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
1997-12-05
2001-04-24
Stole, Einar (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...
C435S252320, C435S320100, C536S023200
Reexamination Certificate
active
06221636
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing L-lysine by cultivating a microorganism obtained by modifying a coryneform bacterium used for fermentative production of amino acid or the like by means of a technique based on genetic engineering.
L-Lysine, which is used as a fodder additive, is usually produced by a fermentative method by using an L-lysine-producing mutant strain belonging to the coryneform bacteria. Various L-lysine-producing bacteria known at present are those created by artificial mutation starting from wild type strains belonging to the coryneform bacteria.
As for the coryneform bacteria, there are disclosed a vector plasmid which is autonomously replicable in bacterial cells and has a drug resistance marker gene (see U.S. Pat. No. 4,514,502), and a method for introducing a gene into bacterial cells (for example, Japanese Patent Application Laid-open No. 2-207791). There is also disclosed a possibility for breeding an L-threonine- or L-isoleucine-producing bacterium by using the techniques as described above (see U.S. Pat. Nos. 4,452,890 and 4,442,208). As for breeding of an L-lysine-producing bacterium, a technique is known, in which a gene participating in L-lysine biosynthesis is incorporated into a vector plasmid to amplify the gene in bacterial cells (for example, Japanese Patent Application Laid-open No. 56-160997).
Known genes for L-lysine biosynthesis include, for example, a dihydrodipicolinate reductase gene (Japanese Patent Application Laid-open No. 7-75578) and a diaminopimelate dehydrogenase gene (Ishino, S. et al.,
Nucleic Acids Res.,
15, 3917 (1987)) in which a gene participating in L-lysine biosynthesis is cloned, as well as a phosphoenolpyruvate carboxylase gene (Japanese Patent Application Laid-open No. 60-87788), a dihydrodipicolinate synthase gene (Japanese Patent Publication No. 6-55149), and a diaminopimelate decarboxylase gene (Japanese Patent Application Laid-open No. 60-62994) in which amplification of a gene affects L-lysine productivity.
As for enzymes participating in L-lysine biosynthesis, a case is known for an enzyme which undergoes feedback inhibition when used as a wild type. In this case, L-lysine productivity is improved by introducing an enzyme gene having such mutation that the feedback inhibition is desensitized. Those known as such a gene specifically include, for example, an aspartokinase gene (International Publication Pamphlet of WO 94/25605).
As described above, certain successful results have been obtained by means of amplification of genes for the L-lysine biosynthesis system, or introduction of mutant genes. For example, a coryneform bacterium, which harbors a mutant aspartokinase gene with desensitized concerted inhibition by lysine and threonine, produces a considerable amount of L-lysine (about 25 g/L). However, this bacterium suffers decrease in growth speed as compared with a bacterium harboring no mutant aspartokinase gene. It is also reported that L-lysine productivity is improved by further introducing a dihydrodipicolinate synthase gene in addition to a mutant aspartokinase gene (
Applied and Environmental Microbiology,
57(6), 1746-1752 (1991)). However, such a bacterium suffers further decrease in growth speed.
No case has been reported in which growth is intended to be improved by enhancing a gene for L-lysine biosynthesis as well. In the present circumstances, no case is known for the coryneform bacteria, in which anyone has succeeded in remarkable improvement in L-lysine yield without restraining growth, by combining a plurality of genes for L-lysine biosynthesis.
SUMMARY OF THE INVENTION
An object of the present invention is to improve the L-lysine yield without restraining the growth of a coryneform bacterium, by enhancing a plurality of genes for L-lysine biosynthesis in combination in the coryneform bacteria.
When an objective substance is produced fermentatively by using a microorganism, the production speed, as well as the yield of the objective substance relative to an introduced material, is an extremely important factor. An objective substance may be produced remarkably inexpensively by increasing the production speed per a unit of fermentation equipment. Accordingly, it is industrially extremely important that the fermentative yield and the production speed are compatible with each other. The present invention proposes a solution for the problem as described above in order to fermentatively produce L-lysine by using a coryneform bacterium.
The principle of the present invention is based on the fact that the growth of a coryneform bacterium can be improved, and the L-lysine-producing speed thereof can be improved by enhancing both of a DNA sequence coding for an aspartokinase in which feedback inhibition by L-lysine and L-threonine is substantially desensitized, and a DNA sequence coding for a diaminopimelate decarboxylase compared with the case in which these DNA sequences are each enhanced singly.
In a first aspect of the present invention, it is provided a recombinant DNA autonomously replicable in cells of coryneform bacteria, comprising a DNA sequence coding for an aspartokinase in which feedback inhibition by L-lysine and L-threonine is substantially desensitized, and a DNA sequence coding for a diaminopimelate decarboxylase. The recombinant DNA further comprising a DNA sequence coding for a phosphoenolpyruvate carboxylase is also provided.
In a second aspect of the present invention, it is provided a coryneform bacterium harboring an aspartokinase in which feedback inhibition by L-lysine and L-threonine is substantially desensitized, and comprising an enhanced DNA sequence coding for a diaminopimelate decarboxylase. The coryneform bacterium further comprising an enhanced DNA sequence coding for a phosphoenolpyruvate carboxylase is also provided.
In a third aspect of the present invention, it is provided a method for producing L-lysine comprising the steps of cultivating any of coryneform bacteria as described in the above in an appropriate medium to allow L-lysine to be produced and accumulated in a culture of the bacterium, and collecting L-lysine from the culture.
Hereinafter, an aspartokinase is referred to as “AK”, a gene coding for AK is referred to as “lysC”, AK which is desensitized in feedback inhibition by L-lysine and L-threonine is referred to as “mutant AK”, and a gene coding for mutant AK is referred to as “mutant lysc”, if necessary. Also, a diaminopimelate decarboxylase is referred to as “DDC”, a gene coding for DDC is referred to as “lysA”, a phosphoenolpyruvate carboxylase is referred to as “PEPC”, and a gene coding for PEPC is referred to as “ppc”, if necessary.
The coryneform bacteria referred to in the present invention are a group of microorganisms as defined in
Beraey's Manual of Determinative Bacteriology,
8th ed., p. 599 (1974), which are aerobic Gram-positive non-acid-fast rods having no spore-forming ability. The coryneform bacteria include bacteria belonging to the genus Corynebacterium, bacteria belonging to the genus Brevibacterium having been hitherto classified into the genus Brevibacterium but united as bacteria belonging to the genus Corynebacterium at present, and bacteria belonging to the genus Brevibacterium closely relative to bacteria belonging to the genus Corynebacterium.
According to the present invention, a production amount and a production speed of L-lysine of coryneform bacteria can be improved.
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patent: 4861722 (1989-08-01), Sano et al.
patent: 4980285 (1990-12-01), Sano et al.
patent: 5688671 (1997-11-01), Sugimoto et al.
patent: 5804414 (1998-09-01), Moriya et al.
patent: 406261766A (1994-09-01), None
patent: 94/25605 (1994-11-01), None
patent: 96/40934 (1996-12-01), None
Rudinger (Jun. 1976) Characteristics of the amino acids as components of a peptide hormone sequence. In: Peptide Hormones. Ed. J. A. Parsons. University Park Press, Baltimore, MD. pp. 1-7.*
Ngo et al. (Jan. 1994) Computational complexity, protein structure prediction, and the ILevinthal paradox. In: The Protein
Hayakawa Atsushi
Nakamatsu Tsuyoshi
Sugimoto Masakazu
Yoshihara Yasuhiko
Ajinomoto Co. Inc.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Stole Einar
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