U.S. patent number RE42,350 [Application Number 12/429,421] was granted by the patent office on 2011-05-10 for method for producing l-glutamic acid by fermentation accompanied by precipitation.
This patent grant is currently assigned to Ajinomoto Co., Inc.. Invention is credited to Yoshihiko Hara, Seiko Hirano, Hisao Ito, Hiroshi Izui, Kazuhiko Matsui, Mika Moriya.
United States Patent |
RE42,350 |
Izui , et al. |
May 10, 2011 |
Method for producing L-glutamic acid by fermentation accompanied by
precipitation
Abstract
A microorganism is provided which can metabolize a carbon source
at a specific pH in a liquid medium containing L-glutamic acid at a
saturation concentration and the carbon source, and which has
ability to accumulate L-glutamic acid in an amount exceeding the
amount corresponding to the saturation concentration in the liquid
medium at the pH. Also provided is a method for producing
L-glutamic acid by fermentation, which comprises culturing the
microorganism in a liquid medium of which pH is adjusted to a pH at
which L-glutamic acid is precipitated, to produce and accumulate
L-glutamic acid and precipitate L-glutamic acid in the medium.
Inventors: |
Izui; Hiroshi (Kawasaki,
JP), Moriya; Mika (Kawasaki, JP), Hirano;
Seiko (Kawasaki, JP), Hara; Yoshihiko (Kawasaki,
JP), Ito; Hisao (Kawasaki, JP), Matsui;
Kazuhiko (Kawasaki, JP) |
Assignee: |
Ajinomoto Co., Inc. (Tokyo,
JP)
|
Family
ID: |
26531768 |
Appl.
No.: |
12/429,421 |
Filed: |
April 24, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
09641892 |
Aug 18, 2000 |
7015010 |
|
|
Reissue of: |
11150265 |
Jun 13, 2005 |
7208296 |
Apr 24, 2007 |
|
|
Foreign Application Priority Data
|
|
|
|
|
Aug 20, 1999 [JP] |
|
|
11-234806 |
Mar 21, 2000 [JP] |
|
|
2000-78771 |
|
Current U.S.
Class: |
435/110;
435/106 |
Current CPC
Class: |
C12N
9/0016 (20130101); C12N 9/88 (20130101); C12P
13/14 (20130101); C12N 1/205 (20210501); C12R
2001/01 (20210501) |
Current International
Class: |
C12P
13/14 (20060101); C12N 15/00 (20060101); C12N
1/20 (20060101); C12N 1/36 (20060101); C12N
15/63 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
0 636 695 |
|
Feb 1995 |
|
EP |
|
0 670 370 |
|
Sep 1995 |
|
EP |
|
0 952 221 |
|
Oct 1999 |
|
EP |
|
62-288 |
|
Jan 1987 |
|
JP |
|
WO 97/08294 |
|
Mar 1997 |
|
WO |
|
Other References
Francoise Gavini, et al., "Transfer of Enterobacter agglomerans
(Beijerinck 1888) Ewing and Fife 1972 to Pantoea gen. nov. as
Pantoea agglomerans Comb. nov. and Description of Pantoea dispersa
sp. nov.," International Journal of Systematic Bacteriology, vol.
39, No. 3, Jul. 1989, pp. 337-345. cited by other .
Mergaert, J., et al., "Transfer of Erwinia ananas (synonym, Erwinia
uredovora) and Erwinia stewartii to the Genus Pantoea emend. as
Pantoea ananas (Serrano 1928) comb. nov. and Pantoea stewartii
(Smith 1898) comb. nov., Respectively, and Description of Pantoea
stewartii subsp. indologenes subsp. nov.," International Journal of
Systematic Bacteriology, vol. 43, No. 1, Jan. 1993, 162-173. cited
by other .
Robacker, D. C., et al., "Purine Metabolizing Capability of
Enterobacter agglomerans Affects Volatiles Production and
Attactiveness to Mexican Fruit Fly," Journal of Chemical Ecology,
vol. 28, No. 8, Aug. 2002, pp. 1549-1563. cited by other .
Moutaouakkil, A., et at "Purification and Partial Characterization
of Azoreductase from Enterobacter agglomerans," Archives of
Biochemistry and Biophysics 413, 2003, pp. 139-146. cited by other
.
Moutaouakkil, A., et al., "Decolorization of Azo Dyes with
Enterobacter agglomerans Immobilized in Different Supports by Using
Fluidized Bed Bioreactor," Current Microbiology, vol. 48, 2004, pp.
124-129. cited by other .
Bergey's Manual of Systematic Bacteriology, 2.sup.nd Edition, vol.
2, published in 2005, Springer Science+Business Media, Inc., New
York, NY, pp. 712-721. cited by other .
U.S. Appl. No. 10/077,999, Feb. 20, 2002, Ueda, et al. cited by
other .
U.S. Appl. No. 12/485,550, Jun. 16, 2009, Ueda, et al. cited by
other .
Kwe-Chao Chao, et al., vol. 77, pp. 715-725, "A Glutamic
Acid-Producing Bacillus", Nove. 10, 1958. cited by other .
R.M. Borichewski, Journal of Bacteriology, vol. 93, No. 2, pp.
597-599, "Keto Acids as Growth-Limiting Factors in Autotrophic
Growth of Thiobacillus thiooxidans", Feb. 1967. cited by other
.
Abstract of Crit. Rev. Biotechnol., vol. 15, No. 1, pp. 73-103,
"Recent Advances in the Physiology and Genetics of Amino
Acid-Producing Bacteria", 1995. cited by other .
Bailey. Toward a science of metabolic engineering. Science 252:
1668-1675. 1991. cited by other .
WPI/DERWENT Abstract, "Manufacturing L-Glutamic Acid by
Fermentation for Foodstuff, Pharmaceutical--Comprises Culturing
Corynebacterium striatum in Culture Medium", JP11009296, Jan. 19,
1999, AN 1999-169864. cited by other .
J. Mergaert, et al., "Transfer of Erwinia ananas (Synonym, Erwinia
uredovora) and Erwinia stewartii to the Genus Patonea Emend. as
Pantoea ananas (Serrano 1928) Comb. Nov. and Pantoea stewartii
(Smith 1898) Comb. Nov., Respectively, and Description of Pantoea
stewartii Subsp. Ingologenes Subsp. Nov.", International Journal of
Systematic Bacteriology, vol. 43, No. 1, Jan. 1993, pp. 162-173.
cited by other .
S.-W. Kwon, et al., "Phylogenetic Analysis of Erwinia Species Based
on 16S RRNA Gene Sequences", International Journal of Systematic
Bacteriology, vol. 47, No. 4, Oct. 1997, pp. 1061-1067. cited by
other .
Kwei-Chao Chao, et al., vol. 77, pp. 715-725, "A Glutamic
Acid-Producing Bacillus", Nov. 10, 1958. cited by other.
|
Primary Examiner: Qian; Celine X
Attorney, Agent or Firm: Oblon, Spivak, McClelland, Maier
& Neustadt, L.L.P.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application is a continuation of U.S. Ser. No.
09/641,892, filed on Aug. 18, 2000 (now U.S. Pat. No. 7,015,010),
which claims priority to Japanese application No. JP 11-234806,
filed on Aug. 20, 1999, and to Japanese application No. JP
2000-78771, filed on Mar. 21, 2000.
Claims
What is claimed is:
1. A method for producing L-glutamic acid by fermentation, which
comprises culturing an isolated microorganism in a liquid medium of
which pH is adjusted to the pH at which L-glutamic acid is
precipitated, to produce and accumulate L-glutamic acid and
precipitate L-glutamic acid in the medium, wherein said
microorganism can metabolize a carbon source at a specific pH in a
liquid medium containing the carbon source and L-glutamic acid at a
saturation concentration, and has the ability to accumulate
L-glutamic acid in an amount exceeding the amount corresponding to
the saturation concentration in the liquid medium at the pH,
wherein said microorganism is .[.Enterobacter agglomerans.].
.Iadd.Pantoea ananatis.Iaddend., and wherein said microorganism has
at least one of the following characteristics: (a) the
microorganism has increased activity, as compared to a
corresponding wild-type microorganism, of an enzyme that catalyzes
a reaction for biosynthesis of L-glutamic acid; and (b) the
microorganism has decreased activity, as compared to a
corresponding wild-type microorganism, or deficient activity of an
enzyme that catalyzes a reaction of a pathway branching from a
biosynthetic pathway of L-glutamic acid and producing a compound
other than L-glutamic acid.
2. The method according to claim 1, wherein said microorganism can
grow in the liquid medium.
3. The method according to claim 1, wherein the pH is not more than
5.0.
4. The method according to claim 1, wherein in said microorganism
an activity of at least one enzyme selected from the group
consisting of citrate synthase, phosphoenolpyruvate carboxylase and
glutamate dehydrogenase, is increased.
5. The method according to claim 1, wherein in said microorganism
the enzyme that catalyzes the reaction of the pathway branching
from the biosynthetic pathway of L-glutamic acid and producing the
compound other than L-glutamic acid is .alpha.-ketoglutarate
dehydrogenase.
6. The method according to claim 1, wherein said microorganism has
a mutation that causes less extracellular secretion of a viscous
material compared with a wild strain when cultured in a medium
containing a saccharide.
.Iadd.7. The method according to claim 1, wherein said
microorganism is Pantoea ananatis AJ13355 strain..Iaddend.
Description
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing L-glutamic
acid by fermentation accompanied by precipitation. L-Glutamic acid
is widely used as a material for seasonings and so forth.
L-Glutamic acid is mainly produced by fermentative methods using
so-called coryneformbacteria producing L-glutamic acid, which
belong to the genus Brevibacterium, Corynebacterium or
Microbacterium, or mutant strains thereof (Amino Acid Fermentation,
pp. 195-215, Gakkai Shuppan Center, 1986). Methods for producing
L-glutamic acid by fermentation using other bacterial strains are
known and include a method using a microorganism belonging to the
genus Bacillus, Streptomyces, Penicillium or the like (U.S. Pat.
No. 3,220,929), a method using a microorganism belonging to the
genus Pseudomonas, Arthrobacter, Serratia, Candida or the like
(U.S. Pat. No. 3,563,857), a method using a microorganism belonging
to the genus Bacillus, Pseudomonas, Serratia, Aerobacter aerogenes
(currently referred to as Enterobacter aerogenes) or the like
(Japanese Patent Publication (Kokoku) No. 32-9393), a method using
a mutant strain of Escherichia coli (Japanese Patent Application
Laid-open (Kokai) No. 5-244970) and so forth. In addition, the
inventors of the present invention have proposed a method for
producing L-glutamic acid by using a microorganism belonging to the
genus Klebsiella, Erwinia or Pantoea (Japanese Patent Application
Laid-open No. 2000-106869).
Further, there have been disclosed various techniques for improving
L-glutamic acid-producing ability by enhancing activities of
L-glutamic acid biosynthetic enzymes through the use of recombinant
DNA techniques. For example, it has been reported that the
introduction of a gene coding for citrate synthase derived from
Escherichia coli or Corynebacterium glutamicum was effective for
the enhancement of L-glutamic acid-producing ability in
Corynebacterium or Brevibacterium bacteria (Japanese Patent
Publication No. 7-121228). In addition, Japanese Patent Application
Laid-open No. 61-268185 discloses a cell harboring recombinant DNA
containing a glutamate dehydrogenase gene derived from
Corynebacterium bacteria. Further, Japanese Patent Application
Laid-open No. 63-214189 discloses a technique for improving
L-glutamic acid-producing ability by amplifying a glutamate
dehydrogenase gene, an isocitrate dehydrogenase gene, an aconitate
hydratase gene and a citrate synthase gene.
Although L-glutamic acid productivity has been considerably
increased by breeding of the aforementioned microorganisms or
improvement of production methods, development of methods for more
efficiently producing L-glutamic acid at a lower cost are still
required to respond to the increasing future demand for L-glutamic
acid.
A method wherein fermentation is performed with crystallizing
L-amino acid accumulated in culture is known (Japanese Patent
Application Laid-open No. 62-288). In this method, the L-amino acid
concentration in the culture is maintained below a certain level by
precipitating the accumulated L-amino acid in the culture.
Specifically, L-tryptophan, L-tyrosine or L-leucine is precipitated
during fermentation by adjusting the temperature and the pH of the
culture or by adding a surface active agent to the medium.
While a fermentative method with precipitating L-amino acid is
known as described above, amino acids suitable for this method are
those of relatively low water solubility. No example exists for
applying the method to highly water-soluble amino acids such as
L-glutamic acid. In addition, the medium must have low pH to
precipitate L-glutamic acid. However, L-glutamic acid-producing
bacteria such as those mentioned above cannot grow under acidic
conditions, and therefore L-glutamic acid fermentation is performed
under neutral conditions (U.S. Pat. Nos. 3,220,929 and 3,032,474;
Chao K. C. & Foster J. W., J. Bacteriol., 77, pp. 715-725
(1959)). Thus, production of L-glutamic acid by fermentation
accompanied by precipitation is not known.
Furthermore, it is known that growth of most acidophile bacteria is
inhibited by organic acids such as acetic acid, lactic acid and
succinic acid (Yasuro Oshima Ed., "Extreme Environment
Microorganism Handbook", p. 231, Science Forum; Borichewski R. M.,
J. Bacteriol., 93, pp. 597-599 (1967) etc.). Therefore, it is
considered that many microorganisms are susceptible to L-glutamic
acid, which is also an organic acid, under acidic conditions. There
exists no report of microorganisms having L-glutamic acid-producing
ability under acidic conditions has been attempted.
SUMMARY OF THE INVENTION
Based on the foregoing, an object of the present invention is to
search and breed a microorganism that produces L-glutamic acid
under low pH conditions and to provide a method for producing
L-glutamic acid using an obtained microorganism by fermentation
with precipitating L-glutamic acid.
The inventors of the present invention considered during the study
for improvement of L-glutamic acid productivity by fermentation
that inhibition of the production by L-glutamic acid accumulated in
a medium at a high concentration was one of obstructions to the
improvement of productivity. For example, cells have an excretory
system and an uptake system for L-glutamic acid. However, if
L-glutamic acid once excreted into the medium is incorporated into
cells again, not only the production efficiency falls, but also the
L-glutamic acid biosynthetic reactions are inhibited as a result.
In order to avoid the inhibition of production by such accumulation
of L-glutamic acid at high concentration, the inventors of the
present invention screened microorganisms that can proliferate
under acidic conditions and in the presence of a high concentration
of L-glutamic acid. As a result, they successfully isolated
microorganisms having such properties from a soil, and thus
accomplished the present invention.
Thus, the present invention provides the following:
(1) A microorganism which can metabolize a carbon source at a
specific pH in a liquid medium containing L-glutamic acid at a
saturation concentration and the carbon source, and has ability to
accumulate L-glutamic acid in an amount exceeding the amount
corresponding to the saturation concentration in the liquid medium
at the pH. (2) The microorganism according to (1), which can grow
in the liquid medium. (3) The microorganism according to (1) or
(2), wherein the pH is not more than 5.0. (4) The microorganism
according to any one of (1) to (3), which has at least one of the
following characteristics: (a) the microorganism is enhanced in
activity of an enzyme that catalyzes a reaction for biosynthesis of
L-glutamic acid; and (b) the microorganism is decreased in or
deficient in activity of an enzyme that catalyzes a reaction
branching from a biosynthetic pathway of L-glutamic acid and
producing a compound other than L-glutamic acid. (5) The
microorganism according to (4), wherein the enzyme that catalyzes
the reaction for biosynthesis of L-glutamic acid is at least one
selected from citrate synthase, phosphoenolpyruvate carboxylase and
glutamate dehydrogenase. (6) The microorganism according to (4) or
(5), wherein the enzyme that catalyzes the reaction branching from
the biosynthetic pathway of L-glutamic acid and producing a
compound other than L-glutamic acid is .alpha.-ketoglutarate
dehydrogenase. (7) The microorganism according to any one of (1) to
(6), wherein the microorganism belongs to the genus
.[.Enterobacter.]. .Iadd.Pantoea.Iaddend.. (8) The microorganism
according to (7), which is .[.Enterobacter agglomerans.].
.Iadd.Pantoea ananatis.Iaddend.. (9) The microorganism according to
(8), which has a mutation that causes less extracellular secretion
of a viscous material compared with a wild strain when cultured in
a medium containing a saccharide. (10) A method for producing
L-glutamic acid by fermentation, which comprises culturing a
microorganism as defined in any one of (1) to (9) in a liquid
medium of which pH is adjusted to a pH at which L-glutamic acid is
precipitated, to produce and accumulate L-glutamic acid and
precipitate L-glutamic acid in the medium. (11) A method for
screening a microorganism suitable for producing L-glutamic acid by
fermentation with precipitating L-glutamic acid in a liquid medium,
which comprises inoculating a sample containing microorganisms into
an acidic medium containing L-glutamic acid at a saturation
concentration and a carbon source, and selecting a strain that can
metabolize the carbon source. (12) The method according to (11),
wherein a strain that can grow in the medium is selected as the
strain that can metabolize the carbon source. (13) The method
according to (11) or (12), wherein a pH of the medium is not more
than 5.0.
According to the method of the present invention, L-glutamic acid
can be produced by fermentation with precipitating L-glutamic acid.
As a result, L-glutamic acid in the medium is maintained below a
certain concentration, and L-glutamic acid can be produced without
suffering from the product inhibition by L-glutamic acid at a high
concentration.
BRIEF EXPLANATION OF THE DRAWINGS
FIG. 1 shows a restriction map of a DNA fragment derived from
.[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis
.Iaddend.pTWVEK101.
FIG. 2A and FIG. 2B show a comparison of the amino acid sequence
deduced from the nucleotide sequence of the sucA gene derived from
.[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis .Iaddend.and
that derived from Escherichia coli. Upper sequence: .[.Enterobacter
agglomerans.]. .Iadd.Pantoea ananatis .Iaddend.(SEQ ID NO: 3),
lower sequence: Escherichia coli (SEQ ID NO: 8) (the same shall
apply hereafter).
FIG. 3 shows comparison of the amino acid sequence deduced from the
nucleotide sequence of the sucB gene derived from .[.Enterobacter
agglomerans.]. .Iadd.Pantoea ananatis .Iaddend.(upper sequence--SEQ
ID NO: 4) and that derived from Escherichia coli (lower
sequence--SEQ ID NO: 9).
FIG. 4 shows comparison of the amino acid sequence deduced from the
nucleotide sequence of the sucC gene derived from .[.Enterobacter
agglomerans.]. .Iadd.Pantoea ananatis .Iaddend.(upper sequence--SEQ
ID NO: 10) and that derived from Escherichia coli (lower
sequence--SEQ ID NO: 11).
FIG. 5 shows comparison of the amino acid sequence deduced from the
nucleotide sequence of the sdhB gene derived from .[.Enterobacter
agglomerans.]. .Iadd.Pantoea ananatis .Iaddend.(upper sequence--SEQ
ID NO: 2) and that derived from Escherichia coli (lower
sequence--SEQ ID NO: 12).
FIG. 6 shows construction of plasmid pMWCPG having a gltA gene, a
ppc gene and a gdhA gene.
FIG. 7 shows construction of plasmid RSF-Tet having the replication
origin of the broad host spectrum plasmid RSF1010 and a
tetracycline resistance gene.
FIG. 8 shows construction of plasmid RSFCPG having the replication
origin of the broad host spectrum plasmid RSF1010 a tetracycline
resistance gene, a gltA gene, a ppc gene and a gdhA gene.
FIG. 9 shows construction of plasmid pSTVCB having a gltA gene.
DETAILED DESCRIPTION OF THE INVENTION
Hereafter, the present invention will be explained in detail.
The microorganism of the present invention is a microorganism that
(1) can metabolize a carbon source at a specific pH in a liquid
medium containing L-glutamic acid at a saturation concentration and
the carbon source and (2) has ability to accumulate L-glutamic acid
in an amount exceeding the amount corresponding to the saturation
concentration in the liquid medium at the pH.
The term "saturation concentration" means a concentration of
L-glutamic acid dissolved in a liquid medium when the liquid medium
is saturated with L-glutamic acid.
Hereafter, a method for screening a microorganism that can
metabolize a carbon source in a liquid medium containing L-glutamic
acid at a saturation concentration and the carbon source at a
specific pH will be described. A sample containing microorganisms
is inoculated into a liquid medium containing L-glutamic acid at a
saturation concentration and a carbon source at a specific pH, and
a strain that can metabolize the carbon source is selected. The
specific pH is not particularly limited, but is usually not more
than about 5.0, preferably not more than about 4.5, more preferably
not more than about 4.3. The microorganism of the present invention
is used to produce L-glutamic acid by fermentation with
precipitating L-glutamic acid. If the pH is too high, it becomes
difficult to allow the microorganism to produce L-glutamic acid
enough for precipitation. Therefore, pH is preferably in the
aforementioned range.
If pH of an aqueous solution containing L-glutamic acid is lowered,
the solubility of L-glutamic acid significantly falls around pKa of
.gamma.-carboxyl group (4.25, 25.degree. C.). The solubility
becomes the lowest at the isoelectric point (pH 3.2) and L-glutamic
acid exceeding the amount corresponding to the saturation
concentration is precipitated. While it depends on the medium
composition, L-glutamic acid is usually dissolved in an amount of
10 to 20 g/L at pH 3.2, 30 to 40 g/L at pH 4.0 and 50 to 60 g/L at
pH 4.7, at about 30.degree. C. Usually pH does not need to be made
below 3.0, because the L-glutamic acid precipitating effect
plateaus when pH goes below a certain value. However, pH may be
below 3.0.
In addition, the expression that a microorganism "can metabolize
the carbon source" means that it can proliferate or can consume the
carbon source even though it cannot proliferate. Therefore, this
phrase indicates that the microorganism catabolizes carbon sources
such as saccharides or organic acids. Specifically, for example, if
a microorganism proliferates when cultured in a liquid medium
containing L-glutamic acid at a saturation concentration at pH 5.0
to 4.0, preferably pH 4.5 to 4.0, more preferably pH 4.3 to 4.0,
still more preferably pH 4.0 at an appropriate temperature, for
example, 28.degree. C., 37.degree. C. or 50.degree. C. for 2 to 4
days, this microorganism can metabolize the carbon source in the
medium.
Further, for example, even if a microorganism does not proliferate
when it is cultured in a liquid medium containing L-glutamic acid
at a saturation concentration at pH 5.0 to 4.0, preferably pH 4.5
to 4.0, more preferably pH 4.3 to 4.0, still more preferably pH 4.0
at an appropriate temperature, for example, 28.degree. C.,
37.degree. C. or 50.degree. C. for 2 to 4 days, the microorganism
which consumes the carbon source in the medium is that can
metabolize the carbon source in the medium.
The microorganism which can metabolize the carbon source includes a
microorganism which can grow in the liquid medium.
The expression that a microorganism "can grow" means that it can
proliferate or can produce L-glutamic acid even though it cannot
proliferate. Specifically, for example, if a microorganism
proliferates when cultured in a liquid medium containing L-glutamic
acid at a saturation concentration at pH 5.0 to 4.0, preferably pH
4.5 to 4.0, more preferably pH 4.3 to 4.0, still more preferably pH
4.0 at an appropriate temperature, for example, 28.degree. C.,
37.degree. C. or 50.degree. C. for 2 to 4 days, this microorganism
can grow in the medium.
Further, for example, even if a microorganism does not proliferate
when it is cultured in a liquid synthetic medium containing
L-glutamic acid at a saturation concentration at pH 5.0 to 4.0,
preferably pH 4.5 to 4.0, more preferably pH 4.3 to 4.0, still more
preferably pH 4.0 at an appropriate temperature, for example,
28.degree. C., 37.degree. C. or 50.degree. C. for 2 to 4 days, the
microorganism which increases the amount of L-glutamic acid in the
medium is that can grow in the medium.
The selection described above may be repeated two or more times
under the same conditions or with changing pH or the concentration
of L-glutamic acid. An initial selection can be performed in a
medium containing L-glutamic acid at a concentration lower than the
saturation concentration, and thereafter a subsequent selection can
be performed in a medium containing L-glutamic acid at a saturation
concentration. Further, strains with favorable properties such as
superior proliferation rate may be selected.
In addition to the property described above, the microorganism of
the present invention has an ability to accumulate L-glutamic acid
in an amount exceeding the amount corresponding to the saturation
concentration of L-glutamic acid in a liquid medium. The pH of the
aforementioned liquid medium is preferably the same as or close to
that of the medium used for screening a microorganism having the
aforementioned property (1). Usually, a microorganism becomes
susceptible to L-glutamic acid at a high concentration as pH
becomes lower. Therefore, it is preferred that pH is not low from
the viewpoint of resistance to L-glutamic acid, but low pH is
preferred from the viewpoint of production of L-glutamic acid with
precipitating it. To satisfy these conditions, pH may be in the
range of 3 to 5, preferably 4 to 5, more preferably 4.0 to 4.7,
still more preferably 4.0 to 4.5, particularly preferably 4.0 to
4.3.
As the microorganism of the present invention or breeding materials
therefor, there can be mentioned, for example, microorganisms
belonging to the genus Enterobacter, Klebsiella, Serratia, Pantoea,
Erwinia, Escherichia, Corynebacterium, Alicyclobacillus, Bacillus,
Saccharomyces or the like. Among these, microorganisms belonging to
the genus Enterobacter are preferred. Hereafter, the microorganism
of the present invention will be explained mainly for
microorganisms belonging to the genus Enterobacter, but the present
invention can be applied to microorganism belonging to other genera
and not limited to the genus Enterobacter.
As microorganisms belonging to the .[.Enterobacter.].
.Iadd.Pantoea.Iaddend., there can be specifically mentioned
.[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis.Iaddend.,
preferably the .[.Enterobacter agglomerans.]. .Iadd.Pantoea
ananatis .Iaddend.AJ13355 strain. This strain was isolated from a
soil in Iwata-shi, Shizuoka, Japan as a strain that can proliferate
in a medium containing L-glutamic acid and a carbon source at low
pH.
The physiological properties of AJ13355 are as follows:
(1) Gram staining: negative
(2) Behavior against oxygen: facultative anaerobic
(3) Catalase: positive
(4) Oxidase: negative
(5) Nitrate-reducing ability: negative
(6) Voges-Proskauer test: positive
(7) Methyl Red test: negative
(8) Urease: negative
(9) Indole production: positive
(10) Motility: motile
(11) H.sub.2S production in TSI medium: weakly active
(12) .beta.-galactosidase: positive
(13) Saccharide-assimilating property:
Arabinose: positive
Sucrose: positive
Lactose: positive
Xylose: positive
Sorbitol: positive
Inositol: positive
Trehalose: positive
Maltose: positive
Glucose: positive
Adonitol: negative
Raffinose: positive
Salicin: negative
Melibiose: positive
(14) Glycerol-assimilating property: positive
(15) Organic acid-assimilating property:
Citric acid: positive
Tartaric acid: negative
Gluconic acid: positive
Acetic acid: positive
Malonic acid: negative
(16) Arginine dehydratase: negative
(17) Ornithine decarboxylase: negative
(18) Lysine decarboxylase: negative
(19) Phenylalanine deaminase: negative
(20) Pigment formation: yellow
(21) Gelatin liquefaction ability: positive
(22) Growth pH: growth is possible at pH 4.0, good growth at pH 4.5
to 7
(23) Growth temperature: good growth at 25.degree. C., good growth
at 30.degree. C., good growth at 37.degree. C., growth is possible
at 42.degree. C., growth is not possible at 45.degree. C.
Based on these bacteriological properties, AJ13355 was determined
as .[.Enterobacter agglomerans.]. .Iadd.Pantoea
ananatis.Iaddend..
The .[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis
.Iaddend.AJ13355 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, Japan)
on Feb. 19, 1998 and received an accession number of FERM P-16644.
It was then transferred to an international deposition under the
provisions of Budapest Treaty on Jan. 11, 1999 and received an
accession number of FERM BP-6614.
The microorganism of the present invention may be a microorganism
originally having L-glutamic acid-producing ability or one having
L-glutamic acid-producing ability imparted or enhanced by breeding
through use of mutation treatment, recombinant DNA techniques or
the like.
L-Glutamic acid-producing ability can be imparted or enhanced by,
for example, increasing activity of an enzyme that catalyzes a
reaction for biosynthesis of L-glutamic acid. L-Glutamic
acid-producing ability can also be enhanced by decreasing activity
of an enzyme that catalyzes a reaction branching from the
biosynthetic pathway of L-glutamic acid and producing a compound
other than L-glutamic acid, or making the activity deficient.
Enzymes that catalyze are action for biosynthesis of L-glutamic
acid, include: glutamate dehydrogenase (hereafter, also referred to
as "GDH"), glutamine synthetase, glutamate synthase, isocitrate
dehydrogenase, aconitate hydratase, citrate synthase (hereafter,
also referred to as "CS"), phosphoenolpyruvate carboxylase
(hereafter, also referred to as "PEPC"), pyruvate dehydrogenase,
pyruvate kinase, enolase, phosphoglyceromutase, phosphoglycerate
kinase, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate
isomerase, fructose bisphosphate aldolase, phosphofructokinase,
glucose phosphate isomerase and so forth. Among these enzymes, one,
two or three of CS, PEPC and GDH are preferred. Further, it is
preferred that the activities of all the three enzymes, CS, PEPC
and GDH, are enhanced in the microorganism of the present
invention. In particular, CS of Brevibacterium lactofermentum is
preferred, because it does not suffer from inhibition by
.alpha.-ketoglutaric acid, L-glutamic acid and NADH.
In order to enhance the activity of CS, PEPC or GDH, for example, a
gene coding for CS, PEPC or GDH may be cloned on an appropriate
plasmid and a host microorganism may be transformed with the
obtained plasmid. The copy number of the gene coding for CS, PEPC
or GDH (hereafter, abbreviated as "gltA gene", "ppc gene" and "gdhA
gene", respectively) in the transformed strain cell increases,
resulting in the increase of the activity of CS, PEPC or GDH.
The cloned gltA gene, ppc gene and gdhA gene are introduced into
the aforementioned starting parent strain solely or in combination
of arbitrary two or three kinds of them. When two or three kinds of
the genes are introduced, two or three kinds of the genes may be
cloned on one kind of plasmid and introduced into the host, or
separately cloned on two or three kinds of plasmids that can
coexist and introduced into the host.
Two or more kinds of genes coding for enzymes of the same kind, but
derived from different microorganisms may be introduced into the
same host.
The plasmids described above are not particularly limited so long
as they are autonomously replicable in cells of a microorganism
belonging to, for example, the genus Enterobacter or the like, but,
for example, there can be mentioned pUC19, pUC18, pBR322, pHSG299,
pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219, pMW218,
pACYC177, pACYC184 and so forth. Besides these, vectors of phage
DNA can also be used.
Transformation can be performed by, for example, the method of D.
M. Morrison (Methods in Enzymology, 68, 326 (1979)), the method
wherein permeability of DNA is increased by treating recipient
bacterium cells with calcium chloride (Mandel M. and Higa A., J.
Mol. Biol., 53, 159 (1970)), the electroporation (Miller J. H., "A
Short Course in Bacterial Genetics", Cold Spring Harbor Laboratory
Press, U.S.A. 1992) or the like.
The activity of CS, PEPC or GDH can also be increased by allowing
multiple copies of a gltA gene, a ppc gene or a gdhA gene to be
present on chromosomal DNA of the aforementioned starting parent
strain to be a host. In order to introduce multiple copies of the
gltA gene, the ppc gene or the gdhA gene on chromosomal DNA of a
microorganism belonging to the genus Enterobacter or the like, a
sequence of which multiple copies are present on the chromosomal
DNA, such as repetitive DNA and inverted repeats present at termini
of a transposable element, can be used. Alternatively, multiple
copies of the genes can be introduced on to chromosomal DNA by
utilizing transfer of a transposon containing the gltA gene, the
ppc gene or the gdhA gene. As a result, the copy number of the gltA
gene, the ppc gene or the gdhA gene in a transformed strain cell is
increased, and thus the activity of CS, PEPC or GDH is
increased.
As organisms to be a source of the gltA gene, the ppc gene or the
gdhA gene of which copy number is increased, any organism can be
used so long as it has activity of CS, PEPC or GDH. Inter alia,
bacteria, which are prokaryotes, for example, those belonging to
the genus Enterobacter, Klebsiella, Erwinia, Pantoea, Serratia,
Escherichia, Corynebacterium, Brevibacterium and Bacillus are
preferred. As specific examples, there can be mentioned Escherichia
coli, Brevibacterium lactofermentum and so forth. The gltA gene,
the ppc gene and the gdhA gene can be obtained from chromosomal DNA
of the microorganisms described above.
The gltA gene, the ppc gene and the gdhA gene can be obtained by
using a mutant strain which is deficient in the activity of CS,
PEPC or GDH to isolate a DNA fragment which complements the
auxotrophy from chromosomal DNA of the aforementioned
microorganisms. Since the nucleotide sequences of these genes of
Escherichia and Corynebacterium bacteria have already been
elucidated (Biochemistry, 22, pp. 5243-5249 (1983); J. Biochem.,
95, pp. 909-916 (1984); Gene, 27, pp. 193-199 (1984); Microbiology,
140, pp. 1817-1828 (1994); Mol. Gen. Genet., 218, pp. 330-339
(1989); Molecular Microbiology, 6, pp. 317-326 (1992)), they can
also be obtained by PCR utilizing primers synthesized based on each
nucleotide sequence and chromosomal DNA as a template.
The activity of CS, PEPC or GDH can also be increased by enhancing
the expression of the gltA gene, the ppc gene or the gdhA gene
besides the aforementioned amplification of the genes. For example,
the expression can be enhanced by replacing a promoter for the gltA
gene, the ppc gene or the gdhA gene with other stronger promoters.
For example, strong promoters are known to include: lac promoter,
trp promoter, trc promoter, tac promoter, P.sub.R promoter and
P.sub.L promoter of the lamda phage and so forth. The gltA gene,
the ppc gene and the gdhA gene of which promoter is replaced are
cloned on a plasmid and introduced into the host microorganism, or
introduced onto the chromosomal DNA of the host microorganism by
using repetitive DNA, inverted repeats, transposon or the like.
The activity of CS, PEPC or GDH can also be enhanced by replacing
the promoter of the gltA gene, the ppc gene or the gdhA gene on the
chromosome with other stronger promoters (see WO 87/03006 and
Japanese Patent Application Laid-open No. 61-268183), or inserting
a strong promoter in the upstream of the coding sequence of each
gene (see Gene, 29, pp. 231-241 (1984)). Specifically, homologous
recombination can be performed between DNA containing the gltA
gene, the ppc gene or the gdhA gene of which promoter is replaced
with a stronger one or a part thereof and the corresponding gene on
the chromosome.
Examples of the enzyme which catalyze a reaction branching from the
biosynthetic pathway of the L-glutamic acid and producing a
compound other than L-glutamic acid include .alpha.-ketoglutarate
dehydrogenase (hereafter, also referred to as ".alpha.KGDH"),
isocitrate lyase, phosphate acetyltransferase, acetate kinase,
acetohydroxy acid synthase, acetolactate synthase, formate
acetyltransferase, lactate dehydrogenase, glutamate decarboxylase,
1-pyrroline dehydrogenase and so forth. Among these enzymes,
.alpha.KGDH is preferred.
In order to obtain a decrease or deficiency of the activity of the
aforementioned enzyme in a microorganism belonging to the genus
Enterobacter or the like, mutation causing decrease or deficiency
of the intracellular activity of the enzyme can be introduced into
the gene of the aforementioned enzyme by a usual mutagenesis or
genetic engineering method.
Examples of the mutagenesis method include, for example, methods
utilizing irradiation with X-ray or ultraviolet ray, methods
utilizing treatment with a mutagenic agent such as
N-methyl-N'-nitro-N-nitrosoguanidine, and so forth. The site where
the mutation is introduced to the gene may be in a coding region
coding for an enzyme protein, or a region for regulating expression
such as a promoter.
Examples of the genetic engineering methods include, for example,
methods utilizing gene recombination, transduction, cell fusion and
so forth. For example, a drug resistance gene is inserted into a
cloned target gene to prepare a gene that has lost its function
(defective gene). Subsequently, this defective gene is introduced
into a cell of a host microorganism, and the target gene on the
chromosome is replaced with the aforementioned defective gene by
utilizing homologous recombination (gene disruption).
A decrease or deficiency of intracellular activity of the target
enzyme and the degree of decrease of the activity can be determined
by measuring the enzyme activity of a cell extract or a purified
fraction thereof obtained from a candidate strain and comparing
with that of a wild strain. For example, the .alpha.KGDH activity
can be measured by the method of Reed et al. (Reed L. J. and
Mukherjee B. B., Methods in Enzymology, 13, pp. 55-61 (1969)).
Depending on the target enzyme, the target mutant strain can be
selected based on the phenotype of the mutant strain. For example,
a mutant strain which is deficient in the .alpha.KGDH activity or
decreases in the .alpha.KGDH activity cannot proliferate or shows a
markedly reduced proliferation rate in a minimal medium containing
glucose or a minimal medium containing acetic acid or L-glutamic
acid as an exclusive carbon source under aerobic conditions.
However, normal proliferation is enabled even under the same
condition by adding succinic acid or lysine, methionine and
diaminopimelic acid to a minimal medium containing glucose. By
utilizing these phenomena as indicators, mutant strains with
decreased .alpha.KGDH activity or deficient in the activity can be
selected.
A method for preparing the .alpha.KGDH gene deficient strain of
Brevibacterium lactofermentum by utilizing homologous recombination
is described in detail in WO 95/34672. Similar methods can be
applied to the other microorganisms.
Further, techniques such as cloning of genes and cleavage and
ligation of DNA, transformation and so forth are described in
detail in Molecular Cloning, 2nd Edition, Cold Spring Harbor Press,
1989 and so forth.
As a specific example of a mutant strain deficient in .alpha.KGDH
activity or with decreased .alpha.KGDH activity obtained as
described above, there can be mentioned .[.Enterobacter
agglomerans.]. .Iadd.Pantoea ananatis .Iaddend.AJ13356.
.[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis
.Iaddend.AJ13356 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, Japan)
on Feb. 19, 1998 and received an accession number of FERM P-16645.
It was then transferred to an international deposition under the
provisions of Budapest Treaty on Jan. 11, 1999 and received an
accession number of FERM BP-6615. The .[.Enterobacter
agglomerans.]. .Iadd.Pantoea ananatis .Iaddend.AJ13356 is deficient
in .alpha.KGDH activity as a result of disruption of the
.alpha.KGDH-E1 subunit gene (sucA).
When .[.Enterobacter agglomerans.]. .Iadd.Pantoea
ananatis.Iaddend., an example of the microorganism used in the
present invention, is cultured in a medium containing a saccharide,
a viscous material is extracellularly secreted, resulting in low
operation efficiency. Therefore, when .[.Enterobacter
agglomerans.]. .Iadd.Pantoea ananatis .Iaddend.having such a
property of secreting the viscous material is used, it is
preferable to use a mutant strain that secretes less the viscous
material compared with a wild strain. Examples of mutagenesis
methods include, for example, methods utilizing irradiation with X
ray or ultraviolet ray, method utilizing treatment with a mutagenic
agent such as N-methyl-N'-nitro-N-nitrosoguanidine and so forth. A
mutant strain with decreased secretion of the viscous material can
be selected by inoculating mutagenized bacterial cells in a medium
containing a saccharide, for example, LB medium plate containing 5
g/L of glucose, culturing them with tilting the plate about 45
degrees and selecting a colony which does not show flowing down of
liquid.
In the present invention, impartation or enhancement of L-glutamic
acid-producing ability and impartation of other favorable
properties such as mutation for less viscous material secretion
described above can be carried out in an arbitrary order.
By culturing the microorganism of the present invention in a liquid
medium of which pH is adjusted to a pH at which L-glutamic acid is
precipitated, L-glutamic acid can be produced and accumulated with
precipitating it in the medium. L-Glutamic acid can also be
precipitated by starting the culture at a neutral pH and then
ending it at a pH at which L-glutamic acid is precipitated.
The pH at which L-glutamic acid is precipitated means one at which
L-glutamic acid is precipitated when the microorganism produces and
accumulates L-glutamic acid.
As the aforementioned medium, a usual nutrient medium containing a
carbon source, a nitrogen source, mineral salts and organic trace
nutrients such as amino acids and vitamins as required can be used
so long as pH is adjusted to a pH at which L-glutamic acid is
precipitated. Either a synthetic medium or a natural medium can be
used. The carbon source and the nitrogen source used in the medium
can be any ones so long as they can be used by the cultured
strain.
As the carbon source, saccharides such as glucose, glycerol,
fructose, sucrose, maltose, mannose, galactose, starch hydrolysate
and molasses are used. In addition, organic acids such as acetic
acid and citric acid may be used each alone or in combination with
another carbon source.
As the nitrogen source, ammonia, ammonium salts such as ammonium
sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate
and ammonium acetate, nitrates and so forth are used.
As the organic trace nutrients, amino acids, vitamins, fatty acids,
nucleic acids, those containing these substances such as peptone,
casamino acid, yeast extract and soybean protein decomposition
products are used. When an auxotrophic mutant strain that requires
an amino acid and so forth for metabolization or growth is used,
the required nutrient must be supplemented.
As mineral salts, phosphates, magnesium salts, calcium salts, iron
salts, manganese salts and so forth are used.
As for the culture method, aeration culture is usually performed
with controlling the fermentation temperature to be 20 to
42.degree. C. and pH to be 3 to 5, preferably 4 to 5, more
preferably 4 to 4.7, particularly preferably 4 to 4.5. Thus, after
about 10 hours to 4 days of culture, a substantial amount of
L-glutamic acid is accumulated in the culture. Accumulated
L-glutamic acid exceeding the amount corresponding to the
saturation concentration is precipitated in the medium.
After completion of the culture, L-glutamic acid precipitated in
the culture can be collected by centrifugation, filtration or the
like. L-Glutamic acid dissolved in the medium can be collected
according to known methods. For example, the L-glutamic acid can be
isolated by concentrating the culture broth to crystallize it or
isolated by ion exchange chromatography or the like. L-Glutamic
acid precipitated in the culture broth may be isolated together
with L-glutamic acid that have been dissolved in the medium after
it is crystallized.
According to the method of the present invention, L-glutamic acid
exceeding the amount corresponding to the saturation concentration
is precipitated, and the concentration of L-glutamic acid dissolved
in the medium is maintained at a constant level. Therefore,
influence of L-glutamic acid at a high concentration on
microorganisms can be reduced. Accordingly, it becomes possible to
breed a microorganism having further improved L-glutamic
acid-producing ability. Further, since L-glutamic acid is
precipitated as crystals, acidification of the culture broth by
accumulation of L-glutamic acid is suppressed, and therefore the
amount of alkali used for maintaining pH of the culture can
significantly be reduced.
EXAMPLES
Hereafter, the present invention will be more specifically
explained with reference to the following examples.
<1> Screening of Microorganism Having L-Glutamic Acid
Resistance in Acidic Environment
Screening of a microorganism having L-glutamic acid resistance in
an acidic environment was performed as follows. Each of about 500
samples obtained from nature including soil, fruits, plant bodies,
river water in an amount of 1 g was suspended in 5 mL of sterilized
water, of which 200 .mu.L, was coated on 20 mL of solid medium of
which pH was adjusted to 4.0 with HCl. The composition of the
medium was as follows: 3 g/L of glucose, 1 g/L of
(NH.sub.4).sub.2SO.sub.4, 0.2 g/L of MgSO.sub.4.7H.sub.2O, 0.5 g/L
of KH.sub.2PO.sub.4, 0.2 g/L of NaCl, 0.1 g/L of
CaCl.sub.2.7H.sub.2O, 0.01 g/L of FeSO.sub.4.7H.sub.2O, 0.01 g/L of
MnSO.sub.4.4H.sub.2O, 0.72 mg/L of ZnSO.sub.4.2H.sub.2O, 0.64 mg/L
of CuSO.sub.4.5H.sub.2O, 0.72 mg/L of CoCl.sub.2.6H.sub.2O, 0.4
mg/L of boric acid, 1.2 mg/L of Na.sub.2MoO.sub.4.2H.sub.2O, 50
.mu.g/L of biotin, 50 .mu.g/L of calcium pantothenate, 50 .mu.g/L
of folic acid, 50 .mu.g/L of inositol, 50 .mu.g/L of niacin, 50
.mu.g/L of p-aminobenzoic acid, 50 .mu.g/L of pyridoxine
hydrochloride, 50 .mu.g/L of riboflavin, 50 .mu.g/L of thiamine
hydrochloride, 50 mg/L of cycloheximide, 20 g/L of agar.
The media plated on which the above samples were plated were
incubated at 28.degree. C., 37.degree. C. or 50.degree. C. for 2 to
4 days and 378 strains each forming a colony were obtained.
Subsequently, each of the strains obtained as described above was
inoculated in a test tube of 16.5 cm in length and 14 mm in a
diameter containing 3 mL of liquid medium (adjusted to pH 4.0 with
HCl) containing a saturation concentration of L-glutamic acid and
cultured at 28.degree. C., 37.degree. C. or 50.degree. C. for 24
hours to 3 days with shaking. Then, the grown strains were
selected. The composition of the aforementioned medium was follows:
40 g/L of glucose, 20 g/L of (NH.sub.4).sub.2SO.sub.4, 0.5 g/L of
MgSO.sub.4.7H.sub.2O, 2 g/L of KH.sub.2PO.sub.4, 0.5 g/L of NaCl,
0.25 g/L of CaCl.sub.2.7H.sub.2O, 0.02 g/L of FeSO.sub.4.7H.sub.2O,
0.02 g/L of MnSO.sub.4.4H.sub.2O, 0.72 mg/L of
ZnSO.sub.4.2H.sub.2O, 0.64 mg/L of CuSO.sub.4.5H.sub.2O, 0.72 mg/L
of CoCl.sub.2.6H.sub.2O, 0.4 mg/L of boric acid, 1.2 mg/L of
Na.sub.2MoO.sub.2.2H.sub.2O, 2 g/L of yeast extract.
Thus, 78 strains of microorganisms having L-glutamic acid
resistance in an acidic environment were successfully obtained.
<2> Selection of Strains with Superior Growth Rate in Acidic
Environment from Microorganisms Having L-glutamic Acid
Resistance
The various microorganisms having L-glutamic acid resistance in an
acidic environment obtained as described above were each inoculated
into a test tube of 16.5 cm in length and 14 mm in diameter
containing 3 mL of medium (adjusted to pH 4.0 with HCl) obtained by
adding 20 g/L of glutamic acid and 2 g/L of glucose to M9 medium
(Sambrook, J., Fritsh, E. F. and Maniatis, T., "Molecular Cloning",
Cold Spring Harbor Laboratory Press, 1989), and the turbidity of
the medium was measured in the time course to select strains with a
favorable growth rate. As a result, as a strain showing favorable
growth, the AJ13355 strain was obtained from a soil in Iwata-shi,
Shizuoka, Japan. This strain was determined as .[.Enterobacter
agglomerans.]. .Iadd.Pantoea ananatis .Iaddend.based on its
bacteriological properties described above.
<3> Acquisition of Strain with Less Viscous Material
Secretion from .[.Enterobacter agglomerans.]. .Iadd.Pantoea
ananatis .Iaddend.AJ13355 Strain
Since the .[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis
.Iaddend.AJ13355 strain extracellularly secretes a viscous material
when cultured in a medium containing a saccharide, operation
efficiency is not favorable. Therefore, a strain with less viscous
material secretion was obtained by the ultraviolet irradiation
method (Miller, J. H. et al., "A Short Course in Bacterial
Genetics; Laboratory Manual", p. 150, Cold Spring Harbor Laboratory
Press, 1992).
The .[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis
.Iaddend.AJ13355 strain was irradiated with ultraviolet ray for 2
minutes at the position 60 cm away from a 60-W ultraviolet lamp and
cultured in LB medium overnight to fix mutation. The mutagenized
strain was diluted and inoculated in LB medium containing 5 g/L of
glucose and 20 g/L of agar so that about 100 colonies per plate
would emerge and cultured at 30.degree. C. overnight with tilting
the plate about 45 degrees, and then 20 colonies showing no flowing
down of the viscous material were selected.
As a strain satisfying conditions that no revertant emerged even
after 5 times of subculture in LB medium containing 5 g/L of
glucose and 20 g/L of agar, and that there should be observed
growth equivalent to the parent strain in LB medium, LB medium
containing 5 g/L of glucose and M9 medium (Sambrook, J. et al.,
Molecular Cloning, 2nd Edition, Cold Spring Harbor Press, 1989) to
which 20 g/L of L-glutamic acid and 2 g/L of glucose were added and
of which pH was adjusted to 4.5 with HCl, SC17 strain was selected
from the strains selected above.
<4> Construction of Glutamic Acid-Producing Bacterium from
.[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis .Iaddend.SC17
Strain
(1) Preparation of .alpha.KGDH Deficient Strain from
.[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis .Iaddend.SC17
Strain
A strain deficient in .alpha.KGDH and with enhanced L-glutamic acid
biosynthetic system was prepared from the .[.Enterobacter
agglomerans.]. .Iadd.Pantoea ananatis .Iaddend.SC17 strain.
(i) Cloning of .alpha.KGDH Gene (Hereafter, Referred to as "sucAB")
of .[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis
.Iaddend.AJ13355 Strain
The sucAB gene of the .[.Enterobacter agglomerans.]. .Iadd.Pantoea
ananatis .Iaddend.AJ13355 strain was cloned by selecting a DNA
fragment complementing the acetic acid-unassimilating property of
the .alpha.KGDH-E1 subunit gene (hereafter, referred to as "sucA")
deficient strain of Escherichia coli from chromosomal DNA of the
.[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis
.Iaddend.AJ13355 strain.
The chromosomal DNA of the .[.Enterobacter agglomerans.].
.Iadd.Pantoea ananatis .Iaddend.AJ13355 strain was isolated by a
method usually employed when chromosomal DNA is extracted from
Escherichia coli (Text for Bioengineering Experiments, Edited by
the Society for Bioscience and Bioengineering, Japan, pp. 97-98,
Baifukan, 1992). The pTWV228 (resistant to ampicillin) used as a
vector was commercially available one from Takara Shuzo Co.,
Ltd.
The chromosomal DNA of the AJ13355 strain digested with EcoT221 and
pTWV228 digested with PstI were ligated by using T4 ligase and used
to transform the sucA deficient Escherichia coli JRG465 strain
(Herbert, J. et al., Mol. Gen. Genetics, 105, 182 (1969)). A strain
growing in an acetate minimal medium was selected from the
transformant strains obtained above, and a plasmid was extracted
from it and designated as pTWVEK101. The Escherichia coil JRG465
strain harboring pTWVEK101 recovered auxotrophy for succinic acid
or L-lysine and L-methionine besides the acetic acid-assimilating
property. This suggests that pTWVEK101 contains the sucA gene of
.[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis.Iaddend..
FIG. 1 shows the restriction map of a DNA fragment derived from
.[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis .Iaddend.in
pTWVEK101. The determined nucleotide sequence of the hatched
portion in FIG. 1 is shown as SEQ ID NO: 1. In this sequence,
nucleotide sequences considered to be two full length ORFs and two
nucleotide sequences considered to be partial sequences of the ORFs
were found. SEQ ID NOS: 2 to 5 show amino acid sequences that can
be encoded by these ORFs or partial sequences in an order from the
5' end. As a result of homology search for these, it was revealed
that the portion of which nucleotide sequences were determined
contained a 3'-end partial sequence of the succinate dehydrogenase
iron-sulfur protein gene (sdhB), full length sucA and
.alpha.KGDH-E2 subunit gene (sucB), and 5'-end partial sequence of
the succinyl CoA synthetase .beta. subunit gene (sucC). The results
of comparison of the amino acid sequences deduced from these
nucleotide sequences with those derived from Escherichia coli (Eur.
J. Biochem., 141, pp. 351-359 (1984); Eur. J. Biochem., 141, pp.
361-374 (1984); Biochemistry, 24, pp. 6245-6252 (1985)) are shown
in FIGS. 2 to 5. Thus, the amino acid sequences each showed very
high homology. In addition, it was found that a cluster of
sdhB-sucA-sucB-sucC was constituted on the chromosome of
.[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis .Iaddend.as
in Escherichia coli is (Eur. J. Biochem., 141, pp. 351-359 (1984);
Eur. J. Biochem., 141, pp. 361-374 (1984); Biochemistry, 24, pp.
6245-6252 (1985)).
(ii) Acquisition of .alpha.KGDH Deficient Strain Derived from
.[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis .Iaddend.SC17
Strain
The homologous recombination was performed by using the sucAB gene
of .[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis
.Iaddend.obtained as described above to obtain an .alpha.KGDH
deficient strain of .[.Enterobacter agglomerans.]. .Iadd.Pantoea
ananatis.Iaddend..
After pTWVEK101 was digested with SphI to excise a fragment
containing sucA, the fragment was blunt-ended with Klenow fragment
(Takara Shuzo Co., Ltd.) and ligated with pBR322 digested with
EcoRI and blunt-ended with Klenow fragment, by using T4 DNA ligase
(Takara Shuzo Co., Ltd.) The obtained plasmid was digested at the
restriction enzyme BglII recognition site positioned substantially
at the center of sucA by using this enzyme, blunt-ended with Klenow
fragment, and then ligated again by using T4 DNA ligase. It was
considered that the sucA gene did not function because a frame
shift mutation was introduced into sucA of the plasmid newly
constructed through the above procedure.
The plasmid constructed as described above was digested with a
restriction enzyme ApaLI, and subjected to agarose gel
electrophoresis to recover a DNA fragment containing sucA into
which the frame shift mutation was introduced and a tetracycline
resistance gene derived from pBR322. The recovered DNA fragment was
ligated again by using T4 DNA ligase to construct a plasmid for
disrupting the .alpha.KGDH gene.
The plasmid for disrupting the .alpha.KGDH gene obtained as
described above was used to transform the .[.Enterobacter
agglomerans.]. .Iadd.Pantoea ananatis .Iaddend.SC17 strain by
electroporation (Miller, J. H., "A Short Course in Bacterial
Genetics; Handbook", p. 279, Cold Spring Harbor Laboratory Press,
U.S.A., 1992), and a strain where in sucA on the chromosome was
replaced with a mutant type one by homologous recombination of the
plasmid was obtained by using the tetracycline resistance as an
indicator. The obtained strain was designated as SC17sucA
strain.
In order to confirm that the SC17sucA strain was deficient in the
.alpha.KGDH activity, the enzyme activity was measured by the
method of Reed et al. (Reed, L. J. and Mukherjee, B. B., Methods in
Enzymology, 13, pp. 55-61, (1969)) by using cells of the strain
cultured in LB medium until the logarithmic growth phase. As a
result, .alpha.KGDH activity of 0.073 (.DELTA.ABS/min/mg protein)
was detected from the SC17 strain, whereas no .alpha.KGDH activity
was detected from the SC17sucA strain, and thus it was confirmed
that the sucA was deficient as purposed.
(2) Enhancement of L-Glutamic Acid Biosynthetic System of
.[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis
.Iaddend.SC17sucA Strain
Subsequently, a citrate synthase gene, a phosphoenolpyruvate
carboxylase gene and a glutamate dehydrogenase gene derived from
Escherichia coli were introduced into the SC17sucA strain.
(i) Preparation of Plasmid Having gltA Gene, ppc Gene and gdhA Gene
Derived from Escherichia coli
The procedures of preparing a plasmid having a gltA gene, a ppc
gene and a gdhA gene will be explained by referring to FIGS. 6 and
7.
A plasmid having a gdhA gene derived from Escherichia coli, pBRGDH
(Japanese Patent Application Laid-open No. 7-203980), was digested
with HindIII and SphI, the both ends were blunt-ended by the T4 DNA
polymerase treatment, and then the DNA fragment having the gdhA
gene was purified and recovered. Separately, a plasmid having a
gltA gene and a ppc gene derived from Escherichia coli, pMWCP (WO
97/08294), was digested with XbaI, and then the both ends were
blunt-ended by using T4 DNA polymerase. This was mixed with the
above purified DNA fragment having the gdhA gene and ligated by
using T4 ligase to obtain a plasmid pMWCPG, which corresponded to
pMWCP further containing the gdhA gene (FIG. 6).
At the same time, the plasmid pVIC40 (Japanese Patent Application
Laid-open No. 8-047397) having the replication origin of the broad
host spectrum plasmid RSF1010 was digested with NotI, treated with
T4 DNA polymerase and digested with PstI. pBR322 was digested with
EcoT14I, treated with T4 DNA polymerase and digested with PstI. The
both products were mixed and ligated by using T4 ligase to obtain a
plasmid RSF-Tet having the replication origin of RSF1010 and a
tetracycline resistance gene (FIG. 7).
Subsequently, 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 recovered. RSF-Tet was similarly digested with EcoRI
and PstI, and a DNA fragment having the replication origin of
RSF1010 was purified and recovered. The both products were mixed
and ligated by using T4 ligase to obtain a plasmid RSFCPG, which
corresponded to RSF-Tet containing the gltA gene, the ppc gene and
the gdhA gene (FIG. 8). It was confirmed that the obtained plasmid
RSFCPG expressed the gltA gene, the ppc gene and the gdhA gene, by
the complementation of the auxotrophy of the gltA, ppc or gdhA gene
deficient strain derived from Escherichia coli and measurement of
each enzyme activity.
(ii) Preparation of Plasmid Having gltA Gene Derived from
Brevibacterium lactofermentum
A plasmid having the gltA gene derived from Brevibacterium
lactofermentum was constructed as follows. PCR was performed by
using the primer DNAs having the nucleotide sequences represented
by SEQ ID NOS: 6 and 7, which were prepared based on the nucleotide
sequence of the Corynebacterium glutamicum gltA gene (Microbiology,
140, pp. 1817-1828 (1994)), and chromosomal DNA of Brevibacterium
lactofermentum ATCC13869 as a template to obtain a gltA gene
fragment of about 3 kb. This fragment inserted into a plasmid
pHSG399 (purchased from Takara Shuzo Co., Ltd.) digested with SmaI
to obtain a plasmid pHSGCB (FIG. 9). Subsequently, pHSGCB was
digested with HindIII, and the excised gltA gene fragment of about
3 kb was inserted into a plasmid pSTV29 (purchased from Takara
Shuzo Co., Ltd.) digested with HindIII to obtain a plasmid pSTVCB
(FIG. 9). It was confirmed that the obtained plasmid pSTVCB
expressed the gltA gene, by measuring the enzyme activity in the
.[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis
.Iaddend.AJ13355 strain.
(iii) Introduction of RSFCPG and pSTVCB into SC17sucA Strain
The .[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis
.Iaddend.SC17sucA strain was transformed with RSFCPG by
electroporation to obtain a transformant SC17sucA/RSFCPG strain
having tetracycline resistance. Further, the SC17sucA/RSFCPG strain
was transformed with pSTVCB by electroporation to obtain a
transformant SC17sucA/RSFCPG+pSTVCB strain having chloramphenicol
resistance.
<4> Acquisition of Strain with Improved Resistance to
L-Glutamic Acid in Low pH Environment
A strain with improved resistance to L-glutamic acid at a high
concentration in a low pH environment (hereafter, also referred to
as "high-concentration Glu-resistant strain at low pH") was
isolated from the .[.Enterobacter agglomerans.]. .Iadd.Pantoea
ananatis .Iaddend.SC17sucA/RSFCPG+pSTVCB strain.
The SC17sucA/RSFCPG+pSTVCB strain was cultured overnight at
30.degree. C. in LBG medium (10 g/L of tryptone, 5 g/L of yeast
extract, 10 g/L of NaCl, 5 g/L of glucose), and the cells washed
with saline was appropriately diluted and plated on an M9-E medium
(4 g/L of glucose, 17 g/L of Na.sub.2HPO.sub.4.12H.sub.2O, 3 g/L of
KH.sub.2PO.sub.4, 0.5 g/L of NaCl, 1 g/L of NH.sub.4Cl, 10 mM of
MgSO.sub.4, 10 .mu.M of CaCl.sub.2, 50 mg/L of L-lysine, 50 mg/L of
L-methionine, 50 mg/L of DL-diaminopimelic acid, 25 mg/L of
tetracycline, 25 mg/L of chloramphenicol, 30 g/L of L-glutamic
acid, adjusted to pH 4.5 with aqueous ammonia) plate. The colony
emerged after culture a 32.degree. C. for 2 days was obtained as a
high-concentration Glu-resistant strain at low pH.
For the obtained strain, growth level in M9-E liquid medium was
measured and L-glutamic acid-producing ability was tested in a
50-ml volume large test tube containing 5 ml of L-glutamic acid
production test medium (40 g/L of glucose, 20 g/L of
(NH.sub.4).sub.2SO.sub.4, 0.5 g/L of MgSO.sub.4.7H.sub.2O, 2 g/L of
KH.sub.2PO.sub.4, 0.5 g/L of NaCl, 0.25 g/L of
CaCl.sub.2.7H.sub.2O, 0.02 g/L of FeSO.sub.4.7H.sub.2O, 0.02 g/L of
MnSO.sub.4.4H.sub.2O, 0.72 mg/L of ZnSO.sub.4.2H.sub.2O, 0.64 mg/L
of CuSO.sub.4.5H.sub.2O, 0.72 mg/L of CoCl.sub.2.6H.sub.2O, 0.4
mg/L of boric acid, 1.2 mg/L of Na.sub.2MoO.sub.4.2H.sub.2O, 2 g/L
of yeast extract, 200 mg/L of L-lysine hydrochloride, 200 mg/L of
L-methionine, 200 mg/L of DL-.alpha.,.epsilon.-diaminopimelic acid,
25 mg/L of tetracycline hydrochloride, 25 mg/L of chloramphenicol).
A strain that exhibited the best growth level and the same
L-glutamic acid producing ability as that of its parent strain, the
SC17/RSFCPG+pSTVCB strain, was designated as .[.Enterobacter
agglomerans.]. .Iadd.Pantoea ananatis .Iaddend.AJ13601. The AJ13601
strain 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, Japan) on
Aug. 18, 1999 and received an accession number of FERM P-17516. It
was then transferred to an international deposition under the
provisions of Budapest Treaty on Jul. 6, 2000 and received an
accession number of FERM BP-7207.
<5> Culture of .[.Enterobacter Agglomerans.]. .Iadd.Pantoea
ananatis .Iaddend.AJ13601 Strain for L-Glutamic Acid Production
(1)
The .[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis
.Iaddend.AJ13601 strain was inoculated into a 1-L jar fermenter
containing 300 ml of medium containing 40 g/L of glucose, 20 g/L of
(NH.sub.4).sub.2SO.sub.4, 0.5 g/L of MgSO.sub.4.7H.sub.2O, 2 g/L of
KH.sub.2PO.sub.4, 0.5 g/L of NaCl, 0.25 g/L of
CaCl.sub.2.7H.sub.2O, 0.02 g/L of FeSO.sub.4.7H.sub.2O, 0.02 g/L of
MnSO.sub.4.4H.sub.2O, 0.72 mg/L of ZnSO.sub.4.2H.sub.2O, 0.64 mg/L
of CuSO.sub.4.5H.sub.2O, 0.72 mg/L of CoCl.sub.2.6H.sub.2O, 0.4
mg/L of boric acid, 1.2 mg/L of Na.sub.2MoO.sub.4.2H.sub.2O, 2 g/L
of yeast extract, 200 mg/L of L-lysine hydrochloride, 200 mg/L of
L-methionine, 200 mg/L of DL-.alpha.,.epsilon.-diaminopimelic acid,
25 mg/L of tetracycline hydrochloride and 25 mg/L of
chloramphenicol, and cultured at 34.degree. C. and pH 6.0 for 14
hours. The culture pH was controlled by introducing ammonia gas
into the medium.
The culture obtained as described above was centrifuged at 5000 rpm
for 10 minutes, and the collected cells were inoculated into a 1-L
jar fermenter containing 300 ml of medium containing 40 g/L of
glucose, 5 g/L of (NH.sub.4).sub.2SO.sub.4, 1.5 g/L of
MgSO.sub.4.7H.sub.2O, 6 g/L of KH.sub.2PO.sub.4, 1.5 g/L of NaCl,
0.75 g/L of CaCl.sub.2.7H.sub.2O, 0.06 g/L of FeSO.sub.4.7H.sub.2O,
0.06 g/L of MnSO.sub.4.4H.sub.2O, 2.16 mg/L of
ZnSO.sub.4.2H.sub.2O, 1.92 mg/L of CuSO.sub.4.5H.sub.2O, 2.16 mg/L
of CoCl.sub.20.6H.sub.2O, 1.2 mg/L of boric acid, 3.6 mg/L of
Na.sub.2MoO.sub.4.2H.sub.2O, 6 g/L of yeast extract, 600 mg/L of
L-lysine hydrochloride, 600 mg/L of L-methionine, 600 mg/L of
DL-.alpha.,.epsilon.-diaminopimelic acid, 25 mg/L of tetracycline
hydrochloride and 25 mg/L of chloramphenicol and cultured at
34.degree. C. and pH 4.5 to perform culture for L-glutamic acid
production. The culture pH was controlled by introducing ammonia
gas into the medium. As the initially added glucose was depleted,
600 g/L of glucose was continuously added.
As a result of the culture for L-glutamic acid production performed
for 50 hours as described above, a substantial amount of L-glutamic
acid crystals were precipitated in the jar fermenter. Table 1 shows
the concentration of L-glutamic acid dissolved in the culture broth
at that time and the L-glutamic acid concentration measured by
dissolving the crystals in 2 M potassium hydroxide. L-Glutamic acid
crystals were collected from the culture by decantation after the
culture was left stood.
TABLE-US-00001 TABLE 1 Concentration of L-glutamic acid 51 g/L
dissolved in culture broth Amount of L-glutamic acid precipitated
67 g/L as crystals Concentration of L-glutamic acid 118 g/L
measured by dissolving crystals
<6> Culture of .[.Enterobacter Agglomerans.]. .Iadd.Pantoea
ananatis .Iaddend.AJ13601 Strain for L-Glutamic Acid Production
(2)
The following experiment was performed in order to confirm that the
.[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis
.Iaddend.AJ13601 strain still had L-glutamic acid-producing ability
even under the condition that L-glutamic acid crystals were
present.
The .[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis
.Iaddend.AJ13601 strain was inoculated into a 1-L jar fermenter
containing 300 ml of medium containing 40 g/L of glucose, 20 g/L of
(NH.sub.4).sub.2SO.sub.4, 0.5 g/L of MgSO.sub.4.7H.sub.2O, 2 g/L of
KH.sub.2PO.sub.4, 0.5 g/L of NaCl, 0.25 g/L of
CaCl.sub.2.7H.sub.2O, 0.02 g/L of FeSO.sub.4.7H.sub.2O, 0.02 g/L of
MnSO.sub.4.4H.sub.2O, 0.72 mg/L of ZnSO.sub.4.2H.sub.2O, 0.64 mg/L
of CuSO.sub.4.5H.sub.2O, 0.72 mg/L of CoCl.sub.2.6H.sub.2O, 0.4
mg/L of boric acid, 1.2 mg/L of Na.sub.2MoO.sub.4.2H.sub.2O, 2 g/L
of yeast extract, 200 mg/L of L-lysine hydrochloride, 200 mg/L of
L-methionine, 200 mg/L of DL-.alpha.,.epsilon.-diaminopimelic acid,
25 mg/L of tetracycline hydrochloride and 25 mg/L of
chloramphenicol, and cultured at 34.degree. C. at pH 6.0 for 14
hours. The culture pH was controlled by bubbling the medium with
ammonia gas. The culture obtained as described above was
centrifuged at 5000 rpm for 10 minutes, and then the collected
cells were cultured in a medium where L-glutamic acid was present
as crystals. The used medium contained 40 g/L of glucose, 5 g/L of
(NH.sub.4).sub.2SO.sub.4, 1.5 g/L of MgSO.sub.4.7H.sub.2O, 6 g/L of
KH.sub.2PO.sub.4, 1.5 g/L of NaCl, 0.75 g/L of
CaCl.sub.2.7H.sub.2O, 0.06 g/L of FeSO.sub.4.7H.sub.2O, 0.06 g/L of
MnSO.sub.4.4H.sub.2O, 2.16 mg/L of ZnSO.sub.4.2H.sub.2O, 1.92 mg/L
of CuSO.sub.4.5H.sub.2O, 2.16 mg/L of CoCl.sub.2.6H.sub.2O, 1.2
mg/L of boric acid, 3.6 mg/L of Na.sub.2MoO.sub.4.2H.sub.2O, 6 g/L
of yeast extract, 600 mg/L of L-lysine hydrochloride, 600 mg/L of
L-methionine, 600 mg/L of DL-.alpha.,.epsilon.-diaminopimelic acid,
25 mg/L of tetracycline hydrochloride and 25 mg/L of
chloramphenicol and L-glutamic acid crystals were added to 40 g/L.
The cells were inoculated in a 1-L jar fermenter containing 300 ml
of this medium and cultured at 34.degree. C. and pH 4.3 to perform
culture for L-glutamic acid production. The culture pH was
controlled by introducing ammonia gas into the medium. As the
initially added glucose was depleted, 600 g/L of glucose was
continuously added. In this medium, only 39 g/L of the added
L-glutamic acid was dissolved at pH 4.3 and the remaining 1 g/L was
present as crystals.
As a result of the culture for L-glutamic acid production performed
for 53 hours as described above, a substantial amount of L-glutamic
acid crystals were precipitated in the jar fermenter. Table 2 shows
the concentration of L-glutamic acid dissolved in the culture
broth, the amount of L-glutamic acid present as crystals at that
time and the L-glutamic acid concentration measured by dissolving
the crystals in 2MKOH solution. L-Glutamic acid crystals were
collected from the culture by decantation after the culture was
left stood. The results showed that the .[.Enterobacter
agglomerans.]. .Iadd.Pantoea ananatis .Iaddend.AJ13101 strain
accumulated L-glutamic acid and precipitated crystals thereof even
under the condition that L-glutamic acid crystals were present.
TABLE-US-00002 TABLE 2 Concentration of L-glutamic acid 39 g/L
dissolved in culture broth Amount of L-glutamic acid precipitated
119 g/L as crystals Concentration of L-glutamic acid 158 g/L
measured by dissolving crystals Amount of L-glutamic acid crystals
118 g/L newly produced by main culture
<7> Culture of .[.Enterobacter agglomerans.]. .Iadd.Pantoea
ananatis .Iaddend.AJ13601 Strain for L-Glutamic Acid Production
(3)
The .[.Enterobacter agglomerans.]. .Iadd.Pantoea ananatis
.Iaddend.AJ13601 strain can grow not only at an acidic pH, but also
at a neutral pH. Therefore, it was confirmed as follows that
L-glutamic acid crystals could also be precipitated by starting the
culture at a neutral pH and allowing production of L-glutamic acid
during the culture so that pH of the culture should spontaneously
be lowered.
Cells of one plate (8.5 cm in diameter) of the .[.Enterobacter
agglomerans.]. .Iadd.Pantoea ananatis .Iaddend.AJ13601 strain,
cultured on LBG agar medium (10 g/ of L tryptone, 5 g/L of yeast
extract, 10 g/L of NaCl, 5 g/L of glucose, 15 g/L of agar)
containing 25 mg/L of tetracycline hydrochloride and 25 mg/L of
chloramphenicol at 30.degree. C. for 14 hours, were inoculated into
a 1-L jar fermenter containing 300 ml of medium containing 40 g/L
of glucose, 5 g/L of (NH.sub.4).sub.2SO.sub.4, 1.5 g/L of
MgSO.sub.4.7H.sub.2O, 6 g/L of KH.sub.2PO.sub.4, 1.5 g/L of NaCl,
0.75 g/L of CaCl.sub.2.7H.sub.2O, 0.06 g/L of FeSO.sub.4.7H.sub.2O,
0.06 g/L of MnSO.sub.4.4H.sub.2O, 2.16 mg/L of
ZnSO.sub.4.2H.sub.2O, 1.92 mg/L of CuSO.sub.4.5H.sub.2O, 2.16 mg/L
of CoCl.sub.2.6H.sub.2O., 1.2 mg/L of boric acid, 3.6 mg/L of
Na.sub.2MoO.sub.4.2H.sub.2O, 6 g/L of yeast extract, 600 mg/L of
L-lysine hydrochloride, 600 mg/L of L-methionine, 600 mg/L of
DL-.alpha.,.epsilon.-diaminopimelic acid, 25 mg/L of tetracycline
hydrochloride and 25 mg/L of chloramphenicol and the culture was
started at 34.degree. C. and pH 7.0. The culture pH was controlled
by introducing ammonia gas into the medium. As the initially added
glucose was depleted, 600 g/L of glucose was continuously
added.
As L-glutamic acid is accumulated, pH lowers spontaneously. The
amount of the introduced ammonia gas was adjusted so that pH should
be gradually lowered from 7.0 to 4.5 during the period between 15
hours and 24 hours after the start of the culture, and 24 hours
after the start of the culture, pH became 4.5. Afterward,
cultivation was continued for 12 hours.
As a result of the culture for L-glutamic acid production conducted
for 36 hours as described above, a substantial amount of L-glutamic
acid crystals were precipitated in the jar fermenter. Table 3 shows
the concentration of L-glutamic acid dissolved in the culture
broth, the amount of L-glutamic acid present as crystals at that
time and the L-glutamic acid concentration measured by dissolving
the crystals in 2 MKOH solution. L-Glutamic acid crystals were
collected from the culture by decantation after the culture was
left stood.
TABLE-US-00003 TABLE 3 Concentration of L-glutamic acid 45 g/L
dissolved in culture broth Amount of L-glutamic acid precipitated
31 g/L as crystals Concentration of L-glutamic acid 76 g/L measured
by dissolving crystals
SEQUENCE LISTINGS
1
1214556DNAEnterobacter
agglomeransCDS(2)..(121)CDS(322)..(3129)CDS(3145)..(4368)CDS(4437)..(4556-
) 1t gca ttc agc gtt ttc cgc tgt cac agc atc atg aac tgt gta agt
gtt 49 Ala Phe Ser Val Phe Arg Cys His Ser Ile Met Asn Cys Val Ser
Val 1 5 10 15tgt cct aaa ggg cta aac ccg acg cgc gct atc ggc cac
att aag tcg 97Cys Pro Lys Gly Leu Asn Pro Thr Arg Ala Ile Gly His
Ile Lys Ser 20 25 30atg ctg ctg caa cgc agc gcg tag ttataccacc
gggaacctca ggttcccggt 151Met Leu Leu Gln Arg Ser Ala 35attttacgga
agcctctgta aacgcggtcc caaccacgtt tacaaaggtt cccttacggg
211ccgggcgcgc gctgcgcaca gtgctcgtat cgctgaactc actacggcaa
accgcgaaag 271cggcaacaaa tgaaacctca aaaaagcata acattgctta
agggatcaca atg cag 327 Met Gln 40aac agc gcg atg aag ccc tgg ctg
gac tcc tcc tgg ctg gcc ggc gcg 375Asn Ser Ala Met Lys Pro Trp Leu
Asp Ser Ser Trp Leu Ala Gly Ala 45 50 55aat cag tct tac ata gag caa
ctc tat gag gat ttc ctg acc gat cct 423Asn Gln Ser Tyr Ile Glu Gln
Leu Tyr Glu Asp Phe Leu Thr Asp Pro 60 65 70gac tct gtg gat gca gtg
tgg cgc tcg atg ttc caa cag tta cca ggc 471Asp Ser Val Asp Ala Val
Trp Arg Ser Met Phe Gln Gln Leu Pro Gly 75 80 85acg gga gtg aaa cct
gag cag ttc cac tcc gca act cgc gaa tat ttc 519Thr Gly Val Lys Pro
Glu Gln Phe His Ser Ala Thr Arg Glu Tyr Phe90 95 100 105cgt cgc ctg
gcg aaa gac gca tct cgt tac acc tcc tca gtt acc gat 567Arg Arg Leu
Ala Lys Asp Ala Ser Arg Tyr Thr Ser Ser Val Thr Asp 110 115 120ccg
gca acc aac tcc aaa caa gtg aaa gtg ctg cag ctg att aac gcg 615Pro
Ala Thr Asn Ser Lys Gln Val Lys Val Leu Gln Leu Ile Asn Ala 125 130
135ttt cgt ttc cgc gga cat cag gaa gca aat ctc gat ccg ctt ggc ctg
663Phe Arg Phe Arg Gly His Gln Glu Ala Asn Leu Asp Pro Leu Gly Leu
140 145 150tgg aaa cag gac cgc gtt gcc gat ctc gat cct gcc ttt cac
gat ctg 711Trp Lys Gln Asp Arg Val Ala Asp Leu Asp Pro Ala Phe His
Asp Leu 155 160 165acc gac gcc gat ttt cag gaa agc ttt aac gta ggt
tct ttt gcc att 759Thr Asp Ala Asp Phe Gln Glu Ser Phe Asn Val Gly
Ser Phe Ala Ile170 175 180 185ggc aaa gaa acc atg aag ctg gcc gat
ctg ttc gac gcg ctg aag cag 807Gly Lys Glu Thr Met Lys Leu Ala Asp
Leu Phe Asp Ala Leu Lys Gln 190 195 200acc tac tgt ggc tcg att ggt
gca gag tat atg cac atc aat aac acc 855Thr Tyr Cys Gly Ser Ile Gly
Ala Glu Tyr Met His Ile Asn Asn Thr 205 210 215gaa gag aaa cgc tgg
atc cag cag cgt atc gaa tcc ggt gcg agc cag 903Glu Glu Lys Arg Trp
Ile Gln Gln Arg Ile Glu Ser Gly Ala Ser Gln 220 225 230acg tca ttc
agt ggc gaa gag aaa aaa ggt ttc ctg aaa gag ctg acc 951Thr Ser Phe
Ser Gly Glu Glu Lys Lys Gly Phe Leu Lys Glu Leu Thr 235 240 245gcg
gca gaa ggg ctg gaa aaa tat ctg ggc gcg aaa ttc ccg ggt gca 999Ala
Ala Glu Gly Leu Glu Lys Tyr Leu Gly Ala Lys Phe Pro Gly Ala250 255
260 265aaa cgt ttc tcg ctg gaa ggc ggt gat gcg ctg gtg ccg atg ctg
cgc 1047Lys Arg Phe Ser Leu Glu Gly Gly Asp Ala Leu Val Pro Met Leu
Arg 270 275 280gag atg att cgt cat gcg ggc aaa agc ggc aca cgt gaa
gtg gta ctg 1095Glu Met Ile Arg His Ala Gly Lys Ser Gly Thr Arg Glu
Val Val Leu 285 290 295ggg atg gcg cac cgt ggc cgt ctt aac gta ctg
att aac gta ctg ggt 1143Gly Met Ala His Arg Gly Arg Leu Asn Val Leu
Ile Asn Val Leu Gly 300 305 310aaa aag cca cag gat ctg ttc gac gaa
ttc tcc ggt aaa cac aaa gag 1191Lys Lys Pro Gln Asp Leu Phe Asp Glu
Phe Ser Gly Lys His Lys Glu 315 320 325cat ctg ggc acc ggt gat gtg
aag tat cac atg ggc ttc tct tcg gat 1239His Leu Gly Thr Gly Asp Val
Lys Tyr His Met Gly Phe Ser Ser Asp330 335 340 345att gaa acc gaa
ggt ggt ctg gtg cat ctg gcg ctg gcg ttt aac ccg 1287Ile Glu Thr Glu
Gly Gly Leu Val His Leu Ala Leu Ala Phe Asn Pro 350 355 360tct cac
ctg gaa att gtc agc ccg gtg gtc atg gga tcg gta cgt gca 1335Ser His
Leu Glu Ile Val Ser Pro Val Val Met Gly Ser Val Arg Ala 365 370
375cgt ctc gat cgt ctg gcc gaa ccg gtc agc aat aaa gtg ttg cct atc
1383Arg Leu Asp Arg Leu Ala Glu Pro Val Ser Asn Lys Val Leu Pro Ile
380 385 390acc att cac ggt gat gcg gcg gtg att ggt cag ggc gtg gtt
cag gaa 1431Thr Ile His Gly Asp Ala Ala Val Ile Gly Gln Gly Val Val
Gln Glu 395 400 405acc ctg aac atg tct cag gcg cgc ggc tac gaa gtg
ggc ggc acg gta 1479Thr Leu Asn Met Ser Gln Ala Arg Gly Tyr Glu Val
Gly Gly Thr Val410 415 420 425cgt atc gtc att aac aac cag gtt ggt
ttt acc acc tcc aac ccg aaa 1527Arg Ile Val Ile Asn Asn Gln Val Gly
Phe Thr Thr Ser Asn Pro Lys 430 435 440gat gcg cgt tca acc ccg tac
tgt act gac atc ggc aag atg gtg ctg 1575Asp Ala Arg Ser Thr Pro Tyr
Cys Thr Asp Ile Gly Lys Met Val Leu 445 450 455gca ccg att ttc cac
gtc aat gct gac gat ccg gaa gcg gtg gcc ttt 1623Ala Pro Ile Phe His
Val Asn Ala Asp Asp Pro Glu Ala Val Ala Phe 460 465 470gtt acc cgc
ctg gcg ctg gac tat cgc aac acc ttc aaa cgc gat gtg 1671Val Thr Arg
Leu Ala Leu Asp Tyr Arg Asn Thr Phe Lys Arg Asp Val 475 480 485ttt
atc gat ctg gtg tgc tat cgc cgt cat ggt cac aac gag gcg gat 1719Phe
Ile Asp Leu Val Cys Tyr Arg Arg His Gly His Asn Glu Ala Asp490 495
500 505gag cca agt gct acc cag ccg ttg atg tac cag aaa atc aaa aag
cat 1767Glu Pro Ser Ala Thr Gln Pro Leu Met Tyr Gln Lys Ile Lys Lys
His 510 515 520ccg acg ccg cgt aaa att tac gcc gat cgt ctg gaa ggc
gaa ggt gtc 1815Pro Thr Pro Arg Lys Ile Tyr Ala Asp Arg Leu Glu Gly
Glu Gly Val 525 530 535gcg tcg cag gaa gat gcc acc gag atg gtg aac
ctg tac cgc gat gcg 1863Ala Ser Gln Glu Asp Ala Thr Glu Met Val Asn
Leu Tyr Arg Asp Ala 540 545 550ctc gat gcg ggc gaa tgc gtg gtg ccg
gaa tgg cgt ccg atg agc ctg 1911Leu Asp Ala Gly Glu Cys Val Val Pro
Glu Trp Arg Pro Met Ser Leu 555 560 565cac tcc ttc acg tgg tcg cct
tat ctg aac cac gaa tgg gat gag cct 1959His Ser Phe Thr Trp Ser Pro
Tyr Leu Asn His Glu Trp Asp Glu Pro570 575 580 585tat ccg gca cag
gtt gac atg aaa cgc ctg aag gaa ctg gca ttg cgt 2007Tyr Pro Ala Gln
Val Asp Met Lys Arg Leu Lys Glu Leu Ala Leu Arg 590 595 600atc agc
cag gtc cct gag cag att gaa gtg cag tcg cgc gtg gcc aag 2055Ile Ser
Gln Val Pro Glu Gln Ile Glu Val Gln Ser Arg Val Ala Lys 605 610
615atc tat aac gat cgc aag ctg atg gcc gaa ggc gag aaa gcg ttc gac
2103Ile Tyr Asn Asp Arg Lys Leu Met Ala Glu Gly Glu Lys Ala Phe Asp
620 625 630tgg ggc ggt gcc gag aat ctg gcg tac gcc acg ctg gtg gat
gaa ggt 2151Trp Gly Gly Ala Glu Asn Leu Ala Tyr Ala Thr Leu Val Asp
Glu Gly 635 640 645att ccg gtt cgc ctc tcg ggt gaa gac tcc ggt cgt
gga acc ttc ttc 2199Ile Pro Val Arg Leu Ser Gly Glu Asp Ser Gly Arg
Gly Thr Phe Phe650 655 660 665cat cgc cac gcg gtc gtg cac aac cag
gct aac ggt tca acc tat acg 2247His Arg His Ala Val Val His Asn Gln
Ala Asn Gly Ser Thr Tyr Thr 670 675 680ccg ctg cac cat att cat aac
agc cag ggc gag ttc aaa gtc tgg gat 2295Pro Leu His His Ile His Asn
Ser Gln Gly Glu Phe Lys Val Trp Asp 685 690 695tcg gtg ctg tct gaa
gaa gcg gtg ctg gcg ttt gaa tac ggt tac gcc 2343Ser Val Leu Ser Glu
Glu Ala Val Leu Ala Phe Glu Tyr Gly Tyr Ala 700 705 710acg gct gag
ccg cgc gtg ctg acc atc tgg gaa gcg cag ttt ggt gac 2391Thr Ala Glu
Pro Arg Val Leu Thr Ile Trp Glu Ala Gln Phe Gly Asp 715 720 725ttt
gcc aac ggt gct cag gtg gtg att gac cag ttc atc agc tct ggc 2439Phe
Ala Asn Gly Ala Gln Val Val Ile Asp Gln Phe Ile Ser Ser Gly730 735
740 745gaa cag aag tgg ggc cgt atg tgt ggc ctg gtg atg ttg ctg ccg
cat 2487Glu Gln Lys Trp Gly Arg Met Cys Gly Leu Val Met Leu Leu Pro
His 750 755 760ggc tac gaa ggt cag gga ccg gaa cac tcc tct gcc cgt
ctg gaa cgc 2535Gly Tyr Glu Gly Gln Gly Pro Glu His Ser Ser Ala Arg
Leu Glu Arg 765 770 775tat ctg caa ctt tgc gcc gag cag aac atg cag
gtt tgc gtc ccg tcg 2583Tyr Leu Gln Leu Cys Ala Glu Gln Asn Met Gln
Val Cys Val Pro Ser 780 785 790acg ccg gct cag gtg tat cac atg ctg
cgc cgt cag gcg ctg cgc ggg 2631Thr Pro Ala Gln Val Tyr His Met Leu
Arg Arg Gln Ala Leu Arg Gly 795 800 805atg cgc cgt ccg ctg gtg gtg
atg tcg ccg aag tcg ctg tta cgc cat 2679Met Arg Arg Pro Leu Val Val
Met Ser Pro Lys Ser Leu Leu Arg His810 815 820 825cca ctg gcg atc
tcg tcg ctg gat gaa ctg gca aac ggc agt ttc cag 2727Pro Leu Ala Ile
Ser Ser Leu Asp Glu Leu Ala Asn Gly Ser Phe Gln 830 835 840ccg gcc
att ggt gag atc gac gat ctg gat ccg cag ggc gtg aaa cgc 2775Pro Ala
Ile Gly Glu Ile Asp Asp Leu Asp Pro Gln Gly Val Lys Arg 845 850
855gtc gtg ctg tgc tcc ggt aag gtt tac tac gat ctg ctg gaa cag cgt
2823Val Val Leu Cys Ser Gly Lys Val Tyr Tyr Asp Leu Leu Glu Gln Arg
860 865 870cgt aaa gac gag aaa acc gat gtt gcc atc gtg cgc atc gaa
cag ctt 2871Arg Lys Asp Glu Lys Thr Asp Val Ala Ile Val Arg Ile Glu
Gln Leu 875 880 885tac ccg ttc ccg cat cag gcg gta cag gaa gca ttg
aaa gcc tat tct 2919Tyr Pro Phe Pro His Gln Ala Val Gln Glu Ala Leu
Lys Ala Tyr Ser890 895 900 905cac gta cag gac ttt gtc tgg tgc cag
gaa gag cct ctg aac cag ggc 2967His Val Gln Asp Phe Val Trp Cys Gln
Glu Glu Pro Leu Asn Gln Gly 910 915 920gcc tgg tac tgt agc cag cat
cat ttc cgt gat gtc gtg ccg ttt ggt 3015Ala Trp Tyr Cys Ser Gln His
His Phe Arg Asp Val Val Pro Phe Gly 925 930 935gcc acc ctg cgt tat
gca ggt cgc ccg gca tcg gct tct ccg gcc gtg 3063Ala Thr Leu Arg Tyr
Ala Gly Arg Pro Ala Ser Ala Ser Pro Ala Val 940 945 950ggt tat atg
tcc gta cac caa caa cag cag caa gac ctg gtt aat gac 3111Gly Tyr Met
Ser Val His Gln Gln Gln Gln Gln Asp Leu Val Asn Asp 955 960 965gca
ctg aac gtc aat taa ttaaaaggaa agata atg agt agc gta gat att
3162Ala Leu Asn Val Asn Met Ser Ser Val Asp Ile970 975 980ctc gtt
ccc gac ctg cct gaa tcg gtt gca gat gcc aca gta gca acc 3210Leu Val
Pro Asp Leu Pro Glu Ser Val Ala Asp Ala Thr Val Ala Thr 985 990
995tgg cac aag aaa cca ggc gat gca gtc agc cgc gat gaa gtc atc
3255Trp His Lys Lys Pro Gly Asp Ala Val Ser Arg Asp Glu Val Ile
1000 1005 1010gtc gaa att gaa act gac aaa gtc gtg ctg gaa gtg ccg
gca tct 3300Val Glu Ile Glu Thr Asp Lys Val Val Leu Glu Val Pro Ala
Ser 1015 1020 1025gcc gat ggc gtg ctg gaa gcc gtg ctg gaa gac gaa
ggg gca acc 3345Ala Asp Gly Val Leu Glu Ala Val Leu Glu Asp Glu Gly
Ala Thr 1030 1035 1040gtt acg tcc cgc cag atc ctg ggt cgc ctg aaa
gaa ggc aac agt 3390Val Thr Ser Arg Gln Ile Leu Gly Arg Leu Lys Glu
Gly Asn Ser 1045 1050 1055gcg ggt aaa gaa agc agt gcc aaa gcg gaa
agc aat gac acc acg 3435Ala Gly Lys Glu Ser Ser Ala Lys Ala Glu Ser
Asn Asp Thr Thr 1060 1065 1070cca gcc cag cgt cag aca gcg tcg ctt
gaa gaa gag agc agc gat 3480Pro Ala Gln Arg Gln Thr Ala Ser Leu Glu
Glu Glu Ser Ser Asp 1075 1080 1085gcg ctc agc ccg gcg atc cgt cgc
ctg att gcg gag cat aat ctt 3525Ala Leu Ser Pro Ala Ile Arg Arg Leu
Ile Ala Glu His Asn Leu 1090 1095 1100gac gct gcg cag atc aaa ggc
acc ggc gta ggc gga cgt tta acg 3570Asp Ala Ala Gln Ile Lys Gly Thr
Gly Val Gly Gly Arg Leu Thr 1105 1110 1115cgt gaa gac gtt gaa aaa
cat ctg gcg aac aaa ccg cag gct gag 3615Arg Glu Asp Val Glu Lys His
Leu Ala Asn Lys Pro Gln Ala Glu 1120 1125 1130aaa gcc gcc gcg cca
gcg gcg ggt gca gca acg gct cag cag cct 3660Lys Ala Ala Ala Pro Ala
Ala Gly Ala Ala Thr Ala Gln Gln Pro 1135 1140 1145gtt gcc aac cgc
agc gaa aaa cgt gtt ccg atg acg cgt tta cgt 3705Val Ala Asn Arg Ser
Glu Lys Arg Val Pro Met Thr Arg Leu Arg 1150 1155 1160aag cgc gtc
gcg gag cgt ctg ctg gaa gcc aag aac agc acc gcc 3750Lys Arg Val Ala
Glu Arg Leu Leu Glu Ala Lys Asn Ser Thr Ala 1165 1170 1175atg ttg
acg acc ttc aac gaa atc aac atg aag ccg att atg gat 3795Met Leu Thr
Thr Phe Asn Glu Ile Asn Met Lys Pro Ile Met Asp 1180 1185 1190ctg
cgt aag cag tac ggc gat gcg ttc gag aag cgt cac ggt gtg 3840Leu Arg
Lys Gln Tyr Gly Asp Ala Phe Glu Lys Arg His Gly Val 1195 1200
1205cgt ctg ggc ttt atg tct ttc tac atc aag gcc gtg gtc gaa gcg
3885Arg Leu Gly Phe Met Ser Phe Tyr Ile Lys Ala Val Val Glu Ala
1210 1215 1220ctg aag cgt tat cca gaa gtc aac gcc tct atc gat ggc
gaa gac 3930Leu Lys Arg Tyr Pro Glu Val Asn Ala Ser Ile Asp Gly Glu
Asp 1225 1230 1235gtg gtg tac cac aac tat ttc gat gtg agt att gcc
gtc tct acg 3975Val Val Tyr His Asn Tyr Phe Asp Val Ser Ile Ala Val
Ser Thr 1240 1245 1250cca cgc gga ctg gtg acg cct gtc ctg cgt gac
gtt gat gcg ctg 4020Pro Arg Gly Leu Val Thr Pro Val Leu Arg Asp Val
Asp Ala Leu 1255 1260 1265agc atg gct gac atc gag aag aaa att aaa
gaa ctg gca gtg aaa 4065Ser Met Ala Asp Ile Glu Lys Lys Ile Lys Glu
Leu Ala Val Lys 1270 1275 1280ggc cgt gac ggc aag ctg acg gtt gac
gat ctg acg ggc ggt aac 4110Gly Arg Asp Gly Lys Leu Thr Val Asp Asp
Leu Thr Gly Gly Asn 1285 1290 1295ttt acc atc acc aac ggt ggt gtg
ttc ggt tcg ctg atg tct acg 4155Phe Thr Ile Thr Asn Gly Gly Val Phe
Gly Ser Leu Met Ser Thr 1300 1305 1310cca atc atc aac ccg cca cag
agc gcg att ctg ggc atg cac gcc 4200Pro Ile Ile Asn Pro Pro Gln Ser
Ala Ile Leu Gly Met His Ala 1315 1320 1325att aaa gat cgt cct atg
gcg gtc aat ggt cag gtt gtg atc ctg 4245Ile Lys Asp Arg Pro Met Ala
Val Asn Gly Gln Val Val Ile Leu 1330 1335 1340cca atg atg tac ctg
gct ctc tcc tac gat cac cgt tta atc gat 4290Pro Met Met Tyr Leu Ala
Leu Ser Tyr Asp His Arg Leu Ile Asp 1345 1350 1355ggt cgt gaa tct
gtc ggc tat ctg gtc gcg gtg aaa gag atg ctg 4335Gly Arg Glu Ser Val
Gly Tyr Leu Val Ala Val Lys Glu Met Leu 1360 1365 1370gaa gat ccg
gcg cgt ctg ctg ctg gat gtc tga ttcatcactg 4378Glu Asp Pro Ala Arg
Leu Leu Leu Asp Val 1375 1380ggcacgcgtt gcgtgcccaa tctcaatact
cttttcagat ctgaatggat agaacatc 4436atg aac tta cac gaa tac cag gct
aaa cag ctg ttt gca cgg tat 4481Met Asn Leu His Glu Tyr Gln Ala Lys
Gln Leu Phe Ala Arg Tyr 1385 1390 1395ggc atg cca gca ccg acc ggc
tac gcc tgt act aca cca cgt gaa 4526Gly Met Pro Ala Pro Thr Gly Tyr
Ala Cys Thr Thr Pro Arg Glu 1400 1405 1410gca gaa gaa gcg gca tcg
aaa atc ggt gca 4556Ala Glu Glu Ala Ala Ser Lys Ile Gly Ala 1415
1420239PRTEnterobacter agglomerans 2Ala Phe Ser Val Phe Arg Cys His
Ser Ile Met Asn Cys Val Ser Val1 5 10 15Cys Pro Lys Gly Leu Asn Pro
Thr Arg Ala Ile Gly His Ile Lys Ser 20 25 30Met Leu Leu Gln
Arg Ser Ala 353935PRTEnterobacter agglomerans 3Met Gln Asn Ser Ala
Met Lys Pro Trp Leu Asp Ser Ser Trp Leu Ala1 5 10 15Gly Ala Asn Gln
Ser Tyr Ile Glu Gln Leu Tyr Glu Asp Phe Leu Thr 20 25 30Asp Pro Asp
Ser Val Asp Ala Val Trp Arg Ser Met Phe Gln Gln Leu 35 40 45Pro Gly
Thr Gly Val Lys Pro Glu Gln Phe His Ser Ala Thr Arg Glu 50 55 60Tyr
Phe Arg Arg Leu Ala Lys Asp Ala Ser Arg Tyr Thr Ser Ser Val65 70 75
80Thr Asp Pro Ala Thr Asn Ser Lys Gln Val Lys Val Leu Gln Leu Ile
85 90 95Asn Ala Phe Arg Phe Arg Gly His Gln Glu Ala Asn Leu Asp Pro
Leu 100 105 110Gly Leu Trp Lys Gln Asp Arg Val Ala Asp Leu Asp Pro
Ala Phe His 115 120 125Asp Leu Thr Asp Ala Asp Phe Gln Glu Ser Phe
Asn Val Gly Ser Phe 130 135 140Ala Ile Gly Lys Glu Thr Met Lys Leu
Ala Asp Leu Phe Asp Ala Leu145 150 155 160Lys Gln Thr Tyr Cys Gly
Ser Ile Gly Ala Glu Tyr Met His Ile Asn 165 170 175Asn Thr Glu Glu
Lys Arg Trp Ile Gln Gln Arg Ile Glu Ser Gly Ala 180 185 190Ser Gln
Thr Ser Phe Ser Gly Glu Glu Lys Lys Gly Phe Leu Lys Glu 195 200
205Leu Thr Ala Ala Glu Gly Leu Glu Lys Tyr Leu Gly Ala Lys Phe Pro
210 215 220Gly Ala Lys Arg Phe Ser Leu Glu Gly Gly Asp Ala Leu Val
Pro Met225 230 235 240Leu Arg Glu Met Ile Arg His Ala Gly Lys Ser
Gly Thr Arg Glu Val 245 250 255Val Leu Gly Met Ala His Arg Gly Arg
Leu Asn Val Leu Ile Asn Val 260 265 270Leu Gly Lys Lys Pro Gln Asp
Leu Phe Asp Glu Phe Ser Gly Lys His 275 280 285Lys Glu His Leu Gly
Thr Gly Asp Val Lys Tyr His Met Gly Phe Ser 290 295 300Ser Asp Ile
Glu Thr Glu Gly Gly Leu Val His Leu Ala Leu Ala Phe305 310 315
320Asn Pro Ser His Leu Glu Ile Val Ser Pro Val Val Met Gly Ser Val
325 330 335Arg Ala Arg Leu Asp Arg Leu Ala Glu Pro Val Ser Asn Lys
Val Leu 340 345 350Pro Ile Thr Ile His Gly Asp Ala Ala Val Ile Gly
Gln Gly Val Val 355 360 365Gln Glu Thr Leu Asn Met Ser Gln Ala Arg
Gly Tyr Glu Val Gly Gly 370 375 380Thr Val Arg Ile Val Ile Asn Asn
Gln Val Gly Phe Thr Thr Ser Asn385 390 395 400Pro Lys Asp Ala Arg
Ser Thr Pro Tyr Cys Thr Asp Ile Gly Lys Met 405 410 415Val Leu Ala
Pro Ile Phe His Val Asn Ala Asp Asp Pro Glu Ala Val 420 425 430Ala
Phe Val Thr Arg Leu Ala Leu Asp Tyr Arg Asn Thr Phe Lys Arg 435 440
445Asp Val Phe Ile Asp Leu Val Cys Tyr Arg Arg His Gly His Asn Glu
450 455 460Ala Asp Glu Pro Ser Ala Thr Gln Pro Leu Met Tyr Gln Lys
Ile Lys465 470 475 480Lys His Pro Thr Pro Arg Lys Ile Tyr Ala Asp
Arg Leu Glu Gly Glu 485 490 495Gly Val Ala Ser Gln Glu Asp Ala Thr
Glu Met Val Asn Leu Tyr Arg 500 505 510Asp Ala Leu Asp Ala Gly Glu
Cys Val Val Pro Glu Trp Arg Pro Met 515 520 525Ser Leu His Ser Phe
Thr Trp Ser Pro Tyr Leu Asn His Glu Trp Asp 530 535 540Glu Pro Tyr
Pro Ala Gln Val Asp Met Lys Arg Leu Lys Glu Leu Ala545 550 555
560Leu Arg Ile Ser Gln Val Pro Glu Gln Ile Glu Val Gln Ser Arg Val
565 570 575Ala Lys Ile Tyr Asn Asp Arg Lys Leu Met Ala Glu Gly Glu
Lys Ala 580 585 590Phe Asp Trp Gly Gly Ala Glu Asn Leu Ala Tyr Ala
Thr Leu Val Asp 595 600 605Glu Gly Ile Pro Val Arg Leu Ser Gly Glu
Asp Ser Gly Arg Gly Thr 610 615 620Phe Phe His Arg His Ala Val Val
His Asn Gln Ala Asn Gly Ser Thr625 630 635 640Tyr Thr Pro Leu His
His Ile His Asn Ser Gln Gly Glu Phe Lys Val 645 650 655Trp Asp Ser
Val Leu Ser Glu Glu Ala Val Leu Ala Phe Glu Tyr Gly 660 665 670Tyr
Ala Thr Ala Glu Pro Arg Val Leu Thr Ile Trp Glu Ala Gln Phe 675 680
685Gly Asp Phe Ala Asn Gly Ala Gln Val Val Ile Asp Gln Phe Ile Ser
690 695 700Ser Gly Glu Gln Lys Trp Gly Arg Met Cys Gly Leu Val Met
Leu Leu705 710 715 720Pro His Gly Tyr Glu Gly Gln Gly Pro Glu His
Ser Ser Ala Arg Leu 725 730 735Glu Arg Tyr Leu Gln Leu Cys Ala Glu
Gln Asn Met Gln Val Cys Val 740 745 750Pro Ser Thr Pro Ala Gln Val
Tyr His Met Leu Arg Arg Gln Ala Leu 755 760 765Arg Gly Met Arg Arg
Pro Leu Val Val Met Ser Pro Lys Ser Leu Leu 770 775 780Arg His Pro
Leu Ala Ile Ser Ser Leu Asp Glu Leu Ala Asn Gly Ser785 790 795
800Phe Gln Pro Ala Ile Gly Glu Ile Asp Asp Leu Asp Pro Gln Gly Val
805 810 815Lys Arg Val Val Leu Cys Ser Gly Lys Val Tyr Tyr Asp Leu
Leu Glu 820 825 830Gln Arg Arg Lys Asp Glu Lys Thr Asp Val Ala Ile
Val Arg Ile Glu 835 840 845Gln Leu Tyr Pro Phe Pro His Gln Ala Val
Gln Glu Ala Leu Lys Ala 850 855 860Tyr Ser His Val Gln Asp Phe Val
Trp Cys Gln Glu Glu Pro Leu Asn865 870 875 880Gln Gly Ala Trp Tyr
Cys Ser Gln His His Phe Arg Asp Val Val Pro 885 890 895Phe Gly Ala
Thr Leu Arg Tyr Ala Gly Arg Pro Ala Ser Ala Ser Pro 900 905 910Ala
Val Gly Tyr Met Ser Val His Gln Gln Gln Gln Gln Asp Leu Val 915 920
925Asn Asp Ala Leu Asn Val Asn 930 9354407PRTEnterobacter
agglomerans 4Met Ser Ser Val Asp Ile Leu Val Pro Asp Leu Pro Glu
Ser Val Ala1 5 10 15Asp Ala Thr Val Ala Thr Trp His Lys Lys Pro Gly
Asp Ala Val Ser 20 25 30Arg Asp Glu Val Ile Val Glu Ile Glu Thr Asp
Lys Val Val Leu Glu 35 40 45Val Pro Ala Ser Ala Asp Gly Val Leu Glu
Ala Val Leu Glu Asp Glu 50 55 60Gly Ala Thr Val Thr Ser Arg Gln Ile
Leu Gly Arg Leu Lys Glu Gly65 70 75 80Asn Ser Ala Gly Lys Glu Ser
Ser Ala Lys Ala Glu Ser Asn Asp Thr 85 90 95Thr Pro Ala Gln Arg Gln
Thr Ala Ser Leu Glu Glu Glu Ser Ser Asp 100 105 110Ala Leu Ser Pro
Ala Ile Arg Arg Leu Ile Ala Glu His Asn Leu Asp 115 120 125Ala Ala
Gln Ile Lys Gly Thr Gly Val Gly Gly Arg Leu Thr Arg Glu 130 135
140Asp Val Glu Lys His Leu Ala Asn Lys Pro Gln Ala Glu Lys Ala
Ala145 150 155 160Ala Pro Ala Ala Gly Ala Ala Thr Ala Gln Gln Pro
Val Ala Asn Arg 165 170 175Ser Glu Lys Arg Val Pro Met Thr Arg Leu
Arg Lys Arg Val Ala Glu 180 185 190Arg Leu Leu Glu Ala Lys Asn Ser
Thr Ala Met Leu Thr Thr Phe Asn 195 200 205Glu Ile Asn Met Lys Pro
Ile Met Asp Leu Arg Lys Gln Tyr Gly Asp 210 215 220Ala Phe Glu Lys
Arg His Gly Val Arg Leu Gly Phe Met Ser Phe Tyr225 230 235 240Ile
Lys Ala Val Val Glu Ala Leu Lys Arg Tyr Pro Glu Val Asn Ala 245 250
255Ser Ile Asp Gly Glu Asp Val Val Tyr His Asn Tyr Phe Asp Val Ser
260 265 270Ile Ala Val Ser Thr Pro Arg Gly Leu Val Thr Pro Val Leu
Arg Asp 275 280 285Val Asp Ala Leu Ser Met Ala Asp Ile Glu Lys Lys
Ile Lys Glu Leu 290 295 300Ala Val Lys Gly Arg Asp Gly Lys Leu Thr
Val Asp Asp Leu Thr Gly305 310 315 320Gly Asn Phe Thr Ile Thr Asn
Gly Gly Val Phe Gly Ser Leu Met Ser 325 330 335Thr Pro Ile Ile Asn
Pro Pro Gln Ser Ala Ile Leu Gly Met His Ala 340 345 350Ile Lys Asp
Arg Pro Met Ala Val Asn Gly Gln Val Val Ile Leu Pro 355 360 365Met
Met Tyr Leu Ala Leu Ser Tyr Asp His Arg Leu Ile Asp Gly Arg 370 375
380Glu Ser Val Gly Tyr Leu Val Ala Val Lys Glu Met Leu Glu Asp
Pro385 390 395 400Ala Arg Leu Leu Leu Asp Val 405540PRTEnterobacter
agglomerans 5Met Asn Leu His Glu Tyr Gln Ala Lys Gln Leu Phe Ala
Arg Tyr Gly1 5 10 15Met Pro Ala Pro Thr Gly Tyr Ala Cys Thr Thr Pro
Arg Glu Ala Glu 20 25 30Glu Ala Ala Ser Lys Ile Gly Ala 35
40630DNAArtificial Sequencemisc_featureArtificial Sequence
synthetic DNA 6gtcgacaata gccygaatct gttctggtcg 30730DNAArtificial
Sequencemisc_featureArtificial Sequence synthetic DNA 7aagcttatcg
acgctcccct ccccaccgtt 308936PRTEscherichia coli 8Met Gln Asn Ser
Ala Leu Lys Ala Trp Leu Asp Ser Ser Tyr Leu Ser1 5 10 15Gly Ala Asn
Gln Ser Trp Glu Ile Glu Gln Leu Tyr Glu Asp Phe Leu 20 25 30Thr Asp
Pro Asp Ser Val Asp Ala Asn Trp Arg Ser Thr Phe Gln Gln 35 40 45Leu
Pro Gly Thr Gly Val Lys Pro Asp Gln Phe His Ser Gln Thr Arg 50 55
60Glu Tyr Phe Arg Arg Leu Ala Lys Asp Ala Ser Arg Tyr Ser Ser Thr65
70 75 80Ile Ser Asp Pro Asp Thr Asn Val Lys Gln Val Lys Val Leu Gln
Leu 85 90 95Ile Asn Ala Tyr Arg Phe Arg Gly His Gln His Ala Asn Leu
Asp Pro 100 105 110Leu Gly Leu Trp Gln Gln Asp Lys Val Ala Asp Leu
Asp Pro Ser Phe 115 120 125His Asp Leu Thr Glu Ala Asp Phe Gln Glu
Thr Phe Asn Val Gly Ser 130 135 140Phe Ala Ser Gly Lys Glu Thr Met
Lys Leu Gly Glu Leu Leu Glu Ala145 150 155 160Leu Lys Gln Thr Tyr
Cys Gly Pro Ile Gly Ala Glu Tyr Met His Ile 165 170 175Thr Ser Thr
Glu Glu Lys Arg Trp Ile Gln Gln Arg Ile Glu Ser Gly 180 185 190Arg
Ala Thr Phe Asn Ser Glu Glu Lys Lys Arg Phe Leu Ser Glu Leu 195 200
205Thr Ala Ala Glu Gly Leu Glu Arg Tyr Leu Gly Ala Lys Phe Pro Gly
210 215 220Ala Lys Arg Phe Ser Leu Glu Gly Gly Asp Ala Leu Ile Pro
Met Leu225 230 235 240Lys Glu Met Ile Arg His Ala Gly Asn Ser Gly
Thr Arg Glu Val Val 245 250 255Leu Gly Met Ala His Arg Gly Arg Leu
Asn Val Leu Asn Val Leu Gly 260 265 270Lys Lys Pro Gln Asp Leu Phe
Asp Glu Phe Ala Gly Lys His Lys Glu 275 280 285His Leu Gly Thr Gly
Asp Val Lys Tyr His Met Gly Phe Ser Ser Asp 290 295 300Phe Gln Thr
Asp Gly Gly Leu Val His Leu Ala Leu Ala Phe Asn Pro305 310 315
320Ser His Leu Glu Ile Val Ser Pro Val Val Ile Gly Ser Val Arg Ala
325 330 335Arg Leu Asp Arg Leu Asp Glu Pro Ser Ser Asn Lys Val Leu
Pro Ile 340 345 350Thr Ile His Gly Asp Ala Ala Val Thr Gly Gln Gly
Val Val Gln Glu 355 360 365Thr Leu Asn Met Ser Lys Ala Arg Gly Tyr
Glu Val Gly Gly Thr Val 370 375 380Arg Ile Val Ile Asn Asn Gln Val
Gly Phe Thr Thr Ser Asn Pro Leu385 390 395 400Asp Ala Arg Ser Thr
Pro Tyr Cys Thr Asp Ile Gly Lys Met Val Gln 405 410 415Ala Pro Ile
Phe His Val Asn Ala Asp Asp Pro Glu Ala Val Ala Phe 420 425 430Val
Thr Arg Leu Ala Leu Asp Phe Arg Asn Thr Phe Lys Arg Asp Val 435 440
445Phe Ile Asp Leu Val Ser Tyr Arg Arg His Gly His Asn Asn Glu Ala
450 455 460Asp Glu Pro Ser Ala Thr Gln Pro Leu Met Tyr Gln Lys Ile
Lys Lys465 470 475 480His Pro Thr Pro Arg Lys Ile Tyr Ala Asp Lys
Leu Glu Gln Glu Lys 485 490 495Val Ala Thr Leu Glu Asp Ala Thr Glu
Met Val Asn Leu Tyr Arg Asp 500 505 510Ala Leu Asp Ala Gly Asp Cys
Val Val Ala Glu Trp Arg Pro Met Asn 515 520 525Met His Ser Phe Thr
Trp Ser Pro Tyr Leu Asn His Glu Trp Asp Glu 530 535 540Glu Tyr Pro
Asn Lys Val Glu Met Lys Arg Leu Gln Glu Leu Ala Lys545 550 555
560Arg Ile Ser Thr Val Pro Glu Ala Val Glu Met Gln Ser Arg Val Ala
565 570 575Lys Ile Tyr Gly Asp Arg Gln Ala Met Ala Ala Gly Glu Lys
Leu Phe 580 585 590Asp Trp Gly Gly Ala Glu Asn Leu Ala Tyr Ala Thr
Leu Val Asp Glu 595 600 605Gly Ile Pro Val Arg Leu Ser Gly Glu Asp
Ser Gly Arg Gly Thr Phe 610 615 620Phe His Arg His Ala Val Ile His
Asn Gln Ser Asn Gly Ser Thr Tyr625 630 635 640Thr Pro Leu Gln His
Ile His Asn Gly Gln Gly Ala Phe Arg Val Trp 645 650 655Asp Ser Val
Leu Ser Glu Glu Ala Val Leu Ala Phe Glu Tyr Gly Tyr 660 665 670Ala
Thr Ala Glu Pro Arg Thr Leu Thr Ile Trp Glu Ala Gln Phe Gly 675 680
685Asp Phe Ala Asn Gly Ala Gln Val Val Ile Asp Gln Phe Ile Ser Ser
690 695 700Gly Glu Gln Lys Trp Gly Arg Met Cys Gly Leu Val Met Leu
Leu Pro705 710 715 720His Gly Tyr Glu Gly Gln Gly Pro Glu His Ser
Ser Ala Arg Leu Glu 725 730 735Arg Tyr Leu Gln Leu Cys Ala Glu Gln
Asn Asn Gln Val Cys Val Pro 740 745 750Ser Thr Pro Ala Gln Val Tyr
His Met Leu Arg Arg Gln Ala Leu Arg 755 760 765Gly Met Arg Arg Pro
Leu Val Val Met Ser Pro Lys Ser Leu Leu Arg 770 775 780His Pro Leu
Ala Val Ser Ser Leu Glu Glu Leu Ala Asn Gly Thr Phe785 790 795
800Leu Pro Ala Ile Gly Glu Glu Ile Asp Glu Leu Asp Pro Lys Gly Val
805 810 815Lys Arg Val Val Met Cys Ser Ser Gly Lys Val Tyr Tyr Asp
Leu Leu 820 825 830Glu Gln Arg Arg Lys Asn Asn Gln His Asp Val Ala
Ile Val Arg Ile 835 840 845Glu Gln Leu Tyr Pro Phe Pro His Lys Ala
Met Gln Glu Val Leu Gln 850 855 860Gln Phe Ala His Val Lys Asp Phe
Val Trp Cys Gln Glu Glu Pro Leu865 870 875 880Asn Gln Gly Ala Trp
Tyr Cys Ser Gln His His Phe Arg Glu Val Ile 885 890 895Pro Phe Gly
Ala Ser Leu Arg Tyr Ala Gly Arg Pro Ala Ser Ala Ser 900 905 910Pro
Ala Val Gly Tyr Met Ser Val His Gln Lys Gln Gln Gln Asp Leu 915 920
925Val Asn Asp Ala Leu Asn Val Glu 930 9359405PRTEscherichia coli
9Met Ser Ser Val Asp Ile Leu Val Pro Asp Leu Pro Glu Ser Val Ala1 5
10 15Asp Ala Thr Val Ala Thr Trp His Lys Lys Pro Gly Asp Ala Val
Val 20 25 30Arg Asp Glu Val Leu Val Glu Ile Glu Thr Asp Lys Val Val
Leu Glu 35 40 45Val Pro Ala Ser Ala Asp Gly Ile Leu Asp Ala Val Leu
Glu Asp Glu 50 55 60Gly Thr Thr Val Thr Ser Arg Gln Ile Leu Gly Arg
Leu Arg Glu Gly65 70 75 80Asn Ser Ala Gly Lys Glu Thr Ser Ala Lys
Ser Glu Glu Lys Ala Ser 85
90 95Thr Pro Ala Gln Arg Gln Gln Ala Ser Leu Glu Glu Gln Asn Asn
Asp 100 105 110Ala Leu Ser Pro Ala Ile Arg Arg Leu Leu Ala Glu His
Asn Leu Asp 115 120 125Ala Ser Ala Ile Lys Gly Thr Gly Val Gly Gly
Arg Leu Thr Arg Glu 130 135 140Asp Val Glu Lys His Leu Ala Lys Ala
Pro Ala Lys Glu Ser Ala Pro145 150 155 160Ala Ala Ala Ala Pro Ala
Ala Gln Pro Ala Leu Ala Ala Arg Ser Glu 165 170 175Lys Arg Val Pro
Met Thr Arg Leu Arg Lys Arg Val Ala Glu Arg Leu 180 185 190Leu Glu
Ala Lys Asn Ser Thr Ala Met Leu Thr Thr Phe Asn Glu Val 195 200
205Asn Met Lys Pro Ile Met Asp Leu Arg Lys Gln Tyr Gly Glu Ala Phe
210 215 220Glu Lys Arg His Gly Ile Arg Leu Gly Phe Met Ser Phe Tyr
Val Lys225 230 235 240Ala Val Val Glu Ala Leu Lys Arg Tyr Pro Glu
Val Asn Ala Ser Ile 245 250 255Asp Gly Asp Asp Val Val Tyr His Asn
Tyr Phe Asp Val Ser Met Ala 260 265 270Val Ser Thr Pro Arg Gly Leu
Val Thr Pro Val Leu Arg Asp Val Asp 275 280 285Thr Leu Gly Met Ala
Asp Ile Glu Lys Lys Ile Lys Glu Leu Ala Val 290 295 300Lys Gly Arg
Asp Gly Lys Leu Thr Val Glu Asp Leu Thr Gly Gly Asn305 310 315
320Phe Thr Ile Thr Asn Gly Gly Val Phe Gly Ser Leu Met Ser Thr Pro
325 330 335Ile Ile Asn Pro Pro Gln Ser Ala Ile Leu Gly Met His Ala
Ile Lys 340 345 350Asp Arg Pro Met Ala Val Asn Gly Gln Val Glu Ile
Leu Pro Met Met 355 360 365Tyr Leu Ala Leu Ser Tyr Asp His Arg Leu
Ile Asp Gly Arg Glu Ser 370 375 380Val Gly Phe Leu Val Thr Ile Lys
Glu Leu Leu Glu Asp Pro Thr Arg385 390 395 400Leu Leu Leu Asp Val
4051041PRTEnterobacter agglomerans 10Met Asn Leu His Glu Tyr Gln
Ala Lys Gln Leu Phe Ala Arg Tyr Gly1 5 10 15Met Pro Ala Pro Thr Gly
Tyr Ala Cys Thr Thr Pro Arg Glu Ala Glu 20 25 30Glu Ala Ala Ser Lys
Ile Gly Ala Gly 35 401160PRTEscherichia coli 11Met Asn Leu His Glu
Tyr Gln Ala Lys Gln Leu Phe Ala Arg Tyr Gly1 5 10 15Leu Pro Ala Pro
Val Gly Tyr Ala Cys Thr Thr Pro Arg Glu Ala Glu 20 25 30Glu Ala Ala
Ser Lys Ile Gly Ala Gly Pro Trp Val Val Lys Cys Gln 35 40 45Val His
Ala Gly Gly Arg Gly Lys Ala Gly Gly Val 50 55 601258PRTEscherichia
coli 12Phe Leu Ile Asp Ser Arg Asp Thr Glu Thr Asp Ser Arg Leu Asp
Gly1 5 10 15Leu Ser Asp Ala Phe Ser Val Phe Arg Cys His Ser Ile Met
Asn Cys 20 25 30Val Ser Val Cys Pro Lys Gly Leu Asn Pro Thr Arg Ala
Ile Gly His 35 40 45Ile Lys Ser Met Leu Leu Gln Arg Asn Ala 50
55
* * * * *