U.S. patent application number 09/494359 was filed with the patent office on 2003-07-03 for carbamoyl-phosphate synthetase gene of coryneform bacteria and method for producing l-arginine.
Invention is credited to Hashiguchi, Kenichi, Ito, Hisao, Kurahashi, Osamu, Kuwabara, Yoko, Mori, Yukiko, Nakamatsu, Tsuyoshi.
Application Number | 20030124685 09/494359 |
Document ID | / |
Family ID | 12130290 |
Filed Date | 2003-07-03 |
United States Patent
Application |
20030124685 |
Kind Code |
A1 |
Kuwabara, Yoko ; et
al. |
July 3, 2003 |
CARBAMOYL-PHOSPHATE SYNTHETASE GENE OF CORYNEFORM BACTERIA AND
METHOD FOR PRODUCING L-ARGININE
Abstract
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 at least the amino acid
sequence of the amino acid numbers 50 to 393 in SEQ ID NO: 2 shown
in Sequence Listing, (b) a polypeptide which has at least the amino
acid sequence of the amino acid numbers 50 to 393 in SEQ ID NO: 2
shown in Sequence Listing 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 the amino acid sequence of SEQ ID NO: 3, (c) a
polypeptide which has the amino acid sequence of SEQ ID NO: 3 shown
in Sequence Listing, (d) a polypeptide which has the amino acid
sequence of SEQ ID NO: 3 shown in Sequence Listing 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 the amino acid sequence of
the amino acid numbers 50 to 393 in SEQ ID NO: 2.
Inventors: |
Kuwabara, Yoko;
(Kawasaki-Shi, JP) ; Hashiguchi, Kenichi;
(Kawasaki-Shi, JP) ; Nakamatsu, Tsuyoshi;
(Kawasaki-Shi, JP) ; Kurahashi, Osamu;
(Kawasaki-Shi, JP) ; Mori, Yukiko; (Kawasaki-Shi,
JP) ; Ito, Hisao; (Kawasaki-Shi, JP) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Family ID: |
12130290 |
Appl. No.: |
09/494359 |
Filed: |
January 31, 2000 |
Current U.S.
Class: |
435/114 ;
435/196; 435/252.3; 435/320.1; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/93 20130101; C12P
13/10 20130101 |
Class at
Publication: |
435/114 ;
435/69.1; 435/196; 435/320.1; 435/252.3; 536/23.2 |
International
Class: |
C12P 013/10; C12N
009/16; C07H 021/04; C12P 021/02; C12N 001/21; C12N 015/74 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 1, 1999 |
JP |
11-24149 |
Claims
What is claimed is:
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 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 comprising the amino acid sequence
of SEQ ID NO: 3.
2. A DNA fragment which encodes a polypeptide defined in the
following (C) or (D): (C) a polypeptide which comprises the amino
acid sequence of SEQ ID NO: 3, (D) a polypeptide which comprises
the amino acid sequence 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 comprises the amino
acid sequence 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 comprising the amino acid sequence
of SEQ ID NO: 3, (c) a polypeptide which comprises the amino acid
sequence of SEQ ID NO: 3, (d) a polypeptide which comprises the
amino acid sequence 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 claim 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 claim 2, which has a nucleotide
sequence comprising at least the nucleotide numbers 1756 to 4809 in
the nucleotide sequence of SEQ ID NO: 1.
7. The DNA fragment according to claim 3, which has a nucleotide
sequence comprising at least the nucleotide numbers 430 to 4809 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 comprising the amino acid sequence
of SEQ ID NO: 3, (c) a polypeptide which comprises the amino acid
sequence of SEQ ID NO: 3, (d) a polypeptide which comprises the
amino acid sequence 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 at least the
amino acid numbers 50 to 393 in SEQ ID NO: 2.
9. A coryneform bacterium which is transformed with a DNA fragment
according to any one of claims 1 to 7.
10. A microorganism which has enhanced intracellular
carbamoyl-phosphate synthetase activity, and has L-arginine
productivity.
11. The microorganism according to claim 10, wherein the enhanced
intracellular carbamoyl-phosphate synthetase activity is obtained
by increasing copy number of DNA encoding carbamoyl-phosphate
synthetase of the microorganism, or by modifying an expression
regulation sequence so that expression of the gene encoding
carbamoyl-phosphate synthetase in the cell should be enhanced.
12. The microorganism according to claim 11, wherein the DNA is a
DNA fragment according to any one of claims 1 to 7.
13. The microorganism according to claim 12, which is a coryneform
bacterium.
14. A method for producing of L-arginine, comprising the steps of
culturing a coryneform bacterium according to any one of claims 10
to 13 in a medium to produce and accumulate L-arginine in the
medium, and collecting the L-arginine from the medium.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to carbamoyl-phosphate
synthetase of coryneform bacteria, and a gene therefor. 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.
[0003] 2. Description of the Related Art
[0004] 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 intermediates of the
pyrimidine biosynthesis system including uridine
5'-monophosphate.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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)).
[0010] On the other hand, any attempts have not been reported to
enhance L-arginine productibity of microorganisms by utilizing a
gene coding for carbamoyl-phosphate synthetase derived from
coryneform bacterium.
SUMMARY OF THE INVENTION
[0011] 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.
[0012] 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 Breveibacterium
lactofermentum by utilizing a carB-deficient strain of Escherichia
coli, and thus accomplished the present invention.
[0013] That is, the present invention provides the followings.
[0014] (1) A DNA fragment which encodes a polypeptide defined in
the following (A) or (B):
[0015] (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,
[0016] (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 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 comprising the amino acid sequence of SEQ ID NO: 3.
[0017] (2) A DNA fragment which encodes a polypeptide defined in
the following (C) or (D):
[0018] (C) a polypeptide which comprises the amino acid sequence of
SEQ ID NO: 3,
[0019] (D) a polypeptide which comprises the amino acid sequence 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.
[0020] (3) A DNA fragment encoding a polypeptide which comprises
the amino acid sequence 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.
[0021] (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):
[0022] (a) a polypeptide which has an amino acid sequence
comprising at least the amino acid numbers 50 to 393 in SEQ ID NO:
2,
[0023] (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 comprising the
amino acid sequence of SEQ ID NO: 3,
[0024] (c) a polypeptide which comprises the amino acid sequence of
SEQ ID NO: 3,
[0025] (d) a polypeptide which comprises the amino acid sequence 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.
[0026] (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.
[0027] (6) The DNA fragment according to (2), which has a
nucleotide sequence comprising at least the nucleotide numbers 1756
to 4809 in the nucleotide sequence of SEQ ID NO: 1.
[0028] (7) The DNA fragment according to (3), which has a
nucleotide sequence comprising at least the nucleotide numbers 430
to 4809 in the nucleotide sequence of SEQ ID NO: 1.
[0029] (8) A protein which comprises a polypeptide defined in the
following (a) or (b), and a polypeptide defined in the following
(c) or (d):
[0030] (a) a polypeptide which has an amino acid sequence
comprising at least the amino acid numbers 50 to 393 in SEQ ID NO:
2,
[0031] (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 comprising the
amino acid sequence of SEQ ID NO: 3,
[0032] (c) a polypeptide which comprises the amino acid sequence of
SEQ ID NO: 3,
[0033] (d) a polypeptide which comprises the amino acid sequence 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 at least the amino acid numbers 50 to 393
in SEQ ID NO: 2.
[0034] (9) A coryneform bacterium which is transformed with a DNA
fragment according to any one of (1) to (7).
[0035] (10) A microorganism which has enhanced intracellular
carbamoyl-phosphate synthetase activity, and has L-arginine
productivity.
[0036] (11) The microorganism according to (10), wherein the
enhanced intracellular carbamoyl-phosphate synthetase activity is
obtained by increasing copy number of DNA encoding
carbamoyl-phosphate synthetase of the microorganism, or by
modifying an expression regulation sequence so that expression of
the gene encoding carbamoyl-phosphate synthetase in the cell should
be enhanced.
[0037] (12) The microorganism according to (11), wherein the DNA is
a DNA fragment according to any one of (1) to (7).
[0038] (13) The microorganism according to (12), which is a
coryneform bacterium.
[0039] (14) A method for producing of L-arginine, comprising the
steps of culturing a coryneform bacterium according to any one of
(10) to (13) in a medium to produce and accumulate L-arginine in
the medium, and collecting the L-arginine from the medium.
[0040] The present invention provides genes coding for the subunits
that constitute carbamoyl-phosphate synthetase. 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. Aditionally,
L-arginine can be produced efficiently according to the present
invention.
BRIEF EXPLANATION OF THE DRAWINGS
[0041] FIG. 1 shows the structure of plasmid p19 containing the
carA gene and carB gene.
[0042] FIG. 2 shows a construction process of plasmid pK1.
[0043] FIG. 3 shows a construction process of plasmid pSFK6.
DETAIL DESCRIPTION OF THE INVENTION
[0044] Hereafter, the present invention will be explained in
detail.
[0045] <1> DNA of the Present Invention
[0046] The DNA of the present invention can be obtained from a
chromosome DNA library of coryneform bacteria prepared with vectors
such as plasmids by selection of the DNA using a microorganism
which is deficient in carA or carB, for example, Escherichia coli
RC50 (carA50, tsx.sup.-273, .lambda..sup.-, rpsL135 (str.sup.R),
malT1 (.lambda.R), xy1A7, thi.sup.-1; Mol. Gen. Genet., 133, 299
(1974)), Escherichia coli JEF8 (thr.sup.-31, .DELTA.carB,
relA.sup.-, metB1, Mol. Gen. Genet., 133, 299 (1974)) and so forth.
Because a microorganism which is deficient in carA or carB exhibits
L-arginine and uracil auxotrophy, a DNA fragment can be obtained by
transforming such a microorganism with a chromosome DNA library,
selecting clones in which the auxotrophy is complemented, and
recovering a recombinant vector from the selected
transformants.
[0047] The coryneform bacteria used for preparing a chromosome DNA
library are not particularly limited, and examples thereof include
bacteria having been hitherto classified into the genus
Brevibacterium but united into the genus Corynebacterium at present
(Int. J. Syst. Bacteriol., 41, 255 (1981)), and include bacteria
belonging to the genus Brevibacterium closely relative to the genus
Corynebacterium, more specifically, wild strains of Breveibacterium
lactofermentum and so forth. Chromosome DNA of coryneform bacteria
can be prepared by, for example, the method of Saito and Miura
(Biochem. Biophys. Acta., 72, 619, (1963)), the method of K. S.
Kirby (Biochem. J., 64, 405, (1956)) and so forth.
[0048] A chromosome DNA library can be obtained by partially
digesting chromosome DNA with suitable restriction enzymes,
ligating each of the obtained DNA fragments to a vector DNA
autonomously replicable in Escherichia coli cells to prepare a
recombinant DNA, and introducing the DNA into Escherichia coli. The
vector is not particularly limited so long as it is a vector
usually used for genetic cloning, and plasmid vectors such as
pUC19, pUC18, pUC118, and pUC119, phage vectors such as .lambda.
phage DNA and so forth can be used. Further, a vector autonomously
replicable in both of Escherichia coli cells and coryneform
bacterium cells may also be used. Such a vector can be constructed
by ligating a vector for Escherichia coli and pAM330, which is a
cryptic plasmid of Breveibacterium lactofermentum (see Japanese
Patent Laid-open No. 58-67699).
[0049] Specific examples of the vector autonomously replicable
within both of Escherichia coli and coryneform bacterium cells
include pSAC4 (see the examples mentioned below), pHK4 (see
Japanese Patent Laid-open No. 5-7491) and so forth. Escherichia
coli HB101 harboring pHK4 was designated as Escherichia coli
AJ13136, and it was deposited at the National Institute of
Bioscience and Human-Technology, Agency of Industrial Science and
Technology, Ministry of International Trade and Industry (postal
code 305-8566, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken,
Japan) on Aug. 1, 1995, and received an accession number of FERM
BP-5186.
[0050] The transformation of Escherichia coli cells can be
performed by, for example, the method of D. A. Morrison (Methods in
Enzymology, 68, 326, 1979), the method of treating recipient cells
with calcium chloride so as to increase the permeability of DNA
(Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)) and so
forth. As for methods for preparation of chromosome DNA library,
preparation of plasmid DNA, and digestion and ligation of DNA, as
well as methods for PCR, preparation of oligonucleotides and
hybridization mentioned hereinafter, conventional methods well
known to those skilled in the art can be used. Such methods are
described in Sambrook, J., Fritsch, E. F. and Maniatis, T.,
"Molecular Cloning, A Laboratory Manual, Second Edition", Cold
Spring Harbor Laboratory Press, (1989) and so forth.
[0051] A nucleotide sequence of a DNA fragment containing carA and
carB obtained as described above is represented as SEQ ID NO: 1 in
Sequence Listing. This sequence contains two open reading frames
(ORF, nucleotide numbers 283 to 1461 and nucleotide numbers 1756 to
4809). The upstream ORF is carA, and the downstream ORF is carB.
The amino acid sequences encoded by these ORFs are shown in SEQ ID
NOS: 2 and 3, respectively. According to the present invention, a
peptide encoded by carA is referred to as a small subunit, and a
peptide encoded by carB is referred to as a large subunit. As for
the coding region of carA, GTG of the nucleotide numbers 283 to 285
is indicated as the initiation codon in Sequence Listing. However,
GTG of the nucleotide numbers 415 to 417 or ATG of the nucleotide
numbers 430 to 432 may possibly be the initiation codon. In any
case, an active small subunit can be obtained by using a longer
open reading frame for the upstream region for the expression of
carA. The amino acid corresponding to the GTG as the initiation
codon is indicated as valine for each subunit, but it may be
methionine, valine or formylmethionine.
[0052] The small subunit of the carbamoyl-phosphate synthetase of
the present invention is, for example, a polypeptide having the
amino acid sequence of the amino acid numbers 50 to 393 in SEQ ID
NO: 2, polypeptide having the amino acid sequence of the amino acid
numbers 45 to 393 in SEQ ID NO: 2, polypeptide having the amino
acid sequence of the amino acid numbers 1 to 393 in SEQ ID NO: 2 or
the like. The large subunit of the carbamoyl-phosphate synthetase
of the present invention is, for example, a polypeptide having the
amino acid sequence shown as SEQ ID NO: 3.
[0053] According to the present invention, the DNA coding for the
small subunit may be one coding for an amino acid sequence which
contains the amino acid sequence of 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, or one coding
for a polypeptide which can constitute a protein having a
carbamoyl-phosphate synthetase activity with the large subunit.
[0054] According to the present invention, the DNA coding for the
large subunit may be one coding for an amino acid sequence which
contains the amino acid sequence of SEQ ID NO: 3 including
substitution, deletion, insertion, addition, or inversion of one or
several amino acids, or one coding for a polypeptide which can
constitute a protein having a carbamoyl-phosphate synthetase
activity with the small subunit. Alternatively, it may be one
coding for a protein which has the amino acid sequence of SEQ ID
NO: 3 including substitution, deletion, insertion, addition, or
inversion of one or several amino acids, and has a
carbamoyl-phosphate synthetase activity.
[0055] Furthermore, a DNA that encodes carbamoyl-phosphate
synthetase containing a mutation or mutations in the small subunit
or the large subunit, or both of them also falls within the scope
of the DNA of the present invention.
[0056] The term "several amino acids" preferably means 1 to 20
amino acids, more preferably 1 to 10 amino acids.
[0057] DNA, which encodes the substantially same peptide as the
small subunit or the large subunit as described above, is obtained,
for example, by modifying the nucleotide sequence of the DNA
encoding the small subunit or the large subunit, for example, by
means of the site-directed mutagenesis method so that one or more
amino acid residues at a specified site of the gene involve
substitution, deletion, insertion, addition, or inversion. DNA
modified as described above may be obtained by the conventionally
known mutation treatment. The mutation treatment includes a method
for treating DNA coding for the small subunit or the large subunit
in vitro, for example, with hydroxylamine, and a method for
treating a microorganism, for example, a bacterium belonging to the
genus Escherichia harboring DNA coding for the small subunit and
the large subunit with ultraviolet irradiation or a mutating agent
such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and nitrous acid
usually used for the mutation treatment.
[0058] The substitution, deletion, insertion, addition, or
inversion of nucleotide as described above also includes mutation
(mutant or variant) which naturally occurs, for example, the
difference in strains, species or genera of the microorganism
having the small subunit and/or the large subunit.
[0059] The DNA, which encodes substantially the same protein as
carbamoyl-phosphate synthetase, is obtained by expressing DNA
having mutation as described above in an appropriate cell, and
investigating the carbamoyl-phosphate synthetase activity of an
expressed product. The carbamoyl-phosphate synthetase activity can
be measured by the known method (Journal of Genral Microbiology,
136, 1177-1183 (1990)). The DNA, which encodes substantially the
same protein as carbamoyl-phosphate synthetase, is also obtained by
isolating DNA which is hybridizable with DNA having, for example, a
nucleotide sequence corresponding to nucleotide numbers of 283 to
1461 or 1756 to 4809 of the nucleotide sequence of SEQ ID NO: 2,
under a stringent condition, and which encodes a protein having the
carbamoyl-phosphate synthetase activity, from DNA coding for
carbamoyl-phosphate synthetase having mutation or from a cell
harboring it. The "stringent condition" referred to herein is a
condition under which so-called specific hybrid is formed, and
non-specific hybrid is not formed. It is difficult to clearly
express this condition by using any numerical value. However, for
example, the stringent condition includes a condition under which
DNA's having high homology, for example, DNA's having homology of
not less than 70%, preferably not less than 80%, more preferably
not less than 90% are hybridized with each other, and DNA's having
homology lower than the above are not hybridized with each other.
Alternatively, the stringent condition is exemplified by a
condition under which DNA's are hybridized with each other at a
salt concentration corresponding to an ordinary condition of
washing in Southern hybridization, i.e., 60.degree. C.,
1.times.SSC, 0.1% SDS, preferably 0.1.times.SSC, 0.1% SDS.
[0060] As a probe, a partial sequence of the nucleotide sequence of
SEQ ID NO: 1 can also be used. Such a probe may be prepared by PCR
using oligonucleotides produced based on the nucleotide sequence of
SEQ ID NO: 1 as primers, and a DNA fragment containing the
nucleotide sequence of SEQ ID NO: 1 as a template. When a DNA
fragment in a length of about 300 bp is used as the probe, the
conditions of washing for the hybridization consist of, for
example, 50.degree. C., 2.times.SSC, and 0.1% SDS.
[0061] Because the nucleotide sequence of the DNA of the present
invention has been elucidated, the DNA of the present invention can
be obtained by amplifying it from coryneform bacterial chromosome
DNA through polymerase chain reaction (PCR: polymerase chain
reaction; see White, T. J. et al., Trends Genet., 5, 185 (1989))
utilizing oligonucleotides prepared based on that nucleotide
sequence as primers, or by selecting it from a coryneform bacterial
chromosome DNA library by hybridization utilizing an
oligonucleotide prepared based on that nucleotide sequence as a
probe. As nucleotide sequences of the primers used for PCR, a
region upstream from the nucleotide number 283, preferably a region
upstream from the nucleotide number 185 of SEQ ID NO: 1 can
suitably be selected as the 5' primer, and a region downstream from
the nucleotide number 4809 of SEQ ID NO: 1 can suitably be selected
as the 3' primer.
[0062] Examples of the host for the expression of the DNA of the
present invention include various bacteria such as Escherichia coli
and coryneform bacteria including Breveibacterium lactofermentum
and Brevibacterium flavum, eukaryotic cells such as those of
Saccharomyces cerevisiae and so forth. In order to introduce the
DNA of the present invention into these hosts, the host cells can
be transformed with a recombinant vector obtained by inserting the
DNA of the present invention into a vector selected according to
the nature of the host in which the DNA is to be expressed. This
procedure can be performed by a method well known to those skilled
in the art. Specific examples of the method include the methods
used for transformation of Escherichia coli mentioned above, the
method in which competent cells are prepared from cells at the
proliferating stage to introduce DNA, as reported for Bacillus
subtilis (Duncan, C. H., Wilson, G. A. and Young, F. E., Gene, 1,
153 (1977)), the method in which DNA recipient cells are allowed to
be in a state of protoplasts or spheroplasts capable of
incorporating recombinant DNA with ease to introduce recombinant
DNA into the DNA recipient cells, as known for Bacillus subtilis,
actinomycetes, and yeasts (Chang, S. and Choen, S. N., Molec. Gen.
Genet., 168, 111 (1979); Bibb, M. J., Ward, J. M. and Hopwood, O.
A., Nature, 274, 398 (1978); Hinnen, A., Hicks, J. B. and Fink, G.
R., Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)), the electric
pulse method useful for cryneform bacteria (refer to Japanese
Patent Publication Laid-Open No. 2-207791) and so forth.
[0063] The DNA to be introduced into the host such as those
mentioned above may be DNA containing either carA or carB, or DNA
containing both of them. Further, in order to attain efficient
expression of these genes, a promoter functioning in the host cells
such as lac, trp and P.sub.L may be ligated at a position upstream
from carA or carB.
[0064] Carbamoyl-phosphate synthetase or its subunits can be
produced by culturing a transformant such as those mentioned above
under a condition that allows the expression of carA or carB. The
DNA of the present invention can also be utilized for breeding of
L-arginine-producing bacteria or nucleic acid-producing bacteria
such as uracil-producing bacteria. That is, a transformant
introduced with the DNA of the present invention, in particular,
one introduced with either carA or carB or both of them, should
have increased carbamoyl-phosphate synthetase activity compared
with non-transformants. Consequently, its productivity for
L-arginine or nucleic acid such as uracil is improved.
[0065] <2> Method for Producing L-Arginine According to the
Present Invention
[0066] L-Arginine can efficiently be produced by culturing a
microorganism that has enhanced intracellular carbamoyl-phosphate
synthetase activity, and has L-arginine productivity in a medium to
produce and accumulate L-arginine in the medium, and collecting the
L-arginine from the medium.
[0067] Specific examples of the microorganism having L-arginine
productivity include coryneform bacteria, bacteria belonging to the
genera Bacillus, Serratia and Escherichia, yeast species belonging
to the genus Saccharomyces or Candida. Of these, coryneform
bacteria are preferred.
[0068] Exemplary specific species include Bacillus subtilis as a
bacterium belonging to the genus Bacillus, Serratia marcescens as a
bacterium belonging to the genus Serratia, Escherichia coli as a
bacterium belonging to the genus Escherichia, Saccharomyces
cerevisiae as a yeast species belonging to the genus Saccharomyces,
Candida tropicalis as a yeast species belonging to the genus
Candida and so forth.
[0069] Exemplary microorganisms having L-arginine productivity
include Bacillus subtilis resistant to 5-azauracil, 6-azauracil,
2-thiouracil, 5-fluorouracil, 5-bromouracil, 5-azacytosine and so
forth, Bacillus subtilis resistant to arginine hydroxamate and
2-thiouracil, Bacillus subtilis resistant to arginine hydroxamate
and 6-azauracil (see Japanese Patent Laid-open No. 49-1268191),
[0070] Bacillus subtilis resistant to histidine analogues or
tryptophan analogues (see Japanese Patent Laid-open No.
52-114092),
[0071] a mutant of Bacillus subtilis exhibiting auxotrophy for at
least one of methionine, histidine, threonine, proline, isoleucine,
lysine, adenine, guanine and uracil (or uracil precursor) (see
Japanese Patent Laid-open No. 52-99289),
[0072] Bacillus subtilis resistant to arginine hydroxamate (see
Japanese Patent Publication No. 51-6754),
[0073] Serratia marcescens exhibiting succinic acid auxotrophy or
resistance to nucleic acid base analogues (Japanese Patent
Laid-open No. 58-9692), Serratia marcescens deficient in ability to
metabolize arginine and exhibiting resistance to arginine
antagonists and canavanine and auxotorophy for lysine (see Japanese
Patent Laid-open No. 52-8729),
[0074] Escherichia coli introduced with the argA gene (see Japanese
Patent Laid-open No. 57-5693),
[0075] Saccharomyces cerevisiae resistant to arginine, arginine
hydroxamate, homoarginine, D-arginine and canavanine, or resistant
to arginine hydroxamate and 6-azauracil (see Japanese Patent
Laid-open No. 53-143288),
[0076] Candida tropicalis resistant to canavanine (see Japanese
Patent Laid-open No. 53-3586) and so forth.
[0077] Coryneform bacteria include those bacteria having been
hitherto classified into the genus Brevibacterium but united into
the genus Corynebacterium at present (Int. J. Syst. Bacteriol., 41,
255 (1981)), and include bacteria belonging to the genus
Brevibacterium closely relative to the genus Corynebacterium.
Examples of such coryneform bacteria are listed below.
[0078] Corynebacterium acetoacidophilum
[0079] Corynebacterium acetoglutamicum
[0080] Corynebacterium alkanolyticum
[0081] Corynebacterium callunae
[0082] Corynebacterium glutamicum
[0083] Corynebacterium lilium (Corynebacterium glutamicum)
[0084] Corynebacterium melassecola
[0085] Corynebacterium thermoaminogenes
[0086] Corynebacterium herculis
[0087] Brevibacterium divaricatum (Corynebacterium glutamicum)
[0088] Brevibacterium flavum (Corynebacterium glutamicum)
[0089] Brevibacterium immariophilum
[0090] Breveibacterium lactofermentum (Corynebacterium
glutamicum)
[0091] Brevibacterium roseum
[0092] Brevibacterium saccharolyticum
[0093] Brevibacterium thiogenitalis
[0094] Brevibacterium album
[0095] Brevibacterium cerinum
[0096] Microbacterium ammoniaphilum
[0097] The coryneform bacteria that have the L-arginine
productivity are not particularly limited so long as they have the
L-arginine productivity. They include, for example, wild-type
strains of coryneform bacteria; coryneform bacteria resistant to
certain agents including sulfa drugs, 2-thiazolealanine,
.alpha.-amino-.beta.-hydroxyvaleric acid and the like; coryneform
bacteria exhibiting L-histidine, L-proline, L-threonine,
L-isoleucine, L-methionine, or L-tryptophan auxotrophy in addition
to the resistance to 2-thiazolealanine (Japanese Patent Laid-open
No. 54-44096); coryneform bacteria resistant to ketomalonic acid,
fluoromalonic acid, or monofluoroacetic acid (Japanese Patent
Laid-open No. 57-18989); coryneform bacteria resistant to argininol
(Japanese Patent Laid-open No. 62-24075); coryneform bacteria
resistant to X-guanidine (X represents a derivative of fatty acid
or aliphatic chain, Japanese Patent Laid-open No. 2-186995) and so
forth.
[0098] Specifically, the following bacterial strains can be
exemplified.
[0099] Brevibacterium flavum AJ11169 (FERM BP-6892)
[0100] Breveibacterium lactofermentum AJ12092 (FERM BP-6906)
[0101] Brevibacterium flavum AJ11336 (FERM BP-6893)
[0102] Brevibacterium flavum AJ11345 (FERM BP-6893)
[0103] Breveibacterium lactofermentum AJ12430 (FERM BP-2228)
[0104] The AJ11169 strain and the AJ12092 strain are the
2-thiazolealanine resistant strains mentioned in Japanese Patent
Laid-open No. 54-44096, the AJ11336 strain is the strain having
argininol resistance and sulfadiazine resistance mentioned in
Japanese Patent Publication No. 62-24075, the AJ11345 strain is the
strain having argininol resistance, 2-thiazolealanine resistance,
sulfaguanidine resistance, and exhibiting histidine auxotrophy
mentioned in Japanese Patent Publication No. 62-24075, and the
AJ12430 strain is the strain having octylguanidine resistance and
2-thiazolealanine resistance mentioned in Japanese Patent Laid-open
No. 2-186995.
[0105] The intracellular carbamoyl-phosphate synthetase activity of
such microorganisms having the L-arginine productivity as mentioned
above can be enhanced by, for example, increasing copy number of a
gene coding for the carbamoyl-phosphate synthetase in the cells of
the aforementioned microorganisms. The enhancement of the
carbamoyl-phosphate synthetase activity can also be achieved by, in
addition to the aforementioned gene amplification, modifying an
expression regulation sequence for the DNA coding for
carbamoyl-phosphate synthetase so that expression of the DNA gene
coding for carbamoyl-phosphate synthetase should be enhanced.
Specifically, an expression regulation sequence such as a promoter
for a gene coding for carbamoyl-phosphate synthetase on the
chromosomal DNA or a plasmid can be replaced with a stronger one
(see Japanese Patent Laid-open No. 1-215280). Strong promoters,
which function in cells of coryneform bacteria, include lac
promoter, tac promoter, trp promoter, of Escherichia coli (Y.
Morinaga, M. Tsuchiya, K. Miwa and K. Sano, J. Biotech., 5, 305-312
(1987)) and the like. In addition, trp promoter of Corynebacterium
bacteria is also a preferable promoter (Japanese Patent Laid-open
No. 62-195294). By the replacement with these promoters the
carbamoyl-phosphate synthetase activity is enhanced. The
modification of expression regulation sequence may be combined with
the increasing of the copy number of DNA coding for
carbamoyl-phosphate synthetase. Further, the intracellular
carbamoyl-phosphate synthetase activity can be enhanced by
introducing one or more mutations into the enzyme protein of
carbamoyl-phosphate synthetase so that the specific activity of the
enzyme should be increased.
[0106] Examples of the DNA coding for carbamoyl-phosphate
synthetase include the aforementioned carA and carB genes of
Breveibacterium lactofermentum and one containing both of them.
[0107] Examples of the vector for introducing DNA coding for
carbamoyl-phosphate synthetase into a microorganism include vectors
autonomously replicable in cells of the microorganism.
Specifically, the aforementioned vectors autonomously replicable in
Escherichia coli cells, and the vectors autonomously replicable in
both of Escherichia coli cells and coryneform bacterium cells.
[0108] The medium used for culturing a microorganism having
enhanced intracellular carbamoyl-phosphate synthetase activity and
L-arginine productivity obtained as described above may be a
well-known medium conventionally used for the production of amino
acids by fermentation. That is, it is a usual medium that contains
a carbon source, nitrogen source, inorganic ions, and other organic
components as required.
[0109] As the carbon source, it is possible to use sugars such as
glucose, sucrose, lactose, galactose, fructose and starch
hydrolysates; alcohols such as glycerol and sorbitol; or organic
acids such as fumaric acid, citric acid and succinic acid and so
forth.
[0110] As the nitrogen source, it is possible to use inorganic
ammonium salts such as ammonium sulfate, ammonium chloride and
ammonium phosphate, organic nitrogen such as soybean hydrolysates,
ammonia gas, aqueous ammonia and so forth.
[0111] The medium preferably contains a suitable amount of required
substance such as vitamin B.sub.1 and L-homoserine, yeast extract
and so forth as trace amount organic nutrients. Other than those
substances, a small amount of potassium phosphate, magnesium
sulfate, iron ions, manganese ions and so forth may be added to the
medium.
[0112] The cultivation is preferably performed under an aerobic
condition for 1-7 days. Cultivation temperature is preferably
24-37.degree. C., and pH of the medium during the cultivation is
preferably 5-9. Inorganic or organic acidic or alkaline substances,
ammonia gas and so forth may be used for adjusting pH. L-Arginine
can usually be recovered from the fermentation medium by a
combination of known techniques such as ion exchange resin
method.
Best Mode for Carrying out the Invention Hereafter, the present
invention will be explained more specifically with reference to the
following examples.
EXAMPLE 1
Cloning of carA and carB of Breveibacterium lactofermentum
[0113] <1> Preparation of Chromosome DNA of Brevibacterium
lactofermentum ATCC13869
[0114] Brevibacterium lactofermentun ATCC13869 was inoculated to
100 ml of T-Y culture medium (1% of Bacto-Trypton (Difco), 0.5% of
Bacto-Yeast Extract (Difco), 0.5% of NaCl (pH 7.2)), and cultured
at a temperature of 31.5.degree. C. for 8 hours to obtain a
culture. The culture was centrifuged at 3,000 r.p.m. for 15 minutes
to obtain 0.5 g of wet bacterial cells, and chromosome DNA was
obtained from the bacterial cells according to the method of Saito
and Miura (Biochem. Biophys. Acta., 72, 619 (1963)). Then, 60 .mu.g
of the chromosome DNA and 3 units of restriction enzyme Sau3AI were
each mixed in 10 mM Tris-HCl buffer (containing 50 mM NaCl, 10 MM
MgSO.sub.4 and 1 mM dithiothreitol (pH 7.4)), and allowed to react
at a temperature of 37.degree. C. for 30 minutes. The reaction
mixture was subjected to phenol extraction and ethanol
precipitation in a conventional manner to obtain 50 .mu.g of
chromosome DNA fragments of Brevibacterium lactofermentum ATCC13869
digested with Sau3AI.
[0115] <2> Preparation of Gene Library of Brevibacterium
lactofermentum ATCC13869 using Plasmid Vector DNA
[0116] As a plasmid vector DNA autonomously replicable in both of
Escherichia coli cells and coryneform bacterium cells, pSAC4 was
used. pSAC4 was prepared as follows. In order to make a vector
pHSG399 for Escherichia coli (Takara Shuzo) autonomously replicable
in coryneform bacterium cells, a replication origin of the
previously obtained plasmid pHM1519 autonomously replicable in
coryneform bacterium cells (Miwa, K. et al., Agric. Biol. Chem., 48
(1984) 2901-2903) was introduced into the vector (Japanese Patent
Laid-open No. 5-7491). Specifically, pHM1519 was digested with
restriction enzymes BamHI and KpnI to obtain a gene fragment
containing the replication origin, and the obtained fragment was
blunt-ended by using Blunting Lit produced by Takara Shuzo, and
inserted into the SalI site of pHSG399 using a SalI linker
(produced by Takara Shuzo) to obtain pSAC4.
[0117] In 50 mM Tris-HCl buffer (containing 100 mM NaCl and 10 mM
magnesium sulfate (pH 7.4)), 20 .mu.g of pSAC4 and 200 units of a
restriction enzyme BamHI were mixed, and allowed to react at a
temperature of 37.degree. C. for 2 hours to obtain a digestion
solution. This solution was subjected to phenol extraction and
ethanol precipitation in a conventional manner. Then, in order to
inhibit re-ligation of the DNA fragments derived from the plasmid
vector, the DNA fragments were dephosphorylated with bacterial
alkaline phosphatase according to the method described in Molecular
Cloning, 2nd Edition (J. Sambrook, E. F. Fritsch and T. Maniatis,
Cold Spring Harbor Laboratory Press, p1.56 (1989)), and subjected
to phenol extraction and ethanol precipitation in a conventional
manner.
[0118] To 66 mM Tris-HCl buffer (pH 7.5) containing 66 mM magnesium
chloride, 10 mM dithiothreitol and 10 mM ATP, 1 .mu.g of the pSAC4
digested with BamHI, 1 .mu.g of the chromosome DNA fragments of
Brevibacterium lactofermentum ATCC13869 digested with Sau3AI
obtained in Example 1, and 2 units of T4 DNA ligase (produced by
Takara Shuzo) were added, and allowed to react at a temperature of
16.degree. C. for 16 hours to ligate the DNA. Then, Escherichia
coli DH5 was transformed with this DNA mixture in a conventional
manner, and plated on an L agar medium containing 170 .mu.g/ml of
chloramphenicol to obtain about 20,000 colonies, which were used as
a gene library.
[0119] <3> Transformation of carB-deficient Strain of
Escherichia coli (JEF8)
[0120] The carB-deficient strain of Escherichia coli, JEF8
(thr.sup.31 31, .DELTA.carB, relA.sup.-, metB1; Mol. Gen. Genet.,
133, 299 (1974)) was transformed with a recombinant DNA mixture of
the aforementioned gene library in a conventional manner.
Transformants of about 15000 strains were obtained as Cm resistant
strains. These transformants were replicated on a minimum medium (5
g/L of glucose, 12.8 g/L of Na.sub.2HPO.sub.4, 3 g/L of
KH.sub.2PO.sub.4, 0.5 g/L of NaCl, 1 g/L of NH.sub.4Cl, 40 .mu.g/ml
of L-threonine, 40 .mu.g/ml of L-methionine) not containing
arginine and uracil, and the minimum medium not containing
L-arginine, but containing only 50 .mu.g/ml of uracil, and screened
for a strain in which arginine auxotrophy and uracil auxotrophy
were restored, or a strain in which arginine auxotrophy was
restored. Strains in which arginine auxotrophy was restored
recovered both of arginine auxotrophy and uracil auxotrophy. A
plasmid harbored in one of such strains was designated as p19, and
the strain harboring it was designated as JEF8/p19. The structure
of p19 is shown in FIG. 1.
[0121] The Escherichia coli JEF8/p19 was designated as Escherichia
coli AJ13574, and it was deposited at the National Institute of
Bioscience and Human-Technology, Agency of Industrial Science and
Technology, Ministry of International Trade and Industry (postal
code 305-8566, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken,
Japan) on Jan. 28, 1999, and received an accession number of FERM
P-17180, and transferred from the original deposit to international
deposit based on Budapest Treaty on Jan. 6, 20000, and has been
deposited as deposition number of FERM BP-6989.
[0122] <4> Acquisition of Plasmid Complementing Arginine and
Uracil Auxotrophy
[0123] A plasmid was prepared from JEF8/p19 in a conventional
manner, and used for re-transformation of the JEF8 strain. The
obtained transformants could grow in the minimum culture medium not
containing L-arginine and uracil, and its auxotrophy for both of
L-arginine and uracil was restored. Therefore, it was found that
that the plasmid contained a gene complementing the auxotrophy for
both of L-arginine and uracil caused by deletion of carB in the
Escherichia coli strain.
[0124] Further, this plasmid was introduced into the carA mutant of
Escherichia coli, RC50 (carA50, tsx.sup.-273, .lambda..sup.-,
rpsL135 (str.sup.R), malT1 (.lambda.R), xylA7, thi.sup.-1; Mol.
Gen. Genet., 133, 299 (1974)). Since the strain introduced with the
plasmid was able to grow in the minimum culture medium not
containing arginine and uracil, the plasmid was also found to have
a gene complementing the auxotrophy for both of L-arginine and
uracil caused by carA mutation of the Escherichia coli strain.
[0125] <5> Nucleotide Sequence Analysis of p19
[0126] Among the DNA sequence of p19, the nucleotide sequence of
about 4.8 kb from the HindIII side of the multi-cloning site of the
vector to the HindIII site contained in the insertion DNA fragment
was determined. The nucleotide sequencing was performed by using
Rohdamin Terminator Cycle Sequencing Kit (produced by ABI)
according to the method of Sanger. The obtained nucleotide sequence
is shown as SEQ ID NO: 1 in Sequence Listing. From analysis of a
consensus sequence which located in the upstream region of this
gene, it was estimated that two open reading frames (open reading
frame from 283rd G to 1461st A and open reading frame from 1756th G
to 4809th T) were contained in this sequence. The nucleotides of
the 162nd (TGCATA) to 194th (TATAAT), the 185th (TGCATA) to 213rd
(TAAACT), the 203rd (TTGAAT) 230th (TATCAA), or the 224th (TTATCA)
to 251st (TAAAAA) can be estimated to be a promoter region for
regulating the transcription.
[0127] The amino acid sequences encoded by these open reading
frames are represented with the nucleotide sequences. The amino
acid sequences were also shown in SEQ ID NOS: 2 and 3. A protein
database (GenBank CDS) was searched for sequences exhibiting
homology with these amino acid sequences. As a result, it was found
that the 5' open reading frame showed high homology (about 40%)
with carA gene products of Escherichia coli, Bacillus subtilis and
so forth, and the 3' open reading frame showed high homology with
known carB gene products of Escherichia coli, Bacillus
stearothermophilus and so forth (about 40 to 50%). Therefore, it
was suggested that these open reading frames coded for carA and
carB, respectively.
[0128] <6> Introduction of carA and carB into Wild-Type
Strain of Coryneform Bacteria
[0129] p19 was introduced into the Brevibacterium flavum wild
strain 2247 (AJ14067) by the electric pulse method (Japanese Patent
Laid-open No. 2-207791). The transformants were selected as
chloramphenicol resistant strains on a CM2G plate medium
(containing 10 g of polypeptone, 10 g of yeast extract, 5 g of
glucose, 5 g of NaCl, 15 g of agar in 1 L of pure water, pH 7.2)
containing 5 .mu.g/ml of chloramphenicol to obtain 2247/p19.
EXAMPLE 2
Production of L-arginine by Coryneform Bacteria Introduced with
carA and carB
[0130] <1> Preparation of Shuttle Vector
[0131] First, a plasmid vector autonomously replicable in both of
Escherichia coli cells and coryneform bacterium cells was newly
produced as a plasmid used for introducing the carA and carB genes
into coryneform bacteria.
[0132] A vector containing a drug resistance gene of Streptococcus
faecalis was constructed first. The kanamycin resistant gene of
Streptococcus faecalis was amplified by PCR from a known plasmid
containing that gene. The nucleotide sequence of the kanamycin
resistant gene of Streptococcus faecalis has already been clarified
(Trieu-Cuot, P. and Courvalin, P., Gene, 23(3), 331-341 (1983)).
The primers shown as SEQ ID NOS: 4 and 5 were synthesized based on
that sequence, and PCR was performed by using pDG783 (Anne-Marie
Guerout-Fleury et al., Gene, 167, 335-337 (1995)) as a template to
amplify a DNA fragment containing the kanamycin resistant gene and
its promoter.
[0133] The obtained DNA fragment was purified by SUPREC02 produced
by the Takara Shuzo, then fully digested with restriction enzymes
HindIII and HincII, and blunt-ended. The blunt-ending was attained
by using Blunting Kit produced by Takara Shuzo. This DNA fragment
was mixed with and ligated to a DNA fragment, which had been
obtained by performing PCR using the primers shown as SEQ ID NOS: 6
and 7 and pHSG399 (see S. Takeshita et al., Gene, 61, 63-74 (1987))
as a template, purifying and blunt-ending the resulted
amplification product. The ligation reaction was performed by DNA
Ligation Kit ver. 2 produced by Takara Shuzo. Competent cells of
Escherichia coli JM109 (produced by Takara Shuzo) were transformed
with the ligated DNA, plated on L madium (10 g/L of Bacto-trypton,
5 g/L of Bacto-yeast extract, 5 g/L of NaCl, 15 g/L of agar, pH
7.2) containing 10 .mu.g/ml of IPTG (isopropyl-.beta.-D-thiogala-
ctopyranoside), 40 .mu.g/ml of X-Gal
(5-bromo-4-chloro-3-indolyl-.beta.-D-- galactoside) and 25 .mu.g/ml
of kanamycin, and cultured overnight. The emerged blue colonies
were picked up, and separated into single colonies to obtain
transformant strains.
[0134] Plasmids were prepared from the transformant strains by the
alkali method (Text for Bioengineering Experiments, Edited by the
Society for Bioscience and Bioengineering, Japan, p.105, Baifukan,
1992), and restriction maps were prepared. One having a restriction
map equivalent to that of FIG. 2 was designated as pK1. This
plasmid is stably retained in Escherichia coli, and imparts
kanamycin resistance to a host. Moreover, since it contains the
lacZ' gene, it is suitably used as a cloning vector.
[0135] The plasmid pAM330 extracted from Brevibacterium
lactofermentum ATCC13869 (see Japanese Patent Laid-open No.
58-67699) was fully digested with a restriction enzyme HindIII, and
blunt-ended. This fragment was ligated to a fragment obtained by
fully digesting the aforementioned pK1 with a restriction enzyme
BsaAI. Breveibacterium lactofermentum ATCC13869 was transformed
with the ligated DNA. The transformation was performed by the
electric pulse method (see Japanese Patent Laid-open No. 2-207791).
Transformants were selected on a M-CM2B plate (10 g/L of
polypeptone, 10 g/L of yeast extract, 5 g/L of NaCl, 10 .mu.g/L of
biotin, 15 g/L of agar, pH 7.2) containing 25 .mu.g/ml of
kanamycin. After cultivation for 2 days, colonies were picked up,
and separated into single colonies to obtain the transformants.
Plasmid DNA was prepared from the transformants, and restriction
maps were prepared. One having the same restriction map as that of
FIG. 3 was designated as pSFK6. This plasmid can autonomously
replicate in both of Escherichia coli and coryneform bacteria, and
imparts kanamycin resistance to a host.
[0136] <2> Introduction of carA and carB Genes into
Coryneform Bacteria and Production of L-arginine
[0137] The aforementioned pSFK6 was digested with SmaI and HindIII.
The product was ligated to carA and carB gene fragments, which had
been obtained by digesting the plasmid p19 prepared from JEF8/p19F
in a conventional manner with a restriction enzyme XbaI,
blunt-ending the product by using Blunting Kit produced by Takara
Shuzo, and further digesting the product with a restriction enzyme
HindIII, to obtain a plasmid pcarAB, which contained the carA and
carB genes and could autonomously replicate in coryneform
bacteria.
[0138] pcarAB was introduced into Brevibacterium flavum AJ11345 and
AJ11336 by the electric pulse method (Japanese Patent Laid-open No.
2-207791). Transformants were selected on a M-CM2B plate (10 g/L of
polypeptone, 10 g/L of yeast extract, 5 g/L of glucose, 5 g/L of
NaCl, 15 g/L of agar, pH 7.2) containing 25 .mu.g/ml of kanamycin
as kanamycin resistant strains. As control, transformants were
obtained by similarly introducing pSFK6 into AJ11345 and
AJ11336.
[0139] Each of the aforementioned transformants was plated on an
agar medium containing 0.5 g/dl of glucose, 1 g/dl of polypeptone,
1 g of yeast extract, 0.5 g/dl of NaCl and 5 .mu.g/l of
chloramphenicol, and cultured at 31.5.degree. C. for 20 hours. One
inoculating loop of the obtained cells were inoculated to a medium
containing 4 g/dl of glucose, 6.5 g/dL of ammonium sulfate, 0.1
g/dl of KH.sub.2PO.sub.4, 0.04 g/dl of MgSO.sub.4, 0.001 g/dl of
FeSO.sub.4, 0.01 g/dl of MnSO.sub.4, 5 .mu.g/dl of VB.sub.1, 5
.mu.g/dl of biotin, 45 mg/dl of soybean hydrolysates (as an amount
of N), and cultured in a flask at 31.5.degree. C. for 50 hours with
shaking. The amounts of L-arginine produced by each strain were
shown in Table 1.
[0140] The strains introduced with the carA and carB gene showed
improved L-arginine productivity compared with the strains
introduced only with the vector.
1 TABLE 1 Strain/plasmid L-arginine (g/dl) AJ11345/pSFK6 1.33
AJ11345/pcarAB 1.39 AJ11336/pSFK6 0.71 AJ11336/pcarAB 0.79
[0141]
Sequence CWU 0
0
* * * * *