CN104342397B - The preparation method of purine substance - Google Patents

Abstract

A kind of preparation method of purine substance is provided, it includes keeping mutant-type yggB gene by cultivating in the medium through modification, so as to Escherichia bacteria, bacillus or corynebacterium ammoniagenes with purine substance production capacity, and purine substance is recycled from the culture medium, prepares purine substance.

Description

The preparation method of purine substance

Technical field

The present invention relates to the fermentation method for producing for the purine substance for having used bacterium.Purine substance as flavouring or The component of pharmaceuticals etc. or its raw material are industrially useful.

Background technology

The purine substance such as purine nucleosides or purine nucleotides, usually by using adenine auxotrophic strains Fermentation carries out industrial production.Resistance of this kind of bacterial strain to reagents such as purine analogue or sulphaguanidines can also further be assigned.Make For the bacterial strain for fermenting and producing purine nucleosides, it is known to for example, belonging to bacterial strain (the patent text of bacillus (Bacillus) category Offer 1-8), belong to bacillus brevis (Brevibacterium) (corynebacteria (Corynebacterium)) category bacterial strain (patent Document 9-10, non-patent literature 1) and belong to Escherichia (Escherichia) category bacterial strain (patent document 11).

The mutant strain such as auxotrophic strain or drug resistance bacterial strain, can typically pass through UV irradiations or N- methyl-N'- Nitro-N nitrosoguanidine (MNNG) processing carrys out Mutation induction, is selected using appropriate Selective agar medium with expected phenotype Mutant strain and obtain.

In addition it is also possible to using technique for gene engineering, the breeding of the bacterial strain of production purine nucleosides is carried out.For example, for bud Spore Bacillus bacteria (patent document 12-21), bacillus brevis (corynebacteria) belong to bacterium (patent document 22) and Escherichia Belong to bacterium (patent document 11), can assign or improve the production capacity of purine nucleosides by technique for gene engineering.Specifically, The enzyme activity in the cell of the biosynthesis of purine nucleosides is participated in for example, by enhancing, reduces and is closed from the biology of purine nucleosides Enzymatic activity in the synthetic reaction of other compounds come out into forehearth limb as catalyst, can assign or improve purine core The production capacity (patent document 11) of glycosides.

The yggB genes of bar shaped bacteria are the homologues (non-patent literature 2,3) of the yggB genes of Escherichia coli, encode power Sensitive channel (mechanosensitive channel) (non-patent literature 4,5).In bar shaped bacteria, it is known that can pass through Increase the expression of yggB genes or keep the mutant-type yggB gene with specific mutation to improve the production capacity of glutamic acid (specially Sharp document 23).

However, yggB genes and the relation for producing the purine substance such as purine nucleosides or purine nucleotides are unknown.

Prior art literature

Patent document

【Patent document 1】Japanese Patent Publication 38-23039 publications (1963)

【Patent document 2】Japanese Patent Publication 54-17033 publications (1979)

【Patent document 3】Japanese Patent Publication 55-2956 publications (1980)

【Patent document 4】Japanese Patent Publication 55-45199 publications (1980)

【Patent document 5】Japanese Unexamined Patent Application 56-162998 publications (1981)

【Patent document 6】Japanese Patent Publication 57-14160 publications (1982)

【Patent document 7】Japanese Patent Publication 57-41915 publications (1982)

【Patent document 8】Japanese Unexamined Patent Application 59-42895 publications (1984)

【Patent document 9】Japanese Patent Publication 51-5075 publications (1976)

【Patent document 10】Japanese Patent Publication 58-17592 publications (1972)

【Patent document 11】No. 99/03988 volume of International Publication No.

【Patent document 12】Japanese Unexamined Patent Application 58-158197 publications (1983)

【Patent document 13】Japanese Unexamined Patent Application 58-175493 publications (1983)

【Patent document 14】Japanese Unexamined Patent Application 59-28470 publications (1984)

【Patent document 15】Japanese Unexamined Patent Application 60-156388 publications (1985)

【Patent document 16】Japanese Unexamined Patent Publication 1-27477 publications (1989)

【Patent document 17】Japanese Unexamined Patent Publication 1-174385 publications (1989)

【Patent document 18】Japanese Unexamined Patent Publication 3-58787 publications (1991)

【Patent document 19】Japanese Unexamined Patent Publication 3-164185 publications (1991)

【Patent document 20】Japanese Unexamined Patent Publication 5-84067 publications (1993)

【Patent document 21】Japanese Unexamined Patent Publication 5-192164 publications (1993)

【Patent document 22】Japanese Unexamined Patent Application 63-248394 (1988)

【Patent document 23】Japanese Unexamined Patent Publication 2007-097573

Non-patent literature

【Non-patent literature 1】Agric.Biol.Chem.,42,399(1978)

【Non-patent literature 2】FEMS Microbiol Lett.2003Jan28；218(2):305-9

【Non-patent literature 3】Mol Microbiol.2004Oct；54(2):420-38.

【Non-patent literature 4】EMBO J.1999,18(7):1730-7

【Non-patent literature 5】Biosci.Biotechnol.Biochem.,74(12),2546-2549,2010

The content of the invention

Problems to be solved by the invention

The problem to be solved in the present invention is the new technology that exploitation improves bacterium production purine substance ability, there is provided effective The method for preparing purine substance.

Solution to the problem

The present inventor to solve the above-mentioned problems, conducts in-depth research, it turns out that making it by modified bacteria The anomaly yggB genes with specific mutation are kept, the ability of bacterium production purine substance can be improved, so as to complete this Invention.

That is, the present invention can be exemplified as follows.

[1] a kind of bacterium with purine substance production capacity,

It is Escherichia (Escherichia) bacterium, bacillus (Bacillus) bacterium or corynebacterium ammoniagenes (Corynebacterium ammoniagenes), and

It has been modified so as to keep mutant-type yggB gene,

Wherein, the mutant-type yggB gene is the yggB genes with mutation, described to be mutated the purine for making above-mentioned bacterium The production capacity of class material is improved.

[2] above-mentioned bacterium, the mutation are selected from following (1)~(3)：

(1) mutation in the region of the amino acid residue of the 419th~533 of encoding wild type YggB protein；

(2) mutation in the region of the transmembrane region of encoding wild type YggB protein；With

(3) combination of above-mentioned mutation.

[3] above-mentioned bacterium, wherein, the mutation (1) is the 419th~533 to encoding wild type YggB protein Base sequence is inserted into the region of amino acid residue.

[4] above-mentioned bacterium, wherein, the mutation (1) is will be present in wild type YggB protein the 419th~533 Proline residue replace with the mutation of other amino acid.

[5] above-mentioned bacterium, wherein, the transmembrane region be selected from the 1st~23 of wild type YggB protein, the 25th~47 Position, the 62nd~84, the amino acid residue of the 86th~108 and the 110th~132.

[6] above-mentioned bacterium, wherein, the mutation (2) is not accompanied by frameshift mutation and nonsense mutation.

[7] above-mentioned bacterium, wherein, the mutation (2) is selected from following (2a)~(2d)：

(2a) is inserted into one between the 14th leucine residue and the 15th trp residue of wild type YggB protein A or more amino acid mutation；

100th alanine residue of wild type YggB protein is replaced with the prominent of other amino acid residues by (2b) Become；

111st alanine residue of wild type YggB protein is replaced with the prominent of other amino acid residues by (2c) Become；With

The combination of (2d) above-mentioned mutation.

[8] above-mentioned bacterium, wherein, it is inserted between the 14th leucine residue and the 15th trp residue Cys-Ser-Leu.

[9] above-mentioned bacterium, wherein, it is residual that the alanine residue of described 100th and/or the 111st are replaced with into threonine Base.

[10] above-mentioned bacterium, wherein, the wild type YggB protein is the protein described in following (A) or (B)：

(A) there is SEQ ID NO:The protein of amino acid sequence shown in 9,11,13 or 15,

(B) have in SEQ ID NO:In amino acid sequence shown in 9,11,13 or 15, comprising replace, missing, insertion or The amino acid sequence of the one or more amino acid residues of addition, and the protein to play a role as power sensitiveness passage.

[11] above-mentioned bacterium, it has been modified so that participating in the activity increase of the enzyme of purine substance biosynthesis.

[12] above-mentioned bacterium, wherein the purine substance is selected from inosine, xanthosine, guanosine and adenosine.

[13] above-mentioned bacterium, wherein the purine substance is selected from inosinicacid, xanthosine monophosphate and guanylic acid.

[14] above-mentioned bacterium is Escherichia coli.

[15] above-mentioned bacterium, it is bacillus subtilis (Bacillus subtilis) or bacillus amyloliquefaciens (Bacillus amyloliquefaciens)。

[16] above-mentioned bacterium is corynebacterium ammoniagenes.

[17] a kind of method for preparing purine substance, it includes cultivating above-mentioned bacterium in the medium, so as to train Support in base and accumulate purine substance, and the purine substance is recycled from the culture medium.

[18] a kind of method for preparing purine nucleosides, it includes cultivating above-mentioned bacterium in the medium, so as to cultivate Purine nucleosides is accumulated in base, and the purine nucleosides is recycled from the culture medium.

[19] a kind of method for preparing purine nucleotides, it includes cultivating above-mentioned bacterium in the medium, so as to train Support in base and accumulate purine nucleotides, and the purine nucleotides is recycled from the culture medium.

[20] a kind of preparation method of purine nucleotides, it includes cultivating above-mentioned bacterium in the medium, so as to cultivate Purine nucleosides is accumulated in base, phosphorylation generation purine nucleotides, and the recycling purine nucleosides are carried out to the purine nucleosides Acid.

The effect of invention

By the invention it is possible to improve the production capacity of the purine substance of bacterium, purines thing can be efficiently prepared Matter.

Embodiment

The more detailed description below present invention.

The bacterium of the ＜ 1 ＞ present invention

The bacterium of the present invention has been modified and has kept the mutant-type yggB gene with " specific mutation ", so that with fast Purine class physical capacity." specific mutation " is as described below.

＜ 1-1 ＞ have the bacterium of purine substance production capacity

In the present invention, when " bacterium with purine substance production capacity " refers to use medium culture, there is production Target product purine substance and the bacterium for running up to the ability of recyclable degree in the medium.Given birth to purine substance The bacterium of production capacity power can accumulate the thin of further amounts of target product purine substance in the medium than not mutated strain Bacterium." not mutated strain " refers to the control strain for not holding mutant-type yggB gene, for example, it may be wild strain or parental strain. In addition, the bacterium with purine substance production capacity can also be can accumulate in the medium such as more than 0.1mM, The bacterium of the target purine class material of the amount of more than 0.5mM, more than 1mM or more than 2mM.

Purine nucleosides and purine nucleotides can be enumerated as purine substance.As purine nucleosides can enumerate inosine, guanosine, Xanthosine and adenosine.5 '-phosphate of purine nucleosides can be enumerated as purine nucleotides.5 '-phosphate as purine nucleosides can Enumerate inosinicacid (inosine -5 '-phosphate；IMP), guanylic acid (guanosine -5 '-phosphate；GMP), xanthosine monophosphate (xanthosine- 5 '-phosphate；) and adenylate (5'-AMP ester XMP；AMP).The bacterium of the present invention, which can have, produces a kind of purine Class physical capacity, it is possible to have the ability of 2 kinds of production or a variety of purine substances.The bacterium of the present invention can have for example raw Produce the ability of one or more purine nucleosides.The bacterium of the present invention can have the energy for for example producing one or more purine nucleotides Power.

The present invention bacterium be Escherichia (Escherichia) belong to bacterium, bacillus (Bacillus) belong to bacterium or Corynebacterium ammoniagenes (Corynebacterium ammoniagenes).

There is no special limitation as Escherichia bacteria, can enumerate according to classifying known to specialist in microbiology and divide Class is the bacterium in Escherichia.Escherichia bacteria, can enumerate the works such as Neidhardt book (Backmann, B.J.1996.Derivations and Genotypes of some mutant derivatives of Escherichia Coli K-12, the 2460-2488 pages.Table 1, In F.D.Neidhardt (ed.), Escherichia coli and Salmonella:Cellular and Molecular Biology, second edition, American Society for Microbiology Press, Washington, D.C.) it is described.As Escherichia bacteria, such as Escherichia coli can be enumerated (Escherichia coli).Escherichia coli can specifically enumerate for example originating from prototype be wild strain K12 Escherichia coli W3110 (ATCC27325) or Escherichia coli MG1655 (ATCC47076).

There is no special limitation as bacillus, can enumerate according to classifying known to specialist in microbiology and divide Class is the bacterium of bacillus.As bacillus, it can arrange and be exemplified below strain.

Bacillus subtilis (Bacillus subtilis)

Bacillus amyloliquefaciens (Bacillus amyloliquefaciens)

Bacillus pumilus (Bacillus pumilus)

Bacillus licheniformis (Bacillus licheniformis)

Bacillus megatherium (Bacillus megaterium)

Bacillus brevis (Bacillus brevis)

Aerobacillus polymyxa Donker (Bacillus polymixa)

Bacillus stearothermophilus (Bacillus stearothermophilus)

As bacillus subtilis, can specifically enumerate for example bacillus subtilis 168Marburg plants (ATCC6051) or withered (plasmid, 1984,12,1-9) bacillus PY79 plants careless.As bacillus amyloliquefaciens, such as solution starch bud can be specifically enumerated Spore bacillus T plants (ATCC23842) or bacillus amyloliquefaciens N plants (ATCC23845).

There is no special limitation as corynebacterium ammoniagenes, can enumerate according to classifying known to specialist in microbiology and be categorized as The bacterium of corynebacterium ammoniagenes.Corynebacterium ammoniagenes further include the bacterium for being categorized as production ammonia bacillus brevis at present.Corynebacterium ammoniagenes can Specifically enumerate for example following bacterial strain.

Corynebacterium ammoniagenes ATCC-6872

Corynebacterium ammoniagenes adenine leaky mutant (Leaky Mutant) (Agr.Bio.Chem., Vol.47 (5), p1035-1041,1983,(KY13102、KY13171、KY13184))。

These bacterial strains can be by such as American type culture collection (american type culture Collection) (address 12301Parklawn Drive, Rockville, Maryland20852P.O.Box1549, Manassas, VA20108, United States of America) sell.That is, number of registration corresponding with each bacterial strain is given, can To receive to sell (referring to http using the number of registration://www.atcc.org/).Number of registration corresponding with each bacterial strain is recorded In the catalogue of American type culture collection.

The bacterium of the present invention can natively have purine substance production capacity, can also have purines through modification Physical capacity.Bacterium with purine substance production capacity can be obtained as below, i.e., for example assign above-mentioned bacterium purine Class physical capacity or the purine substance production capacity of the above-mentioned bacterium of enhancing.

Can be by producing educating for bacterium currently used for purine substances such as bacillus or Escherichia bacterias Kind of method assigns or strengthens purine substance production capacity.

For example, can by assign the auxotropics such as adenine defective or further assign to purine analogue or The resistance of the medicaments such as sulphaguanidine assigns or strengthens the production capacity of purine substance (referring to Japanese Patent Publication 38-23099, spy Public clear 54-17033, examined patent publication 55-45199, examined patent publication 57-14160, examined patent publication 57-41915, examined patent publication 59-42895, US2004-0166575A).Can be by carrying out mutation processing to parental strain or wild-type strain, with appropriate selection culture Base selects the mutant strain with expected phenotype, obtains the auxotroph strain with purine substance production capacity or the resistance to the action of a drug The mutant strains such as strain.Mutation is handled, and can be enumerated such as x-ray bombardment, ultraviolet irradiation, be utilized N- methyl-N '-nitro-N- nitrous The processing of the mutagens such as base guanidine (MNNG), ethylmethane sulfonate (EMS), methyl methylsulfonate (MMS).

Furthermore, it is possible to the activity of the enzyme of purine substance biosynthesis in the cell is participated in by strengthening to assign or strengthen The production capacity of purine substance.The activity of a kind of enzyme can be strengthened, the activity of 2 kinds or a variety of enzymes can also be strengthened.It is described below Zymosthenic method.Strengthen enzymatic activity, for example, can be modified by strengthening the expression for the gene for encoding same enzyme Bacterium carries out.The method of enhancing gene expression is documented in WO00/18935 volumes or European Patent application discloses No. 1010755 Specification etc..

Purine nucleotides is with phosphoribosylpyrophosphate (phosphoribosylpyrophosphate；PRPP in) being used as Between product and biosynthesis.Purine nucleosides be by by purine nucleotides dephosphorylation and biosynthesis.Participate in this kind of fast The enzyme of purine class material biosynthesis, can enumerate such as PRPP synzyme (PRPP synthetase) (prs) or purine operon institute The protein of coding.It should be noted that it is the abbreviation (also identical in record hereafter) for the gene for encoding above-mentioned enzyme in bracket. It is different according to biological species but Gene Name (abbreviation of gene) is an example, there are Gene Name not sometimes The same and situation without the gene.

As purine operon, the purEKBCSQLFMNHD operators (Bacillus of such as bacillus subtilis can be enumerated subtilis and Its Closest Relatives,Editor in Chief:A.L.Sonenshein,ASM Press, Washington D.C., 2002), or pur regulators (Escherichia the and Salmonella, Second of Escherichia coli Edition,Editor in Chief:F.C.Neidhardt,ASM Press,Washington D.C.,1996).For example, can To concentrate the expression of enhancing purine operon, 1 or more in the gene included in purine operon can also be strengthened The expression of a gene.

Wherein, turn for example, it is preferable to strengthen selected from PRPP synzyme (PRPP synthetase) (prs) and PRPP amide groups Move one or more enzymatic activitys of enzyme (PRPP amidotransferase) (purF).

It should be noted that for example, when the enzyme for the biosynthesis for participating in purine substance is subject to feedback inhibition or expression suppression System can strengthen enzymatic activity by reducing or eliminating the regulation and control, improve the production capacity of purine substance when negative regulation (WO99/003988)。

The expression of purine operon is suppressed by the purine aporepressor of purR gene codes.Therefore, can be for example, by drop The activity of low purine repressor, strengthens the expression (U.S. Patent No. 6,284,495) of purine operon.For example, pass through destruction The purR genes of purine aporepressor are encoded, the activity (U.S. Patent No. 6,284,495) of purine repressor can be reduced.This Outside, the expression of purine operon is subject to terminator-anti-terminator (terminator- positioned at promoter downstream Antiterminator) sequence (also referred to as decay subsequence) containment (Ebbole, D.J.and Zalkin, H., J.Biol.Chem.,1987,262,8274-8287、Ebbole,D.J.and Zalkin,H.,J.Biol.Chem.,1988, 263,10894-10902、Ebbole,D.J.and Zalkin,H.,J.Bacteriol.,1989,171,2136-2141).Cause This, for example, the expression of purine operon can be strengthened by making attenuator sequence deletion.The missing of decay subsequence can lead to The method identical with following destruction genes is crossed to carry out.

Feedback inhibition of the PRPP synzyme by ADP.It may be thus possible, for example, to by making bacterium that there is saltant type PRPP synzyme Gene, it encodes desensitization type PRPP synzyme, it reduces or eliminate the feedback inhibition of ADP generations, to strengthen PRPP synzyme Activity, improves the production capacity (WO99/003988) of purine substance.As desensitization type PRPP synzyme, it can enumerate to have and incite somebody to action 128th Asp (D) of wild type PRPP synzyme replace with the mutation of Ala (A) PRPP synzyme (S.G.Bower etc., J.Biol.Chem.,264,10287(1989))。

Feedback inhibition of the PRPP amide transferases by AMP and GMP.It may be thus possible, for example, to by making bacterium keep mutation Type PRPP amidophosphoric acid phosphoribosynltransferase genes, it encodes desensitization type PRPP amide transferases, it reduces or eliminate AMP And/or the feedback inhibition that GMP is produced, to strengthen PRPP amide transferases activity, improve the production capacity of purine substance (WO99/003988).It can be enumerated with following mutation as desensitization type PRPP amide transferases：By wild type PRPP acid amides phosphorus The Lys (K) of the 326th of sour phosphoribosynltransferase replaces with the mutation of Gln (Q), or by wild type PRPP amide transferases The Lys (K) of the 326th replaces with Gln (Q) and the Pro (P) of the 410th is replaced with to mutation (the G.Zhou et of Trp (W) al.,J.Biol.Chem.,269,6784(1994))。

It is branched off from the biosynthesis pathway of purine substance furthermore, it is possible to reduce and generates the synthesis of other compounds In reaction as catalyst enzyme activity, assign or enhancing purine substance production capacity (WO99/003988).It can drop Low a kind of enzymatic activity, can also reduce by 2 kinds or a variety of enzymatic activitys.It is it should be noted that so-called here " from the life of purine substance Thing synthesis forehearth limb comes out the enzyme generated in the synthetic reactions of other compounds as catalyst " in also include participating in decomposition it is fast The enzyme of purine class material.Being described below reduces the method for enzymatic activity.

It is branched off generating from the biosynthesis pathway of purine substance in the synthetic reactions of other compounds as catalysis The enzyme of agent, can enumerate for example purine nucleoside phosphorylase (purine nucleoside phosphorylase) (deoD, PupG), succinyl-AMP synthase (succinyl-AMP synthase) (purA), adenosine deaminase (adenosine Deaminase) (add), inosine-guanosine kinase (inosine-guanosine kinase) (gsk), GMP reductases (GMP Reductase) (guaC), 6-phosphogluconate dehydrogenase (6-phosphogluconate dehydrase) (edd), phosphoric acid Glucose isomerase (phophoglucose isomerase) (pgi), adenine deaminase (adenine deaminase) (yicP), xanthosine phosphorylase (xanthosine phosphorylase) (xapA), IMP dehydrogenase (guaB).Can basis The selections such as the species of target purine class material make the enzyme that activity reduces.

Furthermore, it is possible to the activity by reducing fructose bisphosphatase (fructose1,6-bisphosphatase) (fbp), Assign or strengthen the production capacity (WO2007/125782) of purine substance.

Furthermore, it is possible to the activity by reducing the protein for participating in absorbing purine substance, assigns or strengthens purines thing The production capacity (WO99/003988) of matter.For example, nucleosides permease can be enumerated as the protein for participating in absorbing purine substance (nucleoside permease)(nupG)(WO99/003988)。

Furthermore, it is possible to the activity by strengthening the protein for participating in secretion purine substance, assigns or strengthens purines thing The production capacity of matter.For example, rhtA (ybiF) gene (Russ P can be enumerated as the protein for participating in secretion purine nucleosides No. 2239656), yijE genes (Russian Patent No. 2244003), ydeD genes (Russian Patent No. 2244004), YicM genes (Russian Patent No. 2271391), ydhL genes (special table 2007-530011), nepI genes (FEMS Microbiology Letters, Volume250, Issue1, pages39-47, September2005) coding protein.

Furthermore, it is possible to by assigning resistance of the bacterium to L-Glutamine analog and the resistance to proline analogs, Assign or strengthen inosine acid production ability (special open 2004-516833).As L-Glutamine analog, diazonium silk ammonia can be enumerated Acid or 6- diazonium -5- oxn-l-norieucins (DON).As proline analogs, 3,4- dehydroprolines, L- a word used for translations can be enumerated Fourth pyridine -2- carboxylic acids, L- thiazolidine -4- carboxylic acids, (S) -2,2- dimethyl -4-Oxazolidine carboxylic acid, (S) -5,5- dimethyl -4- thiazoles Alkane carboxylic acid, (4S, 2RS) -2- ethyls -4-thiazolidinecarboxylic acid, (2S, 4S) -4- hydrogen-pyrrolidine 2 carboxylic acid, 2 piperidine carboxylic acid and 2,5- pyrrolidine-diones.

Furthermore, it is possible to by bar-shaped centered on corynebacterium ammoniagenes (Corynebacterium ammmoniagenes) Method used in the xanthosine monophosphate production bacterium breeding of bacterium, assigns or strengthens xanthosine acid production ability.It can be arranged as this kind of method Activity (Unexamined Patent 8-168383) of the citing as strengthened PRPP amide transferases, assigns the resistance (Japan to aliphatic amino acid Unexamined Patent 4-262790), assign to be dehydrated proline resistance (South-Korea state Patent Laid 2003-56490).

The method of above-mentioned imparting or enhancing purine substance production capacity can be used alone, and what can also be combined makes With.

Bacterial strain, specific enumerable such as Escherichia coli FADRaddedd/ are produced as the nucleosides for belonging to Escherichia PKFpurFKQ plants (WO99/003988).The bacterial strain (WO99/003988) is obtained as below：Destroy following gene, i.e. Escherichia coli The purF genes of W3110 plants of coding PRPP amide transferases, the purR genes for encoding purine aporepressor, coding purine nucleosides The deoD genes of phosphorylase, the purA genes for encoding succinyl-AMP synthase, the add genes and volume for encoding adenosine deaminase The edd genes of code 6-Phosphogluconic dehydrogenase；And the plasmid pKFpurFKQ for being equipped with purFKQ genes is imported, it is described PurFKQ gene codes reduce the desensitization type PRPP amidophosphoric acids phosphoribosynltransferase (K326Q) of the feedback inhibition of GMP generations.

Belong to the specific enumerable for example following bacterial strain of inosine production bacterial strain of bacillus.

KMBS16 plants of bacillus subtilis (U.S. Patent Application Publication No. 2004-0166575)

KMBS16-1 plants of bacillus subtilis (Russian Patent No. 2239656)

KMBS310 plants of bacillus subtilis (Japanese Unexamined Patent Publication 2007-117078)

Bacillus subtilis TABS133 plants (WO2007/125782)

AJ12707 plants of bacillus subtilis (FERM P-12951) (the clear 61-13876 publications of Japanese Patent Application)

AJ3772 plants of bacillus subtilis (FERM P-2555) (the clear 62-014794 publications of Japanese Patent Application)

NA-6011 plants of bacillus subtilis (FERM BP-291) (U.S. Patent No. 4,701,413)

NA-6012 plants of bacillus subtilis (FERM BP-292) (U.S. Patent No. 4,701,413)

G1136A plants of bacillus subtilis (AJ1991 plants of bacillus amyloliquefaciens) (VKPM B-8994) (U.S. Patent No. No. 3,575,809)

Gottheil3218 plants of bacillus pumilus (ATCC21005) (U.S. Patent No. 3,616,206)

NA-1102 plants of bacillus pumilus (FERM BP-289)

AS115-7 plants of bacillus amyloliquefaciens (VKPM B-6134) (Russian Patent No. 2003678)

KMBS16 plants be by destroy trpC2 plants of bacillus subtilis coding succinyl-AMP synthase purA bases Bacterial strain obtained from the deoD genes of cause, the purR genes of coding purine repressor and coding purine nucleoside phosphorylase (purA::erm,purR::spc,deoD::Kan) (U.S. Patent Application Publication No. 2004-0166575).KMBS16-1 plants It is by KMBS16 plants of purA::Erm mutation replace with purA::The bacterial strain of cat mutation.KMBS310 plants are obtained as below：Destroy with The purA genes of the coding succinyl-AMP synthase of lower gene, i.e. bacillus subtilis 168Marburg plants (ATCC6051), Encode the purR genes of purine repressor and encode the pupG genes of purine nucleoside phosphorylase；Reduce IMP dehydrogenase (guaB) activity；Modify the promoter region of purine operon and the SD sequences of PRPP synthase genes (prs).

Strain is produced as the guanosine for belonging to bacillus, specifically, can be enumerated for example, bacterial strain IMP dehydrogenase is active The bacillus (Unexamined Patent 3-58787) enhanced, or the gemma bar to adenine-requiring mutant importing carrier Campylobacter bacteria (fairness 4-28357), the carrier are equipped with purine analogue tolerance gene or decoyinine (decoyinine) tolerance gene.

In addition, following bacterium can be enumerated as the bacterium with purine nucleosides acid production ability.Bacterium is produced as inosinicacid, Such as corynebacterium ammoniagenes CJIP009 (KCCM-10226) (Japanese Unexamined Patent Publication 2004-516833) can be enumerated or phosphatase activity lowers Bacillus subtilis inosine production strain (Uchida, K.Et al., Agr.Biol.Chem., 1961,25,804-805； Fujimoto,M.Uchida,K.,Agr.Biol.Chem.,1965,29,249-259).Bacterium is produced as guanylic acid, can be enumerated For example, performance adenine defective and the bacillus guanylic acid production strain with decoyinine or methionine sulfoxide resistance (Japanese Patent Publication 56-12438 publications).

It should be noted that the gene for the production bacterium breeding of above-mentioned purine substance is as long as no the encoded egg of destruction The function of white matter, is just not limited to above-mentioned example gene or gene or its saltant type with known base sequence.Example Such as, the gene for purine substance production bacterium breeding can encode following protein, it has the ammonia in known protein In base acid sequence, substitute, lack, be inserted into or with the addition of the amino acid sequence of one or more amino acid on one or more positions Row.For gene or protein mutant type, it can be adapted for the record below with respect to yggB genes and YggB protein mutant types.

The method that ＜ 1-2 ＞ increase protein active

The method of the protein active of explanation increase below.

" increase protein active " means the activity of the same protein of each cell relative to wild strain or parent bacterium The increase of the non-modified strains such as strain.It should be noted that " increase protein active " is referred to as " protein active is increased ". " increase protein active " is specifically referred to compared with unmodified bacterial strain, the molecular number increase of the same protein of each cell, and/ Or the function increase of each molecule of same protein.That is, " activity " in so-called " protein active increase " is not limited to albumen The catalyst activity of matter, can also presentation code protein the transcription amount (mRNA amounts) of gene or the translation amount (albumen of gene The amount of matter).As long as the activity of protein is than unmodified bacterial strain increase, without special limitation, for example, can be than unmodified Bacterial strain increases by more than 1.5 times, more than 2 times or more than 3 times.In addition, " increase protein active " is not only instigated had target originally The activity increase of target protein described in the active bacterial strain of protein, is additionally included in originally active there is no target protein Bacterial strain in assign the activity of the target protein.In addition, as long as result protein active increase, can make bacterium Target protein activity inherently reduces, and is subsequently introduced suitable protein.

For example, by the expression of increase coding same protein gene, the modification of increase protein active is realized.Need Bright, " the expression increase of gene " is also referred to as " expression of gene is enhanced ".The expression of gene can be than unmodified bacterial strain More than 1.5 times, more than 2 times or more than 3 times of increase.In addition, " the expression increase of gene " not only instigates and expressed target base originally The expression quantity increase of the target gene in the bacterial strain of cause, and the bacterial strain including making not expressing target gene originally expresses institute State target gene.That is, " the expression increase of gene " is included for example, importing the target base into the bacterial strain without target gene Cause, expresses the target gene.

The increase of gene expression can be realized for example, by the copy number of increase gene.

The copy number of increase gene can be realized by importing the target gene in the chromosome to host bacteria.Can With using homologous recombination, quiding gene (MillerI, J.H.Experiments the in Molecular into chromosome Genetics,1972,Cold Spring Harbor Laboratory).Gene can import 1 copy, can also import 2 and copy Shellfish or multicopy.For example, sequence that can be using on chromosome there are multicopy carries out homologous recombination as target, led to chromosome Enter multiple copies of gene.As the sequence on chromosome there are multicopy, reiterated DNA sequences (repetitive can be enumerated DNA), positioned at the inverted repeats at transposons both ends.Further, it is also possible to purine substance is produced into unwanted gene of institute etc. Appropriate sequence carries out homologous recombination as target on chromosome.Homologous recombination can be carried out by the following method, for example, Red Driving integration (Red-driven integration) method (Datsenko, K.A, and Wanner, B.L.Proc.Natl.Acad.Sci.U S A.97:6640-6645 (2000)) etc. using straight chain DNA method, using containing The method of the plasmid of responsive to temperature type replication orgin, use can engage the method for the plasmid of transfer, not have using in host The method of the suicide vector of the replication orgin to work or the transduction method using bacteriophage.Further, it is also possible to use transposons Or Mini-Mu, by gene it is random imported on chromosome (Japanese Unexamined Patent Publication 2-109985 publications, US5,882,888, EP805867B1)。

Determine imported target gene on chromosome can be by the following method：Using with mutually isogenic whole Or the probe of the sequence of partial complementarity carries out blot hybridization (southern hybridization), or by using based on identical Primer prepared by gene order carries out PCR etc..

Furthermore, it is possible to realize the copy number of increase gene by importing the carrier containing target gene to host bacteria. For example, can by by the DNA fragmentation containing target gene with host bacteria functional carrier be connected it is identical to build The expression vector of gene, host bacteria is converted with the expression vector, makes mutually isogenic copy number increase.For example, can with containing There is the genomic DNA of the bacterium of target gene as template, the DNA fragmentation containing target gene is obtained by PCR.As load Body, may be used at the carrier for being capable of autonomous replication into the cell of host bacteria.Carrier is preferably the carrier of multicopy.In addition, it is Selection transformant, carrier preferably have the labels such as antibiotics resistance gene.In addition, in order to express the gene of importing, carrier Promoter and/or terminator can be contained.Carrier, can be the carrier for example originating from bacterial plasmid, the carrier from yeast plasmid, Carrier, clay or phasmid from bacteriophage etc..As can be autonomous in the Escherichia bacterias such as Escherichia coli The carrier of duplication, specific enumerable such as pUC19, pUC18, pHSG299, pHSG399, pHSG398, pACYC184, pBR322, PSTV29 (any all to be bought from TAKARA-BIO (タ カ ラ バ イ オ) company), pMW219 (NIPPON GENE (ニ ッ Port ン ジ ー Application) company), pTrc99A (Pharmacia (Off ァ Le マ シ ア) company), pPROK serial carriers (Clontech (Network ロ Application テ ッ Network) company), pKK233-2 (production of Clontech companies), (Novagen (ノ バ ジ ェ Application) is public for pET serial carriers Department), pQE systems carrier (QIAGEN (キ ア ゲ Application) company), wide host cell RSF1010.As in buds such as bacillus subtilises It is capable of the carrier of autonomous replication, specific enumerable such as pUB110, pC194, pE194 in spore Bacillus bacteria.As in production ammonia It is capable of the carrier of autonomous replication in the bar shaped bacterias such as bar bacterium, specific enumerable such as pHM1519 (Agric, Biol.Chem., 48,2901-2903(1984))；pAM330(Agric.Biol.Chem.,48,2901-2903(1984))；Improve above-mentioned load Body and with drug resistance gene plasmid；Plasmid pCRY30 described in Japanese Unexamined Patent Publication 3-210184 publications；It is Japanese special Open plasmid pCRY21, pCRY2KE described in No. 5,185,262 specification publications of flat 2-72876 publications and United States Patent (USP), PCRY2KX, pCRY31, pCRY3KE and pCRY3KX；Plasmid pCRY2 described in Japanese Unexamined Patent Publication 1-191686 publications and pCRY3；PAJ655, pAJ611 and pAJ1844 described in Japanese Unexamined Patent Application 58-192900 publications；Japanese Unexamined Patent Application 57- PCG1 described in No. 134500 publications；PCG2 described in Japanese Unexamined Patent Application 58-35197 publications；Japanese Unexamined Patent Application 57- PCG4 and pCG11 described in No. 183799 publications.

When quiding gene, as long as keeping gene with can expressing in the bacterium of the present invention.Specifically, import As long as gene by the bacterium of the present invention control of functional promoter sequence expressed.Promoter can be source Promoter from host or the promoter from different plant species.The intrinsic startup of gene that promoter can be introduced into The promoter of son or other genes.The promoter of more strength for example described below can also be utilized as promoter. In addition, for example, the terminator for terminator transcription can be located at the downstream of gene.As long as terminated in the strain of the present invention Son plays a role, and is just not particularly limited.Terminator can be the terminator from host, alternatively, the end from different plant species It is only sub.The terminator of the intrinsic terminator of gene that terminator can be introduced into or other genes.Have as terminator Body can be enumerated, for example, T7 terminators, T4 terminators, fd bacteriophage terminators, tet terminators, and trpA terminators.Specifically Carrier, promoter and terminator in various microorganisms can be in " Fundamental Microbiology Vol.8, Gentic Obtained in Engineering, KYORITSU SHUPPAN CO., LTD, 1987 ".

In addition, when importing 2 or multiple genes, as long as keeping each gene i.e. with can expressing in the bacterium of the present invention Can.For example, each gene can be all held on an expression vector, can also be all held on chromosome.In addition, each base Because can also be kept on multiple expression vectors, can also be kept on single or multiple expression vectors and chromosome On.In addition it is also possible to form operator by 2 or multiple genes.

As long as the gene of importing encodes functional protein in host, without special limitation.The base of importing Because that can be derived from the gene of host or be derived from the gene of different plant species.Can be for example using based on mutually isogenic Base sequence design primer, with mutually isogenic biological genomic DNA or be equipped with mutually isogenic plasmid etc. work For template, imported gene is obtained by PCR.In addition it is also possible to be based on mutually isogenic base sequence, it is fully synthesized and leads The gene entered.

Furthermore, it is possible to the transcriptional efficiency by improving gene, realizes the expression for improving gene.Can be for example, by with stronger The promoter of gene on the promoter substituted dyeing body of effect, realizes the transcriptional efficiency for improving gene." more potent promoter " Refer to the promoter that genetic transcription is more improved than unborn wild-type promoters.As more potent promoter, can arrange Illustrate High-expression promoter T7 promoters as is well known, trp promoters, lac promoters, tac promoters, thr promoters, trc Promoter, tet promoters, araBAD promoters, rpoH promoters, PR promoters and PL promoters.In addition, produce ammonia when using During bar bacterium, can be used artificial design modification P54-6 promoters (Appl.Microbiol.Biotechnolo., 53,674- 679 (2000)), in bar shaped bacteria can pta, aceA, aceB, adh, amyE of the induction such as enough acetic acid, ethanol, pyruvic acid open Mover, high potent promoter cspB, SOD, tuf promoter (the Journal of of expression quantity in bar shaped bacteria Biotechnology104(2003)311-323,Appl Environ Microbiol.2005Dec；71(12):8587-96.) Etc. promoter.In addition, as more potent promoter, the height that original promoter can also be obtained using various reporter genes is living Property type.For example, -35 in promoter region, -10th area are made to improve the activity (International Publication of promoter close to consensus No. 00/18935).As high activity type promoter, various tac samples promoters (Katashkina JI etc., Russia can be enumerated Federation patent application 2006134574) or pnlp8 promoters (WO2010/027045).The paper of Goldstein et al. (Prokaryotic promoters in biotechnology.Biotechnol.Annu.Rev.,1,105-128(1995)) The evaluation method of promoter intensity and the example of potent promoter have been recorded in.

Furthermore, it is possible to the translation efficiency by improving gene, realizes the expression for improving gene.Can be for example, by with stronger Shine-Dalgarno (SD) sequence (also referred to as ribosome bind site of gene on the SD sequence substituted dyeing bodies of effect (RBS)) translation efficiency for improving gene, is realized." more potent SD sequences " refers to than unborn wild type SD sequences more Add the SD sequences for improving mRNA translations.As more potent SD sequences, the RBS of the gene 10 for example originating from phage t7 can be enumerated (Olins P.O.et al,Gene,1988,73,227-235).Further, it is known that the interval between RBS and initiation codon Substitution, insertion or missing in subregion, particularly upstream from start codon and the sequence (5 '-UTR) adjacent with enlightenment codon Several nucleotide, influence the stability and translation efficiency of mRNA, can improve turning over for gene by modifying these nucleotide very much Translate efficiency.

In the present invention, interval subregion between promoter, SD sequences and RBS and initiation codon etc. influences gene The site of expression is referred to as in " expression regulation area ".Can using promoter retrieve the genetic analysis software such as carrier or GENETYX come Determine expression regulation area.Integration method (WO2005/010175) can be driven by using the method or Red of responsive to temperature type carrier To modify these expression regulation areas.

The translation efficiency for improving gene can also be realized for example, by modification codon.In Escherichia coli etc., in mRNA There are obvious codon-bias in the 61 kinds of amino acid codes found in molecular population, the amount of certain tRNA be with it is corresponding Codon frequency of use into directly proportional (Kane, J.F., Curr.Opin.Biotechnol., 6 (5), 494-500 (1995)).That is, if a large amount of the problem of there is the mRNA containing excessive rare codon, translation can be produced.According in recent years Research prompting, especially the cluster of AGG/AGA, CUA, AUA, CGA or CCC codon can reduce the protein of synthesis at the same time Quality and quantity.Such issues that particularly occur in expression heterogenous gene when.Therefore, when carrying out the heterogenous expression of gene, pass through With synonym substituent rare codon because present in of frequency of use higher, the translation efficiency of gene can be improved. Codon substitution can be carried out for example, by importing the mutation site-specific method of targeted mutagenesis to the target site of DNA. As mutation site-specific method, can enumerate using PCR method (Higuchi, R., 61, in PCR technology, Erlich,H.A.Eds.,Stockton press(1989)；Carter,P.,Meth.in Enzymol.,154,382 (1987)) or using bacteriophage method (Kramer, W.and Frits, H.J., Meth.in Enzymol., 154,350 (1987)；Kunkel,T.A.et al,Meth.in Enzymol.,154,367(1987)).In addition it is also possible to it is fully synthesized The substituted genetic fragment of codon." codon usage database " (http://www.kazusa.or.jp/codon； Nakamura, Y. etc., Nucl.Acids Res., 28,292 (2000)) in disclose it is various biology in codon using frequency Rate.

In addition it is also possible to expanding makes the elevated regulator of gene expression or missing or reduction make the tune of gene expression reduction Knot improves the expression of gene to realize.

It is above-mentioned to can be used alone the elevated method of gene expression, it can also be combined and be used.

In addition it is also possible to be lived by the ratio for increasing protein, realization makes the increased modification of protein active.Increase is gone back than living Including reducing or eliminating feedback inhibition.Obtained for example, various biologies can be studied than increased protein living.In addition it is also possible to Mutation is imported into original protein, obtains high activity type.The mutation of importing can be for example at one of protein or Multiple site substitutions, missing, insertion or the one or more amino acid of addition.For example, above-mentioned mutation site-specific can be passed through Method imports mutation.In addition it is also possible to import mutation using mutation processing.Mutation processing can enumerate x-ray bombardment, ultraviolet Irradiation and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethylmethane sulfonate (EMS) and methyl methylsulfonate (MMS) etc. Utilize the processing of mutagens.In addition, directly handling DNA with azanol in vitro, random mutation can also be induced.Can be with than increase living It is used alone, the use that can also be combined with the method for above-mentioned increase gene expression.

Method for transformation does not have special limitation, can use existing known method.It is, for example, possible to use such as Escherichia coli What K-12 was reported, with chlorination Calcium treatment acceptor bacterium cell, increase DNA passabilities method (Mandel, M.and Higa, A., J.Mol.Biol.1970,53,159-162), or as bacillus subtilis is reported, prepare and experience from proliferation period cell State cell, so as to import method (Duncan, C.H., Wilson, the G.A.and Young, F.E.., 1997.Gene1 of DNA: 153-167).Alternatively, can also apply as known in bacillus subtilis, actinomyces and yeast, make the thin of DNA competence Bacterium cell becomes the state for being easy to the protoplast or spheroplast for taking in recombinant DNA, and recombinant DNA is imported NA competent bacterias Method (Chang, S.and Choen, S.N., 1979.Mol.Gen.Genet.168:111-115；Bibb,M.J.,Ward, J.M.and Hopwood,O.A.1978.Nature274:398-400；Hinnen,A.,Hicks,J.B.and Fink, G.R.1978.Proc.Natl.Acad.Sci.USA75:1929-1933).In addition, it is possible to use the electricity of the corynebacteria of report Pulse method (Japanese Patent Laid-open (Kakai) No.2-207791)

The increase of protein active, can be determined by measuring the activity of the protein.

The increase of protein active can also be carried out true by the rise of the expression for the gene for confirming code for said proteins It is fixed.The rise of gene expression can raise or determine the protein by identical gene expression by determining mutually isogenic transcription amount Amount rise confirmed.

Can by the unmodified bacterial strains such as the amount of the mRNA to identical genetic transcription and wild-type strain or parent strain into Row relatively determines the rise of genetic transcription amount.Blot hybridization, RT-PCR etc. can be enumerated by evaluating the method for the amount of mRNA (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 with it is unmodified Bacterial strain, which is compared, can increase such as more than 1.5 times, more than 2 times or more than 3 times.

It can determine that the amount of protein raises by using the Western blotting (Western blotting) of antibody (Molecular cloning(Cold spring Harbor Laboratory Press,Cold spring Harbor (USA),2001)).The amount of protein can increase such as more than 1.5 times, more than 2 times or more than 3 times compared with unmodified bacterial strain.

Make the increased method of above-mentioned protein active can be used for increasing any protein, such as participate in the life of purine substance The activity of the enzyme of thing synthesis, or any gene of increase, such as encode the expression of the gene of these arbitrary protein matter.

The method that ＜ 1-3 ＞ reduce protein active

The method that explanation reduces protein active below.

" reduction protein active " represents the activity of the protein of each cell than wild strain or parent strain etc. Unmodified bacterial strain is reduced, including the situation that activity is wholly absent." reduction protein active " is referred specifically to compared with unmodified bacterial strain, The molecular number of the protein of each cell is reduced, and/or the function of each molecule of the protein reduces.That is, it is so-called " activity " in " reduction protein active " is not limited to the catalyst activity of protein, can also presentation code protein gene Transcription amount (mRNA amounts) or gene translation amount (albumen quality).In addition, " molecular number of the protein of each cell is reduced " Include the situation for being completely absent the protein.It should be noted that " function of each molecule of protein reduces " bag The situation that the function of each molecule of same protein is wholly absent is included.As long as the activity of protein is reduced than unmodified bacterial strain , without special limitation, for example, can be reduced than unmodified bacterial strain to less than 50%, less than 20%, less than 10%, 5% Below or 0%.

Expression for example, by the gene for reducing code for said proteins, realizes the modification for reducing protein active." reduce The expression of gene " includes mutually homogenic situation about not expressing completely.It should be noted that " expression for reducing gene " is also claimed For " weakening gene expression ".The expression of gene can be reduced to than unmodified bacterial strain less than 50%, less than 20%, 10% with Under, less than 5% or 0%.

The reduction of gene expression can be for example reduce transcriptional efficiency or reduce translation efficiency or on State combination.It can come for example, by Expression modulation sequences such as promoter and Shine-Dalgarno (SD) sequences of modifier real The reduction of existing gene expression.When modifying Expression modulation sequence, Expression modulation sequence can preferably more than 1 base, more preferably 2 More than a base, more than particularly preferred 3 bases modified.In addition it is also possible to make the part or all of of Expression modulation sequence Missing.In addition it is also possible to by operating the factor for participating in control table and reaching, the expression for reducing gene is realized.As participation control table The factor reached can enumerate the low molecule (inducing substance, inhibiting substances etc.) for participating in transcription or translation control, protein (transcription factor Deng), nucleic acid (siRNA etc.) etc..

Furthermore, it is possible to the gene by destroying code for said proteins, realizes the modification for reducing protein active.It can lead to The part or all of missing of the gene coding region for example made on chromosome is crossed, realizes the destruction of gene.Also, bag can also be lacked Overall gene containing the gene context on chromosome.As long as realizing reduces protein active, the region of missing can be with It is any region in N-terminal region, interior zone, C-terminal region etc..In general, absent region length can reliably make gene Inactivation.In addition, to be preferably reading frame inconsistent for the sequence before and after absent region.

Furthermore, it is possible to substituted amino acid (missense mutation) is imported in the code area for passing through gene on chromosome, is imported Terminator codon (nonsense mutation) imports addition or has lacked frameshift mutation of 1-2 base etc., realizes and destroys gene (Journal of Biological Chemistry272:8611-8617(1997),Proceedings of the National Academy of Sciences,USA955511-5515(1998),Journal of Biological Chemistry26116,20833-20839(1991))。

Furthermore, it is possible to be inserted into other sequences in code area by gene on chromosome, realize and destroy gene.Insertion Position can be any region of gene, and insetion sequence length reliably can inactivate gene.In addition, before and after insertion position The preferred reading frame of sequence is inconsistent.It is just not special as long as making the protein active of coding reduce or disappear as other sequences Limitation, for example, can enumerate the marker genes such as antibiotics resistance gene or available for production purine substance gene.

Gene on modification chromosome as described above, for example, can by the partial sequence of missing gene, prepare and modify Gene deletion type, it does not produce the protein of normal function, with comprising the gene deletion type recombinant DNA convert bacterium, By making gene deletion type that homologous recombination occur with the wild type gene on chromosome, realize the wild type gene on chromosome It is substituted by gene deletion type.In the case, according to features such as the auxotrophs of host, label base is contained in recombinant DNA Cause, it is easily operated.Even if the protein that generation is encoded by gene deletion type, it also has the solid different from wild-type protein Structure, function are reduced or disappeared.The method that gene is destroyed as described above with the gene substitution of homologous recombination is referred to as it has been established that having " Red driving integrate (Red-driven integration) " method (Datsenko, K.A, and Wanner, B.L.Proc.Natl.Acad.Sci.U S A.97:6640-6645 (2000)), combination Red driving integration methods and come from λ phagocytosis Body cuts out system (Cho, E.H., Gumport, R.I., Gardner, J.F.J.Bacteriol.184:5200-5203 (2002)) method (referring to No. WO2005/010175) etc. is replicated using the method for linear DNA, using containing responsive to temperature type The method of the plasmid of starting point, use can engage the method for the plasmid of transfer, using not having functional duplication in host Method of the suicide vector of starting point etc. (U.S. Patent No. 6303383, Unexamined Patent 05-007491).

In addition it is also possible to handled by mutagenesis, into the modification for exercising protein active reduction.Mutagenesis processing can Enumerate irradiation, ultraviolet irradiation and N- methyl-N '-nitro-N nitrosoguanidine (MNNG), the ethylmethane sulfonate of X-ray (EMS), the mutagens such as methyl methylsulfonate (MMS) are handled.

The reduction of protein active can be determined by measuring the activity of same protein.

It can also be reduced by the expression for the gene for determining coding same protein, to determine the reduction of protein active. It can be come true by determining that mutually isogenic transcription amount reduction, or the amount of the definite protein by identical gene expression reduce Determining the expression of gene reduces.

Can by by from the amount of the mRNA of the genetic transcription compared with unmodified bacterial strain, come determine gene turn Record amount reduces.As the method for the amount of assessment mRNA, (the Molecular cloning such as blot hybridization, RT-PCR can be enumerated (Cold spring Harbor Laboratory Press,Cold spring Harbor(USA),2001)).The amount of mRNA Such as less than 50%, less than 20%, less than 10%, less than 5% or 0% can be reduced to compared with unmodified bacterial strain.

By using the western blot of antibody, decline (the Molecular cloning of the amount of protein can be determined (Cold spring Harbor Laboratory Press,Cold spring Harbor(USA),2001)).Protein Amount can be reduced to such as less than 50%, less than 20%, less than 10%, less than 5% or 0% compared with unmodified bacterial strain.

According to the method for destruction, can determine the part or all of base sequence of gene, restriction map or Total length etc., determines the situation that gene is destroyed.

The above-mentioned method for reducing protein active can be used for the activity for reducing any protein, for example, being catalyzed from purine The enzyme for synthesizing the reaction of other compounds of the biosynthesis pathway branch of class material；Or the expression of any gene is reduced, such as compile The gene of these any protein of code.

＜ 1-4 ＞ import mutant-type yggB gene

The bacterium of the present invention keeps mutant-type yggB gene through modification.It can be made by modification with purine substance life The bacterium of production capacity power keeps mutant-type yggB gene, and obtains the bacterium of the present invention.In addition, carrying out modification to bacterium makes its holding After mutant-type yggB gene, the bacterium of the present invention can be obtained by assigning purine substance production capacity.In the present invention, The modification of the bacterium for building the present invention can be carried out in any order.

YggB genes and YggB protein are illustrated below.YggB genes are coding power sensitive channels The gene of (mechanosensitive channel).In the present invention, there is the yggB gene quilts of following " specific mutation " Referred to as mutant-type yggB gene, its protein encoded are referred to as saltant type YggB protein.In addition, in the present invention, do not have The yggB genes for having following " specific mutation " are referred to as wild type yggB genes, its protein encoded is referred to as wild type YggB protein.In addition, in YggB protein, the change of the amino acid sequence produced by " the specific mutation " in yggB genes Also referred to as " specific mutation ".

As wild type yggB genes, the yggB genes of such as bar shaped bacteria can be enumerated.YggB bases as bar shaped bacteria Cause, specific enumerable ATCC13869 plants of such as corynebacterium glutamicum (Corynebacterium glutamicum), glutamic acid ATCC13032 plants of bar bacterium, ATCC14067 plants of Corynebacterium glutamicum, corynebacterium melassecola (Corynebacterium Melassecola) ATCC17965 plants of yggB genes.The yggB genes and ncbi database of Corynebacterium glutamicum ATCC13032 In with GenBank accession NC_003450 (VERSION NC_003450.3GI:58036263) gene registered The complementary series of the 1336092nd~1337693 bit sequence in group sequence corresponds to.The yggB of Corynebacterium glutamicum ATCC13032 Gene is identical with Cgl1270 implications.In addition, the YggB protein of Corynebacterium glutamicum ATCC13032 is registered as GenBank accession NP_600492(version NP_600492.1GI:19552490th, locus_tag=" NCgl1221 ").SEQ ID NO:8th, 10,12 and 14 base sequence for respectively illustrating these yggB genes.In addition, SEQ ID NO:9th, 11,13 and 15 respectively illustrate the amino acid sequence of the YggB protein of these yggB gene codes.In addition, SEQ ID NO:16 show The conserved sequence of these YggB protein.

In addition, as long as wild type YggB protein does not have following " specific mutation ", and there is the work(of YggB protein The saltant type of energy or above-mentioned YggB protein.It should be noted that sometimes referred to as this kind of saltant type is " conservative variants Type ".As conservative variants type, the homologue of for example above-mentioned YggB protein or artificial trim can be enumerated.In the present invention, " having the function of YggB protein " refers to for example play as power sensitive channel (mechanosensitive channel) Function.In addition, " having the function of YggB protein " refers to for example when expressing rise in bar shaped bacteria, it is bar-shaped thin with making The characteristic (special open 2007-097573) that the Pidolidone production capacity of bacterium improves.

As long as in addition, wild type yggB genes encode above-mentioned YggB protein or its conservative variants type can or The saltant type of above-mentioned yggB genes.

Can be by yggB genes (the SEQ ID NO of for example above-mentioned bar shaped bacteria:8th, 10,12 or search sequence 14) is used as, Using BLAST retrievals or FASTA retrievals, the base for encoding above-mentioned YggB protein homologies thing is easily obtained from public database Cause.Furthermore, it is possible to using the chromosome of such as bar shaped bacteria as template, obtained by PCR and encode above-mentioned YggB protein homologies The gene of thing, the PCR use the oligonucleotides based on these known gene order preparations as primer.

As long as wild type YggB protein has the function of YggB protein without following " specific mutation ", just Can be the protein with following amino acid sequence：Substitute in one or more of above-mentioned amino acid sequence position, lack, The sequence of the amino acid of insertion or addition one or more.It should be noted that above-mentioned " one or more " is residual according to amino acid The species of position or amino acid residue of the base in the stereochemical structure of protein and it is different, but specifically preferably 1~20, more It is preferred that 1~10, further preferred 1~5.

Substitution, missing, insertion or the addition of said one or multiple amino acid are that Protein requirement is normally functioning conservative Property mutation.The representative of conservative mutation is conservative replaces.Conservative replaces refer to, when substitution position is aromatic amino acid When, then the mutation that mutually substitutes between Phe, Trp, Tyr；When it is hydrophobic amino acid to substitute position, then be Leu, Ile, The mutation mutually substituted between Val；When it is polar amino acid to substitute position, then the mutation that mutually substitutes between Gln, Asn； When it is basic amino acid to substitute position, then the mutation that mutually substitutes between Lys, Arg, His；When substitution position is acid ammonia When base is sour, then the mutation that mutually substitutes between Asp, Glu；When it is the amino acid with hydroxyl to substitute position, then be Ser, The mutation mutually substituted between Thr.Be considered as the substitution of conservative replaces, can specifically enumerate Ala substituted by Ser or Thr, Arg is substituted by Gln, His or Lys, Asn is substituted by Glu, Gln, Lys, His or Asp, Asp is substituted by Asn, Glu or Gln, Cys Substituted by Ser or Ala, Gln is substituted by Asn, Glu, Lys, His, Asp or Arg, Glu is taken by Gly, Asn, Gln, Lys or Asp In generation, Gly is substituted by Pro, His is substituted by Asn, Lys, Gln, Arg or Tyr, Ile is substituted by Leu, Met, Val or Phe, Leu quilts Ile, Met, Val or Phe substitute, Lys is substituted by Asn, Glu, Gln, His or Arg, Met is substituted by Ile, Leu, Val or Phe, Phe is substituted by Trp, Tyr, Met, Ile or Leu, Ser is substituted by Thr or Ala, Thr is substituted by Ser or Ala, Trp by Phe or Tyr substitutions, Tyr are substituted by His, Phe or Trp, and Val is substituted by Met, Ile or Leu.In addition, above-mentioned amino acid takes Generation, missing, insertion, addition or inversion etc. are also included by when based on the organisms difference from gene, species difference Saltant type caused by the mutation (mutant or variant) naturally occurred.

Further, as long as wild type YggB protein is without following " specific mutation ", and there is YggB protein Function, can be relative to above-mentioned amino acid sequence total length have more than 80% homology protein, preferably 90% More than, more preferably more than 95%, further preferred more than 97%, particularly preferred more than 99%.In addition, in the present specification, it is " same Source property (homology) " refers to " homogeneity (identity) ".

The wild type YggB protein preferably such as conserved sequence of the YggB protein with above-mentioned bar shaped bacteria.Therefore, Produced in the amino acid residue that conservative variants are not guarded preferably in the YggB protein of for example above-mentioned bar shaped bacteria.It is specific and Speech, for example, can be on one or more of the following amino acid residue selected from wild type YggB protein amino acid residue Produce conservative variants.In addition, the position of the amino acid residue in wild type YggB protein is opposite position as described below.

The Glu (preferably replacing with Arg) of the 48th

The Asp (preferably replacing with Ser) of the 275th

The Glu (preferably replacing with Ala) of the 298th

The Ala (preferably replacing with Val) of the 343rd

The Phe (preferably replacing with Ile) of the 396th

The Ser (preferably replacing with Gly) of the 438th

The Val (preferably replacing with Ala) of the 445th

The Ala (preferably replacing with Val) of the 454th

The Pro (preferably replacing with Ser) of the 457th

The Ser (preferably replacing with Asp) of the 474th

The Val (preferably lacking) of the 517th

The Glu (preferably lacking) of the 518th

The Ala (preferably lacking) of the 519th

The Pro (preferably lacking) of the 520th

In addition, as long as wild type yggB genes do not have following " specific mutation ", and coding has YggB protein The protein of function, can be the probe being prepared by known gene order, for example, under strict conditions with it is above-mentioned The DNA of all or part of complementary sequence hybridization of base sequence." stringent condition " refers to form so-called specificity miscellaneous Zoarium, does not form the condition of non-specific hybrid.Show that an example is then, the high DNA of homology is hybridized, such as is had There are more than 80% homology, preferably more than 90%, more preferably more than 95%, more preferably more than 97%, particularly preferred more than 99%, And the condition that the DNA lower than its homology does not hybridize mutually, or the wash conditions of common blot hybridization can be enumerated, i.e. 60 DEG C, 1 × SSC, 0.1%SDS, preferably 60 DEG C, 0.1 × SSC, 0.1%SDS, more preferably 68 DEG C, 0.1 × SSC, 0.1%SDS Corresponding salinity is washed 1 time, the preferably condition of 2-3 times with a temperature of.

As described above, the above-mentioned probe for hybridization can be a part for gene complement.This kind of probe can be Using the oligonucleotides prepared based on known gene order as primer, using the DNA fragmentation comprising these base sequences as template, pass through Prepared by PCR.It is 300bp's or so when using length for example, the DNA fragmentation with 300bp length can be used as probe When DNA fragmentation is as probe, the wash conditions of hybridization can enumerate 50 DEG C, 2 × SSC, 0.1%SDS.

, can be by any codon in addition, as long as wild type yggB genes encode above-mentioned wild type YggB protein Replace with the codon of equal value with it.For example, can be with the password according to host used by wild type yggB genetic modifications The most suitable codon of sub- frequency of use.

In addition, the record on said gene or the saltant type of protein is suitable for participating in purine substance biosynthesis Any protein such as enzyme and its encoding gene.

Saltant type YggB protein has following " specific prominent in the amino acid sequence of above-mentioned wild type YggB protein Become ".

I.e., in other words, saltant type YggB protein can also be above-mentioned rod for example in addition to following " specific mutation " The YggB protein of shape bacterium or its conservative variants type.

Specifically, saltant type YggB protein can also have SEQ for example in addition to following " specific mutation " ID NO:9th, the amino acid sequence shown in 11,13 or 15.

In addition, specifically, saltant type YggB protein for example in addition to following " specific mutation ", can also be Following protein, it has in SEQ ID NO:9th, in amino acid sequence shown in 11,13 or 15 comprising substitution, missing, insertion or The amino acid sequence of the one or more amino acid of addition.

In addition, specifically, saltant type YggB protein for example in addition to following " specific mutation ", can also be Following protein, it has relative to SEQ ID NO:9th, amino acid sequence shown in 11,13 or 15 has more than 80% homology Amino acid sequence, preferably more than 90%, more preferably more than 95%, more preferably more than 97%, particularly preferred more than 99%.

In addition, in other words, saltant type YggB protein can be under having in the YggB protein of above-mentioned bar shaped bacteria State " specific mutation ", and the position beyond " the specific mutation " also includes the saltant type of conservative mutation.

Specifically, saltant type YggB protein, such as in the protein in SEQ ID NO:9th, shown in 11,13 or 15 In amino acid sequence, have following " specific mutation ", and can also be wrapped with the position beyond " the specific mutation " The amino acid sequence of substitution, missing, insertion or addition containing one or more amino acid.

As long as mutant-type yggB gene encodes above-mentioned saltant type YggB protein, without special limitation.

" the specific mutation " having below to mutant-type yggB gene illustrates.

" specific mutation " improves bacterium life as long as making the amino acid sequence of above-mentioned wild type YggB protein change The ability of purine substance is produced, without special limitation." ability for improving bacterium production purine substance " refers to have Modification type Escherichia bacteria, bacillus or the corynebacterium ammoniagenes of mutant-type yggB gene and unmodified bacterial strain phase Than further amounts of purine substance can be produced." unmodified bacterial strain " can be the control strain without mutant-type yggB gene, For example, wild-type strain or parental strain." further amounts of purine substance is produced compared with unmodified bacterial strain " is as long as refer to fast The yield of purine class material is than unmodified bacterial strain increase, without special limitation, for example, can produce higher than unmodified bacterial strain The purine substance of more than 1.05 times of amount, preferably more than 1.1 times, more preferably more than 1.2 times, particularly preferred more than 1.3 times.

A certain mutation whether improve bacterium production purine substance ability can be carried out as follows it is definite, such as based on belonging to The bacterial strain of Escherichia, bacillus or corynebacterium ammoniagenes makes modified bacterial strain, it holds the coding mutation The gene of YggB protein, the amount for the purine substance that to cultivating the modification bacterial strain in the medium when is produced quantify, Compared with the amount of the purine substance produced when the bacterial strain (unmodified bacterial strain) before modification is cultivated in the medium.

As " specific mutation " specific enumerable such as following mutation (Japanese Unexamined Patent Publication 2007-097573).

(1) C-terminal is mutated

C-terminal mutation be in wild type yggB genes, the 419th~533 of encoding wild type YggB protein ( SEQ ID NO:419-529 in 13) amino acid residue region in mutation.Can be in same area 1 or Multiple sites import C-terminal mutation.The change species of the amino acid sequence as caused by being mutated C-terminal does not have special limitation.C ends It can be by such as amino acid residue substitution (missense mutation), amino acid residue insertion, amino acid residue missing, appearance that distal process, which becomes, What terminator codon (nonsense mutation), frameshift mutation or its combination produced.As C-terminal be mutated, preferably for example insetion sequence (under Text is also referred to as " IS ") or transposons etc. base sequence insertion.

The insertion of (1-1) base sequence

It is mutated, can be enumerated for example at the valine residue of the 419th of encoding wild type YggB protein as C-terminal It is inserted into the mutation (mutation of 2A-1 types) of base sequence.The mutation of 2A-1 types can produce following mutation, such as lack or substitute wild The part or all of amino acid residue of 419-533 of type YggB protein.As the saltant type being mutated with 2A-1 types YggB genes, it is specific enumerable for example in SEQ ID NO:It is inserted into IS after 8 the 1255th " G ", original wild of encoding ratio Type YggB protein (SEQ ID NO:9) the yggB bases of shorter, 423 amino acid residues of total length saltant type YggB protein Because of (special open 2007-222163).

The substitution of (1-2) proline

It is mutated as C-terminal, can enumerate for example will be residual positioned at the proline of 419-533 of wild type YggB protein Base replaces with the mutation of other amino acid.As above-mentioned proline residue, the 424th of wild type YggB protein can be enumerated Position, 437,453,457,462,469,484,489,497,515,529 (SEQ ID NO:In 13 The 525th) and 533 (SEQ ID NO:The 529th in 13) proline residue.Wherein, preferably by the 424th and/ Or the proline residue of 437 replaces with other amino acid.

" other amino acid " as long as natural amino acid beyond proline, just without special limitation." other amino Acid " can enumerate Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Ala、His。

For example, the proline residue of the 424th is preferably carried out with hydrophobic amino acid (Ala, Gly, Val, Leu or Ile) Substitution, is more preferably substituted with branched-chain amino acid (Leu, Val or Ile).Replaced with as the proline residue of the 424th bright The mutation of propylhomoserin, can enumerate SEQ ID NO:14 the 1271st " C " replaces with the mutation (mutation of 66 types) of " T ".

In addition, for example, the proline residue of the 437th preferably has the amino acid (Thr, Ser or Tyr) of hydroxyl with side chain Substituted, more preferably substituted with Ser.The mutation of Ser is replaced with as the proline residue of the 437th, can enumerate by SEQ ID NO:8 the 1309th " C " replaces with the mutation (mutation of 22 types) of " T ".The mutation of 22 types can be with for example by SEQ ID NO:8 the 1624th " C " replaces with the mutation of " T ".

(2) mutation of transmembrane region

Infer that YggB protein has 5 transmembrane regions.Transmembrane region corresponds respectively to the 1st~23 of wild type YggB protein Position (the 1st transmembrane region), the 25th~47 (the 2nd transmembrane region), the 62nd~84 (the 3rd transmembrane region), the 86th~108 (the 4th cross-film Area), the amino acid residue of the 110th~132 (the 5th transmembrane region).The mutation of transmembrane region is encoded in wild type yggB genes Mutation in the region of above-mentioned transmembrane region.The mutation of transmembrane region can be imported in the site of one or more in same area.Cross-film The mutation in area preferably produces substitution, missing, addition, insertion or the inversion of one or more amino acid and dashes forward without frameshit Change and nonsense mutation." one or more " expression preferably 1~20, more preferably 1~10, further preferred 1~5, especially It is preferred that 1~3.

The mutation of (2-1) the 1st transmembrane region

As the mutation of the 1st transmembrane region, can enumerate for example wild type YggB protein the 14th leucine residue and The mutation of the amino acid of one or more is inserted between the trp residue of the 15th.It can be arranged as one or more amino acid Citing such as Cys-Ser-Leu.Mutant-type yggB gene with this kind of mutation is specific enumerable for example in SEQ ID NO:The of 8 The yggB genes (mutation of A1 types) of TTCATTGTG are inserted into after 44 " G ".The base sequence of the mutant-type yggB gene and identical The amino acid sequence of the saltant type YggB protein of gene code is respectively such as SEQ ID NO:Shown in 6 and 7.

The mutation of (2-2) the 4th transmembrane region

As the mutation of the 4th transmembrane region, it can enumerate and for example replace the 100th alanine residue of wild type YggB protein It is changed to the mutation of other amino acid." other amino acid " as long as natural amino acid beyond alanine, just without special limit System.As " other amino acid " can enumerate Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe、Trp、Tyr、Gly、His、Pro.The alanine residue of the 100th preferably with side chain have hydroxyl amino acid (Thr, Ser or Tyr) substitution, more preferably substituted with Thr.It is specific enumerable for example as the mutant-type yggB gene with this kind of mutation SEQ ID NO:8 the 298th " G " is replaced by the yggB genes (mutation of 19 types) of " A ".

The mutation of (2-3) the 5th transmembrane region

As the mutation of the 5th transmembrane region, it can enumerate and for example replace the 111st alanine residue of wild type YggB protein It is changed to the mutation of other amino acid." other amino acid " as long as natural amino acid beyond alanine, just without special limit System.As " other amino acid " can enumerate Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe、Trp、Tyr、Gly、His、Pro.The alanine residue of the 111st preferably with side chain have hydroxyl amino acid (Thr, Ser or Tyr) substitution, more preferably substituted with Thr.The specific enumerable such as SEQ of mutant-type yggB gene with above-mentioned mutation ID NO:8 the 332nd " C " is replaced by the yggB genes (mutation of L30 types) of " T ", or SEQ ID NO:The 331st of 12 " G " is replaced by the yggB genes (mutation of 8 types) of " A ".The base sequence and identical base of mutant-type yggB gene (mutation of L30 types) Because coding saltant type YggB protein amino acid sequence respectively such as SEQ ID NO:Shown in 4 and 5.

In the present invention, " amino acid residue of the X position of wild type YggB protein " without special limitation, represent with SEQ ID NO:The corresponding amino acid residue in X position in 9.In addition, " the X position " in amino acid sequence is represented from identical ammonia The X position that the N-terminal of base acid sequence starts, using the amino acid residue of N-terminal as the 1st amino acids residue.That is, due to above-mentioned ammonia The positional representation of base acid residue is relative position, and therefore, according to the missing of amino acid, insertion or addition etc., its position is sometimes Move back and forth.For example, " amino acid residue of the 419th of wild type YggB protein " represents and SEQ ID NO:In 9 419 corresponding amino acid residues, when only lacking 1 amino acid residue to 419 than N-terminal, by the 418th of N-terminal the Amino acids residue is as " amino acid residue of the 419th of wild type YggB protein ".In addition, work as than N-terminal to 419 When being only inserted into 1 amino acid residue, using the 420th amino acids residue of N-terminal as " the 419th of wild type YggB protein The amino acid residue of position ".

, can be by comparing the arbitrary amino acid sequence and SEQ ID NO in any amino acid sequence:9 ammonia Base acid sequence, it is " with SEQ ID NO to determine which amino acid residue:The corresponding amino acid residue in X position in 9 ".For example, It can be compared using known genetic analysis software.Specific compare can enumerate Hitachi Solutions (Hitachi ソ リ ュ ー シ ョ Application ズ) production DNASIS or GENETYX (ゼ ネ テ ィ ッ Network ス) production the (Elizabeth such as GENETYX C.Tyler et al.,Computers and Biomedical Research,24(1),72-96,1991；Barton GJ et al.,Journal of molecular biology,198(2),327-37.1987)。

It can make it have above-mentioned " specific mutation " by modifying wild type yggB genes, obtain saltant type yggB bases Cause.DNA mutation can be carried out by known method.For example, targeted mutagenesis is imported using mutation site-specific method The target site of DNA, can enumerate using PCR method (Higuchi, R., 61, in PCR technology, Erlich, H.A.Eds.,Stockton press(1989)；Carter, P., Meth.in Enzymol., 154,382 (1987)) or use Method (Kramer, W.and Frits, H.J., Meth.in Enzymol., 154,350 (1987) of bacteriophage；Kunkel, T.A.et al., Meth.in Enzymol., 154,367 (1987)).Furthermore, it is possible to saltant type is obtained by chemical synthesis YggB genes.

The method for being modified to the bacterium with mutant-type yggB gene is illustrated below.

It can realize that modified bacteria makes it have saltant type yggB bases by the way that mutant-type yggB gene is directed into bacterium Cause.Furthermore, it is possible to be handled by mutation or mutagens, mutation is imported in the yggB genes having to bacterium, carrying out modified bacteria makes It is with mutant-type yggB gene.

The method for importing mutant-type yggB gene to bacterium does not have special limitation.In the bacterium of the present invention, saltant type As long as yggB genes remain able to expression under the control of functional promoter in identical bacterium.Promoter can To be derived from the promoter of host or be derived from the promoter of different plant species.Promoter can be the endogenous of yggB genes The promoter of property promoter or other genes.For example, the yggB gene promoters of bar shaped bacteria can be utilized.Big Functional promoter in the Escherichia bacterias such as enterobacteria, specific enumerable such as T7 promoters, trp promoters, trc are opened Mover, lac promoters, tac promoters, tet promoters, araBAD promoters, rpoH promoters, PR promoters and PL start Son.The functional promoter in the bacillus such as bacillus subtilis, specific enumerable such as spac promoters, Xyl promoters, glv promoters, P43 promoters and P2 promoters.In addition, by using various reporter genes, can obtain simultaneously Utilize the promoter than original promoter activity higher.For example, make -35 in promoter region, -10th area close to consensus, The activity (International Publication No. 00/18935) of promoter can be improved.Assess the method for promoter intensity and potent promoter Example is documented in paper (the Prokaryotic promoters in of Goldstein etc. Biotechnology.Biotechnol.Annu.Rev., 1,105-128 (1995)) etc. in.In the bacterium of the present invention, mutation Type yggB genes can reside in plasmid such as and on the carrier of autonomous replication, can also be incorporated into outside chromosome in chromosome.This The bacterium of invention can have the mutant-type yggB gene of 1 copy, it is possible to have the saltant type yggB bases of 2 or multiple copies Cause.The bacterium of the present invention can only have a kind of mutant-type yggB gene, it is possible to have 2 kinds or various mutations type yggB genes. The identical method of above-mentioned increase gene copy number can be used, imports mutant-type yggB gene.

The bacterium of the present invention can have, and can not also have wild type yggB genes.It should be noted that yggB genes It is referred to as such as mscS genes in Escherichia coli, yfkC genes is referred to as in bacillus subtilis.

The bacterium without wild type yggB genes can be obtained by destroying the wild type yggB genes on chromosome. For example, wild type yggB genes can be destroyed by the method for above-mentioned destruction gene.Specifically, for example, missing can be passed through All or part of wild type yggB gene coding regions, destroy wild type yggB genes.

In addition, by the way that the wild type yggB genes on chromosome are replaced with mutant-type yggB gene, can not be had There are wild type yggB genes and the modified bacterium with mutant-type yggB gene.As said gene replace method just like Lower method, such as it is referred to as method (Datsenko, K.A, the and of " (Red-driven integration) is integrated in Red drivings " Wanner,B.L.Proc.Natl.Acad.Sci.U S A.97:6640-6645 (2000)), combination Red driving integration methods and source System (Cho, E.H., Gumport, R.I., Gardner, J.F.J.Bacteriol.184 are cut out in bacteriophage lambda:5200- 5203 (2002)) method (referring to No. WO2005/010175) etc. using the method for linear DNA, using containing responsive to temperature type The method of the plasmid of replication orgin, use can engage the method for the plasmid of transfer, using functional without having in host Method of the suicide vector of replication orgin etc. (U.S. Patent No. 6303383, Unexamined Patent 05-007491).

The preparation method of 2 ＞ purine substances of ＜

The bacterial fermentation production purine substance of the present invention can be utilized.That is, the present invention provides the system of purine substance Preparation Method, it includes cultivating the bacterium of the present invention in the medium, accumulates purine substance in the medium, and from the culture The purine substance is recycled in base.In the present invention, a kind of purine substance can be prepared, 2 kinds or a variety of fast can also be prepared Purine class material.Such as, in the present invention it is possible to prepare one or more purine nucleosides.Such as, in the present invention it is possible to prepare 1 Kind or a variety of purine nucleotides.

As long as the culture medium used can breed the bacterium of the present invention, productive target purine substance, just without special Limitation.As culture medium, the conventional medium for example for Bacteria Culture can be used.As culture medium, can be used if necessary Culture medium containing the component selected from such as carbon source, nitrogen source, source of phosphoric acid, sulphur source, other various organic principles or inorganic constituents.Training The species or concentration for supporting based component can be according to the various bars such as the species of used bacterial species or the purine substance of preparation Part is suitably set.

It is specific enumerable such as glucose, fructose, sucrose, lactose, galactolipin, xylose, arabinose, useless as carbon source Molasses, glucidtemns, the organic acid such as carbohydrate, acetic acid, fumaric acid, citric acid, the nicotinic acid such as hydrolysate of biomass, glycerine, The alcohols such as crude glycerine, ethanol, aliphatic acid.As carbon source, a kind of carbon source can be used, 2 kinds or several kinds of carbon source can also be applied in combination.

As nitrogen source, the specific enumerable ammonium salt such as ammonium sulfate, ammonium chloride, ammonium phosphate, peptone, yeast extract, Organic nitrogen source, ammonium hydroxide, the urea such as meat extract, soybean protein decomposition product.The ammonia or ammonia for being used for adjusting pH can also be used Water is as nitrogen source.As nitrogen source, a kind of nitrogen source can be used, 2 kinds or a variety of nitrogen sources can also be applied in combination.

As source of phosphoric acid, the specific enumerable phosphoric acid such as phosphate, pyrophosphoric acid such as calcium dihydrogen phosphate, dicalcium phosphate gathers Compound.As source of phosphoric acid, a kind of source of phosphoric acid can be used, 2 kinds or a variety of source of phosphoric acid can also be applied in combination.

As sulphur source, the specific enumerable inorganic sulfide compound such as sulfate, thiosulfate, sulphite, half Guang The sulfur-containing amino acid such as propylhomoserin, cystine, glutathione.As sulphur source, a kind of sulphur source can be used, 2 kinds or more can also be applied in combination Kind sulphur source.

As other various organic principles or inorganic constituents, the inorganic salts such as sodium chloride, calcium chloride can be enumerated by having； The trace meters such as iron, manganese, magnesium, calcium；The dimensions such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, niacinamide, vitamin B12 are given birth to Plain class；Amino acids；Nucleic acid；Peptone, casamino acid, yeast extract, soybean protein comprising above-mentioned substance The organic principles such as decomposition product.1 kind of component can be used as these other various organic principles or inorganic constituents, can also combine makes With 2 kinds or Multiple components.

In addition, in the case of needing the auxotrophic mutant of nucleic acid etc. when using propagation, preferably in the medium Nutriment necessary to addition.For example, can be in glands such as culture succinyl-AMP synthase gene (purA) deletion form bacterial strains During Purine auxotroph mutant mutant strain, the organic principle using adenine or comprising adenine.

As long as the bacterium of the present invention, productive target purine substance, to condition of culture just without special limit can be bred System.For example, it can be cultivated in the usual terms for Bacteria Culture.Can be according to the fast of the bacterial species or preparation used The various conditions such as the species of purine class material, rightly set condition of culture.

Aerobic culture can be carried out using fluid nutrient medium.Specifically, can be carried out with ventilation culture or shaken cultivation Culture.Cultivation temperature, can be such as 20~45 DEG C, preferably 25 DEG C~37 DEG C.The pH of culture medium, it can be adjusted to such as 5~ 8.The adjustment pH such as inorganic or organic alkaline matter or ammonia can be used.Incubation time, when can be such as 15 small~90 small When.Batch culture (batch culture), fed-batch culture (Fed-batch culture), continuous culture can be passed through Culture is implemented in (continuous culture) or its combination.Furthermore, it is possible to seed culture and main culture are separately cultivated. In the case, seed culture and the condition of culture of main culture may be the same or different.For example, seed culture and main training Batch culture can all be used by supporting.In addition, for example, seed culture can be carried out with batch culture, and fed-batch culture or continuous Culture carries out main culture.Under such conditions, by cultivating the bacterium of the present invention, purine substance is accumulated in the medium.

It can determine to generate purine substance by the known method for detecting or differentiating compound.As above-mentioned Method, can enumerate such as HPLC, LC/MS, GC/MS, NMR.These methods can rightly be applied in combination.

Generated purine substance can be recycled by isolating and purifying the known method of compound.As the above method, Such as ion-exchange-resin process, membrane processing method, the precipitation method and crystallization can be enumerated.The progress of these methods can rightly be combined Use.When producing purine nucleotides, the purine nucleotides of recycling can be free compound, its salt, and their mixture. I.e., in the present invention, term " purine nucleotides " can mean free purine nucleotides, its salt and their mixture.Its Salt can enumerate sodium salt and sylvite.Inosinate, specific enumerable such as 5 '-IMP disodium salts.Guanosine hydrochlorate, it is specific enumerable Such as 5 '-GMP disodium salts.The purine substance of recycling can also include microorganism, the culture medium in addition to purine substance The components such as component, moisture and the metabolic by-product of bacterium.Purine substance can be purified to preferable degree.Purines thing The purity of matter, can be more than such as 30% (w/w), more than 50% (w/w), more than 70% (w/w), more than 80% (w/w), More than 90% (w/w) or more than 95% (w/w).

The preparation method of 3 ＞ purine nucleotides of ＜

When producing purine nucleosides by the bacterium of the present invention, purine nucleotides can be prepared using the purine nucleosides.That is, The present invention provides the preparation method of purine nucleotides, including this hair with purine nucleosides production capacity is cultivated in the medium Bright bacterium, accumulates purine nucleosides in the medium, and the purine nucleosides phosphorylation is generated purine nucleotides, and recycling institute State purine nucleotides.

As purine nucleotides, 5 '-phosphate of purine nucleosides can be enumerated.As 5 '-phosphate of purine nucleosides, can arrange Elevator muscle thuja acid (inosine -5 '-phosphate；IMP), guanylic acid (guanosine -5 '-phosphate；GMP), xanthosine monophosphate (xanthosine -5 ' - Phosphate；) and adenylate (5'-AMP ester XMP；AMP).In the method, generation and used purine nucleosides phase Corresponding purine nucleotides.That is, for example, inosinicacid can be prepared from inosine respectively, guanylic acid is prepared from guanosine, prepared from xanthosine Xanthosine monophosphate, from adenosine prepare adenylate.In the present invention, a kind of purine nucleotides can be prepared, 2 kinds or a variety of can also be prepared Purine nucleotides.

The purine nucleosides that can be will be contained in culture medium directly carries out phosphorylation, after can also being recycled from culture medium again Carry out phosphorylation.In addition, purine nucleosides can carry out phosphorylation after appropriate pretreatment is carried out.Example can be enumerated as pretreatment Such as, purify, dilute, concentration, crystallization, dry, rupture, dissolving etc..Appropriate combination can also be carried out to these pretreatments.Example Such as, the nutrient solution comprising purine nucleosides can be directly subjected to phosphorylation, or is purified to desired level and carries out phosphoric acid again later Change.

The method for carrying out phosphorylation to purine nucleosides does not have special limitation.Phosphorus can be carried out for example, by known method Acidifying.

Such as chemical phosphorylation can be carried out.Chemical phosphorylation can use the phosphorylation agents such as phosphoryl chloride phosphorus oxychloride (POCl3) (Yoshikawa etc., Studies of phosphorylation, III, Selective phosphorylation of unprotected nucleosides,Bull.Chem.Soc.Jpn.,1969,42:3505-3508)。

Such as microorganism or enzyme can be used to carry out phosphorylation.I.e., it is possible to make that there is generation nucleosides -5'- phosphate abilities Microbial action in purine nucleosides and phosphodonor, generation purine nucleotides (Japanese Unexamined Patent Publication 07-231793).In addition, can So that phosphorylase acts on purine nucleosides and phosphodonor, purine nucleotides is generated.

The specific enumerable for example following microorganism of microorganism with generation nucleosides -5'- phosphate abilities.

Cockroach Escherichia (Escherichia blattae) JCM1650

Serratia ficaria (Serratia ficaria) ATCC33105

Plant raw Klebsiella (Klebsiella planticola) IFO14939 (ATCC33531)

Friedlander's bacillus (Klebsiella pneumoniae) IFO3318 (ATCC8724)

Klebsiella terrigena (Klebsiella terrigena) IFO14941 (ATCC33257)

Morganella morganii strain (Morganella morganii) IFO3168

Clostridium perfringen (Enterobacter aerogenes) IFO12010

Clostridium perfringen (Enterobacter aerogenes) IFO13534 (ATCC13048)

River color bacillus (Chromobacterium fluviatile) IAM13652

Chromobacterium violaceum (Chromobacterium violaceum) IFO12614

Cedecea lapagei (Cedecea lapagei) JCM1684

Cedecea davisae (Cedecea davisiae) JCM1685

Cedecea neteri (Cedecea neteri) JCM5909

Such as phosphatase, nucleoside kinase, nucleoside Phosphate Transferase can be enumerated as phosphorylase.Phosphorylase can be pure It is after change or unpurified.It is, for example, possible to use the culture of the microorganism of production phosphorylase, from the culture The separated culture supernatant of thing, from the separated thalline of the culture, the bacterial disposing thing of the microorganism, above-mentioned object part Purified etc. contains the cut of phosphorylase as phosphorylase.

Such as inosine guanosine kinase can be enumerated as nucleoside kinase.Utilize the specific enumerable example of the method for inosine guanosine kinase Such as purine nucleotides is prepared using the Escherichia bacteria of the gene for the inosine guanosine kinase for having imported encoding E. coli Method (WO91/08286), or using having imported encoding acetyl Exiguobacterium sp (Exiguobacterium acetylicum) The corynebacterium ammoniagenes of the gene of inosine guanosine kinase prepare the method for purine nucleotides (WO96/30501).

Phosphatase can enumerate such as acid phosphatase.As acid phosphatase, such as Japanese Unexamined Patent Publication 2002- can be enumerated Phosphatase disclosed in 000289.In addition, as preferable acid phosphatase, can enumerate for example elevated to the affinity of nucleosides The saltant type acid phosphatase that saltant type acid phosphatase (Japanese Unexamined Patent Publication 10-201481), activity of 5 '-nucleotidase reduce (WO96/37603), the saltant type acid phosphatase (Japanese Unexamined Patent Publication 2001-245676) that phosphate fire-resistant oil activity reduces.

Phosphodonor can enumerate such as polyphosphoric acid, phenyl phosphate, etherophosphoric acid, carbamyl phosphate, ATP, dATP (deoxidation ATP).Such as pyrophosphoric acid, tripolyphosphate, three metaphosphoric acids, four metaphosphoric acids, hexa metaphosphoric acid can be enumerated as polyphosphoric acid.Phosphodonor Can be any educt or its salt or its mixture.Salt can enumerate such as sodium salt or calcium salt.Phosphodonor can root Appropriately selected according to the microorganism used or the species of phosphorylase.In addition, ATP or dATP are being used as phosphodonor When, above-mentioned regenerating system (WO91/08286, WO96/30501) can also be applied in combination.

It can determine to generate purine nucleotides by the known method for detecting or differentiating compound.This kind of method Such as HPLC, LC/MS, GC/MS, NMR can be enumerated.These methods can appropriately be applied in combination.

Generated purine nucleotides can be recycled by the known method for isolating and purifying compound.As on this Method is stated, such as ion-exchange-resin process, membrane processing method, the precipitation method and crystallisation can be enumerated.This appropriately can be applied in combination A little methods.The purine nucleotides of recycling can be free compound, its salt or their compositions.Its salt can enumerate sodium salt with And sylvite.Concrete example as creatinine can be enumerated, for example, 5 '-IMP disodium salts.Concrete example as guanylic acid can be enumerated, example Such as, 5 '-GMP disodium salts.Recycling can also be including the phosphorylase in addition to purine nucleotides, phosphodonor, microorganism, training Support the components such as based component, moisture and the metabolic by-product of bacterium.Purine nucleotides can be purified to preferable degree.Purine core The purity of thuja acid can be, for example, more than 30% (w/w), more than 50% (w/w), more than 70% (w/w), more than 80% (w/w), More than 90% (w/w) or more than 95% (w/w).

Embodiment

Below by the embodiment more specific description present invention.

Embodiment：Inosine is produced using the bacterial strain for importing mutant-type yggB gene

In the present embodiment, inosine production is carried out using the E.coli inosine productions bacterial strain for importing mutant-type yggB gene, Assess influence of the mutant-type yggB gene to inosine production.

FADRaddedd/pMWpurFKQ plants of 1 ＞ of ＜ structure E.coli inosine production strains

With FADRaddedd plants of E.coli (WO99/003988) for parental strain, inosine production bacterial strain is built. FADRaddedd plants are by destroying the purF of W3110 plants of E.coli (amidophosphoric acid phosphoribosynltransferase), purA (adenylos Amber acid enzyme), deoD (purine nucleoside phosphorylase), purR (purine synthesis repressor), add (adenosine deaminase), edd (phosphogluconate dehydrogenase) gene and the bacterial strain obtained.Imported in FADRaddedd plants and express Coded Discharge feedback suppression The saltant type purF genes of the desensitization type amidophosphoric acid phosphoribosynltransferase of system, can build the bacterial strain of efficient production inosine (WO99/003988).Herein, by electroporation (Canadian Journal of Microbiology, 43.197 (1997)) the plasmid pMWpurFKQ (being documented in European Patent publication number 1170370) for expressing saltant type purF genes is imported In FADRaddedd plants.One of transformant of acquisition is FADRaddedd/pMWpurFKQ.

2 ＞ of ＜ build mutant-type yggB gene expression plasmid

Structure is used for the matter that the mutant-type yggB gene of bar shaped bacteria is expressed in Escherichia bacteria in the following order Grain pSTV-yggB (BL2) and pSTV-yggB (BL3).

From passing through 2256 plants of (glutamic acid rods of brevibacterium lactofermentus (Brevibacterium lactofermentum) Bacterium ATCC13869) constructed by bacterial strain with the mutation of L30 types in yggB genes and dash forward with A1 types in yggB genes In the bacterial strain (Japanese Unexamined Patent Publication 2007-97573) of change, chromosomal DNA is extracted respectively.Using the chromosomal DNA of extraction as template, make With SEQ ID NO:1 (primer A；5'-gctaagcttctaaggaggtcttggctcatg-3') and SEQ ID NO:2 (primer B； Primer shown in 5'-gtaggatcctgggacacgtctgtaatcagc-3'), by PCR method (denaturation 98 DEG C -10 seconds, annealing 60 DEG C -15 seconds, 68 DEG C of extension -78 seconds, 30 circulations, TaKaRa PCR Thermal cycler PERSONAL (Takara Bio (タ カ ラ バ イ オ) company)), the DNA fragmentation of about 1.6kb of the amplification comprising each mutant-type yggB gene. PCR reactions use GXL polymerases (production of Takara Bio (タ カ ラ バ イ オ) company).In addition, design of primers is for expanding Increase the region of the ribosome binding sequence (SD sequences) comprising mutant-type yggB gene translation initiation codon upstream.SEQ ID NO:3 show with same primers by the base sequences of the PCR amplified fragments comprising yggB genes and SD sequences obtained (but It is that the yggB genes that identical sequence is shown are wild types).After each amplified fragments have been purified, disappeared with Bam HI and Hind III Change the restriction enzyme site formed at both ends.Use DNA Ligation Kit ver.2.1 (Takara Bio (タ カ ラ バ イ オ) company's production), after connecting Restriction Enzyme Bam HI and Hind the III digestion identical with use of postdigestive each amplified fragments About 3.0kb pSTV29 carriers (production of Takara Bio (タ カ ラ バ イ オ) company) fragment.With the coupled reaction solution Escherichia coli (E.coli JM109 competent cells, the production of Takara Bio (タ カ ラ バ イ オ) company) are converted, are applied to On LB agar mediums containing 50mg/L chloramphenicol, in 37 DEG C of overnight incubations.The bacterium colony that will appear on agar medium connects Kind arrives the LB fluid nutrient mediums containing 50mg/L chloramphenicol, is stayed overnight in 37 DEG C of shaken cultivations.With alkalescence-SDS methods from each culture Plasmid DNA is extracted in liquid.By restricted digestions and measure base sequence, determine the structure of plasmid, obtain pSTV-yggB (BL2) and pSTV-yggB (BL3).PSTV-yggB (BL2) is the matter for the mutant-type yggB gene expression that there is L30 types to be mutated Grain, pSTV-yggB (BL3) are the plasmids for the mutant-type yggB gene expression that there is A1 types to be mutated.Mutant-type yggB gene (L30 Type is mutated) it is by SEQ ID NO:8 " C " of the 332nd replaces with the yggB genes of " T ", encodes SEQ ID NO:The of 9 111 alanine residues replace with the YggB protein of valine.The base sequence of mutant-type yggB gene (mutation of L30 types) with The amino acid sequence of the saltant type YggB protein of identical gene code is expressed as SEQ ID NO:4 and 5.Saltant type yggB Gene (mutation of A1 types) is in SEQ ID NO:The yggB genes of TTCATTGTG are inserted into after 8 " G " of the 44th, are encoded SEQ ID NO:The YggB eggs of Cys-Ser-Leu are inserted between 9 the 14th leucine residue and the 15th trp residue White matter.The base sequence of mutant-type yggB gene (mutation of A1 types) and the amino of the saltant type YggB protein of identical gene code Acid sequence is expressed as SEQ ID NO:6 and 7.In any one plasmid, make the transcriptional orientation of mutant-type yggB gene with The transcriptional orientation of lac promoters possessed by pSTV29 carriers is identical to arrange gene.

3 ＞ of ＜ imported into FADRaddedd/pMWpurFKQ plants of E.coli inosine production strains pSTV-yggB (BL2) and pSTV-yggB(BL3)

Carried by the pSTV-yggB obtained as described above (BL2) and pSTV-yggB (BL3), and as the pSTV29 of control Body, imported into E.coli inosine productions with 2 × TSS methods (Proc.Natl.Acad.Sci.USA, 86,2172 (1989)) respectively In strain FADRaddedd/pMWpurFKQ.Respectively using one in the transformant obtained as FADRaddedd/ pMWpurFKQ/pSTV-yggB(BL2)、FADRaddedd/pMWpurFKQ/pSTV-yggB(BL3)、FADRaddedd/ pMWpurFKQ/pSTV29。

4 ＞ inosine productions of ＜

The bacterial strain being obtained as described above uniformly is applied to LB culture mediums (tryptone 10g/L, ferment containing antibiotic respectively Female extract 10g/L, NaCl10g/L, kanamycins 25mg/L, chloramphenicol 50mg/L) on tablet, in 37 DEG C of overnight incubations.Will The thalline of 1/6 tablet is seeded in the 20mL fermentation mediums in 500mL slope mouth flasks, and (seed is trained when 37 DEG C of cultures 24 are small Support).When the light absorption value of 660nm wavelength is 1.0, the fresh fermentation mediums of 20mL are added to a part for seed culture fluid, Main culture is carried out at 37 DEG C.It is interior when 3 hours are small to 24 after starting main culture to be sampled, measure bag with generally known method The remaining sugar amount and inosine and hypoxanthine amount being contained in nutrient solution.The results are shown in Table 1.Kept with the pSTV29 carriers compareed Strain is compared, and inosine production amount significantly improves in pSTV-yggB (BL2) introduction strains and pSTV-yggB (BL3) introduction strain.

[composition of fermentation medium]

[table 1]

(each numerical value is 2 cultures for inosine and hypoxanthine yield, sugar consumption amount, propagation when the culture of table 1 24 is small Average value)

(sequence table explanation)

SEQ ID NO:1、2：Primer

SEQ ID NO:3：The base sequence of fragment comprising yggB genes and SD sequences

SEQ ID NO:4：The base sequence of the mutant-type yggB gene (BL2) of Corynebacterium glutamicum 2256 (ATCC13869) Row

SEQ ID NO:5：Encoded by the mutant-type yggB gene (BL2) of Corynebacterium glutamicum 2256 (ATCC13869) The amino acid sequence of protein

SEQ ID NO:6：The base sequence of the mutant-type yggB gene (BL3) of Corynebacterium glutamicum 2256 (ATCC13869) Row

SEQ ID NO:7：Encoded by the mutant-type yggB gene (BL3) of Corynebacterium glutamicum 2256 (ATCC13869) The amino acid sequence of protein

SEQ ID NO:8：The base sequence of the yggB genes of Corynebacterium glutamicum 2256 (ATCC13869)

SEQ ID NO:9：The amino acid sequence of the YggB protein of Corynebacterium glutamicum 2256 (ATCC13869)

SEQ ID NO:10：The base sequence of the yggB genes of Corynebacterium glutamicum ATCC13032

SEQ ID NO:11：The amino acid sequence of the YggB protein of Corynebacterium glutamicum ATCC13032

SEQ ID NO:12：The base sequence of the yggB genes of Corynebacterium glutamicum ATCC14067

SEQ ID NO:13：The amino acid sequence of the YggB protein of Corynebacterium glutamicum ATCC14067

SEQ ID NO:14：The base sequence of the yggB genes of corynebacterium melassecola ATCC17965

SEQ ID NO:15：The amino acid sequence of the YggB protein of corynebacterium melassecola ATCC17965

SEQ ID NO:16：The conserved sequence of the wild type YggB protein of bar shaped bacteria

Sequence table

Claims (12)

1. a kind of bacterium with purine substance production capacity,

It is Escherichia (Escherichia) bacterium, bacillus (Bacillus) bacterium or corynebacterium ammoniagenes (Corynebacterium ammoniagenes), and

It has been modified so as to keep mutant-type yggB gene,

Wherein, the mutant-type yggB gene is the yggB genes with mutation, described to be mutated the purines thing for making above-mentioned bacterium The production capacity of matter is improved, and

Wherein described mutation is selected from the group being made of the mutation of following (a)-(c)：

(a) it is inserted into Cys-Ser- between the 14th leucine residue and the 15th trp residue of wild type YggB protein The mutation of Leu；

(b) the 111st alanine residue of wild type YggB protein is replaced with into the mutation of valine residue；With

(c) combination of the mutation of (a) and (b).

2. the bacterium described in claim 1, wherein, the wild type YggB protein is by SEQ ID NO:9,11,13 or 15 The protein of shown amino acid sequence composition.

3. the bacterium described in claim 1 or 2, it has been modified so that participating in the activity of the enzyme of purine substance biosynthesis Increase.

4. bacterium according to any one of claims 1 to 3, wherein the purine substance is selected from inosine, xanthosine, guanosine and gland Glycosides.

5. bacterium according to any one of claims 1 to 3, wherein the purine substance is selected from inosinicacid, xanthosine monophosphate and bird Thuja acid.

6. bacterium according to any one of claims 1 to 5 is Escherichia coli.

7. bacterium according to any one of claims 1 to 5, it is bacillus subtilis (Bacillus subtilis) or solution Bacillus amyloliquefaciens (Bacillus amyloliquefaciens).

8. bacterium according to any one of claims 1 to 5 is corynebacterium ammoniagenes (Corynebacterium ammoniagenes)。

9. a kind of method for preparing purine substance, it includes cultivating in the medium according to any one of claims 1 to 8 Bacterium, so as to accumulate purine substance in the medium, and recycles the purine substance from the culture medium.

10. a kind of method for preparing purine nucleosides, it includes cultivating in the medium according to any one of claims 1 to 8 Bacterium, so as to accumulate purine nucleosides in the medium, and recycles the purine nucleosides from the culture medium.

11. a kind of method for preparing purine nucleotides, it includes cultivating in the medium any one of claim 1~8 Bacterium, so as to accumulate purine nucleotides in the medium, and the purine nucleotides is recycled from the culture medium.

12. a kind of method for preparing purine nucleotides, it includes cultivating in the medium any one of claim 1~8 Bacterium, so as to accumulate purine nucleosides in the medium, phosphorylation is carried out to the purine nucleosides so as to generate purine nucleotides, And the recycling purine nucleotides.