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
1999-09-15
2001-10-16
Prouty, Rebecca E. (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...
C435S115000, C435S116000, C435S252300, C435S320100, C536S023200
Reexamination Certificate
active
06303348
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for producing an amino acid, especially for a method for producing L-homoserine, L-alanine, L-isoleucine, L-valine, or L-threonine using a bacterium belonging to the genus Escherichia.
BACKGROUND ART
The present inventors obtained, with respect to
E. coli
K-12, a mutant having mutation, thrR, (herein referred to as rhtA23) that is concerned in high concentrations of threonine (>40 mg/ml) or homoserine (>5 mg/ml) in a minimal medium (Astaurova, O. B. et al., Appl. Bioch. and Microbiol., 21, 611-616 (1985)). On the basis of rhtA23 mutation an improved threonine-producing strain (SU patent No. 974817), homoserine- and glutamic acid-producing strains (Astaurova et al., Appl. Boch. And Microbiol., 27, 556-561 (1991)) were obtained.
Furthermore, the present inventors has revealed that the rhtA gene exists at 18 min on
E. coli
chromosome and that the rhtA gene is identical to ORF1 between pexB and ompx genes. The unit expressing a protein encoded by the ORF1 has been designated as rhtA (rht: resistance to homoserine and threonine) gene. The rhtA gene includes a 5′-noncoding region including SD sequence, ORF1 and a terminator. Also, the present inventors have found that a wild type rhtA gene participates in resistance to threonine and homoserine if cloned in a multicopy state and that enhancement of expression of the rhtA gene improves amino acid productivity of a bacterium belonging to the genus Escherichia having an ability to produce L-lysine, L-valine or L-threonine (ABSTRACTS of 17th International Congress of Biochemistry and Molecular Biology in conjugation with 1997 Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, Calif. Aug. 24-29, 1997, abstract No. 457).
It is found that at least two different genes which impart homoserine resistance in a multicopy state exist in
E. coli
during cloning of the rhtA gene. One of the genes is the rhtA gene, however the other gene has not been elucidated.
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide a novel gene participating in resistance to homoserine, and a method for producing an amino acid, especially, L-homoserine, L-alanine, L-isoleucine, L-valine and L-threonine with a high yield.
The inventors have found that a region at 86 min on
E. coli
chromosome, when cloned by a multicopy vector, impart resistance to L-homoserine to cells of
E. coli
, and that when the region is amplified, the amino acid productivity of
E. coli
can be improved like the rhtA gene. On the basis of these findings, the present invention have completed.
Thus, the present invention provides:
(1) a DNA coding for a protein as defined in the following (A) or (B):
(A) a protein which comprises an amino acid sequence shown in SEQ ID NO: 2 in Sequence Listing; or
(B) a protein which comprises an amino acid sequence including deletion, substitution, insertion or addition of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 2 in Sequence Listing, and which has an activity of making a bacterium having the protein L-homoserine-resistant,
(2) the DNA according to (1), which is a DNA as defined in the following (a) or (b):
(a) a DNA which comprises a nucleotide sequence corresponding to the nucleotide numbers of 557 to 1171 of a nucleotide sequence shown in SEQ ID NO: 1 in Sequence Listing; or
(b) a DNA which hybridizes with the nucleotide sequence corresponding to the nucleotide numbers of 557 to 1171 of the nucleotide sequence shown in SEQ ID NO: 1 in Sequence Listing under stringent conditions, and which codes for the protein having the activity of making the bacterium having the protein L-homoserine-resistant,
(3) a bacterium belonging to the genus Escherichia, wherein L-homoserine resistance of the bacterium is enhanced by amplifying a copy number of the DNA of (1) in a cell of the bacterium,
(4) the bacterium of (3), wherein the DNA of (1) is carried on a multicopy vector in the cell of the bacterium,
(5) the bacterium of (3), wherein the DNA of (1) is carried on a transposon in the cell of the bacterium,
(6) a method for producing an amino acid, comprising the steps of cultivating the bacterium of any of (3) to (5), which has an ability to produce the amino acid, in a culture medium to produce and accumulate the amino acid in the medium, and recovering the amino acid from the medium, and
(7) the method of (6), wherein the amino acid is at least one selected from the group consisting of L-homoserine, L-alanine, L-isoleucine, L-valine and L-threonine.
The DNA of the present invention may be referred to as “rhtB gene”, a protein coded by the rhtB gene may be referred to as “RhtB protein”, an activity of the RhtB protein which participates in resistance to L-homoserine of a bacterium (i.e. an activity of making a bacterium having the RhtB protein L-homoserine-resistant) may be referred to as “Rh activity”, and a structural gene encoding the RhtB protein in the rhtB gene may be referred to as “rhtB structural gene”. The term “enhancing the Rh activity” means imparting resistance to homoserine to a bacterium or enhance the resistance by means of increasing the number of molecules of the RhtB protein, increasing a specific activity of the RhtB protein, or desensitizing negative regulation against the expression or the activity of the RhtB protein or the like. The terms “DNA coding for a protein” mean a DNA of which one of strands codes for the protein when the DNA is double-stranded. The L-homoserine resistance means a property that a bacterium grows on a minimal medium containing L-homoserine at a concentration at which a wild type strain thereof can not grow, usually at 10 mg/ml. The ability to produce an amino acid means a property that a bacterium produces and accumulates the amino acid in a medium in a larger amount than a wild type strain thereof.
According to the present invention, resistance to homoserine of a high concentration can be imparted to a bacterium belonging to the genus Escherichia. A bacterium belonging to the genus Escherichia, which has increased resistance homoserine and an ability to accumulate an amino acid, especially, L-homoserine, L-alanine, L-isoleucine, L-valine or L-threonine in a medium with a high yield.
The present invention will be explained in detail below.
<1> DNA of the present invention
The DNA of the present invention coding for a protein having the Rh activity and having an amino acid sequence shown in SEQ ID NO: 2 in Sequence Listing. Specifically, the DNA of the present invention may be exemplified by a DNA comprising a nucleotide sequence of the nucleotide numbers 557 to 1171 of a nucleotide sequence shown in SEQ ID NO: 1 in Sequence Listing.
The DNA of the present invention includes a DNA fragment encoding the RhtB protein conferring bacterium
Escherichia coli
resistance to homoserine, which includes the regulatory elements of the rhtB gene and the structural part of rhtB gene, having the nucleotide sequence shown in SEQ ID NO: 1.
The nucleotide sequence shown in SEQ ID NO: 1 corresponds to a part of sequence complement to the sequence of GenBank accession number M87049. SEQ ID NO: 1 includes f138 (nucleotide numbers 61959-61543 of GenBank accession number M87049) which is a known but function-unknown ORF (open reading frame) present at 86 min on
E. coli
chromosome, and 5′-flanking and 3′-flanking regions thereof. The f138, which had only 160 nucleotides in the 5′-flanking region, could not impart the resistance to homoserine. No termination codon is present between the 62160 and 61959 of M87049 (upstream the ORF f138). Hence, the coding region is 201 bp longer. Thus the RhtB protein and the rhtB gene coding for the protein are novel.
The rhtB gene may be obtained, for example, by infecting Mucts lysogenic strain of
E. coli
using a lysate of a lysogenic strain of
E. coli
such as K12 or W3110 according to the method in which mini-Mu d5005 phagemid is used (Groisman, E. A., et al., J. Bacteriol., 168, 357-364 (1986)), and isola
Aleoshin Vladimir Venyamiovich
Balareova Alla Valentinovna
Livshits Vitaly Arkadievich
Tokhmakova Irina Lvovna
Zakataeva Natalya Pavlovna
Ajinomoto Co. Inc.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Prouty Rebecca E.
Rao Manjunath N.
LandOfFree
DNA coding for protein which confers on bacterium... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with DNA coding for protein which confers on bacterium..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and DNA coding for protein which confers on bacterium... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2577949