Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing alpha or beta amino acid or substituted amino acid...
Patent
1996-04-29
1999-03-02
Wax, Robert A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
435107, 435110, 435113, 435115, 435116, 435232, 4352523, 43525233, 4353201, 536 231, 536 232, 536 237, C12P 1304, C12P 1324, C12P 1312, C12P 1308
Patent
active
058769838
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a mutant phosphoenolpyruvate carboxylase, a gene coding for it, and a production method of an amino acid, and in particular relates to a gene having mutation to desensitize feedback inhibition by aspartic acid, and utilization thereof.
BACKGROUND ART
Phosphoenolpyruvate carboxylase is an enzyme which is found in almost all bacteria and all plants. The role of this enzyme resides in biosynthesis of aspartic acid and glutamic acid, and supply of C4 dicarboxylic acid to the citric acid cycle for maintaining its turnover. However, in the fermentative production of an amino acid using a microorganisms, there have been few reports on effects of this enzyme has not been made clear (Atsushi Yokota and Isamu Shiio, Agric. Biol. Chem., 52, 455-463 (1988), Josef Cremer et al., Appl. Environ. Microbiol., 57, 1746-1752 (1991), Petra, G. Peters-Weintisch, FEMS Microbiol. Letters, 112, 269-274 (1993)).
By the way, the amino acid is a compound which universally exists in cells as components of proteins, however, for the sake of economic energy metabolism and substance metabolism, its production is strictly controlled. This control is principally feedback control, in which the final product of a metabolic pathway inhibits the activity of an enzyme which catalyzes the earlier step of the pathway. Phosphoenolpyruvate carboxylase also undergoes various regulations in expression of its activity.
For example, in the case of phosphoenolpyruvate carboxylase of microorganisms belonging to the genus Corynebacterium or the genus Escherichia, the activity is inhibited by aspartic acid. Therefore, the aforementioned amino acid biosynthesis, in which phosphoenolpyruvate carboxylase participates, is also inhibited by aspartic acid.
In the prior art, various techniques have been developed for efficient production in amino acid fermentation, and fermentative production has been carried out for leucine, isoleucine, tryptophan, phenylalanine and the like by using mutant strains converted to be insensitive to feedback control. However, there has been known neither mutant phosphoenolpyruvate carboxylase converted to be insensitive to inhibition by aspartic acid, nor attempt to utilize it for fermentative production of amino acids of the aspartic acid family and the glutamic acid family.
On the other hand, ppc gene, which is a gene coding for phosphoenolpyruvate carboxylase of Escherichia coli, has been already cloned, and also determined for its nucleotide sequence (Fujita, N., Miwa, T., Ishijima, S., Izui, K. and Katsuki, H., J. Biochem., 95, 909-916 (1984)). However, there is no report of a mutant in which inhibition by aspartic acid is desensitized.
The present invention has been made from the aforementioned viewpoint, an object of which is to provide a mutant phosphoenolpyruvate carboxylase with substantially desensitized feedback inhibition by aspartic acid, a gene conding for it, and a utilization method thereof.
DISCLOSURE OF THE INVENTION
As a result of diligent investigation in order to achieve the aforementioned object, the present inventors have found that the inhibition by aspartic acid is substantially desensitized by replacing an amino acid at a specified site of phosphoenolpyruvate carboxylase of Escherichia coli with another amino acid, succeeded in obtaining a gene coding for such a mutant enzyme, and arrived at completion of the present invention.
Namely, the present invention lies in a mutant phosphoenolpyruvate carboxylase, which originates from a microorganism belonging to the genus Escherichia, and has mutation to desensitize feedback inhibition by aspartic acid, and a DNA sequence coding for the mutant phosphoenolpyruvate carboxylase.
The present invention further provides microorganisms belonging to the genus Escherichia or coryneform bacteria harboring the DNA fragment, and a method of producing an amino acid wherein any of these microorganisms is cultivated in a preferable medium, and the amino acid selected from L-lysine, L-threonine, L-methionine, L-isoleucine
REFERENCES:
Biochem. Biophys. Res. Commun., vol. 45, No. 3, 5 Nov. 1971, pp. 689-694.
J. Biochem., vol. 81, No. 5, 1977, pp. 1473-1485.
J. Biochem., vol. 85, No. 2, Feb. 1979, pp. 423-432.
J. Biochem., vol. 84, No. 4, 1978, pp. 795-803.
Fujita et al. "The primary structure of phosphoenolpyruvate carboxylase of Escherichia coli. Nucleotide sequence . . . " J. Biochem. 95, 909-916, 1984.
Izui Katsura
Matsui Hiroshi
Sugimoto Masakazu
Suzuki Tomoko
Ajinomoto Co. Inc.
Nashed Nashaat T.
Wax Robert A.
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