KR100429926B1 - Microorganism overproducing 5’-xanthylic acid - Google Patents

Abstract

The present invention relates to a microorganism producing 5'-xanthyl acid, and more particularly to a variant strain FA120-15-9 of Corynebacterium ammonia genes KFCC 10743 having monofluoroacetate resistance. Variant strain FA120-15-9 according to the present invention exhibits resistance to monofluoroacetate affecting the biosynthesis of 5'-xanthyl acid can produce 5'-xanthyl acid in high yield and high concentration.

Description

Microorganism overproducing 5'-xanthylic acid

The present invention relates to a microorganism producing 5'-xanthyl acid, and more particularly to a variant strain FA120-15-9 of Corynebacterium ammonia genes KFCC 10743 having monofluoroacetate resistance.

Nucleic acid (nucleic acid) is mainly contained in the nucleus of the cell and contains ingredients such as beef flavored inosinate (IMP) and mushroom flavored guanylic acid (guanylate; GMP). . Since this fact is known, a method for mass production has been developed through the initial stage of extracting small amounts of nucleic acid from fish, squid, etc. in order to use the nucleic acid as a raw material in the food or pharmaceutical field. Among them, 5'-xanthylic acid (xanthylate; XMP) as a method for producing the guanylic acid is most widely used. The 5'-xanthyl acid is an intermediate product of the purine nucleotide biosynthetic metabolic system, and is converted to guanylic acid by XMP-glutamine amidotransferase. Therefore, a method of producing 5'-guanylic acid has been used by producing 5'-xanthyl acid and converting it enzymatically to 5'-guanylic acid. However, since a large amount of 5'-xanthyl acid is required for mass production of 5'-guanylic acid, studies are being conducted to mass-produce 5'-xanthyl acid.

Conventional methods for preparing 5'-xanthyl acid include a chemical synthesis method, a method for deaminoating 5'-guanylic acid prepared by decomposing ribonucleic acid in yeast, a method for adding xanthine as a precursor in fermentation broth, Production methods using microbial mutants, production methods by adding antibiotics (Japanese Patent Nos. 42-1477 and 44-4390), and production methods by adding surfactants (Japanese Patents No. 42-3835 and 42-2838) Known. Among them, in particular, a method of directly producing fermentation of 5'-xanthyl acid using microbial mutants is very industrially advantageous. Therefore, the inventors of the present invention, while continuing the research to produce high yield of 5'-xanthyl acid by using corynebacterium ammonia gene, which is an industrial microorganism used for producing nucleic acids such as inosine acid and guanylic acid, the existing corynebacterium The present invention was completed by improving the intrinsic trait possessed by Ammonia Genes KFCC 10743 to develop mutants with increased productivity of 5'-xanthyl acid.

Therefore, it is an object of the present invention to provide microorganisms capable of producing high levels of 5'-xanthyl acid.

In order to achieve the above object, the present invention provides a variant strain FA120-15-9 of Corynebacterium ammonia gene KFCC 10743 having monofluoroacetate resistance.

Hereinafter, the present invention will be described in detail.

In the present invention, after treating the mutagenic agents such as ultraviolet irradiation, N-methyl-N'-nitro-N-nitrosoguanidine (NTG) with Corynebacterium ammonia genes KFCC 10743 as a parent, The mutant strains were grown by adding monofluoroacetate (Sigma, purity 95%) to each concentration of 0 to 200 mg / L in the minimum medium of Table 1 below. Among them, strains growing at 160 mg / l of monofluoroacetate were selected and named FA120-15-9, and were deposited at the Korea Microbial Conservation Center on Dec. 7, 2001 (Accession No .: KCCM 10342).

ingredient content glucose 20g / ℓ Potassium Phosphate 1g / ℓ Dipotassium Phosphate 1g / ℓ Urea 2g / ℓ Ammonium Sulfate 3g / ℓ Magnesium sulfate 1g / ℓ Calcium chloride 100 mg / l Iron sulfate 20 mg / l Manganese sulfate 10mg / l Zinc sulfate 10mg / l Biotin 30 μg / ℓ Thiamate 0.1mg / ℓ Copper sulfate 0.8 mg / l Adenine 20 mg / l Guanine 20 mg / l pH7.2

In addition, the growth of mutant strain FA120-15-9 and parent strain (KFCC 10743) according to the present invention while culturing for 5 days at 30 ° C by adding monofluoroacetate at a concentration of 0 to 200 mg / L to the minimum medium of Table 1 above. The figure was investigated and the results are shown in Table 2 below.

Strain Monofluoroacetate concentration (mg / ml) 0 50 100 120 140 160 180 200 Parent (KFCC 10743) +++ + + - - - - - Mutant strain (FA120-15-9) +++ +++ +++ +++ ++ + - -

+: Growth,-; Lack of growth

As shown in Table 2, the parent strain Corynebacterium ammonia gene KFCC 10743 is resistant to monofluoroacetate 100 mg / L, but no growth occurred at a concentration of 120 mg / L or more. On the other hand, the variant strain FA120-15-9 according to the present invention was found to be resistant to monofluoroacetate 160 mg / l.

Fermentation media that can be used to produce 5'-xanthyl acid using the mutant strain FA120-15-9 according to the present invention include industrially inexpensive saccharide raw materials (glucose, fructose and polysaccharide hydrolysates thereof), nitrogen sources (Organic and inorganic nitrogen sources) and any medium containing inorganic salts, trace elements and vitamins necessary for the growth of microorganisms can be used.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<Example 1>

Glucose 30g / l, peptone 15g / l, yeast extract 15g / l, sodium chloride 2.5g / l, urea 3g / l, sodium chloride 2.5g / l, urea 3g / l, adenine 150mg / l, guanine 150mg / l 5 ml of the constructed seed medium (pH 7.2) were dispensed into a test tube having a diameter of 18 mm and autoclaved, and then inoculated with the mutant strain FA120-15-9 according to the present invention and incubated at 180 rpm for 30 hours at 30 ° C (cultivation). . Then, as a fermentation medium, the main medium (glucose 60g / l, magnesium sulfate 10g / l, iron sulfate 20mg / l, zinc sulfate 10mg / l, manganese sulfate 10mg / l, adenine 30mg / l, guanine 30mg / l, biotin 100 µg / l, copper sulfate 1 mg / l, thiamine hydrochloride 5 mg / l, calcium chloride 10 mg / l, pH 7.2) and starch medium (10 g / l potassium phosphate, 10 g / l potassium phosphate), Urea 7g / l and ammonium sulfate 5g / l) were prepared and autoclaved, and 29ml and 10ml were respectively dispensed into a 500ml volumetric shake flask for autoclaving and inoculated with the seed culture solution 1ml. After incubation for 90 hours at 200rpm, 30 ℃. Thereafter, the accumulation amount of 5'-xanthyl acid in the culture medium was measured and described in Table 3 below (the accumulation concentration of 5'-xanthyl acid is expressed by 5'-sodium xanthyl acid · 7H ₂ O).

Comparative Example 1

The strain was cultured in the same manner as in Example 1 except that Corynebacterium ammonia gene KFCC 10743 was used as the strain, and the accumulation amount of 5'-xanthyl acid in the culture medium was measured and the results are shown in Table 3 below. It was.

Variation FA120-15-9 Chinju KFCC 10743 5'-Xanthyl acid amount (g / l) 26.3 23.8

As shown in Table 3, it can be seen that the 5'-xanthyl acid production of the mutant strain FA120-15-9 (Example 1) according to the present invention is improved by about 10.5% compared to the parent strain KFCC 10743 (Comparative Example 1).

<Example 2>

50 ml of the seed medium in Example 1 was dispensed into a 500 ml shake Erlenmeyer flask, autoclaved and inoculated with the strain FA120-15-9 according to the present invention and incubated at 180 rpm for 30 hours at 30 ° C. Primary seed culture). Secondary seed medium (glucose 60 g / l, potassium first potassium phosphate 2g / l, potassium diphosphate 2g / l, magnesium sulfate 1g / l, iron sulfate 22mg / l, zinc sulfate 15mg / l, manganese sulfate 10mg / l, copper sulfate 1mg / l, calcium chloride 100mg / l, biotin 150µg / l, adenine 150mg / l, guanine 150mg / l, thiamate 5mg / l, antifoam 0.6ml / l, pH 7.2 ) Was autoclaved by dispensing 2 L into a 5 L experimental fermenter, and then inoculated with 50 mL of the primary seed culture medium, and then incubated at 900 rpm and 31 ° C. for 24 hours while supplying air at 0.5 vm. In addition, the culture medium was adjusted to pH 7.3 with ammonia water (secondary species culture).

Fermentation medium (glucose 151g / l, phosphoric acid 32g / l, potassium hydroxide 25g / l, adenine 198mg / l, guanine 119mg / l, iron sulfate 60mg / l, zinc sulfate 42mg / l, manganese sulfate 15 Mg / l, copper sulfate 2.4mg / l, alani salt 22mg / l, NCA 7.5mg / l, biotin 0.4mg / l, magnesium sulfate 15g / l, cystine salt 30mg / l, histidine salt 30mg / l, Calcium chloride 149mg / l, Thiamine salt 15mg / l, Antifoam 0.7ml / l, CSL 27ml / l, Tuna Extract 6g / l, pH7.3) After inoculating 1.5 liters of the secondary seed culture solution, the mixture was incubated at 400 rpm and 33 ° C. while supplying air at 1 vmv. At this time, when the residual sugar concentration during the cultivation was less than 1%, sterilized glucose was supplied to adjust the total sugar content in the fermentation medium to 30%. Subsequently, the accumulation amount of 5'-xanthyl acid in the culture medium in which the culture was completed is shown in Table 4 below (the accumulation concentration of 5'-xanthyl acid is represented by 5'-sodium xanthyl acid · 7H ₂ O).

Comparative Example 2

Except for using the parent strain Corynebacterium ammonia genes KFCC 10743 as a strain was cultured in the same manner as in Example 2, and measured the accumulation amount of 5'- xanthyl acid in the culture medium is completed in the following Table 4 It was.

Variation FA120-15-9 Chinju KFCC 10743 5'-Xanthyl acid amount (g / l) 140 128

As shown in Table 4, it can be seen that the 5'-xanthyl acid production of the mutant strain FA120-15-9 (Example 2) according to the present invention was improved by about 9.4% compared to the parent strain KFCC 10743 (Comparative Example 2).

As described above, the microorganism FA120-15-9 according to the present invention is resistant to monofluoroacetate which affects the biosynthesis of 5'-xanthyl acid, thereby producing 5'-xanthyl acid in high yield and high concentration. have.

Claims (1)

A variant of Corynebacterium ammoniagenes KFCC 10743 with monofluoroacetate resistance, which grows at a concentration of 160 mg / l or less of monofluoroacetate and produces 5'-xanthyl acid. Characterized by microorganism FA120-15-9 (Accession No .: KCCM 10342).