Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-08-10
2003-06-17
Tate, Christopher R. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S006120, C435S106000, C435S115000, C435S169000, C435S252320, C435S252330
Reexamination Certificate
active
06579699
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing a fermentative product. In particular, the present invention relates to a method for producing a useful substance such as an amino acid by fermentation utilizing a microorganism, and a microorganism to which resistance to stress that suppresses growth of the microorganism and/or production of a fermentative product by the microorganism is imparted.
When a cell is exposed to stress, such as high temperature, high osmotic pressure, metabolic inhibition, presence of heavy metal, and viral infection, synthesis of a family of proteins called “heat shock protein” (abbreviated as “HSP” hereinafter) is induced in a short period of time to cause defensive reactions against the stress. These HSPs show homology in a wide range from prokaryotic cells to eukaryotic cells, and they are roughly classified into several groups such as HSP60, HSP70, HSP90, TRiC, and other groups (Hendrick, J. P. and Hartl, F.-V., Annu. Rev. Biochem., 62, 349-384 (1993)).
The mechanism of the stress resistance imparted by the HSP is based on the function of the HSP to form higher-order structure of proteins (folding of proteins). Namely, the HSP can bind to a protein that has been denatured due to stress and become unable to form a correct higher order structure, and restore the normal function of the protein by refolding it into the correct higher order structure.
Because it has been elucidated that such function of the HSP in the formation of higher order structure of proteins serves as a molecular chaperon not only for denatured proteins but also for assembly, transmembrane transport and the like of proteins in normal cells, its importance has been recognized, and is attracting attentions (Ellis, R. J. et al., Science, 250, 954-959 (1990)). The term “chaperon” implies a supporter, and the name is given because the HSP exerts its function by binding to various proteins.
The expression of the HSP is induced when a cell is exposed to the stress as mentioned above. This induction is usually temporary. It is attenuated soon and reaches a new steady state. It has been revealed that this induction of the HSP is caused at transcription level (Cowing, D. C. et al., Proc. Natl. Acad. Sci. USA, 80, 2679-2683 (1985), Zhou, Y. N. et al., J. Bacteriol., 170, 3640-3649 (1988)). It has been known that a group of HSP genes commonly have a promoter structure called heat shock promoter, and that a factor specifically functioning for this heat shock promoter, &sgr;−32 (&sgr;
32
) is present. It has also been known that &sgr;
32
is encoded by rpoH gene, and it is a protein with a very short half life, about 1 minute, and closely relates to the temporary induction of the HSP (Straus, D. B. et al., Nature, 329, 348-351 (1987)). It has been shown that the expression control of &sgr;
32
itself is attained at transcription level and translation level, but its major control is attained at translation level.
The induction of the HSP by heat shock is based on two mechanisms, i.e., increase in synthesized amount and stabilization of &sgr;
32
. Among these mechanisms, as for the increase of synthesis amount of &sgr;
32
, it has been revealed that the structure of &sgr;
32
mRNA modified by heat enhances its translation (Yura, T. et al., Annu. Rev. Microbiol., 47, 321-350 (1993)). As for the stabilization of &sgr;
32
, involvement of an HSP (DnaK etc.) in the decomposition of &sgr;
32
has been shown, and it is considered that feedback control by the HSP works (Tilly, K. et al., Cell, 34, 641-646 (1983), Liberek and K., Proc. Natl. Acad. Sci. USA, 89, 3516-3520 (1994)).
As for
Escherichia coli
(
E. coli
), a relationship between the HSP and growth of cells under the presence of stress have been known (Meury, J. et al., FEMS Microbiol. Lett., 113, 93-100 (1993)), and it has also been known that dnaK and groE affect on the-production of human growth hormones and the secretion of procollagenase, respectively (Hockney, R. C., Trends in Biotechnology, 12, 456 (1994)).
International Publication No. WO96/26289 describes that resistance to stress that inhibits microorganism growth and/or fermentative product production can be imparted to a microorganism by introducing at least one of a gene coding for an HSP and a gene coding for a &sgr; factor which specifically functions for an HSP, into the microorganism to enhance an expression amount of an HSP.
SUMMARY OF THE INVENTION
The object of the present invention is, in the production of a useful substance such as an amino acid by fermentation, to further improve productivity and yield of the fermentative product by reducing the influence of stress that inhibits microorganism growth and/or fermentative product production.
The present inventors conducted studies in order to achieve the aforementioned object. As a result, they sound that the productivity and the growth could be further improved under a high stress condition by introducing a gene coding for HSP derived from a hyperthermophilic archaeon strain KOD-1 into a microorganism to express the HSP, and accomplished the present invention.
Thus, the present invention provides a method for producing a fermentative product by utilizing a microorganism, the method comprising culturing the microorganism in a medium to produce and accumulate the fermentative product in the medium, and collecting the fermentative product, wherein the microorganism expresses an HSP derived from the hyperthermophilic archaeon strain KOD-1 in a cell of the microorganism by introduction of a gene coding for the HSP.
The present invention also provides a microorganism producing a fermentative product, which expresses an HSP derived from the hyperthermophilic archaeon strain KOD-1 in a cell of the microorganism by introduction of a gene coding for the HSP.
In the aforementioned method and-microorganism of the present invention, the fermentative product may be, for example, an amino acid such as L-threonine, L-lysine, L-glutamic acid, L-leucine, L-isoleucine, L-valine, and L-phenylalanine, a nucleic acid or a nucleoside such as guanylic acid, inosine, and inosinic acid, a vitamin, an antibiotic or the like, and an amino acid is preferred.
As the microorganism to which the present invention can be applied, bacteria belonging to the genus Escherichia and coryneform bacteria can be mentioned.
According to the present invention, in the production of useful substances such as amino acids by fermentation, the influence of stress can be further decreased, and productivity and yield can be further improved.
REFERENCES:
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patent: 5188949 (1993-02-01), Tsuchida et al.
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patent: WO 96/26289 (1996-08-01), None
Yan, Z., et al.: “In Vitro Stabilization and In Vivo Solubilization of Foreign Proteins by the Beta Subunit of a Chapernin from the Hyperthermophilic Archaeon Pyrococcus SP. Strain KOD1”, Applied and Environmental Microbiology, U.S. , Washington, D.C., vol. 63, No. 2, Feb. 1, 1997, pp. 785-789, XP000673019.
Y. Izawa, et al: “Cloning and Analysis of the Heat Shock Protein Gene from a New Hyperthermophilic Archaeon, Pyrococcus SP. Strain KOD1”, EMBL Nucleotide Sequence, XX, XX, Apr. 23, 1994, XP002029690.
Kikuchi Yoshimi
Kurahashi Osamu
Tanaka Takashi
Yokozeki Kenzo
Ajinomoto Co. Inc.
Flood Michele C.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Tate Christopher R.
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