Patent application title: AMINO ACID PRODUCING MICROORGANISM AND A METHOD FOR PRODUCING AN AMINO ACID
Inventors:
Masaru Terashita (Kawasaki-Shi, JP)
Yoshihiro Usuda (Kawasaki-Shi, JP)
Kazuhiko Matsui (Kawasaki-Shi, JP)
IPC8 Class: AC12P1322FI
USPC Class:
435108
Class name: Micro-organism, tissue cell culture or enzyme using process to synthesize a desired chemical compound or composition preparing alpha or beta amino acid or substituted amino acid or salts thereof tryptophan; tyrosine; phenylalanine; 3,4 dihydroxyphenylalanine
Publication date: 2010-04-15
Patent application number: 20100093044
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Patent application title: AMINO ACID PRODUCING MICROORGANISM AND A METHOD FOR PRODUCING AN AMINO ACID
Inventors:
Masaru Terashita
Yoshihiro Usuda
Kazuhiko Matsui
Agents:
CERMAK KENEALY VAIDYA & NAKAJIMA LLP;ACS LLC
Assignees:
Origin: ALEXANDRIA, VA US
IPC8 Class: AC12P1322FI
USPC Class:
435108
Patent application number: 20100093044
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+ 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.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, andB)
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, andiii)
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+ reductase by a method selected from the group consisting of:a) increasing the copy number of a gene encoding ferrodoxin-NADP+ reductase,b) modifying an expression control sequence of the gene, andc) 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, andc) 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, andc) 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, andB) 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, andiii) 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, andB) 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, andiii) 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.
§120 of U.S. patent application Ser. No. 12/202,476, filed on Sep.
2, 2008, which claims priority under 35 U.S.C. §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+-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 β-(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 CO2 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+ oxidoreductase. Furthermore, L-amino acid production can be improved by increasing the enzymatic activity of ferredoxin-NADP+ 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+ 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+ 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+ 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+ 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+ oxidoreductase is increased by increasing the copy number of the gene encoding pyruvate synthase or pyruvate:NADP+ 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+ 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+ oxidoreductase activity.
[0033]It is a further aspect of the present invention to provide the aforementioned microorganism, wherein the gene encoding pyruvate:NADP+ 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+ 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+ 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+ oxidoreductase (PNO) gene derived from Euglena gracilis.
[0045]Lane 1: Markers
[0046]Lane 2: Crude enzyme extract obtained from WC196ΔcadAΔldc/pCABD2/pMW-Pthr
[0047]Lane 3: Crude enzyme extract obtained from WC196ΔcadAΔldc/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+ 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+ oxidoreductase is increased" or "to increase the activity of pyruvate synthase or pyruvate:NADP+ oxidoreductase" means that the activity of pyruvate synthase or pyruvate:NADP+ oxidoreductase increases in a microorganism which inherently has pyruvate synthase and/or pyruvate:NADP+ oxidoreductase, or that the activity of pyruvate synthase or pyruvate:NADP+ oxidoreductase is imparted to a microorganism to which pyruvate synthase and pyruvate:NADP+ 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+ 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+ 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 γ-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. γ-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 λ 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 γ-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+=acetaldehyde+NAD++CoA
Acetaldehyde+NADH+H++=ethanol+NAD+
[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), γ-methyllysine, α-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 WC196Δmez/pCABD2 (WO2005/010175), WC196ΔcadAΔldc/pCABD2 (WO2006/078039), and so forth. The WC196Δmez/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 WC196ΔcadAΔldc/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 α and β 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+-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 β-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+ Oxidoreductase Activity
[0140]The microorganism having an L-amino acid-producing ability is modified so that an activity of pyruvate synthase or pyruvate:NADP+ oxidoreductase is increased. The activity of the pyruvate synthase or pyruvate:NADP+ 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+ 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+ oxidoreductase, and then imparted with an L-amino acid-producing ability. In addition, the activity of pyruvate synthase or pyruvate:NADP+ 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+ 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+ oxidoreductase gene by introducing a plasmid containing the pyruvate synthase or pyruvate:NADP+ 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 CO2 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+CO2=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 μmol 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--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 δ-subunit (Genbank Accession No. BAA95604), α-subunit (Genbank Accession No. BAA95605), β-subunit (Genbank Accession No. BAA95606), and γ-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+ oxidoreductase catalyzes the following reaction, which generates pyruvic acid from acetyl CoA and CO2, in the presence of an electron donor such as NADPH or NADH (EC 1.2.1.15). Pyruvate:NADP+ 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+ oxidoreductase. Pyruvate:NADP+ oxidoreductase can use NADPH or NADH as the electron donor.
NADPH+acetyl-CoA+CO2=NADP++pyruvic acid+CoA
[0148]Enhancement of the pyruvate:NADP+ oxidoreductase activity can be confirmed by preparing crude enzyme solutions and measuring the pyruvate:NADP+ oxidoreductase activity in both the microorganism before making the modification to enhance activity, and after making the modification. The activity of pyruvate:NADP+ 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 μmol of methylviologen per 1 minute. When the parent strain has pyruvate:NADP+ 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+ oxidoreductase activity, although it is sufficient that pyruvate:NADP+ oxidoreductase is produced by the introduction of the pyruvate:NADP+ 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+ 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+ 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+ 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+ 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+ reductase is an enzyme that reversibly catalyzes the following reaction (EC 1.18.1.2):
Reduced ferredoxin+NADP+=Oxidized ferredoxin+NADPH+H+
[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+ reductase. Ferredoxin NADP+ 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+ oxidoreductase or NADPH-ferredoxin oxidoreductase.
[0154]Enhancement of the ferredoxin NADP+ reductase activity can be confirmed by preparing crude enzyme solutions and measuring the ferredoxin NADP+ reductase activity in both the microorganism before making the modification to enhance activity, and after making the modification. The activity of ferredoxin NADP+ 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 μmol of NADPH per 1 minute. When the parent strain has ferredoxin NADP+ 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+ 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+ 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+ 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+ 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+ 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+ 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--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 γ-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+ 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+ 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° C., 1×SSC, 0.1% SDS, preferably 60° C., 0.1×SSC, 0.1% SDS, more preferably 68° C., 0.1×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° C., 2×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+ 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+ 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 λ 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 λ 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+ 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+CO2 E1p
CoA+enzyme N6-(S-acetyldihytholipoyblysine=acetyl-CoA+enzyme N6-(dihydrolipoyl)lysine E2p
Protein N6-(dihydrolipoyl)lysine+NAD+=protein N6-(lipoyl)lysine+NADH+H+ 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 λ 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+ 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 C2H5OH, 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 CmHnCOOH. 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 (C17H33COOH) and palmitic acid (C15H31COOH), 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 B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, vitamin B12, 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° C., particularly preferably at 33 to 42° 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::PL-tac adhE Strain
[0236]Substitution of the PL-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 λ 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 λ 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 PL-tac promoter and the cat gene encoding the chloramphenicol resistance (CmR) gene was amplified by PCR using the genome of the Escherichia coli MG1655 PL-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 PL-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 CmR gene in the promoter region of the adhE gene, and this clone was cultured at 37° C. to eliminate the temperature-sensitive plasmid pKD46 and thereby obtain an MG1655::PL-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 (KanR) 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 KmR 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 μg/ml of kanamycin. One of clones confirmed to contain the KmR gene in the adhE gene region was cultured at 37° C. to remove the temperature-sensitive plasmid pKD46 and thereby obtain an MG1655ΔadhE 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::PL-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 CmR gene and the mutant adhE gene downstream of the PL-tac promoter (cat-PL-tacadhE*) was introduced into the MG1655ΔadhE/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::PL-tacadhE*.
[0248]The MG1655Δtdh rhtA* strain was transformed via P1 transduction with P1vir phage (Miller, J. H., 1972, Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, N.Y.) using the Escherichia coli MG1655::PL-tacadhE* strain as a donor, and MG1655Δtdh rhtA* PL-tacadhE* was obtained. The MG1655Δtdh, 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 WC196Δmez Strain
[0250]In order to impart ethanol assimilability to an L-lysine-producing bacterium, the L-lysine-producing bacterium WC196Δmez/pCABD2 strain described in International Patent Publication WO2005/010175 was subjected to P1 transduction using MG1655Δtdh rhtA adhE* as a donor to obtain a WC196Δmez 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° 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 WC196ΔcadAΔldc/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 WC196ΔcadAΔldc/pCABD2/pMW-Pthr-PS, and the control strain was designated WC196ΔcadAΔldc/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° 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 μg 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° 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 MgCl2 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+ Reductase Gene of Escherichia coli, and Ferredoxin Gene of Escherichia coli
[0260]By using the flavodoxin NADP+ 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+ 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+ 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+ 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+ 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+ 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+ Reductase Gene of Escherichia coli, and Ferredoxin Gene of Escherichia coli into the WC196Δmez Strain
[0268]pMW-Pthr-Thr-PS-fdx and the control vector pMW-Pthr were introduced into WC196Δmez/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+ reductase gene of Escherichia coli and the ferredoxin gene of Escherichia coli was designated WC196Δmez/pCABD2/pMW-Pthr-fpr-PS-fdx, and the control strain was designated WC196Δmez/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+ Reductase Gene of Escherichia coli, and Ferredoxin Gene of Escherichia coli Using Oleic Acid as the Carbon Source
[0270]Both WC196Δmez/pCABD2/pMW-Pthr and WC196Δmez/pCABD2/pMW-Pthr-Thr-PS-fdx were inoculated onto the LB plate medium, respectively, and precultured overnight at 37° 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° 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+ 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 MgSO4•7H2O 1.0 g/L (NH4)2SO4 12 g/L KH2PO4 0.5 g/L Yeast extract 1.0 g/L FeSO4•7H2O 0.01 g/L MnSO4•5H2O 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° C., 10 minutes
TABLE-US-00004 TABLE 2 L-lysine Live cell Strain (g/l) count (108/ml) WC196Δmez/pCABD2/pMW-Pthr 1.77 22.3 WC196Δmez/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 WC196ΔcadAΔldc/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 WC196ΔcadAΔldc/pCABD2/pMW-Pthr-ydbK, and the control strain was designated WC196ΔcadAΔldc/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° 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 WC196ΔcadAΔldc/pCABD2/pMW-Pthr, 8.0 U/mg was confirmed for the strain expressing the pyruvate synthase gene of Escherichia coli, WC196ΔcadAΔldc/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+ Reductase Gene of Escherichia coli, and Ferredoxin Gene of Escherichia coli
[0278]The plasmid pMW-Pthr-fpr containing the flavodoxin NADP+ 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+ 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+ 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+ Reductase Gene of Escherichia coli and Ferredoxin Gene of Escherichia coli into WC196Δmez adhE* Strain
[0280]pMW-Pthr-fpr-ydbK-fdx and the control vector pMW-Pthr were introduced into WC196Δmez 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 WC196Δmez adhE*/pCABD2/pMW-Pthr-fpr-ydbK-fdx, and the control strain was designated WC196Δmez 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+ Reductase Gene of Escherichia coli and Ferredoxin Gene of Escherichia coli Using Ethanol as the Carbon Source>
[0282]Both WC196Δmez adhE*/pCABD2/pMW-Pthr and WC196Δmez adhE*/pCABD2/pMW-Pthr-fpr-ydbK-fdx were inoculated onto LB plate medium, respectively, and cultured overnight at 37° 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° 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+ 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 MgSO4•7H2O 1.0 g/L (NH4)2SO4 12 g/L KH2PO4 0.5 g/L Yeast extract 1.0 g/L FeSO4•7H2O 0.01 g/L MnSO4•5H2O 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° C., 10 minutes
TABLE-US-00007 TABLE 4 Lys EtOH Strain (g/l) (V/V %) OD620 WC196Δmez adhE*/pCABD2/pMW-Pthr 2.47 0.00 14.7 WC196Δmez adhE*/pCABD2/pMW-Pthr- 2.89 0.00 9.3 fpr-ydbK-fdx
Example 8
Construction of the Plasmid to Express the Pyruvate:NADP+ Oxidoreductase Gene of Euglena gracilis and Measurement of Activity
[0284]Euglena gracilis is a photosynthetic protist, with an optimum growth temperature of 27° C. The pyruvate:NADP+ 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+ 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+ oxidoreductase gene contained no PCR error over the full length by using BigDye Terminators v1.1 Cycle Sequencing Kit, the pyruvate:NADP+ 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+ oxidoreductase pMW-Pthr-PNO and the control vector pMW-Pthr were introduced into the WC196ΔcadAΔldc/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+ oxidoreductase gene of Euglena gracilis was designated WC196ΔcadAΔldc/pCABD2/pMW-Pthr-PNO, and the control strain was designated WC196ΔcadAΔldc/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° 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° 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 μg 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×, then adding NuPAGE Sample Reducing Agent (Invitrogen) to a final concentration of 10%, and heating the mixture at 70° 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 WC196ΔcadAΔldc/pCABD2/pMW-Pthr-PNO strain, whereas a band was not detected for the control strain WC196ΔcadAΔldc/pCABD2/pMW-Pthr.
Example 9
Construction of the Plasmid to Express the Pyruvate:NADP+ Oxidoreductase Gene of Euglena gracilis
[0289]The pyruvate:NADP+ 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+ Oxidoreductase Gene of Euglena gracilis Using Oleic Acid as the Carbon Source
[0290]<10-1> Introduction of the Plasmid for Amplification of Pyruvate:NADP+ Oxidoreductase Gene of Euglena gracilis into the WC196Δmez Strain
[0291]pMW-Pthr-PNO and the control vector pMW-Pthr were introduced into WC196Δmez/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+ oxidoreductase gene of Euglena gracilis was designated WC196Δmez/pCABD2/pMW-Pthr-PNO, and the control strain was designated WC196Δmez/pCABD2/pMW-Pthr.
[0292]<10-2> Effect on L-Lysine-Producing Ability of the Strain with Enhanced Expression of the Pyruvate:NADP+ Oxidoreductase Gene of Euglena gracilis Using Oleic Acid as the Carbon Source
[0293]Both WC196Δmez/pCABD2/pMW-Pthr and WC196Δmez/pCABD2/pMW-Pthr-PNO were inoculated onto the LB plate medium, respectively, and cultured overnight at 37° 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° 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+ 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 MgSO4•7H2O 1.0 g/L (NH4)2SO4 12 g/L KH2PO4 0.5 g/L Yeast extract 1.0 g/L FeSO4•7H2O 0.01 g/L MnSO4•5H2O 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° C., 10 minutes
TABLE-US-00009 TABLE 5 Lys Live cell Strain (g/l) count (108/ml) WC196Δmez/pCABD2/pMW-Pthr 1.77 22.3 WC196Δmez/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+ Oxidoreductase Gene of Euglena gracilis Using Ethanol as the Carbon Source
[0295]<11-1> Introduction of the Plasmid for Amplification of Pyruvate:NADP+ Oxidoreductase Gene of Euglena gracilis into WC196Δmez adhE*
[0296]pMW-Pthr-PNO and the control vector pMW-Pthr were introduced into WC196Δmez 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+ oxidoreductase gene of Euglena gracilis was designated WC196Δmez adhE*/pCABD2/pMW-Pthr-PNO, and the control strain was designated WC196Δmez adhE*/pCABD2/pMW-Pthr.
[0297]<11-2> Effect on L-Lysine-Producing Ability of the Strain with Enhanced Expression of the Pyruvate:NADP+ Oxidoreductase Gene of Euglena gracilis Using Ethanol as the Carbon Source
[0298]Both WC196Δmez adhE*/pCABD2/pMW-Pthr and WC196Δmez adhE*/pCABD2/pMW-Pthr-PNO were each inoculated onto LB plate medium, and precultured overnight at 37° 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° 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+ oxidoreductase gene of Euglena gracilis, compared with the control.
[0299]Composition of ethanol medium:
TABLE-US-00010 Ethanol 20 ml/L MgSO4•7H2O 1.0 g/L (NH4)2SO4 12 g/L KH2PO4 0.5 g/L Yeast extract 1.0 g/L FeSO4•7H2O 0.01 g/L MnSO4•5H2O 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° C., 10 minutes
TABLE-US-00011 TABLE 6 Lys EtOH Strain (g/l) (V/V %) OD620 WC196Δmez adhE*/pCABD2/pMW-Pthr 2.47 0.00 14.7 WC196Δmez 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+ oxidoreductase gene
[0306]SEQ ID NO: 6: Amino acid sequence of E. gracilis pyruvate: NADP+ oxidoreductase gene
[0307]SEQ ID NO: 7: Nucleotide sequence of E. coli flavodoxin NADP+ 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: PL-tac promoter and chloramphenicol resistance (CmR) gene amplification primer 1
[0324]SEQ ID NO: 24: PL-tac promoter and chloramphenicol resistance (CmR) gene amplification primer 2
[0325]SEQ ID NO: 25: Nucleotide sequence of PL-tac promoter
[0326]SEQ ID NO: 26: PL-tac promoter and chloramphenicol resistance (CmR) gene amplification primer 3
[0327]SEQ ID NO: 27: PL-tac promoter and chloramphenicol resistance (CmR) gene amplification primer 4
[0328]SEQ ID NO: 28: Kanamycin resistance (CmR) gene amplification primer 1
[0329]SEQ ID NO: 29: Kanamycin resistance (CmR) gene amplification primer 2
[0330]SEQ ID NO: 30: Kanamycin resistance (CmR) gene amplification primer 3
[0331]SEQ ID NO: 31: Kanamycin resistance (CmR) 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+ oxidoreductase gene amplification primer 1
[0341]SEQ ID NO: 41: E. gracilis pyruvate:NADP+ oxidoreductase gene amplification primer 2
[0342]SEQ ID NO: 42: E. coli flavodoxin NADP+ reductase gene amplification primer 1
[0343]SEQ ID NO: 43: E. coli flavodoxin NADP+ 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
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
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