Genetically modified microorganism and method for producing oligosaccharide

Abstract

The present invention pertains to a genetically modified microorganism that produces LNT and enhances the activity of at least one protein selected from the group consisting of MhpT, YfcJ, EmrD, YdeE, a GlcNAc permease, a GalNAc permease, a sugar MFS transporter derived from bacteria belonging to the genus Shewanella, Blon_2344, Blon_2343, Blon_2342, Blon_2347, Blon_2346, Blon_2345, Blon_2361, Blon_2360, Blon_2359, a Bifidobacterium breve-derived ABC transporter, and a Bifidobacterium breve-derived galactoside symporter.

Description

Genetically modified microorganism and method for producing oligosaccharides

[0001] The present invention relates to a genetically modified microorganism and a method for producing oligosaccharides.

[0002] Human milk oligosaccharides (HMOs) contained in human breast milk have attracted 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).

[0003] One of the major oligosaccharides in HMO is lacto-N-tetraose (hereinafter also referred to as "LNT") and its fucosylated or sialylated derivatives. LNT is found in relatively high amounts in breast milk, and therefore, the development of an efficient synthesis process for LNT is required to produce powdered milk similar to breast milk. Known methods for producing LNT include microbial fermentation using lactose as an additional substrate and introducing glycosyltransferases such as β-1,3-GlcNAc transferase and β1,3-galactosyl transferase. Chemical and enzymatic synthesis methods for LNT are also known, but have challenges such as process complexity (Non-Patent Document 2).

[0004] In the prior art, it has been reported that E. coli-derived MdfA is involved in the transport of LNT from inside the LNT-producing E. coli to outside the cell, and that overexpression of the gene encoding this protein improves LNT production (Patent Document 1). As transporters of HMOs other than LNT, E. coli-derived SetA protein, MdfA protein, and Neurospora crassa-derived CDT-2 protein, which are involved in the excretion of 2-fucosyllactose and 3-fucosyllactose, have been reported (Patent Documents 2 and 3, and Non-Patent Document 3).

[0005] International Publication No. 2017 / 042382 Japanese Patent No. 5580408 International Publication No. 2021 / 013708

[0006] Int J Pediatr. 2019 Aug 4;2019:2390240. Carbohydr Res. 2019 Dec 1;486:107824. Metab Eng. 2019 Mar;52:232-242.

[0007] As mentioned above, the functionality of HMOs including LNT has been attracting attention in recent years, and there is a need to further explore proteins that contribute to improving LNT productivity as a means of realizing efficient LNT production.

[0008] Therefore, an object of the present invention is to provide a microorganism with excellent LNT productivity and a method for producing oligosaccharides using the microorganism.

[0009] The present inventors have identified MhpT, YfcJ, EmrD, YdeE, GlcNAc permease, GalNAc permease, Shewanella-derived sugar Major Facilitator Superfamily (hereinafter also referred to as "MFS") transporter, Blon_2344, Blon_2343, Blon_2342, Blon_2347, Blon_2346, Blon_2345, Blon_2361, Blon_2360, Blon_2359, Bifidobacterium breve-derived ATP-binding enzyme, and The present inventors have found that a genetically modified microorganism in which the activity of at least one protein selected from the group consisting of an ABC transporter (hereinafter also referred to as "ABC transporter") and a Bifidobacterium breve-derived transporter (Bbr_1551) is enhanced has improved LNT productivity compared to the parent strain, and have completed the present invention.

[0010] That is, the present invention is as follows. <1> MhpT, YfcJ, EmrD, YdeE, GlcNAc permease, GalNAc permease, Shewanella-derived sugar MFS transporter, Blon_2344, Blon_2343, Blon_2342, Blon_2347, Blon_2346, Blon_2345, Blon_2361, Blon_2360, Blon_2359, Bifidobacterium breve-derived ABC transporter, and Bifidobacterium breve-derived galactoside symporter. A genetically modified microorganism that enhances the activity of at least one protein selected from the group consisting of: and produces LNT. <2> The genetically modified microorganism according to <1> above, wherein the GlcNAc permease is NagP derived from bacteria of the genus Shewanella, and the GalNAc permease is AgaP derived from bacteria of the genus Shewanella. <3> The genetically modified microorganism according to <1> or <2> above, in which the activity of the protein described in [1] below is enhanced. [1] A protein consisting of at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 64, 65, 66, 67, 68, 69, 70, 71, 72, 89, 106, 108, 110, and 112. <4> The genetically modified microorganism according to <1> or <2> above, in which the activity of the protein described in [2] or [3] below is enhanced and lacto-N-tetraose (LNT) productivity is improved compared to the parent strain. [2] A mutant protein consisting of an amino acid sequence in which 1 to 20 amino acids have been deleted, substituted, inserted or added in at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 64, 65, 66, 67, 68, 69, 70, 71, 72, 89, 106, 108, 110 and 112. [3] A homologous protein consisting of an amino acid sequence having 60% or more identity to at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 64, 65, 66, 67, 68, 69, 70, 71, 72, 89, 106, 108, 110 and 112.<5> The genetically modified microorganism according to <1> or <2> above, which has been modified by any one of the following [4] to [7]. [4] A modification that enhances the activity of each protein consisting of the amino acid sequences represented by SEQ ID NOs: 64, 65, and 66. [5] A modification that enhances the activity of each protein consisting of the amino acid sequences represented by SEQ ID NOs: 67, 68, and 69. [6] A modification that enhances the activity of each protein consisting of the amino acid sequences represented by SEQ ID NOs: 70, 71, and 72. [7] A modification that enhances the activity of each protein consisting of the amino acid sequences represented by SEQ ID NOs: 106, 108, and 110. <6> The genetically modified microorganism according to <1> or <2> above, which produces an oligosaccharide having an LNT backbone. <7> The genetically modified microorganism according to <6> above, wherein the oligosaccharide having an LNT backbone is at least one of lacto-N-fucopentaose I (LNFPI) and sialyllact-N-tetraose a (LST-a). <8> The genetically modified microorganism according to <1> or <2> above, wherein the genetically modified microorganism is Escherichia coli. <9> A method for producing oligosaccharides, comprising preparing the genetically modified microorganism according to <1> or <2> above, and producing oligosaccharides in at least one of a culture supernatant and intracellularly using the genetically modified microorganism. <10> The method for producing oligosaccharides according to <9> above, wherein the oligosaccharide is at least one selected from the group consisting of lacto-N-tetraose (LNT), lacto-N-fucopentaose I (LNFPI), and sialyllact-N-tetraose a (LST-a).

[0011] The microorganism according to this embodiment can improve LNT productivity by enhancing the activity of a specific protein. Therefore, by using the microorganism according to this embodiment, LNT can be produced efficiently.

[0012] FIG. 1 shows a schematic diagram of the biosynthetic pathway of LNT in a microorganism according to one embodiment of the present invention.

[0013] 1. Genetically modified microorganisms that produce LNT The microorganisms of this embodiment are those that contain MhpT, YfcJ, EmrD, YdeE, GlcNAc permease, GalNAc permease, Shewanella-derived sugar MFS transporter, Blon_2344, Blon_2343, Blon_2342, Blon_2347, Blon_2346, Blon_2345, Blon_2361, Blon_2360, Blon_2359, Bifidobacterium breve-derived ABC transporter, and Bifidobacterium breve-derived ABC transporter. The present invention relates to a genetically modified microorganism that produces LNT, in which the activity of at least one protein selected from the group consisting of L. breve-derived galactoside symporters is enhanced.

[0014] Here, "producing LNT" means producing LNT in the culture of the genetically modified microorganism, i.e., in the culture supernatant (also referred to as extracellular) and / or intracellularly. The amount of LNT produced can be confirmed by detecting LNT in the culture of the genetically modified microorganism using an analytical device or the like described below. The "total amount of LNT produced" is the sum of the quantified amount of extracellular LNT and the amount of intracellular LNT. The "extracellular ratio" refers to the ratio of the amount of extracellular LNT to the total amount of LNT produced (extracellular LNT amount / total LNT production amount). Furthermore, the microorganism of this embodiment may be a genetically modified microorganism that produces LNT in which the activity of at least one of the proteins membrane-bound lytic murein transglycosylase MltF and sodium:proton antiporter is enhanced.

[0015] The genetically modified microorganism is preferably a microorganism that produces LNT and has improved LNT productivity compared to a parent strain. Here, the produced LNT may be a final product or an intermediate product. Because LNT can also be a precursor to other oligosaccharides, the genetically modified microorganism that produces LNT can produce oligosaccharides, such as oligosaccharides having an LNT backbone. For example, the genetically modified microorganism may further produce oligosaccharides having an LNT backbone, which are the final product, using the LNT produced intracellularly as an intermediate.

[0016] <Oligosaccharides> In this specification, oligosaccharides refer to sugar polymers composed of at least three moieties, i.e., trisaccharides, tetrasaccharides, pentasaccharides, etc., and preferably include LNT and oligosaccharides having an LNT backbone.

[0017] As used herein, an oligosaccharide having an LNT backbone is preferably a human milk oligosaccharide containing LNT as the core tetrasaccharide. Note that, as used herein, a human milk oligosaccharide containing LNT as the "core tetrasaccharide" refers to a desired oligosaccharide containing LNT as the tetrasaccharide corresponding to the reducing end, and optionally containing other sugar moieties in addition to the specific tetrasaccharide corresponding to the main portion.

[0018] In this specification, examples of oligosaccharides having an LNT backbone include lacto-N-fucopentaose I (hereinafter also referred to as "LNFPI"), lacto-N-fucopentaose II (hereinafter also referred to as "LNFPII"), lacto-N-fucopentaose V (hereinafter also referred to as "LNFPV"), sialyllacto-N-tetraose-a (hereinafter also referred to as "LST-a"), sialyllacto-N-tetraose-b (hereinafter also referred to as "LST-b"), disialyllacto-N-tetraose (hereinafter also referred to as "DSLNT"), lacto-N-hexaose (hereinafter also referred to as "LNH"), lacto-N-difucosylhexaose I (hereinafter also referred to as "LNDFHI"), and lacto-N-difucosylhexaose II (hereinafter also referred to as "LNDFHII"). It should be noted that oligosaccharides having an LNT backbone do not include LNT.

[0019] In this embodiment, the oligosaccharide having an LNT backbone is preferably at least one of LNFPI and LST-a, and the precursor of LNFPI and LST-a is more preferably LNT.

[0020] In order for the genetically modified microorganism that produces LNT to produce LNFPI, it is preferable that the activity of α1,2-fucosyltransferase (hereinafter referred to as FucT) is further enhanced. Examples of FucT include FucT derived from Neisseriaceae bacterium DSM 100970, FucT derived from Yersinia sp., and FucT derived from Gramella forsetii (WO 2019 / 008133). Furthermore, the activity of at least one protein involved in the GDP-fucose biosynthetic pathway, such as phosphomannomutase (hereinafter referred to as ManB), mannose-1-phosphate guanylyltransferase (hereinafter referred to as ManC), GDP-mannose 4,6 dehydratase (hereinafter referred to as Gmd), or GDP-L-fucose synthetase (hereinafter referred to as Fcl), may be further enhanced. Methods for preparing genetically modified microorganisms in which the activity of proteins required for the biosynthesis of fucosylated oligosaccharides is enhanced include, for example, methods described in Japanese Patent Application Laid-Open No. 2015-529453 and International Publication No. 2019 / 008133.

[0021] In order for the genetically modified microorganism that produces LNT to produce LST-a, it is preferable that the activity of α-2,3-sialyltransferase (hereinafter referred to as siaT) and a protein required for the biosynthesis of sialic acid (also referred to as N-acetylneuraminic acid) be further enhanced. Examples of siaT include siaT derived from Pasteurella multocida PM70 (Japanese Patent No. 4275529) and siaT derived from the genus Photobacterium (Japanese Patent Publication No. 2020-528280). As proteins required for the biosynthesis of sialic acid, at least one activity may be further enhanced, such as UDP-N-acetylglucosamine-2-epimerase (hereinafter referred to as NeuC), N-acetylneuraminic acid synthase (hereinafter referred to as NeuB), or acylneuraminic acid cytidylyltransferase (hereinafter referred to as NeuA). Methods for preparing genetically modified microorganisms in which the activities of proteins required for the biosynthesis of sialic acid and sialylated oligosaccharides are enhanced include, for example, the methods described in Japanese Patent Nos. 4,275,529, 6,441,806, and 3,946,423.

[0022] The various oligosaccharides are also shown in Table 1.

[0023]

[0024] <Proteins with Enhanced Activity> (Escherichia coli Endogenous Transporters: MhpT, YfcJ, EmrD, YdeE) MhpT (3-hydroxyphenylpropionic transporter), YfcJ, EmrD (Multidrug resistance protein D), and YdeE are Escherichia coli endogenous transporters.

[0025] An example of the amino acid sequence of MhpT is the amino acid sequence represented by SEQ ID NO: 2. An example of the base sequence of DNA encoding MhpT is the base sequence represented by SEQ ID NO: 1. An example of the amino acid sequence of YfcJ is the amino acid sequence represented by SEQ ID NO: 4. An example of the base sequence of DNA encoding YfcJ is the base sequence represented by SEQ ID NO: 3. An example of the amino acid sequence of EmrD is the amino acid sequence represented by SEQ ID NO: 6. An example of the base sequence of DNA encoding EmrD is the base sequence represented by SEQ ID NO: 5. An example of the amino acid sequence of YdeE is the amino acid sequence represented by SEQ ID NO: 8. An example of the base sequence of DNA encoding YdeE is the base sequence represented by SEQ ID NO: 7.

[0026] (GlcNAc permease) Examples of GlcNAc permease include NagP. NagP derived from bacteria of the genus Shewanella is preferred. Specific examples of NagP derived from bacteria of the genus Shewanella include NagP derived from Shewanella sp. ANA3 (hereinafter also referred to as "NagP(ANA3)"), NagP derived from Shewanella amazonensis SB2B (hereinafter also referred to as "NagP(Sama)"), and NagP derived from Shewanella sp. MR4 (hereinafter also referred to as "NagP(MR4)"). Furthermore, the GlcNAc permease in this embodiment may have a function as membrane-bound lytic murein transglycosylase MltF or a sodium:proton antiporter.

[0027] An example of the amino acid sequence of NagP derived from Shewanella sp. ANA3 is the amino acid sequence represented by SEQ ID NO: 10. An example of the nucleotide sequence of DNA encoding NagP derived from Shewanella sp. ANA3 is the nucleotide sequence represented by SEQ ID NO: 9. An example of the nucleotide sequence of codon-optimized NagP (ANA3) in which bases have been substituted to provide codons optimal for expression in Escherichia coli is the nucleotide sequence represented by SEQ ID NO: 73. NagP derived from Shewanella sp. ANA3 is also known as membrane-bound lytic murein transglycosylase MltF.

[0028] An example of the amino acid sequence of Shewanella amazonensis SB2B-derived NagP is the amino acid sequence represented by SEQ ID NO: 12. An example of the nucleotide sequence of DNA encoding Shewanella amazonensis SB2B-derived NagP is the nucleotide sequence represented by SEQ ID NO: 11. An example of the nucleotide sequence of codon-optimized NagP (Sama), in which bases have been substituted to provide codons optimal for expression in Escherichia coli, is the nucleotide sequence represented by SEQ ID NO: 74. NagP from Shewanella amazonensis SB2B is also known as sodium:proton antiporter.

[0029] An example of the amino acid sequence of NagP derived from Shewanella sp. MR4 is the amino acid sequence represented by SEQ ID NO: 89. An example of the nucleotide sequence of DNA encoding NagP derived from Shewanella sp. MR4 is the nucleotide sequence represented by SEQ ID NO: 88. An example of the nucleotide sequence of codon-optimized NagP (MR4) in which bases have been substituted to provide codons optimal for expression in Escherichia coli is the nucleotide sequence represented by SEQ ID NO: 126. NagP derived from Shewanella sp. MR4 is also known as sugar MFS transporter.

[0030] (GalNAc permease) Examples of GalNAc permease include AgaP. AgaP derived from bacteria of the genus Shewanella is preferred as AgaP. Specific examples of AgaP derived from bacteria of the genus Shewanella include AgaP derived from Shewanella sp. ANA3 (hereinafter also referred to as "AgaP(ANA3)") and AgaP derived from Shewanella sp. MR-4 (hereinafter also referred to as "AgaP(MR4)").

[0031] An example of the amino acid sequence of AgaP derived from Shewanella sp. ANA3 is the amino acid sequence represented by SEQ ID NO: 14. An example of the nucleotide sequence of DNA encoding AgaP derived from Shewanella sp. ANA3 is the nucleotide sequence represented by SEQ ID NO: 13. An example of the nucleotide sequence of codon-optimized AgaP (ANA3) in which bases have been substituted to provide codons optimal for expression in E. coli is the nucleotide sequence represented by SEQ ID NO: 75.

[0032] An example of the amino acid sequence of AgaP derived from Shewanella sp. MR-4 is the amino acid sequence represented by SEQ ID NO: 16. An example of the nucleotide sequence of DNA encoding AgaP derived from Shewanella sp. MR-4 is the nucleotide sequence represented by SEQ ID NO: 15. An example of the nucleotide sequence of codon-optimized AgaP (MR4) in which bases have been substituted to provide codons optimal for expression in Escherichia coli is the nucleotide sequence represented by SEQ ID NO: 76.

[0033] (Sugar MFS transporter derived from bacteria of the genus Shewanella) Specific examples of sugar MFS transporters derived from bacteria of the genus Shewanella include NagP (MR4), AgaP (ANA3), AgaP (MR4), and the like.

[0034] (Blon_2344, Blon_2343, Blon_2342, Blon_2347, Blon_2346, Blon_2345, Blon_2361, Blon_2360, Blon_2359) Blon_2344, Blon_2343, Blon_2342, Blon_2347, Blon_2346, Blon_2345, Blon_2361, Blon_2360, Blon_2359 are Bifidobacterium longum subsp. Infantis-derived ABC transporter.

[0035] An example of the amino acid sequence of Blon_2344 is the amino acid sequence represented by SEQ ID NO: 64. An example of the nucleotide sequence of DNA encoding Blon_2344 is the nucleotide sequence represented by SEQ ID NO: 77. An example of the amino acid sequence of Blon_2343 is the amino acid sequence represented by SEQ ID NO: 65. An example of the nucleotide sequence of DNA encoding Blon_2343 is the nucleotide sequence represented by SEQ ID NO: 78. An example of the amino acid sequence of Blon_2342 is the amino acid sequence represented by SEQ ID NO: 66. An example of the nucleotide sequence of DNA encoding Blon_2342 is the nucleotide sequence represented by SEQ ID NO: 79. An example of a nucleotide sequence comprising DNA encoding Blon_2344, DNA encoding Blon_2343, and DNA encoding Blon_2342 is the nucleotide sequence represented by SEQ ID NO: 17.

[0036] An example of the amino acid sequence of Blon_2347 is the amino acid sequence represented by SEQ ID NO: 67. An example of the nucleotide sequence of DNA encoding Blon_2347 is the nucleotide sequence represented by SEQ ID NO: 80. An example of the amino acid sequence of Blon_2346 is the amino acid sequence represented by SEQ ID NO: 68. An example of the nucleotide sequence of DNA encoding Blon_2346 is the nucleotide sequence represented by SEQ ID NO: 81. An example of the amino acid sequence of Blon_2345 is the amino acid sequence represented by SEQ ID NO: 69. An example of the nucleotide sequence of DNA encoding Blon_2345 is the nucleotide sequence represented by SEQ ID NO: 82. An example of a nucleotide sequence comprising DNA encoding Blon_2347, DNA encoding Blon_2346, and DNA encoding Blon_2345 is the nucleotide sequence represented by SEQ ID NO: 18.

[0037] An example of the amino acid sequence of Blon_2361 is the amino acid sequence represented by SEQ ID NO: 70. An example of the nucleotide sequence of DNA encoding Blon_2361 is the nucleotide sequence represented by SEQ ID NO: 83. An example of the amino acid sequence of Blon_2360 is the amino acid sequence represented by SEQ ID NO: 71. An example of the nucleotide sequence of DNA encoding Blon_2360 is the nucleotide sequence represented by SEQ ID NO: 84. An example of the amino acid sequence of Blon_2359 is the amino acid sequence represented by SEQ ID NO: 72. An example of the nucleotide sequence of DNA encoding Blon_2359 is the nucleotide sequence represented by SEQ ID NO: 85. An example of a nucleotide sequence comprising DNA encoding Blon_2361, DNA encoding Blon_2360, and DNA encoding Blon_2359 is the nucleotide sequence represented by SEQ ID NO: 19.

[0038] A genetically modified microorganism in which the activity of at least one protein selected from the group consisting of Blon_2344, Blon_2343, Blon_2342, Blon_2347, Blon_2346, Blon_2345, Blon_2361, Blon_2360, and Blon_2359 is enhanced may not have improved productivity of lacto-N-neotetraose (LNnT) compared to the parent strain.

[0039] (ABC Transporters Derived from Bifidobacterium breve) Examples of ABC transporters derived from Bifidobacterium breve include Bbr_1590, Bbr_1589, and Bbr_1588.

[0040] An example of the amino acid sequence of Bbr_1590 is the amino acid sequence represented by SEQ ID NO: 106. An example of the nucleotide sequence of DNA encoding Bbr_1590 is the nucleotide sequence represented by SEQ ID NO: 122. An example of the nucleotide sequence of codon-optimized Bbr_1590 in which bases have been substituted to provide codons optimal for expression in E. coli is the nucleotide sequence represented by SEQ ID NO: 107.

[0041] An example of the amino acid sequence of Bbr_1589 is the amino acid sequence represented by SEQ ID NO: 108. An example of the nucleotide sequence of DNA encoding Bbr_1589 is the nucleotide sequence represented by SEQ ID NO: 123. An example of the nucleotide sequence of codon-optimized Bbr_1589 in which bases have been substituted to provide codons optimal for expression in E. coli is the nucleotide sequence represented by SEQ ID NO: 109.

[0042] An example of the amino acid sequence of Bbr_1588 is the amino acid sequence represented by SEQ ID NO: 110. An example of the nucleotide sequence of DNA encoding Bbr_1588 is the nucleotide sequence represented by SEQ ID NO: 124. An example of the nucleotide sequence of codon-optimized Bbr_1588 in which bases have been substituted to provide codons optimal for expression in E. coli is the nucleotide sequence represented by SEQ ID NO: 111.

[0043] (Galactoside Symporter Derived from Bifidobacterium breve) Examples of the galactoside symporter derived from Bifidobacterium breve include Bbr_1551. An example of the amino acid sequence of Bbr_1551 is the amino acid sequence represented by SEQ ID NO: 112. An example of the base sequence of DNA encoding Bbr_1551 is the base sequence represented by SEQ ID NO: 125. An example of the base sequence of codon-optimized Bbr_1551 in which bases have been substituted to provide codons optimal for expression in Escherichia coli is the base sequence represented by SEQ ID NO: 113.

[0044] <Genetically Modified Microorganism> In this embodiment, the genetically modified microorganism is preferably a genetically modified microorganism in which the activity of the protein described in [1] below is enhanced. Such genetically modified microorganisms have improved LNT productivity compared to the parent strain. [1] A protein consisting of at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 64, 65, 66, 67, 68, 69, 70, 71, 72, 89, 106, 108, 110, and 112.

[0045] In this specification, "a protein consisting of the amino acid sequences represented by SEQ ID NOs: X, Y, and Z" may be "one protein consisting of the amino acid sequence represented by SEQ ID NO: X, the amino acid sequence represented by SEQ ID NO: Y, and the amino acid sequence represented by SEQ ID NO: Z," or may be "a combination of a protein consisting of the amino acid sequence represented by SEQ ID NO: X, a protein consisting of the amino acid sequence represented by SEQ ID NO: Y, and a protein consisting of the amino acid sequence represented by SEQ ID NO: Z." Here, X, Y, and Z are any integers.

[0046] In terms of the amount of extracellular LNT produced, the protein described in [1] above is more preferably a protein consisting of the amino acid sequence represented by SEQ ID NO: 8, a protein consisting of the amino acid sequence represented by SEQ ID NO: 10, a protein consisting of the amino acid sequence represented by SEQ ID NO: 12, a protein consisting of the amino acid sequence represented by SEQ ID NO: 16, a protein consisting of the amino acid sequence represented by SEQ ID NOs: 64 to 66, a protein consisting of the amino acid sequence represented by SEQ ID NOs: 67 to 69, or a protein consisting of the amino acid sequence represented by SEQ ID NOs: 70 to 72. Furthermore, a protein consisting of the amino acid sequence represented by SEQ ID NO: 8, a protein consisting of the amino acid sequence represented by SEQ ID NO: 10, a protein consisting of the amino acid sequence represented by SEQ ID NO: 12, or a protein consisting of the amino acid sequence represented by SEQ ID NOs: 70 to 72 is most preferred.

[0047] In terms of total LNT production volume, the protein described in [1] above is more preferably a protein consisting of the amino acid sequence represented by SEQ ID NO: 4, a protein consisting of the amino acid sequence represented by SEQ ID NO: 6, a protein consisting of the amino acid sequence represented by SEQ ID NO: 8, a protein consisting of the amino acid sequence represented by SEQ ID NO: 10, a protein consisting of the amino acid sequence represented by SEQ ID NO: 12, a protein consisting of the amino acid sequence represented by SEQ ID NO: 16, a protein consisting of the amino acid sequence represented by SEQ ID NO: 64 to 66, a protein consisting of the amino acid sequence represented by SEQ ID NO: 67 to 69, or a protein consisting of the amino acid sequence represented by SEQ ID NO: 70 to 72. Furthermore, a protein consisting of the amino acid sequence represented by SEQ ID NO: 8, a protein consisting of the amino acid sequence represented by SEQ ID NO: 16, a protein consisting of the amino acid sequence represented by SEQ ID NO: 64 to 66, a protein consisting of the amino acid sequence represented by SEQ ID NO: 67 to 69, and a protein consisting of the amino acid sequence represented by SEQ ID NO: 70 to 72 are most preferred.

[0048] Furthermore, the genetically modified microorganism of this embodiment is also preferably a genetically modified microorganism in which the activity of the protein described in [2] or [3] below is enhanced and LNT productivity is improved compared to the parent strain: [2] A mutant protein consisting of an amino acid sequence represented by at least one selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 64, 65, 66, 67, 68, 69, 70, 71, 72, 89, 106, 108, 110, and 112, in which 1 to 20 amino acids have been deleted, substituted, inserted, or added. [3] A homologous protein consisting of an amino acid sequence having 60% or more identity with at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 64, 65, 66, 67, 68, 69, 70, 71, 72, 89, 106, 108, 110, and 112.

[0049] The genetically modified microorganism of this embodiment also includes a genetically modified microorganism having any one of the modifications described in [4] to [7] below. [4] A modification that enhances the activity of each protein consisting of the amino acid sequences represented by SEQ ID NOs: 64, 65, and 66. [5] A modification that enhances the activity of each protein consisting of the amino acid sequences represented by SEQ ID NOs: 67, 68, and 69. [6] A modification that enhances the activity of each protein consisting of the amino acid sequences represented by SEQ ID NOs: 70, 71, and 72. [7] A modification that enhances the activity of each protein consisting of the amino acid sequences represented by SEQ ID NOs: 106, 108, and 110.

[0050] Examples of genetically modified microorganisms of this embodiment include genetically modified microorganisms in which the activity of a protein consisting of at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8, and the activity of a protein consisting of at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, 16, 64, 65, 66, 67, 68, 69, 70, 71, 72, 89, 106, 108, 110, and 112, are both enhanced.

[0051] (Productivity) In this specification, productivity refers to the ability of a microorganism to accumulate a target oligosaccharide produced by the microorganism in a culture of the microorganism, i.e., in the culture supernatant (also referred to as extracellular) and / or intracellular. The productivity of oligosaccharides by a microorganism can be confirmed by detecting at least one of the oligosaccharides in the culture supernatant and intracellular of the microorganism using an analytical device or the like described below. Productivity is also referred to as production amount.

[0052] The genetically modified microorganism of this embodiment preferably has improved LNT productivity, and more preferably has an improved extracellular ratio of the produced LNT, compared to the parent strain.

[0053] Furthermore, a microorganism in which the activity of a protein encoding at least one gene selected from the group consisting of E. coli endogenous transporters EmrD, YdeE, NagP derived from Shewanella sp. ANA3, NagP derived from Shewanella amazonensis SB2B, AgaP derived from Shewanella sp. ANA3, AgaP derived from Shewanella sp. MR4, Blon_2344-2342, Blon_2347-2345, and Blon_2361-2359 genes, has improved extracellular and total LNT productivity and particularly improved extracellular LNT ratio, compared to the parent strain.

[0054] Examples of microorganisms in which the activity of the protein according to any one of [1] to [7] above has been enhanced include microorganisms in which the copy number of the gene is increased compared to that of the parent strain, and which are obtained by transforming a parent strain of a microorganism with a recombinant DNA containing DNA encoding the protein according to any one of [1] to [7] above. In this specification, increasing the copy number of the gene may mean newly introducing the gene into a parent strain that does not have the gene on a chromosome or a plasmid, or may mean additionally introducing the gene into a strain that has the gene on a chromosome or a plasmid.

[0055] As used herein, a mutant protein refers to a protein obtained by artificially deleting or substituting amino acid residues in an original protein, or by inserting or adding amino acid residues into the protein.

[0056] In the mutant protein of [2] above, the deletion, substitution, insertion, or addition of amino acids may mean the deletion, substitution, insertion, or addition of 1 to 20 amino acids at any position within the same sequence. The number of amino acids deleted, substituted, inserted, or added is preferably 1 to 10, more preferably 1 to 8, and most preferably 1 to 5.

[0057] The amino acids to be deleted, substituted, inserted, or added may be naturally occurring or non-naturally occurring. Naturally occurring 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.

[0058] Examples of amino acids that can be substituted for each other are shown below. Amino acids in the same group can be substituted for each other. 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

[0059] The mutant protein preferably has oligosaccharide transport activity.

[0060] As used herein, a homologous protein refers to a protein that is found in organisms that exist in nature and is similar in structure and function to the original protein, such that the gene encoding the protein is considered to have the same evolutionary origin as the gene encoding the original protein.

[0061] Examples of homologous proteins include amino acid sequences that share 60% or more identity with the amino acid sequence of the target protein, preferably 65% ​​or more, 70% or more, 75% or more, 80% or more, 85% or more, more preferably 90% or more, and particularly preferably 95% or more identity. Methods for confirming amino acid sequence identity are described below.

[0062] The homologous protein preferably has oligosaccharide transport activity.

[0063] (Parent strain) As used herein, a parent strain refers to an original strain that is the target of genetic modification, transformation, etc. An original strain that is the target of transformation by gene introduction is also referred to as a host strain.

[0064] In the present specification, the parent strain is preferably a prokaryote or a yeast strain, more preferably a prokaryote belonging to the genus Escherichia such as Escherichia coli, 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 MG1655, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, ...MG1655, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli MG1655, Escherichia coli MG1655, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli MG1655, Escherichia KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli49, Escherichia coli W3110, Escherichia coli W3110S, Escherichia coli NY49, Escherichia coli BL21 codon plus (manufactured by Stratagene), Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium immariophilum ATCC14068, Brevibacterium saccharolyticum ATCC14066, Corynebacterium ammoniagenes, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869, Corynebacterium acetoacidophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354,or Pseudomonas sp. D-0110, or yeast strains such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris, or Candida utilis.

[0065] The parent strain may be a wild-type strain as long as it is a microorganism that produces LNT, or if the wild-type strain does not have the ability to produce LNT, it may be a bred strain that has been artificially imparted with the ability to produce LNT.

[0066] Examples of methods for artificially imparting the ability to produce LNT to a microorganism include the following methods (1a) to (1e), which can be used alone or in combination: (1a) a method for alleviating or deactivating at least one of the mechanisms controlling the biosynthetic pathway for producing LNT, (1b) a method for enhancing the expression of at least one of the enzymes involved in the biosynthetic pathway for producing LNT, (1c) a method for increasing the copy number of at least one of the enzyme genes involved in the biosynthetic pathway for producing LNT, (1d) a method for weakening or blocking at least one of the metabolic pathways branching off from the biosynthetic pathway for producing LNT to a metabolic product other than the target substance, and (1e) a method for selecting a cell line that is more resistant to an LNT analog than a wild-type strain.

[0067] When the genetically modified microorganism of this embodiment produces oligosaccharides having an LNT backbone and LNT, the parent strain may be a wild-type strain as long as it is a microorganism that produces oligosaccharides having an LNT backbone and LNT. If the wild-type strain does not have the ability to produce oligosaccharides having an LNT backbone and LNT, the parent strain may be a bred strain that has been artificially imparted with the ability to produce oligosaccharides having an LNT backbone and LNT.

[0068] Methods for artificially imparting the ability to produce oligosaccharides having an LNT backbone and LNT to a microorganism include the methods described in (2a) to (2e) below, which can be used alone or in combination: (2a) A method for alleviating or canceling at least one of the mechanisms controlling the biosynthetic pathway for producing oligosaccharides having an LNT backbone and LNT; (2b) A method for enhancing the expression of at least one of the enzymes involved in the biosynthetic pathway for producing oligosaccharides having an LNT backbone and LNT; (2c) A method for increasing the copy number of at least one of the enzyme genes involved in the biosynthetic pathway for producing oligosaccharides having an LNT backbone and LNT; (2d) A method for weakening or blocking at least one of the metabolic pathways branching off from the biosynthetic pathway for producing oligosaccharides having an LNT backbone and LNT to metabolites other than the target substance; and (2e) A method for selecting a cell line that is more resistant to oligosaccharides having an LNT backbone and LNT analogs than a wild-type strain.

[0069] Whether a microorganism is capable of producing LNT can be confirmed by transforming the microorganism with a recombinant DNA having a DNA encoding a protein consisting of at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 64, 65, 66, 67, 68, 69, 70, 71, 72, 89, 106, 108, 110, and 112, culturing the transformed microorganism in a medium, and detecting LNT accumulated in at least one of the culture supernatant and the bacterial cells using a sugar analyzer or the like described below. Whether a microorganism is capable of producing oligosaccharides having an LNT backbone and LNT can be confirmed by transforming the microorganism with a recombinant DNA having a DNA encoding a protein consisting of at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 64, 65, 66, 67, 68, 69, 70, 71, 72, 89, 106, 108, 110, and 112, culturing the transformed microorganism in a medium, and detecting the oligosaccharides having an LNT backbone and LNT accumulated in at least one of the culture supernatant and the bacterial cells using a sugar analyzer or the like described below.

[0070] The parent strain preferably has at least one activity selected from the group consisting of lactose permease (hereinafter referred to as LacY) activity, β1,3-N-acetylglucosamine transferase (hereinafter referred to as LgtA) activity, GlmS activity, GlmM activity, GlmU activity, β1,3-galactosyltransferase (hereinafter referred to as GalT) 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 more preferably has an enhanced activity. Among these, it is more preferable that the enzyme has at least one activity selected from the group consisting of LacY, LgtA, and GalT, and it is even more preferable that the activity is enhanced.

[0071] When the genetically modified microorganism of this embodiment produces oligosaccharides having an LNT backbone and LNT, the parent strain may exhibit the LacY activity, the LgtA activity, the GlmS activity, the GlmM activity, the GlmU activity, the GalT activity, the Pgm activity, the GalU activity, the GalE activity, the GalF activity, the Pgi activity, the mannose-6-phosphate isomerase (hereinafter referred to as ManA) activity, the phosphomannomutase (hereinafter referred to as ManB) activity, the mannose-1-phosphate guanylyltransferase (hereinafter referred to as ManC) activity, the GDP-mannose 4,6 dehydratase activity, the ATP ... Preferably, the mutant strain has at least one activity selected from the group consisting of α-1,2-fucosyltransferase (hereinafter referred to as Gmd), GDP-L-fucose synthetase (hereinafter referred to as Fcl), UDP-N-acetylglucosamine-2-epimerase (hereinafter referred to as NeuC), N-acetylneuraminic acid synthase (hereinafter referred to as NeuB), acylneuraminic acid cytidylyltransferase (hereinafter referred to as NeuA), α1,2-fucosyltransferase activity, and α-2,3-sialyltransferase activity, and more preferably, the activity is enhanced. Of these, it is more preferred that the mutant strain has at least one activity selected from the group consisting of LacY, LgtA, GalT, FucT, and SiaT, and even more preferably, the activity is enhanced.

[0072] When the genetically modified microorganism of this embodiment produces oligosaccharides having an LNT backbone, preferably at least one of LNFPI and LST-a, the parent strain preferably has at least one of FucT activity and SiaT activity, and more preferably has enhanced activity.

[0073] As shown in Figure 1, LacY is a membrane protein that takes up lactose, the substrate for LNT, into cells. As shown in Figure 1, LgtA is an enzyme involved in the production of LNTII from lactose and uridine diphosphate-N-acetylglucosamine (hereinafter referred to as UDP-GlcNAc). GlmS, GlmM, and GlmU are enzymes involved in the biosynthetic pathway that produces UDP-GlcNAc, which is involved in the production of LNTII from lactose. GalT is an enzyme involved in the production of LNT from LNTII, as shown in Figure 1. Pgm, GalU, GalE, and GalF are enzymes involved in the pathway that produces uridine diphosphate galactose (hereinafter referred to as UDP-Gal), which is involved in the production of LNT from LNTII. Pgi is an enzyme involved in the pathway that produces LNTII. ManA, ManB, ManC, Gmd, and Fcl are enzymes involved in the supply of GDP-fucose required for the production of LNFPI. NeuC, NeuB, and NeuA are enzymes involved in the supply of CMP-sialic acid required for the production of LST-a or LST-b. FucT is an enzyme involved in the pathway that produces LNFPI from LNT and fucose. SiaT is an enzyme involved in the pathway that produces LST-a or LST-b from LNT.

[0074] Therefore, in this embodiment, preferably, the nucleotide sequence encoding LacY (e.g., accession no. BAE76125), a nucleotide sequence encoding LgtA (e.g., SEQ ID NO: 22), a nucleotide sequence encoding GlmS (e.g., Accession No. BAE77559), a nucleotide sequence encoding GlmM (e.g., Accession No. BAE77220), a nucleotide sequence encoding GlmU (e.g., Accession No. BAE77558), a nucleotide sequence encoding GalT (e.g., SEQ ID NO: 20), a nucleotide sequence encoding Pgm (e.g., Accession No. BAA35337.1), a nucleotide sequence encoding GalU (e.g., Accession No. BAA36104.1), a nucleotide sequence encoding GalE (e.g., Accession No. BAA35421.1), a nucleotide sequence encoding GalF (e.g., Accession No. BAA15896.1), or a nucleotide sequence encoding Pgi (e.g., Accession No. BAE78027.1). In particular, it is more preferable to use a genetically modified microorganism as the parent strain, preferably containing a nucleotide sequence encoding LacY, a nucleotide sequence encoding LgtA, and a nucleotide sequence encoding GalT. In this embodiment, it is preferable that the genetically modified microorganism has increased LNT productivity compared to a non-genetically modified parent strain.

[0075] In addition, in a genetically modified microorganism that produces an oligosaccharide having an LNT backbone and LNT, which is an example of this embodiment, a base sequence encoding LacY (e.g., accession number BAE76125), a nucleotide sequence encoding LgtA (e.g., SEQ ID NO: 22), a nucleotide sequence encoding GlmS (e.g., Accession No. BAE77559), a nucleotide sequence encoding GlmM (e.g., Accession No. BAE77220), a nucleotide sequence encoding GlmU (e.g., Accession No. BAE77558), a nucleotide sequence encoding GalT (e.g., SEQ ID NO: 20), a nucleotide sequence encoding Pgm (e.g., Accession No. BAA35337.1), a nucleotide sequence encoding GalU (e.g., Accession No. BAA36104.1), a nucleotide sequence encoding GalE (e.g., Accession No. BAA35421.1), a nucleotide sequence encoding GalF (e.g., Accession No. BAA15896.1), a nucleotide sequence encoding Pgi (e.g., Accession No. BAE78027.1), a nucleotide sequence encoding ManA ... Preferably, the parent strain is a genetically modified microorganism containing at least one nucleotide sequence selected from the group consisting of a nucleotide sequence encoding ManB (e.g., accession number BAA15361), a nucleotide sequence encoding ManB (e.g., accession number BAA15901.1), a nucleotide sequence encoding ManC (e.g., accession number BAA15905), a nucleotide sequence encoding Gmd (e.g., accession number BAA15909), a nucleotide sequence encoding Fcl (e.g., accession number BAA15908), a nucleotide sequence encoding NeuC (e.g., accession number CAL43338), a nucleotide sequence encoding NeuB (e.g., accession number YP_002344534), a nucleotide sequence encoding NeuA (e.g., accession number AAK02271), a nucleotide sequence encoding FucT (e.g., accession number BBB25221), and a nucleotide sequence encoding SiaT (e.g., accession number BAA25316).

[0076] Known methods may be used for producing Escherichia coli having at least one activity selected from LacY activity, LgtA activity, GlmS activity, GlmM activity, GlmU activity, GalT activity, Pgm activity, GalU activity, GalE activity, GalF activity, and Pgi activity, or in which the activity has been enhanced, and for producing Escherichia coli having at least one activity selected from the group consisting of LacY activity, LgtA activity, GlmS activity, GlmM activity, GlmU activity, GalT activity, Pgm activity, GalU activity, GalE activity, GalF activity, Pgi activity, ManA activity, ManB activity, ManC activity, Gmd activity, Fcl activity, NeuC activity, NeuB activity, NeuA activity, FucT activity, and SiaT activity, or in which the activity has been enhanced. Specific examples include methods using various genetic manipulations (Metabolic Engineering, 2017, 41: 23-38).

[0077] In the parent strain, it is preferred that β-galactosidase (hereinafter referred to as LacZ) activity is reduced or deleted.

[0078] When the genetically modified microorganism of this embodiment produces oligosaccharides having an LNT backbone and LNT, it is preferable that the parent strain has reduced or no WcaJ activity and WcaJM activity in addition to the above-mentioned LacZ activity.

[0079] LacZ is an enzyme that hydrolyzes lactose, the substrate of LNT. Therefore, by inhibiting LacZ activity, the decrease in lactose supply can be suppressed. WcaJ and WcaJM are enzymes involved in the metabolism of GDP-fucose to colanic acid, and by blocking them, the supply of GDP-fucose can be increased.

[0080] Therefore, in this embodiment, it is preferable to use a genetically modified microorganism as a parent strain, which preferably has reduced or deleted LacZ activity, and more preferably does not contain a nucleotide sequence encoding LacZ. In this embodiment, it is preferable that the genetically modified microorganism has increased LNT productivity compared to a non-genetically modified parent strain.

[0081] In this embodiment, in a genetically modified microorganism that produces oligosaccharides having an LNT backbone and LNT, it is preferable to use a genetically modified microorganism as a parent strain in which at least one activity selected from the group consisting of LacZ, WcaJ, and WcaJM is reduced or deleted, and more preferably, the parent strain does not contain at least one base sequence selected from the group consisting of a base sequence encoding LacZ, a base sequence encoding WcaJ, and a base sequence encoding WcaJM.

[0082] A method for producing E. coli in which at least one activity selected from the group consisting of LacZ activity, WcaJ activity, and WcaJM activity has been reduced or eliminated may be performed using a known method, specifically, for example, a method using various genetic manipulations (Metabolic Engineering, 2017, 41:23-38).

[0083] <Production of Genetically Modified Microorganisms> Examples of genetically modified microorganisms in which the activity of a protein described in any one of [1] to [3] above is enhanced compared to a parent strain of a microorganism include microorganisms in which the copy number of the gene is increased compared to the parent strain, and which are obtained by transforming a parent strain of a microorganism with recombinant DNA containing DNA encoding the protein.

[0084] Examples of microorganisms that have an increased copy number of a gene compared to a parent strain, and that can be obtained by transforming a parent strain microorganism with a recombinant DNA containing a DNA encoding a protein according to any one of [1] to [3] above, include microorganisms in which the copy number of the gene on chromosomal DNA has been increased by transforming a parent strain microorganism with a recombinant DNA containing a DNA encoding a protein according to any one of [1] to [3] above, and microorganisms in which the gene is carried outside of chromosomal DNA as plasmid DNA.

[0085] The DNA encoding the protein according to any one of [1] to [3] above may be any DNA encoding a protein having the activity of the protein according to any one of [1] to [3] above, and specifically includes one DNA selected from the group consisting of [8] to

[11] below: [8] DNA encoding the protein according to any one of [1] to [3] above [9] DNA consisting of at least one base sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 18, 19, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 88, 107, 109, 111, 113, 122, 123, 124, 125, and 126

[10] A DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to at least one base sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 18, 19, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 88, 107, 109, 111, 113, 122, 123, 124, 125, and 126, and encodes the homologous protein according to [3].

[11] A DNA encoding the homologous protein according to [3], which comprises a base sequence having 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with at least one base sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 18, 19, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 88, 107, 109, 111, 113, 122, 123, 124, 125, and 126.

[0086] Examples of recombinant DNAs containing DNA encoding the protein according to any one of [1] to [3] above include recombinant DNAs containing DNA according to any one of [8] to

[11] above. The recombinant DNA containing DNA according to any one of [8] to

[11] above refers to recombinant DNA in which the DNA is incorporated into an expression vector that is capable of autonomous replication in a parent strain or integration into a chromosome and contains a promoter at a position where the DNA can be transcribed.

[0087] In the above

[10] , "hybridize" means that DNA hybridizes to DNA having a specific base sequence or a part of the DNA. Therefore, the DNA having the specific base sequence or a part of the DNA can be used as a probe in Northern or Southern blot analysis, and can also be used as an oligonucleotide primer in PCR analysis.

[0088] The DNA used as a probe may be at least 100 bases long, preferably at least 200 bases long, more preferably at least 500 bases long.The DNA used as a primer may be at least 10 bases long, preferably at least 15 bases long.

[0089] Methods for DNA hybridization experiments are well known, and those skilled in the art can determine hybridization conditions according to the present specification, for example, as described in Molecular Cloning, 4th Edition (2012), Methods for General and Molecular Bacteriology, ASM Press (1994), Immunology Methods Manual, Academic Press (1996), and many other standard textbooks.

[0090] Alternatively, DNA that hybridizes under stringent conditions can be obtained by following the instructions provided with a commercially available hybridization kit, such as the Random Primed DNA Labeling Kit (manufactured by Roche Diagnostics), which prepares a probe by the random prime method and hybridizes under stringent conditions.

[0091] The above-mentioned stringent conditions include incubating the DNA-immobilized filter and the probe DNA overnight at 42°C in a solution containing 50% formamide, 5xSSC (750 mmol / L sodium chloride, 75 mmol / L sodium citrate), 50 mmol / L sodium phosphate (pH 7.6), 5xDenhardt's solution, 10% dextran sulfate, and 20 μg / L denatured salmon sperm DNA, followed by washing the filter in a 0.2xSSC solution at approximately 65°C.

[0092] Examples of DNA that can hybridize under the above-mentioned stringent conditions include DNA that has at least 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity to DNA consisting of at least one base sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 18, 19, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 88, 107, 109, 111, 113, 122, 123, 124, 125, and 126, when calculated based on the above-mentioned parameters using, for example, BLAST or FASTA.

[0093] The identity of amino acid sequences and nucleotide 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)]. Based on this algorithm BLAST, programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215, 403 (1990)]. When analyzing a nucleotide sequence using BLASTN based on BLAST, the parameters are, for example, Score = 100 and wordlength = 12. When analyzing an amino acid sequence using BLASTX based on BLAST, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific techniques for these analysis methods are known.

[0094] A microorganism having an increased copy number of the gene compared to the parent strain, which is obtained by transforming a parent strain microorganism with a recombinant DNA containing a DNA encoding the protein according to any one of [1] to [3] above, can be obtained by the following method.

[0095] The DNA encoding the protein of [1] described in [8] above and the DNA of [9] described above can be isolated by, for example, Southern hybridization of a chromosomal DNA library of a microorganism, preferably a microorganism belonging to the genus Escherichia coli, more preferably the Escherichia coli W3110S strain, using a probe DNA that can be designed based on at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 18, 19, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 88, 107, 109, 111, 113, 122, 123, 124, 125, and 126, or PCR using primer DNA that can be designed based on the nucleotide sequence and the chromosomal DNA of the microorganism as a template [PCR Protocols, Academic Press, (1990)].

[0096] The Escherichia coli W3110S strain is available from the National Institute of Technology and Evaluation (NITE) Biological Resource Center.

[0097] The DNA encoding the mutant protein of [2] above can be obtained, for example, by subjecting DNA consisting of at least one base sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 18, 19, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 88, 107, 109, 111, 113, 122, 123, 124, 125, and 126, as a template to error-prone PCR or the like.

[0098] Alternatively, DNA encoding the mutant protein of the above [2] can be obtained by PCR using a pair of PCR primers each having a nucleotide sequence at its 5' end designed to introduce a desired mutation (deletion, substitution, insertion, or addition) [Gene, 77, 51 (1989)].

[0099] Alternatively, the DNA can be obtained by following the instructions provided with a commercially available site-directed mutagenesis kit, such as the PrimeSTAR® Mutagenesis Basal Kit (manufactured by Takara Bio Inc.), which can introduce a mutation (deletion, substitution, insertion, or addition) at the desired site.

[0100] That is, first, a pair of mutagenesis primers is designed with a template of a plasmid having a base sequence designed to introduce the desired mutation (deletion, substitution, insertion, or addition), with a 15-base overlap on the 5' side. The overlapping portion contains the desired mutation. Next, PCR is performed using the mutagenesis primers and a template of a plasmid having the base sequence into which the desired mutation is to be introduced. The amplified fragment obtained is transformed into Escherichia coli, yielding a plasmid having the base sequence into which the desired mutation has been introduced.

[0101] The DNA encoding the homologous protein of [3] above and the DNAs of

[10] and

[11] above can be obtained by searching various gene sequence databases for a base sequence having an identity of 60% or more, preferably 65% ​​or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more, with at least one base sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 18, 19, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 88, 107, 109, 111, 113, 122, 123, 124, 125, and 126, in the following order: A database is searched for amino acid sequences that have 60% or more identity, preferably 65% ​​or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity to at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 64, 65, 66, 67, 68, 69, 70, 71, 72, 89, 106, 108, 110, and 112, in the following order: identities; and 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 the DNA, in a manner similar to the method for obtaining DNA described above.

[0102] Based on the DNA encoding the protein according to any one of [1] to [3] above, preferably one selected from the group consisting of [8] to

[11] above, obtained by the above method, a DNA fragment of an appropriate length containing a portion encoding the protein is prepared as needed. Furthermore, by substituting bases in the base sequence of the portion encoding the protein so that it contains codons optimal for expression in host cells, a transformant with improved productivity can be obtained. Information on codon usage in the parent strain used in the production method of this embodiment is available from public databases.

[0103] The DNA fragment is inserted downstream of a promoter in an appropriate expression vector to prepare a recombinant DNA, which is then transformed into a parent strain to obtain a microorganism in which the copy number of the gene encoding the protein is increased compared to the parent strain.

[0104] When a prokaryote such as a bacterium is used as a parent strain, the recombinant DNA is preferably composed of a promoter, a ribosome binding sequence, the DNA described in any one of [8] to

[11] above, and a transcription termination sequence. A gene that controls the promoter may also be included. It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is the ribosome binding sequence, and the start codon is adjusted to an appropriate distance (e.g., 6 to 18 bases). In the recombinant DNA, a transcription termination sequence is not necessarily required for expression of the DNA, but it is preferable to place a transcription termination sequence immediately downstream of the structural gene.

[0105] When a microorganism belonging to the genus Escherichia is used as the parent strain, 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, pUAKQE31 (Appl. Environ. Microbiol. 2007, 73: 6378-6385), pCDF-1b, pRSF-1b (all manufactured by Novagen), pMAL-c2x, and pMA L-c5x (both manufactured by New England Biolabs), pGEX-4T-1 (manufactured by GE Healthcare Biosciences), pTrcHis (manufactured by Invitrogen), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-30, pQE80L (both manufactured by Qiagen), pET-3, pTrc99A (manufactured by GE Healthcare Biosciences), pET-3 (manufactured by Novagen), pKYP10 (Japanese Patent Laid-Open No. 58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem. , 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. , USA, 82, 4306 (1985)], 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, 20, p. 6378-6385], pPAC31 (WO 1998 / 12343), pUC19 [Gene, 33, 103 (1985)], pSTV28 (Takara Bio), pUC118 (Takara Bio), pPA1 (JP 63-233798 A), pKD46 [Datsenko, K. A., Warner, B. L., Proc. Natl. Acad. Sci., USA, Vol. 97,6640-6645 (2000)], etc.

[0106] When using the above expression vector, any promoter may be used as long as it functions in the cells of a microorganism belonging to the genus Escherichia. For example, the trp promoter (P ｔｒｐ ), lac promoter (P ｌａｃ ), P Ｌ Promoter, P Ｒ Promoter, P ＳＥ Examples of promoters include promoters derived from Escherichia coli or phages, such as the promoter having two Ptrp promoters in tandem, the tac promoter, the lacT7 promoter, and the letI promoter, which are artificially designed and modified.

[0107] When a microorganism belonging to the genus Corynebacterium is used as the parent strain, examples of the expression vector include pCG1 (Japanese Patent Application Laid-Open No. 57-134500), pCG2 (Japanese Patent Application Laid-Open No. 58-35197), pCG4 (Japanese Patent Application Laid-Open No. 57-183799), pCG11 (Japanese Patent Application Laid-Open No. 57-134500), pCG116, pCE54, pCB101 (all Japanese Patent Application Laid-Open No. 58-105999), pCE51, pCE52, and pCE53 (all of which are described in Molecular and General Genetics, 196, 175 (1984)).

[0108] When the above expression vector is used, any promoter may be used as long as it functions in the cells of a microorganism belonging to the genus Corynebacterium. For example, the P54-6 promoter [Appl. Microbiol. Biotechnol., 53, 674-679 (2000)] can be used.

[0109] When a yeast strain is used as the parent strain, examples of the expression vector include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.

[0110] When using the above expression vector, any promoter may be used as long as it functions in the cells of a yeast strain, and examples include the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock polypeptide promoter, MFα1 promoter, and CUP1 promoter.

[0111] The recombinant DNA used in the production method of this embodiment can be prepared by inserting the DNA fragment described in any one of [8] to

[11] above downstream of the promoter of an appropriate expression vector.

[0112] Examples of methods for integrating recombinant DNA into the chromosome of a host cell include homologous recombination. Examples of homologous recombination include a method using a homologous recombination plasmid, which can be prepared by ligating a plasmid DNA carrying a drug resistance gene that cannot autonomously replicate in the host cell to be introduced. Examples of methods using homologous recombination that are frequently used in Escherichia coli include a method in which recombinant DNA is introduced using the lambda phage homologous recombination system [Proc. Natl. Acad. Sci. USA, 97, 6640-6645 (2000)]. Other selection methods include a selection method that utilizes the sucrose sensitivity of Escherichia coli due to Bacillus subtilis levansucrase integrated into the chromosome together with the recombinant DNA, and a selection method that utilizes the streptomycin sensitivity of Escherichia coli achieved by integrating a wild-type rpsL gene into Escherichia coli carrying a streptomycin-resistant mutant rpsL gene [Mol. Microbiol. , 55, 137 (2005), Biosci. Biotechnol. Biochem. , 71, 2905 (2007)], or the like, it is possible to obtain E. coli in which a target region on the chromosomal DNA of the host cell has been replaced with recombinant DNA.

[0113] Whether 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 the gene originally present on the chromosomal DNA of a microorganism cannot be amplified, but an amplified gene introduced by transformation is amplified by PCR to confirm the amplified product. Furthermore, an increase in the transcription level of the DNA or the production level of the protein encoded by the DNA can be confirmed by a method in which the transcription level of the gene in the microorganism is compared with that of the parent strain by Northern blotting, or the production level of the protein in the microorganism is compared with that of the parent strain by Western blotting.

[0114] Whether the microorganism produced by the above method is a microorganism in which the activity of the protein described in any one of [1] to [3] above is enhanced compared to that of the parent strain can be confirmed by culturing the microorganism, appropriately diluting the culture solution, centrifuging it, and analyzing the LNT contained in at least one of the supernatant and the bacterial cells using a sugar analyzer, etc., and comparing it with that of the parent strain. Furthermore, whether the microorganism produced by the above method is a microorganism in which the activity of the protein described in any one of [1] to [3] above is enhanced compared to that of the parent strain can also be confirmed by, for example, measuring the amount of the protein produced by the microorganism and the amount of the protein produced by the parent strain of the microorganism by Western blotting, and comparing the results.

[0115] The above-described microorganisms can improve LNT productivity by enhancing the activity of the protein described in any one of [1] to [3] above compared to the parent strain. Examples of such microorganisms include the CGLY / pSTV29-MhpT strain, which has enhanced expression of the MhpT gene, as described later in the Examples.

[0116] 2. Method for Producing Oligosaccharides The method for producing oligosaccharides according to this embodiment includes the following method: A method for producing oligosaccharides, comprising preparing the genetically modified microorganism described above in 1. and producing oligosaccharides in at least one of a culture supernatant and intracellular space using the genetically modified microorganism.

[0117] <Oligosaccharides> In the method of this embodiment, the oligosaccharide produced is preferably at least one of an oligosaccharide having an LNT backbone and LNT, and more preferably at least one of LNT, LNFPI, and LST-a.

[0118] The method for culturing the microorganisms described above in 1. can be carried out according to a conventional method used for culturing microorganisms.

[0119] The medium for culturing the microorganism may be either a natural medium or a synthetic medium, as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be utilized by the microorganism and allows the transformant to be cultured efficiently.

[0120] The carbon source may be any that can be assimilated by the microorganism, and examples thereof 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.

[0121] Examples of nitrogen sources include ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and 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 bacteria and digested products thereof, and the like.

[0122] Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

[0123] The microorganism used in this embodiment for the method of producing oligosaccharides may be a microorganism capable of producing a carbohydrate that can serve as a substrate for oligosaccharides, such as glucose, lactose, or lactose monohydrate. Such a microorganism may be a microorganism capable of producing glucose and having galactosyltransferase. Alternatively, such a microorganism may be a microorganism capable of producing glucose into which β1,4-galactosyltransferase has been introduced.

[0124] In the method for producing oligosaccharides, glucose, lactose, lactose monohydrate, or the like may be added to the medium during culture. When a microorganism having β1,4-galactosyltransferase is used, glucose may be added to the medium during culture. Alternatively, when β1,4-galactosyltransferase is introduced into the microorganism, glucose may be added to the medium during culture.

[0125] Furthermore, in the method for producing oligosaccharides, instead of adding glucose, lactose, lactose monohydrate, or the like to the medium during cultivation, glucose, lactose, lactose monohydrate, or the like may be supplied to the microorganism of this embodiment by simultaneously culturing a microorganism capable of producing glucose, lactose, lactose monohydrate, or the like from sugar with the microorganism of this embodiment.

[0126] Cultivation is preferably carried out under aerobic conditions, such as shaking culture or submerged aeration and stirring culture. 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 9.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

[0127] By the above-mentioned cultivation, oligosaccharides are produced and accumulated in at least one of the culture supernatant and the bacterial cells, and the oligosaccharides can be collected from at least one of the culture supernatant and the bacterial cells, thereby producing oligosaccharides.

[0128] Typically, oligosaccharides can be collected from the supernatant after centrifugation of the culture. When oligosaccharides accumulate within the cells, the cells can be disrupted, centrifuged to remove the cells, and the resulting supernatant can be collected by an ion exchange resin method or the like.

[0129] That is, the gist of the present invention is as follows. <<1>> A genetically modified microorganism in which the activity of at least one protein selected from the group consisting of MhpT, YfcJ, EmrD, YdeE, GlcNAc permease, GalNAc permease, Shewanella-derived sugar MFS transporter, Blon_2344, Blon_2343, Blon_2342, Blon_2347, Blon_2346, Blon_2345, Blon_2361, Blon_2360, Blon_2359, Bifidobacterium breve-derived ABC transporter, and Bifidobacterium breve-derived galactoside symporter is enhanced and which produces LNT. <<2>> The genetically modified microorganism according to <<1>> above, wherein the GlcNAc permease is NagP derived from bacteria of the genus Shewanella, and the GalNAc permease is AgaP derived from bacteria of the genus Shewanella. <<3>> The genetically modified microorganism according to <<1>> or <<2>> above, wherein the activity of the protein described in [1] below is enhanced. [1] A protein consisting of at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 64, 65, 66, 67, 68, 69, 70, 71, 72, 89, 106, 108, 110, and 112. <<4>> The genetically modified microorganism according to any one of <<1>> to <<3>> above, which has enhanced activity of the protein according to [2] or [3] below and has improved LNT productivity compared to the parent strain: [2] A mutant protein consisting of an amino acid sequence represented by at least one selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 64, 65, 66, 67, 68, 69, 70, 71, 72, 89, 106, 108, 110, and 112, in which 1 to 20 amino acids have been deleted, substituted, inserted, or added. [3] A homologous protein consisting of an amino acid sequence having 60% or more identity with at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 64, 65, 66, 67, 68, 69, 70, 71, 72, 89, 106, 108, 110, and 112.<<5>> A genetically modified microorganism according to any one of <<1>> to <<3>> above, which has been modified according to any one of [4] to [7] below. [4] A modification that enhances the activity of each protein consisting of the amino acid sequences represented by SEQ ID NOs: 64, 65, and 66. [5] A modification that enhances the activity of each protein consisting of the amino acid sequences represented by SEQ ID NOs: 67, 68, and 69. [6] A modification that enhances the activity of each protein consisting of the amino acid sequences represented by SEQ ID NOs: 70, 71, and 72. [7] A modification that enhances the activity of each protein consisting of the amino acid sequences represented by SEQ ID NOs: 106, 108, and 110. <<6>> A genetically modified microorganism according to any one of <<1>> to <<5>> above, which produces oligosaccharides having an LNT backbone. <<7>> The genetically modified microorganism according to <<6>> above, wherein the oligosaccharide having an LNT backbone is at least one of lacto-N-fucopentaose I (LNFPI) and sialyllacto-N-tetraose a (LST-a). <<8>> The genetically modified microorganism according to any one of <<1>> to <<7>> above, wherein the genetically modified microorganism is Escherichia coli. <<9>> A method for producing oligosaccharides, comprising preparing the genetically modified microorganism according to any one of <<1>> to <<8>> above, and producing oligosaccharides in at least one of a culture supernatant and intracellularly using the genetically modified microorganism. <<10>> A method for producing oligosaccharides according to <<9>> above, wherein the oligosaccharide is at least one selected from the group consisting of LNT, LNFPI, and LST-a.

[0130] [Analysis Examples] In the Examples, analysis and quantification of LNT were performed according to the following procedures. After cultivation, the culture solution containing the microorganisms was centrifuged, and the supernatant was collected for quantification of extracellular LNT. Furthermore, the precipitated cells were suspended in the same volume of water as the original culture solution, and an equal volume of chloroform was added to disrupt the cells, followed by centrifugation, and the supernatant aqueous phase was collected as the intracellular fraction. The LNT contained in the supernatant and / or the cells was analyzed using a sugar analyzer ICS-6000 (manufactured by Thermo Fisher Scientific), and the production amount was calculated from a calibration curve prepared using a standard sample. LNT as a standard sample can be obtained, for example, from medchemexpress (product number: HY-N9448).

[0131] [Analysis conditions] Column: CarboPAC PA10 Column temperature: 25°C Mobile phase: (Mobile phase A) Water (Mobile phase B) 500 mmol / L Sodium hydroxide (Mobile phase C) 300 mmol / L Sodium acetate Mixing ratio of mobile phase A, mobile phase B and mobile phase C: (0 to 18 minutes) Gradient from 80:20:0 to 70:20:10 (18 to 20 minutes) 70:20:10 (20 to 25 minutes) 80:20:0 Flow rate: 0.8 mL / min Detector: Pulsed amperometry detector

[0132] Examples of the present invention will be described below, but the present invention is not limited to these examples.

[0133] Example 1: Creation of a microorganism used for producing lacto-N-tetraose (LNT) (1) Obtaining a DNA fragment used as a marker for gene deletion PCR was performed using DNA consisting of the base sequences represented by SEQ ID NOs: 24 and 25 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.

[0134] (2) Construction of Escherichia coli lacking β-galactosidase (LacZ) and lactose permease (LacY) activities Escherichia coli lacking the DNA encoding β-galactosidase (hereinafter referred to as the LacZ gene) and the DNA encoding lactose permease (hereinafter referred to as the LacY gene) were constructed by the following method. Note that LacZ and LacY form an operon on the Escherichia coli genome.

[0135] PCR was carried out using genomic DNA of Escherichia coli W3110S strain prepared by a conventional method as a template and DNA primer sets consisting of the base sequences shown in "Primer set" in Table 2 to amplify each DNA fragment.

[0136]

[0137] LacZ upstream 1 and LacZ upstream 2 comprise the region from the initiation codon of the LacZ gene to about 1000 bp upstream of the initiation codon. LacY downstream 1 and LacY downstream 2 comprise the region from about 50 bp to about 1000 bp downstream of the termination codon of the LacY gene.

[0138] PCR was performed using an equimolar mixture of LacZ upstream 1, LacY downstream 1, and cat-sacB fragments as a template and DNAs consisting of the base sequences shown in SEQ ID NOs: 58 and 61 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 peripheral regions of the LacZ and LacY genes (hereinafter referred to as LacZY::cat-sacB).

[0139] PCR was performed using a mixture of LacZ upstream 2 and LacY downstream 2 in an equimolar ratio as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 58 and 61 as a primer set to obtain a DNA fragment (hereinafter referred to as ΔLacZY) that does not contain LacZY and consists of a sequence in which the upstream LacZ and downstream LacY are directly linked.

[0140] The LacZY::cat-sacB fragment was introduced by electroporation into the W3110S strain harboring the plasmid pKD46 [Datsenko, K. A., Warner, B. L., Proc. Natl. Acad. Sci., USA, Vol. 97, 6640-6645 (2000)], which contains a gene encoding λ recombinase, to obtain a transformant that exhibited chloramphenicol resistance and sucrose sensitivity (a transformant in which the LacZY gene had been replaced with LacZY::cat-sacB).

[0141] The ΔLacZY fragment was introduced into the transformant by electroporation to obtain a transformant exhibiting chloramphenicol sensitivity and sucrose resistance (a transformant in which LacZY::cat-sacB was replaced with ΔLacZY). From these, a transformant exhibiting ampicillin sensitivity (a transformant from which pKD46 had been lost) was further obtained. This transformant was designated W3110SΔLacZY.

[0142] (3) Construction of Expression Vector for LNT Production PCR was performed using DNAs consisting of the base sequences shown in "Primer Set" in Table 3 as a primer set and DNAs shown in "Template" in Table 3 as templates to obtain amplified DNA fragments.

[0143]

[0144] PCR was performed using an equimolar mixture of the cvβ3GalT fragment, the NpLgtA fragment, and the LacY fragment as a template and DNAs consisting of the base sequences represented by SEQ ID NOs: 26 and 31 as a primer set to obtain a DNA fragment in which the three fragments were linked (hereinafter referred to as cvβ3GalT-NpLgtA-LacY).

[0145] PCR was performed using a primer set of DNA consisting of the base sequences shown in SEQ ID NOs: 32 and 33 and plasmid pUAKQE31 (Appl. Environ. Microbiol. 2007, 73: 6378-6385) as a template to obtain a vector fragment of approximately 4.7 kb.

[0146] The DNA represented by SEQ ID NO: 20 encodes a β1,3-Gal transferase derived from Chromobacterium violaceum, and the DNA represented by SEQ ID NO: 22 encodes a β1,3-GlcNAc transferase derived from Neisseria polysaccharea ATCC43768 strain. The DNA sequences of the genes encoding these genes have been codon-optimized for expression in Escherichia coli, and were prepared by artificial synthesis.

[0147] In this case, SEQ ID NO: 26 has a sequence complementary to the 3' end of the pUAKQE fragment added to its 5' region, and SEQ ID NO: 31 has a sequence complementary to the 5' end of the pUAKQE fragment added to its 3' region.

[0148] The cvβ3GalT-NpLgtA-LacY fragment and the vector fragment obtained above were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the expression vector pUAKQE-cvβ3GalT-NpLgtA-LacY.

[0149] The above-constructed W3110SΔLacZY strain was transformed with the expression plasmid pUAKQE-cvβ3GalT-NpLgtA-LacY to construct an E. coli strain carrying pUAKQE-cvβ3GalT-NpLgtA-LacY, which was designated as CGLY strain.

[0150] (4) Construction of expression vector for producing LNnT PCR was performed using DNAs consisting of the base sequences shown in "Primer set" in Table 4 as a primer set and DNAs shown in "Template" in Table 4 as templates to obtain each amplified DNA fragment.

[0151]

[0152] PCR was performed using an equimolar mixture of the HpGalT fragment, the NpLgtA fragment, and the LacY fragment as a template and DNAs consisting of the base sequences represented by SEQ ID NOs: 129 and 134 as a primer set to obtain a DNA fragment in which the three fragments were ligated (hereinafter referred to as HpGalT-NpLgtA-LacY).

[0153] PCR was performed using a primer set consisting of DNAs with the nucleotide sequences shown in SEQ ID NOs: 135 and 136 and plasmid pTK31 (Appl Environ Microbiol. 2007 Oct; 73(20):6378-85.) as a template to obtain a vector fragment of approximately 3.8 kb. The DNA shown in SEQ ID NO: 127 encodes a β1,4-Gal transferase derived from Helicobacter pylori strain, and the DNA shown in SEQ ID NO: 22 encodes a β1,3-GlcNAc transferase derived from Neisseria polysaccharea ATCC43768 strain. These DNAs were prepared by artificial synthesis, with the nucleotide sequences of the genes being codon-optimized for expression in Escherichia coli.

[0154] In this case, SEQ ID NO: 129 has a sequence complementary to the 3' end of the pTK31 fragment added to its 5' region, and SEQ ID NO: 134 has a sequence complementary to the 5' end of the pTK31 fragment added to its 3' region. The HpGalT-NpLgtA-LacY fragment and vector fragment obtained above were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the expression vector pTK31-HpGalT-NpLgtA-LacY.

[0155] The above-constructed W3110SΔLacZY strain was transformed with the expression plasmid pTK31-HpGalT-NpLgtA-LacY to construct an E. coli strain carrying pTK31-HpGalT-NpLgtA-LacY, which was designated as strain HGLY.

[0156] (5) Construction of Microorganisms Having Activities of Various Transporters Escherichia coli carrying a plasmid for expressing a gene encoding various transporters placed under the lac promoter was constructed by the following method.

[0157] PCR was performed using DNAs consisting of the base sequences shown in "Primer set" in Tables 5 and 6 as a primer set and DNAs shown in "Template" in Tables 5 and 6 as templates to obtain each amplified DNA fragment.

[0158]

[0159]

[0160] The DNAs represented by SEQ ID NOs: 1, 3, 5, 7, 86, 90, 92, and 94 were amplified by PCR using genomic DNA of Escherichia coli W3110S strain prepared by a conventional method as a template and DNAs consisting of the base sequences represented by "Primer set" in Table 5 as a primer set.

[0161] The DNA represented by SEQ ID NO: 73 is a DNA in which the base sequence of the gene encoding GlcNAc permease derived from Shewanella sp. ANA3 strain has been codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis. The DNA represented by SEQ ID NO: 74 is a DNA in which the base sequence of the gene encoding GlcNAc permease derived from Shewanella amazonensis SB2B strain has been codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis. The DNA represented by SEQ ID NO: 126 is a DNA in which the base sequence of the gene encoding GlcNAc permease derived from Shewanella sp. MR4 strain has been codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis. PCR was performed on these DNAs using DNAs consisting of the base sequences shown in "Primer Set" in Table 5 as primer sets, and each DNA fragment was amplified.

[0162] The DNA represented by SEQ ID NO: 75 is a DNA in which the nucleotide sequence of the gene encoding GalNAc permease derived from Shewanella sp. ANA3 strain has been codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis. The DNA represented by SEQ ID NO: 76 is a DNA in which the nucleotide sequence of the gene encoding GalNAc permease derived from Shewanella sp. MR4 strain has been codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis. These DNAs were subjected to PCR using DNAs consisting of the nucleotide sequences shown in "Primer Set" in Table 5 as primer sets, and each DNA fragment was amplified.

[0163] The DNA represented by SEQ ID NO: 17 was prepared by PCR using the genomic DNA of a Bifidobacterium longum subsp. Infanti strain prepared by a conventional method, which was used to amplify the nucleotide sequences of the genes encoding the ABC transporters Blon_2344, Blon_2343, and Blon_2342, as well as DNAs consisting of the nucleotide sequences represented by the "Primer Set" in Table 6 as a primer set. This amplified the DNA fragment in a state in which the three genes were linked, thereby obtaining the Blon_2344-2342 fragment.

[0164] The DNA represented by SEQ ID NO: 18 was prepared by PCR using the genomic DNA of a Bifidobacterium longum subsp. Infanti strain prepared by a conventional method, which was used to amplify the nucleotide sequences of the genes encoding the ABC transporters Blon_2347, Blon_2346, and Blon_2345, as well as DNAs consisting of the nucleotide sequences represented by the "Primer Set" in Table 6 as a primer set, thereby amplifying the DNA fragment in a state in which the three genes were linked, thereby obtaining the Blon_2347-2345 fragment.

[0165] The DNA represented by SEQ ID NO: 19 was prepared by PCR using the genomic DNA of a Bifidobacterium longum subsp. Infanti strain prepared by a conventional method, which was used to amplify the base sequences of the genes encoding the ABC transporters Blon_2361, Blon_2360, and Blon_2359, as well as DNAs consisting of the base sequences represented by the "Primer Set" in Table 6 as a primer set. This amplified the DNA fragment in a state in which the three genes were linked, thereby obtaining the Blon_2361-2359 fragment.

[0166] The DNAs represented by SEQ ID NOs: 107, 109, and 111 are DNAs in which the base sequence of a gene encoding an ABC transporter derived from Bifidobacterium breve has been codon-optimized for expression in Escherichia coli, and were prepared by artificial synthesis. PCR was performed on these DNAs using DNAs consisting of the base sequences shown in "Primer set" in Table 6 as a primer set to amplify each DNA fragment. Then, PCR was performed using an equimolar mixture of the Bbr_1590 fragment, the Bbr_1589 fragment, and the Bbr_1588 fragment as a template, and DNAs consisting of the base sequences shown in SEQ ID NOs: 114 and 119 as a primer set to obtain the Bbr_1590-1588 fragment in which the three fragments were linked.

[0167] The DNA represented by SEQ ID NO: 113 is a DNA in which the base sequence of the gene encoding the galactoside symporter derived from Bifidobacterium breve has been codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis. PCR was performed on this DNA using DNAs consisting of the base sequences represented by the "Primer Set" in Table 6 as a primer set, and each DNA fragment was amplified.

[0168] PCR was performed using a primer set consisting of DNAs with the base sequences represented by SEQ ID NOs: 34 and 35 and the plasmid pSTV29 (Takara Bio Inc.) as a template to obtain a vector fragment of approximately 3.0 kb. The various amplified DNA fragments and vector fragments obtained above were ligated using an In-Fusion HD Cloning Kit (Takara Bio Inc.) to generate plasmids expressing various transporters, including pSTV29-MhpT, pSTV29-YfcJ, pSTV29-EmrD, pSTV29-YdeE, pSTV29-SetB, pSTV29-SetC, pSTV29-YnfM, pSTV29-MdtD, pSTV29-NagP(ANA3), and pSTV29-NagP(S ama), pSTV29-NagP(MR4), pSTV29-AgaP(ANA3), pSTV29-AgaP(MR4), pSTV29-(Blon_2344-2342), pSTV29-(Blon_2347-2345), pSTV29-(Blon_2361-2359), pSTV29-(Bbr_1590-1588), and pSTV29-(Bbr_1551) were constructed.

[0169] In this case, SEQ ID NOs: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 96, 98, 100, 102, 104, 114, and 120 have a sequence complementary to the 3' end of the pSTV29 fragment added to the 5' region of the sequence, and SEQ ID NOs: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 97, 99, 101, 103, 105, 119, and 121 have a sequence complementary to the 5' end of the pSTV29 fragment added to the 3' region.

[0170] (6) Construction of E. coli carrying transporter expression plasmids The transporter expression plasmids obtained above, pSTV29-MhpT, pSTV29-YfcJ, pSTV29-EmrD, pSTV29-YdeE, pSTV29-SetB, pSTV29-SetC, pSTV29-YnfM, pSTV29-MdtD, pSTV29-NagP(ANA3), pSTV29-NagP(Sama), pSTV29-NagP(MR4), pSTV29-AgaP(ANA3), pSTV29-AgaP(MR4), pSTV29-(Blon_2344-2342), and pSTV29 The CGLY and HGLY strains constructed in Examples 1(3) and (4) were transformed with pSTV29-(Blon_2347-2345), pSTV29-(Blon_2361-2359), pSTV29-(Bbr_1590-1588), pSTV29-(Bbr_1551), and pSTV29 as a vector control to construct E. coli strains carrying various plasmids, and the strains were respectively CGLY / pSTV29-MhpT, CGLY / pSTV29-YfcJ, CGLY / pSTV29-EmrD, and CGLY / pSTV29-MhpT. 29-YdeE strain, CGLY / pSTV29-SetB strain, CGLY / pSTV29-SetC strain, CGLY / pSTV29-YnfM strain, CGLY / pSTV29-MdtD strain, CGLY / pSTV29-NagP(ANA3) strain, CGLY / pSTV29-NagP(Sama) strain, CGLY / pSTV29-NagP(MR4), CGLY / pSTV29-AgaP(ANA3) strain, CGLY / pSTV29-AgaP(MR4), CGLY / pSTV29-(Blon_2344-2342) strain, CGLY / pSTV29 -(Blon_2347-2345) strain, CGLY / pSTV29-(Blon_2361-2359) strain, CGLY / pSTV29-(Bbr_1590-1588) strain, CGLY / pSTV29-(Bbr_1551) strain, CGLY / Ctrl strain, HGLY / pSTV29-MhpT strain, HGLY / pSTV29-YfcJ strain, HGLY / pSTV29-EmrD strain, HGLY / pSTV29-YdeE strain, HGLY / pSTV29-SetB strain, HGLY / pSTV29-SetC strain, HGLY / pSTV29-YnfM strain,They were named HGLY / pSTV29-MdtD strain, HGLY / pSTV29-(Blon_2344-2342) strain, HGLY / pSTV29-(Blon_2347-2345) strain, HGLY / pSTV29-(Blon_2361-2359) strain, HGLY / pSTV29-(Bbr_1590-1588) strain, HGLY / pSTV29-(Bbr_1551) strain, and HGLY / Ctrl strain.

[0171] [Example 2] Production of lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) The CGLY / pSTV29-MhpT strain, CGLY / pSTV29-YfcJ strain, CGLY / pSTV29-EmrD strain, CGLY / pSTV29-YdeE strain, CGLY / pSTV29-SetB strain, CGLY / pSTV29-SetC strain, CGLY / pSTV29-YnfM strain, CGLY / pSTV29-mdtD strain, CGLY / pSTV29-NagP(ANA3) strain, and CGLY / pSTV29-YfcJ strain obtained in Example 1 were used. 29-NagP (Sama) strain, CGLY / pSTV29-NagP (MR4), CGLY / pSTV29-AgaP (ANA3) strain, CGLY / pSTV29-AgaP (MR4) strain, CGLY / pSTV29- (Blon_2344-2342) strain, CGLY / pSTV29- (Blon_2347-2345) strain, CGLY / pSTV29- (Blon_2361-2359) strain, CGLY / pSTV29- LNT productivity of strains HGLY / pSTV29-(Bbr_1590-1588), CGLY / pSTV29-(Bbr_1551), and CGLY / Ctrl, and strains HGLY / pSTV29-MhpT, HGLY / pSTV29-YfcJ, HGLY / pSTV29-EmrD, HGLY / pSTV29-YdeE, HGLY / pSTV29-SetB, HGLY / pSTV29-SetC, and HGLY / pSTV29-YnfM. The LNnT productivity of the HGLY / pSTV29-MdtD strain, HGLY / pSTV29-(Blon_2344-2342) strain, HGLY / pSTV29-(Blon_2347-2345) strain, HGLY / pSTV29-(Blon_2361-2359) strain, HGLY / pSTV29-(Bbr_1590-1588) strain, HGLY / pSTV29-(Bbr_1551) strain, and HGLY / Ctrl strain was evaluated.

[0172] Each strain was cultured for 16 hours at 30° C. on an LB plate containing 100 mg / L kanamycin and 25 mg / L chloramphenicol, and then inoculated into a test tube containing 2 mL of LB medium containing 100 mg / L kanamycin (and 25 mg / L chloramphenicol for CGLY strain-derived producers) and cultured with shaking for 16 hours at 30° C. The resulting culture was then cultured in a production medium containing 100 mg / L kanamycin and 25 mg / L chloramphenicol [glucose 30 g / L, lactose monohydrate or 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]. (Aqueous solutions containing glucose, lactose monohydrate, and magnesium sulfate heptahydrate were adjusted to pH 7.2 with aqueous sodium hydroxide solution and then autoclaved) (Aqueous solutions containing glucose, lactose monohydrate, and magnesium sulfate heptahydrate were prepared separately, autoclaved, cooled, and then mixed) 0.2 mL of the bacteria was inoculated into a large test tube containing 4 mL of the above solution, and cultured with shaking at 30°C for 29 hours. Six hours after the start of culture, IPTG was added to a final concentration of 1 mM.

[0173] After the culture was completed, the culture medium was centrifuged, diluted appropriately, and the cells were disrupted. The LNT and LNnT contained in the supernatant and intracellular contents were analyzed using a sugar analyzer ICS-6000. The results are shown in Tables 7 to 10. The total LNT amount is the sum of the extracellular (supernatant) LNT amount and the intracellular LNT amount. For the four strains listed in Table 7, the LNT contained in the supernatant and intracellular contents was analyzed simultaneously, and for the six strains listed in Table 9, the LNT contained in the supernatant was analyzed simultaneously. Furthermore, for the four strains listed in Table 8 and the six strains listed in Table 10, the LNnT contained in the supernatant was analyzed simultaneously.

[0174]

[0175]

[0176]

[0177]

[0178] The results shown in Tables 7 and 9 indicate that the CGLY / pSTV29-MhpT, CGLY / pSTV29-YfcJ, CGLY / pSTV29-EmrD, and CGLY / pSTV29-YdeE strains improve extracellular and total LNT production compared to the control strain, CGLY / Ctrl. Among these, the CGLY / pSTV29-EmrD and CGLY / pSTV29-YdeE strains showed a 1.7-fold or greater improvement in total LNT production compared to the control strain, CGLY / Ctrl. Among the strains listed in Table 7, particularly good results were observed when the CCGLY / pSTV29-EmrD and CGLY / pSTV29-YdeE strains were used in terms of extracellular and total LNT production. In addition, the CGLY / pSTV29-EmrD and CGLY / pSTV29-YdeE strains were shown to have improved extracellular and total LNT production amounts as well as an improved extracellular LNT ratio compared to the control strain, CGLY / Ctrl.

[0179] For the six strains listed in Table 11, LNT contained in the supernatant and intracellular space was analyzed simultaneously.

[0180]

[0181] The results shown in Table 11 indicate that the CGLY / pSTV29-NagP(ANA3), CGLY / pSTV29-NagP(Sama), CGLY / pSTV29-NagP(MR4), CGLY / pSTV29-AgaP(ANA3), and CGLY / pSTV29-AgaP(MR4) strains exhibited improved extracellular and total LNT production compared to the control CGLY / Ctrl strain. In particular, the CGLY / pSTV29-NagP(ANA3), CGLY / pSTV29-NagP(Sama), and CGLY / pSTV29-AgaP(MR4) strains exhibited a 1.5-fold or greater improvement in total LNT production compared to the control CGLY / Ctrl strain. Among the strains listed in Table 11, in terms of total LNT production, particularly good results were observed when the CGLY / pSTV29-NagP(ANA3), CGLY / pSTV29-NagP(Sama), and CGLY / pSTV29-AgaP(MR4) strains were used. Furthermore, the CGLY / pSTV29-NagP(ANA3), CGLY / pSTV29-NagP(Sama), and CGLY / pSTV29-AgaP(MR4) strains were shown to produce 1.8-fold or more extracellular LNT compared to the control strain, CGLY / Ctrl. Among the strains listed in Table 11, in terms of the amount of extracellular LNT produced, particularly good results were observed when the CGLY / pSTV29-NagP (ANA3), CGLY / pSTV29-NagP (Sama), and CGLY / pSTV29-AgaP (MR4) strains were used.

[0182] In addition, the CGLY / pSTV29-NagP (ANA3), CGLY / pSTV29-NagP (Sama), CGLY / pSTV29-AgaP (ANA3), and CGLY / pSTV29-AgaP (MR4) strains were shown to improve extracellular and total LNT production and the extracellular ratio of LNT compared to the control strain CGLY / Ctrl. Among the strains listed in Table 11, from the viewpoint of improving the extracellular ratio, particularly good results were shown when using the CGLY / pSTV29-NagP (ANA3), CGLY / pSTV29-NagP (Sama), CGLY / pSTV29-AgaP (ANA3), and CGLY / pSTV29-AgaP (MR4) strains.

[0183] The LNT contained in the supernatant and the cells was analyzed simultaneously for the four strains listed in Table 12. The LNnT contained in the supernatant was analyzed simultaneously for the four strains listed in Table 13.

[0184]

[0185]

[0186] The results shown in Table 12 indicate that the CGLY / pSTV29-(Blon_2344-2342), CGLY / pSTV29-(Blon_2347-2345), and CGLY / pSTV29-(Blon_2361-2359) strains exhibited improved extracellular and total LNT production compared to the control CGLY / Ctrl strain. Furthermore, the CGLY / pSTV29-(Blon_2347-2345) strain exhibited the greatest improvement in total LNT production, with a 1.95-fold increase compared to the control CGLY / Ctrl strain. Among the strains listed in Table 12, in terms of total LNT production, particularly good results were observed when the CGLY / pSTV29-(Blon — 2347-2345) strain was used.

[0187] Furthermore, the CGLY / pSTV29-(Blon_2361-2359) strain showed the greatest improvement in extracellular LNT production, at 2.44 times that of the control strain, CGLY / Ctrl. Among the strains listed in Table 12, particularly good results were observed when the CGLY / pSTV29-(Blon_2361-2359) strain was used in terms of extracellular LNT production.

[0188] The CGLY / pSTV29-(Blon_2344-2342), CGLY / pSTV29-(Blon_2347-2345), and CGLY / pSTV29-(Blon_2361-2359) strains were shown to have improved extracellular and total LNT production and an improved extracellular LNT ratio compared to the control CGLY / Ctrl strain. In particular, the CGLY / pSTV29-(Blon_2361-2359) strain showed the greatest improvement in the extracellular LNT ratio, at 1.3 times that of the control CGLY / Ctrl strain. Among the strains listed in Table 12, in terms of improving the extracellular ratio, particularly good results were observed when the CGLY / pSTV29-(Blon — 2361-2359) strain was used.

[0189] The LNT contained in the supernatant and the cells was analyzed simultaneously for the three strains listed in Table 14. The LNnT contained in the supernatant and the cells was analyzed simultaneously for the three strains listed in Table 15.

[0190]

[0191]

[0192] The results shown in Table 14 indicate that the CGLY / pSTV29-(Bbr_1590-1588) and CGLY / pSTV29-(Bbr_1551) strains showed improved total LNT production compared to the control strain, CGLY. In particular, the CGLY / pSTV29-(Bbr_1551) strain showed a 1.4-fold or greater improvement in total LNT production compared to the control strain, CGLY.

[0193] It is clear that a person skilled in the art can conceive of various modifications and alterations within the scope of the claims, and it is understood that these also naturally fall within the technical scope of the present invention. Furthermore, the components in the above-described embodiments may be combined in any manner as long as they do not deviate from the spirit of the invention.

[0194] This application is based on a Japanese patent application (Patent Application No. 2023-140439) filed on August 30, 2023, the contents of which are incorporated herein by reference.

[0195] SEQ ID NO: 1: Nucleotide sequence of MhpT SEQ ID NO: 2: Amino acid sequence of MhpT (Accession number: WP_000107627.1) SEQ ID NO: 3: Nucleotide sequence of YfcJ SEQ ID NO: 4: Amino acid sequence of YfcJ (Accession number: WP_000127781.1) SEQ ID NO: 5: Nucleotide sequence of EmrD SEQ ID NO: 6: Amino acid sequence of EmrD (Accession number: WP_000828746.1) SEQ ID NO: 7: Nucleotide sequence of YdeE SEQ ID NO: 8: Amino acid sequence of YdeE (Accession number: WP_001054204.1) SEQ ID NO: 9: Nucleotide sequence of NagP (ANA3) SEQ ID NO: 10: Amino acid sequence of NagP (ANA3) (Accession number: WP_011716320.1) SEQ ID NO: 11: Nucleotide sequence of NagP (Sama) SEQ ID NO: 12: Amino acid sequence of NagP (Sama) (Accession number: WP_011759984.1) SEQ ID NO: 13: Nucleotide sequence of AgaP (ANA3) SEQ ID NO: 14: Amino acid sequence of AgaP (ANA3) (Accession number: WP_011717580.1) SEQ ID NO: 15: Nucleotide sequence of AgaP (MR4) SEQ ID NO: 16: Amino acid sequence of AgaP (MR4) (Accession number: WP_011623282.1) SEQ ID NO: 17: Nucleotide sequences of Blon2344, Blon2343, and Blon2342 SEQ ID NO: 18: Nucleotide sequences of Blon2347, Blon2346, and Blon2345 SEQ ID NO: 19: Nucleotide sequences of Blon2361, Blon2360, and Blon2359 SEQ ID NO: 20: Nucleotide sequence of codon-optimized cvβ3GalT SEQ ID NO: 21: Amino acid sequence of codon-optimized cvβ3GalT SEQ ID NO: 22: Nucleotide sequence of codon-optimized NpLgtA SEQ ID NO: 23: Amino acid sequence of NpLgtA derived from ATCC43768 strain SEQ ID NO: 24: Nucleotide sequence of primer F for amplifying catsacB fragment SEQ ID NO: 25: Nucleotide sequence of primer R for amplifying catsacB fragment SEQ ID NO: 26: Nucleotide sequence of primer F for amplifying cvβ3GalT fragment SEQ ID NO: 27: Nucleotide sequence of primer R for amplifying cvβ3GalT fragment SEQ ID NO: 28: Nucleotide sequence of primer F for amplifying NpLgtA fragment SEQ ID NO: 29: Nucleotide sequence of primer R for amplifying NpLgtA fragment SEQ ID NO: 30: Nucleotide sequence of primer F for amplifying LacY fragmentSEQ ID NO: 31: Nucleotide sequence of primer R for amplifying LacY fragment SEQ ID NO: 32: Nucleotide sequence of primer F for amplifying pUAKQE31 fragment SEQ ID NO: 33: Nucleotide sequence of primer R for amplifying pUAKQE31 fragment SEQ ID NO: 34: Nucleotide sequence of primer F for amplifying pSTV29 fragment SEQ ID NO: 35: Nucleotide sequence of primer R for amplifying pSTV29 fragment SEQ ID NO: 36: Nucleotide sequence of primer F for amplifying MhpT fragment SEQ ID NO: 37: Nucleotide sequence of primer R for amplifying MhpT fragment SEQ ID NO: 38: Nucleotide sequence of primer F for amplifying YfcJ fragment SEQ ID NO: 39: Nucleotide sequence of primer R for amplifying YfcJ fragment SEQ ID NO: 40: Nucleotide sequence of primer F for amplifying EmrD fragment SEQ ID NO: 41: Nucleotide sequence of primer R for amplifying EmrD fragment SEQ ID NO: 42: Nucleotide sequence of primer F for amplifying YdeE fragment SEQ ID NO: 43: Nucleotide sequence of primer R for amplifying YdeE fragment SEQ ID NO: 44: Nucleotide sequence of primer F for amplifying a NagP (ANA3) fragment SEQ ID NO: 45: Nucleotide sequence of primer R for amplifying a NagP (ANA3) fragment SEQ ID NO: 46: Nucleotide sequence of primer F for amplifying a NagP (Sama) fragment SEQ ID NO: 47: Nucleotide sequence of primer R for amplifying a NagP (Sama) fragment SEQ ID NO: 48: Nucleotide sequence of primer F for amplifying an AgaP (ANA3) fragment SEQ ID NO: 49: Nucleotide sequence of primer R for amplifying an AgaP (ANA3) fragment SEQ ID NO: 50: Nucleotide sequence of primer F for amplifying an AgaP (MR4) fragment SEQ ID NO: 51: Nucleotide sequence of primer R for amplifying an AgaP (MR4) fragment SEQ ID NO: 52: Nucleotide sequence of primer F for amplifying a Blon_2344-2342 fragment SEQ ID NO: 53: Nucleotide sequence of primer R for amplifying a Blon_2344-2342 fragment SEQ ID NO: 54: Nucleotide sequence of primer F for amplifying Blon_2347-2345 fragment SEQ ID NO: 55: Nucleotide sequence of primer R for amplifying Blon_2347-2345 fragment SEQ ID NO: 56: Nucleotide sequence of primer F for amplifying Blon_2361-2359 fragment SEQ ID NO: 57: Nucleotide sequence of primer R for amplifying Blon_2361-2359 fragment SEQ ID NO: 58: Nucleotide sequence of primer F for amplifying LacZY upstream 1 fragment SEQ ID NO: 59: Nucleotide sequence of primer R for amplifying LacZY upstream 1 fragment SEQ ID NO: 60: Nucleotide sequence of primer F for amplifying LacZY downstream 1 fragmentSEQ ID NO: 61: Nucleotide sequence of primer R for amplifying LacZY downstream 1 fragment SEQ ID NO: 62: Nucleotide sequence of primer R for amplifying LacZY upstream 2 fragment SEQ ID NO: 63: Nucleotide sequence of primer F for amplifying LacZY downstream 2 fragment SEQ ID NO: 64: Amino acid sequence of Blon2344 (Accession number: ACJ53402.1) SEQ ID NO: 65: Amino acid sequence of Blon2343 (Accession number: WP_012578570.1) SEQ ID NO: 66: Amino acid sequence of Blon2342 (Accession number: WP_012578569.1) SEQ ID NO: 67: Amino acid sequence of Blon2347 (Accession number: WP_012578572.1) SEQ ID NO: 68: Amino acid sequence of Blon2346 (Accession number: WP_012578570.1) SEQ ID NO: 69: Amino acid sequence of Blon2345 (Accession number: WP_012578569.1) SEQ ID NO: 70: Amino acid sequence of Blon2361 (Accession number: WP_012578586.1) SEQ ID NO: 71: Amino acid sequence of Blon2360 (Accession number: WP_012578585.1) SEQ ID NO: 72: Amino acid sequence of Blon2359 (Accession number: WP_012578584.1) SEQ ID NO: 73: Nucleotide sequence of codon-optimized NagP (ANA3) SEQ ID NO: 74: Nucleotide sequence of codon-optimized NagP (Sama) SEQ ID NO: 75: Nucleotide sequence of codon-optimized AgaP (ANA3) SEQ ID NO: 76: Nucleotide sequence of codon-optimized AgaP (MR4) SEQ ID NO: 77: Nucleotide sequence of Blon2344 SEQ ID NO: 78: Nucleotide sequence of Blon2343 SEQ ID NO: 79: Nucleotide sequence of Blon2342 SEQ ID NO: 80: Nucleotide sequence of Blon2347 SEQ ID NO: 81: Nucleotide sequence of Blon2346 SEQ ID NO: 82: Nucleotide sequence of Blon2345 SEQ ID NO: 83: Nucleotide sequence of Blon2361 SEQ ID NO: 84: Nucleotide sequence of Blon2360 SEQ ID NO: 85: Nucleotide sequence of Blon2359 SEQ ID NO: 86: Nucleotide sequence of SetB SEQ ID NO: 87: Amino acid sequence of SetB SEQ ID NO: 88: Nucleotide sequence of NagP (MR4) SEQ ID NO: 89: Amino acid sequence of NagP (MR4) (Accession number: WP_011623680.1) SEQ ID NO: 90: Nucleotide sequence of SetC SEQ ID NO: 91: Amino acid sequence of SetCSEQ ID NO: 92: Nucleotide sequence of YnfM SEQ ID NO: 93: Amino acid sequence of YnfM SEQ ID NO: 94: Nucleotide sequence of MdtD SEQ ID NO: 95: Amino acid sequence of MdtD SEQ ID NO: 96: Nucleotide sequence of primer F for amplifying SetB fragment SEQ ID NO: 97: Nucleotide sequence of primer R for amplifying SetB fragment SEQ ID NO: 98: Nucleotide sequence of primer F for amplifying NagP (MR4) fragment SEQ ID NO: 99: Nucleotide sequence of primer R for amplifying NagP (MR4) fragment SEQ ID NO: 100: Nucleotide sequence of primer F for amplifying SetC fragment SEQ ID NO: 101: Nucleotide sequence of primer R for amplifying SetC fragment SEQ ID NO: 102: Nucleotide sequence of primer F for amplifying YnfM fragment SEQ ID NO: 103: Nucleotide sequence of primer R for amplifying YnfM fragment SEQ ID NO: 104: Nucleotide sequence of primer F for amplifying MdtD fragment SEQ ID NO: 105: Nucleotide sequence of primer R for amplifying MdtD fragment SEQ ID NO: 106: Amino acid sequence of Bbr_1590 (Accession number: WP_015439187.1) SEQ ID NO: 107: Nucleotide sequence of Bbr_1590 SEQ ID NO: 108: Amino acid sequence of Bbr_1589 (Accession number: WP_003829947.1) SEQ ID NO: 109: Nucleotide sequence of Bbr_1589 SEQ ID NO: 110: Amino acid sequence of Bbr_1588 (Accession number: WP_003829949.1) SEQ ID NO: 111: Nucleotide sequence of Bbr_1588 SEQ ID NO: 112: Amino acid sequence of Bbr_1551 (Accession number: WP_015439164.1) SEQ ID NO: 113: Nucleotide sequence of Bbr_1551 SEQ ID NO: 114: Nucleotide sequence of primer F for amplifying the Bbr_1590 fragment SEQ ID NO: 115: Nucleotide sequence of primer R for amplifying the Bbr_1590 fragment SEQ ID NO: 116: Nucleotide sequence of primer F for amplifying the Bbr_1589 fragment SEQ ID NO: 117: Nucleotide sequence of primer R for amplifying the Bbr_1589 fragment SEQ ID NO: 118: Nucleotide sequence of primer F for amplifying the Bbr_1588 fragment SEQ ID NO: 119: Nucleotide sequence of primer R for amplifying the Bbr_1588 fragment SEQ ID NO: 120: Nucleotide sequence of primer F for amplifying the Bbr_1551 fragment SEQ ID NO: 121: Nucleotide sequence of primer R for amplifying the Bbr_1551 fragment SEQ ID NO: 122: Nucleotide sequence of Bbr_1590 SEQ ID NO: 123: Nucleotide sequence of Bbr_1589 SEQ ID NO: 124: Nucleotide sequence of Bbr_1588SEQ ID NO: 125: Nucleotide sequence of Bbr_1551 SEQ ID NO: 126: Nucleotide sequence of codon-optimized NagP (MR4) SEQ ID NO: 127: Nucleotide sequence of HpGalT SEQ ID NO: 128: Amino acid sequence of HpGalT SEQ ID NO: 129: Nucleotide sequence of primer F for amplifying the HpGalT fragment SEQ ID NO: 130: Nucleotide sequence of primer R for amplifying the HpGalT fragment SEQ ID NO: 131: Nucleotide sequence of primer F for amplifying the NpLgtA2 fragment SEQ ID NO: 132: Nucleotide sequence of primer R for amplifying the NpLgtA2 fragment SEQ ID NO: 133: Nucleotide sequence of primer F for amplifying the LacY2 fragment SEQ ID NO: 134: Nucleotide sequence of primer R for amplifying the LacY2 fragment SEQ ID NO: 135: Nucleotide sequence of primer F for amplifying the pTK31 fragment SEQ ID NO: 136: Nucleotide sequence of primer R for amplifying the pTK31 fragment

Claims

1. MhpT, YfcJ, EmrD, YdeE, N-acetylglucosamine (hereinafter also referred to as "GlcNAc" ) permease, N-acetylgalactosamine (hereinafter also referred to as "GalNAc" ) permease, Shewanella-derived sugar Major Facilitator Superfamily transporter, Blon_2344, Blon_2343, Blon_2342, Blon_2347, Blon_2346, Blon_2345, Blon_2361, Blon_2360, Blon_2359, Bifidobacterium breve-derived ATP-binding cassette transporter (hereinafter also referred to as "ABC transporter"), and Bifidobacterium A genetically modified microorganism in which the activity of at least one protein selected from the group consisting of galactoside symporters derived from B. breve is enhanced and which produces lacto-N-tetraose (LNT).

2. The genetically modified microorganism according to claim 1, wherein the GlcNAc permease is NagP derived from a bacterium belonging to the genus Shewanella, and the GalNAc permease is AgaP derived from a bacterium belonging to the genus Shewanella.

3. The genetically modified microorganism according to claim 1 or 2, in which the activity of the protein according to the following [1] is enhanced: [1] A protein consisting of at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 64, 65, 66, 67, 68, 69, 70, 71, 72, 89, 106, 108, 110, and 112.

4. The genetically modified microorganism according to claim 1 or 2, in which the activity of the protein according to [2] or [3] below is enhanced and the productivity of lacto-N-tetraose (LNT) is improved as compared to the parent strain. [2] A mutant protein consisting of an amino acid sequence represented by at least one selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 64, 65, 66, 67, 68, 69, 70, 71, 72, 89, 106, 108, 110, and 112, in which 1 to 20 amino acids have been deleted, substituted, inserted, or added. [3] A homologous protein having an amino acid sequence having 60% or more identity with at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 64, 65, 66, 67, 68, 69, 70, 71, 72, 89, 106, 108, 110, and 112.

5. The genetically modified microorganism according to claim 1 or 2, which has been modified as described in any one of [4] to [7] below. [4] A modification that enhances the activity of each protein consisting of the amino acid sequences represented by SEQ ID NOs: 64, 65, and 66. [5] A modification that enhances the activity of each protein consisting of the amino acid sequences represented by SEQ ID NOs: 67, 68, and 69. [6] A modification that enhances the activity of each protein consisting of the amino acid sequences represented by SEQ ID NOs: 70, 71, and 72. [7] A modification that enhances the activity of each protein consisting of the amino acid sequences represented by SEQ ID NOs: 106, 108, and 110.

6. The genetically modified microorganism according to claim 1 or 2, which produces oligosaccharides having an LNT backbone.

7. The genetically modified microorganism according to claim 6, wherein the oligosaccharide having an LNT backbone is at least one of lacto-N-fucopentaose I (LNFPI) and sialyllacto-N-tetraose a (LST-a).

8. The genetically modified microorganism of claim 1 or 2, wherein the genetically modified microorganism is Escherichia coli.

9. A method for producing oligosaccharides, comprising preparing a genetically modified microorganism according to claim 1 or 2, and using the genetically modified microorganism to produce oligosaccharides in at least one of a culture supernatant and intracellular space.

10. The method for producing oligosaccharides according to claim 9, wherein the oligosaccharide is at least one selected from the group consisting of lacto-N-tetraose (LNT), lacto-N-fucopentaose I (LNFPI) and sialyllacto-N-tetraose a (LST-a).

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