Microorganism having ability to produce n-acetylneuraminic acid and / or n-acetylneuraminic acid-containing carbohydrate and method for producing n-acetylneuraminic acid and / or n-acetylneuraminic acid-containing carbohydrate using said microorganism

Abstract

A purpose of the present invention is to provide a microorganism having the ability to produce NeuAc and / or an NeuAc-containing carbohydrate and a method for producing NeuAc and / or an NeuAc-containing carbohydrate efficiently by said microorganism. The present invention relates to a microorganism having the ability to produce NeuAc and / or an NeuAc-containing carbohydrate and having a blocked enterobacterial common antigen (ECA) biosynthesis pathway and to a method for producing NeuAc and / or an NeuAc-containing carbohydrate using said microorganism.

Description

Microorganism capable of producing N-acetylneuraminic acid and / or N-acetylneuraminic acid-containing carbohydrates and method for producing N-acetylneuraminic acid and / or N-acetylneuraminic acid-containing carbohydrates using said microorganisms

[0001] The present invention relates to a microorganism capable of producing N-acetylneuraminic acid (hereinafter also abbreviated as NeuAc) and / or NeuAc-containing carbohydrates, and to an efficient method for producing NeuAc and / or NeuAc-containing carbohydrates using the microorganism.

[0002] N-acetylneuraminic acid (NeuAc) is a type of amino sugar found in the human body and is known to be normally localized as sialic acid at the non-reducing end of glycoproteins and glycolipids. NeuAc is abundantly contained in gangliosides and glycolipids in the brain, and has been suggested to be involved in brain development and cognitive function (Non-Patent Document 1).

[0003] Methods for producing NeuAc have long been used, including extraction from natural resources such as swallow's nests and egg yolks, and polymer hydrolysis, but these methods are complex and have low yields (Non-Patent Document 2).

[0004] Meanwhile, as more efficient methods for producing NeuAc, methods using enzymes and direct fermentation using microorganisms have been developed (Non-Patent Document 2).

[0005] Known examples of production methods using enzymes include a method for producing NeuAc by the reaction of NeuAc aldolase with pyruvic acid and N-acetylmannosamine (hereinafter referred to as ManNAc) as substrates (Non-Patent Documents 3 and 4), a method for producing NeuAc by the reaction of NeuAc aldolase under alkaline conditions with pyruvic acid and N-acetylglucosamine (hereinafter referred to as GlcNAc) as substrates (Patent Document 1), a method for producing NeuAc by the reaction of NeuAc aldolase and N-acetylglucosamine-2-epimerase (hereinafter referred to as GlcNAc epimerase) with pyruvic acid and GlcNAc as substrates (Non-Patent Documents 5 and 6), and a method for producing NeuAc by the reaction of NeuAc synthetase with phosphoenolpyruvate (hereinafter referred to as PEP) and ManNAc as substrates (Patent Document 2 and Non-Patent Document 7). However, the above-mentioned methods for producing NeuAc using enzymes all have the drawback of requiring complicated procedures.

[0006] As a production method by direct fermentation using a microorganism, for example, Patent Document 4 discloses a method in which the neuC gene encoding UDP-GlcNAc epimerase derived from Campylobacter jejuni and the neuB gene encoding sialic acid synthase are introduced into Escherichia coli to convert GlcNAc to NeuAc via ManNAc (this pathway is called the NeuC pathway). Furthermore, Patent Document 5 discloses a method in which GlcNAc is produced from glucosamine hexaphosphate by introducing the GNA1 gene encoding glucosamine acetyltransferase derived from budding yeast, and then the GlcNAc is further produced by the cyanobacterium Synechocystis sp. disclosed in Patent Document 3. A method for producing NeuAc from GlcNAc via ManNAc by introducing the slr1975 gene encoding N-acetylmannosamine epimerase derived from PCC6803 and the neuB gene encoding sialic acid synthase derived from Rhodobacter has been disclosed (this pathway is referred to as the GNA1 pathway).

[0007] Unlike the NeuC pathway, the GNA1 pathway does not involve activated sugar nucleotides such as UDP-GlcNAc and is therefore thought to be advantageous for production. However, as shown in Non-Patent Document 8, there are currently issues with the production efficiency and production costs of NeuAc.

[0008] Escherichia coli has the wecB gene (also known as the yifF gene, nfrC gene, or rffE gene) that encodes UDP-GlcNAc epimerase (WecB), which is responsible for converting UDP-GlcNAc to UDP-ManNAc (Non-Patent Document 9). As mentioned above, UDP-GlcNAc epimerase is an essential enzyme in the NeuC pathway, but it has also been shown that WecB functions complementarily in the GNA1 pathway for NeuAc production (Non-Patent Document 8).

[0009] WecB is also known to function in the biosynthesis of enterobacterial common antigen (hereinafter referred to as ECA) in Escherichia coli (Non-Patent Document 9). The ECA biosynthetic pathway is known, and in addition to WecB, WecA, WecC, and others are known to be involved (Non-Patent Documents 9 and 10).

[0010] However, the relationship between the ECA biosynthetic pathway and the production of NeuAc or NeuAc-containing carbohydrates, and the effect on the production of NeuAc or NeuAc-containing carbohydrates when proteins involved in ECA biosynthesis, such as WecB, are deleted, are not known.

[0011] US Patent No. 5,665,574 Japanese Patent Application Laid-Open No. 10-4961 International Publication No. 2015 / 037698 International Publication No. 2008 / 040717 International Publication No. 2012 / 112777 International Publication No. 2004 / 003175 International Publication No. 2008 / 097366

[0012] Annu. Rev. Nutr. (2009), Vol. 29, p.177-222Biotechnology Advances (2021), Vol.46, 107678J. Am. Chem. Soc. (1988), Vol. 110, p.6481-6486J. Am. Chem. Soc. (1988), Vol. p.7159-7163Angew. Chem. Int. Ed. Eng. (1991), Vol. 30, p.827-828Carbohydrate Research (1998), Vol. 306, p.575-578Glycobiology (1997), Vol. 7, p.697-701Metab. Eng. (2012), Vol. 14, p.623-629FEMS Microbiol. Rev. (1988), Vol. 54, p.195-222J Gen Appl Microbiol (2020), Vol. 66, p.169-174

[0013] An object of the present invention is to provide a microorganism capable of producing NeuAc and / or NeuAc-containing carbohydrates, and a method for efficiently producing NeuAc and / or NeuAc-containing carbohydrates using the microorganism.

[0014] The present inventors have discovered that by using a microorganism capable of producing NeuAc and / or NeuAc-containing carbohydrates and in which the ECA biosynthetic pathway is blocked, NeuAc and / or NeuAc-containing carbohydrates can be produced more efficiently than conventional methods, and have completed the present invention.

[0015] That is, the present invention is as follows: 1. A microorganism having the ability to produce at least one of N-acetylneuraminic acid and N-acetylneuraminic acid-containing carbohydrates, and in which the biosynthetic pathway for enterobacterial common antigen (ECA) is blocked. 2. The microorganism according to 1 above, which is deficient in the activity of at least one protein selected from the following [1] to [3]: [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or a homologous sequence thereof, and having UDP-N-acetylglucosamine-undecaprenylphosphate N-acetylglucosamine phosphotransferase (WecA) activity; [2] A protein consisting of the amino acid sequence represented by SEQ ID NO: 4 or a homologous sequence thereof, and having UDP-N-acetylglucosamine 2-epimerase (WecB) activity; [3] A protein consisting of the amino acid sequence represented by SEQ ID NO: 6 or a homologous sequence thereof, and having UDP-N-acetylmannosamine dehydrogenase (WecC) activity. 3. 3. The microorganism according to 1 or 2 above, which is Escherichia coli. 4. A method for producing at least one of N-acetylneuraminic acid and N-acetylneuraminic acid-containing carbohydrates, which comprises culturing the microorganism according to any one of 1 to 3 above in a medium to produce at least one of N-acetylneuraminic acid and N-acetylneuraminic acid-containing carbohydrates.

[0016] The microorganism of the present invention is a microorganism capable of producing NeuAc and / or NeuAc-containing carbohydrates, and exhibits excellent NeuAc and / or NeuAc-containing carbohydrate production ability due to the blocking of the ECA biosynthetic pathway. By using the microorganism of the present invention, the production amount of NeuAc and / or NeuAc-containing carbohydrates can be increased and impurities can be reduced compared to conventional methods, thereby enabling efficient production of NeuAc and / or NeuAc-containing carbohydrates.

[0017] 1. Microorganism of the present invention and method for producing the same The microorganism of the present invention is a microorganism that has the ability to produce NeuAc and / or NeuAc-containing carbohydrates, and in which the ECA biosynthetic pathway is blocked, thereby improving the efficiency of producing NeuAc and / or NeuAc-containing carbohydrates.

[0018] Examples of the microorganism of the present invention include the following microorganisms (I) and (II): (I) a microorganism in which the ECA biosynthetic pathway of a parent strain of a microorganism that originally has the ability to produce NeuAc and / or NeuAc-containing carbohydrates has been blocked; and (II) a microorganism in which the ability to produce NeuAc and / or NeuAc-containing carbohydrates has been artificially imparted or enhanced to a parent strain of a microorganism in which the ECA biosynthetic pathway has been blocked.

[0019] In the present invention, a parent strain refers to an original strain that is the target of genetic modification, transformation, etc. The original strain that is the target of transformation by gene introduction is also called a host strain.

[0020] The parent strain may be any microorganism, but is preferably a prokaryote or yeast strain, more preferably a prokaryote belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, or Pseudomonas, or a yeast strain belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Siwaniomyces, Pichia, or Candida, and most preferably Escherichia coli BL21 codon plus, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue (all manufactured by Agilent Technologies), Escherichia coli BL21(DE3)pLysS (Merck Millipore), Escherichia coli DH5α, Escherichia coli HST08Premium, Escherichia coli HST02, Escherichia coli HST04 dam- / dcm-, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli CJ236, Escherichia coli BMH71-18 mutS, Escherichia coli MV1184, Escherichia coli TH2 (all Takara Bio Inc.), Escherichia coli W, Escherichia coli JM101, Escherichia coli W3110, Escherichia coli MG1655, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli MP347, Escherichia coli NM522, Escherichia coli ATCC9637, Escherichia coli coli BW25113, 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 Examples of suitable yeast strains include prokaryotes such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris, and Candida utilis.

[0021] 1-1. Microorganisms capable of producing NeuAc and / or NeuAc-containing carbohydrates In this specification, a microorganism capable of producing NeuAc and / or NeuAc-containing carbohydrates refers to a microorganism that has the ability to biosynthesize NeuAc and / or NeuAc-containing carbohydrates when cultured in a medium.

[0022] As microorganisms capable of producing NeuAc and / or NeuAc-containing carbohydrates, bred strains to which the ability to produce NeuAc and / or NeuAc-containing carbohydrates has been artificially imparted or enhanced can also be suitably used.

[0023] The production of NeuAc and / or NeuAc-containing carbohydrates by a microorganism can be confirmed, for example, by culturing the microorganism in a medium and detecting the NeuAc and / or NeuAc-containing carbohydrates accumulated in the culture using HPLC or a carbohydrate analyzer described below.

[0024] Methods for artificially imparting or enhancing the ability to produce NeuAc and / or NeuAc-containing carbohydrates include, for example, the following known methods (a) to (d), which can be used alone or in combination: (a) a method for enhancing the expression of at least one enzyme involved in the biosynthetic pathway that produces NeuAc and / or NeuAc-containing carbohydrates from sugars, (b) a method for increasing the copy number of at least one enzyme gene involved in the biosynthetic pathway that produces NeuAc and / or NeuAc-containing carbohydrates from sugars, (c) a method for relaxing or deactivating at least one mechanism that controls the biosynthetic pathway that produces NeuAc and / or NeuAc-containing carbohydrates from sugars, and (d) a method for weakening or blocking at least one metabolic pathway that branches off from the biosynthetic pathway that produces NeuAc and / or NeuAc-containing carbohydrates from sugars to a metabolite other than the target substance.

[0025] Furthermore, in order to artificially enhance the ability to produce NeuAc-containing carbohydrates, the expression of at least one enzyme involved in the assimilation or decomposition of a metabolite other than the target substance or a compound derived from a medium component may be enhanced. For example, a method may be used in which proteins involved in the uptake of maltose and isomaltose and / or proteins involved in the decomposition of maltose are artificially added or enhanced for the purpose of assimilating maltose or isomaltose derived from a medium component. Specific examples of such methods include those described in WO 2009 / 088049.

[0026] Examples of proteins involved in the uptake of maltose and isomaltose include known proteins such as PTS maltose transporter subunit IIBC MalP derived from Bacillus subtilis strain 168. Examples of proteins involved in the degradation of maltose include known proteins such as maltose-6-phosphate glucosidase MalA derived from Bacillus subtilis strain 168.

[0027] Furthermore, in order to artificially enhance the ability to produce NeuAc and / or NeuAc-containing carbohydrates, consumption of phosphoenolpyruvate, which is necessary for NeuAc production, may be suppressed. For example, in order to suppress consumption of phosphoenolpyruvate, which is necessary for NeuAc production, at least one protein involved in the conversion of pyruvate to oxaloacetate may be artificially added or enhanced. Specific examples of such methods include those described in Vemuri GN et al., Biotechnol Bioeng 2005, 90:64-76.

[0028] Examples of the protein involved in the conversion of pyruvate to oxaloacetate include known proteins such as pyruvate carboxylase Pyc derived from Corynebacterium glutamicum ATCC13032 strain.

[0029] <Microorganism 1 to which the ability to produce NeuAc and / or NeuAc-containing carbohydrates has been artificially imparted or enhanced> Among the microorganisms to which the ability to produce NeuAc has been artificially imparted or enhanced, specific examples of the microorganisms obtainable by the above method (a) or (b) include microorganisms to which the ability to produce at least one protein selected from the following [1] to [3] has been artificially imparted or enhanced: [1] NeuAc synthase [2] GlcNAc epimerase [3] N-acetylglucosamine-6-phosphate-N-acetyltransferase

[0030] Among microorganisms to which the ability to produce NeuAc-containing carbohydrates has been artificially imparted or enhanced, microorganisms obtainable by the above method (a) or (b) specifically include, for example, microorganisms to which the ability to produce at least one protein selected from the following [1] to [5] has been artificially imparted or enhanced: [1] NeuAc synthase [2] GlcNAc epimerase [3] N-acetylglucosamine-6-phosphate-N-acetyltransferase [4] CMP-NeuAc synthase [5] sialyltransferase

[0031] A microorganism that produces the protein can be prepared, for example, by introducing a recombinant DNA into a parent strain, in which a DNA fragment encoding the protein is inserted downstream of a promoter in an appropriate expression vector. When the recombinant DNA can be integrated into the chromosomal DNA of the parent strain, it does not need to contain a promoter.

[0032] 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, DNA containing a target gene, and a transcription termination sequence. A gene that controls the promoter may also be included.

[0033] It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 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.

[0034] The expression vector is not particularly limited as long as it is a suitable nucleic acid molecule for introducing, amplifying, and expressing the target DNA into a host, and not only plasmids but also, for example, artificial chromosomes, vectors using transposons, and cosmids may be used.

[0035] 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.), pMW119 (manufactured by Nippon Gene Co., Ltd.), pET21a, pCOLADuet-1, pCDFDuet-1, pCDF-1b, pRSF-1b (all manufactured by Merck Millipore), pMAL-c5x (manufactured by New England Biolabs), pGEX-4T-1, pTrc99A (all manufactured by GE Healthcare Biosciences), pTrcHis, pSE280 (all manufactured by Thermo Fisher Scientific), pGEMEX-1 (manufactured by Promega), pQE-30, pQE80L (all manufactured by Qiagen), pET-3, and pBluescriptII. SK(+), pBluescriptII KS(-) (both manufactured by Agilent Technologies), pKYP10 (Japanese Patent Laid-Open Publication No. 58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. , USA, 82, 4306 (1985)], pBluescript II SK(+), pBluescript II KS(-) (Stratagene), pTrS30 [prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrS32 [prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)], pTK31 [APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2007, Vol. 73, No. 20, pp. 6378-6385], pPAC31 (WO 98 / 12343), pUC19 [Gene, 33, 103 (1985)], pPA1 (JP 63-233798 A), pKD46 [Proc. Natl. Acad. Sci., USA, 97, 6640-6645 (2000)], and the like.

[0036] When using the above-mentioned expression vectors, any promoter may be used as long as it functions in the cells of a microorganism belonging to the genus Escherichia, and examples of such promoters include promoters of genes involved in amino acid biosynthesis, such as the trp promoter and ilv promoter, and promoters derived from Escherichia coli or phages, such as the uspA promoter, lac promoter, PL promoter, PR promoter, and PSE promoter.Artificially designed and modified promoters, such as a promoter consisting of two trp promoters in tandem, a tac promoter, a trc promoter, a lacT7 promoter, and a letI promoter, can also be used.

[0037] When a coryneform bacterium is used as the parent strain, examples of the expression vector include pCG1 (Japanese Patent Laid-Open No. 57-134500), pCG2 (Japanese Patent Laid-Open No. 58-35197), pCG4 (Japanese Patent Laid-Open No. 57-183799), pCG11 (Japanese Patent Laid-Open No. 57-134500), pCG116, pCE54, pCB101 (all Japanese Patent Laid-Open No. 58-105999), pCE51, pCE52, and pCE53 (all of which are described in Molecular and General Genetics, 196, 175 (1984)).

[0038] When using the above expression vector, any promoter may be used as long as it functions in cells of coryneform bacteria, and an example thereof is the P54-6 promoter [Appl. Microbiol. Biotechnol., 53, pp. 674-679 (2000)].

[0039] 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.

[0040] 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.

[0041] Here, the expression level of the protein encoded by the DNA can be improved by substituting bases in the base sequence of the DNA so that the codons are optimal for expression in the parent strain. Information on codon usage frequencies in the parent strain used in the production method of the present invention is available through public databases.

[0042] Methods for introducing recombinant DNA into a host strain as an autonomously replicable plasmid include, for example, a method using calcium ions [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], the protoplast method (Japanese Patent Laid-Open Publication No. 63-248394), and the electroporation method [Nucleic Acids Res., 16, 6127 (1988)].

[0043] Methods for integrating recombinant DNA into the chromosome of a parent strain include, for example, homologous recombination. Examples of homologous recombination include a method using a plasmid for homologous recombination, 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. Furthermore, a method using homologous recombination that is frequently used in Escherichia coli includes, for example, a method of introducing recombinant DNA using the homologous recombination system of lambda phage [Proc. Natl. Acad. Sci. USA, 97, 6641-6645 (2000)].

[0044] Furthermore, a microorganism in which a target region on the chromosomal DNA of a parent strain has been replaced with recombinant DNA can be obtained using a selection method that utilizes the fact that E. coli becomes sucrose-sensitive due to Bacillus subtilis levansucrase integrated into the chromosome together with recombinant DNA, or a selection method that utilizes the fact that E. coli becomes streptomycin-sensitive by incorporating a wild-type rpsL gene into E. coli having a streptomycin-resistant mutant rpsL gene [Mol. Microbiol., 55, 137 (2005), Biosci. Biotechnol. Biochem., 71, 2905 (2007)]. Here, the chromosomal region to be introduced is not particularly limited, but is preferably a non-essential gene region or a non-gene region upstream of a non-essential gene region.

[0045] The DNAs encoding the proteins selected from the group consisting of [1] NeuAc synthase, [2] GlcNAc epimerase, [3] N-acetylglucosamine-6-phosphate-N-acetyltransferase, [4] CMP-NeuAc synthase, and [5] sialyltransferase may be present separately in any combination in one to four, preferably one to three, more preferably one or two, and most preferably one, type of recombinant DNA.

[0046] <<[1] NeuAc Synthase>> NeuAc synthase refers to a protein having NeuAc synthase activity. NeuAc synthase activity refers to the activity of producing NeuAc using ManNAc and PEP as substrates.

[0047] The NeuAc synthase is not particularly limited as long as it has NeuAc synthase activity, and examples include the NeuAc synthase CjneuB of Campylobacter jejuni ATCC 43438 strain and the protein described in WO 2015 / 037698.

[0048] Whether a target protein has NeuAc synthase activity can be confirmed by preparing a recombinant DNA containing DNA encoding the protein, transforming a microorganism that does not have NeuAc synthase activity, for example, Escherichia coli W3110 strain, with the recombinant DNA, culturing the resulting microorganism, preparing a cell extract containing the protein from the resulting culture, contacting the fraction with the substrates ManNAc and PEP, and detecting the resulting NeuAc by high-performance chromatography or gas chromatography.

[0049] Examples of microorganisms that produce NeuAc synthase include microorganisms that have enhanced NeuAc synthase activity compared to the parent strain, which are obtained by transforming a parent strain with recombinant DNA containing DNA encoding NeuAc synthase.

[0050] The DNA encoding NeuAc synthase is preferably derived from a prokaryote such as a bacterium or yeast, more preferably from a prokaryote, and most preferably from Campylobacter jejuni ATCC 43438 strain, and includes DNA encoding CjneuB (SEQ ID NO: 7).

[0051] A microorganism in which NeuAc synthase activity is enhanced compared to that of a parent strain refers to a microorganism in which the recombinant DNA is introduced into the parent strain as an autonomously replicable plasmid or is integrated into the chromosome of the parent strain, thereby increasing the transcription amount of the DNA or the production amount of the protein encoded by the DNA.

[0052] The increase in the transcription amount of the DNA or the production amount of the protein encoded by the DNA can be confirmed, for example, by comparing the transcription amount of the DNA with that of the parent strain by Northern blotting, or the production amount of the protein with that of the parent strain by Western blotting.

[0053] <<[2] GlcNAc epimerase>> GlcNAc epimerase refers to a protein having GlcNAc epimerase activity. GlcNAc epimerase activity refers to the activity of producing ManNAc through an epimerization reaction using GlcNAc as a substrate.

[0054] Examples of microorganisms that produce GlcNAc epimerase include microorganisms belonging to the genus Synechocystis, and microorganisms that have enhanced GlcNAc epimerase activity compared to a parent strain, which microorganisms are obtained by transforming a parent strain with a recombinant DNA containing DNA encoding GlcNAc epimerase.

[0055] A preferred example of the microorganism belonging to the genus Synechocystis is Synechocystis sp. strain PCC6803 (Pasteur Culture Collection of Cyanobacteria, INSTITUTE PASTEUR).

[0056] Examples of DNA encoding GlcNAc epimerase include DNA (SEQ ID NO: 8) encoding GlcNAc epimerase preferably derived from a prokaryote such as a bacterium or yeast, particularly preferably derived from a prokaryote, and most preferably derived from Synechocystis sp. strain PCC6803.

[0057] For example, DNA encoding GlcNAc epimerase derived from Synechocystis sp. PCC6803 strain can be obtained by the method described in WO 00 / 47730.

[0058] By transforming a parent strain with a recombinant DNA containing a DNA encoding GlcNAc epimerase, a microorganism can be constructed in which the GlcNAc epimerase activity is enhanced compared to that of the parent strain. Whether the microorganism has enhanced GlcNAc epimerase activity compared to that of the parent strain can be confirmed by comparing the transcription amount of the DNA encoding GlcNAc epimerase and the production amount of the protein with those of the parent strain using the above-mentioned method. An example of such a microorganism is W3110 / pRcneuB2.

[0059] <<[3] N-acetylglucosamine-6-phosphate N-acetyltransferase>> N-acetylglucosamine-6-phosphate N-acetyltransferase refers to a protein having N-acetylglucosamine-6-phosphate N-acetyltransferase activity. N-acetylglucosamine-6-phosphate N-acetyltransferase activity refers to the activity of producing N-acetyl-D-glucosamine 6-phosphate from acetyl-CoA and D-glucosamine 6-phosphate.

[0060] Examples of microorganisms that produce N-acetylglucosamine-6-phosphate N-acetyltransferase include microorganisms belonging to the genus Saccharomyces, and microorganisms that have enhanced N-acetylglucosamine-6-phosphate N-acetyltransferase activity compared to a parent strain, which microorganisms are obtained by transforming a parent strain with a recombinant DNA containing DNA encoding N-acetylglucosamine-6-phosphate N-acetyltransferase.

[0061] A preferred example of the microorganism belonging to the genus Saccharomyces is Saccharomyces cerevisiae S288C strain.

[0062] The DNA encoding N-acetylglucosamine-6-phosphate N-acetyltransferase is preferably derived from a prokaryote such as a bacterium or yeast, more preferably from yeast, and most preferably from Saccharomyces cerevisiae S288C strain (SEQ ID NO: 9).

[0063] By transforming a parent strain with recombinant DNA containing DNA encoding N-acetylglucosamine-6-phosphate N-acetyltransferase, a microorganism can be constructed in which N-acetylglucosamine-6-phosphate N-acetyltransferase activity is enhanced compared to that of the parent strain. Whether the microorganism has enhanced N-acetylglucosamine-6-phosphate N-acetyltransferase activity compared to that of the parent strain can be confirmed by comparing the transcription amount of DNA encoding N-acetylglucosamine-6-phosphate N-acetyltransferase and the production amount of the protein with those of the parent strain.

[0064] <<[4] CMP-NeuAc Synthase>> CMP-NeuAc synthase refers to a protein having CMP-NeuAc synthase activity. CMP-NeuAc synthase activity refers to the activity of producing CMP-NeuAc using CTP and NeuAc as substrates.

[0065] Examples of microorganisms that produce CMP-NeuAc synthase include microorganisms belonging to the genus Pasteurella, and microorganisms that are obtained by transforming a parent strain with a recombinant DNA containing DNA encoding CMP-NeuAc synthase and have enhanced CMP-NeuAc synthase activity compared to the parent strain.

[0066] A preferred example of the microorganism belonging to the genus Pasteurella is Pasteurella multocida PM70 strain.

[0067] The DNA encoding the CMP-NeuAc synthase is preferably derived from a prokaryote such as a bacterium or yeast, particularly preferably from a prokaryote, and most preferably from Pasteurella multocida PM70 strain (see JP 2008-5794 A).

[0068] For example, DNA encoding CMP-NeuAc synthase derived from Pasteurella multocida strain PM70 can be obtained by the method described in JP-A-2008-5794.

[0069] By transforming a parent strain with recombinant DNA containing DNA encoding CMP-NeuAc synthase, a microorganism can be constructed in which CMP-NeuAc synthase activity is enhanced compared to the parent strain. Whether the microorganism has enhanced CMP-NeuAc synthase activity compared to the parent strain can be confirmed by comparing the transcription amount of DNA encoding CMP-NeuAc synthase and the production amount of the protein with those of the parent strain. Examples of such microorganisms include Escherichia coli N18-14 / pMnK1 (Japanese Patent Laid-Open Publication No. 2008-5794).

[0070] <<[5] Sialyltransferase>> Sialyltransferase refers to a protein having sialyltransferase activity, which refers to the activity of producing a NeuAc-containing carbohydrate using CMP-NeuAc and an acceptor carbohydrate as substrates.

[0071] Examples of acceptor carbohydrates that serve as substrates for sialyltransferase include oligosaccharides, polysaccharides, and complex carbohydrates such as glycoproteins and glycolipids. Examples of oligosaccharides or polysaccharides that serve as substrates for sialyltransferase include oligosaccharides or polysaccharides having galactose at the non-reducing end, or oligosaccharides or polysaccharides having NeuAc at the non-reducing end.

[0072] Among the above oligosaccharides or polysaccharides, preferred are oligosaccharides having at their non-reducing ends a structure selected from the group consisting of lactose, globotriose, N-acetyllactosamine, lacto-N-tetraose, lacto-N-neotetraose, Lewis a, and Lewis X, or oligosaccharides having at their non-reducing ends a structure selected from the group consisting of NeuAcα2-Galβ1-4Glc and NeuAcα2-3Galβ1-4GlcNAc, and more preferably lactose.

[0073] Examples of complex carbohydrates that can serve as substrates for sialyltransferase include complex carbohydrates in which proteins, lipids, etc. are bound to the above-mentioned oligosaccharides and polysaccharides.

[0074] Examples of NeuAc-containing saccharides include saccharides in which NeuAc is added to the acceptor saccharide, preferably saccharides containing an oligosaccharide having a structure selected from the group consisting of NeuAcα2-3Galβ1-4Glc, NeuAcα2-6Galβ1-4Glc, and NeuAcα2-8NeuAc at the non-reducing end, more preferably 3'-sialyllactose (3'SL), 6'-sialyllactose (6'SL), sialyllactose-N-tetraose a (LST-a), sialyllactose-N-tetraose b (LST-b), sialyllactose-N-tetraose c (LST-c), or disialyllacto-N-tetraose (DSLNT).

[0075] Specific examples of sialyltransferase include known enzymes such as α2,3-sialyltransferase, which produces 3'-sialyllactose using CMP-NeuAc and lactose as substrates, and α2,6-sialyltransferase, which produces 6'-sialyllactose using CMP-NeuAc and lactose as substrates.

[0076] Examples of microorganisms that produce sialyltransferase include microorganisms belonging to the genus Pasteurella, microorganisms belonging to the genus Photobacterium, and microorganisms that have enhanced sialyltransferase activity compared to a parent strain and are obtained by transforming a parent strain with a recombinant DNA containing DNA encoding sialyltransferase.

[0077] A preferred example of the microorganism belonging to the genus Pasteurella is Pasteurella multocida PM70 strain. A preferred example of the microorganism belonging to the genus Photobacterium is Photobacterium damselae JT0160 strain.

[0078] Examples of DNA encoding a sialyltransferase include DNA derived preferably from prokaryotes such as bacteria or yeast, particularly from prokaryotes, and most preferably from Pasteurella multocida PM70 strain (WO 03 / 27297) and DNA encoding α-2,6-sialyltransferase derived from Photobacterium damselae JT0160 strain (GenBank ACCESSION No. BAA25316).

[0079] For example, DNA encoding α-2,3-sialyltransferase derived from Pasteurella multocida PM70 strain and DNA encoding α-2,6-sialyltransferase derived from Photobacterium damselae JT0160 strain can be obtained by the method described in WO 03 / 27297.

[0080] By transforming a parent strain with recombinant DNA containing DNA encoding sialyltransferase, a microorganism can be constructed that has enhanced sialyltransferase activity compared to the parent strain.

[0081] Whether the microorganism constructed by the above method has enhanced sialyltransferase activity compared to the parent strain can be confirmed by comparing the transcription amount of DNA encoding a protein having sialyltransferase activity and the production amount of the protein with those of the parent strain. Examples of such microorganisms include Escherichia coli NM522 / pYP3 (JP 2008-5794 A).

[0082] <Microorganism 2 to which the ability to produce NeuAc and / or NeuAc-containing carbohydrates has been artificially imparted or enhanced> Among microorganisms to which the ability to produce NeuAc and / or NeuAc-containing carbohydrates has been artificially imparted or enhanced, specific examples of microorganisms obtainable by the above method (c) or (d) preferably include microorganisms that are deficient in a transcription factor that negatively regulates the activity of a gene encoding an enzyme involved in NeuAc biosynthesis, and microorganisms with reduced NeuAc decomposition activity.

[0083] <<Microorganism Deficient in Transcription Factors That Negatively Regulate the Activity of Genes That Encode Enzymes Involved in NeuAc Biosynthesis>> Examples of transcription factors that negatively regulate the activity of genes that encode enzymes involved in NeuAc biosynthesis include yhbJ, fruR, and cra. Among these, yhbJ is preferred from the viewpoint of specificity for NeuAc production.

[0084] yhbJ is a transcriptional regulator that negatively regulates the activity of the glmS gene, which encodes L-glutamine-D-fructose-6-phosphate aminotransferase involved in NeuAc biosynthesis.

[0085] Genetic modification that eliminates the function of a transcription factor that negatively regulates the activity of a gene encoding an enzyme involved in NeuAc biosynthesis can be achieved by modifying a portion of the host's genomic DNA that encodes a protein whose function is to be eliminated, thereby reducing or completely terminating the function of the protein encoded by the DNA.

[0086] The form of modification made to the DNA is not particularly limited as long as it is a form that reduces or completely stops the function of the protein encoded by the DNA encoding the portion corresponding to the protein whose function is to be lost, and known methods can be used as appropriate.

[0087] Examples of forms for reducing or completely stopping the function of a protein encoded by a portion corresponding to the protein whose function is to be lost include any one of the following modifications (i) to (iii): (i) removing all or part of the DNA encoding the portion corresponding to the protein whose function is to be lost; (ii) making one or more substitutions, deletions, or additions to the DNA encoding the portion corresponding to the protein whose function is to be lost; or (iii) replacing the DNA encoding the portion corresponding to the protein whose function is to be lost with a DNA sequence that has less than 80% identity to the DNA sequence before modification.

[0088] When the protein whose function is to be lost is yhbJ, the loss of yhbJ function means, for example, that the activity of yhbJ is preferably 20% or less, more preferably 10% or less, and even more preferably 5% or less, compared to an unmodified strain.

[0089] The activity of yhbJ can be confirmed by examining the expression level of GlmS by Western blotting or the like [Kalamorz F. et al., Mol Microbiol. 65(6):1518-33(2007)].

[0090] <<Microorganisms with Reduced NeuAc Degradation Activity>> Examples of microorganisms with reduced NeuAc degrading activity include microorganisms with reduced NeuAc lyase activity. Examples of microorganisms with reduced NeuAc lyase activity include Escherichia coli NAN8-71 (FERM BP-7908, WO 03 / 72783).

[0091] 1-2. Microorganisms in which the enterobacterial common antigen (ECA) biosynthetic pathway is blocked The microorganism of the present invention is a microorganism that has the ability to produce N-acetylneuraminic acid or N-acetylneuraminic acid-containing carbohydrates and in which the ECA biosynthetic pathway is blocked. Examples of methods for blocking the ECA biosynthetic pathway include methods that delete the activity of proteins involved in the ECA biosynthetic pathway.

[0092] Methods for deleting the activity of a protein involved in the ECA biosynthetic pathway include, for example, methods for deleting the function of a specific protein, similar to the method described above in the section "Microorganisms Deficient in Transcription Factors That Negatively Regulate the Activity of Genes Encoding Enzymes Involved in NeuAc Biosynthesis." Specific examples of such methods include knocking out a specific gene using PCR [Baba T. et al., Mol. Systems Biol. (2006)], a method using the lambda phage homologous recombination system [Proc. Natl. Acad. Sci. USA, 97, 6641-6645 (2000)], a method for inhibiting the expression of a specific gene by RNAi, and a method for introducing a mutation into a specific gene and its promoter region using a mutagen such as nitrosoguanidine or ultraviolet light.

[0093] Proteins involved in the ECA biosynthetic pathway include, for example, WecA, WecB, WecC, WecD, WecE, WecF, and WecG.

[0094] WecA refers to a protein having UDP-N-acetylglucosamine-undecaprenylphosphate N-acetylglucosamine phosphotransferase activity, which is involved in the biosynthesis of ECA.

[0095] Examples of WecA include WecA (SEQ ID NO: 2) preferably derived from prokaryotes such as bacteria or yeast, more preferably derived from prokaryotes, particularly preferably derived from Escherichia coli, and most preferably derived from Escherichia coli BW25113 strain.

[0096] The DNA encoding WecA is preferably derived from a prokaryote such as a bacterium or yeast, more preferably derived from a prokaryote, particularly preferably derived from Escherichia coli, and most preferably derived from the Escherichia coli BW25113 strain (SEQ ID NO: 1).

[0097] WecB refers to a protein having UDP-N-acetylglucosamine 2-epimerase activity that is involved in the biosynthesis of ECA. Examples of WecB include WecB (SEQ ID NO: 4) derived preferably from prokaryotes such as bacteria or yeast, more preferably from prokaryotes, particularly preferably from Escherichia coli, and most preferably from Escherichia coli BW25113 strain.

[0098] The DNA encoding WecB is preferably derived from a prokaryote such as a bacterium or yeast, more preferably derived from a prokaryote, particularly preferably derived from Escherichia coli, and most preferably derived from the Escherichia coli BW25113 strain (SEQ ID NO: 3).

[0099] WecC refers to a protein having UDP-N-acetylmannosamine dehydrogenase activity that is involved in the biosynthesis of ECA. Examples of WecC include WecC (SEQ ID NO: 6) derived preferably from prokaryotes such as bacteria or yeast, more preferably from prokaryotes, particularly preferably from Escherichia coli, and most preferably from Escherichia coli BW25113 strain.

[0100] The DNA encoding WecC is preferably derived from a prokaryote such as a bacterium or yeast, more preferably derived from a prokaryote, particularly preferably derived from Escherichia coli, and most preferably derived from the Escherichia coli BW25113 strain (SEQ ID NO: 5).

[0101] WecD refers to a protein having dTDP-4-amino-4,6-dideoxy-D-galactose acyltransferase activity that is involved in the biosynthesis of ECA. Examples of WecD include WecD (SEQ ID NO: 52) derived preferably from prokaryotes such as bacteria or yeast, more preferably from prokaryotes, particularly preferably from Escherichia coli, and most preferably from Escherichia coli BW25113 strain.

[0102] The DNA encoding WecD is preferably derived from a prokaryote such as a bacterium or yeast, more preferably derived from a prokaryote, particularly preferably derived from Escherichia coli, and most preferably derived from the Escherichia coli BW25113 strain (SEQ ID NO: 51).

[0103] WecE refers to a protein having dTDP-4-dehydro-6-deoxy-D-glucose transaminase activity that is involved in the biosynthesis of ECA. Examples of WecE include WecE (SEQ ID NO: 54) derived preferably from prokaryotes such as bacteria or yeast, more preferably from prokaryotes, particularly preferably from Escherichia coli, and most preferably from Escherichia coli BW25113 strain.

[0104] The DNA encoding WecE is preferably derived from a prokaryote such as a bacterium or yeast, more preferably derived from a prokaryote, particularly preferably derived from Escherichia coli, and most preferably derived from the Escherichia coli BW25113 strain (SEQ ID NO: 53).

[0105] WecF refers to a protein having dTDP-N-acetylfucosamine:Lipid II N-acetylfucosaminyltransferase activity, which is involved in the biosynthesis of ECA. Examples of WecF include WecF (SEQ ID NO: 56) derived preferably from prokaryotes such as bacteria or yeast, more preferably from prokaryotes, particularly preferably from Escherichia coli, and most preferably from Escherichia coli BW25113 strain.

[0106] The DNA encoding WecF is preferably derived from a prokaryote such as a bacterium or yeast, more preferably derived from a prokaryote, particularly preferably derived from Escherichia coli, and most preferably derived from the Escherichia coli BW25113 strain (SEQ ID NO: 55).

[0107] WecG refers to a protein having UDP-N-acetyl-D-mannosaminouronate transferase activity that is involved in the biosynthesis of ECA. Examples of WecG include WecG (SEQ ID NO: 58) derived preferably from prokaryotes such as bacteria or yeast, more preferably from prokaryotes, particularly preferably from Escherichia coli, and most preferably from Escherichia coli BW25113 strain.

[0108] The DNA encoding WecG is preferably derived from a prokaryote such as a bacterium or yeast, more preferably derived from a prokaryote, particularly preferably derived from Escherichia coli, and most preferably derived from the Escherichia coli BW25113 strain (SEQ ID NO: 57).

[0109] Specifically, preferred microorganisms in which the ECA biosynthetic pathway is blocked are those lacking the activity of at least one protein selected from the following [1] to [3]: [1] A protein having WecA activity, which consists of the amino acid sequence represented by SEQ ID NO: 2 or a homologous sequence thereof; [2] A protein having WecB activity, which consists of the amino acid sequence represented by SEQ ID NO: 4 or a homologous sequence thereof; [3] A protein having WecC activity, which consists of the amino acid sequence represented by SEQ ID NO: 6 or a homologous sequence thereof.

[0110] The identity of a nucleotide sequence or an amino acid sequence can be determined using the algorithm BLAST (Pro. Nat. Acad. Sci. USA, 90, 5873, 1993) or FASTA (Methods Enzymol., 183, 63, 1990) by Karlin and Altschul. 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 the BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific techniques for these analysis methods are known.

[0111] The identity between the target amino acid sequence (the amino acid sequence represented by SEQ ID NO: 2, 4, or 6) and its homologous sequence is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more.

[0112] The method for obtaining a protein consisting of a homologous sequence (hereinafter also referred to as a homologous protein) is not particularly limited, and any known method can be used. Specifically, for example, the following method can be mentioned. First, various gene sequence databases are searched for a base sequence that has an identity of preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 99% or more with the base sequence encoding the target amino acid sequence. Subsequently, using a probe DNA or primer DNA that can be designed based on the base sequence or amino acid sequence obtained by the search and a microorganism having the DNA, and using a probe that can be designed based on the base sequence of the DNA, the target protein can be obtained by Southern hybridization to a chromosomal DNA library of a microorganism, preferably of the genus Escherichia, more preferably Escherichia coli, and even more preferably Escherichia coli BW25113 strain, or by PCR [PCR Protocols, Academic Press (1990)] using primer DNA that can be designed based on the DNA encoding the target protein and chromosomal DNA of a microorganism, preferably of the genus Escherichia, more preferably Escherichia coli, and even more preferably Escherichia coli BW25113 strain as a template.

[0113] A homologous sequence may be an amino acid sequence obtained by artificially deleting or substituting amino acid residues in the original amino acid sequence, or by artificially inserting or adding amino acid residues into the amino acid sequence. In a homologous protein, "deleted, substituted, inserted, or added amino acids" may mean that 1 to 20 amino acids are deleted, substituted, inserted, or added at any position in the same sequence.

[0114] An alignment of amino acid sequences can be created using, for example, the known alignment program ClustalW [Nucleic Acids Research 22, 4673 (1994)]. When creating an alignment using ClustalW, for example, default values ​​can be used as parameters.

[0115] When substituting, inserting, or adding an amino acid to a target amino acid sequence, the amino acid residue to be substituted, inserted, or added may be naturally occurring or non-naturally occurring. Examples of 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.

[0116] 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

[0117] The activity of a protein involved in the ECA biosynthetic pathway can be confirmed, for example, by examining the expression level of the ECA gene by Northern blotting, Western blotting, etc. Specifically, for example, the WecA activity, WecB activity, or WecC activity of a homologous protein can be confirmed by the method shown below.

[0118] Whether a homologous protein consisting of a sequence homologous to the amino acid sequence represented by SEQ ID NO:2 has WecA activity can be confirmed, for example, by the following method. First, a recombinant DNA is prepared containing DNA encoding the protein whose activity is to be confirmed. Next, a microorganism that does not have WecA activity, such as the Escherichia coli BW25113ΔyhbJΔwecA strain described below, is transformed with the recombinant DNA, and the resulting microorganism is cultured. A cell extract containing the homologous protein is prepared from the resulting culture. The cell extract containing the homologous protein is contacted with an aqueous solution containing the substrates undecaprenyl-1-phosphate and UDP-N-acetylglucosamine, to produce Lipid I in the aqueous solution. Finally, Lipid I in the reaction solution is detected using the SPD-20A analyzer (Shimadzu Corporation) described below, thereby confirming that the homologous protein has WecA activity.

[0119] Whether a homologous protein consisting of a sequence homologous to the amino acid sequence represented by SEQ ID NO:4 has WecB activity can be confirmed, for example, by the following method. First, a recombinant DNA is prepared containing DNA encoding the protein whose activity is to be confirmed. Next, a microorganism lacking WecB activity, such as the Escherichia coli BW25113ΔyhbJΔwecB strain described below, is transformed with the recombinant DNA, and the resulting microorganism is cultured. A cell extract containing the homologous protein is prepared from the resulting culture. The cell extract containing the homologous protein is contacted with an aqueous solution containing the substrate UDP-N-acetylglucosamine, thereby producing UDP-N-acetylmannosamine in the aqueous solution. Finally, the homologous protein can be confirmed to have WecB activity by detecting UDP-N-acetylmannosamine in the reaction solution using the SPD-20A analyzer (Shimadzu Corporation) described below.

[0120] Whether a homologous protein consisting of a sequence homologous to the amino acid sequence represented by SEQ ID NO:6 has WecC activity can be confirmed, for example, by the following method. First, a recombinant DNA is prepared containing DNA encoding the protein whose activity is to be confirmed. Next, a microorganism lacking WecC activity, such as the Escherichia coli BW25113ΔyhbJΔwecC strain described below, is transformed with the recombinant DNA to produce a microorganism, and a cell extract containing the homologous protein is prepared from the resulting culture. The cell extract containing the homologous protein is contacted with an aqueous solution containing the substrate UDP-N-acetylmannosamine to produce UDP-ManNAcA in the aqueous solution. Finally, the homologous protein can be confirmed to have WecC activity by detecting UDP-N-acetyl-D-mannosaminouronate in the reaction solution using the SPD-20A analyzer (Shimadzu Corporation) described below.

[0121] DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, or 6 can be obtained, for example, by Southern hybridization of a chromosomal DNA library of a microorganism, preferably of the genus Escherichia, more preferably Escherichia coli, even more preferably Escherichia coli BW25113 strain, using a probe that can be designed based on the base sequence of DNA encoding the amino acid sequence represented by SEQ ID NO: 2, 4, or 6; or by PCR [PCR Protocols, Academic Press (1990)] using primer DNA that can be designed based on the DNA encoding the amino acid sequence represented by SEQ ID NO: 2, 4, or 6 and chromosomal DNA of a microorganism, preferably of the genus Escherichia, more preferably Escherichia coli, even more preferably Escherichia coli BW25113 strain, as a template.

[0122] A specific example of DNA encoding the amino acid sequence represented by SEQ ID NO: 2 is DNA having the base sequence represented by SEQ ID NO: 1. A specific example of DNA encoding the amino acid sequence represented by SEQ ID NO: 4 is DNA having the base sequence represented by SEQ ID NO: 3. A specific example of DNA encoding the amino acid sequence represented by SEQ ID NO: 6 is DNA having the base sequence represented by SEQ ID NO: 5.

[0123] In the microorganism obtained by the above-mentioned method, the absence of activity of a protein involved in the ECA biosynthetic pathway can be confirmed, for example, by examining the expression level of a gene encoding the protein involved in the above-mentioned ECA biosynthetic pathway by Northern blotting, Western blotting, or the like.

[0124] The fact that the microorganisms constructed by the above-mentioned method have improved NeuAc and / or NeuAc-containing carbohydrate production efficiency compared to the parent strain can be confirmed, for example, by the following method. First, the parent strain and the constructed microorganism are each cultured in a medium. Next, the NeuAc and / or NeuAc-containing carbohydrates accumulated in the culture are detected using HPLC or a carbohydrate analyzer described below, and the production amounts of the parent strain and the constructed microorganism are compared, thereby confirming that the production efficiency of NeuAc and / or NeuAc-containing carbohydrates is improved compared to the parent strain.

[0125] 2. Method for producing NeuAc and / or NeuAc-containing carbohydrates using microorganisms The method for producing NeuAc and / or NeuAc-containing carbohydrates of the present invention is characterized by culturing the above-mentioned microorganisms in a medium and producing NeuAc and / or NeuAc-containing carbohydrates in the culture.

[0126] The microorganisms can be cultured according to a conventional method used for culturing microorganisms.

[0127] As a medium for culturing a microorganism, either a natural medium or a synthetic medium may be used as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be utilized by the microorganism and allows the transformant to be cultured efficiently.

[0128] 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.

[0129] 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.

[0130] Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like.

[0131] Cultivation is preferably carried out under aerobic conditions, such as shaking culture or submerged aeration and stirring culture. The culture temperature is usually 15 to 40°C, and the culture time is usually 5 hours to 7 days. The pH of the culture solution during cultivation is usually maintained at 3.0 to 9.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

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

[0133] When producing NeuAc-containing carbohydrates, it is preferable to include an acceptor carbohydrate such as lactose in the medium. The acceptor carbohydrate may be added at the beginning of the culture or may be added during the culture as needed. The concentration of the acceptor carbohydrate added in the medium is preferably 10 to 300 g / L.

[0134] Furthermore, in the production of NeuAc-containing carbohydrates, when the microorganism used does not have the ability to produce an acceptor carbohydrate such as lactose, instead of adding an acceptor carbohydrate such as lactose to the medium during cultivation, a microorganism capable of producing an acceptor carbohydrate such as lactose from sugar may be cultured simultaneously with the microorganism of the present invention, thereby supplying the acceptor carbohydrate such as lactose to the transformant of the present invention.

[0135] By the above-mentioned cultivation, NeuAc or NeuAc-containing carbohydrates are produced and accumulated in the culture, and by collecting the NeuAc or NeuAc-containing carbohydrates from the culture, NeuAc or NeuAc-containing carbohydrates can be produced.

[0136] NeuAc or NeuAc-containing carbohydrates can be collected from the culture by a combination of known methods such as activated carbon, ion exchange resin, precipitation, etc. When NeuAc or NeuAc-containing carbohydrates accumulate in the cells, the cells can be disrupted by ultrasonication or the like, and the cells can be removed by centrifugation, and the resulting supernatant can be collected by the ion exchange resin method or the like.

[0137] NeuAc or NeuAc-containing carbohydrates can be quantified using a sugar analyzer manufactured by Dionex, a dual wavelength absorbance detector manufactured by Shimadzu Corporation, or the like [Anal. Biochem., 189, 151 (1990)].

[0138] [Analysis Examples] In the Examples, analysis and quantification of NeuAc were carried out according to the following procedures. After cultivation, the culture solution containing the microorganisms was centrifuged, and the supernatant was collected. The NeuAc contained in the supernatant was analyzed using an analyzer SPD-20A (manufactured by Shimadzu Corporation). [Analysis conditions] Column: Shodex RSpak KC-811 Column temperature: 40°C Eluent composition: 1% phosphoric acid aqueous solution Flow rate: 0.5 ml / min Detector: SPD-20A

[0139] Example 1 Construction of Microorganisms Used for NeuAc Production (1) Construction of Plasmids for NeuAc Production Plasmids expressing the NeuAc synthase gene CjneuB (SEQ ID NO: 7) from Campylobacter jejuni ATCC 43438 strain, the N-GluNAc 2-epimerase gene slr1975 (SEQ ID NO: 8) from Synechocystis sp. PCC 6803 strain, and the N-acetylglucosamine-6-phosphate-N-acetyltransferase gene GNA1 (SEQ ID NO: 9) from Saccharomyces cerevisiae S288C strain were constructed by the following procedure.

[0140] Campylobacter jejuni ATCC 43438 strain, Synechocystis sp. PCC 6803 strain, or Saccharomyces cerevisiae S288C strain was cultured by a known culture method, and the chromosomal DNA of each microorganism was isolated and purified. Using the prepared DNA, PCR was performed using DNAs consisting of the base sequences shown in "Primer Set" in Table 1 as a primer set and DNAs shown in "Template" in Table 1 as a template, to obtain each amplified DNA fragment.

[0141]

[0142] PCR was performed using a mixture of the CjneuB fragment, slr1975 fragment, and GNA1 fragment obtained above in an equimolar ratio as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 10 and 15 as a primer set to obtain a DNA fragment of approximately 2.7 kb in which the CjneuB fragment, slr1975 fragment, and GNA1 fragment were ligated (hereinafter referred to as the CjneuB-slr1975-GNA1 fragment).

[0143] The CjneuB-slr1975-GNA1 fragment and the expression vector pTrc99a (GE Healthcare Biosciences) were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the expression plasmid pTrc99a-CjneuB-slr1975-GNA1 (hereinafter referred to as pCSG1).

[0144] (2) Construction of a Host for NeuAc Production <yhbJ Gene-Deficient Escherichia coli> Escherichia coli in which the yhbJ gene (SEQ ID NO: 16), a transcriptional regulator that negatively regulates the activity of the glmS gene encoding L-glutamine-D-fructose-6-phosphate aminotransferase involved in NeuAc biosynthesis, was disrupted by the following procedure.

[0145] Using the BW25113 strain (Keio collection (Systematic single-gene knockout mutants of E. coli K-12)) as a host, a strain in which the yhbJ gene was completely deleted, BW25113ΔyhbJ strain, was obtained by a method similar to that described by Baba et al. [Baba T. et al. (2006) Mol systems Biol].

[0146] <Obtaining DNA fragments to be used as markers for gene deletion> PCR was performed using DNAs consisting of the base sequences shown in "Primer set" in Table 2 as a primer set and DNAs shown in "Template" in Table 2 as a template to obtain each amplified DNA fragment.

[0147]

[0148] Genomic DNA of Bacillus subtilis strain 168 was prepared by a standard method. The amplified DNA fragment cat contains approximately 200 bp upstream to approximately 50 bp downstream of the cat (chloramphenicol acetyltransferase) gene on pHSG396. The amplified DNA fragment sacB contains approximately 300 bp upstream to approximately 100 bp downstream of the sacB (levansucrase) gene on the genomic DNA of Bacillus subtilis strain 168.

[0149] Next, PCR was performed using a mixture of the amplified DNA fragments cat and sacB in an equimolar ratio as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 17 and 20 as a primer set to obtain a DNA fragment containing the cat gene and the sacB gene (hereinafter referred to as cat-sacB).

[0150] <E. coli lacking the wecA gene> Using the BW25113ΔyhbJ strain constructed above as a parent strain, E. coli in which the wecA gene (SEQ ID NO: 1) encoding UDP-N-acetylglucosamine-undecaprenylphosphate N-acetylglucosamine phosphotransferase involved in the biosynthesis of ECA was disrupted was prepared by the following procedure.

[0151] PCR was carried out using chromosomal DNA of the BW25113 strain prepared by a conventional method as a template and DNAs consisting of the base sequences shown in "Primer set" in Table 3 as primer sets to obtain amplified DNA fragments.

[0152]

[0153] wecA upstream 1 and wecA upstream 2 include approximately 1500 bp upstream from the initiation codon of the wecA gene. WecA downstream 1 and wecA downstream 2 include approximately 1300 bp downstream from the termination codon of the wecA gene.

[0154] PCR was performed using an equimolar mixture of wecA upstream 1, wecA downstream 1, and cat-sacB fragment as a template and DNAs consisting of the base sequences shown in SEQ ID NOs: 29 and 30 as a primer set to obtain a DNA fragment (hereinafter referred to as wecA::cat-sacB) consisting of a sequence in which the cat-sacB fragment was inserted into the sequence of the region surrounding the wecA gene.

[0155] PCR was performed using an equimolar mixture of wecA upstream 2 and wecA downstream 2 as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 29 and 30 as a primer set to obtain a DNA fragment (hereinafter referred to as ΔwecA) that does not contain wecA and consists of a sequence in which the upstream and downstream wecA are directly linked.

[0156] The wecA::cat-sacB fragment was introduced by electroporation into the BW25113ΔyhbJ strain harboring the plasmid pKD46 [Datsenko, KA, Warner, BL, 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 wecA gene had been replaced with wecA::cat-sacB).

[0157] The ΔwecA fragment was introduced into the transformant by electroporation to obtain a transformant that exhibited chloramphenicol sensitivity and sucrose resistance (a transformant in which wecA::cat-sacB was replaced with ΔwecA), which was designated BW25113ΔyhbJΔwecA strain.

[0158] <E. coli lacking the wecB gene> Using the BW25113ΔyhbJ strain as a parent strain, E. coli in which the wecB gene (SEQ ID NO: 3) encoding UDP-N-acetylglucosamine-2-epimerase involved in the biosynthesis of ECA was disrupted was prepared by the following procedure.

[0159] PCR was carried out using chromosomal DNA of the BW25113 strain prepared by a conventional method as a template and DNAs consisting of the base sequences shown in "Primer set" in Table 4 as primer sets to obtain amplified DNA fragments.

[0160]

[0161] wecB upstream 1 and wecB upstream 2 include about 1,400 bp upstream from the initiation codon of the wecB gene. WecB downstream 1 and wecB downstream 2 include about 1,400 bp downstream from the termination codon of the wecB gene.

[0162] PCR was performed using an equimolar mixture of wecB upstream 1, wecB downstream 1, and the cat-sacB fragment as a template and DNA consisting of the base sequences shown in SEQ ID NOs: 39 and 40 as a primer set to obtain a DNA fragment (hereinafter referred to as wecB::cat-sacB) consisting of a sequence in which the cat-sacB fragment was inserted into the sequence of the region surrounding the wecB gene.

[0163] PCR was performed using an equimolar mixture of wecB upstream 2 and wecB downstream 2 as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 39 and 40 as a primer set to obtain a DNA fragment (hereinafter referred to as ΔwecB) that does not contain wecB and consists of a sequence in which the upstream and downstream wecB are directly linked.

[0164] The wecB::cat-sacB fragment was introduced by electroporation into the BW25113ΔyhbJ strain harboring the plasmid pKD46 containing the gene encoding the λ recombinase, and a transformant exhibiting chloramphenicol resistance and sucrose sensitivity (a transformant in which the wecB gene had been replaced with wecB::cat-sacB) was obtained.

[0165] The ΔwecB fragment was introduced into the transformant by electroporation to obtain a transformant that was sensitive to chloramphenicol and resistant to sucrose (a transformant in which wecB::cat-sacB was replaced with ΔwecB), which was designated BW25113ΔyhbJΔwecB strain.

[0166] <E. coli lacking the wecC gene> Using the BW25113ΔyhbJ strain as a parent strain, E. coli in which the wecC gene (SEQ ID NO: 5) encoding UDP-N-acetylmannosamine dehydrogenase involved in the biosynthesis of ECA was disrupted was prepared by the following procedure.

[0167] PCR was carried out using chromosomal DNA of the BW25113 strain prepared by a conventional method as a template and DNAs consisting of the base sequences shown in "Primer set" in Table 5 as primer sets to obtain amplified DNA fragments.

[0168]

[0169] WecC upstream 1 and wecC upstream 2 include about 1,400 bp upstream from the initiation codon of the wecC gene. WecC downstream 1 and wecC downstream 2 include about 1,400 bp downstream from the termination codon of the wecC gene.

[0170] PCR was performed using an equimolar mixture of wecC upstream 1, wecC downstream 1, and cat-sacB fragment as a template and DNAs consisting of the base sequences shown in SEQ ID NOs: 49 and 50 as a primer set to obtain a DNA fragment (hereinafter referred to as wecC::cat-sacB) consisting of a sequence in which the cat-sacB fragment was inserted into the sequence of the region surrounding the wecC gene.

[0171] PCR was performed using an equimolar mixture of wecC upstream 2 and wecC downstream 2 as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 49 and 50 as a primer set to obtain a DNA fragment (hereinafter referred to as ΔwecC) that does not contain wecC and consists of a sequence in which the upstream and downstream wecC sequences are directly linked.

[0172] The wecC::cat-sacB fragment was introduced by electroporation into the BW25113ΔyhbJ strain harboring the plasmid pKD46 containing the gene encoding the λ recombinase, and a transformant exhibiting chloramphenicol resistance and sucrose sensitivity (a transformant in which the wecC gene had been replaced with wecC::cat-sacB) was obtained.

[0173] The ΔwecC fragment was introduced into the transformant by electroporation to obtain a transformant that exhibited chloramphenicol sensitivity and sucrose resistance (a transformant in which wecC::cat-sacB was replaced with ΔwecC), which was designated BW25113ΔyhbJΔwecC strain.

[0174] (3) Construction of microorganisms carrying a NeuAc-producing plasmid The plasmid pCSG1 obtained in (1) above was transformed into the BW25113ΔyhbJ strain, BW25113ΔyhbJΔwecA strain, BW25113ΔyhbJΔwecB strain, and BW25113ΔyhbJΔwecC strain constructed in (2) above. For transformation, selection was performed on LB agar medium containing 100 mg / L of sodium ampicillin according to a standard method. The strains obtained by transformation were designated BW25113ΔyhbJ / pCSG1 strain, BW25113ΔyhbJΔwecA / pCSG1 strain, BW25113ΔyhbJΔwecB / pCSG1 strain, and BW25113ΔyhbJΔwecC / pCSG1 strain, respectively.

[0175] Example 2 Production of NeuAc by Fermentation The BW25113ΔyhbJ / pCSG1 strain, BW25113ΔyhbJΔwecA / pCSG1 strain, BW25113ΔyhbJΔwecB / pCSG1 strain, and BW25113ΔyhbJΔwecC / pCSG1 strain obtained in Example 1 were cultured overnight at 30°C on an LB plate containing 100 mg / L of ampicillin, and then inoculated into a wide test tube containing 5 ml of LB medium containing 100 mg / L of ampicillin and cultured at 30°C for 16 hours.

[0176] Thereafter, 0.3 ml of the culture broth was inoculated into a wide test tube containing 3 ml of a production medium containing 100 mg / L of ampicillin [glucose 30 g / L, magnesium sulfate heptahydrate 2.0 g / L, dipotassium hydrogen phosphate 16 g / L, potassium dihydrogen phosphate 14 g / L, ammonium sulfate 2.0 g / L, citric acid monohydrate 1.0 g / L, casamino acids (manufactured by Difco) 5.0 g / L, vitamin B1 10 mg / L, iron sulfate heptahydrate 50 mg / L, manganese sulfate heptahydrate 10 mg / L, pH adjusted to 7.2 with aqueous sodium hydroxide solution, and then autoclaved] [the glucose and magnesium sulfate heptahydrate aqueous solutions were prepared separately, autoclaved, cooled, and mixed], and isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.125 mM. The mixture was then cultured at 30°C for 30 hours with shaking.

[0177] After the cultivation was completed, the culture medium was appropriately diluted and the sample obtained after filter sterilization was subjected to HPLC analysis to quantify the NeuAc concentration in the supernatant and the amount of NeuAc produced per cell mass. The results are shown in Table 6.

[0178] Table 7 also shows the ratio of NeuAc to all peak compounds detected in this analysis, i.e., the HPLC purity of NeuAc.

[0179]

[0180]

[0181] As shown in Tables 6 and 7, it was revealed that disruption of any of the wecA gene, wecB gene, or wecC gene involved in ECA biosynthesis resulted in a higher NeuAc productivity than the parent strain. Furthermore, the increased proportion of NeuAc in the total products contained in the culture broth indicated that this was also effective in reducing impurities. These results demonstrate that the use of a microorganism capable of producing NeuAc or NeuAc-containing carbohydrates and in which the ECA biosynthetic pathway is blocked enables the production of NeuAc or NeuAc-containing carbohydrates more efficiently than conventional methods.

[0182] Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention. This application is based on a Japanese patent application (Patent Application No. 2021-166476) filed on October 8, 2021, the entirety of which is incorporated by reference. All references cited herein are incorporated in their entirety.

[0183] SEQ ID NO: 1: Nucleotide sequence of wecA SEQ ID NO: 2: Amino acid sequence of WecA SEQ ID NO: 3: Nucleotide sequence of wecB SEQ ID NO: 4: Amino acid sequence of WecB SEQ ID NO: 5: Nucleotide sequence of wecC SEQ ID NO: 6: Amino acid sequence of WecC SEQ ID NO: 7: Nucleotide sequence of CjneuB SEQ ID NO: 8: Nucleotide sequence of slr1975 SEQ ID NO: 9: Nucleotide sequence of GNA1 SEQ ID NO: 10: Nucleotide sequence of primer Fw for amplifying the CjneuB fragment SEQ ID NO: 11: Nucleotide sequence of primer Rv for amplifying the CjneuB fragment SEQ ID NO: 12: Nucleotide sequence of primer Fw for amplifying the slr1975 fragment SEQ ID NO: 13: Nucleotide sequence of primer Rv for amplifying the slr1975 fragment SEQ ID NO: 14: Nucleotide sequence of primer Fw for amplifying the GNA1 fragment SEQ ID NO: 15: Nucleotide sequence of primer Rv for amplifying the GNA1 fragment SEQ ID NO: 16: Nucleotide sequence of yhbJ SEQ ID NO: 17: Nucleotide sequence of primer Fw for amplifying cat fragment SEQ ID NO: 18: Nucleotide sequence of primer Rv for amplifying cat fragment SEQ ID NO: 19: Nucleotide sequence of primer Fw for amplifying sacB fragment SEQ ID NO: 20: Nucleotide sequence of primer Rv for amplifying sacB fragment SEQ ID NO: 21: Nucleotide sequence of primer Fw for amplifying wecA upstream 1 fragment SEQ ID NO: 22: Nucleotide sequence of primer Rv for amplifying wecA upstream 1 fragment SEQ ID NO: 23: Nucleotide sequence of primer Fw for amplifying wecA downstream 1 fragment SEQ ID NO: 24: Nucleotide sequence of primer Rv for amplifying wecA downstream 1 fragment SEQ ID NO: 25: Nucleotide sequence of primer Fw for amplifying wecA upstream 2 fragment SEQ ID NO: 26: Nucleotide sequence of primer Rv for amplifying wecA upstream 2 fragment SEQ ID NO: 27: Nucleotide sequence of primer Fw for amplifying wecA downstream 2 fragment SEQ ID NO: 28: Nucleotide sequence of primer Rv for amplifying wecA downstream 2 fragment SEQ ID NO: 29: Nucleotide sequence of primer Fw for generating a wecA disruption fragment SEQ ID NO: 30: Nucleotide sequence of primer Rv for generating a wecA disruption fragment SEQ ID NO: 31: Nucleotide sequence of primer Fw for amplifying the wecB upstream 1 fragment SEQ ID NO: 32: Nucleotide sequence of primer Rv for amplifying the wecB upstream 1 fragment SEQ ID NO: 33: Nucleotide sequence of primer Fw for amplifying the wecB downstream 1 fragment SEQ ID NO: 34: Nucleotide sequence of primer Rv for amplifying the wecB downstream 1 fragment SEQ ID NO: 35: Nucleotide sequence of primer Fw for amplifying the wecB upstream 2 fragment SEQ ID NO: 36: Nucleotide sequence of primer Rv for amplifying the wecB upstream 2 fragmentSEQ ID NO: 37: Nucleotide sequence of primer Fw for amplifying wecB downstream 2 fragment SEQ ID NO: 38: Nucleotide sequence of primer Rv for amplifying wecB downstream 2 fragment SEQ ID NO: 39: Nucleotide sequence of primer Fw for generating a wecB disruption fragment SEQ ID NO: 40: Nucleotide sequence of primer Rv for generating a wecB disruption fragment SEQ ID NO: 41: Nucleotide sequence of primer Fw for amplifying wecC upstream 1 fragment SEQ ID NO: 42: Nucleotide sequence of primer Rv for amplifying wecC upstream 1 fragment SEQ ID NO: 43: Nucleotide sequence of primer Fw for amplifying wecC downstream 1 fragment SEQ ID NO: 44: Nucleotide sequence of primer Rv for amplifying wecC downstream 1 fragment SEQ ID NO: 45: Nucleotide sequence of primer Fw for amplifying wecC upstream 2 fragment SEQ ID NO: 46: Nucleotide sequence of primer Rv for amplifying wecC upstream 2 fragment SEQ ID NO: 47: Nucleotide sequence of primer Fw for amplifying wecC downstream 2 fragment SEQ ID NO: 48: Nucleotide sequence of primer_Rv for amplifying 2 downstream fragments of wecC SEQ ID NO: 49: Nucleotide sequence of primer_Fw for creating a fragment for disrupting wecC SEQ ID NO: 50: Nucleotide sequence of primer_Rv for creating a fragment for disrupting wecC SEQ ID NO: 51: Nucleotide sequence of wecD SEQ ID NO: 52: Amino acid sequence of WecD SEQ ID NO: 53: Nucleotide sequence of wecE SEQ ID NO: 54: Amino acid sequence of WecE SEQ ID NO: 55: Nucleotide sequence of wecF SEQ ID NO: 56: Amino acid sequence of WecF SEQ ID NO: 57: Nucleotide sequence of wecG SEQ ID NO: 58: Amino acid sequence of WecG

Claims

**Claim 1** A microorganism having the ability to produce at least one of N - acetylneuraminic acid and N - acetylneuraminic acid - containing carbohydrates, and in which the biosynthetic pathway of enterobacterial common antigen (ECA) is blocked. **Claim 2** The microorganism according to claim 1, lacking the activity of at least one protein selected from the following [1] to [3]. [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or a homologous sequence thereof, and having the activity of UDP - N - acetylglucosamine - undecaprenyl phosphate N - acetylglucosamine phosphotransferase (WecA). [2] A protein consisting of the amino acid sequence represented by SEQ ID NO: 4 or a homologous sequence thereof, and having the activity of UDP - N - acetylglucosamine 2 - epimerase (WecB). [3] A protein consisting of the amino acid sequence represented by SEQ ID NO: 6 or a homologous sequence thereof, and having the activity of UDP - N - acetylmannosamine dehydrogenase (WecC). **Claim 3** The microorganism according to claim 1 or 2, which is Escherichia coli. **Claim 4** A method for producing at least one of N - acetylneuraminic acid and N - acetylneuraminic acid - containing carbohydrates, comprising culturing the microorganism according to claim 1 or 2 in a medium to produce at least one of N - acetylneuraminic acid and N - acetylneuraminic acid - containing carbohydrates.