Corynebacterium ammoniagenes KCCM 10340 for producing...

Details Corynebacterium ammoniagenes KCCM 10340 for producing... Corynebacterium ammoniagenes KCCM 10340 for producing...

2002-12-19

2004-11-23

Naff, David (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S843000, C435S136000

Reexamination Certificate

active

06821768

ABSTRACT:

TECHNICAL FIELD
The invention relates to a microorganism overproducing 5′-xanthylic acid. More specifically, the invention relates to
Corynebacterium ammoniagenes
CJXPK001 which is a mutant strain of
Corynebacterium ammoniagenes
KFCC 10743 having a resistance to 3,4-dehydro-DL-proline.
BACKGROUND ART
Nucleic acid is mostly found in the nucleus of a cell and it includes ingredients such as inosinic acid (inosinate; IMP) and guanylic acid (guanylate; GMP), which have the taste of beef and mushroom respectively.
Since this has been known to the world, and after going through the early stage of extracting small amount of nucleic acid from squids or other fish in order to use it as raw material in food or pharmaceutical sectors, mass production methods were developed. Among those methods to produce the above-mentioned guanylic acid, the use of 5′-xanthylic acid (XMP) is most popular. The above-mentioned 5′-xanthylic acid is an intermediary product of purine nucleotide biosynthesis process and, by means of XMP-glutamine amidotransferase, it is converted into guanylic acid. Therefore the method which produces 5′-xanthylic acid first and converts it into 5′-guanylic acid enzymatically is used in producing 5′-guanylic acid. However in order to mass-produce 5′-guanylic acid, corresponding amount of 5′-xanthylic acid is necessary. Therefore methods to mass-produce 5′-xanthylic acid are underway.
Conventional methods to produce 5′-xanthylic acid are chemosynthesis, deaminization of 5′-guanylic acid which is produced as a result of decomposition of ribonucleic acid in yeast, a method to add xanthine, as precursor material, in fermenting medium, use of a mutant strain of microorganism, a method to add antibiotic material (JP 1477/67 and JP 20390/69), a method to add surfactant (JP 3835/67 and JP 3838/67) and so on. Among these, a direct fermentation method to use a mutant strain of microorganism to produce 5′-xanthylic acid is quite advantageous in terms of industrial aspect. While we developed into the research to produce 5′-xanthylic acid at a large yield rate by using
Corynebacterium ammoniagenes
which is industrially utilized to produce nucleic acids such as inosinic acid and guanylic acid, we were able to, by modifying the existing character of
Corynebacterium ammoniagenes
KFCC 10743, develop a mutant strain with increased productivity of 5′-xanthylic acid and accomplished in this invention.
DISCLOSURE OF THE INVENTION
The purpose of this invention is to procure microorganism capable of producing 5′-xanthylic acid at a large yield rate.
In order to achieve the above-mentioned object, this invention is aimed at providing 3,4-dehydroproline resistant CJXPK001, a mutant strain of
Corynebacterium ammoniagenes
KFCC 10743.
Henceforth the present invention will be explained in more detail.
In order to avoid dehydration by external osmotic pressure, most microorganisms increase their internal osmotic pressure by accumulating organic solute such as ionized potassium and osmolytes in their body. The above-mentioned osmolytes include proline, betaine and carnitine (Beumer, R. R., et al.,
Appl. Environ. Microbiol
. 60:1359-1363, 1994), and among them, proline is known as the most important factor in controlling osmotic pressure. Further, in
Brevibacterium lactofermentum
, it is reported that, under increased external osmotic pressure caused by 5′-xanthylic acid outside the bacterial cell, as the activity of pyrroline-5-carboxylate reductase which is an important enzyme in the biosynthetic process increases, proline is accumulated in the bacterial cell (Yoshio K. et al.,
Agr. Bio. Chem
., 53(9): 2475-2479, 1989).
It is also reported that, in case of
E. coli, Salmonella typhimurium, Serratia marcescens
and others, L-proline is accumulated in their cells and it is controlled by external osmotic pressure (V. J. Dunlap et al.,
J. Bacteriol
., 163: 296, 1985).
By providing existing
Corynebacterium ammoniagenes
KFCC 10743 with resistance to L-proline analogue and by suppressing the inhibition of L-proline synthetic process by feedback inhibition, reinforced L-proline synthesis capability was resulted, which further increased osmotic pressure resistant character of microorganism. Finally we were able to develop a mutant strain to produce 5′-xanthylic acid at a large yield rate.
To be more specific, in this innovative research, we adopted
Corynebacterium ammoniagenes
KFCC 10743 as parent strain and treated it with UV radiation and mutation derivatives such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) according to ordinary procedure and, by adding 0 to 50 mg/L of proline analogue 3,4-dehydroproline (Sigma Company) in the minimum medium shown in the following Table 1, we developed growing mutant strains. From them, a strain which grows in 30 mg/L 3,4-dehydroproline was separated, named CJXPK001. And, it was deposited under the Budapest Treaty to the Korea Culture Center of Microorganisms on Dec. 7, 2001 with accession Number KCCM 10340.
TABLE 1
Ingredient
Content
Glucose
20
g/L
Potassium phosphate monobasic
1
g/L
Potassium phosphate dibasic
1
g/L
Urea
2
g/L
Ammonium sulfate
3
g/L
Magnesium sulfate
1
g/L
Calcium chloride
100
mg/L
Ferrous sulfate
20
mg/L
Manganese sulfate
10
mg/L
Zinc sulfate
10
mg/L
Biotin
30
ug/L
Thiamine hydrochloride
0.1
mg/L
Copper sulfate
0.8
mg/L
Adenine
20
mg/L
Guanine
20
mg/L
pH 7.2
Further, after adding 0 to 50 mg/L of 3,4-dehydroproline in the minimum medium shown in the above Table 1 according to concentration levels, the medium was fermented at 30° C. for 5 days. And the whole process was scrutinized to find the growth rates of the mutant strain CJXPK001 and the parent strain KFCC 10743. The result is shown in the following Table 2.
TABLE 2
3,4-dehydroproline concentration (mg/L)
Strain
0
5
10
20
30
40
50
Parent strain
+++
+
−
−
−
−
−
KFCC 10743
Mutant strain
+++
+++
+++
++
+
−
−
CJXPK001
+: growth, −: no growth
As shown in the above Table 2, the parent strain
Corynebacterium ammoniagenes
KFCC 10743 showed resistance in 5 mg/L of 3,4-dehydroproline of proline analogue but there was no growth observed at the concentration level above 10 mg/L. However the mutant strain CJXK200101 which was obtained in the present invention showed resistance in 3,4-dehydroproline with the concentration level of up to 30 mg/L.
As for the fermentation medium candidates which can be adopted to produce 5′-xanthylic acid with the help of the mutant strain CJXPK001 of the present invention, any medium made of cheap industrial level sugar source material (glucose, fructose and/or other hydrolyzed material of polysaccharide which contains the preceding two), nitrogen source (organic or inorganic), inorganic mineral salt necessary for the growth of microorganisms, rare elements and vitamins will foot the bill.
The present invention will be specifically explained by the following examples.
However the following examples merely give some examples of the present invention and the contents of the present invention are not limited to the following examples.


REFERENCES:
patent: 2002/0192746 (2002-12-01), Takano et al.
patent: 60-156399 (1985-08-01), None
patent: 04-262790 (1992-09-01), None
Teshiba, S. et al;Production of Nucleotides and Nucleosides by Fermentation; Gordon and Breach Sci. Pub.; 1989; pp 53-63.

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