KR20160078694A - Mutant corynebacterium ammoniagenes strain with enhanced 5'-inosinic acid production and method for preparing of 5'-inosinic acid using the same - Google Patents

Abstract

The present invention relates to a method for producing Corynebacterium ammoniagenes mutant strain having the productivity of 5′-inosinic acid (IMP) having reinforced activities of transketolase, transaldolase, glucose-6-phosphate 1-dehydrogenase, hetero multimeric protein, and 6-phosphogluconolactonase, and to a method for producing IMP by using the same. The strain according to the present invention not only reinforces a promoter of a major gene so as to enhance metabolism of a pentose sugar circuit but also preferably reduces an activity of a glucose isomerase of a central metabolism route which is a competition circuit, so IMP productivity is increased compared to Corynebacterium ammoniagenes which is a base strain.

Description

[0001] The present invention relates to a 5'-inosinic acid, a 5'-inosinic acid, a 5'-inosinic acid, a 5'-inosinic acid, a 5'-inosinic acid, a 5'-inosinic acid, the Same}

The present invention relates to a Corynebacterium ammoniagenes mutant strain having the ability to produce 5'-inosinic acid (IMP) enhanced in pentose cycle, a process for producing the same, and a process for producing 5'-inosinic acid (IMP) using the same.

5'-inosinic acid (5'-inosinic acid or inosine monophosphate (IMP)) is an intermediate of the nucleic acid biosynthetic metabolic pathway and plays an important physiological role in the body of plants and animals. And it is one of the nucleic acid-based seasoning which is widely used as a seasoning with high synergy of taste when it is used with sodium glutamate in particular.

Examples of the method for producing 5'-inosinic acid include a method of enzymatically degrading ribonucleic acid extracted from yeast cells (Japanese Patent Publication No. 1614/1957), a method of chemically phosphorylating inosine produced by fermentation (Agri. Biol. Chem., 36, 1511 (1972)), and a method of culturing a microorganism capable of producing 5'-inosinic acid and recovering 5'-inosinic acid accumulated in the medium.

Of these methods, the most widely used method is the method of producing 5'-inosinic acid by using microorganisms. For example, a method of producing Corynebacterium ammoniagenes by fermentation of a strain of the genus Corynebacterium , which is used for producing 5'-inosine acid (Korean Patent Publication No. 2003-42972) , Or a glucose-6-phosphate isomerase is enhanced in the activity of 5'-inosine acid (Korean Patent Laid-Open Publication No. 2009-0095138 And the like are known.

Meanwhile, such conventional 5'-inosine monophosphate (IMP) high-producing strains amplify genes of de novo pathway, which is a purine-based nucleic acid material biosynthesis pathway, to increase nucleic acid production such as IMP, which is a final product. The biosynthetic pathway can be roughly divided into a precursor transport step through a 5-cell cycle and a nucleic acid biosynthetic step. The latter, which is the active stage of the latter, has reached a limit in improving productivity, It is necessary to develop more advanced nucleic acid producing strains by activation.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made efforts to develop a Corynebacterium ammoniagenesis strain capable of efficiently producing 5'-inosinic acid (IMP). As a result, the transketolase, transaldolase, glucose-6-phosphate 1-dehydrogenase, heteromeric protein (hetero- multimeric protein and 6-phosphogluconolactonase to increase the ability to produce 5'-inosinic acid (IMP), thereby completing the present invention.

Accordingly, an object of the present invention is to provide a Corynebacterium ammoniagenes mutant strain having the ability to produce 5'-inosinic acid (IMP).

Another object of the present invention is to provide a method for producing the Corynebacterium ammoniagenes mutant strain of the present invention described above.

It is another object of the present invention to provide a process for preparing 5'-inosinic acid (IMP).

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a transketolase, a transaldolase, a glucose-6-phosphate 1-dehydrogenase, a heterologous protein (IMP) having an enhanced activity of 6-phosphogluconolactonase and a heteromultimeric protein capable of producing 5'-inosinic acid (IMP). The present invention also provides a mutant strain of Corynebacterium ammoniagenes.

The present inventors have made efforts to develop a Corynebacterium ammoniagenesis strain capable of efficiently producing 5'-inosinic acid (IMP). As a result, the transketolase, transaldolase, glucose-6-phosphate 1-dehydrogenase, heteromeric protein (hetero- multinomial protein and 6-phosphogluconolactonase, the productivity of 5'-inosinic acid (IMP) can be increased.

According to one embodiment of the present invention, said activity is selected from the group consisting of a tkt gene encoding transketolase, a tal gene encoding transaldolase, a zwf gene encoding glucose-6-phosphate 1-dehydrogenase, Encoding opcA gene and the 6-phosphogluconolactonase-encoding pgl gene, respectively, are enhanced with increased expression.

As used herein, the expression "increase in expression" means that the expression level of the gene is increased over the original expression level. If there is no gene to increase expression in the microorganism prior to mutagenesis, one or more of the genes may be introduced into the microorganism to increase the expression, and a gene to increase expression in the microorganism before mutation is present , One or more genes may be introduced into the microorganism in the same manner or may be genetically engineered so that the expression level of an existing gene is increased. For example, when a gene that increases expression is present in a microorganism to be mutated, the expression of the gene is increased by replacing a native promoter that drives the expression of the gene with a strong promoter .

According to another embodiment of the present invention, the increase of the expression is caused by the fact that the promoter of the gene cluster constituting the operon with the tkt, tal, zwf, opcA and pgl genes encodes superoxide dismutase (sod) (Sod promoter).

As used herein, the expression " attenuation of activity " means attenuating expression of a protein through nucleotide substitution, insertion, deletion, or a combination thereof that encodes the gene.

According to another embodiment of the present invention, the strain further comprises an activity-impaired glucose isomerase, more preferably the strain is a pgi nucleotide sequence which is a gene encoding glucose isomerase The activity of glucose isomerase is weakened through substitution, insertion, deletion, or a combination thereof. More preferably, the strain has a deletion initiation codon for the pgi gene substituted with GTG in ATG to weaken the activity of glucose isomerase Lt; / RTI >

Therefore, the Corynebacterium ammoniagenesis mutant strain of the present invention not only enhances the promoter of the major gene for enhancing the metabolic flow of the pentose cycle, but also preferably weakens the expression of the pgi gene in the central metabolic pathway, Step was added to enhance the flow of the pentacene circuit, which is the beginning of nucleic acid metabolism (Figure 4).

According to another embodiment of the present invention, the strain having enhanced activity of the 5-valent main enzyme has a yield of 5'-inosinic acid (IMP) of 3-15% as compared with that of the parent strain of Corynebacterium ammoniagenes. , And more preferably by 5-10%.

According to another embodiment of the present invention, the activity of the pentasaccharide main enzyme is enhanced and the strain which weakens the activity of the glucose oxidase (glucose isomerase) in the central metabolic pathway, which is a competing circuit, is the parent corynebacterium ammonia The production amount of 5'-inosinic acid (IMP) is increased by 5-30%, more preferably by 8-25%, and even more preferably by 10-20%, compared with the genetic strain.

According to another aspect of the present invention, the present invention provides a method for the production of transketolase, transaldolase, glucose-6-phosphate 1-dehydrogenase, (IMP) comprising the step of enhancing the activity of 6-phosphogluconolactonase, which is a heteromultimeric protein, and a method for producing a Corynebacterium ammoniagenes mutant strain having the ability to produce 5'-inosinic acid (IMP) .

The method for producing the Corynebacterium ammoniagenesis mutant strains having the ability to produce 5'-inosinic acid (IMP) of the present invention is characterized in that the Corynebacterium ammonia genus strain having the ability to produce 5'-inosinic acid (IMP) Since mutation strains are to be produced, the description common to both is omitted in order to avoid the excessive complexity of the present specification.

According to another embodiment of the present invention, the method further comprises the step of attenuating the activity of the phosphoglucose isomerase of the Corynebacterium ammoniagenes mutant strain.

Strengthening and weakening the activity in the method of the present invention can be carried out through known methods. For example, CaCl ₂ method (Cohen, SN et al, Proc Natl Acac Sci USA, 9:..... 2110-2114 (1973))..., One method (Cohen, SN et al, Proc Natl Acac. (Dower, WJ et al., Nucleic Acids (1983)), and Hanahan, D., J. Mol. Biol., 166: 557-580 Res., 16: 6127-6145 (1988)).

According to another embodiment of the present invention, the method comprises isolating and obtaining the Corynebacterium ammoniagenes mutant strain transformed from the untransformed Corynebacterium ammoniagenes strain.

In the present invention, a selectively labeled vector may be used in carrying out the step of isolating and obtaining the Corynebacterium ammoniagenes mutant strain transformed from the Corynebacterium ammoniagenesis strain which has not been transformed.

In addition, the vector used in the present invention includes, but is not limited to, a plasmid or a phage.

The selectable marker used in the present invention includes an antibiotic resistance gene commonly used in the art and includes, for example, antibiotic resistance genes such as ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline There is a resistance gene for resistance to the disease, preferably a resistance gene for ampicillin or kanamycin in view of cost.

According to another aspect of the present invention, the present invention provides a method for producing 5'-inosinic acid (IMP) comprising the steps of: (a) culturing the mutant strain of Corynebacterium ammoniagenes, Culturing in a medium; And (b) recovering 5'-inosinic acid (IMP) from the medium.

The strain used in the present invention can be cultured through a known culture method. As the medium, natural medium or synthetic medium can be used. Examples of the carbon source of the medium include glucose, sucrose, dextrin, glycerol and starch. Examples of the nitrogen source include peptone, meat extract, yeast extract, dried yeast, soybean cake, urea, thiourea, Rate and other organic or inorganic nitrogen-containing compounds may be used, but are not limited to these components.

Examples of inorganic salts contained in the medium include, but are not limited to, phosphates such as magnesium, manganese, potassium, calcium and iron, nitrates, carbonates, chlorides and the like.

Amino acids, vitamins, nucleic acids and related compounds may be added to the medium in addition to the carbon source, the nitrogen source and the components of the inorganic salt.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides a Corynebacterium ammoniagenes mutant strain having the ability to produce 5'-inosinic acid (IMP), a method for producing the same and a method for producing 5'-inosinic acid (IMP) using the same.

(ii) The Corynebacterium ammoniagenes mutant strain of the present invention not only enhances the promoter of the major gene for enhancing the metabolic flow of the pentose cycle, but also preferably enhances the glucose uptake of glucose isomerase of the central metabolic pathway Activity, resulting in increased production of 5'-inosinic acid (IMP) as compared to wild-type Corynebacterium ammoniagenes.

Figure 1 shows the structure of the plasmid pK19mobsacB according to an embodiment of the present invention.
2 shows the structure of the plasmid pK19msbpgi (A1G) according to another embodiment of the present invention.
Figure 3 shows the structure of the plasmid pK19msbPPP according to another embodiment of the present invention.
FIG. 4 shows the production route of 5'-inosinic acid (IMP) of Corynebacterium ammoniagenes mutant strain according to another embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

In the examples below, methods not specifically mentioned use commonly used molecular biological methods. For example, plasmid DNA isolation, restriction enzyme treatment, ligation, and standard transformation of E. coli are described by Sambrook et al. (Molecular Cloning. A Laboratory Manual (1989) Cold Spring Harbor Laboratories, USA, which is incorporated herein by reference.

Example

Example 1:

1.1 Preparation of pK19msbPPP vector for recombination

In order to enhance the expression of tkt, tal, zwf, opcA and pgl, the major genes of the pentose cycle on the genome of the nucleic acid producing strain IP1G, the upper native promoter in which these genes exist as operons was replaced by the sod promoter . To replace the native promoter 150 bp and the sod promoter in the genome of coryneform bacteria, the gene fragment of the sod promoter was amplified and cloned into the recombinant vector pK19mobsacB. At this time, 500 bp of each was amplified for the recombination in the native promoter region and cloned into a recombinant vector by overlapping PCR method with the sod promoter.

This plasmid is named pK19sodPPPP and is shown in Fig. To construct this plasmid, each gene fragment was amplified by the PCR method according to the following conditions using the primer shown in Table 1 below and then cloned.

primer Remarks sodP fragment amplification primer Forward 5'-accctacttagctgccaatt-3 ' First sequence Reverse 5'-gttctcctttcgtaggtttc-3 ' Second sequence Left homologous arm fragment amplification primer Forward 5'-GAACCAAAGGACGCGCGCGT-3 ' Third sequence Reverse 5'-CTTATACGTAGACAAAAGTT-3 ' Fourth sequence Right homology arm fragment amplification primer Forward 5'-GTGTCTTCTTCGAACCTCTC-3 ' Fifth sequence Reverse 5'-TCGCCGGCTGGAGTCTCTGG-3 ' 6th sequence

The above primers were used to carry out the PCR reaction under the following conditions. (DATP, dCTP, dGTP, dTTP) was added to each 100 μM deoxynucleotide triphosphate using a thermocycler (TP600, TAKARA BIO Inc., JAPAN) 10 ng of ATCC 6872 chromosomal DNA was used as a template in 25-30 cycles in the presence of 1 unit of pfu-X DNA polymerase mixture (bioneer).

The PCR reaction was carried out at 94 ° C for 30 seconds, at 54 ° C for 30 seconds, and at 72 ° C for 1 minute to 3 minutes (giving a polymerization time of 2 minutes per 1 kb).

The thus-prepared gene fragment was cloned into pK19mobsacB using appropriate restriction enzymes, transformed into E. coli DH5a, plated on an LB-agar plate containing 50 mu g / ml of kanamycin, and the resulting colonies were separated to obtain inserts insert was precisely present in the vector, this vector was separated and used for the recombination of Corynebacterium ammoniagenes IP1G.

1.2 Preparation of pK19pgi (A1G) vector for recombination

In addition, a recombinant vector was prepared to replace the initiation codon of the pgi gene with GTG in ATG to enhance the pentose cycle of the IP1G strain and attenuate the central metabolic pathway (corresponding process). On the IP1G genome, the 500 bp region of the left arm and the 500 bp region of the right arm were amplified by PCR using an over-repetition PCR method and cloned into a recombinant vector pK19mobsacB. This plasmid is named pK19msbpgi (A1G) and is shown in Fig. To construct this plasmid, each gene fragment was amplified by the PCR method according to the following conditions using the primer shown in Table 2 below and then cloned.

primer Remarks pK19mobsacB vector amplification primer Forward 5'-gggcggttttatggacagcaagcga-3 ' Seventh sequence Reverse 5'-ggcgagcggtatcagctcactcaaa-3 ' Eighth sequence pgi left homology arm fragment amplification primer
(Initiation codon substitution) Forward 5'-aaccgtattaccgcctttgagtgagctgatacc
gctcgccAGGCGGCCGCGGCCGCAGCG-3 ' Ninth sequence Reverse 5'-GGTTGTTGCGTTATTTCCACGATTGACCTTT
CGATACCAG-3 ' Tenth sequence pgi right homology arm fragment amplification primer
(Initiation codon substitution) Forward 5'-GTGGAAATAACGCAACAACC-3 ' Eleventh sequence Reverse 5'-gctggcaattccggttcgcttgctgtccataaaa
ccgccc / CTGCGCAGTGCCTGGGCTGC-3 ' 12th sequence

The above primers were used to carry out the PCR reaction under the following conditions. (DATP, dCTP, dGTP, dTTP) was added to each 100 μM deoxynucleotide triphosphate using a thermocycler (TP600, TAKARA BIO Inc., JAPAN) 10 ng of ATCC 6872 chromosomal DNA was used as a template in 25-30 cycles in the presence of 1 unit of pfu-X DNA polymerase mixture (Solgent).

PCR was carried out under conditions of 30 seconds at 94 ° C, 30 seconds at 54 ° C, and 1 minute to 2 minutes at 72 ° C (giving a polymerization time of 2 minutes per 1 kb).

The thus-prepared gene fragment was cloned into pK19mobsacB using appropriate restriction enzymes, transformed into E. coli DH5a, plated on an LB-agar plate containing 50 mu g / ml of kanamycin, and the resulting colonies were separated to obtain inserts insert was precisely present in the vector, this vector was separated and used for the recombination of Corynebacterium ammoniagenes IP1G.

As a common procedure in the above method, the amplification of the corresponding genes was amplified by PCR method from C. ammoniagenes ATCC 6872 genomic DNA, digested with appropriate restriction enzymes according to the strategy, and then amplified by pK19mobsacB And selected from E. coli top10F 'or JM110. Optionally, cloning was performed in pK19mobsacB using the Gibson Assembly Cloning Kit (NEB). Chromosomal site direct mutagenesis (base substitution, promoter switch) was performed by individually amplifying the fragment gene and preparing a target DNA fragment by an overlapping PCR method. Ex Taq Polymerase (Takara) and Pfu Polymerase (Solgent) were used as PCR amplification enzymes. Various restriction enzymes and DNA modifying enzymes were used according to the supplied buffer and protocols.

Example 2:

2.1 Preparation of strain IP2G

The IP2G strain was prepared using the pk19 msbPPP vector mentioned above. This vector was prepared to have a final concentration of 1 ug / ul or more, and primary recombination was induced in Corynebacterium ammoniagenes IP1G strain by electroporation (FEMS Microbiology Lettes. 123: 343-347, 1994). At this time, the electroporated strain was plated on an LB agar plate containing 20 ug / ul of kanamycin, and the colonies were separated and confirmed by PCR and sequencing analysis to confirm that they were properly inserted into the genome-derived site. The thus-isolated strain was further cultured overnight in a LB agar medium containing 10% sucrose to induce secondary recombination, and the colonies were isolated on a sucrose agar plate of the same concentration. After confirming the resistance of the antibiotic kanamycin among the finally isolated colonies, it was confirmed by nucleotide sequencing that the sod promoter was replaced at a desired position among strains without antibiotic resistance (Gene, 145 (1994) 69-73].

2.2 Preparation of strain IP3G

IP3G strains were prepared by the same procedure as above for the final confirmed strain of IP2G. Pk19msbpgi (A1G) vector to a final concentration of 1 ug / ul or more, and then subjected to electroporation (FEMS Microbiology Lettes. 123: 343-347, 1994) on Corynebacterium ammoniagenes IP2G strain to obtain a first recombinant Lt; / RTI > At this time, the electroporated strain was plated on an LB agar plate at 20 ug / ul and the colonies were separated and confirmed by PCR and sequencing analysis that they were properly inserted into the genome-derived site. The thus isolated strain was further cultured overnight in an LB agar medium containing 10% sucrose to induce secondary recombination, and the colonies were separated by streaking on an LB agar plate containing sucrose at the same concentration. After confirming the resistance of the antibiotic kanamycin among the finally isolated colonies, it was confirmed by nucleotide sequencing that ATG, which is the initiation codon of pgi gene, was subsitituted by GTG among strains without antibiotic resistance (Gene, 145 (1994) 69-73.

Example 3:

3.1 Preparation of 5'-Inosine Acid

In order to determine the improvement of the ability to produce 5'-inosinic acid, the productivity of the Corynebacterium ammoniagenes IP2G obtained in Example 2.1 was measured. To this end, Corynebacterium ammoniagenes IP3G strain was cultured on an agar plate for 24 hours and seeded in a preliminary culture medium (seed medium) (10 ml liquid amount / 100 ml Erlenmeyer flask).

The preliminary cultures were cultured in a rotary shaker at 180 rpm at 30 DEG C for 20 hours. 1 ml of the preliminary culture was transferred to the main medium prepared in advance and inoculated. At this time, 9 ml medium was prepared in the same 100 ml baffle flask so that the inoculation ratio was 10% (1 ml / 9 ml). The ideal preliminary medium and the production medium are shown in Table 3. The main culture is cultured in the same rotary shaker at 180 rpm at 30 DEG C for 48 hours. Finally, the content of 5'-inosinic acid in the supernatant of the culture was measured. The experimental results are shown in Table 4.

The method for determining whether the 5'-inosinic acid production ability of the Corynebacterium ammoniagenes IP3G obtained in Example 2.2 was improved was also performed in the same manner as in the measurement of the productivity of the above IP2G, and the results of the experiment are shown in Table 5 .

ingredient Seed (reserve) medium content Main (production) medium content H ₃ PO ₄ (75%) 5 g / L 11.71 g / L KOH (45%) 5 g / L 12.89 g / L NaOH (33%) 3 g / L 6.97 g / L glucose 40 g / L 50 g / L fruit sugar - 50 g / L Corn dip 5 g / L 3% (w / v) (NH _{4) 2} SO ₄ - 2 g / L Urea 1 g / L 2.5 g / L L-Cysteine 15 mg / L 20 mg / L β-Alanine 15 mg / L 20 mg / L Thi-HCl 5 mg 5 mg Nicotinic acid 15 mg 5 mg Biotin 60 ug 50 ug Mono-sodium glutamate - 1 g / L CaCl ₂ 1g 100 mg / L MgSO ₄ 5 mg / L 5 g / L MnSO ₄ 5 mg / L 10 mg / L ZnSO ₄ 1 mg / L 10 mg / L FeSO ₄ - 5 mg / L Adenine 100 mg / L 85 mg / L Guanine 100 mg / L 45 mg / L KOH (45%) 5 g / L 12.89 g / L CaCO ₃ - 5 g / L

Strain OD610 5 'inosine (g / L) IP1G 64.1 19.2 IP2G 65.2 20.4

Strain OD610 5 'inosine (g / L) IP1G 62.8 19.5 IP2G 64.8 20.8 IP3G 71.2 22.6

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> DAESANG Corporation <120> Mutant Corynebacterium ammoniagenes Strain with Enhanced          5'-inosinic acid Production and Method for Preparing          5'-inosinic acid Using the Same <130> PN140607 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplifying sodP fragment <400> 1 accctactta gctgccaatt 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for amplifying sodP fragment <400> 2 gttctccttt cgtaggtttc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplifying left homology arm fragment <400> 3 gaaccaaagg acgcgcgcgt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for amplifying left homology arm fragment <400> 4 cttatacgta gacaaaagtt 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplifying right homology arm fragment <400> 5 gtgtcttctt cgaacctctc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for amplifying right homology arm fragment <400> 6 tcgccggctg gagtctctgg 20 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplifying pK19mobsacB vector <400> 7 gggcggtttt atggacagca agcga 25 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for amplifying pK19mobsacB vector <400> 8 ggcgagcggt atcagctcac tcaaa 25 <210> 9 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplifying pgi left homology arm fragment <400> 9 aaccgtatta ccgcctttga gtgagctgat accgctcgcc aggcggccgc ggccgcagcg 60                                                                           60 <210> 10 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for amplifying pgi left homology arm fragment <400> 10 ggttgttgcg ttatttccac gattgacctt tcgataccag 40 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplifying pgi right homology arm fragment <400> 11 gtggaaataa cgcaacaacc 20 <210> 12 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for amplification <400> 12 gctggcaatt ccggttcgct tgctgtccat aaaaccgccc ctgcgcagtg cctgggctgc 60                                                                           60

Claims (9)

Transketolase, transaldolase, glucose-6-phosphate 1-dehydrogenase, heteromimeric protein, and 6-phosphoglucono A mutant strain of Corynebacterium ammoniagenes having the ability to produce 5'-inosinic acid (IMP) enhanced in activity of 6-phosphogluconolactonase.
The method of claim 1, wherein the activity is selected from the group consisting of a tkt gene encoding transketolase, a tal gene encoding transaldolase, a zwf gene encoding glucose-6-phosphate 1-dehydrogenase, opcA And the pgl gene encoding the 6-phosphogluconolactonase are each enhanced by increased expression, and the Corynebacterium ammoniagenes mutant strain.
2. The method according to claim 1, wherein the increase of the expression is caused by a promoter of a gene cluster constituting the operon with the tkt, tal, zwf, opcA and pgl genes being a promoter of a gene encoding superoxide dismutase (sod) Wherein the amino acid sequence of the Corynebacterium ammoniagenes mutant strain is the amino acid sequence of SEQ ID NO:
2. The Corynebacterium ammoniagenes mutant strain according to claim 1, wherein the strain further comprises glucose-isomerase (glucose-isomerase) whose activity is weakened.
5. The method according to claim 4, wherein the strain is selected from the group consisting of Corynebacterium ammonia, which is weakened in activity of glucose isomerase through substitution, insertion, deletion, or a combination of nucleotide sequences of pgi coding for glucose isomerase Geneis mutant strains.
[Claim 6] The Corynebacterium ammoniagenes mutant strain according to claim 5, wherein the strain is a strain in which the detoxification initiation codon of the pgi gene is replaced with GTG in ATG to weaken the activity of glucose isomerase.
The method according to claim 1, wherein the strain has an increased production of 5'-inosinic acid (IMP) by 3-15% as compared to a parent strain of Corynebacterium ammoniagenes. Strain.
5. The method according to claim 4, wherein the strain has an increased production of 5'-inosinic acid (IMP) by 5-30% as compared to that of the parent strain of Corynebacterium ammoniagenes (Corynebacterium ammoniagenes) Strain.
A process for preparing 5'-inosinic acid (IMP) comprising the steps of:
(a) culturing the Corynebacterium ammoniagenes mutant strain of claim 1 in a medium; And
(b) recovering 5'-inosinic acid (IMP) from the medium.

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