U.S. patent application number 11/785643 was filed with the patent office on 2010-05-20 for bacterium producing l-glutamic acid and method for producing l-glutamic acid.
This patent application is currently assigned to Ajinomoto Co., Inc.. Invention is credited to Hiroshi Izui, Tsuyoshi Nakamatsu, Jun Nakamura, Hiromi Ohtaki.
Application Number | 20100124777 11/785643 |
Document ID | / |
Family ID | 18701550 |
Filed Date | 2010-05-20 |
United States Patent
Application |
20100124777 |
Kind Code |
A1 |
Ohtaki; Hiromi ; et
al. |
May 20, 2010 |
Bacterium producing L-glutamic acid and method for producing
L-glutamic acid
Abstract
L-Glutamic acid is produced by culturing a coryneform bacterium
having L-glutamic acid producing ability, in which trehalose
synthesis ability is decreased or deleted by, for example,
disrupting a gene coding for trehalose-6-phosphate synthase, a gene
coding for maltooligosyltrehalose synthase, or both of these genes
to produce and accumulate L-glutamic acid in the medium, and
collecting the L-glutamic acid from the medium.
Inventors: |
Ohtaki; Hiromi;
(Kawasaki-shi, JP) ; Nakamura; Jun; (Kawasaki-shi,
JP) ; Izui; Hiroshi; (Kawasaki-shi, JP) ;
Nakamatsu; Tsuyoshi; (Kawasaki-shi, JP) |
Correspondence
Address: |
THE NATH LAW GROUP
112 South West Street
Alexandria
VA
22314
US
|
Assignee: |
Ajinomoto Co., Inc.
Tokyo
JP
|
Family ID: |
18701550 |
Appl. No.: |
11/785643 |
Filed: |
April 19, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10637551 |
Aug 11, 2003 |
7307160 |
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11785643 |
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09895382 |
Jul 2, 2001 |
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10637551 |
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Current U.S.
Class: |
435/252.3 ;
536/23.2 |
Current CPC
Class: |
C12P 13/14 20130101;
C12R 1/13 20130101; C12R 1/15 20130101; C12N 9/1051 20130101; C12N
9/90 20130101 |
Class at
Publication: |
435/252.3 ;
536/23.2 |
International
Class: |
C12N 1/21 20060101
C12N001/21; C07H 21/04 20060101 C07H021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 5, 2000 |
JP |
2000-204256 |
Claims
1-7. (canceled)
8. An isolated DNA coding for a protein defined in the following
(A) or (B): (A) a protein having the amino acid sequence of SEQ ID
NO: 32, (B) a protein having the amino acid sequence of SEQ ID NO:
32 including substitution, deletion, insertion or addition of 1-20
amino acid residues and having maltooligosyltrehalose synthase
activity.
9. The isolated DNA according to claim 8, which is a DNA defined in
the following (a) or (b): (a) a DNA comprising at least the
nucleotide residues 82-2514 in the nucleotide sequence of SEQ ID
NO: 31, or (b) a DNA which is hybridizable with a nucleotide
sequence complementary to the nucleotide sequence comprising at
least the nucleotide residues 82-2514 in the nucleotide sequence of
SEQ ID NO: 31 under a stringent condition, and which codes for a
protein having maltooligosyltrehalose synthase activity, wherein
the stringent condition is 1.times.SSC, 0.1% SDS, at 60.degree. C.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a novel L-glutamic acid
producing bacterium and a method for producing L-glutamic acid by
fermentation utilizing it. L-glutamic acid is an important amino
acid as foodstuffs, drugs and so forth.
[0003] 2. Description of the Related Art
[0004] Conventionally, L-glutamic acid is mainly produced by
fermentative methods using so-called L-glutamic acid producing
coryneform bacteria belonging to the genus Brevibacterium,
Corynebacterium or Microbacterium, or mutant strains thereof (Amino
Acid Fermentation, pp. 195-215, Gakkai Shuppan Center, 1986).
[0005] It is known that, in the production of L-glutamic acid by
fermentation, trehalose is also produced as a secondary product.
Therefore, techniques have been developed for decomposing or
metabolizing the produced trehalose. Such techniques include the
method of producing an amino acid by fermentation using a
coryneform bacterium in which proliferation ability on trehalose is
induced (Japanese Patent Laid-open (Kokai) No. 5-276935) and the
method of producing amino acid by fermentation using a coryneform
bacterium in which a gene coding for trehalose catabolic enzyme is
amplified (Korean Patent Publication (B1) No. 165836). However, it
is not known how to suppress the formation of trehalose itself in
an L-glutamic acid producing bacterium.
[0006] In Escherichia coli, the synthesis of trehalose is catalyzed
by trehalose-6-phosphate synthase. This enzyme is known to be
encoded by otsA gene. Further, it has been also reported that an
otsA gene-disrupted strain of Escherichia coli can scarcely grow in
a hyperosmotic medium (H. M. Glaever, et al., J. Bacteriol.,
170(6), 2841-2849 (1998)). However, the relationship between
disruption of otsA gene and production of substances has not been
known.
[0007] On the other hand, although the treY gene is known for
Brevibacterium helvolum among bacteria belonging to the genus
Brevibacterium bacteria, any otsA gene is not known for them. As
for bacteria belonging to the genus Mycobacterium bacteria, there
is known a pathway via a reaction catalyzed by a product encoded by
treS gene (trehalose synthase (TreS)), which gene is different from
the otsA gene and treY gene, as a gene coding for a enzyme in
trehalose biosynthesis pathway (De Smet K. A., et al.,
Microbiology, 146 (1), 199-208 (2000)). However, this pathway
utilizes maltose as a substrate and does not relate to usual
L-glutamic acid fermentation that utilizes glucose, fructose or
sucrose as a starting material.
SUMMARY OF THE INVENTION
[0008] An object of the present invention is to improve production
efficiency of L-glutamic acid in L-glutamic acid production by
fermentation using coryneform bacteria through suppression of the
production of trehalose as a secondary product.
[0009] The inventors of the present invention assiduously studied
in order to achieve the aforementioned object. As a result, they
found that bacterium belonging to the genus Brevibacterium
contained otsA gene and trey gene like Mycobacterium tuberculosis,
and the production efficiency of L-glutamic acid was improved by
disrupting at least one of these genes. Thus, they accomplished the
present invention.
[0010] That is, the present invention provides the followings.
[0011] (1) A coryneform bacterium having L-glutamic acid producing
ability, wherein trehalose synthesis ability is decreased or
deleted in the bacterium.
[0012] (2) The coryneform bacteria according to (1), wherein the
trehalose synthesis ability is decreased or deleted by introducing
a mutation into a chromosomal gene coding for an enzyme in a
trehalose synthesis pathway or disrupting the gene.
[0013] (3) The coryneform bacteria according to (2), wherein the
gene coding for the enzyme in trehalose synthesis pathway consists
of a gene coding for trehalose-6-phosphate synthase, a gene coding
for maltooligosyltrehalose synthase, or both of these genes.
[0014] (4) The coryneform bacteria according to (3), wherein the
gene coding for trehalose-6-phosphate synthase codes for the amino
acid sequence of SEQ ID NO: 30, and the gene coding for
maltooligosyltrehalose synthase codes for the amino acid sequence
of SEQ ID NO: 32.
[0015] (5) A method for producing L-glutamic acid comprising
culturing a coryneform bacterium according to any one of (1) to (4)
in a medium to produce and accumulate L-glutamic acid in the
medium, and collecting the L-glutamic acid from the medium.
[0016] (6) A DNA coding for a protein defined in the following (A)
or (B):
[0017] (A) a protein having the amino acid sequence of SEQ ID NO:
30,
[0018] (B) a protein having an amino acid sequence of SEQ ID NO: 30
including substitution, deletion, insertion or addition of one or
several amino acid residues and having trehalose-6-phosphate
synthase activity.
[0019] (7) A DNA according to (6), which is a DNA defined in the
following (a) or (b):
[0020] (a) a DNA containing a nucleotide sequence comprising at
least the residues of nucleotide numbers 484-1938 in the nucleotide
sequence of SEQ ID NO: 29,
[0021] (b) a DNA hybridizable with a nucleotide sequence comprising
at least the residues of nucleotide numbers 484-1938 in the
nucleotide sequence of SEQ ID NO: 29 under a stringent condition,
showing homology of 55% or more to the foregoing nucleotide
sequence, and coding for a protein having trehalose-6-phosphate
synthase activity.
[0022] (8) A DNA coding for a protein defined in the following (A)
or (B):
[0023] (A) a protein having the amino acid sequence of SEQ ID NO:
32,
[0024] (B) a protein having an amino acid sequence of SEQ ID NO: 32
including substitution, deletion, insertion or addition of one or
several amino acid residues and having maltooligosyltrehalose
synthase activity.
[0025] (9) A DNA according to (8), which is a DNA defined in the
following (a) or (b):
[0026] (a) a DNA containing a nucleotide sequence comprising at
least the residues of nucleotide numbers 82-2514 in the nucleotide
sequence of SEQ ID NO: 31,
[0027] (b) a DNA hybridizable with a nucleotide sequence comprising
at least the residues of nucleotide numbers 82-2514 in the
nucleotide sequence of SEQ ID NO: 31 under a stringent condition,
showing homology of 60% or more to the foregoing nucleotide
sequence, and coding for a protein having maltooligosyltrehalose
synthase activity.
[0028] The trehalose-6-phosphate synthase activity means an
activity to catalyze a reaction in which
.alpha.,.alpha.-trehalose-6-phosphate and UDP are produced from
UDP-glucose and glucose-6-phosphate, and the maltooligosyltrehalose
synthase activity means an activity to catalyze a reaction in which
maltotriosyltrehalose is produced from maltopentose.
[0029] According to the present invention, production efficiency of
L-glutamic acid in L-glutamic acid production by fermentation using
coryneform bacteria can be improved through inhibition of the
production of trehalose as a secondary product.
PREFERRED EMBODIMENTS OF THE INVENTION
[0030] Hereafter, the present invention will be explained in
detail.
[0031] The coryneform bacterium of the present invention is a
coryneform bacterium having L-glutamic acid producing ability, in
which trehalose synthesis ability is decreased or deleted.
[0032] The coryneform bacteria referred to in the present invention
include the group of microorganisms defined in Bergey's Manual of
Determinative Bacteriology, 8th edition, p. 599 (1974), which are
aerobic Gram-positive rods having no acid resistance and no
spore-forming ability aerobic. They have hitherto been classified
into the genus Brevibacterium, but united into the genus
Corynebacterium at present (Int. J. Syst. Bacteriol., 41, 255
(1981)), and include bacteria belonging to the genus Brevibacterium
or Microbacterium closely relative to the genus Corynebacterium.
Examples of such coryneform bacteria are mentioned below.
[0033] Corynebacterium acetoacidophilum
[0034] Corynebacterium acetoglutamicum
[0035] Corynebacterium alkanolyticum
[0036] Corynebacterium callunae
[0037] Corynebacterium glutamicum
[0038] Corynebacterium ilium (Corynebacterium glutamicum)
[0039] Corynebacterium melassecola
[0040] Corynebacterium thermoaminogenes
[0041] Corynebacterium herculis
[0042] Brevibacterium divaricatum (Corynebacterium glutamicum)
[0043] Brevibacterium flavum (Corynebacterium glutamicum)
[0044] Brevibacterium immariophilum
[0045] Brevibacterium lactofermentum (Corynebacterium
glutamicum)
[0046] Brevibacterium roseum
[0047] Brevibacterium saccharolyticum
[0048] Brevibacterium thiogenitalis
[0049] Brevibacterium ammoniagenes (Corynebacterium
ammoniagenes)
[0050] Brevibacterium album
[0051] Brevibacterium cerium
[0052] Microbacterium ammoniaphilum
[0053] Specifically, the following strains can be exemplified.
[0054] Corynebacterium acetoacidophilum ATCC 13870
[0055] Corynebacterium acetoglutamicum ATCC 15806
[0056] Corynebacterium alkanolyticum ATCC21511
[0057] Corynebacterium callunae ATCC 15991
[0058] Corynebacterium glutamicum ATCC 13020, 13032, 13060
[0059] Corynebacterium lilium (Corynebacterium glutamicum) ATCC
15990
[0060] Corynebacterium melassecola ATCC 17965
[0061] Corynebacterium thermoaminogenes AJ12340 (FERN BP-1539)
[0062] Corynebacterium herculis ATCC13868
[0063] Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC
14020
[0064] Brevibacterium flavum (Corynebacterium glutamicum) ATCC
13826, ATCC 14067
[0065] Brevibacterium immariophilum ATCC 14068
[0066] Brevibacterium lactofermentum (Corynebacterium glutamicum)
ATCC 13665, ATCC 13869
[0067] Brevibacterium roseum ATCC 13825
[0068] Brevibacterium saccharolyticum ATCC 14066
[0069] Brevibacterium thiogenitalis ATCC 19240
[0070] Brevibacterium ammoniagenes (Corynebacterium ammoniagenes)
ATCC 6871
[0071] Brevibacterium album ATCC 15111
[0072] Brevibacterium cerium ATCC 15112
[0073] Microbacterium ammoniaphilum ATCC 15354
[0074] The trehalose synthesis ability of such coryneform bacteria
as mentioned above can be decreased or deleted by mutagenizing or
disrupting a gene coding for an enzyme in trehalose synthesis
pathway using mutagenesis treatment or genetic recombination
technique. Such a mutation may be a mutation that suppresses
transcription or translation of the gene coding for the enzyme in
trehalose synthesis pathway, or a mutation that causes elimination
or decrease of an enzyme in trehalose synthesis pathway. The enzyme
in trehalose synthesis pathway may be exemplified by, for example,
trehalose-6-phosphate synthase, maltooligosyltrehalose synthases,
or both of these.
[0075] The disruption of a gene coding for an enzyme in trehalose
synthesis pathway can be performed by gene substitution utilizing
homologous recombination. A gene on a chromosome of a coryneform
bacterium can be disrupted by transforming the coryneform bacterium
with DNA containing a gene coding for an enzyme in trehalose
synthesis pathway modified so that a part thereof should be deleted
and hence the enzyme in trehalose synthesis pathway should not
normally function (deletion type gene), and allowing recombination
between the deletion type gene and a normal gene on the chromosome.
Such gene disruption by homologous recombination has already been
established. To this end, there can be mentioned a method utilizing
a linear DNA or a cyclic DNA that does not replicate in coryneform
bacteria and a method utilizing a plasmid containing a temperature
sensitive replication origin. However, a method utilizing a cyclic
DNA that does not replicate in coryneform bacteria or a plasmid
containing a temperature sensitive replication origin is
preferred.
[0076] The gene coding for an enzyme in trehalose synthesis pathway
may be exemplified by, for example, the otsA gene or treY gene, or
it may consist of both of these. Since the nucleotide sequences of
the otsA gene and treY gene of Brevibacterium lactofermentum and
flanking regions thereof have been elucidated by the present
invention, those genes can be easily obtained by preparing primers
based on the sequences and performing PCR (polymerase chain
reaction, see White, T. J. et al., Trends Genet., 5, 185 (1989))
using the primers and chromosomal DNA of Brevibacterium
lactofermentum as a template.
[0077] The nucleotide sequence comprising the otsA gene and the
nucleotide sequence comprising the treY gene of Brevibacterium
lactofermentum obtained in the examples described later are shown
in SEQ ID NOS: 29 and 31, respectively. Further, the amino acid
sequences encoded by these nucleotide sequences are shown in SEQ ID
NOS: 30 and 32, respectively.
[0078] The otsA gene and treY gene each may be one coding for a
protein including substitution, deletion, insertion or addition of
one or several amino acids at one or a plurality of positions,
provided that the activity of trehalose-6-phosphate synthase or
maltooligosyltrehalose synthase encoded thereby is not
deteriorated. While the number of "several" amino acids differs
depending on positions or types of amino acid residues in the
three-dimensional structure of the protein, it is preferably 1-40,
more preferably 1-20, further preferably 1-10.
[0079] A DNA coding for the substantially same protein as
trehalose-6-phosphate synthase or maltooligosyltrehalose synthase
described above can be obtained by, for example, modifying each of
the nucleotide sequences by, for example, the site-directed
mutagenesis method so that one or more amino acid residues at a
specified site should involve substitution, deletion, insertion,
addition or inversion. Such a DNA modified as described above may
also be obtained by a conventionally known mutation treatment. The
mutation treatment includes a method of treating DNA coding for
trehalose-6-phosphate synthase or maltooligosyltrehalose in vitro,
for example, with hydroxylamine, and a method for treating a
microorganism, for example, a bacterium belonging to the genus
Escherichia harboring a DNA coding for trehalose-6-phosphate
synthase or maltooligosyltrehalose with ultraviolet irradiation or
a mutating agent usually used for mutation treatment such as
N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and nitrous acid.
[0080] The substitution, deletion, insertion, addition, or
inversion of nucleotide as described above also includes a
naturally occurring mutant or variant on the basis of, for example,
individual difference or difference in species or genus of
microorganisms that harbor trehalose-6-phosphate synthase or
maltooligosyltrehalose.
[0081] A DNA coding for the substantially same protein as
trehalose-6-phosphate synthase or maltooligosyltrehalose synthase
described above can be obtained by expressing such a DNA having a
mutation as described above in a suitable and examining the
trehalose-6-phosphate synthase activity or maltooligosyltrehalose
synthase activity of the expression product.
[0082] A DNA coding for substantially the same protein as
trehalose-6-phosphate synthase can also be obtained by isolating a
DNA hybridizable with a DNA having, for example, a nucleotide
sequence corresponding to nucleotide numbers of 484-1938 of the
nucleotide sequence shown in SEQ ID NO: 29 or a probe that can be
prepared from the nucleotide sequence under a stringent condition,
showing homology of 55% or more, preferably 65% or more, more
preferably 75% or more, to the foregoing nucleotide sequence, and
having trehalose-6-phosphate synthase activity from a DNA coding
for trehalose-6-phosphate synthase having a mutation or from a cell
harboring it. Similarly, a DNA coding for substantially the same
protein as maltooligosyltrehalose synthase can also be obtained by
isolating a DNA hybridizable with a DNA having, for example, a
nucleotide sequence corresponding to nucleotide numbers of 82-2514
of the nucleotide sequence shown in SEQ ID NO: 31 or a probe that
can be prepared from the nucleotide sequence under a stringent
condition, showing homology of 60% or more, preferably 70% or more,
more preferably 80% or more, to the foregoing nucleotide sequence,
and having maltooligosyltrehalose synthase activity from a DNA
coding for maltooligosyltrehalose synthase having a mutation or
from a cell harboring it.
[0083] The "stringent condition" referred to herein is a condition
under which so-called specific hybrid is formed, and non-specific
hybrid is not formed. It is difficult to clearly express this
condition by using any numerical value. However, for example, the
stringent condition includes a condition under which DNA's having
high homology, for example, DNA's having homology of not less than
55%, preferably not less than 60%, are hybridized with each other,
and DNA's having homology lower than the above level are not
hybridized with each other. Alternatively, the stringent condition
is exemplified by a condition under which DNA's are hybridized with
each other at a salt concentration corresponding to an ordinary
condition of washing in Southern hybridization, i.e., 1.times.SSC,
0.1% SDS, preferably 0.1.times.SSC, 0.1% SDS, at 60.degree. C.
[0084] As the probe, a partial sequence of each gene can also be
used. Such a probe can be produced by PCR using oligonucleotides
produced based on the nucleotide sequence of each gene as primers
and a DNA fragment containing each gene as a template. When a DNA
fragment in a length of about 300 by is used as the probe, the
washing conditions for the hybridization may consists of 50.degree.
C., 2.times.SSC and 0.1% SDS.
[0085] Genes hybridizable under such conditions as described above
include those having a stop codon generated in a coding region of
the genes, and those having no activity due to mutation of active
center. However, such mutants can be easily removed by ligating
each of the genes with a commercially available expression vector,
and measuring trehalose-6-phosphate synthase activity or
maltooligosyltrehalose synthase activity.
[0086] When an otsA gene or treY gene is used for the disruption of
these genes on chromosomes of coryneform bacteria, the encoded
trehalose-6-phosphate synthase or maltooligosyltrehalose synthase
are not required to have their activities. Further, the otsA gene
or treY gene used for the gene disruption may be a gene derived
from another microorganism, so long as they can undergo homologous
recombination with these genes of coryneform bacteria. For example,
an otsA gene of bacterium belonging to the genus Escherichia or
Mycobacterium, treY gene of bacterium belonging to the genus
Arthrobacter, Brevibacterium helvolum, or bacterium belonging to
the genus Rhizobium can be mentioned.
[0087] A deletion type gene of the otsA gene or treY gene can be
prepared by excising a certain region with restriction enzyme(s)
from a DNA fragment containing one of these genes or a part of them
to delete at least a part of coding region or an expression
regulatory sequence such as promoter.
[0088] Further, a deletion type gene can also be obtained by
performing PCR using primers designed so that a part of gene should
be deleted. Furthermore, a deletion type gene may be one obtained
by single nucleotide mutation, for example, a frame shift
mutation.
[0089] Gene disruption of the otsA gene will be explained
hereafter. Gene disruption of the treY gene can be performed
similarly.
[0090] An otsA gene on a host chromosome can be replaced with a
deletion type otsA gene as follows. That is, a deletion type otsA
gene and a marker gene for resistance to a drug, such as kanamycin,
chloramphenicol, tetracycline and streptomycin, are inserted into a
plasmid that cannot autonomously replicate in coryneform bacteria
to prepare a recombinant DNA. A coryneform bacterium can be
transformed with the recombinant DNA, and the transformant strain
can be cultured in a medium containing the drug to obtain a
transformant strain in which the recombinant DNA was introduced
into chromosomal DNA. Alternatively, such a transformant strain can
be obtained by using a temperature sensitive plasmid as the
plasmid, and culturing the transformants at a temperature at which
the temperature sensitive plasmid cannot replicate.
[0091] In a strain in which the recombinant DNA is incorporated
into a chromosome as described above, the recombinant DNA causes
recombination with an otsA gene sequence that originally exists on
the chromosome, and two of fused genes comprising the chromosomal
otsA gene and the deletion type otsA gene are inserted into the
chromosome so that other portions of the recombinant DNA (vector
portion and drug resistance marker gene) should be interposed
between them.
[0092] Then, in order to leave only the deletion type otsA gene on
the chromosomal DNA, one copy of the otsA gene is eliminated from
the chromosomal DNA together with the vector portion (including the
drug resistance marker gene) by recombination of two of the otsA
genes. In that case, the normal otsA gene is left on the
chromosomal DNA and the deletion type otsA gene is excised, or
conversely, the deletion type otsA gene is left on the chromosomal
DNA and the normal otsA gene is excised. It can be confirmed which
type of the gene is left on the chromosomal DNA by investigating
structure of the otsA gene on the chromosome by PCR, hybridization
or the like.
[0093] The coryneform bacterium used for the present invention may
have enhanced activity of an enzyme that catalyzes the biosynthesis
of L-glutamic acid in addition to the deletion or decrease of
trehalose synthesis ability. Examples of the enzyme that catalyzes
the biosynthesis of L-glutamic acid include glutamate
dehydrogenase, glutamine synthetase, glutamate synthase, isocitrate
dehydrogenase, aconitate hydratase, citrate synthase, pyruvate
carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate
synthase, enolase, phosphoglyceromutase, phosphoglycerate kinase,
glyceraldehyde-3-phosphate dehydrogenase, triosephosphate
isomerase, fructose bisphosphate aldolase, phosphofructokinase,
glucose phosphate isomerase and so forth.
[0094] Further, in the coryneform bacterium used for the present
invention, an enzyme that catalyzes a reaction for generating a
compound other than L-glutamic acid by branching off from the
biosynthetic pathway of L-glutamic acid may be declined or made
deficient. Examples of such an enzyme include .alpha.-ketoglutarate
dehydrogenase, isocitrate lyase, phosphate acetyltransferase,
acetate kinase, acetohydroximate synthase, acetolactate synthase,
formate acetyltransferase, lactate dehydrogenase, L-glutamate
decarboxylase, 1-pyrroline dehydrogenase and so forth.
[0095] Furthermore, by introducing a temperature sensitive mutation
for a biotin activity inhibiting substance such as surface active
agents into a coryneform bacterium having L-glutamic acid producing
ability, the bacterium becomes to be able to produce L-glutamic
acid in a medium containing an excessive amount of biotin in the
absence of a biotin activity inhibiting substance (see WO96/06180).
As such a coryneform bacterium, the Brevibacterium lactofermentum
AJ13029 strain disclosed in WO96/06180 can be mentioned. The
AJ13029 strain was deposited at the National Institute of
Bioscience and Human-Technology, Agency of Industrial Science and
Technology (currently, the independent administrative corporation,
National Institute of Advanced Industrial Science and Technology,
International Patent Organism Depositary (Chuo Dai-6, 1-1 Higashi
1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, postal code: 305-5466) on
Sep. 2, 1994, and received an accession number of FERM P-14501.
Then, it was transferred to an international deposit under the
provisions of the Budapest Treaty on Aug. 1, 1995, and received an
accession number of FERM BP-5189.
[0096] When a coryneform bacterium having L-glutamic acid producing
ability, in which trehalose synthesis ability is decreased or
deleted, is cultured in a suitable medium, L-glutamic acid is
accumulated in the medium.
[0097] The medium used for producing L-glutamic acid is a usual
medium that contains a carbon source, a nitrogen source, inorganic
ions and other organic trace nutrients as required. As the carbon
source, it is possible to use sugars such as glucose, lactose,
galactose, fructose, sucrose, maltose, blackstrap molasses and
starch hydrolysate; alcohols such as ethanol and inositol; or
organic acids such as acetic acid, fumaric acid, citric acid and
succinic acid.
[0098] As the nitrogen source, there can be used inorganic ammonium
salts such as ammonium sulfate, ammonium nitrate, ammonium
chloride, ammonium phosphate and ammonium acetate, ammonia, organic
nitrogen such as peptone, meat extract, yeast extract, corn steep
liquor and soybean hydrolysate, ammonia gas, aqueous ammonia and so
forth.
[0099] As the inorganic ions (or sources thereof), added is a small
amount of potassium phosphate, magnesium sulfate, iron ions,
manganese ions and so forth. As for the organic trace nutrients, it
is desirable to add required substances such as vitamin B.sub.1,
yeast extract and so forth in a suitable amount as required.
[0100] The culture is preferably performed under an aerobic
condition performed by shaking, stirring for aeration or the like
for 16 to 72 hours. The culture temperature is controlled to be at
30.degree. C. to 45.degree. C., and pH is controlled to be 5 to 9
during the culture. For such adjustment of pH, inorganic or organic
acidic or alkaline substances, ammonia gas and so forth can be
used.
[0101] Collection of L-glutamic acid from fermentation broth can be
performed by, for example, methods utilizing ion exchange resins,
crystallization and so forth. Specifically, L-glutamic acid can be
adsorbed on an anion exchange resin and isolated from it, or
crystallized by neutralization.
EXAMPLES
[0102] Hereafter, the present invention will be explained more
specifically with reference to the following examples.
Example 1
Construction of otsA Gene-Disrupted Strain of Brevibacterium
lactofermentum
[0103] <1> Cloning of otsA Gene
[0104] Since otsA gene of Brevibacterium lactofermentum was not
known, it was obtained by utilizing a nucleotide sequence of otsA
gene of another microorganism for reference. The otsA genes of
Escherichia and Mycobacterium had been hitherto elucidated for
their entire nucleotide sequences (Kaasen I., et al., Gene, 145
(1), 9-15 (1994); De Smet K. A., et al., Microbiology, 146 (1),
199-208 (2000)). Therefore, referring to an amino acid sequence
deduced from these nucleotide sequences, DNA primers P1 (SEQ ID NO:
1) and P2 (SEQ ID NO: 2) for PCR were synthesized first. The DNA
primers P1 and P2 corresponded to the regions of the nucleotide
numbers of 1894-1913 and 2531-2549 of the nucleotide sequence of
the otsA gene of Escherichia coli (GenBank accession X69160),
respectively. They also corresponded to the regions of the
nucleotide numbers 40499-40518 and 41166-41184 of the otsA gene of
Mycobacterium tuberculosis (GenBank accession Z95390),
respectively.
[0105] Then, PCR was performed by using the primers P1 and P2 and
chromosomal DNA of Brevibacterium lactofermentum ATCC 13869 as a
template with a cycle consisting of reactions at 94.degree. C. for
0.5 minute, 50.degree. C. for 0.5 minute and 72.degree. C. for 4
minutes, which was repeated for 30 cycles. As a result, a
substantially single kind of amplified fragment of about 0.6 kbp
was obtained. This amplified fragment was cloned into a plasmid
vector pCR2.1 by using "Original TA Cloning Kit" produced by
Invitrogen to obtain pCotsA. Then, the nucleotide sequence of the
cloned fragment was determined.
[0106] Based on the nucleotide sequence of the partial fragment of
otsA gene obtained as described above, DNA primers P10 (SEQ ID NO:
8) and P12 (SEQ ID NO: 10) were newly synthesized, and unknown
regions flanking to the partial fragment was amplified by "inverse
PCR" (Triglia, T. et al., Nucleic Acids Res., 16, 81-86 (1988);
Ochman H., et al., Genetics, 120, 621-623 (1988)). The chromosomal
DNA of Brevibacterium lactofermentum ATCC 13869 was digested with a
restriction enzyme BamHI, BglII, ClaI, HindIII, KpnI, MluI, MunL,
SalI or XhoI, and self-ligated by using T4 DNA ligase (Takara
Shuzo). By using resultant DNA as a template and the DNA primers
P10 and P12, PCR was performed with a cycle consisting of reactions
at 94.degree. C. for 0.5 minute, 55.degree. C. for 1 minute and
72.degree. C. for 4 minutes, which was repeated for 30 cycles. As a
result, when ClaI or BglII was used as the restriction enzyme, an
amplified fragment of 4 kbp was obtained for each case. The
nucleotide sequences of these amplified fragments were directly
determined by using the DNA primers P5 to P9 (SEQ ID NOS: 3-7) and
P11 to P15 (SEQ ID NOS: 9-11). Thus, the entire nucleotide sequence
of otsA gene of Brevibacterium lactofermentum ATCC 13869 was
determined as shown in SEQ ID NO: 29. The amino acid sequence
encoded by this nucleotide sequence is shown in SEQ ID NOS: 29 and
30.
[0107] When homology of the sequence of the aforementioned otsA
gene was determined with respect to the otsA gene of Escherichia
coli (GenBank accession X69160) and the otsA gene of Mycobacterium
tuberculosis (GenBank accession Z95390), the nucleotide sequence
showed homologies of 46.3% and 55.9%, respectively, and the amino
acid sequence showed homologies of 30.9% and 51.7%, respectively.
The homologies were calculated by using software, "GENETIX-WIN"
(Software Development), based on the Lipman-Person method (Science,
227, 1435-1441 (1985)).
<2> Preparation of Plasmid for otsA Gene Disruption
[0108] In order to examine presence or absence of improvement
effect in L-glutamic acid productivity by disruption of a gene
coding for an enzyme in trehalose biosynthesis pathway in
coryneform bacteria, a plasmid for otsA gene disruption was
produced. A plasmid for otsA gene disruption was produced as
follows. PCR was performed by using the plasmid pCotsA previously
constructed in the cloning of the otsA gene as a template and the
primers P29 (SEQ ID NO: 33) and P30 (SEQ ID NO: 34) comprising ClaI
site with a cycle consisting of reactions at 94.degree. C. for 0.5
minute, 55.degree. C. for 0.5 minute and 72.degree. C. for 8
minutes, which was repeated for 30 cycles. The amplified fragment
was digested with ClaI, blunt-ended by using T4 DNA polymerase
(Takara Shuzo), and self-ligated by using T4 ligase (Takara Shuzo)
to construct a plasmid pCotsAC containing the otsA gene having a
frame shift mutation (1258-1300th nucleotides of SEQ ID NO: 29 were
deleted) at an approximately central part thereof.
<3> Preparation of otsA Gene-Disrupted Strain
[0109] By using the plasmid pCotsAC for gene disruption, a
L-glutamic acid producing bacterium, Breibacterium lactofermentum
ATCC 13869, was transformed by the electric pulse method, and
transformants were selected as to the ability to grow in CM2B
medium containing 20 mg/L of kanamycin. Because the plasmid pCotsAC
for otsA gene disruption did not have a replication origin that
could function in Brevibacterium lactofermentum, resultant
transformants obtained by using the plasmid suffered homologous
recombination occurred between the otsA genes on the chromosome of
Brevibacterium lactofermentum and the plasmid pCotsAC for gene
disruption. From the homologous recombinant strains obtained as
described above, strains in which the vector portion of the plasmid
pCotsAC for gene disruption was eliminated due to re-occurrence of
homologous recombination were selected based on acquired kanamycin
sensitivity as a marker.
[0110] From the strains obtained as described above, a strain
introduced with the desired frame shift mutation was selected.
Selection of such a strain was performed by PCR using chromosomal
DNA extracted from a strain that became kanamycin sensitive as a
template and the DNA primers P8 (SEQ ID NO: 14) and P13 (SEQ ID NO:
11) with a cycle consisting of reactions at 94.degree. C. for 0.5
minute, 55.degree. C. for 0.5 minute and 72.degree. C. for 1
minutes, which was repeated for 30 cycles, and sequencing of the
obtained amplified fragment using the DNA primer P8 to confirm
disfunction of the otsA gene due to introduction of frame shift
mutation. The strain obtained as described above was designated as
.DELTA.OA strain.
Example 2
Construction of treY Gene-Disrupted Strain
[0111] <1> Cloning of treY Gene
[0112] Since treY gene of Brevibacterium lactofermentum was not
known, it was obtained by using nucleotide sequences of treY genes
of the other microorganisms for reference. The nucleotide sequences
of treY genes were hitherto elucidated for the genera Arthrobacter,
Brevibacterium and Rhizobium (Maruta K., et al., Biochim. Biophys.
Acta, 1289 (1), 10-13 (1996); Genbank accession AF039919; Maruta
K., et al., Biosci. Biotechnol. Biochem., 60 (4), 717-720 (1996)).
Therefore, referring to an amino acid sequence deduced from these
nucleotide sequences, the PCR DNA primers P3 (SEQ ID NO: 14) and P4
(SEQ ID NO: 15) were synthesized first. The DNA primers P3 and P4
correspond to the regions of the nucleotide numbers of 975-992 and
2565-2584 of the nucleotide sequence of the treY gene of
Arthrobacter species (GenBank accession D63343), respectively.
Further, they correspond to the regions of the nucleotide numbers
893-910 and 2486-2505 of the treY gene of Brevibacterium helvolum
(GenBank accession AF039919), respectively. Furthermore, they
correspond to the regions of the nucleotide numbers of 862-879 and
2452-2471 of treY gene of Rhizobium species (GenBank accession
D78001).
[0113] Then, PCR was performed by using the primers P3 and P4 and
chromosomal DNA of Brevibacterium lactofermentum ATCC13869 as a
template with a cycle consisting of reactions at 94.degree. C. for
0.5 minute, 55.degree. C. for 0.5 minute and 72.degree. C. for 2
minutes, which was repeated for 30 cycles. As a result, a
substantially single kind of an amplified fragment of about 1.6 kbp
was obtained. This amplified fragment was cloned into a plasmid
vector pCR2.1 by using "Original TA Cloning Kit" produced by
Invitrogen. Then, the nucleotide sequence was determined for about
0.6 kb.
[0114] Based on the nucleotide sequence of the partial fragment of
treY gene obtained as described above, the DNA primers P16 (SEQ ID
NO: 16) and P26 (SEQ ID NO: 26) were newly synthesized, and unknown
regions flanking to the partial fragment was amplified by "inverse
PCR" (Triglia, T. et al., Nucleic Acids Res., 16, 81-86 (1988);
Ochman H., et al., Genetics, 120, 621-623 (1988)). The chromosomal
DNA of Brevibacterium lactofermentum ATCC 13869 was digested with a
restriction enzyme BamHI, HindIII, SalI or XhoI, and self-ligated
by using T4 DNA ligase (Takara Shuzo). By using this as a template
and the DNA primers P16 and P26, PCR was performed with a cycle
consisting of reactions at 94.degree. C. for 0.5 minute, 55.degree.
C. for 1 minute and 72.degree. C. for 4 minutes, which was repeated
for 30 cycles. As a result, when HindIII or SalI was used as the
restriction enzyme, an amplified fragment of 0.6 kbp or 1.5 kbp was
obtained, respectively. The nucleotide sequences of these amplified
fragments were directly determined by using the DNA primers P16 to
P28 (SEQ ID NOS: 16-28). Thus, the entire nucleotide sequence of
treY gene of Brevibacterium lactofermentum ATCC 13869 was
determined as shown in SEQ ID NO: 31. The amino acid sequence
encoded by this nucleotide sequence is shown in SEQ ID NOS: 31 and
32.
[0115] When homology of the sequence of the aforementioned treY
gene was determined with respect to the treY gene of Arthrobacter
sp. (GenBank accession D63343), treY gene of Brevibacterium
helvolum (GenBank accession AF039919) and treY gene of Rhizobium
sp. (GenBank accession D78001), the nucleotide sequence showed
homologies of 52.0%, 52.3% and 51.9%, respectively, and the amino
acid sequence showed homologies of 40.9%, 38.5% and 39.8%,
respectively. The homologies were calculated by using software,
"GENETIX-WIN" (Software Development), based on the Lipman-Person
method (Science, 227, 1435-1441 (1985)).
<2> Preparation of Plasmid for treY Gene Disruption
[0116] In order to examine presence or absence of improvement
effect in L-glutamic acid productivity by disruption of the gene
coding for the enzyme in trehalose biosynthesis pathway in
coryneform bacteria, a plasmid for treY gene disruption was
produced. First, PCR was performed by using the primers P17 (SEQ ID
NO: 17) and P25 (SEQ ID NO: 25) and the chromosomal DNA of ATCC
13869 as a template with a cycle consisting of reactions at
94.degree. C. for 0.5 minute, 60.degree. C. for 0.5 minute and
72.degree. C. for 2 minutes, which was repeated for 30 cycles. The
amplified fragment was digested with EcoRI and ligated to pHSG299
(Takara Shuzo) digested with EcoRI by using T4 DNA ligase (Takara
Shuzo) to obtain a plasmid pHtreY. Further, this pHtreY was
digested with AflII (Takara Shuzo), blunt-ended by using T4 DNA
polymerase (Takara Shuzo), and self-ligated by using T4 ligase
(Takara Shuzo) to construct a plasmid pHtreYA containing the treY
gene having a frame shift mutation (four nucleotides were inserted
after the 1145th nucleotide in the sequence of SEQ ID NO: 31) at an
approximately central part thereof.
<3> Preparation of treY Gene-Disrupted Strain
[0117] By using the plasmid pCtreYA for gene disruption, a
L-glutamic acid producing bacterium, Brevibacterium lactofermentum
ATCC 13869, was transformed by the electric pulse method, and
transformants were selected as to the ability to grow in CM2B
medium containing 20 mg/L of kanamycin. Because the plasmid pCtreYA
for treY gene disruption does not have a replication origin that
could function in Brevibacterium lactofermentum, the transformants
obtained by using the plasmid suffered recombination occurred
between the treY genes on the Brevibacterium lactofermentum
chromosome and the plasmid pCtreYA for gene disruption. From the
homologous recombinant strains obtained as described above, strains
in which the vector portion of the plasmid pCtreYA for gene
disruption was eliminated due to re-occurrence of homologous
recombination were selected based on acquired kanamycin sensitivity
as a marker.
[0118] From the strains obtained as described above, a strain
introduced with the desired frame shift mutation was selected.
Selection of such a strain was performed by PCR using the DNA
primers P19 (SEQ ID NO: 19) and P25 (SEQ ID NO: 25) with a cycle
consisting of reactions at 94.degree. C. for 0.5 minute, 55.degree.
C. for 0.5 minute and 72.degree. C. for 1.5 minutes, which was
repeated for 30 cycles, and sequencing the obtained fragment using
the DNA primer P21 or P23 to confirm dysfunction of the treY gene
due to introduction of frame shift mutation. The strain obtained as
described above was designated as ATA strain.
Example 3
Evaluation of L-Glutamic Acid Producing Ability of .DELTA.OA Strain
and ATA Strain
[0119] The ATCC 13869 strain, .DELTA.OA strain and .DELTA.TA strain
were each cultured for producing L-glutamic acid as follows. Each
of these strains was refreshed by culturing it on a CM2B plate
medium, and each refreshed strain was cultured in a medium
containing 80 g of glucose, 1 g of KH.sub.2PO.sub.4, 0.4 g of
MgSO.sub.4, 30 g of (NH.sub.4).sub.2SO.sub.4, 0.01 g of
FeSO.sub.4.7H.sub.2O, 0.01 g MnSO.sub.4.7H.sub.2O, 15 ml of soybean
hydrolysate solution, 200 .mu.g of thiamin hydrochloride, 3 .mu.g
of biotin and 50 g of CaCO.sub.3 in 1 L of pure water (adjusted to
pH 8.0 with KOH) at 31.5.degree. C. After the culture, amount of
L-glutamic acid accumulated in the medium and absorbance at 620 nm
of the culture broth diluted 51 times were measured. The results
are shown in Table 1.
[0120] The Brevibacterium lactofermentum strains of which otsA gene
or treY gene was disrupted showed growth in a degree similar to
that of the parent strain, and in addition, increased L-glutamic
acid production compared with the parent strain.
TABLE-US-00001 TABLE 1 Strain OD.sub.620 (x51) L-Glutamic acid
(g/L) Yield (%) ATCC 13869 0.930 40.2 48.4 .DELTA.OA 1.063 43.8
52.8 .DELTA.TA 0.850 45.6 54.9
(Explanation of Sequence Listing)
[0121] SEQ. ID NO: 1: Primer P1 for amplification of otsA SEQ ID
NO: 2: Primer P2 for amplification of otsA
SEQ ID NO: 3: Primer P5
SEQ ID NO: 4: Primer P6
SEQ ID NO: 5: Primer P7
SEQ ID NO: 6: Primer P8
SEQ ID NO: 7: Primer P9
SEQ ID NO: 8: Primer P10
SEQ ID NO: 9: Primer P11
SEQ ID NO: 10: Primer P12
SEQ ID NO: 11: Primer P13
SEQ ID NO: 12: Primer P14
SEQ ID NO: 13: Primer P15
[0122] SEQ ID NO: 14: Primer P3 for amplification of treY SEQ ID
NO: 15: Primer P4 for amplification of trey
SEQ ID NO: 16: Primer P16
SEQ ID NO: 17: Primer P17
SEQ ID NO: 18: Primer P18
SEQ ID NO: 19: Primer P19
SEQ ID NO: 20: Primer P20
SEQ ID NO: 21: Primer P21
SEQ ID NO: 22: Primer P22
SEQ ID NO: 23: Primer P23
SEQ ID NO: 24: Primer P24
SEQ ID NO: 25: Primer P25
SEQ ID NO: 26: Primer P26
SEQ ID NO: 27: Primer P27
SEQ ID NO: 28: Primer P28
[0123] SEQ ID NO: 29: Nucleotide sequence of otsA gene SEQ ID NO:
30: Amino acid sequence of OtsA SEQ ID NO: 31: Nucleotide sequence
of treY gene SEQ ID NO: 32: Amino acid sequence of TreY
SEQ ID NO: 33: Primer P29
[0124] SEQ ID NO: 34: Primer P30
Sequence CWU 1
1
34120DNAArtificialprimer 1canathggnt tyttyytnca
20219DNAArtificialprimer 2canarrttca tnccrtcnc
19323DNAArtificialprimer 3gaatcatcca tataagatcc ggc
23424DNAArtificialprimer 4tagctttgta gttgttgcta accg
24524DNAArtificialprimer 5agcgaacttg aggtttactt cccg
24624DNAArtificialprimer 6tgctggttcc tggcattttg cgcc
24720DNAArtificialprimer 7tcgaacaatc tcttcacgcc
20821DNAArtificialprimer 8gaatcccacc aaatctgcgc c
21920DNAArtificialprimer 9tgatgttgaa atgtttgggg
201020DNAArtificialprimer 10gatgtcatgc tggttacgcc
201122DNAArtificialprimer 11caaagcacca gtgccgtcgc gg
221224DNAArtificialprimer 12tgttcgtttt cattcgcgtt gccg
241324DNAArtificialprimer 13atagtttcct ggattgtttg gcgc
241418DNAArtificialprimer 14caraayccnt ggtggtgg
181520DNAArtificialprimer 15ggncgncgrt trtcnggrtc
201620DNAArtificialprimer 16cgagctcttc attgatggcg
201720DNAArtificialprimer 17gcagctacac acgagttggg
201820DNAArtificialprimer 18gcaacaccta aatggttggg
201920DNAArtificialprimer 19gcaagaagtc tacaagcgcc
202016DNAArtificialprimer 20gccaacgtat tcacgg
162120DNAArtificialprimer 21tgatgaacca ctcgatcccc
202220DNAArtificialprimer 22aagacaccac cttctaccgc
202320DNAArtificialprimer 23caagtggaat tctgcagcgg
202421DNAArtificialprimer 24cctcctacaa aacctgctgg g
212520DNAArtificialprimer 25tcgcggatag cttttagggc
202620DNAArtificialprimer 26tgagttttta gaagactccc
202720DNAArtificialprimer 27cgcttcagtg gtgttgtccc
202824DNAArtificialprimer 28cgtaccactc cacggaaatt cccg
24292369DNABrevibacterium lactofermentumCDS(484)..(1938)
29acagaatcag cgccggcaga gaaacgtcca aagactaatc agagattcgg tataaaggta
60aaaatcaacc tgcttaggcg tctttcgctt aaatagcgta gaatatcggg tcgatcgctt
120ttaaacactc aggaggatcc ttgccggcca aaatcacgga cactcgtccc
accccagaat 180cccttcacgc tgttgaagag gaaaccgcag ccggtgcccg
caggattgtt gccacctatt 240ctaaggactt cttcgacggc gtcactttga
tgtgcatgct cggcgttgaa cctcagggcc 300tgcgttacac caaggtcgct
tctgaacacg aggaagctca gccaaagaag gctacaaagc 360ggactcgtaa
ggctaccagc taagaaggct gctgctaaga aaacgaccaa gaagaccact
420aagaaaacta ctaaaaagac caccgcaaag aagaccacaa agaagtctta
agccggatct 480tat atg gat gat tcc aat agc ttt gta gtt gtt gct aac
cgt ctg cca 528 Met Asp Asp Ser Asn Ser Phe Val Val Val Ala Asn Arg
Leu Pro 1 5 10 15gtg gat atg act gtc cac cca gat ggt agc tat agc
atc tcc ccc agc 576Val Asp Met Thr Val His Pro Asp Gly Ser Tyr Ser
Ile Ser Pro Ser 20 25 30ccc ggt ggc ctt gtc acg ggg ctt tcc ccc gtt
ctg gaa caa cat cgt 624Pro Gly Gly Leu Val Thr Gly Leu Ser Pro Val
Leu Glu Gln His Arg 35 40 45gga tgt tgg gtc gga tgg cct gga act gta
gat gtt gca ccc gaa cca 672Gly Cys Trp Val Gly Trp Pro Gly Thr Val
Asp Val Ala Pro Glu Pro 50 55 60ttt cga aca gat acg ggt gtt ttg ctg
cac cct gtt gtc ctc act gca 720Phe Arg Thr Asp Thr Gly Val Leu Leu
His Pro Val Val Leu Thr Ala 65 70 75agt gac tat gaa ggc ttc tac gag
ggc ttt tca aac gca acg ctg tgg 768Ser Asp Tyr Glu Gly Phe Tyr Glu
Gly Phe Ser Asn Ala Thr Leu Trp 80 85 90 95cct ctt ttc cac gat ctg
att gtt act ccg gtg tac aac acc gat tgg 816Pro Leu Phe His Asp Leu
Ile Val Thr Pro Val Tyr Asn Thr Asp Trp 100 105 110tgg cat gcg ttt
cgg gaa gta aac ctc aag ttc gct gaa gcc gtg agc 864Trp His Ala Phe
Arg Glu Val Asn Leu Lys Phe Ala Glu Ala Val Ser 115 120 125caa gtg
gcg gca cac ggt gcc act gtg tgg gtg cag gac tat cag ctg 912Gln Val
Ala Ala His Gly Ala Thr Val Trp Val Gln Asp Tyr Gln Leu 130 135
140ttg ctg gtt cct ggc att ttg cgc cag atg cgc ctt gat ttg aag atc
960Leu Leu Val Pro Gly Ile Leu Arg Gln Met Arg Leu Asp Leu Lys Ile
145 150 155ggt ttc ttc ctc cac att ccc ttc cct tcc cct gat ctg ttc
cgt cag 1008Gly Phe Phe Leu His Ile Pro Phe Pro Ser Pro Asp Leu Phe
Arg Gln160 165 170 175ctg ccg tgg cgt gaa gag att gtt cga ggc atg
ctg ggc gca gat ttg 1056Leu Pro Trp Arg Glu Glu Ile Val Arg Gly Met
Leu Gly Ala Asp Leu 180 185 190gtg gga ttc cat ttg gtt caa aac gca
gaa aac ttc ctt gcg tta acc 1104Val Gly Phe His Leu Val Gln Asn Ala
Glu Asn Phe Leu Ala Leu Thr 195 200 205cag cag gtt gcc ggc act gcc
ggg tct cat gtg ggt cag ccg gac acc 1152Gln Gln Val Ala Gly Thr Ala
Gly Ser His Val Gly Gln Pro Asp Thr 210 215 220ttg cag gtc agt ggt
gaa gca ttg gtg cgt gag att ggc gct cat gtt 1200Leu Gln Val Ser Gly
Glu Ala Leu Val Arg Glu Ile Gly Ala His Val 225 230 235gaa acc gct
gac gga agg cga gtt agc gtc ggg gcg ttc ccg atc tcg 1248Glu Thr Ala
Asp Gly Arg Arg Val Ser Val Gly Ala Phe Pro Ile Ser240 245 250
255att gat gtt gaa atg ttt ggg gag gcg tcg aaa agc gcc gtt ctt gat
1296Ile Asp Val Glu Met Phe Gly Glu Ala Ser Lys Ser Ala Val Leu Asp
260 265 270ctt tta aaa acg ctc gac gag ccg gaa acc gta ttc ctg ggc
gtt gac 1344Leu Leu Lys Thr Leu Asp Glu Pro Glu Thr Val Phe Leu Gly
Val Asp 275 280 285cga ctg gac tac acc aag ggc att ttg cag cgc ctg
ctt gcg ttt gag 1392Arg Leu Asp Tyr Thr Lys Gly Ile Leu Gln Arg Leu
Leu Ala Phe Glu 290 295 300gaa ctg ctg gaa tcc ggc gcg ttg gag gcc
gac aaa gct gtg ttg ctg 1440Glu Leu Leu Glu Ser Gly Ala Leu Glu Ala
Asp Lys Ala Val Leu Leu 305 310 315cag gtc gcg acg cct tcg cgt gag
cgc att gat cac tat cgt gtg tcg 1488Gln Val Ala Thr Pro Ser Arg Glu
Arg Ile Asp His Tyr Arg Val Ser320 325 330 335cgt tcg cag gtc gag
gaa gcc gtc ggc cgt atc aat ggt cgt ttc ggt 1536Arg Ser Gln Val Glu
Glu Ala Val Gly Arg Ile Asn Gly Arg Phe Gly 340 345 350cgc atg ggg
cgt ccc gtg gtg cat tat cta cac agg tca ttg agc aaa 1584Arg Met Gly
Arg Pro Val Val His Tyr Leu His Arg Ser Leu Ser Lys 355 360 365aat
gat ctc cag gtg ctg tat acc gca gcc gat gtc atg ctg gtt acg 1632Asn
Asp Leu Gln Val Leu Tyr Thr Ala Ala Asp Val Met Leu Val Thr 370 375
380cct ttt aaa gac ggt atg aac ttg gtg gct aaa gaa ttc gtg gcc aac
1680Pro Phe Lys Asp Gly Met Asn Leu Val Ala Lys Glu Phe Val Ala Asn
385 390 395cac cgc gac ggc act ggt gct ttg gtg ctg tcc gaa ttt gcc
ggc gcg 1728His Arg Asp Gly Thr Gly Ala Leu Val Leu Ser Glu Phe Ala
Gly Ala400 405 410 415gcc act gag ctg acc ggt gcg tat tta tgc aac
cca ttt gat gtg gaa 1776Ala Thr Glu Leu Thr Gly Ala Tyr Leu Cys Asn
Pro Phe Asp Val Glu 420 425 430tcc atc aaa cgg caa atg gtg gca gct
gtc cat gat ttg aag cac aat 1824Ser Ile Lys Arg Gln Met Val Ala Ala
Val His Asp Leu Lys His Asn 435 440 445ccg gaa tct gcg gca acg cga
atg aaa acg aac agc gag cag gtc tat 1872Pro Glu Ser Ala Ala Thr Arg
Met Lys Thr Asn Ser Glu Gln Val Tyr 450 455 460acc cac gac gtc aac
gtg tgg gct aat agt ttc ctg gat tgt ttg gcg 1920Thr His Asp Val Asn
Val Trp Ala Asn Ser Phe Leu Asp Cys Leu Ala 465 470 475cag tcg gga
gaa aac tca tgaaccgcgc acgaatcgcg accataggcg 1968Gln Ser Gly Glu
Asn Ser480 485ttcttccgct tgctttactg ctggcgtcct gtggttcaga
caccgtggaa atgacagatt 2028ccacctggtt ggtgaccaat atttacaccg
atccagatga gtcgaattcg atcagtaatc 2088ttgtcatttc ccagcccagc
ttagattttg gcaattcttc cctgtctggt ttcactggct 2148gtgtgccttt
tacggggcgt gcggaattct tccaaaatgg tgagcaaagc tctgttctgg
2208atgccgatta tgtgaccttg tcttccctgg atttcgataa acttcccgat
gattgccaag 2268gacaagaact caaagttcat aacgagctgg ttgatcttct
gcctggttct tttgaaatct 2328ccaggacttc tggttcagaa atcttgctga
ctagcgatgt c 236930485PRTBrevibacterium lactofermentum 30Met Asp
Asp Ser Asn Ser Phe Val Val Val Ala Asn Arg Leu Pro Val 1 5 10
15Asp Met Thr Val His Pro Asp Gly Ser Tyr Ser Ile Ser Pro Ser Pro
20 25 30Gly Gly Leu Val Thr Gly Leu Ser Pro Val Leu Glu Gln His Arg
Gly 35 40 45Cys Trp Val Gly Trp Pro Gly Thr Val Asp Val Ala Pro Glu
Pro Phe 50 55 60Arg Thr Asp Thr Gly Val Leu Leu His Pro Val Val Leu
Thr Ala Ser 65 70 75 80Asp Tyr Glu Gly Phe Tyr Glu Gly Phe Ser Asn
Ala Thr Leu Trp Pro 85 90 95Leu Phe His Asp Leu Ile Val Thr Pro Val
Tyr Asn Thr Asp Trp Trp 100 105 110His Ala Phe Arg Glu Val Asn Leu
Lys Phe Ala Glu Ala Val Ser Gln 115 120 125Val Ala Ala His Gly Ala
Thr Val Trp Val Gln Asp Tyr Gln Leu Leu 130 135 140Leu Val Pro Gly
Ile Leu Arg Gln Met Arg Leu Asp Leu Lys Ile Gly145 150 155 160Phe
Phe Leu His Ile Pro Phe Pro Ser Pro Asp Leu Phe Arg Gln Leu 165 170
175Pro Trp Arg Glu Glu Ile Val Arg Gly Met Leu Gly Ala Asp Leu Val
180 185 190Gly Phe His Leu Val Gln Asn Ala Glu Asn Phe Leu Ala Leu
Thr Gln 195 200 205Gln Val Ala Gly Thr Ala Gly Ser His Val Gly Gln
Pro Asp Thr Leu 210 215 220Gln Val Ser Gly Glu Ala Leu Val Arg Glu
Ile Gly Ala His Val Glu225 230 235 240Thr Ala Asp Gly Arg Arg Val
Ser Val Gly Ala Phe Pro Ile Ser Ile 245 250 255Asp Val Glu Met Phe
Gly Glu Ala Ser Lys Ser Ala Val Leu Asp Leu 260 265 270Leu Lys Thr
Leu Asp Glu Pro Glu Thr Val Phe Leu Gly Val Asp Arg 275 280 285Leu
Asp Tyr Thr Lys Gly Ile Leu Gln Arg Leu Leu Ala Phe Glu Glu 290 295
300Leu Leu Glu Ser Gly Ala Leu Glu Ala Asp Lys Ala Val Leu Leu
Gln305 310 315 320Val Ala Thr Pro Ser Arg Glu Arg Ile Asp His Tyr
Arg Val Ser Arg 325 330 335Ser Gln Val Glu Glu Ala Val Gly Arg Ile
Asn Gly Arg Phe Gly Arg 340 345 350Met Gly Arg Pro Val Val His Tyr
Leu His Arg Ser Leu Ser Lys Asn 355 360 365Asp Leu Gln Val Leu Tyr
Thr Ala Ala Asp Val Met Leu Val Thr Pro 370 375 380Phe Lys Asp Gly
Met Asn Leu Val Ala Lys Glu Phe Val Ala Asn His385 390 395 400Arg
Asp Gly Thr Gly Ala Leu Val Leu Ser Glu Phe Ala Gly Ala Ala 405 410
415Thr Glu Leu Thr Gly Ala Tyr Leu Cys Asn Pro Phe Asp Val Glu Ser
420 425 430Ile Lys Arg Gln Met Val Ala Ala Val His Asp Leu Lys His
Asn Pro 435 440 445Glu Ser Ala Ala Thr Arg Met Lys Thr Asn Ser Glu
Gln Val Tyr Thr 450 455 460His Asp Val Asn Val Trp Ala Asn Ser Phe
Leu Asp Cys Leu Ala Gln465 470 475 480Ser Gly Glu Asn Ser
485312956DNABrevibacterium
lactofermentumCDS(82)..(2514)misc_feature(2953)n=a or g or c or t
31ttttcccacg cagggaaggc gtgaacacta agatcgagga cgtaccgcac gattttgcct
60aacttttaag ggtgtttcat c atg gca cgt cca att tcc gca acg tac agg
111 Met Ala Arg Pro Ile Ser Ala Thr Tyr Arg 1 5 10ctt caa atg cga
gga cct caa gca gat agc gcc ggg cgt ttc ttt ggt 159Leu Gln Met Arg
Gly Pro Gln Ala Asp Ser Ala Gly Arg Phe Phe Gly 15 20 25ttt gcg cag
gcc aaa gcc cag ctt ccc tat ctg aag aag cta ggc atc 207Phe Ala Gln
Ala Lys Ala Gln Leu Pro Tyr Leu Lys Lys Leu Gly Ile 30 35 40agc cac
ctg tac ctc tcc cct att ttt acg gcc atg cca gat tcc aat 255Ser His
Leu Tyr Leu Ser Pro Ile Phe Thr Ala Met Pro Asp Ser Asn 45 50 55cat
ggc tac gat gtc att gat ccc acc gcc atc aat gaa gag ctc ggt 303His
Gly Tyr Asp Val Ile Asp Pro Thr Ala Ile Asn Glu Glu Leu Gly 60 65
70ggc atg gag ggt ctt cga gat ctt gct gca gct aca cac gag ttg ggc
351Gly Met Glu Gly Leu Arg Asp Leu Ala Ala Ala Thr His Glu Leu Gly
75 80 85 90atg ggc atc atc att gat att gtt ccc aac cat tta ggt gtt
gcc gtt 399Met Gly Ile Ile Ile Asp Ile Val Pro Asn His Leu Gly Val
Ala Val 95 100 105cca cat ttg aat cct tgg tgg tgg gat gtt cta aaa
aac ggc aaa gat 447Pro His Leu Asn Pro Trp Trp Trp Asp Val Leu Lys
Asn Gly Lys Asp 110 115 120tcc gct ttt gag ttc tat ttc gat att gac
tgg cac gaa gac aac ggt 495Ser Ala Phe Glu Phe Tyr Phe Asp Ile Asp
Trp His Glu Asp Asn Gly 125 130 135tct ggt ggc aag ctg ggc atg ccg
att ctg ggt gct gaa ggc gat gaa 543Ser Gly Gly Lys Leu Gly Met Pro
Ile Leu Gly Ala Glu Gly Asp Glu 140 145 150gac aag ctg gaa ttc gcg
gag ctt gat gga gag aaa gtg ctc aaa tat 591Asp Lys Leu Glu Phe Ala
Glu Leu Asp Gly Glu Lys Val Leu Lys Tyr155 160 165 170ttt gac cac
ctc ttc cca atc gcg cct ggt acc gaa gaa ggg aca ccg 639Phe Asp His
Leu Phe Pro Ile Ala Pro Gly Thr Glu Glu Gly Thr Pro 175 180 185caa
gaa gtc tac aag cgc cag cat tac cgc ctg cag ttc tgg cgc gac 687Gln
Glu Val Tyr Lys Arg Gln His Tyr Arg Leu Gln Phe Trp Arg Asp 190 195
200ggc gtg atc aac ttc cgt cgc ttc ttt tcc gtg aat acg ttg gct ggc
735Gly Val Ile Asn Phe Arg Arg Phe Phe Ser Val Asn Thr Leu Ala Gly
205 210 215atc agg caa gaa gat ccc ttg gtg ttt gaa cat act cat cgt
ctg ctg 783Ile Arg Gln Glu Asp Pro Leu Val Phe Glu His Thr His Arg
Leu Leu 220 225 230cgc gaa ttg gtg gcg gaa gac ctc att gac ggc gtg
cgc gtc gat cac 831Arg Glu Leu Val Ala Glu Asp Leu Ile Asp Gly Val
Arg Val Asp His235 240 245 250ccc gac ggg ctt tcc gat cct ttt gga
tat ctg cac aga ctc cgc gac 879Pro Asp Gly Leu Ser Asp Pro Phe Gly
Tyr Leu His Arg Leu Arg Asp 255 260 265ctc att gga cct gac cgc tgg
ctg atc atc gaa aag atc ttg agc gtt 927Leu Ile Gly Pro Asp Arg Trp
Leu Ile Ile Glu Lys Ile Leu Ser Val 270 275 280gat gaa cca ctc gat
ccc cgc ctg gcc gtt gat ggc acc act ggc tac 975Asp Glu Pro Leu Asp
Pro Arg Leu Ala Val Asp Gly Thr Thr Gly Tyr 285 290 295gac ccc ctc
cgt gaa ctc gac ggc gtg ttt atc tcc cga gaa tct gag 1023Asp Pro Leu
Arg Glu Leu Asp Gly Val Phe Ile Ser Arg Glu Ser Glu 300 305 310gac
aaa ttc tcc atg ttg gcg ctg acc cac agt gga tcc acc tgg gat 1071Asp
Lys Phe Ser Met Leu Ala Leu Thr His Ser Gly Ser Thr Trp Asp315 320
325 330gaa cgc gcc cta aaa tcc acg gag gaa agc ctc aaa cga gtc gtc
gcg 1119Glu Arg Ala Leu Lys Ser Thr Glu Glu Ser Leu Lys Arg Val Val
Ala 335 340 345caa caa gaa ctc gca gcc gaa atc tta agg ctc gcc cgc
gcc atg cgc 1167Gln Gln Glu Leu Ala Ala Glu Ile Leu Arg Leu Ala Arg
Ala Met Arg 350 355 360cgc gat aac ttc tcc acc gca ggc acc aac gtc
acc gaa gac aaa ctt 1215Arg Asp Asn Phe Ser Thr Ala Gly Thr Asn Val
Thr Glu Asp Lys Leu 365 370 375agc gaa acc atc atc gaa tta gtc gcc
gcc atg ccc gtc tac cgc gcc 1263Ser Glu Thr Ile Ile Glu Leu Val Ala
Ala Met Pro Val Tyr Arg Ala 380 385
390gac tac atc tcc ctc tca cgc acc acc gcc acc gtc atc gcg gag atg
1311Asp Tyr Ile Ser Leu Ser Arg Thr Thr Ala Thr Val Ile Ala Glu
Met395 400 405 410tcc aaa cgc ttc ccc tcc cgg cgc gac gca ctc gac
ctc atc tcg gcc 1359Ser Lys Arg Phe Pro Ser Arg Arg Asp Ala Leu Asp
Leu Ile Ser Ala 415 420 425gcc cta ctt ggc aat ggc gag gcc aaa atc
cgc ttc gcc caa gtc tgc 1407Ala Leu Leu Gly Asn Gly Glu Ala Lys Ile
Arg Phe Ala Gln Val Cys 430 435 440ggc gcc gtc atg gcc aaa ggt gtg
gaa gac acc acc ttc tac cgc gca 1455Gly Ala Val Met Ala Lys Gly Val
Glu Asp Thr Thr Phe Tyr Arg Ala 445 450 455tct agg ctc gtt gca ctg
caa gaa gtc ggt ggc gcg ccg ggc agg ttc 1503Ser Arg Leu Val Ala Leu
Gln Glu Val Gly Gly Ala Pro Gly Arg Phe 460 465 470ggc gtc tcc gct
gca gaa ttc cac ttg ctg cag gaa gaa cgc agc ctg 1551Gly Val Ser Ala
Ala Glu Phe His Leu Leu Gln Glu Glu Arg Ser Leu475 480 485 490ctg
tgg cca cgc acc atg acc acc ttg tcc acg cac gac acc aaa cgc 1599Leu
Trp Pro Arg Thr Met Thr Thr Leu Ser Thr His Asp Thr Lys Arg 495 500
505ggc gaa gat acc cgc gcc cgc atc atc tcc ctg tcc gaa gtc ccc gat
1647Gly Glu Asp Thr Arg Ala Arg Ile Ile Ser Leu Ser Glu Val Pro Asp
510 515 520atg tac tcc gag ctg gtc aat cgt gtt ttc gca gtg ctc ccc
gcg cca 1695Met Tyr Ser Glu Leu Val Asn Arg Val Phe Ala Val Leu Pro
Ala Pro 525 530 535gac ggc gca acg ggc agt ttc ctc cta caa aac ctg
ctg ggc gta tgg 1743Asp Gly Ala Thr Gly Ser Phe Leu Leu Gln Asn Leu
Leu Gly Val Trp 540 545 550ccc gcc gac ggc gtg atc acc gat gcg ctg
cgc gat cga ttc agg gaa 1791Pro Ala Asp Gly Val Ile Thr Asp Ala Leu
Arg Asp Arg Phe Arg Glu555 560 565 570tac gcc cta aaa gct atc cgc
gaa gca tcc aca aaa acc acg tgg gtg 1839Tyr Ala Leu Lys Ala Ile Arg
Glu Ala Ser Thr Lys Thr Thr Trp Val 575 580 585gac ccc aac gag tcc
ttc gag gct gcg gtc tgc gat tgg gtg gaa gcg 1887Asp Pro Asn Glu Ser
Phe Glu Ala Ala Val Cys Asp Trp Val Glu Ala 590 595 600ctt ttc gac
gga ccc tcc acc tca tta atc acc gaa ttt gtc tcc cac 1935Leu Phe Asp
Gly Pro Ser Thr Ser Leu Ile Thr Glu Phe Val Ser His 605 610 615atc
aac cgt ggc tct gtg aat atc tcc tta ggt agg aaa ctg ctg caa 1983Ile
Asn Arg Gly Ser Val Asn Ile Ser Leu Gly Arg Lys Leu Leu Gln 620 625
630atg gtg ggc gct gga atc ccc gac act tac caa gga act gag ttt tta
2031Met Val Gly Ala Gly Ile Pro Asp Thr Tyr Gln Gly Thr Glu Phe
Leu635 640 645 650gaa gac tcc ctg gta gat ccc gat aac cga cgc ttt
gtt gat tac acc 2079Glu Asp Ser Leu Val Asp Pro Asp Asn Arg Arg Phe
Val Asp Tyr Thr 655 660 665gcc aga gaa caa gtc ctg gag cgc ctg caa
acc tgg gat tgg acg cag 2127Ala Arg Glu Gln Val Leu Glu Arg Leu Gln
Thr Trp Asp Trp Thr Gln 670 675 680gtt aat tcg gta gaa gac ttg gtg
gat aac gcc gac atc gcc aaa atg 2175Val Asn Ser Val Glu Asp Leu Val
Asp Asn Ala Asp Ile Ala Lys Met 685 690 695gcc gtg gtc cat aaa tcc
ctc gag ttg cgt gct gaa ttt cgt gca agc 2223Ala Val Val His Lys Ser
Leu Glu Leu Arg Ala Glu Phe Arg Ala Ser 700 705 710ttt gtt ggt gga
gat cat cag gca gta ttt ggc gaa ggt cgc gca gaa 2271Phe Val Gly Gly
Asp His Gln Ala Val Phe Gly Glu Gly Arg Ala Glu715 720 725 730tcc
cac atc atg ggc atc gcc cgc ggt aca gac cga aac cac ctc aac 2319Ser
His Ile Met Gly Ile Ala Arg Gly Thr Asp Arg Asn His Leu Asn 735 740
745atc att gct ctt gct acc cgt cga cca ctg atc ttg gaa gac cgt ggc
2367Ile Ile Ala Leu Ala Thr Arg Arg Pro Leu Ile Leu Glu Asp Arg Gly
750 755 760gga tgg tat gac acc acc gtc acg ctt cct ggt gga caa tgg
gaa gac 2415Gly Trp Tyr Asp Thr Thr Val Thr Leu Pro Gly Gly Gln Trp
Glu Asp 765 770 775agg ctc acc ggg caa cgc ttc agt ggt gtt gtc cca
gcc acc gat ttg 2463Arg Leu Thr Gly Gln Arg Phe Ser Gly Val Val Pro
Ala Thr Asp Leu 780 785 790ttc tca cat tta ccc gta tct ttg ttg gtt
tta gta ccc gat agt gag 2511Phe Ser His Leu Pro Val Ser Leu Leu Val
Leu Val Pro Asp Ser Glu795 800 805 810ttt tgatccctgc acaggaaagt
tagcggcgct actatgaacg atcgatatgt 2564Phectgacaacac tctctcccaa
tttggcagtt actaccacga attccgacgt gcccatccca 2624tggccgacgt
cgaattcctc ctagcaattg aagaattact cacagacggt ggtgtcacct
2684tcgatcgcgt caccacacgc atcaaagaat ggtcaagcct gaaagccaag
gctcgcaagc 2744gtcgcgacga tggctcgttg atctaccctg atccgcgcaa
agacatccac gacatgatcg 2804gtgttcggat caccacgtac cactccacgg
aaattcccgt ggccttaaaa gtgctccaag 2864actccttcat cgtccacaaa
tccgtagaca aagccgctga aactcgcatc tcaggcggct 2924ttggttacgg
ctcccaccac caaggattnt ag 295632811PRTBrevibacterium lactofermentum
32Met Ala Arg Pro Ile Ser Ala Thr Tyr Arg Leu Gln Met Arg Gly Pro 1
5 10 15Gln Ala Asp Ser Ala Gly Arg Phe Phe Gly Phe Ala Gln Ala Lys
Ala 20 25 30Gln Leu Pro Tyr Leu Lys Lys Leu Gly Ile Ser His Leu Tyr
Leu Ser 35 40 45Pro Ile Phe Thr Ala Met Pro Asp Ser Asn His Gly Tyr
Asp Val Ile 50 55 60Asp Pro Thr Ala Ile Asn Glu Glu Leu Gly Gly Met
Glu Gly Leu Arg 65 70 75 80Asp Leu Ala Ala Ala Thr His Glu Leu Gly
Met Gly Ile Ile Ile Asp 85 90 95Ile Val Pro Asn His Leu Gly Val Ala
Val Pro His Leu Asn Pro Trp 100 105 110Trp Trp Asp Val Leu Lys Asn
Gly Lys Asp Ser Ala Phe Glu Phe Tyr 115 120 125Phe Asp Ile Asp Trp
His Glu Asp Asn Gly Ser Gly Gly Lys Leu Gly 130 135 140Met Pro Ile
Leu Gly Ala Glu Gly Asp Glu Asp Lys Leu Glu Phe Ala145 150 155
160Glu Leu Asp Gly Glu Lys Val Leu Lys Tyr Phe Asp His Leu Phe Pro
165 170 175Ile Ala Pro Gly Thr Glu Glu Gly Thr Pro Gln Glu Val Tyr
Lys Arg 180 185 190Gln His Tyr Arg Leu Gln Phe Trp Arg Asp Gly Val
Ile Asn Phe Arg 195 200 205Arg Phe Phe Ser Val Asn Thr Leu Ala Gly
Ile Arg Gln Glu Asp Pro 210 215 220Leu Val Phe Glu His Thr His Arg
Leu Leu Arg Glu Leu Val Ala Glu225 230 235 240Asp Leu Ile Asp Gly
Val Arg Val Asp His Pro Asp Gly Leu Ser Asp 245 250 255Pro Phe Gly
Tyr Leu His Arg Leu Arg Asp Leu Ile Gly Pro Asp Arg 260 265 270Trp
Leu Ile Ile Glu Lys Ile Leu Ser Val Asp Glu Pro Leu Asp Pro 275 280
285Arg Leu Ala Val Asp Gly Thr Thr Gly Tyr Asp Pro Leu Arg Glu Leu
290 295 300Asp Gly Val Phe Ile Ser Arg Glu Ser Glu Asp Lys Phe Ser
Met Leu305 310 315 320Ala Leu Thr His Ser Gly Ser Thr Trp Asp Glu
Arg Ala Leu Lys Ser 325 330 335Thr Glu Glu Ser Leu Lys Arg Val Val
Ala Gln Gln Glu Leu Ala Ala 340 345 350Glu Ile Leu Arg Leu Ala Arg
Ala Met Arg Arg Asp Asn Phe Ser Thr 355 360 365Ala Gly Thr Asn Val
Thr Glu Asp Lys Leu Ser Glu Thr Ile Ile Glu 370 375 380Leu Val Ala
Ala Met Pro Val Tyr Arg Ala Asp Tyr Ile Ser Leu Ser385 390 395
400Arg Thr Thr Ala Thr Val Ile Ala Glu Met Ser Lys Arg Phe Pro Ser
405 410 415Arg Arg Asp Ala Leu Asp Leu Ile Ser Ala Ala Leu Leu Gly
Asn Gly 420 425 430Glu Ala Lys Ile Arg Phe Ala Gln Val Cys Gly Ala
Val Met Ala Lys 435 440 445Gly Val Glu Asp Thr Thr Phe Tyr Arg Ala
Ser Arg Leu Val Ala Leu 450 455 460Gln Glu Val Gly Gly Ala Pro Gly
Arg Phe Gly Val Ser Ala Ala Glu465 470 475 480Phe His Leu Leu Gln
Glu Glu Arg Ser Leu Leu Trp Pro Arg Thr Met 485 490 495Thr Thr Leu
Ser Thr His Asp Thr Lys Arg Gly Glu Asp Thr Arg Ala 500 505 510Arg
Ile Ile Ser Leu Ser Glu Val Pro Asp Met Tyr Ser Glu Leu Val 515 520
525Asn Arg Val Phe Ala Val Leu Pro Ala Pro Asp Gly Ala Thr Gly Ser
530 535 540Phe Leu Leu Gln Asn Leu Leu Gly Val Trp Pro Ala Asp Gly
Val Ile545 550 555 560Thr Asp Ala Leu Arg Asp Arg Phe Arg Glu Tyr
Ala Leu Lys Ala Ile 565 570 575Arg Glu Ala Ser Thr Lys Thr Thr Trp
Val Asp Pro Asn Glu Ser Phe 580 585 590Glu Ala Ala Val Cys Asp Trp
Val Glu Ala Leu Phe Asp Gly Pro Ser 595 600 605Thr Ser Leu Ile Thr
Glu Phe Val Ser His Ile Asn Arg Gly Ser Val 610 615 620Asn Ile Ser
Leu Gly Arg Lys Leu Leu Gln Met Val Gly Ala Gly Ile625 630 635
640Pro Asp Thr Tyr Gln Gly Thr Glu Phe Leu Glu Asp Ser Leu Val Asp
645 650 655Pro Asp Asn Arg Arg Phe Val Asp Tyr Thr Ala Arg Glu Gln
Val Leu 660 665 670Glu Arg Leu Gln Thr Trp Asp Trp Thr Gln Val Asn
Ser Val Glu Asp 675 680 685Leu Val Asp Asn Ala Asp Ile Ala Lys Met
Ala Val Val His Lys Ser 690 695 700Leu Glu Leu Arg Ala Glu Phe Arg
Ala Ser Phe Val Gly Gly Asp His705 710 715 720Gln Ala Val Phe Gly
Glu Gly Arg Ala Glu Ser His Ile Met Gly Ile 725 730 735Ala Arg Gly
Thr Asp Arg Asn His Leu Asn Ile Ile Ala Leu Ala Thr 740 745 750Arg
Arg Pro Leu Ile Leu Glu Asp Arg Gly Gly Trp Tyr Asp Thr Thr 755 760
765Val Thr Leu Pro Gly Gly Gln Trp Glu Asp Arg Leu Thr Gly Gln Arg
770 775 780Phe Ser Gly Val Val Pro Ala Thr Asp Leu Phe Ser His Leu
Pro Val785 790 795 800Ser Leu Leu Val Leu Val Pro Asp Ser Glu Phe
805 8103330DNAArtificialprimer 33ccaaaatcga taacatcaat cgagatcggg
303430DNAArtificialprimer 34cttgatcgat taaaaacgct cgacgagccg 30
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