U.S. patent application number 11/738617 was filed with the patent office on 2008-02-14 for l-amino acid-producing bacterium and method for producing l-amino acid.
Invention is credited to Yoshiya Gunji, Yuri Miyata, Manami Oba, Megumi Shimaoka, Shinichi Sugimoto, Nobuharu Tsujimoto, Hisashi Yasueda.
Application Number | 20080038825 11/738617 |
Document ID | / |
Family ID | 27309903 |
Filed Date | 2008-02-14 |
United States Patent
Application |
20080038825 |
Kind Code |
A1 |
Gunji; Yoshiya ; et
al. |
February 14, 2008 |
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) |
Correspondence
Address: |
CERMAK & KENEALY LLP;ACS LLC
515 EAST BRADDOCK ROAD
SUITE B
ALEXANDRIA
VA
22314
US
|
Family ID: |
27309903 |
Appl. No.: |
11/738617 |
Filed: |
April 23, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09926299 |
Oct 9, 2001 |
7223572 |
|
|
PCT/JP00/02295 |
Apr 7, 2000 |
|
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11738617 |
Apr 23, 2007 |
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Current U.S.
Class: |
435/471 ;
435/252.1 |
Current CPC
Class: |
C12N 9/88 20130101; C12R
1/01 20130101; C12P 13/08 20130101; C12N 9/1217 20130101; C12P
13/04 20130101 |
Class at
Publication: |
435/471 ;
435/252.1 |
International
Class: |
C12N 15/74 20060101
C12N015/74; C12N 1/20 20060101 C12N001/20 |
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 cells of a Methylophilus methylotrophus
strain with an increased content of an L-amino acid comprising
culturing a Methylophilus methylotrophus strain which is able to
produce the L-amino acid in a medium.
2. The method according to claim 1, wherein the L-amino acid is
selected from the group consisting of L-lysine, L-valine,
L-leucine, L-isoleucine, and L-threonine.
3. The method according to claim 1, wherein said strain is able to
produce L-lysine, and is modified to enhance dihydrodipicolinate
synthase activity as compared to a wild-type Methylophilus
methylotrophus strain, and wherein said dihydrodipicolinate
synthase is selected from the group consisting of: (a) a protein
encoded by a DNA comprising nucleotides 1268 to 2155 of SEQ ID NO:
9; and (b) a protein having dihydrodipicolinate synthase activity
and encoded by a DNA comprising nucleotides 1268 to 2155 of SEQ ID
NO: 9, except that substitution, deletion, or addition of one to 10
amino acids is present in the amino acid sequence of said protein,
and wherein said activity is enhanced by a method selected from the
group consisting of: i) increasing the copy number of said DNA in
said strain, ii) placing multiple copies of said DNA on the
chromosome of said strain, and iii) replacing a native promoter
with a stronger promoter upstream of said DNA.
4. The method according to claim 3, wherein said strain is further
modified to enhance an activity or activities of one, two or three
enzymes selected from the group consisting of aspartic acid
semialdehyde dehydrogenase, dihydrodipicolinate reductase, and
diaminopimelate decarboxylase as compared to a wild-type
Methylophilus methylotrophus strain by a method selected from the
group consisting of: i) increasing the copy number(s) of a DNA(s)
encoding said one, two, or three enzyme(s) in said strain, ii)
placing multiple copies of said DNA(s) on the chromosome of said
strain, and iii) replacing a native promoter with a stronger
promoter upstream of said DNA(s).
5. The method according to claim 1, wherein said strain has
L-lysine-producing ability, and is modified to enhance
dihydrodipicolinate synthase activity and aspartokinase activity as
compared to a wild-type Methylophilus methylotrophus strain, and
wherein said dihydrodipicolinate synthase is selected from the
group consisting of: (a) a protein encoded by a DNA comprising
nucleotides 1268 to 2155 of SEQ ID NO: 9; and (b) a protein having
dihydrodipicolinate synthase activity and encoded by a DNA
comprising nucleotides 1268 to 2155 of SEQ ID NO: 9, except that
substitution, deletion, or addition of one to 10 amino acids is
present in the amino acid sequence of said protein, and wherein
said aspartokinase is selected from the group consisting of: (a) a
protein encoded by a DNA comprising nucleotides 510 to 1736 of SEQ
ID NO: 5; and (b) a protein having aspartokinase activity and
encoded by a DNA comprising nucleotides 510 to 1736 of SEQ ID NO:
5, except that substitution, deletion, or addition of one to 10
amino acids is present in the amino acid sequence of said protein,
and wherein said activities are enhanced by a method selected from
the group consisting of: i) increasing the copy number of said DNAs
in said strain, ii) placing multiple copies of said DNAs on the
chromosome of said strain, and iii) replacing a native promoter
with a stronger promoter upstream of said DNAs.
6. The method according to claim 5, wherein said strain is further
modified to enhance an activity or activities of one, two, or three
enzymes selected from the group consisting of aspartic acid
semialdehyde dehydrogenase, dihydrodipicolinate reductase and
diaminopimelate decarboxylase as compared to a wild-type
Methylophilus methylotrophus strain by a method selected from the
group consisting of: i) increasing the copy number(s) of a DNA(s)
encoding said one, two, or three enzyme(s) in said strain, ii)
placing multiple copies of said DNA(s) on the chromosome of said
strain, and iii) replacing a native promoter with a stronger
promoter upstream of said DNA(s).
7. The method according to claim 5, wherein the dihydrodipicolinate
synthase activity and the aspartokinase activity are enhanced as
compared to a wild-type Methylophilus methylotrophus strain by
transformation with a DNA coding for said dihydrodipicolinate
synthase and a DNA coding for said aspartokinase.
8. The method according to claim 1, wherein said strain is modified
to enhance aspartokinase activity as compared to a wild-type
Methylophilus methylotrophus strain, and wherein said aspartokinase
is selected from the group consisting of: (a) a protein encoded by
a DNA comprising nucleotides 510 to 1736 of SEQ ID NO: 5; and (b) a
protein having aspartokinase activity and encoded by a DNA
comprising nucleotide numbers 510 to 1736 of SEQ ID NO: 5, except
that substitution, deletion, or addition of one to 10 amino acids
is present in the amino acid sequence of said protein, and wherein
said activity is enhanced by a method selected from the group
consisting of: i) increasing the copy number of said DNA in said
strain, ii) placing multiple copies of said DNA on the chromosome
of said strain, and iii) replacing a native promoter with a
stronger promoter upstream of said DNA.
9. The isolated strain according to claim 8, wherein said strain is
further modified to enhance activities of homoserine dehydrogenase,
homoserine kinase and threonine synthase as compared to a wild-type
Methylophilus methylotrophus strain by a method selected from the
group consisting of: i) increasing the copy numbers of DNAs
encoding homoserine dehydrogenase, homoserine kinase and threonine
synthase in said strain, ii) placing multiple copies of said DNAs
on the chromosome of said strain, and iii) replacing a native
promoter with a stronger promoter upstream of said DNAs, and
wherein said strain has L-threonine-producing ability.
10. The method according to claim 8, wherein the L-amino acid is
L-lysine.
11. The method according to claim 10, wherein said strain is
further modified to enhance an activity or activities of one, two
or three enzymes selected from the group consisting of aspartic
acid semialdehyde dehydrogenase, dihydrodipicolinate reductase and
diaminopimelate decarboxylase as compared to a wild-type
Methylophilus methylotrophus strain by a method selected from the
group consisting of: i) increasing the copy number(s) of a DNA(s)
encoding said one, two, or three enzyme(s) in said strain, ii)
placing multiple copies of said DNA(s) on the chromosome of said
strain, and iii) replacing a native promoter with a stronger
promoter upstream of said DNA(s).
Description
[0001] This application claims priority under 35 U.S.C. .sctn.120
as a divisional of U.S. patent application Ser. No. 09/926,299,
filed Oct. 9, 2001, now allowed, 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, Japanese Patent Application 11-368097, filed Dec.
24, 1999, and PCT Application No. PCT/JP00/02295, filed Apr. 7,
2000. 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 DNA
as 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 achieved. 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 pathway may 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.quadrature.131397/1980, 59.quadrature.31691/1984 and
56.quadrature.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.quadrature.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 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 U.S. 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 (NCIMB10515),
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, Conn., 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.times.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 steam-sterilized at 120.degree. C.
for 20 minutes
[0154] L agar medium: TABLE-US-00002 L medium Bacto agar (DIFCO) 15
g/L steam-sterilized at 120.degree. C. for 20 minutes
[0155] 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
[0156] 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.
[0157] 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
[0158] The constituents except for methanol were steam-sterilized
at 121.degree. C. for 15 minutes. After the constituents
sufficiently cooled, methanol was added.
[0159] 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
[0160] 121M1 Agar medium: TABLE-US-00006 121M1 medium Bacto agar
(DIFCO) 15 g/L
[0161] The constituents except for methanol were steam-sterilized
at 121.degree. C. for 15 minutes. After the constituents
sufficiently cooled, methanol was added.
[0162] 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.2O 246.48
mg/L Glucose 2 g/L pH 7.0
[0163] 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.
[0164] M9 minimal agar medium: TABLE-US-00008 M9 minimal medium
Bacto agar (DIFCO) 15 g/L
Example 1
Creation of L-Lysine-Producing Bacterium: (I)
[0165] (1) Introduction of Mutant lysC and Mutant dapA into
Methylophilus Bacterium
[0166] 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.
[0167] 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.
[0168] 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.
[0169] 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).
[0170] The RSFD80 plasmid as described above was introduced into
the Methylophilus methylotrophus AS1 strain (NCIMB10515) 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.
[0171] (2) AKIII Activity of Methylophilus Bacterium Containing
Mutant lysC and Mutant dapA Derived from E. coli
[0172] 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.
[0173] 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.
[0174] 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.
[0175] 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 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
[0176] TABLE-US-00010 TABLE 1 AK activity Desensitization (Specific
Specific activity degree of Strain activity*.sup.1) with 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
[0177] 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).
[0178] (3) Production of L-Lysine by Methylophilus Bacterium
Containing Mutant lysC and Mutant dapA Derived from E. coli
[0179] 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
Creation of L-Lysine-Producing Bacterium (II)
[0180] (1) Introduction of the tac Promoter Region into a Broad
Host Spectrum Vector
[0181] 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.
[0182] 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.
[0183] (2) Preparation of dapA Gene (Dihydrodipicolinate Synthase
Gene) Expression Plasmid pRS-dapA24 and lysC Gene (Aspartokinase
Gene) Expression Plasmid pRS-lysC80
[0184] A mutant gene (dapA*24) coding for dihydrodipicolinate
synthase with partially desensitized feedback inhibition by Lys was
introduced into the plasmid pRS-tac, which was prepared as
described above (1).
[0185] 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 Sse83871 and XbaI. pRS-tac was also treated
with Sse83871 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.
[0186] 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 Sse83871 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.
[0187] (3) Introduction of pRS-dapA24 or pRS-lysC80 into
Methylophilus methylotrophus And Evaluation of the Culture
[0188] pRS-dapA24 and pRS-lysC80 obtained as described above were
each separately introduced into the Methylophilus methylotrophus
AS1 strain (NCIMB10515) 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
Creation of L-Lysine-Producing Bacterium (III)
[0189] 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.
[0190] 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
[0191] 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
Creation of L-Threonine-Producing Bacterium
[0192] (1) Introduction of Threonine Operon Plasmid into a
Methylophilus Bacterium
[0193] 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 (NCIMB10515) 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.
[0194] (2) Production of L-Threonine by Methylophilus Bacterium
Containing the Threonine Operon Derived from E. coli
[0195] The AS1/pVIC40 and AS1/pRS strains were each inoculated into
121 production medium containing 20 mg/L of streptomycin, 1 .mu.l
of L-valine and 1 .mu.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
Creation of Branched Chain Amino Acid-Producing Bacterium
[0196] 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 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 C.sub.1-38 strain.
[0197] The Methylophilus methylotrophus C.sub.1-38 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.
[0198] The parent strain (AS1 strain) and the C.sub.1-38 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
Preparation of Chromosomal DNA Library of Methylophilus
methylotrophus AS1 Strain
[0199] (1) Preparation of Chromosome DNA of Methylophilus
methylotrophus AS1 Strain
[0200] One platinum loop of the Methylophilus methylotrophus AS1
strain (NCIMB10515) 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.
[0201] 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)).
[0202] (2) Preparation of the Gene Library
[0203] 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 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.
[0204] 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 PI, 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.
[0205] 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).
[0206] 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).
[0207] 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 PI 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.
[0208] 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
Cloning of the Lysine Biosynthesis Gene of Methylophilus
methylotrophus AS1 Strain
[0209] (1) Cloning of the Gene Coding for Aspartokinase (AK)
[0210] 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.
[0211] 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.
[0212] 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.
[0213] (2) Cloning of Gene Coding for Aspartic Acid Semialdehyde
Dehydrogenase (ASD)
[0214] 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.
[0215] 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).
[0216] 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.
[0217] (3) Cloning of Gene Coding for Dihydrodipicolinate Synthase
(DDPS)
[0218] 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.
[0219] 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).
[0220] 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.
[0221] (4) Cloning of Gene Coding for Dihydrodipicolinate Reductase
(DDPR)
[0222] 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.
[0223] 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).
[0224] 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.
[0225] (5) Cloning of Gene Coding for Diaminopimelate Decarboxylase
(DPDC)
[0226] 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.
[0227] 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).
[0228] 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
[0229] 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.
[0230] 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
20 1 1197 DNA Escherichia coli CDS (272)..(1147) 1 ccaggcgact
gtcttcaata ttacagccgc aactactgac atgacgggtg atggtgttca 60
caattccacg gcgatcggca cccaacgcag tgatcaccag ataatgtgtt gcgatgacag
120 tgtcaaactg gttattcctt taaggggtga gttgttctta aggaaagcat
aaaaaaaaca 180 tgcatacaac aatcagaacg gttctgtctg cttgctttta
atgccatacc aaacgtacca 240 ttgagacact tgtttgcaca gaggatggcc c atg
ttc acg gga agt att gtc 292 Met Phe Thr Gly Ser Ile Val 1 5 gcg att
gtt act ccg atg gat gaa aaa ggt aat gtc tgt cgg gct agc 340 Ala Ile
Val Thr Pro Met Asp Glu Lys Gly Asn Val Cys Arg Ala Ser 10 15 20
ttg aaa aaa ctg att gat tat cat gtc gcc agc ggt act tcg gcg atc 388
Leu Lys Lys Leu Ile Asp Tyr His Val Ala Ser Gly Thr Ser Ala Ile 25
30 35 gtt tct gtt ggc acc act ggc gag tcc gct acc tta aat cat gac
gaa 436 Val Ser Val Gly Thr Thr Gly Glu Ser Ala Thr Leu Asn His Asp
Glu 40 45 50 55 cat gct gat gtg gtg atg atg acg ctg gat ctg gct gat
ggg cgc att 484 His Ala Asp Val Val Met Met Thr Leu Asp Leu Ala Asp
Gly Arg Ile 60 65 70 ccg gta att gcc ggg acc ggc gct aac gct act
gcg gaa gcc att agc 532 Pro Val Ile Ala Gly Thr Gly Ala Asn Ala Thr
Ala Glu Ala Ile Ser 75 80 85 ctg acg cag cgc ttc aat gac agt ggt
atc gtc ggc tgc ctg acg gta 580 Leu Thr Gln Arg Phe Asn Asp Ser Gly
Ile Val Gly Cys Leu Thr Val 90 95 100 acc cct tac tac aat cgt ccg
tcg caa gaa ggt ttg tat cag cat ttc 628 Thr Pro Tyr Tyr Asn Arg Pro
Ser Gln Glu Gly Leu Tyr Gln His Phe 105 110 115 aaa gcc atc gct gag
cat act gac ctg ccg caa att ctg tat aat gtg 676 Lys Ala Ile Ala Glu
His Thr Asp Leu Pro Gln Ile Leu Tyr Asn Val 120 125 130 135 ccg tcc
cgt act ggc tgc gat ctg ctc ccg gaa acg gtg ggc cgt ctg 724 Pro Ser
Arg Thr Gly Cys Asp Leu Leu Pro Glu Thr Val Gly Arg Leu 140 145 150
gcg aaa gta aaa aat att atc gga atc aaa gag gca aca ggg aac tta 772
Ala Lys Val Lys Asn Ile Ile Gly Ile Lys Glu Ala Thr Gly Asn Leu 155
160 165 acg cgt gta aac cag atc aaa gag ctg gtt tca gat gat ttt gtt
ctg 820 Thr Arg Val Asn Gln Ile Lys Glu Leu Val Ser Asp Asp Phe Val
Leu 170 175 180 ctg agc ggc gat gat gcg agc gcg ctg gac ttc atg caa
ttg ggc ggt 868 Leu Ser Gly Asp Asp Ala Ser Ala Leu Asp Phe Met Gln
Leu Gly Gly 185 190 195 cat ggg gtt att tcc gtt acg act aac gtc gca
gcg cgt gat atg gcc 916 His Gly Val Ile Ser Val Thr Thr Asn Val Ala
Ala Arg Asp Met Ala 200 205 210 215 cag atg tgc aaa ctg gca gca gaa
gaa cat ttt gcc gag gca cgc gtt 964 Gln Met Cys Lys Leu Ala Ala Glu
Glu His Phe Ala Glu Ala Arg Val 220 225 230 att aat cag cgt ctg atg
cca tta cac aac aaa cta ttt gtc gaa ccc 1012 Ile Asn Gln Arg Leu
Met Pro Leu His Asn Lys Leu Phe Val Glu Pro 235 240 245 aat cca atc
ccg gtg aaa tgg gca tgt aag gaa ctg ggt ctt gtg gcg 1060 Asn Pro
Ile Pro Val Lys Trp Ala Cys Lys Glu Leu Gly Leu Val Ala 250 255 260
acc gat acg ctg cgc ctg cca atg aca cca atc acc gac agt ggt cgt
1108 Thr Asp Thr Leu Arg Leu Pro Met Thr Pro Ile Thr Asp Ser Gly
Arg 265 270 275 gag acg gtc aga gcg gcg ctt aag cat gcc ggt ttg ctg
taaagtttag 1157 Glu Thr Val Arg Ala Ala Leu Lys His Ala Gly Leu Leu
280 285 290 ggagatttga tggcttactc tgttcaaaag tcgcgcctgg 1197 2 292
PRT Escherichia coli 2 Met Phe Thr Gly Ser Ile Val Ala Ile Val Thr
Pro Met Asp Glu Lys 1 5 10 15 Gly Asn Val Cys Arg Ala Ser Leu Lys
Lys Leu Ile Asp Tyr His Val 20 25 30 Ala Ser Gly Thr Ser Ala Ile
Val Ser Val Gly Thr Thr Gly Glu Ser 35 40 45 Ala Thr Leu Asn His
Asp Glu His Ala Asp Val Val Met Met Thr Leu 50 55 60 Asp Leu Ala
Asp Gly Arg Ile Pro Val Ile Ala Gly Thr Gly Ala Asn 65 70 75 80 Ala
Thr Ala Glu Ala Ile Ser Leu Thr Gln Arg Phe Asn Asp Ser Gly 85 90
95 Ile Val Gly Cys Leu Thr Val Thr Pro Tyr Tyr Asn Arg Pro Ser Gln
100 105 110 Glu Gly Leu Tyr Gln His Phe Lys Ala Ile Ala Glu His Thr
Asp Leu 115 120 125 Pro Gln Ile Leu Tyr Asn Val Pro Ser Arg Thr Gly
Cys Asp Leu Leu 130 135 140 Pro Glu Thr Val Gly Arg Leu Ala Lys Val
Lys Asn Ile Ile Gly Ile 145 150 155 160 Lys Glu Ala Thr Gly Asn Leu
Thr Arg Val Asn Gln Ile Lys Glu Leu 165 170 175 Val Ser Asp Asp Phe
Val Leu Leu Ser Gly Asp Asp Ala Ser Ala Leu 180 185 190 Asp Phe Met
Gln Leu Gly Gly His Gly Val Ile Ser Val Thr Thr Asn 195 200 205 Val
Ala Ala Arg Asp Met Ala Gln Met Cys Lys Leu Ala Ala Glu Glu 210 215
220 His Phe Ala Glu Ala Arg Val Ile Asn Gln Arg Leu Met Pro Leu His
225 230 235 240 Asn Lys Leu Phe Val Glu Pro Asn Pro Ile Pro Val Lys
Trp Ala Cys 245 250 255 Lys Glu Leu Gly Leu Val Ala Thr Asp Thr Leu
Arg Leu Pro Met Thr 260 265 270 Pro Ile Thr Asp Ser Gly Arg Glu Thr
Val Arg Ala Ala Leu Lys His 275 280 285 Ala Gly Leu Leu 290 3 2147
DNA Escherichia coli CDS (584)..(1930) 3 tcgaagtgtt tctgtagtgc
ctgccaggca gcggtctgcg ttggattgat gtttttcatt 60 agcaatactc
ttctgatttt gagaattgtg actttggaag attgtagcgc cagtcacaga 120
aaaatgtgat ggttttagtg ccgttagcgt aatgttgagt gtaaaccctt agcgcagtga
180 agcatttatt agctgaacta ctgaccgcca ggagtggatg aaaaatccgc
atgaccccat 240 cgttgacaac cgccccgctc accctttatt tataaatgta
ctacctgcgc tagcgcaggc 300 cagaagaggc gcgttgccca agtaacggtg
ttggaggagc cagtcctgtg ataacacctg 360 agggggtgca tcgccgaggt
gattgaacgg ctggccacgt tcatcatcgg ctaagggggc 420 tgaatcccct
gggttgtcac cagaagcgtt cgcagtcggg cgtttcgcaa gtggtggagc 480
acttctgggt gaaaatagta gcgaagtatc gctctgcgcc cacccgtctt ccgctcttcc
540 cttgtgccaa ggctgaaaat ggatcccctg acacgaggta gtt atg tct gaa att
595 Met Ser Glu Ile 1 gtt gtc tcc aaa ttt ggc ggt acc agc gta gct
gat ttt gac gcc atg 643 Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala
Asp Phe Asp Ala Met 5 10 15 20 aac cgc agc gct gat att gtg ctt tct
gat gcc aac gtg cgt tta gtt 691 Asn Arg Ser Ala Asp Ile Val Leu Ser
Asp Ala Asn Val Arg Leu Val 25 30 35 gtc ctc tcg gct tct gct ggt
atc act aat ctg ctg gtc gct tta gct 739 Val Leu Ser Ala Ser Ala Gly
Ile Thr Asn Leu Leu Val Ala Leu Ala 40 45 50 gaa gga ctg gaa cct
ggc gag cga ttc gaa aaa ctc gac gct atc cgc 787 Glu Gly Leu Glu Pro
Gly Glu Arg Phe Glu Lys Leu Asp Ala Ile Arg 55 60 65 aac atc cag
ttt gcc att ctg gaa cgt ctg cgt tac ccg aac gtt atc 835 Asn Ile Gln
Phe Ala Ile Leu Glu Arg Leu Arg Tyr Pro Asn Val Ile 70 75 80 cgt
gaa gag att gaa cgt ctg ctg gag aac att act gtt ctg gca gaa 883 Arg
Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr Val Leu Ala Glu 85 90
95 100 gcg gcg gcg ctg gca acg tct ccg gcg ctg aca gat gag ctg gtc
agc 931 Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp Glu Leu Val
Ser 105 110 115 cac ggc gag ctg atg tcg acc ctg ctg ttt gtt gag atc
ctg cgc gaa 979 His Gly Glu Leu Met Ser Thr Leu Leu Phe Val Glu Ile
Leu Arg Glu 120 125 130 cgc gat gtt cag gca cag tgg ttt gat gta cgt
aaa gtg atg cgt acc 1027 Arg Asp Val Gln Ala Gln Trp Phe Asp Val
Arg Lys Val Met Arg Thr 135 140 145 aac gac cga ttt ggt cgt gca gag
cca gat ata gcc gcg ctg gcg gaa 1075 Asn Asp Arg Phe Gly Arg Ala
Glu Pro Asp Ile Ala Ala Leu Ala Glu 150 155 160 ctg gcc gcg ctg cag
ctg ctc cca cgt ctc aat gaa ggc tta gtg atc 1123 Leu Ala Ala Leu
Gln Leu Leu Pro Arg Leu Asn Glu Gly Leu Val Ile 165 170 175 180 acc
cag gga ttt atc ggt agc gaa aat aaa ggt cgt aca acg acg ctt 1171
Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg Thr Thr Thr Leu 185
190 195 ggc cgt gga ggc agc gat tat acg gca gcc ttg ctg gcg gag gct
tta 1219 Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu Ala Glu
Ala Leu 200 205 210 cac gca tct cgt gtt gat atc tgg acc gac gtc ccg
ggc atc tac acc 1267 His Ala Ser Arg Val Asp Ile Trp Thr Asp Val
Pro Gly Ile Tyr Thr 215 220 225 acc gat cca cgc gta gtt tcc gca gca
aaa cgc att gat gaa atc gcg 1315 Thr Asp Pro Arg Val Val Ser Ala
Ala Lys Arg Ile Asp Glu Ile Ala 230 235 240 ttt gcc gaa gcg gca gag
atg gca act ttt ggt gca aaa gta ctg cat 1363 Phe Ala Glu Ala Ala
Glu Met Ala Thr Phe Gly Ala Lys Val Leu His 245 250 255 260 ccg gca
acg ttg cta ccc gca gta cgc agc gat atc ccg gtc ttt gtc 1411 Pro
Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile Pro Val Phe Val 265 270
275 ggc tcc agc aaa gac cca cgc gca ggt ggt acg ctg gtg tgc aat aaa
1459 Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Thr Leu Val Cys Asn
Lys 280 285 290 act gaa aat ccg ccg ctg ttc cgc gct ctg gcg ctt cgt
cgc aat cag 1507 Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu
Arg Arg Asn Gln 295 300 305 act ctg ctc act ttg cac agc ctg aat atg
ctg cat tct cgc ggt ttc 1555 Thr Leu Leu Thr Leu His Ser Leu Asn
Met Leu His Ser Arg Gly Phe 310 315 320 ctc gcg gaa gtt ttc ggc atc
ctc gcg cgg cat aat att tcg gta gac 1603 Leu Ala Glu Val Phe Gly
Ile Leu Ala Arg His Asn Ile Ser Val Asp 325 330 335 340 tta atc acc
acg tca gaa gtg agc gtg gca tta acc ctt gat acc acc 1651 Leu Ile
Thr Thr Ser Glu Val Ser Val Ala Leu Thr Leu Asp Thr Thr 345 350 355
ggt tca acc tcc act ggc gat acg ttg ctg acg caa tct ctg ctg atg
1699 Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln Ser Leu Leu
Met 360 365 370 gag ctt tcc gca ctg tgt cgg gtg gag gtg gaa gaa ggt
ctg gcg ctg 1747 Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu
Gly Leu Ala Leu 375 380 385 gtc gcg ttg att ggc aat gac ctg tca aaa
gcc tgc ggc gtt ggc aaa 1795 Val Ala Leu Ile Gly Asn Asp Leu Ser
Lys Ala Cys Gly Val Gly Lys 390 395 400 gag gta ttc ggc gta ctg gaa
ccg ttc aac att cgc atg att tgt tat 1843 Glu Val Phe Gly Val Leu
Glu Pro Phe Asn Ile Arg Met Ile Cys Tyr 405 410 415 420 ggc gca tcc
agc cat aac ctg tgc ttc ctg gtg ccc ggc gaa gat gcc 1891 Gly Ala
Ser Ser His Asn Leu Cys Phe Leu Val Pro Gly Glu Asp Ala 425 430 435
gag cag gtg gtg caa aaa ctg cat agt aat ttg ttt gag taaatactgt 1940
Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe Glu 440 445
atggcctgga agctatattt cgggccgtat tgattttctt gtcactatgc tcatcaataa
2000 acgagcctgt actctgttaa ccagcgtctt tatcggagaa taattgcctt
taattttttt 2060 atctgcatct ctaattaatt atcgaaagag ataaatagtt
aagagaaggc aaaatgaata 2120 ttatcagttc tgctcgcaaa ggaattc 2147 4 449
PRT Escherichia coli 4 Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly
Thr Ser Val Ala Asp 1 5 10 15 Phe Asp Ala Met Asn Arg Ser Ala Asp
Ile Val Leu Ser Asp Ala Asn 20 25 30 Val Arg Leu Val Val Leu Ser
Ala Ser Ala Gly Ile Thr Asn Leu Leu 35 40 45 Val Ala Leu Ala Glu
Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu 50 55 60 Asp Ala Ile
Arg Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr 65 70 75 80 Pro
Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr 85 90
95 Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp
100 105 110 Glu Leu Val Ser His Gly Glu Leu Met Ser Thr Leu Leu Phe
Val Glu 115 120 125 Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe
Asp Val Arg Lys 130 135 140 Val Met Arg Thr Asn Asp Arg Phe Gly Arg
Ala Glu Pro Asp Ile Ala 145 150 155 160 Ala Leu Ala Glu Leu Ala Ala
Leu Gln Leu Leu Pro Arg Leu Asn Glu 165 170 175 Gly Leu Val Ile Thr
Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg 180 185 190 Thr Thr Thr
Leu Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu 195 200 205 Ala
Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro 210 215
220 Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile
225 230 235 240 Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr
Phe Gly Ala 245 250 255 Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala
Val Arg Ser Asp Ile 260 265 270 Pro Val Phe Val Gly Ser Ser Lys Asp
Pro Arg Ala Gly Gly Thr Leu 275 280 285 Val Cys Asn Lys Thr Glu Asn
Pro Pro Leu Phe Arg Ala Leu Ala Leu 290 295 300 Arg Arg Asn Gln Thr
Leu Leu Thr Leu His Ser Leu Asn Met Leu His 305 310 315 320 Ser Arg
Gly Phe Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn 325 330 335
Ile Ser Val Asp Leu Ile Thr Thr Ser Glu Val Ser Val Ala Leu Thr 340
345 350 Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr
Gln 355 360 365 Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu
Val Glu Glu 370 375 380 Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp
Leu Ser Lys Ala Cys 385 390 395 400 Gly Val Gly Lys Glu Val Phe Gly
Val Leu Glu Pro Phe Asn Ile Arg 405 410 415 Met Ile Cys Tyr Gly Ala
Ser Ser His Asn Leu Cys Phe Leu Val Pro 420 425 430 Gly Glu Asp Ala
Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe 435 440 445 Glu 5
1981 DNA Methylophilus methylotrophus CDS (510)..(1736) 5
gtttaacgcg gccagtgaat ttgactcggt cccctgcctg gcaaaatcgc acaggtgatg
60 gacaacgtga aatcgcttga aaaagaattg gcacgcctca agtccaagct
ggcctcctca 120 cagggggatg acctcgcgac gcaagcgcag gacgtcaacg
gcgccaaagt actggcagcc 180 accctcgacg gggcggatgc caatgccttg
cgtgaaacca tggataagct caaagataaa 240 ctcaaatctg cagtcattgt
gctggcgagc gtggctgacg gtaaagtcag cctggctgcg 300 ggtgtcacta
ctgacttgac tggcaaggtc aaagcaggcg aagttggtca atcatgtggc 360
tggtcaggtc ggtggcaaag gtggtggtaa accggatatg gcgatggcag gtggtactga
420 gcccgctaat ttgccgcagg ctttggcaag tgtgaaggct tgggtagaaa
caaaactaaa 480 ttaatttaat tgattaacag agcgaaata atg gca tta atc gta
caa aaa tat 533 Met Ala Leu Ile Val Gln Lys Tyr 1 5 ggt ggt acc tcg
gtg gct aat ccc gag cgt atc cgt aat gtg gcg cgt 581 Gly Gly Thr Ser
Val Ala Asn Pro Glu Arg Ile Arg Asn Val Ala Arg 10 15 20 cgc gtg
gcg cgt tac aag gca ttg ggc cac cag gtg gtg gtt gtg gta 629 Arg Val
Ala Arg Tyr Lys Ala Leu Gly His Gln Val Val Val Val Val 25 30 35 40
tcc gca atg tct ggt gaa acc aac cgg ttg atc tca ctg gcc aag gaa 677
Ser Ala Met Ser Gly Glu Thr Asn Arg Leu Ile Ser Leu Ala Lys Glu 45
50 55 atc atg caa gac cct gat cca cgt gag ctg gat gtg atg gta tca
acc 725 Ile Met Gln Asp Pro Asp Pro Arg Glu Leu Asp Val Met Val Ser
Thr 60 65 70 ggt gag cag gtc acc atc ggc atg acg gcc ctg gca ctg
atg gag ctt 773 Gly Glu Gln Val Thr Ile Gly Met Thr Ala Leu Ala Leu
Met Glu Leu 75 80 85 ggc att aag gca aaa agc tat acc ggt acc cag
gtt aag atc ttg act 821 Gly Ile Lys Ala Lys Ser Tyr Thr Gly Thr Gln
Val Lys Ile Leu Thr 90 95 100 gac gat gct ttt acc aag gca cgt att
ctg gat atc gac gaa cat aac 869 Asp Asp Ala Phe Thr Lys Ala Arg Ile
Leu Asp Ile Asp Glu His Asn 105 110
115 120 ctg aaa aaa gac ctg gat gat ggc tat gtc tgc gtg gtg gct ggg
ttc 917 Leu Lys Lys Asp Leu Asp Asp Gly Tyr Val Cys Val Val Ala Gly
Phe 125 130 135 cag ggc gtg gat gcc aat ggc aat att acg acc ttg ggc
cgt ggc ggc 965 Gln Gly Val Asp Ala Asn Gly Asn Ile Thr Thr Leu Gly
Arg Gly Gly 140 145 150 tca gat act act ggt gta gca ctg gct gcg gcg
tta aag gcg gat gaa 1013 Ser Asp Thr Thr Gly Val Ala Leu Ala Ala
Ala Leu Lys Ala Asp Glu 155 160 165 tgt cag att tat acc gat gtc gat
ggc gtt tac acc acc gat ccg cgt 1061 Cys Gln Ile Tyr Thr Asp Val
Asp Gly Val Tyr Thr Thr Asp Pro Arg 170 175 180 gtg gtg cct gag gca
cgc cgc ttg gat aaa att acc ttt gaa gaa atg 1109 Val Val Pro Glu
Ala Arg Arg Leu Asp Lys Ile Thr Phe Glu Glu Met 185 190 195 200 ttg
gaa ctg gct tca cag ggc tcc aaa gta ttg caa att cgc tcg gtt 1157
Leu Glu Leu Ala Ser Gln Gly Ser Lys Val Leu Gln Ile Arg Ser Val 205
210 215 gag ttt gcc ggt aaa tac aaa gtc aaa tta cgt gtg ctg tcc agc
ttc 1205 Glu Phe Ala Gly Lys Tyr Lys Val Lys Leu Arg Val Leu Ser
Ser Phe 220 225 230 gaa gag gag ggc gac ggt aca ctg atc aca ttc gaa
gaa aat gag gaa 1253 Glu Glu Glu Gly Asp Gly Thr Leu Ile Thr Phe
Glu Glu Asn Glu Glu 235 240 245 aac atg gaa gaa cca att atc tcc ggc
atc gcc ttt aac cgc gat gag 1301 Asn Met Glu Glu Pro Ile Ile Ser
Gly Ile Ala Phe Asn Arg Asp Glu 250 255 260 gcg aaa att acc gtg acg
ggc gtg ccc gac aaa cca gga att gcc tat 1349 Ala Lys Ile Thr Val
Thr Gly Val Pro Asp Lys Pro Gly Ile Ala Tyr 265 270 275 280 cag att
ttg ggc ccg gtg gca gac gcc aat att gat gtg gat atg att 1397 Gln
Ile Leu Gly Pro Val Ala Asp Ala Asn Ile Asp Val Asp Met Ile 285 290
295 atc cag aac gtc ggt gcg gat ggt acg act gac ttc acc ttt acc gta
1445 Ile Gln Asn Val Gly Ala Asp Gly Thr Thr Asp Phe Thr Phe Thr
Val 300 305 310 cat aaa aat gag atg aac aaa gcc ctg agc att ctt aga
gat aaa gtg 1493 His Lys Asn Glu Met Asn Lys Ala Leu Ser Ile Leu
Arg Asp Lys Val 315 320 325 cag ggc cat atc cag gca cgt gaa atc agc
ggc gac gac aag att gcc 1541 Gln Gly His Ile Gln Ala Arg Glu Ile
Ser Gly Asp Asp Lys Ile Ala 330 335 340 aaa gtc tct gtg gtt ggg gtg
ggt atg cgc tca cat gta ggg atc gcc 1589 Lys Val Ser Val Val Gly
Val Gly Met Arg Ser His Val Gly Ile Ala 345 350 355 360 agc cag atg
ttc cgt acg ctg gcc gaa gaa ggg atc aat att caa atg 1637 Ser Gln
Met Phe Arg Thr Leu Ala Glu Glu Gly Ile Asn Ile Gln Met 365 370 375
atc tca acc agc gaa att aaa att gca gtc gtg atc gaa gag aag tac
1685 Ile Ser Thr Ser Glu Ile Lys Ile Ala Val Val Ile Glu Glu Lys
Tyr 380 385 390 atg gaa ctg gct gta cgc gtg ttg cat aaa gca ttc ggc
ctc gaa aac 1733 Met Glu Leu Ala Val Arg Val Leu His Lys Ala Phe
Gly Leu Glu Asn 395 400 405 gca taatcgccaa cggacgaata aagaaataaa
acattcttct tttttgcgtt 1786 Ala gatttttgaa gggttttcac gtagtatggc
agcccttcga tgcagtagca atgctgcaaa 1846 gagaacagca tgccgctgtg
ttggtactat taaaacttca ttgttttaat aaggtgaggg 1906 ggatcctcta
gagtcgacct gcaggcatgc aagcttggcc gtaatccatg gtcatagctg 1966
tttcctggtg tgaaa 1981 6 409 PRT Methylophilus methylotrophus 6 Met
Ala Leu Ile Val Gln Lys Tyr Gly Gly Thr Ser Val Ala Asn Pro 1 5 10
15 Glu Arg Ile Arg Asn Val Ala Arg Arg Val Ala Arg Tyr Lys Ala Leu
20 25 30 Gly His Gln Val Val Val Val Val Ser Ala Met Ser Gly Glu
Thr Asn 35 40 45 Arg Leu Ile Ser Leu Ala Lys Glu Ile Met Gln Asp
Pro Asp Pro Arg 50 55 60 Glu Leu Asp Val Met Val Ser Thr Gly Glu
Gln Val Thr Ile Gly Met 65 70 75 80 Thr Ala Leu Ala Leu Met Glu Leu
Gly Ile Lys Ala Lys Ser Tyr Thr 85 90 95 Gly Thr Gln Val Lys Ile
Leu Thr Asp Asp Ala Phe Thr Lys Ala Arg 100 105 110 Ile Leu Asp Ile
Asp Glu His Asn Leu Lys Lys Asp Leu Asp Asp Gly 115 120 125 Tyr Val
Cys Val Val Ala Gly Phe Gln Gly Val Asp Ala Asn Gly Asn 130 135 140
Ile Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Gly Val Ala Leu 145
150 155 160 Ala Ala Ala Leu Lys Ala Asp Glu Cys Gln Ile Tyr Thr Asp
Val Asp 165 170 175 Gly Val Tyr Thr Thr Asp Pro Arg Val Val Pro Glu
Ala Arg Arg Leu 180 185 190 Asp Lys Ile Thr Phe Glu Glu Met Leu Glu
Leu Ala Ser Gln Gly Ser 195 200 205 Lys Val Leu Gln Ile Arg Ser Val
Glu Phe Ala Gly Lys Tyr Lys Val 210 215 220 Lys Leu Arg Val Leu Ser
Ser Phe Glu Glu Glu Gly Asp Gly Thr Leu 225 230 235 240 Ile Thr Phe
Glu Glu Asn Glu Glu Asn Met Glu Glu Pro Ile Ile Ser 245 250 255 Gly
Ile Ala Phe Asn Arg Asp Glu Ala Lys Ile Thr Val Thr Gly Val 260 265
270 Pro Asp Lys Pro Gly Ile Ala Tyr Gln Ile Leu Gly Pro Val Ala Asp
275 280 285 Ala Asn Ile Asp Val Asp Met Ile Ile Gln Asn Val Gly Ala
Asp Gly 290 295 300 Thr Thr Asp Phe Thr Phe Thr Val His Lys Asn Glu
Met Asn Lys Ala 305 310 315 320 Leu Ser Ile Leu Arg Asp Lys Val Gln
Gly His Ile Gln Ala Arg Glu 325 330 335 Ile Ser Gly Asp Asp Lys Ile
Ala Lys Val Ser Val Val Gly Val Gly 340 345 350 Met Arg Ser His Val
Gly Ile Ala Ser Gln Met Phe Arg Thr Leu Ala 355 360 365 Glu Glu Gly
Ile Asn Ile Gln Met Ile Ser Thr Ser Glu Ile Lys Ile 370 375 380 Ala
Val Val Ile Glu Glu Lys Tyr Met Glu Leu Ala Val Arg Val Leu 385 390
395 400 His Lys Ala Phe Gly Leu Glu Asn Ala 405 7 1452 DNA
Methylophilus methylotrophus CDS (98)..(1207) misc_feature
(839)..(839) n = a, c, or g 7 gcatgcccgc aggtcgactc tagaggatcc
ccctgttcaa aaatcttcca aataatcact 60 gtaatgccgg gttgtccggc
tgaaatatcg agtcact atg tta aaa gta ggg ttt 115 Met Leu Lys Val Gly
Phe 1 5 gta ggc tgg cgt ggc atg gtt gga tcc gtg cta atg cag cgc atg
atg 163 Val Gly Trp Arg Gly Met Val Gly Ser Val Leu Met Gln Arg Met
Met 10 15 20 cag gaa aac gat ttt gcg gat att gaa ccg caa ttc ttt
acg acc tca 211 Gln Glu Asn Asp Phe Ala Asp Ile Glu Pro Gln Phe Phe
Thr Thr Ser 25 30 35 caa acg gga ggg gct gcg cct aaa gtt gga aaa
gat act cct gcg ctg 259 Gln Thr Gly Gly Ala Ala Pro Lys Val Gly Lys
Asp Thr Pro Ala Leu 40 45 50 aaa gat gcc aag gat att gat gct ttg
cgc cag atg gat gtg att gtg 307 Lys Asp Ala Lys Asp Ile Asp Ala Leu
Arg Gln Met Asp Val Ile Val 55 60 65 70 acc tgc cag ggt ggc gat tac
acg agt gac gtc ttc cca caa ttg cgc 355 Thr Cys Gln Gly Gly Asp Tyr
Thr Ser Asp Val Phe Pro Gln Leu Arg 75 80 85 gca acc ggc tgg agc
ggc cac tgg att gac gcg gcc tct acc tta cgc 403 Ala Thr Gly Trp Ser
Gly His Trp Ile Asp Ala Ala Ser Thr Leu Arg 90 95 100 atg gaa aaa
gac tcc gtg atc att tta gac ccg gtg aac atg cat gtg 451 Met Glu Lys
Asp Ser Val Ile Ile Leu Asp Pro Val Asn Met His Val 105 110 115 att
aaa gat gca ttg tcc aat ggc ggc aaa aac tgg atc ggc ggc aac 499 Ile
Lys Asp Ala Leu Ser Asn Gly Gly Lys Asn Trp Ile Gly Gly Asn 120 125
130 tgt acc gtc tca ctt atg ttg atg gcg ctg aat ggc ctg ttt aag gct
547 Cys Thr Val Ser Leu Met Leu Met Ala Leu Asn Gly Leu Phe Lys Ala
135 140 145 150 gac ctg gtc gag tgg gcc act tcc atg acc tac cag gcg
gct tca ggc 595 Asp Leu Val Glu Trp Ala Thr Ser Met Thr Tyr Gln Ala
Ala Ser Gly 155 160 165 gca ggc gcg cag aat atg cgt gaa ctg att agc
cag atg ggc gta gtg 643 Ala Gly Ala Gln Asn Met Arg Glu Leu Ile Ser
Gln Met Gly Val Val 170 175 180 aat gcc tcc gtg gct gat ttg ctg gcg
gat cca gct tct gcc att ttg 691 Asn Ala Ser Val Ala Asp Leu Leu Ala
Asp Pro Ala Ser Ala Ile Leu 185 190 195 cag atc gat aaa aca gtg gcg
gat acc atc cgt agc gaa gag ttg cct 739 Gln Ile Asp Lys Thr Val Ala
Asp Thr Ile Arg Ser Glu Glu Leu Pro 200 205 210 aaa tct aac ttt ggt
gtg cca ttg gcg ggc agt ctg atc cca tgg atc 787 Lys Ser Asn Phe Gly
Val Pro Leu Ala Gly Ser Leu Ile Pro Trp Ile 215 220 225 230 gac aag
gac tta ggg aat ggt caa agt aaa gaa gaa tgg aag ggc ggc 835 Asp Lys
Asp Leu Gly Asn Gly Gln Ser Lys Glu Glu Trp Lys Gly Gly 235 240 245
gta nag acc aat aag att tta ggt cgt gaa gcg aac ccg att gtg att 883
Val Xaa Thr Asn Lys Ile Leu Gly Arg Glu Ala Asn Pro Ile Val Ile 250
255 260 gac ggt ttg tgt gta cgt atc ggc gcc atg cgt tgc cat tca caa
gcg 931 Asp Gly Leu Cys Val Arg Ile Gly Ala Met Arg Cys His Ser Gln
Ala 265 270 275 ttg act atc aag ctg cgc aag gat gtg ccg ctg gat gaa
atc aat cag 979 Leu Thr Ile Lys Leu Arg Lys Asp Val Pro Leu Asp Glu
Ile Asn Gln 280 285 290 atg ctg gct gaa gcg aac gac tgg gct aaa gtc
att ccc aat gag cgt 1027 Met Leu Ala Glu Ala Asn Asp Trp Ala Lys
Val Ile Pro Asn Glu Arg 295 300 305 310 gag gtc agt atg cgg gaa ctc
acc ccg gca gcg att acc ggc agt ctg 1075 Glu Val Ser Met Arg Glu
Leu Thr Pro Ala Ala Ile Thr Gly Ser Leu 315 320 325 gcg acg cca gta
ggg cgt ttg cgc aaa ctg gcg atg ggt ggt gaa tac 1123 Ala Thr Pro
Val Gly Arg Leu Arg Lys Leu Ala Met Gly Gly Glu Tyr 330 335 340 ttg
tcg gca ttt acc gta ggt gac cag ttg tta tgg ggc gct gcc gaa 1171
Leu Ser Ala Phe Thr Val Gly Asp Gln Leu Leu Trp Gly Ala Ala Glu 345
350 355 cct ttg cgc aga atg ttg agg att ctg gtc gaa tct taagtaattg
1217 Pro Leu Arg Arg Met Leu Arg Ile Leu Val Glu Ser 360 365 370
tttaagtagc agcccgtaaa gctatgattt atcaataaaa tcatggtctt ttcgggcttt
1277 tgcttttggt gcaatcctgt ttaatggtta ttgtagcctc aaatcctgta
tttattgctc 1337 tcaagccgcc tgggtgcgct tgcgtggctg ggtgaatgat
gctattttga caaacgccat 1397 gaattactaa gggttaatcg gtgagtaaat
ttcaattaaa aaaaatagcc tttgc 1452 8 370 PRT Methylophilus
methylotrophus misc_feature (248)..(248) The 'Xaa' at location 248
stands for Lys, Glu, or Gln. misc_feature (839)..(839) n = a, c, or
g 8 Met Leu Lys Val Gly Phe Val Gly Trp Arg Gly Met Val Gly Ser Val
1 5 10 15 Leu Met Gln Arg Met Met Gln Glu Asn Asp Phe Ala Asp Ile
Glu Pro 20 25 30 Gln Phe Phe Thr Thr Ser Gln Thr Gly Gly Ala Ala
Pro Lys Val Gly 35 40 45 Lys Asp Thr Pro Ala Leu Lys Asp Ala Lys
Asp Ile Asp Ala Leu Arg 50 55 60 Gln Met Asp Val Ile Val Thr Cys
Gln Gly Gly Asp Tyr Thr Ser Asp 65 70 75 80 Val Phe Pro Gln Leu Arg
Ala Thr Gly Trp Ser Gly His Trp Ile Asp 85 90 95 Ala Ala Ser Thr
Leu Arg Met Glu Lys Asp Ser Val Ile Ile Leu Asp 100 105 110 Pro Val
Asn Met His Val Ile Lys Asp Ala Leu Ser Asn Gly Gly Lys 115 120 125
Asn Trp Ile Gly Gly Asn Cys Thr Val Ser Leu Met Leu Met Ala Leu 130
135 140 Asn Gly Leu Phe Lys Ala Asp Leu Val Glu Trp Ala Thr Ser Met
Thr 145 150 155 160 Tyr Gln Ala Ala Ser Gly Ala Gly Ala Gln Asn Met
Arg Glu Leu Ile 165 170 175 Ser Gln Met Gly Val Val Asn Ala Ser Val
Ala Asp Leu Leu Ala Asp 180 185 190 Pro Ala Ser Ala Ile Leu Gln Ile
Asp Lys Thr Val Ala Asp Thr Ile 195 200 205 Arg Ser Glu Glu Leu Pro
Lys Ser Asn Phe Gly Val Pro Leu Ala Gly 210 215 220 Ser Leu Ile Pro
Trp Ile Asp Lys Asp Leu Gly Asn Gly Gln Ser Lys 225 230 235 240 Glu
Glu Trp Lys Gly Gly Val Xaa Thr Asn Lys Ile Leu Gly Arg Glu 245 250
255 Ala Asn Pro Ile Val Ile Asp Gly Leu Cys Val Arg Ile Gly Ala Met
260 265 270 Arg Cys His Ser Gln Ala Leu Thr Ile Lys Leu Arg Lys Asp
Val Pro 275 280 285 Leu Asp Glu Ile Asn Gln Met Leu Ala Glu Ala Asn
Asp Trp Ala Lys 290 295 300 Val Ile Pro Asn Glu Arg Glu Val Ser Met
Arg Glu Leu Thr Pro Ala 305 310 315 320 Ala Ile Thr Gly Ser Leu Ala
Thr Pro Val Gly Arg Leu Arg Lys Leu 325 330 335 Ala Met Gly Gly Glu
Tyr Leu Ser Ala Phe Thr Val Gly Asp Gln Leu 340 345 350 Leu Trp Gly
Ala Ala Glu Pro Leu Arg Arg Met Leu Arg Ile Leu Val 355 360 365 Glu
Ser 370 9 3098 DNA Methylophilus methylotrophus CDS (1268)..(2155)
9 cgtgccaact tgcatgcctg ccggtcgctc tagaggatca attgctggca acatttgagt
60 acattattcg cctttgcatg gtaaaggcct atggtcttga tgtaactttc
aagacctgcc 120 agccccaaat ccaggatagc ctgcggtgtg ttggccacct
tgaacaattt gcgggtggca 180 atattgacac ctttgtctgt cgcctgtgca
gacaagatga cggcaatcag taattcgaac 240 gtggagctat gctccagctc
agtggttgga ttggggatgg cttgggccag ccgctcaaat 300 atcgccagtc
ttttttgtgc attcataaaa cggtttcaat cataggtcac agggtcaacc 360
tgtcttttgc gctttgacgc gcgccatggc tgcggcaatg gcatttttct tgagcacctc
420 agttgagggt gtctcggtcg tagcaagcgt ctggttgcgt ttgctgtagg
tttgggcggt 480 ctcccgtttt tcaagggcga ggcgagaaag gcgttgctgg
tggcgttgtc tcgctaccgc 540 ggcttcagct tcattcatgg cggtagcccg
accgggaatc gtttgcatct gtatgcagtc 600 caccgggcag ggcggtaaac
atagctcaca gccagtgcat tcctgggaaa tcaccgtatg 660 catcagtttg
gatgcgccca aaatggcatc aacgggacag gcctgtatac acagggtgca 720
gccgatgcat gtttcctcat caatcaaggc caccgctttg ggtttggtga tgccgtgggc
780 cggatttaat gcctggaaag gacgttgcag taatttggca agcgcatgaa
tgcccgcttc 840 tcctccaggc ggacattggt tgatattggc ctctccgcgg
gcgatcgctt cagcataagg 900 tttgcatccc tcgtaaccgc attggcggca
ttgagtttgc ggtaataccg cgtcgatctt 960 tgcaatgagg tcgacaaagc
gttctggcag ctcaggcgca gtcccttcga cttcaatcat 1020 gtgatggcag
gtgagtctgc attcggtcct ggctaaatag ccgtttaaga tgggttgcta 1080
agagttttat tataaccgaa accttgcttt tcctttggcc gggagctagg cggaaaaagc
1140 ttgccgcagt tgggtgccag tgattttgcc gccgtcttgc gcttgtatcc
gtccagatac 1200 agcaagtagg cgcgttcttt ggcgttagac cggataatca
gttaaaatat tcgctttatt 1260 cttaaag 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 10 acc ccc atg ttt gaa gat gga cgt ttg gat
ctg gac gcc ctc aaa aag 1357 Thr Pro Met Phe Glu Asp Gly Arg Leu
Asp Leu Asp Ala Leu Lys Lys 15 20 25 30 ctg gtc gac ttt cat gta gag
gca ggg aca gat ggt att gtc atc gtt 1405 Leu Val Asp Phe His Val
Glu Ala Gly Thr Asp Gly Ile Val Ile Val 35 40 45 ggc acg act ggc
gag tcg ccc acg gtg gat gta gat gag cat tgt ctg 1453 Gly Thr Thr
Gly Glu Ser Pro Thr Val Asp Val Asp Glu His Cys Leu 50 55 60 ctg
atc aaa acc acg atc gag cat gtc gcc aag cgc gtg cca gtc att 1501
Leu Ile Lys Thr Thr Ile Glu His Val Ala Lys Arg Val Pro Val Ile 65
70 75 gcc ggt act ggc gca aat tcc act gct gaa gcc att gaa ctg act
gcc 1549 Ala Gly Thr Gly Ala Asn Ser Thr Ala Glu Ala Ile Glu Leu
Thr Ala 80 85 90 aag gcc aag gcg ctt ggc gca gac gcc tgc ctg ctg
gtg gca ccg tat 1597 Lys Ala Lys Ala Leu Gly Ala Asp Ala Cys Leu
Leu Val Ala Pro Tyr 95 100 105 110 tac aac aag ccc tcg caa gag ggt
ttg tac cag cac ttt aaa gcc gtg 1645 Tyr Asn Lys Pro Ser Gln Glu
Gly Leu Tyr Gln His Phe Lys Ala Val 115 120 125 gct gag gcg gtc gat
att ccg caa att ctc tat aat gtg cca ggc cgc 1693 Ala Glu Ala Val
Asp Ile Pro Gln Ile Leu Tyr Asn Val Pro Gly Arg 130 135 140
acc ggt tgc gac ttg tct aac gac acc gta ttg cgc ctg gcg cag att
1741 Thr Gly Cys Asp Leu Ser Asn Asp Thr Val Leu Arg Leu Ala Gln
Ile 145 150 155 cgc aac att gtc ggg att aag gat gcg act gga ggg att
gag cgc ggt 1789 Arg Asn Ile Val Gly Ile Lys Asp Ala Thr Gly Gly
Ile Glu Arg Gly 160 165 170 acc gat ttg ttg ttg cgt gca cca gct gat
ttc gcc att tac agc ggg 1837 Thr Asp Leu Leu Leu Arg Ala Pro Ala
Asp Phe Ala Ile Tyr Ser Gly 175 180 185 190 gat gat gcc act gcg ctg
gcc ctg atg tta tta ggg ggg aaa ggc gtg 1885 Asp Asp Ala Thr Ala
Leu Ala Leu Met Leu Leu Gly Gly Lys Gly Val 195 200 205 att tcg gtc
acg gcc aat gtc gcg ccc aaa tta atg cat gaa atg tgc 1933 Ile Ser
Val Thr Ala Asn Val Ala Pro Lys Leu Met His Glu Met Cys 210 215 220
gag cat gct ttg aat ggc aac ctg gcc gca gcc aaa gcg gcc aat gcc
1981 Glu His Ala Leu Asn Gly Asn Leu Ala Ala Ala Lys Ala Ala Asn
Ala 225 230 235 aaa ctg ttt gca ttg cac cag aag ttg ttt gta gaa gcg
aac ccg att 2029 Lys Leu Phe Ala Leu His Gln Lys Leu Phe Val Glu
Ala Asn Pro Ile 240 245 250 cca gtg aaa tgg gta tta caa caa atg gga
atg att gcc act ggc atc 2077 Pro Val Lys Trp Val Leu Gln Gln Met
Gly Met Ile Ala Thr Gly Ile 255 260 265 270 cgt ttg ccg ctg gtc aat
tta tcc agc caa tat cat gaa gta ttg cgc 2125 Arg Leu Pro Leu Val
Asn Leu Ser Ser Gln Tyr His Glu Val Leu Arg 275 280 285 aac gcc atg
aag cag gca gaa att gcc gct tgatcggcta aaactaattt 2175 Asn Ala Met
Lys Gln Ala Glu Ile Ala Ala 290 295 agggtgaaac aagtgaaata
catgagtcat gtttggttac aacgtttggt gctggccagt 2235 ctggtcacag
cgctttcagc gtgcgattcc atcccgttta ttgataatag ttctgactac 2295
aagggcgcag gtcgctccag gccacttgaa gtgccgccag acctgaccgc ggtgcgtacc
2355 agcagtactt acaatgtgcc tggtagcacc agttactctg cctatagcca
gaaccaggaa 2415 gtgcaagagc agaatggtcc acagcctgtg ctcgcagata
tgaaaaacgt gcgcatggtg 2475 aaagcaggcc agcagcgttg gctggtggtc
aatgcgcctc cggaaaaaat ctggccgatt 2535 gtgcgtgatt tctggctgga
tcaaggcttt gctgtcaggg tagagaatcc tgagcttggc 2595 gtgattgaaa
ccgagtggtt gcaatctgat gccatcaagc ctaaggaaga taaccgtggc 2655
tatggtgaaa agtttgatgc ctggctggat aaactttctg gttttgccga caggcgtaaa
2715 ttccgtacgc gtctggaacg tggggagaaa gacggcacca ccgaaatcta
tatgacgcac 2775 cgtactgtcg ccggtgcacc ggatgatggc aaaaattatg
tgcagaccca attgggtgtc 2835 attgataccg gttatcgccc caacgcggct
gaaaacaaga acaatgccgg taaagagttt 2895 gatgctgact tggatgcaga
attactccgt cgaatgatgg tgaaattagg tctggatgag 2955 cagaaagcag
accaggtgat ggcacaatct gcttcagaca agcgtgcaga tgtggtcaag 3015
gagtctgacc agagcgtcac cttgaagttg aatgagccgt ttgaccgtgc ctggcgccgt
3075 gtggcctggc ctggatcccc ggg 3098 10 296 PRT Methylophilus
methylotrophus 10 Met Ala Leu Gly Met Leu Thr Gly Ser Leu Val Ala
Ile Val Thr Pro 1 5 10 15 Met Phe Glu Asp Gly Arg Leu Asp Leu Asp
Ala Leu Lys Lys Leu Val 20 25 30 Asp Phe His Val Glu Ala Gly Thr
Asp Gly Ile Val Ile Val Gly Thr 35 40 45 Thr Gly Glu Ser Pro Thr
Val Asp Val Asp Glu His Cys Leu Leu Ile 50 55 60 Lys Thr Thr Ile
Glu His Val Ala Lys Arg Val Pro Val Ile Ala Gly 65 70 75 80 Thr Gly
Ala Asn Ser Thr Ala Glu Ala Ile Glu Leu Thr Ala Lys Ala 85 90 95
Lys Ala Leu Gly Ala Asp Ala Cys Leu Leu Val Ala Pro Tyr Tyr Asn 100
105 110 Lys Pro Ser Gln Glu Gly Leu Tyr Gln His Phe Lys Ala Val Ala
Glu 115 120 125 Ala Val Asp Ile Pro Gln Ile Leu Tyr Asn Val Pro Gly
Arg Thr Gly 130 135 140 Cys Asp Leu Ser Asn Asp Thr Val Leu Arg Leu
Ala Gln Ile Arg Asn 145 150 155 160 Ile Val Gly Ile Lys Asp Ala Thr
Gly Gly Ile Glu Arg Gly Thr Asp 165 170 175 Leu Leu Leu Arg Ala Pro
Ala Asp Phe Ala Ile Tyr Ser Gly Asp Asp 180 185 190 Ala Thr Ala Leu
Ala Leu Met Leu Leu Gly Gly Lys Gly Val Ile Ser 195 200 205 Val Thr
Ala Asn Val Ala Pro Lys Leu Met His Glu Met Cys Glu His 210 215 220
Ala Leu Asn Gly Asn Leu Ala Ala Ala Lys Ala Ala Asn Ala Lys Leu 225
230 235 240 Phe Ala Leu His Gln Lys Leu Phe Val Glu Ala Asn Pro Ile
Pro Val 245 250 255 Lys Trp Val Leu Gln Gln Met Gly Met Ile Ala Thr
Gly Ile Arg Leu 260 265 270 Pro Leu Val Asn Leu Ser Ser Gln Tyr His
Glu Val Leu Arg Asn Ala 275 280 285 Met Lys Gln Ala Glu Ile Ala Ala
290 295 11 3390 DNA Methylophilus methylotrophus CDS (2080)..(2883)
11 ccgcaggtcg ctctagagga tcagagttgg acggacaagc tgaagttttg
ggagtctgaa 60 gaagctgcgg gcgaagtgat aaagcagctg aatcaactgt
agccactgca agcgacgaat 120 gaaagcaaag gcgctgcact cgctaaggat
gaggcagccg aatctcagaa aaccacgtca 180 gagcctgtca aggccgagca
agaggtattg ccctcggcca ctgcaacaaa taattcagct 240 gctgcagcga
cattggctga agaagaagtg gttccctaca ttccggaggg ggagtatcag 300
gctgcaccca ctccagaaga gatggccaag ggtaatctgg atgtcagtga aaaccaggtt
360 actgaggcta aggcacatcc agtgaatgaa aaggaaatgg ctgcccaaat
tgcagatacg 420 gttgagccac cacccgtttt tcagcaggaa ccgatggcag
aacctattgt agcggctgaa 480 cccgaacccg tattgccacc gcccgtaaaa
gccgaaccag ctgtgaagaa tatcacagcg 540 ccagttgttg ccgcagccac
tgttgcagcg gcggcaacca agactgctga atctgagtca 600 gttaaatcca
aacctgttga tcctaagcct gtggaagcaa aaaccgctgt atcaaaaact 660
gaagtacaaa cacccgcggc acaggcacct gctgcggcag cggccgttga agatgacgag
720 gtcattccat atattcccga aggtgaatat gtggctcctg tcattcctag
tgaggccgaa 780 atggttaaag gcaatatggc ggaggcaaat gcacctgcga
ctgatgctca agcgcgccag 840 gtaactgaaa aaggggtggc acccacatcg
gatgcggcag cagagccatc accgacattt 900 gtcgctgagc aattgccaga
accagagcca gaacctgaat tgccaccgcc gcctccgcca 960 tccgtcagca
agcctgttgt gagagaggta gcgccagtgg ctgcgctggc agcagaagaa 1020
gagaaaccag tcgctgcgca gcctgagact gagcagccgg ctgccaaggt tgttgagcct
1080 gcatcggtcg cctcccctgt ggcgacgcca gaagcgccag ctggtgatgc
tgaaatcaac 1140 caggctgtgg cggcatgggc acaagcttgg cgcagcaagg
acattaaaaa ctacctcgct 1200 gcatatgccc ctgacttcat gccagaaggg
ttgccttcca gaaaggcatg ggagtcgcaa 1260 cgcaaacagc gtttatctgc
aggccagggt gcgattacac tcgtactaaa taatgtgcag 1320 attcagcgtg
acggtaccac tgtcgccgtg cagtttgagc aaaaatatgc tgctaaagtt 1380
tataaagatg aattggtcaa aacactggaa atgcgttacg agccaacgca gaaacgttgg
1440 ttgatcacac gtgaacgtgt tgccccttta accggtttgc cagtagcgag
tgtgccaacg 1500 acccgtctgc cagcagtcgc tgcagcgtca tccaatacgg
atgtggtcga gtcagctgtg 1560 ccaccgacac aatcgacatc atctgcgcct
gtagcggaag tgagtgttga atcagcgatt 1620 gacgcctggg cacaggcttg
gcgcagtaaa aacatcaatg cttactttgc ggcgtattct 1680 ccagaatttg
tgccggaggg attgccaaac agaggtgtct gggaagcgca acgtaaaaag 1740
cgcttgtccc cacagcaggg caagatcagc ctggatgtca cgaatgtaag cgtgagccgc
1800 gaaggagaaa cagccgtggc cacctttagg cagaaatatg cgtctaaggc
ctatcgtgat 1860 gaagtagtga agcgtctaca gttaaaactg gatgctgcaa
gcaatcgctg gctgattgtg 1920 cgtgaaagta ccggtagtga ggcagaagtg
ccaatgggca agcagtcagt gagtgcgcca 1980 gaagagagct cggaacatca
ggatggtgct ctggagccga tcggatttta atggtctgct 2040 gatgtcgtgg
tttaagtatt aaaaataatt gagtgagtt atg ttg aaa gta gtg 2094 Met Leu
Lys Val Val 1 5 att gct ggc gtg tct ggt cgt atg gga cat gcc tta ctg
gat gga gtt 2142 Ile Ala Gly Val Ser Gly Arg Met Gly His Ala Leu
Leu Asp Gly Val 10 15 20 ttt tct gat aac ggc ttg cag ttg cac gcg
gca ctc gat cgt gct gaa 2190 Phe Ser Asp Asn Gly Leu Gln Leu His
Ala Ala Leu Asp Arg Ala Glu 25 30 35 agc gcc atg ata ggg cgg gat
gca ggc gag cag ttt ggc aag gtc agt 2238 Ser Ala Met Ile Gly Arg
Asp Ala Gly Glu Gln Phe Gly Lys Val Ser 40 45 50 ggc gtg aaa atc
acg gct gac atc cat gcc gca ttg gtc ggt gcc gat 2286 Gly Val Lys
Ile Thr Ala Asp Ile His Ala Ala Leu Val Gly Ala Asp 55 60 65 gtg
ctg gtg gat ttc acg cgg ccg gaa gcc agt atg caa tat tta caa 2334
Val Leu Val Asp Phe Thr Arg Pro Glu Ala Ser Met Gln Tyr Leu Gln 70
75 80 85 gcc tgc cag caa gcc aac gtt aaa tta gtg att ggt act acc
ggg ttt 2382 Ala Cys Gln Gln Ala Asn Val Lys Leu Val Ile Gly Thr
Thr Gly Phe 90 95 100 agt gag gca gaa aag gcc agt att gag gct gcg
tcc aaa aat atc ggt 2430 Ser Glu Ala Glu Lys Ala Ser Ile Glu Ala
Ala Ser Lys Asn Ile Gly 105 110 115 atc gta ttt gct cca aac atg agc
gta ggg gtc acc ctc ttg att aac 2478 Ile Val Phe Ala Pro Asn Met
Ser Val Gly Val Thr Leu Leu Ile Asn 120 125 130 ctg gtt gag caa gcc
gca cgg gtg ctc aat gaa ggc tat gat att gag 2526 Leu Val Glu Gln
Ala Ala Arg Val Leu Asn Glu Gly Tyr Asp Ile Glu 135 140 145 gtg gtt
gaa atg cat cac cgc cat aag gtg gat gcg cct tca ggc acg 2574 Val
Val Glu Met His His Arg His Lys Val Asp Ala Pro Ser Gly Thr 150 155
160 165 gct tta cgg ttg ggt gag gct gcg gca aaa ggg att gat aaa gcg
ctt 2622 Ala Leu Arg Leu Gly Glu Ala Ala Ala Lys Gly Ile Asp Lys
Ala Leu 170 175 180 aaa gat tgt gct gtg tat gcg cgc gaa ggc gtg act
ggt gaa cgc gaa 2670 Lys Asp Cys Ala Val Tyr Ala Arg Glu Gly Val
Thr Gly Glu Arg Glu 185 190 195 gcg ggc acg att ggt ttt gca acc tta
cgt ggt ggg gat gtg gtc ggt 2718 Ala Gly Thr Ile Gly Phe Ala Thr
Leu Arg Gly Gly Asp Val Val Gly 200 205 210 gac cat acg gtg gtt ctg
gct ggt gtg ggt gag cga gta gag tta acg 2766 Asp His Thr Val Val
Leu Ala Gly Val Gly Glu Arg Val Glu Leu Thr 215 220 225 cat aaa gca
tca agc cgt gcc aca ttt gca caa ggt gcg tta cgt gcg 2814 His Lys
Ala Ser Ser Arg Ala Thr Phe Ala Gln Gly Ala Leu Arg Ala 230 235 240
245 gct aaa ttt ctg gct gat aaa ccc aag gga ttg ttt gat atg cgt gat
2862 Ala Lys Phe Leu Ala Asp Lys Pro Lys Gly Leu Phe Asp Met Arg
Asp 250 255 260 gtg ttg gga ttt gaa aag aac tgatctttag taggcgatcc
cgtctggcta 2913 Val Leu Gly Phe Glu Lys Asn 265 aggtctggca
ggaatcgtct gatgcttctg agttgccctt gagtgggctg tcaatgtacg 2973
ctataatgct gtaattctga aacgggaaga gtcgaacaag cttttcccgt tttgcacatc
3033 tattcactgc agcttgaatt tcacttccag ccatggtgaa ccctctaaaa
gatgtgtttc 3093 gtgtcaaact taaggagcta aaggtgtcaa aaacaattcc
agcgattctc gtgttagcag 3153 atggaactgt ttttaagggc attagcattg
gcgcttccgg tcatacggta ggtgaggtgg 3213 tgtttaatac ctccatcacc
ggttatcagg agattcttac cgatccttcc tataccgaac 3273 aaatcgtgac
actgacctat ccgcacattg gtaactacgg gaccaatcgt gaagatggga 3333
gtcaggtaaa gtctatgctg cgggtctgat ccccgggacc gagccgggtt cgtaaag 3390
12 268 PRT Methylophilus methylotrophus 12 Met Leu Lys Val Val Ile
Ala Gly Val Ser Gly Arg Met Gly His Ala 1 5 10 15 Leu Leu Asp Gly
Val Phe Ser Asp Asn Gly Leu Gln Leu His Ala Ala 20 25 30 Leu Asp
Arg Ala Glu Ser Ala Met Ile Gly Arg Asp Ala Gly Glu Gln 35 40 45
Phe Gly Lys Val Ser Gly Val Lys Ile Thr Ala Asp Ile His Ala Ala 50
55 60 Leu Val Gly Ala Asp Val Leu Val Asp Phe Thr Arg Pro Glu Ala
Ser 65 70 75 80 Met Gln Tyr Leu Gln Ala Cys Gln Gln Ala Asn Val Lys
Leu Val Ile 85 90 95 Gly Thr Thr Gly Phe Ser Glu Ala Glu Lys Ala
Ser Ile Glu Ala Ala 100 105 110 Ser Lys Asn Ile Gly Ile Val Phe Ala
Pro Asn Met Ser Val Gly Val 115 120 125 Thr Leu Leu Ile Asn Leu Val
Glu Gln Ala Ala Arg Val Leu Asn Glu 130 135 140 Gly Tyr Asp Ile Glu
Val Val Glu Met His His Arg His Lys Val Asp 145 150 155 160 Ala Pro
Ser Gly Thr Ala Leu Arg Leu Gly Glu Ala Ala Ala Lys Gly 165 170 175
Ile Asp Lys Ala Leu Lys Asp Cys Ala Val Tyr Ala Arg Glu Gly Val 180
185 190 Thr Gly Glu Arg Glu Ala Gly Thr Ile Gly Phe Ala Thr Leu Arg
Gly 195 200 205 Gly Asp Val Val Gly Asp His Thr Val Val Leu Ala Gly
Val Gly Glu 210 215 220 Arg Val Glu Leu Thr His Lys Ala Ser Ser Arg
Ala Thr Phe Ala Gln 225 230 235 240 Gly Ala Leu Arg Ala Ala Lys Phe
Leu Ala Asp Lys Pro Lys Gly Leu 245 250 255 Phe Asp Met Arg Asp Val
Leu Gly Phe Glu Lys Asn 260 265 13 2566 DNA Methylophilus
methylotrophus CDS (751)..(1995) misc_feature (2467)..(2467) n = a,
c, g, or t 13 tgctttaggg ggaacctaga ggatccccct acccgaggaa
gaagtgagcc aacatgtact 60 tccagtcgta ccatcaaaag tagaagtttt
cggcgttatc ctgattcaca gtaaacgaaa 120 aattgcccat attctgaccg
gatttaccgg tggcttttaa ggtataagtg gtcgctgact 180 ggttctcaat
gctgtaatca aaaaatttgg catcactggg gacacaggca aatcccacat 240
atgtgaagtt gtcctgataa aactgttcgg cctgcacacg gcaattggca agattggcag
300 gcgcttccgc ggcattaccg cttttgatgt aatcctgata gcctggtatg
gcgatgctgg 360 ccaagatacc cataatggcc accacgacca tgacttctat
caggctgaat ccgtactgat 420 ttgaggactt cattatcaaa ccccttttta
gatagcctta tcatgcaaac aggcagctgt 480 catgtccagc atcagccgac
caatggtcag gattacccga cgaacggtca aaccactaaa 540 acgcccagtc
actggtgcca tgagcaactg caggtttaat gataaaatgg cactcaattt 600
acattggact gtgaacatgt tttccttcta tacgagatta ttggcggttg ccctgctatt
660 ggcacaattg agtgcctgtg gtctcaaagg ggacctgtat attcctgagc
gccaataccc 720 tcaaacgcct caacaagata agtcttcatc gtg acc gct ttt tca
atc caa caa 774 Val Thr Ala Phe Ser Ile Gln Gln 1 5 ggc cta cta cat
gcc gag aat gta gcc ctg cgt gac att gca caa acg 822 Gly Leu Leu His
Ala Glu Asn Val Ala Leu Arg Asp Ile Ala Gln Thr 10 15 20 cat caa
acg ccc act tac gtc tat tca cgt gcc gcc ttg acg act gct 870 His Gln
Thr Pro Thr Tyr Val Tyr Ser Arg Ala Ala Leu Thr Thr Ala 25 30 35 40
ttc gag cgt ttt cag gca ggc ctg act gga cat gac cat ttg atc tgc 918
Phe Glu Arg Phe Gln Ala Gly Leu Thr Gly His Asp His Leu Ile Cys 45
50 55 ttt gct gtc aaa gcc aac cca agc ctg gcc att ctc aac ctg ttt
gcg 966 Phe Ala Val Lys Ala Asn Pro Ser Leu Ala Ile Leu Asn Leu Phe
Ala 60 65 70 cga atg gga gcg ggc ttt gat att gtg tcc ggt ggt gag
ctg gca cgc 1014 Arg Met Gly Ala Gly Phe Asp Ile Val Ser Gly Gly
Glu Leu Ala Arg 75 80 85 gtc ttg gcc gca ggt ggc gac ccg aaa aaa
gtg gtg ttt tct ggt gtg 1062 Val Leu Ala Ala Gly Gly Asp Pro Lys
Lys Val Val Phe Ser Gly Val 90 95 100 ggc aaa tcc cat gcg gaa atc
aaa gcc gcg ctt gaa gcg ggc att ctt 1110 Gly Lys Ser His Ala Glu
Ile Lys Ala Ala Leu Glu Ala Gly Ile Leu 105 110 115 120 tgc ttc aac
gtg gaa tca gtg aat gag cta gac cgc atc cag cag gtg 1158 Cys Phe
Asn Val Glu Ser Val Asn Glu Leu Asp Arg Ile Gln Gln Val 125 130 135
gcg gcc agc ctg ggc aaa aaa gcg cct att tcc ctg cgc gtg aac ccc
1206 Ala Ala Ser Leu Gly Lys Lys Ala Pro Ile Ser Leu Arg Val Asn
Pro 140 145 150 aat gtg gat gcc aaa aca cat ccc tat att tcc cac ccg
gct ctc aaa 1254 Asn Val Asp Ala Lys Thr His Pro Tyr Ile Ser His
Pro Ala Leu Lys 155 160 165 aac aat aaa ttt ggt gtg gca ttt gaa gat
gcc ttg ggc ctc tat gaa 1302 Asn Asn Lys Phe Gly Val Ala Phe Glu
Asp Ala Leu Gly Leu Tyr Glu 170 175 180 aaa gcg gcg caa ctg cca aac
atc gag gta cac ggc gta gat tgc cat 1350 Lys Ala Ala Gln Leu Pro
Asn Ile Glu Val His Gly Val Asp Cys His 185 190 195 200 atc ggc tcg
caa atc act gag ctg tca cct ttc ctc gat gcc ttg gat 1398 Ile Gly
Ser Gln Ile Thr Glu Leu Ser Pro Phe Leu Asp Ala Leu Asp 205 210 215
aaa gta ttg ggc ctg gta gat gca ttg gcc gcc aaa ggc att cat atc
1446 Lys Val Leu Gly Leu Val Asp Ala Leu Ala Ala Lys Gly Ile His
Ile 220 225 230 cag cat ata gac gtt ggc ggc ggt gtc ggt att act tac
agc gac gaa 1494 Gln His Ile Asp Val Gly Gly Gly Val Gly Ile Thr
Tyr Ser Asp Glu 235 240 245 acg cca cca gac ttt gca gcc tac act gca
gcg att ctt aaa aag ctg 1542 Thr Pro Pro Asp Phe Ala Ala Tyr Thr
Ala Ala Ile Leu Lys Lys Leu 250 255 260 gca ggc agg aat gta aaa gtg
ttg ttt gag ccc ggc cgt gcc ctg gtg 1590 Ala Gly Arg Asn Val Lys
Val Leu Phe Glu Pro Gly Arg Ala Leu Val 265 270 275
280 ggt aac gcc ggt gtg ctg ctg acc aag gtc gaa tac ctg aaa cct ggc
1638 Gly Asn Ala Gly Val Leu Leu Thr Lys Val Glu Tyr Leu Lys Pro
Gly 285 290 295 gaa acc aaa aac ttt gcg att gtc gat gcc gcc atg aac
gac ctc atg 1686 Glu Thr Lys Asn Phe Ala Ile Val Asp Ala Ala Met
Asn Asp Leu Met 300 305 310 cgc ccg gct ttg tat gat gct ttc cac aac
att acg acc att gcc act 1734 Arg Pro Ala Leu Tyr Asp Ala Phe His
Asn Ile Thr Thr Ile Ala Thr 315 320 325 tct gca gcc ccc gca caa atc
tat gag atc gtt ggc ccg gtt tgc gag 1782 Ser Ala Ala Pro Ala Gln
Ile Tyr Glu Ile Val Gly Pro Val Cys Glu 330 335 340 agt ggt gac ttt
tta ggc cat gac cgt aca ctt gcg atc gaa gaa ggt 1830 Ser Gly Asp
Phe Leu Gly His Asp Arg Thr Leu Ala Ile Glu Glu Gly 345 350 355 360
gat tac ctg gcg att cac tcc gca ggc gct tat ggc atg agc atg gcc
1878 Asp Tyr Leu Ala Ile His Ser Ala Gly Ala Tyr Gly Met Ser Met
Ala 365 370 375 agc aac tac aac acg cgc gcc cgt gcc gca gag gta ttg
gtt gat ggt 1926 Ser Asn Tyr Asn Thr Arg Ala Arg Ala Ala Glu Val
Leu Val Asp Gly 380 385 390 gac cag gtg cat gtg atc cgt gaa cgt gaa
caa att gcc gac ctg ttt 1974 Asp Gln Val His Val Ile Arg Glu Arg
Glu Gln Ile Ala Asp Leu Phe 395 400 405 aaa ctg gag cgt acg ctg cca
taacattgac ggcaacccct aataaaaaaa 2025 Lys Leu Glu Arg Thr Leu Pro
410 415 ccgaagccgc caagcttcgg ttttttatta atagcgcatc ctttaatcaa
agatcacggt 2085 cttgttcgcg tagagcaaga ttctatgctc aatatgccag
cgcacggctt tggaaagcac 2145 aacacgctcc aggtcacggc ctttctggat
caggtcttcc acctgatcgc ggtgtgaaat 2205 gcgcgccaag tcctgctcaa
taatcggccc ctcatccaac acctctgtca cataatgact 2265 ggtcgcaccg
atcagtttca cgccacgctc aaacgcacgg tggtaaggac gtgcgccgat 2325
aaatgctggc aggaatgagt ggtggtgaat gttgataatc cgctgaggat accgtgcgac
2385 aaaatctggt gacagaatct gcatgtagcg tgccagcaca atcaggtcaa
tcttgtgttg 2445 atcaaacagg gcaaactgct gngcctctac ctctgccttg
gtttaccttg gtcatcggta 2505 aatagtgaaa cgggatgcca taaaactgcg
ccagggggat cctctgggtc cccctaaagc 2565 a 2566 14 415 PRT
Methylophilus methylotrophus misc_feature (2467)..(2467) n = a, c,
g, or t 14 Val Thr Ala Phe Ser Ile Gln Gln Gly Leu Leu His Ala Glu
Asn Val 1 5 10 15 Ala Leu Arg Asp Ile Ala Gln Thr His Gln Thr Pro
Thr Tyr Val Tyr 20 25 30 Ser Arg Ala Ala Leu Thr Thr Ala Phe Glu
Arg Phe Gln Ala Gly Leu 35 40 45 Thr Gly His Asp His Leu Ile Cys
Phe Ala Val Lys Ala Asn Pro Ser 50 55 60 Leu Ala Ile Leu Asn Leu
Phe Ala Arg Met Gly Ala Gly Phe Asp Ile 65 70 75 80 Val Ser Gly Gly
Glu Leu Ala Arg Val Leu Ala Ala Gly Gly Asp Pro 85 90 95 Lys Lys
Val Val Phe Ser Gly Val Gly Lys Ser His Ala Glu Ile Lys 100 105 110
Ala Ala Leu Glu Ala Gly Ile Leu Cys Phe Asn Val Glu Ser Val Asn 115
120 125 Glu Leu Asp Arg Ile Gln Gln Val Ala Ala Ser Leu Gly Lys Lys
Ala 130 135 140 Pro Ile Ser Leu Arg Val Asn Pro Asn Val Asp Ala Lys
Thr His Pro 145 150 155 160 Tyr Ile Ser His Pro Ala Leu Lys Asn Asn
Lys Phe Gly Val Ala Phe 165 170 175 Glu Asp Ala Leu Gly Leu Tyr Glu
Lys Ala Ala Gln Leu Pro Asn Ile 180 185 190 Glu Val His Gly Val Asp
Cys His Ile Gly Ser Gln Ile Thr Glu Leu 195 200 205 Ser Pro Phe Leu
Asp Ala Leu Asp Lys Val Leu Gly Leu Val Asp Ala 210 215 220 Leu Ala
Ala Lys Gly Ile His Ile Gln His Ile Asp Val Gly Gly Gly 225 230 235
240 Val Gly Ile Thr Tyr Ser Asp Glu Thr Pro Pro Asp Phe Ala Ala Tyr
245 250 255 Thr Ala Ala Ile Leu Lys Lys Leu Ala Gly Arg Asn Val Lys
Val Leu 260 265 270 Phe Glu Pro Gly Arg Ala Leu Val Gly Asn Ala Gly
Val Leu Leu Thr 275 280 285 Lys Val Glu Tyr Leu Lys Pro Gly Glu Thr
Lys Asn Phe Ala Ile Val 290 295 300 Asp Ala Ala Met Asn Asp Leu Met
Arg Pro Ala Leu Tyr Asp Ala Phe 305 310 315 320 His Asn Ile Thr Thr
Ile Ala Thr Ser Ala Ala Pro Ala Gln Ile Tyr 325 330 335 Glu Ile Val
Gly Pro Val Cys Glu Ser Gly Asp Phe Leu Gly His Asp 340 345 350 Arg
Thr Leu Ala Ile Glu Glu Gly Asp Tyr Leu Ala Ile His Ser Ala 355 360
365 Gly Ala Tyr Gly Met Ser Met Ala Ser Asn Tyr Asn Thr Arg Ala Arg
370 375 380 Ala Ala Glu Val Leu Val Asp Gly Asp Gln Val His Val Ile
Arg Glu 385 390 395 400 Arg Glu Gln Ile Ala Asp Leu Phe Lys Leu Glu
Arg Thr Leu Pro 405 410 415 15 39 DNA Artificial Sequence Synthetic
DNA 15 agggaattcc ccgttctgga taatgttttt tgcgccgac 39 16 58 DNA
Artificial Sequence Synthetic DNA 16 cggatgcatc tagagttaac
ctgcagggtg aaattgttat ccgctcacaa ttccacac 58 17 35 DNA Artificial
Sequence Synthetic DNA 17 tgacctgcag gtttgcacag aggatggccc atgtt 35
18 36 DNA Artificial Sequence Synthetic DNA 18 cattctagat
ccctaaactt tacagcaaac cggcat 36 19 35 DNA Artificial Sequence
Synthetic DNA 19 gaacctgcag gccctgacac gaggtagatt atgtc 35 20 55
DNA Artificial Sequence Synthetic DNA 20 ctttcggcta gaagagcgag
atgcagataa aaaaattaaa ggcaattatt ctccg 55
* * * * *