Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-06-08
2001-09-04
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091100
Reexamination Certificate
active
06284495
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for producing nucleic acid substances by fermentation. Nucleic acid substances are industrially useful as materials of seasonings and the like.
BACKGROUND ART
In the production of nucleic acid substances such as inosine, guanosine and bases thereof (hypoxanthine and guanine and the like) by fermentation, there have conventionally been used mutant strains imparted with adenine auxotropy and nucleic acid analogue resistance (Japanese Patent Publication (KOKOKU) Nos. Sho 55-2956 and Sho 55-45199) with a limited amount of adenine substances in their culture medium.
Mutant strains obtained by usual mutagenesis procedures are often introduced with mutations in their genes other than the target gene. In addition, because complicated regulation mechanisms are involved in biosynthetic pathways of nucleic acid substances, it is difficult to obtain a microorganism that produces a significant amount of nucleic acid substance. Therefore, mutant strains obtained by conventional breeding methods have not necessarily been satisfactory ones.
On the other hand, there has been disclosed a method for producing inosine or guanosine by utilizing a bacterium belonging to the genus Bacillus exhibiting enhanced purine operon expression obtained by modification of a gene sequence encoding enzymes involved in the purine biosynthesis (purine operon) (Japanese Patent Unexamined Publication (KOKAI) No. Hei 3-164185). This method is characterized in that a promoter or operator of the purine operon is modified to increase the expression level of the operon, thereby increasing the production of inosine or guanosine.
Expression of the purine operon of
Bacillus subtilis
(purEKBC(ORF)OLFMNHD where ORF represents an open reading frame of unknown function) is suppressed by an excessive amount of adenine, and also regulated by attenuation caused by guanine. Further, a repressor protein binding to the 5′ flanking region of the purine operon and a gene encoding the protein (purR) have been isolated, and it has been reported that, in a
Bacillus subtilis
strain whose purR gene was disrupted, suppression by adenine as for the expression of purC-lacZ fusion gene integrated into the purine operon was reduced to about {fraction (1/10)} (
Proc. Natl. Acad. Sci. USA,
92, 7455-7549 (1995)).
In
Bacillus subtilis
, the repressor protein has been known to regulate the expression of, in addition to the genes of the purine operon, purA gene involved in the biosynthesis of adenine and genes of pyrimidine operon involved in the pyrimidine biosynthesis (1997,
J. Bacteriol.
179, 7394-7402, H. Zalkin).
On the other hand, in
Escherichia coli
, it has been reported that the purine operon repressor also affects the expression of glyA gene involved in the biosynthesis of glycine, which is a substance of 5′-IMP (inosinic acid) biosynthesis (1990,
J. Bacteriol.
172, 3799-3803, H. Zalkin et al.), and the expression of gcv operon genes involved in the production of C
1
and CO
2
supplied from glycine (1993,
J. Bacteriol.
175, 5129-5134, G. V. Stauffer et al.) in addition to the genes of the purine operon.
As described above, several reports have been made about the relationship between the purine operon and inosine or guanosine production. However, there are various biosynthesis pathways involving the purR gene. Further, the purine operon of
Bacillus subtilis
encodes ten enzymes, and involved in many reactions. Thus, the biosynthesis pathways of nucleic acid substances are very complicated, and the relationship between the purR gene and accumulation of nucleic acid substances has scarcely been known.
DESCRIPTION OF THE INVENTION
The object of the present invention is to provide a method for more advantageously producing nucleic acid substances compared with conventional methods in view of the industrial importance of nucleic acid substances.
The present inventors earnestly conducted studies about the function of purR gene in order to achieve the aforementioned object, and as a result they found that
Bacillus subtilis
strain whose purR gene had been disrupted accumulated a nucleic acid substance. Thus, the present invention has been accomplished.
That is, the present invention provides:
(1) a method for producing a nucleic acid substance comprising steps of: cultivating in a culture medium a microorganism whose repressor protein for purine operon does not function in a normal manner to produce and accumulate the nucleic acid substance in the culture medium, and collecting the substance from the culture medium;
(2) the method defined in the above (1), whrerin the repressor protein does not function in a normal manner because a gene encoding the repressor protein on a chromosome of the microorganism is disrupted;
(3) the method defined in the above (1), wherein the repressor protein has the amino acid sequence shown in SEQ ID NO: 6;
(4) the method defined in the above (1), wherein the microorganism is a bacterium belonging to the genus Bacillus;
(5) The method defined in the above (4), wherein the microorganism is
Bacillus subtilis;
(6) the method defined in the above (1), wherein the nucleic acid substance is a nucleic acid base, nucleoside or nucleotide; and
(7) the method defined in the above (6), wherein the nucleic acid substance is selected from the group consisting of hypoxanthine, uracil, guanine and adenine.
For the purpose of the present invention, the expression “repressor protein for purine operon does not function in a normal manner” means that the repressor protein binds to the 5′ flanking region of the purine operon, and hence the function for suppressing transcription of the operon is reduced compared with the normal level, or it is substantially eliminated.
In the present invention, the nucleic acid substance includes nucleic acid bases such as hypoxanthine, adenine, guanine, uracil, thymine and cytosine, nucleosides such as inosine, adenosine, guanosine, uridine, thymidine and cytidine, nucleotides such as inosinic acid, adenylic acid, guanylic acid, uridylic acid, thymidylic acid and cytidylic acid, and compounds composed of any one of those nucleosides or nucleotides whose ribose is replaced with deoxyribose. Among these, the nucleic acid bases are preferred. In the present invention, the nucleic acid substance includes both of purine compounds and pyrimidine compounds. Such purine compounds include purine base, and nucleosides and nucleotides having purine base. The pyrimidine compounds include pyrimidine base, and nucleosides and nucleotides having pyrimidine base. In the present invention, hypoxanthine, adenine and guanine are preferred as the purine compounds, and uracil is preferred as the pyrimidine compound
REFERENCES:
patent: 3-164185 (1991-07-01), None
Meng et al.,Eur. J. Biochem.,vol. 187, (1990), pp. 373-379.*
Weng et al.,PNAS,vol. 92, (Aug. 1995) pp. 7455-7459.*
Rebecca L. Wilson et al, Journal of Bacteriology, “Roles of the GcvA and PurR Proteins in Negative Regulation ofEscherichia coliGlycine Cleavage Enzyme System” Aug. 1993, pp. 5129-5134.
John G. Steiert et al, Journal of Bacteriology, Regulation of theEscherichia coliglyA Gene by the purR Gene Product, Jul. 1990, pp. 3799-3803.
Byung Sik Shin et al, Journal of Bacteriology, “Interaction ofBacillus subtilisPurine Repressor with DNA” Dec., 1997, pp. 7394-7402.
Sato Katsuaki
Usuda Yoshihiro
Ajinomoto Co. Inc.
Ketter James
Oblon, Spivak, McClelland, Maier & Neustadt
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