JP2015029474A - Production method of purine-derived substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a production method of a purine-derived substance.SOLUTION: Escherichia bacteria, Bacillus bacteria, or Corynebacterium ammoniagenes modified to hold a variant yggB gene and having purine-derived substance production ability are cultured in culture medium, and a purine-derived substance is collected from the culture medium, to produce the purine-derived substance.

Description

  The present invention relates to a method for fermentative production of purine-based substances using bacteria. Purine-based substances are industrially useful as ingredients for seasonings and pharmaceuticals, or as raw materials thereof.

  Purine substances such as purine nucleosides and purine nucleotides are usually produced industrially by fermentation using an adenine auxotrophic strain. Such strains may further be given resistance to drugs such as purine analogs and sulfaguanidines. Examples of strains used for the fermentation production of purine nucleosides include, for example, strains belonging to the genus Bacillus (Patent Documents 1 to 8), strains belonging to the genus Brevibacterium (Corynebacterium) (patents) Documents 9 to 10, Non-Patent Document 1), and strains belonging to the genus Escherichia (Patent Document 11) are known.

  Variants such as auxotrophic strains and drug resistant strains are typically mutagenized by UV irradiation or N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment and the appropriate selective medium. Can be obtained by selecting a mutant strain having a desired phenotype.

  Breeding of purine nucleoside production strains using genetic engineering techniques is also being carried out. For example, the ability to produce purine nucleoside by genetic engineering technology for Bacillus bacteria (Patent Documents 12 to 21), Brevibacterium (corynebacterium) bacteria (Patent Document 22), and Escherichia bacteria (Patent Document 11) Or enhancements have been made. Specifically, for example, the intracellular activity of an enzyme involved in purine nucleoside biosynthesis, or the activity of an enzyme that catalyzes a reaction that branches from the purine nucleoside biosynthesis pathway to produce other compounds. By reducing the value, purine nucleoside-producing ability can be imparted or enhanced (Patent Document 11).

  The yggB gene of the coryneform bacterium is a homologue of the yggB gene of Escherichia coli (Non-Patent Documents 2 and 3) and encodes a mechanosensitive channel (Non-Patent Documents 4 and 5). In coryneform bacteria, it is known that glutamic acid-producing ability is improved by increasing the expression of the yggB gene or retaining a mutant yggB gene having a specific mutation (Patent Document 23).

  However, the relationship between the yggB gene and the production of purine substances such as purine nucleosides and purine nucleotides is not known.

Japanese Examined Patent Publication No. 38-23039 (1963) Japanese Patent Publication No.54-17033 (1979) Japanese Examined Patent Publication No. 55-2956 (1980) Japanese Patent Publication No. 55-45199 (1980) JP 56-162998 (1981) Japanese Patent Publication No.57-14160 (1982) Japanese Examined Patent Publication No.57-41915 (1982) JP 59-42895 (1984) Japanese Patent Publication No.51-5075 (1976) Japanese Examined Patent Publication No. 58-17592 (1972) International Publication No. 99/03988 Pamphlet JP 58-158197 (1983) JP 58-175493 A (1983) JP 59-28470 (1984) JP-A-60-156388 (1985) JP-A-1-27477 (1989) JP-A-1-174385 (1989) JP-A-3-58787 (1991) JP-A-3-164185 (1991) JP-A-5-84067 (1993) JP-A-5-192164 (1993) JP 63-248394 (1988) JP2007-097573

Agric. Biol. Chem., 42, 399 (1978) FEMS Microbiol Lett. 2003 Jan 28; 218 (2): 305-9. Mol Microbiol. 2004 Oct; 54 (2): 420-38. EMBO J. 1999, 18 (7): 1730-7 Biosci. Biotechnol. Biochem., 74 (12), 2546-2549, 2010

  An object of the present invention is to develop a novel technique for improving the ability of bacteria to produce purine substances and to provide an efficient method for producing purine substances.

  As a result of intensive studies to solve the above problems, the present inventor improves the purine-based substance production ability of bacteria by modifying the bacteria so as to retain the mutant yggB gene having a specific mutation. The present invention has been completed.

That is, the present invention can be exemplified as follows.
[1]
A bacterium having the ability to produce purine substances,
Escherichia bacteria, Bacillus bacteria, or Corynebacterium ammoniagenes,
Modified to retain the mutant yggB gene,
The bacterium, wherein the mutant yggB gene is a yggB gene having a mutation that improves the ability of the bacterium to produce purine substances.
[2]
The bacterium, wherein the mutation is selected from the group consisting of the following (1) to (3):
(1) a mutation in the region encoding the amino acid residues at positions 419 to 533 of the wild type YggB protein;
(2) mutations in the region encoding the transmembrane region of wild-type YggB protein; and (3) combinations thereof.
[3]
The bacterium as described above, wherein the mutation (1) is insertion of a base sequence into a region encoding amino acid residues at positions 419 to 533 of the wild type YggB protein.
[4]
The bacterium as described above, wherein the mutation (1) is a mutation that substitutes a proline residue at positions 419 to 533 of the wild-type YggB protein with another amino acid.
[5]
The bacterium as described above, wherein the transmembrane region is selected from amino acid residues at positions 1 to 23, 25 to 47, 62 to 84, 86 to 108, and 110 to 132 of the wild type YggB protein.
[6]
Said bacterium, wherein said mutation (2) is not accompanied by a frameshift mutation and a nonsense mutation. [7]
Wherein the mutation (2) is selected from the group consisting of (2a) to (2d) below: (2a) a leucine residue at position 14 and a tryptophan residue at position 15 of the wild-type YggB protein Mutations between which one or several amino acids are inserted;
(2b) a mutation that substitutes the alanine residue at position 100 of the wild-type YggB protein with another amino acid;
(2c) a mutation that replaces the alanine residue at position 111 of the wild-type YggB protein with another amino acid; and (2d) a combination thereof.
[8]
The bacterium, wherein Cys-Ser-Leu is inserted between the leucine residue at position 14 and the tryptophan residue at position 15.
[9]
Said bacterium, wherein said alanine residue at position 100 and / or 111 is replaced by threonine.
[10]
The bacterium, wherein the wild-type YggB protein is a protein described in (A) or (B) below:
(A) a protein having the amino acid sequence shown in SEQ ID NO: 9, 11, 13, or 15,
(B) a mechanosensitive channel having an amino acid sequence including substitution, deletion, insertion, or addition of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 9, 11, 13, or 15 Protein that functions as.
[11]
The bacterium as described above, which has been modified to increase the activity of an enzyme involved in the biosynthesis of purine substances.
[12]
The bacterium as described above, wherein the purine substance is selected from the group consisting of inosine, xanthosine, guanosine, and adenosine.
[13]
The bacterium as described above, wherein the purine substance is selected from the group consisting of inosinic acid, xanthylic acid, and guanylic acid.
[14]
The bacterium as described above, which is Escherichia coli.
[15]
The bacterium as described above, which is Bacillus subtilis or Bacillus amyloliquefaciens.
[16]
Said bacterium which is Corynebacterium ammoniagenes.
[17]
A method for producing a purine material, comprising culturing the bacterium in a medium, accumulating the purine substance in the medium, and recovering the purine substance from the medium.
[18]
A method for producing a purine nucleoside comprising culturing the bacterium in a medium, accumulating the purine nucleoside in the medium, and recovering the purine nucleoside from the medium.
[19]
A method for producing a purine nucleotide, comprising culturing the bacterium in a medium, accumulating the purine nucleotide in the medium, and recovering the purine nucleotide from the medium.
[20]
A method for producing a purine nucleotide, comprising culturing the bacterium in a medium and accumulating a purine nucleoside in the medium, phosphorylating the purine nucleoside to produce a purine nucleotide, and recovering the purine nucleotide.

  According to the present invention, the ability of bacteria to produce purine substances can be improved, and purine substances can be produced efficiently.

  Hereinafter, the present invention will be described in detail.

<1> Bacteria of the Present Invention The bacterium of the present invention is a bacterium having the ability to produce purine substances modified so as to retain a mutant yggB gene having a “specific mutation”. The “specific mutation” will be described later.

<1-1> Bacteria having purine-based substance-producing ability In the present invention, “bacteria having purine-based substance-producing ability” refers to the extent that a desired purine-based substance can be produced and recovered when cultured in a medium. Refers to bacteria having the ability to accumulate in the culture medium. The bacterium having the ability to produce a purine substance may be a bacterium that can accumulate a larger amount of the purine substance in the medium than the unmodified strain. An “unmodified strain” refers to a control strain that does not carry a mutant yggB gene, and may be, for example, a wild strain or a parent strain. The bacterium having the ability to produce purine substances is, for example, a bacterium capable of accumulating the target purine substance in an amount of 0.1 mM or more, 0.5 mM or more, 1 mM or more, or 2 mM or more in the medium. May be.

  Examples of purine substances include purine nucleosides and purine nucleotides. Purine nucleosides include inosine, guanosine, xanthosine, and adenosine. Purine nucleotides include 5'-phosphate esters of purine nucleosides. Examples of 5'-phosphate esters of purine nucleosides include inosinic acid (inosine-5'-phosphate ester; IMP), guanylic acid (guanosine-5'-phosphate ester; GMP), xanthylic acid (xanthosine-5'-). Phosphate ester; XMP), and adenylic acid (adenosine-5′-phosphate ester; AMP). The bacterium of the present invention may have the ability to produce one kind of purine substance, or may have the ability to produce two or more kinds of purine substances. The bacterium of the present invention may have, for example, the ability to produce one or more purine nucleosides. The bacterium of the present invention may have, for example, the ability to produce one or more purine nucleotides.

  The bacterium of the present invention is an Escherichia bacterium, a Bacillus genus bacterium, or a Corynebacterium ammoniagenes.

Examples of the genus Escherichia include, but are not particularly limited to, bacteria classified into the genus Escherichia by classification known to specialists in microbiology. Examples of Escherichia bacteria include, for example, Neidhardt et al. (Backmann, BJ 1996. Derivations and Genotypes of some mutant derivatives of Escherichia coli K-12, p. 2460-2488. Table 1. In FD Neidhardt (ed.), Escherichia coli and Salmonella: Cellular and Molecular Biolo
gy, Second Edition, American Society for Microbiology Press, Washington, DC). Examples of bacteria belonging to the genus Escherichia include Escherichia coli. Specific examples of Escherichia coli include Escherichia coli W3110 (ATCC 27325) and Escherichia coli MG1655 (ATCC 47076) derived from the prototype wild type K12.

Although it does not restrict | limit especially as a bacterium of the genus Bacillus, The bacteria classified into the genus Bacillus by the classification | category known to the expert of microbiology are mentioned. Examples of Bacillus bacteria include the following species.
Bacillus subtilis
Bacillus amyloliquefaciens
Bacillus pumilus
Bacillus licheniformis
Bacillus megaterium
Bacillus brevis
Bacillus polymixa
Bacillus stearothermophilus

  Specific examples of Bacillus subtilis include Bacillus subtilis 168 Marburg strain (ATCC 6051) and Bacillus subtilis PY79 strain (Plasmid, 1984, 12, 1-9). Specific examples of Bacillus amyloliquefaciens include Bacillus amyloliquefaciens T strain (ATCC 23842) and Bacillus amyloliquefaciens N strain (ATCC 23845).

Corynebacterium ammoniagenes is not particularly limited, but includes bacteria classified as Corynebacterium ammoniagenes by classification known to microbiologists. Corynebacterium ammoniagenes includes bacteria previously classified as Brevibacterium ammoniagenes. Specific examples of Corynebacterium ammoniagenes include the following strains:
Corynebacterium ammoniagenes ATCC-6872
Corynebacterium ammoniagenes adenine leaking mutant (Leaky Mutant) (Agr.
Bio. Chem., Vol. 47 (5), p1035-1041, 1983, (KY13102, KY13171, KY13184))

  These strains can be sold, for example, from the American Type Culture Collection (address 12301 Parklawn Drive, Rockville, Maryland 20852 P.O. Box 1549, Manassas, VA 20108, United States of America). That is, a registration number corresponding to each strain is given, and it is possible to receive a sale using this registration number (see http://www.atcc.org/). The registration number corresponding to each strain is described in the catalog of American Type Culture Collection.

  The bacterium of the present invention may inherently have a purine-based substance-producing ability or may be modified so as to have a purine-based substance-producing ability. A bacterium having a purine substance-producing ability can be obtained, for example, by imparting a purine substance-producing ability to the bacterium as described above or by enhancing the purine substance-producing ability of the bacterium as described above. .

  Conventionally, the ability to produce purine-based substances can be imparted or enhanced by methods that have been adopted for breeding purine-based substance-producing bacteria such as Bacillus bacteria and Escherichia bacteria.

For example, the ability to produce purine substances can be imparted or enhanced by imparting auxotrophy such as adenine requirement or by imparting resistance to drugs such as purine analogs and sulfaguanidine ( (See JP-B-38-23099, JP-B-54-17033, JP-B-55-45199, JP-B-57-14160, JP-B-57-41915, JP-B-59-42895, US2004-0166575A). Mutants such as auxotrophic strains and drug-resistant strains having the ability to produce purine substances are subjected to mutation treatment of the parent strain or wild strain, and a mutant strain having a desired phenotype is selected using an appropriate selection medium. Can be obtained. Examples of the mutation treatment include X-ray irradiation, ultraviolet irradiation, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ethylmethanesulfonate (EMS), methylmethanesulfonate (MMS). ) And the like.

  In addition, the ability to produce purine substances can be imparted or enhanced by enhancing the intracellular activity of enzymes involved in the biosynthesis of purine substances. The activity of one enzyme may be enhanced, and the activity of two or more enzymes may be enhanced. A method for enhancing the enzyme activity will be described later. Enhancing enzyme activity can be performed, for example, by modifying bacteria so that expression of a gene encoding the enzyme is enhanced. Techniques for enhancing gene expression are described in WO00 / 18935 pamphlet and European Patent Application Publication No. 1010755.

  Purine nucleotides are biosynthesized using phosphoribosylpyrophosphate (PRPP) as an intermediate. Purine nucleosides are biosynthesized by dephosphorylation of purine nucleotides. Examples of the enzyme involved in the biosynthesis of these purine substances include PRPP synthetase (prs) and proteins encoded by the purine operon. The parentheses are abbreviations for genes encoding the enzymes (the same applies to the following description). However, the gene name (abbreviated symbol of the gene) is an example, and depending on the species, the gene name may be different or may not have the corresponding gene.

Examples of the purine operon include Bacillus subtilis and Its Closest Relatives, Editor in Chief: AL Sonenshein,
ASM Press, Washington DC, 2002) and Escherichia coli pur regulon (Escherichia and Salmonella, Second Edition, Editor in Chief: FC Neidhardt, ASM Press, Washington DC, 1996). For example, the expression of the purine operon may be enhanced together, or the expression of one or more genes selected from genes contained in the purine operon may be enhanced.

  Among these, it is preferable to enhance the activity of one or more enzymes selected from, for example, PRPP synthetase (prs) and PRPP amidotransferase (purF).

  In addition, for example, when an enzyme involved in the biosynthesis of purine-based substances is subjected to negative regulation such as feedback inhibition or expression suppression, the enzyme activity is enhanced by reducing or canceling the regulation, and purine Substance production ability can be improved (WO99 / 003988).

The expression of the purine operon is suppressed by the purine repressor encoded by the purR gene. Thus, purine operon expression can be enhanced, for example, by reducing the activity of the purine repressor (US Pat. No. 6,284,495). Purine repressor activity can be reduced, for example, by disrupting the purR gene encoding the purine repressor (US Pat. No. 6,284,495). The expression of the purine operon is controlled by a terminator-antiterminator sequence (also referred to as an attenuator sequence) located downstream of the promoter (Ebbole, DJ and Zalkin, H., J. Biol. Chem., 1987, 262). , 8274-8287, Ebbole, DJ and Zalkin, H., J. Biol. Chem., 1988, 263, 10894-1
0902, Ebbole, DJ and Zalkin, H., J. Bacteriol., 1989, 171, 2136-2141). Thus, the expression of the purine operon can be enhanced, for example, by deleting the attenuator sequence. The deletion of the attenuator sequence can be performed by the same technique as that for the gene destruction described later.

  PRPP synthetase is subject to feedback inhibition by ADP. Therefore, for example, by retaining a mutant PRPP synthetase gene encoding a desensitized PRPP synthetase in which feedback inhibition by ADP is reduced or eliminated, the PRPP synthetase activity is enhanced and the purine substance production ability is improved. (WO99 / 003988). Desensitized PRPP synthetases include those having a mutation in which Asp (D) at position 128 of wild type PRPP synthetase is replaced with Ala (A) (SG Bower et al., J. Biol. Chem. , 264, 10287 (1989)).

  PRPP amide transferase is subject to feedback inhibition by AMP and GMP. Thus, for example, by allowing bacteria to retain a mutant PRPP amide transferase gene encoding a desensitized PRPP amide transferase with reduced or eliminated feedback inhibition by AMP and / or GMP, the PRPP amide transferase activity is enhanced, Purine-based substance production ability can be improved (WO99 / 003988). Examples of desensitized PRPP amide transferase include those having a mutation in which Lys (K) at position 326 of wild-type PRPP amide transferase is replaced with Gln (Q), and Lys (K at position 326 of wild-type PRPP amide transferase). ) Is substituted with Gln (Q) and Pro (P) at position 410 is substituted with Trp (W) (G. Zhou et al., J. Biol. Chem., 269). 6784 (1994)).

  The purine substance production ability can be imparted or enhanced by reducing the activity of an enzyme that catalyzes a reaction that branches from the biosynthetic pathway of the purine substance to produce another compound (WO99 / 003988). ). The activity of one enzyme may be reduced, and the activity of two or more enzymes may be reduced. The “enzyme that catalyzes the reaction of branching from the biosynthetic pathway of purine-based substances to produce other compounds” includes enzymes involved in the degradation of purine-based substances. A method for reducing the enzyme activity will be described later.

  Examples of the enzyme that catalyzes the reaction of branching from the biosynthetic pathway of purine substances to produce other compounds include purine nucleoside phosphorylase (deoD, pupG), succinyl-AMP synthase (succinyl-AMP synthase) ) (PurA), adenosine deaminase (add), inosine-guanosine kinase (gsk), GMP reductase (guaC), 6-phosphogluconate dehydrase (6-phosphogluconate dehydrase) ) (Edd), phosphophoglucose isomerase (pgi), adenine deaminase (yicP), xanthosine phosphorylase (xapA), IMP dehydrogenase (guaB). The enzyme that decreases the activity may be selected depending on the type of the purine substance of interest.

  The purine substance production ability can be imparted or enhanced by reducing the activity of fructose 1,6-bisphosphatase (fbp) (WO2007 / 125782).

  The purine substance production ability can be imparted or enhanced by reducing the activity of a protein involved in the incorporation of purine substances (WO99 / 003988). For example, a protein involved in purine nucleoside uptake includes nucleoside permiase (nupG) (WO99 / 003988).

  The purine substance production ability can be imparted or enhanced by enhancing the activity of proteins involved in the purine substance discharge. For example, proteins involved in purine nucleoside excretion include rhtA (ybiF) gene (Russian Patent No. 2239656), yijE gene (Russian Patent No. 2244003), ydeD gene (Russian Patent No. 2244004), yicM Examples include proteins encoded by genes (Russian Patent No. 2271391), ydhL gene (Special Table 2007-530011), nepI gene (FEMS Microbiology Letters, Volume 250, Issue 1, pages 39-47, September 2005).

  The inosinic acid producing ability can be imparted or enhanced by imparting resistance to an analog of L-glutamine and resistance to an analog of proline to a bacterium (Japanese Patent Laid-Open No. 2004-516833). L-glutamine analogs include azaserine and 6-diazo-5-oxo-L-norleucine (DON). Examples of proline analogs include 3,4-dehydroproline, L-azetidine-2-carboxylic acid, L-thiazolidine-4-carboxylic acid, (S) -2,2-dimethyl-4-oxazolide carboxylic acid, ( S) -5,5-Dimethyl-4-thiazolide carboxylic acid, (4S, 2RS) -2-ethyl-4-thiazolidine-carboxylic acid, (2S, 4S) -4-hydroxy-2-pyrroline-carboxylic acid , 2-piperidinecarboxylic acid, and 2,5-pyrrolidinedione.

  Moreover, the ability to produce xanthylic acid can be imparted or enhanced by a method used for breeding xanthylic acid-producing bacteria of coryneform bacteria centered on Corynebacterium ammmoniagenes. Such methods include, for example, enhancement of PRPP amidotransferase activity (Japanese Patent Laid-Open No. 8-168383), imparting resistance to aliphatic amino acids (Japanese Patent Laid-Open No. 4-262790), imparting resistance to dehydroproline (Korean Patent Publication No. 2003- 56490).

  The methods for imparting or enhancing the purine substance production ability as described above may be used alone or in any combination.

  Specific examples of the nucleoside producing strain belonging to the genus Escherichia include, for example, Escherichia coli FADRaddedd / pKFpurFKQ strain (WO99 / 003988). This strain is obtained from the Escherichia coli W3110 strain by purF gene encoding PRPP amide transferase, purR gene encoding purine repressor, deoD gene encoding purine nucleoside phosphorylase, purA gene encoding succinyl-AMP synthase, and adenosine deaminase. A plasmid carrying a purFKQ gene encoding a desensitized PRPP amide transferase (K326Q) in which an add gene encoding and an edd gene encoding 6-phosphogluconate dehydrase are disrupted and feedback inhibition by GMP is reduced It is a strain obtained by introducing pKFpurFKQ (WO99 / 003988).

Specific examples of inosine-producing strains belonging to the genus Bacillus include the following.
Bacillus subtilis KMBS16 strain (US Patent Application Publication No. 2004-0166575)
Bacillus subtilis KMBS16-1 (Russian Patent No. 2239656)
Bacillus subtilis KMBS310 strain (JP 2007-117078)
Bacillus subtilis TABS133 strain (WO2007 / 125782)
Bacillus subtilis AJ12707 strain (FERM P-12951) (Japanese Patent Application No. 61-13876)
Bacillus subtilis AJ3772 strain (FERM P-2555) (Japanese Patent Application No. 62-014794)
Bacillus subtilis NA-6011 strain (FERM BP-291) (US Pat. No. 4,701,413)
Bacillus subtilis NA-6012 strain (FERM BP-292) (US Pat. No. 4,701,413)
Bacillus subtilis G1136A strain (Bacillus amyloliquefaciens AJ1991 strain) (VKPM B-8994)
(US Pat. No. 3,575,809)
Bacillus pumilus Gottheil 3218 strain (ATCC 21005) (US Pat. No. 3,616,206)
Bacillus pumilus NA-1102 strain (FERM BP-289)
Bacillus amyloliquefaciens AS115-7 strain (VKPM B-6134) (Russian Patent No. 2003678)

  The KMBS16 strain was obtained by destroying the purA gene encoding succinyl-AMP synthase, the purR gene encoding purine repressor, and the deoD gene encoding purine nucleoside phosphorylase from the Bacillus subtilis trpC2 strain (purA: : erm, purR :: spc, deoD :: kan) (US Patent Application Publication No. 2004-0166575). The KMBS16-1 strain is a strain obtained by replacing the purA :: erm mutation of the KMBS16 strain with a purA :: cat mutation. The KMBS310 strain destroyed the purA gene encoding the succinyl-AMP synthase, the purR gene encoding the purine repressor, and the pupG gene encoding the purine nucleoside phosphorylase from the Bacillus subtilis 168 Marburg strain (ATCC 6051). guaB) is a strain obtained by reducing the activity and modifying the purine operon promoter region and the SD sequence of the PRPP synthetase gene (prs).

  Specific examples of guanosine-producing strains belonging to the genus Bacillus include, for example, a Bacillus bacterium having enhanced IMP dehydrogenase activity (JP-A-3-58787) and a vector carrying a purine analog resistance gene or a decoinin resistance gene. Examples include Bacillus bacteria (Japanese Patent Publication No. 4-28357) introduced into mutant strains.

Examples of bacteria having purine nucleotide-producing ability include the following bacteria. Examples of inosinic acid producing bacteria include Corynebacterium ammoniagenes CJIP009 (KCCM-10226) (Japanese Patent Laid-Open No. 2004-516833), and an inosine producing strain of Bacillus subtilis (Uchida, K. et al. , Agr. Biol. Chem., 1961, 25, 804-805;
Fujimoto, M. Uchida, K., Agr. Biol. Chem., 1965, 29, 249-259). Examples of the guanylate-producing bacterium include a guanylate-producing strain belonging to the genus Bacillus that exhibits adenine requirement and is resistant to decoinin or methionine sulfoxide (Japanese Patent Publication No. 56-12438).

  The gene used for breeding the above purine substance-producing bacteria is not limited to the above-exemplified genes or genes having a known base sequence, as long as the function of the encoded protein is not impaired. May be. For example, a gene used for breeding a purine substance-producing bacterium has an amino acid sequence in which one or several amino acids at one or several positions are substituted, deleted, inserted or added in the amino acid sequence of a known protein. It may be a gene encoding a protein having a sequence. Regarding gene and protein variants, the descriptions regarding the yggB gene and YggB protein variants described below can be applied mutatis mutandis.

<1-2> Technique for Increasing Protein Activity A technique for increasing protein activity is described below.

“Protein activity is increased” means that the activity of the protein is increased relative to a non-modified strain such as a wild strain or a parent strain. Note that “increasing protein activity” is also referred to as “enhancing protein activity”. “Protein activity increases” specifically means that the number of molecules per cell of the protein is increased and / or the function per molecule of the protein compared to an unmodified strain. Is increasing. That is, “activity” in the case of “increasing the activity of a protein” is not limited to the catalytic activity of the protein, and may mean the transcription amount (mRNA amount) of the gene encoding the protein or the amount of the protein. The activity of the protein is not particularly limited as long as it is increased compared to the non-modified strain. For example, the protein activity is increased 1.5 times or more, 2 times or more, or 3 times or more compared to the non-modified strain. Good. In addition, “the protein activity increases” means not only to increase the activity of the protein in a strain that originally has the activity of the target protein, but also to the activity of the protein in a strain that does not originally have the activity of the target protein. Including granting. As a result, as long as the activity of the protein is increased, a suitable protein may be introduced after reducing the activity of the target protein originally possessed by the bacterium.

  Modifications that increase the activity of the protein are achieved, for example, by increasing the expression of the gene encoding the protein. Note that “increasing gene expression” is also referred to as “enhanced gene expression”. The expression of the gene may be increased 1.5 times or more, 2 times or more, or 3 times or more, for example, as compared to the unmodified strain. In addition, “increasing gene expression” means not only increasing the expression level of a target gene in a strain that originally expresses the target gene, but also in a strain that originally does not express the target gene. Including expressing a gene. That is, “increasing gene expression” includes, for example, introducing the gene into a strain that does not have the target gene and expressing the gene.

  An increase in gene expression can be achieved, for example, by increasing the copy number of the gene.

Increasing the copy number of the gene can be achieved by introducing the gene into the chromosome of the host bacterium. Introduction of a gene into a chromosome can be performed, for example, using homologous recombination (Miller I, JH Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Only one copy of the gene may be introduced, or two copies or more may be introduced. For example, multiple copies of a gene can be introduced into a chromosome by performing homologous recombination with a sequence having multiple copies on the chromosome as a target. Examples of sequences having multiple copies on a chromosome include repetitive DNA sequences and inverted repeats present at both ends of a transposon. Alternatively, homologous recombination may be performed by targeting an appropriate sequence on a chromosome such as a gene unnecessary for purine-based material production. Homologous recombination is, for example, the Red-driven integration method (Datsenko,
K. A, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97: 6640-6645 (2000)), a method using a plasmid containing a temperature-sensitive replication origin, conjugation It can be carried out by a method using a transmissible plasmid, a method using a suicide vector that does not have an origin of replication and functions in a host, or a transduction method using a phage. The gene can also be randomly introduced onto the chromosome using transposon or Mini-Mu (Japanese Patent Laid-Open No. 2-109985, US Pat. No. 5,882,888, EP805867B1).

  Confirmation of the introduction of the target gene on the chromosome was performed using Southern hybridization using a probe having a sequence complementary to all or part of the gene, or a primer prepared based on the sequence of the gene. It can be confirmed by PCR.

An increase in the copy number of the gene can also be achieved by introducing a vector containing the target gene into the host bacterium. For example, a DNA fragment containing a target gene is linked to a vector that functions in the host bacterium to construct an expression vector for the gene, and the host bacterium is transformed with the expression vector to increase the copy number of the gene. be able to. A DNA fragment containing a target gene can be obtained, for example, by PCR using a bacterial genomic DNA having the target gene as a template. As the vector, a vector capable of autonomous replication in a host bacterial cell can be used. The vector is preferably a multicopy vector. In order to select a transformant, the vector preferably has a marker such as an antibiotic resistance gene. The vector may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a cosmid, or a phagemid. Specific examples of vectors capable of autonomous replication in Escherichia bacteria such as Escherichia coli include, for example, pUC19, pUC18, pHSG299, pHSG399, pHSG398, pACYC184, pBR322, and pSTV29 (all available from Takara Bio Inc.), pMW219 (Nippon Gene), pTrc99A (Pharmacia), pPROK vector (Clontech), pKK233-2 (Clontech), pET vector (Novagen), pQE vector (Qiagen), wide host range vector RSF1010 Can be mentioned. Specific examples of vectors capable of autonomous replication in Bacillus bacteria such as Bacillus subtilis include pUB110, pC194, and pE194. Specific examples of vectors capable of autonomous replication in coryneform bacteria such as Corynebacterium ammoniagenes include, for example, pHM1519 (Agric, Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol Chem., 48, 2901-2903 (1984)); plasmids having improved drug resistance genes; plasmid pCRY30 described in JP-A-3-210184; JP-A-2-72876 and US Pat. No. 5,185,262 Plasmids pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE and pCRY3KX described in the specification gazette; plasmids pCRY2 and pCRY3 described in JP-A-1-91686; pAJ655, pAJ611 and pAJ1844 described in JP-A-58-192900 PCG1 described in JP-A-57-134500; pCG2 described in JP-A-58-35197; pCG4 and pCG11 described in JP-A-57-183799.

  When a gene is introduced, the gene only needs to be retained in the bacterium of the present invention so that it can be expressed. Specifically, the gene may be introduced so as to be expressed under the control of a promoter sequence that functions in the bacterium of the present invention. The promoter may be a host-derived promoter or a heterologous promoter. The promoter may be a native promoter of a gene to be introduced or a promoter of another gene. As the promoter, for example, a stronger promoter as described later may be used.

  Moreover, when introducing two or more genes, each gene should just be hold | maintained in the bacterium of this invention so that expression is possible. For example, all the genes may be held on a single expression vector, or all may be held on a chromosome. Moreover, each gene may be separately hold | maintained on several expression vector, and may be separately hold | maintained on the single or several expression vector and chromosome. Further, an operon may be constructed by introducing two or more genes.

  The introduced gene is not particularly limited as long as it encodes a protein that functions in the host. The introduced gene may be a host-derived gene or a heterologous gene. The gene to be introduced can be obtained by PCR using, for example, a primer designed based on the base sequence of the gene, and using a genomic DNA of an organism having the gene or a plasmid carrying the gene as a template. Moreover, you may synthesize | combine the gene to introduce | transduce based on the base sequence of the same gene, for example.

Moreover, the increase in gene expression can be achieved by improving the transcription efficiency of the gene. Improvement of gene transcription efficiency can be achieved, for example, by replacing a promoter of a gene on a chromosome with a stronger promoter. By “stronger promoter” is meant a promoter that improves transcription of the gene over the native wild-type promoter. Examples of stronger promoters include the known high expression promoters T7 promoter, trp promoter, lac promoter, tac promoter, thr promoter, trc promoter, and PL promoter. In addition, when using Corynebacterium ammoniagenes, artificially modified P54-6 promoter (Appl. Microbiol. Biotechnolo., 53, 674-679 (2000)), acetic acid in coryneform bacteria, Pta, aceA, aceB, adh, amyE promoter that can be induced by ethanol, pyruvate, etc., cspB, SOD, tuf promoters (Journal of Biotechnology 104 (2003) 311-323) , Appl Environ Microbiol. 2005 Dec; 71 (12): 8587-96.) And the like can be used. As a more powerful promoter, a highly active promoter of a conventional promoter may be obtained by using various reporter genes. For example, the promoter activity can be increased by bringing the -35 and -10 regions in the promoter region closer to the consensus sequence (WO 00/18935). Examples of highly active promoters include various tac-like promoters (Katashkina JI et al. Russian Federation Patent application 2006134574) and pnlp8 promoter (WO2010 / 027045). Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)).

  Moreover, the increase in gene expression can be achieved by improving the translation efficiency of the gene. Improvement of gene translation efficiency can be achieved, for example, by replacing the Shine-Dalgarno (SD) sequence (also referred to as ribosome binding site (RBS)) of the gene on the chromosome with a stronger SD sequence. By “a stronger SD sequence” is meant an SD sequence in which the translation of mRNA is improved over the originally existing wild-type SD sequence. An example of a stronger SD sequence is RBS of gene 10 derived from phage T7 (Olins P. O. et al, Gene, 1988, 73, 227-235). Furthermore, the substitution, insertion or deletion of several nucleotides in the spacer region between the RBS and the start codon, particularly in the sequence immediately upstream of the start codon (5′-UTR), greatly contributes to mRNA stability and translation efficiency. It is known that the translation efficiency of genes can be improved by modifying them.

  In the present invention, sites that affect gene expression, such as a promoter, an SD sequence, and a spacer region between the RBS and the start codon, are collectively referred to as an “expression control region”. The expression regulatory region can be determined using a promoter search vector or gene analysis software such as GENETYX. These expression control regions can be modified by, for example, a method using a temperature sensitive vector or a Red driven integration method (WO2005 / 010175).

  Improvement of gene translation efficiency can also be achieved, for example, by codon modification. In Escherichia coli, etc., there is a clear codon bias among the 61 amino acid codons found in the population of mRNA molecules, and the abundance of a tRNA seems to be directly proportional to the frequency of use of the corresponding codon. (Kane, JF, Curr. Opin. Biotechnol., 6 (5), 494-500 (1995)). That is, if a large amount of mRNA containing an excessive rare codon is present, translation problems may occur. Recent studies suggest that, inter alia, clusters of AGG / AGA, CUA, AUA, CGA, or CCC codons can reduce both the amount and quality of the synthesized protein. Such a problem can occur particularly during the expression of heterologous genes. Therefore, when performing heterologous expression of a gene, the translation efficiency of the gene can be improved by replacing rare codons present in the gene with synonymous codons that are used more frequently. Codon substitution can be performed, for example, by a site-specific mutagenesis method in which a target mutation is introduced into a target site of DNA. As a site-specific mutation method, a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press (1989); Carter, P., Meth. In Enzymol., 154, 382 (1987)) and methods using phage (Kramer, W. and Frits, HJ, Meth. In Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. In Enzymol., 154, 367 ( 1987)). Alternatively, gene fragments in which codons have been replaced may be fully synthesized. The frequency of codon usage in various organisms can be found in the “Codon Usage Database” (http://www.kazusa.or.jp/codon; Nakamura, Y. et al, Nucl. Acids Res., 28, 292 (2000)). Is disclosed.

  An increase in gene expression can also be achieved by amplifying a regulator that increases gene expression, or by deleting or weakening a regulator that decreases gene expression.

  The methods for increasing gene expression as described above may be used alone or in any combination.

  Modifications that increase protein activity can also be achieved, for example, by enhancing the specific activity of the protein. Specific activity enhancement also includes the reduction and elimination of feedback inhibition. Proteins with enhanced specific activity can be obtained by searching for various organisms, for example. Alternatively, a highly active protein may be obtained by introducing a mutation into a conventional protein. The introduced mutation may be, for example, a substitution, deletion, insertion or addition of one or several amino acids at one or several positions of the protein. Mutation can be introduced by, for example, the site-specific mutation method as described above. Moreover, you may introduce | transduce a variation | mutation by a mutation process, for example. Mutation treatments include X-ray irradiation, ultraviolet irradiation, and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ethylmethanesulfonate (EMS), and methylmethanesulfonate (MMS). ) And the like. Alternatively, DNA may be treated directly with hydroxylamine in vitro to induce random mutations. The enhancement of specific activity may be used alone or in any combination with the above-described method for enhancing gene expression.

The method for transformation is not particularly limited, and a conventionally known method can be used. For example, a method for increasing DNA permeability by treating recipient cells with calcium chloride as reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol. 1970, 53,
159-162) and methods for introducing DNA by preparing competent cells from proliferating cells as reported for Bacillus subtilis (Duncan, CH, Wilson, GA and Young, FE., 1997. Gene 1: 153-167) can be used. Alternatively, DNA-receptive cells, such as those known for Bacillus subtilis, actinomycetes, and yeast, can be made into protoplasts or spheroplasts that readily incorporate recombinant DNA into recombinant DNA. Introduction method (Chang, S. and Choen, SN, 1979. Mol. Gen. Genet. 168: 111-115; Bibb, MJ, Ward, JM and Hopwood, OA 1978. Nature 274: 398-400; Hinnen, A Hicks, JB and Fink, GR 1978. Proc. Natl. Acad. Sci. USA 75: 1929-1933).

  An increase in protein activity can be confirmed by measuring the activity of the protein.

  The increase in the activity of the protein can also be confirmed by confirming that the expression of the gene encoding the protein has increased. An increase in gene expression can be confirmed by confirming that the transcription amount of the gene has increased, or by confirming that the amount of protein expressed from the gene has increased.

  Confirmation that the amount of gene transcription has increased can be carried out by comparing the amount of mRNA transcribed from the gene with an unmodified strain such as a wild strain or a parent strain. Methods for assessing the amount of mRNA include Northern hybridization, RT-PCR, etc. (Sambrook, J., et al., Molecular Cloning A Laboratory Manual / Third Edition, Cold spring Harbor Laboratory Press, Cold spring Harbor (USA ), 2001). The amount of mRNA may be increased by, for example, 1.5 times or more, 2 times or more, or 3 times or more, compared to the unmodified strain.

  Confirmation that the amount of the protein has increased can be performed by Western blotting using an antibody (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). The amount of protein may be increased by, for example, 1.5 times or more, 2 times or more, or 3 times or more as compared to the unmodified strain.

  The above-described methods for increasing the activity of a protein are used to enhance the activity of an arbitrary protein, for example, an enzyme involved in the biosynthesis of purine substances, and to enhance the expression of an arbitrary gene, for example, a gene encoding the arbitrary protein. Available.

<1-3> Technique for reducing protein activity A technique for reducing protein activity is described below.

  “Protein activity decreases” means that the activity of the protein is reduced as compared to a non-modified strain such as a wild strain or a parent strain, and includes a case where the activity is completely lost. Specifically, “the activity of the protein is decreased” means that the number of molecules per cell of the protein is decreased and / or the function per molecule of the protein compared to the unmodified strain. Means that it is decreasing. That is, “activity” in the case of “decrease in protein activity” is not limited to the catalytic activity of the protein, and may mean the transcription amount (mRNA amount) of the gene encoding the protein or the amount of the protein. Note that “the number of molecules per cell of the protein is decreased” includes a case where the protein does not exist at all. Moreover, “the function per molecule of the protein is reduced” includes the case where the function per molecule of the protein is completely lost. The activity of the protein is not particularly limited as long as it is lower than that of the non-modified strain. For example, it is 50% or less, 20% or less, 10% or less, 5% or less, or 0, compared to the non-modified strain. %.

  The modification that decreases the activity of the protein is achieved, for example, by decreasing the expression of a gene encoding the protein. “Gene expression decreases” includes the case where the gene is not expressed at all. In addition, “the expression of the gene is reduced” is also referred to as “the expression of the gene is weakened”. Gene expression may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% compared to an unmodified strain.

  The decrease in gene expression may be due to, for example, a decrease in transcription efficiency, a decrease in translation efficiency, or a combination thereof. Reduction of gene expression can be achieved, for example, by modifying an expression regulatory sequence such as a gene promoter or Shine-Dalgarno (SD) sequence. In the case of modifying the expression control sequence, the expression control sequence is preferably modified by 1 base or more, more preferably 2 bases or more, particularly preferably 3 bases or more. Further, part or all of the expression regulatory sequence may be deleted. In addition, reduction of gene expression can be achieved, for example, by manipulating factors involved in expression control. Factors involved in expression control include small molecules (such as inducers and inhibitors) involved in transcription and translation control, proteins (such as transcription factors), nucleic acids (such as siRNA), and the like.

  Moreover, the modification that decreases the activity of the protein can be achieved, for example, by destroying a gene encoding the protein. Gene disruption can be achieved, for example, by deleting part or all of the coding region of the gene on the chromosome. Furthermore, the entire gene including the sequences before and after the gene on the chromosome may be deleted. The region to be deleted may be any region such as an N-terminal region, an internal region, or a C-terminal region as long as a decrease in protein activity can be achieved. Usually, the longer region to be deleted can surely inactivate the gene. Moreover, it is preferable that the reading frames of the sequences before and after the region to be deleted do not match.

In addition, gene disruption is, for example, introducing an amino acid substitution (missense mutation) into a coding region of a gene on a chromosome, introducing a stop codon (nonsense mutation), or adding or deleting 1 to 2 bases. It can also be achieved by introducing a frameshift mutation (Journal of Biological Chemistry 272: 8611-8617 (1997), Proceedings of the Na
tional Academy of Sciences, USA 95 5511-5515 (1998), Journal of Biological Chemistry 26 116, 20833-20839 (1991)).

  In addition, gene disruption can be achieved, for example, by inserting another sequence into the coding region of the gene on the chromosome. The insertion site may be any region of the gene, but the longer the inserted sequence, the more reliably the gene can be inactivated. Moreover, it is preferable that the reading frames of the sequences before and after the insertion site do not match. Other sequences are not particularly limited as long as they reduce or eliminate the activity of the encoded protein, and examples thereof include marker genes such as antibiotic resistance genes and genes useful for purine-based substance production.

  Modifying a gene on a chromosome as described above includes, for example, deleting a partial sequence of the gene and preparing a deleted gene modified so as not to produce a normally functioning protein. By transforming bacteria with recombinant DNA containing, and causing homologous recombination between the deleted gene and the wild-type gene on the chromosome to replace the wild-type gene on the chromosome with the deleted gene Can be achieved. At this time, the recombinant DNA can be easily manipulated by including a marker gene in accordance with a trait such as auxotrophy of the host. Even if the protein encoded by the deletion-type gene is produced, it has a three-dimensional structure different from that of the wild-type protein, and its function is reduced or lost. Gene disruption by gene replacement using such homologous recombination has already been established, and a method called “Red-driven integration” (Datsenko, KA, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97: 6640-6645 (2000)), Red-driven integration method and excision system derived from λ phage (Cho, EH, Gumport, RI, Gardner, JFJ Bacteriol. 184: 5200-5203 (2002) ) And the like (see WO2005 / 010175), a method using linear DNA, a method using a plasmid containing a temperature-sensitive replication origin, a method using a plasmid capable of conjugation transfer, replication functioning in the host There is a method using a suicidal vector having no origin (US Pat. No. 6,303,383, Japanese Patent Laid-Open No. 05-007491).

  Further, the modification that decreases the activity of the protein may be performed by, for example, a mutation treatment. Mutation treatments include X-ray irradiation, ultraviolet irradiation, and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ethylmethanesulfonate (EMS), and methylmethanesulfonate (MMS). ) And the like.

  The decrease in protein activity can be confirmed by measuring the activity of the protein.

  The decrease in the activity of the protein can also be confirmed by confirming that the expression of the gene encoding the protein has decreased. The decrease in gene expression can be confirmed by confirming that the transcription amount of the gene has decreased, or confirming that the amount of protein expressed from the gene has decreased.

  Confirmation that the amount of gene transcription has been reduced can be confirmed by comparing the amount of mRNA transcribed from the same gene with an unmodified strain. Examples of methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR and the like (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). The amount of mRNA may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% compared to the unmodified strain.

Confirmation that the amount of protein has decreased can be performed by Western blotting using an antibody (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). The amount of protein is, for example, 50% compared to the unmodified strain
Hereinafter, it may be reduced to 20% or less, 10% or less, 5% or less, or 0%.

  Whether or not a gene has been destroyed can be confirmed by determining part or all of the base sequence, restriction enzyme map, full length, etc. of the gene according to the means used for the destruction.

  The above-described techniques for reducing the activity of a protein include reducing the activity of any protein, for example, an enzyme that catalyzes a reaction that generates other compounds by branching from the biosynthetic pathway of purine substances, or any gene such as those It can be used to reduce the expression of a gene encoding any protein.

<1-4> Introduction of mutant yggB gene The bacterium of the present invention has been modified to retain the mutant yggB gene. The bacterium of the present invention can be obtained by modifying a bacterium having a purine substance-producing ability so as to retain a mutant yggB gene. The bacterium of the present invention can also be obtained by imparting purine-based substance-producing ability after modifying the bacterium so as to retain the mutant yggB gene. In the present invention, the modification for constructing the bacterium of the present invention can be performed in any order.

  Hereinafter, the yggB gene and the YggB protein will be described. The yggB gene encodes a mechanosensitive channel. In the present invention, a yggB gene having a “specific mutation” described later is also referred to as a mutant yggB gene, and a protein encoded thereby is also referred to as a mutant YggB protein. In the present invention, a yggB gene having no “specific mutation” described later is also referred to as a wild-type yggB gene, and a protein encoded thereby is also referred to as a wild-type YggB protein. In the YggB protein, a change in amino acid sequence caused by a “specific mutation” in the yggB gene is also referred to as a “specific mutation”.

  Examples of the wild type yggB gene include the yggB gene of coryneform bacteria. Specific examples of the coryneform bacterium yggB gene include, for example, the corynebacterium glutamicum ATCC13869 strain, the Corynebacterium glutamicum ATCC13032 strain, the Corynebacterium glutamicum ATCC14967 strain, the Corynebacterium melassecola ATCC17965 strain yggB Gene. The yggB gene of Corynebacterium glutamicum ATCC13032 corresponds to a complementary sequence of the positions 1336092 to 1336993 in the genome sequence registered as GenBank accession NC_003450 (VERSION NC_003450.3 GI: 58036263) in the NCBI database. The yggB gene of Corynebacterium glutamicum ATCC13032 is synonymous with Cgl1270. Further, the YggB protein of Corynebacterium glutamicum ATCC13032 is registered as GenBank accession NP_600492 (version NP_600492.1 GI: 19552490, locus_tag = "NCgl1221"). The base sequences of these yggB genes are shown in SEQ ID NOs: 8, 10, 12, and 14, respectively. In addition, the amino acid sequences of YggB proteins encoded by these yggB genes are shown in SEQ ID NOs: 9, 11, 13, and 15, respectively. The conserved sequence of these YggB proteins is shown in SEQ ID NO: 16.

The wild-type YggB protein may be a variant of the YggB protein as long as it does not have a “specific mutation” described later and has a function as a YggB protein. Such variants may be referred to as “conservative variants”. Examples of the conservative variant include a homologue of the YggB protein and an artificially modified variant. In the present invention, “having a function as a YggB protein” may mean, for example, functioning as a mechanosensitive channel. Further, “having a function as a YggB protein” may mean, for example, having a property of improving L-glutamic acid-producing ability of coryneform bacteria when expression is increased in coryneform bacteria (special features). Open 2007-097573).

  The wild type yggB gene may be a variant of the yggB gene as long as it encodes the YggB protein or a conservative variant thereof.

  The gene encoding the homologue of the YggB protein can be easily obtained from a public database by BLAST search or FASTA search using the yggB gene (SEQ ID NO: 8, 10, 12, or 14) of the coryneform bacterium as a query sequence. Can be acquired. Moreover, the gene encoding the homologue of the YggB protein can be obtained, for example, by PCR using a chromosome of a coryneform bacterium as a template and oligonucleotides prepared based on these known gene sequences as primers.

  As long as the wild-type YggB protein does not have the “specific mutation” described below and has a function as a YggB protein, one or several amino acids at one or several positions in the amino acid sequence are substituted. It may be a protein having an amino acid sequence deleted, inserted or added. The “one or several” is different depending on the position of the protein in the three-dimensional structure of the amino acid residue and the type of the amino acid residue, but specifically preferably 1 to 20, more preferably 1 to 10 More preferably, it means 1 to 5.

  One or several amino acid substitutions, deletions, insertions or additions described above are conservative mutations in which the function of the protein is maintained normally. A typical conservative mutation is a conservative substitution. Conservative substitution is a polar amino acid between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid. In this case, between Gln and Asn, when it is a basic amino acid, between Lys, Arg, and His, when it is an acidic amino acid, between Asp and Glu, when it is an amino acid having a hydroxyl group Is a mutation that substitutes between Ser and Thr. Specifically, substitutions considered as conservative substitutions include substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys, substitution from Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp Substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val or Phe substitution, Substitution from Lys to Asn, Glu, Gln, His or Arg, substitution from Met to Ile, Leu, Val or Phe, substitution from Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala Substitution, substitution from Thr to Ser or Ala, substitution from Trp to Phe or Tyr, substitution from Tyr to His, Phe or Trp, and substitution from Val to Met, Ile or Leu. In addition, amino acid substitutions, deletions, insertions, additions, or inversions as described above include naturally occurring mutations (mutants or variants) such as those based on individual differences or species differences in the bacteria from which the gene is derived. Also included by

  Furthermore, as long as the wild-type YggB protein does not have a “specific mutation” described later and has a function as a YggB protein, it is 80% or more, preferably 90% or more, more than the entire amino acid sequence. It may be a protein having a homology of preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more. In the present specification, “homology” may refer to “identity”.

The wild-type YggB protein preferably has a conserved sequence of the above-mentioned coryneform bacterium YggB protein, for example. Therefore, it is preferable that the conservative mutation occurs at an amino acid residue that is not conserved in the YggB protein of the coryneform bacterium, for example. Specifically, for example, a conservative mutation may occur in one or more amino acid residues selected from the following amino acid residues of the wild-type YggB protein. In addition, the position of the amino acid residue in the wild type YggB protein is relative as described later.
Glu at position 48 (preferably substituted with Arg)
Asp at position 275 (preferably replaced with Ser)
Glu at position 298 (preferably substituted with Ala)
Ala at position 343 (preferably substituted with Val)
Phe at position 396 (preferably replaced by Ile)
Ser at position 438 (preferably replaced by Gly)
Val at position 445 (preferably substituted with Ala)
Ala at position 454 (preferably substituted with Val)
Pro at position 457 (preferably replaced with Ser)
Ser at position 474 (preferably replaced with Asp)
Val 517 (preferably deletion)
Glu at position 518 (preferably deleted)
Ala at position 519 (preferably deleted)
Pro at position 520 (preferably deletion)

  In addition, the wild-type yggB gene does not have a “specific mutation” described later and encodes a protein having a function as a YggB protein. For example, a probe that can be prepared from a known gene sequence, It may be DNA that hybridizes under stringent conditions with a complementary sequence in whole or in part. “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, highly homologous DNAs, for example, 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more between DNAs having homology. Is hybridized and DNAs of lower homology do not hybridize with each other, or washing conditions for normal Southern hybridization, 60 ° C, 1 × SSC, 0.1% SDS, preferably 60 ° C, 0.1 × SSC , 0.1% SDS, more preferably 68 ° C., 0.1 × SSC, a salt concentration and temperature corresponding to 0.1% SDS, and conditions for washing once, preferably 2-3 times.

  As described above, the probe used for the hybridization may be a part of a complementary sequence of a gene. Such a probe can be prepared by PCR using an oligonucleotide prepared on the basis of a known gene sequence as a primer and a DNA fragment containing these base sequences as a template. For example, when a DNA fragment having a length of about 300 bp is used as a probe, hybridization washing conditions include 50 ° C., 2 × SSC, and 0.1% SDS.

  In addition, the wild type yggB gene may be a gene in which an arbitrary codon is replaced with an equivalent codon as long as it encodes the wild type YggB protein as described above. For example, the wild type yggB gene may be modified to have an optimal codon depending on the codon usage frequency of the host to be used.

  In addition, the description regarding the said gene or protein variant can be applied mutatis mutandis to any protein such as an enzyme involved in biosynthesis of purine-based substances and genes encoding them.

  The mutant YggB protein has a “specific mutation” described later in the amino acid sequence of the wild-type YggB protein as described above.

  That is, in other words, the mutant YggB protein may be the above-mentioned coryneform bacterium YggB protein or a conservative variant thereof except that it has a “specific mutation” described later.

  Specifically, for example, the mutant YggB protein may be a protein having the amino acid sequence shown in SEQ ID NO: 9, 11, 13, or 15 except that it has a “specific mutation” described later.

  Specifically, for example, the mutant YggB protein has one or several amino acids in the amino acid sequence shown in SEQ ID NO: 9, 11, 13, or 15 except that it has a “specific mutation” described later. It may be a protein having an amino acid sequence containing substitutions, deletions, insertions, or additions.

  Specifically, for example, the mutant YggB protein has 80% or more, preferably 80% or more of the amino acid sequence shown in SEQ ID NO: 9, 11, 13, or 15 except that it has a “specific mutation” described later. May be a protein having an amino acid sequence having homology of 90% or more, more preferably 95% or more, more preferably 97% or more, and particularly preferably 99% or more.

  Moreover, in other words, the mutant YggB protein has the “specific mutation” described later in the above-mentioned coryneform bacterium YggB protein, and further includes a conservative mutation at a place other than the “specific mutation”. It may be a variant.

  Specifically, for example, the mutant YggB protein has a “specific mutation” described later in the amino acid sequence shown in SEQ ID NO: 9, 11, 13, or 15, and other than the “specific mutation”. It may be a protein having an amino acid sequence further including substitution, deletion, insertion, or addition of one or several amino acids at a position.

  The mutant yggB gene is not particularly limited as long as it encodes the mutant YggB protein as described above.

  Hereinafter, “specific mutation” of the mutant yggB gene will be described.

  The “specific mutation” is not particularly limited as long as it is a mutation that changes the amino acid sequence of the wild-type YggB protein as described above and improves the purine substance production ability of bacteria. “Improve the ability of bacteria to produce purine substances” means that the amount of Escherichia bacterium, Bacillus bacterium, or Corynebacterium ammoniagenes modified to retain the mutant yggB gene is higher than that of the unmodified strain. This means that it is possible to produce purine substances. An “unmodified strain” refers to a control strain that does not carry a mutant yggB gene, and may be, for example, a wild strain or a parent strain. “To produce a purine substance in a larger amount than the unmodified strain” is not particularly limited as long as the production amount of the purine substance is increased as compared with the non-modified strain. 1.05 times or more, preferably 1.1 times or more, more preferably 1.2 times or more, particularly preferably 1.3 times or more.

  Whether a mutation is a mutation that improves the ability of bacteria to produce purine substances, for example, encodes a YggB protein having the mutation based on a strain belonging to the genus Escherichia, Bacillus, or Corynebacterium ammoniagenes. When a strain that has been modified to retain a gene is prepared, the amount of purine substances produced when the modified strain is cultured in a medium, and the strain before modification (non-modified strain) is cultured in the medium It can be confirmed by comparing with the amount of purine-based substance produced.

  Specific examples of the “specific mutation” include the following mutations (JP 2007-097573).

(1) C-terminal mutation
The C-terminal mutation is a mutation in a region encoding the amino acid residue at positions 419 to 533 (positions 419 to 529 in SEQ ID NO: 13) of the wild type YggB protein in the wild type yggB gene. C-terminal mutations may be introduced at one or more points in the same region. The type of amino acid sequence change caused by the C-terminal mutation is not particularly limited. C-terminal mutations cause, for example, amino acid residue substitution (missense mutation), amino acid residue insertion, amino acid residue deletion, stop codon appearance (nonsense mutation), frameshift mutation, or combinations thereof It may be a thing. As the C-terminal side mutation, for example, insertion of a nucleotide sequence such as an insertion sequence (hereinafter also referred to as “IS”) or a transposon is preferable.

(1-1) Insertion of nucleotide sequence
Examples of the C-terminal mutation include a mutation (2A-1 type mutation) in which a base sequence is inserted at a position encoding the 419th valine residue of the wild type YggB protein. The 2A-1 type mutation may cause, for example, deletion or substitution of part or all of amino acid residues at positions 419 to 533 of the wild type YggB protein. Specifically, as a mutant yggB gene having 2A-1 type mutation, for example, IS is inserted after “G” at position 1255 of SEQ ID NO: 8, and the original wild type YggB protein (SEQ ID NO: 9) Also, a yggB gene encoding a mutant YggB protein having a short full-length 423 amino residues can be mentioned (Japanese Patent Laid-Open No. 2007-222163).

(1-2) Replacement of proline
Examples of the C-terminal mutation include a mutation that substitutes a proline residue at positions 419 to 533 of the wild-type YggB protein with another amino acid. Examples of such proline residues include wild type YggB protein at positions 424, 437, 453, 457, 462, 469, 484, 489, 497, 515, 529 (SEQ ID NO: 13). And the proline residue at position 533 (position 529 in SEQ ID NO: 13). Among them, it is preferable to substitute the proline residue at position 424 and / or 437 with another amino acid.

  The “other amino acid” is not particularly limited as long as it is a natural amino acid other than proline. “Other amino acids” include Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Ala, His.

  For example, the proline residue at position 424 may be preferably replaced with a hydrophobic amino acid (Ala, Gly, Val, Leu, or Ile), more preferably with a branched chain amino acid (Leu, Val, or Ile). It's okay. Examples of the mutation that replaces the proline residue at position 424 with leucine include a mutation that replaces “C” at position 1271 of SEQ ID NO: 14 with “T” (type 66 mutation).

  Also, for example, the proline residue at position 437 may be preferably substituted with an amino acid (Thr, Ser, or Tyr) having a hydroxyl group in the side chain, and more preferably with Ser. Examples of the mutation that replaces the proline residue at position 437 with Ser include a mutation that replaces “C” at position 1309 of SEQ ID NO: 8 with “T” (type 22 mutation). The type 22 mutation may be accompanied by, for example, a mutation in which “C” at position 1624 of SEQ ID NO: 8 is replaced with “T”.

(2) Mutation of transmembrane region
YggB protein is presumed to have 5 transmembrane regions. The transmembrane regions are 1 to 23 positions (first transmembrane region), 25 to 47 positions (second transmembrane region), 62 to 84 positions (third transmembrane region), and 86 to 108, respectively, of the wild-type YggB protein. It corresponds to the amino acid residue at position (fourth transmembrane region), positions 110 to 132 (fifth transmembrane region). Mutations in the transmembrane region are mutations in the region encoding these transmembrane regions in the wild-type yggB gene. Mutations in the transmembrane region may be introduced at one or more locations in the same region. The mutation in the transmembrane region is preferably one that causes substitution, deletion, addition, insertion, or inversion of one or several amino acids, and is not accompanied by a frameshift mutation or a nonsense mutation. “One or several” means preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5, particularly preferably 1 to 3.

(2-1) Mutation in the first transmembrane region Examples of the mutation in the first transmembrane region include one or several between the leucine residue at position 14 and the tryptophan residue at position 15 of the wild-type YggB protein. Mutations in which the amino acid is inserted. Examples of one or several amino acids include Cys-Ser-Leu. Specific examples of the mutant yggB gene having such a mutation include the yggB gene (A1 mutation) in which TTCATTGTG is inserted after “G” at position 44 of SEQ ID NO: 8. The nucleotide sequence of this mutant yggB gene and the amino acid sequence of the mutant YggB protein encoded by this gene are shown in SEQ ID NOs: 6 and 7, respectively.

(2-2) Mutation in the fourth transmembrane region Examples of the mutation in the fourth transmembrane region include a mutation that substitutes the alanine residue at position 100 of the wild-type YggB protein with another amino acid. The “other amino acid” is not particularly limited as long as it is a natural amino acid other than alanine. “Other amino acids” include Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, His, Pro. The alanine residue at position 100 may be preferably substituted with an amino acid (Thr, Ser, or Tyr) having a hydroxyl group in the side chain, and more preferably with Thr. Specific examples of the mutant yggB gene having such a mutation include the yggB gene (19 mutation) in which “G” at position 298 in SEQ ID NO: 8 is replaced with “A”.

(2-3) Mutation in the fifth transmembrane region Examples of the mutation in the fifth transmembrane region include a mutation that substitutes the alanine residue at position 111 of the wild-type YggB protein with another amino acid. The “other amino acid” is not particularly limited as long as it is a natural amino acid other than alanine. “Other amino acids” include Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, His, Pro. The alanine residue at position 111 may be preferably substituted with an amino acid having a hydroxyl group in the side chain (Thr, Ser, or Tyr), more preferably with Thr. Specific examples of the mutant yggB gene having such a mutation include, for example, the yggB gene (L30 mutation) in which “C” at position 332 of SEQ ID NO: 8 is replaced with “T”, and 331 of SEQ ID NO: 12. The yggB gene (type 8 mutation) in which the “G” at the position is replaced with “A”. The nucleotide sequence of the mutant yggB gene (L30 mutation) and the amino acid sequence of the mutant YggB protein encoded by the same gene are shown in SEQ ID NOs: 4 and 5, respectively.

  In the present invention, the “amino acid residue at the X position of the wild-type YggB protein” means an amino acid residue corresponding to the X position in SEQ ID NO: 9, unless otherwise specified. The “X position” in the amino acid sequence means the X position from the N terminal of the amino acid sequence, and the amino acid residue at the N terminal is the first amino acid residue. That is, the position of the amino acid residue indicates a relative position, and the position may be moved back and forth by amino acid deletion, insertion, or addition. For example, “the amino acid residue at position 419 of the wild-type YggB protein” means an amino acid residue corresponding to position 419 in SEQ ID NO: 9, and one amino acid residue on the N-terminal side from position 419 is deleted. The 418th amino acid residue from the N-terminal is “the amino acid residue at position 419 of the wild-type YggB protein”. When one amino acid residue is inserted at the N-terminal side from the 419th position, the 420th amino acid residue from the N-terminal is assumed to be “the 419th amino acid residue of the wild-type YggB protein”.

Which amino acid residue in any amino acid sequence is the “amino acid residue corresponding to position X in SEQ ID NO: 9” can be determined by aligning the arbitrary amino acid sequence with the amino acid sequence of SEQ ID NO: 9 . The alignment can be performed using, for example, known gene analysis software. Specific software includes DNA Solutions from Hitachi Solutions and GENETYX from GENETICS (Elizabeth C. Tyler et al., Computers and Biomedical Research, 24 (1), 72-96, 1991; Barton GJ et al. al., Journal of molecular biology, 198 (2), 327-37. 1987).

The mutant yggB gene can be obtained by modifying the wild-type yggB gene so as to have the above-mentioned “specific mutation”. Modification of DNA can be performed by a known method. Specifically, for example, as a site-specific mutation method for introducing a target mutation into a target site of DNA, a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press) (1989); Carter, P., Meth. In Enzymol., 154, 382 (1987)) and methods using phage (Kramer, W. and Frits, HJ, Meth. In Enzymol., 154, 350 (1987) Kunkel, T.
A. et al., Meth. In Enzymol., 154, 367 (1987)). The mutant yggB gene can also be obtained by chemical synthesis.

  Hereinafter, a technique for modifying bacteria so as to have a mutant yggB gene will be described.

  Modifying a bacterium to have a mutated yggB gene can be achieved by introducing the mutated yggB gene into the bacterium. Further, modification of a bacterium so as to have a mutant yggB gene can also be achieved by introducing a mutation into the yggB gene of the bacterium by natural mutation or mutagen treatment.

  The method for introducing the mutant yggB gene into bacteria is not particularly limited. In the bacterium of the present invention, the mutant yggB gene only needs to be retained so that it can be expressed under the control of a promoter that functions in the bacterium. The promoter may be a host-derived promoter or a heterologous promoter. The promoter may be a native promoter of the yggB gene or a promoter of another gene. For example, the promoter of the coryneform bacterium yggB gene may be used. Specific examples of promoters that function in Escherichia bacteria such as Escherichia coli include, for example, T7 promoter, trp promoter, trc promoter, lac promoter, tac promoter, tet promoter, araBAD promoter, PR promoter, and PL promoter. It is done. Specific examples of promoters that function in Bacillus bacteria such as Bacillus subtilis include the spac promoter, xyl promoter, glv promoter, P43 promoter, and P2 promoter. Moreover, as a promoter, you may acquire and utilize the highly active type of a conventional promoter by using various reporter genes. For example, the promoter activity can be increased by bringing the -35 and -10 regions in the promoter region closer to the consensus sequence (WO 00/18935). Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)). In the bacterium of the present invention, the mutant yggB gene may be present on a vector that autonomously propagates outside the chromosome, such as a plasmid, or may be integrated on the chromosome. The bacterium of the present invention may have only one copy of the mutant yggB gene, or may have two or more copies. The bacterium of the present invention may have only one type of mutant yggB gene, or may have two or more types of mutant yggB genes. The introduction of the mutant yggB gene can be performed, for example, in the same manner as the above-described method for increasing the copy number of the gene.

  The bacterium of the present invention may or may not have a wild type yggB gene. The yggB gene is also called, for example, the mscS gene in Escherichia coli and the yfkC gene in Bacillus subtilis.

Bacteria that do not have a wild-type yggB gene can be obtained by disrupting the wild-type yggB gene on the chromosome. The destruction of the wild-type yggB gene can be performed, for example, by a technique for destroying the above-described gene. Specifically, for example, the wild-type yggB gene can be destroyed by deleting part or all of the coding region of the wild-type yggB gene.

  Further, by replacing the wild-type yggB gene on the chromosome with the mutant yggB gene, a bacterium that does not have the wild-type yggB gene and is modified to have the mutant yggB gene can be obtained. As a method for performing such gene replacement, for example, a method called “Red-driven integration” (Datsenko, K. A, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97 : 6640-6645 (2000)), a combination of Red driven integration method and λ phage-derived excision system (Cho, EH, Gumport, RI, Gardner, JFJ Bacteriol. 184: 5200-5203 (2002)) Method using linear DNA, such as a method using a plasmid containing a temperature-sensitive replication origin, a method using a plasmid capable of conjugation transfer, or a suicide vector that does not have a replication origin and functions in the host. (US Pat. No. 6,303,383, Japanese Patent Laid-Open No. 05-007491).

<2> Method for Producing Purine Substances Using the bacteria of the present invention, a purine substance can be produced by fermentation. That is, the present invention provides a method for producing a purine material, comprising culturing the bacterium of the present invention in a medium, accumulating the purine substance in the medium, and recovering the purine substance from the medium. In the present invention, one type of purine-based material may be manufactured, or two or more types of purine-based materials may be manufactured. In the present invention, for example, one or more purine nucleosides may be produced. In the present invention, for example, one or more purine nucleotides may be produced.

  The medium to be used is not particularly limited as long as the bacterium of the present invention can grow and the target purine substance is produced. As the medium, for example, a normal medium used for bacterial culture can be used. As the medium, for example, a medium containing a carbon source, a nitrogen source, a phosphate source, a sulfur source, and other components selected from various organic components and inorganic components as necessary can be used. The type and concentration of the medium component may be appropriately set according to various conditions such as the type of bacteria to be used and the type of purine substance to be produced.

  Specific examples of carbon sources include glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, waste molasses, starch hydrolysate, biomass hydrolyzate, and other sugars, acetic acid, fumaric acid, citric acid, Examples thereof include organic acids such as succinic acid, alcohols such as glycerol and ethanol, and fatty acids. As the carbon source, one type of carbon source may be used, or two or more types of carbon sources may be used in combination.

  Specific examples of the nitrogen source include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen sources such as peptone, yeast extract, meat extract, and soybean protein degradation product, ammonia, and urea. Ammonia gas or ammonia water used for pH adjustment may be used as a nitrogen source. As the nitrogen source, one kind of nitrogen source may be used, or two or more kinds of nitrogen sources may be used in combination.

  Specific examples of the phosphoric acid source include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphoric acid. As the phosphoric acid source, one type of phosphoric acid source may be used, or two or more types of phosphoric acid sources may be used in combination.

Specific examples of the sulfur source include inorganic sulfur compounds such as sulfate, thiosulfate, and sulfite, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. As the sulfur source, one kind of sulfur source may be used, or two or more kinds of sulfur sources may be used in combination.

  Other various organic and inorganic components include, for example, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium and calcium; vitamin B1, vitamin B2, vitamin B6 and nicotine Examples include vitamins such as acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components such as peptone, casamino acid, yeast extract, and soybean protein degradation products containing these. As other various organic components and inorganic components, one component may be used, or two or more components may be used in combination.

  In addition, when an auxotrophic mutant strain that requires a nucleic acid or the like for growth is used, it is preferable to supplement nutrients required for the medium. For example, adenine or an organic component containing the same can be used for culturing an adenine-requiring mutant such as a succinyl-AMP synthase gene (purA) -deficient strain.

  The culture conditions are not particularly limited as long as the bacterium of the present invention can grow and the desired purine substance is produced. The culture can be performed, for example, under ordinary conditions used for bacterial culture. The culture conditions may be appropriately set according to various conditions such as the type of bacteria to be used and the type of purine substance to be produced.

  Culturing can be performed aerobically using a liquid medium. Specifically, the culture can be performed by aeration culture or shaking culture. The culture temperature may be, for example, 20 to 45 ° C, preferably 25 to 37 ° C. The pH of the medium may be adjusted to 5 to 8, for example. For pH adjustment, an inorganic or organic acidic or alkaline substance, ammonia gas, or the like can be used. The culture period may be, for example, 15 hours to 90 hours. The culture can be performed by batch culture, fed-batch culture, continuous culture, or a combination thereof. Moreover, culture | cultivation may be performed by dividing into seed culture and main culture. In that case, the culture conditions of the seed culture and the main culture may or may not be the same. For example, both seed culture and main culture may be performed by batch culture. Further, for example, seed culture may be performed by batch culture, and main culture may be performed by fed-batch culture or continuous culture. By culturing the bacterium of the present invention under such conditions, purine substances accumulate in the medium.

  The generation of the purine substance can be confirmed by a known method used for detection or identification of the compound. Examples of such a method include HPLC, LC / MS, GC / MS, and NMR. These methods can be used in appropriate combination.

  The produced purine-based material can be recovered by a known method used for separation and purification of compounds. Examples of such a method include an ion exchange resin method, a membrane treatment method, a precipitation method, and a crystallization method. These methods can be used in appropriate combination. The recovered purine-based material may contain components such as bacterial cells, medium components, moisture, and bacterial metabolic byproducts in addition to the purine-based material. The purine-based material may be purified to a desired degree. Purine substances have a purity of, for example, 30% (w / w) or higher, 50% (w / w) or higher, 70% (w / w) or higher, 80% (w / w) or higher, 90% (w / w) w) or more, or 95% (w / w) or more.

<3> Method for Producing Purine Nucleotide When purine nucleoside is produced by the bacterium of the present invention, the purine nucleotide can be produced using the purine nucleoside. That is, the present invention comprises culturing the bacterium of the present invention having purine nucleoside-producing ability in a medium, accumulating the purine nucleoside in the medium, phosphorylating the purine nucleoside to produce a purine nucleotide, and recovering the purine nucleotide To provide a method for producing purine nucleotides.

  Purine nucleotides include 5'-phosphate esters of purine nucleosides. Examples of 5'-phosphate esters of purine nucleosides include inosinic acid (inosine-5'-phosphate ester; IMP), guanylic acid (guanosine-5'-phosphate ester; GMP), xanthylic acid (xanthosine-5'-). Phosphate ester; XMP), and adenylic acid (adenosine-5′-phosphate ester; AMP). In this method, purine nucleotides corresponding to the purine nucleoside used are generated. That is, for example, inosinic acid can be produced from inosine, guanylic acid from guanosine, xanthylic acid from xanthosine, and adenylic acid from adenosine. In the present invention, one kind of purine nucleotide may be produced, or two or more kinds of purine nucleotides may be produced.

  Purine nucleoside may be subjected to phosphorylation while contained in the medium, or may be recovered from the medium and then subjected to phosphorylation. The purine nucleoside may be subjected to phosphorylation after appropriate pretreatment. Examples of the pretreatment include purification, dilution, concentration, crystallization, drying, crushing, and dissolution. These pretreatments may be appropriately combined. For example, a culture solution containing purine nucleoside may be subjected to phosphorylation as it is or after being purified to a desired degree.

  The technique for phosphorylating purine nucleosides is not particularly limited. Phosphorylation can be performed, for example, by a known method.

Phosphorylation can be performed chemically, for example. Chemical phosphorylation can be performed using a phosphorylating agent such as phosphoryl chloride (POCl ₃ ) (Yoshikawa et. Al., Studies of
phosphorylation, III, Selective phosphorylation of unprotected nucleosides, Bull. Chem. Soc. Jpn., 1969, 42: 3505-3508).

  Phosphorylation can be performed using, for example, microorganisms or enzymes. That is, purine nucleotides can be produced by allowing a microorganism having the ability to produce nucleoside-5′-phosphate esters to act on purine nucleosides and phosphate donors (Japanese Patent Laid-Open No. 07-231793). In addition, purine nucleotides can be generated by causing a phosphorylating enzyme to act on purine nucleosides and phosphate donors.

Specific examples of microorganisms having the ability to produce nucleoside-5′-phosphate esters include the following (Japanese Patent Laid-Open No. 07-231793).
Escherichia blattae JCM 1650
Serratia ficaria ATCC 33105
Klebsiella planticola IFO 14939 (ATCC 33531)
Klebsiella pneumoniae IFO 3318 (ATCC 8724)
Klebsiella terrigena IFO 14941 (ATCC 33257)
Morganella morganii IFO 3168
Enterobacter aerogenes IFO 12010
Enterobacter aerogenes IFO 13534 (ATCC 13048)
Chromobacterium fluviatile IAM 13652
Chromobacterium violaceum IFO 12614
Cedecea lapagei JCM 1684
Cedecea davisiae JCM 1685
Cedecea neteri JCM 5909

  Examples of the phosphorylase include phosphatase, nucleoside kinase, and nucleoside phosphotransferase. The phosphorylase may or may not be purified. For example, phosphorylation of a culture of a microorganism producing phosphorylation enzyme, a culture supernatant separated from the culture, a cell separated from the culture, a treated product of the microorganism, a partially purified product thereof, etc. A fraction containing an enzyme may be used as a phosphorylating enzyme.

  Examples of the nucleoside kinase include inosine anosine kinase. As a method of using inosine anosine kinase, specifically, for example, a method for producing purine nucleotides using a bacterium belonging to the genus Escherichia into which a gene encoding inosine anosine kinase of Escherichia coli has been introduced (WO91 / 08286), And a method for producing purine nucleotides using Corynebacterium ammoniagenes into which a gene encoding inosine anosine kinase of Um acetylicum has been introduced (WO96 / 30501).

  Examples of the phosphatase include acid phosphatase. Examples of the acid phosphatase include those disclosed in JP-A-2002-000289. Preferred acid phosphatases include, for example, mutant acid phosphatases with increased affinity for nucleosides (Japanese Patent Laid-Open No. 10-201481), mutant acid phosphatases with decreased nucleotidase activity (WO96 / 37603), phosphate hydrolysis A mutant acid phosphatase having a reduced activity (JP 2001-245676 A) can be mentioned.

  Examples of the phosphoric acid donor include polyphosphoric acid, phenyl phosphoric acid, acetyl phosphoric acid, carbamyl phosphoric acid, ATP, and dATP (deoxy ATP). Examples of polyphosphoric acid include pyrophosphoric acid, tripolyphosphoric acid, trimetaphosphoric acid, tetrametaphosphoric acid, and hexametaphosphoric acid. Any phosphoric acid donor may be a free form or a salt thereof, or a mixture thereof. Examples of the salt include sodium salt and potassium salt. The phosphate donor can be appropriately selected according to the type of microorganism used, the phosphorylating enzyme, and the like. Further, when ATP or dATP is used as a phosphate donor, these regeneration systems can be used in combination (WO91 / 08286, WO96 / 30501).

  The generation of a purine nucleotide can be confirmed by a known method used for detection or identification of a compound. Examples of such a method include HPLC, LC / MS, GC / MS, and NMR. These methods can be used in appropriate combination.

  The generated purine nucleotide can be recovered by a known method used for separation and purification of compounds. Examples of such a method include an ion exchange resin method, a membrane treatment method, a precipitation method, and a crystallization method. These methods can be used in appropriate combination. In addition to purine nucleotides, the recovered product may contain components such as a phosphorylating enzyme, a phosphate donor, bacterial cells, medium components, moisture, and bacterial metabolic byproducts. Purine nucleotides may be purified to the desired extent. Purine nucleotide purity is, for example, 30% (w / w) or higher, 50% (w / w) or higher, 70% (w / w) or higher, 80% (w / w) or higher, 90% (w / w) ) Or more, or 95% (w / w) or more.

  Hereinafter, the present invention will be described more specifically with reference to examples.

Example: Inosine production using a mutant yggB gene-introduced strain In this example, inosine production is carried out using an inosine-producing strain of E. coli introduced with a mutant yggB gene, and the mutant yggB gene gives inosine production. The impact was evaluated.

<1> Construction of E. coli inosine production strain FADRaddedd / pMWpurFKQ
An inosine production strain was constructed using the E. coli FADRaddedd strain (WO99 / 003988) as a parent strain. FADRaddedd strains are from E. coli W3110 strain, purF (amidophosphoribosyltransferase), purA (adenylosuccinate synthetase), deoD (purine nucleoside phosphorylase), purR (purine synthesis repressor), add (adenosine deaminase), It is a strain obtained by disrupting the edd (phosphogluconate dehydratase) gene. By introducing and expressing a mutant purF gene encoding a desensitized amidophosphoribosyltransferase in which feedback inhibition has been released into FADRaddedd strain, it is possible to construct a strain that produces inosine with high efficiency (WO99 / 003988). Therefore, a plasmid pMWpurFKQ (described in European Patent Publication No. 1170370) expressing a mutant purF gene was introduced into the FADRaddedd strain by electroporation (Canadian Journal of Microbiology, 43. 197 (1997)). One of the obtained transformants was designated as FADRaddedd / pMWpurFKQ.

<2> Construction of mutant yggB gene expression plasmid Plasmids pSTV-yggB (BL2) and pSTV-yggB (BL3) for expressing the mutant yggB gene of coryneform bacteria in Escherichia bacteria are as follows. It was constructed.

Chromosomal DNA from strains having L30 type mutation in yggB gene and strain having A1 type mutation in yggB gene (JP 2007-97573) constructed from Brevibacterium lactofermentum strain 2256 (Corynebacterium glutamicum ATCC13869) Extracted. PCR method using modified chromosomal DNA as a template and primers shown in SEQ ID NO: 1 (primer A; 5'-gctaagcttctaaggaggtcttggctcatg-3 ') and SEQ ID NO: 2 (primer B; 5'-gtaggatcctgggacacgtctgtaatcagc-3') ℃ -10 seconds, annealing 60 ℃ -15 seconds, extension reaction 68 ℃ -78 seconds, 30 cycles, TaKaRa PCR Thermal cycler PERSONAL (Takara Bio Inc.)) About 1.6 kb DNA fragment containing each mutant yggB gene Was amplified. For the PCR reaction, GXL polymerase (manufactured by Takara Bio Inc.) was used. The primers were designed to amplify a region containing a ribosome binding sequence (SD sequence) upstream from the translation start codon of the mutant yggB gene. The base sequence of the amplified fragment containing the yggB gene and SD sequence obtained by PCR using the same primers is shown in SEQ ID NO: 3 (however, the yggB gene shown in the same sequence is a wild type). After each amplified fragment was purified, restriction enzyme sites formed at both ends were digested with Bam HI and Hind III. Using the DNA Ligation Kit ver.2.1 (manufactured by Takara Bio Inc.), the digested amplified fragments and the fragments of about 3.0 kb pSTV29 vector (Takara Bio Inc.) digested with the restriction enzymes Bam HI and Hind III are used. Connected. Escherichia coli (E. coli JM109 competent cells, manufactured by Takara Bio Inc.) was transformed with this ligation reaction solution, applied to an LB agar medium containing 50 mg / L chloramphenicol, and incubated at 37 ° C overnight. did. Colonies that appeared on the agar medium were inoculated into an LB liquid medium containing 50 mg / L chloramphenicol and cultured overnight at 37 ° C. with shaking. Plasmid DNA was extracted from each culture solution by the alkali-SDS method. The plasmid structure was confirmed by restriction enzyme digestion and nucleotide sequence determination to obtain pSTV-yggB (BL2) and pSTV-yggB (BL3). pSTV-yggB (BL2) is an expression plasmid for a mutant yggB gene having an L30 mutation, and pSTV-yggB (BL3) is an expression plasmid for a mutant yggB gene having an A1 mutation. The mutant yggB gene (L30 mutation) is a yggB gene in which “C” at position 332 in SEQ ID NO: 8 is replaced with “T”, and the alanine residue at position 111 in SEQ ID NO: 9 is replaced with valine. Encodes YggB protein. The nucleotide sequence of the mutant yggB gene (L30 mutation) and the amino acid sequence of the mutant YggB protein encoded by the same gene are shown in SEQ ID NOs: 4 and 5, respectively. The mutant yggB gene (A1 type mutation) is a yggB gene in which TTCATTGTG is inserted after “G” at position 44 in SEQ ID NO: 8, and the leucine residue at position 14 and the tryptophan residue at position 15 in SEQ ID NO: 9. It encodes the YggB protein with Cys-Ser-Leu inserted between the base. The nucleotide sequence of the mutant yggB gene (A1 mutation) and the amino acid sequence of the mutant YggB protein encoded by the same gene are shown in SEQ ID NOs: 6 and 7, respectively. In any plasmid, the same gene is arranged such that the transcription direction of the mutant yggB gene is the same as the transcription direction of the lac promoter of the pSTV29 vector.

<3> Introduction of pSTV-yggB (BL2) and pSTV-yggB (BL3) into E. coli inosine-producing strain FADRaddedd / pMWpurFKQ pSTV-yggB (BL2) and pSTV-yggB (BL3 ), And pSTV29 vector as a control were introduced into E. coli inosine producing strain FADRaddedd / pMWpurFKQ by 2 × TSS method (Proc. Natl. Acad. Sci. USA, 86, 2172 (1989)), respectively. One of the obtained transformants was designated as FADRaddedd / pMWpurFKQ / pSTV-yggB (BL2), FADRaddedd / pMWpurFKQ / pSTV-yggB (BL3), FADRaddedd / pMWpurFKQ / pSTV29.

<4> Inosine production Each of the strains obtained as described above was added to an LB medium containing antibiotics (tryptone 10 g / L, yeast extract 10 g / L, NaCl 10 g / L, kanamycin 25 mg / L, (Chloramphenicol 50 mg / L) was evenly spread on the plate and cultured at 37 ° C. overnight. 1/6 plate cells were inoculated into 20 mL of fermentation medium in a 500 mL Sakaguchi flask and cultured at 37 ° C. for 24 hours (seed culture). A part of the seed culture was added to 20 mL of fresh fermentation medium so that the absorbance at a wavelength of 660 nm was 1.0, and main culture was performed at 37 ° C. Sampling was performed every 3 hours up to 24 hours after the start of main culture, and the amount of residual sugar, inosine and hypoxanthine contained in the culture solution were measured by a known method. The results are shown in Table 1. A significant increase in inosine production was observed in the pSTV-yggB (BL2) -introduced strain and the pSTV-yggB (BL3) -introduced strain as compared with the control pSTV29 vector-bearing strain.

[Fermentation medium composition]
Glucose 40 g / L
(NH ₄ ) ₂ SO ₄ 16 g / L
KH ₂ PO ₄ 1 g / L
MgSO ₄・ 7H ₂ O 1 g / L
FeSO ₄・ 7H ₂ O 0.01 g / L
MnSO ₄・ 5H ₂ O 0.01 g / L
Yeast extract 8 g / L
Kanamycin 25 mg / L
Chloramphenicol 50 mg / L
Isopropyl-β-D-galactopyranoside 0.1 M
Calcium carbonate 30 g / L

[Explanation of Sequence Listing]
SEQ ID NOs: 1, 2: Primer SEQ ID NO: 3: Base sequence of fragment containing yggB gene and SD sequence SEQ ID NO: 4: Base sequence of mutant yggB gene (BL2) of Corynebacterium glutamicum 2256 (ATCC13869) SEQ ID NO: 5: Corynebacterium glutamicum 2256 The amino acid sequence of the protein encoded by the mutant yggB gene (BL2) of (ATCC13869) SEQ ID NO: 6: The nucleotide sequence of the mutant yggB gene (BL3) of Corynebacterium glutamicum 2256 (ATCC13869) SEQ ID NO: 7: Corynebacterium glutamicum 2256 (ATCC13869) The amino acid sequence of the protein encoded by the mutant yggB gene (BL3) SEQ ID NO: 8: The nucleotide sequence of the yggB gene of Corynebacterium glutamicum 2256 (ATCC13869) SEQ ID NO: 9: The amino acid sequence of the YggB protein of Corynebacterium glutamicum 2256 (ATCC13869) Nucleotide sequence of yggB gene of Corynebacterium glutamicum ATCC13032 SEQ ID NO: 11: YggB tag of Corynebacterium glutamicum ATCC13032 Protein amino acid sequence SEQ ID NO: 12: nucleotide sequence of yggB gene of Corynebacterium glutamicum ATCC14967 SEQ ID NO: 13: amino acid sequence sequence of YggB protein of Corynebacterium glutamicum ATCC14967 SEQ ID NO: 14: nucleotide sequence sequence of yggB gene of Corynebacterium melassecola ATCC17965: Corynebacterium melassecola Amino acid sequence of YggB protein of ATCC17965 SEQ ID NO: 16: Conserved sequence of wild-type YggB protein of coryneform bacterium

Claims (20)

A bacterium having the ability to produce purine substances,
Escherichia bacteria, Bacillus bacteria, or Corynebacterium ammoniagenes,
Modified to retain the mutant yggB gene,
The bacterium, wherein the mutant yggB gene is a yggB gene having a mutation that improves the ability of the bacterium to produce purine substances. The bacterium according to claim 1, wherein the mutation is selected from the group consisting of the following (1) to (3):
(1) a mutation in the region encoding the amino acid residues at positions 419 to 533 of the wild type YggB protein;
(2) mutations in the region encoding the transmembrane region of wild-type YggB protein; and (3) combinations thereof.   The bacterium according to claim 2, wherein the mutation (1) is an insertion of a base sequence into a region encoding the amino acid residues at positions 419 to 533 of the wild type YggB protein.   The bacterium according to claim 2, wherein the mutation (1) is a mutation that substitutes another amino acid for a proline residue present at positions 419 to 533 of the wild-type YggB protein.   The transmembrane region is selected from amino acid residues at positions 1 to 23, 25 to 47, 62 to 84, 86 to 108, and 110 to 132 of the wild type YggB protein. The bacterium according to any one of the above.   The bacterium according to any one of claims 2 to 5, wherein the mutation (2) is not accompanied by a frameshift mutation and a nonsense mutation. The bacterium according to any one of claims 2 to 6, wherein the mutation (2) is selected from the group consisting of the following (2a) to (2d):
(2a) a mutation in which one or several amino acids are inserted between the leucine residue at position 14 and the tryptophan residue at position 15 of the wild-type YggB protein;
(2b) a mutation that substitutes the alanine residue at position 100 of the wild-type YggB protein with another amino acid;
(2c) a mutation that replaces the alanine residue at position 111 of the wild-type YggB protein with another amino acid; and (2d) a combination thereof.   The bacterium according to claim 7, wherein Cys-Ser-Leu is inserted between the 14th leucine residue and the 15th tryptophan residue.   The bacterium according to claim 7 or 8, wherein the alanine residue at position 100 and / or position 111 is substituted with threonine. The bacterium according to any one of claims 1 to 9, wherein the wild-type YggB protein is a protein described in (A) or (B) below:
(A) a protein having the amino acid sequence shown in SEQ ID NO: 9, 11, 13, or 15,
(B) having an amino acid sequence including substitution, deletion, insertion, or addition of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 9, 11, 13, or 15;
A protein that functions as a mechanosensitive channel.   The bacterium according to any one of claims 1 to 10, which has been modified to increase the activity of an enzyme involved in the biosynthesis of purine substances.   The bacterium according to any one of claims 1 to 11, wherein the purine substance is selected from the group consisting of inosine, xanthosine, guanosine, and adenosine.   The bacterium according to any one of claims 1 to 11, wherein the purine substance is selected from the group consisting of inosinic acid, xanthylic acid, and guanylic acid.   The bacterium according to any one of claims 1 to 13, which is Escherichia coli.   The bacterium according to any one of claims 1 to 13, which is Bacillus subtilis or Bacillus amyloliquefaciens.   The bacterium according to any one of claims 1 to 13, which is Corynebacterium ammoniagenes.   A method for producing a purine-based substance, comprising culturing the bacterium according to any one of claims 1 to 16 in a medium, accumulating the purine-based substance in the medium, and recovering the purine-based substance from the medium. .   A method for producing a purine nucleoside comprising culturing the bacterium according to any one of claims 1 to 16 in a medium, accumulating purine nucleosides in the medium, and recovering the purine nucleosides from the medium.   A method for producing a purine nucleotide, comprising culturing the bacterium according to any one of claims 1 to 16 in a medium, accumulating the purine nucleotide in the medium, and recovering the purine nucleotide from the medium.   Culturing the bacterium according to any one of claims 1 to 16 in a medium and accumulating a purine nucleoside in the medium, phosphorylating the purine nucleoside to produce a purine nucleotide, and recovering the purine nucleotide And a method for producing a purine nucleotide.