Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-10-25
2002-02-05
Tate, Christopher R. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S252300, C435S253600, C435S832000, C435S836000, C435S839000
Reexamination Certificate
active
06344344
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a fermentative method for producing uridine-5′-monophosphate, and to microorganisms for use in such methods.
Uridine-5′-monophosphate is useful as a biochemical reagent or as a starting material for synthesizing pharmaceuticals, see WO 95/16785; EP Patent Specification 0 149 775 B1; US Pat. No. 5,422,343; Clin. Exp. Pharmacol. Physiol. 14(3):253 (1987); and Chim-Pharm. J. (Russia) 27(7):27 (1993).
BACKGROUND ART
Methods of enzymatic production of uridine-5′-monophosphate using uracil and orotic acid as substrates known, so far are described by Agr. Bio 1. Chem. 35:518 (1971) and Biosci. Biotech. Biochem. 61(6):956 (1997).
Known methods for producing uridine-5′-monophosphate by direct fermentative processes include a method using various strains belonging to genus of Streptomyces (Japanese Patent laid-Open Publication No.49-031117) and a method using Micromonosporum resistant to analogues of uracil, uracil ribosides or uracil ribotides, (Japanese Patent laid-Open Publication No.57-018873).
A further process that uses strains of the genus Brevibacterium endowed with a resistance to purine analogues is disclosed in Japanese Patent laid-Open Publication No.57-30476.
A method for producing uridine-5′-monophosphate using strains of Corynebacterium (Brevibacterium) ammoniagenes (hereafter referred to as
Corynebacterium ammoniagenes
) having resistance to pyrimidine analogues is proposed in SU Patent 923186. Yet another later process using strains of
Corynebacterium ammoniagenes
endowed with resistance to pyrimidine analogues is described in Japanese Patent laid-Open Publication No. 4-158792.
A process using strains
Corynebacterium ammoniagenes
obtained from an inosine-5′-monophosphate producer, which strains take on an ability to produce significant amounts of uridine-5′-monophosphate due to sensitivity to the bacteriostatic action of adenine, is proposed in SU Patent 1446927. These strains were characterized by a high resistance to pyrimidine analogues.
Further attempts to increase the productivity of uridine-5′-monophosphate producing strains
Corynebacterium ammoniagenes
by imparting additional properties to them also have been made. For example, it has been found that the productivity of uridine-5′-monophosphate producing strains
Corynebacterium ammoniagenes
can be greatly improved by endowing the adenine-sensitive mutants with resistance to sulfaguanidine, SU Patent 1782028, RU Patent Application 95112366, or to arsenate, SU Patent 1832127. The highest level of uridine-5′-monophosphate accumulation (22.0 g/1) was achieved by using the strain VKPM B-6307 described in RU Patent Application 95112366.
Although the processes exemplified result in improved yields of uridine-5′-monophosphate, from the standpoint of commercial application the production yields of such processes are comparatively low. Thus, a need exists for a process for producing uridine-5′-monophosphate in higher yields at low cost.
A method of producing uridine using a microorganism which belongs to the genus Bacillus, which is deficient in uridine nucleoside phosphorylase activity and which is resistant to pyrimidine analogues, is known, U.S. Pat. No. 4880736. However, it was not known that coryneform bacteria having properties similar to the above microorganism would efficiently produce uridine-5′-monophosphate.
DISCLOSURE OF THE INVENTION
The present invention has been made taking the aforementioned viewpoints into consideration. An object of the present invention is to provide a more efficient method for the production of uridine-5′-monophosphate in high yields for industrial purposes and microorganisms which can be used in such a method.
To this end, the present inventors after many studies on bacteria producing uridine-5′-monophosphate, found that microorganisms belonging to
Corynebacterium ammoniagenes
and having resistance to pyrimidine analogues produce and accumulate a considerable quantity of uridine-5′-monophosphate in a medium. The investigation of a series of mutants showed the direct correlation between resistance to pyrimidine analogues and accumulation of uridine-5′-monophosphate. The higher this resistance: the more the uridine-5′-monophosphate yield. Moreover, it has now been found that certain newly discovered mutants of the known strains, which are resistant to at least one pyrimidine analogue and which are deficient in uridine degrading ability, produce higher yields of uridine-5′-monophosphate and exhibit decreased by-production of uracil than the previously known strains.
Heretofore, it was not recognized that the productivity of uridine-5′-monophosphate could be improved by endowing an uridine-5′-monophosphate producing microorganism with such traits.
Therefore work was continued on the basis of this finding to complete the present invention.
Thus, the present invention is as follows.
(1) Coryneform bacterium which has a resistance to growth inhibition by pyrimidine analogues in a minimal medium containing guanine, arginine and casamino acids, and has an activity to produce uridine-5′-monophosphate.
(2) The bacterium according to (1), wherein the pyrimidine analogue is selected from the group consisting of 6-azauracil, 2-thiouracil, 5-fluorouracil, a riboside thereof or a ribotide thereof.
(3) The bacterium according to (2), wherein the pyrimidine analogues is 5-fluouridine.
(4) The bacterium according to (1), wherein the bacterium belongs to
Corynebacterium ammoniagenes.
(5) The bacterium according to (4), wherein the bacterium is
Corynebacterium ammoniagenes
LK
40-2
(VKPM B-7811).
(6) A coryneform bacterium which has a resistance to growth inhibition by pyrimidine analogues and has a weaker uridine degrading activity, and has an activity to produce uridine-5′-monophosphate.
(7) The bacterium according to (6), wherein the pyrimidine analogue is selected from the group consisting of 6-azauracil, 2-thiouracil, 5-fluorouracil, a riboside thereof or a ribotide thereof.
(8) The bacterium according to (7), wherein the pyrimidine analogues is 5-fluouridine.
(9) The bacterium according to (6), wherein the bacterium belongs to
Corynebacterium ammoniagenes.
The bacterium according (9), wherein the bacterium is
Corynebacterium ammoniagenes
LK 75-15 (VKPM B-7812) or LK 75-66 (VKPM B-7813). (11) A method for producing uridine-5′-monophosphate by fermentation comprising the steps of cultivating the bacterium illustrated in any one of above (1) to (10) in a medium to produce and accumulate uridine-5′-monophosphate in the culture, and recovering the uridine-5′-monophosphate therefrom.
The present invention will be explained in detail below.
The microorganisms of the present invention may be obtained from microorganisms inherently having an ability to produce uridine-5′-monophosphate by imparting thereto the specified resistance or the weaker uridine degrading activity.
The term “microorganism which is resistant to a pyrimidine analogue” means a microorganism derived from a strain of bacteria belonging to coryneform bacteria as the parent strain and that has been modified in its genetic properties such that it can grow in medium containing a pyrimidine analogue. The term “pyrimidine analogue” means a substance similar in structure to a pyrimidine base, such as uracil, for example 6-azauracil, 2-thiouracil, 5-hydroxyuracil, 5-fluorouracil, ribosides of these or ribotides of these. It is sufficient that a pyrimidine analogue-resistant microorganism has resistance to one pyrimidine analogue. Usually, a mutant resistant to one type of pyrimidine analogue is resistant to at least one additional pyrimidine analogue.
Thus, as used herein the term “resistant to growth inhibition by pyrimidine analogue” means that mutant is capable of growing in nutrient medium containing pyrimidine analogue as an inhibitor in an amount which would inhibit the grow of the parent strains. As an amount of th
Kazarinova Lyudmila Anatolievna
Livshits Vitaliy Arkadievich
Preobrazhenskaya Ekaterina Sergeevna
Starovoytova Irina Mikhailovna
Ajinomoto Co. Inc.
Srivastava Kailash C.
Tate Christopher R.
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