Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing alpha or beta amino acid or substituted amino acid...
Reexamination Certificate
2000-08-08
2002-01-15
Achutamurthy, Ponnathapur (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
C435S108000, C435S110000, C435S115000, C435S128000, C435S132000, C435S170000, C435S171000
Reexamination Certificate
active
06338956
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method of producing a fermentative product. In particular, the present invention relates to a method of fermentatively producing useful substances such as amino acids by utilizing microorganisms, and to microorganisms having added resistance to stress which would otherwise restrain growth of the microorganisms and/or production of the fermentative products.
BACKGROUND ART
When cells are exposed to stress such as high temperature, high osmotic pressure, metabolic inhibition, presence of heavy metal, and viral infection, a family of proteins called “heat shock proteins” (hereinafter referred to as “HSP”) are induced and synthesized in a short period of time to cause a defense reaction against the stress. HSP presents broad homology ranging from procaryotic cells to eucaryotic cells, and it is roughly divided into several groups (HSP 60 group, HSP 70 group, HSP 90 group, TRiC group, and miscellaneous group) (Hendrick, J. P. and Hartl, F. V.,
Annu. Rev. Biochem
., 62, 349-384 (1993)).
The mechanism of stress resistance exhibited by HSP resides in the function of HSP to form higher-order structures of proteins (folding of proteins). Namely, when a protein is denatured due to stress, and becomes incapable of forming a correct higher-order structure, HSP binds to the protein, and the protein is subjected to refolding into the correct higher-order structure. Thus the protein can be returned to have its normal function.
HSP, which functions for the formation of higher-order structures of proteins as described above, has been revealed to serve as a molecular chaperon not only for denatured proteins but also for cells in a normal state through the process of protein folding, assembly, membrane transport and so on. Accordingly, its importance is recognized and widely noticed (Ellis, R. J. et al.,
Science
, 250, 954-959 (1990)). The term “chaperon” means a supporter. This designation results from the fact that HSP binds to various proteins, and it exhibits its function.
Expression of HSP is induced when cells are exposed to stress as described above. The induction is usually temporary. It attenuates soon, and a new steady state is achieved. It has been revealed that the induction of HSP is made at the transcription level (Cowing, D. C. et al.,
Proc. Natl. Acad. Sci. USA
, 80, 2679-2683 (198); Zhou, Y. N. et al.,
J. Bacteriol
., 170, 3640-3049 (1988)). It is known that each of the family of HSP genes has a promoter structure called “heat shock promoter”, and sigma-32 (&sgr;
32
) is present which is a &sgr; (sigma) factor to specifically function for the heat shock promoter. It is known that &sgr;
32
is a protein encoded by a rpoH gene, having an extremely short half-life life of about 1 minute, and it closely relates to the temporary induction of HSP (Straus, D. B. et al.,
Nature
, 329, 348-351 (1987)). It has been revealed that expression control for &sgr;
32
itself is made at the transcription level and at the translation level, however, major control is made at the translation level.
The induction of HSP by heat shock is caused by two mechanisms of increase in synthetic amount of &sgr;
32
and stabilization thereof. Among them, as for the increase in synthetic amount of &sgr;
32
, it has been already revealed that the structure of &sgr;
32
changes due to heat, and thus translation is accelerated (Yura, T. et al.,
Annu. Rev. Microbiol
., 47, 321-350 (1993)). As for the stabilization of &sgr;
32
, it has been shown that HSP (DnaK or the like) participates in degradation of &sgr;
32
, assuming that feedback control by HSP functions (Tilly, K. et al.,
Cell
, 34, 641-646 (1983); Liberek, K.,
Proc. Natl. Acad. Sci. USA
, 89, 3516-3520 (1994)).
As for
Escherichia coli
(
E. coli
), it is known that the growth of cells relates to HSP in the presence of stress as described above (Meury, J. et al.,
FEMS Microbiol. Lett
., 113, 93-100 (1993)). It is also known that production of human growth hormone is affected by dnaK, and secretion of procollagenase is affected by aroE (Hockney, R. C.,
Trends in Biotechnology
, 12, 456 (1994)). However, no relationship is known between HSP and productivity of fermentative products such as amino acids and nucleic acids and the like. As for coryneform bacteria, no relationship is known between HSP and growth, and no relationship is also known between HSP and productivity of fermentative products.
DISCLOSURE OF THE INVENTION
An object of the present invention is to clarify the relationships between HSP and growth of microorganisms and between HSP and productivity of fermentative products in order to decrease the influence of stress which restrains growth of microorganisms and/or production of fermentative products so that the productivity and the yield are improved instead of being lowered in production of useful substances such as amino acids by fermentation.
As a result of diligent investigations by the present inventors in order to achieve the object described above, it has been found out that the productivity and the growth can be improved by introducing, into a microorganism, a gene coding for HSP or a gene coding for a &sgr; factor which specifically functions for the HSP gene, and enhancing expression of HSP. Thus the present invention has been completed.
Namely, the present invention lies in a method of producing a fermentative product by utilizing a microorganism, comprising the steps of cultivating the microorganism in a medium to allow the fermentative product to be produced and accumulated in the medium, and collecting the fermentative product, wherein the microorganism is modified by introduction of at least one of a gene coding for HSP and a gene coding for a &sgr; factor which specifically functions for the HSP gene to enhance expression amount of HSP in cells, whereby the microorganism is allowed to have added resistance to stress which would otherwise restrain growth of the microorganism and/or production of the fermentative product.
In another aspect, the present invention lies in a microorganism for producing a fermentative product, wherein the microorganism is modified by introduction of at least one of a gene coding for HSP and a gene coding for a &sgr; factor which specifically functions for the HSP gene to enhance expression amount of HSP in cells, whereby the microorganism is allowed to have added resistance to stress which would otherwise restrain growth of the microorganism and/or production of the fermentative product.
In a preferred embodiment, the method and the microorganism according to the present invention deal with various fermentative products including, for example, amino acids such as L-threoriine, L-lysine, L-glutamic acid, L-leucine, L-isoleucine, L-valine, and L-phenylalanine; nucleic acids or nucleosides such as guanylic acid, inosine, and inosinic acid; and other substances such as vitamins and antibiotics.
In another preferred embodiment, the stress includes temperature, osmotic pressure of the medium, and high concentration of the fermentative product which are not preferable for the growth of the microorganism.
In still another preferred embodiment, the gene coding for the heat shock protein specifically includes groE, and the gene coding for the &sgr; factor specifically includes rpoH.
In still another preferred embodiment, the microorganism to which the present invention is applied includes bacteria belonging to the genus Escherichia, and coryneform bacteria.
The present invention will be described in detail below.
The fermentative product to which the present invention is applied is not specifically limited provided that it is those produced by fermentation by using any microorganism. The fermentative product includes those produced by microorganisms including, for example, various L-amino acids such as L-threonine, L-lysine, L-glutamic acid, L-leucine, L-isoleucine, L-valine, and L-phenylalanine; nucleic acids or nucleosides such as guanylic acid, inosine, and inosinic acid; and other substances such as vitamins and antibiotics. Even in the case of subs
Goto Shinya
Kawahara Yoshio
Kikuchi Yoshimi
Kimura Eiichiro
Kurahashi Osamu
Achutamurthy Ponnathapur
Ajinomoto Co. Inc.
Kerr Kat
LandOfFree
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