WO2024253084A1 - PROTÉINE PRÉSENTANT UNE ACTIVITÉ DE α1,2-FUCOSYLTRANSFERASE ET PROCÉDÉ DE PRODUCTION DE LACTO-N-FUCOPENTAOSE I (LNFPI) - Google Patents

PROTÉINE PRÉSENTANT UNE ACTIVITÉ DE α1,2-FUCOSYLTRANSFERASE ET PROCÉDÉ DE PRODUCTION DE LACTO-N-FUCOPENTAOSE I (LNFPI) Download PDF

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WO2024253084A1
WO2024253084A1 PCT/JP2024/020349 JP2024020349W WO2024253084A1 WO 2024253084 A1 WO2024253084 A1 WO 2024253084A1 JP 2024020349 W JP2024020349 W JP 2024020349W WO 2024253084 A1 WO2024253084 A1 WO 2024253084A1
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position corresponding
amino acid
seq
acid sequence
valine
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駿 遠藤
和貴 中村
智惇 杉田
彩花 釜井
史 山▲崎▼
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Kirin Holdings Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals

Definitions

  • the present invention relates to a protein having ⁇ 1,2-fucosyltransferase activity and a method for producing lacto-N-fucopentaose I (LNFPI).
  • Non-Patent Document 1 The oligosaccharides contained in human breast milk (Human Milk Oligosaccharides, HMOs) have been attracting attention as prebiotic materials, and have been shown to be effective in improving cognitive function development, infection prevention, and intestinal environment in infants (Non-Patent Document 1).
  • Lacto-N-fucopentaose I (hereafter referred to as LNFPI) is a type of HMO, a pentasaccharide HMO in which fucose is ⁇ 1,2-linked to the 2-position of galactose in lacto-N-tetraose (hereafter referred to as LNT).
  • LNFPI is the third most abundant sugar in breast milk after 2'-fucosyllactose (hereafter referred to as 2'FL) and lacto-N-difucohexaose (hereafter referred to as LNDFHI), and is known to be present in a higher amount in breast milk than lacto-N-fucopentaose II (hereafter referred to as LNFPII) and lacto-N-fucopentaose III (hereafter referred to as LNFPIII), which are also pentasaccharides and are known to be isomers of LNFPI (Non-Patent Document 2).
  • LNFPI is known to have functions such as an inhibitory effect against meningitis-causing group B Streptococcus (GBS) and norovirus (Non-Patent Documents 3 and 4).
  • GFS meningitis-causing group B Streptococcus
  • Non-Patent Documents 3 and 4 Bifidobacterium infantis, which has a high occupancy rate in the intestines of newborns, has been found to grow preferentially in LNFPI, and its prebiotic function has also attracted attention (Non-Patent Document 5).
  • Non-patent documents 4, 5, and 6 disclose a method for producing oligosaccharides such as LNFPI by overexpressing ⁇ 1,2-fucosyltransferase derived from microorganisms such as Thermosynechococcus elongatus, Sideroxydans lithotrophicus, or Helicobacter pylori in Escherichia coli and using LNT and GDP-fucose as substrates through fermentation or continuous enzyme reaction.
  • Methods for reducing by-products include an enzyme reaction method that uses highly purified LNT as a substrate (Non-Patent Document 5) and a method for producing LNFPI by inducing the expression of ⁇ 1,2-fucosyltransferase when the initial raw material lactose is depleted (Non-Patent Document 6).
  • the present invention therefore aims to provide a protein having ⁇ 1,2-fucosyltransferase activity with excellent LNFPI productivity and a method for producing LNFPI.
  • LNFPI lipoprotein kinase
  • a microorganism capable of producing a protein with ⁇ 1,2-fucosyltransferase activity consisting of a specific amino acid sequence LNFPI can be produced more efficiently than by conventional methods, and thus completed the present invention.
  • the present invention is as follows. 1. A protein having a fucosyl transfer activity to lacto-N-tetraose (LNT), A protein into which at least one of the following mutations [1] and [2] has been introduced (excluding those in which the amino acid sequence constituting the protein before and after the mutation is identical): [1] A mutation in which an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 98 is inserted. [2] A mutation in which an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 99 is inserted. 2.
  • LNT lacto-N-tetraose
  • the protein according to 1 which is a protein in which at least one of the mutations described in [1] and [2] above is introduced into an amino acid sequence having a full-length sequence identity of 50% or more to at least any one of the amino acid sequences represented by SEQ ID NOs: 63, 94, and 95. 3.
  • the mutation of [1] above is a mutation introduced at a site corresponding to a region contributing to interaction with a substrate in a protein having the amino acid sequence represented by SEQ ID NO: 101 when aligned with the amino acid sequence represented by SEQ ID NO: 101;
  • the mutation of [2] is a mutation introduced at a site corresponding to a region contributing to interaction with a substrate in a protein having the amino acid sequence represented by SEQ ID NO: 131 when aligned with the amino acid sequence represented by SEQ ID NO: 131.
  • 4. The protein according to 3 above, wherein the region contributing to the interaction with the substrate is an amino acid sequence corresponding to positions 162 to 176 of the amino acid sequence shown in SEQ ID NO:101 when aligned therewith. 5.
  • the mutation of [1] above is a mutation in which, when aligned with the amino acid sequence represented by SEQ ID NO: 101, the amino acid sequence corresponding to positions 162 to 176 is replaced with an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 98;
  • the mutation of [2] is a mutation in which, when aligned with the amino acid sequence represented by SEQ ID NO: 131, the amino acid sequence corresponding to positions 168 to 184 is replaced with an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 99. 5.
  • the mutation of [1] is at least one mutation selected from the following [1a] to [1o] at a position corresponding to positions 162 to 176 when aligned with the amino acid sequence represented by SEQ ID NO: 101:
  • the mutation of [2] is at least one mutation selected from the following [2a] to [2q] at positions corresponding to positions 168 to 184 when aligned with the amino acid sequence represented by SEQ ID NO: 131: 6.
  • the protein according to any one of 1 to 5.
  • [2b] is lanine; at position 170, [2c] is valine; at position 170, [2d] is glutamine; at position 171, [2e] is aspartic acid; at position 172, [2f] is proline; at position 173, [2g] is valine; at position 174, [2h] is valine; at position 175, [2i] is arginine; at position 176, [2i] is arginine.
  • a protein having fucosyl transfer activity to LNT A protein in which at least one mutation selected from the following (1) to (31) has been introduced at each of the positions corresponding to positions 7, 42, 94, 95, 159 to 173, 259, 260, and 262 to 270 when aligned with the amino acid sequence represented by SEQ ID NO:63, and which has a higher fucosyl group transfer activity to LNT than the protein before the introduction of the mutation.
  • the protein according to 7 above which is an amino acid sequence having a full-length sequence identity of 50% or more with at least any one of the amino acid sequences represented by SEQ ID NO:63, 94, and 95, in which at least one mutation selected from (1) to (31) above has been introduced at a position corresponding to each of positions 7, 42, 94, 95, 159 to 173, 259, 260, and 262 to 270 of the amino acid sequence represented by SEQ ID NO:63 when aligned with the amino acid sequence represented by SEQ ID NO:63. 9.
  • the protein according to 7 or 8, wherein the mutations introduced at positions corresponding to positions 7, 42, 94, 95, 159 to 173, 259, 260, and 262 to 270 of the amino acid sequence shown in SEQ ID NO:63 are any one of the following [3] to [12]: [3] a phenylalanine at the position corresponding to position 159; At position 160, there is a valine. At the position corresponding to position 161, it is glutamine; At the position corresponding to position 162, it is aspartic acid; At position 164, it is a valine. At position 165, there is a valine.
  • the transformant according to 13 above, wherein the microorganism is Escherichia coli.
  • a method for producing a fucose-containing saccharide comprising preparing the transformant according to any one of 13 to 14 above, and producing the fucose-containing saccharide in a culture using the transformant.
  • the protein of the present invention is composed of a specific amino acid sequence and has ⁇ 1,2-fucosyltransferase activity that allows sugar transfer to the non-reducing terminal galactose site of LNT.
  • FIG. 1 shows the biosynthetic pathway of LNFPI in one embodiment of the present invention.
  • 2 shows the amino acid sequences of FucT (FsFucT) derived from Francisella sp. FSC1006 strain, ⁇ 1,2-fucosyltransferase of S3, and ⁇ 1,2-fucosyltransferase derived from Amphritea japonica.
  • FIG. 3 is a graph showing the total amount of LNFP1 (g/L).
  • FIG. 4 is a graph showing the total amount of LNFP1 (g/L).
  • FIG. 5 is a graph showing the total amount of LNFP1 (g/L).
  • the present invention will be described below with reference to the following embodiments.
  • the embodiments include embodiment 1 and embodiment 2.
  • the identity of amino acid sequences and base sequences can be determined using the algorithm BLAST by Karlin and Altschul [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] or FASTA [Methods Enzymol., 183, 63 (1990)].
  • BLAST Altschul
  • FASTA Method Enzymol., 183, 63 (1990)
  • programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215, 403 (1990)].
  • the default parameters of each program are used. The specific techniques for these analysis methods are publicly known.
  • the alignment of amino acid sequences can be created using known alignment programs such as ClustalW [Nucleic Acids Research 22, 4673, (1994)], MAFTT [Nucleic Acids Research 30, 3059, (2002)], Clustal Omega, etc.
  • ClustalW Nucleic Acids Research 22, 4673, (1994)
  • MAFTT Nucleic Acids Research 30, 3059, (2002)
  • Clustal Omega etc.
  • These alignment programs are available from http://www.ebi.ac.uk/clustalw/ (European Bioinformatics Institute). For example, default values are used as parameters when creating an alignment using these alignment programs.
  • the fucosyl transfer activity to LNT refers to the activity of transferring a fucose residue from the donor substrate GDP-fucose to the galactose hydroxyl group of the acceptor substrate carbohydrate (hereinafter referred to as the "acceptor carbohydrate"), LNT.
  • ⁇ 1,2-fucosyltransferase activity refers to the activity of transferring a fucose residue from the donor substrate GDP-fucose to the galactose hydroxyl group of an acceptor saccharide via an ⁇ 1,2-bond to generate a fucose-containing saccharide.
  • the acceptor saccharide is preferably LNT.
  • the fucose-containing saccharide is preferably LNFPI.
  • the protein having the activity of transferring a fucosyl group to LNT is not particularly limited as long as it has the activity of transferring a fucose residue from the donor substrate GDP-fucose to the galactose hydroxyl group of LNT, and examples of such proteins include ⁇ 1,2-fucosyltransferase. Specific examples of such proteins include ⁇ 1,2-fucosyltransferase derived from Francisella sp.
  • FSC1006 strain ⁇ 1,2-fucosyltransferase derived from Sideroxydans lithotrophicus ES-11, ⁇ 1,2-fucosyltransferase derived from Neisseriaceae bacterium DSM 100970 strain, and ⁇ 1,2-fucosyltransferase derived from Gramella sp.
  • ⁇ 1,2-fucosyltransferase derived from MAR_2010_147 strain ⁇ 1,2-fucosyltransferase derived from Methylobacter tundripaludum strain, ⁇ 1,2-fucosyltransferase derived from Amphritea japonica strain, ⁇ 1,2-fucosyltransferase derived from Sterolibacteriaceae bacterium J5B strain -ase, ⁇ 1,2-fucosyltransferase derived from Neisseriales bacterium strain, ⁇ 1,2-fucosyltransferase derived from Helicobacter mustelae ATCC43772 strain, ⁇ 1,2-fucosyltransferase derived from Pseudohalocynthiibacter aestuariivvens strain, Pedobacter sp.
  • Examples include ⁇ 1,2-fucosyltransferase derived from the CF074 strain, ⁇ 1,2-fucosyltransferase derived from the Candidatus Methylobacter favarea strain, ⁇ 1,2-fucosyltransferase derived from the Escherichia coli O86 strain, and ⁇ 1,2-fucosyltransferase derived from the Escherichia coli O127 strain.
  • each mutation may be substituted with the amino acids which can be substituted for each other as shown below.
  • 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
  • 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
  • LNFPI Biosynthetic pathway of LNFPI> The biosynthetic pathway of LNFPI in this embodiment is shown in Figure 1.
  • LNFPI is produced by the transfer of a fucose residue from GDP-fucose to the galactose hydroxyl group.
  • the present inventors have confirmed that by replacing a portion of the base sequence encoding ⁇ 1,2-fucosyltransferase derived from Francisella sp. FSC1006 with at least one of the corresponding portions of the base sequence encoding ⁇ 1,2-fucosyltransferase derived from Amphritea japonica and the corresponding portion of the base sequence encoding ⁇ 1,2-fucosyltransferase derived from Sterolibacteriaceae bacterium J5B, the by-production of 2'FL can be reduced compared to the enzyme before replacement. In other words, they have newly discovered an ⁇ 1,2-fucosyltransferase protein that has higher substrate selectivity for LNT.
  • the inventors also confirmed that the production of 2'FL by-product was reduced by replacing a portion of the base sequence encoding the ⁇ 1,2-fucosyltransferase derived from Sideroxydans lithotrophicus ES-11 (WO 2019/008133) and the ⁇ 1,2-fucosyltransferase derived from Neisseriaceae bacterium DSM 100970 with the corresponding base sequence of the base sequence encoding the ⁇ 1,2-fucosyltransferase derived from Amphritea japonica.
  • amino acid sequences encoded by the base sequences used for the above-mentioned substitutions in the ⁇ 1,2-fucosyltransferase derived from Amphritea japonica and the ⁇ 1,2-fucosyltransferase derived from Sterolibacteriaceae bacterium J5B contribute to the reduction of 2'FL as a by-product in the ⁇ 1,2-fucosyltransferase homologs, i.e., to the improvement of substrate selectivity for LNT.
  • the protein of embodiment 1 is a protein having fucosyl transfer activity to LNT (hereinafter also simply referred to as "fucosyl transfer activity"), and is a protein into which at least one of the following mutations [1] and [2] has been introduced.
  • [1] A mutation that inserts an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO:98.
  • [2] A mutation that inserts an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO:99.
  • mutations do not include those in which the amino acid sequence constituting the protein is identical before and after the mutation.
  • Examples of the insertion include mutations by insertion and mutations by substitution.
  • the protein of embodiment 1 is preferably a protein in which the substrate selectivity for LNT of the protein after the mutation is improved compared to the protein before the mutation is introduced. It is more preferable that the protein of embodiment 1 is a protein in which, in addition to improved substrate selectivity for LNT, the fucosyl transfer activity of the protein after the mutation is improved compared to the protein before the mutation is introduced.
  • the substrate selectivity of a protein for LNT can be confirmed, for example, by the following procedures (A1) to (A3).
  • A1 A recombinant DNA is prepared having a DNA encoding a protein whose activity is to be confirmed.
  • A2 A parent strain is transformed with the recombinant DNA to produce a transformant, and the amounts of LNFPI and 2'FL produced and accumulated in the culture medium of the transformant are measured.
  • A3 It is determined that the smaller the ratio of the amount of 2'FL to the amount of LNFPI, the higher the substrate selectivity for LNT.
  • the transfucosylation activity of a protein can be confirmed, for example, by the following procedures (B1) to (B3).
  • B1 A recombinant DNA is prepared having a DNA encoding a protein whose activity is to be confirmed.
  • B2 A parent strain is transformed with the recombinant DNA to produce a transformant, and the amount of fucose-containing carbohydrate produced and accumulated in the culture medium of the transformant is measured.
  • the transfucosylation activity is evaluated based on the amount of the fucose-containing saccharide.
  • a specific example of the fucose-containing saccharide is LNFPI.
  • the protein of embodiment 1 is preferably a protein in which at least one of the mutations [1] and [2] has been introduced into an amino acid sequence having a full-length sequence identity of 50% or more with at least one of the amino acid sequences represented by SEQ ID NOs: 63, 94, and 95.
  • the full-length sequence identity is preferably 50% or more, more preferably 60% or more, 70% or more, 80% or more, 90% or more, even more preferably 95% or more, particularly preferably 97% or more, and most preferably 100%.
  • the amino acid sequence shown in SEQ ID NO:63 is the amino acid sequence of FucT derived from Francisella sp. FSC1006 (hereinafter also abbreviated as FsFucT).
  • the amino acid sequence shown by SEQ ID NO:94 is the amino acid sequence of FucT derived from Sideroxydans lithotrophicus ES-11 (hereinafter also abbreviated as FucT54).
  • the amino acid sequence represented by SEQ ID NO: 95 is the amino acid sequence of FucT derived from Neisseriaceae bacterium DSM 100970 (hereinafter also abbreviated as NbFucT1).
  • the amino acid sequence of SEQ ID NO:98 is a partial sequence of the amino acid sequence constituting the amino acid sequence of ⁇ 1,2-fucosyltransferase derived from Amphritea japonica (SEQ ID NO:101), and is an amino acid sequence corresponding to the amino acid sequence from positions 162 to 176 of the amino acid sequence represented by SEQ ID NO:101.
  • the amino acid sequence inserted in [1] has an identity of 80% or more with the amino acid sequence of SEQ ID NO: 98.
  • the identity is preferably 90% or more, more preferably 93% or more, even more preferably 95%, particularly preferably 97% or more, and most preferably 99% or more.
  • the mutation in [1] is preferably a mutation introduced at a position corresponding to a region that contributes to interaction with a substrate in a protein having the amino acid sequence represented by SEQ ID NO: 101 when aligned with the amino acid sequence represented by SEQ ID NO: 101.
  • the region that contributes to interaction with the substrate consists of a binding site with the substrate by hydrogen bonds or the like and a region in the vicinity thereof.
  • An example of a region that contributes to interaction with the substrate is a region that forms a helix structure.
  • the region that contributes to interaction with the substrate is preferably a region that corresponds to positions 140 to 280 when aligned with the amino acid sequence represented by SEQ ID NO: 101, more preferably a region that corresponds to positions 150 to 200, even more preferably a region that corresponds to positions 160 to 180, and particularly preferably a region that corresponds to positions 162 to 176.
  • the mutation in [1] is preferably a mutation in which, when aligned with the amino acid sequence represented by SEQ ID NO:101, the amino acid sequence corresponding to positions 140 to 280, more preferably positions 150 to 200, even more preferably positions 160 to 180, and particularly preferably positions 162 to 176, is replaced with an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO:98.
  • the mutation [1] is preferably at least one selected from the following mutations [1a] to [1o] at positions corresponding to positions 162 to 176 when aligned with the amino acid sequence represented by SEQ ID NO: 101.
  • [1e] At position 166 it is alanine;
  • the amino acid sequence of SEQ ID NO: 99 is a partial sequence of the amino acid sequence constituting the amino acid sequence of SbFucT derived from Sterolibacteriaceae bacterium J5B (SEQ ID NO: 131), and is an amino acid sequence corresponding to the amino acid sequence from positions 168 to 184 of the amino acid sequence represented by SEQ ID NO: 131.
  • the amino acid sequence inserted in [2] has an identity of 80% or more with the amino acid sequence of SEQ ID NO: 99.
  • the identity is preferably 90% or more, more preferably 93% or more, even more preferably 95%, particularly preferably 97% or more, and most preferably 99% or more.
  • the mutation [2] is preferably a mutation introduced at a position corresponding to a region that contributes to interaction with a substrate in a protein having the amino acid sequence represented by SEQ ID NO: 131 when aligned with the amino acid sequence represented by SEQ ID NO: 131.
  • the region that contributes to interaction with the substrate can be a region that forms a helix structure.
  • the region that contributes to interaction with the substrate is preferably a region that corresponds to positions 168 to 184 when aligned with the amino acid sequence represented by SEQ ID NO: 131, more preferably a region that corresponds to positions 150 to 200, even more preferably positions 160 to 190, and particularly preferably positions 168 to 184.
  • the mutation in [2] is preferably a mutation in which, when aligned with the amino acid sequence represented by SEQ ID NO:131, the amino acid sequence corresponding to positions 140 to 280, more preferably positions 150 to 200, even more preferably positions 160 to 190, and particularly preferably positions 168 to 184, is replaced with an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO:99.
  • the mutation [2] is preferably at least one selected from the following mutations [2a] to [2q] at positions corresponding to positions 168 to 184 when aligned with the amino acid sequence represented by SEQ ID NO: 131.
  • [2a] At the position corresponding to position 168, it is phenylalanine;
  • [2b] At the position corresponding to position 169, it is valine;
  • [2c] At the position corresponding to position 170, it is glutamine;
  • [2d] At the position corresponding to position 171, it is aspartic acid;
  • [2e] At the position corresponding to position 172, it is proline;
  • [2f] At the position corresponding to position 173, it is valine;
  • [2g] At the position corresponding to position 174, it is valine;
  • [2h] At the position corresponding to position 175, it is arginine;
  • [2i] At the position corresponding to position 176 In terms of position, [2j] is
  • amino acid sequences constituting the protein of embodiment 1 include the amino acid sequences represented by SEQ ID NOs: 66, 96, and 97.
  • the second embodiment is a protein having fucosyl transfer activity to LNT, in which at least one mutation selected from the following (1) to (31) has been introduced at each of positions 7, 42, 94, 95, 159 to 173, 259, 260, and 262 to 270 when aligned with the amino acid sequence represented by SEQ ID NO: 63, and which has higher fucosyl transfer activity to LNT than the protein before the introduction of the mutation.
  • the protein has the mutation introduced at a position corresponding to at least one position selected from the group consisting of positions 7, 42, 94, 95, 159 to 173, 259, 260, and 262 to 270 when aligned with the amino acid sequence represented by SEQ ID NO: 63.
  • the full-length sequence identity is preferably 50% or more, more preferably 60% or more, 70% or more, 80% or more, 90% or more, even more preferably 95% or more, particularly preferably 97% or more, and most preferably 100%.
  • amino acid sequences represented by SEQ ID NOs: 63, 94, and 95 are the same as those described above in the section entitled "Embodiment 1: Protein having fucosyl transfer activity to LNT.”
  • Each mutation in embodiment 2 is preferably any one of the following [3] to [12].
  • At the position corresponding to position 169 it is a histidine; At the position corresponding to position 170, it is glycine; At position 171, there is a valine. At the position corresponding to position 172, it is aspartic acid; At the position corresponding to position 173, it is a tyrosine; Between the positions corresponding to positions 172 and 173, leucine, tyrosine and alanine are inserted. [4] a histidine at the position corresponding to position 94; At the position corresponding to position 95, it is a tyrosine; At the position corresponding to position 159, it is a phenylalanine; At position 160, there is a valine.
  • amino acid sequences constituting the protein of embodiment 2 include the amino acid sequences represented by any one of SEQ ID NOs: 64 to 78. Among these, the amino acid sequences represented by any one of SEQ ID NOs: 64 to 66, 68, and 72 to 77 are particularly preferred.
  • the DNA of this embodiment is a DNA encoding the above-mentioned protein of this embodiment.
  • Examples of the DNA encoding the protein of embodiment 1 include DNA having the base sequence shown in SEQ ID NO: 49, 92, or 93.
  • Examples of the DNA encoding the protein of embodiment 2 include DNA having the base sequence shown in any one of SEQ ID NOs: 47 to 61.
  • the DNA of this embodiment can be used as is or after cleavage with an appropriate restriction enzyme or the like, incorporated into a vector by standard methods, and the resulting recombinant DNA can be introduced into a host cell.
  • the DNA can then be analyzed using a standard base sequence analysis method, such as the dideoxy method [Proc. Natl. Acad. Sci., USA, 74, 5463 (1977)] or a base sequence analyzer such as an Applied Biosystems 3500 Genetic Analyzer or an Applied Biosystems 3730 DNA Analyzer (both manufactured by Thermo Fisher Scientific), to determine the base sequence of the DNA.
  • a standard base sequence analysis method such as the dideoxy method [Proc. Natl. Acad. Sci., USA, 74, 5463 (1977)] or a base sequence analyzer such as an Applied Biosystems 3500 Genetic Analyzer or an Applied Biosystems 3730 DNA Analyzer (both manufactured by Thermo Fisher Scientific), to determine the base sequence of the DNA.
  • Any host cell can be used when determining the base sequence of the DNA, so long as it can grow after the vector is introduced into it.
  • Examples of such cells include Escherichia coli DH5 ⁇ , 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,
  • Examples of such strains include Escherichia coli MV1184, Escherichia coli TH2 (both manufactured by Takara Bio Inc.), Escherichia coli XL1-Blue, Escherichia coli XL2-Blue (both manufactured by Agilent Technologies), Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli W31
  • Examples of the vector 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).
  • Any method for introducing recombinant DNA into a host cell can be used, such as the calcium ion method [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], the protoplast method (JP Patent Publication 63-248394), and the electroporation method [Nucleic Acids Res., 16, 6127 (1988)].
  • full-length DNA can be obtained by Southern hybridization or other methods using the partial length DNA as a probe against a chromosomal DNA library.
  • the desired DNA can be prepared by chemical synthesis using a Nippon Techno Service Co., Ltd. NTS M Series DNA synthesizer or similar.
  • the recombinant DNA containing the DNA encoding the protein of this embodiment refers to recombinant DNA that is capable of autonomous replication in a parent strain or of being integrated into a chromosome, and that is integrated into an expression vector that contains a promoter at a position where the DNA can be transcribed.
  • the recombinant DNA is capable of being integrated into a chromosome, it does not need to contain a promoter.
  • a microorganism having an increased copy number of the gene compared to the parent strain, obtained by transforming a parent strain microorganism with recombinant DNA containing DNA encoding the protein of this embodiment, can be obtained by the following method.
  • a DNA fragment of an appropriate length containing a portion encoding the protein is prepared as necessary.
  • a transformant with improved productivity can be obtained.
  • the DNA fragment is inserted downstream of the promoter of an appropriate expression vector to produce recombinant DNA.
  • a microorganism By transforming a parent strain with the recombinant DNA, a microorganism can be obtained in which the copy number of the gene encoding the protein is increased compared to the parent strain.
  • the recombinant DNA is preferably a recombinant DNA composed of a promoter, a ribosome binding sequence, DNA encoding the protein of this embodiment, and a transcription termination sequence.
  • a gene that controls the promoter may also be included.
  • a transcription termination sequence is not necessarily required for the expression of the DNA, but it is preferable to place a transcription termination sequence immediately downstream of the structural gene.
  • the expression level of the protein having ⁇ 1,2-fucosyltransferase activity can be improved.
  • Information on the codon usage frequency in the parent strain used in this embodiment can be obtained through public databases.
  • the expression vector is not particularly limited as long as it is an appropriate nucleic acid molecule for introducing the target DNA into a host and allowing it to grow and express.
  • plasmids for example, artificial chromosomes, vectors using transposons, and cosmids may also be used.
  • examples of the expression vector include pColdI, pSTV28, pSTV29, pUC118 (all manufactured by Takara Bio Inc.), pMW118, pMW119 (all manufactured by Nippon Gene Co., Ltd.), pET21a, pCOLADuet-1, pCDFDuet-1, pCDF-1b, pRSF-1b (all manufactured by Merck Millipore), pMAL-c5x (manufactured by New England Biolabs), pGEX-4T-1, and pTrc99A (all manufactured by GE Healthcare Biosciences, Inc.).
  • pTrcHis pSE280 (all manufactured by Thermo Fisher Scientific), pGEMEX-1 (manufactured by Promega), pQE-30, pQE80L (all manufactured by Qiagen), pET-3, pBluescriptII SK(+), pBluescriptII KS(-) (all manufactured by Agilent Technologies), pUAKQE31 (Appl. Environ. Microbiol. 2007, 73: 6378-6385), pKYP10 (JP Patent Publication 1983-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem.
  • pGEL1 Proc. Natl. Acad. Sci. , USA, 82, 4306 (1985)]
  • pBluescript II SK(+), pBluescript II KS(-) (Stratagene)
  • pTrS30 prepared from Escherichia coli JM109/pTrS30 (FERM BP-5407)
  • pTrS32 prepared from Escherichia coli JM109/pTrS32 (FERM BP-5408)
  • pTK31 [APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2007, Vol. 73, No. 20, p. 6378-6385]
  • pPAC31 International Publication No.
  • any promoter may be used as long as it functions in the cells of a microorganism belonging to the genus Escherichia.
  • promoters of genes involved in amino acid biosynthesis such as the trp promoter and ilv promoter, and promoters derived from Escherichia coli or phages, such as the uspA promoter, lac promoter, PL promoter, PR promoter, and PSE promoter, can be used.
  • Other examples include artificially designed and modified promoters, such as a promoter with two trp promoters in series, the tac promoter, the trc promoter, the lacT7 promoter, and the letI promoter.
  • examples of the expression vector include pCG1 (JP Patent Publication No. 57-134500), pCG2 (JP Patent Publication No. 58-35197), pCG4 (JP Patent Publication No. 57-183799), pCG11 (JP Patent Publication No. 57-134500), pCG116, pCE54, pCB101 (all JP Patent Publication No. 58-105999), pCE51, pCE52, and pCE53 (all Molecular and General Genetics, 196, 175 (1984)).
  • any promoter that functions in the cells of a microorganism belonging to the genus Corynebacterium may be used, and an example of such a promoter is the P54-6 promoter [Appl. Microbiol. Biotechnol., 53, 674-679 (2000)].
  • examples of the expression vector include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15, etc.
  • any promoter that functions in the cells of a yeast strain may be used, and examples include the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock polypeptide promoter, MF ⁇ 1 promoter, CUP1 promoter, etc.
  • the transformant of this embodiment can be obtained by transforming a host cell with the recombinant DNA of this embodiment.
  • the term "parent strain” refers to the original strain to be transformed.
  • the parent strain is preferably a prokaryote or a yeast strain, more preferably a prokaryote belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, or Pseudomonas, or a yeast strain belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Siwaniomyces, Pichia, or Candida, and most preferably Escherichia coli.
  • yeast strains such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris, or Candida utilis.
  • the parent strain may be a wild-type strain, so long as it is a microorganism that produces GDP-fucose and/or LNT. If the wild-type strain does not have the ability to produce GDP-fucose and/or LNT, the parent strain may be a bred strain that has been artificially endowed with the ability to supply GDP-fucose and/or LNT.
  • microorganisms that can be used as parent strains include the following 1) and 2).
  • 1) and 2) are described below.
  • Microorganisms used as parent strains in which the ability to supply GDP-fucose, which is a reaction substrate for ⁇ 1,2-fucosyltransferase, has been artificially imparted or enhanced.
  • the parent strain is preferably a microorganism in which the ability to supply GDP-fucose, which is a reaction substrate for ⁇ 1,2-fucosyltransferase, has been artificially imparted or enhanced.
  • Specific examples of methods for imparting or enhancing the ability to supply GDP-fucose in a microorganism used as a parent strain include known methods such as various genetic engineering methods (Metabolic Engineering (2017) 41: 23-38).
  • An example of the ability to supply GDP-fucose is the ability to produce GDP-fucose from sugar.
  • Methods for artificially imparting or enhancing the ability to produce GDP-fucose from sugar to a microorganism used as a parent strain include, for example, the following methods (1a) to (1d). These methods may be used alone or in combination.
  • mechanisms that control the biosynthetic pathway that produces GDP-fucose from sugars include known mechanisms, such as a control mechanism by a transcriptional regulatory factor (e.g., RcsA) involved in the control of the biosynthetic pathway.
  • RcsA is a regulatory factor that upregulates the entire colanic acid biosynthetic pathway, which uses GDP-fucose as an intermediate.
  • RcsA is a regulatory factor that upregulates the entire colanic acid biosynthetic pathway, which uses GDP-fucose as an intermediate.
  • enzymes involved in the biosynthetic pathway that produces GDP-fucose from sugars include known enzymes such as mannose-6-phosphate isomerase, phosphomannomutase, mannose-1-phosphate guanylyltransferase, GDP mannose-4,6-dehydratase, and GDP-L-fucose synthase.
  • WcaJ, WzxC, WcaK, WcaL, or WcaM which are pathways downstream of GDP-fucose in the colanic acid biosynthetic pathway, the supply of GDP-fucose can be increased.
  • the microorganism used as the parent strain may be modified to promote the transfer of exogenous L-fucose across its cell membrane. For example, by expressing or overexpressing a base sequence encoding FucP (accession number AIZ90162), the uptake of exogenous L-fucose across the cell membrane can be improved, thereby increasing the amount of fucose for producing GDP-fucose.
  • FucP accession number AIZ90162
  • the microorganism used as the parent strain may be deleted in the genes fucI and/or fucK encoding L-fucose isomerase and L-fuculose kinase, respectively, and the nucleotide sequences of fucI and/or fucK may be altered so as to irreversibly inactivate the enzymatic activity of the corresponding polypeptide, or may be modified so as to impair the expression of fucI and/or fucK. Eliminating the intracellular synthesis of FucI and/or FucK eliminates fucose metabolism in the cell, thereby enabling an increase in the amount of fucose for producing GDP-fucose.
  • a microorganism used as a parent strain to which the ability to supply LNT, a reaction substrate of ⁇ 1,2-fucosyltransferase, has been artificially imparted or enhanced.
  • methods for artificially imparting the ability to supply LNT to a microorganism used as a parent strain include the following methods (2a) to (2h), and these methods may be used alone or in combination.
  • enzymes involved in the biosynthetic pathway that produces LNT from sugar include known enzymes such as enzymes with ⁇ 1,4-galactosyltransferase (hereinafter referred to as galT) activity and enzymes with ⁇ 1,3-N-acetylglucosaminetransferase (hereinafter referred to as lgtA) activity, which are involved in the biosynthetic pathway that produces LNT from glucose and lactose.
  • galT ⁇ 1,4-galactosyltransferase
  • lgtA enzymes with ⁇ 1,3-N-acetylglucosaminetransferase
  • Mechanisms for decomposing LNT or its substrate sugars include known enzymes such as ⁇ -galactosidase, which catalyzes the hydrolysis of lactose, the substrate of LNT, to produce glucose and galactose.
  • ⁇ -galactosidase which catalyzes the hydrolysis of lactose, the substrate of LNT, to produce glucose and galactose.
  • lacZ ⁇ -galactosidase
  • enzymes involved in the cellular uptake of LNT or its substrate sugar include known enzymes such as lactose permease, which is involved in the cellular uptake of lactose, which is a substrate for LNT.
  • lacY lactose permease
  • galT ⁇ 1,4-galactosyltransferase
  • glmA glutamine fructose-6-phosphate transaminase
  • glmM phosphoglucosamine mutase
  • the enzyme has at least one activity selected from the group consisting of phosphate acetyltransferase (hereinafter referred to as glmU) activity, phosphoglucomutase (hereinafter referred to as pgm) activity, UTP glucose-1-phosphate uridylyltransferase (hereinafter referred to as galU) activity, UDP glucose-4-epimerase (hereinafter referred to as galE) activity, UTP glucose-1-phosphate uridylyltransferase (hereinafter referred to as galF) activity, and glucose-6-phosphate isomerase (hereinafter referred to as pgi) activity, and it is more preferable that the activity is enhanced.
  • glmU phosphate acetyltransferase
  • pgm phosphoglucomutase
  • galU UTP glucose-1-phosphate uridylyltransferase
  • galF UDP glucose-4-epime
  • lacY, galT and lgtA activities are present, and it is even more preferable that these activities are enhanced.
  • lacY is a membrane protein that takes up lactose, the substrate for LNT, into the cell.
  • galT is an enzyme involved in the production of LNT from lacto-N-triose II (LNTII).
  • LNT is the precursor of LNFPI.
  • LgtA is an enzyme involved in the production of LNTII from lactose and uridine diphosphate-N-acetylglucosamine (hereafter referred to as UDP-GlcNAc).
  • LNTII is the precursor of LNT.
  • glmS, glmM, and glmU are enzymes involved in the biosynthetic pathway that produces LNTII.
  • Pgm, galU, galE, and galF are enzymes involved in the pathway that produces uridine diphosphate galactose (hereafter referred to as UDP-Gal).
  • Pgi is an enzyme involved in the pathway that produces LNTII.
  • Whether a microorganism is capable of producing GDP-fucose and/or LNT can be confirmed by culturing the microorganism in a medium and detecting the GDP-fucose and/or LNT accumulated in the culture using a general method such as a sugar analyzer or high performance liquid chromatograph mass spectrometer described below.
  • a general method such as a sugar analyzer or high performance liquid chromatograph mass spectrometer described below.
  • commercially available samples can be used as appropriate as standard samples.
  • the microorganism used as the parent strain of the present invention is preferably a microorganism to which the ability to supply GDP-fucose and/or LNT, which are reaction substrates for ⁇ 1,2-fucosyltransferase, has been artificially imparted or enhanced. Therefore, in one embodiment of the present invention, a nucleotide sequence encoding rcsA (Accession No. BAA15776.1), a nucleotide sequence encoding mannose-6-phosphate isomerase (Accession No. BAA15361.1), a nucleotide sequence encoding phosphomannomutase (Accession No.
  • BAA15901.1 a nucleotide sequence encoding mannose-1-phosphate guanylyltransferase (Accession No. BAA15905.1), a nucleotide sequence encoding GDP mannose-4,6-dehydratase (Accession No. BAA15909.1), a nucleotide sequence encoding GDP-L-fucose synthase (Accession No. BAA15908.1), a nucleotide sequence encoding lacY (Accession No. BAE76125.1), a nucleotide sequence encoding galT (Accession No. BAE76125.1), a nucleotide sequence encoding galT (Accession No.
  • BAE76125.1 a nucleotide sequence encoding galT (Accession No. BAE76125.1), a nucleotide sequence encoding galT (Accession No. BAA1590 ... 29), a base sequence encoding lgtA (SEQ ID NO: 31), a base sequence encoding glmS (accession number BAE77559.1), a base sequence encoding glmM (accession number BAE77220.1), a base sequence encoding glmU (accession number BAE77558.1), a base sequence encoding Pgm (accession number BAA35337.1), a base sequence encoding galU (accession number BAA36104.1), a base sequence encoding galE (accession number BAA35421.1), a base sequence encoding galF (accession number BAA15896.1), and a base sequence encoding pgi (accession number BAE78027.1) are
  • a genetically modified microorganism as the parent strain, the genetically modified microorganism preferably including a base sequence encoding lacY, a base sequence encoding rcsA, a base sequence encoding galT, and a base sequence encoding lgtA.
  • the genetically modified microorganism has an increased ability to produce GDP-fucose and/or LNT compared to a parent strain that has not been genetically modified.
  • a method for producing a microorganism having at least one activity selected from lacY activity, rcsA activity, galT activity, lgtA activity, glmS activity, glmM activity, glmU activity, pgm activity, galU activity, galE activity, galF activity, and pgi activity, or having enhanced activity may be used by using a known method. Specific examples include methods using various genetic manipulations (Syst Microbiol Biomanufact, 2021, 1, 291), etc.
  • the parent strain has reduced or absent lacZ activity and/or colanic acid synthesis activity.
  • a genetically modified microorganism as a parent strain, which preferably has reduced or absent lacZ activity and/or colanic acid synthesis activity, and more preferably does not contain a base sequence encoding lacZ and/or a base sequence encoding the wcaJ, wzxC, wcaK, wcaL or wcaM genes, which are base sequences encoding proteins related to colanic acid production.
  • a method for producing E. coli with reduced or lost ⁇ -galactosidase activity and/or colanic acid synthesis activity may be used that is publicly known. Specific examples include methods using various genetic manipulations (Metabolic Engineering, 2017, 41: 23-38), etc.
  • An example of a microorganism in which the activity of the protein of this embodiment is enhanced compared to a parent strain of a microorganism is a microorganism obtained by transforming a parent strain of a microorganism with recombinant DNA containing DNA encoding the protein, and in which the copy number of the recombinant DNA is increased compared to the parent strain.
  • Microorganisms in which the copy number of recombinant DNA is increased compared to the parent strain, obtained by transforming a parent strain microorganism with recombinant DNA containing DNA encoding the protein of this embodiment include microorganisms in which the copy number of recombinant DNA on the chromosomal DNA is increased by transforming a parent strain microorganism with recombinant DNA containing DNA encoding the protein of this embodiment, and microorganisms in which the gene is carried outside the chromosomal DNA as plasmid DNA.
  • Methods for introducing recombinant DNA into a parent strain as an autonomously replicable plasmid include, for example, the calcium ion method [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], the protoplast method (JP Patent Publication 63-248394), and the electroporation method [Nucleic Acids Res., 16, 6127 (1988)].
  • Methods for incorporating recombinant DNA into the chromosome of a host cell include, for example, homologous recombination.
  • homologous recombination include a method using a plasmid for homologous recombination that can be produced by linking with plasmid DNA having a drug resistance gene that cannot autonomously replicate in the host cell to be introduced.
  • An example of a method using homologous recombination that is frequently used in Escherichia coli is a method that uses the homologous recombination system of lambda phage to introduce recombinant DNA [Proc. Natl. Acad. Sci. USA, 97, 6640-6645 (2000)].
  • E. coli in which a target region on the chromosomal DNA of a host cell has been replaced with recombinant DNA using a selection method that utilizes the fact that E. coli becomes sucrose sensitive due to Bacillus subtilis levan sucrase that has been incorporated on the chromosome together with recombinant DNA, or a selection method that utilizes the fact that E. coli becomes streptomycin sensitive by incorporating a wild-type rpsL gene into E. coli that has a mutant rpsL gene that is resistant to streptomycin [Mol. Microbiol., 55, 137 (2005); Biosci. Biotechnol. Biochem., 71, 2905 (2007)], etc.
  • That the recombinant DNA has been introduced into the parent strain as an autonomously replicable plasmid or incorporated into the chromosome of the parent strain can be confirmed, for example, by a method in which a gene that is originally present on the chromosomal DNA of a microorganism cannot be amplified, but an amplified product is confirmed by PCR using a primer set that can amplify the gene introduced by transformation.
  • an increase in the amount of transcription of the DNA or the amount of production of the protein encoded by the DNA can be confirmed by a method in which the amount of transcription of the gene in the microorganism is compared with that of the parent strain by Northern blotting, or the amount of production of the protein in the microorganism is compared with that of the parent strain by Western blotting.
  • the microorganism created by the above method is a microorganism in which the activity of the protein of this embodiment is enhanced and the productivity of LNFPI is improved compared to the parent strain
  • culturing the microorganism diluting the culture solution appropriately, centrifuging it, and analyzing the LNFPI contained in the supernatant or the cells using a sugar analyzer or high-performance liquid chromatograph mass spectrometer described below, and comparing it with that of the parent strain.
  • a commercially available sample can be used as the standard sample.
  • the method for producing the fucose-containing saccharide of this embodiment includes a method for producing the fucose-containing saccharide, which includes preparing the transformant of this embodiment described above and producing oligosaccharides in a culture using the transformant.
  • the fucose-containing saccharide is preferably LNFPI.
  • the above-mentioned transformants can be cultured according to the usual method used for culturing microorganisms.
  • a medium for culturing the transformants either a natural medium or a synthetic medium may be used as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the microorganism and allows the transformants to be cultured efficiently.
  • the carbon source may be anything that can be assimilated by the microorganism, and examples of such carbon sources include sugars such as glucose, fructose, sucrose, molasses containing these, starch, and starch hydrolysates, organic acids such as acetic acid and propionic acid, and alcohols such as glycerol, ethanol, and propanol.
  • sugars such as glucose, fructose, sucrose, molasses containing these, starch, and starch hydrolysates
  • organic acids such as acetic acid and propionic acid
  • alcohols such as glycerol, ethanol, and propanol.
  • Nitrogen sources include, for example, ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other ammonium salts of inorganic or organic acids, as well as other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal, soybean meal hydrolysate, various fermentation bacteria and their digested products, etc.
  • inorganic salts include potassium dibasic phosphate, potassium dibasic phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc.
  • the transformant used in this production method may be a microorganism capable of producing glucose, lactose, lactose monohydrate, or the like.
  • glucose, lactose, lactose monohydrate, etc. may be added to the medium during cultivation.
  • GDP-fucose and/or LNT may be added to the medium.
  • a microorganism capable of producing glucose, lactose, lactose monohydrate, LNT, or the like from sugar may be simultaneously cultured with the transformant of the present invention, thereby supplying glucose, lactose, lactose monohydrate, LNT, or the like to the transformant of the present invention.
  • ⁇ -galactosidase and WcaJ are not present in the medium.
  • Cultivation is preferably carried out under aerobic conditions, such as by shaking culture, deep aeration stirring culture, or jar fermenter.
  • the culture temperature is usually 30 to 37°C, and the culture time is usually 24 hours to 3 days.
  • the pH of the culture solution during cultivation is usually maintained at 6.0 to 8.0.
  • the pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.
  • fucose-containing carbohydrates can be produced in the culture by generating fucose-containing carbohydrates.
  • fucose-containing carbohydrates can be collected from the supernatant. If fucose-containing carbohydrates accumulate within the cells, the cells can be disrupted, for example, by ultrasonication, and the cells can be removed by centrifugation. The fucose-containing carbohydrates can then be collected from the resulting supernatant by an ion exchange resin method or the like.
  • the desired fucose-containing carbohydrate can also be produced by adding other sugars to the fucose-containing carbohydrate in the culture or to the collected fucose-containing carbohydrate.
  • Example 1 Construction of microorganisms expressing various ⁇ 1,2-fucosyltransferases (1) Construction of host strain ⁇ Obtaining DNA fragments to be used as markers for gene deletion> PCR was performed using DNA consisting of the base sequences represented by SEQ ID NOs: 102 and 103 as a primer set and pCatSac (Appl Environ Microbiol (2013) 79, 3033-3039) as a template to obtain a cat-sacB fragment containing a chloramphenicol-resistant cat gene and a sucrose-sensitive sacB gene.
  • lacZ and lacY (hereinafter referred to as lacZY), and wcaJ, wzxC, wcaK, wcaL, and wcaM (hereinafter referred to as wcaJ-wzxC-wcaKLM) each form an operon on the Escherichia coli genome.
  • PCR was performed using DNA consisting of the base sequence shown in "Primer Set" in Table 1 as a primer set to amplify each DNA fragment.
  • lacZ upstream 1 and lacZ upstream 2 include the region from the start codon of the lacZ gene to approximately 1000 bp upstream of the start codon.
  • lacY downstream 1 and lacY downstream 2 include the region from approximately 50 bp downstream of the stop codon of the lacY gene to approximately 1000 bp downstream.
  • a PCR was performed using an equimolar mixture of lacZ upstream 1, lacY downstream 1, and cat-sacB fragments as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 105 and 107 as a primer set to obtain a DNA fragment (hereinafter referred to as lacZY::cat-sacB) consisting of a sequence in which the cat-sacB fragment was inserted into the sequence of the region surrounding the lacZ and lacY genes.
  • a PCR was performed using an equimolar mixture of lacZ upstream 2 and lacY downstream 2 as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 105 and 107 as a primer set to obtain a DNA fragment (hereinafter referred to as ⁇ lacZY) that does not contain lacZY and consists of a sequence in which the upstream of lacZ and the downstream of lacY are directly linked.
  • ⁇ lacZY DNA fragment that does not contain lacZY and consists of a sequence in which the upstream of lacZ and the downstream of lacY are directly linked.
  • the lacZY::cat-sacB fragment was introduced by electroporation into the W3110S3GK strain (NBRC114657) carrying the plasmid pKD46 [Datsenko, K. A., Warner, B. L., Proc. Natl. Acad. Sci., USA, Vol. 97, 6640-6645 (2000)], which contains a gene encoding ⁇ recombinase, to obtain a transformant that exhibited chloramphenicol resistance and sucrose sensitivity (a transformant in which the lacZY gene was replaced with lacZY::cat-sacB).
  • the ⁇ lacZY 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 lacZY::cat-sacB was replaced by ⁇ lacZY). From these, a transformant that was sensitive to ampicillin (a transformant from which pKD46 had been lost) was further obtained.
  • the transformant was named W3110S3GK ⁇ lacZY.
  • PCR was performed using the genomic DNA of the W3110 strain as a template and DNA consisting of the base sequence shown in "Primer set" in Table 2 as a primer set, to obtain each amplified DNA fragment.
  • wcaJ upstream 1 and wcaJ upstream 2 include the region from the start codon of the wcaJ gene to approximately 1000 bp upstream of the start codon.
  • wcaM downstream 1 and wcaM downstream 2 include the region from the stop codon of the wcaM gene to approximately 1000 bp downstream of the stop codon.
  • wcaJ upstream 1, wcaM downstream 1 and cat-sacB fragments was used as a template, and PCR was performed using DNA consisting of the base sequences represented by SEQ ID NOs: 111 and 113 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 region surrounding the wcaJ-wzxC-wcaKLM operon (hereinafter referred to as wcaJ-wzxC-wcaKLM::cat-sacB).
  • ⁇ wcaJ-wzxC-wcaKLM DNA consisting of the base sequences represented by SEQ ID NOs: 111 and 113 as a primer set to obtain a DNA fragment (hereinafter referred to as ⁇ wcaJ-wzxC-wcaKLM) that does not contain wcaJ-wzxC-wcaKLM and consists of a sequence in which the upstream of wcaJ and the downstream of wcaM are directly linked.
  • the wcaJ-wzxC-wcaKLM::cat-sacB fragment was introduced into the W3110S3GK ⁇ lacZY constructed above by electroporation, and a transformant that showed chloramphenicol resistance and sucrose sensitivity (a transformant in which wcaJ-wzxC-wcaKLM was replaced with wcaJ-wzxC-wcaKLM::cat-sacB) was obtained.
  • the ⁇ wcaJ-wzxC-wcaKLM 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 had been replaced with ⁇ wcaJ-wzxC-wcaKLM).
  • a transformant that was sensitive to ampicillin was obtained.
  • the transformant was named W3110S3GK ⁇ lacZY ⁇ wcaJM.
  • PCR was performed using DNA consisting of the base sequence shown in “Primer set” in Table 3 as a primer set and DNA shown in “Template” in Table 3 as a template to obtain each amplified DNA fragment.
  • the DNA represented by SEQ ID NO:122 is a codon-optimized DNA for expressing in Escherichia coli the base sequence of the gene encoding ⁇ 1,3-galactosyltransferase Cv ⁇ 3GalT derived from Chromobacterium violaceum ATCC553 strain, described in ACS Catal. 2019, 9(12), 10721-10726, and was prepared by artificial synthesis.
  • the DNA represented by SEQ ID NO:123 is a DNA in which the base sequence of the gene encoding ⁇ 1,3-N-acetylglucosamine transferase NpLgtA derived from Neisseria polysaccharea ATCC43768 strain represented by SEQ ID NO:125 has been codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis.
  • the base sequences represented by SEQ ID NOs:117 and 118, and SEQ ID NOs:119 and 120 each contain a complementary sequence at the 5' end.
  • Cv ⁇ 3galT fragment, NplgtA fragment and lacY fragment were mixed in an equimolar ratio as a template, and PCR was performed using DNA consisting of the base sequences represented by SEQ ID NOs: 116 and 121 as a primer set to obtain a DNA fragment in which the three fragments were linked (hereinafter referred to as Cv ⁇ 3galT-NplgtA-lacY).
  • PCR was performed to obtain a vector fragment of approximately 4.7 kb.
  • the base sequences represented by SEQ ID NOs: 116 and 126, and SEQ ID NOs: 121 and 127 each contain a complementary sequence at the 5' end.
  • the Cv ⁇ 3galT-NplgtA-lacY fragment and vector fragment obtained above were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the expression plasmid pUAKQE-Cv ⁇ 3galT-NplgtA-lacY.
  • the expression plasmid pUAKQE-Cv ⁇ 3galT-NplgtA-lacY was used to transform the W3110S3GK ⁇ lacZY ⁇ wcaJM strain constructed above to construct an E. coli strain carrying pUAKQE-Cv ⁇ 3galT-NplgtA-lacY, which was named the NNN strain.
  • PCR was performed using the plasmid pSTV29 (manufactured by Takara Bio Inc.) as a template and DNA represented by SEQ ID NOs: 134 and 135 as a primer set to obtain a vector fragment of about 2.9 kb.
  • the base sequences represented by SEQ ID NOs: 132 and 134, and SEQ ID NOs: 133 and 135 each contain a complementary sequence at their 5' ends.
  • the rcsA fragment and vector fragment obtained above were ligated using In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.) to obtain an expression vector pSTV-rcsA.
  • FsFucT ⁇ 1,2-fucosyltransferase derived from Francisella sp. FSC1006 strain.
  • the expression vector pFsFucT serving as a template was prepared as follows: PCR was performed using DNA consisting of the base sequences shown in "Primer set” in Table 4 as a primer set and DNA shown in "Template” in Table 4 as a template to obtain an amplified DNA fragment.
  • the DNA represented by SEQ ID NO:46 is a DNA in which the base sequence of the gene encoding ⁇ 1,2-fucosyltransferase FsFucT derived from Francisella sp. FSC1006 strain represented by SEQ ID NO:63 has been codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis.
  • the base sequences represented by SEQ ID NOs: 128 and 129 each contain a complementary sequence at the 5' end.
  • the amplified DNA fragment and vector fragment were ligated using In-Fusion HD Cloning Kit (Takara Bio) to create the plasmid pFsFucT expressing ⁇ 1,2-fucosyltransferase.
  • the DNA consisting of the base sequences shown in the various primer sets in Table 5 was used as a primer set to perform PCR using pFsFucT as a template.
  • the resulting vector fragments were ligated using In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.) to obtain the various constructed plasmids shown in Table 5 in which point mutations were introduced into the gene sequence of FsFucT, i.e., various plasmids expressing ⁇ 1,2-fucosyltransferase.
  • Y159F This indicates that Y at the position corresponding to position 159 is replaced with F. Mutations at other positions are represented in the same manner.
  • D172_Y173insLTA D at position corresponding to position 172 and Y at position corresponding to position 173, indicating that L, T and A are inserted between positions corresponding to positions 172 and 173. Mutations at other positions are represented similarly.
  • D167_I168insIHG D at the position corresponding to position 167 and I at the position corresponding to position 168, indicating that I, H and G are inserted between the positions corresponding to positions 167 and 168.
  • I267del This indicates that I at the position corresponding to position 267 is deleted.
  • 0.2 mL of the obtained culture solution was inoculated into a large test tube containing 4 mL of production medium containing 100 mg/L kanamycin and 25 mg/L chloramphenicol [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 acids 5 g/L, thiamine hydrochloride 10 mg/L, ferrous sulfate heptahydrate 50 mg/L, manganese sulfate pentahydrate 10 mg/L (pH was adjusted to 7.2 with aqueous sodium hydroxide solution for all components except glucose, lactose monohydrate, and magnesium sulfate heptahydrate, and then autoclaved) (aqueous solutions
  • the S3 and S9 strains which showed significant inhibition of 2'FL production and had LNFPI production equivalent to that of the NNN/pFsFucT strain, were selected as candidates for ⁇ 1,2-fucosyltransferase effective in LNFPI production.
  • Example 2 Jar evaluation The S3 and S9 strains selected in ⁇ Productivity evaluation of LNFPI and 2'FL (test tube)> in Example 1 and the control strain NNN/pFsFucT strain were each cultured on an LB plate containing 100 mg/L kanamycin and 25 mg/L chloramphenicol at 30°C for 24 hours, and then inoculated into a 300 mL baffled Erlenmeyer flask containing 65 mL of a medium [yeast extract 5 g/L, peptone 10 g/L, sodium chloride 5 g/L] containing 100 mg/L kanamycin and 25 mg/L chloramphenicol, and cultured with shaking at 30°C for 16 hours.
  • a medium containing 100 mg/L kanamycin and 25 mg/L chloramphenicol
  • both the S3 and S9 strains suppressed 2'FL production and increased LNFPI production.
  • the S3 strain produced 0.2% of the amount of 2'FL produced relative to the amount of LNFPI produced, significantly reducing the by-production of 2'FL, suggesting that LNT can be used preferentially as a substrate.
  • Figure 2 shows the amino acid sequences of Francisella sp. FSC1006-derived FucT (FsFucT), S3 ⁇ 1,2-fucosyltransferase, and Amphritea japonica-derived ⁇ 1,2-fucosyltransferase.
  • the amino acid sequence of S3 ⁇ 1,2-fucosyltransferase is one in which the 161st to 172nd amino acid residues of FsFucT have been replaced with the sequence motif of Amphritea japonica-derived ⁇ 1,2-fucosyltransferase (shown in shaded areas in Figure 2) ( Figure 2). From this, it was found that the sequence motif of Amphritea japonica-derived ⁇ 1,2-fucosyltransferase contributes to improved LNT selectivity.
  • the DNA represented by SEQ ID NO:91 is a DNA in which the base sequence of the gene encoding ⁇ 1,2-fucosyltransferase (NbFucT1) derived from Neisseriaceae bacterium DSM 100970 represented by SEQ ID NO:95 has been codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis.
  • NbFucT1 ⁇ 1,2-fucosyltransferase
  • each of the obtained amplified DNA fragments was linked to a vector fragment amplified using the expression vector pSTV29-rcsA as a template using In-Fusion HD Cloning Kit (Takara Bio Inc.) to construct plasmids pNbFucT1 and pFucT54 expressing various ⁇ 1,2-fucosyltransferases.
  • the obtained amplified DNA fragment and linearized vector fragment were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain plasmids expressing each ⁇ 1,2-fucosyltransferase shown in "Constructed Plasmid” in Table 11.
  • N170T indicates that N at position 170 in the amino acid sequence represented by SEQ ID NO:94 is substituted with T. Mutations at other positions are represented similarly.
  • F_H helix indicates that N at position 160 in the amino acid sequence represented by SEQ ID NO:95 is substituted with T. Mutations at other positions are represented similarly.
  • F_H helix N160_H161insDLSA indicates that DLSA has been inserted between N160 and H161.
  • G165del indicates that G165 has been deleted.
  • the A_H helix strain, the F_H helix strain, the S3 strain constructed in Example 1, and the control strains NNN/pFsFucT strain, NNN/pNbFucT1 strain, and NNN/pFucT54 strain were each cultured on an LB plate containing 100 mg/L kanamycin and 25 mg/L chloramphenicol at 30° C.
  • IPTG final concentration 1 mM
  • 480 g/L glucose solution and 40 g/L lactose monohydrate were added at a rate of 5 to 7 mL/h.
  • the culture medium was centrifuged and appropriately diluted, and the 2'FL and LNFP1 contained in the supernatant and the cells were analyzed. The results are shown in Table 13. The total amount of LNFP1 (g/L) is shown in a graph in Figure 5.
  • SEQ ID NOs: 1 to 37 Base sequences of primers SEQ ID NO:38: Base sequence of No. 1 SEQ ID NO:39: Base sequence of No. 2 SEQ ID NO:40: Base sequence of No. 3 SEQ ID NO:41: Base sequence of No. 4 SEQ ID NO:42: Base sequence of No. 5 SEQ ID NO:43: Base sequence of No. 6 SEQ ID NO:44: Base sequence of No. 7 SEQ ID NO:45: Base sequence of No.

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Abstract

La présente invention a pour but de procurer : une protéine présentant une activité α1,2-fucosyltransférase et une excellente productivité en LNFPI ; et un procédé de production de LNFPI. La présente invention concerne une protéine présentant une activité de transfert d'un groupe fucosyl dans le lacto-N-tétraose (LNT) et dans laquelle une mutation spécifique a été introduite (à l'exclusion d'une protéine présentant une propriété telle qu'une séquence d'acides aminés pour la protéine dans laquelle une mutation n'a pas encore été introduite et une séquence d'acides aminés pour la protéine dans laquelle la mutation a été introduite sont identiques l'une à l'autre).
PCT/JP2024/020349 2023-06-05 2024-06-04 PROTÉINE PRÉSENTANT UNE ACTIVITÉ DE α1,2-FUCOSYLTRANSFERASE ET PROCÉDÉ DE PRODUCTION DE LACTO-N-FUCOPENTAOSE I (LNFPI) Pending WO2024253084A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015529453A (ja) * 2012-07-25 2015-10-08 グリコシン リミテッド ライアビリティー カンパニー フコシル化オリゴ糖の生産における使用に適したアルファ（１，２）フコシルトランスフェラーゼ
US20180371432A1 (en) * 2015-12-18 2018-12-27 The Regents Of The University Of California Te2ft enzyme for enzymatic synthesis of alpha1-2-fucosides
JP2020526200A (ja) * 2017-07-07 2020-08-31 イェネヴァイン ビオテヒノロギー ゲーエムベーハー フコシル基転移酵素及びフコシル化オリゴ糖の生産におけるその使用
WO2023182528A1 (fr) * 2022-03-25 2023-09-28 キリンホールディングス株式会社 Protéine ayant une activité alpha 1,2-fucosyltransférase et procédé de production de lacto-n-fucopentaose i (lnfpi)

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015529453A (ja) * 2012-07-25 2015-10-08 グリコシン リミテッド ライアビリティー カンパニー フコシル化オリゴ糖の生産における使用に適したアルファ（１，２）フコシルトランスフェラーゼ
US20180371432A1 (en) * 2015-12-18 2018-12-27 The Regents Of The University Of California Te2ft enzyme for enzymatic synthesis of alpha1-2-fucosides
JP2020526200A (ja) * 2017-07-07 2020-08-31 イェネヴァイン ビオテヒノロギー ゲーエムベーハー フコシル基転移酵素及びフコシル化オリゴ糖の生産におけるその使用
WO2023182528A1 (fr) * 2022-03-25 2023-09-28 キリンホールディングス株式会社 Protéine ayant une activité alpha 1,2-fucosyltransférase et procédé de production de lacto-n-fucopentaose i (lnfpi)

Non-Patent Citations (2)

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Title
DATABASE GenBank 13 April 2019 (2019-04-13), .: "alpha-1,2-fucosyltransferase [Sulfuricystis multivorans]", XP093246026, Database accession no. WP_126446643 *
DATABASE GenBank 9 December 2016 (2016-12-09), .: "Alpha-1, 2-fucosyltransferase [Amphritea japonica]. NCBI Reference Sequence: WP_019622926.1. ", XP093246015, Database accession no. WP_019622926.1 *

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