U.S. patent application number 09/784208 was filed with the patent office on 2001-09-06 for l-glutamic acid-producing bacterium and method for producing l-glutamic acid.
This patent application is currently assigned to AJINOMOTO CO., INC. Invention is credited to Hara, Yoshihiko, Ito, Hisao, Izui, Hiroshi, Matsui, Kazuhiko, Moriya, Mika, Ono, Eiji.
Application Number | 20010019836 09/784208 |
Document ID | / |
Family ID | 26410250 |
Filed Date | 2001-09-06 |
United States Patent
Application |
20010019836 |
Kind Code |
A1 |
Moriya, Mika ; et
al. |
September 6, 2001 |
L-glutamic acid-producing bacterium and method for producing
L-glutamic acid
Abstract
L-Glutamic acid is produced by culturing in a liquid culture
medium a microorganism belonging to the genus Enterobacter or
Serratia and having an ability to produce L-glutamic acid, which
increases in an activity of enzyme catalyzing a reaction for
L-glutamic acid biosynthesis, or which decreases in or is deficient
in an activity of an enzyme catalyzing a reaction branching from a
pathway for L-glutamic acid biosynthesis and producing a compound
other than L-glutamic acid, and collecting produced L-glutamic acid
from the culture medium.
Inventors: |
Moriya, Mika; (Kawasaki-shi,
JP) ; Izui, Hiroshi; (Kawasaki-shi, JP) ; Ono,
Eiji; (Kawasaki-shi, JP) ; Matsui, Kazuhiko;
(Kawasaki-shi, JP) ; Ito, Hisao; (Kawasaki-shi,
JP) ; Hara, Yoshihiko; (Kawasaki-shi, JP) |
Correspondence
Address: |
OBLON SPIVAK MCCLELLAND MAIER & NEUSTADT PC
FOURTH FLOOR
1755 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Assignee: |
AJINOMOTO CO., INC
15-1, Kyobashi 1-chome, Chuo-Ku
Tokyo
JP
|
Family ID: |
26410250 |
Appl. No.: |
09/784208 |
Filed: |
February 16, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09784208 |
Feb 16, 2001 |
|
|
|
09271438 |
Mar 18, 1999 |
|
|
|
Current U.S.
Class: |
435/110 ;
435/252.1; 435/252.8 |
Current CPC
Class: |
C12R 2001/01 20210501;
C12N 9/0016 20130101; C12R 2001/425 20210501; Y10S 435/822
20130101; C12P 13/14 20130101; C12N 1/205 20210501; Y10S 435/88
20130101; C12N 9/88 20130101 |
Class at
Publication: |
435/110 ;
435/252.8; 435/252.1 |
International
Class: |
C12N 001/20; C12P
013/14 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 18, 1998 |
JP |
10-69068 |
Oct 19, 1998 |
JP |
10-297129 |
Claims
What is claimed is:
1. A microorganism belonging to the genus Enterobacter or Serratia
and having an ability to produce L-glutamic acid and at least one
of the following properties: (a) the microorganism increases in an
activity of an enzyme catalyzing a reaction for the L-glutamic acid
biosynthesis; and (b) the microorganism decreases in or is
deficient in an activity of an enzyme catalyzing a reaction
branching from a pathway for L-glutamic acid biosynthesis and
producing a compound other than L-glutamic acid.
2. A microorganism according to claim 1 wherein the enzyme
catalyzing the reaction for the L-glutamic acid biosynthesis is at
least one selected from the group consisting of citrate synthase,
phosphoenolpyruvate carboxylase, and glutamate dehydrogenase.
3. A microorganism according to claim 2 wherein the enzyme
catalyzing the reaction for the L-glutamic acid biosynthesis
includes all of citrate synthase, phosphoenolpyruvate carboxylase,
and glutamate dehydrogenase.
4. A microorganism according to any one of claims 1 to 3 wherein
the enzyme catalyzing the reaction branching from the pathway for
L-glutamic acid biosynthesis and producing the compound other than
L-glutamic acid is .alpha.-ketoglutarate dehydrogenase.
5. A microorganism according to any one of claims 1 to 4 which is
Enterobacter agglomerans or Serratia liquefacience.
6. A method for producing L-glutamic acid which comprises culturing
the microorganism as defined in any one of claims 1 to 5 in a
liquid culture medium to produce and accumulate L-glutamic acid in
the culture medium, and collecting the L-glutamic acid from the
culture medium.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to a novel L-glutamic
acid-producing bacterium and a method for producing L-glutamic acid
by fermentation using the same. L-Glutamic acid is an important
amino acid as food, drugs and the like.
[0002] L-Glutamic acid has conventionally been produced by
fermentation methods utilizing the so-called coryneform L-glutamic
acid-producing bacteria which principally belong to the genera
Brevibacterium, Corynebacterium, and Microbacterium or variants
thereof ("Amino Acid Fermentation", Gakkai Shuppan Center,
pp.195-215, 1986). As methods for producing L-glutamic acid by
fermentation utilizing other bacterial strains, there have been
known the methods utilizing microorganisms of the genera Bacillus,
Streptomyces, Penicillium and the like (U.S. Pat. No. 3,220,929),
the methods utilizing microorganisms of the genera Pseudomonas,
Arthrobacter, Serratia, Candida and the like (U.S. Pat. No.
3,563,857), the methods utilizing microorganisms of the genera
Bacillus, Pseudomonas, Serratia and the like or Aerobacter
aerogenes (currently referred to as Enterobacter aerogenes)
(Japanese Patent Publication (KOKOKU) No. 32-9393(1957)), the
method utilizing variant strains of Escherichia coli (Japanese
Patent Application Laid-Open (KOKAI) No. 5-244970(1993)) and the
like.
[0003] Though the productivity of L-glutamic acid has considerably
been improved by breeding of such microorganisms as mentioned above
or improvements of production methods, it is still desired to
develop a more inexpensive and more efficient method for producing
L-glutamic acid in order to meet the expected markedly increasing
future demand of the amino acid.
SUMMARY OF THE INVENTION
[0004] The object of the present invention is to find a novel
L-glutamic acid-producing bacterium having a high ability to
produce L-glutamic acid, thereby developing a more inexpensive and
more efficient method for producing L-glutamic acid.
[0005] To achieve the aforementioned object, the present inventors
intensively searched for and studied microorganisms having the
ability to produce L-glutamic acid that are different from the
previously reported microorganisms. As a result, they found that
certain strains derived from microorganisms belonging to the genus
Enterobacter or Serratia had a high ability to produce L-glutamic
acid, and have completed the present invention.
[0006] Thus, the present invention provide:
[0007] (1) a microorganism belonging to the genus Enterobacter or
Serratia and having an ability to produce L-glutamic acid and at
least one of the following properties:
[0008] (a) the microorganism increases in an activity of an enzyme
catalyzing a reaction for L-glutamic acid biosynthesis; and
[0009] (b) the microorganism decreases in or is deficient in an
activity of an enzyme catalyzing a reaction branching from a
pathway for L-glutamic acid biosynthesis and producing a compound
other than L-glutamic acid;
[0010] (2) a microorganism of the above (1) wherein the enzyme
catalyzing the reaction for the L-glutamic acid biosynthesis is at
least one selected from the group consisting of citrate synthase
(abbreviated as "CS" hereinafter), phosphoenolpyruvate carboxylase
(abbreviated as "PEPC" hereinafter), and glutamate dehydrogenase
(abbreviated as "GDH" hereinafter);
[0011] (3) a microorganism of the above (2) wherein the enzyme
catalyzing the reaction for the L-glutamic acid biosynthesis
includes all of CS, PEPC and GDH;
[0012] (4) a microorganism of any one of the above (1) to (3)
wherein the enzyme catalyzing the reaction branching from the
pathway for L-glutamic acid biosynthesis and producing the compound
other than L-glutamic acid is .alpha.-ketoglutarate dehydrogenase
(abbreviated as ".alpha.KGDH" hereinafter);
[0013] (5) a microorganism of any one of the above (1) to (4) which
is Enterobacter agglomerans or Serratia liquefacience; and
[0014] (6) a method for producing L-glutamic acid which comprises
culturing the microorganism as defined in any one of the above (1)
to (5) in a liquid culture medium to produce and accumulate
L-glutamic acid in the culture medium, and collecting the
L-glutamic acid from the culture medium.
[0015] Because the microorganism of the present invention have a
high ability to produce L-glutamic acid, it is considered to be
possible to impart a further higher production ability to the
microorganism by using the breeding techniques previously known for
the coryneform L-glutamic acid-producing bacteria and the like, and
it is expected to contribute to development of a more inexpensive
and more efficient method for producing L-glutamic acid by
appropriately selecting culture conditions and the like.
BRIEF EXPLANATION OF THE DRAWINGS
[0016] FIG. 1 shows construction of a plasmid pMWCPG having a gltA
gene, a ppc gene and a gdha gene.
[0017] FIG. 2 shows construction of a plasmid pSTVG having the gdhA
gene.
[0018] FIG. 3 shows construction of a plasmid RSF-Tet having a
replication origin of a wide-host-range plasmid RSF1010 and a
tetracycline resistance gene.
[0019] FIG. 4 shows construction of a plasmid RSFCPG having the
replication origin of the wide-host-range plasmid RSF1010, the
tetracycline resistance gene, the gltA gene, the ppc gene and the
gdhA gene.
[0020] FIG. 5 shows construction of a plasmid pMWCB having the gltA
gene.
[0021] FIG. 6 shows a restriction map of a DNA fragment of
pTWVEK101 derived from Enterobacter agglomerans.
[0022] FIG. 7 shows comparison of an amino acid sequence deduced
from a nucleotide sequence of a sucA gene derived from Enterobacter
agglomerans with one derived from Escherichia coli. The upper
sections: Enterobacter agglomerans, the lower sections: Escherichia
coli (the same shall apply hereinafter).
[0023] FIG. 8 shows comparison of an amino acid sequence deduced
from a nucleotide sequence of a sucB gene derived from Enterobacter
agglomerans with one derived from Escherichia coli.
[0024] FIG. 9 shows comparison of an amino acid sequence deduced
from a nucleotide sequence of a sdhB gene derived from Enterobacter
agglomerans with one derived from Escherichia coli.
[0025] FIG. 10 shows comparison of an amino acid sequence deduced
from a nucleotide sequence of a sucC gene derived from Enterobacter
agglomerans with one derived from Escherichia coli.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention will be explained in detail
hereinafter.
[0027] The microorganism of the present invention is a
microorganism belonging to the genus Enterobacter or Serratia, and
having at least one of the following properties:
[0028] (a) the microorganism increases in an activity of an enzyme
catalyzing a reaction for L-glutamic acid biosynthesis; and
[0029] (b) the microorganism decreases in or is deficient in an
activity of an enzyme catalyzing a reaction branching from a
pathway for L-glutamic acid biosynthesis and producing a compound
other than L-glutamic acid.
[0030] Such a microorganism can be obtained by using a
microorganism belonging to the genus Enterobacter or the genus
Serratia as a parent strain, and imparting the properties of the
above (a) and/or (b) to the microorganism. Examples of the
microorganism belonging to the genus Enterobacter or the genus
Serratia that can be used as the parent strain are listed
below:
[0031] Enterobacter agglomerans
[0032] Enterobacter aerogenes
[0033] Enterobacter amnigenus
[0034] Enterobacter asburiae
[0035] Enterobacter cloacae
[0036] Enterobacter dissolvens
[0037] Enterobacter gergoviae
[0038] Enterobacter hormaechei
[0039] Enterobacter intermedius
[0040] Enterobacter nimipressuralis
[0041] Enterobacter sakazakii
[0042] Enterobacter taylorae
[0043] Serratia liquefacience
[0044] Serratia entomophila
[0045] Serratia ficaria
[0046] Serratia fonticola
[0047] Serratia grimesii
[0048] Serratia proteamaculans
[0049] Serratia odorifera
[0050] Serratia plymuthica
[0051] Serratia rubidaea
[0052] More preferably, those bacterial strains listed below can be
mentioned:
[0053] Enterobacter agglomerans ATCC 12287
[0054] Enterobacter agglomerans AJ13355
[0055] Serratia liquefacience ATCC 14460
[0056] The Enterobacter agglomerans AJ13355 was deposited at the
National Institute of Bioscience and Human-Technology, Agency of
Industrial Science and Technology, Ministry of International Trade
and Industry on Feb. 19, 1998, and received an accession number of
FERM P-16644, and then transferred to an international deposition
under the Budapest Treaty on Jan. 11, 1999, and received an
accession number of FERM BP-6614. The Enterobacter agglomerans ATCC
12287, and the Serratia liquefacience ATCC 14460 are available from
ATCC.
[0057] The Enterobacter agglomerans AJ13355 is a strain isolated
from soil in Iwata-shi, Shizuoka, Japan.
[0058] Physiological properties of AJ13355 are as follows:
[0059] (1) Gram stain: Negative
[0060] (2) Behavior for oxygen: Facultative anaerobe
[0061] (3) Catalase: Negative
[0062] (4) Oxidase: Positive
[0063] (5) Nitrate reduction: Negative
[0064] (6) voges-Proskauer reaction: Positive
[0065] (7) Methyl Red test: Negative
[0066] (8) Urease: Negative
[0067] (9) Indole production: Positive
[0068] (10) Motility: Present
[0069] (11) Hydrogen sulfide production in TSI culture medium:
Slightly active
[0070] (12) .beta.-Galactosidase: Positive
[0071] (13) Sugar assimilability:
[0072] Arabinose: Positive
[0073] Sucrose: Positive
[0074] Lactose: Positive
[0075] Xylose: Positive
[0076] Sorbitol: Positive
[0077] Inositol: Positive
[0078] Trehalose: Positive
[0079] Maltose: Positive
[0080] Melibiose: Positive
[0081] Adonitol: Negative
[0082] Raffinose: Positive
[0083] Salicin: Negative
[0084] Melibiose: Positive
[0085] (14) Glycerose assimilability: Positive
[0086] (15) Organic acid assimilability:
[0087] Citric acid: Positive
[0088] Tartaric acid: Negative
[0089] Gluconic acid: Positive
[0090] Acetic acid: Positive
[0091] Malonic acid: Negative
[0092] (16) Arginine dehydratase: Negative
[0093] (17) Ornithine decarboxylase: Negative
[0094] (18) Lysine decarboxylase: Negative
[0095] (19) Phenylalanine deaminase: Negative
[0096] (20) Pigment formation: Yellow
[0097] (21) Gelatin liquefaction: Positive
[0098] (22) Growth pH: Not good growth at pH 4, good growth at pH
4.5 to 7
[0099] (23) Growth temperature: Good growth at 25.degree. C., good
growth at 30.degree. C., good growth at 37.degree. C., growth
possible at 42.degree. C., no growth at 45.degree. C.
[0100] From these bacteriological properties, AJ13355 is determined
to be Enterobacter agglomerans.
[0101] In the working examples described hereinafter, Enterobacter
agglomerans ATCC12287, Enterobacter agglomerans AJ13355, and
Serratia liquefacience ATCC14460 were used as starting parent
strains for obtaining strains which increase in the activity of the
enzyme catalyzing the reactions for the L-glutamic acid
biosynthesis, or strains which decrease in or are deficient in the
activity of the enzyme catalyzing the reaction branching from the
pathway for L-glutamic acid biosynthesis and producing the compound
other than L-glutamic acid. However, the sugar metabolism by any of
bacteria belonging to the genera Enterobacter and Serratia is
achieved via the Embden-Meyerhof pathway, and pyruvate produced in
the pathway is oxidized in the tricarboxylic acid cycle under
aerobic conditions. L-Glutamic acid is biosynthesized from
.alpha.-ketoglutaric acid which is an intermediate of the
tricarboxylic acid cycle by GDH or glutamine synthetase/glutamate
synthase. Thus, these microorganisms share the same biosynthetic
pathway for L-glutamic acid, and microorganism belonging to the
genera Enterobacter and Serratia are encompassed within a single
concept according to the present invention. Therefore,
microorganisms belonging to the genera Enterobacter and Serratia
other than species and strains specifically mentioned above also
fall within the scope of the present invention.
[0102] The microorganism of the present invention is a
microorganism belonging to the genus Enterobacter or the genus
Serratia and having an ability to produce L-glutamic acid. The
expression "having an ability to produce L-glutamic acid" as herein
used means to have an ability to accumulate L-glutamic acid in a
culture medium during cultivation. According to the present
invention, the ability to produce L-glutamic acid is imparted by
giving either one or both of the following characteristics:
[0103] (a) the microorganism increases in the activity of the
enzyme catalyzing the reaction for the L-glutamic acid
biosynthesis; and
[0104] (b) the microorganism decreases in or is deficient in the
activity of the enzyme catalyzing the reaction branching from the
pathway for L-glutamic acid biosynthesis and producing the compound
other than L-glutamic acid.
[0105] As examples of the enzyme catalyzing the reaction for
L-glutamic acid biosynthesis of microorganisms of the genus
Enterobacter or Serratia, there can be mentioned GDH, glutamine
synthetase, glutamate synthase, isocitrate dehydrogenase, aconitate
hydratase, CS, PEPC, pyruvate dehydrogenase, pyruvate kinase,
enolase, phosphoglyceromutase, phosphoglycerate kinase,
glyceraldehyde-3-phosphate dehydrogenase, triosephosphate
isomerase, fructose bisphosphate aldolase, phosphofructokinase,
glucose phosphate isomerase and the like. Among these enzymes, one
or two or three kinds of CS, PEPC and GDH are preferred. As for the
microorganism of the present invention, it is further preferred
that activities of all of the three kinds of enzymes, CS, PEPC and
GDH, are increased. Whether a microorganism increases in an
activity of a target enzyme, and degree of the increase of the
activity can be determined by measuring the enzyme activity of a
bacterial cell extract or a purified fraction, and comparing it
with that of a wild type strain or a parent strain.
[0106] The microorganism of the present invention, which belongs to
the genus Enterobacter or Serratia, and increases in the activity
of the enzyme catalyzing the reaction for L-glutamic acid
biosynthesis, can be obtained as, for example, a variant where
mutation has been made in a gene encoding the enzyme or a genetic
recombinant strain by using any of the microorganisms mentioned
above as a starting parent strain.
[0107] To enhance the activity of CS, PEPC or GDH, for example, a
gene encoding CS, PEPC or GDH can be cloned in a suitable plasmid,
and the aforementioned starting parent strain as a host can be
transformed with the resulting plasmid. This can increase the copy
number of each of the genes encoding CS, PEPC and GDH (hereinafter
abbreviated as "gltA gene", "ppc gene", and "gdhA gene",
respectively), and as a result the activities of CS, PEPC and GDH
can be increased.
[0108] One or two or three kinds selected from the cloned gltA
gene, ppc gene and gdhA gene in any combination are introduced into
the starting parent strain mentioned above. When two or three kinds
of the genes are introduced, either the two or three kinds of the
genes are cloned in one kind of plasmid, and introduced into the
host, or they are separately cloned in two or three kinds of
plasmids that can exist in the same host, and introduced into the
host.
[0109] The plasmid is not particularly limited so long as it can
autonomously replicate in a microorganism belonging to the genus
Enterobacter or Serratia. Examples of the plasmid include, for
example, pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398,
RSF1010, pMW119, pMW118, pMW219, pMW218 and the like. Other than
these plasmids, phage DNA vectors can also be utilized.
[0110] Transformation can be achieved by, for example, the method
of D. M. Morrison (Methods in Enzymology 68, 326 (1979)), the
method by increasing permeability of recipient cells for DNA with
calcium chloride (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159
(1970)), or the like.
[0111] The activities of CS, PEPC and GDH can also be increased by
using multiple copies of the gltA gene, the ppc gene and/or the gdh
gene present on the chromosome DNA of the starting parent strain as
a host. In order to introduce multiple copies of the gltA gene, the
ppc gene and/or the gdhA gene into a chromosome DNA of a
microorganism belonging to the genus Enterobacter or Serratia,
sequences present on chromosome DNA in a multiple copy number such
as repetitive DNA, and inverted repeats present at an end of
transposition factors can be utilized. Alternatively, multiple
copies of the genes can also be introduced into a chromosome DNA by
utilizing transposition of transposons carrying the gltA gene, the
ppc gene, or the gdhA gene. These techniques can increase the copy
number of the gltA gene, the ppc gene, and the gdhA gene in
transformant cells, and as a result increase the activities of CS,
PEPC and GDH.
[0112] Any organisms can be used as a source of the gltA gene, the
ppc gene and the gdhA gene used for increasing copy numbers, so
long as the organisms have the CS, PEPC and GDH activities. Among
such organisms, bacteria, i.e., prokaryotes, such as those bacteria
belonging to the genera Enterobacter, Klebsiella, Erwinia, Pantoea,
Serratia, Escherichia, Corynebacterium, Brevibacterium, and
Bacillus are preferred. As a specific example, Escherichia coli can
be mentioned. The gltA gene, the ppc gene and the gdhA gene can be
obtained from a chromosome DNA of such microorganisms as mentioned
above.
[0113] The gltA gene, the ppc gene and the gdhA gene can each be
obtained from a chromosome DNA of any of the aforementioned
microorganisms by isolating a DNA fragment complementing auxotrophy
of a variant strain lacking the CS, PEPC or GDH activity.
Alternatively, because the nucleotide sequences of these genes of
bacteria of the genus Escherichia or Corynebacterium have already
been elucidated (Biochemistry, Vol. 22, pp.5243-5249, 1983; J.
Biochem. Vol. 95, pp.909-916, 1984; Gene, Vol. 27, pp.193-199,
1984; Microbiology, Vol. 140, pp.1817-1828, 1994; Mol. Gen. Genet.
Vol. 218, pp.330-339, 1989; and Molecular Microbiology, Vol. 6,
pp.317-326, 1992), the genes can be obtained by PCR using primers
synthesized based on each of the elucidated nucleotide sequences,
and the chromosome DNA as a template.
[0114] The activity of CS, PEPC or GDH can also be increased by,
other than by the gene amplification mentioned above, enhancing
expression of the gltA gene, the ppc gene or the gdhA gene. For
example, the expression is enhanced by replacing the promoter of
the gltA gene, the ppc gene, or the gdhA gene with another stronger
promoter. Examples of such a strong promoter include, for example,
a lac promoter, a trp promoter, a trc promoter, a tac promoter, a
P.sub.R promoter and a P.sub.L promoter of lambda phage and the
like. The gltA gene, the ppc gene, or the gdhA gene of which
promoter has been substituted is cloned into a plasmid and
introduced into a host microorganism, or introduced into a
chromosome DNA of host microorganism using a repetitive DNA,
inverted repeat, transposon or the like.
[0115] The activities of CS, PEPC or GDH can also be increased by
replacing the promoter of the gltA gene, the ppc gene, or the gdhA
gene on a chromosome with another stronger promoter (see
WO87/03006, and Japanese Patent Application Laid-Open (KOKAI) No.
61-268183(1986)), or inserting a strong promoter at the upstream of
each coding sequence of the genes (see Gene, 29, pp. 231-241,
1984). Specifically, these are achieved by homologous recombination
between the gltA gene, the ppc gene, or the gdhA gene of which
promoter is replaced with a stronger promoter or DNA containing a
part of them, and a corresponding gene on the chromosome.
[0116] Specific examples of the microorganism belonging to the
genus Enterobacter or Serratia of which CS, PEPC or GDH activity is
increased include, for example, Enterobacter agglomerans
ATCC12287/RSFCPG, Enterobacter agglomerans AJ13355/RSFCPG, and
Serratia liquefacience ATCC14460/RSFCPG.
[0117] Examples of the enzyme catalyzing the reaction branching
from the pathway of L-glutamic acid biosynthesis and producing the
compound other than L-glutamic acid include, for example,
.alpha.KGDH, isocitrate lyase, phosphate acetyltransferase, acetate
kinase, acetohydroxy acid synthase, acetolactate synthase, formate
acetyltransferase, lactate dehydrogenase, glutamate decarboxylase,
1-pyrroline dehydrogenase and the like. Among these enzymes,
.alpha.KGDH is preferred.
[0118] In order to obtain such decrease or deficiency of enzyme
activity as mentioned above in a microorganism belonging to the
genus Enterobacter or Serratia, a mutation causing the decrease or
deficiency of the enzyme activity can be introduced into a gene
encoding the enzyme by a conventional mutagenesis technique or
genetic engineering technique.
[0119] Examples of the mutagenesis technique include, for example,
the method utilizing irradiation of X-ray or ultraviolet light, the
method utilizing treatment with a mutagenic agent such as
N-methyl-N'-nitro-N-nitrosoguanidine and the like. The site of gene
to which a mutation is introduced may be a coding region encoding
an enzyme protein, or an expression regulatory region such as a
promoter.
[0120] Examples of the genetic engineering technique include, for
example, genetic recombination, genetic transduction, cell fusion
and the like. For example, a drug resistance gene is inserted into
a target gene to produce a functionally inactivated gene (defective
gene). Then, this defective gene is introduced into a cell of a
microorganism belonging to the genus Enterobacter or Serratia, and
the target gene on a chromosome is replaced with the defective gene
by homologous recombination (gene disruption).
[0121] Whether a microorganism decreases in an activity of a target
enzyme or is deficient in the activity, and degree of the decrease
of the activity can be determined by measuring the enzyme activity
of a bacterial cell extract or a purified fraction of a candidate
strain, and comparing it with that of a wild type strain or a
parent strain. The .alpha.KGDH enzymatic activity can be measured
by, for example, the method of Reed et al. (L. J. Reed and B. B.
Mukherjee, Methods in Enzymology 1969, 13, p.55-61).
[0122] Depending on the target enzyme, a target variant can be
selected based on a phenotype of the variant. For example, a
variant which is deficient in the .alpha.KGDH activity or decreases
in the activity cannot grow on a minimal medium containing glucose,
or a minimal medium containing acetic acid or L-glutamic acid as an
exclusive carbon source, or shows markedly reduced growth rate
therein under aerobic conditions. However, even under the same
condition, it can exhibit normal growth by addition of succinic
acid or lysine, methionine and diaminopimelate to the minimal
medium containing glucose. Based on these phenomena, a variant that
is deficient in the .alpha.KGDH activity or decreases in the
activity can be selected.
[0123] A method for producing a Brevibacterium lactofermentum
strain lacking the .alpha.KGDH gene based on homogenous
recombination is detailed in WO95/34672, and a similar method can
be used for microorganisms belonging to the genera Enterobacter and
Serratia.
[0124] In addition, procedures of genetic cloning, cleavage and
ligation of DNA, transformation and the like are detailed in
Molecular Cloning, 2nd edition, Cold Spring Harbor Press (1989) and
the like.
[0125] An example of the variant strain that is deficient in the
.alpha.KGDH activity or decreases in the activity obtained as
described above is Enterobacter agglomerans AJ13356. The
Enterobacter agglomerans AJ13356 was deposited at the National
Institute of Bioscience and Human-Technology, Agency of Industrial
Science and Technology, Ministry of International Trade and
Industry on Feb. 19, 1998, received an accession number of FERM
P-16645, and then transferred to an international deposition under
the Budapest Treaty on Jan. 11, 1999, and received an accession
number of FERM BP-6615.
[0126] The microorganism belonging to the genus Enterobacter or
Serratia, and having at least one of the properties (a) and (b) and
an ability to produce L-glutamic acid can be cultured in a liquid
medium to produce and accumulate L-glutamic acid in the medium.
[0127] The culture medium may be an ordinary nutrient medium
containing a carbon source, a nitrogen source, and inorganic salts,
as well as organic trace nutrients such as amino acids, vitamins
and the like, as required. It can be a synthetic medium or a
natural medium. Any carbon sources and nitrogen sources can be used
for the culture medium so long as they can be utilized by the
microorganism to be cultured.
[0128] The carbon source may be a saccharide such as glucose,
glycerol, fructose, sucrose, maltose, mannose, galactose, starch
hydrolysates, molasses and the like. Further, an organic acid such
as acetic acid and citric acid may also be used alone or in
combination with other carbon sources.
[0129] The nitrogen source may be ammonia, ammonium salts such as
ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium
phosphate, and ammonium acetate, nitrates and the like.
[0130] As organic trace nutrients, amino acids, vitamins, fatty
acids, nucleic acids, materials containing them such as peptone,
casamino acid, yeast extract, and soybean protein decomposition
products and the like are used, and when an auxotrophic variant
which requires an amino acid or the like for its growth is used, it
is necessary to complement the nutrient required.
[0131] As the inorganic salt, phosphates, magnesium salts, calcium
salts, iron salts, manganese salts and the like are used.
[0132] As for the culture conditions, cultivation may be performed
under aerobic conditions at a temperature of 20 to 42.degree. C.
and a pH of 4 to 8. The cultivation can be continued for 10 hours
to 4 days to accumulate a considerable amount of L-glutamic acid in
the liquid culture medium.
[0133] After the completion of the cultivation, L-glutamic acid
accumulated in the culture medium may be collected by a known
method. For example, it can be isolated by a method comprising
concentrating the medium after removing the cells to crystallize
the product, ion exchange chromatography or the like.
[0134] Examples
[0135] The present invention will be explained more specifically
with reference to the following examples.
[0136] (1) Construction of plasmid having gltA gene, ppc gene and
gdhA gene
[0137] Procedure for construction of a plasmid having a gltA gene,
a ppc gene and a gdhA gene will be explained by referring to FIG. 1
to FIG. 5.
[0138] A plasmid pBRGDH having a gdhA gene derived from Escherichia
coli (Japanese Patent Application Laid-Open (KOKAI) No.
7-203980(1995)) was digested with HindIII and SphI, and the both
ends were blunt-ended by a treatment with T4 DNA polymerase. Then,
a DNA fragment containing the gdhA gene was purified and collected.
On the other hand, a plasmid pMWCP having a gltA gene and a ppc
gene derived from Escherichia coli (WO97/08294) was digested with
XbaI, and the both ends were blunt-ended by a treatment with T4 DNA
polymerase. This was mixed with the DNA fragment having the gdhA
gene purified above, and ligated with T4 ligase, giving a plasmid
pMWCPG, which corresponds to the pMWCP further carrying the gdhA
gene (FIG. 1).
[0139] A DNA fragment having the gdhA gene obtained by digesting
the pBRGDH with HindIII and SalI was purified and collected, and
introduced into the HindIII-SalI site of a plasmid pSTV29
(purchased from Takara Shuzo) to obtain a plasmid pSTVG (FIG.
2).
[0140] At the same time, a product obtained by digesting a plasmid
pVIC40 having a replication origin of a wide-host-range plasmid
RSF1010 (Japanese Patent Application Laid-Open (KOKAI) No.
8-047397(1996)) with NotI, followed by T4 DNA polymerase treatment
and PstI digestion, and a product obtained by digesting pBR322 with
EcoT141, followed by T4 DNA polymerase treatment and PstI
digestion, were mixed and ligated with T4 ligase to obtain a
plasmid RSF-Tet having the replication origin of RSF1010 and a
tetracycline resistance gene (FIG. 3).
[0141] Then, the pMWCPG was digested with EcoRI and PstI, and a DNA
fragment having the gltA gene, the ppc gene and the gdhA gene was
purified and collected. Similarly, the RSF-Tet was digested with
EcoRI and PstI, and a DNA fragment having the replication origin of
RSF1010 was purified and collected. Those DNA fragments were mixed
and ligated with T4 ligase to obtain a plasmid RSFCPG composed of
RSF-Tet carrying the gltA gene, the ppc gene and the gdhA gene
(FIG. 4). Expression of the gitA gene, the ppc gene and the gdhA
gene by the resulting plasmid RSFCPG, and expression of the gdhA
gene by the pSTVG were confirmed based on complementation of
auxotrophy of Escherichia coli strains lacking the gltA gene, the
ppc gene or the gdhA gene, and measurement of each enzyme
activity.
[0142] A plasmid having a gltA gene derived from Brevibacterium
lactofermentum was constructed as follows. PCR was performed by
using primers having the nucleotide sequences represented in SEQ ID
NOS: 6 and 7 selected based on the nucleotide sequence of the gltA
gene of Corynebacterium glutamicum (Microbiology, 140, 1817-1828,
1994), and a chromosome DNA of Brevibacteriuin lactofermentum ATCC
13869 as a template to obtain a gltA gene fragment of about 3 kb.
This fragment was inserted into a plasmid pHSG399 (purchased from
Takara Shuzo) digested with SmaI to obtain a plasmid pHSGCB (FIG.
5). Then, the pHSGCB was digested with HindIII, and an excised gltA
gene fragment of about 3 kb was inserted into a plasmid pMW218
(purchased from Nippon Gene) digested with HindIII to obtain a
plasmid pMWCB (FIG. 5). Expression of the gltA gene by the
resulting plasmid pMWCB was confirmed by determination of enzyme
activity in the Enterobacter agglomerans AJ13355.
[0143] (2) Introduction of RSFCPG, pMWCB and pSTVG into
Enterobacter agglomerans or Serratia liquefacience, and evaluation
of L-glutamic acid productivity
[0144] The Enterobacter agglomerans ATCC 12287, the Enterobacter
agglomerans AJ13355 and the Serratia liquefacience ATCC 14460 were
transformed with the RSFCPG, pMWCB and pSTVG by electroporation
(Miller J. H., "A Short Course in Bacterial Genetics; Handbook"
Cold Spring Harbor Laboratory Press, USA, 1992) to obtain
transformants exhibiting tetracycline resistance.
[0145] Each of the resulting transformants and the parent strains
was inoculated into 50 ml-volume large size test tube containing 5
ml of a culture medium comprising 40 g/L glucose, 20 g/L ammonium
sulfate, 0.5 g/L magnesium sulfate heptahydrate, 2 g/L potassium
dihydrogenphosphate, 0.5 g/L sodium chloride, 0.25 g/L calcium
chloride heptahydrate, 0.02 g/L ferrous sulfate heptahydrate, 0.02
g/L manganese sulfate tetrahydrate, 0.72 mg/L zinc sulfate
dihydrate, 0.64 mg/L copper sulfate pentahydrate, 0.72 mg/L cobalt
chloride hexahydrate, 0.4 mg/L boric acid, 1.2 mg/L sodium
molybdate dihydrate, 2 g/L yeast extract, and 30 g/L calcium
carbonate, and cultured at 37.degree. C. with shaking until the
glucose contained in the culture medium was consumed. However, as
for the AJ13355/pMWCB strain and the AJ13355/pSTVG strain, the
cultivation was stopped when about 10 g/L of glucose was consumed,
i.e., cultivated for 15 hours like the parent strain AJ13355,
because their glucose consumption rates were low. To the culture
medium of the transformants, 25 mg/L of tetracycline was added.
After the cultivation was completed, L-glutamic acid accumulated in
the culture medium was measured. The results are shown in Table
1.
1TABLE 1 Accumulated amount of L-glutamic acid Accumulated amount
of L- Bacterial strain glutamic acid ATCC12287 0.0 g/L
ATCC12287/RSFCPG 6.1 AJ13355 0.0 AJ13355/RSFCPG 3.3 AJ13355/pMWCB
0.8 AJ13355/pSTVG 0.8 ATCC14460 0.0 ATCC14460/RSFCPG 2.8 Culture
medium alone 0.2
[0146] While the Enterobacter agglomerans ATCC12287, the
Enterobacter agglomerans AJ13355 and the Serratia liquefacience
ATCC14460 did not accumulate L-glutamic acid, the strains whose CS,
PEPC and GDH activities were amplified by introducing RSFCPG
accumulated 6.1 g/L, 3.3 g/L, and 2.8 g/L of L-glutamic acid,
respectively. The AJ13355 strain of which CS activity alone was
amplified accumulated 0.8 g/L of L-glutamic acid, and the strain of
which GDH activity alone was amplified also accumulated 0.8 g/L of
L-glutamic acid.
[0147] (3) Cloning of .alpha.KGDH gene (referred to as "sucAB"
hereinafter) of Enterobacter agglomerans AJ13355
[0148] The sucAB gene of the Enterobacter agglomerans AJ13355 was
cloned by selecting a DNA fragment complementing acetate
non-assimilation of an Escherichia coli strain lacking the
.alpha.KGDH-E1 subunit gene (referred to as "sucA" hereinafter)
from the chromosome DNA of the Enterobacter agglomerans
AJ13355.
[0149] The chromosome DNA of the Enterobacter agglomerans AJ13355
strain was isolated by the same method as conventionally used for
extracting chromosome DNA from Escherichia coli (Seibutsu Kogaku
Jikken-sho (Textbook of Bioengineering Experiments), Ed. by the
Society of Fermentation and Bioengineering, Japan, p.97-98,
Baifukan, 1992). The pTWV228 used as the vector (ampicillin
resistant) was a marketed product from Takara Shuzo.
[0150] Products obtained by digesting the chromosome DNA of the
AJ13355 strain with EcoT221 and products obtained by digesting the
pTWV228 with PstI were ligated by T4 ligase, and the Escherichia
coli JRG465 lacking sucA (Herbert J. et al., Mol. Gen. Genetics,
1969, 105, p.182) was transformed with them. Strains grown on the
acetic acid minimal medium were selected from the transformants
obtained as described above, and a plasmid extracted from them was
designated as pTWVEK101. The Escherichia coli JRG465 carrying the
pTWVEK101 recovered the characteristics of acetate
non-assimilability as well as auxotrophy for succinic acid or
L-lysine and L-methionine. This suggests that the pTWVEK101
contains the sucA gene of Enterobacter agglomerans.
[0151] A restriction map of Enterobacter agglomerans-derived DNA
fragment of pTWVEK101 is shown in FIG. 6. The result of nucleotide
sequencing of the hatched portion in FIG. 6 is shown in SEQ ID NO:
1. In this sequence, two full length ORFs and two nucleotides
sequences considered as partial sequences of ORFs were found. Amino
acid sequences that can be encoded by these ORFs and the partial
sequences thereof are shown in SEQ ID NOS: 2 to 5 in order from the
5' ends. As a result of homology analysis of these sequences, it
was found that the portion of which nucleotide sequence had been
determined contained a 3' partial sequence of succinate
dehydrogenase iron-sulfur protein gene (sdhB), full length sucA and
.alpha.KGDH-E2 subunit gene (sucB gene), and 5' partial sequence of
succinyl-CoA synthetase .beta. subunit gene (sucC gene). Comparison
of the amino acid sequences deduced from these nucleotide sequences
with those of Escherichia coli. (Eur. J. Biochem., 141, 351-359
(1984), Eur. J. Biochem., 141, 361-374 (1984), and Biochemistry,
24, 6245-6252 (1985)) is shown in FIGS. 7 to 9. As shown by these
results, the amino acid sequences exhibited markedly high homology.
It was also found that a cluster of sdhB-sucA-sucB-sucC is formed
on the Enterobacter agglomerans chromosome like Escherichia coli
(Eur. J. Biochem., 141, 351-359 (1984), Eur. J. Biochem., 141,
361-374 (1984), and Biochemistry, 24, 6245-6252 (1985)).
[0152] (4) Acquisition of strain deficient in .alpha.KGDH derived
from Enterobacter agglomerans AJ13355
[0153] Using the sucAB gene of Enterobacter agglomerans obtained as
described above, a strain lacking .alpha.KGDH of Enterobacter
agglomerans was obtained by homologous recombination.
[0154] First, pTWVEK101 was digested with BglII to remove the
C-terminus region corresponding to about half of the sucA gene and
the full length of the sucB gene. To this site, a chloramphenicol
resistance gene fragment cut out from the pHSG399 (Takara Shuzo)
with AccI was then inserted. The region of
sdhB-.DELTA.sucAB::Cm.sup.r-sucC obtained above was cut out with
AflII and SacI. The resulting DNA fragment was used to transform
the Enterobacter agglomerans AJ13355 strain by electroporation to
obtain a chloramphenicol resistant strain, and thus a Enterobacter
agglomerans AJ13356 strain lacking the sucAB gene where the sucAB
gene on the chromosome was replaced by sucAB::Cm.sup.r was
obtained.
[0155] To confirm that the AJ13356 strain obtained as described
above was deficient in the .alpha.KGDH activity, its enzymatic
activity was determined by the method of Reed (L. J. Reed and B. B.
Mukherjee, Methods in Enzymology 1969, 13, p.55-61). As a result,
the .alpha.KGDH activity could not be detected in the AJ13356
strain as shown in Table 2, and thus it was confirmed that the
strain lacked the sucAB as desired.
2TABLE 2 .alpha.KGDH activity .alpha.KGDH activity
(.DELTA.ABS/min/mg Bacterial strain protein) AJ13355 0.481 AJ13356
<0.0001
[0156] (5) Evaluation of L-glutamic acid productivity of
Enterobacter agglomerans strain deficient in .alpha.KGDH
[0157] Each of the AJ13355 and AJ13356 strains was inoculated into
a 500 ml-volume flask containing 20 ml of a culture medium
comprising 40 g/L glucose, 20 g/L ammonium sulfate, 0.5 g/L
magnesium sulfate heptahydrate, 2 g/L potassium
dihydrogenphosphate, 0.5 g/L sodium chloride, 0.25 g/L calcium
chloride heptahydrate, 0.02 g/L ferrous sulfate heptahydrate, 0.02
g/L manganese sulfate tetrahydrate, 0.72 mg/L zinc sulfate
dihydrate, 0.64 mg/L copper sulfate pentahydrate, 0.72 mg/L cobalt
chloride hexahydrate, 0.4 mg/L boric acid, 1.2 mg/L sodium
molybdate dihydrate, 2 g/L yeast extract, 30 g/L calcium carbonate,
200 mg/L L-lysine monohydrochloride, 200 mg/L L-methionine and 200
mg/L DL-.alpha.,.epsilon.-diaminopimelic acid (DAP), and cultured
at 37.degree. C. with shaking until the glucose contained in the
culture medium was consumed. After the cultivation was completed,
L-glutamic acid and .alpha.-ketoglutaric acid (abbreviated as
".alpha.KG" hereinafter) accumulated in the culture medium were
measured. The results are shown in Table 3.
3TABLE 3 Accumulated amounts of L-glutamic acid and .alpha.KG
Accumulated Bacterial amount of L- Accumulated strain glutamic acid
amount of .alpha.KG AJ13355 0.0 g/L 0.0 g/L AJ13356 1.5 3.2
[0158] The AJ13356 strain deficient in the .alpha.KGDH activity
accumulated 1.5 g/L of L-glutamic acid, and simultaneously
accumulated 3.2 g/L of .alpha.KG.
[0159] (6) Introduction of RSFCPG into Enterobacter agglomerans
strain lacking .alpha.KGDH and evaluation of L-glutamic acid
productivity
[0160] The AJ13356 strain was transformed with the RSFCPG, and the
resulting strain introduced with the RSFCPG, AJ13356/RSFCPG, was
inoculated into a 500 ml-volume flask containing 20 ml of a culture
medium comprising 40 g/L glucose, 20 g/L ammonium sulfate, 0.5 g/L
magnesium sulfate heptahydrate, 2 g/L potassium
dihydrogenphosphate, 0.5 g/L sodium chloride, 0.25 g/L calcium
chloride heptahydrate, 0.02 g/L ferrous sulfate heptahydrate, 0.02
g/L manganese sulfate tetrahydrate, 0.72 mg/L zinc sulfate
dihydrate, 0.64 mg/L copper sulfate pentahydrate, 0.72 mg/L cobalt
chloride hexahydrate, 0.4 mg/L boric acid, 1.2 mg/L sodium
molybdate dihydrate, 2 g/L yeast extract, 25 mg/L tetracycline, 30
g/L calcium carbonate, 200 mg/L L-lysine monohydrochloride, 200
mg/L L-methionine and 200 mg/L DL-.alpha.,.epsilon.-DAP, and
cultured at 37.degree. C. with shaking until the glucose contained
in the culture medium was consumed. After the cultivation was
completed, L-glutamic acid and .alpha.KG accumulated in the culture
medium were measured. The results are shown in Table 4.
4TABLE 4 Accumulated amounts of L-glutamic acid and .alpha.KG
Accumulated Bacterial amount of L- Accumulated strain glutamic acid
amount of .alpha.KG AJ13356 1.4 g/L 2.9 g/L AJ13356/RSFCPG 5.1
0.0
[0161] In the strain of which CS, PEPC and GDH activities were
amplified by the introduction of RSFCPG, the accumulated amount of
.alpha.KG was reduced, and the accumulated amount of L-glutamic
acid was further improved.
* * * * *