U.S. patent application number 12/635323 was filed with the patent office on 2010-04-15 for amino acid producing microorganism and a method for producing an amino acid.
Invention is credited to Kazuhiko Matsui, Masaru Terashita, Yoshihiro Usuda.
Application Number | 20100093044 12/635323 |
Document ID | / |
Family ID | 40432274 |
Filed Date | 2010-04-15 |
United States Patent
Application |
20100093044 |
Kind Code |
A1 |
Terashita; Masaru ; et
al. |
April 15, 2010 |
AMINO ACID PRODUCING MICROORGANISM AND A METHOD FOR PRODUCING AN
AMINO ACID
Abstract
A microorganism is provided which has an ability to produce an
L-amino acid such as L-lysine, L-tryptophan, L-phenylalanine,
L-valine, L-leucine, L-isoleucine and L-serine, and has been
modified to increase the activity of pyruvate synthase or
pyruvate:NADP.sup.+ oxidoreductase. This microorganism is cultured
in a medium containing ethanol or an aliphatic acid as the carbon
source to produce and accumulate the L-amino acid in the medium or
cells, and the L-amino acid is collected from the medium or the
cells.
Inventors: |
Terashita; Masaru;
(Kawasaki-shi, JP) ; Usuda; Yoshihiro;
(Kawasaki-shi, JP) ; Matsui; Kazuhiko;
(Kawasaki-shi, JP) |
Correspondence
Address: |
CERMAK KENEALY VAIDYA & NAKAJIMA LLP;ACS LLC
515 EAST BRADDOCK ROAD, SUITE B
ALEXANDRIA
VA
22314
US
|
Family ID: |
40432274 |
Appl. No.: |
12/635323 |
Filed: |
December 10, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12202476 |
Sep 2, 2008 |
|
|
|
12635323 |
|
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|
|
Current U.S.
Class: |
435/108 ;
435/115; 435/116 |
Current CPC
Class: |
C12P 13/227 20130101;
C12P 13/06 20130101; C12P 13/08 20130101; C12P 13/222 20130101;
C12N 9/0008 20130101 |
Class at
Publication: |
435/108 ;
435/115; 435/116 |
International
Class: |
C12P 13/22 20060101
C12P013/22; C12P 13/08 20060101 C12P013/08; C12P 13/06 20060101
C12P013/06 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 4, 2007 |
JP |
2007-228733 |
Claims
1. A method for producing an L-amino acid selected from the group
consisting of L-lysine, L-tryptophan, L-phenylalanine, L-valine,
L-leucine, L-isoleucine, and L-serine comprising: A) culturing in a
medium a microorganism which has an ability to produce the L-amino
acid, and B) collecting the L-amino acid from the medium or the
microorganism, wherein said microorganism has been modified to
increase an activity of NADH+ oxidoreductase by a method selected
from the group consisting of: i) increasing the copy number of a
gene encoding NADH+ oxidoreductase, ii) modifying an expression
control sequence of the gene, and iii) combinations thereof; and
wherein NADH+ oxidoreductase is selected from the group consisting
of: (a) a polypeptide comprising the amino acid sequence shown in
SEQ ID NO: 6, and (b) a polypeptide comprising the amino acid
sequence shown in SEQ ID NO: 6, but which includes between 1 and 20
substitutions, deletions, insertions, or additions, and has NADH+
oxidoreductase activity.
2. The method according to claim 1, wherein the medium contains
ethanol or an aliphatic acid as the carbon source.
3. The method according to claim 1, wherein the gene encoding NADH+
oxidoreductase comprises a DNA selected from the group consisting
of: (a) a DNA comprising the nucleotide sequence shown in SEQ ID
NO: 5, and (b) a DNA which is able to hybridize with a sequence
complementary to the nucleotide sequence shown in SEQ ID NO: 5
under stringent conditions comprising washing at 68.degree. C.,
0.1.times.SSC, 0.1% SDS and encoding a polypeptide having NADH+
oxidoreductase activity.
4. The method according to claim 1, wherein the microorganism has
been further modified to increase the activity of
ferredoxin-NADP.sup.+ reductase by a method selected from the group
consisting of: a) increasing the copy number of a gene encoding
ferrodoxin-NADP.sup.+ reductase, b) modifying an expression control
sequence of the gene, and c) combinations thereof.
5. The method according to claim 1, wherein the microorganism has
been further modified to increase production of ferredoxin or
flavodoxin by a method selected from the group consisting of: a)
increasing the copy number of a gene encoding ferredoxin or
flavodoxin, b) modifying an expression control sequence of the
gene, and c) combinations thereof.
6. The method according to claim 1, wherein the microorganism has
been further modified to decrease pyruvate dehydrogenase activity
by a method selected from the group consisting of: a) introducing a
deletion or mutation into a gene encoding pyruvate dehydrogenase,
b) introducing a deletion or mutation into an expression control
sequence of the gene, and c) combinations thereof.
7. The method according to claim 1, wherein the microorganism has
been further modified so that it can aerobically assimilate
ethanol.
8. The method according to claim 1, wherein the microorganism is a
bacterium belonging to a genus selected from the group consisting
of Escherichia, Enterobacter, Pantoea, Klebsiella, and
Serratia.
9. The method according to claim 1, wherein the microorganism is a
coryneform bacterium.
10. The method according to claim 1, wherein the microorganism is
Escherichia coli.
11. A method for producing an L-amino acid selected from the group
consisting of L-lysine, L-tryptophan, L-phenylalanine, L-valine,
L-leucine, L-isoleucine and L-serine comprising: A) culturing in a
medium a microorganism which has an ability to produce the L-amino
acid, and B) collecting the L-amino acid from the medium or the
microorganism, wherein said microorganism has been modified to
increase an activity of NADH+ oxidoreductase by a method selected
from the group consisting of: i) increasing the copy number of a
gene encoding NADH+ oxidoreductase, ii) modifying an expression
control sequence of the gene, and iii) combinations thereof; and
wherein NADH+ oxidoreductase comprises the amino acid sequence
shown in SEQ ID NO: 6.
12. A method for producing an L-amino acid selected from the group
consisting of L-lysine, L-tryptophan, L-phenylalanine, L-valine,
L-leucine, L-isoleucine and L-serine comprising: A) culturing in a
medium a microorganism which has an ability to produce the L-amino
acid, and B) collecting the L-amino acid from the medium or the
microorganism, wherein said microorganism has been modified to
increase an activity of NADH+ oxidoreductase by method selected
from the group consisting of: i) increasing the copy number of a
gene encoding NADH+ oxidoreductase, ii) modifying an expression
control sequence of the gene, and iii) combinations thereof; and
wherein the gene encoding NADH+ oxidoreductase is a DNA comprising
the nucleotide sequence shown in SEQ ID NO: 5.
Description
[0001] This application is a divisional application under 35 U.S.C.
.sctn.120 of U.S. patent application Ser. No. 12/202,476, filed on
Sep. 2, 2008, which claims priority under 35 U.S.C. .sctn.119 to
Japanese Patent Application No. 2007-228733, filed Sep. 4, 2007,
both of which are incorporated by reference in their entireties.
The Sequence Listing in electronic format filed herewith is also
hereby incorporated by reference in its entirety (File Name:
US-372D_Seq_List; File Size: 210 KB; Date Created: Dec. 10,
2009).
TECHNICAL FIELD
[0002] The present invention relates to a microorganism which
produces an L-amino acid and a method for producing an L-amino
acid. L-lysine and L-tryptophan are widely used as feed additives,
etc. L-phenylalanine is used as a raw material in the production of
sweeteners. L-valine, L-leucine, and L-isoleucine are used for
amino acid infusions or supplements. L-serine is useful as a food
additive and a raw material in the production of cosmetics,
etc.
BACKGROUND ART
[0003] Methods for production of a target substance, such as an
L-amino acid, by fermentation of a microorganism have been
reported. The microorganisms used for this purpose include
wild-type microorganisms (wild-type strain), auxotrophic strains
derived from wild-type strains, metabolic regulation mutant strains
derived from wild-type strains which are resistant to various
drugs, strains which act as both auxotrophic and metabolic
regulation mutants, and so forth.
[0004] In recent years, recombinant DNA techniques have been used
in the production of target substances by fermentation. For
example, it is well-known that L-amino acid productivity of a
microorganism can be improved by enhancing expression of a gene
encoding an L-amino acid biosynthetic enzyme or by enhancing uptake
of a carbon source to the L-amino acid biosynthesis system.
[0005] For example, known methods include, for L-lysine, enhancing
expression of genes encoding enzymes such as dihydrodipicolinate
synthase, aspartokinase, dihydrodipicolinate reductase,
diaminopimelate decarboxylase, and diaminopimelate dehydrogenase
(U.S. Pat. No. 6,040,160), reducing the activities of homoserine
dehydrogenase and lysine decarboxylase (U.S. Pat. No. 5,827,698),
reducing the activity of the malic enzyme (WO2005/010175), and so
forth.
[0006] For L-tryptophan, desensitization to the feedback inhibition
of phosphoglycerate dehydrogenase and anthranilate synthase (U.S.
Pat. No. 6,180,373), deletion of tryptophanase (U.S. Pat. No.
4,371,614), and so forth are known.
[0007] For L-phenylalanine, desensitization to the feedback
inhibition of chorismate mutase-prephenate dehydratase (U.S. Pat.
No. 5,354,672), deletion of chorismate mutase-prephenate
dehydrogenase and tyrosine repressor (WO03/044191), and so forth
are known.
[0008] For L-valine, a mutant strain requiring lipoic acid for its
growth and/or which is deficient in H.sup.+-ATPase (U.S. Pat. No.
5,888,783), and so forth are known. For L-leucine, desensitization
to the feedback inhibition of isopropyl malate synthase (U.S. Pat.
No. 6,403,342) and so forth are known, and for L-isoleucine,
increasing the expression of genes encoding threonine deaminase and
acetohydroxy acid synthase (U.S. Pat. No. 5,998,178), and so forth
are known.
[0009] For L-serine, a strain containing 3-phosphoglycerate
dehydrogenase which is desensitized to feedback inhibition by
serine (U.S. Pat. No. 5,618,716), a bacterium having
L-serine-producing ability and at least phosphoserine phosphatase
activity, phosphoserine transaminase activity, or both, is
enhanced, a bacterium deficient in L-serine decomposition ability
(U.S. Pat. No. 6,037,154), a bacterium resistant to azaserine or
.beta.-(2-thienyl)-DL-alanine and having L-serine-producing ability
(U.S. Pat. No. 6,258,573), and so forth are known.
SUMMARY OF THE INVENTION
[0010] The present invention provides a bacterial strain which can
efficiently produce an L-amino acid. A method is also provided for
efficiently producing an L-amino acid using such a strain.
[0011] Conventional L-amino acid production is mainly based on
maintaining the supply of acetyl-CoA to the TCA cycle by pyruvate
dehydrogenase using sugar as the carbon source. However, since the
reaction catalyzed by pyruvate dehydrogenase is accompanied by
decathoxylation, one molecule of CO.sub.2 is inevitably released.
Therefore, in order to further increase the productivity, it is
necessary to decrease this decathoxylation. As a result, ethanol
and aliphatic acids can be used as the carbon source which provides
acetyl-CoA. Also, the enzymatic activity of pyruvate synthase can
be increased. This enzyme catalyzes carbon dioxide fixation, or
pyruvate:NADP.sup.+ oxidoreductase. Furthermore, L-amino acid
production can be improved by increasing the enzymatic activity of
ferredoxin-NADP.sup.+ reductase, which reduces ferredoxin or
flavodoxin from the oxidized proteins, and is required for the
enzymatic activity of pyruvate synthase. Also, the ability to
produce ferredoxin or flavodoxin can be increased.
[0012] It is an aspect of the present invention to provide a
microorganism which has an ability to produce an L-amino acid
selected from the group consisting of L-lysine, L-tryptophan,
L-phenylalanine, L-valine, L-leucine, L-isoleucine and L-serine,
and has been modified to increase the activity of pyruvate synthase
or pyruvate:NADP.sup.+ oxidoreductase.
[0013] It is a further aspect of the present invention to provide
the aforementioned microorganism, which is modified to increase the
activity of pyruvate synthase.
[0014] It is a further aspect of the present invention to provide
the aforementioned microorganism, which is modified to increase the
activity of pyruvate:NADP.sup.+ oxidoreductase.
[0015] It is a further aspect of the present invention to provide
the aforementioned microorganism, wherein the activity of pyruvate
synthase or pyruvate:NADP.sup.+ oxidoreductase is increased by a
method selected from the group consisting of [0016] A) increasing
expression of the gene encoding pyruvate synthase or
pyruvate:NADP.sup.+ oxidoreductase, [0017] b) increasing
translation of the gene, and [0018] c) combinations thereof.
[0019] It is a further aspect of the present invention to provide
the aforementioned microorganism, wherein the activity of pyruvate
synthase or pyruvate:NADP.sup.+ oxidoreductase is increased by
increasing the copy number of the gene encoding pyruvate synthase
or pyruvate:NADP.sup.+ oxidoreductase, or by modifying an
expression control sequence of the gene.
[0020] It is a further aspect of the present invention to provide
the aforementioned microorganism, wherein pyruvate synthase is
selected from the group consisting of:
[0021] (A) a polypeptide comprising the amino acid sequence shown
in SEQ ID NO: 2,
[0022] (B) a polypeptide comprising the amino acid sequence shown
in SEQ ID NO: 2, but which includes one or more substitutions,
deletions, insertions, or additions of one or several amino acid
residues, and having pyruvate synthase activity,
[0023] (C) a polypeptide comprising the amino acid sequence shown
in SEQ ID NO: 4,
[0024] (D) a polypeptide comprising the amino acid sequence shown
in SEQ ID NO: 4, but which includes one or more substitutions,
deletions, insertions, or additions of one or several amino acid
residues and having pyruvate synthase activity.
[0025] It is a further aspect of the present invention to provide
the aforementioned microorganism, wherein the gene encoding
pyruvate synthase is selected from the group consisting of:
[0026] (a) a DNA comprising the nucleotide sequence shown in SEQ ID
NO: 1,
[0027] (b) a DNA which is able to hybridize with a sequence
complementary to the nucleotide sequence shown in SEQ ID NO: 1, or
a probe which is prepared from the nucleotide sequence, under
stringent conditions, and encoding a polypeptide having pyruvate
synthase activity,
[0028] (c) a DNA comprising the nucleotide sequence shown in SEQ ID
NO: 3,
[0029] (d) a DNA which is able to hybridize with a sequence
complementary to the nucleotide sequence shown in SEQ ID NO: 3, or
a probe which can be prepared from the nucleotide sequence, under
stringent conditions, and encoding a polypeptide having pyruvate
synthase activity.
[0030] (8) The aforementioned microorganism, wherein NADP.sup.+
oxidoreductase is selected from the group consisting of:
[0031] (A) a polypeptide comprising the amino acid sequence shown
in SEQ ID NO: 6,
[0032] (B) a polypeptide comprising the amino acid sequence shown
in SEQ ID NO: 6, but which includes one or more substitutions,
deletions, insertions or addition of one or several amino acid
residues and having pyruvate:NADP.sup.+ oxidoreductase
activity.
[0033] It is a further aspect of the present invention to provide
the aforementioned microorganism, wherein the gene encoding
pyruvate:NADP.sup.+ oxidoreductase is selected from the group
consisting of:
[0034] (a) a DNA comprising the nucleotide sequence shown in SEQ ID
NO: 5,
[0035] (b) a DNA which is able to hybridize with a sequence
complementary to the nucleotide sequence shown in SEQ ID NO: 5, or
a probe which can be prepared from the nucleotide sequence, under
stringent conditions, and encoding a polypeptide having
pyruvate:NADP.sup.+ oxidoreductase activity.
[0036] It is a further aspect of the present invention to provide
the aforementioned microorganism, which has been modified to
increase the activity of ferredoxin-NADP.sup.+ reductase.
[0037] It is a further aspect of the present invention to provide
the aforementioned microorganism, which has been modified to
improve the ability of said microorganism to produce ferredoxin or
flavodoxin.
[0038] It is a further aspect of the present invention to provide
the aforementioned microorganism, which has been modified to
decrease pyruvate dehydrogenase activity.
[0039] It is a further aspect of the present invention to provide
the aforementioned microorganism, which has been modified so that
it can aerobically assimilate ethanol.
[0040] It is a further aspect of the present invention to provide
the aforementioned microorganism, wherein said microorganism is a
bacterium belonging to a genus selected from the group consisting
of Escherichia, Enterobacter, Pantoea, Klebsiella and Serratia.
[0041] It is a further aspect of the present invention to provide
the aforementioned microorganism, which is a coryneform
bacterium.
[0042] It is a further aspect of the present invention to provide a
method for producing an L-amino acid comprising culturing the
aforementioned microorganism in a medium to produce an L-amino acid
selected from the group consisting of L-lysine, L-tryptophan,
L-phenylalanine, L-valine, L-leucine, L-isoleucine, and L-serine,
and collecting the L-amino acid from the medium or the
microorganism.
[0043] It is a further aspect of the present invention to provide
the aforementioned method, wherein the medium contains ethanol or
an aliphatic acid as the carbon source.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] FIG. 1 is a photograph showing the result of Western
blotting showing expression of the pyruvate:NADP.sup.+
oxidoreductase (PNO) gene derived from Euglena gracilis.
[0045] Lane 1: Markers
[0046] Lane 2: Crude enzyme extract obtained from
WC196.DELTA.cadA.DELTA.ldc/pCABD2/pMW-Pthr
[0047] Lane 3: Crude enzyme extract obtained from
WC196.DELTA.cadA.DELTA.ldc/pCABD2/pMW-Pthr-PNO.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0048] Hereinafter, the present invention will be explained in
detail.
[0049] <1> Microorganism
[0050] The microorganism has the ability to produced an L-amino
acid, such as L-lysine, L-tryptophan, L-phenylalanine, L-valine,
L-leucine, L-isoleucine, and L-serine, and has been modified to
increase an activity of pyruvate synthase or pyruvate:NADP.sup.+
oxidoreductase.
[0051] The "L-amino acid" means L-lysine, L-tryptophan,
L-phenylalaine, L-valine, L-leucine, L-isoleucine, and L-serine,
unless specifically mentioned otherwise.
[0052] The phrase "ability to produce an L-amino acid (L-amino
acid-producing ability)" refers to the ability to produce an
L-amino acid and cause accumulation of the L-amino acid in the
cells of the microorganism or into the medium to such a degree that
the L-amino acid can be collected from the cells or medium when the
microorganism is cultured in the medium. One or more amino acids
may be produced by the microorganism. The microorganism may
inherently have the ability to produce the L-amino acid, or the
ability may be imparted by modifying the microorganism using
mutagenesis or recombinant DNA techniques, or by introducing the
gene described herein to the microorganism.
[0053] The expression "activity of pyruvate synthase or
pyruvate:NADP.sup.+ oxidoreductase is increased" or "to increase
the activity of pyruvate synthase or pyruvate:NADP.sup.+
oxidoreductase" means that the activity of pyruvate synthase or
pyruvate:NADP.sup.+ oxidoreductase increases in a microorganism
which inherently has pyruvate synthase and/or pyruvate:NADP.sup.+
oxidoreductase, or that the activity of pyruvate synthase or
pyruvate:NADP.sup.+ oxidoreductase is imparted to a microorganism
to which pyruvate synthase and pyruvate:NADP.sup.+ oxidoreductase
are not native.
[0054] <1-1> Imparting the Ability to Produce an L-Amino
Acid
[0055] The microorganism can be obtained by modifying a parent
strain which is able to produce an L-amino acid so that the
activity of pyruvate synthase or pyruvate:NADP.sup.+
oxidoreductase, or both, is increased. The microorganism can also
be obtained by modifying a parent strain to have increased activity
of pyruvate synthase or pyruvate:NADP.sup.+ oxidoreductase, and
then imparting or enhancing the ability to produce L-amino
acids.
[0056] Methods for imparting the L-amino acid-producing ability to
a microorganism, and microorganisms imparted with L-amino
acid-producing ability, will be exemplified below, but the methods
are not limited to these.
[0057] Microorganisms belonging to .gamma.-Proteobacteria such as
bacteria belonging to the genera Escherichia, Enterobacter,
Pantoea, Klebsiella, Serratia, Erwinia, Salmonella, Morganella,
etc.; coryneform bacteria such as bacteria belonging to the genera
Brevibacterium, Corynebacterium, and Microbacterium; and
microorganisms belonging to the genera Alicyclobacillus, Bacillus,
and Saccharomyces can be used. .gamma.-proteobacteria include those
classified according to the NCBI (National Center for Biotechnology
Information) taxonomy database and can be used.
[0058] Examples of Escherichia bacteria include Escherichia coli
and so forth. When Escherichia coli strains are bred by using
genetic engineering techniques, the E. coli K12 strain and
derivatives thereof, the Escherichia coli MG1655 strain (ATCC
47076), and the W3110 strain (ATCC 27325) can be used. The
Escherichia coli K12 strain was isolated at Stanford University in
1922. This strain is a lysogenic bacterium of .lamda. phage and has
the F-factor. This strain is a highly versatile strain from which
genetic recombinants can be constructed by conjugation or the like.
Furthermore, the genome sequence of the Escherichia coli K12 strain
has been determined, and the genetic information can be used
freely. The Escherichia coli K12 strain and derivatives thereof are
available from the American Type Culture Collection (ATCC, Address:
P.O. Box 1549, Manassas, Va. 20108, United States of America).
[0059] In particular, Pantoea bacteria, Erwinia bacteria, and
Enterobacter bacteria are classified as .gamma.-proteobacteria, and
they are taxonomically very close to one another (J. Gen. Appl.
Microbiol., 1997, 43, 355-361; Int. J. Syst. Bacteriol., 1997, 43,
1061-1067). In recent years, some bacteria belonging to the genus
Enterobacter were reclassified as Pantoea agglomerans, Pantoea
dispersa, or the like, on the basis of DNA-DNA hybridization
experiments etc. (International Journal of Systematic Bacteriology,
July 1989, 39:337-345). Furthermore, some bacteria belonging to the
genus Erwinia were reclassified as Pantoea ananas or Pantoea
stewartii (refer to Int. J. Syst. Bacteriol., 1993,
43:162-173).
[0060] Examples of the Enterobacter bacteria include, but are not
limited to, Enterobacter agglomerans, Enterobacter aerogenes, and
so forth. Specifically, the strains exemplified in European Patent
Publication No. 952221 can be used. A typical strain of the genus
Enterobacter is the Enterobacter agglomeranses ATCC 12287
strain.
[0061] Typical strains of the Pantoea bacteria include, but are not
limited to, Pantoea ananatis, Pantoea stewartii, Pantoea
agglomerans, and Pantoea citrea. Specific examples include the
following strains:
[0062] Pantoea ananatis AJ13355 (PERM BP-6614, European Patent
Publication No. 0952221)
[0063] Pantoea ananatis AJ13356 (FERM BP-6615, European Patent
Publication No. 0952221)
[0064] Although these strains are described as Enterobacter
agglomerans in European Patent Publication No. 0952221, they are
currently classified as Pantoea ananatis on the basis of nucleotide
sequence analysis of the 16S rRNA etc., as described above.
[0065] Examples of the Erwinia bacteria include, but are not
limited to, Erwinia amylovora and Erwinia carotovora, and examples
of the Klebsiella bacteria include Klebsiella planticola. Specific
examples include the following strains:
[0066] Erwinia amylovora ATCC 15580
[0067] Erwinia carotovora ATCC 15713
[0068] Klebsiella planticola AJ13399 (FERM BP-6600, European Patent
Publication No. 955368)
[0069] Klebsiella planticola AJ13410 (FERM BP-6617, European Patent
Publication No. 955368).
[0070] The coryneform bacteria are a group of microorganisms
defined in Bergey's Manual of Determinative Bacteriology, 8th Ed.,
p. 599, 1974, and include aerobic, Gram-positive, and nonacid-fast
bacilli which are not able to sporulate, and which were originally
classified into the genus Brevibacterium, but are now recognized as
being in the genus Corynebacterium (Liebl, W., Ehrmann, M., Ludwig,
W., and Schleifer, K. H., 1991, Int. J. Syst. Bacteriol.
41:255-260). These bacteria also include bacteria belonging to the
genus Brevibacterium or Microbacterium, which are closely related
to the genus Corynebacterium.
[0071] Specific examples of coryneform bacteria which are used to
produce amino acids of the L-glutamic acid family include the
following:
[0072] Corynebacterium acetoacidophilum
[0073] Corynebacterium acetoglutamicum
[0074] Corynebacterium alkanolyticum
[0075] Corynebacterium callunae
[0076] Corynebacterium glutamicum
[0077] Corynebacterium lilium (Corynebacterium glutamicum)
[0078] Corynebacterium melassecola
[0079] Corynebacterium thermoaminogenes (Corynebacterium
efficiens)
[0080] Corynebacterium herculis
[0081] Brevibacterium divaricatum (Corynebacterium glutamicum)
[0082] Brevibacterium flavum (Corynebacterium glutamicum)
[0083] Brevibacterium immariophilum
[0084] Brevibacterium lactofermentum (Corynebacterium
glutamicum)
[0085] Brevibacterium roseum
[0086] Brevibacterium saccharolyticum
[0087] Brevibacterium thiogenitalis
[0088] Brevibacterium ammoniagenes (Corynebacterium
ammoniagenes)
[0089] Brevibacterium album
[0090] Brevibacterium cerinum
[0091] Microbacterium ammoniaphilum
[0092] Specifically, the following strains can be mentioned:
[0093] Corynebacterium thermoaminogenes AJ12340 (FIRM BP-1539)
[0094] Corynebacterium glutamicum ATCC 13032
[0095] Brevibacterium flavum (Corynebacterium glutamicum) ATCC
13826, ATCC 14067
[0096] Brevibacterium lactofermentum (Corynebacterium glutamicum)
ATCC 13665, ATCC 13869
[0097] Brevibacterium ammoniagenes (Corynebacterium ammoniagenes)
ATCC 6871
[0098] The bacterium may be able to assimilate ethanol. The
bacterium may inherently be able to assimilate ethanol, or the
ability to assimilate ethanol may be imparted or increased
recombinantly. Escherichia coli is known to have AdhE, which has
activities of acetaldehyde dehydrogenase and alcohol dehydrogenase,
which are enzymes which can generate ethanol under anaerobic
conditions, and catalyze the reactions described below.
Acetyl-CoA+NADH+H.sup.+=acetaldehyde+NAD.sup.++CoA
Acetaldehyde+NADH+H.sup.++=ethanol+NAD.sup.+
[0099] Although Escherichia coli cannot assimilate ethanol under
aerobic conditions, the mutation of AdhE results in Escherichia
coli to be able to aerobically assimilate ethanol (Clark D. P., and
Cronan, J. E. Jr., 1980, J. Bacteriol., 144:179-184;
Membrillo-Hernandez, J. et al., 2000, J. Biol. Chem.,
275:33869-33875). The specific mutation is that the glutamic acid
at position 569 in Escherichia coli AdhE is replaced with an amino
acid other than glutamic acid and aspartic acid, such as lysine
(Glu568Lys or E568K).
[0100] The aforementioned AdhE mutant may further include the
following additional mutations:
[0101] A) Replacement of the glutamic acid at position 560 with
another amino acid, such as lysine,
[0102] B) Replacement of the phenylalanine at position 566 with
another amino acid,
[0103] C) Replacement of the glutamic acid at position 22,
methionine at position 236, tyrosine at position 461, isoleucine at
position 554, and alanine at position 786, with glycine, valine,
cysteine, serine, and valine, respectively, or
[0104] D) a combination of the aforementioned mutations.
[0105] It is known that Corynebacterium glutamicum has two or more
kinds of alcohol dehydrogenases, and can aerobically assimilate
ethanol (Pelechova J, Smekal F, Koura V, Plachy J and Krumphanzl V,
1980, Folia Microbiol (Praha) 25:341-346).
[0106] The bacterium may be able to assimilate fat, oil, or an
aliphatic acid. The bacterium may inherently be able to assimilate
fat, oil, or aliphatic acids, or the ability can be imparted or
increased recombinantly. Escherichia coli is known to be able to
assimilate long chain aliphatic acids having a length of 12 or
longer (Clark D. P. and Cronan J. E., 1996, In Escherichia coli and
Salmonella: Cellular and Molecular Biology/Second Edition
(Neidhardt, F. C. Ed.) pp. 343-357). Furthermore, Escherichia coli
strains which were mutated to assimilate short- to medium-chain
aliphatic acids are known (Nunn, W. D. et al., 1979, J. Biol.
Chem., 254:9130-9134; Salanitro, J. P. and Wegener, W. S., 1971, J.
Bacteriol., 108:885-892).
[0107] A bacterium which is able to produce an L-amino acid means
that the bacterium can produce and cause accumulation of an L-amino
acid in the medium in such an amount that the L-amino acid can be
collected from the medium when the bacterium is cultured in the
medium. The target L-amino acid can accumulate in the medium in an
amount not less than 0.5 g/L, more preferably not less than 1.0
g/L. The "L-amino acid" encompasses L-lysine, L-tryptophan,
L-phenylalanine, L-valine, L-leucine, L-isoleucine, and L-serine.
L-Lysine and L-tryptophan are especially preferred.
[0108] Hereinafter, methods for imparting an L-amino acid-producing
ability to such bacteria as mentioned above, or methods for
enhancing an L-amino acid-producing ability of such bacteria as
described above, are described.
[0109] To impart the ability to produce an L-amino acid, methods
conventionally employed in the breeding of coryneform bacteria or
bacteria of the genus Escherichia (see "Amino Acid Fermentation",
Gakkai Shuppan Center (Ltd.), 1st Edition, published May 30, 1986,
pp. 77-100) can be used. Such methods include by acquiring the
properties of an auxotrophic mutant, an analogue-resistant strain,
or a metabolic regulation mutant, or by constructing a recombinant
strain so that it overexpresses an L-amino acid biosynthesis
enzyme. Here, in the breeding of an L-amino acid-producing
bacteria, one or more of the above described properties may be
imparted. The expression of L-amino acid biosynthesis enzyme(s) can
be enhanced alone or in combinations of two or more. Furthermore,
the methods of imparting properties such as an auxotrophic
mutation, analogue resistance, or metabolic regulation mutation may
be combined with the methods of enhancing the biosynthesis
enzymes.
[0110] An auxotrophic mutant strain, L-amino acid
analogue-resistant strain, or metabolic regulation mutant strain
with the ability to produce an L-amino acid can be obtained by
subjecting a parent strain or wild-type strain to conventional
mutatagenesis, such as exposure to X-rays or UV irradiation, or
treatment with a mutagen such as
N-methyl-N-nitro-N-nitrosoguanidine, etc., then selecting those
which exhibit autotrophy, analogue resistance, or a metabolic
regulation mutation and which also have the ability to produce an
L-amino acid.
[0111] Moreover, L-amino acid-producing ability can also be
imparted or enhanced by enhancing an enzymatic activity by gene
recombination. Examples of the method for enhancing enzymatic
activity include, for example, modifying the bacterium to increase
expression of a gene encoding an enzyme involved in the
biosynthesis of an L-amino acid. Gene expression can also be
increased by introducing an amplification plasmid prepared by
introducing a DNA fragment containing the gene into an appropriate
plasmid, for example, a plasmid vector containing at least a gene
responsible for replication and proliferation of the plasmid in the
microorganism, increasing the copy number of the gene on the
chromosome by conjugation, transfer or the like, or introducing a
mutation into the promoter region of the gene (refer to
International Patent Publication WO95/34672).
[0112] When a target gene is introduced into the aforementioned
amplification plasmid or chromosome, any promoter may be used to
express the gene so long as the chosen promoter functions in the
L-amino acid-producing bacterium. The promoter may be inherent to
the gene, or may be a modified form. Expression of the gene can
also be controlled by suitably choosing a promoter that potently
functions in the L-amino acid-producing bacterium, or by
approximating the -35 and -10 regions of the promoter close to the
consensus sequence. The methods for enhancing expression of genes
encoding the target enzymes are described in WO00/18935, European
Patent Publication No. 1010755, and so forth.
[0113] Examples of methods for imparting L-amino acid-producing
ability to a bacterium and bacteria imparted with an L-amino
acid-producing ability will be described below.
[0114] L-Lysine-Producing Bacteria
[0115] Examples of L-lysine-producing Escherichia bacteria include
mutants which are resistant to L-lysine analogues. L-lysine
analogues inhibit growth of the bacteria, but this inhibition is
fully or partially desensitized when L-lysine is present in the
medium. Examples of the L-lysine analogues include, but are not
limited to, oxalysine, lysine hydroxamate,
S-(2-aminoethyl)-L-cysteine (AEC), .gamma.-methyllysine,
.alpha.-chlorocaprolactam, and so forth. Mutants which are
resistant to these lysine analogues can be obtained by subjecting
the bacteria to a conventional artificial mutagenesis treatment.
Specific examples of bacterial strains useful for producing
L-lysine include Escherichia coli AJ11442 (PERM BP-1543,
NRRLB-12185; see U.S. Pat. No. 4,346,170) and Escherichia coli
VL611. In these microorganisms, feedback inhibition of
aspartokinase by L-lysine is desensitized.
[0116] The WC196 strain is an L-lysine-producing Escherichia coli
bacterium. This bacterial strain was bred by conferring AEC
resistance to the W3110 strain, which was derived from Escherichia
coli K-12. The resulting strain was designated Escherichia coli
AJ13069 and was deposited at the National Institute of Bioscience
and Human-Technology, Agency of Industrial Science and Technology
(currently National Institute of Advanced Industrial Science and
Technology, International Patent Organism Depositary, Tsukuba
Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken,
305-8566, Japan) on Dec. 6, 1994 and received an accession number
of PERM P-14690. Then, it was converted to an international deposit
under the provisions of the Budapest Treaty on Sep. 29, 1995, and
received an accession number of PERM BP-5252 (U.S. Pat. No.
5,827,698).
[0117] Examples of L-lysine-producing bacteria and parent strains
which can be used to derive L-lysine-producing bacteria also
include strains in which expression is increased of one or more
genes encoding an L-lysine biosynthetic enzyme. Examples of such
enzymes include, but are not limited to, dihydrodipicolinate
synthase (dapA), aspartokinase (lysC), dihydrodipicolinate
reductase (dapB), diaminopimelate decarboxylase (lysA),
diaminopimelate dehydrogenase (ddh) (U.S. Pat. No. 6,040,160),
phosphoenolpyrvate carboxylase (ppc), aspartate semialdehyde
dehydrogenase (asd), diaminopimelate epimerase (dapF),
tetrahydrodipicolinate succinylase (dapD), succinyl diaminopimelate
deacylase (dapE), and aspartase (aspA) (EP 1253195 A). The
abbreviations in parentheses are the gene names which correspond to
the enzymes, and this convention is used throughout this
specification. Dihydrodipicolinate reductase, diaminopimelate
decathoxylase, diaminopimelate dehydrogenase, phosphoenolpyrvate
carboxylase, aspartate aminotransferase, diaminopimelate epimerase,
aspartate semialdehyde dehydrogenase, tetrahydrodipicolinate
succinylase, and succinyl diaminopimelate deacylase are especially
preferred. In addition, the chosen parent strains may overexpress
the cyo gene, which is involved in energy efficiency (EP 1170376
A), the gene encoding nicotinamide nucleotide transhythogenase
(pntAB) (U.S. Pat. No. 5,830,716), the ybjE gene (WO2005/073390),
or combinations thereof.
[0118] Examples of L-lysine-producing bacteria and parent strains
which can be used to derive L-lysine-producing bacteria also
include strains with decreased or no activity of an enzyme that
catalyzes a reaction which produces a compound other than L-lysine
via a biosynthetic pathway which branches off from the biosynthetic
pathway of L-lysine. Examples of these enzymes include homoserine
dehydrogenase, lysine decathoxylase (U.S. Pat. No. 5,827,698), and
the malic enzyme (WO2005/010175).
[0119] Preferred examples of L-lysine-producing bacteria include
Escherichia coli WC196.DELTA.mez/pCABD2 (WO2005/010175),
WC196.DELTA.cadA.DELTA.ldc/pCABD2 (WO2006/078039), and so forth.
The WC196.DELTA.mez/pCABD2 strain is obtained by introducing the
plasmid pCABD2, which is disclosed in U.S. Pat. No. 6,040,160, into
the WC196 strain with disrupted sfcA and b2463 genes, which encode
the malic enzyme. The nucleotide sequences of the sfcA and b2463
genes and the amino acid sequences encoded by these genes are shown
in SEQ ID NOS: 52 to 55.
[0120] The WC196.DELTA.cadA.DELTA.ldc/pCABD2 strain is obtained by
introducing the plasmid pCABD2, which is disclosed in U.S. Pat. No.
6,040,160, into a WC196 strain with disrupted cadA and ldcC genes,
which encode lysine decarboxylase. The pCABD2 plasmid contains a
mutant Escherichia coli dapA gene encoding a dihydrodipicolinate
synthase (DDPS) which is desensitized to feedback inhibition by
L-lysine, a mutant Escherichia coli lysC gene which encodes
aspartokinase III which is desensitized to feedback inhibition by
L-lysine, the Escherichia coli dapB gene encoding
dihythodipicolinate reductase, and the Brevibacterium
lactofermentum ddh gene encoding diaminopimelate dehydrogenase.
[0121] L-Tryptophan-Producing Bacteria
[0122] Examples of L-tryptophan-producing bacteria and parent
strains which can be used to derive L-tryptophan-producing bacteria
include, but are not limited to, strains belonging to the genus
Escherichia, such as E. coli JP4735/pMU3028 (DSM10122) and
JP6015/pMU91 (DSM10123), which is deficient in tryptophanyl-tRNA
synthetase encoded by a mutant bpS gene (U.S. Pat. No. 5,756,345);
E. coli SV164 (pGH5) having a serA allele encoding phosphoglycerate
dehydrogenase not subject to feedback inhibition by serine and a
trpE allele encoding anthranilate synthase not subject to feedback
inhibition by tryptophan (U.S. Pat. No. 6,180,373); E. coli AGX17
(pGX44) (NRRLB-12263) and AGX6(pGX50)aroP (NRRL B-12264) deficient
in the enzyme tryptophanase (U.S. Pat. No. 4,371,614); E. coli
AGX17/pGX50,pACKG4-pps with enhanced phosphoenolpyruvate-producing
ability (WO97/08333, U.S. Pat. No. 6,319,696), and so forth.
L-typtophan-producing Escherichia bacteria with enhanced activity
of the protein encoded by the yedA or yddG genes may also be used
(U.S. Published Patent Applications 2003/0148473 A1 and
2003/0157667 A1).
[0123] Examples of L-tryptophan-producing bacteria and parent
strains which can be used to derive L-tryptophan-producing bacteria
also include strains with enhanced activity of one or more enzymes
such as anthranilate synthase (trpE), phosphoglycerate
dehydrogenase (serA), 3-deoxy-D-arabinoheptulosonate-7-phosphate
synthase (aroG), 3-dehythoquinate synthase (aroB), shikidmate
dehydrogenase (aroE), shikimate kinase (aroL),
5-enolpyruvylshikidmate-3-phosphate synthase (aroA), chorismate
synthase (aroC), prephenate dehydratase, chorismate mutase, and
tryptophan synthase (trpAB). Prephenate dehydratase and chorismate
mutase are encoded by the pheA gene as a bifunctional enzyme
(CM-PD). Phosphoglycerate dehydrogenase,
3-deoxy-D-arabinoheptulosonate-7-phosphate synthase,
3-dehydroquinate synthase, shikimate dehydratase, shikimate kinase,
5-enolpyruvylshikimate-3-phosphate synthase, chorismate synthase,
prephenate dehydratase, chorismate mutase-prephenate dehydratase
are especially preferred. The anthranilate synthase and
phosphoglycerate dehydrogenase are both subject to feedback
inhibition by L-tryptophan and L-serine, and therefore a mutation
desensitizing the feedback inhibition may be introduced into these
enzymes. Specific examples of strains having such a mutation
include E. coli SV164 which harbors desensitized anthranilate
synthase, and a transformant strain obtained by introducing pGH5
(WO 94/08031) into E. coli SV164, which contains a mutant serA gene
encoding feedback inhibition-desensitized phosphoglycerate
dehydrogenase.
[0124] Examples of L-tryptophan-producing bacteria and parent
strains which can be used to derive L-tryptophan-producing bacteria
also include strains transformed with the tryptophan operon which
contains a gene encoding desensitized anthranilate synthase (JP
57-71397 A, JP 62-244382 A, U.S. Pat. No. 4,371,614). Moreover,
L-tryptophan-producing ability may be imparted by enhancing the
expression of the gene which encodes tryptophan synthase, which is
part of the tryptophan operon (trpBA). The tryptophan synthase
consists of .alpha. and .beta. subunits which are encoded by trpA
and trpB, respectively. In addition, L-tryptophan-producing ability
may be improved by enhancing expression of the isocitrate
lyase-malate synthase operon (WO2005/103275).
[0125] L-Phenylalanine-Producing Bacteria
[0126] Examples of L-phenylalanine-producing bacteria and parent
strains which can be used to derive L-phenylalanine-producing
bacteria include, but are not limited to, strains belonging to the
genus Escherichia, such as E. coli AJ12739 (tyrA::Tn10, tyrR) (VKPM
B-8197) (WO03/044191), E. coli HW 1089 (ATCC 55371) harboring the
pheA34 gene encoding chorismate mutase-prephenate dehydratase
desensitized to the feedback inhibition (U.S. Pat. No. 5,354,672),
E. coli MWEC101-b (KR8903681), E. coli NRRL B-12141, NRRL B-12145,
NRRL B-12146, and NRRL B-12147 (U.S. Pat. No. 4,407,952). Also, as
a parent strain, E. coli K-12 [W3110 (tyrA)/pPHAB (FERM BP-3566)
having a gene encoding chorismate mutase-prephenate dehydratase
desensitized to feedback inhibition, E. coli K-12 [W3110
(tyrA)/pPHAD] (FERM BP-12659), E. coli K-12 [W3110 (tyrA)/pPHATerm]
(FERM BP-12662) and E. coli K-12 [W3110 (tyrA)/pBR-aroG4, pACMAB],
also called AJ12604 (FERM BP-3579) may be used (EP 488-424 B1).
Furthermore, L-phenylalanine-producing Escherichia bacteria with
enhanced activity of the protein encoded by the yedA or yddG genes
may also be used (U.S. Published Patent Applications 2003/0148473
A1 and 2003/0157667 A1, WO03/044192).
[0127] L-Valine-Producing Bacteria
[0128] Examples of L-valine-producing bacteria and parent strains
which can be used to derive L-valine-producing bacteria include,
but are not limited to, strains which have been modified to
overexpress the ilvGMEDA operon (U.S. Pat. No. 5,998,178). The
region in the ilvGMEDA operon which is required for attenuation can
be removed so that expression of the operon is not attenuated by
the L-valine that is produced. Furthermore, the ilvA gene in the
operon can be disrupted so that threonine deaminase activity is
decreased.
[0129] Examples of L-valine-producing bacteria and parent strains
which can be used to derive L-valine-producing bacteria also
include strains with amino-acyl t-RNA synthetase mutants (U.S. Pat.
No. 5,658,766). For example, E. coli VL1970, which has a mutation
in the ileS gene, which encodes isoleucine tRNA synthetase, can be
used. E. coli VL1970 was deposited at the Russian National
Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd.,
1 Moscow 117545, Russia) on Jun. 24, 1988 under accession number
VKPM B-4411.
[0130] Furthermore, mutants requiring lipoic acid for growth and/or
lacking H.sup.+-ATPase can also be used as parent strains
(WO96/06926, U.S. Pat. No. 5,888,783).
[0131] L-Leucine-Producing Bacteria
[0132] Examples of L-leucine-producing bacteria and parent strains
which can be used to derive L-leucine-producing bacteria include,
but are not limited to, strains belonging to the genus Escherichia,
such as E. coli strains resistant to leucine (for example, the
strain 57 (VKPM B-7386, U.S. Pat. No. 6,124,121)) or leucine
analogues including .beta.-2-thienylalanine, 3-hydroxyleucine,
4-azaleucine and 5,5,5-trifluoroleucine (JP 62-34397 B and JP
8-70879 A); E. coli strains obtained by the genetic engineering
method described in WO96/06926; and E. coli H-9068 (JP 8-70879
A).
[0133] The bacterium may be improved by enhancing the expression of
one or more genes which encode proteins involved in L-leucine
biosynthesis. Examples of such genes include genes of the leuABCD
operon, such as a mutant leuA gene encoding isopropylmalate
synthase which is not subject to feedback inhibition by L-leucine
(U.S. Pat. No. 6,403,342). In addition, the bacterium may be
improved by enhancing the expression of one or more genes encoding
proteins which promote secretion of L-amino acids from the
bacterial cell. Examples of such genes include b2682 and b2683
(ygaZH genes) (EP 1239041 A2).
[0134] L-Isoleucine-Producing Bacteria
[0135] Examples of L-isoleucine-producing bacteria and parent
strains which can be used to derive L-isoleucine-producing bacteria
include, but are not limited to, mutants having resistance to
6-dimethylaminopurine (JP 5-304969 A), mutants having resistance to
an isoleucine analogue such as thiaisoleucine and isoleucine
hydroxamate, and mutants additionally having resistance to
DL-ethionine and/or arginine hydroxamate (JP 5-130882 A). In
addition, recombinant strains transformed with genes encoding
proteins involved in L-isoleucine biosynthesis, such as threonine
deaminase and acetohydroxy acid synthase, can also be used as
parent strains (JP 2-458 A, FR 0356739, and U.S. Pat. No.
5,998,178).
[0136] L-Serine-Producing Bacteria
[0137] Examples of L-serine-producing bacteria and parent strains
which can be used to derive L-serine-producing bacteria include
Escherichia coli which are desensitized to feedback inhibition of
3-phosphoglycerate dehydrogenase by serine (Japanese Patent No.
2584409, U.S. Pat. No. 5,618,716). Moreover, coryneform bacteria
which are able to produce L-serine and have increased activity of
at least one of phosphoserine phosphatase and phosphoserine
transaminase, coryneform bacteria which cannot decompose L-serine
(JP 11-253187 A, U.S. Pat. No. 6,037,154), and coryneform bacteria
which is resistant to azaserine or (3-(2-thienyl)-DL-alanine and is
able to produce L-serine (JP 11-266881 A, U.S. Pat. No. 6,258,573)
can also be used.
[0138] When the aforementioned L-amino acid-producing bacteria are
bred by gene recombination, the chosen genes are not limited to
genes having the genetic information described above or genes
having known sequences, but genes having conservative mutations
such as homologues or artificially modified genes can also be used,
so long as the functions of the encoded proteins are not degraded.
That is, the chosen genes may encode a known amino acid sequence
including substitution, deletion, insertion, addition or the like
of one or several amino acid residues at one or several positions.
As for the "conservative mutation", the descriptions concerning
pyruvate synthase etc. described below are also applied to the
aforementioned genes.
[0139] <1-2> Enhancement of Pyruvate Synthase or
Pyruvate:NADP.sup.+ Oxidoreductase Activity
[0140] The microorganism having an L-amino acid-producing ability
is modified so that an activity of pyruvate synthase or
pyruvate:NADP.sup.+ oxidoreductase is increased. The activity of
the pyruvate synthase or pyruvate:NADP.sup.+ oxidoreductase
activity is increased so that it is higher as compared to that of
the parent strain, for example, a wild-type strain or a
non-modified strain. In addition, this is true when the pyruvate
synthase activity is not native to the microorganism, for example,
the pyruvate synthase or pyruvate:NADP.sup.+ oxidoreductase
activity of the microorganism, which has been modified to have that
enzymatic activity, is increased as compared with a non-modified
strain.
[0141] The bacterium may be modified first to increase the
enzymatic activity of pyruvate synthase or pyruvate:NADP.sup.+
oxidoreductase, and then imparted with an L-amino acid-producing
ability. In addition, the activity of pyruvate synthase or
pyruvate:NADP.sup.+ oxidoreductase can be increased by increasing
the expression of a gene as described above. That is, enzyme
activity may be increased by increasing expression of the
endogenous pyruvate synthase or pyruvate:NADP.sup.+ oxidoreductase
genes by modifying the expression control regions such as the
promoter or the like, or by enhancing expression of an exogenous
pyruvate synthase gene or pyruvate:NADP.sup.+ oxidoreductase gene
by introducing a plasmid containing the pyruvate synthase or
pyruvate:NADP.sup.+ oxidoreductase gene into the bacterium,
introducing these genes into the chromosome of the bacterium, or
the like.
[0142] Pyruvate synthase catalyzes the following reaction, which
generates pyruvic acid from acetyl-CoA and CO.sub.2 in the presence
of an electron donor such as ferredoxin and flavodoxin (EC
1.2.7.1). Pyruvate synthase may be abbreviated as "PS", and may be
also be called pyruvate oxidoreductase, pyruvate ferredoxin
oxidoreductase, pyruvate flavodoxin oxidoreductase, or pyruvate
oxidoreductase. As the electron donor, ferredoxin or flavodoxin can
be used.
Reduced ferredoxin+acetyl-CoA+CO.sub.2=oxidized ferredoxin+pyruvic
acid+CoA
[0143] Enhancement of the pyruvate synthase activity can be
confirmed by preparing crude enzyme solutions and measuring the
pyruvate synthase activity in both the microorganism before making
the modification to enhance activity, and after making the
modification. The activity of pyruvate synthase can be measured by,
for example, the method of Yoon et al. (Yoon, K. S. Ishii, M.,
Kodama, T., and Igarashi, Y. 1997. Arch. Microbiol. 167:275-279,
1997). For example, pyruvic acid is added to a reaction mixture
containing oxidized methylviologen which acts as an electron
acceptor, CoA, and crude enzyme solution, and spectroscopically
measuring the amount of reduced methylviologen, which increases due
to the decarboxylation of pyruvic acid. One unit (U) of the
enzymatic activity is defined as the activity of reducing 1 .mu.mol
of methylviologen per 1 minute. When the parent strain has pyruvate
synthase activity, the activity desirably increases, for example,
preferably 1.5 times or more, more preferably 2 times or more,
still more preferably 3 times or more, compared with that of the
parent strain. When the parent strain does not have pyruvate
synthase activity, although it is sufficient that pyruvate synthase
is produced by the introduction of the pyruvate synthase gene, the
activity is preferably enhanced to such an extent that the
enzymatic activity can be measured, and the activity is preferably
0.001 U/mg (cell protein) or higher, more preferably 0.005 U/mg or
higher, still more preferably 0.01 U/mg or higher. Pyruvate
synthase is sensitive to oxygen, and activity expression and
measurement are often generally difficult (Buckel, W. and Golding,
B. T., 2006, Ann. Rev. of Microbiol., 60:27-49). Therefore, as
described in the examples, the enzymatic activity is measured
preferably under reduced oxygen concentration in the reaction
vessel.
[0144] The gene encoding pyruvate synthase may be derived from, or
native to, bacteria with the reductive TCA cycle, and includes
pyruvate synthase genes from Chlorobium tepidum and Hydrogenobacter
thermophilus.
[0145] Specific examples include the pyruvate synthase gene having
the nucleotide sequence located at nucleotide numbers from 1534432
to 1537989 of the genome sequence of Chlorobium tepidum (Genbank
Accession No. NC.sub.--002932) and shown in SEQ ID NO: 1. The amino
acid sequence encoded by this gene is shown in SEQ ID NO: 2
(Genbank Accession No. AAC76906). Furthermore, the pyruvate
synthase from Hydrogenobacter thermophilus forms a complex of four
subunits, the .delta.-subunit (Genbank Accession No. BAA95604),
.alpha.-subunit (Genbank Accession No. BAA95605), .beta.-subunit
(Genbank Accession No. BAA95606), and .gamma.-subunit (Genbank
Accession No. BAA95607) (Ikeda, T. Ochiai, T., Morita, S.,
Nishiyama, A., Yamada, E., Arai, H., Ishii, M. and Igarashi, Y.
2006, Biochem. Biophys. Res. Commun., 340:76-82). The pyruvate
synthase gene may also include the four genes HP1108, HP1109, HP
1110, and HP1111, located at nucleotide numbers from 1170138 to
1173296 in the genome sequence of Helicobacter pylori (GenBank
Accession No. NC 000915), and the pyruvate synthase gene encoded by
the four genes SSO1208, SSO7412, SSO1207, and SSO1206, identified
by nucleotide numbers from 1047593 to 1044711 in the genome
sequence of Sulfolobus solfataricus (GenBank Accession No. NC
002754). Furthermore, the pyruvate synthase gene may be cloned from
Chlorobium, Desulfobacter, Aquifex, Hydrogenobacter, Thermoproteus,
Pyrobaculum bacteria, or the like on the basis of homology to the
genes exemplified above.
[0146] The Escherichia coli ydbK gene (b1378), which is shown in
SEQ ID NO: 3, is located at nucleotide numbers from 1435284 to
1438808 in the genome sequence of the K-12 strain (GenBank
Accession No. U00096). This gene is predicted to encode pyruvate
flavodoxin oxidoreductase, that is, pyruvate synthase, on the basis
of homology of the sequences. The amino acid sequence encoded by
this gene is shown in SEQ ID NO: 4 (GenBank Accession No.
AAC76906). As demonstrated in the example section, it was verified
that this gene product has pyruvate synthase activity, and
enhancing expression of this gene improves the ability to produce
an L-amino acid.
[0147] Pyruvate:NADP.sup.+ oxidoreductase catalyzes the following
reaction, which generates pyruvic acid from acetyl CoA and
CO.sub.2, in the presence of an electron donor such as NADPH or
NADH (EC 1.2.1.15). Pyruvate:NADP.sup.+ oxidoreductase may be
abbreviated as "PNO", and may also be called pyruvate
dehydrogenase. However, pyruvate dehydrogenase activity is the
activity of catalyzing the oxidative decathoxylation of pyruvic
acid to generate acetyl-CoA, as described later, and pyruvate
dehydrogenase (PDH) which catalyses this reaction is different from
pyruvate:NADP.sup.+ oxidoreductase. Pyruvate:NADP.sup.+
oxidoreductase can use NADPH or NADH as the electron donor.
NADPH+acetyl-CoA+CO.sub.2=NADP.sup.++pyruvic acid+CoA
[0148] Enhancement of the pyruvate:NADP.sup.+ oxidoreductase
activity can be confirmed by preparing crude enzyme solutions and
measuring the pyruvate:NADP.sup.+ oxidoreductase activity in both
the microorganism before making the modification to enhance
activity, and after making the modification. The activity of
pyruvate:NADP.sup.+ oxidoreductase can be measured by, for example,
the method of Inui et al. (Inui, H., Ono, K., Miyatake, K, Nakano,
Y., and Kitaoka, S., 1987, J. Biol. Chem., 262:9130-9135). For
example, pyruvic acid is added to a reaction mixture containing
oxidized methylviologen which acts as an electron acceptor, CoA,
and crude enzyme solution, and spectroscopically measuring the
amount of reduced methylviologen, which increases due to the
decathoxylation of pyruvic acid. One unit (U) of the enzymatic
activity is defined as the activity of reducing 1 .mu.mol of
methylviologen per 1 minute. When the parent strain has
pyruvate:NADP.sup.+ oxidoreductase activity, the activity
increases, for example, preferably 1.5 times or more, more
preferably 2 times or more, still more preferably 3 times or more,
as compared to that of the parent strain. When the parent strain
does not have pyruvate:NADP.sup.+ oxidoreductase activity, although
it is sufficient that pyruvate:NADP.sup.+ oxidoreductase is
produced by the introduction of the pyruvate:NADP.sup.+
oxidoreductase gene, the activity is preferably enhanced to such an
extent that the enzymatic activity can be measured, and the
activity is preferably 0.001 U/mg (cell protein) or higher, more
preferably 0.005 U/mg or higher, still more preferably 0.01 U/mg or
higher. Pyruvate:NADP.sup.+ oxidoreductase is sensitive to oxygen,
and activity expression and measurement are often generally
difficult (Inui, H., Ono, K., Miyatake, K, Nakano, Y., and Kitaoka,
S., 1987, J. Biol. Chem., 262: 9130-9135; Rotte, C., Stejskal, F.,
Zhu, G., Keithly, J. S., and Martin, W., 2001, Mol. Biol. Evol,
18:710-720). When the activity cannot be measured due to
inactivation or the like, it is still possible to confirm
expression of the protein by Western blotting or the like, as
described in the examples section.
[0149] The gene encoding pyruvate:NADP.sup.+ oxidoreductase may be
derived from, or native to, Euglena gracilis, which is a
photosynthetic eukaryotic microorganism and is also classified into
protozoans (Nakazawa, M., Inui, H. Yamaji R., Yamamoto, T.,
Takenaka, S., Ueda, M., Nakano, Y., Miyatake, K, 2000, FEBS Lett,
479:155-156), and the protist Cryptosporidium parvum (Rotte, C.,
Stejskal, F., Zhu, G., Keithly, J. S., and Martin, W., 2001, Mol.
Biol. Evol., 18:710-720). Furthermore, it is known that a
homologous gene also exists in Tharassiosira pseudonana which
belongs to Bacillariophyta (Ctrnacta, V., Ault, J. G., Stejskal,
F., and Keithly, J. S., 2006, J. Eukaryot. Microbiol.,
53:225-231).
[0150] Specifically, the pyruvate:NADP.sup.+ oxidoreductase gene
from Euglena gracilis has the nucleotide sequence shown in SEQ ID
NO: 5 (GenBank Accession No. AB021127). The amino acid sequence
encoded by this gene is shown in SEQ ID NO: 6 (GenBank Accession
No. BAB 12024).
[0151] The microorganism may be modified so that the pyruvate
synthase activity is increased by increasing the activity of
recycling the oxidized electron donor to a reduced electron donor,
which is required for pyruvate synthase activity, as compared to a
parent strain, for example, a wild-type strain or a non-modified
strain. An example of the activity for recycling the oxidized
electron donor to a reduced electron donor is ferredoxin NADP.sup.+
reductase activity. Furthermore, the microorganism may be modified
so that the activity of pyruvate synthase is increased, in addition
to enhancing the electron donor recycling activity. The gene
encoding the electron donor recycling activity may be native to the
parent strain, or may be introduced into the parent strain to
impart the activity, and the ability to produce an L-amino acid is
improved.
[0152] The ferredoxin NADP.sup.+ reductase is an enzyme that
reversibly catalyzes the following reaction (EC 1.18.1.2):
Reduced ferredoxin+NADP.sup.+=Oxidized ferredoxin+NADPH+H.sup.+
[0153] This reaction is reversible, and can generate the reduced
ferredoxin in the presence NADPH and the oxidized ferredoxin.
Ferredoxin can be replaced with flavodoxin, and the enzyme is a
functional equivalent to flavodoxin NADP.sup.+ reductase.
Ferredoxin NADP.sup.+ reductase has been confirmed to be present in
a wide variety of organisms ranging from microorganisms to higher
organisms (refer to Carrillo, N. and Ceccarelli, E. A., 2004, Eur.
J. Biochem., 270:1900-1915; Ceccarelli, E. A. Arakaki, A. K,
Cortez, N., and Carrillo, N. 2004, Biochim Biophys. Acta,
1698:155-165), and it is also known as ferredoxin NADP.sup.+
oxidoreductase or NADPH-ferredoxin oxidoreductase.
[0154] Enhancement of the ferredoxin NADP.sup.+ reductase activity
can be confirmed by preparing crude enzyme solutions and measuring
the ferredoxin NADP.sup.+ reductase activity in both the
microorganism before making the modification to enhance activity,
and after making the modification. The activity of ferredoxin
NADP.sup.+ reductase can be measured by, for example, the method of
Blaschkowski et at (Blaschkowski, H. P., Neuer, G., Ludwig-Festl,
M., and Knappe, J. 1989, Eur. J. Biochem., 123:563-569). For
example, the activity can be measured by using ferredoxin as a
substrate to spectroscopically measure the decrease of the amount
of NADPH. One unit (U) of the enzymatic activity is defined as the
activity of oxidizing 1 .mu.mol of NADPH per 1 minute. When the
parent strain has ferredoxin NADP.sup.+ reductase activity, and the
activity of the parent strain is sufficiently high, it is not
necessary to enhance the activity. However, the enzymatic activity
is desirably increased preferably 1.5 times or more, more
preferably 2 times or more, still more preferably 3 times or more,
compared with that of the parent strain.
[0155] Genes encoding ferredoxin NADP.sup.+ reductase are found in
many biological species, and any that have activity in the chosen
L-amino acid producing strain can be used. In Escherichia coli, the
fpr gene has been identified as the gene encoding flavodoxin NADP
reductase (Bianchi, V. Reichard, P., Eliasson, R., Pontis, E.,
Krook, M., Jomvall, H., and Haggard-Ljungquist, E. 1993,
175:1590-1595). Moreover, it is known that, in Pseudomonas putida,
the NADPH-putidaredoxin reductase gene and the putidaredoxin gene
are present as an operon (Koga, H., Yamaguchi, E., Matsunaga, K,
Aramaki, H., and Horiuchi, T. 19089, J. Biochem. (Tokyo),
106:831-836).
[0156] The flavodoxin NADP.sup.+ reductase gene from Escherichia
coli (fpr gene) is located at nucleotide numbers from 4111749 to
4112495 (complementary strand) in the genome sequence of the
Escherichia coli K-12 strain (Genbank Accession No. U00096) and is
shown in SEQ ID NO: 7. The amino acid sequence of Fpr is shown in
SEQ ID NO: 8 (Genbank Accession No. AAC76906). Moreover, the
ferredoxin NADP.sup.+ reductase gene (Genbank Accession No.
BAB99777) is found at the nucleotide numbers from 2526234 to
2527211 of the genome sequence of Corynebacterium glutamicum
(Genbank Accession No. BA00036).
[0157] The pyruvate synthase activity requires the presence of
ferredoxin or flavodoxin, which acts as an electron donor.
Therefore, the microorganism may be modified so that the activity
of pyruvate synthase is increased by improving the production of
ferredoxin or flavodoxin.
[0158] Moreover, the microorganism may also be modified to improve
the production of ferredoxin or flavodoxin, in addition to being
modified to enhance pyruvate synthase activity alone, or enhance
both the activities of flavodoxin NADP.sup.+ reductase and pyruvate
synthase.
[0159] "Ferredoxin" refers to a protein containing nonheme iron
atoms (Fe) and sulfur atoms bound with an iron-sulfur cluster
called 4Fe-4S, 3Fe-4S or 2Fe-2S, and which functions as a
one-electron carrier. "Flavodoxin" refers to a protein containing
FMN (flavin-mononucleotide) as a prosthetic group and which
functions as a one- or two-electron carrier. Ferredoxin and
flavodoxin are described in McLean et al. (McLean K. J., Sabri, M.,
Marshall, K. R, Lawson, R. J., Lewis, D. G., Clift, D., Balding, P.
R., Dunford, A. J., Warman, A. J., McVey, J. P., Quinn, A. M.,
Sutcliffe, M. J., Scrutton, N. S., and Munro, A. W. 2005, Biochem.
Soc. Trans., 33:796-801).
[0160] Ferredoxin or flavodoxin may be native to the parent strains
which are used to derive the modified microorganism described
herein, or a gene encoding ferredoxin or flavodoxin may be
introduced into the parent strains to impart the activity to
produce ferredoxin or flavodoxin, and to improve L-glutamic
producing ability.
[0161] An improvement in the ability to produce ferredoxin or
flavodoxin as compared with the parent strain, such as a wild-type
or non-modified strain, can be confirmed by, for example, comparing
the amount of mRNA for ferredoxin or flavodoxin with that in a
wild-type strain or non-modified strain. The expression amount can
be confirmed by, for example, Northern hybridization and RT-PCR
(Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989, Molecular
Cloning A Laboratory Manual/Second Edition, Cold Spring Harbor
Laboratory Press, New York). The degree of the increase of the
expression is not particularly limited so long as it is increased
compared with that of a wild-type strain or non-modified strain.
However, it is increased, for example, 1.5 times or more,
preferably 2 times or more, more preferably 3 times or more,
compared with that of a wild-type strain or non-modified
strain.
[0162] Whether the ability to produce ferredoxin or flavodoxin is
improved as compared with a parent strain, for example, a wild-type
strain or a non-modified strain, can be detected by SDS-PAGE,
two-dimensional electrophoresis, or Western blotting using
antibodies (Sambrook J., Fritsch, E. F., and Maniatis, T. 1989,
Molecular Cloning A Laboratory Manual/Second Edition, Cold Spring
Harbor Laboratory Press, New York). The degree of improvement is
not particularly limited so long as it is increased as compared
with that of a wild-type strain or non-modified strain. However, it
is increased, for example, 1.5 times or more, preferably 2 times or
more, more preferably 3 times or more, compared with that of a
wild-type strain or non-modified strain.
[0163] The activities of ferredoxin and flavodoxin can be measured
by adding them to a suitable oxidation-reduction reaction system.
For example, reducing ferredoxin with ferredoxin NADP.sup.+
reductase and quantifying the reduction of cytochrome C by the
reduced ferredoxin is disclosed by Boyer et al. (Boyer, M. E. et
al., 2006, Biotechnol. Bioeng., 94:128-138). Furthermore, the
activity of flavodoxin can be measured by the same method, but
using flavodoxin NADP.sup.+ reductase.
[0164] Genes encoding ferredoxin or flavodoxin are known from many
species, and any of these can be used so long as the ferredoxin or
flavodoxin encoded by the genes can be utilized by pyruvate
synthase and an electron donor recycling system. For example, in
Escherichia coli, the fdx gene encodes ferredoxin which has a
2Fe-2S cluster (Ta, D. T. and Vickery, L. E., 1992, J. Biol. Chem.,
267:11120-11125), and the yfhL gene encodes ferredoxin which has a
4Fe-4S cluster. Furthermore, the fldA gene (Osborne C. et al.,
1991, J. Bacteriol., 173:1729-1737) and the fldB gene (Gaudu, P.
and Weiss, B., 2000, J. Bacteriol., 182:1788-1793) are known to
encode flavodoxin. In the genome sequence of Corynebacterium
glutamicum (Genbank Accession No. BA00036), multiple ferredoxin
genes were found at nucleotide numbers from 562643 to 562963
(fdx--Genbank Accession No. BAB97942), and nucleotide numbers from
1148953 to 1149270 (fer--Genbank Accession No. BAB98495).
Furthermore, in Chlorobium tepidum, many ferredoxin genes have been
identified, for example, ferredoxin I and ferredoxin II are of the
4Fe-4S type, which acts as the electron acceptor for pyruvate
synthase (Yoon, K. S., Bobst, C., Hemann, C F., Hille, R, and
Tabita, F. R 2001, J. Biol. Chem., 276:44027-44036). Ferredoxin or
flavodoxin native to or derived from bacteria having the reductive
TCA cycle, such as the ferredoxin gene of Hydrogenobacter
thermophilus, can also be used.
[0165] The ferredoxin gene of Escherichia coli includes the fdx
gene at nucleotide numbers from 2654770 to 2655105 (complementary
strand) in the genome sequence of the Escherichia coli K-12 strain
(Genbank Accession No. U00096) and shown in SEQ ID NO: 9, and the
yfhL gene at nucleotide numbers from 2697685 to 2697945 also from
K-12, and shown in SEQ ID NO: 11. The amino acid sequences of Fdx
and YfhL are shown in SEQ ID NOS: 10 and 12 (Genbank Accession Nos.
AAC75578 and AAC75615, respectively). The flavodoxin gene of
Escherichia coli includes the fldA gene at nucleotide numbers from
710688 to 710158 (complementary strand) in the genome sequence of
the Escherichia coli K-12 strain (Genbank Accession No. U00096) and
shown in SEQ ID NO: 13, and the fldB gene at nucleotide numbers
from 3037877 to 3038398 also from K-12, and shown in SEQ ID NO: 15.
The amino acid sequences encoded by the fldA gene and the fldB gene
are shown in SEQ ID NOS: 14 and 16 (Genbank Accession Nos. AAC73778
and AAC75933, respectively).
[0166] The ferredoxin gene of Chlorobium tepidum includes the
ferredoxin I gene at nucleotide numbers from 1184078 to 1184266 in
the genome sequence of Chlorobium tepidum (Genbank Accession No.
NC.sub.--002932) and shown in SEQ ID NO: 17, and the ferredoxin II
gene at nucleotide numbers from 1184476 to 1184664 also from
Chlorobium tepidum and shown in SEQ ID NO: 19. The amino acid
sequences of ferredoxin I and ferredoxin II are shown in SEQ ID
NOS: 18 and 20 (Genbank Accession Nos. AAM72491 and AAM72490,
respectively). Examples further include the ferredoxin gene of
Hydrogenobacter thermophilus (Genbank Accession No. BAE02673) and
the ferredoxin gene of Sulfolobus solfataricus, which is present at
nucleotide numbers from 2345414 to 2345728 in the genome of
Sulfolobus solfataricus. Furthermore, the gene may be those cloned
from Chlorobium, Desulfobacter, Aquifex, Hydrogenobacter,
Thermoproteus, Pyrobaculum bacteria, or the like on the basis of
homology to the genes exemplified above, or those cloned from
.gamma.-proteobacteria such as those of the genus Enterobacter,
Klebsiella, Serratia, Erwinia, and Yersinia, coryneform bacteria
such as Corynebacterium glutamicum and Brevibacterium
lactofermentum, Pseudomonas bacteria such as Pseudomonas
aeruginosa, Mycobacterium bacteria such as Mycobacterium
tuberculosis, and so forth.
[0167] Any of the genes described herein may have conservative
mutations, and may be homologues or artificially modified genes so
long as the functions of the encoded proteins are not degraded.
That is, the genes described herein may encode a conservative
variant of the proteins having amino acid sequences of the known
proteins or wild-type proteins, and may include one or more
substitutions, deletions, insertions, or additions of one or
several amino acid residues at one or several positions. Although
the number of the "one or several" amino acid residues may differ
depending on their position in the three-dimensional structure or
the types of amino acid residues of the proteins, it is preferably
1 to 20, more preferably 1 to 10, particularly preferably 1 to
5.
[0168] These substitutions are preferably conservative
substitutions that are neutral mutations so to preserve the
function of the protein. A conservative mutation is a mutation
wherein substitution takes place mutually among Phe, Trp and Tyr,
if the substitution site is an aromatic amino acid; among Leu, Ile
and Val, if the substitution site is a hydrophobic amino acid;
between Gln and Asn, if it is a polar amino acid; among Lys, Arg
and His, if it is a basic amino acid; between Asp and Glu, if it is
an acidic amino acid; and between Ser and Thr, if it is an amino
acid having a hydroxyl group.
[0169] Specific examples of conservative substitutions include:
substitution of Ser or Thr for Ala; substitution of Gln, His or Lys
for Arg; substitution of Glu, Gln, Lys, His or Asp for Asn;
substitution of Asn, Glu or Gln for Asp; substitution of Ser or Ala
for Cys; substitution of Asn, Glu, Lys, His, Asp or Arg for Gln;
substitution of Gly, Asn, Gln, Lys or Asp for Glu; substitution of
Pro for Gly; substitution of Asn, Lys, Gln, Arg or Tyr for His;
substitution of Leu, Met, Val or Phe for Ile; substitution of Be,
Met, Val or Phe for Leu; substitution of Asn, Glu, Gln, His or Arg
for Lys; substitution of Be, Leu, Val or Phe for Met; substitution
of Tip, Tyr, Met, Be or Leu for Phe; substitution of Thr or Ala for
Ser; substitution of Ser or Ala for Thr; substitution of Phe or Tyr
for Trp; substitution of His, Phe or Tip for Tyr; and substitution
of Met, Be or Leu for Val. The above-mentioned amino acid
substitution, deletion, insertion, addition, inversion etc. may be
the result of a naturally-occurring mutation or variation due to an
individual difference, or a difference of species of a
bacterium.
[0170] Furthermore, a gene may be used which has codon
substitutions that can be easily used in the chosen host into which
the gene is introduced. Similarly, so long as the gene maintains
its function, it may be extended or shortened at either the
N-terminus and/or C-terminus by, for example, 50 or less,
preferably 20 or less, more preferably 10 or less, particularly
preferably 5 or less, of the number of amino acid residues.
[0171] A gene encoding a conservative variant can be obtained by,
for example, modifying the nucleotide sequence by site-specific
mutagenesis so that the encoded protein includes substitutions,
deletions, insertions, or additions of amino acid residues at
specific sites. Furthermore, it can also be obtained by the
conventionally known mutagenesis techniques, such as by treating
the gene with hydroxylamine or the like in vitro and irradiating
the microorganism containing the gene with ultraviolet light, or
treating the microorganism with a known mutagen such as
N-methyl-N-nitro-N-nitrosoguanidine (NTG) or ethyl methanesulfonate
(EMS). Moreover, the substitutions, deletions, insertions,
additions, inversions etc. of amino acid residues as described
above include those due to a naturally occurring mutation or
variation based on the difference of individuals or species of the
microorganism containing the gene. Whether the gene(s) encodes
pyruvate synthase, ferredoxin-NADP.sup.+ reductase, ferredoxin, or
flavodoxin can be confirmed by, for example, introducing each gene
into a microorganism, and measuring the activity of each
protein.
[0172] The gene may be a DNA which hybridizes with a DNA having any
one of the aforementioned nucleotide sequences, or a probe prepared
from a DNA which has any one of these nucleotide sequences, under
stringent conditions and which encodes pyruvate synthase,
ferredoxin-NADP.sup.+ reductase, ferredoxin, or flavodoxin.
[0173] The term "stringent conditions" refers to conditions when a
so-called specific hybrid is formed and a non-specific hybrid is
not formed. Examples thereof include conditions where DNAs having
high homology, for example, at least 70%, preferably 80%, more
preferably 90%, and further more preferably 95% homology, hybridize
with each other and DNAs having homology less than the value do not
hybridize with each other; and specifically include conditions
corresponding to a salt concentration and temperature of washing
which are typical of Southern hybridization, e.g., washing at
60.degree. C., 1.times.SSC, 0.1% SDS, preferably 60.degree. C.,
0.1.times.SSC, 0.1% SDS, more preferably 68.degree. C.,
0.1.times.SSC, 0.1% SDS, once or preferably twice or three
times.
[0174] The probe may have a partial sequence of the gene. Such a
probe can be prepared by PCR using oligonucleotides prepared based
on the nucleotide sequence of each gene as primers according to a
method well known to a person skilled in the art, and a DNA
fragment containing each gene as the template. When a DNA fragment
of a length of about 300 by is used as the probe, the conditions of
washing after hybridization can be, for example, 50.degree. C.,
2.times.SSC, and 0.1% SDS.
[0175] The aforementioned descriptions concerning the conservative
variant is also applied to the enzymes and genes described above
which are used to impart L-amino acid-producing ability.
[0176] The modification for enhancing expression of the gene can be
performed in the same manner as that described to enhance the
expression of a target gene which is used to impart the L-amino
acid-producing ability. The gene can be obtained by PCR using the
chromosomal DNA of the microorganism as the template.
[0177] For example, the pyruvate synthase gene of Chlorobium
tepidum can be obtained by PCR (polymerase chain reaction) (see
White, T. J., Arnheim, N., and Erlich, H. A. 1989, Trends Genet,
5:185-189) using primers prepared on the basis of the nucleotide
sequence of SEQ ID NO: 1, for example, the primers shown in SEQ ID
NOS: 35 and 36, and using the chromosomal DNA of Chlorobium tepidum
as the template.
[0178] The pyruvate synthase gene of Escherichia coli can be
obtained by PCR using primers prepared on the basis of the
nucleotide sequence of SEQ ID NO: 3, for example, the primers shown
in SEQ ID NOS: 38 and 39, and the chromosomal DNA of Escherichia
coli as the template.
[0179] The NADP.sup.+ oxidoreductase gene of Euglena gracilis can
be obtained by PCR using primers prepared on the basis of the
nucleotide sequence of SEQ ID NO: 5, for example, the primers shown
in SEQ ID NOS: 40 and 41, and the chromosomal DNA of Euglena
gracilis as the template.
[0180] The flavodoxin NADP.sup.+ reductase gene of Escherichia coli
can be obtained by PCR using primers prepared on the basis of the
nucleotide sequence of SEQ ID NO: 7, for example, the primers shown
in SEQ ID NOS: 42 and 43, and the chromosomal DNA of Escherichia
coli as the template.
[0181] The ferredoxin gene fdx of Escherichia coli can be obtained
by PCR using primers prepared on the basis of the nucleotide
sequence of SEQ ID NO: 9, for example, the primers shown in SEQ ID
NOS: 44 and 45, and the chromosomal DNA of Escherichia coli as the
template.
[0182] The flavodoxin gene fldA of Escherichia coli can be obtained
by PCR using primers prepared on the basis of the nucleotide
sequence of SEQ ID NO: 13, and the flavodoxin gene fldB of
Escherichia coli can be obtained by PCR using primers prepared on
the basis of the nucleotide sequence of SEQ ID NO: 15, and the
chromosomal DNA of Escherichia coli as the template,
respectively.
[0183] Furthermore, the ferredoxin I gene of Chlorobium tepidum can
be obtained by PCR using primers prepared on the basis of the
nucleotide sequence of SEQ ID NO: 17, and the ferredoxin II gene of
Chlorobium tepidum can be obtained by PCR using primers prepared on
the basis of the nucleotide sequence of SEQ ID NO: 19, with using
the chromosomal DNA of Chlorobium tepidum as the template in both
cases.
[0184] Genes derived from other microorganisms can also be obtained
from the chromosomal DNA or a chromosomal DNA library from the
chosen microorganism by PCR using, as primers, oligonucleotides
prepared based on the sequences of the aforementioned gene or
sequences of genes or proteins known in the chosen microorganism;
or hybridization using an oligonucleotide prepared based on such
sequence as mentioned above as a probe. A chromosomal DNA can be
prepared from a microorganism that serves as a DNA donor by the
method of Saito and Miura (Saito H. and Miura K., 1963, Biochem.
Biophys. Acta, 72:619-629; Experiment Manual for Biotechnology,
edited by The Society for Biotechnology, Japan, p97-98, Baifukan
Co., Ltd., 1992) or the like.
[0185] The expression of the gene and genes of L-amino acid
synthesis systems can be increased by increasing the copy number of
the gene by transformation or homologous recombination, or
modifying an expression control sequence of the gene as described
above. Furthermore, the expression of the gene can also be
increased by amplifying an activator which increases expression of
the gene, and/or by eliminating or attenuating a regulator which
reduces expression of the gene.
[0186] Methods for increasing gene expression will be explained
below.
[0187] To increase the copy number of a target gene, for example,
the gene can be cloned on an appropriate vector and then used to
transform a host microorganism.
[0188] The vector used for transformation may be a plasmid which
autonomously replicates in the host microorganism. Examples of a
plasmid which is able to autonomously replicate in
Enterobacteriaceae include pUC19, pUC18, pBR322, RSF1010, pHSG299,
pHSG298, pHSG399, pHSG398, pSTV28, pSTV29 (pHSG and pSTV vectors
are available from Takara Bio Inc.), pMW119, pMW118, pMW219, pMW218
(pMW vectors are available from Nippon Gene Co., Ltd.), and so
forth. Furthermore, plasmids for coryneform bacteria include pAM330
(Japanese Patent Laid-open No. 58-67699), pHM1519 (Japanese Patent
Laid-open No. 58-77895), pSFK6 (Japanese Patent Laid-open No.
2000-262288), pVK7 (USP2003-0175912A), pAJ655, pAJ611, pAJ1844
(Japanese Patent Laid-open No. 58-192900), pCG1 (Japanese Patent
Laid-open No. 57-134500), pCG2 (Japanese Patent Laid-open No.
58-35197), pCG4, pCG11 (Japanese Patent Laid-open No. 57-183799),
pHK4 (Japanese Patent Laid-open No. 5-7491), and so forth.
Moreover, a DNA fragment which is able to impart the ability to
autonomously replicate to a plasmid in a coryneform bacterium can
be cut from these vectors and inserted into the aforementioned
vectors for Escherichia coli, and then can be used as a so-called
shuttle vector which is able to autonomously replicate in both
Escherichia coli and coryneform bacteria. In addition, a phage DNA
may also be used as the vector instead of a plasmid.
[0189] Examples of transformation methods include treating
recipient cells with calcium chloride so to increase permeability
of the DNA, which has been reported for Escherichia coli K-12
(Mandel, M. and Higa, A., 1970, J. Mol. Biol., 53:159-162), and
preparing competent cells from cells which are at the growth phase,
followed by transformation with DNA, which has been reported for
Bacillus subtilis (Duncan, C. H., Wilson, G. A. and Young, F. E.
1977, Gene, 1:153-167). Alternatively, a method of making
DNA-recipient cells into protoplasts or spheroplasts, which can
easily take up recombinant DNA, followed by introducing the
recombinant DNA into the cells, which is known to be applicable to
Bacillus subtilis, actinomycetes and yeasts (Chang, S, and Choen,
S. N., 1979, Mol. Gen. Genet, 168:111-115; Bibb, M. J. et al.,
1978, Nature, 274:398-400; Hinnen, A., Hicks, J. B. and Fink, G. R.
1978, Proc. Natl. Sci., USA, 75:1929-1933) can also be employed. In
addition, microorganisms can also be transformed by the electric
pulse method (Japanese Patent Laid-open No. 2-207791).
[0190] The copy number of the target gene can also be increased by
introducing multiple copies of the gene into the chromosomal DNA of
the microorganism by homologous recombination (Milled, J. H.
Experiments in Molecular Genetics, 1972, Cold Spring Harbor
Laboratory) using multiple copies of a sequence as targets in the
chromosomal DNA. Sequences present in multiple copies on the
chromosomal DNA include, but are not limited to, repetitive DNAs,
and inverted repeats present at the end of a transposable element.
Also, as disclosed in Japanese Patent Laid-open No. 2-109985, it is
possible to incorporate the target gene into a transposon, and
allow it to be transferred to introduce multiple copies of the gene
into the chromosomal DNA. The target gene can also be introduced
into the bacterial chromosome by Mu phage (Japanese Patent
Laid-open No. 2-109985), or the like. Transfer of a target gene to
a chromosome can be confirmed by Southern hybridization using a
part of the gene as a probe.
[0191] When the copy number of a gene is increased, the copy number
is not particularly limited so long as activity of the product of
the target gene is enhanced. However, when the target gene is
native to the chosen microorganism, the copy number is preferably 2
or more. When the target gene is not native to the chosen
microorganism, the copy number of the gene may be 1, but it may
also be 2 or more.
[0192] Expression of the target gene may also be increased by
replacing an expression regulatory sequence of the target gene,
such as promoter, on the chromosomal DNA or plasmid with a promoter
which has an appropriate strength. For example, the thr promoter,
lac promoter, tip promoter, trc promoter, pL promoter, tac
promoter, etc., are known as promoters frequently used to increase
expression of a target gene. Examples of strong promoters and
methods for evaluating the strength of promoters are described in
an article by Goldstein and Doi (Goldstein, M. A. and Doi R. H.,
1995, Biotechnol. Annu. Rev., 1:105-128), etc.
[0193] Moreover, it is also possible to substitute several
nucleotides in the promoter region of a gene, so that the promoter
has an appropriate strength, as disclosed in International Patent
Publication WO00/18935. Substitution of the expression regulatory
sequence can be performed, for example, in the same manner as in
gene substitution using a temperature-sensitive plasmid. Examples
of vectors having a temperature-sensitive replication origin which
can be used for Escherichia coli or Pantoea ananatis include, for
example, the temperature-sensitive plasmid pMAN997 described in
International Publication WO99/03988, its derivatives, and so
forth. Furthermore, substitution of an expression regulatory
sequence can also be performed by methods which employ linear DNA,
such as "Red-driven integration" using Red recombinase of .lamda.
phage (Datsenko, K. A. and Wanner, B. L., 2000, Proc. Natl. Acad.
Sci. USA., 97:6640-6645), by combining the Red-driven integration
method and the .lamda. phage excision system (Cho, E. H., Gumport,
R. I., Gardner, J. F. 2002, J. Bacteriol., 184:5200-5203)
(WO2005/010175), and so forth. The modification of an expression
regulatory sequence can be combined with increasing gene copy
number described above.
[0194] Furthermore, it is known that substitution of several
nucleotides in a spacer between the ribosome binding site (RBS) and
the start codon, in particular, the sequences immediately upstream
of the start codon, profoundly affects the mRNA translatability.
Translation can be enhanced by modifying these sequences.
[0195] When pyruvate synthase consists of multiple subunits, the
expression of the genes encoding the subunits may be individually
enhanced, or may be simultaneously enhanced as a polycistron.
Furthermore, when the genes are introduced into a microorganism by
using a vector, the genes encoding the subunits may be carried on a
single vector molecule, or may be separately carried on different
vector molecules. Also when the genes encoding the subunits are
inserted into the chromosome, the genes may be simultaneously
inserted into the same site on the genome, or may be separately
inserted at different sites.
[0196] Furthermore, pyruvate dehydrogenase activity may be reduced,
in addition to enhancing pyruvate synthase activity or
pyruvate:NADH.sup.+ oxidoreductase activity. Pyruvate dehydrogenase
(henceforth also referred to as "PDH") activity means an activity
for catalyzing the reaction of oxidatively decarboxylating pyruvic
acid to produce acetyl-CoA. The aforementioned reaction is
catalyzed by three kinds of enzymes, PDH (E1p, pyruvate
dehydrogenase, EC:1.2.4.1, aceE gene, SEQ ID NO: 46), dihydrolipoyl
transacetylase (E2p, EC:2.3.1.12, aceF gene, SEQ ID NO: 48), and
dihydrolipoamide dehydrogenase (E3, EC:1.8.1.4, lpdA gene, SEQ ID
NO: 50). That is, these three subunits catalyze the following
reactions, respectively, and the activity for catalyzing the total
reaction resulting from these three reactions is called PDH
activity. PDH activity can be measured according to the method of
Visser and Strafing (Visser, J. and Strafing, M., 1982, Methods
Enzymol., 89:391-399).
Pyruvate+[dihydrolipoyllysine-residue
succinyltransferase]lipoyllysine=[dihydrolipoyllysine-residue
acetyltransferase]S-acetyldihydrolipoyllysine+CO.sub.2 E1p
CoA+enzyme N6-(S-acetyldihytholipoyblysine=acetyl-CoA+enzyme
N6-(dihydrolipoyl)lysine E2p
Protein N6-(dihydrolipoyl)lysine+NAD.sup.+=protein
N6-(lipoyl)lysine+NADH+H.sup.+ E3
[0197] To decrease or eliminate enzyme activity, for example, a
part of or the entire coding region may be deleted from one or more
of the aceE, aceF and lpdA genes, or an expression control sequence
such as a promoter or Shine Dargarno (SD) sequence can be modified,
or the like. The expression can also be reduced by modifying a
non-translation region other than expression control regions.
Furthermore, the entire gene, including the upstream and downstream
regions of the genes on the chromosome, may be deleted. In
addition, an amino acid substitution (missense mutation), a stop
codon (nonsense mutation), or a frame shift mutation which adds or
deletes one or two nucleotides may be introduced into the enzyme
coding region on the chromosome by genetic recombination (Journal
of Biological Chemistry, 272:8611-8617 (1997), Proceedings of the
National Academy of Sciences, USA, 95 5511-5515 (1998), Journal of
Biological Chemistry, 266, 20833-20839 (1991)).
[0198] To reduce the intracellular enzymatic activity, a part or
all of an expression control sequence such as promoter region, a
coding region or a non-coding region of the gene on the chromosome
may be deleted, or another sequence may be inserted into these
regions by homologous recombination. However, these modifications
may be accomplished by known mutatagenesis techniques, such as
exposure to X-rays or UV irradiation, or treatment with a mutagen
such as N-methyl-N'-nitro-N-nitrosoguanidine, etc., so long as the
PDH activity is reduced by the modification.
[0199] The expression control sequence is preferably modified by
one or more nucleotides, more preferably two or more nucleotides,
particularly preferably three or more nucleotides. When a coding
region is deleted, it may be in the N-terminus region, an internal
region, or the C-terminus region, or even the entire coding region,
so long as the function of the enzyme protein is reduced. Deletion
of a longer region will usually ensure inactivation of the gene.
Furthermore, the reading frames upstream and downstream of the
deleted region are not preferably the same.
[0200] Also, when another sequence is inserted into the coding
region, the sequence may be inserted anywhere, and inserting a
longer region will usually ensure inactivation of the gene. The
reading frames upstream and downstream of the insertion site are
not preferably the same. The other sequence is not particularly
limited so long as the sequence reduces or deletes the function of
the enzyme protein, and examples include a transposon carrying an
antibiotic resistance gene or a gene useful for L-amino acid
production.
[0201] A gene on the chromosome can be modified as described above
by, for example, preparing a deletion-type version of the gene in
which a partial sequence of the gene is deleted, and transforming a
bacterium with a DNA containing the deletion-type gene to cause
homologous recombination between the deletion-type gene and the
native gene on the chromosome, and thereby substitute the
deletion-type gene for the gene on the genome. The enzyme protein
encoded by the deletion-type gene has a conformation different from
that of the wild-type enzyme protein, if it is even produced, and
thus the function is reduced or deleted. These types of gene
disruption can be performed by methods using a linear DNA such as
Red-driven integration, and Red-driven integration in combination
with an excision system derived from .lamda. phage, or by using a
plasmid containing a temperature-sensitive replication origin, or a
plasmid capable of conjugative transfer, utilizing a suicide vector
which does not have a replication origin usable in the chosen host
(U.S. Pat. No. 6,303,383, JP 05-007491 A) etc.
[0202] The aforementioned description concerning reduction of the
PDH activity is also applied to "reduction of activity" of the
other enzymes described above, or "destruction" of the other genes
described above.
[0203] When the microorganism is cultured under anaerobic or
microaerobic conditions, it may be already have been modified so
that it does not produce any organic acid or ethanol under the
anaerobic or microaerobic conditions, in addition to enhancing the
pyruvate synthase activity or pyruvate: NADH.sup.+ oxidoreductase
activity. Examples of the organic acids include lactic acid, formic
acid, and acetic acid. The method for modifying a microorganism so
that organic acid or ethanol is not produced include by disrupting
the gene encoding lactate dehydrogenase (Verumi, G. N. et al.,
2002, J. Industrial Microbiol. Biotechnol., 28:325-332; Japanese
Patent Laid-open No. 2005-95169).
[0204] <2> Method for Producing an L-Amino Acid
[0205] The microorganism is cultured in a medium to produce and
cause accumulation of an L-amino acid in the medium or cells, and
collecting the L-amino acid from the medium or cells.
[0206] A batch culture, fed-batch culture, and/or continuous
culture may be used. Ethanol or an aliphatic acid may be added to
the starting medium or feed medium, or both.
[0207] A fed-batch culture refers to a culture method in which the
medium is continuously or intermittently fed into the culture
vessel, and the medium is not extracted until the end of the
culture. A continuous culture means a method in which the medium is
continuously or intermittently fed into the culture vessel, and the
medium is extracted from the vessel (usually in a volume equivalent
to the volume of the fed medium) at the same time. A starting
medium indicates the medium used in the batch culture, the
fed-batch culture, or continuous culture before feeding the feed
medium, that is, the medium used at the start of the culture. A
feed medium indicates the medium which is supplied to the
fermentation tank in the fed-batch culture or continuous culture. A
batch culture means a method in which fresh medium is prepared for
every culture, and the strain is inoculated into the medium, and
the medium is not added until harvest.
[0208] A substance from which acetyl-CoA can be produced without a
decarboxylation reaction is preferred as the carbon source, and
specific examples include ethanol, aliphatic acids, aliphatic acid
esters including fats and oils which generate an aliphatic acid
upon decomposition, and so forth. Examples of using ethanol or an
aliphatic acid as the carbon source will be described below.
[0209] Ethanol is a monohydric alcohol represented by the molecular
formula C.sub.2H.sub.5OH, and may be used alone, or may be present
as a mixture in the medium, such as the ethanol which is produced
in ethanol fermentation in the medium etc.
[0210] Aliphatic acids are monovalent carboxylic acids represented
by the general formula C.sub.mH.sub.nCOOH. So long as it is able to
be assimilated by the bacteria having L-amino acid-producing
ability, it may be of any length, and may contain aliphatic acids
of any length at any ratio. Preferred aliphatic acids are oleic
acid (C.sub.17H.sub.33COOH) and palmitic acid
(C.sub.15H.sub.31COOH), and oleic acid is particularly preferred. A
mixture of long chain aliphatic acids containing oleic acid can be
obtained by hydrolysis of fats and oils. Oleic acid can be obtained
as a hydrolysate of fats and oils such as palm oil, and oleic acid
extracted from animal oils, vegetable oils, waste cooking oils,
other blended fats and oils, or foodstuffs containing fats such as
chocolate may be used. The aliphatic acid may be a free acid, or an
alkali metal salt, such as sodium salts and potassium salts, or an
ammonium salt.
[0211] Ethanol or aliphatic acids may be present in the medium at
any concentration so long as the chosen bacterium can assimilate it
as the carbon source. When it is used as the sole carbon source in
the medium, it is present in an amount of 20% w/v or less, more
preferably 10% w/v or less, still more preferably 2% w/v or less.
Furthermore, ethanol or aliphatic acids may be present in the
medium at any concentration so long as it can be assimilated as the
carbon source by the chosen bacterium. When it is used as the sole
carbon source in the medium, it is desirably present in the medium
in an amount of 0.001% w/v or more, preferably 0.05% w/v or more,
more preferably 0.1% w/v or more.
[0212] As for the feed medium, when ethanol or aliphatic acid is
used as the sole carbon source, it is preferably present in the
medium in an amount of 10% w/v or less, more preferably 5% w/v or
less, still more preferably 1% w/v or less, and it is preferably
present in the medium in an amount of 0.001% w/v or more, more
preferably 0.05% w/v or more, still more preferably 0.1% w/v or
more.
[0213] Although the concentration of ethanol can be measured by
various methods, the enzymatic method is convenient and common
(Swift R., 2003, Addiction, 98:73-80). The concentration of
aliphatic acid can be measured by known methods such as gas
chromatography and HPLC (TrAC Trends Anal. Chem., 2002, 21:686-697;
Lin J. T., Snyder L. R., and McKeon, T. A., 1998, J. Chromatogr.
A., 808:43-49).
[0214] Furthermore, the medium may contain a mixture of ethanol and
an aliphatic acid. The concentrations of ethanol and aliphatic acid
which are added may be any concentration so long as the chosen
bacterium can assimilate them as the carbon source. However, when a
mixture of ethanol and an aliphatic acid is used as the sole carbon
source in the medium, it is preferably present in an amount of 20%
w/v or less, more preferably 10% w/v or less, still more preferably
2% w/v or less, in terms of the total concentration. Furthermore, a
mixture of ethanol and an aliphatic acid may be present in the
medium at any concentration so long as it can be assimilated as the
carbon source by the bacterium. However, when a mixture of ethanol
and an aliphatic acid is used as the sole carbon source in the
medium, it is desirably contained in the medium in an amount of
0.001% w/v or more, preferably 0.05% w/v or more, more preferably
0.1% w/v or more, in terms of the total concentration of ethanol
and oleic acid.
[0215] Any ratio of ethanol and aliphatic acid may be present so
long as they are at such concentrations that the chosen bacteria
can assimilate them as the carbon source. However, the aliphatic
acid is generally mixed at a ratio of about 2 or less, preferably
about 1.5 or less, preferably about 1 or less, based on ethanol,
which is taken as 1. Although the lower limit of the mixing ratio
of the aliphatic acid is not particularly limited in the case of
mixing the aliphatic acid, the aliphatic acid is preferably mixed
at a ratio of 0.05 or more, desirably 0.1 or more, based on
ethanol, which is taken as 1.
[0216] In addition to ethanol or aliphatic acid, or both, other
carbon sources may also be added to the medium, for example, such
as saccharides such as glucose, fructose, sucrose, lactose,
galactose, blackstrap molasses, and starch hydrolysate, polyhydric
alcohols such as glycerol, and organic acids such as fumaric acid,
citric acid, and succinic acid. Glucose, sucrose, fructose, and
glycerol are especially preferred. As glycerol, crude glycerol
produced in biodiesel fuel production can also be used. The carbon
source may be one kind of substance or a mixture of two or more
kinds of substances. When other carbon sources are used, the ratio
of ethanol, aliphatic acid, or a mixture of ethanol and aliphatic
acid in the carbon source is preferably 10% by weight or more, more
preferably 30% by weight or more, still more preferably 50% by
weight or more.
[0217] Ethanol or aliphatic acid may be present at a certain
constant concentration throughout the culture process, or it may be
added only to the starting medium or the feed medium. If other
carbon sources are sufficient, there may be a period when ethanol
or aliphatic acid temporarily runs short. The term "temporarily"
means that, for example, the aliphatic acid may run short for a
period corresponding to 10%, 20%, or 30% at most, of the entire
fermentation period.
[0218] As for the other components to be added to the medium,
typical media ingredients such as a nitrogen source, inorganic
ions, and if needed, other organic components in addition to the
carbon source can be used. Examples of the nitrogen source present
in the medium include ammonia, ammonium salts such as ammonium
sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate,
ammonium acetate and urea, nitrates, and so forth. Ammonia gas and
aqueous ammonia used to adjust the pH can also be utilized as the
nitrogen source. Furthermore, peptone, yeast extract, meat extract,
malt extract, corn steep liquor, soybean hydrolysate, and so forth
can also be utilized. The medium may contain one or more of these
nitrogen sources. These nitrogen sources can also be used for both
the starting medium and the feed medium. Furthermore, the same
nitrogen source can be used for both the starting medium and the
feed medium, or the nitrogen source of the feed medium may be
different from that of the starting medium.
[0219] The medium preferably contains a phosphoric acid source and
a sulfur source in addition to the carbon source, the nitrogen
source, and sulfur. As the phosphoric acid source, potassium
dihydrogenphosphate, dipotassium hydrogenphosphate, phosphate
polymers such as pyrophosphoric acid and so forth can be utilized.
Although the sulfur source may be any substance containing sulfur
atoms, sulfuric acid salts such as sulfates, thiosulfates and
sulfites, and sulfur-containing amino acids such as cysteine,
cystine and glutathione are desirable, and ammonium sulfate is
especially desirable.
[0220] Furthermore, the medium may contain a growth promoting
factor, such as a nutrient with a growth promoting effect, in
addition to the carbon source, nitrogen source and sulfur. As the
growth promoting factor, trace metals, amino acids, vitamins,
nucleic acids as well as peptone, casamino acid, yeast extract,
soybean protein degradation product and so forth containing the
foregoing substances can be used. Examples of the trace metals
include iron, manganese, magnesium, calcium, and so forth. Examples
of the vitamins include vitamin B.sub.1, vitamin B.sub.2, vitamin
B.sub.6, nicotinic acid, nicotinamide, vitamin B.sub.12, and so
forth. These growth promoting factors may be present in the
starting medium or the feed medium.
[0221] Furthermore, when an auxotrophic mutant that requires an
amino acid or the like for growth thereof is used, it is preferable
to supplement the required nutrient to the medium. In particular,
since the L-lysine biosynthetic pathway is enhanced and L-lysine
degrading ability is often attenuated in L-lysine-producing
bacteria, one or more of L-threonine, L-homoserine, L-isoleucine,
and L-methionine are preferably added. The starting medium and the
feed medium may have the same or different medium composition.
Furthermore, when the feed medium is fed at multiple stages, the
compositions of the feed medium fed at the various stages may be
the same or different.
[0222] The culture is preferably performed as an aeration culture
at a fermentation temperature of 20 to 45.degree. C., particularly
preferably at 33 to 42.degree. C. The oxygen concentration is
adjusted to 5 to 50%, desirably about 10%. Furthermore, the
aeration culture is preferably performed with the pH adjusted to 5
to 9. If pH is lowered during the culture, for example, calcium
carbonate or an alkali such as ammonia gas and aqueous ammonia is
added to neutralize the culture. When culture is performed under
such conditions preferably for about 10 to 120 hours, a marked
amount of L-amino acid accumulates in the culture medium. Although
the concentration of L-amino acid which accumulates is not limited
so long as it is higher than that observed with wild-type strains
and the L-amino acid can be isolated and collected from the medium,
it may be 50 g/L or higher, desirably 75 g/L or higher, more
desirably 100 g/L or higher.
[0223] When the target amino acid is a basic amino acid, the
fermentation is performed with the pH of the medium controlled to
be 6.5 to 9.0 during the culture and to be 7.2 to 9.0 at the end of
the culture. Furthermore, the internal pressure in the fermentation
tank is controlled to be positive during the fermentation, or
carbon dioxide or a mixed gas containing carbon dioxide is supplied
to the medium so that there is a culture period that bicarbonate
ions and/or carbonate ions are present in an amount of 2 g/L or
larger in the medium, and thereby the bicarbonate ions and/or
carbonate ions can be used as counter ions of cations mainly
consisting of the basic amino acid (refer to JP 2002-065287 A).
[0224] The L-amino acid can be collected by a known collection
method from the culture medium after the culture. For example, the
L-amino acid can be collected by an ion exchange resin method or
precipitation method, or after the bacterial cells are removed from
the culture medium by centrifugation or the like, the L-amino acid
is collected by concentration for crystallization.
[0225] The culture of the microorganism may be performed as a seed
culture and a main culture in order to ensure accumulation of the
L-amino acid higher than a certain level. The seed culture may be
performed as a shaking culture using a flask or the like, or batch
culture, and the main culture may be performed as fed-batch culture
or continuous culture. Alternatively, both the seed culture and the
main culture may be performed as batch culture.
[0226] When a fed-batch culture or continuous culture is performed,
the feed medium may be intermittently fed so that the supply of
ethanol, aliphatic acid or other carbon sources is temporarily
stopped. The supply of the feed medium is preferably stopped for,
at maximum, 30% or less, desirably 20% or less, particularly
desirably 10% or less, of the feeding time. When the feed medium is
intermittently fed, the feed medium may be initially added over a
predetermined time, and the second and following additions may be
controlled so that it is started when pH increases or the dissolved
oxygen concentration is detected by a computer upon depletion of
the carbon source in the fermentation medium during an
addition-stopped period prior to a certain medium-addition period,
and thus the substrate concentration in the culture tank is always
automatically maintained at a low level (U.S. Pat. No.
5,912,113).
[0227] The feed medium used for the fed-batch culture preferably
contains ethanol or an aliphatic acid, another carbon source, and a
nutrient having a growth promoting effect (growth promoting
factor), and may be controlled so that the concentration of the
aliphatic acid in the fermentation medium is at a predetermined
concentration or lower. The expression "predetermined concentration
or lower" means that the medium is prepared so that the aliphatic
acid concentration in the medium becomes 10% w/v or lower,
preferably 5% w/v or lower, more preferably 1% w/v or lower.
[0228] As the other carbon source, glucose, sucrose, fructose and
glycerol are preferred. As the growth promoting factor, nitrogen
sources, phosphoric acid, amino acids and so forth are preferred.
As the nitrogen source, ammonia, ammonium salts such as ammonium
sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate,
ammonium acetate and urea, nitrates and so forth can be used.
Furthermore, as the phosphoric acid source, potassium
dihydrogenphosphate and dipotassium hydrogenphosphate can be used.
As for the amino acids, when an auxotrophic mutant strain is used,
it is preferable to supplement the required nutrient. Furthermore,
the feed medium may consist of one type of medium, or a mixture of
two or more types of media. When two or more types of feed media
are used, the media may be mixed and fed by using one feed can, or
the media may be separately fed by using two or more feed cans.
[0229] When the continuous culture method is used for the present
invention, the medium may be extracted and fed simultaneously, or a
part of the medium may be extracted, and then the medium may be
fed. Furthermore, the method may also be a continuous culture
method in which the culture medium containing L-amino acids and
bacterial cells is extracted, and only the cells are returned to
the fermenter for reuse (French Patent No. 2669935). As the method
for continuously or intermittently feeding a nutrient source, the
same method as used in the fed-batch culture is used.
[0230] The continuous culture method reusing bacterial cells
intermittently or continuously extracts the fermentation medium
when the amino acid concentration reaches a predetermined level,
extracting only L-amino acid and re-circulating filtration residues
containing bacterial cells into the fermenter, and it can be
performed by referring to, for example, French Patent No.
2669935.
[0231] When the culture medium is intermittently extracted, it is
preferred that some of the L-amino acid is extracted when the
L-amino acid concentration reaches a predetermined level, and a
fresh medium is fed to continue the culture. Furthermore, as for
the volume of the medium to be added, the culture is preferably
performed so that the final volume of the medium after the addition
of the medium is equal to the volume of the culture medium before
the extraction. The term "equal" used herein means that the volume
after the addition of the medium corresponds to about 93 to 107% of
the volume of the medium before the extraction.
[0232] When the culture medium is continuously extracted, the
extraction is preferably started at the same time as, or after the
feeding of, the nutrient medium. For example, within 5 hours,
desirably 3 hours, more desirably 1 hour, after the start of the
feeding, the extraction is started. Furthermore, the extraction
volume of the culture medium is preferably equal to the volume of
the fed medium.
EXAMPLES
[0233] Hereinafter, the present invention will be more specifically
explained with reference to the following non-limiting
examples.
Example 1
Construction of Alcohol Dehydrogenase (AdhE) Mutated Strain Derived
from Escherichia coli
[0234] An Escherichia coli strain having mutant alcohol
dehydrogenase AdhE was constructed so as to obtain an aerobically
ethanol assimilable Escherichia coli strain. The nucleotide
sequence of the wild-type AdhE gene (adhE) derived from Escherichia
coli and the encoded amino acid sequence are shown in SEQ ID NOS:
21 and 22, respectively.
[0235] <1-1> Construction of Escherichia coli
MG1655::P.sub.L-tac adhE Strain
[0236] Substitution of the P.sub.L-tac promoter for the promoter
region of the Escherichia coli adhE gene was performed by
"Red-driven integration", which was developed by Datsenko and
Wanner (Datsenko, K. A. and Wanner, B. L., 2000, Proc. Natl. Acad.
Sci. USA., 97:6640-6645) using the excision system derived from
.lamda. phage (Cho, E. H., Gumport, R. I., and Gardner, J. F.,
2002, J. Bacteriol., 184:5200-5203).
[0237] By this technique, a genetic recombinant strain can be
constructed in one step using a PCR product obtained by using
primers designed so as to contain a part of a target gene at the 5'
end and a part of antibiotic resistance gene at the 3' end. By
further using the excision system derived from .lamda. phage in
combination, it is possible to eliminate the antibiotic resistance
gene which had been integrated into the genetic recombinant
strain.
[0238] A fragment containing the P.sub.L-tac promoter and the cat
gene encoding the chloramphenicol resistance (Cm.sup.R) gene was
amplified by PCR using the genome of the Escherichia coli MG1655
P.sub.L-tacxylE strain described in WO2006/043730 as the template
and the primers shown in SEQ ID NOS: 23 and 24. The primer of SEQ
ID NO: 23 has a sequence complementary to the upstream region of
the adhE gene, and the primer of SEQ ID NO: 23 has a sequence
complementary to a 5' region of the adhE gene.
[0239] The sequence of the P.sub.L-tac promoter is shown in SEQ ID
NO: 25. For PCR, Gene Amp PCR System 2700 Amplificatory (Applied
Biosystems) and Taq DNA polymerase (Fermentas) were used. The
amplified fragment was purified and collected by agarose gel
electrophoresis. This fragment was introduced into the Escherichia
coli MG1655/pKD46 strain harboring the plasmid pKD46 having a
temperature-sensitive replication ability by electroporation.
[0240] The strain was grown on M9 medium plates (Sambrook J.,
Fritsch, E. F., and Maniatis, T, 1989, Molecular Cloning A
Laboratory Manual/Second Edition, Cold Spring Harbor Laboratory
Press, New York) containing 2% ethanol for 36 hours, and about 100
clones appeared. PCR amplification was performed using the primers
shown in SEQ ID NOS: 26 and 27, and then the nucleotide sequence of
the amplified product was determined. It was confirmed that one of
the clones contained the Cm.sup.R gene in the promoter region of
the adhE gene, and this clone was cultured at 37.degree. C. to
eliminate the temperature-sensitive plasmid pKD46 and thereby
obtain an MG1655::P.sub.L-tacadhE strain.
[0241] <1-2> Construction of Escherichia coli MG1655AadhE
Strain
[0242] The adhE gene of wild-type Escherichia coli MG1655 (ATCC
700926) was replaced with an inactivated adhE gene by the method
developed by Datsenko and Wanner. A fragment containing the kan
gene encoding the kanamycin resistance (Kan.sup.R) marker was
amplified by PCR using the plasmid pACYC177 (GenBank/EMBL accession
number X06402, Fermentas) as the template and the primers shown in
SEQ ID NOS: 28 and 29. The primer of SEQ ID NO: 28 has a sequence
of 40 bases complementary to the region 318 by upstream of the adhE
gene, and the primer of SEQ ID NO: 29 has the sequence of 41 bases
complementary to the region on the 3' side of the adhE gene. For
PCR, Gene Amp PCR System 2700 Amplificatory (Applied Biosystems)
and Taq DNA Polymerase (Fermentas) were used. The amplified
fragment was purified and collected by agarose gel electrophoresis.
This fragment was introduced into the Escherichia coli MG1655/pKD46
strain harboring the plasmid pKD46 by electroporation.
[0243] PCR amplification was performed by using the primers shown
in SEQ ID NOS: 30 and 31 to confirm the presence of the Km.sup.R
gene in clones grown on the LB plate medium (Sambrook, J., Fritsch,
E. F., and Maniatis, T., 1989, Molecular Cloning A Laboratory
Manual/Second Edition, Cold Spring Harbor Laboratory Press, New
York) containing 20 .mu.g/ml of kanamycin. One of clones confirmed
to contain the Km.sup.R gene in the adhE gene region was cultured
at 37.degree. C. to remove the temperature-sensitive plasmid pKD46
and thereby obtain an MG1655.DELTA.adhE strain.
[0244] <1-3> Construction of Mutant Alcohol Dehydrogenase
(AdhE*)
[0245] In order to introduce the Glu568Lys (E568K) mutation into
AdhE, PCR was performed using the primer of SEQ ID NO: 32 which is
complementary to nucleotide sequences of 1662 to 1701 and 1703 to
1730 of the adhE gene and containing a g->a mutation at the
nucleotide of position 1702, the primer of SEQ ID NO: 33 which is
homologous to the 3' end region of the adhE gene, and the genome of
the Escherichia coli MG1655 strain as the template. For PCR, Gene
Amp PCR System 2700 Amplificatory (Applied Biosystems) and Pyrobest
DNA Polymerase (Takara Shuzo) were used. The amplification fragment
of 1.05 kbp was purified and collected by agarose gel
electrophoresis.
[0246] PCR was performed using the genome of the Escherichia coli
MG1655::P.sub.L-tacadhE strain as the template, the primer shown in
SEQ ID NO: 34 and the 1.05 kbp fragment having the mutation as
another primer. The primer of SEQ ID NO: 34 corresponds to the
sequence from 402 to 425 by upstream from the start codon of the
adhE gene. For PCR, Gene Amp PCR System 2700 Amplificatory (Applied
Biosystems) and TaKaRa LA DNA Polymerase (Takara Shuzo) were used.
The amplification fragment of 4.7 kbp was purified and collected by
agarose gel electrophoresis.
[0247] In order to replace the wild-type adhE gene with the mutant
adhE gene, the 4.7 kbp fragment containing the Cm.sup.R gene and
the mutant adhE gene downstream of the P.sub.L-tac promoter
(cat-P.sub.L-tacadhE*) was introduced into the
MG1655.DELTA.adhE/pKD46 strain by electroporation according to the
method of Datsenko and Wanner. The clones were selected on the M9
plate medium containing 2% ethanol as the sole carbon source. By
sequencing the adhE gene of the grown clone, Glu568Lys (gag-aag),
Ile554Ser (atc-agc), Glu22Gly (gaa-gga), Met236Val (atg-gtg),
Tyr461Cys (tac-tgc) and Ala786Val (gca-gta) were identified, and
this clone was designated MG1655::P.sub.L-tacadhE*.
[0248] The MG1655.DELTA.tdh rhtA* strain was transformed via P1
transduction with P1.sub.vir phage (Miller, J. H., 1972,
Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press,
Plainview, N.Y.) using the Escherichia coli
MG1655::P.sub.L-tacadhE* strain as a donor, and MG1655.DELTA.tdh
rhtA* P.sub.L-tacadhE* was obtained. The MG1655.DELTA.tdh, rhtA*
strain corresponds to the MG1655 strain, but the Oh gene encoding
threonine dehydrogenase is disrupted by the method of Datsenko and
Wanner and a rhtA23 mutation is introduced therein, which imparts
resistance to high concentrations of threonine in a minimal medium
to the rhtA gene (Livshits, V. A., Zakataeva, N. P., Aleshin, V.
V., Vitushkina, M. V., 2003, Res. Microbiol., 154:123-135).
[0249] <1-4> Construction of Alcohol Dehydrogenase (AdhE)
Mutated Strain Derived from Escherichia coli WC196.DELTA.mez
Strain
[0250] In order to impart ethanol assimilability to an
L-lysine-producing bacterium, the L-lysine-producing bacterium
WC196.DELTA.mez/pCABD2 strain described in International Patent
Publication WO2005/010175 was subjected to P1 transduction using
MG1655.DELTA.tdh rhtA adhE* as a donor to obtain a WC196.DELTA.mez
adhE*/pCABD2 strain. pCABD2 is the plasmid described in U.S. Pat.
No. 6,040,160, and has the dapA* gene which imparts resistance to
feedback inhibition by L-lysine, the lysC* gene which imparts
resistance to feedback inhibition by L-lysine, the dapB gene, and
ddh gene.
Example 2
Construction of a Plasmid to Express the Pyruvate Synthase Gene of
Chlorobium tepidum and Measurement of the Activity
[0251] <2-1> Construction of a Plasmid to Express the
Pyruvate Synthase Gene of Chlorobium tepidum
[0252] Chlorobium tepidum is a meso- to thermophilic autotrophic
bacterium, and its optimum growth temperature is 48.degree. C. The
genome sequence of the Chlorobium tepidum TLS strain has been
elucidated by Eisen et al. (Eisen, J. A. et al., 2002, Proc. Natl.
Acad. Sci. USA, 99:9509-9514). The pyruvate synthase gene was
isolated from this strain, and a plasmid expressing it was
constructed.
[0253] <2-2> Measurement of Pyruvate Synthase Activity in a
Strain Expressing the Pyruvate Synthase Gene of Chlorobium
tepidum
[0254] PCR was performed using the chromosomal DNA of the
Chlorobium tepidum TLS strain (ATCC 49652) as the template and the
oligonucleotides shown in SEQ ID NOS: 35 and 36 to amplify a
pyruvate synthase gene fragment. The gene fragment was digested
with Sad, and inserted into the Sad site of pSTV28 (Takara Bio) to
construct a plasmid, which was designated pSTV-PS. After it was
confirmed that the pyruvate synthase gene contained no PCR error
over the full length by using BigDye Terminators v1.1 Cycle
Sequencing Kit, the pyruvate synthase gene was excised from pSTV-PS
with Sad, and inserted into the Sad site of pMW-Pthr to construct
plasmid pMW-Pthr-PS. pMW-Pthr corresponds to the vector pMW219
(Nippon Gene) having the promoter region (Pthr) of the threonine
operon (thrABC) of the Escherichia coli K-12 strain between the
HindIII site and the XbaI site and which is capable of expressing
the gene cloned downstream of the promoter. The promoter sequence
of the chosen threonine operon is shown in SEQ ID NO: 37.
[0255] pMW-Pthr-PS and the control vector pMW-Pthr were introduced
into the WC196.DELTA.cadA.DELTA.ldc/pCABD2 strain by
electroporation, respectively, and transformants were obtained on
the basis of the kanamycin resistance, and the presence of the
plasmids was confirmed. The strain expressing the pyruvate synthase
gene of Chlorobium tepidum was designated
WC196.DELTA.cadA.DELTA.ldc/pCABD2/pMW-Pthr-PS, and the control
strain was designated
WC196.DELTA.cadA.DELTA.ldc/pCABD2/pMW-Pthr.
[0256] The aforementioned strains were each inoculated into LB
medium containing 20 mg/l of streptomycin and 40 mg/l of kanamycin,
and cultured overnight at 37.degree. C. with shaking. The cells
were collected by centrifugation and suspended in a 50 mM HEPES
buffer (pH 8.0). The cells in the suspension were disrupted by
using an ultrasonicator, the suspension was centrifuged at 15000
rpm for 15 minutes, and the supernatant was used as a crude enzyme
solution.
[0257] Protein concentration in the crude enzyme solution was
measured by using Protein Assay CBB Solution (Nakalai Tesque), and
the crude enzyme solution containing 250 .mu.g of the total protein
was used to measure the activity.
[0258] The activity was measured as follows. 2 ml of the following
reaction solution was added to the crude enzyme solution. The
reaction solution containing all the ingredients except for pyruvic
acid was first added to a cell for spectrometry, and the cell was
sealed with a rubber stopper and an aluminum cap. The oxygen
concentration was reduced in the cell by injecting argon gas into
the cell for 5 minutes using a syringe, and then the cell was set
on a spectrophotometer (U-3210 Spectrophotometer, Hitachi). A
pyruvic acid solution was added by using a syringe to start the
reaction. The reaction continued at 37.degree. C. for 30 minutes,
and absorbance was periodically measured at 578 nm to examine the
change in the reduced methylviologen amount. The results are shown
in Table 1. In the table, the unit of the specific activity is U/mg
protein. One unit is defined as activity for reducing 1 nmol of
methylviologen per 1 minute.
[0259] Reaction mixture:
TABLE-US-00001 MgCl.sub.2 1 mM Dithiothreitol 1 mM Methylviologen 5
mM CoA 0.25 mM Pyruvic acid 10 mM (added immediately before start
of measurement) HEPES (pH 8.0) 50 mM
TABLE-US-00002 TABLE 1 Plasmid Specific activity pMW-Pthr 0.0
pMW-Pthr-PS 1.2
Example 3
Construction of a Plasmid to Express the Pyruvate Synthase Gene of
Chlorobium tepidum, Flavodoxin NADP.sup.+ Reductase Gene of
Escherichia coli, and Ferredoxin Gene of Escherichia coli
[0260] By using the flavodoxin NADP.sup.+ reductase gene of
Escherichia coli (fpr) and the ferredoxin gene of Escherichia coli
(fdx) as coenzyme regenerating systems, a plasmid simultaneously
expressing all three genes, including the pyruvate synthase gene,
was constructed.
[0261] <3-1> Construction of a Vector to Amplify the
Flavodoxin NADP.sup.+ Reductase Gene of E. coli
[0262] PCR was performed using the chromosomal DNA of the E. coli
MG1655 strain as the template and the oligonucleotides shown in SEQ
ID NOS: 42 and 43. The gene fragment was digested with SmaI and
inserted into the SmaI site of pMW-Pthr to construct a plasmid for
amplifying the flavodoxin NADP.sup.+ reductase gene, which was
designated pMW-Pthr-fpr.
<3-3> Construction of a Plasmid to Amplify the Ferredoxin
(fdx) Gene of E. coli
[0263] PCR was performed using the chromosomal DNA of the E. coli
MG1655 strain as the template and the oligonucleotides shown in SEQ
ID NOS: 44 and 45. The gene fragment was digested with EcoRI, and
inserted into the EcoRI site of pMW-Pthr to construct a plasmid to
amplify the ferredoxin (fdx) gene, pMW-Pthr-fdx.
[0264] <3-4> Construction of a Plasmid to Amplify the
Pyruvate Synthase Gene of C. tepidum, the Flavodoxin NADP.sup.+
Reductase Gene, and the Ferredoxin (fdx) Gene of E. coli
[0265] pMW-Pthr-Pthr was digested with SmaI, and the fpr gene
fragment was ligated with pMW-Pthr-fdx which had been treated with
SmaI to obtain pMW-Pthr-fpr-fdx. Then, pMW-Pthr-PS was digested
with Sad, and the PS gene fragment was ligated with pMWPthr-fpr-fdx
which had been treated with Sad to construct a plasmid to express
the pyruvate synthase gene of C. tepidum and enhance expression of
the flavodoxin NADP.sup.+ reductase and the ferredoxin (fdx) genes
of E. coli, and was named pMW-Pthr-fpr-PS-fdx.
[0266] In the aforementioned plasmids, the pyruvate synthase gene
of C. tepidum is transcribed from Pthr, and the other genes are
also transcribed by read through from Pthr.
Example 4
Effect on the L-Lysine-Producing Ability of a Strain with Enhanced
Expression of the Pyruvate Synthase Gene of Chlorobium tepidum,
Flavodoxin NADP.sup.+ Reductase Gene of Escherichia coli, and
Ferredoxin Gene of Escherichia coli, Using Oleic Acid as the Carbon
Source
[0267] <4-1> Introduction of the Plasmid to Amplify the
Pyruvate Synthase Gene of Chlorobium tepidum, Flavodoxin NADP.sup.+
Reductase Gene of Escherichia coli, and Ferredoxin Gene of
Escherichia coli into the WC196.DELTA.mez Strain
[0268] pMW-Pthr-Thr-PS-fdx and the control vector pMW-Pthr were
introduced into WC196.DELTA.mez/pCABD2 by electroporation,
respectively, and transformants were obtained on the basis of the
kanamycin resistance, and introduction of the plasmids were
confirmed. The strain expressing the pyruvate synthase gene of
Chlorobium tepidum, the flavodoxin NADP.sup.+ reductase gene of
Escherichia coli and the ferredoxin gene of Escherichia coli was
designated WC196.DELTA.mez/pCABD2/pMW-Pthr-fpr-PS-fdx, and the
control strain was designated WC196.DELTA.mez/p CABD2/pMW-Pthr.
[0269] <4-2> Effect on L-Lysine-Producing Ability of the
Strain with Enhanced Expression of the Pyruvate Synthase Gene of
Chlorobium tepidum, Flavodoxin NADP.sup.+ Reductase Gene of
Escherichia coli, and Ferredoxin Gene of Escherichia coli Using
Oleic Acid as the Carbon Source
[0270] Both WC196.DELTA.mez/pCABD2/pMW-Pthr and
WC196.DELTA.mez/pCABD2/pMW-Pthr-Thr-PS-fdx were inoculated onto the
LB plate medium, respectively, and precultured overnight at
37.degree. C. The cells corresponding to 1/8 of the plate were
inoculated into 20 ml of the oleic acid medium having the following
composition in a 500 ml-volume Sakaguchi flask, and aerobically
cultured at a stirring rate of 120 mm at 37.degree. C. for 72
hours. The L-lysine that accumulated in the medium was measured by
using Biosensor BF-5 (Oji Scientific Instruments). The live cell
count in the medium was also measured. Averages of the values
obtained in the culture performed in duplicate are shown in Table
2. Improvement in L-lysine accumulation was observed for the strain
in which expression of pyruvate synthase gene of Chlorobium
tepidum, flavodoxin NADP.sup.+ reductase gene of Escherichia coli
and ferredoxin gene of Escherichia coli were enhanced, compared
with the control.
[0271] Composition of oleic acid medium:
TABLE-US-00003 Sodium oleate 20 g/L MgSO.sub.4.cndot.7H.sub.2O 1.0
g/L (NH.sub.4).sub.2SO.sub.4 12 g/L KH.sub.2PO.sub.4 0.5 g/L Yeast
extract 1.0 g/L FeSO.sub.4.cndot.7H.sub.2O 0.01 g/L
MnSO.sub.4.cndot.5H.sub.2O 0.01 g/L Kanamycin 40 mg/L Streptomycin
20 mg/L Calcium carbonate 30 g/L pH 7.0 (adjusted with KOH)
Sterilization conditions: 115.degree. C., 10 minutes
TABLE-US-00004 TABLE 2 L-lysine Live cell Strain (g/l) count
(10.sup.8/ml) WC196.DELTA.mez/pCABD2/pMW-Pthr 1.77 22.3
WC196.DELTA.mez/pCABD2/pMW-Pthr- 2.35 13.6 fpr-PS-fdx
Example 5
Construction of a Plasmid to Express the Pyruvate Synthase Gene of
Escherichia coli and Measurement of Activity
[0272] An expression plasmid for the ydbK gene, which is homologous
to the pyruvate synthase gene found in the genome of Escherichia
coli MG1655 strain, was constructed, and the activity was
measured.
[0273] <5-1> Construction of a Plasmid to Express the
Pyruvate Synthase Gene of Escherichia coli
[0274] PCR was performed using the chromosomal DNA of the
Escherichia coli MG1655 strain as the template and the
oligonucleotides shown in SEQ ID NOS: 38 and 39. The gene fragment
was digested with KpnI, and the digested fragment was inserted into
the KpnI site of pSTV28 (Takara Bio) to construct a plasmid, which
was designated pSTV-ydbK After it was confirmed that the pyruvate
synthase gene contained no PCR error over the full length by using
BigDye Terminators v1.1 Cycle Sequencing Kit, the pyruvate synthase
gene was excised from pSTV-ydbK with KpnI, and inserted into the
KpnI site of pMW-Pthr to construct the plasmid pMW-Pthr-ydbK.
[0275] <5-2> Measurement of Pyruvate Synthase Activity in a
Strain Expressing the Pyruvate Synthase Gene of Escherichia
coli
[0276] pMW-Pthr-ydbK and the control vector pMW-Pthr were
introduced into the WC196.DELTA.cadA.DELTA.ldc/pCABD2 strain by
electroporation, respectively, and transformants were obtained on
the basis of the kanamycin resistance, and introduction of the
plasmids was confirmed. The strain expressing the pyruvate synthase
gene of Escherichia coli was designated
WC196.DELTA.cadA.DELTA.ldc/pCABD2/pMW-Pthr-ydbK, and the control
strain was designated
WC196.DELTA.cadA.DELTA.ldc/pCABD2/pMW-Pthr.
[0277] The aforementioned strains were each inoculated into LB
medium containing 20 mg/l of streptomycin and 40 mg/l of kanamycin,
and cultured overnight at 37.degree. C. with shaking. The cells
were collected by centrifugation, and the activity was measured in
the same manner as that for the strain expressing the pyruvate
synthase gene of Chlorobium tepidum described in Example 2. The
results are shown in Table 3. Whereas the activity of pyruvate
synthase was not confirmed for the control strain
WC196.DELTA.cadA.DELTA.ldc/pCABD2/pMW-Pthr, 8.0 U/mg was confirmed
for the strain expressing the pyruvate synthase gene of Escherichia
coli, WC196.DELTA.cadA.DELTA.ldc/pCABD2/pMW-Pthr-ydbK. The results
are shown in Table 3. The unit of the specific activity is the same
as that used in Table 1.
TABLE-US-00005 TABLE 3 Plasmid Specific activity pMW-Pthr 0.0
pMW-Pthr-ydbK 8.0
Example 6
Construction of a Plasmid to Express the Pyruvate Synthase Gene of
Escherichia coli, Flavodoxin NADP.sup.+ Reductase Gene of
Escherichia coli, and Ferredoxin Gene of Escherichia coli
[0278] The plasmid pMW-Pthr-fpr containing the flavodoxin
NADP.sup.+ reductase gene of Escherichia coli described in Example
3 was digested with SmaI, and the obtained fpr gene fragment was
ligated with the plasmid pMW-Pthr-fdx containing the ferredoxin
gene of Escherichia coli treated with SmaI to obtain
pMW-Pthr-Thr-fdx. Then, pMW-Pthr-ydbK was digested with KpnI, and
the ydbK gene fragment was ligated with pMW-Pthr-Thr-fdx treated
with KpnI to construct a plasmid to enhance expression of the
pyruvate synthase gene of Escherichia coli, flavodoxin NADP.sup.+
reductase gene of Escherichia coli and ferredoxin fdx gene,
pMW-Pthr-fpr-ydbK-fdx.
Example 7
Effect on L-Lysine-Producing Ability of a Strain with Enhanced
Expression of the Pyruvate Synthase Gene of Escherichia coli,
Flavodoxin NADP.sup.+ Reductase Gene of Escherichia coli and
Ferredoxin Gene of Escherichia coli Using Ethanol as the Carbon
Source
[0279] <7-1> Introduction of the Plasmid to Amplify the
Pyruvate Synthase Gene of Escherichia coli, Flavodoxin NADP.sup.+
Reductase Gene of Escherichia coli and Ferredoxin Gene of
Escherichia coli into WC196.DELTA.mez adhE* Strain
[0280] pMW-Pthr-fpr-ydbK-fdx and the control vector pMW-Pthr were
introduced into WC196.DELTA.mez adhE*/pCABD2 by electroporation,
respectively, and transformants were obtained on the basis of the
kanamycin resistance, and introduction of the plasmids were
confirmed. The strain expressing the pyruvate synthase gene of
Escherichia coli was designated WC196.DELTA.mez
adhE*/pCABD2/pMW-Pthr-fpr-ydbK-fdx, and the control strain was
designated WC196.DELTA.mez adhE*/pCABD2/pMW-Pthr.
[0281] <7-2> Effect on L-Lysine-Producing Ability of the
Strain with Enhanced Expression of the Pyruvate Synthase Gene of
Escherichia coli, Flavodoxin NADP.sup.+ Reductase Gene of
Escherichia coli and Ferredoxin Gene of Escherichia coli Using
Ethanol as the Carbon Source>
[0282] Both WC196.DELTA.mez adhE*/pCABD2/pMW-Pthr and
WC196.DELTA.mez adhE*/pCABD2/pMW-Pthr-fpr-ydbK-fdx were inoculated
onto LB plate medium, respectively, and cultured overnight at
37.degree. C. The cells corresponding to 1/8 of the plate were
inoculated into 20 ml of the ethanol medium having the following
composition in a 500 ml-volume Sakaguchi flask, and aerobically
cultured at a stirring rate of 120 rpm at 37.degree. C. for 96
hours. L-lysine which accumulated in the medium and residual
ethanol were measured by using a Biosensor BF-5 (Oji Scientific
Instruments). The turbidity of the medium was also measured.
Averages of the values obtained in the culture performed in
duplicate are shown in Table 4. Improved production of L-lysine was
observed for the strain with enhanced expression of the pyruvate
synthase gene of Escherichia coli, flavodoxin NADP.sup.+ reductase
gene of Escherichia coli and ferredoxin gene of Escherichia coli,
compared with the control.
[0283] Composition of ethanol medium:
TABLE-US-00006 Ethanol 20 ml/L MgSO.sub.4.cndot.7H.sub.2O 1.0 g/L
(NH.sub.4).sub.2SO.sub.4 12 g/L KH.sub.2PO.sub.4 0.5 g/L Yeast
extract 1.0 g/L FeSO.sub.4.cndot.7H.sub.2O 0.01 g/L
MnSO.sub.4.cndot.5H.sub.2O 0.01 g/L Kanamycin 40 mg/L Streptomycin
20 mg/L Calcium carbonate 30 g/L pH 7.0 (adjusted with KOH)
Sterilization conditions: 115.degree. C., 10 minutes
TABLE-US-00007 TABLE 4 Lys EtOH Strain (g/l) (V/V %) OD620
WC196.DELTA.mez adhE*/pCABD2/pMW-Pthr 2.47 0.00 14.7
WC196.DELTA.mez adhE*/pCABD2/pMW-Pthr- 2.89 0.00 9.3
fpr-ydbK-fdx
Example 8
Construction of the Plasmid to Express the Pyruvate:NADP.sup.+
Oxidoreductase Gene of Euglena gracilis and Measurement of
Activity
[0284] Euglena gracilis is a photosynthetic protist, with an
optimum growth temperature of 27.degree. C. The pyruvate:NADP.sup.+
oxidoreductase gene was isolated from this organism, and a plasmid
expressing this gene was constructed.
[0285] <8-1> Construction of the Plasmid to Express the
Pyruvate:NADP.sup.+ Oxidoreductase Gene of Euglena gracilis
[0286] PCR was performed by using the chromosomal DNA of Euglena
gracilis as the template and the oligonucleotides shown in SEQ ID
NOS: 40 and 41. The gene fragment was digested with KpnI, and the
digested fragment was inserted into the KpnI site of pUC19 (Takara
Bio) to construct a plasmid, which was designated pUC-PNO. After it
was confirmed that the pyruvate:NADP.sup.+ oxidoreductase gene
contained no PCR error over the full length by using BigDye
Terminators v1.1 Cycle Sequencing Kit, the pyruvate:NADP.sup.+
oxidoreductase gene was excised from pUC-PNO with KpnI, and
inserted into the KpnI site of pMW-Pthr to construct the plasmid
pMW-Pthr-PNO. <8-2> Confirmation of expression of
pyruvate:NADP.sup.+ oxidoreductase pMW-Pthr-PNO and the control
vector pMW-Pthr were introduced into the
WC196.DELTA.cadA.DELTA.ldc/pCABD2 strain by electroporation,
respectively, and transformants were obtained on the basis of the
kanamycin resistance, and introduction of the plasmids was
confirmed. The strain expressing the pyruvate:NADP.sup.+
oxidoreductase gene of Euglena gracilis was designated
WC196.DELTA.cadA.DELTA.ldc/pCABD2/pMW-Pthr-PNO, and the control
strain was designated
WC196.DELTA.cadA.DELTA.ldc/pCABD2/pMW-Pthr.
[0287] The aforementioned strains were each inoculated into LB
medium containing 20 mg/l of streptomycin and 40 mg/l of kanamycin,
and cultured overnight at 37.degree. C. with shaking. 1 ml of the
medium was inoculated into 20 ml of LB medium containing 20 mg/l of
streptomycin and 40 mg/l of kanamycin, and cultured at 37.degree.
C. for 5 hours with shaking. The cells were collected by
centrifugation and suspended in 1 ml of PBS. The cells in the
suspension were disrupted by using an ultrasonicator, the
suspension was centrifuged at 15000 rpm for 15 minutes, and the
supernatant was used as a crude extract. Protein concentration in
the crude extract was measured by using Protein Assay CBB Solution
(Nakalai Tesque), and the crude extract containing 10 .mu.g of
protein was used to prepare the samples. Each sample was prepared
by adding NuPAGE LDS Sample Buffer (Invitrogen) to the crude
extract at a concentration of 1.1.times., then adding NuPAGE Sample
Reducing Agent (Invitrogen) to a final concentration of 10%, and
heating the mixture at 70.degree. C. for 10 minutes. The prepared
sample was subjected to electrophoresis using NuPAGE Tris-Acetate
Gel 3-8% (Invitrogen). MagicMark XP Western Protein Standard
(Invitrogen) was used as markers.
[0288] The gel after electrophoresis was transferred to a membrane
by using iBlot Gel Transfer Device (Invitrogen). After the
transfer, the process from blocking to detection were performed by
using WesternBreeze Chemiluminescent Western Blot Immunodetection
Kit (Invitrogen). First, the membrane was subjected to a blocking
treatment for 30 minutes, washed twice with purified water and
incubated in an anti-PNO serum solution diluted 1000 times for 1
hour. The membrane was washed 3 times with a washing solution, and
incubated in a second antibody solution for 30 minutes. The
membrane was washed 3 times with a washing solution and further
twice with purified water, sprinkled with a detection reagent, and
subjected to detection using Lumino-image Analyzer LAS-1000 (Fuji
Photo Film). The results are shown in FIG. 1. A band presumed to be
PNO was detected around 200 kD for the
WC196.DELTA.cadA.DELTA.ldc/pCABD2/pMW-Pthr-PNO strain, whereas a
band was not detected for the control strain
WC196.DELTA.cadA.DELTA.ldc/pCABD2/pMW-Pthr.
Example 9
Construction of the Plasmid to Express the Pyruvate:NADP.sup.+
Oxidoreductase Gene of Euglena gracilis
[0289] The pyruvate:NADP.sup.+ oxidoreductase gene fragment was
excised from the plasmid pUC-PNO described in Example 8 with KpnI,
and inserted into the KpnI site of pMW-Pthr to construct the
plasmid pMW-Pthr-PNO.
Example 10
Effect on L-Lysine-Producing Ability of the Strain with Enhanced
Expression of the Pyruvate:NADP.sup.+ Oxidoreductase Gene of
Euglena gracilis Using Oleic Acid as the Carbon Source
[0290] <10-1> Introduction of the Plasmid for Amplification
of Pyruvate:NADP.sup.+ Oxidoreductase Gene of Euglena gracilis into
the WC196.DELTA.mez Strain
[0291] pMW-Pthr-PNO and the control vector pMW-Pthr were introduced
into WC196.DELTA.mez/pCABD2 by electroporation, respectively, and
transformants were obtained on the basis of the kanamycin
resistance, and introduction of the plasmids were confirmed. The
strain expressing the pyruvate:NADP.sup.+ oxidoreductase gene of
Euglena gracilis was designated
WC196.DELTA.mez/pCABD2/pMW-Pthr-PNO, and the control strain was
designated WC196.DELTA.mez/pCABD2/pMW-Pthr.
[0292] <10-2> Effect on L-Lysine-Producing Ability of the
Strain with Enhanced Expression of the Pyruvate:NADP.sup.+
Oxidoreductase Gene of Euglena gracilis Using Oleic Acid as the
Carbon Source
[0293] Both WC196.DELTA.mez/pCABD2/pMW-Pthr and
WC196.DELTA.mez/pCABD2/pMW-Pthr-PNO were inoculated onto the LB
plate medium, respectively, and cultured overnight at 37.degree. C.
The cells corresponding to 1/8 of the plate were inoculated into 20
ml of the oleic acid medium having the following composition in a
500 ml-volume Sakaguchi flask, and aerobically cultured at a
stirring rate of 120 rpm at 37.degree. C. for 72 hours. L-lysine
which accumulated in the medium was measured by using a Biosensor
BF-5 (Oji Scientific Instruments). The live cell count in the
medium was also measured. Averages of the values obtained in the
culture performed in duplicate are shown in Table 5. Improvement in
the production of L-lysine was observed for the strain in which
expression of pyruvate:NADP.sup.+ oxidoreductase gene of Euglena
gracilis was enhanced, compared with the control.
[0294] Composition of oleic acid medium:
TABLE-US-00008 Sodium oleate 20 g/L MgSO.sub.4.cndot.7H.sub.2O 1.0
g/L (NH.sub.4).sub.2SO.sub.4 12 g/L KH.sub.2PO.sub.4 0.5 g/L Yeast
extract 1.0 g/L FeSO.sub.4.cndot.7H.sub.2O 0.01 g/L
MnSO.sub.4.cndot.5H.sub.2O 0.01 g/L Kanamycin 40 mg/L Streptomycin
20 mg/L Calcium carbonate 30 g/L pH 7.0 (adjusted with KOH)
Sterilization conditions: 115.degree. C., 10 minutes
TABLE-US-00009 TABLE 5 Lys Live cell Strain (g/l) count
(10.sup.8/ml) WC196.DELTA.mez/pCABD2/pMW-Pthr 1.77 22.3
WC196.DELTA.mez/pCABD2/pMW-Pthr-PNO 2.41 15.9
Example 11
Effect on L-Lysine-Producing Ability of the Strain with Enhanced
Expression of the Pyruvate:NADP.sup.+ Oxidoreductase Gene of
Euglena gracilis Using Ethanol as the Carbon Source
[0295] <11-1> Introduction of the Plasmid for Amplification
of Pyruvate:NADP.sup.+ Oxidoreductase Gene of Euglena gracilis into
WC196.DELTA.mez adhE*
[0296] pMW-Pthr-PNO and the control vector pMW-Pthr were introduced
into WC196.DELTA.mez adhE*/pCABD2 by electroporation, respectively,
and transformants were obtained on the basis of the kanamycin
resistance, and introduction of the plasmids was confirmed. The
strain expressing the pyruvate:NADP.sup.+ oxidoreductase gene of
Euglena gracilis was designated WC196.DELTA.mez
adhE*/pCABD2/pMW-Pthr-PNO, and the control strain was designated
WC196.DELTA.mez adhE*/pCABD2/pMW-Pthr.
[0297] <11-2> Effect on L-Lysine-Producing Ability of the
Strain with Enhanced Expression of the Pyruvate:NADP.sup.+
Oxidoreductase Gene of Euglena gracilis Using Ethanol as the Carbon
Source
[0298] Both WC196.DELTA.mez adhE*/pCABD2/pMW-Pthr and
WC196.DELTA.mez adhE*/pCABD2/pMW-Pthr-PNO were each inoculated onto
LB plate medium, and precultured overnight at 37.degree. C. The
cells corresponding to 1/8 of the plate were inoculated into 20 ml
of the ethanol medium having the following composition in a 500
ml-volume Sakaguchi flask, and aerobically cultured at a stirring
rate of 120 rpm at 37.degree. C. for 96 hours. After 96 hours, 1 ml
of the medium was sampled, and L-lysine which had accumulated in
the medium was measured by using a Biosensor BF-5 (Oji Scientific
Instruments). The turbidity of the medium was also measured.
Averages of the values obtained in the culture performed in
duplicate are shown in Table 6. Improvement in the production of
L-lysine was observed in the strain with enhanced expression of the
pyruvate:NADP.sup.+ oxidoreductase gene of Euglena gracilis,
compared with the control.
[0299] Composition of ethanol medium:
TABLE-US-00010 Ethanol 20 ml/L MgSO.sub.4.cndot.7H.sub.2O 1.0 g/L
(NH.sub.4).sub.2SO.sub.4 12 g/L KH.sub.2PO.sub.4 0.5 g/L Yeast
extract 1.0 g/L FeSO.sub.4.cndot.7H.sub.2O 0.01 g/L
MnSO.sub.4.cndot.5H.sub.2O 0.01 g/L Kanamycin 40 mg/L Streptomycin
20 mg/L Calcium carbonate 30 g/L pH 7.0 (adjusted with KOH)
Sterilization conditions: 115.degree. C., 10 minutes
TABLE-US-00011 TABLE 6 Lys EtOH Strain (g/l) (V/V %) OD620
WC196.DELTA.mez adhE*/pCABD2/pMW-Pthr 2.47 0.00 14.7
WC196.DELTA.mez adhE*/pCABD2/pMW-Pthr- 2.89 0.00 9.3
fpr-ydbK-fdx
[0300] Explanation of Sequence Listing:
[0301] SEQ ID NO: 1: Nucleotide sequence of C. tepidum pyruvate
synthase gene
[0302] SEQ ID NO: 2: Amino acid sequence of C. tepidum pyruvate
synthase
[0303] SEQ ID NO: 3: Nucleotide sequence of E. coli pyruvate
synthase gene
[0304] SEQ ID NO: 4: Amino acid sequence of E. coli pyruvate
synthase
[0305] SEQ ID NO: 5: Nucleotide sequence of E. gracilis pyruvate:
NAPD.sup.+ oxidoreductase gene
[0306] SEQ ID NO: 6: Amino acid sequence of E. gracilis pyruvate:
NADP.sup.+ oxidoreductase gene
[0307] SEQ ID NO: 7: Nucleotide sequence of E. coli flavodoxin
NADP.sup.+ reductase(fpr) gene
[0308] SEQ ID NO: 8: Amino acid sequence encoded by E. coli E. coli
flavodoxin NADP reductase(fpr) gene
[0309] SEQ ID NO: 9: Nucleotide sequence of E. coli ferredoxin(fdx)
gene
[0310] SEQ ID NO: 10: Amino acid sequence encoded by E. coli
ferredoxin(fdx) gene
[0311] SEQ ID NO: 11: Nucleotide sequence of E. coli
ferredoxin(yfhL) gene
[0312] SEQ ID NO: 12: Amino acid sequence encoded by E. coli
ferredoxin(yfhL) gene
[0313] SEQ ID NO: 13: Nucleotide sequence of E. coli
flavodoxin(fldA) gene
[0314] SEQ ID NO: 14: Amino acid sequence encoded by E. coli
flavodoxin(fldA) gene
[0315] SEQ ID NO: 15: Nucleotide sequence of E. coli
flavodoxin(fldB) gene
[0316] SEQ ID NO: 16: Amino acid sequence encoded by E. coli
flavodoxin(fldB) gene
[0317] SEQ ID NO: 17: Nucleotide sequence of C. tepidum ferredoxin
I gene
[0318] SEQ ID NO: 18: Amino acid sequence encoded by C. tepidum
ferredoxin I gene
[0319] SEQ ID NO: 19: Nucleotide sequence of C. tepidum ferredoxin
II gene
[0320] SEQ ID NO: 20: Amino acid sequence encoded by C. tepidum
ferredoxin II gene
[0321] SEQ ID NO: 21: Nucleotide sequence of E. coli alcohol
dehydrogenase gene
[0322] SEQ ID NO: 22: Amino acid sequence encoded by E. coli
alcohol dehydrogenase gene
[0323] SEQ ID NO: 23: P.sub.L-tac promoter and chloramphenicol
resistance (Cm.sup.R) gene amplification primer 1
[0324] SEQ ID NO: 24: P.sub.L-tac promoter and chloramphenicol
resistance (Cm.sup.R) gene amplification primer 2
[0325] SEQ ID NO: 25: Nucleotide sequence of P.sub.L-tac
promoter
[0326] SEQ ID NO: 26: P.sub.L-tac promoter and chloramphenicol
resistance (Cm.sup.R) gene amplification primer 3
[0327] SEQ ID NO: 27: P.sub.L-tac promoter and chloramphenicol
resistance (Cm.sup.R) gene amplification primer 4
[0328] SEQ ID NO: 28: Kanamycin resistance (Cm.sup.R) gene
amplification primer 1
[0329] SEQ ID NO: 29: Kanamycin resistance (Cm.sup.R) gene
amplification primer 2
[0330] SEQ ID NO: 30: Kanamycin resistance (Cm.sup.R) gene
amplification primer 3
[0331] SEQ ID NO: 31: Kanamycin resistance (Cm.sup.R) gene
amplification primer 4
[0332] SEQ ID NO: 32: E. coli mutant alcohol dehydrogenase gene
amplification primer 1
[0333] SEQ ID NO: 33: E. coli mutant alcohol dehydrogenase gene
amplification primer 2
[0334] SEQ ID NO: 34: E. coli mutant alcohol dehydrogenase gene
amplification primer 3
[0335] SEQ ID NO: 35: C. tepidum pyruvate synthase gene
amplification primer 1
[0336] SEQ ID NO: 36: C. tepidum pyruvate synthase gene
amplification primer 2
[0337] SEQ ID NO: 37: Threonine operon promoter sequence
[0338] SEQ ID NO: 38: E. coli pyruvate synthase gene amplification
primer 1
[0339] SEQ ID NO: 39: E. coli pyruvate synthase gene amplification
primer 2
[0340] SEQ ID NO: 40: E. gracilis pyruvate:NADP.sup.+
oxidoreductase gene amplification primer 1
[0341] SEQ ID NO: 41: E. gracilis pyruvate:NADP.sup.+
oxidoreductase gene amplification primer 2
[0342] SEQ ID NO: 42: E. coli flavodoxin NADP.sup.+ reductase gene
amplification primer 1
[0343] SEQ ID NO: 43: E. coli flavodoxin NADP.sup.+ reductase gene
amplification primer 2
[0344] SEQ ID NO: 44: E. coli fdx gene amplification primer 1
[0345] SEQ ID NO: 45: E. coli fdx gene amplification primer 2
[0346] SEQ ID NO: 46: Nucleotide sequence of E. coli pyruvate
dehydrogenase Ep1 subunit gene (aceE)
[0347] SEQ ID NO: 47: Amino acid sequence of E. coli pyruvate
dehydrogenase Ep1 subunit
[0348] SEQ ID NO: 48: Nucleotide sequence of E. coli pyruvate
dehydrogenase E2 subunit gene (aceF)
[0349] SEQ ID NO: 49: Amino acid sequence of E. coli pyruvate
dehydrogenase E2 subunit
[0350] SEQ ID NO: 50: Nucleotide sequence of E. coli pyruvate
dehydrogenase E3 subunit gene (lpdA)
[0351] SEQ ID NO: 51: Amino acid sequence of E. coli pyruvate
dehydrogenase E3 subunit
[0352] SEQ ID NO: 52: Nucleotide sequence of gene (sfcA) encoding
E. coli malic enzyme
[0353] SEQ ID NO: 53: Amino acid sequence of malic enzyme encoded
by E. coli sfcA gene
[0354] SEQ ID NO: 54: Nucleotide sequence of gene (b2463) encoding
E. coli malic enzyme
[0355] SEQ ID NO: 55: Amino acid sequence of malic enzyme encoded
by E. coli b2463 gene
INDUSTRIAL APPLICABILITY
[0356] By using the microorganism of the present invention, an
L-amino acid can be efficiently produced by fermentation. Moreover,
according to a preferred embodiment of the method of the present
invention, the method of the present invention is an
environmentally-friendly method which can reduce carbon dioxide
emission by suppressing decarboxylation and utilizing carbon
dioxide fixation.
[0357] While the invention has been described in detail with
reference to preferred embodiments thereof, it will be apparent to
one skilled in the art that various changes can be made, and
equivalents employed, without departing from the scope of the
invention. Each of the aforementioned documents is incorporated by
reference herein in its entirety,
Sequence CWU 1
1
5513558DNAChlorobium tepidumCDS(1)..(3558) 1atg acc cgg aca ttc aag
aca atg gag ggg aat gaa gct ctt gct cat 48Met Thr Arg Thr Phe Lys
Thr Met Glu Gly Asn Glu Ala Leu Ala His1 5 10 15gtc gcc tat cgc act
aat gaa gtc atc tcg ata tac ccg att acc ccg 96Val Ala Tyr Arg Thr
Asn Glu Val Ile Ser Ile Tyr Pro Ile Thr Pro 20 25 30gca tct ccg atg
gga gag tac tcc gac gca tgg gcc gct gtc gat gta 144Ala Ser Pro Met
Gly Glu Tyr Ser Asp Ala Trp Ala Ala Val Asp Val 35 40 45aaa aat atc
tgg ggt acc gtg cca ctc gtc aat gag atg cag agc gaa 192Lys Asn Ile
Trp Gly Thr Val Pro Leu Val Asn Glu Met Gln Ser Glu 50 55 60gcc ggt
gcc gcc gcc gcc gtt cac ggc gcg ttg cag acc ggc gcg ctg 240Ala Gly
Ala Ala Ala Ala Val His Gly Ala Leu Gln Thr Gly Ala Leu65 70 75
80acg acc acc ttc acg gcc tct cag ggt ctc tta ctg atg atc ccg aac
288Thr Thr Thr Phe Thr Ala Ser Gln Gly Leu Leu Leu Met Ile Pro Asn
85 90 95atg tac aag atc gcc ggt gaa ctg acc ccc tgc gtg att cac gtg
tca 336Met Tyr Lys Ile Ala Gly Glu Leu Thr Pro Cys Val Ile His Val
Ser 100 105 110 gcc cgt tcg ctg gcc gcg cag gcg ctc tcg ata ttc tgc
gac cac ggt 384Ala Arg Ser Leu Ala Ala Gln Ala Leu Ser Ile Phe Cys
Asp His Gly 115 120 125gac gtg atg tcg gtc agg ggc acc ggc ttc gcg
ctg ctc gct tcc tgt 432Asp Val Met Ser Val Arg Gly Thr Gly Phe Ala
Leu Leu Ala Ser Cys 130 135 140tcg gta cag gag gta atg gac atg gcg
ctg att tcg cag gcc gca acg 480Ser Val Gln Glu Val Met Asp Met Ala
Leu Ile Ser Gln Ala Ala Thr145 150 155 160ctc gaa tcg cgc gtg cca
ttc ctg cac ttc ttc gac ggc ttc cgc acg 528Leu Glu Ser Arg Val Pro
Phe Leu His Phe Phe Asp Gly Phe Arg Thr 165 170 175tcg cac gaa atc
tcg aaa atc gag gtg ctc tcg gac gaa cag att cgc 576Ser His Glu Ile
Ser Lys Ile Glu Val Leu Ser Asp Glu Gln Ile Arg 180 185 190 tcg atg
atc aac gac gag ctg gtc ttc gca cac cgc atg cgc cgc atg 624Ser Met
Ile Asn Asp Glu Leu Val Phe Ala His Arg Met Arg Arg Met 195 200
205tcg cct gat gca ccg atc atc cgc ggt acc tcg cag aat ccg gac gtc
672Ser Pro Asp Ala Pro Ile Ile Arg Gly Thr Ser Gln Asn Pro Asp Val
210 215 220tat ttc cag gca cgc gag agc gtc aac aaa tat tat gag gcc
tgc ccg 720Tyr Phe Gln Ala Arg Glu Ser Val Asn Lys Tyr Tyr Glu Ala
Cys Pro225 230 235 240tca atc acc cag aag gcg atg gac cag ttc gcc
aaa ctg act ggg cgc 768Ser Ile Thr Gln Lys Ala Met Asp Gln Phe Ala
Lys Leu Thr Gly Arg 245 250 255agc tat aaa ctt tac cag tac tac ggc
gct ccg gat gcc gac cgt atc 816Ser Tyr Lys Leu Tyr Gln Tyr Tyr Gly
Ala Pro Asp Ala Asp Arg Ile 260 265 270 atc atc atg atg ggg tca ggt
gcc gag acc gct ctc gaa act gtc gaa 864Ile Ile Met Met Gly Ser Gly
Ala Glu Thr Ala Leu Glu Thr Val Glu 275 280 285tac ctc aac aac cac
ggc gaa aag gtc ggt ctg gtc aag gta cgc ctt 912Tyr Leu Asn Asn His
Gly Glu Lys Val Gly Leu Val Lys Val Arg Leu 290 295 300ttc agg cca
ttc gac gtt gca acc ttc atc gca tcg cta cca tcg agc 960Phe Arg Pro
Phe Asp Val Ala Thr Phe Ile Ala Ser Leu Pro Ser Ser305 310 315
320gtg aag agt atc gcg gtg ctc gac cgt gtc aag gaa cca ggc agc gct
1008Val Lys Ser Ile Ala Val Leu Asp Arg Val Lys Glu Pro Gly Ser Ala
325 330 335ggc gaa ccg ctc tat ctc gat gta gtc aac gcc gta gcc gaa
tcg tac 1056Gly Glu Pro Leu Tyr Leu Asp Val Val Asn Ala Val Ala Glu
Ser Tyr 340 345 350 cag gaa ggc aaa tgc gct tcg atg cca agc gtt ttg
ggt ggg cgc tat 1104Gln Glu Gly Lys Cys Ala Ser Met Pro Ser Val Leu
Gly Gly Arg Tyr 355 360 365ggc ctg tcg tcg aag gag ttc act ccg gcg
atg gtc aag gcg atc ttc 1152Gly Leu Ser Ser Lys Glu Phe Thr Pro Ala
Met Val Lys Ala Ile Phe 370 375 380gac aat atg aac gcg gaa tct cca
aag aat cac ttc acc gtt ggc atc 1200Asp Asn Met Asn Ala Glu Ser Pro
Lys Asn His Phe Thr Val Gly Ile385 390 395 400gac gat gac gta acc
aag aag agc ctc gcc tac gac gag acc ttc tcg 1248Asp Asp Asp Val Thr
Lys Lys Ser Leu Ala Tyr Asp Glu Thr Phe Ser 405 410 415att gag ccg
gac tcg gtc ttc cgc gcc ctc ttc tac ggc ctc ggt tca 1296Ile Glu Pro
Asp Ser Val Phe Arg Ala Leu Phe Tyr Gly Leu Gly Ser 420 425 430 gac
ggc acg gtc ggt gca aac aag aac tcg atc aag atc att ggc gaa 1344Asp
Gly Thr Val Gly Ala Asn Lys Asn Ser Ile Lys Ile Ile Gly Glu 435 440
445aac acc gac aac tac gcg cag ggc ttc ttc gtc tac gac tcc aag aaa
1392Asn Thr Asp Asn Tyr Ala Gln Gly Phe Phe Val Tyr Asp Ser Lys Lys
450 455 460gcc ggt tcg atc acg acc tcg cac ctg cgg ttc ggc ccg gag
cag atc 1440Ala Gly Ser Ile Thr Thr Ser His Leu Arg Phe Gly Pro Glu
Gln Ile465 470 475 480cgc tcg acc tac ctc atc acc gag gcg cag ttc
gtc ggc tgc cac cac 1488Arg Ser Thr Tyr Leu Ile Thr Glu Ala Gln Phe
Val Gly Cys His His 485 490 495tgg gtc ttt ctc gaa atg atc gac gtt
gcc aag aac ctc aag cag ggt 1536Trp Val Phe Leu Glu Met Ile Asp Val
Ala Lys Asn Leu Lys Gln Gly 500 505 510 ggt acg ctg ctc atc aac tcg
gcc tat gcg ccg gat gtg gtg tgg agc 1584Gly Thr Leu Leu Ile Asn Ser
Ala Tyr Ala Pro Asp Val Val Trp Ser 515 520 525aag ctc ccg cgt ccg
gtg cag cag cac ttg atc gac aag cag gcg aag 1632Lys Leu Pro Arg Pro
Val Gln Gln His Leu Ile Asp Lys Gln Ala Lys 530 535 540ctc tac acc
atc gat gcc tac aag gtc gcc cac gaa agc ggc atg ggt 1680Leu Tyr Thr
Ile Asp Ala Tyr Lys Val Ala His Glu Ser Gly Met Gly545 550 555
560cag cgc atc aac act atc atg cag gcc tgt ttc ttc gcc att tcg ggc
1728Gln Arg Ile Asn Thr Ile Met Gln Ala Cys Phe Phe Ala Ile Ser Gly
565 570 575gtg ctg ccg cgt gaa gag gca atc gaa aag atc aag gac gcg
atc cgc 1776Val Leu Pro Arg Glu Glu Ala Ile Glu Lys Ile Lys Asp Ala
Ile Arg 580 585 590 cac acc tac ggc aaa aag ggc gat gag gtc gtt cag
cag aac atc aag 1824His Thr Tyr Gly Lys Lys Gly Asp Glu Val Val Gln
Gln Asn Ile Lys 595 600 605gca gtt gac aac acg ctt gcc aac ctg cat
gaa gtg aaa atc ggc gct 1872Ala Val Asp Asn Thr Leu Ala Asn Leu His
Glu Val Lys Ile Gly Ala 610 615 620gtg gca gac agc acc aag gag ctg
cgc tcg ccc atc gtt ggc gac gcg 1920Val Ala Asp Ser Thr Lys Glu Leu
Arg Ser Pro Ile Val Gly Asp Ala625 630 635 640cca gag ttc gtc tgt
aac gtg ctg gca aag att att gcc ggc gag ggc 1968Pro Glu Phe Val Cys
Asn Val Leu Ala Lys Ile Ile Ala Gly Glu Gly 645 650 655gac tcg att
ccg gtc agc aag ctg cct gcc gat gga acc tat ccg ctc 2016Asp Ser Ile
Pro Val Ser Lys Leu Pro Ala Asp Gly Thr Tyr Pro Leu 660 665 670 ggc
acc acg aag ttc gag aaa cgc aac ctc gcg cag gag att ccg gtc 2064Gly
Thr Thr Lys Phe Glu Lys Arg Asn Leu Ala Gln Glu Ile Pro Val 675 680
685tgg gct ccg gag ctg tgc atc gag tgt ggc aag tgc tcg atg gtc tgc
2112Trp Ala Pro Glu Leu Cys Ile Glu Cys Gly Lys Cys Ser Met Val Cys
690 695 700ccg cac gct gcc atc cgc atc aag gtt tac gag ccg aag cac
ctc gaa 2160Pro His Ala Ala Ile Arg Ile Lys Val Tyr Glu Pro Lys His
Leu Glu705 710 715 720aac gcc ccg gca acc ttc aag agc ctc gat gcg
aaa gca aaa aac tgg 2208Asn Ala Pro Ala Thr Phe Lys Ser Leu Asp Ala
Lys Ala Lys Asn Trp 725 730 735gag ggc atg cgc tat acg gtt cag att
gca ccg gaa gat tgt acc ggc 2256Glu Gly Met Arg Tyr Thr Val Gln Ile
Ala Pro Glu Asp Cys Thr Gly 740 745 750 tgc caa ctc tgc gtc aac gcc
tgc ccc gca aga gac aag cag gtt gaa 2304Cys Gln Leu Cys Val Asn Ala
Cys Pro Ala Arg Asp Lys Gln Val Glu 755 760 765ggc cgc aaa gcg ctc
aac atg cac gag cag gct ccg ctg cgc gaa acc 2352Gly Arg Lys Ala Leu
Asn Met His Glu Gln Ala Pro Leu Arg Glu Thr 770 775 780gaa tct gcc
tgc tgg agc ttc ttc atc aat ctc ccg gaa ttc gac cgc 2400Glu Ser Ala
Cys Trp Ser Phe Phe Ile Asn Leu Pro Glu Phe Asp Arg785 790 795
800aac aag atc aac cag cgc ctc atc aaa gag cag cag ctt cag cag cca
2448Asn Lys Ile Asn Gln Arg Leu Ile Lys Glu Gln Gln Leu Gln Gln Pro
805 810 815ctc ttc gag ttc tcg ggc gca tgc tcg ggc tgc ggc gaa acg
cca tac 2496Leu Phe Glu Phe Ser Gly Ala Cys Ser Gly Cys Gly Glu Thr
Pro Tyr 820 825 830 gtc aag ctg atg act cag ctc ttc ggt gat cgc ctc
gtt atc ggc aac 2544Val Lys Leu Met Thr Gln Leu Phe Gly Asp Arg Leu
Val Ile Gly Asn 835 840 845gcc acc ggc tgc tcg tcg atc tac ggc ggc
aac ctg ccg acc acg ccg 2592Ala Thr Gly Cys Ser Ser Ile Tyr Gly Gly
Asn Leu Pro Thr Thr Pro 850 855 860tat gca gcc aac ccg cag ggc ctt
ggg cca acg tgg tcg aac tcg ctt 2640Tyr Ala Ala Asn Pro Gln Gly Leu
Gly Pro Thr Trp Ser Asn Ser Leu865 870 875 880ttc gag gac acg gca
gag ttc gcg ctt ggt ttc cgg ata tcg atc gac 2688Phe Glu Asp Thr Ala
Glu Phe Ala Leu Gly Phe Arg Ile Ser Ile Asp 885 890 895aag cag cag
caa ttt gcc aaa gag ctg gtc aaa aag ctc gct ggt gac 2736Lys Gln Gln
Gln Phe Ala Lys Glu Leu Val Lys Lys Leu Ala Gly Asp 900 905 910 atc
ggt gaa aac ctt gcc acc gcc att ctc aac gcc acg cag aac agt 2784Ile
Gly Glu Asn Leu Ala Thr Ala Ile Leu Asn Ala Thr Gln Asn Ser 915 920
925gaa ccg gag att ttc gag cag cgt gag cgc gtg gcc gtg ctg aag gat
2832Glu Pro Glu Ile Phe Glu Gln Arg Glu Arg Val Ala Val Leu Lys Asp
930 935 940aag ctc cag cag atg aaa tcc gac gat gcc aag aac ctg ctt
gct gtg 2880Lys Leu Gln Gln Met Lys Ser Asp Asp Ala Lys Asn Leu Leu
Ala Val945 950 955 960gct gac atg ctg gtc aag aag agc gtg tgg gct
gtc ggc ggc gac ggc 2928Ala Asp Met Leu Val Lys Lys Ser Val Trp Ala
Val Gly Gly Asp Gly 965 970 975tgg gcc tac gat atc ggt tac ggg ggt
ctc gac cac gtc acc gca tcg 2976Trp Ala Tyr Asp Ile Gly Tyr Gly Gly
Leu Asp His Val Thr Ala Ser 980 985 990 ggc aag aac gtc aac atg ctc
gtg ctc gac acc gag gtc tat tcc aat 3024Gly Lys Asn Val Asn Met Leu
Val Leu Asp Thr Glu Val Tyr Ser Asn 995 1000 1005acc ggc ggt cag
gcc tcc aag gct acg ccg aaa gcc gcg atc gcc 3069Thr Gly Gly Gln Ala
Ser Lys Ala Thr Pro Lys Ala Ala Ile Ala 1010 1015 1020aag ttt gcc
gct gcg ggg cgc atc gct acc aag aaa gac ctt ggt 3114Lys Phe Ala Ala
Ala Gly Arg Ile Ala Thr Lys Lys Asp Leu Gly 1025 1030 1035ctg atc
tcg atg agc tac ggc aat gcc tat gtg gcc agt gtt gca 3159Leu Ile Ser
Met Ser Tyr Gly Asn Ala Tyr Val Ala Ser Val Ala 1040 1045 1050ctt
ggc gca cgt gac gag cag aca ctc aga gct ttc atc gaa gcc 3204Leu Gly
Ala Arg Asp Glu Gln Thr Leu Arg Ala Phe Ile Glu Ala 1055 1060
1065gag gcg tac gat ggc ccg tcg att atc atc gcc tac tcg cac tgc
3249Glu Ala Tyr Asp Gly Pro Ser Ile Ile Ile Ala Tyr Ser His Cys
1070 1075 1080att gca cac ggc ttt gac ttg tct atg ggt ctg gag cac
cag aaa 3294Ile Ala His Gly Phe Asp Leu Ser Met Gly Leu Glu His Gln
Lys 1085 1090 1095gca gcg gtc gat tcc ggc cac tgg ctg ctg tat cgc
tac aat ccc 3339Ala Ala Val Asp Ser Gly His Trp Leu Leu Tyr Arg Tyr
Asn Pro 1100 1105 1110gac aga ctc aag gag gga ctg aat ccg ctg cag
ctc gac tcc aaa 3384Asp Arg Leu Lys Glu Gly Leu Asn Pro Leu Gln Leu
Asp Ser Lys 1115 1120 1125aag ccg aaa atg ccg gtc gcg gag ttc ctg
aac atg gag aac cgc 3429Lys Pro Lys Met Pro Val Ala Glu Phe Leu Asn
Met Glu Asn Arg 1130 1135 1140ttc aga ata ctg aag aag acc cac ccc
gat ctg gcc aag aag tac 3474Phe Arg Ile Leu Lys Lys Thr His Pro Asp
Leu Ala Lys Lys Tyr 1145 1150 1155ttc gag gca atc cag cac gag gtc
aat gcc cgc tgg gca cac tac 3519Phe Glu Ala Ile Gln His Glu Val Asn
Ala Arg Trp Ala His Tyr 1160 1165 1170gaa cac ctc gcc aac cgt tcg
att gaa ggc gaa gca taa 3558Glu His Leu Ala Asn Arg Ser Ile Glu Gly
Glu Ala 1175 1180 118521185PRTChlorobium tepidum 2Met Thr Arg Thr
Phe Lys Thr Met Glu Gly Asn Glu Ala Leu Ala His1 5 10 15Val Ala Tyr
Arg Thr Asn Glu Val Ile Ser Ile Tyr Pro Ile Thr Pro 20 25 30Ala Ser
Pro Met Gly Glu Tyr Ser Asp Ala Trp Ala Ala Val Asp Val 35 40 45Lys
Asn Ile Trp Gly Thr Val Pro Leu Val Asn Glu Met Gln Ser Glu 50 55
60Ala Gly Ala Ala Ala Ala Val His Gly Ala Leu Gln Thr Gly Ala Leu65
70 75 80Thr Thr Thr Phe Thr Ala Ser Gln Gly Leu Leu Leu Met Ile Pro
Asn 85 90 95Met Tyr Lys Ile Ala Gly Glu Leu Thr Pro Cys Val Ile His
Val Ser 100 105 110Ala Arg Ser Leu Ala Ala Gln Ala Leu Ser Ile Phe
Cys Asp His Gly 115 120 125Asp Val Met Ser Val Arg Gly Thr Gly Phe
Ala Leu Leu Ala Ser Cys 130 135 140Ser Val Gln Glu Val Met Asp Met
Ala Leu Ile Ser Gln Ala Ala Thr145 150 155 160Leu Glu Ser Arg Val
Pro Phe Leu His Phe Phe Asp Gly Phe Arg Thr 165 170 175Ser His Glu
Ile Ser Lys Ile Glu Val Leu Ser Asp Glu Gln Ile Arg 180 185 190Ser
Met Ile Asn Asp Glu Leu Val Phe Ala His Arg Met Arg Arg Met 195 200
205Ser Pro Asp Ala Pro Ile Ile Arg Gly Thr Ser Gln Asn Pro Asp Val
210 215 220Tyr Phe Gln Ala Arg Glu Ser Val Asn Lys Tyr Tyr Glu Ala
Cys Pro225 230 235 240Ser Ile Thr Gln Lys Ala Met Asp Gln Phe Ala
Lys Leu Thr Gly Arg 245 250 255Ser Tyr Lys Leu Tyr Gln Tyr Tyr Gly
Ala Pro Asp Ala Asp Arg Ile 260 265 270Ile Ile Met Met Gly Ser Gly
Ala Glu Thr Ala Leu Glu Thr Val Glu 275 280 285Tyr Leu Asn Asn His
Gly Glu Lys Val Gly Leu Val Lys Val Arg Leu 290 295 300Phe Arg Pro
Phe Asp Val Ala Thr Phe Ile Ala Ser Leu Pro Ser Ser305 310 315
320Val Lys Ser Ile Ala Val Leu Asp Arg Val Lys Glu Pro Gly Ser Ala
325 330 335Gly Glu Pro Leu Tyr Leu Asp Val Val Asn Ala Val Ala Glu
Ser Tyr 340 345 350Gln Glu Gly Lys Cys Ala Ser Met Pro Ser Val Leu
Gly Gly Arg Tyr 355 360 365Gly Leu Ser Ser Lys Glu Phe Thr Pro Ala
Met Val Lys Ala Ile Phe 370 375 380Asp Asn Met Asn Ala Glu Ser Pro
Lys Asn His Phe Thr Val Gly Ile385 390 395 400Asp Asp Asp Val Thr
Lys Lys Ser Leu Ala Tyr Asp Glu Thr Phe Ser 405 410 415Ile Glu Pro
Asp Ser Val Phe Arg Ala Leu Phe Tyr Gly Leu Gly Ser 420 425 430Asp
Gly Thr Val Gly Ala Asn Lys Asn Ser Ile Lys Ile Ile Gly Glu 435 440
445Asn Thr Asp Asn Tyr Ala Gln Gly Phe Phe Val Tyr Asp Ser Lys Lys
450 455 460Ala Gly Ser Ile Thr Thr Ser His Leu Arg Phe Gly Pro Glu
Gln Ile465 470 475 480Arg Ser Thr Tyr Leu Ile Thr Glu Ala Gln Phe
Val Gly Cys His His 485 490 495Trp Val Phe Leu Glu Met Ile Asp Val
Ala Lys Asn Leu Lys Gln Gly 500 505 510Gly Thr Leu Leu Ile Asn Ser
Ala Tyr Ala Pro Asp Val Val Trp Ser 515 520 525Lys Leu Pro Arg Pro
Val Gln Gln His Leu Ile Asp Lys Gln Ala Lys 530 535 540Leu Tyr Thr
Ile Asp Ala Tyr Lys Val Ala
His Glu Ser Gly Met Gly545 550 555 560Gln Arg Ile Asn Thr Ile Met
Gln Ala Cys Phe Phe Ala Ile Ser Gly 565 570 575Val Leu Pro Arg Glu
Glu Ala Ile Glu Lys Ile Lys Asp Ala Ile Arg 580 585 590His Thr Tyr
Gly Lys Lys Gly Asp Glu Val Val Gln Gln Asn Ile Lys 595 600 605Ala
Val Asp Asn Thr Leu Ala Asn Leu His Glu Val Lys Ile Gly Ala 610 615
620Val Ala Asp Ser Thr Lys Glu Leu Arg Ser Pro Ile Val Gly Asp
Ala625 630 635 640Pro Glu Phe Val Cys Asn Val Leu Ala Lys Ile Ile
Ala Gly Glu Gly 645 650 655Asp Ser Ile Pro Val Ser Lys Leu Pro Ala
Asp Gly Thr Tyr Pro Leu 660 665 670Gly Thr Thr Lys Phe Glu Lys Arg
Asn Leu Ala Gln Glu Ile Pro Val 675 680 685Trp Ala Pro Glu Leu Cys
Ile Glu Cys Gly Lys Cys Ser Met Val Cys 690 695 700Pro His Ala Ala
Ile Arg Ile Lys Val Tyr Glu Pro Lys His Leu Glu705 710 715 720Asn
Ala Pro Ala Thr Phe Lys Ser Leu Asp Ala Lys Ala Lys Asn Trp 725 730
735Glu Gly Met Arg Tyr Thr Val Gln Ile Ala Pro Glu Asp Cys Thr Gly
740 745 750Cys Gln Leu Cys Val Asn Ala Cys Pro Ala Arg Asp Lys Gln
Val Glu 755 760 765Gly Arg Lys Ala Leu Asn Met His Glu Gln Ala Pro
Leu Arg Glu Thr 770 775 780Glu Ser Ala Cys Trp Ser Phe Phe Ile Asn
Leu Pro Glu Phe Asp Arg785 790 795 800Asn Lys Ile Asn Gln Arg Leu
Ile Lys Glu Gln Gln Leu Gln Gln Pro 805 810 815Leu Phe Glu Phe Ser
Gly Ala Cys Ser Gly Cys Gly Glu Thr Pro Tyr 820 825 830Val Lys Leu
Met Thr Gln Leu Phe Gly Asp Arg Leu Val Ile Gly Asn 835 840 845Ala
Thr Gly Cys Ser Ser Ile Tyr Gly Gly Asn Leu Pro Thr Thr Pro 850 855
860Tyr Ala Ala Asn Pro Gln Gly Leu Gly Pro Thr Trp Ser Asn Ser
Leu865 870 875 880Phe Glu Asp Thr Ala Glu Phe Ala Leu Gly Phe Arg
Ile Ser Ile Asp 885 890 895Lys Gln Gln Gln Phe Ala Lys Glu Leu Val
Lys Lys Leu Ala Gly Asp 900 905 910Ile Gly Glu Asn Leu Ala Thr Ala
Ile Leu Asn Ala Thr Gln Asn Ser 915 920 925Glu Pro Glu Ile Phe Glu
Gln Arg Glu Arg Val Ala Val Leu Lys Asp 930 935 940Lys Leu Gln Gln
Met Lys Ser Asp Asp Ala Lys Asn Leu Leu Ala Val945 950 955 960Ala
Asp Met Leu Val Lys Lys Ser Val Trp Ala Val Gly Gly Asp Gly 965 970
975Trp Ala Tyr Asp Ile Gly Tyr Gly Gly Leu Asp His Val Thr Ala Ser
980 985 990Gly Lys Asn Val Asn Met Leu Val Leu Asp Thr Glu Val Tyr
Ser Asn 995 1000 1005Thr Gly Gly Gln Ala Ser Lys Ala Thr Pro Lys
Ala Ala Ile Ala 1010 1015 1020Lys Phe Ala Ala Ala Gly Arg Ile Ala
Thr Lys Lys Asp Leu Gly 1025 1030 1035Leu Ile Ser Met Ser Tyr Gly
Asn Ala Tyr Val Ala Ser Val Ala 1040 1045 1050Leu Gly Ala Arg Asp
Glu Gln Thr Leu Arg Ala Phe Ile Glu Ala 1055 1060 1065Glu Ala Tyr
Asp Gly Pro Ser Ile Ile Ile Ala Tyr Ser His Cys 1070 1075 1080Ile
Ala His Gly Phe Asp Leu Ser Met Gly Leu Glu His Gln Lys 1085 1090
1095Ala Ala Val Asp Ser Gly His Trp Leu Leu Tyr Arg Tyr Asn Pro
1100 1105 1110Asp Arg Leu Lys Glu Gly Leu Asn Pro Leu Gln Leu Asp
Ser Lys 1115 1120 1125Lys Pro Lys Met Pro Val Ala Glu Phe Leu Asn
Met Glu Asn Arg 1130 1135 1140Phe Arg Ile Leu Lys Lys Thr His Pro
Asp Leu Ala Lys Lys Tyr 1145 1150 1155Phe Glu Ala Ile Gln His Glu
Val Asn Ala Arg Trp Ala His Tyr 1160 1165 1170Glu His Leu Ala Asn
Arg Ser Ile Glu Gly Glu Ala 1175 1180 118533525DNAEscherichia
coliCDS(1)..(3525) 3atg att act att gac ggt aat ggc gcg gtt gct tcg
gtc gca ttt cgc 48Met Ile Thr Ile Asp Gly Asn Gly Ala Val Ala Ser
Val Ala Phe Arg1 5 10 15acc agt gaa gtt atc gcc atc tac cct att acc
ccc agt tcc acg atg 96Thr Ser Glu Val Ile Ala Ile Tyr Pro Ile Thr
Pro Ser Ser Thr Met 20 25 30gca gaa cag gct gat gcc tgg gcc gga aac
ggc tta aag aac gtt tgg 144Ala Glu Gln Ala Asp Ala Trp Ala Gly Asn
Gly Leu Lys Asn Val Trp 35 40 45gga gac aca cca cgc gtg gtt gaa atg
cag tcg gaa gcg ggt gct atc 192Gly Asp Thr Pro Arg Val Val Glu Met
Gln Ser Glu Ala Gly Ala Ile 50 55 60gct acc gtg cat ggc gct ttg cag
acg ggt gcc ctt tca aca tcg ttt 240Ala Thr Val His Gly Ala Leu Gln
Thr Gly Ala Leu Ser Thr Ser Phe65 70 75 80acg tca tcg cag ggt ttg
ctg ctg atg atc ccg acg ctg tac aaa ctg 288Thr Ser Ser Gln Gly Leu
Leu Leu Met Ile Pro Thr Leu Tyr Lys Leu 85 90 95gca ggc gaa cta aca
ccg ttt gtc ctg cat gta gcg gca cgt acc gtt 336Ala Gly Glu Leu Thr
Pro Phe Val Leu His Val Ala Ala Arg Thr Val 100 105 110gcc aca cat
gca ctc tct att ttt ggc gat cat tcc gac gtt atg gcg 384Ala Thr His
Ala Leu Ser Ile Phe Gly Asp His Ser Asp Val Met Ala 115 120 125gtg
cgc cag acg ggt tgc gcg atg ttg tgt gca gca aac gtc cag gaa 432Val
Arg Gln Thr Gly Cys Ala Met Leu Cys Ala Ala Asn Val Gln Glu 130 135
140gcg caa gac ttt gct ctc att tcg caa atc gcg acg ctg aaa agc cgc
480Ala Gln Asp Phe Ala Leu Ile Ser Gln Ile Ala Thr Leu Lys Ser
Arg145 150 155 160gtg cca ttt att cat ttc ttt gat ggt ttc cgc acg
tcc cac gaa atc 528Val Pro Phe Ile His Phe Phe Asp Gly Phe Arg Thr
Ser His Glu Ile 165 170 175aat aaa att gtc ccg ctg gcc gat gac acg
att ctt gat ctc atg ccg 576Asn Lys Ile Val Pro Leu Ala Asp Asp Thr
Ile Leu Asp Leu Met Pro 180 185 190cag gtc gaa att gat gct cat cgc
gcc cgg gca ctc aac ccg gaa cat 624Gln Val Glu Ile Asp Ala His Arg
Ala Arg Ala Leu Asn Pro Glu His 195 200 205ccg gtg atc cgc ggt acg
tcc gcc aat cct gac act tat ttc cag tct 672Pro Val Ile Arg Gly Thr
Ser Ala Asn Pro Asp Thr Tyr Phe Gln Ser 210 215 220cgc gaa gcc acc
aac cca tgg tac aac gcg gtc tat gac cat gtt gaa 720Arg Glu Ala Thr
Asn Pro Trp Tyr Asn Ala Val Tyr Asp His Val Glu225 230 235 240cag
gcg atg aat gat ttc tct gcc gcg aca ggt cgt cag tat cag ccg 768Gln
Ala Met Asn Asp Phe Ser Ala Ala Thr Gly Arg Gln Tyr Gln Pro 245 250
255ttt gaa tat tac ggg cat ccg caa gcg gaa cgg gtg att atc ctg atg
816Phe Glu Tyr Tyr Gly His Pro Gln Ala Glu Arg Val Ile Ile Leu Met
260 265 270ggc tct gcc att ggc acc tgt gaa gaa gtg gtt gat gaa ttg
cta acc 864Gly Ser Ala Ile Gly Thr Cys Glu Glu Val Val Asp Glu Leu
Leu Thr 275 280 285cgt ggc gaa aaa gtt ggc gtg ctg aaa gtt cgc ctg
tac cgc ccc ttc 912Arg Gly Glu Lys Val Gly Val Leu Lys Val Arg Leu
Tyr Arg Pro Phe 290 295 300tcc gct aaa cat tta ctg caa gct ctg ccg
gga tcc gta cgc agc gtg 960Ser Ala Lys His Leu Leu Gln Ala Leu Pro
Gly Ser Val Arg Ser Val305 310 315 320gcg gta ctg gac aga acc aaa
gaa ccc ggt gcc cag gca gaa ccg ctc 1008Ala Val Leu Asp Arg Thr Lys
Glu Pro Gly Ala Gln Ala Glu Pro Leu 325 330 335tat ctg gat gta atg
acc gca ctg gca gaa gcc ttt aat aat ggc gag 1056Tyr Leu Asp Val Met
Thr Ala Leu Ala Glu Ala Phe Asn Asn Gly Glu 340 345 350cgc gaa act
ctg ccc cgt gtc att ggt ggg cgc tat ggt ctt tca tcc 1104Arg Glu Thr
Leu Pro Arg Val Ile Gly Gly Arg Tyr Gly Leu Ser Ser 355 360 365aaa
gaa ttt ggc cca gac tgt gta ctg gcg gta ttt gcc gag ctc aac 1152Lys
Glu Phe Gly Pro Asp Cys Val Leu Ala Val Phe Ala Glu Leu Asn 370 375
380gcg gct aaa ccg aaa gcg cgc ttt acg gtt ggt att tac gat gat gtg
1200Ala Ala Lys Pro Lys Ala Arg Phe Thr Val Gly Ile Tyr Asp Asp
Val385 390 395 400acc aat ctg tca ctg ccg ttg ccg gaa aac acc ctg
cca aac tcg gcg 1248Thr Asn Leu Ser Leu Pro Leu Pro Glu Asn Thr Leu
Pro Asn Ser Ala 405 410 415aaa ctg gaa gcc ttg ttt tat ggc ctt ggt
agt gat ggc agc gtt tcc 1296Lys Leu Glu Ala Leu Phe Tyr Gly Leu Gly
Ser Asp Gly Ser Val Ser 420 425 430gcg acc aaa aac aat atc aag att
atc ggt aat tcc acg ccg tgg tac 1344Ala Thr Lys Asn Asn Ile Lys Ile
Ile Gly Asn Ser Thr Pro Trp Tyr 435 440 445gca cag ggc tat ttt gtt
tac gac tcc aaa aag gcg ggc ggc ctg acg 1392Ala Gln Gly Tyr Phe Val
Tyr Asp Ser Lys Lys Ala Gly Gly Leu Thr 450 455 460gtt tct cac ctt
cga gtg agc gaa cag ccg att cgt tcc gct tat ctc 1440Val Ser His Leu
Arg Val Ser Glu Gln Pro Ile Arg Ser Ala Tyr Leu465 470 475 480att
tcc cag gct gat ttt gtt ggc tgc cac cag ttg cag ttt atc gat 1488Ile
Ser Gln Ala Asp Phe Val Gly Cys His Gln Leu Gln Phe Ile Asp 485 490
495aaa tat cag atg gct gag cgt tta aaa cct ggc ggc att ttc ctg ctc
1536Lys Tyr Gln Met Ala Glu Arg Leu Lys Pro Gly Gly Ile Phe Leu Leu
500 505 510aac acg ccg tac agc gca gat gaa gtg tgg tcg cgc ttg ccg
caa gaa 1584Asn Thr Pro Tyr Ser Ala Asp Glu Val Trp Ser Arg Leu Pro
Gln Glu 515 520 525gtt cag gcc gtg tta aac cag aaa aaa gcg cgc ttc
tat gtg att aac 1632Val Gln Ala Val Leu Asn Gln Lys Lys Ala Arg Phe
Tyr Val Ile Asn 530 535 540gcg gcg aaa atc gcc cgc gaa tgt ggc ctg
gcg gcc cgt att aat acc 1680Ala Ala Lys Ile Ala Arg Glu Cys Gly Leu
Ala Ala Arg Ile Asn Thr545 550 555 560gtc atg cag atg gct ttt ttc
cat ctg acg caa att ctg cct ggc gat 1728Val Met Gln Met Ala Phe Phe
His Leu Thr Gln Ile Leu Pro Gly Asp 565 570 575agc gcc ctc gca gaa
ttg cag ggt gcg att gcc aaa agt tac agt agc 1776Ser Ala Leu Ala Glu
Leu Gln Gly Ala Ile Ala Lys Ser Tyr Ser Ser 580 585 590aaa ggc cag
gat ctg gtg gaa cgc aac tgg cag gct ctg gcg ctg gcg 1824Lys Gly Gln
Asp Leu Val Glu Arg Asn Trp Gln Ala Leu Ala Leu Ala 595 600 605cgt
gaa tcc gta gaa gaa gtt ccg ttg caa ccg gta aat ccg cac agc 1872Arg
Glu Ser Val Glu Glu Val Pro Leu Gln Pro Val Asn Pro His Ser 610 615
620gcc aat cga ccg cca gtg gtt tcc gat gcc gcc cct gat ttc gtg aaa
1920Ala Asn Arg Pro Pro Val Val Ser Asp Ala Ala Pro Asp Phe Val
Lys625 630 635 640acc gta acc gct gcg atg ctc gcc ggg ctt ggt gac
gcc ctc ccc gtt 1968Thr Val Thr Ala Ala Met Leu Ala Gly Leu Gly Asp
Ala Leu Pro Val 645 650 655tcg gcg ctg ccg cca gac ggc acc tgg ccg
atg ggc act acg cgc tgg 2016Ser Ala Leu Pro Pro Asp Gly Thr Trp Pro
Met Gly Thr Thr Arg Trp 660 665 670gaa aaa cgc aat atc gcc gaa gag
atc ccc atc tgg aaa gag gaa ctc 2064Glu Lys Arg Asn Ile Ala Glu Glu
Ile Pro Ile Trp Lys Glu Glu Leu 675 680 685tgt acc caa tgt aac cac
tgc gtt gcc gct tgc cca cac tca gct att 2112Cys Thr Gln Cys Asn His
Cys Val Ala Ala Cys Pro His Ser Ala Ile 690 695 700cgc gca aaa gtg
gtg ccg cct gaa gcg atg gaa aac gcc cct gcc agc 2160Arg Ala Lys Val
Val Pro Pro Glu Ala Met Glu Asn Ala Pro Ala Ser705 710 715 720ctg
cat tcg ctg gat gtg aaa tcg cgt gat atg cgc ggg cag aaa tat 2208Leu
His Ser Leu Asp Val Lys Ser Arg Asp Met Arg Gly Gln Lys Tyr 725 730
735gtc ttg cag gtg gca ccg gaa gat tgc acc ggt tgt aac ctg tgc gtc
2256Val Leu Gln Val Ala Pro Glu Asp Cys Thr Gly Cys Asn Leu Cys Val
740 745 750gaa gtt tgc ccg gcg aaa gac cgt cag aat cca gag att aaa
gcc atc 2304Glu Val Cys Pro Ala Lys Asp Arg Gln Asn Pro Glu Ile Lys
Ala Ile 755 760 765aat atg atg tct cgc ctg gaa cat gtc gaa gaa gag
aaa atc aat tac 2352Asn Met Met Ser Arg Leu Glu His Val Glu Glu Glu
Lys Ile Asn Tyr 770 775 780gat ttc ttc ctc aac ctg cca gaa atc gac
cgt agc aaa ctg gaa cgt 2400Asp Phe Phe Leu Asn Leu Pro Glu Ile Asp
Arg Ser Lys Leu Glu Arg785 790 795 800att gat att cgt aca tcg cag
ctg att aca ccg ctg ttt gaa tat tca 2448Ile Asp Ile Arg Thr Ser Gln
Leu Ile Thr Pro Leu Phe Glu Tyr Ser 805 810 815ggt gct tgc tcc ggt
tgt ggc gag acg ccg tat att aaa tta ctg act 2496Gly Ala Cys Ser Gly
Cys Gly Glu Thr Pro Tyr Ile Lys Leu Leu Thr 820 825 830cag ctc tat
ggc gac cgg atg ttg atc gct aac gcc act ggc tgt tct 2544Gln Leu Tyr
Gly Asp Arg Met Leu Ile Ala Asn Ala Thr Gly Cys Ser 835 840 845tca
att tat ggc ggt aac ctg ccc tct aca ccg tat acc acc gat gcc 2592Ser
Ile Tyr Gly Gly Asn Leu Pro Ser Thr Pro Tyr Thr Thr Asp Ala 850 855
860aac ggt cgt ggg ccg gca tgg gcg aac tct cta ttt gaa gat aat gcc
2640Asn Gly Arg Gly Pro Ala Trp Ala Asn Ser Leu Phe Glu Asp Asn
Ala865 870 875 880gaa ttt ggc ctt ggt ttc cgc ctg acg gtc gat caa
cac cgt gtc cgc 2688Glu Phe Gly Leu Gly Phe Arg Leu Thr Val Asp Gln
His Arg Val Arg 885 890 895gtg ctg cgt ctg ctg gat caa ttt gcc gat
aaa atc ccg gcg gaa tta 2736Val Leu Arg Leu Leu Asp Gln Phe Ala Asp
Lys Ile Pro Ala Glu Leu 900 905 910ctg acg gcg ttg aaa tca gac gcc
acg cca gag gtt cgt cgt gaa cag 2784Leu Thr Ala Leu Lys Ser Asp Ala
Thr Pro Glu Val Arg Arg Glu Gln 915 920 925gtt gca gct tta cgc cag
caa ctc aac gat gtt gcc gaa gca cat gaa 2832Val Ala Ala Leu Arg Gln
Gln Leu Asn Asp Val Ala Glu Ala His Glu 930 935 940ctg cta cgt gat
gca gat gca ctg gtg gaa aaa tca atc tgg ctg att 2880Leu Leu Arg Asp
Ala Asp Ala Leu Val Glu Lys Ser Ile Trp Leu Ile945 950 955 960ggt
ggt gat ggc tgg gct tac gat atc ggc ttt ggc ggt ctg gat cat 2928Gly
Gly Asp Gly Trp Ala Tyr Asp Ile Gly Phe Gly Gly Leu Asp His 965 970
975gta ttg agt ttg acg gaa aac gtc aac att ctg gtg ctg gat acg caa
2976Val Leu Ser Leu Thr Glu Asn Val Asn Ile Leu Val Leu Asp Thr Gln
980 985 990tgc tat tcc aac acc ggt ggt cag gcg tcg aaa gcg aca ccg
ctg ggt 3024Cys Tyr Ser Asn Thr Gly Gly Gln Ala Ser Lys Ala Thr Pro
Leu Gly 995 1000 1005gca gta act aaa ttt ggc gag cac ggc aaa cgt
aaa gcg cgt aaa gat 3072Ala Val Thr Lys Phe Gly Glu His Gly Lys Arg
Lys Ala Arg Lys Asp 1010 1015 1020ctt ggc gtc agt atg atg atg tac
ggt cat gtt tat gtg gcg cag att 3120Leu Gly Val Ser Met Met Met Tyr
Gly His Val Tyr Val Ala Gln Ile1025 1030 1035 1040tct ctc ggc gcg
cag ctg aac cag acg gtg aaa gcg att cag gaa gcg 3168Ser Leu Gly Ala
Gln Leu Asn Gln Thr Val Lys Ala Ile Gln Glu Ala 1045 1050 1055gaa
gcg tat ccg ggg cca tcg ctg atc att gct tat agc ccg tgt gaa 3216Glu
Ala Tyr Pro Gly Pro Ser Leu Ile Ile Ala Tyr Ser Pro Cys Glu 1060
1065 1070gag cat ggt tac gat ctg gca ctc agc cac gac cag atg cgc
caa ctc 3264Glu His Gly Tyr Asp Leu Ala Leu Ser His Asp Gln Met Arg
Gln Leu 1075 1080 1085aca gct acc ggc ttc tgg ccg cta tat cgc ttt
gat ccg cgt cgt gcc 3312Thr Ala Thr Gly Phe Trp Pro Leu Tyr Arg Phe
Asp Pro Arg Arg Ala 1090 1095 1100gat gaa ggc aaa ctg ccg ctg gcc
ttg gat tca cgc ccg ccg tca gaa 3360Asp Glu Gly Lys Leu Pro Leu Ala
Leu Asp Ser Arg Pro Pro Ser Glu1105 1110 1115 1120gca ccg gaa gaa
acg tta ctt cac gag caa cgt ttc cgt cgg ctg aat 3408Ala Pro Glu Glu
Thr Leu Leu His Glu Gln Arg Phe Arg Arg Leu Asn 1125 1130 1135tcg
cag cag cca gaa
gtg gca gaa cag tta tgg aaa gat gct gca gct 3456Ser Gln Gln Pro Glu
Val Ala Glu Gln Leu Trp Lys Asp Ala Ala Ala 1140 1145 1150gat ttg
caa aaa cgc tat gac ttc ctg gca caa atg gcc gga aaa gcg 3504Asp Leu
Gln Lys Arg Tyr Asp Phe Leu Ala Gln Met Ala Gly Lys Ala 1155 1160
1165gaa aaa agc aac acc gat taa 3525Glu Lys Ser Asn Thr Asp
117041174PRTEscherichia coli 4Met Ile Thr Ile Asp Gly Asn Gly Ala
Val Ala Ser Val Ala Phe Arg1 5 10 15Thr Ser Glu Val Ile Ala Ile Tyr
Pro Ile Thr Pro Ser Ser Thr Met 20 25 30Ala Glu Gln Ala Asp Ala Trp
Ala Gly Asn Gly Leu Lys Asn Val Trp 35 40 45Gly Asp Thr Pro Arg Val
Val Glu Met Gln Ser Glu Ala Gly Ala Ile 50 55 60Ala Thr Val His Gly
Ala Leu Gln Thr Gly Ala Leu Ser Thr Ser Phe65 70 75 80Thr Ser Ser
Gln Gly Leu Leu Leu Met Ile Pro Thr Leu Tyr Lys Leu 85 90 95Ala Gly
Glu Leu Thr Pro Phe Val Leu His Val Ala Ala Arg Thr Val 100 105
110Ala Thr His Ala Leu Ser Ile Phe Gly Asp His Ser Asp Val Met Ala
115 120 125Val Arg Gln Thr Gly Cys Ala Met Leu Cys Ala Ala Asn Val
Gln Glu 130 135 140Ala Gln Asp Phe Ala Leu Ile Ser Gln Ile Ala Thr
Leu Lys Ser Arg145 150 155 160Val Pro Phe Ile His Phe Phe Asp Gly
Phe Arg Thr Ser His Glu Ile 165 170 175Asn Lys Ile Val Pro Leu Ala
Asp Asp Thr Ile Leu Asp Leu Met Pro 180 185 190Gln Val Glu Ile Asp
Ala His Arg Ala Arg Ala Leu Asn Pro Glu His 195 200 205Pro Val Ile
Arg Gly Thr Ser Ala Asn Pro Asp Thr Tyr Phe Gln Ser 210 215 220Arg
Glu Ala Thr Asn Pro Trp Tyr Asn Ala Val Tyr Asp His Val Glu225 230
235 240Gln Ala Met Asn Asp Phe Ser Ala Ala Thr Gly Arg Gln Tyr Gln
Pro 245 250 255Phe Glu Tyr Tyr Gly His Pro Gln Ala Glu Arg Val Ile
Ile Leu Met 260 265 270Gly Ser Ala Ile Gly Thr Cys Glu Glu Val Val
Asp Glu Leu Leu Thr 275 280 285Arg Gly Glu Lys Val Gly Val Leu Lys
Val Arg Leu Tyr Arg Pro Phe 290 295 300Ser Ala Lys His Leu Leu Gln
Ala Leu Pro Gly Ser Val Arg Ser Val305 310 315 320Ala Val Leu Asp
Arg Thr Lys Glu Pro Gly Ala Gln Ala Glu Pro Leu 325 330 335Tyr Leu
Asp Val Met Thr Ala Leu Ala Glu Ala Phe Asn Asn Gly Glu 340 345
350Arg Glu Thr Leu Pro Arg Val Ile Gly Gly Arg Tyr Gly Leu Ser Ser
355 360 365Lys Glu Phe Gly Pro Asp Cys Val Leu Ala Val Phe Ala Glu
Leu Asn 370 375 380Ala Ala Lys Pro Lys Ala Arg Phe Thr Val Gly Ile
Tyr Asp Asp Val385 390 395 400Thr Asn Leu Ser Leu Pro Leu Pro Glu
Asn Thr Leu Pro Asn Ser Ala 405 410 415Lys Leu Glu Ala Leu Phe Tyr
Gly Leu Gly Ser Asp Gly Ser Val Ser 420 425 430Ala Thr Lys Asn Asn
Ile Lys Ile Ile Gly Asn Ser Thr Pro Trp Tyr 435 440 445Ala Gln Gly
Tyr Phe Val Tyr Asp Ser Lys Lys Ala Gly Gly Leu Thr 450 455 460Val
Ser His Leu Arg Val Ser Glu Gln Pro Ile Arg Ser Ala Tyr Leu465 470
475 480Ile Ser Gln Ala Asp Phe Val Gly Cys His Gln Leu Gln Phe Ile
Asp 485 490 495Lys Tyr Gln Met Ala Glu Arg Leu Lys Pro Gly Gly Ile
Phe Leu Leu 500 505 510Asn Thr Pro Tyr Ser Ala Asp Glu Val Trp Ser
Arg Leu Pro Gln Glu 515 520 525Val Gln Ala Val Leu Asn Gln Lys Lys
Ala Arg Phe Tyr Val Ile Asn 530 535 540Ala Ala Lys Ile Ala Arg Glu
Cys Gly Leu Ala Ala Arg Ile Asn Thr545 550 555 560Val Met Gln Met
Ala Phe Phe His Leu Thr Gln Ile Leu Pro Gly Asp 565 570 575Ser Ala
Leu Ala Glu Leu Gln Gly Ala Ile Ala Lys Ser Tyr Ser Ser 580 585
590Lys Gly Gln Asp Leu Val Glu Arg Asn Trp Gln Ala Leu Ala Leu Ala
595 600 605Arg Glu Ser Val Glu Glu Val Pro Leu Gln Pro Val Asn Pro
His Ser 610 615 620Ala Asn Arg Pro Pro Val Val Ser Asp Ala Ala Pro
Asp Phe Val Lys625 630 635 640Thr Val Thr Ala Ala Met Leu Ala Gly
Leu Gly Asp Ala Leu Pro Val 645 650 655Ser Ala Leu Pro Pro Asp Gly
Thr Trp Pro Met Gly Thr Thr Arg Trp 660 665 670Glu Lys Arg Asn Ile
Ala Glu Glu Ile Pro Ile Trp Lys Glu Glu Leu 675 680 685Cys Thr Gln
Cys Asn His Cys Val Ala Ala Cys Pro His Ser Ala Ile 690 695 700Arg
Ala Lys Val Val Pro Pro Glu Ala Met Glu Asn Ala Pro Ala Ser705 710
715 720Leu His Ser Leu Asp Val Lys Ser Arg Asp Met Arg Gly Gln Lys
Tyr 725 730 735Val Leu Gln Val Ala Pro Glu Asp Cys Thr Gly Cys Asn
Leu Cys Val 740 745 750Glu Val Cys Pro Ala Lys Asp Arg Gln Asn Pro
Glu Ile Lys Ala Ile 755 760 765Asn Met Met Ser Arg Leu Glu His Val
Glu Glu Glu Lys Ile Asn Tyr 770 775 780Asp Phe Phe Leu Asn Leu Pro
Glu Ile Asp Arg Ser Lys Leu Glu Arg785 790 795 800Ile Asp Ile Arg
Thr Ser Gln Leu Ile Thr Pro Leu Phe Glu Tyr Ser 805 810 815Gly Ala
Cys Ser Gly Cys Gly Glu Thr Pro Tyr Ile Lys Leu Leu Thr 820 825
830Gln Leu Tyr Gly Asp Arg Met Leu Ile Ala Asn Ala Thr Gly Cys Ser
835 840 845Ser Ile Tyr Gly Gly Asn Leu Pro Ser Thr Pro Tyr Thr Thr
Asp Ala 850 855 860Asn Gly Arg Gly Pro Ala Trp Ala Asn Ser Leu Phe
Glu Asp Asn Ala865 870 875 880Glu Phe Gly Leu Gly Phe Arg Leu Thr
Val Asp Gln His Arg Val Arg 885 890 895Val Leu Arg Leu Leu Asp Gln
Phe Ala Asp Lys Ile Pro Ala Glu Leu 900 905 910Leu Thr Ala Leu Lys
Ser Asp Ala Thr Pro Glu Val Arg Arg Glu Gln 915 920 925Val Ala Ala
Leu Arg Gln Gln Leu Asn Asp Val Ala Glu Ala His Glu 930 935 940Leu
Leu Arg Asp Ala Asp Ala Leu Val Glu Lys Ser Ile Trp Leu Ile945 950
955 960Gly Gly Asp Gly Trp Ala Tyr Asp Ile Gly Phe Gly Gly Leu Asp
His 965 970 975Val Leu Ser Leu Thr Glu Asn Val Asn Ile Leu Val Leu
Asp Thr Gln 980 985 990Cys Tyr Ser Asn Thr Gly Gly Gln Ala Ser Lys
Ala Thr Pro Leu Gly 995 1000 1005Ala Val Thr Lys Phe Gly Glu His
Gly Lys Arg Lys Ala Arg Lys 1010 1015 1020Asp Leu Gly Val Ser Met
Met Met Tyr Gly His Val Tyr Val Ala 1025 1030 1035Gln Ile Ser Leu
Gly Ala Gln Leu Asn Gln Thr Val Lys Ala Ile 1040 1045 1050 Gln Glu
Ala Glu Ala Tyr Pro Gly Pro Ser Leu Ile Ile Ala Tyr 1055 1060
1065Ser Pro Cys Glu Glu His Gly Tyr Asp Leu Ala Leu Ser His Asp
1070 1075 1080Gln Met Arg Gln Leu Thr Ala Thr Gly Phe Trp Pro Leu
Tyr Arg 1085 1090 1095Phe Asp Pro Arg Arg Ala Asp Glu Gly Lys Leu
Pro Leu Ala Leu 1100 1105 1110Asp Ser Arg Pro Pro Ser Glu Ala Pro
Glu Glu Thr Leu Leu His 1115 1120 1125Glu Gln Arg Phe Arg Arg Leu
Asn Ser Gln Gln Pro Glu Val Ala 1130 1135 1140Glu Gln Leu Trp Lys
Asp Ala Ala Ala Asp Leu Gln Lys Arg Tyr 1145 1150 1155Asp Phe Leu
Ala Gln Met Ala Gly Lys Ala Glu Lys Ser Asn Thr 1160 1165
1170Asp55412DNAEuglena gracilisCDS(1)..(5412) 5atg aag cag tct gtc
cgc cca att att tcc aat gta ctg cgc aag gag 48Met Lys Gln Ser Val
Arg Pro Ile Ile Ser Asn Val Leu Arg Lys Glu1 5 10 15gtt gct ctg tac
tca aca atc att gga caa gac aag ggg aag gaa cca 96Val Ala Leu Tyr
Ser Thr Ile Ile Gly Gln Asp Lys Gly Lys Glu Pro 20 25 30act ggt cga
aca tac acc agt ggc cca aaa ccg gca tct cac att gaa 144Thr Gly Arg
Thr Tyr Thr Ser Gly Pro Lys Pro Ala Ser His Ile Glu 35 40 45gtt ccc
cat cat gtg act gtg cct gcc act gac cgc acc ccg aat ccc 192Val Pro
His His Val Thr Val Pro Ala Thr Asp Arg Thr Pro Asn Pro 50 55 60gat
gct caa ttc ttt cag tct gta gat ggg tca caa gcc acc agt cac 240Asp
Ala Gln Phe Phe Gln Ser Val Asp Gly Ser Gln Ala Thr Ser His65 70 75
80gtt gcg tac gct ctg tct gac aca gcg ttc att tac cca att aca ccc
288Val Ala Tyr Ala Leu Ser Asp Thr Ala Phe Ile Tyr Pro Ile Thr Pro
85 90 95agt tct gtg atg ggc gag ctg gct gat gtt tgg atg gct caa ggg
agg 336Ser Ser Val Met Gly Glu Leu Ala Asp Val Trp Met Ala Gln Gly
Arg 100 105 110aag aac gcc ttt ggt cag gtt gtg gat gtc cgt gag atg
caa tct gag 384Lys Asn Ala Phe Gly Gln Val Val Asp Val Arg Glu Met
Gln Ser Glu 115 120 125gct gga gcc gca ggc gcc ctg cat ggg gca ctg
gct gct gga gct att 432Ala Gly Ala Ala Gly Ala Leu His Gly Ala Leu
Ala Ala Gly Ala Ile 130 135 140gct aca acc ttc act gcc tct caa ggg
ttg ttg ttg atg att ccc aac 480Ala Thr Thr Phe Thr Ala Ser Gln Gly
Leu Leu Leu Met Ile Pro Asn145 150 155 160atg tat aag att gca ggt
gag ctg atg ccc tct gtc atc cac gtt gca 528Met Tyr Lys Ile Ala Gly
Glu Leu Met Pro Ser Val Ile His Val Ala 165 170 175gcc cga gag ctt
gca ggc cac gct ctg tcc att ttt gga gga cac gct 576Ala Arg Glu Leu
Ala Gly His Ala Leu Ser Ile Phe Gly Gly His Ala 180 185 190gat gtc
atg gct gtc cgc caa aca gga tgg gct atg ctg tgc tcc cac 624Asp Val
Met Ala Val Arg Gln Thr Gly Trp Ala Met Leu Cys Ser His 195 200
205aca gtg cag cag tct cac gac atg gct ctc atc tcc cac gtg gcc acc
672Thr Val Gln Gln Ser His Asp Met Ala Leu Ile Ser His Val Ala Thr
210 215 220ctc aag tcc agc atc ccc ttc gtt cac ttc ttt gat ggt ttc
cgc aca 720Leu Lys Ser Ser Ile Pro Phe Val His Phe Phe Asp Gly Phe
Arg Thr225 230 235 240agc cac gaa gtg aac aaa atc aaa atg ctg cct
tat gca gaa ctg aag 768Ser His Glu Val Asn Lys Ile Lys Met Leu Pro
Tyr Ala Glu Leu Lys 245 250 255aaa ctg gtg cct cct ggc acc atg gaa
cag cac tgg gct cgt tcg ctg 816Lys Leu Val Pro Pro Gly Thr Met Glu
Gln His Trp Ala Arg Ser Leu 260 265 270aac ccc atg cac ccc acc atc
cga gga aca aac cag tct gca gac atc 864Asn Pro Met His Pro Thr Ile
Arg Gly Thr Asn Gln Ser Ala Asp Ile 275 280 285tac ttc cag aat atg
gaa agt gca aac cag tac tac act gat ctg gcc 912Tyr Phe Gln Asn Met
Glu Ser Ala Asn Gln Tyr Tyr Thr Asp Leu Ala 290 295 300gag gtc gtt
cag gag aca atg gac gaa gtt gca cca tac atc ggt cgc 960Glu Val Val
Gln Glu Thr Met Asp Glu Val Ala Pro Tyr Ile Gly Arg305 310 315
320cac tac aag atc ttt gag tat gtt ggt gca cca gat gca gaa gaa gtg
1008His Tyr Lys Ile Phe Glu Tyr Val Gly Ala Pro Asp Ala Glu Glu Val
325 330 335aca gtg ctc atg ggt tct ggt gca acc aca gtc aac gag gca
gtg gac 1056Thr Val Leu Met Gly Ser Gly Ala Thr Thr Val Asn Glu Ala
Val Asp 340 345 350ctt ctt gtg aag cgt gga aag aag gtt ggt gca gtc
ttg gtg cac ctc 1104Leu Leu Val Lys Arg Gly Lys Lys Val Gly Ala Val
Leu Val His Leu 355 360 365tac cga cca tgg tca aca aag gca ttt gaa
aag gtc ctg ccc aag aca 1152Tyr Arg Pro Trp Ser Thr Lys Ala Phe Glu
Lys Val Leu Pro Lys Thr 370 375 380gtg aag cgc att gct gct ctg gat
cgc tgc aag gag gtg act gca ctg 1200Val Lys Arg Ile Ala Ala Leu Asp
Arg Cys Lys Glu Val Thr Ala Leu385 390 395 400ggt gag cct ctg tat
ctg gat gtg tcg gca act ctg aat ttg ttc ccg 1248Gly Glu Pro Leu Tyr
Leu Asp Val Ser Ala Thr Leu Asn Leu Phe Pro 405 410 415gaa cgc cag
aat gtg aaa gtc att gga gga cgt tac gga ttg ggc tca 1296Glu Arg Gln
Asn Val Lys Val Ile Gly Gly Arg Tyr Gly Leu Gly Ser 420 425 430aag
gat ttc atc ccg gag cat gcc ctg gca att tac gcc aac ttg gcc 1344Lys
Asp Phe Ile Pro Glu His Ala Leu Ala Ile Tyr Ala Asn Leu Ala 435 440
445agc gag aac ccc att caa aga ttc act gtg ggt atc aca gat gat gtc
1392Ser Glu Asn Pro Ile Gln Arg Phe Thr Val Gly Ile Thr Asp Asp Val
450 455 460act ggc aca tcc gtt cct ttc gtc aac gag cgt gtt gac acg
ttg ccc 1440Thr Gly Thr Ser Val Pro Phe Val Asn Glu Arg Val Asp Thr
Leu Pro465 470 475 480gag ggc acc cgc cag tgt gtc ttc tgg gga att
ggt tca gat gga aca 1488Glu Gly Thr Arg Gln Cys Val Phe Trp Gly Ile
Gly Ser Asp Gly Thr 485 490 495gtg gga gcc aat cgc tct gcc gtg aga
atc att gga gac aac agc gat 1536Val Gly Ala Asn Arg Ser Ala Val Arg
Ile Ile Gly Asp Asn Ser Asp 500 505 510ttg atg gtt cag gcc tac ttc
caa ttt gat gct ttc aag tca ggt ggt 1584Leu Met Val Gln Ala Tyr Phe
Gln Phe Asp Ala Phe Lys Ser Gly Gly 515 520 525gtc act tcc tcg cat
ctc cgt ttt gga cca aag ccc atc aca gcg caa 1632Val Thr Ser Ser His
Leu Arg Phe Gly Pro Lys Pro Ile Thr Ala Gln 530 535 540tac ctt gtt
acc aat gct gac tac atc gcg tgc cac ttc cag gag tat 1680Tyr Leu Val
Thr Asn Ala Asp Tyr Ile Ala Cys His Phe Gln Glu Tyr545 550 555
560gtc aag cgc ttt gac atg ctt gat gcc atc cgt gag ggg ggc acc ttt
1728Val Lys Arg Phe Asp Met Leu Asp Ala Ile Arg Glu Gly Gly Thr Phe
565 570 575gtt ctc aat tct cgg tgg acc acg gag gac atg gag aag gag
att ccg 1776Val Leu Asn Ser Arg Trp Thr Thr Glu Asp Met Glu Lys Glu
Ile Pro 580 585 590gct gac ttc cgg cgc aag ctg gca cag aag aag gtc
cgc ttc tac aat 1824Ala Asp Phe Arg Arg Lys Leu Ala Gln Lys Lys Val
Arg Phe Tyr Asn 595 600 605gtg gat gct cga aag atc tgt gac agt ttt
ggt ctt ggg aag cgc atc 1872Val Asp Ala Arg Lys Ile Cys Asp Ser Phe
Gly Leu Gly Lys Arg Ile 610 615 620aat atg ctg atg cag gct tgt ttc
ttc aag ctg tct ggg gtg ctc cca 1920Asn Met Leu Met Gln Ala Cys Phe
Phe Lys Leu Ser Gly Val Leu Pro625 630 635 640ctg gcc gaa gct cag
cgg ctg ctg aac gag tcc att gtg cat gag tat 1968Leu Ala Glu Ala Gln
Arg Leu Leu Asn Glu Ser Ile Val His Glu Tyr 645 650 655gga aag aag
ggt ggc aag gtg gtg gag atg aac caa gca gtg gtg aat 2016Gly Lys Lys
Gly Gly Lys Val Val Glu Met Asn Gln Ala Val Val Asn 660 665 670gct
gtc ttt gct ggt gac ctg ccc cag gaa gtt caa gtc cct gcc gcc 2064Ala
Val Phe Ala Gly Asp Leu Pro Gln Glu Val Gln Val Pro Ala Ala 675 680
685tgg gca aac gca gtt gat aca tcc acc cgt acc ccc acc ggg att gag
2112Trp Ala Asn Ala Val Asp Thr Ser Thr Arg Thr Pro Thr Gly Ile Glu
690 695 700ttt gtt gac aag atc atg cgc ccg ctg atg gat ttc aag ggt
gac cag 2160Phe Val Asp Lys Ile Met Arg Pro Leu Met Asp Phe Lys Gly
Asp Gln705 710 715 720ctc cca gtc agt gtg atg act cct ggt gga acc
ttc cct gtc ggg aca 2208Leu Pro Val Ser Val Met Thr Pro Gly Gly Thr
Phe Pro Val Gly Thr 725 730 735aca cag tat gcc aag cgt gca att gct
gct ttc att ccc cag tgg att 2256Thr Gln Tyr Ala Lys Arg Ala Ile Ala
Ala Phe Ile Pro Gln Trp Ile 740 745 750cct gcc aac tgc aca cag tgc
aac tat tgt tcg tat gtt tgc ccc cac 2304Pro Ala Asn Cys Thr Gln Cys
Asn Tyr Cys Ser Tyr Val Cys Pro His
755 760 765gcc acc atc cga cct ttc gtg ctg aca gac cag gag gtg cag
ctg gcc 2352Ala Thr Ile Arg Pro Phe Val Leu Thr Asp Gln Glu Val Gln
Leu Ala 770 775 780ccg gag agc ttt gtg aca cgc aag gcg aag ggt gat
tac cag ggg atg 2400Pro Glu Ser Phe Val Thr Arg Lys Ala Lys Gly Asp
Tyr Gln Gly Met785 790 795 800aat ttc cgc atc caa gtt gct cct gag
gat tgc act ggc tgc cag gtg 2448Asn Phe Arg Ile Gln Val Ala Pro Glu
Asp Cys Thr Gly Cys Gln Val 805 810 815tgc gtg gag acg tgc ccc gat
gat gcc ctg gag atg acc gac gct ttc 2496Cys Val Glu Thr Cys Pro Asp
Asp Ala Leu Glu Met Thr Asp Ala Phe 820 825 830acc gcc acc cct gtg
caa cgc acc aac tgg gag ttc gcc atc aag gtg 2544Thr Ala Thr Pro Val
Gln Arg Thr Asn Trp Glu Phe Ala Ile Lys Val 835 840 845ccc aac cgc
ggc acc atg acg gac cgc tac tcc ctg aag ggc agc cag 2592Pro Asn Arg
Gly Thr Met Thr Asp Arg Tyr Ser Leu Lys Gly Ser Gln 850 855 860ttc
cag cag ccc ctc ctg gag ttc tcc ggg gcc tgc gag ggc tgc ggc 2640Phe
Gln Gln Pro Leu Leu Glu Phe Ser Gly Ala Cys Glu Gly Cys Gly865 870
875 880gag acc cca tat gtc aag ctg ctc acc cag ctc ttc ggc gag cgg
acg 2688Glu Thr Pro Tyr Val Lys Leu Leu Thr Gln Leu Phe Gly Glu Arg
Thr 885 890 895gtc atc gcc aac gcc acc ggc tgc agt tcc atc tgg ggt
ggc act gcc 2736Val Ile Ala Asn Ala Thr Gly Cys Ser Ser Ile Trp Gly
Gly Thr Ala 900 905 910ggc ctg gcg ccg tac acc acc aac gcc aag ggc
cag ggc ccg gcc tgg 2784Gly Leu Ala Pro Tyr Thr Thr Asn Ala Lys Gly
Gln Gly Pro Ala Trp 915 920 925ggc aac agc ctg ttc gag gac aac gcc
gag ttc ggc ttt ggc att gca 2832Gly Asn Ser Leu Phe Glu Asp Asn Ala
Glu Phe Gly Phe Gly Ile Ala 930 935 940gtg gcc aac gcc cag aag agg
tcc cgc gtg agg gac tgc atc ctg cag 2880Val Ala Asn Ala Gln Lys Arg
Ser Arg Val Arg Asp Cys Ile Leu Gln945 950 955 960gca gtg gag aag
aag gtc gcc gat gag ggt ttg acc aca ttg ttg gcg 2928Ala Val Glu Lys
Lys Val Ala Asp Glu Gly Leu Thr Thr Leu Leu Ala 965 970 975caa tgg
ctg cag gat tgg aac aca gga gac aag acc ttg aag tac caa 2976Gln Trp
Leu Gln Asp Trp Asn Thr Gly Asp Lys Thr Leu Lys Tyr Gln 980 985
990gac cag atc att gca ggg ctg gca cag cag cgc agc aag gat ccc ctt
3024Asp Gln Ile Ile Ala Gly Leu Ala Gln Gln Arg Ser Lys Asp Pro Leu
995 1000 1005ctg gag cag atc tat ggc atg aag gac atg ctg cct aac
atc agc cag 3072Leu Glu Gln Ile Tyr Gly Met Lys Asp Met Leu Pro Asn
Ile Ser Gln 1010 1015 1020tgg atc att ggt ggt gat ggc tgg gcc aac
gac att ggt ttc ggt ggg 3120Trp Ile Ile Gly Gly Asp Gly Trp Ala Asn
Asp Ile Gly Phe Gly Gly1025 1030 1035 1040ctg gac cac gtg ctg gcc
tct ggg cag aac ctc aac gtc ctg gtg ctg 3168Leu Asp His Val Leu Ala
Ser Gly Gln Asn Leu Asn Val Leu Val Leu 1045 1050 1055gac acc gag
atg tac agc aac acc ggt ggg cag gcc tcc aag tcc acc 3216Asp Thr Glu
Met Tyr Ser Asn Thr Gly Gly Gln Ala Ser Lys Ser Thr 1060 1065
1070cac atg gcc tct gtg gcc aag ttt gcc ctg gga ggg aag cgc acc aac
3264His Met Ala Ser Val Ala Lys Phe Ala Leu Gly Gly Lys Arg Thr Asn
1075 1080 1085aag aag aac ttg acg gag atg gca atg agc tat ggc aac
gtc tat gtg 3312Lys Lys Asn Leu Thr Glu Met Ala Met Ser Tyr Gly Asn
Val Tyr Val 1090 1095 1100gcc acc gtc tcc cat ggc aac atg gcc cag
tgc gtc aag gcg ttt gtg 3360Ala Thr Val Ser His Gly Asn Met Ala Gln
Cys Val Lys Ala Phe Val1105 1110 1115 1120gag gct gag tct tat gat
gga cct tcg ctc att gtt ggc tat gcg cca 3408Glu Ala Glu Ser Tyr Asp
Gly Pro Ser Leu Ile Val Gly Tyr Ala Pro 1125 1130 1135tgc atc gag
cat ggt ctg cgt gct ggt atg gca agg atg gtt caa gag 3456Cys Ile Glu
His Gly Leu Arg Ala Gly Met Ala Arg Met Val Gln Glu 1140 1145
1150tct gag gct gcc atc gcc acg gga tac tgg ccc ctg tac cgc ttt gac
3504Ser Glu Ala Ala Ile Ala Thr Gly Tyr Trp Pro Leu Tyr Arg Phe Asp
1155 1160 1165ccc cgc ctg gcg acc gag ggc aag aac ccc ttc cag ctg
gac tcc aag 3552Pro Arg Leu Ala Thr Glu Gly Lys Asn Pro Phe Gln Leu
Asp Ser Lys 1170 1175 1180cgc atc aag ggc aac ctg cag gag tac ctg
gac cgc cag aac cgg tat 3600Arg Ile Lys Gly Asn Leu Gln Glu Tyr Leu
Asp Arg Gln Asn Arg Tyr1185 1190 1195 1200gtc aac ctg aag aag aac
aac ccg aag ggt gcg gat ctg ctg aag tct 3648Val Asn Leu Lys Lys Asn
Asn Pro Lys Gly Ala Asp Leu Leu Lys Ser 1205 1210 1215cag atg gcc
gac aac atc acc gcc cgg ttc aac cgc tac cga cgc atg 3696Gln Met Ala
Asp Asn Ile Thr Ala Arg Phe Asn Arg Tyr Arg Arg Met 1220 1225
1230ttg gag ggc ccc aat aca aaa gcc gcc gcc ccc agc ggc aac cat gtg
3744Leu Glu Gly Pro Asn Thr Lys Ala Ala Ala Pro Ser Gly Asn His Val
1235 1240 1245acc atc ctg tac ggc tcc gaa act ggc aac agt gag ggt
ctg gca aag 3792Thr Ile Leu Tyr Gly Ser Glu Thr Gly Asn Ser Glu Gly
Leu Ala Lys 1250 1255 1260gag ctg gcc acc gac ttc gag cgc cgg gag
tac tcc gtc gca gtg cag 3840Glu Leu Ala Thr Asp Phe Glu Arg Arg Glu
Tyr Ser Val Ala Val Gln1265 1270 1275 1280gct ttg gat gac atc gac
gtt gct gac ttg gag aac atg ggc ttc gtg 3888Ala Leu Asp Asp Ile Asp
Val Ala Asp Leu Glu Asn Met Gly Phe Val 1285 1290 1295gtc att gcg
gtg tcc acc tgt ggg cag gga cag ttc ccc cgc aac agc 3936Val Ile Ala
Val Ser Thr Cys Gly Gln Gly Gln Phe Pro Arg Asn Ser 1300 1305
1310cag ctg ttc tgg cgg gag ctg cag cgg gac aag cct gag ggc tgg ctg
3984Gln Leu Phe Trp Arg Glu Leu Gln Arg Asp Lys Pro Glu Gly Trp Leu
1315 1320 1325aag aac ttg aag tac act gtc ttc ggg ctg ggc gac agc
aca tac tac 4032Lys Asn Leu Lys Tyr Thr Val Phe Gly Leu Gly Asp Ser
Thr Tyr Tyr 1330 1335 1340ttc tac tgc cac acc gcc aag cag atc gac
gct cgc ctg gcc gcc ttg 4080Phe Tyr Cys His Thr Ala Lys Gln Ile Asp
Ala Arg Leu Ala Ala Leu1345 1350 1355 1360ggc gct cag cgg gtg gtg
ccc att ggc ttc ggc gac gat ggg gat gag 4128Gly Ala Gln Arg Val Val
Pro Ile Gly Phe Gly Asp Asp Gly Asp Glu 1365 1370 1375gac atg ttc
cac acc ggc ttc aac aac tgg atc ccc agt gtg tgg aat 4176Asp Met Phe
His Thr Gly Phe Asn Asn Trp Ile Pro Ser Val Trp Asn 1380 1385
1390gag ctc aag acc aag act ccg gag gaa gcg ctg ttc acc ccg agc atc
4224Glu Leu Lys Thr Lys Thr Pro Glu Glu Ala Leu Phe Thr Pro Ser Ile
1395 1400 1405gcc gtg cag ctc acc ccc aac gcc acc ccg cag gat ttc
cat ttc gcc 4272Ala Val Gln Leu Thr Pro Asn Ala Thr Pro Gln Asp Phe
His Phe Ala 1410 1415 1420aag tcc acc cca gtg ctg tcc atc acc ggt
gcc gaa cgc atc acg ccg 4320Lys Ser Thr Pro Val Leu Ser Ile Thr Gly
Ala Glu Arg Ile Thr Pro1425 1430 1435 1440gca gac cac acc cgc aac
ttc gtc act atc cga tgg aag acc gat ttg 4368Ala Asp His Thr Arg Asn
Phe Val Thr Ile Arg Trp Lys Thr Asp Leu 1445 1450 1455tcg tac cag
gtg ggt gac tct ctt ggt gtc ttc cct gag aac acc cgg 4416Ser Tyr Gln
Val Gly Asp Ser Leu Gly Val Phe Pro Glu Asn Thr Arg 1460 1465
1470tca gtg gtg gag gag ttc ctg cag tat tac ggc ttg aac ccc aag gac
4464Ser Val Val Glu Glu Phe Leu Gln Tyr Tyr Gly Leu Asn Pro Lys Asp
1475 1480 1485gtc atc acc atc gaa aac aag ggc agc cgg gag ttg ccc
cac tgc atg 4512Val Ile Thr Ile Glu Asn Lys Gly Ser Arg Glu Leu Pro
His Cys Met 1490 1495 1500gct gtt ggg gat ctc ttc acg aag gtg ttg
gac atc ttg ggc aaa ccc 4560Ala Val Gly Asp Leu Phe Thr Lys Val Leu
Asp Ile Leu Gly Lys Pro1505 1510 1515 1520aac aac cgg ttc tac aag
acc ctt tct tac ttt gca gtg gac aag gcc 4608Asn Asn Arg Phe Tyr Lys
Thr Leu Ser Tyr Phe Ala Val Asp Lys Ala 1525 1530 1535gag aag gag
cgc ttg ttg aag atc gcc gag atg ggg ccg gag tac agc 4656Glu Lys Glu
Arg Leu Leu Lys Ile Ala Glu Met Gly Pro Glu Tyr Ser 1540 1545
1550aac atc ctg tct gag acg tac cac tac gcg gac atc ttc cac atg ttc
4704Asn Ile Leu Ser Glu Thr Tyr His Tyr Ala Asp Ile Phe His Met Phe
1555 1560 1565ccg tcc gcc cgg ccc acg ctg cag tac ctc atc gag atg
atc ccc aac 4752Pro Ser Ala Arg Pro Thr Leu Gln Tyr Leu Ile Glu Met
Ile Pro Asn 1570 1575 1580atc aag ccc cgg tac tac tcc atc tcc tcc
gcc ccc atc cac acc cct 4800Ile Lys Pro Arg Tyr Tyr Ser Ile Ser Ser
Ala Pro Ile His Thr Pro1585 1590 1595 1600ggc gag gtc cac agc ctg
gtg ctc atc gac acc tgg atc acg ctg tcc 4848Gly Glu Val His Ser Leu
Val Leu Ile Asp Thr Trp Ile Thr Leu Ser 1605 1610 1615ggc aag cac
cgc acg ggg ctg acc tgc acc atg ctg gag cac ctg cag 4896Gly Lys His
Arg Thr Gly Leu Thr Cys Thr Met Leu Glu His Leu Gln 1620 1625
1630gcg ggc cag gtg gtg gat ggc tgc atc cac ccc acg gcg atg gag ttc
4944Ala Gly Gln Val Val Asp Gly Cys Ile His Pro Thr Ala Met Glu Phe
1635 1640 1645ccc gac cac gag aag ccg gtg gtg atg tgc gcc atg ggc
agt ggc ctg 4992Pro Asp His Glu Lys Pro Val Val Met Cys Ala Met Gly
Ser Gly Leu 1650 1655 1660gca ccg ttc gtt gct ttc ctg cgc gac ggc
tcc acg ctg cgg aag cag 5040Ala Pro Phe Val Ala Phe Leu Arg Asp Gly
Ser Thr Leu Arg Lys Gln1665 1670 1675 1680ggc aag aag acc ggg aac
atg gca ttg tac ttc ggc aac agg tat gag 5088Gly Lys Lys Thr Gly Asn
Met Ala Leu Tyr Phe Gly Asn Arg Tyr Glu 1685 1690 1695aag acg gag
ttc ctg atg aag gag gag ctg aag ggt cac atc aac gat 5136Lys Thr Glu
Phe Leu Met Lys Glu Glu Leu Lys Gly His Ile Asn Asp 1700 1705
1710ggt ttg ctg aca ctt cga tgc gct ttc agc cga gat gac ccc aag aag
5184Gly Leu Leu Thr Leu Arg Cys Ala Phe Ser Arg Asp Asp Pro Lys Lys
1715 1720 1725aag gtg tat gtg cag gac ctt atc aag atg gac gaa aag
atg atg tac 5232Lys Val Tyr Val Gln Asp Leu Ile Lys Met Asp Glu Lys
Met Met Tyr 1730 1735 1740gat tac ctc gtg gtg cag aag ggt tct atg
tat tgc tgt gga tcc cgc 5280Asp Tyr Leu Val Val Gln Lys Gly Ser Met
Tyr Cys Cys Gly Ser Arg1745 1750 1755 1760agt ttc atc aag cct gtc
cag gag tca ttg aaa cat tgc ttc atg aaa 5328Ser Phe Ile Lys Pro Val
Gln Glu Ser Leu Lys His Cys Phe Met Lys 1765 1770 1775gct ggt ggg
ctg act gca gag caa gct gag aac gag gtc atc gat atg 5376Ala Gly Gly
Leu Thr Ala Glu Gln Ala Glu Asn Glu Val Ile Asp Met 1780 1785
1790ttc acg acc ggg cgg tac aat atc gag gca tgg taa 5412Phe Thr Thr
Gly Arg Tyr Asn Ile Glu Ala Trp 1795 180061803PRTEuglena gracilis
6Met Lys Gln Ser Val Arg Pro Ile Ile Ser Asn Val Leu Arg Lys Glu1 5
10 15Val Ala Leu Tyr Ser Thr Ile Ile Gly Gln Asp Lys Gly Lys Glu
Pro 20 25 30Thr Gly Arg Thr Tyr Thr Ser Gly Pro Lys Pro Ala Ser His
Ile Glu 35 40 45Val Pro His His Val Thr Val Pro Ala Thr Asp Arg Thr
Pro Asn Pro 50 55 60Asp Ala Gln Phe Phe Gln Ser Val Asp Gly Ser Gln
Ala Thr Ser His65 70 75 80Val Ala Tyr Ala Leu Ser Asp Thr Ala Phe
Ile Tyr Pro Ile Thr Pro 85 90 95Ser Ser Val Met Gly Glu Leu Ala Asp
Val Trp Met Ala Gln Gly Arg 100 105 110Lys Asn Ala Phe Gly Gln Val
Val Asp Val Arg Glu Met Gln Ser Glu 115 120 125Ala Gly Ala Ala Gly
Ala Leu His Gly Ala Leu Ala Ala Gly Ala Ile 130 135 140Ala Thr Thr
Phe Thr Ala Ser Gln Gly Leu Leu Leu Met Ile Pro Asn145 150 155
160Met Tyr Lys Ile Ala Gly Glu Leu Met Pro Ser Val Ile His Val Ala
165 170 175Ala Arg Glu Leu Ala Gly His Ala Leu Ser Ile Phe Gly Gly
His Ala 180 185 190Asp Val Met Ala Val Arg Gln Thr Gly Trp Ala Met
Leu Cys Ser His 195 200 205Thr Val Gln Gln Ser His Asp Met Ala Leu
Ile Ser His Val Ala Thr 210 215 220Leu Lys Ser Ser Ile Pro Phe Val
His Phe Phe Asp Gly Phe Arg Thr225 230 235 240Ser His Glu Val Asn
Lys Ile Lys Met Leu Pro Tyr Ala Glu Leu Lys 245 250 255Lys Leu Val
Pro Pro Gly Thr Met Glu Gln His Trp Ala Arg Ser Leu 260 265 270Asn
Pro Met His Pro Thr Ile Arg Gly Thr Asn Gln Ser Ala Asp Ile 275 280
285Tyr Phe Gln Asn Met Glu Ser Ala Asn Gln Tyr Tyr Thr Asp Leu Ala
290 295 300Glu Val Val Gln Glu Thr Met Asp Glu Val Ala Pro Tyr Ile
Gly Arg305 310 315 320His Tyr Lys Ile Phe Glu Tyr Val Gly Ala Pro
Asp Ala Glu Glu Val 325 330 335Thr Val Leu Met Gly Ser Gly Ala Thr
Thr Val Asn Glu Ala Val Asp 340 345 350Leu Leu Val Lys Arg Gly Lys
Lys Val Gly Ala Val Leu Val His Leu 355 360 365Tyr Arg Pro Trp Ser
Thr Lys Ala Phe Glu Lys Val Leu Pro Lys Thr 370 375 380Val Lys Arg
Ile Ala Ala Leu Asp Arg Cys Lys Glu Val Thr Ala Leu385 390 395
400Gly Glu Pro Leu Tyr Leu Asp Val Ser Ala Thr Leu Asn Leu Phe Pro
405 410 415Glu Arg Gln Asn Val Lys Val Ile Gly Gly Arg Tyr Gly Leu
Gly Ser 420 425 430Lys Asp Phe Ile Pro Glu His Ala Leu Ala Ile Tyr
Ala Asn Leu Ala 435 440 445Ser Glu Asn Pro Ile Gln Arg Phe Thr Val
Gly Ile Thr Asp Asp Val 450 455 460Thr Gly Thr Ser Val Pro Phe Val
Asn Glu Arg Val Asp Thr Leu Pro465 470 475 480Glu Gly Thr Arg Gln
Cys Val Phe Trp Gly Ile Gly Ser Asp Gly Thr 485 490 495Val Gly Ala
Asn Arg Ser Ala Val Arg Ile Ile Gly Asp Asn Ser Asp 500 505 510Leu
Met Val Gln Ala Tyr Phe Gln Phe Asp Ala Phe Lys Ser Gly Gly 515 520
525Val Thr Ser Ser His Leu Arg Phe Gly Pro Lys Pro Ile Thr Ala Gln
530 535 540Tyr Leu Val Thr Asn Ala Asp Tyr Ile Ala Cys His Phe Gln
Glu Tyr545 550 555 560Val Lys Arg Phe Asp Met Leu Asp Ala Ile Arg
Glu Gly Gly Thr Phe 565 570 575Val Leu Asn Ser Arg Trp Thr Thr Glu
Asp Met Glu Lys Glu Ile Pro 580 585 590Ala Asp Phe Arg Arg Lys Leu
Ala Gln Lys Lys Val Arg Phe Tyr Asn 595 600 605Val Asp Ala Arg Lys
Ile Cys Asp Ser Phe Gly Leu Gly Lys Arg Ile 610 615 620Asn Met Leu
Met Gln Ala Cys Phe Phe Lys Leu Ser Gly Val Leu Pro625 630 635
640Leu Ala Glu Ala Gln Arg Leu Leu Asn Glu Ser Ile Val His Glu Tyr
645 650 655Gly Lys Lys Gly Gly Lys Val Val Glu Met Asn Gln Ala Val
Val Asn 660 665 670Ala Val Phe Ala Gly Asp Leu Pro Gln Glu Val Gln
Val Pro Ala Ala 675 680 685Trp Ala Asn Ala Val Asp Thr Ser Thr Arg
Thr Pro Thr Gly Ile Glu 690 695 700Phe Val Asp Lys Ile Met Arg Pro
Leu Met Asp Phe Lys Gly Asp Gln705 710 715 720Leu Pro Val Ser Val
Met Thr Pro Gly Gly Thr Phe Pro Val Gly Thr 725 730 735Thr Gln Tyr
Ala Lys Arg Ala Ile Ala Ala Phe Ile Pro Gln Trp Ile 740 745 750Pro
Ala Asn Cys Thr Gln Cys Asn Tyr Cys Ser Tyr Val Cys Pro His 755 760
765Ala Thr Ile Arg Pro Phe Val Leu Thr Asp Gln Glu Val Gln Leu Ala
770 775 780Pro Glu Ser Phe Val Thr Arg Lys
Ala Lys Gly Asp Tyr Gln Gly Met785 790 795 800Asn Phe Arg Ile Gln
Val Ala Pro Glu Asp Cys Thr Gly Cys Gln Val 805 810 815Cys Val Glu
Thr Cys Pro Asp Asp Ala Leu Glu Met Thr Asp Ala Phe 820 825 830Thr
Ala Thr Pro Val Gln Arg Thr Asn Trp Glu Phe Ala Ile Lys Val 835 840
845Pro Asn Arg Gly Thr Met Thr Asp Arg Tyr Ser Leu Lys Gly Ser Gln
850 855 860Phe Gln Gln Pro Leu Leu Glu Phe Ser Gly Ala Cys Glu Gly
Cys Gly865 870 875 880Glu Thr Pro Tyr Val Lys Leu Leu Thr Gln Leu
Phe Gly Glu Arg Thr 885 890 895Val Ile Ala Asn Ala Thr Gly Cys Ser
Ser Ile Trp Gly Gly Thr Ala 900 905 910Gly Leu Ala Pro Tyr Thr Thr
Asn Ala Lys Gly Gln Gly Pro Ala Trp 915 920 925Gly Asn Ser Leu Phe
Glu Asp Asn Ala Glu Phe Gly Phe Gly Ile Ala 930 935 940Val Ala Asn
Ala Gln Lys Arg Ser Arg Val Arg Asp Cys Ile Leu Gln945 950 955
960Ala Val Glu Lys Lys Val Ala Asp Glu Gly Leu Thr Thr Leu Leu Ala
965 970 975Gln Trp Leu Gln Asp Trp Asn Thr Gly Asp Lys Thr Leu Lys
Tyr Gln 980 985 990Asp Gln Ile Ile Ala Gly Leu Ala Gln Gln Arg Ser
Lys Asp Pro Leu 995 1000 1005Leu Glu Gln Ile Tyr Gly Met Lys Asp
Met Leu Pro Asn Ile Ser Gln 1010 1015 1020Trp Ile Ile Gly Gly Asp
Gly Trp Ala Asn Asp Ile Gly Phe Gly Gly1025 1030 1035 1040Leu Asp
His Val Leu Ala Ser Gly Gln Asn Leu Asn Val Leu Val Leu 1045 1050
1055Asp Thr Glu Met Tyr Ser Asn Thr Gly Gly Gln Ala Ser Lys Ser Thr
1060 1065 1070His Met Ala Ser Val Ala Lys Phe Ala Leu Gly Gly Lys
Arg Thr Asn 1075 1080 1085Lys Lys Asn Leu Thr Glu Met Ala Met Ser
Tyr Gly Asn Val Tyr Val 1090 1095 1100Ala Thr Val Ser His Gly Asn
Met Ala Gln Cys Val Lys Ala Phe Val1105 1110 1115 1120Glu Ala Glu
Ser Tyr Asp Gly Pro Ser Leu Ile Val Gly Tyr Ala Pro 1125 1130
1135Cys Ile Glu His Gly Leu Arg Ala Gly Met Ala Arg Met Val Gln Glu
1140 1145 1150Ser Glu Ala Ala Ile Ala Thr Gly Tyr Trp Pro Leu Tyr
Arg Phe Asp 1155 1160 1165Pro Arg Leu Ala Thr Glu Gly Lys Asn Pro
Phe Gln Leu Asp Ser Lys 1170 1175 1180Arg Ile Lys Gly Asn Leu Gln
Glu Tyr Leu Asp Arg Gln Asn Arg Tyr1185 1190 1195 1200Val Asn Leu
Lys Lys Asn Asn Pro Lys Gly Ala Asp Leu Leu Lys Ser 1205 1210
1215Gln Met Ala Asp Asn Ile Thr Ala Arg Phe Asn Arg Tyr Arg Arg Met
1220 1225 1230Leu Glu Gly Pro Asn Thr Lys Ala Ala Ala Pro Ser Gly
Asn His Val 1235 1240 1245Thr Ile Leu Tyr Gly Ser Glu Thr Gly Asn
Ser Glu Gly Leu Ala Lys 1250 1255 1260Glu Leu Ala Thr Asp Phe Glu
Arg Arg Glu Tyr Ser Val Ala Val Gln1265 1270 1275 1280Ala Leu Asp
Asp Ile Asp Val Ala Asp Leu Glu Asn Met Gly Phe Val 1285 1290
1295Val Ile Ala Val Ser Thr Cys Gly Gln Gly Gln Phe Pro Arg Asn Ser
1300 1305 1310Gln Leu Phe Trp Arg Glu Leu Gln Arg Asp Lys Pro Glu
Gly Trp Leu 1315 1320 1325Lys Asn Leu Lys Tyr Thr Val Phe Gly Leu
Gly Asp Ser Thr Tyr Tyr 1330 1335 1340Phe Tyr Cys His Thr Ala Lys
Gln Ile Asp Ala Arg Leu Ala Ala Leu1345 1350 1355 1360Gly Ala Gln
Arg Val Val Pro Ile Gly Phe Gly Asp Asp Gly Asp Glu 1365 1370
1375Asp Met Phe His Thr Gly Phe Asn Asn Trp Ile Pro Ser Val Trp Asn
1380 1385 1390Glu Leu Lys Thr Lys Thr Pro Glu Glu Ala Leu Phe Thr
Pro Ser Ile 1395 1400 1405Ala Val Gln Leu Thr Pro Asn Ala Thr Pro
Gln Asp Phe His Phe Ala 1410 1415 1420Lys Ser Thr Pro Val Leu Ser
Ile Thr Gly Ala Glu Arg Ile Thr Pro1425 1430 1435 1440Ala Asp His
Thr Arg Asn Phe Val Thr Ile Arg Trp Lys Thr Asp Leu 1445 1450
1455Ser Tyr Gln Val Gly Asp Ser Leu Gly Val Phe Pro Glu Asn Thr Arg
1460 1465 1470Ser Val Val Glu Glu Phe Leu Gln Tyr Tyr Gly Leu Asn
Pro Lys Asp 1475 1480 1485Val Ile Thr Ile Glu Asn Lys Gly Ser Arg
Glu Leu Pro His Cys Met 1490 1495 1500Ala Val Gly Asp Leu Phe Thr
Lys Val Leu Asp Ile Leu Gly Lys Pro1505 1510 1515 1520Asn Asn Arg
Phe Tyr Lys Thr Leu Ser Tyr Phe Ala Val Asp Lys Ala 1525 1530
1535Glu Lys Glu Arg Leu Leu Lys Ile Ala Glu Met Gly Pro Glu Tyr Ser
1540 1545 1550Asn Ile Leu Ser Glu Thr Tyr His Tyr Ala Asp Ile Phe
His Met Phe 1555 1560 1565Pro Ser Ala Arg Pro Thr Leu Gln Tyr Leu
Ile Glu Met Ile Pro Asn 1570 1575 1580Ile Lys Pro Arg Tyr Tyr Ser
Ile Ser Ser Ala Pro Ile His Thr Pro1585 1590 1595 1600Gly Glu Val
His Ser Leu Val Leu Ile Asp Thr Trp Ile Thr Leu Ser 1605 1610
1615Gly Lys His Arg Thr Gly Leu Thr Cys Thr Met Leu Glu His Leu Gln
1620 1625 1630Ala Gly Gln Val Val Asp Gly Cys Ile His Pro Thr Ala
Met Glu Phe 1635 1640 1645Pro Asp His Glu Lys Pro Val Val Met Cys
Ala Met Gly Ser Gly Leu 1650 1655 1660Ala Pro Phe Val Ala Phe Leu
Arg Asp Gly Ser Thr Leu Arg Lys Gln1665 1670 1675 1680Gly Lys Lys
Thr Gly Asn Met Ala Leu Tyr Phe Gly Asn Arg Tyr Glu 1685 1690
1695Lys Thr Glu Phe Leu Met Lys Glu Glu Leu Lys Gly His Ile Asn Asp
1700 1705 1710Gly Leu Leu Thr Leu Arg Cys Ala Phe Ser Arg Asp Asp
Pro Lys Lys 1715 1720 1725Lys Val Tyr Val Gln Asp Leu Ile Lys Met
Asp Glu Lys Met Met Tyr 1730 1735 1740Asp Tyr Leu Val Val Gln Lys
Gly Ser Met Tyr Cys Cys Gly Ser Arg1745 1750 1755 1760Ser Phe Ile
Lys Pro Val Gln Glu Ser Leu Lys His Cys Phe Met Lys 1765 1770
1775Ala Gly Gly Leu Thr Ala Glu Gln Ala Glu Asn Glu Val Ile Asp Met
1780 1785 1790Phe Thr Thr Gly Arg Tyr Asn Ile Glu Ala Trp 1795
18007747DNAEscherichia coliCDS(1)..(747) 7atg gct gat tgg gta aca
ggc aaa gtc act aaa gtg cag aac tgg acc 48Met Ala Asp Trp Val Thr
Gly Lys Val Thr Lys Val Gln Asn Trp Thr1 5 10 15gac gcc ctg ttt agt
ctc acc gtt cac gcc ccc gtg ctt ccg ttt acc 96Asp Ala Leu Phe Ser
Leu Thr Val His Ala Pro Val Leu Pro Phe Thr 20 25 30gcc ggg caa ttt
acc aag ctt ggc ctt gaa atc gac ggc gaa cgc gtc 144Ala Gly Gln Phe
Thr Lys Leu Gly Leu Glu Ile Asp Gly Glu Arg Val 35 40 45cag cgc gcc
tac tcc tat gta aac tcg ccc gat aat ccc gat ctg gag 192Gln Arg Ala
Tyr Ser Tyr Val Asn Ser Pro Asp Asn Pro Asp Leu Glu 50 55 60ttt tac
ctg gtc acc gtc ccc gat ggc aaa tta agc cca cga ctg gcg 240Phe Tyr
Leu Val Thr Val Pro Asp Gly Lys Leu Ser Pro Arg Leu Ala65 70 75
80gca ctg aaa cca ggc gat gaa gtg cag gtg gtt agc gaa gcg gca gga
288Ala Leu Lys Pro Gly Asp Glu Val Gln Val Val Ser Glu Ala Ala Gly
85 90 95ttc ttt gtg ctc gat gaa gtg ccg cac tgc gaa acg cta tgg atg
ctg 336Phe Phe Val Leu Asp Glu Val Pro His Cys Glu Thr Leu Trp Met
Leu 100 105 110gca acc ggt aca gcg att ggc cct tat tta tcg att ctg
caa cta ggt 384Ala Thr Gly Thr Ala Ile Gly Pro Tyr Leu Ser Ile Leu
Gln Leu Gly 115 120 125aaa gat tta gat cgc ttc aaa aat ctg gtc ctg
gtg cac gcc gca cgt 432Lys Asp Leu Asp Arg Phe Lys Asn Leu Val Leu
Val His Ala Ala Arg 130 135 140tat gcc gcc gac tta agc tat ttg cca
ctg atg cag gaa ctg gaa aaa 480Tyr Ala Ala Asp Leu Ser Tyr Leu Pro
Leu Met Gln Glu Leu Glu Lys145 150 155 160cgc tac gaa gga aaa ctg
cgc att cag acg gtg gtc agt cgg gaa acg 528Arg Tyr Glu Gly Lys Leu
Arg Ile Gln Thr Val Val Ser Arg Glu Thr 165 170 175gca gcg ggg tcg
ctc acc gga cgg ata ccg gca tta att gaa agt ggg 576Ala Ala Gly Ser
Leu Thr Gly Arg Ile Pro Ala Leu Ile Glu Ser Gly 180 185 190gaa ctg
gaa agc acg att ggc ctg ccg atg aat aaa gaa acc agc cat 624Glu Leu
Glu Ser Thr Ile Gly Leu Pro Met Asn Lys Glu Thr Ser His 195 200
205gtg atg ctg tgc ggc aat cca cag atg gtg cgc gat aca caa cag ttg
672Val Met Leu Cys Gly Asn Pro Gln Met Val Arg Asp Thr Gln Gln Leu
210 215 220ctg aaa gag acc cgg cag atg acg aaa cat tta cgt cgc cga
ccg ggc 720Leu Lys Glu Thr Arg Gln Met Thr Lys His Leu Arg Arg Arg
Pro Gly225 230 235 240cat atg aca gcg gag cat tac tgg taa 747His
Met Thr Ala Glu His Tyr Trp 2458248PRTEscherichia coli 8Met Ala Asp
Trp Val Thr Gly Lys Val Thr Lys Val Gln Asn Trp Thr1 5 10 15 Asp
Ala Leu Phe Ser Leu Thr Val His Ala Pro Val Leu Pro Phe Thr 20 25
30Ala Gly Gln Phe Thr Lys Leu Gly Leu Glu Ile Asp Gly Glu Arg Val
35 40 45Gln Arg Ala Tyr Ser Tyr Val Asn Ser Pro Asp Asn Pro Asp Leu
Glu 50 55 60Phe Tyr Leu Val Thr Val Pro Asp Gly Lys Leu Ser Pro Arg
Leu Ala65 70 75 80Ala Leu Lys Pro Gly Asp Glu Val Gln Val Val Ser
Glu Ala Ala Gly 85 90 95 Phe Phe Val Leu Asp Glu Val Pro His Cys
Glu Thr Leu Trp Met Leu 100 105 110Ala Thr Gly Thr Ala Ile Gly Pro
Tyr Leu Ser Ile Leu Gln Leu Gly 115 120 125Lys Asp Leu Asp Arg Phe
Lys Asn Leu Val Leu Val His Ala Ala Arg 130 135 140Tyr Ala Ala Asp
Leu Ser Tyr Leu Pro Leu Met Gln Glu Leu Glu Lys145 150 155 160Arg
Tyr Glu Gly Lys Leu Arg Ile Gln Thr Val Val Ser Arg Glu Thr 165 170
175 Ala Ala Gly Ser Leu Thr Gly Arg Ile Pro Ala Leu Ile Glu Ser Gly
180 185 190Glu Leu Glu Ser Thr Ile Gly Leu Pro Met Asn Lys Glu Thr
Ser His 195 200 205Val Met Leu Cys Gly Asn Pro Gln Met Val Arg Asp
Thr Gln Gln Leu 210 215 220Leu Lys Glu Thr Arg Gln Met Thr Lys His
Leu Arg Arg Arg Pro Gly225 230 235 240His Met Thr Ala Glu His Tyr
Trp 2459336DNAEscherichia coliCDS(1)..(336) 9atg cca aag att gtt
att ttg cct cat cag gat ctc tgc cct gat ggc 48Met Pro Lys Ile Val
Ile Leu Pro His Gln Asp Leu Cys Pro Asp Gly1 5 10 15gct gtt ctg gaa
gct aat agc ggt gaa acc att ctc gac gca gct ctg 96Ala Val Leu Glu
Ala Asn Ser Gly Glu Thr Ile Leu Asp Ala Ala Leu 20 25 30cgt aac ggt
atc gag att gaa cac gcc tgt gaa aaa tcc tgt gct tgc 144Arg Asn Gly
Ile Glu Ile Glu His Ala Cys Glu Lys Ser Cys Ala Cys 35 40 45acc acc
tgc cac tgc atc gtt cgt gaa ggt ttt gac tca ctg ccg gaa 192Thr Thr
Cys His Cys Ile Val Arg Glu Gly Phe Asp Ser Leu Pro Glu 50 55 60agc
tca gag cag gaa gac gac atg ctg gac aaa gcc tgg gga ctg gag 240Ser
Ser Glu Gln Glu Asp Asp Met Leu Asp Lys Ala Trp Gly Leu Glu65 70 75
80ccg gaa agc cgt tta agc tgc cag gcg cgc gtt acc gac gaa gat tta
288Pro Glu Ser Arg Leu Ser Cys Gln Ala Arg Val Thr Asp Glu Asp Leu
85 90 95gta gtc gaa atc ccg cgt tac act atc aac cat gcg cgt gag cat
taa 336Val Val Glu Ile Pro Arg Tyr Thr Ile Asn His Ala Arg Glu His
100 105 11010111PRTEscherichia coli 10Met Pro Lys Ile Val Ile Leu
Pro His Gln Asp Leu Cys Pro Asp Gly1 5 10 15Ala Val Leu Glu Ala Asn
Ser Gly Glu Thr Ile Leu Asp Ala Ala Leu 20 25 30 Arg Asn Gly Ile
Glu Ile Glu His Ala Cys Glu Lys Ser Cys Ala Cys 35 40 45Thr Thr Cys
His Cys Ile Val Arg Glu Gly Phe Asp Ser Leu Pro Glu 50 55 60Ser Ser
Glu Gln Glu Asp Asp Met Leu Asp Lys Ala Trp Gly Leu Glu65 70 75
80Pro Glu Ser Arg Leu Ser Cys Gln Ala Arg Val Thr Asp Glu Asp Leu
85 90 95Val Val Glu Ile Pro Arg Tyr Thr Ile Asn His Ala Arg Glu His
100 105 11011261DNAEscherichia coliCDS(1)..(261) 11atg gcg ttg tta
atc act aaa aaa tgc atc aat tgt gat atg tgt gaa 48Met Ala Leu Leu
Ile Thr Lys Lys Cys Ile Asn Cys Asp Met Cys Glu1 5 10 15ccc gaa tgc
ccg aat gag gcg att tca atg gga gat cat atc tac gag 96Pro Glu Cys
Pro Asn Glu Ala Ile Ser Met Gly Asp His Ile Tyr Glu 20 25 30att aac
agc gat aag tgt acc gaa tgc gta ggg cac tac gag aca cca 144Ile Asn
Ser Asp Lys Cys Thr Glu Cys Val Gly His Tyr Glu Thr Pro 35 40 45acc
tgc cag aag gtg tgc ccg atc ccc aat act att gtg aaa gat ccg 192Thr
Cys Gln Lys Val Cys Pro Ile Pro Asn Thr Ile Val Lys Asp Pro 50 55
60gcg cat gtc gag aca gaa gaa cag ttg tgg gat aaa ttt gtg ctg atg
240Ala His Val Glu Thr Glu Glu Gln Leu Trp Asp Lys Phe Val Leu
Met65 70 75 80cac cac gcg gat aaa att taa 261His His Ala Asp Lys
Ile 851286PRTEscherichia coli 12Met Ala Leu Leu Ile Thr Lys Lys Cys
Ile Asn Cys Asp Met Cys Glu1 5 10 15Pro Glu Cys Pro Asn Glu Ala Ile
Ser Met Gly Asp His Ile Tyr Glu 20 25 30Ile Asn Ser Asp Lys Cys Thr
Glu Cys Val Gly His Tyr Glu Thr Pro 35 40 45Thr Cys Gln Lys Val Cys
Pro Ile Pro Asn Thr Ile Val Lys Asp Pro 50 55 60Ala His Val Glu Thr
Glu Glu Gln Leu Trp Asp Lys Phe Val Leu Met65 70 75 80His His Ala
Asp Lys Ile 8513531DNAEscherichia coliCDS(1)..(531) 13atg gct atc
act ggc atc ttt ttc ggc agc gac acc ggt aat acc gaa 48Met Ala Ile
Thr Gly Ile Phe Phe Gly Ser Asp Thr Gly Asn Thr Glu1 5 10 15aat atc
gca aaa atg att caa aaa cag ctt ggt aaa gac gtt gcc gat 96Asn Ile
Ala Lys Met Ile Gln Lys Gln Leu Gly Lys Asp Val Ala Asp 20 25 30gtc
cat gac att gca aaa agc agc aaa gaa gat ctg gaa gct tat gac 144Val
His Asp Ile Ala Lys Ser Ser Lys Glu Asp Leu Glu Ala Tyr Asp 35 40
45att ctg ctg ctg ggc atc cca acc tgg tat tac ggc gaa gcg cag tgt
192Ile Leu Leu Leu Gly Ile Pro Thr Trp Tyr Tyr Gly Glu Ala Gln Cys
50 55 60gac tgg gat gac ttc ttc ccg act ctc gaa gag att gat ttc aac
ggc 240Asp Trp Asp Asp Phe Phe Pro Thr Leu Glu Glu Ile Asp Phe Asn
Gly65 70 75 80aaa ctg gtt gcg ctg ttt ggt tgt ggt gac cag gaa gat
tac gcc gaa 288Lys Leu Val Ala Leu Phe Gly Cys Gly Asp Gln Glu Asp
Tyr Ala Glu 85 90 95 tat ttc tgc gac gca ttg ggc acc atc cgc gac
atc att gaa ccg cgc 336Tyr Phe Cys Asp Ala Leu Gly Thr Ile Arg Asp
Ile Ile Glu Pro Arg 100 105 110ggt gca acc atc gtt ggt cac tgg cca
act gcg ggc tat cat ttc gaa 384Gly Ala Thr Ile Val Gly His Trp Pro
Thr Ala Gly Tyr His Phe Glu 115 120 125gca tca aaa ggt ctg gca gat
gac gac cac ttt gtc ggt ctg gct atc 432Ala Ser Lys Gly Leu Ala Asp
Asp Asp His Phe Val Gly Leu Ala Ile 130 135 140gac gaa gac cgt cag
ccg gaa ctg acc gct gaa cgt gta gaa aaa tgg 480Asp Glu Asp Arg Gln
Pro Glu Leu Thr Ala Glu Arg Val Glu Lys Trp145
150 155 160gtt aaa cag att tct gaa gag ttg cat ctc gac gaa att ctc
aat gcc 528Val Lys Gln Ile Ser Glu Glu Leu His Leu Asp Glu Ile Leu
Asn Ala 165 170 175tga 53114176PRTEscherichia coli 14Met Ala Ile
Thr Gly Ile Phe Phe Gly Ser Asp Thr Gly Asn Thr Glu1 5 10 15Asn Ile
Ala Lys Met Ile Gln Lys Gln Leu Gly Lys Asp Val Ala Asp 20 25 30Val
His Asp Ile Ala Lys Ser Ser Lys Glu Asp Leu Glu Ala Tyr Asp 35 40
45Ile Leu Leu Leu Gly Ile Pro Thr Trp Tyr Tyr Gly Glu Ala Gln Cys
50 55 60Asp Trp Asp Asp Phe Phe Pro Thr Leu Glu Glu Ile Asp Phe Asn
Gly65 70 75 80Lys Leu Val Ala Leu Phe Gly Cys Gly Asp Gln Glu Asp
Tyr Ala Glu 85 90 95Tyr Phe Cys Asp Ala Leu Gly Thr Ile Arg Asp Ile
Ile Glu Pro Arg 100 105 110Gly Ala Thr Ile Val Gly His Trp Pro Thr
Ala Gly Tyr His Phe Glu 115 120 125Ala Ser Lys Gly Leu Ala Asp Asp
Asp His Phe Val Gly Leu Ala Ile 130 135 140Asp Glu Asp Arg Gln Pro
Glu Leu Thr Ala Glu Arg Val Glu Lys Trp145 150 155 160Val Lys Gln
Ile Ser Glu Glu Leu His Leu Asp Glu Ile Leu Asn Ala 165 170
17515522DNAEscherichia coliCDS(1)..(522) 15atg aat atg ggt ctt ttt
tac ggt tcc agc acc tgt tac acc gaa atg 48Met Asn Met Gly Leu Phe
Tyr Gly Ser Ser Thr Cys Tyr Thr Glu Met1 5 10 15gcg gca gaa aaa atc
cgc gat att atc ggc cca gaa ctg gtg acc tta 96Ala Ala Glu Lys Ile
Arg Asp Ile Ile Gly Pro Glu Leu Val Thr Leu 20 25 30cat aac ctc aag
gac gac tcc ccg aaa tta atg gag cag tac gat gtg 144His Asn Leu Lys
Asp Asp Ser Pro Lys Leu Met Glu Gln Tyr Asp Val 35 40 45ctc att ctg
ggt atc ccg acc tgg gat ttt ggt gaa atc cag gaa gac 192Leu Ile Leu
Gly Ile Pro Thr Trp Asp Phe Gly Glu Ile Gln Glu Asp 50 55 60tgg gaa
gcc gtc tgg gat cag ctc gac gac ctg aac ctt gaa ggt aaa 240Trp Glu
Ala Val Trp Asp Gln Leu Asp Asp Leu Asn Leu Glu Gly Lys65 70 75
80att gtt gcg ctg tat ggg ctt ggc gat caa ctg gga tac ggc gag tgg
288Ile Val Ala Leu Tyr Gly Leu Gly Asp Gln Leu Gly Tyr Gly Glu Trp
85 90 95ttc ctc gat gcg ctc ggt atg ctg cat gac aaa ctc tcg acc aaa
ggc 336Phe Leu Asp Ala Leu Gly Met Leu His Asp Lys Leu Ser Thr Lys
Gly 100 105 110gtg aag ttc gtc ggc tac tgg cca acg gaa gga tat gaa
ttt acc agc 384Val Lys Phe Val Gly Tyr Trp Pro Thr Glu Gly Tyr Glu
Phe Thr Ser 115 120 125ccg aaa ccg gtg att gct gac ggg caa ctg ttc
gtg ggt ctg gcg ctg 432Pro Lys Pro Val Ile Ala Asp Gly Gln Leu Phe
Val Gly Leu Ala Leu 130 135 140gat gaa act aac cag tat gac ctt agc
gac gag cgt att cag agc tgg 480Asp Glu Thr Asn Gln Tyr Asp Leu Ser
Asp Glu Arg Ile Gln Ser Trp145 150 155 160tgc gag caa atc ctc aac
gaa atg gca gag cat tac gcc tga 522Cys Glu Gln Ile Leu Asn Glu Met
Ala Glu His Tyr Ala 165 17016173PRTEscherichia coli 16Met Asn Met
Gly Leu Phe Tyr Gly Ser Ser Thr Cys Tyr Thr Glu Met1 5 10 15Ala Ala
Glu Lys Ile Arg Asp Ile Ile Gly Pro Glu Leu Val Thr Leu 20 25 30His
Asn Leu Lys Asp Asp Ser Pro Lys Leu Met Glu Gln Tyr Asp Val 35 40
45Leu Ile Leu Gly Ile Pro Thr Trp Asp Phe Gly Glu Ile Gln Glu Asp
50 55 60Trp Glu Ala Val Trp Asp Gln Leu Asp Asp Leu Asn Leu Glu Gly
Lys65 70 75 80Ile Val Ala Leu Tyr Gly Leu Gly Asp Gln Leu Gly Tyr
Gly Glu Trp 85 90 95Phe Leu Asp Ala Leu Gly Met Leu His Asp Lys Leu
Ser Thr Lys Gly 100 105 110Val Lys Phe Val Gly Tyr Trp Pro Thr Glu
Gly Tyr Glu Phe Thr Ser 115 120 125 Pro Lys Pro Val Ile Ala Asp Gly
Gln Leu Phe Val Gly Leu Ala Leu 130 135 140Asp Glu Thr Asn Gln Tyr
Asp Leu Ser Asp Glu Arg Ile Gln Ser Trp145 150 155 160Cys Glu Gln
Ile Leu Asn Glu Met Ala Glu His Tyr Ala 165 17017522DNAChlorobium
tepidumCDS(1)..(522) 17atg aat atg ggt ctt ttt tac ggt tcc agc acc
tgt tac acc gaa atg 48Met Asn Met Gly Leu Phe Tyr Gly Ser Ser Thr
Cys Tyr Thr Glu Met1 5 10 15gcg gca gaa aaa atc cgc gat att atc ggc
cca gaa ctg gtg acc tta 96Ala Ala Glu Lys Ile Arg Asp Ile Ile Gly
Pro Glu Leu Val Thr Leu 20 25 30cat aac ctc aag gac gac tcc ccg aaa
tta atg gag cag tac gat gtg 144His Asn Leu Lys Asp Asp Ser Pro Lys
Leu Met Glu Gln Tyr Asp Val 35 40 45ctc att ctg ggt atc ccg acc tgg
gat ttt ggt gaa atc cag gaa gac 192Leu Ile Leu Gly Ile Pro Thr Trp
Asp Phe Gly Glu Ile Gln Glu Asp 50 55 60tgg gaa gcc gtc tgg gat cag
ctc gac gac ctg aac ctt gaa ggt aaa 240Trp Glu Ala Val Trp Asp Gln
Leu Asp Asp Leu Asn Leu Glu Gly Lys65 70 75 80att gtt gcg ctg tat
ggg ctt ggc gat caa ctg gga tac ggc gag tgg 288Ile Val Ala Leu Tyr
Gly Leu Gly Asp Gln Leu Gly Tyr Gly Glu Trp 85 90 95ttc ctc gat gcg
ctc ggt atg ctg cat gac aaa ctc tcg acc aaa ggc 336Phe Leu Asp Ala
Leu Gly Met Leu His Asp Lys Leu Ser Thr Lys Gly 100 105 110gtg aag
ttc gtc ggc tac tgg cca acg gaa gga tat gaa ttt acc agc 384Val Lys
Phe Val Gly Tyr Trp Pro Thr Glu Gly Tyr Glu Phe Thr Ser 115 120
125ccg aaa ccg gtg att gct gac ggg caa ctg ttc gtg ggt ctg gcg ctg
432Pro Lys Pro Val Ile Ala Asp Gly Gln Leu Phe Val Gly Leu Ala Leu
130 135 140gat gaa act aac cag tat gac ctt agc gac gag cgt att cag
agc tgg 480Asp Glu Thr Asn Gln Tyr Asp Leu Ser Asp Glu Arg Ile Gln
Ser Trp145 150 155 160tgc gag caa atc ctc aac gaa atg gca gag cat
tac gcc tga 522Cys Glu Gln Ile Leu Asn Glu Met Ala Glu His Tyr Ala
165 17018173PRTChlorobium tepidum 18Met Asn Met Gly Leu Phe Tyr Gly
Ser Ser Thr Cys Tyr Thr Glu Met1 5 10 15Ala Ala Glu Lys Ile Arg Asp
Ile Ile Gly Pro Glu Leu Val Thr Leu 20 25 30His Asn Leu Lys Asp Asp
Ser Pro Lys Leu Met Glu Gln Tyr Asp Val 35 40 45Leu Ile Leu Gly Ile
Pro Thr Trp Asp Phe Gly Glu Ile Gln Glu Asp 50 55 60Trp Glu Ala Val
Trp Asp Gln Leu Asp Asp Leu Asn Leu Glu Gly Lys65 70 75 80Ile Val
Ala Leu Tyr Gly Leu Gly Asp Gln Leu Gly Tyr Gly Glu Trp 85 90 95Phe
Leu Asp Ala Leu Gly Met Leu His Asp Lys Leu Ser Thr Lys Gly 100 105
110Val Lys Phe Val Gly Tyr Trp Pro Thr Glu Gly Tyr Glu Phe Thr Ser
115 120 125Pro Lys Pro Val Ile Ala Asp Gly Gln Leu Phe Val Gly Leu
Ala Leu 130 135 140Asp Glu Thr Asn Gln Tyr Asp Leu Ser Asp Glu Arg
Ile Gln Ser Trp145 150 155 160Cys Glu Gln Ile Leu Asn Glu Met Ala
Glu His Tyr Ala 165 17019189DNAChlorobium tepidumCDS(1)..(189)
19atg gca ctg tat atc acc gaa gaa tgc acc tac tgc ggt gct tgc gaa
48Met Ala Leu Tyr Ile Thr Glu Glu Cys Thr Tyr Cys Gly Ala Cys Glu1
5 10 15ccc gaa tgc ccg acc aac gct atc tcc gct ggc agc gag atc tac
gtt 96Pro Glu Cys Pro Thr Asn Ala Ile Ser Ala Gly Ser Glu Ile Tyr
Val 20 25 30atc gat gcc gca tcc tgc aac gag tgc gcc ggt ttt gct gac
tct cct 144Ile Asp Ala Ala Ser Cys Asn Glu Cys Ala Gly Phe Ala Asp
Ser Pro 35 40 45gct tgc gtt gct gtc tgc ccg gca gag tgc atc gtt cag
ggc tga 189Ala Cys Val Ala Val Cys Pro Ala Glu Cys Ile Val Gln Gly
50 55 602062PRTChlorobium tepidum 20Met Ala Leu Tyr Ile Thr Glu Glu
Cys Thr Tyr Cys Gly Ala Cys Glu1 5 10 15Pro Glu Cys Pro Thr Asn Ala
Ile Ser Ala Gly Ser Glu Ile Tyr Val 20 25 30Ile Asp Ala Ala Ser Cys
Asn Glu Cys Ala Gly Phe Ala Asp Ser Pro 35 40 45Ala Cys Val Ala Val
Cys Pro Ala Glu Cys Ile Val Gln Gly 50 55 60212676DNAEscherichia
coliCDS(1)..(2676) 21atg gct gtt act aat gtc gct gaa ctt aac gca
ctc gta gag cgt gta 48Met Ala Val Thr Asn Val Ala Glu Leu Asn Ala
Leu Val Glu Arg Val1 5 10 15aaa aaa gcc cag cgt gaa tat gcc agt ttc
act caa gag caa gta gac 96Lys Lys Ala Gln Arg Glu Tyr Ala Ser Phe
Thr Gln Glu Gln Val Asp 20 25 30aaa atc ttc cgc gcc gcc gct ctg gct
gct gca gat gct cga atc cca 144Lys Ile Phe Arg Ala Ala Ala Leu Ala
Ala Ala Asp Ala Arg Ile Pro 35 40 45ctc gcg aaa atg gcc gtt gcc gaa
tcc ggc atg ggt atc gtc gaa gat 192Leu Ala Lys Met Ala Val Ala Glu
Ser Gly Met Gly Ile Val Glu Asp 50 55 60aaa gtg atc aaa aac cac ttt
gct tct gaa tat atc tac aac gcc tat 240Lys Val Ile Lys Asn His Phe
Ala Ser Glu Tyr Ile Tyr Asn Ala Tyr65 70 75 80aaa gat gaa aaa acc
tgt ggt gtt ctg tct gaa gac gac act ttt ggt 288Lys Asp Glu Lys Thr
Cys Gly Val Leu Ser Glu Asp Asp Thr Phe Gly 85 90 95acc atc act atc
gct gaa cca atc ggt att att tgc ggt atc gtt ccg 336Thr Ile Thr Ile
Ala Glu Pro Ile Gly Ile Ile Cys Gly Ile Val Pro 100 105 110acc act
aac ccg act tca act gct atc ttc aaa tcg ctg atc agt ctg 384Thr Thr
Asn Pro Thr Ser Thr Ala Ile Phe Lys Ser Leu Ile Ser Leu 115 120
125aag acc cgt aac gcc att atc ttc tcc ccg cac ccg cgt gca aaa gat
432Lys Thr Arg Asn Ala Ile Ile Phe Ser Pro His Pro Arg Ala Lys Asp
130 135 140gcc acc aac aaa gcg gct gat atc gtt ctg cag gct gct atc
gct gcc 480Ala Thr Asn Lys Ala Ala Asp Ile Val Leu Gln Ala Ala Ile
Ala Ala145 150 155 160ggt gct ccg aaa gat ctg atc ggc tgg atc gat
caa cct tct gtt gaa 528Gly Ala Pro Lys Asp Leu Ile Gly Trp Ile Asp
Gln Pro Ser Val Glu 165 170 175ctg tct aac gca ctg atg cac cac cca
gac atc aac ctg atc ctc gcg 576Leu Ser Asn Ala Leu Met His His Pro
Asp Ile Asn Leu Ile Leu Ala 180 185 190act ggt ggt ccg ggc atg gtt
aaa gcc gca tac agc tcc ggt aaa cca 624Thr Gly Gly Pro Gly Met Val
Lys Ala Ala Tyr Ser Ser Gly Lys Pro 195 200 205gct atc ggt gta ggc
gcg ggc aac act cca gtt gtt atc gat gaa act 672Ala Ile Gly Val Gly
Ala Gly Asn Thr Pro Val Val Ile Asp Glu Thr 210 215 220gct gat atc
aaa cgt gca gtt gca tct gta ctg atg tcc aaa acc ttc 720Ala Asp Ile
Lys Arg Ala Val Ala Ser Val Leu Met Ser Lys Thr Phe225 230 235
240gac aac ggc gta atc tgt gct tct gaa cag tct gtt gtt gtt gtt gac
768Asp Asn Gly Val Ile Cys Ala Ser Glu Gln Ser Val Val Val Val Asp
245 250 255tct gtt tat gac gct gta cgt gaa cgt ttt gca acc cac ggc
ggc tat 816Ser Val Tyr Asp Ala Val Arg Glu Arg Phe Ala Thr His Gly
Gly Tyr 260 265 270ctg ttg cag ggt aaa gag ctg aaa gct gtt cag gat
gtt atc ctg aaa 864Leu Leu Gln Gly Lys Glu Leu Lys Ala Val Gln Asp
Val Ile Leu Lys 275 280 285aac ggt gcg ctg aac gcg gct atc gtt ggt
cag cca gcc tat aaa att 912Asn Gly Ala Leu Asn Ala Ala Ile Val Gly
Gln Pro Ala Tyr Lys Ile 290 295 300gct gaa ctg gca ggc ttc tct gta
cca gaa aac acc aag att ctg atc 960Ala Glu Leu Ala Gly Phe Ser Val
Pro Glu Asn Thr Lys Ile Leu Ile305 310 315 320ggt gaa gtg acc gtt
gtt gat gaa agc gaa ccg ttc gca cat gaa aaa 1008Gly Glu Val Thr Val
Val Asp Glu Ser Glu Pro Phe Ala His Glu Lys 325 330 335ctg tcc ccg
act ctg gca atg tac cgc gct aaa gat ttc gaa gac gcg 1056Leu Ser Pro
Thr Leu Ala Met Tyr Arg Ala Lys Asp Phe Glu Asp Ala 340 345 350gta
gaa aaa gca gag aaa ctg gtt gct atg ggc ggt atc ggt cat acc 1104Val
Glu Lys Ala Glu Lys Leu Val Ala Met Gly Gly Ile Gly His Thr 355 360
365tct tgc ctg tac act gac cag gat aac caa ccg gct cgc gtt tct tac
1152Ser Cys Leu Tyr Thr Asp Gln Asp Asn Gln Pro Ala Arg Val Ser Tyr
370 375 380ttc ggt cag aaa atg aaa acg gcg cgt atc ctg att aac acc
cca gcg 1200Phe Gly Gln Lys Met Lys Thr Ala Arg Ile Leu Ile Asn Thr
Pro Ala385 390 395 400tct cag ggt ggt atc ggt gac ctg tat aac ttc
aaa ctc gca cct tcc 1248Ser Gln Gly Gly Ile Gly Asp Leu Tyr Asn Phe
Lys Leu Ala Pro Ser 405 410 415ctg act ctg ggt tgt ggt tct tgg ggt
ggt aac tcc atc tct gaa aac 1296Leu Thr Leu Gly Cys Gly Ser Trp Gly
Gly Asn Ser Ile Ser Glu Asn 420 425 430gtt ggt ccg aaa cac ctg atc
aac aag aaa acc gtt gct aag cga gct 1344Val Gly Pro Lys His Leu Ile
Asn Lys Lys Thr Val Ala Lys Arg Ala 435 440 445gaa aac atg ttg tgg
cac aaa ctt ccg aaa tct atc tac ttc cgc cgt 1392Glu Asn Met Leu Trp
His Lys Leu Pro Lys Ser Ile Tyr Phe Arg Arg 450 455 460ggc tcc ctg
cca atc gcg ctg gat gaa gtg att act gat ggc cac aaa 1440Gly Ser Leu
Pro Ile Ala Leu Asp Glu Val Ile Thr Asp Gly His Lys465 470 475
480cgt gcg ctc atc gtg act gac cgc ttc ctg ttc aac aat ggt tat gct
1488Arg Ala Leu Ile Val Thr Asp Arg Phe Leu Phe Asn Asn Gly Tyr Ala
485 490 495gat cag atc act tcc gta ctg aaa gca gca ggc gtt gaa act
gaa gtc 1536Asp Gln Ile Thr Ser Val Leu Lys Ala Ala Gly Val Glu Thr
Glu Val 500 505 510ttc ttc gaa gta gaa gcg gac ccg acc ctg agc atc
gtt cgt aaa ggt 1584Phe Phe Glu Val Glu Ala Asp Pro Thr Leu Ser Ile
Val Arg Lys Gly 515 520 525gca gaa ctg gca aac tcc ttc aaa cca gac
gtg att atc gcg ctg ggt 1632Ala Glu Leu Ala Asn Ser Phe Lys Pro Asp
Val Ile Ile Ala Leu Gly 530 535 540ggt ggt tcc ccg atg gac gcc gcg
aag atc atg tgg gtt atg tac gaa 1680Gly Gly Ser Pro Met Asp Ala Ala
Lys Ile Met Trp Val Met Tyr Glu545 550 555 560cat ccg gaa act cac
ttc gaa gag ctg gcg ctg cgc ttt atg gat atc 1728His Pro Glu Thr His
Phe Glu Glu Leu Ala Leu Arg Phe Met Asp Ile 565 570 575cgt aaa cgt
atc tac aag ttc ccg aaa atg ggc gtg aaa gcg aaa atg 1776Arg Lys Arg
Ile Tyr Lys Phe Pro Lys Met Gly Val Lys Ala Lys Met 580 585 590atc
gct gtc acc acc act tct ggt aca ggt tct gaa gtc act ccg ttt 1824Ile
Ala Val Thr Thr Thr Ser Gly Thr Gly Ser Glu Val Thr Pro Phe 595 600
605gcg gtt gta act gac gac gct act ggt cag aaa tat ccg ctg gca gac
1872Ala Val Val Thr Asp Asp Ala Thr Gly Gln Lys Tyr Pro Leu Ala Asp
610 615 620tat gcg ctg act ccg gat atg gcg att gtc gac gcc aac ctg
gtt atg 1920Tyr Ala Leu Thr Pro Asp Met Ala Ile Val Asp Ala Asn Leu
Val Met625 630 635 640gac atg ccg aag tcc ctg tgt gct ttc ggt ggt
ctg gac gca gta act 1968Asp Met Pro Lys Ser Leu Cys Ala Phe Gly Gly
Leu Asp Ala Val Thr 645 650 655cac gcc atg gaa gct tat gtt tct gta
ctg gca tct gag ttc tct gat 2016His Ala Met Glu Ala Tyr Val Ser Val
Leu Ala Ser Glu Phe Ser Asp 660 665 670ggt cag gct ctg cag gca ctg
aaa ctg ctg aaa gaa tat ctg cca gcg 2064Gly Gln Ala Leu Gln Ala Leu
Lys Leu Leu Lys Glu Tyr Leu Pro Ala 675 680 685tcc tac cac gaa ggg
tct aaa aat ccg gta gcg cgt gaa cgt gtt cac 2112Ser Tyr His Glu Gly
Ser Lys Asn Pro Val Ala Arg Glu Arg Val His 690 695 700agt gca gcg
act atc gcg ggt atc gcg ttt gcg aac gcc ttc ctg ggt 2160Ser Ala Ala
Thr Ile Ala Gly Ile Ala Phe Ala Asn Ala Phe Leu Gly705 710
715 720gta tgt cac tca atg gcg cac aaa ctg ggt tcc cag ttc cat att
ccg 2208Val Cys His Ser Met Ala His Lys Leu Gly Ser Gln Phe His Ile
Pro 725 730 735cac ggt ctg gca aac gcc ctg ctg att tgt aac gtt att
cgc tac aat 2256His Gly Leu Ala Asn Ala Leu Leu Ile Cys Asn Val Ile
Arg Tyr Asn 740 745 750gcg aac gac aac ccg acc aag cag act gca ttc
agc cag tat gac cgt 2304Ala Asn Asp Asn Pro Thr Lys Gln Thr Ala Phe
Ser Gln Tyr Asp Arg 755 760 765ccg cag gct cgc cgt cgt tat gct gaa
att gcc gac cac ttg ggt ctg 2352Pro Gln Ala Arg Arg Arg Tyr Ala Glu
Ile Ala Asp His Leu Gly Leu 770 775 780agc gca ccg ggc gac cgt act
gct gct aag atc gag aaa ctg ctg gca 2400Ser Ala Pro Gly Asp Arg Thr
Ala Ala Lys Ile Glu Lys Leu Leu Ala785 790 795 800tgg ctg gaa acg
ctg aaa gct gaa ctg ggt att ccg aaa tct atc cgt 2448Trp Leu Glu Thr
Leu Lys Ala Glu Leu Gly Ile Pro Lys Ser Ile Arg 805 810 815gaa gct
ggc gtt cag gaa gca gac ttc ctg gcg aac gtg gat aaa ctg 2496Glu Ala
Gly Val Gln Glu Ala Asp Phe Leu Ala Asn Val Asp Lys Leu 820 825
830tct gaa gat gca ttc gat gac cag tgc acc ggc gct aac ccg cgt tac
2544Ser Glu Asp Ala Phe Asp Asp Gln Cys Thr Gly Ala Asn Pro Arg Tyr
835 840 845ccg ctg atc tcc gag ctg aaa cag att ctg ctg gat acc tac
tac ggt 2592Pro Leu Ile Ser Glu Leu Lys Gln Ile Leu Leu Asp Thr Tyr
Tyr Gly 850 855 860cgt gat tat gta gaa ggt gaa act gca gcg aag aaa
gaa gct gct ccg 2640Arg Asp Tyr Val Glu Gly Glu Thr Ala Ala Lys Lys
Glu Ala Ala Pro865 870 875 880gct aaa gct gag aaa aaa gcg aaa aaa
tcc gct taa 2676Ala Lys Ala Glu Lys Lys Ala Lys Lys Ser Ala 885
89022891PRTEscherichia coli 22Met Ala Val Thr Asn Val Ala Glu Leu
Asn Ala Leu Val Glu Arg Val1 5 10 15Lys Lys Ala Gln Arg Glu Tyr Ala
Ser Phe Thr Gln Glu Gln Val Asp 20 25 30Lys Ile Phe Arg Ala Ala Ala
Leu Ala Ala Ala Asp Ala Arg Ile Pro 35 40 45Leu Ala Lys Met Ala Val
Ala Glu Ser Gly Met Gly Ile Val Glu Asp 50 55 60Lys Val Ile Lys Asn
His Phe Ala Ser Glu Tyr Ile Tyr Asn Ala Tyr65 70 75 80Lys Asp Glu
Lys Thr Cys Gly Val Leu Ser Glu Asp Asp Thr Phe Gly 85 90 95Thr Ile
Thr Ile Ala Glu Pro Ile Gly Ile Ile Cys Gly Ile Val Pro 100 105
110Thr Thr Asn Pro Thr Ser Thr Ala Ile Phe Lys Ser Leu Ile Ser Leu
115 120 125Lys Thr Arg Asn Ala Ile Ile Phe Ser Pro His Pro Arg Ala
Lys Asp 130 135 140Ala Thr Asn Lys Ala Ala Asp Ile Val Leu Gln Ala
Ala Ile Ala Ala145 150 155 160Gly Ala Pro Lys Asp Leu Ile Gly Trp
Ile Asp Gln Pro Ser Val Glu 165 170 175Leu Ser Asn Ala Leu Met His
His Pro Asp Ile Asn Leu Ile Leu Ala 180 185 190Thr Gly Gly Pro Gly
Met Val Lys Ala Ala Tyr Ser Ser Gly Lys Pro 195 200 205Ala Ile Gly
Val Gly Ala Gly Asn Thr Pro Val Val Ile Asp Glu Thr 210 215 220Ala
Asp Ile Lys Arg Ala Val Ala Ser Val Leu Met Ser Lys Thr Phe225 230
235 240Asp Asn Gly Val Ile Cys Ala Ser Glu Gln Ser Val Val Val Val
Asp 245 250 255Ser Val Tyr Asp Ala Val Arg Glu Arg Phe Ala Thr His
Gly Gly Tyr 260 265 270Leu Leu Gln Gly Lys Glu Leu Lys Ala Val Gln
Asp Val Ile Leu Lys 275 280 285Asn Gly Ala Leu Asn Ala Ala Ile Val
Gly Gln Pro Ala Tyr Lys Ile 290 295 300Ala Glu Leu Ala Gly Phe Ser
Val Pro Glu Asn Thr Lys Ile Leu Ile305 310 315 320Gly Glu Val Thr
Val Val Asp Glu Ser Glu Pro Phe Ala His Glu Lys 325 330 335Leu Ser
Pro Thr Leu Ala Met Tyr Arg Ala Lys Asp Phe Glu Asp Ala 340 345
350Val Glu Lys Ala Glu Lys Leu Val Ala Met Gly Gly Ile Gly His Thr
355 360 365Ser Cys Leu Tyr Thr Asp Gln Asp Asn Gln Pro Ala Arg Val
Ser Tyr 370 375 380Phe Gly Gln Lys Met Lys Thr Ala Arg Ile Leu Ile
Asn Thr Pro Ala385 390 395 400Ser Gln Gly Gly Ile Gly Asp Leu Tyr
Asn Phe Lys Leu Ala Pro Ser 405 410 415Leu Thr Leu Gly Cys Gly Ser
Trp Gly Gly Asn Ser Ile Ser Glu Asn 420 425 430Val Gly Pro Lys His
Leu Ile Asn Lys Lys Thr Val Ala Lys Arg Ala 435 440 445Glu Asn Met
Leu Trp His Lys Leu Pro Lys Ser Ile Tyr Phe Arg Arg 450 455 460Gly
Ser Leu Pro Ile Ala Leu Asp Glu Val Ile Thr Asp Gly His Lys465 470
475 480Arg Ala Leu Ile Val Thr Asp Arg Phe Leu Phe Asn Asn Gly Tyr
Ala 485 490 495Asp Gln Ile Thr Ser Val Leu Lys Ala Ala Gly Val Glu
Thr Glu Val 500 505 510Phe Phe Glu Val Glu Ala Asp Pro Thr Leu Ser
Ile Val Arg Lys Gly 515 520 525Ala Glu Leu Ala Asn Ser Phe Lys Pro
Asp Val Ile Ile Ala Leu Gly 530 535 540Gly Gly Ser Pro Met Asp Ala
Ala Lys Ile Met Trp Val Met Tyr Glu545 550 555 560His Pro Glu Thr
His Phe Glu Glu Leu Ala Leu Arg Phe Met Asp Ile 565 570 575Arg Lys
Arg Ile Tyr Lys Phe Pro Lys Met Gly Val Lys Ala Lys Met 580 585
590Ile Ala Val Thr Thr Thr Ser Gly Thr Gly Ser Glu Val Thr Pro Phe
595 600 605Ala Val Val Thr Asp Asp Ala Thr Gly Gln Lys Tyr Pro Leu
Ala Asp 610 615 620Tyr Ala Leu Thr Pro Asp Met Ala Ile Val Asp Ala
Asn Leu Val Met625 630 635 640Asp Met Pro Lys Ser Leu Cys Ala Phe
Gly Gly Leu Asp Ala Val Thr 645 650 655His Ala Met Glu Ala Tyr Val
Ser Val Leu Ala Ser Glu Phe Ser Asp 660 665 670Gly Gln Ala Leu Gln
Ala Leu Lys Leu Leu Lys Glu Tyr Leu Pro Ala 675 680 685Ser Tyr His
Glu Gly Ser Lys Asn Pro Val Ala Arg Glu Arg Val His 690 695 700Ser
Ala Ala Thr Ile Ala Gly Ile Ala Phe Ala Asn Ala Phe Leu Gly705 710
715 720Val Cys His Ser Met Ala His Lys Leu Gly Ser Gln Phe His Ile
Pro 725 730 735His Gly Leu Ala Asn Ala Leu Leu Ile Cys Asn Val Ile
Arg Tyr Asn 740 745 750Ala Asn Asp Asn Pro Thr Lys Gln Thr Ala Phe
Ser Gln Tyr Asp Arg 755 760 765Pro Gln Ala Arg Arg Arg Tyr Ala Glu
Ile Ala Asp His Leu Gly Leu 770 775 780Ser Ala Pro Gly Asp Arg Thr
Ala Ala Lys Ile Glu Lys Leu Leu Ala785 790 795 800Trp Leu Glu Thr
Leu Lys Ala Glu Leu Gly Ile Pro Lys Ser Ile Arg 805 810 815Glu Ala
Gly Val Gln Glu Ala Asp Phe Leu Ala Asn Val Asp Lys Leu 820 825
830Ser Glu Asp Ala Phe Asp Asp Gln Cys Thr Gly Ala Asn Pro Arg Tyr
835 840 845Pro Leu Ile Ser Glu Leu Lys Gln Ile Leu Leu Asp Thr Tyr
Tyr Gly 850 855 860Arg Asp Tyr Val Glu Gly Glu Thr Ala Ala Lys Lys
Glu Ala Ala Pro865 870 875 880Ala Lys Ala Glu Lys Lys Ala Lys Lys
Ser Ala 885 8902369DNAArtificialprimer 23cgttattgtt atctagttgt
gcaaaacatg ctaatgtagc attacgcccc gccctgccac 60tcatcgcag
692458DNAArtificialprimer 24attagtaaca gccataatgc tctcctgata
atgttaaacc gctcacaatt ccacacat 5825183DNAArtificialhybrid promoter
25ctagatctct cacctaccaa acaatgcccc cctgcaaaaa ataaattcat aaaaaacata
60cagataacca tctgcggtga taaattatct ctggcggtgt tgacaattaa tcatcggctc
120gtataatgtg tggaattgtg agcggtttaa cattatcagg agagcattat
ggctgttact 180aat 1832624DNAArtificialprimer 26acttgttctt
gagtgaaact ggca 242722DNAArtificialprimer 27aagacgcgct gacaatacgc
ct 222869DNAArtificialprimer 28cgttattgtt atctagttgt gcaaaacatg
ctaatgtagc atcagaaaaa ctcatcgagc 60atcaaatga
692965DNAArtificialprimer 29agccggagca gcttctttct tcgctgcagt
ttcaccttct acgttgtgtc tcaaaatctc 60tgatg 653024DNAArtificialprimer
30aagacgcgct gacaatacgc cttt 243124DNAArtificialprimer 31aaggggccgt
ttatgttgcc agac 243269DNAArtificialprimer 32catgtgggtt atgtacgaac
atccggaaac tcacttcgaa aagctggcgc tgcgctttat 60ggatatccg
693324DNAArtificialprimer 33aaggggccgt ttatgttgcc agac
243424DNAArtificialprimer 34aagacgcgct gacaatacgc cttt
243551DNAArtificialprimer 35ttttctagat aaggaggaat gacgtatgac
ccggacattc aagacaatgg a 513639DNAArtificialprimer 36aaatctagat
tcagcttatg cttcgccttc aatcgaacg 3937344DNAEscherichia coli
37aagctttacg cgaacgagcc atgacattgc tgacgactct ggcagtggca gatgacataa
60aactggtcga ctggttacaa caacgcctgg ggcttttaga gcaacgagac acggcaatgt
120tgcaccgttt gctgcatgat attgaaaaaa atatcaccaa ataaaaaacg
ccttagtaag 180tatttttcag cttttcattc tgactgcaac gggcaatatg
tctctgtgtg gattaaaaaa 240agagtgtctg atagcagctt ctgaactggt
tacctgccgt gagtaaatta aaattttatt 300gacttaggtc actaaatact
ttaaccaata taggcgactc taga 3443853DNAArtificialprimer 38ttttctggta
cctaaggagg aatgacgtat gattactatt gacggtaatg gcg
533940DNAArtificialprimer 39gagagaccgg gtaccttaat cggtgttgct
tttttccgct 404049DNAArtificialprimer 40aattggtacc taaggaggaa
tgacgtatga ccagtggccc aaaaccggc 494142DNAArtificialprimer
41gcgacgccta gcttagggta ccttaccatg cctcgatatt gt
424235DNAArtificialprimer 42ctctctggcc ccgggctgat tgatttgatc gattg
354333DNAArtificialprimer 43gagagacccc gggttaccag taatgctccg ctg
334430DNAArtificialprimer 44gcgcgaattc gggcgatgat gttgacgcca
304530DNAArtificialprimer 45gcgcgaattc gtcccatact aacctctgtt
30462664DNAEscherichia coliCDS(1)..(2664) 46atg tca gaa cgt ttc cca
aat gac gtg gat ccg atc gaa act cgc gac 48Met Ser Glu Arg Phe Pro
Asn Asp Val Asp Pro Ile Glu Thr Arg Asp1 5 10 15tgg ctc cag gcg atc
gaa tcg gtc atc cgt gaa gaa ggt gtt gag cgt 96Trp Leu Gln Ala Ile
Glu Ser Val Ile Arg Glu Glu Gly Val Glu Arg 20 25 30gct cag tat ctg
atc gac caa ctg ctt gct gaa gcc cgc aaa ggc ggt 144Ala Gln Tyr Leu
Ile Asp Gln Leu Leu Ala Glu Ala Arg Lys Gly Gly 35 40 45gta aac gta
gcc gca ggc aca ggt atc agc aac tac atc aac acc atc 192Val Asn Val
Ala Ala Gly Thr Gly Ile Ser Asn Tyr Ile Asn Thr Ile 50 55 60ccc gtt
gaa gaa caa ccg gag tat ccg ggt aat ctg gaa ctg gaa cgc 240Pro Val
Glu Glu Gln Pro Glu Tyr Pro Gly Asn Leu Glu Leu Glu Arg65 70 75
80cgt att cgt tca gct atc cgc tgg aac gcc atc atg acg gtg ctg cgt
288Arg Ile Arg Ser Ala Ile Arg Trp Asn Ala Ile Met Thr Val Leu Arg
85 90 95gcg tcg aaa aaa gac ctc gaa ctg ggc ggc cat atg gcg tcc ttc
cag 336Ala Ser Lys Lys Asp Leu Glu Leu Gly Gly His Met Ala Ser Phe
Gln 100 105 110tct tcc gca acc att tat gat gtg tgc ttt aac cac ttc
ttc cgt gca 384Ser Ser Ala Thr Ile Tyr Asp Val Cys Phe Asn His Phe
Phe Arg Ala 115 120 125cgc aac gag cag gat ggc ggc gac ctg gtt tac
ttc cag ggc cac atc 432Arg Asn Glu Gln Asp Gly Gly Asp Leu Val Tyr
Phe Gln Gly His Ile 130 135 140tcc ccg ggc gtg tac gct cgt gct ttc
ctg gaa ggt cgt ctg act cag 480Ser Pro Gly Val Tyr Ala Arg Ala Phe
Leu Glu Gly Arg Leu Thr Gln145 150 155 160gag cag ctg gat aac ttc
cgt cag gaa gtt cac ggc aat ggc ctc tct 528Glu Gln Leu Asp Asn Phe
Arg Gln Glu Val His Gly Asn Gly Leu Ser 165 170 175tcc tat ccg cac
ccg aaa ctg atg ccg gaa ttc tgg cag ttc ccg acc 576Ser Tyr Pro His
Pro Lys Leu Met Pro Glu Phe Trp Gln Phe Pro Thr 180 185 190gta tct
atg ggt ctg ggt ccg att ggt gct att tac cag gct aaa ttc 624Val Ser
Met Gly Leu Gly Pro Ile Gly Ala Ile Tyr Gln Ala Lys Phe 195 200
205ctg aaa tat ctg gaa cac cgt ggc ctg aaa gat acc tct aaa caa acc
672Leu Lys Tyr Leu Glu His Arg Gly Leu Lys Asp Thr Ser Lys Gln Thr
210 215 220gtt tac gcg ttc ctc ggt gac ggt gaa atg gac gaa ccg gaa
tcc aaa 720Val Tyr Ala Phe Leu Gly Asp Gly Glu Met Asp Glu Pro Glu
Ser Lys225 230 235 240ggt gcg atc acc atc gct acc cgt gaa aaa ctg
gat aac ctg gtc ttc 768Gly Ala Ile Thr Ile Ala Thr Arg Glu Lys Leu
Asp Asn Leu Val Phe 245 250 255gtt atc aac tgt aac ctg cag cgt ctt
gac ggc ccg gtc acc ggt aac 816Val Ile Asn Cys Asn Leu Gln Arg Leu
Asp Gly Pro Val Thr Gly Asn 260 265 270ggc aag atc atc aac gaa ctg
gaa ggc atc ttc gaa ggt gct ggc tgg 864Gly Lys Ile Ile Asn Glu Leu
Glu Gly Ile Phe Glu Gly Ala Gly Trp 275 280 285aac gtg atc aaa gtg
atg tgg ggt agc cgt tgg gat gaa ctg ctg cgt 912Asn Val Ile Lys Val
Met Trp Gly Ser Arg Trp Asp Glu Leu Leu Arg 290 295 300aag gat acc
agc ggt aaa ctg atc cag ctg atg aac gaa acc gtt gac 960Lys Asp Thr
Ser Gly Lys Leu Ile Gln Leu Met Asn Glu Thr Val Asp305 310 315
320ggc gac tac cag acc ttc aaa tcg aaa gat ggt gcg tac gtt cgt gaa
1008Gly Asp Tyr Gln Thr Phe Lys Ser Lys Asp Gly Ala Tyr Val Arg Glu
325 330 335cac ttc ttc ggt aaa tat cct gaa acc gca gca ctg gtt gca
gac tgg 1056His Phe Phe Gly Lys Tyr Pro Glu Thr Ala Ala Leu Val Ala
Asp Trp 340 345 350act gac gag cag atc tgg gca ctg aac cgt ggt ggt
cac gat ccg aag 1104Thr Asp Glu Gln Ile Trp Ala Leu Asn Arg Gly Gly
His Asp Pro Lys 355 360 365aaa atc tac gct gca ttc aag aaa gcg cag
gaa acc aaa ggc aaa gcg 1152Lys Ile Tyr Ala Ala Phe Lys Lys Ala Gln
Glu Thr Lys Gly Lys Ala 370 375 380aca gta atc ctt gct cat acc att
aaa ggt tac ggc atg ggc gac gcg 1200Thr Val Ile Leu Ala His Thr Ile
Lys Gly Tyr Gly Met Gly Asp Ala385 390 395 400gct gaa ggt aaa aac
atc gcg cac cag gtt aag aaa atg aac atg gac 1248Ala Glu Gly Lys Asn
Ile Ala His Gln Val Lys Lys Met Asn Met Asp 405 410 415ggt gtg cgt
cat atc cgc gac cgt ttc aat gtg ccg gtg tct gat gca 1296Gly Val Arg
His Ile Arg Asp Arg Phe Asn Val Pro Val Ser Asp Ala 420 425 430gat
atc gaa aaa ctg ccg tac atc acc ttc ccg gaa ggt tct gaa gag 1344Asp
Ile Glu Lys Leu Pro Tyr Ile Thr Phe Pro Glu Gly Ser Glu Glu 435 440
445cat acc tat ctg cac gct cag cgt cag aaa ctg cac ggt tat ctg cca
1392His Thr Tyr Leu His Ala Gln Arg Gln Lys Leu His Gly Tyr Leu Pro
450 455 460agc cgt cag ccg aac ttc acc gag aag ctt gag ctg ccg agc
ctg caa 1440Ser Arg Gln Pro Asn Phe Thr Glu Lys Leu Glu Leu Pro Ser
Leu Gln465 470 475 480gac ttc ggc gcg ctg ttg gaa gag cag agc aaa
gag atc tct acc act 1488Asp Phe Gly Ala Leu Leu Glu Glu Gln Ser Lys
Glu Ile Ser Thr Thr 485 490 495atc gct ttc gtt cgt gct ctg aac gtg
atg ctg aag aac aag tcg atc 1536Ile Ala Phe Val Arg Ala Leu Asn Val
Met Leu Lys Asn Lys Ser Ile 500 505 510aaa gat cgt ctg gta ccg atc
atc gcc gac gaa gcg cgt act ttc ggt 1584Lys Asp Arg Leu Val Pro Ile
Ile Ala Asp Glu Ala Arg Thr Phe Gly 515 520 525atg gaa ggt ctg ttc
cgt cag att ggt att tac agc ccg aac ggt cag 1632Met Glu
Gly Leu Phe Arg Gln Ile Gly Ile Tyr Ser Pro Asn Gly Gln 530 535
540cag tac acc ccg cag gac cgc gag cag gtt gct tac tat aaa gaa gac
1680Gln Tyr Thr Pro Gln Asp Arg Glu Gln Val Ala Tyr Tyr Lys Glu
Asp545 550 555 560gag aaa ggt cag att ctg cag gaa ggg atc aac gag
ctg ggc gca ggt 1728Glu Lys Gly Gln Ile Leu Gln Glu Gly Ile Asn Glu
Leu Gly Ala Gly 565 570 575tgt tcc tgg ctg gca gcg gcg acc tct tac
agc acc aac aat ctg ccg 1776Cys Ser Trp Leu Ala Ala Ala Thr Ser Tyr
Ser Thr Asn Asn Leu Pro 580 585 590atg atc ccg ttc tac atc tat tac
tcg atg ttc ggc ttc cag cgt att 1824Met Ile Pro Phe Tyr Ile Tyr Tyr
Ser Met Phe Gly Phe Gln Arg Ile 595 600 605ggc gat ctg tgc tgg gcg
gct ggc gac cag caa gcg cgt ggc ttc ctg 1872Gly Asp Leu Cys Trp Ala
Ala Gly Asp Gln Gln Ala Arg Gly Phe Leu 610 615 620atc ggc ggt act
tcc ggt cgt acc acc ctg aac ggc gaa ggt ctg cag 1920Ile Gly Gly Thr
Ser Gly Arg Thr Thr Leu Asn Gly Glu Gly Leu Gln625 630 635 640cac
gaa gat ggt cac agc cac att cag tcg ctg act atc ccg aac tgt 1968His
Glu Asp Gly His Ser His Ile Gln Ser Leu Thr Ile Pro Asn Cys 645 650
655atc tct tac gac ccg gct tac gct tac gaa gtt gct gtc atc atg cat
2016Ile Ser Tyr Asp Pro Ala Tyr Ala Tyr Glu Val Ala Val Ile Met His
660 665 670gac ggt ctg gag cgt atg tac ggt gaa aaa caa gag aac gtt
tac tac 2064Asp Gly Leu Glu Arg Met Tyr Gly Glu Lys Gln Glu Asn Val
Tyr Tyr 675 680 685tac atc act acg ctg aac gaa aac tac cac atg ccg
gca atg ccg gaa 2112Tyr Ile Thr Thr Leu Asn Glu Asn Tyr His Met Pro
Ala Met Pro Glu 690 695 700ggt gct gag gaa ggt atc cgt aaa ggt atc
tac aaa ctc gaa act att 2160Gly Ala Glu Glu Gly Ile Arg Lys Gly Ile
Tyr Lys Leu Glu Thr Ile705 710 715 720gaa ggt agc aaa ggt aaa gtt
cag ctg ctc ggc tcc ggt tct atc ctg 2208Glu Gly Ser Lys Gly Lys Val
Gln Leu Leu Gly Ser Gly Ser Ile Leu 725 730 735cgt cac gtc cgt gaa
gca gct gag atc ctg gcg aaa gat tac ggc gta 2256Arg His Val Arg Glu
Ala Ala Glu Ile Leu Ala Lys Asp Tyr Gly Val 740 745 750ggt tct gac
gtt tat agc gtg acc tcc ttc acc gag ctg gcg cgt gat 2304Gly Ser Asp
Val Tyr Ser Val Thr Ser Phe Thr Glu Leu Ala Arg Asp 755 760 765ggt
cag gat tgt gaa cgc tgg aac atg ctg cac ccg ctg gaa act ccg 2352Gly
Gln Asp Cys Glu Arg Trp Asn Met Leu His Pro Leu Glu Thr Pro 770 775
780cgc gtt ccg tat atc gct cag gtg atg aac gac gct ccg gca gtg gca
2400Arg Val Pro Tyr Ile Ala Gln Val Met Asn Asp Ala Pro Ala Val
Ala785 790 795 800tct acc gac tat atg aaa ctg ttc gct gag cag gtc
cgt act tac gta 2448Ser Thr Asp Tyr Met Lys Leu Phe Ala Glu Gln Val
Arg Thr Tyr Val 805 810 815ccg gct gac gac tac cgc gta ctg ggt act
gat ggc ttc ggt cgt tcc 2496Pro Ala Asp Asp Tyr Arg Val Leu Gly Thr
Asp Gly Phe Gly Arg Ser 820 825 830gac agc cgt gag aac ctg cgt cac
cac ttc gaa gtt gat gct tct tat 2544Asp Ser Arg Glu Asn Leu Arg His
His Phe Glu Val Asp Ala Ser Tyr 835 840 845gtc gtg gtt gcg gcg ctg
ggc gaa ctg gct aaa cgt ggc gaa atc gat 2592Val Val Val Ala Ala Leu
Gly Glu Leu Ala Lys Arg Gly Glu Ile Asp 850 855 860aag aaa gtg gtt
gct gac gca atc gcc aaa ttc aac atc gat gca gat 2640Lys Lys Val Val
Ala Asp Ala Ile Ala Lys Phe Asn Ile Asp Ala Asp865 870 875 880aaa
gtt aac ccg cgt ctg gcg taa 2664Lys Val Asn Pro Arg Leu Ala
88547887PRTEscherichia coli 47Met Ser Glu Arg Phe Pro Asn Asp Val
Asp Pro Ile Glu Thr Arg Asp1 5 10 15Trp Leu Gln Ala Ile Glu Ser Val
Ile Arg Glu Glu Gly Val Glu Arg 20 25 30Ala Gln Tyr Leu Ile Asp Gln
Leu Leu Ala Glu Ala Arg Lys Gly Gly 35 40 45Val Asn Val Ala Ala Gly
Thr Gly Ile Ser Asn Tyr Ile Asn Thr Ile 50 55 60Pro Val Glu Glu Gln
Pro Glu Tyr Pro Gly Asn Leu Glu Leu Glu Arg65 70 75 80Arg Ile Arg
Ser Ala Ile Arg Trp Asn Ala Ile Met Thr Val Leu Arg 85 90 95Ala Ser
Lys Lys Asp Leu Glu Leu Gly Gly His Met Ala Ser Phe Gln 100 105
110Ser Ser Ala Thr Ile Tyr Asp Val Cys Phe Asn His Phe Phe Arg Ala
115 120 125Arg Asn Glu Gln Asp Gly Gly Asp Leu Val Tyr Phe Gln Gly
His Ile 130 135 140Ser Pro Gly Val Tyr Ala Arg Ala Phe Leu Glu Gly
Arg Leu Thr Gln145 150 155 160Glu Gln Leu Asp Asn Phe Arg Gln Glu
Val His Gly Asn Gly Leu Ser 165 170 175Ser Tyr Pro His Pro Lys Leu
Met Pro Glu Phe Trp Gln Phe Pro Thr 180 185 190Val Ser Met Gly Leu
Gly Pro Ile Gly Ala Ile Tyr Gln Ala Lys Phe 195 200 205Leu Lys Tyr
Leu Glu His Arg Gly Leu Lys Asp Thr Ser Lys Gln Thr 210 215 220Val
Tyr Ala Phe Leu Gly Asp Gly Glu Met Asp Glu Pro Glu Ser Lys225 230
235 240Gly Ala Ile Thr Ile Ala Thr Arg Glu Lys Leu Asp Asn Leu Val
Phe 245 250 255Val Ile Asn Cys Asn Leu Gln Arg Leu Asp Gly Pro Val
Thr Gly Asn 260 265 270Gly Lys Ile Ile Asn Glu Leu Glu Gly Ile Phe
Glu Gly Ala Gly Trp 275 280 285Asn Val Ile Lys Val Met Trp Gly Ser
Arg Trp Asp Glu Leu Leu Arg 290 295 300Lys Asp Thr Ser Gly Lys Leu
Ile Gln Leu Met Asn Glu Thr Val Asp305 310 315 320Gly Asp Tyr Gln
Thr Phe Lys Ser Lys Asp Gly Ala Tyr Val Arg Glu 325 330 335His Phe
Phe Gly Lys Tyr Pro Glu Thr Ala Ala Leu Val Ala Asp Trp 340 345
350Thr Asp Glu Gln Ile Trp Ala Leu Asn Arg Gly Gly His Asp Pro Lys
355 360 365Lys Ile Tyr Ala Ala Phe Lys Lys Ala Gln Glu Thr Lys Gly
Lys Ala 370 375 380Thr Val Ile Leu Ala His Thr Ile Lys Gly Tyr Gly
Met Gly Asp Ala385 390 395 400Ala Glu Gly Lys Asn Ile Ala His Gln
Val Lys Lys Met Asn Met Asp 405 410 415Gly Val Arg His Ile Arg Asp
Arg Phe Asn Val Pro Val Ser Asp Ala 420 425 430Asp Ile Glu Lys Leu
Pro Tyr Ile Thr Phe Pro Glu Gly Ser Glu Glu 435 440 445His Thr Tyr
Leu His Ala Gln Arg Gln Lys Leu His Gly Tyr Leu Pro 450 455 460Ser
Arg Gln Pro Asn Phe Thr Glu Lys Leu Glu Leu Pro Ser Leu Gln465 470
475 480Asp Phe Gly Ala Leu Leu Glu Glu Gln Ser Lys Glu Ile Ser Thr
Thr 485 490 495Ile Ala Phe Val Arg Ala Leu Asn Val Met Leu Lys Asn
Lys Ser Ile 500 505 510Lys Asp Arg Leu Val Pro Ile Ile Ala Asp Glu
Ala Arg Thr Phe Gly 515 520 525Met Glu Gly Leu Phe Arg Gln Ile Gly
Ile Tyr Ser Pro Asn Gly Gln 530 535 540Gln Tyr Thr Pro Gln Asp Arg
Glu Gln Val Ala Tyr Tyr Lys Glu Asp545 550 555 560Glu Lys Gly Gln
Ile Leu Gln Glu Gly Ile Asn Glu Leu Gly Ala Gly 565 570 575Cys Ser
Trp Leu Ala Ala Ala Thr Ser Tyr Ser Thr Asn Asn Leu Pro 580 585
590Met Ile Pro Phe Tyr Ile Tyr Tyr Ser Met Phe Gly Phe Gln Arg Ile
595 600 605Gly Asp Leu Cys Trp Ala Ala Gly Asp Gln Gln Ala Arg Gly
Phe Leu 610 615 620Ile Gly Gly Thr Ser Gly Arg Thr Thr Leu Asn Gly
Glu Gly Leu Gln625 630 635 640His Glu Asp Gly His Ser His Ile Gln
Ser Leu Thr Ile Pro Asn Cys 645 650 655Ile Ser Tyr Asp Pro Ala Tyr
Ala Tyr Glu Val Ala Val Ile Met His 660 665 670Asp Gly Leu Glu Arg
Met Tyr Gly Glu Lys Gln Glu Asn Val Tyr Tyr 675 680 685Tyr Ile Thr
Thr Leu Asn Glu Asn Tyr His Met Pro Ala Met Pro Glu 690 695 700Gly
Ala Glu Glu Gly Ile Arg Lys Gly Ile Tyr Lys Leu Glu Thr Ile705 710
715 720Glu Gly Ser Lys Gly Lys Val Gln Leu Leu Gly Ser Gly Ser Ile
Leu 725 730 735Arg His Val Arg Glu Ala Ala Glu Ile Leu Ala Lys Asp
Tyr Gly Val 740 745 750Gly Ser Asp Val Tyr Ser Val Thr Ser Phe Thr
Glu Leu Ala Arg Asp 755 760 765Gly Gln Asp Cys Glu Arg Trp Asn Met
Leu His Pro Leu Glu Thr Pro 770 775 780Arg Val Pro Tyr Ile Ala Gln
Val Met Asn Asp Ala Pro Ala Val Ala785 790 795 800Ser Thr Asp Tyr
Met Lys Leu Phe Ala Glu Gln Val Arg Thr Tyr Val 805 810 815Pro Ala
Asp Asp Tyr Arg Val Leu Gly Thr Asp Gly Phe Gly Arg Ser 820 825
830Asp Ser Arg Glu Asn Leu Arg His His Phe Glu Val Asp Ala Ser Tyr
835 840 845Val Val Val Ala Ala Leu Gly Glu Leu Ala Lys Arg Gly Glu
Ile Asp 850 855 860Lys Lys Val Val Ala Asp Ala Ile Ala Lys Phe Asn
Ile Asp Ala Asp865 870 875 880Lys Val Asn Pro Arg Leu Ala 885
481893DNAEscherichia coliCDS(1)..(1893) 48atg gct atc gaa atc aaa
gta ccg gac atc ggg gct gat gaa gtt gaa 48Met Ala Ile Glu Ile Lys
Val Pro Asp Ile Gly Ala Asp Glu Val Glu1 5 10 15atc acc gag atc ctg
gtc aaa gtg ggc gac aaa gtt gaa gcc gaa cag 96Ile Thr Glu Ile Leu
Val Lys Val Gly Asp Lys Val Glu Ala Glu Gln 20 25 30tcg ctg atc acc
gta gaa ggc gac aaa gcc tct atg gaa gtt ccg tct 144Ser Leu Ile Thr
Val Glu Gly Asp Lys Ala Ser Met Glu Val Pro Ser 35 40 45ccg cag gcg
ggt atc gtt aaa gag atc aaa gtc tct gtt ggc gat aaa 192Pro Gln Ala
Gly Ile Val Lys Glu Ile Lys Val Ser Val Gly Asp Lys 50 55 60acc cag
acc ggc gca ctg att atg att ttc gat tcc gcc gac ggt gca 240Thr Gln
Thr Gly Ala Leu Ile Met Ile Phe Asp Ser Ala Asp Gly Ala65 70 75
80gca gac gct gca cct gct cag gca gaa gag aag aaa gaa gca gct ccg
288Ala Asp Ala Ala Pro Ala Gln Ala Glu Glu Lys Lys Glu Ala Ala Pro
85 90 95gca gca gca cca gcg gct gcg gcg gca aaa gac gtt aac gtt ccg
gat 336Ala Ala Ala Pro Ala Ala Ala Ala Ala Lys Asp Val Asn Val Pro
Asp 100 105 110atc ggc agc gac gaa gtt gaa gtg acc gaa atc ctg gtg
aaa gtt ggc 384Ile Gly Ser Asp Glu Val Glu Val Thr Glu Ile Leu Val
Lys Val Gly 115 120 125gat aaa gtt gaa gct gaa cag tcg ctg atc acc
gta gaa ggc gac aag 432Asp Lys Val Glu Ala Glu Gln Ser Leu Ile Thr
Val Glu Gly Asp Lys 130 135 140gct tct atg gaa gtt ccg gct ccg ttt
gct ggc acc gtg aaa gag atc 480Ala Ser Met Glu Val Pro Ala Pro Phe
Ala Gly Thr Val Lys Glu Ile145 150 155 160aaa gtg aac gtg ggt gac
aaa gtg tct acc ggc tcg ctg att atg gtc 528Lys Val Asn Val Gly Asp
Lys Val Ser Thr Gly Ser Leu Ile Met Val 165 170 175ttc gaa gtc gcg
ggt gaa gca ggc gcg gca gct ccg gcc gct aaa cag 576Phe Glu Val Ala
Gly Glu Ala Gly Ala Ala Ala Pro Ala Ala Lys Gln 180 185 190gaa gca
gct ccg gca gcg gcc cct gca cca gcg gct ggc gtg aaa gaa 624Glu Ala
Ala Pro Ala Ala Ala Pro Ala Pro Ala Ala Gly Val Lys Glu 195 200
205gtt aac gtt ccg gat atc ggc ggt gac gaa gtt gaa gtg act gaa gtg
672Val Asn Val Pro Asp Ile Gly Gly Asp Glu Val Glu Val Thr Glu Val
210 215 220atg gtg aaa gtg ggc gac aaa gtt gcc gct gaa cag tca ctg
atc acc 720Met Val Lys Val Gly Asp Lys Val Ala Ala Glu Gln Ser Leu
Ile Thr225 230 235 240gta gaa ggc gac aaa gct tct atg gaa gtt ccg
gcg ccg ttt gca ggc 768Val Glu Gly Asp Lys Ala Ser Met Glu Val Pro
Ala Pro Phe Ala Gly 245 250 255gtc gtg aag gaa ctg aaa gtc aac gtt
ggc gat aaa gtg aaa act ggc 816Val Val Lys Glu Leu Lys Val Asn Val
Gly Asp Lys Val Lys Thr Gly 260 265 270tcg ctg att atg atc ttc gaa
gtt gaa ggc gca gcg cct gcg gca gct 864Ser Leu Ile Met Ile Phe Glu
Val Glu Gly Ala Ala Pro Ala Ala Ala 275 280 285cct gcg aaa cag gaa
gcg gca gcg ccg gca ccg gca gca aaa gct gaa 912Pro Ala Lys Gln Glu
Ala Ala Ala Pro Ala Pro Ala Ala Lys Ala Glu 290 295 300gcc ccg gca
gca gca cca gct gcg aaa gcg gaa ggc aaa tct gaa ttt 960Ala Pro Ala
Ala Ala Pro Ala Ala Lys Ala Glu Gly Lys Ser Glu Phe305 310 315
320gct gaa aac gac gct tat gtt cac gcg act ccg ctg atc cgc cgt ctg
1008Ala Glu Asn Asp Ala Tyr Val His Ala Thr Pro Leu Ile Arg Arg Leu
325 330 335gca cgc gag ttt ggt gtt aac ctt gcg aaa gtg aag ggc act
ggc cgt 1056Ala Arg Glu Phe Gly Val Asn Leu Ala Lys Val Lys Gly Thr
Gly Arg 340 345 350aaa ggt cgt atc ctg cgc gaa gac gtt cag gct tac
gtg aaa gaa gct 1104Lys Gly Arg Ile Leu Arg Glu Asp Val Gln Ala Tyr
Val Lys Glu Ala 355 360 365atc aaa cgt gca gaa gca gct ccg gca gcg
act ggc ggt ggt atc cct 1152Ile Lys Arg Ala Glu Ala Ala Pro Ala Ala
Thr Gly Gly Gly Ile Pro 370 375 380ggc atg ctg ccg tgg ccg aag gtg
gac ttc agc aag ttt ggt gaa atc 1200Gly Met Leu Pro Trp Pro Lys Val
Asp Phe Ser Lys Phe Gly Glu Ile385 390 395 400gaa gaa gtg gaa ctg
ggc cgc atc cag aaa atc tct ggt gcg aac ctg 1248Glu Glu Val Glu Leu
Gly Arg Ile Gln Lys Ile Ser Gly Ala Asn Leu 405 410 415agc cgt aac
tgg gta atg atc ccg cat gtt act cac ttc gac aaa acc 1296Ser Arg Asn
Trp Val Met Ile Pro His Val Thr His Phe Asp Lys Thr 420 425 430gat
atc acc gag ttg gaa gcg ttc cgt aaa cag cag aac gaa gaa gcg 1344Asp
Ile Thr Glu Leu Glu Ala Phe Arg Lys Gln Gln Asn Glu Glu Ala 435 440
445gcg aaa cgt aag ctg gat gtg aag atc acc ccg gtt gtc ttc atc atg
1392Ala Lys Arg Lys Leu Asp Val Lys Ile Thr Pro Val Val Phe Ile Met
450 455 460aaa gcc gtt gct gca gct ctt gag cag atg cct cgc ttc aat
agt tcg 1440Lys Ala Val Ala Ala Ala Leu Glu Gln Met Pro Arg Phe Asn
Ser Ser465 470 475 480ctg tcg gaa gac ggt cag cgt ctg acc ctg aag
aaa tac atc aac atc 1488Leu Ser Glu Asp Gly Gln Arg Leu Thr Leu Lys
Lys Tyr Ile Asn Ile 485 490 495ggt gtg gcg gtg gat acc ccg aac ggt
ctg gtt gtt ccg gta ttc aaa 1536Gly Val Ala Val Asp Thr Pro Asn Gly
Leu Val Val Pro Val Phe Lys 500 505 510gac gtc aac aag aaa ggc atc
atc gag ctg tct cgc gag ctg atg act 1584Asp Val Asn Lys Lys Gly Ile
Ile Glu Leu Ser Arg Glu Leu Met Thr 515 520 525att tct aag aaa gcg
cgt gac ggt aag ctg act gcg ggc gaa atg cag 1632Ile Ser Lys Lys Ala
Arg Asp Gly Lys Leu Thr Ala Gly Glu Met Gln 530 535 540ggc ggt tgc
ttc acc atc tcc agc atc ggc ggc ctg ggt act acc cac 1680Gly Gly Cys
Phe Thr Ile Ser Ser Ile Gly Gly Leu Gly Thr Thr His545 550 555
560ttc gcg ccg att gtg aac gcg ccg gaa gtg gct atc ctc ggc gtt tcc
1728Phe Ala Pro Ile Val Asn Ala Pro Glu Val Ala Ile Leu Gly Val Ser
565 570 575aag tcc gcg atg gag ccg gtg tgg aat ggt aaa gag ttc gtg
ccg cgt 1776Lys Ser Ala Met Glu Pro Val Trp Asn Gly Lys Glu Phe Val
Pro Arg 580 585 590ctg atg ctg ccg att tct ctc tcc ttc gac cac cgc
gtg atc gac ggt 1824Leu Met Leu Pro Ile Ser Leu Ser Phe Asp His Arg
Val Ile Asp Gly 595 600 605gct gat ggt gcc cgt ttc att acc atc att
aac aac acg ctg tct gac 1872Ala Asp Gly Ala Arg Phe Ile Thr Ile Ile
Asn Asn Thr Leu Ser Asp 610 615 620att cgc cgt ctg gtg atg taa
1893Ile Arg Arg Leu Val Met625 63049630PRTEscherichia coli 49Met
Ala Ile Glu Ile Lys Val Pro Asp Ile Gly Ala Asp Glu Val Glu1 5 10
15Ile Thr Glu Ile Leu Val Lys Val Gly Asp Lys Val Glu Ala Glu Gln
20 25 30Ser Leu Ile Thr Val Glu Gly Asp Lys Ala Ser Met Glu Val Pro
Ser 35 40 45Pro Gln Ala Gly Ile Val Lys Glu Ile Lys Val Ser Val Gly
Asp Lys 50 55 60Thr Gln Thr Gly Ala Leu Ile Met Ile Phe Asp Ser Ala
Asp Gly Ala65 70 75 80Ala Asp Ala Ala Pro Ala Gln Ala Glu Glu Lys
Lys Glu Ala Ala Pro 85 90 95Ala Ala Ala Pro Ala Ala Ala Ala Ala Lys
Asp Val Asn Val Pro Asp 100 105 110Ile Gly Ser Asp Glu Val Glu Val
Thr Glu Ile Leu Val Lys Val Gly 115 120 125Asp Lys Val Glu Ala Glu
Gln Ser Leu Ile Thr Val Glu Gly Asp Lys 130 135 140Ala Ser Met Glu
Val Pro Ala Pro Phe Ala Gly Thr Val Lys Glu Ile145 150 155 160Lys
Val Asn Val Gly Asp Lys Val Ser Thr Gly Ser Leu Ile Met Val 165 170
175Phe Glu Val Ala Gly Glu Ala Gly Ala Ala Ala Pro Ala Ala Lys Gln
180 185 190Glu Ala Ala Pro Ala Ala Ala Pro Ala Pro Ala Ala Gly Val
Lys Glu 195 200 205Val Asn Val Pro Asp Ile Gly Gly Asp Glu Val Glu
Val Thr Glu Val 210 215 220Met Val Lys Val Gly Asp Lys Val Ala Ala
Glu Gln Ser Leu Ile Thr225 230 235 240Val Glu Gly Asp Lys Ala Ser
Met Glu Val Pro Ala Pro Phe Ala Gly 245 250 255Val Val Lys Glu Leu
Lys Val Asn Val Gly Asp Lys Val Lys Thr Gly 260 265 270Ser Leu Ile
Met Ile Phe Glu Val Glu Gly Ala Ala Pro Ala Ala Ala 275 280 285Pro
Ala Lys Gln Glu Ala Ala Ala Pro Ala Pro Ala Ala Lys Ala Glu 290 295
300Ala Pro Ala Ala Ala Pro Ala Ala Lys Ala Glu Gly Lys Ser Glu
Phe305 310 315 320Ala Glu Asn Asp Ala Tyr Val His Ala Thr Pro Leu
Ile Arg Arg Leu 325 330 335Ala Arg Glu Phe Gly Val Asn Leu Ala Lys
Val Lys Gly Thr Gly Arg 340 345 350Lys Gly Arg Ile Leu Arg Glu Asp
Val Gln Ala Tyr Val Lys Glu Ala 355 360 365Ile Lys Arg Ala Glu Ala
Ala Pro Ala Ala Thr Gly Gly Gly Ile Pro 370 375 380Gly Met Leu Pro
Trp Pro Lys Val Asp Phe Ser Lys Phe Gly Glu Ile385 390 395 400Glu
Glu Val Glu Leu Gly Arg Ile Gln Lys Ile Ser Gly Ala Asn Leu 405 410
415Ser Arg Asn Trp Val Met Ile Pro His Val Thr His Phe Asp Lys Thr
420 425 430Asp Ile Thr Glu Leu Glu Ala Phe Arg Lys Gln Gln Asn Glu
Glu Ala 435 440 445Ala Lys Arg Lys Leu Asp Val Lys Ile Thr Pro Val
Val Phe Ile Met 450 455 460Lys Ala Val Ala Ala Ala Leu Glu Gln Met
Pro Arg Phe Asn Ser Ser465 470 475 480Leu Ser Glu Asp Gly Gln Arg
Leu Thr Leu Lys Lys Tyr Ile Asn Ile 485 490 495Gly Val Ala Val Asp
Thr Pro Asn Gly Leu Val Val Pro Val Phe Lys 500 505 510Asp Val Asn
Lys Lys Gly Ile Ile Glu Leu Ser Arg Glu Leu Met Thr 515 520 525Ile
Ser Lys Lys Ala Arg Asp Gly Lys Leu Thr Ala Gly Glu Met Gln 530 535
540Gly Gly Cys Phe Thr Ile Ser Ser Ile Gly Gly Leu Gly Thr Thr
His545 550 555 560Phe Ala Pro Ile Val Asn Ala Pro Glu Val Ala Ile
Leu Gly Val Ser 565 570 575Lys Ser Ala Met Glu Pro Val Trp Asn Gly
Lys Glu Phe Val Pro Arg 580 585 590Leu Met Leu Pro Ile Ser Leu Ser
Phe Asp His Arg Val Ile Asp Gly 595 600 605Ala Asp Gly Ala Arg Phe
Ile Thr Ile Ile Asn Asn Thr Leu Ser Asp 610 615 620Ile Arg Arg Leu
Val Met625 630501425DNAEscherichia coliCDS(1)..(1425) 50atg agt act
gaa atc aaa act cag gtc gtg gta ctt ggg gca ggc ccc 48Met Ser Thr
Glu Ile Lys Thr Gln Val Val Val Leu Gly Ala Gly Pro1 5 10 15gca ggt
tac tcc gct gcc ttc cgt tgc gct gat tta ggt ctg gaa acc 96Ala Gly
Tyr Ser Ala Ala Phe Arg Cys Ala Asp Leu Gly Leu Glu Thr 20 25 30gta
atc gta gaa cgt tac aac acc ctt ggc ggt gtt tgc ctg aac gtc 144Val
Ile Val Glu Arg Tyr Asn Thr Leu Gly Gly Val Cys Leu Asn Val 35 40
45ggc tgt atc cct tct aaa gca ctg ctg cac gta gca aaa gtt atc gaa
192Gly Cys Ile Pro Ser Lys Ala Leu Leu His Val Ala Lys Val Ile Glu
50 55 60gaa gcc aaa gcg ctg gct gaa cac ggt atc gtc ttc ggc gaa ccg
aaa 240Glu Ala Lys Ala Leu Ala Glu His Gly Ile Val Phe Gly Glu Pro
Lys65 70 75 80acc gat atc gac aag att cgt acc tgg aaa gag aaa gtg
atc aat cag 288Thr Asp Ile Asp Lys Ile Arg Thr Trp Lys Glu Lys Val
Ile Asn Gln 85 90 95ctg acc ggt ggt ctg gct ggt atg gcg aaa ggc cgc
aaa gtc aaa gtg 336Leu Thr Gly Gly Leu Ala Gly Met Ala Lys Gly Arg
Lys Val Lys Val 100 105 110gtc aac ggt ctg ggt aaa ttc acc ggg gct
aac acc ctg gaa gtt gaa 384Val Asn Gly Leu Gly Lys Phe Thr Gly Ala
Asn Thr Leu Glu Val Glu 115 120 125ggt gag aac ggc aaa acc gtg atc
aac ttc gac aac gcg atc att gca 432Gly Glu Asn Gly Lys Thr Val Ile
Asn Phe Asp Asn Ala Ile Ile Ala 130 135 140gcg ggt tct cgc ccg atc
caa ctg ccg ttt att ccg cat gaa gat ccg 480Ala Gly Ser Arg Pro Ile
Gln Leu Pro Phe Ile Pro His Glu Asp Pro145 150 155 160cgt atc tgg
gac tcc act gac gcg ctg gaa ctg aaa gaa gta cca gaa 528Arg Ile Trp
Asp Ser Thr Asp Ala Leu Glu Leu Lys Glu Val Pro Glu 165 170 175cgc
ctg ctg gta atg ggt ggc ggt atc atc ggt ctg gaa atg ggc acc 576Arg
Leu Leu Val Met Gly Gly Gly Ile Ile Gly Leu Glu Met Gly Thr 180 185
190gtt tac cac gcg ctg ggt tca cag att gac gtg gtt gaa atg ttc gac
624Val Tyr His Ala Leu Gly Ser Gln Ile Asp Val Val Glu Met Phe Asp
195 200 205cag gtt atc ccg gca gct gac aaa gac atc gtt aaa gtc ttc
acc aag 672Gln Val Ile Pro Ala Ala Asp Lys Asp Ile Val Lys Val Phe
Thr Lys 210 215 220cgt atc agc aag aaa ttc aac ctg atg ctg gaa acc
aaa gtt acc gcc 720Arg Ile Ser Lys Lys Phe Asn Leu Met Leu Glu Thr
Lys Val Thr Ala225 230 235 240gtt gaa gcg aaa gaa gac ggc att tat
gtg acg atg gaa ggc aaa aaa 768Val Glu Ala Lys Glu Asp Gly Ile Tyr
Val Thr Met Glu Gly Lys Lys 245 250 255gca ccc gct gaa ccg cag cgt
tac gac gcc gtg ctg gta gcg att ggt 816Ala Pro Ala Glu Pro Gln Arg
Tyr Asp Ala Val Leu Val Ala Ile Gly 260 265 270cgt gtg ccg aac ggt
aaa aac ctc gac gca ggc aaa gca ggc gtg gaa 864Arg Val Pro Asn Gly
Lys Asn Leu Asp Ala Gly Lys Ala Gly Val Glu 275 280 285gtt gac gac
cgt ggt ttc atc cgc gtt gac aaa cag ctg cgt acc aac 912Val Asp Asp
Arg Gly Phe Ile Arg Val Asp Lys Gln Leu Arg Thr Asn 290 295 300gta
ccg cac atc ttt gct atc ggc gat atc gtc ggt caa ccg atg ctg 960Val
Pro His Ile Phe Ala Ile Gly Asp Ile Val Gly Gln Pro Met Leu305 310
315 320gca cac aaa ggt gtt cac gaa ggt cac gtt gcc gct gaa gtt atc
gcc 1008Ala His Lys Gly Val His Glu Gly His Val Ala Ala Glu Val Ile
Ala 325 330 335ggt aag aaa cac tac ttc gat ccg aaa gtt atc ccg tcc
atc gcc tat 1056Gly Lys Lys His Tyr Phe Asp Pro Lys Val Ile Pro Ser
Ile Ala Tyr 340 345 350acc gaa cca gaa gtt gca tgg gtg ggt ctg act
gag aaa gaa gcg aaa 1104Thr Glu Pro Glu Val Ala Trp Val Gly Leu Thr
Glu Lys Glu Ala Lys 355 360 365gag aaa ggc atc agc tat gaa acc gcc
acc ttc ccg tgg gct gct tct 1152Glu Lys Gly Ile Ser Tyr Glu Thr Ala
Thr Phe Pro Trp Ala Ala Ser 370 375 380ggt cgt gct atc gct tcc gac
tgc gca gac ggt atg acc aag ctg att 1200Gly Arg Ala Ile Ala Ser Asp
Cys Ala Asp Gly Met Thr Lys Leu Ile385 390 395 400ttc gac aaa gaa
tct cac cgt gtg atc ggt ggt gcg att gtc ggt act 1248Phe Asp Lys Glu
Ser His Arg Val Ile Gly Gly Ala Ile Val Gly Thr 405 410 415aac ggc
ggc gag ctg ctg ggt gaa atc ggc ctg gca atc gaa atg ggt 1296Asn Gly
Gly Glu Leu Leu Gly Glu Ile Gly Leu Ala Ile Glu Met Gly 420 425
430tgt gat gct gaa gac atc gca ctg acc atc cac gcg cac ccg act ctg
1344Cys Asp Ala Glu Asp Ile Ala Leu Thr Ile His Ala His Pro Thr Leu
435 440 445cac gag tct gtg ggc ctg gcg gca gaa gtg ttc gaa ggt agc
att acc 1392His Glu Ser Val Gly Leu Ala Ala Glu Val Phe Glu Gly Ser
Ile Thr 450 455 460gac ctg ccg aac ccg aaa gcg aag aag aag taa
1425Asp Leu Pro Asn Pro Lys Ala Lys Lys Lys465
47051474PRTEscherichia coli 51Met Ser Thr Glu Ile Lys Thr Gln Val
Val Val Leu Gly Ala Gly Pro1 5 10 15Ala Gly Tyr Ser Ala Ala Phe Arg
Cys Ala Asp Leu Gly Leu Glu Thr 20 25 30Val Ile Val Glu Arg Tyr Asn
Thr Leu Gly Gly Val Cys Leu Asn Val 35 40 45Gly Cys Ile Pro Ser Lys
Ala Leu Leu His Val Ala Lys Val Ile Glu 50 55 60Glu Ala Lys Ala Leu
Ala Glu His Gly Ile Val Phe Gly Glu Pro Lys65 70 75 80Thr Asp Ile
Asp Lys Ile Arg Thr Trp Lys Glu Lys Val Ile Asn Gln 85 90 95Leu Thr
Gly Gly Leu Ala Gly Met Ala Lys Gly Arg Lys Val Lys Val 100 105
110Val Asn Gly Leu Gly Lys Phe Thr Gly Ala Asn Thr Leu Glu Val Glu
115 120 125Gly Glu Asn Gly Lys Thr Val Ile Asn Phe Asp Asn Ala Ile
Ile Ala 130 135 140Ala Gly Ser Arg Pro Ile Gln Leu Pro Phe Ile Pro
His Glu Asp Pro145 150 155 160Arg Ile Trp Asp Ser Thr Asp Ala Leu
Glu Leu Lys Glu Val Pro Glu 165 170 175Arg Leu Leu Val Met Gly Gly
Gly Ile Ile Gly Leu Glu Met Gly Thr 180 185 190Val Tyr His Ala Leu
Gly Ser Gln Ile Asp Val Val Glu Met Phe Asp 195 200 205Gln Val Ile
Pro Ala Ala Asp Lys Asp Ile Val Lys Val Phe Thr Lys 210 215 220Arg
Ile Ser Lys Lys Phe Asn Leu Met Leu Glu Thr Lys Val Thr Ala225 230
235 240Val Glu Ala Lys Glu Asp Gly Ile Tyr Val Thr Met Glu Gly Lys
Lys 245 250 255Ala Pro Ala Glu Pro Gln Arg Tyr Asp Ala Val Leu Val
Ala Ile Gly 260 265 270Arg Val Pro Asn Gly Lys Asn Leu Asp Ala Gly
Lys Ala Gly Val Glu 275 280 285Val Asp Asp Arg Gly Phe Ile Arg Val
Asp Lys Gln Leu Arg Thr Asn 290 295 300Val Pro His Ile Phe Ala Ile
Gly Asp Ile Val Gly Gln Pro Met Leu305 310 315 320Ala His Lys Gly
Val His Glu Gly His Val Ala Ala Glu Val Ile Ala 325 330 335Gly Lys
Lys His Tyr Phe Asp Pro Lys Val Ile Pro Ser Ile Ala Tyr 340 345
350Thr Glu Pro Glu Val Ala Trp Val Gly Leu Thr Glu Lys Glu Ala Lys
355 360 365Glu Lys Gly Ile Ser Tyr Glu Thr Ala Thr Phe Pro Trp Ala
Ala Ser 370 375 380Gly Arg Ala Ile Ala Ser Asp Cys Ala Asp Gly Met
Thr Lys Leu Ile385 390 395 400Phe Asp Lys Glu Ser His Arg Val Ile
Gly Gly Ala Ile Val Gly Thr 405 410 415Asn Gly Gly Glu Leu Leu Gly
Glu Ile Gly Leu Ala Ile Glu Met Gly 420 425 430Cys Asp Ala Glu Asp
Ile Ala Leu Thr Ile His Ala His Pro Thr Leu 435 440 445His Glu Ser
Val Gly Leu Ala Ala Glu Val Phe Glu Gly Ser Ile Thr 450 455 460Asp
Leu Pro Asn Pro Lys Ala Lys Lys Lys465 470521698DNAEscherichia
coliCDS(1)..(1698) 52atg gaa cca aaa aca aaa aaa cag cgt tcg ctt
tat atc cct tac gct 48Met Glu Pro Lys Thr Lys Lys Gln Arg Ser Leu
Tyr Ile Pro Tyr Ala1 5 10 15ggc cct gta ctg ctg gaa ttt ccg ttg ttg
aat aaa ggc agt gcc ttc 96Gly Pro Val Leu Leu Glu Phe Pro Leu Leu
Asn Lys Gly Ser Ala Phe 20 25 30agc atg gaa gaa cgc cgt aac ttc aac
ctg ctg ggg tta ctg ccg gaa 144Ser Met Glu Glu Arg Arg Asn Phe Asn
Leu Leu Gly Leu Leu Pro Glu 35 40 45gtg gtc gaa acc atc gaa gaa caa
gcg gaa cga gca tgg atc cag tat 192Val Val Glu Thr Ile Glu Glu Gln
Ala Glu Arg Ala Trp Ile Gln Tyr 50 55 60cag gga ttc aaa acc gaa atc
gac aaa cac atc tac ctg cgt aac atc 240Gln Gly Phe Lys Thr Glu Ile
Asp Lys His Ile Tyr Leu Arg Asn Ile65 70 75 80cag gac act aac gaa
acc ctc ttc tac cgt ctg gta aac aat cat ctt 288Gln Asp Thr Asn Glu
Thr Leu Phe Tyr Arg Leu Val Asn Asn His Leu 85 90 95gat gag atg atg
cct gtt att tat acc cca acc gtc ggc gca gcc tgt 336Asp Glu Met Met
Pro Val Ile Tyr Thr Pro Thr Val Gly Ala Ala Cys 100 105 110gag cgt
ttt tct gag atc tac cgc cgt tca cgc ggc gtg ttt atc tct 384Glu Arg
Phe Ser Glu Ile Tyr Arg Arg Ser Arg Gly Val Phe Ile Ser 115 120
125tac cag aac cgg cac aat atg gac gat att ctg caa aac gtg ccg aac
432Tyr Gln Asn Arg His Asn Met Asp Asp Ile Leu Gln Asn Val Pro Asn
130 135 140cat aat att aaa gtg att gtg gtg act gac ggt gaa cgc att
ctg ggg 480His Asn Ile Lys Val Ile Val Val Thr Asp Gly Glu Arg Ile
Leu Gly145 150 155 160ctt ggt gac cag ggc atc ggc ggg atg ggc att
ccg atc ggt aaa ctg 528Leu Gly Asp Gln Gly Ile Gly Gly Met Gly Ile
Pro Ile Gly Lys Leu 165 170 175tcg ctc tat acc gcc tgt ggc ggc atc
agc ccg gcg tat acc ctt ccg 576Ser Leu Tyr Thr Ala Cys Gly Gly Ile
Ser Pro Ala Tyr Thr Leu Pro 180 185 190gtg gtg ctg gat gtc gga acg
aac aac caa cag ctg ctt aac gat ccg 624Val Val Leu Asp Val Gly Thr
Asn Asn Gln Gln Leu Leu Asn Asp Pro 195 200 205ctg tat atg ggc tgg
cgt aat ccg cgt atc act gac gac gaa tac tat 672Leu Tyr Met Gly Trp
Arg Asn Pro Arg Ile Thr Asp Asp Glu Tyr Tyr 210 215 220gaa ttc gtt
gat gaa ttt atc cag gct gtg aaa caa cgc tgg cca gac 720Glu Phe Val
Asp Glu Phe Ile Gln Ala Val Lys Gln Arg Trp Pro Asp225 230 235
240gtg ctg ttg cag ttt gaa gac ttt gct caa aaa aat gcg atg ccg tta
768Val Leu Leu Gln Phe Glu Asp Phe Ala Gln Lys Asn Ala Met Pro Leu
245 250 255ctt aac cgc tat cgc aat gaa att tgt tct ttt aac gat gac
att cag 816Leu Asn Arg Tyr Arg Asn Glu Ile Cys Ser Phe Asn Asp Asp
Ile Gln 260 265 270ggc act gcg gcg gta aca gtc ggc aca ctg atc gca
gca agc cgc gcg 864Gly Thr Ala Ala Val Thr Val Gly Thr Leu Ile Ala
Ala Ser Arg Ala 275 280 285gca ggt ggt cag tta agc gag aaa aaa atc
gtc ttc ctt ggc gca ggt 912Ala Gly Gly Gln Leu Ser Glu Lys Lys Ile
Val Phe Leu Gly Ala Gly 290 295 300tca gcg gga tgc ggc att gcc gaa
atg atc atc tcc cag acc cag cgc 960Ser Ala Gly Cys Gly Ile Ala Glu
Met Ile Ile Ser Gln Thr Gln Arg305 310 315 320gaa gga tta agc gag
gaa gcg gcg cgg cag aaa gtc ttt atg gtc gat 1008Glu Gly Leu Ser Glu
Glu Ala Ala Arg Gln Lys Val Phe Met Val Asp 325 330 335cgc ttt ggc
ttg ctg act gac aag atg ccg aac ctg ctg cct ttc cag 1056Arg Phe Gly
Leu Leu Thr Asp Lys Met Pro Asn Leu Leu Pro Phe Gln 340 345 350acc
aaa ctg gtg cag aag cgc gaa aac ctc agt gac tgg gat acc gac 1104Thr
Lys Leu Val Gln Lys Arg Glu Asn Leu Ser Asp Trp Asp Thr Asp
355 360 365agc gat gtg ctg tca ctg ctg gat gtg gtg cgc aat gta aaa
cca gat 1152Ser Asp Val Leu Ser Leu Leu Asp Val Val Arg Asn Val Lys
Pro Asp 370 375 380att ctg att ggc gtc tca gga cag acc ggg ctg ttt
acg gaa gag atc 1200Ile Leu Ile Gly Val Ser Gly Gln Thr Gly Leu Phe
Thr Glu Glu Ile385 390 395 400atc cgt gag atg cat aaa cac tgt ccg
cgt ccg atc gtg atg ccg ctg 1248Ile Arg Glu Met His Lys His Cys Pro
Arg Pro Ile Val Met Pro Leu 405 410 415tct aac ccg acg tca cgc gtg
gaa gcc aca ccg cag gac att atc gcc 1296Ser Asn Pro Thr Ser Arg Val
Glu Ala Thr Pro Gln Asp Ile Ile Ala 420 425 430tgg acc gaa ggt aac
gcg ctg gtc gcc acg ggc agc ccg ttt aat cca 1344Trp Thr Glu Gly Asn
Ala Leu Val Ala Thr Gly Ser Pro Phe Asn Pro 435 440 445gtg gta tgg
aaa gat aaa atc tac cct atc gcc cag tgt aac aac gcc 1392Val Val Trp
Lys Asp Lys Ile Tyr Pro Ile Ala Gln Cys Asn Asn Ala 450 455 460ttt
att ttc ccg ggc atc ggc ctg ggt gtt att gct tcc ggc gcg tca 1440Phe
Ile Phe Pro Gly Ile Gly Leu Gly Val Ile Ala Ser Gly Ala Ser465 470
475 480cgt atc acc gat gag atg ctg atg tcg gca agt gaa acg ctg gcg
cag 1488Arg Ile Thr Asp Glu Met Leu Met Ser Ala Ser Glu Thr Leu Ala
Gln 485 490 495tat tca cca ttg gtg ctg aac ggc gaa ggt atg gta ctg
ccg gaa ctg 1536Tyr Ser Pro Leu Val Leu Asn Gly Glu Gly Met Val Leu
Pro Glu Leu 500 505 510aaa gat att cag aaa gtc tcc cgc gca att gcg
ttt gcg gtt ggc aaa 1584Lys Asp Ile Gln Lys Val Ser Arg Ala Ile Ala
Phe Ala Val Gly Lys 515 520 525atg gcg cag cag caa ggc gtg gcg gtg
aaa acc tct gcc gaa gcc ctg 1632Met Ala Gln Gln Gln Gly Val Ala Val
Lys Thr Ser Ala Glu Ala Leu 530 535 540caa cag gcc att gac gat aat
ttc tgg caa gcc gaa tac cgc gac tac 1680Gln Gln Ala Ile Asp Asp Asn
Phe Trp Gln Ala Glu Tyr Arg Asp Tyr545 550 555 560cgc cgt acc tcc
atc taa 1698Arg Arg Thr Ser Ile 56553565PRTEscherichia coli 53Met
Glu Pro Lys Thr Lys Lys Gln Arg Ser Leu Tyr Ile Pro Tyr Ala1 5 10
15Gly Pro Val Leu Leu Glu Phe Pro Leu Leu Asn Lys Gly Ser Ala Phe
20 25 30Ser Met Glu Glu Arg Arg Asn Phe Asn Leu Leu Gly Leu Leu Pro
Glu 35 40 45Val Val Glu Thr Ile Glu Glu Gln Ala Glu Arg Ala Trp Ile
Gln Tyr 50 55 60Gln Gly Phe Lys Thr Glu Ile Asp Lys His Ile Tyr Leu
Arg Asn Ile65 70 75 80Gln Asp Thr Asn Glu Thr Leu Phe Tyr Arg Leu
Val Asn Asn His Leu 85 90 95Asp Glu Met Met Pro Val Ile Tyr Thr Pro
Thr Val Gly Ala Ala Cys 100 105 110Glu Arg Phe Ser Glu Ile Tyr Arg
Arg Ser Arg Gly Val Phe Ile Ser 115 120 125Tyr Gln Asn Arg His Asn
Met Asp Asp Ile Leu Gln Asn Val Pro Asn 130 135 140His Asn Ile Lys
Val Ile Val Val Thr Asp Gly Glu Arg Ile Leu Gly145 150 155 160Leu
Gly Asp Gln Gly Ile Gly Gly Met Gly Ile Pro Ile Gly Lys Leu 165 170
175Ser Leu Tyr Thr Ala Cys Gly Gly Ile Ser Pro Ala Tyr Thr Leu Pro
180 185 190Val Val Leu Asp Val Gly Thr Asn Asn Gln Gln Leu Leu Asn
Asp Pro 195 200 205Leu Tyr Met Gly Trp Arg Asn Pro Arg Ile Thr Asp
Asp Glu Tyr Tyr 210 215 220Glu Phe Val Asp Glu Phe Ile Gln Ala Val
Lys Gln Arg Trp Pro Asp225 230 235 240Val Leu Leu Gln Phe Glu Asp
Phe Ala Gln Lys Asn Ala Met Pro Leu 245 250 255Leu Asn Arg Tyr Arg
Asn Glu Ile Cys Ser Phe Asn Asp Asp Ile Gln 260 265 270Gly Thr Ala
Ala Val Thr Val Gly Thr Leu Ile Ala Ala Ser Arg Ala 275 280 285Ala
Gly Gly Gln Leu Ser Glu Lys Lys Ile Val Phe Leu Gly Ala Gly 290 295
300Ser Ala Gly Cys Gly Ile Ala Glu Met Ile Ile Ser Gln Thr Gln
Arg305 310 315 320Glu Gly Leu Ser Glu Glu Ala Ala Arg Gln Lys Val
Phe Met Val Asp 325 330 335Arg Phe Gly Leu Leu Thr Asp Lys Met Pro
Asn Leu Leu Pro Phe Gln 340 345 350Thr Lys Leu Val Gln Lys Arg Glu
Asn Leu Ser Asp Trp Asp Thr Asp 355 360 365Ser Asp Val Leu Ser Leu
Leu Asp Val Val Arg Asn Val Lys Pro Asp 370 375 380Ile Leu Ile Gly
Val Ser Gly Gln Thr Gly Leu Phe Thr Glu Glu Ile385 390 395 400Ile
Arg Glu Met His Lys His Cys Pro Arg Pro Ile Val Met Pro Leu 405 410
415Ser Asn Pro Thr Ser Arg Val Glu Ala Thr Pro Gln Asp Ile Ile Ala
420 425 430Trp Thr Glu Gly Asn Ala Leu Val Ala Thr Gly Ser Pro Phe
Asn Pro 435 440 445Val Val Trp Lys Asp Lys Ile Tyr Pro Ile Ala Gln
Cys Asn Asn Ala 450 455 460Phe Ile Phe Pro Gly Ile Gly Leu Gly Val
Ile Ala Ser Gly Ala Ser465 470 475 480Arg Ile Thr Asp Glu Met Leu
Met Ser Ala Ser Glu Thr Leu Ala Gln 485 490 495Tyr Ser Pro Leu Val
Leu Asn Gly Glu Gly Met Val Leu Pro Glu Leu 500 505 510Lys Asp Ile
Gln Lys Val Ser Arg Ala Ile Ala Phe Ala Val Gly Lys 515 520 525Met
Ala Gln Gln Gln Gly Val Ala Val Lys Thr Ser Ala Glu Ala Leu 530 535
540Gln Gln Ala Ile Asp Asp Asn Phe Trp Gln Ala Glu Tyr Arg Asp
Tyr545 550 555 560Arg Arg Thr Ser Ile 565542280DNAEscherichia
coliCDS(1)..(2280) 54atg gat gac cag tta aaa caa agt gca ctt gat
ttc cat gaa ttt cca 48Met Asp Asp Gln Leu Lys Gln Ser Ala Leu Asp
Phe His Glu Phe Pro1 5 10 15gtt cca ggg aaa atc cag gtt tct cca acc
aag cct ctg gca aca cag 96Val Pro Gly Lys Ile Gln Val Ser Pro Thr
Lys Pro Leu Ala Thr Gln 20 25 30cgc gat ctg gcg ctg gcc tac tca cca
ggc gtt gcc gca cct tgt ctt 144Arg Asp Leu Ala Leu Ala Tyr Ser Pro
Gly Val Ala Ala Pro Cys Leu 35 40 45gaa atc gaa aaa gac ccg tta aaa
gcc tac aaa tat acc gcc cga ggt 192Glu Ile Glu Lys Asp Pro Leu Lys
Ala Tyr Lys Tyr Thr Ala Arg Gly 50 55 60aac ctg gtg gcg gtg atc tct
aac ggt acg gcg gtg ctg ggg tta ggc 240Asn Leu Val Ala Val Ile Ser
Asn Gly Thr Ala Val Leu Gly Leu Gly65 70 75 80aac att ggc gcg ctg
gca ggc aaa ccg gtg atg gaa ggc aag ggc gtt 288Asn Ile Gly Ala Leu
Ala Gly Lys Pro Val Met Glu Gly Lys Gly Val 85 90 95ctg ttt aag aaa
ttc gcc ggg att gat gta ttt gac att gaa gtt gac 336Leu Phe Lys Lys
Phe Ala Gly Ile Asp Val Phe Asp Ile Glu Val Asp 100 105 110gaa ctc
gac ccg gac aaa ttt att gaa gtt gtc gcc gcg ctc gaa cca 384Glu Leu
Asp Pro Asp Lys Phe Ile Glu Val Val Ala Ala Leu Glu Pro 115 120
125acc ttc ggc ggc atc aac ctc gaa gac att aaa gcg cca gaa tgt ttc
432Thr Phe Gly Gly Ile Asn Leu Glu Asp Ile Lys Ala Pro Glu Cys Phe
130 135 140tat att gaa cag aaa ctg cgc gag cgg atg aat att ccg gta
ttc cac 480Tyr Ile Glu Gln Lys Leu Arg Glu Arg Met Asn Ile Pro Val
Phe His145 150 155 160gac gat cag cac ggc acg gca att atc agc act
gcc gcc atc ctc aac 528Asp Asp Gln His Gly Thr Ala Ile Ile Ser Thr
Ala Ala Ile Leu Asn 165 170 175ggc ttg cgc gtg gtg gag aaa aac atc
tcc gac gtg cgg atg gtg gtt 576Gly Leu Arg Val Val Glu Lys Asn Ile
Ser Asp Val Arg Met Val Val 180 185 190tcc ggc gcg ggt gcc gca gca
atc gcc tgt atg aac ctg ctg gta gcg 624Ser Gly Ala Gly Ala Ala Ala
Ile Ala Cys Met Asn Leu Leu Val Ala 195 200 205ctg ggt ctg caa aaa
cat aac atc gtg gtt tgc gat tca aaa ggc gtt 672Leu Gly Leu Gln Lys
His Asn Ile Val Val Cys Asp Ser Lys Gly Val 210 215 220 atc tat cag
ggc cgt gag cca aac atg gcg gaa acc aaa gcc gca tat 720Ile Tyr Gln
Gly Arg Glu Pro Asn Met Ala Glu Thr Lys Ala Ala Tyr225 230 235
240gcg gtg gtg gat gac ggc aaa cgt acc ctc gat gat gtg att gaa ggc
768Ala Val Val Asp Asp Gly Lys Arg Thr Leu Asp Asp Val Ile Glu Gly
245 250 255gcg gat att ttc ctg ggc tgt tcc ggc ccg aaa gtg ctg acc
cag gaa 816Ala Asp Ile Phe Leu Gly Cys Ser Gly Pro Lys Val Leu Thr
Gln Glu 260 265 270atg gtg aag aaa atg gct cgt gcg cca atg atc ctg
gcg ctg gcg aac 864Met Val Lys Lys Met Ala Arg Ala Pro Met Ile Leu
Ala Leu Ala Asn 275 280 285ccg gaa ccg gaa att ctg ccg ccg ctg gcg
aaa gaa gtg cgt ccg gat 912Pro Glu Pro Glu Ile Leu Pro Pro Leu Ala
Lys Glu Val Arg Pro Asp 290 295 300gcc atc att tgc acc ggt cgt tct
gac tat ccg aac cag gtg aac aac 960Ala Ile Ile Cys Thr Gly Arg Ser
Asp Tyr Pro Asn Gln Val Asn Asn305 310 315 320gtc ctg tgc ttc ccg
ttc atc ttc cgt ggc gcg ctg gac gtt ggc gca 1008Val Leu Cys Phe Pro
Phe Ile Phe Arg Gly Ala Leu Asp Val Gly Ala 325 330 335acc gcc atc
aac gaa gag atg aaa ctg gcg gcg gta cgt gcg att gca 1056Thr Ala Ile
Asn Glu Glu Met Lys Leu Ala Ala Val Arg Ala Ile Ala 340 345 350gaa
ctc gcc cat gcg gaa cag agc gaa gtg gtg gct tca gcg tat ggc 1104Glu
Leu Ala His Ala Glu Gln Ser Glu Val Val Ala Ser Ala Tyr Gly 355 360
365gat cag gat ctg agc ttt ggt ccg gaa tac atc att cca aaa ccg ttt
1152Asp Gln Asp Leu Ser Phe Gly Pro Glu Tyr Ile Ile Pro Lys Pro Phe
370 375 380gat ccg cgc ttg atc gtt aag atc gct cct gcg gtc gct aaa
gcc gcg 1200Asp Pro Arg Leu Ile Val Lys Ile Ala Pro Ala Val Ala Lys
Ala Ala385 390 395 400atg gag tcg ggc gtg gcg act cgt ccg att gct
gat ttc gac gtc tac 1248Met Glu Ser Gly Val Ala Thr Arg Pro Ile Ala
Asp Phe Asp Val Tyr 405 410 415atc gac aag ctg act gag ttc gtt tac
aaa acc aac ctg ttt atg aag 1296Ile Asp Lys Leu Thr Glu Phe Val Tyr
Lys Thr Asn Leu Phe Met Lys 420 425 430ccg att ttc tcc cag gct cgc
aaa gcg ccg aag cgc gtt gtt ctg ccg 1344Pro Ile Phe Ser Gln Ala Arg
Lys Ala Pro Lys Arg Val Val Leu Pro 435 440 445gaa ggg gaa gag gcg
cgc gtt ctg cat gcc act cag gaa ctg gta acg 1392Glu Gly Glu Glu Ala
Arg Val Leu His Ala Thr Gln Glu Leu Val Thr 450 455 460ctg gga ctg
gcg aaa ccg atc ctt atc ggt cgt ccg aac gtg atc gaa 1440Leu Gly Leu
Ala Lys Pro Ile Leu Ile Gly Arg Pro Asn Val Ile Glu465 470 475
480atg cgc att cag aaa ctg ggc ttg cag atc aaa gcg ggc gtt gat ttt
1488Met Arg Ile Gln Lys Leu Gly Leu Gln Ile Lys Ala Gly Val Asp Phe
485 490 495gag atc gtc aat aac gaa tcc gat ccg cgc ttt aaa gag tac
tgg acc 1536Glu Ile Val Asn Asn Glu Ser Asp Pro Arg Phe Lys Glu Tyr
Trp Thr 500 505 510gaa tac ttc cag atc atg aag cgt cgc ggc gtc act
cag gaa cag gcg 1584Glu Tyr Phe Gln Ile Met Lys Arg Arg Gly Val Thr
Gln Glu Gln Ala 515 520 525cag cgg gcg ctg atc agt aac ccg aca gtg
atc ggc gcg atc atg gtt 1632Gln Arg Ala Leu Ile Ser Asn Pro Thr Val
Ile Gly Ala Ile Met Val 530 535 540cag cgt ggg gaa gcc gat gca atg
att tgc ggt acg gtg ggt gat tat 1680Gln Arg Gly Glu Ala Asp Ala Met
Ile Cys Gly Thr Val Gly Asp Tyr545 550 555 560cat gaa cat ttt agc
gtg gtg aaa aat gtc ttt ggt tat cgc gat ggc 1728His Glu His Phe Ser
Val Val Lys Asn Val Phe Gly Tyr Arg Asp Gly 565 570 575gtt cac acc
gca ggt gcc atg aac gcg ctg ctg ctg ccg agt ggt aac 1776Val His Thr
Ala Gly Ala Met Asn Ala Leu Leu Leu Pro Ser Gly Asn 580 585 590acc
ttt att gcc gat aca tat gtt aat gat gaa ccg gat gca gaa gag 1824Thr
Phe Ile Ala Asp Thr Tyr Val Asn Asp Glu Pro Asp Ala Glu Glu 595 600
605ctg gcg gag atc acc ttg atg gcg gca gaa act gtc cgt cgt ttt ggt
1872Leu Ala Glu Ile Thr Leu Met Ala Ala Glu Thr Val Arg Arg Phe Gly
610 615 620att gag ccg cgc gtt gct ttg ttg tcg cac tcc aac ttt ggt
tct tct 1920Ile Glu Pro Arg Val Ala Leu Leu Ser His Ser Asn Phe Gly
Ser Ser625 630 635 640gac tgc ccg tcg tcg agc aaa atg cgt cag gcg
ctg gaa ctg gtc agg 1968Asp Cys Pro Ser Ser Ser Lys Met Arg Gln Ala
Leu Glu Leu Val Arg 645 650 655gaa cgt gca cca gaa ctg atg att gat
ggt gaa atg cac ggc gat gca 2016Glu Arg Ala Pro Glu Leu Met Ile Asp
Gly Glu Met His Gly Asp Ala 660 665 670gcg ctg gtg gaa gcg att cgc
aac gac cgt atg ccg gac agc tct ttg 2064Ala Leu Val Glu Ala Ile Arg
Asn Asp Arg Met Pro Asp Ser Ser Leu 675 680 685aaa ggt tcc gcc aat
att ctg gtg atg ccg aac atg gaa gct gcc cgc 2112Lys Gly Ser Ala Asn
Ile Leu Val Met Pro Asn Met Glu Ala Ala Arg 690 695 700att agt tac
aac tta ctg cgt gtt tcc agc tcg gaa ggt gtg act gtc 2160Ile Ser Tyr
Asn Leu Leu Arg Val Ser Ser Ser Glu Gly Val Thr Val705 710 715
720ggc ccg gtg ctg atg ggt gtg gcg aaa ccg gtt cac gtg tta acg ccg
2208Gly Pro Val Leu Met Gly Val Ala Lys Pro Val His Val Leu Thr Pro
725 730 735atc gca tcg gtg cgt cgt atc gtc aac atg gtg gcg ctg gcc
gtg gta 2256Ile Ala Ser Val Arg Arg Ile Val Asn Met Val Ala Leu Ala
Val Val 740 745 750gaa gcg caa acc caa ccg ctg taa 2280Glu Ala Gln
Thr Gln Pro Leu 75555759PRTEscherichia coli 55Met Asp Asp Gln Leu
Lys Gln Ser Ala Leu Asp Phe His Glu Phe Pro1 5 10 15Val Pro Gly Lys
Ile Gln Val Ser Pro Thr Lys Pro Leu Ala Thr Gln 20 25 30Arg Asp Leu
Ala Leu Ala Tyr Ser Pro Gly Val Ala Ala Pro Cys Leu 35 40 45Glu Ile
Glu Lys Asp Pro Leu Lys Ala Tyr Lys Tyr Thr Ala Arg Gly 50 55 60Asn
Leu Val Ala Val Ile Ser Asn Gly Thr Ala Val Leu Gly Leu Gly65 70 75
80Asn Ile Gly Ala Leu Ala Gly Lys Pro Val Met Glu Gly Lys Gly Val
85 90 95Leu Phe Lys Lys Phe Ala Gly Ile Asp Val Phe Asp Ile Glu Val
Asp 100 105 110Glu Leu Asp Pro Asp Lys Phe Ile Glu Val Val Ala Ala
Leu Glu Pro 115 120 125Thr Phe Gly Gly Ile Asn Leu Glu Asp Ile Lys
Ala Pro Glu Cys Phe 130 135 140Tyr Ile Glu Gln Lys Leu Arg Glu Arg
Met Asn Ile Pro Val Phe His145 150 155 160Asp Asp Gln His Gly Thr
Ala Ile Ile Ser Thr Ala Ala Ile Leu Asn 165 170 175Gly Leu Arg Val
Val Glu Lys Asn Ile Ser Asp Val Arg Met Val Val 180 185 190Ser Gly
Ala Gly Ala Ala Ala Ile Ala Cys Met Asn Leu Leu Val Ala 195 200
205Leu Gly Leu Gln Lys His Asn Ile Val Val Cys Asp Ser Lys Gly Val
210 215 220Ile Tyr Gln Gly Arg Glu Pro Asn Met Ala Glu Thr Lys Ala
Ala Tyr225 230 235 240Ala Val Val Asp Asp Gly Lys Arg Thr Leu Asp
Asp Val Ile Glu Gly 245 250 255Ala Asp Ile Phe Leu Gly Cys Ser Gly
Pro Lys Val Leu Thr Gln Glu 260 265 270Met Val Lys Lys Met Ala Arg
Ala Pro Met Ile Leu Ala Leu Ala Asn 275 280 285Pro Glu Pro Glu Ile
Leu Pro Pro Leu Ala Lys Glu Val Arg Pro Asp 290 295 300Ala Ile Ile
Cys Thr Gly Arg Ser Asp Tyr Pro Asn Gln Val Asn Asn305 310 315
320Val Leu Cys Phe Pro Phe Ile Phe Arg Gly Ala Leu Asp Val Gly Ala
325 330 335Thr Ala Ile Asn Glu Glu Met Lys Leu Ala Ala Val Arg Ala
Ile Ala
340 345 350Glu Leu Ala His Ala Glu Gln Ser Glu Val Val Ala Ser Ala
Tyr Gly 355 360 365Asp Gln Asp Leu Ser Phe Gly Pro Glu Tyr Ile Ile
Pro Lys Pro Phe 370 375 380Asp Pro Arg Leu Ile Val Lys Ile Ala Pro
Ala Val Ala Lys Ala Ala385 390 395 400Met Glu Ser Gly Val Ala Thr
Arg Pro Ile Ala Asp Phe Asp Val Tyr 405 410 415Ile Asp Lys Leu Thr
Glu Phe Val Tyr Lys Thr Asn Leu Phe Met Lys 420 425 430Pro Ile Phe
Ser Gln Ala Arg Lys Ala Pro Lys Arg Val Val Leu Pro 435 440 445Glu
Gly Glu Glu Ala Arg Val Leu His Ala Thr Gln Glu Leu Val Thr 450 455
460Leu Gly Leu Ala Lys Pro Ile Leu Ile Gly Arg Pro Asn Val Ile
Glu465 470 475 480Met Arg Ile Gln Lys Leu Gly Leu Gln Ile Lys Ala
Gly Val Asp Phe 485 490 495Glu Ile Val Asn Asn Glu Ser Asp Pro Arg
Phe Lys Glu Tyr Trp Thr 500 505 510Glu Tyr Phe Gln Ile Met Lys Arg
Arg Gly Val Thr Gln Glu Gln Ala 515 520 525Gln Arg Ala Leu Ile Ser
Asn Pro Thr Val Ile Gly Ala Ile Met Val 530 535 540Gln Arg Gly Glu
Ala Asp Ala Met Ile Cys Gly Thr Val Gly Asp Tyr545 550 555 560His
Glu His Phe Ser Val Val Lys Asn Val Phe Gly Tyr Arg Asp Gly 565 570
575Val His Thr Ala Gly Ala Met Asn Ala Leu Leu Leu Pro Ser Gly Asn
580 585 590Thr Phe Ile Ala Asp Thr Tyr Val Asn Asp Glu Pro Asp Ala
Glu Glu 595 600 605Leu Ala Glu Ile Thr Leu Met Ala Ala Glu Thr Val
Arg Arg Phe Gly 610 615 620Ile Glu Pro Arg Val Ala Leu Leu Ser His
Ser Asn Phe Gly Ser Ser625 630 635 640Asp Cys Pro Ser Ser Ser Lys
Met Arg Gln Ala Leu Glu Leu Val Arg 645 650 655Glu Arg Ala Pro Glu
Leu Met Ile Asp Gly Glu Met His Gly Asp Ala 660 665 670Ala Leu Val
Glu Ala Ile Arg Asn Asp Arg Met Pro Asp Ser Ser Leu 675 680 685Lys
Gly Ser Ala Asn Ile Leu Val Met Pro Asn Met Glu Ala Ala Arg 690 695
700Ile Ser Tyr Asn Leu Leu Arg Val Ser Ser Ser Glu Gly Val Thr
Val705 710 715 720Gly Pro Val Leu Met Gly Val Ala Lys Pro Val His
Val Leu Thr Pro 725 730 735Ile Ala Ser Val Arg Arg Ile Val Asn Met
Val Ala Leu Ala Val Val 740 745 750Glu Ala Gln Thr Gln Pro Leu
755
* * * * *