Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-10-25
2004-08-17
Prouty, Rebecca (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S252300, C435S252330, C435S320100, C435S088000, C536S023200
Reexamination Certificate
active
06777208
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for producing a 2′-deoxyribonucleosides such as 2′-deoxyguanosine and a microorganism suitably used for the method. 2′-Deoxyribonucleosides are useful as raw materials of drugs, intermediate thereof and so forth.
2. Description of the Related Art
As methods for producing 2′-deoxyribonucleosides, there are known chemical synthesis methods, methods of extracting them from hydrolysates of DNA and biochemical production methods.
As the biochemical methods, there are known methods of producing 2′-deoxyribofuranosylpurine and 2′-deoxyribofuranosylthioguanine (Japanese Patent Laid-open Publication (Kokai) No. 58-63393), 2′-deoxycytidine (Japanese Patent Laid-open Publication No. 01-060396) and 2′-deoxythymidine (Japanese Patent Laid-open Publication No. 01-104190) by using nucleoside phosphorylases of microorganisms.
Further, as methods that utilize microorganisms, there have been disclosed a method of producing 2′-deoxyadenosine from deoxyribose-1-phosphate or a salt thereof and adenine, adenosine or 5′-adenylic acid, a method of producing 2′-deoxyadenosine from 2′-deoxyuridine or thymidine and adenine, adenosine or 5′-adenylic acid in the presence of inorganic phosphoric acid or a salt thereof, a method of producing 2′-deoxyguanosine from 2′-deoxyribose-1-phosphate or a salt thereof and guanine, guanosine or 5′-guanylic acid, and a method of producing 2′-deoxyguanosine from 2′-deoxyuridine or thymidine and guanine, guanosine or 5′-guanylic acid in the presence of inorganic phosphoric acid or a salt thereof (Japanese Patent Laid-open Publication No. 11-137290).
There has also been reported a method of producing a 2′-deoxyribonucleoside-5′-phosphate from a ribonucleotide as a raw material in the presence of a reducing agent such as dithiothreitol by using a recombinant type enzyme, which is obtained by isolating a gene for ribonucleoside triphosphate reductase of a
Lactobacillus bacterium
and expressing this gene in
Escherichia coli
(Brunella, A. et al.,
Journal of Molecular Catalysis B: Enzymatic,
10, 215-222 (2000)).
Furthermore, there have been reported a method of producing thymine or thymidine by culture utilizing viable microbial cells (Japanese Patent Laid-open Publication No. 2-39894).
DISCLOSURE OF THE INVENTION
Objects of the present invention is to provide a method for efficiently producing a 2′-deoxyribonucleoside such as 2′-deoxyguanosine by using a microorganism and a microorganism used for the method.
The inventors of the present invention assiduously studied in order to achieve the aforementioned objects. As a result, they found that a deoxyribonucleoside could be efficiently produced from a carbon source, ribonucleoside or base by using a microorganism having increased ribonucleotide reductase activity and decreased deoxyribonucleoside degradation activity, and thus accomplished the present invention.
That is, the present invention provides the followings.
(1) A method for producing a 2′-deoxyribonucleoside, which comprises culturing a microorganism, which is transformed with a gene encoding a ribonucleotide reductase and in which 2′-deoxyribonucleoside degradation activity is decreased or eliminated, in a medium in which the microorganism can grow to produce the 2′-deoxyribonucleoside.
(2) The method for producing a 2′-deoxyribonucleoside according to (1), wherein a ribonucleoside or base corresponding to the 2′-deoxyribonucleoside is added to the medium.
(3) The method for producing a 2′-deoxyribonucleoside according to (1) or (2), wherein the 2′-deoxyribonucleoside is 2′-deoxyguanosine.
(4) The method for producing a 2′-deoxyribonucleoside according to any one of (1) to (3), wherein the 2′-deoxyribonucleoside degradation activity of the microorganism is decreased or eliminated by disrupting a gene encoding a purine nucleoside phosphorylase on chromosomal DNA.
(5) The method for producing a 2′-deoxyribonucleoside according to any one of (1) to (4), wherein the ribonucleotide reductase does not suffer from feedback inhibition by a deoxyribonucleotide.
(6) The method for producing a 2′-deoxyribonucleoside according to any one of (1) to (5), wherein the ribonucleotide reductase is a ribonucleoside diphosphate reductase.
(7) The method for producing a 2′-deoxyribonucleoside according to any one of (1) to (6), wherein the microorganism is a bacterium belonging to the genus Escherichia.
(8) A microorganism, which is transformed with a gene encoding a ribonucleotide reductase, in which a gene encoding a purine nucleoside phosphorylase on chromosomal DNA is disrupted, and which has an ability to produce a 2′-deoxyribonucleoside.
(9) The microorganism according to (8), wherein the ribonucleotide reductase is a ribonucleoside diphosphate reductase.
According to the present invention, a 2′-deoxyribonucleoside such as 2′-deoxyguanosine can be produced by using a microorganism.
Hereafter, the present invention will be explained in detail.
The microorganism used for the present invention is a microorganism which is transformed with a gene encoding a ribonucleotide reductase, in which 2′-deoxyribonucleoside degradation activity is decreased or eliminated, and which has an ability to produce a 2′-deoxyribonucleoside from a carbon source, ribonucleoside or base.
The microorganism of the present invention will be explained hereafter.
In the present invention, preferred microorganisms include microorganisms having an ability to supply reducing power. The term “ability to supply reducing power” means an ability to supply a reducing substance (for example, reducing type of glutaredoxin) in an amount sufficient for advance of the reaction for reducing a ribonucleoside diphosphate to convert it into a 2′-deoxyribonucleoside diphosphate, which is catalyzed by a ribonucleotide reductase. As such microorganisms having ability to supply reducing power referred to in the present invention, bacteria belonging to the genus Escherichia can be mentioned, for example. Examples of such bacteria belonging to the genus Escherichia include
Escherichia coli.
The microorganism of the present invention can be obtained by decreasing or eliminating the 2′-deoxyribonucleoside degradation activity of a microorganism and then transforming it with a ribonucleotide reductase gene. The microorganism of the present invention can also be obtained by transforming a microorganism with a ribonucleotide reductase gene and then decreasing or eliminating the 2′-deoxyribonucleoside degradation activity of the transformant strain.
In order to transform a microorganism with a ribonucleotide reductase gene, specifically, a gene fragment encoding a ribonucleotide reductase can be ligated to a vector functioning in the microorganism, preferably a multi-copy type vector to produce a recombinant DNA, and it can be introduced into the microorganism to transform it.
The source of the ribonucleotide reductase gene is not particularly limited so long as it is a microorganism containing a ribonucleotide reductase. For example, there can be mentioned
Escherlchia coli, Corynebacterium ammoniagenes, Saccharomyces cerevisae, Lactobacillus leichmannii
and so forth.
In the present invention, the ribonucleotide reductase is preferably one that does not suffer from feedback inhibition by a deoxyribonucleotide. Examples of a ribonucleotide reductase include ribonucleoside diphosphate reductases and ribonucleoside triphosphate reductases.
In
Escherichia coli
, there are known three types of ribonucleoside diphosphate reductases, NrdAB, NrdDG, and NrdEF, and it has been reported that NrdAB and NrdDG suffer from feedback inhibition by a deoxyribonucleotide such as 2′-dATP, whereas NrdEF does not suffer from such feedback inhibition (
J. Biol. Chem.,
271 (43), 26582-26587 (
Hara Seiichi
Tonouchi Naoto
Yokozeki Kenzo
Ajinomoto Co. Inc.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Prouty Rebecca
Swope S
LandOfFree
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