Genetically modified microorganism and method for culturing same

Abstract

The present disclosure provides a genetically modified microorganism in which the productivity of acetic acid is reduced. Provided is a genetically modified microorganism in which pyruvate carboxylase activity is enhanced compared to the parent strain, and the productivity of acetic acid under aerobic conditions is lower than that of the parent strain.

Description

Genetically modified microorganisms and their cultivation methods

[0001] The present disclosure relates to genetically modified microorganisms with enhanced pyruvate carboxylase activity and methods for culturing the same.

[0002] Facultative anaerobic microorganisms, such as Escherichia coli, produce energy by metabolizing glucose to carbon dioxide through aerobic respiration under aerobic conditions, i.e., in the presence of oxygen, but under anaerobic conditions, they produce energy by converting glucose into organic acids through fermentation (Non-Patent Document 1).

[0003] In substance production using facultative anaerobic microorganisms, they are generally cultured under aerobic conditions due to their energy generation efficiency. However, due to the nature of facultative anaerobic microorganisms, it is known that organic acids such as acetic acid are produced and accumulated even under aerobic conditions due to changes in metabolic balance, etc. (Non-patent document 2).

[0004] The accumulation of acetate is undesirable because it wastes energy in the production of target substances other than acetate, complicates the purification process, and reduces the final purity of the target substance (Non-Patent Document 2). In response to this, attempts have been made to reduce acetate accumulation by genetic modification that alters sugar uptake (Non-Patent Documents 3 and 4) and by devising cultivation processes (Non-Patent Documents 5 and 6).

[0005] Pyruvate carboxylase is an enzyme that irreversibly carboxylates pyruvate and converts it into oxaloacetate, and is present in many organisms, but not in some organisms such as Escherichia coli. Since oxaloacetate is a compound in the TCA cycle, it has been reported that the introduction of pyruvate carboxylase is advantageous for the production of succinic acid, which is also a compound in the TCA cycle (Non-Patent Document 7).

[0006] On the other hand, it has been reported that the introduction of pyruvate carboxylase into Escherichia coli increases the ability to produce acetate under anaerobic conditions (Non-Patent Document 8).

[0007] PLoS One. 2016, 11, e0158711Eng Life Sci. 2019, 19, p. 770-780Biotechnol. Bioeng. 1994, 44, p. 952-960Biotechnol. Progr. 1999, 15, p. 140-145Biotechnol. Bioeng. 2001, 73, p. 223-230Biotechnol. Bioeng. 2003, 84, p. 314-323J. Biotechnol. 2011, 155, p. 236-243Appl Environ Microbiol. 2000, 66, p. 1844-1850

[0008] As described above, there have been reports of attempts to reduce acetic acid production in substance production using microorganisms, but the effects have not been sufficient, and further improvements are needed. The present disclosure relates to providing a genetically modified microorganism with reduced acetic acid production ability and a culture method thereof.

[0009] As a result of investigating the above-mentioned problems, the present inventors found that the acetic acid-producing ability of a microorganism can be reduced by enhancing the expression of pyruvate carboxylase in the microorganism, and completed the present invention based on this finding.

[0010] The present disclosure includes the following: 1. A genetically modified microorganism having enhanced pyruvate carboxylase activity compared to a parent strain and having reduced acetic acid production ability under aerobic conditions compared to the parent strain. 2. The genetically modified microorganism according to 1 above, wherein the acetic acid production ability under aerobic conditions is 90% or less of the acetic acid production ability of the parent strain under aerobic conditions. 3. The genetically modified microorganism according to 1 above, which is Escherichia coli. 4. The genetically modified microorganism according to 1 above, wherein the pyruvate carboxylase is derived from the genus Sinorhizobium (Ensifer). 5. The genetically modified microorganism according to 1 above, wherein the activity of any one protein selected from the following [1] to [3] is enhanced compared to the parent strain. [1] A protein comprising the amino acid sequence represented by SEQ ID NO: 2; [2] A protein comprising an amino acid sequence in which 1 to 10 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and having pyruvate carboxylase activity; [3] A protein having 70% or more identity to the amino acid sequence represented by SEQ ID NO: 2, and having pyruvate carboxylase activity. 6. A method for producing acidic sugars, comprising culturing the genetically modified microorganism described in any one of 1 to 5 above under the aerobic conditions, and producing acidic sugars in the culture. 7. The production method described in 6 above, comprising culturing the genetically modified microorganism under conditions in which the sugar loading rate defined below is 3% or more. Sugar loading rate: A value expressed as a percentage of the amount of sugar added to the amount of sugar consumed per unit volume of culture solution per unit time. 8. The production method described in 6 above, wherein the acidic sugar is sialic acid or human milk oligosaccharide. 9. A method for culturing a genetically modified microorganism, comprising the following (a1) and (a2): (a1) preparing a genetically modified microorganism having enhanced pyruvate carboxylase activity compared to a parent strain; (a2) culturing the genetically modified microorganism prepared in (a1) under aerobic conditions; 10. The method for culturing a genetically modified microorganism according to 9 above, wherein in (a2) above, the genetically modified microorganism is cultured under conditions where the sugar loading rate defined below is 3% or more: Sugar loading rate: a value expressed as a percentage of the amount of sugar added to the amount of sugar consumed per unit volume of culture solution per unit time; 11. A method for reducing acetic acid in a culture obtained by culturing a microorganism, the method comprising the following (b1) and (b2):(b1) preparing a genetically modified microorganism in which pyruvate carboxylase activity is enhanced compared to a parent strain, and (b2) culturing the genetically modified microorganism prepared in (b1) under aerobic conditions. 12. The method according to 11 above, wherein in (b2), the genetically modified microorganism is cultured under conditions where the sugar loading rate defined below is 3% or more: Sugar loading rate: A value expressed as a percentage of the amount of sugar added to the amount of sugar consumed per unit volume of culture solution per unit time.

[0011] The present disclosure provides a genetically modified microorganism that has enhanced pyruvate carboxylase activity compared to a parent strain and reduced acetic acid production ability under aerobic conditions compared to the parent strain, and a culture method thereof. The genetically modified microorganism and culture method thereof of the present disclosure can efficiently and stably produce a target substance.

[0012] [Genetically Modified Microorganism] The genetically modified microorganism of this embodiment is characterized by having enhanced pyruvate carboxylase activity compared to a parent strain and reduced acetic acid production ability under aerobic conditions compared to the parent strain. In this embodiment, pyruvate carboxylase (EC 6.4.1.1) is an enzyme that irreversibly carboxylates pyruvate and converts it to oxaloacetate. In this specification, pyruvate carboxylase may be abbreviated as Pyc, and the gene encoding pyruvate carboxylase may be abbreviated as pyc.

[0013] In this embodiment, aerobic conditions refer to a state in which oxygen is present.

[0014] In this embodiment, the origin of pyruvate carboxylase is not particularly limited. Many organisms are known to possess pyruvate carboxylase, including mammals including humans, birds such as chickens, reptiles, amphibians, fish, insects such as fruit flies, nematodes, some green algae such as Chlamydomonas, fungi such as budding yeast, bacteria, and archaea. In this embodiment, pyruvate carboxylases derived from these organisms can be used.

[0015] In particular, pyruvate carboxylases derived from α-proteobacteria including rhizobia and gram-positive bacteria are preferred from the viewpoint of introduction into Escherichia coli. Among these, pyruvate carboxylases derived from the genera Rhizobium, Sinorhizobium (Ensifer), and Bradirhizobium, and the gram-positive genus Corynebacterium are preferred.

[0016] The parent strain is a type strain (i.e., a standard strain of the species to which the microorganism belongs), a strain available from a public bank or a reagent company, or a strain to which genetic modification has been added to produce a target substance, and is a strain that is the target of genetic modification to enhance pyruvate carboxylase activity.

[0017] The parent strain may be a wild-type strain as long as it is a microorganism that produces the target substance, or if the wild-type strain does not have the ability to produce the target substance, it may be a bred strain that has been artificially imparted with the ability to produce the target substance. The target substance is preferably an acidic sugar, more preferably sialic acid or acidic human milk oligosaccharide.

[0018] Methods for artificially imparting the ability to produce a target substance to a microorganism include, for example, the following methods (1a) to (1e), which may be used alone or in combination: (1a) a method for alleviating or canceling at least one of the mechanisms controlling the biosynthetic pathway for producing the target substance; (1b) a method for enhancing the expression of at least one enzyme involved in the biosynthetic pathway for producing the target substance; (1c) a method for increasing the copy number of at least one enzyme gene involved in the biosynthetic pathway for producing the target substance; (1d) a method for weakening or blocking at least one metabolic pathway branching off from the biosynthetic pathway for producing the target substance to a metabolic product other than the target substance; and (1e) a method for selecting a cell line that has a higher degree of resistance to an analogue of the target substance compared to a wild-type strain.

[0019] Examples of target substances include acidic sugars, neutral sugars, organic acids, amino acids, nucleic acids, vitamins, and lipids, among which polar compounds are preferred. Acidic sugars, organic acids, and acidic amino acids are preferred from the viewpoint of the difficulty of separation from acetic acid and the load on the ion exchange resin, while basic amino acids and nucleic acids are preferred from the viewpoint of the load on the ion exchange resin due to the alkali added for neutralization.

[0020] Examples of organic acids and amino acids include succinic acid, pyruvic acid, itaconic acid, lactic acid, citric acid, gluconic acid, 5-keto-D-gluconic acid, 2,5-furfural dicarboxylic acid, 3-hydroxypropionic acid, glucaric acid, levulinic acid, aspartic acid, and glutamic acid.

[0021] Neutral sugars refer to sugars that exhibit neutrality. Examples include glucose, galactose, lactose, sucrose, and neutral human milk oligosaccharides. Neutral human milk oligosaccharides include 2'-fucosyllactose, 3-fucosyllactose, lacto-N-triose II (LNTII), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-fucopentaose I (LNFPI), lacto-N-fucopentaose II (LNFPI), and lacto-N-fucopentaose III (LNFPIII).

[0022] Acidic sugars refer to sugars that have a carboxyl group and exhibit acidity. Examples of acidic sugars include sialic acid and acidic oligosaccharides. Examples of sialic acids include N-acetylneuraminic acid, N-glycolylneuraminic acid, glucuronic acid, N-acetylgalactosamine 6-sulfate, and mannose 6-phosphate, with N-acetylneuraminic acid being preferred. Examples of acidic oligosaccharides include acidic human milk oligosaccharides (HMOs), sialyllacto-N-biose (SLNB), and sialyllacto-N-acetylglucosamine (SLNac). Human milk oligosaccharides are a general term for oligosaccharides contained in breast milk. Human milk oligosaccharides are prebiotics that serve as nutrients for beneficial bacteria such as bifidobacteria. HMOs are not metabolized by human digestive enzymes and reach the large intestine, where they are metabolized by intestinal bacteria and can exert various physiological functions.

[0023] Acidic human milk oligosaccharides include sialyllactose, sialyllactose-N-tetraose (LST), disialyllacto-N-tetraose (DSLNT), 3-fucosyl-sialyllactose (F-SL), fucosyl-LSTb (F-LSTb), and the like.

[0024] Sialyllactose is an acidic oligosaccharide in which sialic acid is bound to lactose. Examples of sialyllactose include 6'-sialyllactose [O-(N-acetyl-α-neuraminosyl)-(2→6)-O-β-D-galactopyranosyl-(1→4)-D-glucose] (6'SL) and 3'-sialyllactose [O-(N-acetyl-α-neuraminosyl)-(2→3)-O-β-D-galactopyranosyl-(1→4)-D-glucose] (3'SL).

[0025] Sialyl lacto-N-biose is an acidic oligosaccharide in which sialic acid is bound to lacto-N-biose, and examples of sialyl lacto-N-biose include 6'-sialyllacto-N-biose (6SLNB) and 3'-sialyllacto-N-biose (3SLNB).

[0026] Sialyl lacto-N-acetylglucosamine is an acidic oligosaccharide in which sialic acid is bound to lacto-N-acetylglucosamine, and examples of sialylacto-N-acetylglucosamine include 6'-sialylacto-N-acetylglucosamine (6SLNAc) and 3'-sialylacto-N-acetylglucosamine (3SLNAc).

[0027] Sialyl lacto-N-tetraose is an acidic oligosaccharide in which sialic acid is bound to lacto-N-tetraose or lacto-N-neotetraose. Examples of sialyllacto-N-tetraose include LST-a, LST-b, and LST-c.

[0028] Disialyl lacto-N-tetraose is an acidic oligosaccharide in which two molecules of sialic acid are bound to lacto-N-tetraose.

[0029] In this embodiment, the parent strain is preferably a prokaryote or a yeast strain, more preferably a prokaryote belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Salmonella, Enterobacter, Klebsiella, Citrobacter, or Pseudomonas, or a yeast strain belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Siwaniomyces, Pichia, or Candida, even more preferably the genus Escherichia, and particularly preferably Escherichia coli.

[0030] The parent strain may be a microorganism having pyc as an endogenous gene, or may be a microorganism not having pyc as an endogenous gene. From the viewpoint of further improving the effect of reducing acetic acid by introducing pyc, it is preferable that the parent strain not have pyc as an endogenous gene. Not having pyc as an endogenous gene can further improve the effect of reducing acetic acid. Examples of microorganisms not having pyc as an endogenous gene include those of the genus Escherichia, Salmonella, Enterobacter, Klebsiella, and Citrobacter, with Escherichia coli of the genus Escherichia being particularly preferred.

[0031] As the parent strain, more specifically, for example, Escherichia coli MG1655, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No. 49, 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 acetophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354, or Pseudomonas sp. D-0110 and other prokaryotes, or yeast strains such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvium, Pichia pastoris, or Candida utilis can be mentioned.

[0032] When the target substance is sialic acid or sialyllactose, the genetic modification for producing the target substance can be performed using, for example, the methods described in International Publication No. 2019 / 228993, Japanese Patent Application Publication No. 2021-505170, and Japanese Patent Application Publication No. 2020-531039.

[0033] Whether a microorganism is capable of producing a target substance can be confirmed, for example, by transforming the microorganism with recombinant DNA having DNA encoding a protein for producing the target substance, culturing the transformed microorganism in a medium, and detecting the target substance accumulated in the culture using, for example, HPLC.

[0034] Pyruvate carboxylase activity refers to the activity of irreversibly carboxylating pyruvate as a substrate and converting it to oxaloacetate. A microorganism with enhanced pyruvate carboxylase activity compared to a parent strain can be confirmed, for example, by culturing the parent strain and the genetically modified microorganism, treating the culture with ultrasound or the like, adding pyruvate and other substrates to the treated product, and detecting and comparing the resulting oxaloacetate by high-performance chromatography or gas chromatography.

[0035] Whether a microorganism has a reduced ability to produce acetic acid under aerobic conditions compared to the parent strain can be confirmed, for example, by culturing the parent strain and the genetically modified microorganism under the same conditions, sampling each culture, and then analyzing and comparing the acetic acid concentrations of the cultures or processed products of the cultures using, for example, HPLC.

[0036] In one aspect of this embodiment, the acetic acid concentration in the culture or treated culture obtained by culturing the genetically modified microorganism under aerobic conditions is preferably 98% or less, more preferably 95% or less, 90% or less, or 85% or less, even more preferably 80% or less or 75% or less, and particularly preferably 70% or less, of the acetic acid concentration in the culture or treated culture obtained by culturing the parent strain.

[0037] The genetically modified microorganism of this embodiment preferably has an acetic acid productivity under aerobic conditions of 90% or less, more preferably 85% or less, even more preferably 80% or less or 75% or less, and particularly preferably 70% or less, of the acetic acid productivity of the parent strain under aerobic conditions.

[0038] The ratio of the acetic acid production ability of a genetically modified microorganism under aerobic conditions to the acetic acid production ability of a parent strain under aerobic conditions can be confirmed by the following procedure: x1) The parent strain and the genetically modified microorganism are cultured under the same conditions, and the acetic acid concentration in the culture is measured after at least 8 hours of culture from the start of acetic acid production in the culture solution. x2) The ratio of the acetic acid concentration in the culture of the genetically modified microorganism to the acetic acid concentration in the culture of the parent strain is determined.

[0039] In one aspect of this embodiment, a genetically modified microorganism is cultured under aerobic conditions, and the acetic acid concentration in the culture after 55 hours from the start of culture is preferably 7.2 g / L or less, more preferably 7.0 g / L or less, 6.7 g / L or less, 6.5 g / L or less, or 6.3 g / L or less, and even more preferably 6.0 g / L or less. More specific culture conditions for obtaining the culture include, for example, the culture conditions described in Example 3.

[0040] In one aspect of this embodiment, a genetically modified microorganism is cultured under aerobic conditions, and the acetic acid concentration in the culture after 71 hours from the start of culture is preferably 20.0 g / L or less, more preferably 18.0 g / L or less, 16.0 g / L or less, or 15.0 g / L or less, and even more preferably 14.5 g / L or less. More specific culture conditions for obtaining the culture include, for example, the culture conditions described in Example 3.

[0041] Examples of microorganisms having enhanced pyruvate carboxylase activity compared to a parent strain and reduced acetic acid-producing ability under aerobic conditions compared to the parent strain include microorganisms having enhanced activity of any one of proteins selected from the following [1] to [3] compared to the parent strain. [1] A protein comprising the amino acid sequence represented by SEQ ID NO: 2. [2] A protein comprising an amino acid sequence represented by SEQ ID NO: 2 in which 1 to 10 amino acids are deleted, substituted or added, and having pyruvate carboxylase activity. [3] A protein having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having pyruvate carboxylase activity. The above [1] and [2] may be the following [1'] and [2'], respectively. [1'] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2. [2'] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2 in which 1 to 10 amino acids are deleted, substituted or added, and having pyruvate carboxylase activity.

[0042] As used herein, a mutant protein refers to a protein obtained by artificially deleting or substituting amino acid residues in a parent protein, or by inserting or adding amino acid residues into the protein. In this embodiment, an example of a mutant protein is the protein described in [2] above.

[0043] In the mutant protein of [2] above, the deletion, substitution, or addition of amino acids may mean the deletion, substitution, or addition of 1 to 10 amino acids at any position within the same sequence. The number of deleted, substituted, or added amino acids is 1 to 10, preferably 1 to 8, and most preferably 1 to 5.

[0044] 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.

[0045] 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

[0046] As used herein, a homologous protein refers to a protein that is structurally and functionally similar to an original protein, such that the gene encoding the protein is considered to have the same evolutionary origin as the gene encoding the original protein, and is found in a naturally occurring organism. In this embodiment, an example of a homologous protein is the protein described in [3] above.

[0047] The homologous protein of [3] above is a protein consisting of an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 2. The identity is preferably 80% or more, more preferably 90% or more, 95% or more, 97% or more, 98% or more, in that order, and even more preferably 99% or more.

[0048] In this embodiment, the percentage of sequence identity between two amino acid sequences or two nucleotide sequences is calculated as the ratio of matching residues when the two sequences are aligned so that the residues contained in the two sequences are most identical. For example, the percentage of sequence identity can be determined using a mathematical algorithm.

[0049] Examples of such mathematical algorithms include the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482, the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, the similarity search method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448, and the similarity search method of Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877, and an improved version of the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, although mathematical algorithms are not limited to these examples.

[0050] Alignment for determining the percentage of sequence identity can be performed using programs based on these mathematical algorithms, and the programs can be executed by a computer, if appropriate. Examples of such programs include, but are not limited to, the PC / Gene program CLUSTAL (available from Intelligenetics, Mountain View, Calif.), MAFFT (Katoh, K., Misawa, K., Kuma, K., & Miyata, T. (2002), 30(14), 3059-3066., http: / / mafft.cbrc.jp / alignment / server / ), and MUSCLE (Edgar R. C. (2004). Nucleic acids research, 32(5), 1792-1797., http: / / www.ebi.ac.uk / Tools / msa / muscle / ), BLAST, FASTA, and TFASTA.

[0051] Alignment using these programs can be performed, for example, using default parameters. The CLUSTAL program is described in Higgins et al. (1988) Gene 73:237-244, Higgins et al. (1989) CABIOS 5:151-153, Corpet et al. (1988) Nucleic Acids Res. 16:10881-90, Huang et al. (1992) CABIOS 8:155-65, and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. BLAST is described in Altschul, S. F. , Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). 215(3), 403-410. , Mount D. W. (2007). CSH protocols, 2007, pdb. top17. Programs called BLASTP and BLASTN have been developed based on BLAST, and these programs can be used with default settings to calculate the percentage of sequence identity.

[0052] Examples of microorganisms in which the activity of any one of the proteins selected from [1] to [3] above is enhanced compared to that of a parent strain include the microorganisms (2a) and (2b) below. (2a) Microorganisms i) and ii) below, which are obtained by modifying a gene encoding any one of the proteins selected from [1] to [3] above on the chromosomal DNA of a parent strain: i) A microorganism in which the specific activity of the protein is enhanced compared to that of the parent strain microorganism; ii) A microorganism in which the transcription amount of the gene or the production amount of the protein is increased compared to that of the parent strain microorganism; and (2b) A microorganism in which the copy number of the gene is increased compared to that of the parent strain, which is obtained by transforming a parent strain microorganism with a recombinant DNA containing a gene encoding any one of the proteins selected from [1] to [3] above.

[0053] (2a) A microorganism obtained by modifying a gene encoding any one of the proteins selected from [1] to [3] above on the chromosomal DNA of a parent strain, i) in which the specific activity of the protein is enhanced compared to that of the parent strain, can be obtained, for example, by enhancing the specific activity of pyruvate carboxylase of the parent strain using a conventional mutation treatment method, a gene substitution method using recombinant DNA technology, or the like.

[0054] Examples of the mutation treatment method include a method using N-methyl-N'-nitro-N-nitrosoguanidine (NTG) (Microorganism Experiment Manual, 1986, p. 131, Kodansha Scientific Co.), ultraviolet irradiation, and the like.

[0055] Examples of gene replacement methods using recombinant DNA technology include a method in which a mutation is introduced into DNA encoding a protein having pyruvate carboxylase activity by subjecting the DNA to in vitro mutagenesis using a mutagen or error-prone PCR, and then replacing the gene encoding pyruvate carboxylase present in the chromosomal DNA of the parent strain by homologous recombination.

[0056] DNA encoding a protein having pyruvate carboxylase activity can be obtained, for example, by a method using PCR, using a probe DNA that can be designed based on the base sequence shown in SEQ ID NO: 1.

[0057] 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 cells to be introduced. Methods using homologous recombination that are frequently used in Escherichia coli include a method in which recombinant DNA is introduced using the homologous recombination system of lambda phage [Proc. Natl. Acad. Sci. USA, 97, 6641-6645 (2000)].

[0058] 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, for example, a selection method utilizing the fact that Escherichia coli becomes sucrose-sensitive due to Bacillus subtilis levansucrase integrated into the chromosome together with recombinant DNA, or a selection method utilizing the fact that Escherichia coli becomes streptomycin-sensitive by incorporating a wild-type rpsL gene into Escherichia coli having a mutant rpsL gene that is resistant to streptomycin [Mol. Microbiol., 55, 137 (2005), Biosci. Biotechnol. Biochem., 71, 2905 (2007)].

[0059] (2a) Microorganisms obtained by modifying a gene encoding any one of the proteins selected from [1] to [3] above, which are present on the chromosomal DNA of a parent strain, and ii) which have an increased transcription level of the gene or an increased production level of pyruvate carboxylase compared to the parent strain, include microorganisms having a promoter region in which one or more bases, preferably 1 to 10 bases, more preferably 1 to 5 bases, and even more preferably 1 to 3 bases, have been substituted in the base sequence of the transcription regulatory region or promoter region of the gene encoding any one of the proteins selected from [1] to [3] above, which is present on the chromosomal DNA of the parent strain, and which have an increased expression level of the gene compared to the parent strain, and microorganisms obtained by substituting the promoter region of the gene present on the chromosomal DNA of the parent strain with a known strong promoter sequence, which have an increased expression level of the gene.

[0060] (a) A microorganism obtained by modifying a gene encoding any one of the proteins selected from [1] to [3] above, which is present on the chromosomal DNA of a parent strain, and ii) in which the transcription amount of the gene or the production amount of the protein is increased compared to that of the parent strain, can be obtained, for example, by enhancing the transcription amount of the gene encoding the protein of the parent strain or the production amount of the protein using a conventional mutation treatment method, a gene replacement method using recombinant DNA technology, etc. Examples of mutation treatment methods include the methods described above.

[0061] Examples of gene replacement methods using recombinant DNA techniques include a method in which a transcriptional regulatory region and a promoter region of a gene encoding any one of the proteins selected from [1] to [3] above, which is contained in a parent strain, is subjected to in vitro mutagenesis or error-prone PCR or the like to introduce a mutation into the DNA, and then the gene encoding the protein present in the chromosomal DNA of the parent strain is replaced by the above-mentioned homologous recombination method.

[0062] Alternatively, a microorganism having improved production of a protein selected from the above [1] to [3] can be obtained by replacing the promoter region of a gene encoding the protein of the parent strain with a known strong promoter sequence.

[0063] When a microorganism belonging to the genus Escherichia is used as the parent strain, examples of such promoters include promoters derived from Escherichia coli or phages, such as the trp promoter (Ptrp), lac promoter (Plac), ilv promoter (Pilv), PL promoter, PR promoter, and PSE promoter, which function in Escherichia coli. Other examples include artificially constructed promoters such as a promoter with two Ptrp promoters in tandem, the tac promoter, the lacT7 promoter, and the letI promoter. When a microorganism belonging to the genus Bacillus is used as the parent strain, examples of such promoters include the SPO1 promoter, SPO2 promoter, and penP promoter, which function in Bacillus subtilis.

[0064] When a coryneform bacterium is used as the parent strain, examples of promoters include the P54-6 promoter [Appl. Microbiol. Biotechnol., 53, 674-679 (2000)].

[0065] When a yeast strain is used as the parent strain, examples of promoters include the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock polypeptide promoter, MFα1 promoter, and CUP1 promoter.

[0066] Whether the microorganism obtained by the above method is a microorganism in which the transcription level of the gene encoding any one of the proteins selected from [1] to [3] above or the production level of the protein is increased compared to the parent strain can be confirmed, for example, by comparing the transcription level of the gene in the microorganism with that of the parent strain by Northern blotting or the production level of pyruvate carboxylase in the microorganism with that of the parent strain by Western blotting.

[0067] (b) Examples of microorganisms that have an increased copy number of a gene compared to the parent strain, and that can be obtained by transforming a parent strain of a microorganism with a recombinant DNA containing a gene encoding any one of the proteins selected from [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 of a microorganism with a recombinant DNA containing a gene encoding any one of the proteins selected from [1] to [3] above, and microorganisms in which the gene is carried outside of chromosomal DNA as plasmid DNA.

[0068] Specific examples of DNA encoding any one of the proteins selected from [1] to [3] above include DNA selected from the group consisting of [4] to [7] below: [4] DNA encoding any one of the proteins described in [1] to [3] above; [5] DNA comprising the nucleotide sequence represented by SEQ ID NO: 1; [6] DNA that hybridizes under stringent conditions with DNA comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1 and encodes a homologous protein having pyruvate carboxylase activity; [7] DNA that comprises a nucleotide sequence having 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 and encodes a homologous protein having pyruvate carboxylase activity.

[0069] In this embodiment, recombinant DNA refers to recombinant DNA that is capable of autonomous replication in a parent strain or of being integrated into a chromosome and that has been incorporated into an expression vector containing a promoter at a position where the DNA can be transcribed.

[0070] In the above [6], "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.

[0071] 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.

[0072] Methods for DNA hybridization experiments are well known, and those skilled in the art can determine hybridization conditions according to the present specification. The hybridization conditions can be determined according to the methods described in Molecular Cloning, 2nd and 3rd Editions (2001), Methods for General and Molecular Bacteriology, ASM Press (1994), Immunology Methods Manual, Academic Press (1996), and many other standard textbooks.

[0073] 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.

[0074] 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.

[0075] 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 the base sequence represented by SEQ ID NO: 50 or 51.

[0076] (b) 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.

[0077] A DNA fragment of an appropriate length containing a portion encoding the protein according to any one of [1] to [3] above is prepared as needed. Furthermore, by substituting bases in the nucleotide sequence of the portion encoding the protein so that it contains optimal codons for expression in host cells, a transformant with improved productivity can be obtained.

[0078] 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.

[0079] When a prokaryote such as a bacterium is used as a parent strain, the recombinant DNA is preferably a recombinant DNA comprising a promoter, a ribosome binding sequence, the DNA described in any one of [4] to [7] above, and a transcription termination sequence. A gene that controls the promoter may also be included.

[0080] 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.

[0081] Furthermore, by substituting bases in the nucleotide sequence of the portion encoding any one of the proteins selected from [1] to [3] above so that the codons are optimal for expression in the host, the expression level of the protein can be improved. Information on codon usage in the parent strain used in the production method of the present invention can be obtained from public databases.

[0082] When a microorganism belonging to the genus Escherichia is used as the parent strain, examples of the expression vector include pColdI (Takara Bio Inc.), pCDF-1b, pRSF-1b (all manufactured by Novagen), pMAL-c2x (New England Biolabs), pGEX-4T-1 (GE Healthcare Biosciences), pTrcHis (Invitrogen), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-30 (Qiagen), pET-3 (Novagen), pKYP10 (JP 58-110600 A), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], and pLSA1 [Agric. Biol. Chem., 48, 669 (1984)]. , 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, p. 6378-6385] pPAC31 (WO 1998 / 12343), pUC19 [Gene, 33, 103 (1985)], pSTV28 (Takara Bio), pUC118 (Takara Bio), pPA1 (JP 63-233798 A), and the like.

[0083] When using the above-mentioned expression vector, any promoter may be used as long as it functions in the cells of a microorganism belonging to the genus Escherichia, and examples of promoters that can be used include promoters derived from Escherichia coli or phages, such as the trp promoter (Ptrp), lac promoter (Plac), ilv promoter (Pilv), PL promoter, PR promoter, and PSE promoter. Artificially designed and modified promoters such as a promoter with two Ptrp promoters in tandem, the tac promoter, the lacT7 promoter, and the letI promoter can also be used.

[0084] 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)).

[0085] When the above expression vector is used, any promoter may be used as long as it functions in the cells of coryneform bacteria. For example, the P54-6 promoter [Appl. Microbiol. Biotechnol., 53, 674-679 (2000)] can be used.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] The DNA encoding the protein of [1] above and the DNA of [5] above can be obtained, for example, by Southern hybridization of a chromosomal DNA library of a microorganism, preferably a microorganism belonging to the genus Sinorhizobium meliloti (also known as Ensifer meliloti), using a probe DNA that can be designed based on the nucleotide sequence represented by SEQ ID NO: 1, or by PCR [PCR Protocols, Academic Press (1990)] using primer DNA that can be designed based on the nucleotide sequence and the chromosomal DNA of the microorganism as a template.

[0090] The DNA encoding the mutant protein of [2] above can be obtained, for example, by subjecting the DNA consisting of the base sequence represented by SEQ ID NO: 1 to error-prone PCR or the like using the DNA as a template.

[0091] Alternatively, the DNA of [2] above 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)].

[0092] That is, first, PCR is performed using the DNA as a template with a sense primer corresponding to the 5' end of the DNA and an antisense primer corresponding to the sequence immediately (5' side) of the mutation introduction site, which has a sequence complementary to the mutation sequence at its 5' end, to amplify fragment A from the 5' end of the DNA to the mutation introduction site (with a mutation introduced at the 3' end). Next, PCR is performed using the DNA as a template with a sense primer corresponding to the sequence immediately (3' side) of the mutation introduction site, which has a mutation sequence at its 5' end, and an antisense primer corresponding to the 3' end of the DNA, to amplify fragment B from the mutation introduction site to the 3' end of the DNA with a mutation introduced at its 5' end. After purifying these amplified fragments, they are mixed and subjected to PCR without adding a template or primer. Since the sense strand of amplified fragment A and the antisense strand of amplified fragment B share the mutation introduction site, they hybridize and act as both primers and templates, allowing the PCR reaction to proceed and amplify the mutated DNA.

[0093] The DNA encoding the homologous protein of [3] above, and the DNAs of [6] and [7] above can be obtained, for example, by searching various gene sequence databases for a base sequence having an identity of 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more with the base sequence represented by SEQ ID NO: 1, or by searching various protein sequence databases for an amino acid sequence having an identity of 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more with the amino acid sequence represented by SEQ ID NO: 2, and 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, in a manner similar to the method for obtaining the above-mentioned DNA.

[0094] The obtained DNA according to any one of the above [4] to [7] can be used as is or cleaved with an appropriate restriction enzyme or the like, and then inserted into a vector by a conventional method. The resulting recombinant DNA is then introduced into a host cell, and the DNA can be analyzed using a commonly used base sequence analysis method, for example, the dideoxy method [Proc. Natl. Acad. Sci., USA, 74, 5463 (1977)] or a base sequence analyzer such as a 3700 DNA Analyzer (manufactured by Applied Biosystems), to determine the base sequence of the DNA.

[0095] Examples of the host cells include Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, and Escherichia coli No. 49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli MP347 and Escherichia coli NM522.

[0096] Examples of the vector include pBluescriptIIKS(+) (Stratagene), pDIRECT [Nucleic Acids Res., 18, 6069 (1990)], pCR-Script Amp SK(+) (Stratagene), pT7Blue (Novagen), pCR II (Invitrogen), and pCR-TRAP (Gene Hunter).

[0097] Any method for introducing recombinant DNA into a host cell can be used, and examples thereof include a method using calcium ions [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], a protoplast method (Japanese Patent Laid-Open Publication No. 63-248394), and an electroporation method [Nucleic Acids Res., 16, 6127 (1988)].

[0098] If the result of determining the base sequence shows that the obtained DNA is a partial length, the full-length DNA can be obtained by Southern hybridization or the like against a chromosomal DNA library using the partial length DNA as a probe.

[0099] Furthermore, the desired DNA can be prepared by chemical synthesis using a DNA synthesizer such as the 8905 model manufactured by Perceptive Biosystems, based on the determined DNA base sequence.

[0100] The recombinant DNA used in the production method of the present invention can be prepared by inserting the DNA fragment downstream of a promoter in an appropriate expression vector, such as pMW219-Smpyc, which will be described later in the Examples.

[0101] Methods for introducing recombinant DNA into a parent 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)].

[0102] The recombinant DNA can be integrated into the chromosome of the parent strain by substitution using the homologous recombination method described above.

[0103] Whether the microorganism constructed 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 the parent strain can be confirmed by comparing the transcription amount of the DNA described in any one of [4] to [7] above, the production amount of the protein, or the specific activity of the protein with that of the parent strain using the above method.

[0104] An example of such a microorganism is W3110S / pMW219-Ptrp-Smpyc, which will be described later in the Examples section.

[0105] [Culturing Method of Genetically Modified Microorganisms] The culturing method of genetically modified microorganisms of this embodiment (hereinafter also referred to as the culturing method of this embodiment) is characterized by comprising the following steps (a1) and (a2): (a1) preparing a genetically modified microorganism having enhanced pyruvate carboxylase activity compared to a parent strain and having reduced acetic acid-producing ability under aerobic conditions compared to the parent strain; and (a2) culturing the genetically modified microorganism prepared in (a1) under aerobic conditions.

[0106] In the culture method of this embodiment, the genetically modified microorganism in (a1) may be the genetically modified microorganism of this embodiment.

[0107] In the culture method of this embodiment, the genetically modified microorganism can be cultured according to a conventional method. The medium for culturing the genetically modified microorganism may be either a natural medium or a synthetic medium, and may be either a liquid medium or a solid medium, as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the genetically modified microorganism and allows the genetically modified microorganism to be cultured efficiently.

[0108] The carbon source may be any that can be utilized by the genetically modified microorganism, and examples thereof include carbohydrates 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 ethanol and propanol.

[0109] Examples of nitrogen sources that can be used 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 and soybean meal hydrolysate, various fermentation bacteria, and digested products thereof.

[0110] Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

[0111] In the culture method of this embodiment, a genetically modified microorganism is cultured under aerobic conditions. Examples of culture under aerobic conditions include conventional shaking culture and submerged aeration and stirring culture. The culture temperature is preferably 15 to 40°C, and the culture time is usually 5 hours to 7 days. The pH during culture is preferably maintained at 3.0 to 9.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

[0112] In the culture method of this embodiment, it is preferable to culture the genetically modified microorganism under conditions where the sugar loading rate, as defined below, is 3% or more. Specifically, if the sugar consumption rate of the microorganism is 10 g / L / h per 1 L of culture solution, the sugar loading rate will be 100% when sugar is added at a rate of 10 g / L / h, and 50% when sugar is added at a rate of 5 g / L / h. Sugar loading rate: A value expressed as a percentage of the amount of sugar added (g / L / h) to the sugar consumption per unit volume and unit time of culture solution (the value obtained by multiplying the sugar consumption per unit time per bacterial cell by the number of bacteria) (g / L / h).

[0113] Here, the sugar consumption per unit volume and unit time of the culture medium can be calculated, for example, by multiplying the bacterial cell weight per unit volume by the sugar consumption per unit time per unit bacterial cell weight. The bacterial cell weight per unit volume can be calculated from the OD value based on the description in, for example, Annals of the New York Academy of Sciences, 1994, 721, pp. 30-42. The sugar consumption per unit bacterial cell weight can be calculated, for example, from the glucose consumption per unit bacterial cell weight of Escherichia coli described in the literature (Microb Cell Fact. 2022; 21: 189).

[0114] In the culture method of this embodiment, by setting the sugar loading rate at 3% or more, the effect of reducing acetic acid production ability is more easily achieved, and the production efficiency of the target substance can be increased. The sugar loading rate is more preferably 4% or more, even more preferably 5% or more, particularly preferably 6% or more, and most preferably 7% or more. The upper limit of the sugar loading rate is not particularly limited, and is, for example, 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, or 28% or less.

[0115] If necessary, antibiotics such as ampicillin or tetracycline may be added to the medium during culture. When culturing a microorganism transformed with an expression vector using an inducible promoter, an inducer may be added to the medium if necessary.

[0116] For example, when a microorganism transformed with an expression vector using a lac promoter is cultured, isopropyl-β-D-thiogalactopyranoside or the like may be added to the medium, and when a microorganism transformed with an expression vector using a trp promoter is cultured, indoleacrylic acid or the like may be added to the medium.

[0117] [Method for Producing Acidic Sugars] The method for producing acidic sugars of this embodiment (hereinafter also referred to as the production method of this embodiment) comprises culturing the genetically modified microorganism of this embodiment described above under aerobic conditions to produce acidic sugars in the culture. The method for culturing the genetically modified microorganism of this embodiment is the same as the culture method described in the section [Method for Cultivating Genetically Modified Microorganisms].

[0118] The description in [Genetically Modified Microorganisms] can be applied to the acidic sugars in this embodiment. Because acetic acid and acidic sugars have similar properties, reducing acetic acid is a particular challenge in the production of acidic sugars, due to the difficulty of separating them in the purification process and the increased load on the purification process using ion exchange resins. In this production method, by using a genetically modified microorganism in which pyruvate carboxylase activity is enhanced compared to the parent strain and the ability to produce acetic acid under aerobic conditions is reduced, the accumulation of acetic acid in the culture can be reduced, and acidic sugars can be produced efficiently.

[0119] The target substance can be produced by collecting acidic sugars from the culture of the genetically modified microorganism obtained by the above-mentioned culture or from a processed product of the culture.

[0120] The culture contains a medium in which a genetically modified microorganism has been cultured for a predetermined period of time. The culture may contain, but preferably does not contain, the genetically modified microorganism. The predetermined period of time is not particularly limited, and may be, for example, 1 hour or more, 5 hours or more, 10 hours or more, 20 hours or more, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 1 week or more. The predetermined period of time may be 24 hours, 31 hours, 47 hours, 55 hours, or 71 hours. Examples of processed culture products include concentrates of the above cultures, dried products of the cultures, supernatants obtained by centrifuging the cultures, and sonicated products of the cultures, but it is preferable that the genetically modified microorganisms are not contained in the culture.

[0121] When the target substance is an acidic sugar, the amount of the acidic sugar can be determined using a sugar analyzer manufactured by Dionex (Anal. Biochem., 189, 151 (1990)). The acidic sugar can be collected by a conventional method using activated carbon, ion exchange resin, or the like.

[0122] [Method for reducing acetic acid in a microbial culture] The method for reducing acetic acid in a culture obtained by culturing a microorganism according to this embodiment is characterized by comprising the following steps (b1) and (b2): (b1) preparing a genetically modified microorganism in which pyruvate carboxylase activity is enhanced compared to a parent strain; and (b2) culturing the genetically modified microorganism prepared in (b1) under aerobic conditions.

[0123] The microorganism may be a facultative anaerobic microorganism. The genetically modified microorganism in (b1) is the same as that described in the section [Genetically modified microorganism]. The culture method in (b2) is the same as that described in the section [Culturing method of genetically modified microorganism].

[0124] As explained above, the present specification discloses the following configurations: 1. A genetically modified microorganism in which pyruvate carboxylase activity is enhanced compared to a parent strain and in which the ability to produce acetic acid under aerobic conditions is reduced compared to the parent strain. 2. The genetically modified microorganism according to 1 above, in which the ability to produce acetic acid under aerobic conditions is 90% or less of the ability of the parent strain to produce acetic acid under aerobic conditions. 3. The genetically modified microorganism according to 1 or 2 above, which is Escherichia coli. 4. The genetically modified microorganism according to any one of 1 to 3 above, in which the pyruvate carboxylase is derived from the genus Sinorhizobium (Ensifer). 5. The genetically modified microorganism according to any one of 1 to 4 above, in which the activity of any one protein selected from the following [1] to [3] is enhanced compared to the parent strain. [1] A protein comprising the amino acid sequence represented by SEQ ID NO: 2; [2] A protein comprising an amino acid sequence in which 1 to 10 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and having pyruvate carboxylase activity; [3] A protein having 70% or more identity to the amino acid sequence represented by SEQ ID NO: 2, and having pyruvate carboxylase activity. 6. A method for producing acidic sugars, comprising culturing the genetically modified microorganism described in any one of 1 to 5 above under the aerobic conditions, and producing acidic sugars in the culture. 7. The production method described in 6 above, comprising culturing the genetically modified microorganism under conditions in which the sugar loading rate defined below is 3% or more. Sugar loading rate: A value expressed as a percentage of the amount of sugar added to the amount of sugar consumed per unit volume of culture solution per unit time. 8. The production method described in 6 or 7 above, wherein the acidic sugar is sialic acid or human milk oligosaccharide. 9. A method for culturing a genetically modified microorganism, comprising the following (a1) and (a2): (a1) preparing a genetically modified microorganism having enhanced pyruvate carboxylase activity compared to a parent strain; and (a2) culturing the genetically modified microorganism prepared in (a1) under aerobic conditions. 10. The method for culturing a genetically modified microorganism according to 9 above, wherein in (a2), the genetically modified microorganism is cultured under conditions where the sugar loading rate defined below is 3% or more.Sugar loading rate: A value expressed as a percentage of the amount of sugar added relative to the amount of sugar consumed per unit volume of the culture solution per unit time 11. A method for reducing acetic acid in a culture obtained by culturing a microorganism, the method comprising the following (b1) and (b2): (b1) preparing a genetically modified microorganism in which pyruvate carboxylase activity is enhanced compared to a parent strain; and (b2) culturing the genetically modified microorganism prepared in (b1) under aerobic conditions. 12. The method according to 11, wherein in (b2), the genetically modified microorganism is cultured under conditions where the sugar loading rate defined below is 3% or more. Sugar loading rate: A value expressed as a percentage of the amount of sugar added relative to the amount of sugar consumed per unit volume of the culture solution per unit time.

[0125] [Analysis Example] In the Examples, the concentration of acetic acid contained in the culture medium was analyzed according to the following procedure. After cultivation, the culture medium containing the microorganism was centrifuged, and the supernatant was collected. Sodium acetate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used as a standard, and the acetic acid contained in the supernatant was quantified using an HPLC analyzer SPD-20A (manufactured by Shimadzu Corporation).

[0126] [Analysis conditions] Column: RSpak KC-811 (Shodex) Column temperature: 50°C Mobile phase: 5 mM sulfuric acid Mobile phase flow rate: 0.6 ml / min Detection wavelength: 210 nm Detection limit: 0.650 mg / L Quantitation limit: 1.97 mg / L

[0127] Examples of the present invention will be described below, but the present invention is not limited to these examples.

[0128] Example 1: Construction of a pyc-enhanced plasmid Genomic DNA was extracted from Sinorhizobium meliloti (also known as Ensifer meliloti) by a standard method, and PCR was performed using the extracted DNA as a template and primers of SEQ ID NOs: 3 and 4 to amplify the nucleotide sequence (SEQ ID NO: 1) of the gene (hereinafter referred to as Smpyc) encoding pyruvate carboxylase (SEQ ID NO: 2) derived from Sinorhizobium meliloti, thereby obtaining an amplified Smpyc fragment. Note that the nucleotide sequence of the start codon in the Smpyc-encoding sequence had been substituted from TTG to ATG to optimize codon expression in E. coli.

[0129] PCR was performed using the trp promoter-containing expression vector pTK31 for Escherichia coli (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2007, Vol. 73, No. 20, pp. 6378-6385) as a template and primers of SEQ ID NOs: 5 and 6 to obtain an amplified pTK31 fragment.

[0130] To introduce Smpyc into pTK31, the amplified Smpyc fragment and the amplified pTK31 fragment were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the expression plasmid pTK31-Smpyc.

[0131] Furthermore, to clone the obtained expression plasmid into a low-copy vector, pMW219 (manufactured by Nippon Gene Co., Ltd.), PCR was performed using pMW219 as a template and primers of SEQ ID NOs: 7 and 8 to obtain an amplified pMW219 fragment. PCR was performed using pTK31-Smpyc as a template and primers of SEQ ID NOs: 9 and 10 to amplify Smpyc linked to the tryptophan promoter, thereby obtaining a Ptrp-Smpyc fragment.

[0132] To introduce Ptrp-Smpyc into pMW219, the amplified pMW219 fragment and the trp-Smpyc fragment were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the expression plasmid pMW219-Smpyc.

[0133] Example 2 Construction of Smpyc-Expressing Strain The E. coli strain W3110S (W3110, ilVG:: (functional ilvG from E. coli B), pyr:: (functional pyrE from E. coli B, from the NBRP collection) was transformed with pMW219-Smpyc obtained in Example 1 to obtain W3110S / pMW219-Smpyc. As a control, a strain W3110S / pMW219 was also constructed, into which an empty vector not containing Smpyc had been introduced.

[0134] Example 3 Culturing of Smpyc-Expressing Strains W3110S / pMW219-Smpyc and BW3110S / pMW219 were spread on LB agar medium containing 50 mg / L of kanamycin [Bactotryptone (manufactured by Difco) 10 g / L, yeast extract (manufactured by Difco) 5 g / L, sodium chloride 10 g / L, agar 15 g] and cultured overnight at 30°C.

[0135] The grown cells were inoculated into a 2 L baffled Erlenmeyer flask containing 300 ml of LB medium [10 g / L bactotryptone (manufactured by Difco), 5 g / L yeast extract (manufactured by Difco), 10 g / L sodium chloride] containing 50 mg / L kanamycin, and cultured at 30°C for 18 hours.

[0136] 80 mL of the culture broth was inoculated into a 6 L jar fermenter (NBC series, manufactured by Mitsuwa Frontech Co., Ltd.) containing 1.6 L of a main culture medium (disodium hydrogen phosphate 6 g / L, potassium dihydrogen phosphate 3 g / L, sodium chloride 5 g / L, ammonium chloride 1 g / L, yeast extract (Difco) 5 g / L, manganese (II) sulfate pentahydrate 10 mg / L, thiamine hydrochloride 10 mg / L, glucose 20 g / L, iron (II) sulfate heptahydrate 200 mg / L, magnesium sulfate heptahydrate 2 g / L) containing 50 mg / L of kanamycin.

[0137] Cultivation was carried out at 30 ° C., and the pH was controlled with aqueous ammonia to 7.0. The culture was controlled by applying a stirring cascade to achieve aerobic conditions with a dissolved oxygen concentration of 1 ppm. After confirming that the glucose contained in the culture solution was depleted, a 60% by mass glucose solution was added to the culture solution at a flow rate of 10 mL / h. The culture solution was sampled at culture times of 0 h, 24 h, 31 h, 47 h, 55 h, and 71 h, and the concentration of acetic acid contained in each sample was analyzed by HPLC.

[0138] Since the OD value during cultivation was approximately 100, the bacterial weight was calculated to be 36 g / L based on the description in the literature (Annals of the New York Academy of Sciences, 1994, 721, pp. 30-42). Since the glucose consumption rate per unit bacterial cell weight of E. coli is 0.35 to 1.52 g / g / h (Microb Cell Fact. 2022; 21: 189), the sugar consumption capacity per 1 L of culture solution was calculated to be 13 to 55 g / L / h.

[0139] Furthermore, since a 60% glucose solution was added to 1.6 L of culture medium at a flow rate of 10 mL / h, the amount of glucose added per unit volume and unit time was calculated to be 3.75 g / L / h. Therefore, the glucose loading rate was calculated as 3.75 g / L / h ÷ 13 to 55 g / L / h × 100, which is 7 to 29%.

[0140] The results of the analysis are shown in Table 1.

[0141]

[0142] As shown in Table 1, the acetic acid concentration in the culture medium of W3110S / pMW219-Ptrp-Smpyc expressing Smpyc after 71 hours of culture was approximately 70% of the acetic acid concentration in the culture medium of W3110S / pMW219 not expressing Smpyc after 71 hours of culture.

[0143] That is, it was shown that E. coli into which Smyc was introduced had a reduced ability to produce acetate under aerobic conditions. Therefore, it was shown that the expression of pyruvate carboxylase in E. coli reduces the acetate producing ability of the E. coli and reduces the accumulation of acetate in the culture.

[0144] 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. 2024-076100) filed on May 8, 2024, the entirety of which is incorporated by reference. All references cited herein are incorporated in their entirety.

[0145] SEQ ID NO: 1: Nucleotide sequence of Smpyc gene SEQ ID NO: 2: Amino acid sequence of Smpyc SEQ ID NO: 3: Nucleotide sequence of primer for Smpyc cloning SEQ ID NO: 4: Nucleotide sequence of primer for Smpyc cloning SEQ ID NO: 5: Nucleotide sequence of primer for pTK31 amplification SEQ ID NO: 6: Nucleotide sequence of primer for pTK31 amplification SEQ ID NO: 7: Nucleotide sequence of primer for pMW219 amplification SEQ ID NO: 8: Nucleotide sequence of primer for pMW219 amplification SEQ ID NO: 9: Nucleotide sequence of primer for Ptrp-Smpyc cloning SEQ ID NO: 10: Nucleotide sequence of primer for Ptrp-Smpyc cloning

Claims

1. A genetically modified microorganism in which pyruvate carboxylase activity is enhanced compared to that of a parent strain and the ability to produce acetate under aerobic conditions is reduced compared to that of the parent strain.

2. The genetically modified microorganism according to claim 1, wherein the ability to produce acetic acid under aerobic conditions is 90% or less of the ability of the parent strain to produce acetic acid under aerobic conditions.

3. The genetically modified microorganism of claim 1, which is Escherichia coli.

4. The genetically modified microorganism of claim 1, wherein the pyruvate carboxylase is derived from the genus Sinorhizobium (Ensifer).

5. The genetically modified microorganism according to claim 1, wherein the activity of any one of the proteins selected from the following [1] to [3] is enhanced compared to the parent strain: [1] a protein comprising the amino acid sequence represented by SEQ ID NO: 2; [2] a protein comprising the amino acid sequence represented by SEQ ID NO: 2 in which 1 to 10 amino acids have been deleted, substituted, or added, and having pyruvate carboxylase activity; or [3] a protein having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having pyruvate carboxylase activity.

6. A method for producing acidic sugars, comprising culturing the genetically modified microorganism according to any one of claims 1 to 5 under the aerobic conditions and producing acidic sugars in the culture.

7. The production method according to claim 6, which comprises culturing the genetically modified microorganism under conditions where the sugar loading rate is 3% or more, as defined below: Sugar loading rate: A value expressed as a percentage of the amount of sugar added to the amount of sugar consumed per unit volume of culture medium per unit time.

8. The method of claim 6, wherein the acidic sugar is sialic acid or a human milk oligosaccharide.

9. A method for culturing a genetically modified microorganism, comprising the following steps (a1) and (a2): (a1) preparing a genetically modified microorganism having enhanced pyruvate carboxylase activity compared to a parent strain; and (a2) culturing the genetically modified microorganism prepared in (a1) under aerobic conditions.

10. The method for culturing a genetically modified microorganism according to claim 9, wherein in (a2), the genetically modified microorganism is cultured under conditions where the sugar loading rate is 3% or more, as defined below: Sugar loading rate: A value expressed as a percentage of the amount of sugar added to the amount of sugar consumed per unit volume of culture liquid per unit time.

11. A method for reducing acetic acid in a culture obtained by culturing a microorganism, the method comprising the following steps (b1) and (b2): (b1) preparing a genetically modified microorganism in which pyruvate carboxylase activity is enhanced compared to a parent strain; and (b2) culturing the genetically modified microorganism prepared in (b1) under aerobic conditions.

12. The method according to claim 11, wherein in (b2), the genetically modified microorganism is cultured under conditions in which the sugar loading rate defined below is 3% or more. Sugar loading rate: The ratio of the amount of sugar added to the amount of sugar consumed per unit volume of culture medium per unit time, expressed as a percentage.

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