) was obtained. This transformant was named W3110ΔlacZY.

[0117] Similarly, PCR was performed using the genomic DNA of strain W3110 as a template, and the DNA consisting of the base sequences shown in "Primer Set" in Table 3 was used as the primer set to obtain each amplified DNA fragment.

[0118] [Table 3]

[0119] wcaJ upstream 1 and wcaJ upstream 2 include the region of the wcaJ gene from the start codon to approximately 1000 bp upstream of the start codon. wcaM downstream 1 and wcaM downstream 2 include the region of the wcaM gene from the stop codon to approximately 1000 bp downstream of the stop codon.

[0120] Using a template made by mixing wcaJ upstream 1, wcaM downstream 1, and cat-sacB fragments in equimolar proportions, PCR was performed using a primer set consisting of DNA with the nucleotide sequences represented by SEQ ID NOs: 19 and 21 to obtain a DNA fragment (hereinafter referred to as wcaJ-wzxC-wcaKLM::cat-sacB) in which the cat-sacB fragment was inserted into the sequence surrounding the wcaJ-wzxC-wcaKLM operon.

[0121] Using a template made by mixing wcaJ upstream 2 and wcaM downstream 2 in equimolar ratios, PCR was performed using a primer set consisting of DNA with the nucleotide sequences represented by SEQ ID NOs. 21, and a DNA fragment (hereinafter referred to as ΔwcaJ-wzxC-wcaKLM) was obtained that did not contain wcaJ-wzxC-wcaKLM and consisted of a sequence in which wcaJ upstream and wcaM downstream were directly linked.

[0122] The wcaJ-wzxC-wcaKLM::cat-sacB fragment was introduced into the W3110ΔlacZY strain created above by electroporation, yielding a transformant that was chloramphenicol resistant and sucrose sensitive (a transformant in which wcaJ-wzxC-wcaKLM was replaced with wcaJ-wzxC-wcaKLM::cat-sacB).

[0123] The ΔwcaJM fragment was introduced into the transformant by electroporation to obtain a transformant that was sensitive to chloramphenicol and resistant to sucrose (a transformant in which wcaJ-wzxC-wcaKLM::cat-sacB was replaced with ΔwcaJ-wzxC-wcaKLM). Furthermore, a transformant that was sensitive to ampicillin (a transformant in which pKD46 was removed) was obtained. This transformant was named T166.

[0124] (3) Creation of microorganisms possessing α1,3-fucosyltransferase activity PCR was performed using DNA consisting of the base sequences shown in "Primer Set" in Table 4 as the primer set, and the DNA described in "Template" in Table 4 as the template, to obtain each amplified DNA fragment.

[0125] [Table 4]

[0126] The genomic DNA of Escherichia coli strain W3110 and Bacteroides reticulotermitis strain JCM10512 was prepared by conventional methods. The DNA consisting of the nucleotide sequence represented by Sequence ID No. 7 is a codon-optimized DNA for expression in E. coli, which is a partial sequence of the gene encoding α1,3-fucosyltransferase from Bacteroides fragilis strain ATCC25285. In addition, the nucleotide sequences represented by Sequence ID Nos. 25 and 26, Sequence ID Nos. 28 and 29, Sequence ID Nos. 30 and 31, and Sequence ID Nos. 27 and 32 each contain complementary sequences at their respective 5' ends.

[0127] First, a mixture of three fragments—upstream, midstream, and downstream of cBrFucT—in equimolar proportions was used as a template. PCR was performed using a primer set consisting of DNA with the base sequences represented by SEQ ID NOs. 26 and 27 to obtain a DNA fragment (hereinafter referred to as the cBrFucT fragment) formed by ligating the three fragments.

[0128] Using a template made by mixing rcsA, cBrFucT, and lacY fragments in equimolar proportions, PCR was performed using a primer set consisting of DNA with the nucleotide sequences represented by SEQ ID NOs. 34 and 35 to obtain a DNA fragment (hereinafter referred to as rcsA-cBrFucT-lacY) formed by ligating the three fragments.

[0129] Using DNA consisting of the nucleotide sequences represented by SEQ ID NOs. 36 and 37 as a primer set, PCR was performed on plasmid pPE167 (Appl. Environ. Microbiol. 2007, 73:6378-6385) as a template to obtain a vector fragment of approximately 4.4 kb. At this time, the nucleotide sequences represented by SEQ ID NOs. 34 and 37, and SEQ ID NOs. 35 and 36, each contain a complementary sequence at their respective 5' ends.

[0130] The rcsA-cBrFucT-lacY fragment and vector fragment obtained above were ligated using the In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain an α1,3-fucosyltransferase expression plasmid.

[0131] By transforming the T166 strain created in (2) above using the α1,3-fucosyltransferase expression plasmid described above, a microorganism expressing α1,3-fucosyltransferase was created and named the FucT strain.

[0132] (4) Creation of microorganisms with enhanced expression of transporter genes derived from Escherichia coli Escherichia coli-derived transporter gene (ydeA) belonging to the drug:H+ antiporter-1 family was placed under the uspA promoter, and Escherichia coli-containing plasmids for gene expression were created using the following method.

[0133] Using the expression vector pSTV29 (manufactured by Takara Bio Inc.) as a template, PCR was performed using oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs. 38 and 39 as a primer set to obtain the pSTV29 vector fragment.

[0134] Using chromosomal DNA from Escherichia coli strain ATCC9637 as a template, PCR was performed using oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs. 40 and 41 as primer sets to amplify DNA fragments containing the uspA promoter. At this time, the nucleotide sequences represented by SEQ ID NOs. 38 and 41, and SEQ ID NOs. 39 and 40, each contain a complementary sequence at their respective 5' ends.

[0135] The obtained DNA fragment containing the uspA promoter was ligated to the pSTV29 vector fragment using the In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the expression vector pSTV.

[0136] Using chromosomal DNA from Escherichia coli strain W3110 as a template, PCR was performed using oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs. 43 and 44 as primer sets to amplify the DNA fragment encoding the YdeA protein (a protein having the amino acid sequence represented by SEQ ID NOs. 2). At this time, the nucleotide sequences represented by SEQ ID NOs. 41 and 43, and SEQ ID NOs. 42 and 44, each contain a complementary sequence at their respective 5' ends.

[0137] The DNA fragment encoding YdeA obtained above was ligated to a pSTV vector fragment using the In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the expression vector pSTV_YdeA.

[0138] Next, Escherichia coli-derived E. coli-derived transporter gene (mdfA) belonging to the drug:H+ antiporter-1 family was placed under the lac promoter, and E. coli strains containing a plasmid for gene expression were created using the following method.

[0139] Using the expression vector pMW118 (manufactured by Nippon Gene Co., Ltd.) as a template, PCR was performed using oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs. 45 and 46 as a primer set to obtain the pMW118 vector fragment.

[0140] Using chromosomal DNA from Escherichia coli strain W3110 as a template, PCR was performed using oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs. 47 and 48 as primer sets to amplify the DNA fragment encoding the MdfA protein (a protein having the amino acid sequence represented by SEQ ID NOs. 4). At this time, the nucleotide sequences represented by SEQ ID NOs. 45 and 48, and SEQ ID NOs. 46 and 47, each contain a complementary sequence at their respective 5' ends.

[0141] The obtained DNA fragment encoding MdfA was ligated to the pMW118 vector fragment using the In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the expression vector pMW118_MdfA.

[0142] Using the expression vectors pSTV and pMW118, and the expression plasmids pSTV_YdeA and pMW118_MdfA obtained above, the FucT strain created in Example 1(3) was transformed to obtain the FucT / pSTV strain, FucT / pMW118 strain, FucT / pSTV_YdeA strain, and FucT / pMW118_MdfA strain.

[0143] [Example 2] Production of 3-fucosyl lactose by fermentation using microorganisms that strongly express transporters The FucT / pSTV strain, FucT / pMW118, FucT / pSTV_YdeA strain, and FucT / pMW118_MdfA strain obtained in Example 1 were cultured on LB plates at 30°C for 24 hours. These cells were then inoculated into large test tubes containing 4 mL of LB medium with 100 mg / L kanamycin and 25 mg / L chloramphenicol (pSTV29 plasmid-expressing strain) or 100 mg / L ampicillin (pMW118 plasmid-expressing strain), and cultured with shaking at 30°C for 18 hours.

[0144] Subsequently, the resulting culture medium is prepared by adding 100 mg / L kanamycin and 25 mg / L chloramphenicol (pSTV29 plasmid expression strain) or 100 mg / L ampicillin (pMW118 plasmid expression strain) to a production medium containing [glucose 30 g / L, lactose monohydrate 10 g / L, magnesium sulfate heptahydrate 2 g / L, dipotassium hydrogen phosphate 16 g / L, potassium dihydrogen phosphate 14 g / L, ammonium sulfate 2 g / L, citric acid 1 g / L, casamino acid 5 g / L, thiamine]. 0.2 mL of a large test tube containing 4 mL of a solution of 10 mg / L hydrochloride, 50 mg / L ferrous sulfate heptahydrate, and 10 mg / L manganese sulfate pentahydrate (except for glucose, lactose monohydrate, and magnesium sulfate heptahydrate, the solutions were adjusted to pH 7.2 with sodium hydroxide aqueous solution and then autoclaved) (the aqueous solutions containing glucose, lactose monohydrate, and magnesium sulfate heptahydrate were prepared separately, autoclaved, cooled, and then mixed) was inoculated and cultured with shaking at 30°C for 28 hours. For FucT / pMW118 strain and FucT / pMW118_MdfA strain, IPTG was added to a concentration of 1 mM 5 hours after the start of culture.

[0145] After the culture was completed, the culture medium was diluted as needed and centrifuged, and the 3-fucosyl lactose contained in the supernatant was analyzed using a glucose analyzer ICS-5000. The results are shown in Table 5.

[0146] [Table 5]

[0147] As shown in Table 5, the FucT / pSTV_YdeA and FucT / pMW118_MdfA strains showed significantly higher 3-fucosyl lactose productivity compared to the FucT / pSTV and FucT / pMW118 strains.

[0148] Therefore, it was found that overexpression of YdeA or MdfA, which belong to the drug:H+ antiporter-1 family, improves the productivity of 3-fucosyl lactose.

[0149] Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications are possible without departing from the spirit and scope of the invention. This application is based on Japanese Patent Application No. 2021-018484, filed on 8 February 2021, which is incorporated by reference in its entirety. All references incorporated herein are incorporated as a whole. [Sequence Listing Free Text]

[0150] Sequence ID 1: Base sequence of ydeA derived from E. coli W3110 Sequence ID 2: Amino acid sequence of YdeA derived from E. coli W3110 Sequence ID 3: Base sequence of mdfA derived from E. coli W3110 Sequence ID 4: Amino acid sequence of MdfA derived from E. coli W3110 Sequence ID 5: Base sequence of chimeric FucT Sequence ID 6: Amino acid sequence of chimeric FucT Sequence ID 7: Subsequence of codon-optimized BfFucT Sequence IDs 8 and 9: Base sequences of CAT amplification primers Sequence IDs 10 and 11: Base sequences of primers for sacB amplification. Sequence IDs 12 and 13: Base sequences of primers for upstream 1 amplification of lacZ Sequence IDs 14 and 15: Base sequences of primers for lacY downstream 1 amplification. Sequence ID 16: Base sequence of primer for upstream 2 amplification of lacZ Sequence ID 17: Base sequence of primer for lacY downstream 2 amplification Sequence IDs 18 and 19: Base sequences of primers for upstream 1 amplification of wcaJ Sequence IDs 20 and 21: Base sequences of primers for wcaM downstream 1 amplification. Sequence ID 22: Base sequence of primer for upstream 2 amplification of wcaJ Sequence ID 23: Base sequence of primers for wcaM downstream 2 amplification Sequence IDs 24 and 25: Base sequences of primers for rcsA amplification. Sequence IDs 26 and 28: Base sequences of primers for upstream amplification of cBrFucT. Sequence IDs 27 and 31: Base sequences of primers for downstream amplification of cBrFucT Sequence IDs 29 and 30: Base sequences of primers for midstream amplification of cBrFucT Sequence IDs 32 and 33: Base sequences of primers for lacY amplification. Sequence IDs 34 and 35: Base sequences of primers for rcsA-cBrFucT-lacY amplification. Sequence IDs 36, 37: Base sequences of primers for pPE167 amplification Sequence IDs 38 and 39: Base sequences of primers for pSTV29 amplification. Sequence IDs 40 and 41: Base sequences of primers for PuspA amplification. Sequence ID 42: Base sequence of primer for pSTV-PuspA amplification Sequence IDs 43 and 44: Base sequences of primers for ydeA amplification. Sequence IDs 45 and 46: Base sequences of primers for pMW118 amplification. Sequence IDs 47 and 48: Base sequences of mdfA amplification primers

Claims

1. A method for producing 3-fucosyl lactose, This includes culturing the transformant in a culture medium to produce the 3-fucosyl lactose in the culture obtained, The transformed organism is a transformed E. coli organism having the ability to produce 3-fucosyl lactose. By expressing the gene encoding α1,3-fucosyltransferase, the ability to produce the aforementioned 3-fucosyl lactose is conferred. The aforementioned E. coli transformant is modified to express a protein consisting of the amino acid sequence represented by Sequence ID No. 2, A method for producing 3-fucosyl lactose, wherein the modification is achieved by introducing recombinant DNA encoding the protein into the parent strain as an autonomously replicating plasmid, or by incorporating it into the chromosome of the parent strain, thereby increasing the amount of DNA transcribed or the production of the protein encoded by the DNA.

2. A transformed E. coli, It has the ability to produce 3-fucosyl lactose, By expressing the gene encoding α1,3-fucosyltransferase, the ability to produce the aforementioned 3-fucosyl lactose is conferred. The aforementioned E. coli transformant is modified to express a protein consisting of the amino acid sequence represented by Sequence ID No. 2, A transformed E. coli in which the modification is achieved by introducing recombinant DNA encoding the protein as an autonomously replicating plasmid in the parental strain, or by incorporating it into the chromosome of the parental strain, thereby increasing the amount of DNA transcribed or the production of the protein encoded by the DNA.

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