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
2000-07-31
2001-07-03
Prouty, Rebecca E. (Department: 1632)
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, C435S006120, C435S320100
Reexamination Certificate
active
06255086
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to carbamoyl-phosphate synthetase of coryneform bacteria, and a gene therefore. The gene can be utilized for production of carbamoyl-phosphate synthetase and subunits thereof, breeding of L-arginine-producing bacteria and nucleic acid-producing bacteria and so forth.
2. Description of the Related Art
Carbamoyl-phosphate synthetase is an enzyme that catalyzes the reactions producing carbamoyl phosphate from carbonic acid, ATP and glutamine. Carbamoyl phosphate produced by these reactions serves as a source of carbamoyl group required for the reaction producing citrulline from ornithine in the L-arginine biosynthetic pathway. Furthermore, carbamoyl aspartate produced from aspartic acid and carbamoyl phosphate is one of the intermnediates of the pyrimidine biosynthesis system including uridine 5′-monophosphate.
Carbamoyl-phosphate synthetase consists of two subunits, and it has been known for bacteria belonging to the genus Escherichia or Bacillus that those subunits are encoded by carA and carB genes.
However, as for coryneform bacteria, there have been no findings about the carbamoyl-phosphate synthetase activity and enzymes therefor, and any genes therefor have not been elucidated.
Incidentally, it has been reported that when a transformant of
Escherichia coli
to which introduced a plasmid harboring the genes carA, carB, argI and arg box was cultured in the medium added with glutamine which is substrate of carbamoyl-phosphate synthetase, the concentration of intracellular L-arginine was the same as that of a control strain to which only the vector was introduced. However, when the transformant was cultured in a medium added with glutamine accompanied with ornithine which is a substrate of ArgI together with carbamoyl phosphate, the concentration of intracellular L-arginine was higher than that of the control strain (Malamy M. et al.,
Applied Environmental Microbiology
, 63(1), 33 (1997)). From these result, it was suggested that the rate-determining step of synthesis of L-arginine is supply of ornithine.
There was thought to be a possibility that the rate-determining step of supply of ornithine is N-acetylglutamine synthetase (ArgA). ArgA suffers feedback inhibition by the final product, L-arginine, in the biosynthesis pathway of
Escherichia coli.
AS for the strain in which argA gene coding for feedback inhibition-desensitized ArgA was amplified by plasmid, the concentration of intracellular L-arginine was increased even in a medium added with only glutamine as well as in a medium added with both glutamine and ornithine. However, farther increase of concentration of intracellular L-arginine was not observed in the case that the strain was cultured with addition of glutamine, or glutamine and ornithin, also in the case that the both of carA and carB genes were further amplified in the strain (Malamy M. et al.,
Applied Environmental Microbiology
, 64(5), 1805 (1998)).
On the other hand, any attempts have not been reported to enhance L-arginine productivity of microorganisms by utilizing a gene coding for carbamoyl-phosphate synthetase derived from coryneform bacterium.
SUMMARY OF THE INVENTION
An object of the present invention is to provide carbamoyl-phosphate synthetase of coryneform bacteria, a gene coding for it, and a method for producing L-arginine with a microorganism utilizing the gene.
The inventors of the present invention eagerly studied in order to achieve the aforementioned object. As a result, the inventors successfully obtained a DNA fragment containing the carA gene and the carB gene from a wild strain of
Brevibacterium lactofermentum
by utilizing a carB-deficient strain of
Escherichia coli
, and thus accomplished the present invention.
That is, the present invention provides the followings.
(1) A DNA fragment which encodes a polypeptide defined in the following (A) or (B):
(A) a polypeptide which has an amino acid sequence comprises at least the amino acid numbers 50 to 393 of the amino acid sequence of SEQ ID NO: 2,
(B) a polypeptide which has an amino acid sequence comprises at least the amino acid numbers 50 to 393 of the amino acid sequence of SED ID NO: 2 including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and can constitute a protein having a carbamoyl-phosphate synthetase activity with a large subunit of carbamoyl-phosphate synthetase having an amino acid sequence comprising at least the amino acid numbers 55 to 1113 of SEQ ID NO: 3.
(2) A DNA fragment which encodes a polypeptide defined in the following (C) or (D):
(C) a polypeptide which has an amino acid sequence comprising at least the amino acid numbers 55 to 1113 of SEQ ID NO: 3,
(D) a polypeptide which has an amino acid sequence comprising at least the amino acid numbers 55 to 1113 of SEQ ID NO: 3 including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and can constitute a protein having a carbamoyl-phosphate synthetase activity with a small subunit of carbamoyl-phosphate synthetase having an amino acid sequence comprises at least the amino acid numbers 50 to 393 of the amino acid sequence of SEQ ID No: 2.
(3) A DNA fragment encoding a polypeptide which has an amino acid sequence comprising at least the amino acid numbers 55 to 1113 of SEQ ID NO: 3 including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and can constitute a protein having a carbamoyl-phosphate synthetase activity.
(4) A DNA fragment which encodes a polypeptide defined in the following (a) or (b), and a polypeptide defined in the following (c) or (d):
(a) a polypeptide which has an amino acid sequence comprising at least the amino acid numbers 50 to 393 in SEQ ID NO: 2,
(b) a polypeptide which has an amino acid sequence comprising at least the amino acid numbers 50 to 393 in SEQ ID NO: 2 including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and can constitute a protein having a carbamoyl-phosphate synthetase activity with a large subunit of carbamoyl-phosphate synthetase having an amino acid sequence comprising at least the amino acid numbers 55 to 1113 of SEQ ID NO; 3,
(c) a polypeptide which has an amino acid sequence comprising at least the amino acid numbers 55 to 1113 of SEQ ID NO. 3,
(d) a polypeptide which has an amino acid sequence comprising at least the amino acid numbers 55 to 1113 of SEQ ID NO: 3 including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and can constitute a protein having a carbamoyl-phosphate synthetase activity with a small subunit of carbamoyl-phosphate synthetase having an amino acid sequence comprising the amino acid numbers 50 to 393 in SEQ ID NO: 2.
(5) The DNA fragment according to (1), which has a nucleotide sequence comprising at least the nucleotide numbers 430 to 1461 in the nucleotide sequence of SEQ ID NO: 1.
(6) The DNA fragment according to (2), which has a nucleotide sequence comprising at least the nucleotide numbers 1756 to 4808 in the nucleotide sequence of SEQ ID NO: 1.
(7) The DNA fragment according to (3), which has a nucleotide sequence comprising at least the nucleotide numbers 430 to 4808 in the nucleotide sequence of SEQ ID NO: 1.
(8) A protein which comprises a polypeptide defined in the following (a) or (b), and a polypeptide defined in the following (c) or (d):
(a) a polypeptide which has an amino acid sequence comprising at least the amino acid numbers 50 to 393 in SEQ ID NO: 2,
(b) a polypeptide which has an amino acid sequence comprising at least the amino acid numbers 50 to 393 in SEQ ID NO: 2 including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and can constitute a protein having a carbamoyl-phosphate synthetase activity with a large subunit of carbamoyl-phosphate synthetase having an amino acid sequence comprising at least the amino acid numbers 55 to 1113 of SEQ ID NO: 3,
(c) a polypeptide which has an amino a
Hashiguchi Ken-ichi
Ito Hisao
Kurahashi Osamu
Kuwabara Yoko
Mori Yukiko
Ajinomoto Co. Inc.
Monshipouri M.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Prouty Rebecca E.
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