U.S. patent application number 13/772959 was filed with the patent office on 2013-07-04 for l-amino acid-producing bacterium and method for producing l-amino acid.
This patent application is currently assigned to AJINOMOTO CO., INC.. The applicant listed for this patent is Ajinomoto Co., Inc.. Invention is credited to Yoshiya Gunji, Yuri Miyata, Manami Oba, Megumi Shimaoka, Shinichi Sugimoto, Nobuharu Tsujimoto, Hisashi Yasueda.
Application Number | 20130171700 13/772959 |
Document ID | / |
Family ID | 27309903 |
Filed Date | 2013-07-04 |
United States Patent
Application |
20130171700 |
Kind Code |
A1 |
Gunji; Yoshiya ; et
al. |
July 4, 2013 |
L-Amino Acid-Producing Bacterium and Method for Producing L-Amino
Acid
Abstract
An L-amino acid is produced by culturing a Methylophilus
bacterium which can grow by using methanol as the main carbon
source and has L-amino acid-producing ability, for example, a
Methylophilus bacterium in which dihydrodipicolinate synthase
activity and aspartokinase activity are enhanced by transformation
of cells with a DNA coding for dihydrodipicolinate synthase that is
desensitized to feedback inhibition by L-lysine and a DNA coding
for aspartokinase that is desensitized to feedback inhibition by
L-lysine, or a Methylophilus bacterium which is casamino acid
auxotrophic, in a medium containing methanol as a main carbon
source, to produce and accumulate an L-amino acid in culture, and
collecting the L-amino acid from the culture.
Inventors: |
Gunji; Yoshiya;
(Kawasaki-shi, JP) ; Yasueda; Hisashi;
(Kawasaki-shi, JP) ; Sugimoto; Shinichi;
(Kawasaki-shi, JP) ; Tsujimoto; Nobuharu;
(Kawasaki-shi, JP) ; Shimaoka; Megumi;
(Kawasaki-shi, JP) ; Miyata; Yuri; (Kawasaki-shi,
JP) ; Oba; Manami; (Kawasaki-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ajinomoto Co., Inc.; |
Tokyo |
|
JP |
|
|
Assignee: |
AJINOMOTO CO., INC.
Tokyo
JP
|
Family ID: |
27309903 |
Appl. No.: |
13/772959 |
Filed: |
February 21, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11738617 |
Apr 23, 2007 |
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13772959 |
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09926299 |
Oct 9, 2001 |
7223572 |
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PCT/JP00/02295 |
Apr 7, 2000 |
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11738617 |
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Current U.S.
Class: |
435/115 |
Current CPC
Class: |
C12N 9/88 20130101; C12R
1/01 20130101; C12N 9/1217 20130101; C12P 13/08 20130101; C12P
13/04 20130101 |
Class at
Publication: |
435/115 |
International
Class: |
C12P 13/08 20060101
C12P013/08 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 9, 1999 |
JP |
11-103143 |
Jun 16, 1999 |
JP |
11-169447 |
Dec 24, 1999 |
JP |
11-368097 |
Claims
1. A method for producing L-lysine comprising culturing a
Methylophilus bacterium in a medium containing methanol to produce
and accumulate L-lysine in the medium, and collecting L-lysine from
the medium, wherein said Methylophilus bacterium has been modified
to have enhanced activities of dihydrodipicolinate synthase and
aspartokinase by transformation with a DNA from Escherichia coli
that encodes dihydrodipicolinate synthase resistant to feedback
inhibition by L-lysine and with a DNA from Escherichia coli that
encodes aspartokinase resistant to feedback inhibition by
L-lysine.
2. The method according to claim 1, wherein said DNA from
Escherichia coli that encodes dihydrodipicolinate synthase encodes
dihydrodipicolinate synthase comprising replacement of histidine at
position 118 in SEQ ID NO: 2 with tyrosine.
3. The method according to claim 1, wherein said DNA from
Escherichia coli that encodes aspartokinase encodes aspartokinase
comprising replacement of threonine at position 352 in SEQ ID NO: 4
with isoleucine.
Description
[0001] This application is a Continuation of, and claims priority
under 35 U.S.C. .sctn.120 to, U.S. patent application Ser. No.
11/738,617, filed Apr. 23, 2007, which was a Divisional of, and
claims priority under U.S.C. .sctn.120 to, U.S. patent application
Ser. No. 09/926,299, filed Oct. 9, 2001, now U.S. Pat. No.
7,223,572, issued May 29, 2007, which was a U.S. national phase
filing under 35 U.S.C. .sctn.371 of PCT Patent Application No.
PCT/JP00/02295, filed Apr. 7, 2000, which in turn claimed priority
under 35 U.S.C. .sctn.119 to Japanese Patent Application 11-103143,
filed Apr. 9, 1999, Japanese Patent Application 11-169447, filed
Jun. 16, 1999, and Japanese Patent Application 11-368097, filed
Dec. 24, 1999. These applications are hereby incorporated by
reference in their entireties.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to techniques useful in the
microbial industry. In particular, the present invention relates to
a method for producing an L-amino acid by fermentation, and a
microorganism which can be used in the method.
[0004] 2. Brief Description of the Related Art
[0005] Amino acids such as L-lysine, L-glutamic acid, L-threonine,
L-leucine, L-isoleucine, L-valine, and L-phenylalanine are
typically produced in industry by fermentation using microorganisms
that belong to the genus Brevibacterium, Corynebacterium, Bacillus,
Escherichia, Streptomyces, Pseudomonas, Arthrobacter, Serratia,
Penicillium, Candida, or the like. In order to improve
productivity, microorganism strains isolated from nature or
artificial mutants thereof have typically been used. To increase
the ability to produce L-glutamic acid, various techniques have
been disclosed to enhance or increase the activities of L-glutamic
acid biosynthetic enzymes using recombinant DNA techniques.
[0006] The production of L-amino acids has been considerably
increased by breeding microorganisms such as those mentioned above.
However, in order to meet increased demand in the future,
development of more efficient production methods at lower cost are
desirable.
[0007] Methanol is a raw material useful in fermentation since it
is readily available and inexpensive. Known methods using methanol
in fermentation typically use microorganisms that belong to the
genus Achromobacter or Pseudomonas (Japanese Patent Publication
(Kokoku) No. 45-25273/1970), Protaminobacter (Japanese Patent
Application Laid-open (Kokai) No. 49-125590/1974), Protaminobacter
or Methanomonas (Japanese Patent Application Laid-open (Kokai) No.
50-25790/1975), Microcyclus (Japanese Patent Application Laid-open
(Kokai) No. 52-18886/1977), Methylobacillus (Japanese Patent
Application Laid-open (Kokai) No. 4-91793/1992), Bacillus (Japanese
Patent Application Laid-open (Kokai) No. 3-505284/1991), and so
forth.
[0008] To date, however, the use of Methylophilus bacteria in
production of L-amino acids has not been reported. Although the
methods described in EP 0 035 831 A, EP 0 037 273 A, and EP 0 066
994 A are methods for transforming Methylophilus bacteria using
recombinant DNA, the use of such techniques to improve the amino
acid productivity of Methylophilus bacteria has not been
reported.
SUMMARY OF THE INVENTION
[0009] The object of the present invention is to provide a novel
bacterium which is able to produce L-amino acids, and a method for
producing an L-amino acid by using the L-amino acid-producing
bacterium.
[0010] As a result of the present inventors' efforts to achieve the
aforementioned objectives, is was found that Methylophilus bacteria
were suitable for producing L-amino acids. Furthermore, although it
has conventionally been considered difficult to obtain auxotrophic
mutants of Methylophilus bacteria (FEMS Microbiology Rev. 39,
235-258 (1986) and Antonie van Leeuwenhoek 53, 47-53 (1987)), the
present inventors have succeeded in obtaining auxotrophic mutants
of said bacteria. Thus, the present invention has been
accomplished.
[0011] That is, the present invention provides the following.
[0012] It is an object of the present invention to provide a
Methylophilus bacterium having L-amino acid-producing ability.
[0013] It is an object of the present invention to provide the
Methylophilus bacterium as described above, wherein the L-amino
acid is L-lysine, L-valine, L-leucine, L-isoleucine, or
L-threonine.
[0014] It is an object of the present invention to provide the
Methylophilus bacterium as described above, which has resistance to
an L-amino acid analogue or L-amino acid auxotrophy.
[0015] It is an object of the present invention to provide the
Methylophilus bacterium as described above, wherein L-amino acid
biosynthetic enzyme activity is enhanced.
[0016] It is an object of the present invention to provide the
Methylophilus bacterium as described above, wherein
dihydrodipicolinate synthase activity and aspartokinase activity
are enhanced, and the bacterium has L-lysine-producing ability.
[0017] It is an object of the present invention to provide the
Methylophilus bacterium as described above, wherein
dihydrodipicolinate synthase activity is enhanced, and the
bacterium has L-lysine-producing ability.
[0018] It is an object of the present invention to provide the
Methylophilus bacterium as described above, wherein aspartokinase
activity is enhanced, and the bacterium has L-lysine-producing
ability.
[0019] It is an object of the present invention to provide the
Methylophilus bacterium as described above, wherein an activity or
activities of one, two, or three enzymes selected from aspartic
acid semialdehyde dehydrogenase, dihydrodipicolinate reductase, and
diaminopimelate decarboxylase is/are enhanced.
[0020] It is an object of the present invention to provide the
Methylophilus bacterium as described above, wherein the
dihydrodipicolinate synthase activity and the aspartokinase
activity are enhanced by transformation through introduction into
cells, of a DNA coding for dihydrodipicolinate synthase that is not
subject to feedback inhibition by L-lysine and a DNA coding for
aspartokinase that is not subject to feedback inhibition by
L-lysine.
[0021] It is an object of the present invention to provide the
bacterium as described above, wherein activities of aspartokinase,
homoserine dehydrogenase, homoserine kinase, and threonine
synthase, is/are enhanced, and the bacterium has
L-threonine-producing ability.
[0022] It is an object of the present invention to provide the
bacterium as described above, wherein the Methylophilus bacterium
is Methylophilus methylotrophus.
[0023] It is an object of the present invention to provide a method
for producing an L-amino acid, which comprises culturing a
Methylophilus bacterium as described above in a medium to produce
and accumulate an L-amino acid in culture and collecting the
L-amino acid from the culture.
[0024] It is an object of the present invention to provide the
method as described above, wherein the medium contains methanol as
a main carbon source.
[0025] It is an object of the present invention to provide a method
for producing bacterial cells of a Methylophilus bacterium with an
increased content of an L-amino acid, which comprises culturing a
Methylophilus bacterium as described above in a medium to produce
and accumulate an L-amino acid in bacterial cells of the
bacterium.
[0026] It is an object of the present invention to provide the
method for producing bacterial cells of the Methylophilus bacterium
as described above, wherein the L-amino acid is L-lysine, L-valine,
L-leucine, L-isoleucine or L-threonine.
[0027] It is an object of the present invention to provide a DNA
which codes for a protein defined in the following (A) or (B):
[0028] (A) a protein which has the amino acid sequence of SEQ ID
NO: 6, or
[0029] (B) a protein which has an amino acid sequences of SEQ ID
NO: 6 including substitution, deletion, insertion, addition or
inversion of one or several amino acids, and has aspartokinase
activity.
[0030] It is an object of the present invention to provide the DNA
as described above, which is a DNA defined in the following (a) or
(b):
[0031] (a) a DNA which has a nucleotide sequence comprising the
nucleotide sequence of the nucleotide numbers 510 to 1736 of SEQ ID
NO: 5; or
[0032] (b) a DNA which is hybridizable with a probe having the
nucleotide sequence of the nucleotide numbers 510 to 1736 of SEQ ID
NO: 5 or a part thereof under a stringent condition, and codes for
a protein having aspartokinase activity.
[0033] It is an object of the present invention to provide a DNA
which codes for a protein defined in the following (C) or (D):
[0034] (C) a protein which has the amino acid sequence of SEQ ID
NO: 8, or
[0035] (D) a protein which has an amino acid sequences of SEQ ID
NO: 8 including substitution, deletion, insertion, addition or
inversion of one or several amino acids, and has aspartic acid
semialdehyde dehydrogenase activity.
[0036] It is an object of the present invention to provide the DNA
as described above, which is a DNA defined in the following (c) or
(d):
[0037] (c) a DNA which has a nucleotide sequence comprising the
nucleotide sequence of the nucleotide numbers 98 to 1207 of SEQ ID
NO: 7; or
[0038] (d) a DNA which is hybridizable with a probe having the
nucleotide sequence of the nucleotide numbers 98 to 1207 of SEQ ID
NO: 7 or a part thereof under a stringent condition, and codes for
a protein having aspartic acid semialdehyde dehydrogenase
activity.
[0039] It is an object of the present invention to provide a DNA
which codes for a protein defined in the following (E) or (F):
[0040] (E) a protein which has the amino acid sequence of SEQ ID
NO: 10, or
[0041] (F) a protein which has an amino acid sequences of SEQ ID
NO: 10 including substitution, deletion, insertion, addition or
inversion of one or several amino acids, and has
dihydrodipicolinate synthase activity.
[0042] It is an object of the present invention to provide the DNA
as described above, which is a DNA defined in the following (e) or
(f):
[0043] (e) a DNA which has a nucleotide sequence comprising the
nucleotide sequence of the nucleotide numbers 1268 to 2155 of SEQ
ID NO: 9; or
[0044] (f) a DNA which is hybridizable with a probe having the
nucleotide sequence of the nucleotide numbers 1268 to 2155 of SEQ
ID NO: 9 or a part thereof under a stringent condition, and codes
for a protein having dihydrodipicolinate synthase activity.
[0045] It is an object of the present invention to provide a DNA
which codes for a protein defined in the following (G) or (H):
[0046] (G) a protein which has the amino acid sequence of SEQ ID
NO: 12, or
[0047] (H) a protein which has an amino acid sequences of SEQ ID
NO: 12 including substitution, deletion, insertion, addition or
inversion of one or several amino acids, and has
dihydrodipicolinate reductase activity.
[0048] It is an object of the present invention to provide the DNA
as described above, which is a DNA defined in the following (g) or
(h):
[0049] (g) a DNA which has a nucleotide sequence comprising the
nucleotide sequence of the nucleotide numbers 2080 to 2883 of SEQ
ID NO: 11; or
[0050] (h) a DNA which is hybridizable with a probe having the
nucleotide sequence of the nucleotide numbers 2080 to 2883 of SEQ
ID NO: 11 or a part thereof under a stringent condition, and codes
for a protein having dihydrodipicolinate reductase activity.
[0051] It is an object of the present invention to provide a DNA
which codes for a protein defined in the following (I) or (J):
[0052] (I) a protein which has the amino acid sequence of SEQ ID
NO: 14, or
[0053] (J) a protein which has an amino acid sequences of SEQ ID
NO: 14 including substitution, deletion, insertion, addition or
inversion of one or several amino acids, and has diaminopimelate
decarboxylase activity.
[0054] It is an object of the present invention to provide the
DNAas described above, which is a DNA defined in the following (i)
or (j):
[0055] (i) a DNA which has a nucleotide sequence comprising the
nucleotide sequence of the nucleotide numbers 751 to 1995 of SEQ ID
NO: 13; or
[0056] (j) a DNA which is hybridizable with a probe having the
nucleotide sequence of the nucleotide numbers 751 to 1995 of SEQ ID
NO: 13 or a part thereof under a stringent condition, and codes for
a protein having diaminopimelate decarboxylase activity.
[0057] In the present specification, "L-amino acid-producing
ability" refers to the ability to accumulate a significant amount
of an L-amino acid in a medium or to increase the amino acid
content in the microbial cells when a microorganism of the present
invention is cultured in the medium.
BRIEF DESCRIPTION OF THE DRAWINGS
[0058] FIG. 1 shows the production process for the plasmid RSF24P,
which includes the mutant dapA. The term "dapA*24" refers to the
mutant dapA that codes for the mutant DDPS, wherein the
118-histidine residue is replaced with a tyrosine residue.
[0059] FIG. 2 shows the production process for the plasmid RSFD80
which includes the mutant dapA and mutant lysC. The term "lysC*80"
refers to the mutant lysC that codes for the mutant AKIII, wherein
the 352-threonine residue is replaced with an isoleucine
residue.
[0060] FIG. 3 shows the aspartokinase activity of E. coli strains
transformed with the ask gene.
[0061] FIG. 4 shows the aspartic acid semialdehyde dehydrogenase
activity of E. coli strains transformed with the asd gene.
[0062] FIG. 5 shows the dihydrodipicolinate synthase activity of E.
coli strains transformed with the dapA gene.
[0063] FIG. 6 shows the dihydrodipicolinate reductase activity of
an E. coli strain transformed with the dapB gene.
[0064] FIG. 7 shows the diaminopimelate decarboxylase activity of
E. coli strains transformed with the lysA gene.
DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS
[0065] <1> Microorganism of the Present Invention
[0066] The microorganism of the present invention is a bacterium
belonging to the genus Methylophilus which is able to produce
L-amino acids. The Methylophilus bacterium of the present invention
includes, for example, Methylophilus methylotrophus AS1 strain
(NCIMB10515) and so forth. This strain is available from the
National Collections of Industrial and Marine Bacteria (Address:
NCIMB Lts., Torry Research Station 135, Abbey Road, Aberdeen AB9
8DG, United Kingdom).
[0067] L-amino acids which can be produced according to the present
invention include L-lysine, L-glutamic acid, L-threonine, L-valine,
L-leucine, L-isoleucine, L-tryptophan, L-phenylalanine, L-tyrosine,
and so forth. One or more types of such amino acids may be
produced.
[0068] Methylophilus bacteria which are able to produce L-amino
acids can be obtained by imparting this L-amino acid-producing
ability to wild-type strains of the Methylophilus bacteria. In
order to impart L-amino acid-producing ability, methods
conventionally used for breeding coryneform bacteria, Escherichia
bacteria, or the like, may be used. These methods include breeding
auxotrophic mutant strains, strains resistant to L-amino acid
analogues or metabolic control mutant strains, and methods for
producing recombinant strains wherein L-amino acid biosynthetic
enzyme activities are enhanced (see "Amino Acid Fermentation", the
Japan Scientific Societies Press [Gakkai Shuppan Center], 1st
Edition, published on May 30, 1986, pp. 77 to 100). When breeding
amino acid-producing bacteria, characteristics such as auxotrophy,
L-amino acid analogue resistance, and metabolic control mutations
may be imparted alone or in combination. One or more L-amino acid
biosynthetic enzymes may be enhanced, and/or one or more of the
methods mentioned above may be combined with enhancing one or more
of the biosynthetic enzymes. For example, bacteria which produce
L-lysine are bred to be auxotrophic for L-homoserine or
L-threonine, and L-methionine (Japanese Patent Publication (Kokoku)
Nos. 48-28078/1973 and 56-6499/1981), inositol or acetic acid
(Japanese Patent Application Laid-open (Kokai) Nos. 55-9784/1980
and 56-8692/1981). These bacteria have also been bred to be
resistant to oxalysine, lysine hydroxamate,
S-(2-aminoethyl)-cysteine, .beta.-methyllysine,
.alpha.-chlorocaprolactam, DL-.alpha.-amino-.epsilon.-caprolactam,
.alpha.-amino-lauryllactam, aspartic acid analogues, sulfa drugs,
quinoid, or N-lauroylleucine.
[0069] Furthermore, L-glutamic acid-producing bacteria can be bred
as mutants which are auxotrophic for oleic acid or the like.
Bacteria which produce L-threonine can be bred to be resistant to
.alpha.-amino-.beta.-hydroxyvaleric acid. Bacteria which produce
L-homoserine can be bred to be auxotrophic for L-threonine, or to
be resistant to L-phenylalanine analogues. Bacteria which produce
L-phenylalanine can be bred to be auxotrophic for L-tyrosine.
Bacteria which produce L-isoleucine can be bred to be auxotrophic
for L-leucine. Bacteria which produce L-proline can be bred to be
auxotrophic for L-isoleucine.
[0070] Furthermore, strains that produce one or more kinds of
branched-chain amino acids (L-valine, L-leucine, and L-isoleucine)
can be bred to be auxotrophic for casamino acid.
[0071] In order to obtain mutants of Methylophilus bacteria, the
optimal mutagenesis conditions were examined using emergence
frequency of streptomycin-resistant strains as an index. As a
result, the maximum emergence frequency of streptomycin resistant
strains was obtained when the survival rate after mutagenesis was
about 0.5%. Therefore, auxotrophic strains were obtained under
these conditions. Auxotrophic strains were also obtained by
screening mutants on a large scale, which had been previously
reported to be difficult, as compared with that previously reported
for E. coli and so forth.
[0072] As described above, since mutants can be obtained by
mutagenizing Methylophilus bacteria under suitable conditions, it
is possible to readily obtain desirable mutants by suitably setting
conditions that result in a survival rate after mutagenesis of
about 0.5%, depending on the mutagenesis method.
[0073] Mutagenesis methods for obtaining mutants of Methylophilus
bacteria include UV irradiation and treatments with typical
mutagenesis agents, such as N-methyl-N'-nitro-N-nitrosoguanidine
(NTG) and nitrous acid. Methylophilus bacteria which are able to
produce L-amino acids can also be obtained by selecting naturally
occurring mutants of Methylophilus bacteria.
[0074] Mutants resistant to L-amino acid analogues can be obtained
by, for example, inoculating mutagenized Methylophilus bacteria
into agar medium containing an L-amino acid analogue at a variety
of concentrations, and selecting for bacterial colonies.
[0075] Auxotrophic mutants can be obtained by allowing
Methylophilus bacteria to form colonies on an agar medium
containing a target nutrient (for example, an L-amino acid),
replicating the colonies on an agar medium which does not contain
said nutrient, and selecting strains that cannot grow on this agar
medium.
[0076] Methods for imparting, enhancing, and/or increasing the
ability to produce L-amino acids by enhancing L-amino acid
biosynthetic enzyme activity is exemplified below.
[0077] L-lysine
[0078] The ability to produce L-lysine can be imparted by, for
example, enhancing dihydrodipicolinate synthase activity and/or
aspartokinase activity.
[0079] The dihydrodipicolinate synthase and/or the aspartokinase
activity in Methylophilus bacteria can be enhanced by ligating a
gene fragment coding for dihydrodipicolinate synthase and/or a gene
fragment coding for aspartokinase with a vector that functions in
Methylophilus bacteria, preferably a multiple-copy type vector, to
create a recombinant DNA. This recombinant DNA is then used to
transform a Methylophilus bacterium. As a result of the increase in
the copy number of the gene coding for dihydrodipicolinate synthase
and/or the gene coding for aspartokinase in the transformant
strain, the activity or activities thereof is/are enhanced.
Hereinafter, dihydrodipicolinate synthase, aspartokinase, and
aspartokinase III are also referred to as DDPS, AK, and AKIII,
respectively.
[0080] Any microorganism can be used to provide the genes encoding
for DDPS and/or AK, so long as the chosen microorganism has genes
enabling expression of DDPS activity and AK activity in
Methylophilus microorganisms. Such microorganisms may be wild-type
strains or mutant strains derived therefrom. Specifically, examples
of such microorganisms include E. coli (Escherichia coli) K-12
strain, Methylophilus methylotrophus AS1 strain (NCIMB10515), and
so forth. Since the nucleotide sequences of the gene coding for
DDPS (dapA, Richaud, F. et al., J. Bacteriol., 297, (1986)) and the
gene coding for AKIII (lysC, Cassan, M., Parsot, C., Cohen, G. N.
and Patte, J. C., J. Biol. Chem., 261, 1052 (1986)) native to and
derived from Escherichia bacteria have both been reported, these
genes can be obtained by PCR using primers synthesized based on the
nucleotide sequences of these genes and chromosomal DNA of a
microorganism such as E. coli K-12, or the like, as the template.
As specific examples, dapA and lysC derived from E. coli will be
explained below. However, genes used in the present invention are
not limited to these.
[0081] It is preferred that DDPS and AK are not inhibited by
L-lysine, i.e. feedback inhibition. It has been reported that
wild-type DDPS derived from E. coli is subject to such feedback
inhibition by L-lysine, and that wild-type AKIII derived from E.
coli is also subject to suppression and feedback inhibition by
L-lysine. Therefore, the dapA and lysC which are used to transform
Methylophilus bacteria preferably code for DDPS and AKIII which
have a mutation that desensitizes this feedback inhibition.
Hereinafter, the DDPS which has a mutation that desensitizes
feedback inhibition by L-lysine is also referred to as "mutant
DDPS", and the DNA coding for this mutant DDPS is also referred to
as "mutant dapA". AKIII derived from E. coli which has a mutation
that desensitizes feedback inhibition by L-lysine is also referred
to as "mutant AKIII", and the DNA coding for this mutant AKIII is
also referred to as "mutant lysC".
[0082] According to the present invention, DDPS and AK are not
necessarily required to be a mutated as such. It is known that, for
example, DDPS native to Corynebacterium bacteria does not suffer
feedback inhibition by L-lysine.
[0083] The nucleotide sequence of wild-type dapA native to E. coli
is shown in SEQ ID NO: 1. The amino acid sequence of wild-type DDPS
coded by this nucleotide sequence is shown in SEQ ID NO: 2. The
nucleotide sequence of wild-type lysC native to E. coli is shown in
SEQ ID NO: 3. The amino acid sequence of wild-type ATIII coded by
this nucleotide sequence is shown in SEQ ID NO: 4.
[0084] The DNA coding for mutant DDPS that is not subject to
feedback inhibition by L-lysine includes the DNA coding for DDPS
having the amino acid sequence shown in SEQ ID NO: 2, but wherein
the 118-histidine residue is replaced with a tyrosine residue. The
DNA coding for mutant AKIII that is not subject to feedback
inhibition by L-lysine includes a DNA coding for AKIII having the
amino sequence shown in SEQ ID NO: 4, but wherein the 352-threonine
residue is replaced with an isoleucine residue.
[0085] Any plasmid may be used for gene cloning, so long as it can
replicate in microorganisms such as Escherichia bacteria or the
like. Specifically, useful plasmids may include pBR322, pTWV228,
pMW119, pUC19, and so forth.
[0086] Vectors that function in Methylophilus bacteria include, for
example, plasmids that can autonomously replicate in Methylophilus
bacteria. Specifically, RSF1010, which is a broad host spectrum
vector, and derivatives thereof, for example, pAYC32 (Chistorerdov,
A. Y., Tsygankov, Y. D. Plasmid, 16, 161-167, (1986)), pMFY42
(Gene, 44, 53, (1990)), pRP301, pTB70 (Nature, 287, 396, (1980)),
and so forth, may be used.
[0087] The chosen vector may be digested with a restriction enzyme
that corresponds to the terminus of the DNA fragments containing
dapA and lysC. The vector is then ligated to the gene fragments,
and ligation is usually performed with a ligase such as T4 DNA
ligase. dapA and lysC may be individually ligated into separate
vectors or into a single vector.
[0088] The plasmid containing mutant dapA coding for mutant DDPS
and mutant lysC coding for mutant AKIII has been reported. This
plasmid is the broad host spectrum plasmid RSFD80 (WO95/16042). E.
coli JM109 strain transformed with this plasmid was designated
AJ12396, and deposited at the National Institute of Bioscience and
Human-Technology, Agency of Industrial Science and Technology,
Ministry of International Trade and Industry (postal code 305-8566,
1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on Oct. 28,
1993 and received an accession number of FERM P-13936, and it was
converted to an international deposit under the provisions of the
Budapest Treaty on Nov. 1, 1994, and received an accession number
of FERM BP-4859. RSFD80 can be obtained from the AJ12396 strain
using known techniques.
[0089] The mutant dapA from RSFD80 has a substitution of the C at
nucleotide 597 in the wild-type dapA (SEQ ID NO: 1), with a T. As a
result, the encoded mutant DDPS has a tyrosine at position 118 in
SEQ ID NO: 2 instead of the native histidine. The mutant lysC from
RSFD80 has a substitution of the C at nucleotide 1638 in the
wild-type lysC (SEQ ID NO: 3), with a T. As a result, the encoded
mutant AKIII has an isoleucine at position 352 instead of the
native threonine.
[0090] Any method can be used to introduce the recombinant DNA
prepared as described above into the Methylophilus bacteria, so
long as sufficient transformation efficiency is acheived. For
example, electroporation can be used (Canadian Journal of
Microbiology, 43, 197 (1997)).
[0091] The DDPS activity and/or the AK activity can also be
enhanced by the presence of multiple copies of dapA and/or lysC on
the chromosomal DNA of Methylophilus bacteria. Homologous
recombination can be used to introduce multiple copies of dapA
and/or lysC into chromosomal DNA of Methylophilus bacteria. As a
target, a sequence that is present in multiple copies on the
chromosomal DNA of Methylophilus bacteria can be used, such as
repetitive DNA, inverted repeats present at the end of a
transposable element, or the like. Alternatively, as disclosed in
Japanese Patent Application Laid-open (Kokai) No. 2-109985/1990,
multiple copies of dapA and/or lysC can be transfered to the
chromosomal DNA using a transposon. In both of these methods, the
DDPS and AK activities should be increased by increasing the copy
numbers of dapA and/or lysC.
[0092] Other than increasing the gene copy numbers, the DDPS
activity and/or the AK activity can be amplified by replacing an
expression control sequence such as the promoters of dapA and/or
lysC with stronger ones (Japanese Patent Application Laid-open
(Kokai) No. 1-215280/1989). Strong promoters are known in the art
and include, for example, the lac promoter, trp promoter, trc
promoter, tac promoter, P.sub.R promoter and P.sub.L promoter of
lambda phage, tet promoter, amyE promoter, spac promoter, and so
forth. Substitution of native promoters with these stronger
promoters enhances the expression of dapA and/or lysC, and thus the
DDPS activity and the AK activity are amplified. Enhancing the
expression control sequences can be combined with increasing the
copy numbers of dapA and/or lysC.
[0093] In order to prepare a recombinant DNA of a gene fragment and
a vector, the vector is digested with a restriction enzyme
corresponding to the terminus of the gene fragment, and ligation is
performed using a ligase such as T4 ligase. The methods used for
digestion, ligation, preparation of chromosomal DNA, PCR,
preparation of plasmid DNA, transformation, design of
oligonucleotides used as primers and so forth, can be typical
methods well known to those skilled in the art. Such methods are
described in Sambrook, J., Fritsch, E. F., and Maniatis, T.,
"Molecular Cloning: A Laboratory Manual, 2nd Edition", Cold Spring
Harbor Laboratory Press, (1989) and so forth.
[0094] In addition to enhancing the DDPS activity and/or the AK
activity, the activities of other enzymes involved in the L-lysine
biosynthesis may also be enhanced. Such enzymes include
diaminopimelate pathway enzymes such as dihydrodipicolinate
reductase, diaminopimelate decarboxylase, diaminopimelate
dehydrogenase (WO96/40934 for all of the foregoing enzymes),
phosphoenolpyruvate carboxylase (Japanese Patent Application
Laid-open (Kokai) No. 60-87788/1985), aspartate aminotransferase
(Japanese Patent Publication (Kokoku) No. 6-102028/1994),
diaminopimelate epimerase, aspartic acid semialdehyde dehydrogenase
and so forth, or aminoadipate pathway enzymes such as homoaconitate
hydratase and so forth. Preferably, the activity of at least
aspartic acid semialdehyde dehydrogenase, dihydrodipicolinate
reductase, and/or diaminopimelate decarboxylase is/are
enhanced.
[0095] Aspartokinase, aspartic acid semialdehyde dehydrogenase,
dihydrodipicolinate synthase, dihydrodipicolinate reductase, and
diaminopimelate decarboxylase derived from Methylophilus
methylotrophus will be described later.
[0096] Furthermore, the activity of an enzyme that catalyzes a
reaction that generates a compound other than L-lysine via a branch
of the L-lysine biosynthetic pathway may be decreased in the chosen
host microorganism. An example of such an enzyme is homoserine
dehydrogenase (see WO95/23864).
[0097] The aforementioned techniques for enhancing enzyme activity
can be similarly used for other amino acids, as mentioned
below.
[0098] L-glutamic acid
[0099] The ability to produce L-glutamic acid can be imparted to
Methylophilus bacteria by, for example, introducing a DNA that
codes for any one of the following enzymes: glutamate dehydrogenase
(Japanese Patent Application Laid-open (Kokai) 61-268185/1986),
glutamine synthetase, glutamate synthase, isocitrate dehydrogenase
(Japanese Patent Application Laid-open (Kokai) Nos. 62-166890/1987
and 63-214189/1988), aconitate hydratase (Japanese Patent
Application Laid-open (Kokai) No. 62-294086/1987), citrate synthase
(Japanese Patent Application Laid-open (Kokai) Nos. 62-201585/1987
and 63-119688/1988), phosphoenolpyruvate carboxylase (Japanese
Patent Application Laid-open (Kokai) Nos. 60-87788/1985 and
62-55089/1987), pyruvate dehydrogenase, pyruvate kinase,
phosphoenolpyruvate synthase, enolase, phosphoglyceromutase,
phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase,
triose phosphate isomerase, fructose bisphosphate aldolase,
phosphofructokinase (Japanese Patent Application Laid-open (Kokai)
No. 63-102692/1988), glucose phosphate isomerase,
glutamine-oxoglutarate aminotransferase (WO99/07853), and so
forth.
[0100] Furthermore, the activity of an enzyme that catalyzes a
reaction that generates a compound other than L-glutamic acid via a
branch of the L-glutamic acid biosynthetic pathwaymay be decreased
in the chosen host. Examples of such enzymes are
.alpha.-ketoglutarate dehydrogenase (.alpha.KGDH), isocitrate
lyase, phosphate acetyltransferase, acetate kinase, acetohydroxy
acid synthase, acetolactate synthase, formate acetyltransferase,
lactate dehydrogenase, glutamate decarboxylase, 1-pyrroline
dehydrogenase, and so forth.
[0101] L-threonine
[0102] The ability to produce L-threonine can be imparted or
increased by, for example, enhancing the activities of
aspartokinase, homoserine dehydrogenase, homoserine kinase, and
threonine synthase. The activities of these enzymes can be enhanced
by, for example, transforming Methylophilus bacteria with a
recombinant plasmid containing the threonine operon (Japanese
Patent Application Laid-open (Kokai) Nos. 55-131397/1980,
59-31691/1984 and 56-15696/1981 and Japanese Patent Application
Laid-open (Kohyo) No. 3-501682/1991).
[0103] Production of L-threonine can also be imparted or enhanced
by amplifying or introducing a threonine operon which includes the
gene coding for aspartokinase which has been desensitized to
feedback inhibition by L-threonine (Japanese Patent Publication
(Kokoku) No. 1-29559/1989), the gene coding for homoserine
dehydrogenase (Japanese Patent Application Laid-open (Kokai) No.
60-012995/1985), or the genes coding for homoserine kinase and
homoserine dehydrogenase (Japanese Patent Application Laid-open
(Kokai) No. 61-195695/1986).
[0104] Furthermore, the production of L-threonine can be improved
by introducing a DNA coding for a mutant phosphoenolpyruvate
carboxylase which has been desensitized to feedback inhibition by
aspartic acid.
[0105] L-valine
[0106] The ability to produce L-valine can be imparted by, for
example, introducing into Methylophilus bacteria an L-valine
biosynthesis gene with a substantially desensitized control
mechanism. A mutation that substantially desensitizes the control
mechanism of an L-valine biosynthesis gene in Methylophilus
bacteria may also be introduced.
[0107] Examples of the L-valine biosynthesis gene include, for
example, the ilvGMEDA operon of E. coli. Threonine deaminase
encoded by the ilvA gene catalyzes the deamination reaction which
results in conversion of L-threonine to 2-ketobutyric acid, which
is the rate-determining step of L-isoleucine biosynthesis.
Therefore, in order for efficient progression of the L-valine
synthesis reactions, it is preferable to use an operon that does
not express threonine deaminase activity. Examples of such an
ilvGMEDA operon include the ilvGMEDA operon with an inactive ilvA
gene, whether the gene is mutated, disrupted, or deleted. Since
L-valine, L-isoleucine, and/or L-leucine attenuate expression of
the ilvGMEDA operon, the region causing the attenuation is
preferably removed or mutated so to desensitize this attenuation by
L-valine.
[0108] An ilvGMEDA operon which does not express threonine
deaminase activity and and with desensitized attenuation as
described above can be obtained by subjecting the wild-type
ilvGMEDA operon to mutagenesis or modifying it using gene
recombination techniques (see WO96/06926).
[0109] L-leucine
[0110] The ability to produce L-leucine is imparted or enhanced by,
for example, introducing into a Methylophilus bacteria an L-leucine
biosynthesis gene with a substantially desensitized control
mechanism, in addition to the above-described manipulations
required for the production of L-valine. It is also possible to
eliminate the control mechanism of an L-leucine biosynthesis gene
in Methylophilus via mutation. A mutation in the leuA gene which
substantially eliminates inhibition by L-leucine is one
example.
[0111] L-isoleucine
[0112] The ability to produce L-isoleucine can be imparted by, for
example, substantially desensitizing the host microorganism to
inhibition by L-threonine by introducing the thrABC operon with the
E. coli thrA gene coding for aspartokinase I/homoserine
dehydrogenase I. Also, inhibition by L-isoleucine can be
substantially desensitized in the host by removing the region of
the ilvA gene required for attenuation in the ilvGMEDA operon
(Japanese Patent Application Laid-open (Kokai) No.
8-47397/1996).
[0113] Other Amino Acids:
[0114] Biosyntheses of L-tryptophan, L-phenylalanine, L-tyrosine,
L-threonine, and L-isoleucine can be enhanced by increasing the
ability of the Methylophilus bacteria to produce
phosphoenolpyruvate (WO97/08333).
[0115] The ability to produce L-phenylalanine and L-tyrosine can be
improved by amplifying or introducing a desensitized chorismate
mutase-prephenate dehydratase (CM-PDT) gene (Japanese Patent
Application Laid-open (Kokai) Nos. 5-236947/1993 and
62-130693/1987) and a desensitized
3-deoxy-D-arabinoheptulonate-7-phosphate synthase (DS) gene
(Japanese Patent Application Laid-open (Kokai) Nos. 5-236947/1993
and 61-124375/1986).
[0116] The ability to produce L-tryptophan can be improved by
amplifying or introducing a tryptophan operon with a gene coding
for desensitized anthranilate synthase (Japanese Patent Application
Laid-open (Kokai) Nos. 57-71397/1982, 62-244382/1987 and US Pat.
No. 4,371,614).
[0117] In the present specification, the expression that the
"enzyme activity is enhanced" usually means that the intracellular
activity of the enzyme is higher than that in a wild-type strain.
Furthermore, when the activity of the enzyme is enhanced by
modification using gene recombinant techniques or the like, the
intracellular activity of the enzyme is higher than that in the
strain before the modification. The expression that "enzyme
activity is decreased" usually means that the intracellular
activity of the enzyme is lower than that in a wild-type strain.
Similarly, when the activity of the enzyme is decreased by
modification using gene recombinant techniques or the like, the
intracellular activity of the enzyme is lower than that in the
strain before the modification.
[0118] L-amino acids can be produced by culturing the Methylophilus
bacteria obtained as described above in a medium under conditions
which allow for production and accumulation of the L-amino acids in
the medium, and collecting the L-amino acids from the medium.
[0119] Methylophilus bacteria with an increased amount of L-amino
acid as compared with wild-type strains of Methylophilus bacteria
can be produced by culturing Methylophilus bacteria with an ability
to produce L-amino acids in a medium under conditions which allow
for production and accumulation of the L-amino acids.
[0120] Microorganisms used in the present invention can be cultured
by methods which are typically used for culturing
methanol-assimilating microorganisms. The medium for the culture
may be a natural or synthetic medium so long as it contains a
carbon source, a nitrogen source, inorganic ions, and other trace
organic components as required.
[0121] By using methanol as the main source of carbon, L-amino
acids can be inexpensively produced. 0.001 to 30% of methanol is
typically necessary in the culture medium. Ammonium sulfate or the
like can be used as the nitrogen source. Otherwise, small amounts
of the trace components such as potassium phosphate, sodium
phosphate, magnesium sulfate, ferrous sulfate, and manganese
sulfate may be added to the medium.
[0122] The culture is usually performed under aerobic conditions,
for example, shaking or stirring for aeration, at pH 5 to 9 and a
temperature of 20 to 45.degree. C., and it is usually completed
within 24 to 120 hours.
[0123] Collection of the L-amino acids from the culture can be
attained by a combination of known methods, such as by using ion
exchange resin, precipitation, and others.
[0124] Furthermore, the Methylophilus bacterial cells can be
separated from the medium by typical methods which are know in the
art for separating microbial cells.
[0125] <2> Gene of the Present Invention
[0126] The DNA of the present invention is a gene which codes for
one of the following enzymes: aspartokinase (henceforth also
abbreviated as "AK"), aspartic acid semialdehyde dehydrogenase
(henceforth also abbreviated as "ASD"), dihydrodipicolinate
synthase (henceforth also abbreviated as "DDPS"),
dihydrodipicolinate reductase (henceforth also abbreviated as
"DDPR"), and diaminopimelate decarboxylase (henceforth also
abbreviated as "DPDC") derived from Methylophilus
methylotrophus.
[0127] The DNA of the present invention can be obtained by, for
example, transforming a mutant strain of a microorganism which is
deficient in AK, ASD, DDPS, DDPR, or DPDC using a gene library from
Methylophilus methylotrophus, and selecting a clone in which the
auxotrophy is recovered.
[0128] A gene library of Methylophilus methylotrophus can be
produced as follows, for example. First, total chromosomal DNA is
prepared from a Methylophilus methylotrophus wild-type strain, for
example, the Methylophilus methylotrophus AS1 strain (NCIMB 10515),
by the method of Saito et al. (Saito, H. and Miura, K., Biochem.
Biophys. Acta 72, 619-629, (1963)) or the like, and partially
digested with a suitable restriction enzyme, for example, Sau3AI or
AluI, to obtain a mixture of various fragments. By controlling the
degree of the digestion by adjusting the digestion reaction time
and so forth, a wide range of restriction enzymes can be used.
[0129] Subsequently, the digested chromosomal DNA fragments are
ligated to vector DNA which is able to autonomously replicate in
Escherichia coli cells to produce recombinant DNA. Specifically, a
restriction enzyme producing the same terminal nucleotide sequence
as that produced by the restriction enzyme used for the digestion
of chromosomal DNA is allowed to act on the vector DNA to fully
digest and cleave the vector. Then, the mixture of chromosome DNA
fragments and the digested and cleaved vector DNA are ligated with
a ligase, preferably T4 DNA ligase, to obtain recombinant DNA.
[0130] A gene library solution can be obtained by transforming
Escherichia coli, for example, the Escherichia coli JM109 strain or
the like, with the recombinant DNA, and preparing recombinant DNA
from the culture broth of the transformant. This transformation can
be performed by the method of D. M. Morrison (Methods in Enzymology
68, 326 (1979)), the method of treating recipient cells with
calcium chloride so as to increase the permeability of DNA (Mandel,
M. and Higa, A., J. Mol. Biol., 53, 159 (1970)), and so forth. In
the examples mentioned hereinafter, electroporation was used.
[0131] Vectors which can be used as described above include pUC19,
pUC18, pUC118, pUC119, pBR322, pHSG299, pHSG298, pHSG399, pHSG398,
RSF1010, pMW119, pMW118, pMW219, pMW218, pSTV28, pSTV29, and so
forth. Phage vectors can also be used. Since pUC118 and pUC119
contain an ampicillin resistance gene, and pSTV28 and pSTV29
contain a chloramphenicol resistance gene, for example, only
transformants with the vector or the recombinant DNA can be grown
in a medium containing ampicillin or chloramphenicol.
[0132] The alkali SDS method and the like can be used to culture
the transformants and collect the recombinant DNA from the
bacterial cells.
[0133] A mutant microbial strain which does not contain AK, ASD,
DDPS, DDPR, or DPDC is transformed by using the gene library
solution of Methylophilus methylotrophus as described above, and
clones whose auxotrophy is recovered are selected.
[0134] Examples of a mutant microbial strain deficient in AK
include E. coli GT3 deficient in three different genes coding for
AK (thrA, metLM, lysC). Examples of a mutant microbial strain
deficient in ASD include E. coli Hfr3000 U482 (CGSC 5081 strain).
Examples of a mutant microbial strain deficient in DDPS include E.
coli AT997 (CGSC 4547 strain). Examples of a mutant microbial
strain deficient in DDPR include E. coli AT999 (CGSC 4549 strain).
Examples of a mutant microbial strain deficient in DPDC include E.
coli AT2453 (CGSC 4505 strain). These mutant strains can be
obtained from the E. coli Genetic Stock Center (the Yale
University, Department of Biology, Osborn Memorial Labs., P.O. Box
6666, New Haven 06511-7444, Connecticut, U.S.).
[0135] Although all of the aforementioned mutant strains cannot
grow in M9 minimal medium, transformant strains which contain a
gene coding for AK, ASD, DDPS, DDPR, or DPDC can grow in M9 minimal
medium because these genes are able to function in the
transformants. Therefore, by selecting transformant strains that
can grow in the minimal medium and collecting recombinant DNA from
the strains, DNA fragments containing a gene that codes for each
enzyme can be obtained. E. coli AT999 (CGSC 4549 strain) shows an
extremely slow growth rate even in a complete medium such as L
medium when diaminopimelic acid is not added to the medium.
However, normal growth is observed for the transformant strains
which contain the gene coding for DDPR derived from Methylophilus
methylotrophus, because of the function of the gene. Therefore, a
transformant strain that contains the gene coding for DDPR can also
be obtained by selecting a transformant strain which can normally
grow in L medium.
[0136] By extracting an insert DNA fragment from the recombinant
DNA and determining its nucleotide sequence, the amino acid
sequence of each enzyme and nucleotide sequence coding for it can
be determined.
[0137] The gene coding for AK of the present invention (henceforth
also referred to "ask") codes for AK which has the amino acid
sequence of SEQ ID NO: 6. As a specific example of the ask gene,
the DNA having the nucleotide sequence of SEQ ID NO: 5 can be used.
The ask gene of the present invention may have a sequence in which
the codon corresponding to each of the amino acids is replaced with
an equivalent codon so long as it codes for the same amino acid
sequence as shown in SEQ ID NO: 6.
[0138] The gene which codes for ASD of the present invention
(henceforth also referred to as "asd") codes for ASD which has the
amino acid sequence of SEQ ID NO: 8. As a specific example of the
asd gene, the DNA which contains the nucleotide sequence of
nucleotide 98-1207 shown in SEQ ID NO: 7 can be used. The asd gene
of the present invention may have a sequence in which the codon
corresponding to each of the amino acids is replaced with an
equivalent codon so long as it codes for the same amino acid
sequence as shown in SEQ ID NO: 8.
[0139] The gene which codes for DDPS of the present invention
(henceforth also referred to as "dapA") codes for DDPS which has
the amino acid sequence of SEQ ID NO: 10. As a specific example of
the dapA gene, the DNA which has the nucleotide sequence of
nucleotide 1268-2155 in SEQ ID NO: 9 can be used. The dapA gene of
the present invention may have a sequence in which the codon
corresponding to each of the amino acids is replaced with an
equivalent codon so long as it codes for the same amino acid
sequence as shown in SEQ ID NO: 10.
[0140] The gene which codes for DDBR of the present invention
(henceforth also referred to as "dapB") codes for DDBR which has
the amino acid sequence of SEQ ID NO: 12. As a specific example of
the dapB gene, the DNA which has the nucleotide sequence of numbers
2080-2883 in SEQ ID NO: 11 can be used. The dapB gene of the
present invention may have a sequence in which the codon
corresponding to each of the amino acids is replaced with an
equivalent codon so long as it codes for the same amino acid
sequence as shown in SEQ ID NO: 12.
[0141] The gene which codes for DPDC of the present invention
(henceforth also referred to as "lysA") codes for DPDC which has
the amino acid sequence of SEQ ID NO: 14. As a specific example of
the lysA gene, the DNA which has the nucleotide sequence of numbers
751-1995 in SEQ ID NO: 13 can be used. The lysA gene of the present
invention may have a sequence in which the codon corresponding to
each of the amino acids is replaced with an equivalent codon so
long as it codes for the same amino acid sequence as shown in SEQ
ID NO: 14.
[0142] The enzymes of the present invention may have the amino acid
sequences of SEQ ID NO: 6, 8, 10, 12, or 14, and these sequences
may include substitutions, deletions, insertions, additions, or
inversions of one or several amino acids, as long as the activity
of the enzyme is maintained. The expression "one or several" used
herein preferably means 1 to 10, more preferably 1 to 5, and more
preferably 1 to 2.
[0143] The DNA which codes for the substantially same protein as
AK, ASD, DDPS, DDPR, or DPDC such as those described above can be
obtained by modifying each nucleotide sequence so that the encoded
amino acid sequence contains substitutions, deletions, insertions,
additions, or inversions of an amino acid(s) at a particular site
by, for example, site-specific mutagenesis. This modified DNA may
also be obtained using conventional mutagenesis treatments.
Examples of mutagenesis treatments include in vitro treatment of
DNA coding for AK, ASD, DDPS, DDPR or DPDC with hydroxylamine or
the like, treatment of a microorganism such as Escherichia bacteria
containing a gene coding for AK, ASD, DDPS, DDPR or DPDC with UV
irradiation or with typical mutagenesis agents such as
N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and nitrous acid.
[0144] The aforementioned substitution, deletion, insertion,
addition, or inversion of nucleotides and/or amino acids includes
naturally occurring mutations (mutant or variant) such as those
between species or strains of microorganisms containing AK, ASD,
DDPS, DDPR, or DPDC, and so forth.
[0145] The DNA which codes for substantially the same protein as
AK, ASD, DDPS, DDPR or DPDC can be obtained by expressing a DNA
having such a mutation as described above in a suitable cell, and
examining the AK, ASD, DDPS, DDPR, or DPDC activity of the
expression product. The DNA which codes for substantially the same
protein as AK, ASD, DDPS, DDPR or DPDC can also be obtained by
isolating, a DNA which is able to hybridize to a probe containing a
nucleotide sequence of numbers 510-1736 of SEQ ID NO: 5, a
nucleotide sequence of numbers 98-1207 of SEQ ID NO: 7, a
nucleotide sequence of numbers 1268-2155 of SEQ ID NO: 9, a
nucleotide sequence of numbers 2080-2883 of SEQ ID NO: 11, or a
nucleotide sequence of numbers 751-1995 of SEQ ID NO: 13, or
portions of these nucleotide sequences under stringent conditions,
and coding for a protein having AK, ASD, DDPS, DDPR or DPDC
activity. In the present specification, to have a nucleotide
sequence or a portion thereof means to have the nucleotide sequence
or the portion thereof, or a nucleotide complementary thereto.
[0146] The term "stringent conditions" used herein means conditions
that allow for formation of a so-called specific hybrid and does
not allow for formation of a non-specific hybrid. These conditions
may vary depending on the nucleotide sequence and length of the
probe. However, for example, conditions that allow for
hybridization of highly homologous DNA such as DNA having homology
of 40% or higher, but does not allow for hybridization of DNA of
lower homology than defined above, or conditions that allow for
hybridization using washing conditions typical in Southern
hybridization, of a temperature of 60.degree. C. and salt
concentrations corresponding to 1.times.SSC and 0.1% SDS,
preferably 0.1 x SSC and 0.1% SDS.
[0147] A partial sequence of each gene can also be used as the
probe. Such a probe can be produced by PCR (polymerase chain
reaction) using oligonucleotides produced based on the nucleotide
sequence of each gene as primers and a DNA fragment containing each
gene as the template. When a DNA fragment having a length of about
300 bp is used as the probe, washing conditions for hybridization
may be, for example, 50.degree. C., 2.times.SSC and 0.1% SDS.
[0148] Genes that hybridize under conditions as described above
also include those having a stop codon in its sequence and those
encoding an enzyme which has lost its activity due to a mutation in
the active center. However, such genes can readily be eliminated by
ligating the genes to a commercially available activity expression
vector, and measuring AK, ASD, DDPS, DDPR or DPDC activity.
[0149] Since the nucleotide sequences of the genes that code for
AK, ASD, DDPS, DDPR and DPDC derived from Methylophilus
methylotrophus are first described herein, DNA sequences which code
for AK, ASD, DDPS, DDPR, and DPDC can be obtained from a
Methylophilus methylotrophus gene library by hybridization using
oligonucleotide probes produced based on the reported sequences.
Moreover, DNA sequences which code for these enzymes can also be
obtained by amplifying them from Methylophilus methylotrophus
chromosomal DNA by PCR using oligonucleotide primers produced based
on the aforementioned nucleotide sequences.
[0150] The aforementioned genes can suitably be utilized to enhance
the ability of Methylophilus bacteria to produce L-lysine.
EXAMPLES
[0151] The present invention will further specifically be explained
with reference to the following non-limiting examples.
[0152] The reagents used were obtained from Wako Pure Chemicals or
Nakarai Tesque unless otherwise indicated. The compositions of the
media used in each example are shown below. pH was adjusted with
NaOH or HCl for all media.
[0153] L Medium:
TABLE-US-00001 Bacto trypton (DIFCO) 10 g/L Yeast extract (DIFCO) 5
g/L NaCl 5 g/L
[0154] steam-sterilized at 120.degree. C. for 20 minutes
[0155] L Agar Medium:
TABLE-US-00002 L medium Bacto agar (DIFCO) 15 g/L
[0156] steam-sterilized at 120.degree. C. for 20 minutes
[0157] SOC Medium:
TABLE-US-00003 Bacto trypton (DIFCO) 20 g/L Yeast extract (DIFCO) 5
g/L 10 mM NaCl 2.5 mM KCl 10 mM MgSO.sub.4 10 mM MgCl.sub.2 20 mM
Glucose
[0158] The constituents except for the magnesium solution and
glucose were steam-sterilized (120.degree. C., 20 minutes), then 2
M magnesium stock solution (1 M MgSO.sub.4, 1 M MgCl.sub.2) and 2 M
glucose solution, which had been passed through a 0.22-.mu.m
filter, were added thereto, and the mixture was passed through a
0.22-.mu.m filter again.
[0159] 121M1 Medium:
TABLE-US-00004 K.sub.2HPO.sub.4 1.2 g/L KH.sub.2PO.sub.4 0.62 g/L
NaCl 0.1 g/L (NH.sub.4).sub.2SO.sub.4 0.5 g/L
MgSO.sub.4.cndot.7H.sub.2O 0.2 g/L CaCl.sub.2.cndot.6H.sub.2O 0.05
g/L FeCl.sub.3.cndot.6H.sub.2O 1.0 mg/L H.sub.3BO.sub.3 10 .mu.g/L
CuSO.sub.4.cndot.5H.sub.2O 5 .mu.g/L MnSO.sub.4.cndot.5H.sub.2O 10
.mu.g/L ZnSO.sub.4.cndot.7H.sub.2O 70 .mu.g/L
NaMoO.sub.4.cndot.2H.sub.2O 10 .mu.g/L CoCl.sub.2.cndot.6H.sub.2O 5
.mu.g/L Methanol 1% (vol/vol), pH 7.0
[0160] The constituents except for methanol were steam-sterilized
at 121.degree. C. for 15 minutes. After the constituents
sufficiently cooled, methanol was added.
[0161] Composition of 121 production medium:
TABLE-US-00005 Methanol 2% Dipotassium phosphate 0.12% Potassium
phosphate 0.062% Calcium chloride hexahydrate 0.005% Magnesium
sulfate heptahydrate 0.02% Sodium chloride 0.01% Ferric chloride
hexahydrate 1.0 mg/L Ammonium sulfate 0.3% Cupric sulfate
pentahydrate 5 .mu.g/L Manganous sulfate pentahydrate 10 .mu.g/L
Sodium molybdate dihydrate 10 .mu.g/L Boric acid 10 .mu.g/L Zinc
sulfate heptahydrate 70 .mu.g/L Cobaltous chloride hexahydrate 5
.mu.g/L Calcium carbonate (Kanto Kagaku) 3% pH 7.0
[0162] 121M1 Agar Medium:
TABLE-US-00006 121M1 medium 15 g/L Bacto agar (DIFCO)
[0163] The constituents except for methanol were steam-sterilized
at 121.degree. C. for 15 minutes. After the constituents
sufficiently cooled, methanol was added.
[0164] M9 Minimal Medium:
TABLE-US-00007 Na.sub.2HPO.sub.4.cndot.12H.sub.2O 16 g/L
KH.sub.2PO.sub.4 3 g/L NaCl 0.5 g/L NH.sub.4Cl 1 g/L
MgSO.sub.4.cndot.7H.sub.20 246.48 mg/L Glucose 2 g/L pH 7.0
[0165] MgSO.sub.4 and glucose were separately sterilized
(120.degree. C., 20 minutes) and added. A suitable amount of amino
acids and vitamins were added as required.
[0166] M9 Minimal Agar Medium:
TABLE-US-00008 M9 minimal medium 15 g/L Bacto agar (DIFCO)
Example 1
[0167] Creation of L-lysine-Producing Bacterium: (I)
[0168] (1) Introduction of Mutant lysC and Mutant dapA into
Methylophilus Bacterium
[0169] A Methylophilus bacterium was transformed with plasmid
RSFD80 (see WO95/16042) which contained a mutant lysC and a mutant
dapA. RSFD80 is plasmid pVIC40 (International Publication
WO90/04636, Japanese Patent Application Laid-open (Kohyo) No.
3-501682/1991) derived from the broad host spectrum vector plasmid
pAYC32 (Chistorerdov, A. Y., Tsygankov, Y. D., Plasmid, 16,
161-167, (1986)), which is a derivative of RSF1010. RSF1010
contains a mutant dapA and a mutant lysC derived from E. coli
located downstream of the promoter (tetP) of the tetracycline
resistance gene of pVIC40 in this order, so that the transcription
directions of the genes are ordinary with respect to tetP. The
mutant dapA codes for a mutant DDPS with a tyrosine in place of the
histidine at position 118. The mutant lysC codes for a mutant AKIII
with an isoleucine in place of the threonine at position 352.
[0170] RSFD80 was constructed as follows. The mutant dapA on
plasmid pdapAS24 was ligated to pVIC40 downstream of the promoter
of the tetracycline resistance gene to obtain RSF24P as shown in
FIG. 1. Then, the plasmid RSFD80 which had the mutant dapA and a
mutant lysC was prepared from RSF24P and pLLC*80 containing the
mutant lysC as shown in FIG. 2. That is, while pVIC40 contains a
threonine operon, this threonine operon is replaced with a DNA
fragment containing the mutant dapA and a DNA fragment containing
the mutant lysC in RSFD80.
[0171] The E. coli JM109 strain transformed with the RSFD80 plasmid
was designated AJ12396, and deposited at the National Institute of
Bioscience and Human-Technology, Agency of Industrial Science and
Technology, Ministry of International Trade and Industry (postal
code 305-8566, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken,
Japan) on Oct. 28, 1993 and received an accession number of FERM
P-13936, and it was converted to an international deposit under the
provisions of the Budapest Treaty on Nov. 1, 1994, and received an
accession number of FERM BP-4859.
[0172] The E. coli AJ1239 strain was cultured in 30 ml of LB medium
containing 20 mg/L of streptomycin at 30.degree. C. for 12 hours,
then the RSFD80 plasmid was purified from the cells using
Wizard.RTM. Plus Midipreps DNA Purification System (sold by
Promega).
[0173] The RSFD80 plasmid as described above was introduced into
the Methylophilus methylotrophus AS1 strain (NCIMB 10515) by
electroporation (Canadian Journal of Microbiology, 43, 197 (1997)).
As a control, the DNA region coding for the threonine operon was
deleted from the pVIC40 plasmid from which the RSFD80 plasmid was
derived, to produce a pRS plasmid having only the vector region
(see Japanese Patent Application Laid-open (Kohyo) No.
3-501682/1991). The pRS plasmid was introduced into the AS1 strain
in the same manner as that used for RSFD80.
[0174] (2) AKIII Activity of Methylophilus Bacterium Containing
mutant lysC and Mutant dapA Derived from E. coli
[0175] Cell-free extracts were prepared from the Methylophilus
methylotrophus AS1 strain containing the RSFD80 plasmid (also
referred to as "AS1/RSFD80" hereinafter) and the Methylophilus
methylotrophus AS1 strain containing the pRS plasmid (also referred
to as "AS1/pRS" hereinafter), and AK activity was measured. The
cell-free extracts (crude enzyme solutions) were prepared as
follows. The AS1/RSFD80 strain and AS1/pRS strain were each
inoculated into the above-described 121 production medium
containing 20 mg/L of streptomycin, cultured at 37.degree. C. for
34 hours with shaking, and then calcium carbonate was removed and
cells were harvested.
[0176] The bacterial cells obtained as described above were washed
with 0.2% KCl at 0.degree. C., suspended in 20 mM potassium
phosphate buffer (pH 7) containing 10 mM MgSO.sub.4, 0.8 M
(NH.sub.4).sub.2SO.sub.4 and 0.03 M .beta.-mercaptoethanol, and
disrupted by sonication (0.degree. C., 200 W, 10 minutes). The
sonicated cell suspension was centrifuged at 33,000 rpm for 30
minutes at 0.degree. C., and the supernatant was removed. To the
supernatant, ammonium sulfate was added to 80% saturation, and the
mixture was left at 0.degree. C. for 1 hour, and centrifuged. The
pellet was dissolved in 20 mM potassium phosphate buffer (pH 7)
containing 10 mM MgSO.sub.4, 0.8 M (NH.sub.4).sub.2SO.sub.4 and
0.03 M .beta.-mercaptoethanol.
[0177] AK activity was measured in accordance with the method of
Stadtman (Stadtman, E. R., Cohen, G. N., LeBras, G., and
Robichon-Szulmajster, H., J. Biol. Chem., 236, 2033 (1961)). That
is, a following reaction solution was incubated at 30.degree. C.
for 45 minutes, resulting in color development (2.8 N HCl: 0.4 ml,
12% TCA: 0.4 ml, 5% FeCl.sub.3.6H.sub.2O/0.1 N HCl: 0.7 ml). The
reaction solution was centrifuged, and absorbance of the
supernatant was measured at 540 nm. The activity was expressed in
terms of the amount of hydroxamic acid produced in 1 minute (1 U=1
.mu.mol/minute). The molar extinction coefficient was set at 600. A
reaction solution without potassium aspartate was used as a blank.
When the enzymatic activity was measured, L-lysine was added to the
enzymatic reaction solution at various concentrations to examine
the degree of inhibition by L-lysine. The results are shown in
Table 1.
[0178] Composition of Reaction Solution:
TABLE-US-00009 Reaction mixture .sup.*1 0.3 ml Hydroxylamine
solution .sup.*2 0.2 ml 0.1 M Potassium aspartate (pH 7.0) 0.2 ml
Enzyme solution 0.1 ml Water (balance) Total 1 ml .sup.*1 M
Tris-HCl (pH 8.1): 9 ml, 0.3 M MgSO.sub.4: 0.5 ml and 0.2 M ATP (pH
7.0): 5 ml .sup.*2 8 M Hydroxylamine solution neutralized with KOH
immediately before use
TABLE-US-00010 TABLE 1 AK activity Specific Desensitization
(Specific activity with degree of Strain activity.sup.*1) 5 mM
L-lysine inhibition.sup.*2 (%) AS1/pRS 7.93 9.07 114 AS1/RSFD80
13.36 15.33 115 .sup.*1nmol/minute/mg protein .sup.*2Activity
retention ratio in the presence of 5 mM L-lysine
[0179] As shown in Table 1, AK activity was increased by about 1.7
times by the introduction of the RSFD80 plasmid. Furthermore, it
was confirmed that the inhibition by L-lysine was completely
desensitized in E. coli AK encoded by the RSFD80 plasmid. Moreover,
it was found that AK that was originally retained by the AS1 strain
was not inhibited by L-lysine alone. The inventors of the present
invention have discovered that the AK derived from the AS1 strain
was inhibited by 100% when 2 mM each of L-lysine and L-threonine
were present in the reaction solution (concerted inhibition).
[0180] (3) Production of L-lysine by Methylophilus Bacterium
Containing Mutant lysC and Mutant dapA Derived from E. coli
[0181] Then, the AS1/RSFD80 strain and the AS1/pRS strain were
inoculated into 121 production medium containing 20 mg/L of
streptomycin, and cultured at 37.degree. C. for 34 hours with
shaking. After the culture was completed, the bacterial cells and
calcium carbonate were removed by centrifugation, and L-lysine
concentration in the culture supernatant was measured by an amino
acid analyzer (JASCO Corporation [Nihon Bunko], high performance
liquid chromatography). The results are shown in Table 2.
TABLE-US-00011 TABLE 2 Production amount of L-lysine Strain
hydrochloride (g/L) AS1/pRS 0 AS1/RSFD80 0.3
Example 2
[0182] Creation of L-lysine-Producing Bacterium (II)
[0183] (1) Introduction of the Tac Promoter Region into a Broad
Host Spectrum Vector
[0184] In order to produce a large amount of a L-lysine
biosynthetic enzyme in Methylophilus methylotrophus, the tac
promoter was used for gene expression of this target enzyme. This
promoter is frequently used in E. coli.
[0185] The tac promoter region was obtained by amplification
through PCR using pKK233-3 (Pharmacia) as the template, DNA
fragments having the nucleotide sequences of SEQ ID NOS: 15 and 16
as primers, and a heat-resistant DNA polymerase. The PCR was
performed with cycles of 94.degree. C. for 20 seconds, 60.degree.
C. for 30 seconds, and 72.degree. C. for 60 seconds, repeated 30
times. Then, the amplified DNA fragment was collected and treated
with restriction enzymes EcoRI and PstI. The broad host spectrum
vector pRS (see Japanese Patent Application Laid-open (Kohyo) No.
3-501682/1991) was also digested with the same restriction enzymes,
and the aforementioned DNA fragment containing the tac promoter
region was introduced into the restriction enzyme digestion termini
to construct pRS-tac.
[0186] (2) Preparation of dapA Gene (Dihydrodipicolinate Synthase
Gene) Expression Plasmid pRS-dapA24 and lysC Gene (Aspartokinase
Gene) Expression Plasmid pRS-lysC80
[0187] A mutant gene (dapA*24) coding for dihydrodipicolinate
synthase with partially desensitized feedback inhibition by Lys was
was introduced into the plasmid pRS-tac, which was prepared as
described above (1).
[0188] First, the dapA*24 gene region was obtained by amplification
through PCR using RSFD80 (see Example 1) as the template, and DNA
fragments having the nucleotide sequences of SEQ ID NOS: 17 and 18
as primers. The PCR was performed with cycles of 94.degree. C. for
20 seconds, 60.degree. C. for 30 seconds, and 72.degree. C. for 90
seconds, repeated 30 times. Then, the fragment was treated with
restriction enzymes Sse8387I and XbaI. pRS-tac was also treated
with Sse8387I and partially digested with XbaI in the same manner
as described above. To this digested plasmid, the aforementioned
dapA*24 gene fragment was ligated with T4 ligase to obtain
pRS-dapA24.
[0189] Similarly, the gene (lysC*80) coding for aspartokinase with
partially desensitized feedback inhibition by Lys was obtained by
PCR using RSFD80 as the template, and DNA fragments having the
nucleotide sequences of SEQ ID NOS: 19 and 20 as primers. The PCR
was performed with cycles of 94.degree. C. for 20 seconds,
60.degree. C. for 30 seconds, and 72.degree. C. for 90 seconds,
repeated 30 times. Then, the DNA fragment was treated with
restriction enzymes Sse8387I and SapI. The vector pRS-tac was also
treated with Sse8387I and SapI. To this digested plasmid, the
aforementioned lysC*80 gene fragment was ligated with T4 ligase to
obtain pRS-lysC80.
[0190] (3) Introduction of pRS-dapA24 or pRS-lysC80 into
Methylophilus methylotrophus and Evaluation of the Culture
[0191] pRS-dapA24 and pRS-lysC80 obtained as described above were
each separately introduced into the Methylophilus methylotrophus
AS1 strain (NCIMB 10515) by electroporation to obtain
AS1/pRS-dapA24 and AS1/pRS-lysC80, respectively. Each strain was
inoculated into 121 production medium containing 20 mg/L of
streptomycin, and cultured at 37.degree. C. for 48 hours with
shaking. As a control strain, AS1 strain harboring pRS was also
cultured in a similar manner. After the culture was completed, the
cells and calcium carbonate were removed by centrifugation, and the
L-lysine concentration in the culture supernatant was measured by
an amino acid analyzer (JASCO Corporation [Nihon Bunko], high
performance liquid chromatography). The results are shown in Table
3.
TABLE-US-00012 TABLE 3 Production amount of L-lysine Strain
hydrochloride (g/L) AS1/pRS <0.01 AS1/pRS-lysC80 0.06
AS1/pRS-dapA24 0.13
Example 3
[0192] Creation of L-lysine-Producing Bacterium (III)
[0193] The Methylophilus methylotrophus AS1 strain (NCIMB10515) was
inoculated into 121M1 medium and cultured at 37.degree. C. for 15
hours. The obtained bacterial cells were treated with NTG in a
conventional manner (NTG concentration: 100 mg/L, 37.degree. C., 5
minutes), and spread onto 121M1 agar medium containing 7 g/L of
S-(2-aminoethyl)-cysteine (AEC) and 3 g/L of L-threonine. The cells
were cultured at 37.degree. C. for 2 to 8 days, and the colonies
which formed were picked up to obtain AEC-resistant strains.
[0194] The aforementioned AEC-resistant strains were inoculated
into 121 production medium, and cultured at 37.degree. C. for 38
hours under aerobic conditions. After the culture was completed,
the cells and calcium carbonate were removed from the medium by
centrifugation, and the L-lysine concentration in the culture
supernatant was measured by an amino acid analyzer (JASCO
Corporation [Nihon Bunko], high performance liquid chromatography).
The strain with improved L-lysine-producing ability as compared
with the parent strain was selected, and designated Methylophilus
methylotrophus AR-166 strain. The L-lysine production amounts of
the parent strain (AS1 strain) and the AR-166 strain are shown in
Table 4.
TABLE-US-00013 TABLE 4 Production amount of L-lysine Strain
hydrochloride (mg/L) AS1 5.8 AR-166 80
[0195] The Methylophilus methylotrophus AR-166 strain was given a
private number of AJ13608, and was deposited at the National
Institute of Bioscience and Human-Technology, Agency of Industrial
Science and Technology, Ministry of International Trade and
Industry (postal code 305-8566, 1-3 Higashi 1-chome, Tsukuba-shi,
Ibaraki-ken, Japan) on Jun. 10, 1999 and received an accession
number of FERM P-17416, and it was converted to an international
deposit under the provisions of the Budapest Treaty on Mar. 31,
2000, and received an accession number of FERM BP-7112.
Example 4
[0196] Creation of L-threonine-Producing Bacterium
[0197] (1) Introduction of Threonine Operon Plasmid into a
Methylophilus Bacterium
[0198] Plasmid pVIC40 (International Publication WO90/04636,
Japanese Patent Application Laid-open (Kohyo) No. 3-501682/1991)
containing a threonine operon derived from E. coli was introduced
into the Methylophilus methylotrophus AS1 strain (NCIMB 10515) by
electroporation (Canadian Journal of Microbiology, 43, 197 (1997))
to obtain AS1/pVIC40 strain. As a control, pRS (Japanese Patent
Application Laid-open (Kohyo) No. 3-501682/1991) with only the
vector region was obtained by deleting the DNA region coding for
the threonine operon from the pVIC40 plasmid, and it was introduced
into the AS1 strain in the same manner as for pVIC40 to obtain
AS1/pRS strain.
[0199] (2) Production of L-threonine by Methylophilus Bacterium
Containing the Threonine Operon Derived from E. coli
[0200] The AS1/pVIC40 and AS1/pRS strains were each inoculated into
121 production medium containing 20 mg/L of streptomycin, 1 g/l of
L-valine and 1 g/l of L-leucine, and cultured at 37.degree. C. for
50 hours with shaking. After the culture was completed, the cells
and calcium carbonate were removed by centrifugation, and the
L-threonine concentration in the culture supernatant was measured
by an amino acid analyzer (JASCO Corporation [Nihon Bunko], high
performance liquid chromatography). The results are shown in Table
5.
TABLE-US-00014 TABLE 5 Production amount of Strain L-threonine
(mg/L) AS1/pRS 15 AS1/pVIC40 30
Example 5
[0201] Creation of Branched Chain Amino Acid-Producing
Bacterium
[0202] The Methylophilus methylotrophus AS1 strain (NCIMB 10515)
was inoculated into 121M1 medium and cultured at 37.degree. C. for
15 hours. The obtained bacterial cells were treated with NTG in a
conventional manner (NTG concentration: 100 mg/L, 37.degree. C., 5
minutes), and spread onto 121M1 agar medium containing 0.5% of
casamino acid (DIFCO). The cells were cultured at 37.degree. C. for
2 to 8 days, and allowed to form colonies. The colonies were picked
up, and inoculated into 121M1 agar medium and 121M1 agar medium
containing 0.5% casamino acid. Strains exhibiting better growth on
the latter medium compared with on the former medium were selected
as casamino acid auxotrophic strains. In this way, 9 leaky casamino
acid auxotrophic strains were obtained from the NTG-treated 500
strains. From these casamino acid auxotrophic strains, one strain
produced more L-valine, L-leucine, and L-isoleucine in the medium
as compared with its parent strain. This strain was designated
Methylophilus methylotrophus C138 strain.
[0203] The Methylophilus methylotrophus C138 strain was given a
private number of AJ13609, and was deposited at the National
Institute of Bioscience and Human-Technology, Agency of Industrial
Science and Technology, Ministry of International Trade and
Industry (postal code 305-8566, 1-3 Higashi 1-chome, Tsukuba-shi,
Ibaraki-ken, Japan) on Jun. 10, 1999 and received an accession
number of FERM P-17417, and it was converted to an international
deposit under the provisions of the Budapest Treaty on Mar. 31,
2000, and received an accession number of FERM BP-7113.
[0204] The parent strain (AS1 strain) and the C138 strain were
inoculated into 121 production medium, and cultured at 37.degree.
C. for 34 hours under aerobic conditions. After the culture was
completed, the cells and calcium carbonate were removed from the
medium by centrifugation, and the concentrations of L-valine,
L-leucine, and L-isoleucine in the culture supernatant were
measured by an amino acid analyzer (JASCO Corporation [Nihon
Bunko], high performance liquid chromatography). The results are
shown in Table 6.
TABLE-US-00015 TABLE 6 Strain L-valine (mg/L) L-leucine (mg/L)
L-isoleucine (mg/L) AS1 7.5 5.0 2.7 C138 330 166 249
Example 6
[0205] Preparation of Chromosomal DNA Library of Methylophilus
methylotrophus AS1 Strain
[0206] (1) Preparation of Chromosome DNA of Methylophilus
methylotrophus AS1 Strain
[0207] One platinum loop of the Methylophilus methylotrophus AS1
strain (NCIMB 10515) was inoculated into 5 ml of 121M1 medium in a
test tube, and cultured at 37.degree. C. overnight with shaking.
Then, the culture broth was inoculated into 50 ml of 121M1 medium
in a 500 ml-volume Sakaguchi flask to 1%, and cultured at
37.degree. C. overnight with shaking. Then, the cells were
harvested by centrifugation, and suspended in 50 ml of TEN solution
(solution containing 50 mM Tris-HCl (pH 8.0), 10 mM EDTA and 20 mM
NaCl (pH 8.0)). The cells were collected by centrifugation, and
suspended again in 5 ml of the TEN solution containing 5 mg/ml of
lysozyme and 10 .mu.g/ml of RNase A. The suspension was maintained
at 37.degree. C. for 30 minutes, and then proteinase K and sodium
laurylsulfate were added thereto to final concentrations of 10
.mu.g/ml and 0.5% (wt/vol), respectively.
[0208] The suspension was maintained at 70.degree. C. for 2 hours,
and then an equal amount of a saturated solution of phenol (phenol
solution saturated with 10 mM Tris-HCl (pH 8.0)) was added and
mixed. The suspension was centrifuged, and the supernatant was
collected. An equal amount of phenol/chloroform solution
(phenol:chloroform:isoamyl alcohol=25:24:1) was added and mixed,
and the mixture was centrifuged. The supernatant was collected, and
an equal amount of chloroform solution (chloroform:isoamyl
alcohol=24:1) was added thereto to repeat the same extraction
procedure. To the supernatant, a 1/10 volume of 3 M sodium acetate
(pH 4.8) and 2.5-fold volume of ethanol were added to precipitate
the chromosomal DNA. The precipitates were collected by
centrifugation, washed with 70% ethanol, dried under reduced
pressure, and dissolved in a suitable amount of TE solution (10 mM
Tris-HCl, 1 mM EDTA (pH 8.0)).
[0209] (2) Preparation of the Gene Library
[0210] A 50 .mu.l portion of the chromosomal DNA (1 .mu.g/.mu.l)
obtained in the above (1), 20 .mu.l of H buffer (500 mM Tris-HCl,
100 mM MgCl.sub.2, 10 mM dithiothreitol, 1000 mM NaCl (pH
.sup.7.5)) and 8 units of a restriction enzyme Sau3AI (Takara
Shuzo) were allowed to react at 37.degree. C. for 10 minutes in a
total volume of 200 .mu.l, and then 200 .mu.l of the
phenol/chloroform solution was added and mixed to stop the
reaction. The reaction mixture was centrifuged, and the upper layer
was collected and separated on a 0.8% agarose gel. DNA
corresponding to 2 to 5 kilobase pair (henceforth abbreviated as
"kbp") was collected by using Concert.TM. Rapid Gel Extraction
System (DNA collecting kit, GIBCO BRL Co.). In this way, 50 .mu.l
of a solution of DNA with fractionated sizes was obtained.
[0211] 2.5 .mu.g of plasmid pUC118 (Takara Shuzo), 2 .mu.l of K
buffer (200 mM Tris-HCl, 100 mM MgCl.sub.2, 10 mM dithiothreitol,
1000 mM KCl (pH 8.5)) and 10 units of restriction enzyme BamHI
(Takara Shuzo) were allowed to react at 37.degree. C. for 2 hours
in a total volume of 20 .mu.l, then 20 units of calf small
intestine alkaline phosphatase (Takara Shuzo) was added and mixed,
and the mixture was allowed to react for an additional 30 minutes.
The reaction mixture was mixed with an equal amount of the
phenol/chloroform solution, and the mixture was centrifuged. The
supernatant was collected, and an equal amount of the chloroform
solution was added thereto to repeat a similar extraction
procedure. To the supernatant, a 1/10 volume of 3 M sodium acetate
(pH 4.8) and 2.5-fold volume of ethanol were added to precipitate
DNA. The DNA was collected by centrifugation, washed with 70%
ethanol, dried under reduced pressure, and dissolved in a suitable
amount of TE solution.
[0212] A Sau3AI digestion product of the chromosomal DNA prepared
as described above and a BamHI digestion product of pUC118 were
ligated by using a Ligation Kit ver. 2 (Takara Shuzo). To the
reaction mixture, a 1/10 volume of 3 M sodium acetate (pH 4.8) and
2.5-fold volume of ethanol were added to precipitate DNA. The DNA
was collected by centrifugation, washed with 70% ethanol, dried
under reduced pressure, and dissolved in TE solution (Ligase
solution A).
[0213] In the same manner as in the above procedure, fragments
obtained by partial digestion of the chromosomal DNA with a
restriction enzyme AluI (Takara Shuzo) and a SmaI digestion product
of plasmid pSTV29 (Takara Shuzo) were ligated (Ligase solution
B).
[0214] One platinum loop of E. coli JM109 was inoculated into 5 ml
of L medium in a test tube, and cultured at 37.degree. C. overnight
with shaking. Then, the culture broth was inoculated into 50 ml of
L medium in a 500 ml-volume Sakaguchi flask to 1%, cultured at
37.degree. C. until OD.sub.660 of the culture became 0.5 to 0.6,
and cooled on ice for 15 minutes. Then, the cells were harvested by
centrifugation at 4.degree. C. The cells were suspended in 50 ml of
ice-cooled water and centrifuged to wash the cells. This operation
was repeated once again, and the cells were suspended in 50 ml of
ice-cooled 10% glycerol solution, and centrifuged to wash the
cells. The cells were suspended in an equal volume of 10% glycerol
solution, and divided into 50 .mu.l aliquots. To the cells in the
50 .mu.l volume, 1 .mu.l of Ligase solution A or Ligase solution B
prepared above was added. Then, the mixture was put into a special
cuvette (0.1 cm width, preliminarily ice-cooled) for an
electroporation apparatus of BioRad.
[0215] The setting of the apparatus was 1.8 kV and 25 .mu.F, and
the setting of pulse controller was 200 ohms. The cuvette was
mounted on the apparatus and pulses were applied thereto.
Immediately after the application of pulse, 1 ml of ice-cooled SOC
medium was added thereto, and the mixture was transferred to a
sterilized test tube, and cultured at 37.degree. C. for 1 hour with
shaking. Each cell culture broth was spread onto L agar medium
containing an antibiotic (100 .mu.g/ml of ampicillin when Ligase
solution A was used, or 20 .mu.g/ml of chloramphenicol when Ligase
solution B was used), and incubated at 37.degree. C. overnight. The
colonies which grew on each agar medium were scraped, inoculated
into 50 ml of L medium containing respective antibiotic in a 500
ml-volume Sakaguchi flask, and cultured at 37.degree. C. for 2
hours with shaking. Plasmid DNA was extracted from each culture
broth by the alkali SDS method to form Gene library solution A and
Gene library solution B, respectively.
Example 7
[0216] Cloning of the Lysine Biosynthesis Gene of Methylophilus
methylotrophus AS1 Strain
[0217] (1) Cloning of the Gene Coding for Aspartokinase (AK)
[0218] E. coli GT3 deficient in the three genes coding for AK
(thrA, metLM and lysC) was transformed with Gene library solution B
by the same electroporation procedure as mentioned above. SOC
medium containing 20 .mu.g/ml of diaminopimelic acid was added to
the transformation solution, and cultured at 37.degree. C. with
shaking. Then, the culture broth was spread onto L medium
containing 20 .mu.g/ml of diaminopimelic acid and 20 .mu.g/ml of
chloramphenicol, and colonies grew. This was replicated as a master
plate to M9 agar medium containing 20 .mu.g/ml of chloramphenicol,
and the replicate was incubated at 37.degree. C. for 2 to 3 days.
The host could not grow in M9 minimal medium without diaminopimelic
acid since it did not have AK activity. In contrast, it was
expected that the transformant strain that contained the gene
coding for AK derived from Methylophilus methylotrophus could grow
in M9 minimal medium because of the function of the gene.
[0219] Two transformants out of about 3000 transformants formed
colonies on M9 medium. Plasmids were extracted from the colonies
which emerged on M9 medium and analyzed. As a result, the presence
of an inserted fragment on the plasmids was confirmed. The plasmids
were designated pMMASK-1 and pMMASK-2, respectively. By using these
plasmids, E. coli GT3 was transformed again. The obtained
transformants grew on M9 minimal medium. Furthermore, the
transformant which contained each of these plasmids was cultured
overnight in L medium containing 20 .mu.g/ml of chloramphenicol,
and the cells were collected by centrifugation of the culture
broth. Cell-free extracts were prepared by sonicating the cells,
and AK activity was measured according to the method of Miyajima et
al. (Journal of Biochemistry (Tokyo), vol. 63, 139-148 (1968))
(FIG. 3: pMMASK-1, pMMASK-2). In addition, a GT3 strain harboring
the vector pSTV29 was similarly cultured in L medium containing 20
.mu.g/ml of diaminopimelic acid and 20 .mu.g/ml of chloramphenicol,
and AK activity was measured (FIG. 3: Vector). As a result,
increase in AK activity was observed in two of the clones
containing the inserted fragments compared with the transformant
harboring only the vector. Therefore, it was confirmed that the
gene that could be cloned on pSTV29 was the gene coding for AK
derived from Methylophilus methylotrophus. This gene was designated
as ask.
[0220] The DNA nucleotide sequence of the ask gene was determined
by the dideoxy method. It was found that pMMASK-1 and pMMASK-2
contained a common fragment. The nucleotide sequence of the DNA
fragment containing the ask gene derived from Methylophilus
methylotrophus is shown in SEQ ID NO: 5. The amino acid sequence
that can be encoded by the nucleotide sequence is shown in SEQ ID
NOS: 5 and 6.
[0221] (2) Cloning of Gene Coding for Aspartic Acid Semialdehyde
Dehydrogenase (ASD)
[0222] E. coli Hfr3000 U482 (CGSC 5081 strain) deficient in the asd
gene was transformed by electroporation using Gene library solution
B in the same manner as described above. To the transformation
solution, SOC medium containing 20 .mu.g/ml of diaminopimelic acid
was added and the mixture was cultured at 37.degree. C. with
shaking. The cells were harvested by centrifugation. The cells were
washed by suspending them in L medium and centrifuging the
suspension. The same washing operation was repeated once again, and
the cells were suspended in L medium. Then, the suspension was
spread onto L agar medium containing 20 .mu.g/ml of
chloramphenicol, and incubated overnight at 37.degree. C. The host
grew extremely slowly in L medium without diaminopimelic acid since
it was deficient in the asd gene. In contrast, it was expected that
normal growth would be observed for a transformant strain which
contained the gene coding for ASD derived from Methylophilus
methylotrophus even in L medium because of the function of the
gene. Furthermore, the host E. coli could not grow in M9 minimal
medium, but a transformant strain that contained the gene coding
for ASD derived from Methylophilus methylotrophus was expected to
be able to grow in M9 minimal medium because of the function of the
gene. Therefore, colonies of transformants that normally grew on L
medium were picked up, streaked and cultured on M9 agar medium. As
a result, growth was observed. Thus, it was confirmed that the gene
coding for ASD functioned in these transformant strains as
expected.
[0223] Plasmids were extracted from the three transformant strains
which emerged on M9 medium, and the presence of an inserted
fragment in the plasmids was confirmed. The plasmids were
designated pMMASD-1, pMMASD-2 and pMMASD-3, respectively. When the
E. coli Hfr3000 U482 was transformed again with these plasmids,
each transformant grew in M9 minimal medium. Furthermore, each
transformant was cultured overnight in L medium containing 20
.mu.g/ml of chloramphenicol, and the cells were collected by
centrifugation of the culture broth. The cells were sonicated to
prepare a crude enzyme solution, and ASD activity was measured
according to the method of Boy et al. (Journal of Bacteriology,
vol. 112 (1), 84-92 (1972)) (FIG. 4: pMMASD-1, pMMASD-2, pMMASD-3).
In addition, the host harboring the vector was similarly cultured
in L medium containing 20 .mu.g/ml of diaminopimelic acid and 20
.mu.g/ml of chloramphenicol, and ASD activity was measured as a
control experiment (FIG. 4: Vector). As a result, the enzymatic
activity could not be detected for the transformant harboring only
the vector, whereas the ASD activity could be detected in three of
the clones having an insert fragment. Therefore, it was confirmed
that the obtained gene was a gene coding for ASD derived from
Methylophilus methylotrophus (designated as asd).
[0224] The DNA nucleotide sequence of the asd gene was determined
by the dideoxy method. It was found that all of the three obtained
clones contained a common fragment. The nucleotide sequence of the
DNA fragment containing the asd gene derived from Methylophilus
methylotrophus is shown in SEQ ID NO: 7. The amino acid sequence
that can be encoded by the nucleotide sequence is shown in SEQ ID
NOS: 7 and 8.
[0225] (3) Cloning of Gene Coding for Dihydrodipicolinate Synthase
(DDPS)
[0226] E. coli AT997 (CGSC 4547 strain) deficient in the dapA gene
was transformed by the same electroporation procedure using Gene
library solution A. To the transformation solution, SOC medium
containing 20 .mu.g/ml of diaminopimelic acid was added, and the
mixture was cultured at 37.degree. C. with shaking. Then, the
culture broth was spread onto L medium containing 20 .mu.g/ml of
diaminopimelic acid and 100 .mu.g/ml of ampicillin, and colonies
grew. This was replicated as a master plate to M9 minimal agar
medium containing 100 .mu.g/ml of ampicillin, and the replicate was
incubated at 37.degree. C. for 2 to 3 days. The host could not grow
in M9 minimal medium that did not contain diaminopimelic acid since
it was deficient in dapA gene. In contrast, it was expected that a
transformant strain that contained the gene coding for DDPS derived
from Methylophilus methylotrophus could grow in M9 minimal medium
because of the function of that gene.
[0227] Plasmids were extracted from the colonies of two strains
emerged on M9 medium, and analyzed. As a result, the presence of
the inserted fragment in the plasmids was confirmed. The plasmids
were designated pMMDAPA-1 and pMMDAP-2, respectively. When E. coli
AT997 was transformed again with these plasmids, each transformant
was grown in M9 minimal medium. Furthermore, each transformant
containing each plasmid was cultured overnight in L medium
containing 100 .mu.g/ml of ampicillin, and the cells were collected
by centrifugation of the culture broth. The cells were sonicated to
prepare a cell extract, and DDPS activity was measured according to
the method of Yugari et al. (Journal of Biological Chemistry, vol.
240, and p. 4710 (1965)) (FIG. 5: pMMDAPA-1, pMMDAPA-2). In
addition, the host harboring the vector was similarly cultured in L
medium containing 20 .mu.g/ml of diaminopimelic acid and 100
.mu.g/ml of ampicillin, and DDPS activity was measured as a control
experiment (FIG. 5: Vector). As a result, the enzymatic activity
could not be detected for the transformant harboring only the
vector, whereas the DDPS activity could be detected in each of the
transformants harboring the plasmids having the insert fragment.
Therefore, it was confirmed that this gene was the gene coding for
DDPS derived from Methylophilus methylotrophus (designated as
dapA).
[0228] The DNA nucleotide sequence of the dapA gene was determined
by the dideoxy method. It was found that two of the inserted
fragments contained a common fragment. The nucleotide sequence of
the DNA fragment containing the dapA gene derived from
Methylophilus methylotrophus is shown in SEQ ID NO: 9. The amino
acid sequence that can be encoded by the nucleotide sequence is
shown in SEQ ID NOS: 9 and 10.
[0229] (4) Cloning of Gene Coding for Dihydrodipicolinate Reductase
(DDPR)
[0230] E. coli AT999 (CGSC 4549 strain) deficient in the dapB gene
was transformed by the same electroporation procedure as described
above using Gene library solution A. To the transformation
solution, SOC medium containing 20 .mu.g/ml of diaminopimelic acid
was added, and the mixture was cultured at 37.degree. C. with
shaking. Then, the cells were harvested by centrifugation. The
cells were washed by suspending them in L medium and centrifuging
the suspension. The same washing operation was repeated once again,
and the cells were suspended in L medium. Then, the suspension was
spread onto L agar medium containing 100 .mu.g/ml of ampicillin,
and incubated overnight at 37.degree. C. The host grew extremely
slowly in L medium not containing diaminopimelic acid since it was
deficient in the dapB gene. In contrast, it was expected that
normal growth would be observed for the transformant strain that
contained the gene coding for DDPR derived from Methylophilus
methylotrophus even in L medium because of the function of the
gene. Furthermore, the host E. coli could not grow in M9 minimal
medium, but it was expected that a transformant strain which
contained the gene coding for DDPR derived from Methylophilus
methylotrophus would grow in M9 minimal medium because of the
function of the gene.
[0231] Therefore, a colony of the transformant that grew normally
on L medium was streaked and cultured on M9 agar medium. Then,
growth was also observed on M9 medium. Thus, it was confirmed that
the gene coding for DDPR functioned in the transformant strain. A
plasmid was extracted from the colony which grew on M9 medium, and
the presence of an inserted fragment in the plasmid was confirmed.
When E. coli AT999 was transformed again by using the plasmid
(pMMDAPB), the transformant grew in M9 minimal medium. Furthermore,
the transformant containing the plasmid was cultured overnight in L
medium, and the cells were collected by centrifugation of the
culture broth. The cells were sonicated to prepare a cell extract,
and DDPR activity was measured according to the method of Tamir et
al. (Journal of Biological Chemistry, vol. 249, p. 3034 (1974))
(FIG. 6: pMMDAPB). In addition, the host harboring the vector was
similarly cultured in L medium containing 20 .mu.g/ml
diaminopimelic acid and 100 .mu.g/ml of ampicillin, and DDPR
activity was measured as a control experiment (FIG. 6: Vector). As
a result, the enzymatic activity could not be detected for the
transformant harboring only the vector, whereas the DDPR activity
could be detected for the transformant harboring pMMDAPB.
Therefore, it was confirmed that this gene was the gene coding for
DDPR derived from Methylophilus methylotrophus (designated as
dapB).
[0232] The DNA nucleotide sequence of the dapB gene was determined
by the dideoxy method. The nucleotide sequence of the DNA fragment
containing the dapB gene derived from Methylophilus methylotrophus
is shown in SEQ ID NO: 11. The amino acid sequence that can be
encoded by the nucleotide sequence is shown in SEQ ID NOS: 11 and
12.
[0233] (5) Cloning of Gene Coding for Diaminopimelate Decarboxylase
(DPDC)
[0234] E. coli AT2453 (CGSC 4505 strain) deficient in the lysA gene
was transformed by the same electroporation procedure as described
above using Gene library solution A. To the transformation
solution, SOC medium was added, and the mixture was cultured at
37.degree. C. with shaking. The cells were harvested by
centrifugation. The cells were washed by suspending them in 5 ml of
sterilized water and centrifuging the suspension. The same washing
operation was repeated once again, and the cells were suspended in
500 .mu.l of sterilized water. Then, the suspension was spread onto
M9 minimal agar medium containing 20 .mu.g/ml of chloramphenicol,
and incubated at 37.degree. C. for 2 to 3 days. The host did not
grow in M9 minimal medium without lysine since it was deficient in
the lysA gene. In contrast, it was expected that a transformant
strain that contained the gene coding for DPDC derived from
Methylophilus methylotrophus would grow in M9 minimal medium
because of the function of the gene.
[0235] Therefore, plasmids were extracted from the three
transformant strains which grew on M9 medium, and analyzed. As a
result, the presence of an inserted fragment in the plasmids was
confirmed. The plasmids were designated pMMLYSA-1, pMMLYSA-2 and
pMMLYSA-3, respectively. When E. coli AT2453 was transformed again
by using each of these plasmids, each transformant grew in M9
minimal medium. Furthermore, each transformant containing each
plasmid was cultured overnight in L medium containing 20 .mu.g/ml
of chloramphenicol, and the cells were collected by centrifugation
of the culture broth. The cells were sonicated to prepare a cell
extract, and DPDC activity was measured according to the method of
Cremer et al. (Journal of General Microbiology, vol. 134, 3221-3229
(1988)) (FIG. 7: pMMLYSA-1, pMMLYSA-2, pMMLYSA-3). In addition, the
host harboring the vector was similarly cultured in L medium
containing 20 .mu.g/ml of chloramphenicol, and DPDC activity was
measured as a control experiment (FIG. 7: Vector). As a result, the
enzymatic activity could not be detected for the transformant
harboring only the vector, whereas the DPDC activity could be
detected in three of the clones having an insert fragment.
Therefore, it was confirmed that this gene was the gene coding for
DPDC derived from Methylophilus methylotrophus (designated as
lysA).
[0236] The DNA nucleotide sequence of the lysA gene was determined
by the dideoxy method. It was found that all of the three inserted
fragments contained a common DNA fragment. The nucleotide sequence
of the DNA fragment containing the lysA gene derived from
Methylophilus methylotrophus is shown in SEQ ID NO: 13. The amino
acid sequence that can be encoded by the nucleotide sequence is
shown in SEQ ID NOS: 13 and 14.
INDUSTRIAL APPLICABILITY
[0237] According to the present invention, a Methylophilus
bacterium having the ability to produce L-amino acids, a method for
producing an L-amino acid using the Methylophilus bacterium, and
Methylophilus bacterial cells with increased content of an L-amino
acid are provided. By the method of the present invention, L-amino
acids can be produced using methanol as a raw material. Moreover,
novel L-lysine biosynthesis enzyme genes derived from Methylophilus
bacteria are provided.
[0238] While the invention has been described in detail with
reference to preferred embodiments thereof, it will be apparent to
one skilled in the art that various changes can be made, and
equivalents employed, without departing from the scope of the
invention. Each of the aforementioned documents is incorporated by
reference herein in its entirety.
Sequence CWU 1
1
2011197DNAEscherichia coliCDS(272)..(1147) 1ccaggcgact gtcttcaata
ttacagccgc aactactgac atgacgggtg atggtgttca 60caattccacg gcgatcggca
cccaacgcag tgatcaccag ataatgtgtt gcgatgacag 120tgtcaaactg
gttattcctt taaggggtga gttgttctta aggaaagcat aaaaaaaaca
180tgcatacaac aatcagaacg gttctgtctg cttgctttta atgccatacc
aaacgtacca 240ttgagacact tgtttgcaca gaggatggcc c atg ttc acg gga
agt att gtc 292 Met Phe Thr Gly Ser Ile Val 1 5gcg att gtt act ccg
atg gat gaa aaa ggt aat gtc tgt cgg gct agc 340Ala Ile Val Thr Pro
Met Asp Glu Lys Gly Asn Val Cys Arg Ala Ser 10 15 20ttg aaa aaa ctg
att gat tat cat gtc gcc agc ggt act tcg gcg atc 388Leu Lys Lys Leu
Ile Asp Tyr His Val Ala Ser Gly Thr Ser Ala Ile 25 30 35gtt tct gtt
ggc acc act ggc gag tcc gct acc tta aat cat gac gaa 436Val Ser Val
Gly Thr Thr Gly Glu Ser Ala Thr Leu Asn His Asp Glu40 45 50 55cat
gct gat gtg gtg atg atg acg ctg gat ctg gct gat ggg cgc att 484His
Ala Asp Val Val Met Met Thr Leu Asp Leu Ala Asp Gly Arg Ile 60 65
70ccg gta att gcc ggg acc ggc gct aac gct act gcg gaa gcc att agc
532Pro Val Ile Ala Gly Thr Gly Ala Asn Ala Thr Ala Glu Ala Ile Ser
75 80 85ctg acg cag cgc ttc aat gac agt ggt atc gtc ggc tgc ctg acg
gta 580Leu Thr Gln Arg Phe Asn Asp Ser Gly Ile Val Gly Cys Leu Thr
Val 90 95 100acc cct tac tac aat cgt ccg tcg caa gaa ggt ttg tat
cag cat ttc 628Thr Pro Tyr Tyr Asn Arg Pro Ser Gln Glu Gly Leu Tyr
Gln His Phe 105 110 115aaa gcc atc gct gag cat act gac ctg ccg caa
att ctg tat aat gtg 676Lys Ala Ile Ala Glu His Thr Asp Leu Pro Gln
Ile Leu Tyr Asn Val120 125 130 135ccg tcc cgt act ggc tgc gat ctg
ctc ccg gaa acg gtg ggc cgt ctg 724Pro Ser Arg Thr Gly Cys Asp Leu
Leu Pro Glu Thr Val Gly Arg Leu 140 145 150gcg aaa gta aaa aat att
atc gga atc aaa gag gca aca ggg aac tta 772Ala Lys Val Lys Asn Ile
Ile Gly Ile Lys Glu Ala Thr Gly Asn Leu 155 160 165acg cgt gta aac
cag atc aaa gag ctg gtt tca gat gat ttt gtt ctg 820Thr Arg Val Asn
Gln Ile Lys Glu Leu Val Ser Asp Asp Phe Val Leu 170 175 180ctg agc
ggc gat gat gcg agc gcg ctg gac ttc atg caa ttg ggc ggt 868Leu Ser
Gly Asp Asp Ala Ser Ala Leu Asp Phe Met Gln Leu Gly Gly 185 190
195cat ggg gtt att tcc gtt acg act aac gtc gca gcg cgt gat atg gcc
916His Gly Val Ile Ser Val Thr Thr Asn Val Ala Ala Arg Asp Met
Ala200 205 210 215cag atg tgc aaa ctg gca gca gaa gaa cat ttt gcc
gag gca cgc gtt 964Gln Met Cys Lys Leu Ala Ala Glu Glu His Phe Ala
Glu Ala Arg Val 220 225 230att aat cag cgt ctg atg cca tta cac aac
aaa cta ttt gtc gaa ccc 1012Ile Asn Gln Arg Leu Met Pro Leu His Asn
Lys Leu Phe Val Glu Pro 235 240 245aat cca atc ccg gtg aaa tgg gca
tgt aag gaa ctg ggt ctt gtg gcg 1060Asn Pro Ile Pro Val Lys Trp Ala
Cys Lys Glu Leu Gly Leu Val Ala 250 255 260acc gat acg ctg cgc ctg
cca atg aca cca atc acc gac agt ggt cgt 1108Thr Asp Thr Leu Arg Leu
Pro Met Thr Pro Ile Thr Asp Ser Gly Arg 265 270 275gag acg gtc aga
gcg gcg ctt aag cat gcc ggt ttg ctg taaagtttag 1157Glu Thr Val Arg
Ala Ala Leu Lys His Ala Gly Leu Leu280 285 290ggagatttga tggcttactc
tgttcaaaag tcgcgcctgg 11972292PRTEscherichia coli 2Met Phe Thr Gly
Ser Ile Val Ala Ile Val Thr Pro Met Asp Glu Lys1 5 10 15Gly Asn Val
Cys Arg Ala Ser Leu Lys Lys Leu Ile Asp Tyr His Val 20 25 30Ala Ser
Gly Thr Ser Ala Ile Val Ser Val Gly Thr Thr Gly Glu Ser 35 40 45Ala
Thr Leu Asn His Asp Glu His Ala Asp Val Val Met Met Thr Leu 50 55
60Asp Leu Ala Asp Gly Arg Ile Pro Val Ile Ala Gly Thr Gly Ala Asn65
70 75 80Ala Thr Ala Glu Ala Ile Ser Leu Thr Gln Arg Phe Asn Asp Ser
Gly 85 90 95Ile Val Gly Cys Leu Thr Val Thr Pro Tyr Tyr Asn Arg Pro
Ser Gln 100 105 110Glu Gly Leu Tyr Gln His Phe Lys Ala Ile Ala Glu
His Thr Asp Leu 115 120 125Pro Gln Ile Leu Tyr Asn Val Pro Ser Arg
Thr Gly Cys Asp Leu Leu 130 135 140Pro Glu Thr Val Gly Arg Leu Ala
Lys Val Lys Asn Ile Ile Gly Ile145 150 155 160Lys Glu Ala Thr Gly
Asn Leu Thr Arg Val Asn Gln Ile Lys Glu Leu 165 170 175Val Ser Asp
Asp Phe Val Leu Leu Ser Gly Asp Asp Ala Ser Ala Leu 180 185 190Asp
Phe Met Gln Leu Gly Gly His Gly Val Ile Ser Val Thr Thr Asn 195 200
205Val Ala Ala Arg Asp Met Ala Gln Met Cys Lys Leu Ala Ala Glu Glu
210 215 220His Phe Ala Glu Ala Arg Val Ile Asn Gln Arg Leu Met Pro
Leu His225 230 235 240Asn Lys Leu Phe Val Glu Pro Asn Pro Ile Pro
Val Lys Trp Ala Cys 245 250 255Lys Glu Leu Gly Leu Val Ala Thr Asp
Thr Leu Arg Leu Pro Met Thr 260 265 270Pro Ile Thr Asp Ser Gly Arg
Glu Thr Val Arg Ala Ala Leu Lys His 275 280 285Ala Gly Leu Leu
29032147DNAEscherichia coliCDS(584)..(1930) 3tcgaagtgtt tctgtagtgc
ctgccaggca gcggtctgcg ttggattgat gtttttcatt 60agcaatactc ttctgatttt
gagaattgtg actttggaag attgtagcgc cagtcacaga 120aaaatgtgat
ggttttagtg ccgttagcgt aatgttgagt gtaaaccctt agcgcagtga
180agcatttatt agctgaacta ctgaccgcca ggagtggatg aaaaatccgc
atgaccccat 240cgttgacaac cgccccgctc accctttatt tataaatgta
ctacctgcgc tagcgcaggc 300cagaagaggc gcgttgccca agtaacggtg
ttggaggagc cagtcctgtg ataacacctg 360agggggtgca tcgccgaggt
gattgaacgg ctggccacgt tcatcatcgg ctaagggggc 420tgaatcccct
gggttgtcac cagaagcgtt cgcagtcggg cgtttcgcaa gtggtggagc
480acttctgggt gaaaatagta gcgaagtatc gctctgcgcc cacccgtctt
ccgctcttcc 540cttgtgccaa ggctgaaaat ggatcccctg acacgaggta gtt atg
tct gaa att 595 Met Ser Glu Ile 1gtt gtc tcc aaa ttt ggc ggt acc
agc gta gct gat ttt gac gcc atg 643Val Val Ser Lys Phe Gly Gly Thr
Ser Val Ala Asp Phe Asp Ala Met5 10 15 20aac cgc agc gct gat att
gtg ctt tct gat gcc aac gtg cgt tta gtt 691Asn Arg Ser Ala Asp Ile
Val Leu Ser Asp Ala Asn Val Arg Leu Val 25 30 35gtc ctc tcg gct tct
gct ggt atc act aat ctg ctg gtc gct tta gct 739Val Leu Ser Ala Ser
Ala Gly Ile Thr Asn Leu Leu Val Ala Leu Ala 40 45 50gaa gga ctg gaa
cct ggc gag cga ttc gaa aaa ctc gac gct atc cgc 787Glu Gly Leu Glu
Pro Gly Glu Arg Phe Glu Lys Leu Asp Ala Ile Arg 55 60 65aac atc cag
ttt gcc att ctg gaa cgt ctg cgt tac ccg aac gtt atc 835Asn Ile Gln
Phe Ala Ile Leu Glu Arg Leu Arg Tyr Pro Asn Val Ile 70 75 80cgt gaa
gag att gaa cgt ctg ctg gag aac att act gtt ctg gca gaa 883Arg Glu
Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr Val Leu Ala Glu85 90 95
100gcg gcg gcg ctg gca acg tct ccg gcg ctg aca gat gag ctg gtc agc
931Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp Glu Leu Val Ser
105 110 115cac ggc gag ctg atg tcg acc ctg ctg ttt gtt gag atc ctg
cgc gaa 979His Gly Glu Leu Met Ser Thr Leu Leu Phe Val Glu Ile Leu
Arg Glu 120 125 130cgc gat gtt cag gca cag tgg ttt gat gta cgt aaa
gtg atg cgt acc 1027Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys
Val Met Arg Thr 135 140 145aac gac cga ttt ggt cgt gca gag cca gat
ata gcc gcg ctg gcg gaa 1075Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp
Ile Ala Ala Leu Ala Glu 150 155 160ctg gcc gcg ctg cag ctg ctc cca
cgt ctc aat gaa ggc tta gtg atc 1123Leu Ala Ala Leu Gln Leu Leu Pro
Arg Leu Asn Glu Gly Leu Val Ile165 170 175 180acc cag gga ttt atc
ggt agc gaa aat aaa ggt cgt aca acg acg ctt 1171Thr Gln Gly Phe Ile
Gly Ser Glu Asn Lys Gly Arg Thr Thr Thr Leu 185 190 195ggc cgt gga
ggc agc gat tat acg gca gcc ttg ctg gcg gag gct tta 1219Gly Arg Gly
Gly Ser Asp Tyr Thr Ala Ala Leu Leu Ala Glu Ala Leu 200 205 210cac
gca tct cgt gtt gat atc tgg acc gac gtc ccg ggc atc tac acc 1267His
Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro Gly Ile Tyr Thr 215 220
225acc gat cca cgc gta gtt tcc gca gca aaa cgc att gat gaa atc gcg
1315Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile Asp Glu Ile Ala
230 235 240ttt gcc gaa gcg gca gag atg gca act ttt ggt gca aaa gta
ctg cat 1363Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala Lys Val
Leu His245 250 255 260ccg gca acg ttg cta ccc gca gta cgc agc gat
atc ccg gtc ttt gtc 1411Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp
Ile Pro Val Phe Val 265 270 275ggc tcc agc aaa gac cca cgc gca ggt
ggt acg ctg gtg tgc aat aaa 1459Gly Ser Ser Lys Asp Pro Arg Ala Gly
Gly Thr Leu Val Cys Asn Lys 280 285 290act gaa aat ccg ccg ctg ttc
cgc gct ctg gcg ctt cgt cgc aat cag 1507Thr Glu Asn Pro Pro Leu Phe
Arg Ala Leu Ala Leu Arg Arg Asn Gln 295 300 305act ctg ctc act ttg
cac agc ctg aat atg ctg cat tct cgc ggt ttc 1555Thr Leu Leu Thr Leu
His Ser Leu Asn Met Leu His Ser Arg Gly Phe 310 315 320ctc gcg gaa
gtt ttc ggc atc ctc gcg cgg cat aat att tcg gta gac 1603Leu Ala Glu
Val Phe Gly Ile Leu Ala Arg His Asn Ile Ser Val Asp325 330 335
340tta atc acc acg tca gaa gtg agc gtg gca tta acc ctt gat acc acc
1651Leu Ile Thr Thr Ser Glu Val Ser Val Ala Leu Thr Leu Asp Thr Thr
345 350 355ggt tca acc tcc act ggc gat acg ttg ctg acg caa tct ctg
ctg atg 1699Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln Ser Leu
Leu Met 360 365 370gag ctt tcc gca ctg tgt cgg gtg gag gtg gaa gaa
ggt ctg gcg ctg 1747Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu
Gly Leu Ala Leu 375 380 385gtc gcg ttg att ggc aat gac ctg tca aaa
gcc tgc ggc gtt ggc aaa 1795Val Ala Leu Ile Gly Asn Asp Leu Ser Lys
Ala Cys Gly Val Gly Lys 390 395 400gag gta ttc ggc gta ctg gaa ccg
ttc aac att cgc atg att tgt tat 1843Glu Val Phe Gly Val Leu Glu Pro
Phe Asn Ile Arg Met Ile Cys Tyr405 410 415 420ggc gca tcc agc cat
aac ctg tgc ttc ctg gtg ccc ggc gaa gat gcc 1891Gly Ala Ser Ser His
Asn Leu Cys Phe Leu Val Pro Gly Glu Asp Ala 425 430 435gag cag gtg
gtg caa aaa ctg cat agt aat ttg ttt gag taaatactgt 1940Glu Gln Val
Val Gln Lys Leu His Ser Asn Leu Phe Glu 440 445atggcctgga
agctatattt cgggccgtat tgattttctt gtcactatgc tcatcaataa
2000acgagcctgt actctgttaa ccagcgtctt tatcggagaa taattgcctt
taattttttt 2060atctgcatct ctaattaatt atcgaaagag ataaatagtt
aagagaaggc aaaatgaata 2120ttatcagttc tgctcgcaaa ggaattc
21474449PRTEscherichia coli 4Met Ser Glu Ile Val Val Ser Lys Phe
Gly Gly Thr Ser Val Ala Asp1 5 10 15Phe Asp Ala Met Asn Arg Ser Ala
Asp Ile Val Leu Ser Asp Ala Asn 20 25 30Val Arg Leu Val Val Leu Ser
Ala Ser Ala Gly Ile Thr Asn Leu Leu 35 40 45Val Ala Leu Ala Glu Gly
Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu 50 55 60Asp Ala Ile Arg Asn
Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr65 70 75 80Pro Asn Val
Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr 85 90 95Val Leu
Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp 100 105
110Glu Leu Val Ser His Gly Glu Leu Met Ser Thr Leu Leu Phe Val Glu
115 120 125Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe Asp Val
Arg Lys 130 135 140Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu
Pro Asp Ile Ala145 150 155 160Ala Leu Ala Glu Leu Ala Ala Leu Gln
Leu Leu Pro Arg Leu Asn Glu 165 170 175Gly Leu Val Ile Thr Gln Gly
Phe Ile Gly Ser Glu Asn Lys Gly Arg 180 185 190Thr Thr Thr Leu Gly
Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu 195 200 205Ala Glu Ala
Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro 210 215 220Gly
Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile225 230
235 240Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly
Ala 245 250 255Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg
Ser Asp Ile 260 265 270Pro Val Phe Val Gly Ser Ser Lys Asp Pro Arg
Ala Gly Gly Thr Leu 275 280 285Val Cys Asn Lys Thr Glu Asn Pro Pro
Leu Phe Arg Ala Leu Ala Leu 290 295 300Arg Arg Asn Gln Thr Leu Leu
Thr Leu His Ser Leu Asn Met Leu His305 310 315 320Ser Arg Gly Phe
Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn 325 330 335Ile Ser
Val Asp Leu Ile Thr Thr Ser Glu Val Ser Val Ala Leu Thr 340 345
350Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln
355 360 365Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val
Glu Glu 370 375 380Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu
Ser Lys Ala Cys385 390 395 400Gly Val Gly Lys Glu Val Phe Gly Val
Leu Glu Pro Phe Asn Ile Arg 405 410 415Met Ile Cys Tyr Gly Ala Ser
Ser His Asn Leu Cys Phe Leu Val Pro 420 425 430Gly Glu Asp Ala Glu
Gln Val Val Gln Lys Leu His Ser Asn Leu Phe 435 440
445Glu51981DNAMethylophilus methylotrophusCDS(510)..(1736)
5gtttaacgcg gccagtgaat ttgactcggt cccctgcctg gcaaaatcgc acaggtgatg
60gacaacgtga aatcgcttga aaaagaattg gcacgcctca agtccaagct ggcctcctca
120cagggggatg acctcgcgac gcaagcgcag gacgtcaacg gcgccaaagt
actggcagcc 180accctcgacg gggcggatgc caatgccttg cgtgaaacca
tggataagct caaagataaa 240ctcaaatctg cagtcattgt gctggcgagc
gtggctgacg gtaaagtcag cctggctgcg 300ggtgtcacta ctgacttgac
tggcaaggtc aaagcaggcg aagttggtca atcatgtggc 360tggtcaggtc
ggtggcaaag gtggtggtaa accggatatg gcgatggcag gtggtactga
420gcccgctaat ttgccgcagg ctttggcaag tgtgaaggct tgggtagaaa
caaaactaaa 480ttaatttaat tgattaacag agcgaaata atg gca tta atc gta
caa aaa tat 533 Met Ala Leu Ile Val Gln Lys Tyr 1 5ggt ggt acc tcg
gtg gct aat ccc gag cgt atc cgt aat gtg gcg cgt 581Gly Gly Thr Ser
Val Ala Asn Pro Glu Arg Ile Arg Asn Val Ala Arg 10 15 20cgc gtg gcg
cgt tac aag gca ttg ggc cac cag gtg gtg gtt gtg gta 629Arg Val Ala
Arg Tyr Lys Ala Leu Gly His Gln Val Val Val Val Val25 30 35 40tcc
gca atg tct ggt gaa acc aac cgg ttg atc tca ctg gcc aag gaa 677Ser
Ala Met Ser Gly Glu Thr Asn Arg Leu Ile Ser Leu Ala Lys Glu 45 50
55atc atg caa gac cct gat cca cgt gag ctg gat gtg atg gta tca acc
725Ile Met Gln Asp Pro Asp Pro Arg Glu Leu Asp Val Met Val Ser Thr
60 65 70ggt gag cag gtc acc atc ggc atg acg gcc ctg gca ctg atg gag
ctt 773Gly Glu Gln Val Thr Ile Gly Met Thr Ala Leu Ala Leu Met Glu
Leu 75 80 85ggc att aag gca aaa agc tat acc ggt acc cag gtt aag atc
ttg act 821Gly Ile Lys Ala Lys Ser Tyr Thr Gly Thr Gln Val Lys Ile
Leu Thr 90 95 100gac gat gct ttt acc aag gca cgt att ctg gat atc
gac gaa cat aac 869Asp Asp Ala Phe Thr Lys Ala Arg Ile Leu Asp Ile
Asp Glu His Asn105 110 115 120ctg aaa aaa gac ctg gat gat ggc tat
gtc tgc gtg gtg gct ggg ttc 917Leu Lys Lys Asp Leu Asp Asp Gly Tyr
Val Cys Val Val Ala Gly Phe 125
130 135cag ggc gtg gat gcc aat ggc aat att acg acc ttg ggc cgt ggc
ggc 965Gln Gly Val Asp Ala Asn Gly Asn Ile Thr Thr Leu Gly Arg Gly
Gly 140 145 150tca gat act act ggt gta gca ctg gct gcg gcg tta aag
gcg gat gaa 1013Ser Asp Thr Thr Gly Val Ala Leu Ala Ala Ala Leu Lys
Ala Asp Glu 155 160 165tgt cag att tat acc gat gtc gat ggc gtt tac
acc acc gat ccg cgt 1061Cys Gln Ile Tyr Thr Asp Val Asp Gly Val Tyr
Thr Thr Asp Pro Arg 170 175 180gtg gtg cct gag gca cgc cgc ttg gat
aaa att acc ttt gaa gaa atg 1109Val Val Pro Glu Ala Arg Arg Leu Asp
Lys Ile Thr Phe Glu Glu Met185 190 195 200ttg gaa ctg gct tca cag
ggc tcc aaa gta ttg caa att cgc tcg gtt 1157Leu Glu Leu Ala Ser Gln
Gly Ser Lys Val Leu Gln Ile Arg Ser Val 205 210 215gag ttt gcc ggt
aaa tac aaa gtc aaa tta cgt gtg ctg tcc agc ttc 1205Glu Phe Ala Gly
Lys Tyr Lys Val Lys Leu Arg Val Leu Ser Ser Phe 220 225 230gaa gag
gag ggc gac ggt aca ctg atc aca ttc gaa gaa aat gag gaa 1253Glu Glu
Glu Gly Asp Gly Thr Leu Ile Thr Phe Glu Glu Asn Glu Glu 235 240
245aac atg gaa gaa cca att atc tcc ggc atc gcc ttt aac cgc gat gag
1301Asn Met Glu Glu Pro Ile Ile Ser Gly Ile Ala Phe Asn Arg Asp Glu
250 255 260gcg aaa att acc gtg acg ggc gtg ccc gac aaa cca gga att
gcc tat 1349Ala Lys Ile Thr Val Thr Gly Val Pro Asp Lys Pro Gly Ile
Ala Tyr265 270 275 280cag att ttg ggc ccg gtg gca gac gcc aat att
gat gtg gat atg att 1397Gln Ile Leu Gly Pro Val Ala Asp Ala Asn Ile
Asp Val Asp Met Ile 285 290 295atc cag aac gtc ggt gcg gat ggt acg
act gac ttc acc ttt acc gta 1445Ile Gln Asn Val Gly Ala Asp Gly Thr
Thr Asp Phe Thr Phe Thr Val 300 305 310cat aaa aat gag atg aac aaa
gcc ctg agc att ctt aga gat aaa gtg 1493His Lys Asn Glu Met Asn Lys
Ala Leu Ser Ile Leu Arg Asp Lys Val 315 320 325cag ggc cat atc cag
gca cgt gaa atc agc ggc gac gac aag att gcc 1541Gln Gly His Ile Gln
Ala Arg Glu Ile Ser Gly Asp Asp Lys Ile Ala 330 335 340aaa gtc tct
gtg gtt ggg gtg ggt atg cgc tca cat gta ggg atc gcc 1589Lys Val Ser
Val Val Gly Val Gly Met Arg Ser His Val Gly Ile Ala345 350 355
360agc cag atg ttc cgt acg ctg gcc gaa gaa ggg atc aat att caa atg
1637Ser Gln Met Phe Arg Thr Leu Ala Glu Glu Gly Ile Asn Ile Gln Met
365 370 375atc tca acc agc gaa att aaa att gca gtc gtg atc gaa gag
aag tac 1685Ile Ser Thr Ser Glu Ile Lys Ile Ala Val Val Ile Glu Glu
Lys Tyr 380 385 390atg gaa ctg gct gta cgc gtg ttg cat aaa gca ttc
ggc ctc gaa aac 1733Met Glu Leu Ala Val Arg Val Leu His Lys Ala Phe
Gly Leu Glu Asn 395 400 405gca taatcgccaa cggacgaata aagaaataaa
acattcttct tttttgcgtt 1786Alagatttttgaa gggttttcac gtagtatggc
agcccttcga tgcagtagca atgctgcaaa 1846gagaacagca tgccgctgtg
ttggtactat taaaacttca ttgttttaat aaggtgaggg 1906ggatcctcta
gagtcgacct gcaggcatgc aagcttggcc gtaatccatg gtcatagctg
1966tttcctggtg tgaaa 19816409PRTMethylophilus methylotrophus 6Met
Ala Leu Ile Val Gln Lys Tyr Gly Gly Thr Ser Val Ala Asn Pro1 5 10
15Glu Arg Ile Arg Asn Val Ala Arg Arg Val Ala Arg Tyr Lys Ala Leu
20 25 30Gly His Gln Val Val Val Val Val Ser Ala Met Ser Gly Glu Thr
Asn 35 40 45Arg Leu Ile Ser Leu Ala Lys Glu Ile Met Gln Asp Pro Asp
Pro Arg 50 55 60Glu Leu Asp Val Met Val Ser Thr Gly Glu Gln Val Thr
Ile Gly Met65 70 75 80Thr Ala Leu Ala Leu Met Glu Leu Gly Ile Lys
Ala Lys Ser Tyr Thr 85 90 95Gly Thr Gln Val Lys Ile Leu Thr Asp Asp
Ala Phe Thr Lys Ala Arg 100 105 110Ile Leu Asp Ile Asp Glu His Asn
Leu Lys Lys Asp Leu Asp Asp Gly 115 120 125Tyr Val Cys Val Val Ala
Gly Phe Gln Gly Val Asp Ala Asn Gly Asn 130 135 140Ile Thr Thr Leu
Gly Arg Gly Gly Ser Asp Thr Thr Gly Val Ala Leu145 150 155 160Ala
Ala Ala Leu Lys Ala Asp Glu Cys Gln Ile Tyr Thr Asp Val Asp 165 170
175Gly Val Tyr Thr Thr Asp Pro Arg Val Val Pro Glu Ala Arg Arg Leu
180 185 190Asp Lys Ile Thr Phe Glu Glu Met Leu Glu Leu Ala Ser Gln
Gly Ser 195 200 205Lys Val Leu Gln Ile Arg Ser Val Glu Phe Ala Gly
Lys Tyr Lys Val 210 215 220Lys Leu Arg Val Leu Ser Ser Phe Glu Glu
Glu Gly Asp Gly Thr Leu225 230 235 240Ile Thr Phe Glu Glu Asn Glu
Glu Asn Met Glu Glu Pro Ile Ile Ser 245 250 255Gly Ile Ala Phe Asn
Arg Asp Glu Ala Lys Ile Thr Val Thr Gly Val 260 265 270Pro Asp Lys
Pro Gly Ile Ala Tyr Gln Ile Leu Gly Pro Val Ala Asp 275 280 285Ala
Asn Ile Asp Val Asp Met Ile Ile Gln Asn Val Gly Ala Asp Gly 290 295
300Thr Thr Asp Phe Thr Phe Thr Val His Lys Asn Glu Met Asn Lys
Ala305 310 315 320Leu Ser Ile Leu Arg Asp Lys Val Gln Gly His Ile
Gln Ala Arg Glu 325 330 335Ile Ser Gly Asp Asp Lys Ile Ala Lys Val
Ser Val Val Gly Val Gly 340 345 350Met Arg Ser His Val Gly Ile Ala
Ser Gln Met Phe Arg Thr Leu Ala 355 360 365Glu Glu Gly Ile Asn Ile
Gln Met Ile Ser Thr Ser Glu Ile Lys Ile 370 375 380Ala Val Val Ile
Glu Glu Lys Tyr Met Glu Leu Ala Val Arg Val Leu385 390 395 400His
Lys Ala Phe Gly Leu Glu Asn Ala 40571452DNAMethylophilus
methylotrophusCDS(98)..(1207) 7gcatgcccgc aggtcgactc tagaggatcc
ccctgttcaa aaatcttcca aataatcact 60gtaatgccgg gttgtccggc tgaaatatcg
agtcact atg tta aaa gta ggg ttt 115 Met Leu Lys Val Gly Phe 1 5gta
ggc tgg cgt ggc atg gtt gga tcc gtg cta atg cag cgc atg atg 163Val
Gly Trp Arg Gly Met Val Gly Ser Val Leu Met Gln Arg Met Met 10 15
20cag gaa aac gat ttt gcg gat att gaa ccg caa ttc ttt acg acc tca
211Gln Glu Asn Asp Phe Ala Asp Ile Glu Pro Gln Phe Phe Thr Thr Ser
25 30 35caa acg gga ggg gct gcg cct aaa gtt gga aaa gat act cct gcg
ctg 259Gln Thr Gly Gly Ala Ala Pro Lys Val Gly Lys Asp Thr Pro Ala
Leu 40 45 50aaa gat gcc aag gat att gat gct ttg cgc cag atg gat gtg
att gtg 307Lys Asp Ala Lys Asp Ile Asp Ala Leu Arg Gln Met Asp Val
Ile Val55 60 65 70acc tgc cag ggt ggc gat tac acg agt gac gtc ttc
cca caa ttg cgc 355Thr Cys Gln Gly Gly Asp Tyr Thr Ser Asp Val Phe
Pro Gln Leu Arg 75 80 85gca acc ggc tgg agc ggc cac tgg att gac gcg
gcc tct acc tta cgc 403Ala Thr Gly Trp Ser Gly His Trp Ile Asp Ala
Ala Ser Thr Leu Arg 90 95 100atg gaa aaa gac tcc gtg atc att tta
gac ccg gtg aac atg cat gtg 451Met Glu Lys Asp Ser Val Ile Ile Leu
Asp Pro Val Asn Met His Val 105 110 115att aaa gat gca ttg tcc aat
ggc ggc aaa aac tgg atc ggc ggc aac 499Ile Lys Asp Ala Leu Ser Asn
Gly Gly Lys Asn Trp Ile Gly Gly Asn 120 125 130tgt acc gtc tca ctt
atg ttg atg gcg ctg aat ggc ctg ttt aag gct 547Cys Thr Val Ser Leu
Met Leu Met Ala Leu Asn Gly Leu Phe Lys Ala135 140 145 150gac ctg
gtc gag tgg gcc act tcc atg acc tac cag gcg gct tca ggc 595Asp Leu
Val Glu Trp Ala Thr Ser Met Thr Tyr Gln Ala Ala Ser Gly 155 160
165gca ggc gcg cag aat atg cgt gaa ctg att agc cag atg ggc gta gtg
643Ala Gly Ala Gln Asn Met Arg Glu Leu Ile Ser Gln Met Gly Val Val
170 175 180aat gcc tcc gtg gct gat ttg ctg gcg gat cca gct tct gcc
att ttg 691Asn Ala Ser Val Ala Asp Leu Leu Ala Asp Pro Ala Ser Ala
Ile Leu 185 190 195cag atc gat aaa aca gtg gcg gat acc atc cgt agc
gaa gag ttg cct 739Gln Ile Asp Lys Thr Val Ala Asp Thr Ile Arg Ser
Glu Glu Leu Pro 200 205 210aaa tct aac ttt ggt gtg cca ttg gcg ggc
agt ctg atc cca tgg atc 787Lys Ser Asn Phe Gly Val Pro Leu Ala Gly
Ser Leu Ile Pro Trp Ile215 220 225 230gac aag gac tta ggg aat ggt
caa agt aaa gaa gaa tgg aag ggc ggc 835Asp Lys Asp Leu Gly Asn Gly
Gln Ser Lys Glu Glu Trp Lys Gly Gly 235 240 245gta nag acc aat aag
att tta ggt cgt gaa gcg aac ccg att gtg att 883Val Xaa Thr Asn Lys
Ile Leu Gly Arg Glu Ala Asn Pro Ile Val Ile 250 255 260gac ggt ttg
tgt gta cgt atc ggc gcc atg cgt tgc cat tca caa gcg 931Asp Gly Leu
Cys Val Arg Ile Gly Ala Met Arg Cys His Ser Gln Ala 265 270 275ttg
act atc aag ctg cgc aag gat gtg ccg ctg gat gaa atc aat cag 979Leu
Thr Ile Lys Leu Arg Lys Asp Val Pro Leu Asp Glu Ile Asn Gln 280 285
290atg ctg gct gaa gcg aac gac tgg gct aaa gtc att ccc aat gag cgt
1027Met Leu Ala Glu Ala Asn Asp Trp Ala Lys Val Ile Pro Asn Glu
Arg295 300 305 310gag gtc agt atg cgg gaa ctc acc ccg gca gcg att
acc ggc agt ctg 1075Glu Val Ser Met Arg Glu Leu Thr Pro Ala Ala Ile
Thr Gly Ser Leu 315 320 325gcg acg cca gta ggg cgt ttg cgc aaa ctg
gcg atg ggt ggt gaa tac 1123Ala Thr Pro Val Gly Arg Leu Arg Lys Leu
Ala Met Gly Gly Glu Tyr 330 335 340ttg tcg gca ttt acc gta ggt gac
cag ttg tta tgg ggc gct gcc gaa 1171Leu Ser Ala Phe Thr Val Gly Asp
Gln Leu Leu Trp Gly Ala Ala Glu 345 350 355cct ttg cgc aga atg ttg
agg att ctg gtc gaa tct taagtaattg 1217Pro Leu Arg Arg Met Leu Arg
Ile Leu Val Glu Ser 360 365 370tttaagtagc agcccgtaaa gctatgattt
atcaataaaa tcatggtctt ttcgggcttt 1277tgcttttggt gcaatcctgt
ttaatggtta ttgtagcctc aaatcctgta tttattgctc 1337tcaagccgcc
tgggtgcgct tgcgtggctg ggtgaatgat gctattttga caaacgccat
1397gaattactaa gggttaatcg gtgagtaaat ttcaattaaa aaaaatagcc tttgc
14528370PRTMethylophilus methylotrophusmisc_feature(248)..(248)The
'Xaa' at location 248 stands for Lys, Glu, or Gln. 8Met Leu Lys Val
Gly Phe Val Gly Trp Arg Gly Met Val Gly Ser Val1 5 10 15Leu Met Gln
Arg Met Met Gln Glu Asn Asp Phe Ala Asp Ile Glu Pro 20 25 30Gln Phe
Phe Thr Thr Ser Gln Thr Gly Gly Ala Ala Pro Lys Val Gly 35 40 45Lys
Asp Thr Pro Ala Leu Lys Asp Ala Lys Asp Ile Asp Ala Leu Arg 50 55
60Gln Met Asp Val Ile Val Thr Cys Gln Gly Gly Asp Tyr Thr Ser Asp65
70 75 80Val Phe Pro Gln Leu Arg Ala Thr Gly Trp Ser Gly His Trp Ile
Asp 85 90 95Ala Ala Ser Thr Leu Arg Met Glu Lys Asp Ser Val Ile Ile
Leu Asp 100 105 110Pro Val Asn Met His Val Ile Lys Asp Ala Leu Ser
Asn Gly Gly Lys 115 120 125Asn Trp Ile Gly Gly Asn Cys Thr Val Ser
Leu Met Leu Met Ala Leu 130 135 140Asn Gly Leu Phe Lys Ala Asp Leu
Val Glu Trp Ala Thr Ser Met Thr145 150 155 160Tyr Gln Ala Ala Ser
Gly Ala Gly Ala Gln Asn Met Arg Glu Leu Ile 165 170 175Ser Gln Met
Gly Val Val Asn Ala Ser Val Ala Asp Leu Leu Ala Asp 180 185 190Pro
Ala Ser Ala Ile Leu Gln Ile Asp Lys Thr Val Ala Asp Thr Ile 195 200
205Arg Ser Glu Glu Leu Pro Lys Ser Asn Phe Gly Val Pro Leu Ala Gly
210 215 220Ser Leu Ile Pro Trp Ile Asp Lys Asp Leu Gly Asn Gly Gln
Ser Lys225 230 235 240Glu Glu Trp Lys Gly Gly Val Xaa Thr Asn Lys
Ile Leu Gly Arg Glu 245 250 255Ala Asn Pro Ile Val Ile Asp Gly Leu
Cys Val Arg Ile Gly Ala Met 260 265 270Arg Cys His Ser Gln Ala Leu
Thr Ile Lys Leu Arg Lys Asp Val Pro 275 280 285Leu Asp Glu Ile Asn
Gln Met Leu Ala Glu Ala Asn Asp Trp Ala Lys 290 295 300Val Ile Pro
Asn Glu Arg Glu Val Ser Met Arg Glu Leu Thr Pro Ala305 310 315
320Ala Ile Thr Gly Ser Leu Ala Thr Pro Val Gly Arg Leu Arg Lys Leu
325 330 335Ala Met Gly Gly Glu Tyr Leu Ser Ala Phe Thr Val Gly Asp
Gln Leu 340 345 350Leu Trp Gly Ala Ala Glu Pro Leu Arg Arg Met Leu
Arg Ile Leu Val 355 360 365Glu Ser 37093098DNAMethylophilus
methylotrophusCDS(1268)..(2155) 9cgtgccaact tgcatgcctg ccggtcgctc
tagaggatca attgctggca acatttgagt 60acattattcg cctttgcatg gtaaaggcct
atggtcttga tgtaactttc aagacctgcc 120agccccaaat ccaggatagc
ctgcggtgtg ttggccacct tgaacaattt gcgggtggca 180atattgacac
ctttgtctgt cgcctgtgca gacaagatga cggcaatcag taattcgaac
240gtggagctat gctccagctc agtggttgga ttggggatgg cttgggccag
ccgctcaaat 300atcgccagtc ttttttgtgc attcataaaa cggtttcaat
cataggtcac agggtcaacc 360tgtcttttgc gctttgacgc gcgccatggc
tgcggcaatg gcatttttct tgagcacctc 420agttgagggt gtctcggtcg
tagcaagcgt ctggttgcgt ttgctgtagg tttgggcggt 480ctcccgtttt
tcaagggcga ggcgagaaag gcgttgctgg tggcgttgtc tcgctaccgc
540ggcttcagct tcattcatgg cggtagcccg accgggaatc gtttgcatct
gtatgcagtc 600caccgggcag ggcggtaaac atagctcaca gccagtgcat
tcctgggaaa tcaccgtatg 660catcagtttg gatgcgccca aaatggcatc
aacgggacag gcctgtatac acagggtgca 720gccgatgcat gtttcctcat
caatcaaggc caccgctttg ggtttggtga tgccgtgggc 780cggatttaat
gcctggaaag gacgttgcag taatttggca agcgcatgaa tgcccgcttc
840tcctccaggc ggacattggt tgatattggc ctctccgcgg gcgatcgctt
cagcataagg 900tttgcatccc tcgtaaccgc attggcggca ttgagtttgc
ggtaataccg cgtcgatctt 960tgcaatgagg tcgacaaagc gttctggcag
ctcaggcgca gtcccttcga cttcaatcat 1020gtgatggcag gtgagtctgc
attcggtcct ggctaaatag ccgtttaaga tgggttgcta 1080agagttttat
tataaccgaa accttgcttt tcctttggcc gggagctagg cggaaaaagc
1140ttgccgcagt tgggtgccag tgattttgcc gccgtcttgc gcttgtatcc
gtccagatac 1200agcaagtagg cgcgttcttt ggcgttagac cggataatca
gttaaaatat tcgctttatt 1260cttaaag atg gcg cta ggt atg tta acg ggc
agt ttg gtc gca atc gtg 1309 Met Ala Leu Gly Met Leu Thr Gly Ser
Leu Val Ala Ile Val 1 5 10acc ccc atg ttt gaa gat gga cgt ttg gat
ctg gac gcc ctc aaa aag 1357Thr Pro Met Phe Glu Asp Gly Arg Leu Asp
Leu Asp Ala Leu Lys Lys15 20 25 30ctg gtc gac ttt cat gta gag gca
ggg aca gat ggt att gtc atc gtt 1405Leu Val Asp Phe His Val Glu Ala
Gly Thr Asp Gly Ile Val Ile Val 35 40 45ggc acg act ggc gag tcg ccc
acg gtg gat gta gat gag cat tgt ctg 1453Gly Thr Thr Gly Glu Ser Pro
Thr Val Asp Val Asp Glu His Cys Leu 50 55 60ctg atc aaa acc acg atc
gag cat gtc gcc aag cgc gtg cca gtc att 1501Leu Ile Lys Thr Thr Ile
Glu His Val Ala Lys Arg Val Pro Val Ile 65 70 75gcc ggt act ggc gca
aat tcc act gct gaa gcc att gaa ctg act gcc 1549Ala Gly Thr Gly Ala
Asn Ser Thr Ala Glu Ala Ile Glu Leu Thr Ala 80 85 90aag gcc aag gcg
ctt ggc gca gac gcc tgc ctg ctg gtg gca ccg tat 1597Lys Ala Lys Ala
Leu Gly Ala Asp Ala Cys Leu Leu Val Ala Pro Tyr95 100 105 110tac
aac aag ccc tcg caa gag ggt ttg tac cag cac ttt aaa gcc gtg 1645Tyr
Asn Lys Pro Ser Gln Glu Gly Leu Tyr Gln His Phe Lys Ala Val 115 120
125gct gag gcg gtc gat att ccg caa att ctc tat aat gtg cca ggc cgc
1693Ala Glu Ala Val Asp Ile Pro Gln Ile Leu Tyr Asn Val Pro Gly Arg
130 135 140acc ggt tgc gac ttg tct aac gac acc gta ttg cgc ctg gcg
cag att 1741Thr Gly Cys Asp Leu Ser Asn Asp Thr Val Leu Arg Leu Ala
Gln Ile 145 150 155cgc aac att gtc ggg att aag gat gcg act gga ggg
att gag cgc ggt 1789Arg Asn Ile Val Gly Ile Lys Asp Ala Thr Gly Gly
Ile Glu Arg Gly 160 165 170acc gat ttg ttg ttg cgt gca cca gct gat
ttc gcc att tac agc ggg 1837Thr Asp Leu Leu Leu
Arg Ala Pro Ala Asp Phe Ala Ile Tyr Ser Gly175 180 185 190gat gat
gcc act gcg ctg gcc ctg atg tta tta ggg ggg aaa ggc gtg 1885Asp Asp
Ala Thr Ala Leu Ala Leu Met Leu Leu Gly Gly Lys Gly Val 195 200
205att tcg gtc acg gcc aat gtc gcg ccc aaa tta atg cat gaa atg tgc
1933Ile Ser Val Thr Ala Asn Val Ala Pro Lys Leu Met His Glu Met Cys
210 215 220gag cat gct ttg aat ggc aac ctg gcc gca gcc aaa gcg gcc
aat gcc 1981Glu His Ala Leu Asn Gly Asn Leu Ala Ala Ala Lys Ala Ala
Asn Ala 225 230 235aaa ctg ttt gca ttg cac cag aag ttg ttt gta gaa
gcg aac ccg att 2029Lys Leu Phe Ala Leu His Gln Lys Leu Phe Val Glu
Ala Asn Pro Ile 240 245 250cca gtg aaa tgg gta tta caa caa atg gga
atg att gcc act ggc atc 2077Pro Val Lys Trp Val Leu Gln Gln Met Gly
Met Ile Ala Thr Gly Ile255 260 265 270cgt ttg ccg ctg gtc aat tta
tcc agc caa tat cat gaa gta ttg cgc 2125Arg Leu Pro Leu Val Asn Leu
Ser Ser Gln Tyr His Glu Val Leu Arg 275 280 285aac gcc atg aag cag
gca gaa att gcc gct tgatcggcta aaactaattt 2175Asn Ala Met Lys Gln
Ala Glu Ile Ala Ala 290 295agggtgaaac aagtgaaata catgagtcat
gtttggttac aacgtttggt gctggccagt 2235ctggtcacag cgctttcagc
gtgcgattcc atcccgttta ttgataatag ttctgactac 2295aagggcgcag
gtcgctccag gccacttgaa gtgccgccag acctgaccgc ggtgcgtacc
2355agcagtactt acaatgtgcc tggtagcacc agttactctg cctatagcca
gaaccaggaa 2415gtgcaagagc agaatggtcc acagcctgtg ctcgcagata
tgaaaaacgt gcgcatggtg 2475aaagcaggcc agcagcgttg gctggtggtc
aatgcgcctc cggaaaaaat ctggccgatt 2535gtgcgtgatt tctggctgga
tcaaggcttt gctgtcaggg tagagaatcc tgagcttggc 2595gtgattgaaa
ccgagtggtt gcaatctgat gccatcaagc ctaaggaaga taaccgtggc
2655tatggtgaaa agtttgatgc ctggctggat aaactttctg gttttgccga
caggcgtaaa 2715ttccgtacgc gtctggaacg tggggagaaa gacggcacca
ccgaaatcta tatgacgcac 2775cgtactgtcg ccggtgcacc ggatgatggc
aaaaattatg tgcagaccca attgggtgtc 2835attgataccg gttatcgccc
caacgcggct gaaaacaaga acaatgccgg taaagagttt 2895gatgctgact
tggatgcaga attactccgt cgaatgatgg tgaaattagg tctggatgag
2955cagaaagcag accaggtgat ggcacaatct gcttcagaca agcgtgcaga
tgtggtcaag 3015gagtctgacc agagcgtcac cttgaagttg aatgagccgt
ttgaccgtgc ctggcgccgt 3075gtggcctggc ctggatcccc ggg
309810296PRTMethylophilus methylotrophus 10Met Ala Leu Gly Met Leu
Thr Gly Ser Leu Val Ala Ile Val Thr Pro1 5 10 15Met Phe Glu Asp Gly
Arg Leu Asp Leu Asp Ala Leu Lys Lys Leu Val 20 25 30Asp Phe His Val
Glu Ala Gly Thr Asp Gly Ile Val Ile Val Gly Thr 35 40 45Thr Gly Glu
Ser Pro Thr Val Asp Val Asp Glu His Cys Leu Leu Ile 50 55 60Lys Thr
Thr Ile Glu His Val Ala Lys Arg Val Pro Val Ile Ala Gly65 70 75
80Thr Gly Ala Asn Ser Thr Ala Glu Ala Ile Glu Leu Thr Ala Lys Ala
85 90 95Lys Ala Leu Gly Ala Asp Ala Cys Leu Leu Val Ala Pro Tyr Tyr
Asn 100 105 110Lys Pro Ser Gln Glu Gly Leu Tyr Gln His Phe Lys Ala
Val Ala Glu 115 120 125Ala Val Asp Ile Pro Gln Ile Leu Tyr Asn Val
Pro Gly Arg Thr Gly 130 135 140Cys Asp Leu Ser Asn Asp Thr Val Leu
Arg Leu Ala Gln Ile Arg Asn145 150 155 160Ile Val Gly Ile Lys Asp
Ala Thr Gly Gly Ile Glu Arg Gly Thr Asp 165 170 175Leu Leu Leu Arg
Ala Pro Ala Asp Phe Ala Ile Tyr Ser Gly Asp Asp 180 185 190Ala Thr
Ala Leu Ala Leu Met Leu Leu Gly Gly Lys Gly Val Ile Ser 195 200
205Val Thr Ala Asn Val Ala Pro Lys Leu Met His Glu Met Cys Glu His
210 215 220Ala Leu Asn Gly Asn Leu Ala Ala Ala Lys Ala Ala Asn Ala
Lys Leu225 230 235 240Phe Ala Leu His Gln Lys Leu Phe Val Glu Ala
Asn Pro Ile Pro Val 245 250 255Lys Trp Val Leu Gln Gln Met Gly Met
Ile Ala Thr Gly Ile Arg Leu 260 265 270Pro Leu Val Asn Leu Ser Ser
Gln Tyr His Glu Val Leu Arg Asn Ala 275 280 285Met Lys Gln Ala Glu
Ile Ala Ala 290 295113390DNAMethylophilus
methylotrophusCDS(2080)..(2883) 11ccgcaggtcg ctctagagga tcagagttgg
acggacaagc tgaagttttg ggagtctgaa 60gaagctgcgg gcgaagtgat aaagcagctg
aatcaactgt agccactgca agcgacgaat 120gaaagcaaag gcgctgcact
cgctaaggat gaggcagccg aatctcagaa aaccacgtca 180gagcctgtca
aggccgagca agaggtattg ccctcggcca ctgcaacaaa taattcagct
240gctgcagcga cattggctga agaagaagtg gttccctaca ttccggaggg
ggagtatcag 300gctgcaccca ctccagaaga gatggccaag ggtaatctgg
atgtcagtga aaaccaggtt 360actgaggcta aggcacatcc agtgaatgaa
aaggaaatgg ctgcccaaat tgcagatacg 420gttgagccac cacccgtttt
tcagcaggaa ccgatggcag aacctattgt agcggctgaa 480cccgaacccg
tattgccacc gcccgtaaaa gccgaaccag ctgtgaagaa tatcacagcg
540ccagttgttg ccgcagccac tgttgcagcg gcggcaacca agactgctga
atctgagtca 600gttaaatcca aacctgttga tcctaagcct gtggaagcaa
aaaccgctgt atcaaaaact 660gaagtacaaa cacccgcggc acaggcacct
gctgcggcag cggccgttga agatgacgag 720gtcattccat atattcccga
aggtgaatat gtggctcctg tcattcctag tgaggccgaa 780atggttaaag
gcaatatggc ggaggcaaat gcacctgcga ctgatgctca agcgcgccag
840gtaactgaaa aaggggtggc acccacatcg gatgcggcag cagagccatc
accgacattt 900gtcgctgagc aattgccaga accagagcca gaacctgaat
tgccaccgcc gcctccgcca 960tccgtcagca agcctgttgt gagagaggta
gcgccagtgg ctgcgctggc agcagaagaa 1020gagaaaccag tcgctgcgca
gcctgagact gagcagccgg ctgccaaggt tgttgagcct 1080gcatcggtcg
cctcccctgt ggcgacgcca gaagcgccag ctggtgatgc tgaaatcaac
1140caggctgtgg cggcatgggc acaagcttgg cgcagcaagg acattaaaaa
ctacctcgct 1200gcatatgccc ctgacttcat gccagaaggg ttgccttcca
gaaaggcatg ggagtcgcaa 1260cgcaaacagc gtttatctgc aggccagggt
gcgattacac tcgtactaaa taatgtgcag 1320attcagcgtg acggtaccac
tgtcgccgtg cagtttgagc aaaaatatgc tgctaaagtt 1380tataaagatg
aattggtcaa aacactggaa atgcgttacg agccaacgca gaaacgttgg
1440ttgatcacac gtgaacgtgt tgccccttta accggtttgc cagtagcgag
tgtgccaacg 1500acccgtctgc cagcagtcgc tgcagcgtca tccaatacgg
atgtggtcga gtcagctgtg 1560ccaccgacac aatcgacatc atctgcgcct
gtagcggaag tgagtgttga atcagcgatt 1620gacgcctggg cacaggcttg
gcgcagtaaa aacatcaatg cttactttgc ggcgtattct 1680ccagaatttg
tgccggaggg attgccaaac agaggtgtct gggaagcgca acgtaaaaag
1740cgcttgtccc cacagcaggg caagatcagc ctggatgtca cgaatgtaag
cgtgagccgc 1800gaaggagaaa cagccgtggc cacctttagg cagaaatatg
cgtctaaggc ctatcgtgat 1860gaagtagtga agcgtctaca gttaaaactg
gatgctgcaa gcaatcgctg gctgattgtg 1920cgtgaaagta ccggtagtga
ggcagaagtg ccaatgggca agcagtcagt gagtgcgcca 1980gaagagagct
cggaacatca ggatggtgct ctggagccga tcggatttta atggtctgct
2040gatgtcgtgg tttaagtatt aaaaataatt gagtgagtt atg ttg aaa gta gtg
2094 Met Leu Lys Val Val 1 5att gct ggc gtg tct ggt cgt atg gga cat
gcc tta ctg gat gga gtt 2142Ile Ala Gly Val Ser Gly Arg Met Gly His
Ala Leu Leu Asp Gly Val 10 15 20ttt tct gat aac ggc ttg cag ttg cac
gcg gca ctc gat cgt gct gaa 2190Phe Ser Asp Asn Gly Leu Gln Leu His
Ala Ala Leu Asp Arg Ala Glu 25 30 35agc gcc atg ata ggg cgg gat gca
ggc gag cag ttt ggc aag gtc agt 2238Ser Ala Met Ile Gly Arg Asp Ala
Gly Glu Gln Phe Gly Lys Val Ser 40 45 50ggc gtg aaa atc acg gct gac
atc cat gcc gca ttg gtc ggt gcc gat 2286Gly Val Lys Ile Thr Ala Asp
Ile His Ala Ala Leu Val Gly Ala Asp 55 60 65gtg ctg gtg gat ttc acg
cgg ccg gaa gcc agt atg caa tat tta caa 2334Val Leu Val Asp Phe Thr
Arg Pro Glu Ala Ser Met Gln Tyr Leu Gln70 75 80 85gcc tgc cag caa
gcc aac gtt aaa tta gtg att ggt act acc ggg ttt 2382Ala Cys Gln Gln
Ala Asn Val Lys Leu Val Ile Gly Thr Thr Gly Phe 90 95 100agt gag
gca gaa aag gcc agt att gag gct gcg tcc aaa aat atc ggt 2430Ser Glu
Ala Glu Lys Ala Ser Ile Glu Ala Ala Ser Lys Asn Ile Gly 105 110
115atc gta ttt gct cca aac atg agc gta ggg gtc acc ctc ttg att aac
2478Ile Val Phe Ala Pro Asn Met Ser Val Gly Val Thr Leu Leu Ile Asn
120 125 130ctg gtt gag caa gcc gca cgg gtg ctc aat gaa ggc tat gat
att gag 2526Leu Val Glu Gln Ala Ala Arg Val Leu Asn Glu Gly Tyr Asp
Ile Glu 135 140 145gtg gtt gaa atg cat cac cgc cat aag gtg gat gcg
cct tca ggc acg 2574Val Val Glu Met His His Arg His Lys Val Asp Ala
Pro Ser Gly Thr150 155 160 165gct tta cgg ttg ggt gag gct gcg gca
aaa ggg att gat aaa gcg ctt 2622Ala Leu Arg Leu Gly Glu Ala Ala Ala
Lys Gly Ile Asp Lys Ala Leu 170 175 180aaa gat tgt gct gtg tat gcg
cgc gaa ggc gtg act ggt gaa cgc gaa 2670Lys Asp Cys Ala Val Tyr Ala
Arg Glu Gly Val Thr Gly Glu Arg Glu 185 190 195gcg ggc acg att ggt
ttt gca acc tta cgt ggt ggg gat gtg gtc ggt 2718Ala Gly Thr Ile Gly
Phe Ala Thr Leu Arg Gly Gly Asp Val Val Gly 200 205 210gac cat acg
gtg gtt ctg gct ggt gtg ggt gag cga gta gag tta acg 2766Asp His Thr
Val Val Leu Ala Gly Val Gly Glu Arg Val Glu Leu Thr 215 220 225cat
aaa gca tca agc cgt gcc aca ttt gca caa ggt gcg tta cgt gcg 2814His
Lys Ala Ser Ser Arg Ala Thr Phe Ala Gln Gly Ala Leu Arg Ala230 235
240 245gct aaa ttt ctg gct gat aaa ccc aag gga ttg ttt gat atg cgt
gat 2862Ala Lys Phe Leu Ala Asp Lys Pro Lys Gly Leu Phe Asp Met Arg
Asp 250 255 260gtg ttg gga ttt gaa aag aac tgatctttag taggcgatcc
cgtctggcta 2913Val Leu Gly Phe Glu Lys Asn 265aggtctggca ggaatcgtct
gatgcttctg agttgccctt gagtgggctg tcaatgtacg 2973ctataatgct
gtaattctga aacgggaaga gtcgaacaag cttttcccgt tttgcacatc
3033tattcactgc agcttgaatt tcacttccag ccatggtgaa ccctctaaaa
gatgtgtttc 3093gtgtcaaact taaggagcta aaggtgtcaa aaacaattcc
agcgattctc gtgttagcag 3153atggaactgt ttttaagggc attagcattg
gcgcttccgg tcatacggta ggtgaggtgg 3213tgtttaatac ctccatcacc
ggttatcagg agattcttac cgatccttcc tataccgaac 3273aaatcgtgac
actgacctat ccgcacattg gtaactacgg gaccaatcgt gaagatggga
3333gtcaggtaaa gtctatgctg cgggtctgat ccccgggacc gagccgggtt cgtaaag
339012268PRTMethylophilus methylotrophus 12Met Leu Lys Val Val Ile
Ala Gly Val Ser Gly Arg Met Gly His Ala1 5 10 15Leu Leu Asp Gly Val
Phe Ser Asp Asn Gly Leu Gln Leu His Ala Ala 20 25 30Leu Asp Arg Ala
Glu Ser Ala Met Ile Gly Arg Asp Ala Gly Glu Gln 35 40 45Phe Gly Lys
Val Ser Gly Val Lys Ile Thr Ala Asp Ile His Ala Ala 50 55 60Leu Val
Gly Ala Asp Val Leu Val Asp Phe Thr Arg Pro Glu Ala Ser65 70 75
80Met Gln Tyr Leu Gln Ala Cys Gln Gln Ala Asn Val Lys Leu Val Ile
85 90 95Gly Thr Thr Gly Phe Ser Glu Ala Glu Lys Ala Ser Ile Glu Ala
Ala 100 105 110Ser Lys Asn Ile Gly Ile Val Phe Ala Pro Asn Met Ser
Val Gly Val 115 120 125Thr Leu Leu Ile Asn Leu Val Glu Gln Ala Ala
Arg Val Leu Asn Glu 130 135 140Gly Tyr Asp Ile Glu Val Val Glu Met
His His Arg His Lys Val Asp145 150 155 160Ala Pro Ser Gly Thr Ala
Leu Arg Leu Gly Glu Ala Ala Ala Lys Gly 165 170 175Ile Asp Lys Ala
Leu Lys Asp Cys Ala Val Tyr Ala Arg Glu Gly Val 180 185 190Thr Gly
Glu Arg Glu Ala Gly Thr Ile Gly Phe Ala Thr Leu Arg Gly 195 200
205Gly Asp Val Val Gly Asp His Thr Val Val Leu Ala Gly Val Gly Glu
210 215 220Arg Val Glu Leu Thr His Lys Ala Ser Ser Arg Ala Thr Phe
Ala Gln225 230 235 240Gly Ala Leu Arg Ala Ala Lys Phe Leu Ala Asp
Lys Pro Lys Gly Leu 245 250 255Phe Asp Met Arg Asp Val Leu Gly Phe
Glu Lys Asn 260 265132566DNAMethylophilus
methylotrophusCDS(751)..(1995) 13tgctttaggg ggaacctaga ggatccccct
acccgaggaa gaagtgagcc aacatgtact 60tccagtcgta ccatcaaaag tagaagtttt
cggcgttatc ctgattcaca gtaaacgaaa 120aattgcccat attctgaccg
gatttaccgg tggcttttaa ggtataagtg gtcgctgact 180ggttctcaat
gctgtaatca aaaaatttgg catcactggg gacacaggca aatcccacat
240atgtgaagtt gtcctgataa aactgttcgg cctgcacacg gcaattggca
agattggcag 300gcgcttccgc ggcattaccg cttttgatgt aatcctgata
gcctggtatg gcgatgctgg 360ccaagatacc cataatggcc accacgacca
tgacttctat caggctgaat ccgtactgat 420ttgaggactt cattatcaaa
ccccttttta gatagcctta tcatgcaaac aggcagctgt 480catgtccagc
atcagccgac caatggtcag gattacccga cgaacggtca aaccactaaa
540acgcccagtc actggtgcca tgagcaactg caggtttaat gataaaatgg
cactcaattt 600acattggact gtgaacatgt tttccttcta tacgagatta
ttggcggttg ccctgctatt 660ggcacaattg agtgcctgtg gtctcaaagg
ggacctgtat attcctgagc gccaataccc 720tcaaacgcct caacaagata
agtcttcatc gtg acc gct ttt tca atc caa caa 774 Val Thr Ala Phe Ser
Ile Gln Gln 1 5ggc cta cta cat gcc gag aat gta gcc ctg cgt gac att
gca caa acg 822Gly Leu Leu His Ala Glu Asn Val Ala Leu Arg Asp Ile
Ala Gln Thr 10 15 20cat caa acg ccc act tac gtc tat tca cgt gcc gcc
ttg acg act gct 870His Gln Thr Pro Thr Tyr Val Tyr Ser Arg Ala Ala
Leu Thr Thr Ala25 30 35 40ttc gag cgt ttt cag gca ggc ctg act gga
cat gac cat ttg atc tgc 918Phe Glu Arg Phe Gln Ala Gly Leu Thr Gly
His Asp His Leu Ile Cys 45 50 55ttt gct gtc aaa gcc aac cca agc ctg
gcc att ctc aac ctg ttt gcg 966Phe Ala Val Lys Ala Asn Pro Ser Leu
Ala Ile Leu Asn Leu Phe Ala 60 65 70cga atg gga gcg ggc ttt gat att
gtg tcc ggt ggt gag ctg gca cgc 1014Arg Met Gly Ala Gly Phe Asp Ile
Val Ser Gly Gly Glu Leu Ala Arg 75 80 85gtc ttg gcc gca ggt ggc gac
ccg aaa aaa gtg gtg ttt tct ggt gtg 1062Val Leu Ala Ala Gly Gly Asp
Pro Lys Lys Val Val Phe Ser Gly Val 90 95 100ggc aaa tcc cat gcg
gaa atc aaa gcc gcg ctt gaa gcg ggc att ctt 1110Gly Lys Ser His Ala
Glu Ile Lys Ala Ala Leu Glu Ala Gly Ile Leu105 110 115 120tgc ttc
aac gtg gaa tca gtg aat gag cta gac cgc atc cag cag gtg 1158Cys Phe
Asn Val Glu Ser Val Asn Glu Leu Asp Arg Ile Gln Gln Val 125 130
135gcg gcc agc ctg ggc aaa aaa gcg cct att tcc ctg cgc gtg aac ccc
1206Ala Ala Ser Leu Gly Lys Lys Ala Pro Ile Ser Leu Arg Val Asn Pro
140 145 150aat gtg gat gcc aaa aca cat ccc tat att tcc cac ccg gct
ctc aaa 1254Asn Val Asp Ala Lys Thr His Pro Tyr Ile Ser His Pro Ala
Leu Lys 155 160 165aac aat aaa ttt ggt gtg gca ttt gaa gat gcc ttg
ggc ctc tat gaa 1302Asn Asn Lys Phe Gly Val Ala Phe Glu Asp Ala Leu
Gly Leu Tyr Glu 170 175 180aaa gcg gcg caa ctg cca aac atc gag gta
cac ggc gta gat tgc cat 1350Lys Ala Ala Gln Leu Pro Asn Ile Glu Val
His Gly Val Asp Cys His185 190 195 200atc ggc tcg caa atc act gag
ctg tca cct ttc ctc gat gcc ttg gat 1398Ile Gly Ser Gln Ile Thr Glu
Leu Ser Pro Phe Leu Asp Ala Leu Asp 205 210 215aaa gta ttg ggc ctg
gta gat gca ttg gcc gcc aaa ggc att cat atc 1446Lys Val Leu Gly Leu
Val Asp Ala Leu Ala Ala Lys Gly Ile His Ile 220 225 230cag cat ata
gac gtt ggc ggc ggt gtc ggt att act tac agc gac gaa 1494Gln His Ile
Asp Val Gly Gly Gly Val Gly Ile Thr Tyr Ser Asp Glu 235 240 245acg
cca cca gac ttt gca gcc tac act gca gcg att ctt aaa aag ctg 1542Thr
Pro Pro Asp Phe Ala Ala Tyr Thr Ala Ala Ile Leu Lys Lys Leu 250 255
260gca ggc agg aat gta aaa gtg ttg ttt gag ccc ggc cgt gcc ctg gtg
1590Ala Gly Arg Asn Val Lys Val Leu Phe Glu Pro Gly Arg Ala Leu
Val265 270 275 280ggt aac gcc ggt gtg ctg ctg acc aag gtc gaa tac
ctg aaa cct ggc 1638Gly Asn Ala Gly Val Leu Leu Thr Lys Val Glu Tyr
Leu Lys Pro Gly 285 290 295gaa acc aaa aac ttt gcg att gtc gat gcc
gcc atg aac gac ctc atg 1686Glu Thr Lys Asn Phe Ala Ile Val Asp Ala
Ala Met Asn Asp Leu Met 300 305 310cgc ccg gct ttg tat gat gct ttc
cac aac att acg acc att gcc act 1734Arg Pro Ala Leu Tyr Asp Ala Phe
His Asn Ile Thr Thr Ile Ala Thr 315 320 325tct gca gcc ccc gca caa
atc tat gag atc gtt ggc ccg gtt tgc gag 1782Ser Ala Ala Pro Ala Gln
Ile
Tyr Glu Ile Val Gly Pro Val Cys Glu 330 335 340agt ggt gac ttt tta
ggc cat gac cgt aca ctt gcg atc gaa gaa ggt 1830Ser Gly Asp Phe Leu
Gly His Asp Arg Thr Leu Ala Ile Glu Glu Gly345 350 355 360gat tac
ctg gcg att cac tcc gca ggc gct tat ggc atg agc atg gcc 1878Asp Tyr
Leu Ala Ile His Ser Ala Gly Ala Tyr Gly Met Ser Met Ala 365 370
375agc aac tac aac acg cgc gcc cgt gcc gca gag gta ttg gtt gat ggt
1926Ser Asn Tyr Asn Thr Arg Ala Arg Ala Ala Glu Val Leu Val Asp Gly
380 385 390gac cag gtg cat gtg atc cgt gaa cgt gaa caa att gcc gac
ctg ttt 1974Asp Gln Val His Val Ile Arg Glu Arg Glu Gln Ile Ala Asp
Leu Phe 395 400 405aaa ctg gag cgt acg ctg cca taacattgac
ggcaacccct aataaaaaaa 2025Lys Leu Glu Arg Thr Leu Pro 410
415ccgaagccgc caagcttcgg ttttttatta atagcgcatc ctttaatcaa
agatcacggt 2085cttgttcgcg tagagcaaga ttctatgctc aatatgccag
cgcacggctt tggaaagcac 2145aacacgctcc aggtcacggc ctttctggat
caggtcttcc acctgatcgc ggtgtgaaat 2205gcgcgccaag tcctgctcaa
taatcggccc ctcatccaac acctctgtca cataatgact 2265ggtcgcaccg
atcagtttca cgccacgctc aaacgcacgg tggtaaggac gtgcgccgat
2325aaatgctggc aggaatgagt ggtggtgaat gttgataatc cgctgaggat
accgtgcgac 2385aaaatctggt gacagaatct gcatgtagcg tgccagcaca
atcaggtcaa tcttgtgttg 2445atcaaacagg gcaaactgct gngcctctac
ctctgccttg gtttaccttg gtcatcggta 2505aatagtgaaa cgggatgcca
taaaactgcg ccagggggat cctctgggtc cccctaaagc 2565a
256614415PRTMethylophilus methylotrophusmisc_feature(2467)..(2467)n
= a, c, g, or t 14Val Thr Ala Phe Ser Ile Gln Gln Gly Leu Leu His
Ala Glu Asn Val1 5 10 15Ala Leu Arg Asp Ile Ala Gln Thr His Gln Thr
Pro Thr Tyr Val Tyr 20 25 30Ser Arg Ala Ala Leu Thr Thr Ala Phe Glu
Arg Phe Gln Ala Gly Leu 35 40 45Thr Gly His Asp His Leu Ile Cys Phe
Ala Val Lys Ala Asn Pro Ser 50 55 60Leu Ala Ile Leu Asn Leu Phe Ala
Arg Met Gly Ala Gly Phe Asp Ile65 70 75 80Val Ser Gly Gly Glu Leu
Ala Arg Val Leu Ala Ala Gly Gly Asp Pro 85 90 95Lys Lys Val Val Phe
Ser Gly Val Gly Lys Ser His Ala Glu Ile Lys 100 105 110Ala Ala Leu
Glu Ala Gly Ile Leu Cys Phe Asn Val Glu Ser Val Asn 115 120 125Glu
Leu Asp Arg Ile Gln Gln Val Ala Ala Ser Leu Gly Lys Lys Ala 130 135
140Pro Ile Ser Leu Arg Val Asn Pro Asn Val Asp Ala Lys Thr His
Pro145 150 155 160Tyr Ile Ser His Pro Ala Leu Lys Asn Asn Lys Phe
Gly Val Ala Phe 165 170 175Glu Asp Ala Leu Gly Leu Tyr Glu Lys Ala
Ala Gln Leu Pro Asn Ile 180 185 190Glu Val His Gly Val Asp Cys His
Ile Gly Ser Gln Ile Thr Glu Leu 195 200 205Ser Pro Phe Leu Asp Ala
Leu Asp Lys Val Leu Gly Leu Val Asp Ala 210 215 220Leu Ala Ala Lys
Gly Ile His Ile Gln His Ile Asp Val Gly Gly Gly225 230 235 240Val
Gly Ile Thr Tyr Ser Asp Glu Thr Pro Pro Asp Phe Ala Ala Tyr 245 250
255Thr Ala Ala Ile Leu Lys Lys Leu Ala Gly Arg Asn Val Lys Val Leu
260 265 270Phe Glu Pro Gly Arg Ala Leu Val Gly Asn Ala Gly Val Leu
Leu Thr 275 280 285Lys Val Glu Tyr Leu Lys Pro Gly Glu Thr Lys Asn
Phe Ala Ile Val 290 295 300Asp Ala Ala Met Asn Asp Leu Met Arg Pro
Ala Leu Tyr Asp Ala Phe305 310 315 320His Asn Ile Thr Thr Ile Ala
Thr Ser Ala Ala Pro Ala Gln Ile Tyr 325 330 335Glu Ile Val Gly Pro
Val Cys Glu Ser Gly Asp Phe Leu Gly His Asp 340 345 350Arg Thr Leu
Ala Ile Glu Glu Gly Asp Tyr Leu Ala Ile His Ser Ala 355 360 365Gly
Ala Tyr Gly Met Ser Met Ala Ser Asn Tyr Asn Thr Arg Ala Arg 370 375
380Ala Ala Glu Val Leu Val Asp Gly Asp Gln Val His Val Ile Arg
Glu385 390 395 400Arg Glu Gln Ile Ala Asp Leu Phe Lys Leu Glu Arg
Thr Leu Pro 405 410 4151539DNAArtificial SequenceSynthetic DNA
15agggaattcc ccgttctgga taatgttttt tgcgccgac 391658DNAArtificial
SequenceSynthetic DNA 16cggatgcatc tagagttaac ctgcagggtg aaattgttat
ccgctcacaa ttccacac 581735DNAArtificial SequenceSynthetic DNA
17tgacctgcag gtttgcacag aggatggccc atgtt 351836DNAArtificial
SequenceSynthetic DNA 18cattctagat ccctaaactt tacagcaaac cggcat
361935DNAArtificial SequenceSynthetic DNA 19gaacctgcag gccctgacac
gaggtagatt atgtc 352055DNAArtificial SequenceSynthetic DNA
20ctttcggcta gaagagcgag atgcagataa aaaaattaaa ggcaattatt ctccg
55
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