KR100768750B1 - 5'- 5'- A microorganism whose biotin carboxylase activity is enhanced for producing 5'-inosinic acid and method for producing 5'-inonsinic acid using the same microorganism - Google Patents

Abstract

A microorganism mutant is provided to increase the activity of biotic carboxylase, thereby increasing production concentration and yield of 5'-inosinic acid. A method for producing 5'-inosinic acid using the same is provided. A Corynebacterium ammoniagenes mutant producing 5'-inosinic acid is characterized in that an endogenous promoter of biotin carboxylase gene is substituted by a CJ1 promoter consisting of a sequence described as SEQ ID : NO. 1. The Corynebacterium ammoniagenes is Corynebacterium ammoniagenes CJKTS93 deposited as a deposition number of KCCM 10779. To produce 5'-inosinic acid, the Corynebacterium ammoniagenes mutant is cultured.

Description

A 'microorganism whose biotin carboxylase activity is enhanced for producing 5'-inosinic acid and method for producing 5'-inonsinic acid using the same microorganism}

1 is a diagram showing the results of the development of the Corynebacterium ammonia genes cell extract sample after two-dimensional electrophoresis analysis and silver staining and the function of the identified protein.

2 is a diagram showing the function of biotin carboxylase.

3 is a view showing a manufacturing process of the recombinant vector p119-bc.

4 is a diagram illustrating a manufacturing process of the recombinant vector pcj1-bc.

FIG. 5 is a diagram illustrating a process for preparing a genetically modified strain in which a promoter of biotin carboxylase is replaced with a cj1 promoter.

The present invention relates to a microorganism producing 5'-inosinic acid and a method of producing 5'-inosinic acid using the same. More specifically, the present invention Corynebacterium ammonia gene ( Coynebacterium ammoniagenes ) (formerly Brevibacterium ) A genetically engineered strain of CJHB100 (KCCM 10330), which produces 5'-inosinic acid at high concentration and high yield by direct fermentation, compared to CJHB100 strains. CJKTS93 (KCCM 10779) and a method for producing 5'-inosinic acid using the same.

5'-inosinic acid is an intermediate of the nucleic acid biosynthetic metabolic system, which not only has a physiological significance in the body of plants and animals, but is also used in various fields for food, medicine, and various medical purposes. In particular, when used together with sodium glutamate, It is one of nucleic acid-based seasonings that has a great synergistic effect and has been spotlighted as a seasoning seasoning.

Microorganisms having 5'-inosine acid production ability and methods for producing 5'-inosine acid by direct fermentation using them are well known. The most important thing in the method of producing 5'-inosine acid through microbial fermentation is the yield and economics of 5'-inosine acid production.

To date, the development of microorganisms producing high concentrations and high yields of 5'-inosine acid has relied on methods of selecting strains that are mainly resistant to various chemical analogues, with an emphasis on the final product 5'-inosine acid. However, the method of imparting resistance to these chemical analogues is to randomly induce modifications to the genes of the microbial genome and to select strains that have become resistant by this. Specifically, the yield of production of 5'-inosinic acid by It is not known whether it has been improved, and it may be accompanied by side effects such as decreased microbial activity and delayed fermentation time.

Accordingly, the present inventors measure the proteome change according to the culture stage of the 5'-inosinic acid producing strain, to select the protein whose activity is inhibited as the fermentation progresses, and based on the nucleotide sequence information The promoter was replaced with a stronger promoter to enhance the activity of the gene. As a result, the present invention was completed by developing a microorganism producing high concentration and high yield of 5'-inosinic acid by direct fermentation.

An object of the present invention is to increase the activity of biotin carboxylase to provide microbial mutants with increased production concentration and yield of 5'-inosinic acid.

Still another object of the present invention is to provide a method for producing 5'-inosinic acid comprising culturing a microbial mutant strain having increased activity of the biotin carboxylase.

In order to achieve the above object, the present invention provides a Corynebacterium ammonia genes strain that produces 5'-inosinic acid in which the intrinsic promoter of the biotin carboxylase gene is substituted with the CJ1 promoter.

Corynebacterium ammonia gene strain according to the present invention as shown in Figure 2, biotin carboxyla, an enzyme that catalyzes the reaction of fixing carbon dioxide to biotin, a kind of vitamin which is the first gateway in the biosynthetic pathway of fatty acids The 5'-inosine acid is produced in high yield and high concentration by replacing the promoter of the aze with a stronger promoter to increase its expression.

In one aspect of the invention, the intrinsic promoter of biotin carboxylase is derived from Corynebacterium ammonia genes CJHB100 (KCCM 10330).

According to one aspect of the invention, the step of separating the chromosome of Corynebacterium ammonia genes CJHB100 (KCCM 10330), the isolated chromosome as a template and the primer pair of SEQ ID NO: 1 and 2 and SEQ ID NO: 3 and 4, respectively Amplifying a portion of the CJ1 promoter and biotin carboxylase gene by performing PCR using the same; cloning the fragments of the amplified CJ1 promoter and biotin carboxylase gene into the pCR2.1-T0PO vector, respectively, Cloning the cloning vector of the CJ1 promoter and the cloning vector of the biotin carboxylase into SalI and EcoRV and SalI and BamH1 , respectively, and conjugating with T4 ligase, and the conjugated product to E. coli by electroshock. Transforming, separating the recombinant plasmid from the transformed Escherichia coli and the isolated recombinant plasmid Corynebacter which produces 5'-inosinic acid in which the promoter of the biotin carboxylase gene is substituted with the CJ1 promoter by a recombinant microorganism production method consisting of transforming Corynebacterium, a 5'-inosinic acid producing strain with mid. Prepare aium ammonia genes strain.

In one aspect of the invention, the CJ1 promoter of biotin carboxylase consists of the sequence of SEQ ID NO: 7.

In one aspect of the invention, the Corynebacterium ammonia genes strain is Corynebacterium ammonia genes CJKTS93 (KCCM 10779).

The microorganism Corynebacterium ammonia genes CJKTS93 (KCCM 10779) of the present invention is a genetic variant of Corynebacterium ammonia genes CJHB100 (KCCM 10330). Corynebacterium ammonia genes CJHB100 (KCCM 10330) [see Patent Publication No. 10-2003-0042972] used as a parent strain in the present invention is a variant of Brevibacterium ammonia genes ATCC6872 Leeum ammonia genes CJIP009 (KCCM 10226) (see Korean Patent Publication No. 10-0397321). Therefore, the microorganism according to the present invention shares the following main characteristics and effects of Corynebacterium ammonia genes CJHB100 (KCCM 10330). In other words,

i) properties of xanthine and guanine leaky mutants that are adenine-required and xanthine and guanine non-constitutive but promote growth upon addition of xanthine or guanine;

ii) urease defect properties capable of magnetizing urea;

iii) having a high sensitivity to lysozyme, a cell wall degrading enzyme, and partially deficient in cell wall synthesis ability to secrete a large amount of 5'-inosinic acid produced in the cell to the outside of the cell;

iv) resistance to streptomycin, which allows for effective response to contamination during fermentation;

v) The normal physiological activity of microorganisms is inhibited by osmotic pressure outside the cells caused by the accumulation of high concentrations of glucose and other carbon sources in the culture medium, or by the accumulation of 5'-inosinic acid produced in the culture, resulting in slowing of cell growth and consequently In order to prevent the degradation of 5'-inosinic acid production, analogs of proline to increase the intracellular concentration of proline (L-proline), which can play an important role in osmotic pressure regulation and confer osmotic resistance traits Endows resistance to osmotic pressure;

vi) properties of resistance to analogs of glutamine, essentially required for purine biosynthesis, such as azaserine and DON (6-diazo-5-oxo-L-norleucine);

vii) analogs of valine, leucine and isoleucine known to promote purine biosynthesis upon addition of fermentation medium, such as valine hydroxamate, norvaline, methylvaline, thiisoleucine, isoleucine hydroxamate, Properties with resistance to leucine hydroxamate and leucine methyl ester;

viii) has properties of resistance to analogs of tetrahydrofolate, known as monocarbon carriers, in the purine biosynthesis system, for example, trimethoprim, pyrimetamine, methotrexate and aminopterin.

In addition, Corynebacterium ammonia genes CJKTS93 (KCCM 10779) according to the present invention is an enzyme that catalyzes the reaction of fixing carbon dioxide to biotin, a kind of vitamin which is the first gateway in the biosynthetic pathway of fatty acids, in addition to the above properties. The expression of biotin carboxylase can be increased to directly accumulate 5'-inosinic acid in culture with high yield and high concentration.

Unlike conventional methods that focus on the final product 5'-inosine acid, the microorganism according to the present invention increases the sugar consumption rate and cell mass of the 5'-inosine acid producing strain, leading to high efficiency and high concentration production of 5'-inosine acid. As shown in Figure 1 in order to obtain the analysis of the expression of the entire protein by the fermentation stage of the 5'-inosinic acid producing strain, the change with the passage of the fermentation time was observed. Based on the overall comparison data, the target genes contribute to 5'-inosinic acid production by identifying, comparing and analyzing proteins by metabolic pathway. They are involved in fatty acid synthesis and carbon dioxide fixation, which are essential for maintaining cell activity. The biotin carboxylase was found to decrease as the expression level increases.

As a method for increasing the expression level of a specific gene, there is a method of increasing the number of genes of one microorganism. For this purpose, a plasmid in which the number of copies per microorganism is maintained is usually used. In other words, by inserting the desired gene into the plasmid and transforming the microorganism with the recombinant plasmid can be expected to increase the gene by the number of copies of the plasmid per one microorganism. In this way attempts to improve the productivity of 5'-inosinic acid have been reported in part success. However, the technique using such plasmids causes a large burden on the host microorganism by overexpressing only a specific gene in most cases, and causes problems such as plasmid stability such that plasmids are lost during cultivation of recombinant strains.

Therefore, in the present invention, a method of inserting a specific gene into the chromosomal DNA was used to compensate for this disadvantage. That is, as shown in Fig. 5, in the Corynebacterium ammonia genes, the gene that exchanged the intrinsic promoter of biotin carboxylase with a stronger promoter (CJ1 promoter of SEQ ID NO: 7) is replaced with the same gene present in the chromosome The technique was applied.

As mentioned in the description and examples of the present invention, the nutrient medium (Note 1), the minimal medium (Note 2), the seed medium (Note 3), the flask fermentation medium (Note) used for the cultivation of microorganisms in the present invention 4), fermenter seed medium (Note 5), fermenter This medium (Note 6) means a medium consisting of the composition as shown in Table 1, respectively. However, the medium composition is not limited to those shown in Table 1, and any culture medium suitable for the culture of Corynebacterium ammonia genes can be used.

Medium composition used in the present invention Badge type Badge composition Note 1 1% peptone, 1% gravy, 0.25% sodium chloride, 1% yeast extract, 2% agar (pH 7.2) Note 2 Glucose 2%, Sodium sulfate 0.3%, Potassium phosphate 0.1%, Dipotassium phosphate 0.3%, Magnesium sulfate 0.3%, Calcium chloride 10mg / l, Iron sulfate 10mg / l, Zinc sulfate 1mg / l, Manganese chloride 3.6mg / l , L-cysteine 20mg / l, calcium pantothenate 10mg / l, thiamine hydrochloride 5mg / l, biotin 30μg / l, adenine 20mg / l, guanine 20mg / l, (pH 7.3) Note 3 Glucose 5%, Peptone 0.5%, Juicy 0.5%, Yeast Extract 1%, Sodium Chloride 0.25%, Adenine 100mg / l, Guanine 100mg / l (pH 7.2) Note 4 Sodium glutamate 0.1%, ammonium chloride 1%, magnesium sulfate 1.2%, calcium chloride 0.01%, iron sulfate 20mg / l, manganese sulfate 20mg / l, zinc sulfate 20mg / l, copper sulfate 5mg / l, L-cysteine 23mg / l, alanine 24mg / l, nicotinic acid 8mg / l, biotin 45ug / l, thiamine hydrochloride 5mg / l, adenine 30mg / l, phosphoric acid (85%) 1.9%, fructose, glucose and molasses are added to reduce the sugar to 8% (pH 7.2) Week 5 Glucose 5.4%, Peptone 1%, Yeast extract 2%, Potassium phosphate 0.1%, Dipotassium phosphate 0.1%, Magnesium sulfate 0.1%, Ammonium sulfate 0.5%, Iron sulfate 80mg / l, Zinc sulfate 40mg / l, Sulfuric acid Manganese 40 mg / l, L-cysteine 80 mg / l, calcium pantothenate 60 mg / l, thiamine hydrochloride 20 mg / l, biotin 240 μg / l, adenine 1200 mg / l, guanine 1200 mg / l (pH 7.2) Week 6 Calcium chloride 120mg / l, copper sulfate 8mg / l, magnesium sulfate 1.5%, iron sulfate 24mg / l, zinc sulfate 24mg / l, manganese sulfate mg / l, L-cysteine 26.4mg / l, sodium glutamate 0.12%, thiamine hydrochloride 6mg / l, Biotin 40μg / l, Nicotinic Acid 50mg / l, Alanine 145mg / l, Adenine 200mg / l, Phosphoric Acid (85%) 4.3%, Fructose, Glucose and Molasses mixed to 32% as reducing sugar (pH 7.2 )

The present invention also provides a method of producing 5'-inosinic acid comprising culturing a Corynebacterium ammonia genes variant in which the intrinsic promoter of the biotin carboxylase gene is substituted with the CJ1 promoter.

Corynebacterium ammonia genes CJKTS93 (KCCM 10779) according to the present invention is incubated in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins and the like under aerobic conditions while controlling the temperature, pH and the like.

As the carbon source, carbohydrates such as glucose, fructose, sterilized pretreated molasses (ie, molasses converted to reducing sugars) and the like can be used. As nitrogen sources, various inorganic nitrogen sources such as ammonia, ammonium chloride, ammonium sulfate and peptone, NZ-amine, Organic nitrogen sources such as meat extracts, yeast extracts, corn steep liquors, casein hydrolysates, fish or degradation products thereof, skim soy cakes, or degradation products thereof can be used.

As the inorganic compound, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and the like may be used. In addition, vitamins and nutrient-containing bases may be added as necessary.

The culturing is carried out under aerobic conditions, for example by shaking culture or aeration stirred culture, preferably at a temperature of 20 to 40 ° C. The pH of the medium is preferably maintained near neutral during incubation, the incubation lasted for 5-6 days, and the 5'-inosinic acid accumulated by direct fermentation was analyzed according to conventional methods.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated concretely based on an Example. However, it does not mean that the scope of the present invention is limited by the embodiment in any form.

Example

In an embodiment of the present invention, by culturing Corynebacterium ammonia genes CJHB100 (KCCM 10330) to prepare a cell extract according to the culture step, by performing two-dimensional electrophoresis to select the overexpressed protein at each culture period, selection The peptide protein was digested to analyze peptide sequences, and the obtained peptide sequence data was used to identify genes of the proteins, and the identified proteins were analyzed by metabolic pathways to find target genes contributing to 5'-inosinic acid production.

Example  One : Corynebacterium Ammonia Genes CJHB100  ( KCCM  10330) Comparison of protein expression level and target gene selection according to culture and time of culture

(1) Cell culture

Corynebacterium ammonia genes CJHB100 (KCCM 10330) was incubated in a medium containing raw hydrolyzate (50% glucose + 50% fructose). The concentration of cells in the culture was measured, culture samples were collected at the initial and stationary phases, centrifuged to remove the supernatant, and the obtained cells were crushed in crushing buffer to obtain 100 μl of cell extract.

(2) 2D electrophoresis analysis

460 μl of rehydration solution (6M urea, 2M thiourea, 4% CHAPS, 0.4% DTT, 1.9% IPG buffer and 0.8% bromophenol blue (BPB)) containing the cell extract from the cell extract obtained in (1) Was prepared. The rehydration solution was Immobiline DryStrip ^TM Reswelling Tray added to the slot of the (Amersham Bioscience, USA) and placed on the 24 cm, Immobiline DryStrip ^TM pH gradient gel (pH 3-10, nonlinear, Amersham Science , USA) of 2ml cover Liquid (Amersham Bioscience, USA) was added and rehydrated at room temperature for about 24 hours. The rehydrated strip gel was adjusted to 1 h at 0-100 V, 1 h at 300 V, 1 h at 600 V, and 8000 V at a time of about 43-97 kVhr (pH 4-7: 43.4 kVhr, pH 4.5-5.5, pH 5.5-6.7: 97 kVhr), Isoelectric Focusing (IEF) was performed at 20 ° C using a Multiphor II isoelectric focusing device (Amersham Bioscience, USA).

Upon completion of the IEF, each strip gel was equilibrated for 15 minutes in a solution containing 20 mM Tris-HCl, 6M urea, 2% SDS, 20% glycerol, 2.5% acrylamide and 5 mM TBP at pH 8.8. Each equilibrated strip was placed on a two-dimensional gel (9-16% gradient) and sutured with SDS solution containing 0.5% low boiling agarose and 0.001% BPB, and electrophoresis was performed at 100 V for about 19 hours.

After electrophoresis, the gel was fixed with 45% methanol and 5% acetic acid solution, and then acetic acid was washed with distilled water for 1 hour. It was sensitized with 0.02% sodium thiosulfate for 2 minutes and washed with distilled water. Next, the gel was reacted with 0.1% silver nitrate for 20 minutes and then washed again with distilled water. It was then developed with a solution containing 2% (w / v) sodium carbonate and 0.04% (v / v) formaldehyde to stop the reaction with 1% acetic acid when spots of the desired intensity appeared. Finally the gel was washed with distilled water, stored in a sealed plastic bag and stored at 4 ° C.

In case of Coomassie staining, the gel was taken out, fixed with 30% methanol and 10% acetic acid solution for 1 hour, washed briefly with distilled water, and stained with colloidal Coomassie Brilliant G-250 for 24 hours. Destained for 4 hours with% acetic acid solution.

(3) Preparation of peptide sample for mass spectrometry from spot

Separation of peptides from the spot was used by modifying known methods (Shevchenko et al. Anal. Chem., 68 (5), 850-8, 1996).

First, protein spots were cut from the gel, destained in 120 μl of a 1: 1 mixture of 30 mM potassium ferricyanide and 100 mM sodium thiosulfate, washed with distilled water and then again 120 μl of 50% acetonitrile / 25 mM Washed with ammonium bicarbonate, pH 7.8 for 10 minutes. It was reacted with 50 μl of 100% acetonitrile for about 5 minutes until white, followed by vacuum drying.

10 μl of 0.02 μg / μl two-dimensional electrophoretic trypsin was added to each dried spot for 45 minutes on ice, followed by addition of 50 mM ammonium bicarbonate buffer, pH 7.8 and reaction at 37 ° C. for 12 to 14 hours. I was. Peptides were extracted by three sonication methods in 10 μl 0.5% TFA, 50% acetonitrile for 10 minutes.

(4) mass spectrometry

Peptides extracted as above were analyzed by HPLC-MS / MS. HPLC-MS / MS used a 1100 series HPLC system (Agilient, USA) using a Finnigan LCQ DECA ion-trap mass spectrometery (ThermoQuest, USA) equipped with a nano spray ionization source. HPLC uses a C18 microprobe reversed phase column, flowing 0.1% formic acid (solvent A) and 90% (v / v) acetonitrile and 0.1% formic acid solution (solvent B) in a linear gradient (flow rate 1 μl / min) Peptides were separated.

Detection of peptides was based on the following nano spray ionization (NSI) criteria:

Spray voltage: 1.8 kV; Capillary temperature: 200 ° C .; Capillary voltage: 34 V; Tubular lens offset: 40 V; Electron multiplier: -60 V.

All measurements were collected in centroid mode after 3 measurements, and after a full MS scan of 400-2000 Da, the threshold was fixed at 1x10 ⁵ counts to scan the most powerful ions at high resolution. And then subjected to collision-induced dissociation (CID) MS / MS. Sequences of uninterpreted CID spectra were identified using TurboQuest software (Thermo Finnigan, USA), and SEQUEST search results were confirmed by cross correlation and ΔCn (delta normalized correlation).

Identification and identification of the amino acid sequence of the peptides analyzed in this way were analyzed for amino acid sequences using Q-star Pulsar LC MS / MS (Applied Biosystems, USA).

As a result, 50 proteins were identified, and among them, proteins essential for carbon dioxide fixation and fatty acid synthesis were selected. 1 is a view showing the results of development after performing a two-dimensional electrophoresis analysis of the sample of the present embodiment and silver staining. As shown in Figure 1, as the fermentation proceeds it can be seen that the amount of expression of the target gene is significantly reduced. The function of the biotin carboxylase thus identified protein is shown in FIG. The function of these proteins was confirmed by comparing the obtained peptide sequences with amino acid sequences of known NCBI gene bank databases.

The protein identification process and the function confirmation process are shown in FIG. ORF sites were selected by analyzing the gene sequence of biotin carboxylase, the target protein identified as described above.

Example  2 : cj1  Promoter and Biotin  Of the recombinant gene of the carboxylase gene Cloning

(1) Amplification of the cj1 promoter sequence from the genome of Corynebacterium ammonia genes CJHB100 and a portion of the biotin carboxylase gene ORF sequence

According to the method of Eikmann et al. (Gene, 102, 93-98, 1991), 500 µg of chromosomal DNA was isolated from 25 ml of Corynebacterium ammonia genes CJHB100 cultured for 1 day. Primer set using the isolated chromosome as a template and for amplifying the CJ1 promoter and biotin carboxylase of SEQ ID NO: 7 (for CJ1 promoter: SEQ ID NOs: 1 and 2, for biotin carboxylase: SEQ ID NOs: 3 and 4) PCR was performed using 30 cycles of 1 cycle consisting of denaturation at 94 ° C. for 30 seconds, annealing at 55 ° C. for 30 seconds, extension at 72 ° C. for 30 seconds, and part of the cj1 promoter sequence and the biotin carboxylase ORF sequence. Was amplified.

(2) Construction of recombinant vector

Each fragment obtained through the process of (1) was cloned into the pCR2.1-TOPO vector (Invitrogen, USA) as shown in Figs. 3 and 4, and then again to Sal I, EcoR V for the cj1 promoter, Biotin carboxylase gene fragment was digested with EcoR V and BamH I, respectively, to determine the Sal I, EcoR V and EcoR V and BamH I of pECCG119 (Korean Patent Publication No. 2006-0079297). It was conjugated to each other using T4 DNA ligase in place. Conjugated DNA was transformed into E. coli GM2929 by electric field shock to recover single colonies grown in LB solid medium containing antibiotics to 50mg of kanamycin per liter. Determine the size of the included to remove the plasmid from the obtained by culturing in the same antibiotic of the recovered colonies was added LB medium cells were plasmid as the primary and the double-cut back to Sal I, BamH I as the secondary this fragment of 1kb size comes out By confirming the recombinant plasmid Pcj1-BC containing a part of the biotin carboxylase gene was produced.

Example  3: Construction and Selection of Chromosome Insertion Strains of Recombinant Plasmids

First, 5'-inosinic acid-producing strain Corynebacterium CJHB100 (KCCM 10330) was transformed with recombinant plasmid Pcj1-BC isolated from Escherichia coli GM2929 and added to 10 mg / L kanamycin added to CM solid medium (see Note 1). Single colonies that were grown to spread were recovered. The recovered colonies were cultured in the CM medium to which the same antibiotic was added, and the plasmids were separated from the cells and treated with Nhe I to remove the origin of replication of Corynebacterium from the recombinant plasmids. The DNA fragment from which the origin of replication was removed was recovered through a 1% agarose gel, and T4 DNA ligase was used to join both ends to form a circular DNA from which the replication term was removed, and then to 5'-inosine producing strain. The cells were transformed by electric field bombardment and plated onto CM solid medium containing 10 mg of kanamycin per liter, and single colonies were recovered.

The chromosomal DNA was isolated from the cells obtained by culturing the recovered colonies in a CM medium to which the same antibiotic was added, and then PCR was carried out using primers of SEQ ID NOs: 5 and 6 using the isolated chromosomes as templates (for 30 seconds at 94 ° C). One cycle consisting of denaturation, annealing for 30 seconds at 55 ° C., and extension for 2 minutes at 72 ° C. was repeated 30 times). Colonies in which the CJ1 promoter and the biotin carboxylase were expressively inserted were selected. Thereby, recombinant strains were finally selected in which the endogenous promoter of the biotin carboxylase was substituted with the CJ1 promoter (SEQ ID NO: 7). The process is shown in FIG.

Example  4 variant strains Fermentation potency  Experiment

(1) Fermentation titer experiment in 500 ml Erlenmeyer flask

Use strain: CJKTS93 (KCCM 10779)

Species: See note 3 in table 1

Flask Fermentation Medium: See Note 4 in Table 1

Fermentation method: 3 ml of the seed medium was dispensed into a 18 cm diameter test tube and inoculated with the strain used after autoclaving according to the conventional method, followed by shaking culture at 30 ° C. for 24 hours, and used as a seed culture solution. 27 ml of fermentation medium was dispensed into a 500 ml shake flask for 3 minutes, sterilized at 120 ° C. for 10 minutes, inoculated with 3 ml of the seed culture solution, and then cultured for 5 to 6 days. The rotation speed was adjusted to 200 times per minute at a temperature of 32 ° C. and pH 7.2. As a result of the culture, the accumulation amount of 5'-inosinic acid in the medium was 15.25 g / l. This was better than the result of incubating the parent strain CJHB100 under the same conditions (14.65g / l), the cell mass also showed a 15% increase than the parent strain.

(2) Fermentation titer test in 5-L fermenter

Use strain: CJKTS93 (KCCM 10779)

Species medium: same as Example 3

Fermenter Seed Medium: See Note 5 in Table 1.

Fermenter Main Medium: See Note 6 in Table 1

Fermentation method: 50 ml of the seed medium was dispensed into a 500 ml shaking Erlenmeyer flask and inoculated using strain after autoclaving according to the conventional method, and shake cultured at 30 ° C. for 24 hours to use as a seed culture solution. 1000 ml of the fermenter seed medium were placed in a 2.5 L-fermentation tank and autoclaved at 15 ° C. for 15 minutes to inoculate 50 ml of the seed culture solution, followed by incubation for 1-2 days. Rotation speed was adjusted to 900 times per minute, temperature 28 to 34 ℃, pH 7.2.

Fermenter 1250ml of this medium was added to a 5L-fermenter and autoclaved at 120 ° C. for 15 minutes to inoculate 250ml of fermenter seed culture medium.Then, when the reducing sugar was 2% in the culture medium, the primary, secondary, tertiary, Fourth additional sugar was mixed with fructose, glucose and molasses and added to be 32% as the final reducing sugar, followed by incubation for 5 to 6 days. Rotation speed was adjusted to 900 times per minute, temperature 30 ℃, pH 7.2. At this time, the accumulation amount of 5'-inosinic acid in the medium was 73.9g / l. The culture of parent strain CJHB100 under the same conditions was better than that of the result (71.9 g / l), and the final fermentation time was also shortened by 3 hours compared to the parent strain.

As described above, when 5'-inosinic acid is produced using the Corynebacterium ammonia genes variant according to the present invention, 5'-inosinic acid can be economically produced at a higher concentration and higher yield by direct fermentation. have.

Attach sequence list electronic file  

Claims (3)

A Corynebacterium ammonia genes strain that produces 5'-inosinic acid, wherein the intrinsic promoter of the biotin carboxylase gene is substituted with a CJ1 promoter consisting of the sequence of SEQ ID NO: 7. The Corynebacterium ammonia gene strain of claim 1, which is Corynebacterium ammonia genes CJKTS93 (KCCM 10779). A method for producing 5'-inosinic acid comprising the step of culturing the Corynebacterium ammonia genes strain according to claim 1 or claim 2.

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