U.S. patent application number 09/927395 was filed with the patent office on 2002-05-16 for dna coding for protein which confers on bacterium escherichia coli resistance to l-homoserine, and method for producing l-amino acids.
This patent application is currently assigned to AJINOMOTO CO., INC.. Invention is credited to Aleoshin, Vladimir Venyamiovich, Balareova, Alla Valentinovna, Livshits, Vitaly Arkadievich, Tokhmakova, Irina Lvovna, Zakataeva, Natalya Pavlovna.
Application Number | 20020058314 09/927395 |
Document ID | / |
Family ID | 20211138 |
Filed Date | 2002-05-16 |
United States Patent
Application |
20020058314 |
Kind Code |
A1 |
Livshits, Vitaly Arkadievich ;
et al. |
May 16, 2002 |
DNA coding for protein which confers on bacterium escherichia coli
resistance to L-homoserine, and method for producing L-amino
acids
Abstract
A bacterium which has an ability to produce an amino acid and in
which a novel gene (rhtB) coding for a protein having an activity
of making a bacterium having the protein L-homoserine-resistant is
enhanced, is cultivated in a culture medium to produce and
accumulate the amino acid in the medium, and the amino acid is
recovered from the medium.
Inventors: |
Livshits, Vitaly Arkadievich;
(Moscow, RU) ; Zakataeva, Natalya Pavlovna;
(Moscow, RU) ; Aleoshin, Vladimir Venyamiovich;
(Moscow, RU) ; Balareova, Alla Valentinovna;
(Moscow, RU) ; Tokhmakova, Irina Lvovna; (Moscow,
RU) |
Correspondence
Address: |
OBLON SPIVAK MCCLELLAND MAIER & NEUSTADT PC
FOURTH FLOOR
1755 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Assignee: |
AJINOMOTO CO., INC.
15-1, Kyobashi 1-chome, chuo-ku
Tokyo
JP
|
Family ID: |
20211138 |
Appl. No.: |
09/927395 |
Filed: |
August 13, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09927395 |
Aug 13, 2001 |
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09396357 |
Sep 15, 1999 |
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6303348 |
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Current U.S.
Class: |
435/106 ;
435/193; 435/252.3; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12P 13/08 20130101;
C07K 14/245 20130101; C12P 13/06 20130101; C12P 13/04 20130101 |
Class at
Publication: |
435/106 ;
435/252.3; 435/193; 536/23.2; 435/69.1 |
International
Class: |
C12P 013/04; C12N
009/10; C07H 021/04; C12N 001/21; C12P 021/02 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 13, 1998 |
RU |
98118425 |
Claims
What is claimed is:
1. A DNA coding for a protein as defined in the following (A) or
(B): (A) a protein which comprises an amino acid sequence shown in
SEQ ID NO:2 in Sequence Listing; or (B) a protein which comprises
an amino acid sequence including deletion, substitution, insertion
or addition of one or several amino acids in the amino acid
sequence shown in SEQ ID NO:2 in Sequence Listing, and which has an
activity of making a bacterium having the protein
L-homoserine-resistant.
2. The DNA according to claim 1, which is a DNA as defined in the
following (a) or (b): (a) a DNA which comprises a nucleotide
sequence of the nucleotide numbers of 557 to 1171 of a nucleotide
sequence shown in SEQ ID NO:1 in Sequence Listing; or (b) a DNA
which hybridizes with the nucleotide sequence of the nucleotide
numbers of 557 to 1171 of the nucleotide sequence shown in SEQ ID
NO:1 in Sequence Listing under stringent conditions, and which
codes for the protein having the activity of making the bacterium
having the protein L-homoserine-resistant.
3. A bacterium belonging to the genus Escherichia, wherein
L-homoserine resistance of said bacterium is enhanced by amplifying
a copy number of the DNA as defined in claim 1 in a cell of said
bacterium.
4. The bacterium according to claim 3, wherein the DNA as defined
in claim 1 is carried on a multicopy vector in the cell of said
bacterium.
5. The bacterium according to claim 3, wherein the DNA as defined
in claim 1 is carried on a transposon in the cell of said
bacterium.
6. A method for producing an amino acid, comprising the steps of:
cultivating the bacterium as defined in any one of claims 3 to 5,
which has an ability to produce the amino acid, in a culture
medium, to produce and accumulate the amino acid in the medium, and
recovering the amino acid from the medium.
7. The method according to the claim 6, wherein said amino acid is
at least one selected from the group consisting of L-homoserine,
L-alanine, L-isoleucine, L-valine and L-threonine.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for producing an
amino acid, especially for a method for producing L-homoserine,
L-alanine, L-isoleucine, L-valine, or L-threonine using a bacterium
belonging to the genus Escherichia.
BACKGROUND ART
[0002] The present inventors obtained, with respect to E. coli
K-12, a mutant having mutation, thrR, (herein referred to as
rhtA23) that is concerned in high concentrations of threonine
(>40 mg/ml) or homoserine (>5 mg/ml) in a minimal medium
(Astaurova, O. B. et al., Appl. Bioch. and Microbiol., 21, 611-616
(1985)). On the basis of rhtA23 mutation an improved
threonine-producing strain (SU patent No. 974817), homoserine- and
glutamic acid-producing strains (Astaurova et al., Appl. Boch. And
Microbiol., 27, 556-561 (1991)) were obtained.
[0003] Furthermore, the present inventors has revealed that the
rhtA gene exists at 18 min on E. coli chromosome and that the rhtA
gene is identical to ORF1 between pexB and ompX genes. The unit
expressing a protein encoded by the ORF1 has been designated as
rhtA (rht: resistance to homoserine and threonine) gene. The rhtA
gene includes a 5'-noncoding region including SD sequence, ORF1 and
a terminator. Also, the present inventors have found that a wild
type rhtA gene participates in resistance to threonine and
homoserine if cloned in a multicopy state and that enhancement of
expression of the rhtA gene improves amino acid productivity of a
bacterium belonging to the genus Escherichia having an ability to
produce L-lysine, L-valine or L-threonine (ABSTRACTS of 17th
International Congress of Biochemistry and Molecular Biology in
conjugation with 1997 Annual Meeting of the American Society for
Biochemistry and Molecular Biology, San Francisco, Calif. August
24-29, 1997, abstract No. 457).
[0004] It is found that at least two different genes which impart
homoserine resistance in a multicopy state exist in E. coli during
cloning of the rhtA gene. One of the genes is the rhtA gene,
however the other gene has not been elucidated.
DISCLOSURE OF THE INVENTION
[0005] An object of the present invention is to provide a novel
gene participating in resistance to homoserine, and a method for
producing an amino acid, especially, L-homoserine, L-alanine,
L-isoleucine, L-valine and L-threonine with a high yield.
[0006] The inventors have found that a region at 86 min on E. coli
chromosome, when cloned by a multicopy vector, impart resistance to
L-homoserine to cells of E. coli, and that when the region is
amplified, the amino acid productivity of E. coli can be improved
like the rhtA gene. On the basis of these findings, the present
invention have completed.
[0007] Thus, the present invention provides:
[0008] (1) a DNA coding for a protein as defined in the following
(A) or (B):
[0009] (A) a protein which comprises an amino acid sequence shown
in SEQ ID NO: 2 in Sequence Listing; or
[0010] (B) a protein which comprises an amino acid sequence
including deletion, substitution, insertion or addition of one or
several amino acids in the amino acid sequence shown in SEQ ID NO:
2 in Sequence Listing, and which has an activity of making a
bacterium having the protein L-homoserine-resistant,
[0011] (2) the DNA according to (1), which is a DNA as defined in
the following (a) or (b):
[0012] (a) a DNA which comprises a nucleotide sequence
corresponding to the nucleotide numbers of 557 to 1171 of a
nucleotide sequence shown in SEQ ID NO: 1 in Sequence Listing;
or
[0013] (b) a DNA which hybridizes with the nucleotide sequence
corresponding to the nucleotide numbers of 557 to 1171 of the
nucleotide sequence shown in SEQ ID NO: 1 in Sequence Listing under
stringent conditions, and which codes for the protein having the
activity of making the bacterium having the protein
L-homoserine-resistant,
[0014] (3) a bacterium belonging to the genus Escherichia, wherein
L-homoserine resistance of the bacterium is enhanced by amplifying
a copy number of the DNA of (1) in a cell of the bacterium,
[0015] (4) the bacterium of (3), wherein the DNA of (1) is carried
on a multicopy vector in the cell of the bacterium,
[0016] (5) the bacterium of (3), wherein the DNA of (1) is carried
on a transposon in the cell of the bacterium,
[0017] (6) a method for producing an amino acid, comprising the
steps of cultivating the bacterium of any of (3) to (5), which has
an ability to produce the amino acid, in a culture medium to
produce and accumulate the amino acid in the medium, and recovering
the amino acid from the medium, and
[0018] (7) the method of (6), wherein the amino acid is at least
one selected from the group consisting of L-homoserine, L-alanine,
L-isoleucine, L-valine and L-threonine.
[0019] The DNA of the present invention may be referred to as "rhtB
gene", a protein coded by the rhtB gene may be referred to as "RhtB
protein", an activity of the RhtB protein which participates in
resistance to L-homoserine of a bacterium (i.e. an activity of
making a bacterium having the RhtB protein L-homoserine-resistant)
may be referred to as "Rh activity", and a structural gene encoding
the RhtB protein in the rhtB gene may be referred to as "rhtB
structural gene". The term "enhancing the Rh activity" means
imparting resistance to homoserine to a bacterium or enhance the
resistance by means of increasing the number of molecules of the
RhtB protein, increasing a specific activity of the RhtB protein,
or desensitizing negative regulation against the expression or the
activity of the RhtB protein or the like. The terms "DNA coding for
a protein" mean a DNA of which one of strands codes for the protein
when the DNA is double-stranded. The L-homoserine resistance means
a property that a bacterium grows on a minimal medium containing
L-homoserine at a concentration at which a wild type strain thereof
can not grow, usually at 10 mg/ml. The ability to produce an amino
acid means a property that a bacterium produces and accumulates the
amino acid in a medium in a larger amount than a wild type strain
thereof.
[0020] According to the present invention, resistance to homoserine
of a high concentration can be imparted to a bacterium belonging to
the genus Escherichia. A bacterium belonging to the genus
Escherichia, which has increased resistance homoserine and an
ability to accumulate an amino acid, especially, L-homoserine,
L-alanine, L-isoleucine, L-valine or L-threonine in a medium with a
high yield.
[0021] The present invention will be explained in detail below.
[0022] <1>DNA of the Present Invention
[0023] The DNA of the present invention coding for a protein having
the Rh activity and having an amino acid sequence shown in SEQ ID
NO: 2 in Sequence Listing. Specifically, the DNA of the present
invention may be exemplified by a DNA comprising a nucleotide
sequence of the nucleotide numbers 557 to 1171 of a nucleotide
sequence shown in SEQ ID NO: 1 in Sequence Listing.
[0024] The DNA of the present invention includes a DNA fragment
encoding the RhtB protein conferring bacterium Escherichia coli
resistance to homoserine, which includes the regulatory elements of
the rhtB gene and the structural part of rhtB gene, having the
nucleotide sequence shown in SEQ ID NO: 1.
[0025] The nucleotide sequence shown in SEQ ID NO: 1 corresponds to
a part of sequence complement to the sequence of GenBank accession
number M87049. SEQ ID NO: 1 includes f138 (nucleotide numbers
61959-61543 of GenBank accession number M87049) which is a known
but function-unknown ORF (open reading frame) present at 86 min on
E. coli chromosome, and 5'-flanking and 3'-flanking regions
thereof. The f138, which had only 160 nucleotides in the
5'-flanking region, could not impart the resistance to homoserine.
No termination codon is present between the 62160 and 61959 of
M87049 (upstream the ORF f138). Hence, the coding region is 201 bp
longer. Thus the RhtB protein and the rhtB gene coding for the
protein are novel.
[0026] The rhtB gene may be obtained, for example, by infecting
Mucts lysogenic strain of E. coli using a lysate of a lysogenic
strain of E. coli such as K12 or W3110 according to the method in
which mini-Mu d5005 phagemid is used (Groisman, E. A., et al., J.
Bacteriol., 168, 357-364 (1986)), and isolating plasmid DNAs from
colonies growing on a minimal medium containing kanamycin (40
.mu.g/ml) and L-homoserine (10 mg/ml). As illustrated in the
Example described below, the rhtB gene was mapped at 86 min on the
chromosome of E. coli. Therefore, the DNA fragment including the
rhtB gene may be obtained from the chromosome of E. Coli by colony
hybridization or PCR (polymerase chain reaction, refer to White, T.
J. et al, Trends Genet. 5, 185(1989)) using oligonucleotide(s)
which has a sequence corresponding to the region near the portion
of 86 min on the chromosome of E. coli. Alternatively, the
oligonucleotide may be designed according to the nucleotide
sequence shown in SEQ ID NO: 1. By using oligonucleotides having
nucleotide sequences corresponding to a upstream region from the
nucleotide number 557 and a downstream region from the nucleotide
number 1171 in SEQ ID NO: 1 as the primers for PCR, the entire
coding region can be amplified.
[0027] Synthesis of the oligonucleotides can be performed by an
ordinary method such as a phosphoamidite method (see Tetrahedron
Letters, 22, 1859 (1981)) by using a commercially available DNA
synthesizer (for example, DNA Synthesizer Model 380B produced by
Applied Biosystems). Further, the PCR can be performed by using a
commercially available PCR apparatus (for example, DNA Thermal
Cycler Model PJ2000 produced by Takara Shuzo Co., Ltd.), using Taq
DNA polymerase (supplied by Takara Shuzo Co., Ltd.) in accordance
with a method designated by the supplier.
[0028] The DNA coding for the RhtB protein of the present invention
may code for RhtB protein including deletion, substitution,
insertion, or addition of one or several amino acids at one or a
plurality of positions, provided that the Rh activity of RhtB
protein encoded thereby is not deteriorated. The DNA, which codes
for the substantially same protein as the RhtB protein as described
above, may be obtained, for example, by modifying the nucleotide
sequence, for example, by means of the site-directed mutagenesis
method so that one or more amino acid residues at a specified site
involve deletion, substitution, insertion or addition. DNA modified
as described above may be obtained by the conventionally known
mutation treatment. The mutation treatment includes a method for
treating a DNA coding for the RhtB protein in vitro, for example,
with hydroxylamine, and a method for treating a microorganism, for
example, a bacterium belonging to the genus Escherichia harboring a
DNA coding for the RhtB protein with ultraviolet irradiation or a
mutating agent such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG)
and nitrous acid usually used for the mutation treatment.
[0029] The DNA, which codes for substantially the same protein as
the RhtB protein, can be obtained by expressing a DNA subjected to
in vitro mutation treatment as described above in multicopy in an
appropriate cell, investigating the resistance to homoserine, and
selecting the DNA which increase the resistance. Also, it is
generally known that an amino acid sequence of a protein and a
nucleotide sequence coding for it may be slightly different between
species, strains, mutants or variants, and therefore the DNA, which
codes for substantially the same protein, can be obtained from
L-homoserine-resistant species, strains, mutants and variants
belonging to the genus Escherichia. Specifically, the DNA, which
codes for substantially the same protein as the RhtB protein, can
be obtained by isolating a DNA which hybridizes with DNA having,
for example, a nucleotide sequence of the nucleotide numbers 557 to
1171 of the nucleotide sequence shown in SEQ ID NO: 1 in Sequence
Listing under stringent conditions, and which codes for a protein
having the Rh activity, from a bacterium belonging to the genus
Escherichia which is subjected to mutation treatment, or a
spontaneous mutant or a variant of a bacterium belonging to the
genus Escherichia. The term "stringent conditions" referred to
herein is a condition under which so-called specific hybrid is
formed, and non-specific hybrid is not formed. It is difficult to
clearly express this condition by using any numerical value.
However, for example, the stringent conditions include a condition
under which DNAs having high homology, for example, DNAs having
homology of not less than 70% with each other are hybridized, and
DNAs having homology lower than the above with each other are not
hybridized.
[0030] <2>Bacterium Belonging to the Genus Escherichia of the
Present Invention
[0031] The bacterium belonging the genus Escherichia of the present
invention is a bacterium belonging to the genus Escherichia of
which the Rh activity is enhanced. A bacterium belonging to the
genus Escherichia is exemplified by Escherichia Coli. The Rh
activity can be enhanced by, for example, amplification of the copy
number of the rhtB structural gene in a cell, or transformation of
a bacterium belonging to the genus Escherichia with a recombinant
DNA in which a DNA fragment including the rhtB structural gene
encoding the RhtB protein is ligated with a promoter sequence which
functions efficiently in a bacterium belonging to the genus
Escherichia. The Rh activity can be also enhanced by substitution
of the promoter sequence of the rhtB gene on a chromosome with a
promoter sequence which functions efficiently in a bacterium
belonging to the genus Escherichia.
[0032] The amplification of the copy number of the rhtB structural
gene in a cell can be performed by introduction of a multicopy
vector which carries the rhtB structural gene into a cell of a
bacterium belonging to the genus Escherichia. Specifically, the
copy number can be increased by introduction of a plasmid, a phage
or a transposon (Berg, D. E. and Berg, C. M., Bio/Technol., 1, 417
(1983)) which carries the rhtB structural gene into a cell of a
bacterium belonging to the genus Escherichia.
[0033] The multicopy vector is exemplified by plasmid vectors such
as pBR322, pMW118, pUC19 or the like, and phage vectors such as
.lambda.1059, .lambda.BF101, M13mp9 or the like. The transposon is
exemplified by Mu, Tn10, Tn5 or the like.
[0034] The introduction of a DNA into a bacterium belonging to the
genus Escherichia can be performed, for example, by a method of D.
M. Morrison (Methods in Enzymology 68, 326 (1979)) or a method in
which recipient bacterial cells are treated with calcium chloride
to increase permeability of DNA (Mandel, M. and Higa, A., J. Mol.
Biol., 53, 159 (1970)) and the like.
[0035] If the Rh activity is enhanced in an amino acid-producing
bacterium belonging to the genus Escherichia as described above, a
produced amount of the amino acid can be increased. As the
bacterium belonging to the genus Escherichia which is to be the Rh
activity is enhanced, strains which have abilities to produce
desired amino acids are used. Besides, an ability to produce an
amino acid may be imparted to a bacterium in which the Rh activity
is enhanced. Examples of amino acid-producing bacteria belonging to
the genus Escherichia are described below.
[0036] (1) L-threonine-producing Bacteria
[0037] The L-threonine-producing bacteria belonging to the genus
Escherichia may be exemplified by strain MG442 (Guayatiner et al.,
Genetika (in Russian), 14, 947-956 (1978)).
[0038] (2) L-homoserine-producing Bacteria
[0039] The L-homoserine-producing bacteria belonging to the genus
Escherichia may be exemplified by strain NZ10 (thrB). This strain
was derived from the known strain C600 (thrB, leuB) (Appleyard R.
K., Genetics, 39, 440-452 (1954)) as Leu.sup.+revertant.
[0040] On the basis of the rhtB DNA fragment, new amino
acid-producing strains E. coli NZ10/pAL4,pRhtB; E. coli
MG422/pVIC40,pRhtB; and E. coli MG442/pRhtB were obtained which are
used for the production of amino acids by fermentation.
[0041] The new strains have been deposited (according to
international deposition based on Budapest Treaty) in the Russian
National Collection of Industrial Microorganisms (VKPM) on Oct. 6,
1998. The strain E. coli NZ10/pAL4, pRhtB has been deposited as an
accession number of VKPM B-7658; the strain E. coli MG442/pRhtB has
been deposited as an accession number of VKPM B-7659; and the
strain E. coli MG442/pVIC40,pRhtB has been deposited as an
accession number of VKPM B-7660.
[0042] The strain E. coli NZ10/pAL4, pRhtB (VKPM B-7658) exhibits
the following cultural-morphological and biochemical features.
[0043] Cytomorphology. Gram-negative weakly-motile rods having
rounded ends. Longitudinal size, 1.5 to 2 .mu.m.
[0044] Cultural features:
[0045] Beef-extract agar. After 24-hour growth at 37.degree. C.,
produces round whitish semitransparent colonies 1.5 to 3 mm in
diameter, featuring a smooth surface, regular or slightly wavy
edges, the center is slightly raised, homogeneous structure,
pastelike consistency, readily emulsifiable.
[0046] Luria's agar. After a 24-hour growth at 37.degree. C.,
develops whitish semitranslucent colonies 1.5 to 2.5 mm in diameter
having a smooth surface, homogeneous structure, pastelike
consistency, readily emulsifiable.
[0047] Minimal agar-doped medium M9. After 40 to 48 hours of growth
at 37.degree. C., forms colonies 0.5 to 1.5 mm in diameter, which
are colored greyish-white, semitransparent, slightly convex, with a
lustrous surface.
[0048] Growth in a beef-extract broth. After 24-hour growth at
37.degree. C., exhibits strong uniform cloudiness, has a
characteristic odor.
[0049] Physiological and biochemical features:
[0050] Grows upon thrust inoculation in a beef-extract agar.
Exhibits good growth throughout the inoculated area. The
microorganism proves to be a facultative anaerobe.
[0051] It does not liquefy gelatin.
[0052] Features a good growth on milk, accompanied by milk
coagulation.
[0053] Does not produce indole.
[0054] Temperature conditions. Grows on beef-extract broth at
20-42.degree. C., an optimum temperature lying within 33-37.degree.
C.
[0055] pH value of culture medium. Grows on liquid media having the
pH value from 6 to 8, an optimum value being 7.2.
[0056] Carbon sources. Exhibits good growth on glucose, fructose,
lactose, mannose, galactose, xylose, glycerol, and mannitol to
produce an acid and gas. Nitrogen sources. Assimilates nitrogen in
the form of ammonium, nitric acid salts, as well as from some
organic compounds.
[0057] Resistant to ampicillin, kanamycin and L-homoserine.
L-Threonine is used as a growth factor.
[0058] Content of plasmids. The cells contain multicopy hybrid
plasmid pAL4 ensuring resistance to ampicillin and carrying the
gene thrA of the threonine operon, which codes for aspartate
kinase-homoserine dehydrogenase I responsible for the increased
homoserine biosynthesis. Besides, the cells contain a multicopy
hybrid plasmid pRhtB ensuring resistance to kanamycin and carrying
the rhtB gene which confers resistance to homoserine (10 mg/l).
[0059] The strain E. coli MG442/pRhtB (VKPM B-7659) has the same
cultural-morphological and biochemical features as the strain
NZ10/pAL4,pRhtB except for L-isoleucine is used as a growth factor
instead of L-threonine. However, the strain can grow slowly without
isoleucine. Besides, the cells of the strain contain only one
multicopy hybrid plasmid pRhtB ensuring resistance to kanamycin and
carrying the rhtB gene which confers resistance to homoserine (10
mg/l).
[0060] The strain E. coli MG442/pVIC40,pRhtB (VKPM B-7660) has the
same cultural-morphological and biochemical features as the strain
NZ10/pAL4,pRhtB except for L-isoleucine is used as a growth factor
instead of L-threonine. However, the strain can grow slowly without
isoleucine. The cells of the strain contain multicopy hybrid
plasmid pVIC40 ensuring resistance to streptomycin and carrying the
genes of the threonine operon. Besides, they contain multicopy
hybrid plasmid pRhtB ensuring resistance to kanamycin and carrying
the rhtB gene which confers resistance to homoserine (10 mg/l).
[0061] <3>Method for Producing an Amino Acid
[0062] An amino acid can be efficiently produced by cultivating the
bacterium in which the Rh activity is enhanced by amplifying a copy
number of the rhtB gene as described above, and which has an
ability to produce the amino acid, in a culture medium, producing
and accumulating the amino acid in the medium, and recovering the
amino acid from the medium. The amino acid is exemplified
preferably by L-homoserine, L-alanine, L-isoleucine, L-valine and
L-threonine.
[0063] In the method of present invention, the cultivation of the
bacterium belonging to the genus Escherichia, the collection and
purification of amino acid from the liquid medium may be performed
in a manner similar to those of the conventional method for
producing an amino acid by fermentation using a bacterium. A medium
used in cultivation may be either a synthetic medium or a natural
medium, so long as the medium includes a carbon and a nitrogen
source and minerals and, if necessary, nutrients which the
bacterium used requires for growth in appropriate amounts. The
carbon source may include various carbohydrates such as glucose and
sucrose, and various organic acids. Depending on assimilatory
ability of the used bacterium, alcohol including ethanol and
glycerol may be used. As the nitrogen source, ammonia, various
ammonium salts such as ammonium sulfate, other nitrogen compounds
such as amines, a natural nitrogen source such as peptone, soybean
hydrolyte and digested fermentative microbe are used. As minerals,
monopotassium phosphate, magnesium sulfate, sodium chloride,
ferrous sulfate, manganese sulfate, calcium carbonate are used.
[0064] The cultivation is preferably culture under an aerobic
condition such as a shaking culture, and an aeration and stirring
culture. The temperature of culture is usually 20 to 40.degree. C.,
preferably 30 to 38.degree. C. The pH of the culture is usually
between 5 and 9, preferably between 6.5 and 7.2. The pH of the
culture can be adjusted with ammonia, calcium carbonate, various
acids, various bases, and buffers. Usually, a 1 to 3-day
cultivation leads to the accumulation of the target amino acid in
the medium.
[0065] Recovering the amino acid can be performed by removing
solids such as cells from the medium by centrifugation or membrane
filtration after cultivation, and then collecting and purifying the
target amino acid by ion exchange, concentration and crystalline
fraction methods and the like.
Brief Explanation of Drawing
[0066] FIG. 1 shows cloning, identification and inactivation of the
rhtB gene.
[0067] FIG. 2 shows the amino acid sequence of the RhtB
protein.
EXAMPLES
[0068] The present invention will be more concretely explained
below with reference to Examples. In the Examples, an amino acid is
of L-configuration unless otherwise noted.
Example 1
Obtaining of rhtB DNA Fragment
[0069] (1) Cloning of rhtB Gene into Mini-Mu Phagemid
[0070] The wild-type rhtB gene was cloned in vivo using mini-Mu
d5005 phagemid (Groisman, E. A., et al., J. Bacteriol., 168,
357-364 (1986)). MuCts62 lysogen of the strain MG442 was used as a
donor. Freshly prepared lysates were used to infect a Mucts
lysogenic derivative of a strain VKPM B-513 (Hfr K10 metB). The
cells were plated on M9 glucose minimal medium with methionine (50
.mu.g/ml), kanamycin (40 .mu.g/ml) and homoserine (10 mg/ml).
Colonies which appeared after 48 hr were picked and isolated.
Plasmid DNA was isolated and used to transform the strain VKPM
B-513 by standard techniques. Transformants were selected on
L-broth agar plates with kanamycin as above. Plasmid DNA was
isolated from those which were resistant to homoserine, and
analyzed by restriction mapping of the structure of the inserted
fragments. It appeared that two types of inserts belonging to
different chromosome regions had been cloned from the donor. Thus,
at least two different genes that is in multicopy and imparts
resistance to homoserine exist in E. Coli. One of the two type of
inserts is the rhtA gene which has already reported (ABSTRACTS of
17th International Congress of Biochemistry and Molecular Biology
in conjugation with 1997 Annual Meeting of the American Society for
Biochemistry and Molecular Biology, San Francisco, Calif. August
24-29, 1997). Among the other of the two types of inserts, a
fragment of a minimum length which imparts the resistance to
homoserine is of 0.8 kb (FIG. 1).
[0071] (2) Identification of rhtB Gene
[0072] The insert fragment was sequenced by the dideoxy chain
termination method of Sanger. Both DNA strands were sequenced in
their entirety and all junctions were overlapped. The sequencing
showed that the insert fragment included f138 (nucleotide numbers
61543 to 61959 of GenBank accession number M87049) which was a
known but function-unknown ORF (open reading frame) present at 86
min of E. coli chromosome and 201 bp of an upstream region thereof
(downstream region in the sequence of M87049). The f138 which had
only 160 nucleotides in the 5'-flanking region could not impart the
resistance to homoserine. No termination codon is present upstream
the ORF f138 between 62160 and 61959 nucleotides of M87049.
Furthermore, one ATG following a sequence predicted as a ribosome
binding site is present in the sequence. The larger ORF (nucleotide
numbers 62160 to 61546) is designated as rhtB gene. The RhtB
protein deduced from the gene is highly hydrophobic and contains 5
possible transmembrane segments.
Example 2
Production of homoserine-producing Strain
[0073] Strain NZ10 of E. coli was transformed by a plasmid pAL4
which was a pBR322 vector into which the thrA gene coding for
aspartokinase-homoserine dehydrogenase I was inserted, to obtain
the strains NZ10/pAL4. The strain NZ10 is a leuB.sup.+-reverted
mutant (thrB) obtained from the E. coli strain C600 (thrB, leuB)
(Appleyard, Genetics, 39, 440-452 (1954)).
[0074] The rhtB gene was inserted to a plasmid pUK21 which is a
known plasmid pUC19 in which a kanamycin resistance gene
substituted for an ampicillin resistance gene (Vieira, J. and
Messing, J., Gene, 100, 189-194 (1991)), to obtain pRhtB.
[0075] The strain NZ10/pAL4 was transformed with pUK21 or pRhtB to
obtain strains NZ10/pAL4,pUK21 and NZ10/pAL4,pRhtB.
[0076] The thus obtained transformants were each cultivated at
37.degree. C. for 18 hours in a nutrient broth with 50 mg/l
kanamycin and 100 mg/l ampicillin, and 0.3 ml of the obtained
culture was inoculated into 3 ml of a fermentation medium having
the following composition and containing 50 mg/l kanamycin and 100
mg/l ampicillin, in a 20.times.200 mm test tube, and cultivated at
37.degree. C. for 46 hours with a rotary shaker. After the
cultivation, an accumulated amount of homoserine in the medium and
an absorbance at 560 nm of the medium were determined by known
methods. Fermentation medium composition (g/L)
1 Glucose 80 (NH.sub.4).sub.2SO.sub.4 22 K.sub.2HPO.sub.4 2 NaCl
0.8 MgSO.sub.4.7H.sub.2O 0.8 FeSO.sub.4.7H.sub.2O 0.02
MnSO.sub.4.5H.sub.2O 0.02 Thiamine hydrochloride 0.0002 Yeast
Extract 1.0 CaCO.sub.3 30 (CaCO.sub.3 was separately
sterilized.)
[0077] The results are shown in Table 1. As shown in Table 1, the
strain NZ10/pAL4,pRhtB accumulated homoserine in a larger amount
than the strains NZ10/pAL4 and NZ10/pAL4,pUK21 in which the rhtB
gene was not enhanced.
2 TABLE 1 Accumulated amount of Strain OD.sub.560 homoserine (g/L)
NZ10/pAL4 16.4 3.1 NZ10/pAL4,pUK21 14.3 3.3 NZ10/pAL4,pRhtB 15.6
6.4
Example 3
Production of Alanine. Valine and Isoleucine with pRhtB-introduced
Strain
[0078] E. coli strain MG442 is a known strain (Gusyatiner, et al.,
1978, Genetika (in Russian), 14:947-956).
[0079] The strain MG442 was transformed with the plasmids pUK21 and
pRhtB to obtain strains MG442/pUK21 and MG442/pRhtB.
[0080] The thus obtained transformants were each cultivated at
37.degree. C. for 18 hours in a nutrient broth with 50 mg/l
kanamycin, and 0.3 ml of the obtained culture was inoculated into 3
ml of the fermentation medium described in Example 3 and containing
50 mg/l kanamycin, in a 20.times.200 mm test tube, and cultivated
at 37.degree. C. for 40 hours with a rotary shaker. After the
cultivation, accumulated amounts of alanine, valine and isoleucine
in the medium and an absorbance at 560 nm of the medium were
determined by known methods.
[0081] The results are shown in Table 2. As shown in Table 2, the
strain MG442/pRhtB accumulated each of alanine, valine and
isoleucine in a larger amount than the strain MG442/pUK21 in which
the rhtB gene was not enhanced.
3 TABLE 2 Accumulated amount (g/L) Strain OD.sub.560 Alanine Valine
Isoleucine MG442/pUK21 13.4 0.2 0.2 0.3 MG442/pRhtB 13.7 0.7 0.5
0.5
Example 4
Production of threonine-producing Strain
[0082] The strain MG442 (Example 3) was transformed by introducing
a known plasmid pVIC40 (U.S. Pat. No. 5,175,107 (1992)) by an
ordinary transformation method. Transformants were selected on LB
agar plates containing 0.1 mg/ml streptomycin. Thus a novel strain
MG422/pVIC40 was obtained.
[0083] The strain MG442/pVIC40 was transformed with pUK21 or pRhtB
to obtain strains MG442/pVIC40, pUK21 and MG442/pVIC40, pRhtB.
[0084] The thus obtained transformants were each cultivated at
37.degree. C. for 18 hours in a nutrient broth with 50 mg/l
kanamycin and 100 mg/l streptomycin, and 0.3 ml of the obtained
culture was inoculated into 3 ml of the fermentation medium
described in Example 3 and containing 50 mg/l kanamycin and 100
mg/l streptomycin, in a 20.times.200 mm test tube, and cultivated
at 37.degree. C. for 46 hours with a rotary shaker. After the
cultivation, an accumulated amount of threonine in the medium and
an absorbance at 560 nm of the medium were determined by known
methods.
[0085] The results are shown in Table 3. As shown in Table 3, the
strain MG442/pVIC40,pRhtB accumulated threonine in a larger amount
than the strains MG442/pVIC40 and MG442/pVIC40, pUK21 in which the
rhtB gene was not enhanced.
4 TABLE 3 Accumulated amount of Strain OD.sub.560 threonine (g/L)
MG442/pVIC40 17 13.6 MG442/pVIC40,pUK21 16.3 12.9
MG442/pVIC40,pRhtB 15.2 16.3
Example 5
Effect of rhtB Gene Inactivation and Amplification on Bacterium E.
coli Resistance to Some Amino Acids and Amino Acid Analogues
[0086] To inactivate the chromosomal rhtB gene the plasmid pNPZ46
was constructed (FIG. 1) on the basis of PUK21 vector. It harbors a
DNA fragment from 86 min of E. coli chromosome, with the rhtB gene
and 5'-flanking and 3'-flanking regions thereof. Then the
ClaI-Eco47III fragment of the pNPZ46 plasmid rhtB gene was
substituted for AsuII-BsrBI fragment containing cat (Cm.sup.R) gene
of pACYC184 plasmid (Chang and Cohen, J. Bacteriol., 134,
1141-1156, 1978) giving the pNPZ47 plasmid (FIG. 1). To introduce
the obtained insertionally inactivated rhtB gene into the
chromosome of the E. coli strain N99 (the streptomycin-resistant
derivative of the known strain W3350 (Campbell, virology, 14,
22-33, 1961)), the method of Parker and Marinus was used (Parker,
B. and Marinus, M. G., Gene, 73, 531-535, 1988). The substitution
of the wild type allele for the inactivated one was proved by phage
P1 transduction and by Southern hybridization (Southern, E. M., J.
Mol. Biol., 98, 503-517, 1975).
[0087] Then the susceptibility of the thus obtained E. coli strain
N99 rhtB::cat, of the initial strain N99 (rhtB.sup.-) and of its
derivative transformed with pRhtB plasmid, N99/pRhtB, to some amino
acids and amino acid analogues was tested. Overnight cultures of
the strains grown in M9 minimal medium at 37.degree. C. with a
rotary shaker (10.sup.9 cfu/ml) were diluted 1:100 and grown for 5
hours under the same conditions. Then the log phase cultures thus
obtained were diluted and about 10.sup.4 of alive cells were
applied to well-dried test plates with M9 agar containing doubling
increments of amino acids or analogues. The minimum inhibitory
concentration (MIC) of these compounds were examined after 40-46 h
cultivation. The results are shown in Table 4.
5 TABLE 4 MIC (.mu.g/ml) N99 Substrate N99(rhtB.sup.+) N99/pRhtB
rhtB::cat 1. L-homoserine 250 30000 125 2. L-threonine 30000 50000
30000 3. L-serine 5000 10000 5000 4. L-valine 0.5 1 0.5 5. AHVA 50
2000 25 6. AEC 10 25 10 7. 4-aza-DL-leucine 40 100 40
[0088] It follows from the Table 4 that multiple copies of rhtB
besides homoserine confered upon cells increased resistance to
threonine, serine, valine, .alpha.-amino-.beta.-hydroxyvaleric-acid
(AHVA), S-(2-aminoethyl)-L-cysteine (AEC), and 4-aza-DL-leucine.
The inactivation of the rhtB gene, on the contrary, increased the
cell sensitivity to homoserine and AHVA. These results in
conjunction with the data on homology of the RhtB protein to LysE
lysine efflux transporter of Corynebacterium glutamicum (Vrljic et
al., Mol. Microbiol., 22, 815-826, 1996) indicate the analogues
function for the rhtB gene product. The presumed efflux
transporters, RhtB, has specificity to several substrates (amino
acids), or may show non-specific effects as a result of
amplification.
Sequence CWU 1
1
2 1 1200 DNA Escherichia coli CDS (557)..(1171) 1 agaaataatg
tggagatcgc accgcccatc gaatgtgcca gtatatagcg tttacgccac 60
ggaccgggct gaacctcctg ctgccagaat gccgccagat catcaacata atcattaaag
120 cgattaacat gcccgagatg cggatcggct aacaggcgac cggaacgtcc
ctgcccgcga 180 tggtcgatga ttaagacatc aaaccccaaa tggaacaggt
cataggccag ttccgcatat 240 tttacgtagc tctcaatacg ccccgggcag
atgactacca cccggtcatg gtgctgtgcg 300 cgaaaacgga caaagcgcac
cggaatgtca tccacaccag taaactctgc ttcatcacgc 360 tgacgccaga
aatcagtcag cggtcccatg gtaaaagcag caaacgcgtt ttctcttgtt 420
tcccagtctt tttgctgctg aaacatcggg taatctgcct cttaaaccac gtaaaatcgt
480 tttttttagc gtgcctgaca caacgctgcg acagtagcgt attgtggcac
aaaaatagac 540 acaccgggag ttcatc atg acc tta gaa tgg tgg ttt gcc
tac ctg ctg aca 592 Met Thr Leu Glu Trp Trp Phe Ala Tyr Leu Leu Thr
1 5 10 tcg atc att tta acg ctg tcg cca ggc tct ggt gca atc aac act
atg 640 Ser Ile Ile Leu Thr Leu Ser Pro Gly Ser Gly Ala Ile Asn Thr
Met 15 20 25 acc acc tcg ctc aac cac ggt tat ccg gcc ggt ggc gtc
tat tgc tgg 688 Thr Thr Ser Leu Asn His Gly Tyr Pro Ala Gly Gly Val
Tyr Cys Trp 30 35 40 gct tca gac cgg act ggc gat tca tat tgt gct
ggt tgg cgt ggg gtt 736 Ala Ser Asp Arg Thr Gly Asp Ser Tyr Cys Ala
Gly Trp Arg Gly Val 45 50 55 60 ggg acg cta ttt tcc cgc tca gtg att
gcg ttt gaa gtg ttg aag tgg 784 Gly Thr Leu Phe Ser Arg Ser Val Ile
Ala Phe Glu Val Leu Lys Trp 65 70 75 gca ggc gcg gct tac ttg att
tgg ctg gga atc cag cag tgg cgc gcc 832 Ala Gly Ala Ala Tyr Leu Ile
Trp Leu Gly Ile Gln Gln Trp Arg Ala 80 85 90 gct ggt gca att gac
ctt aaa tcg ctg gcc tct act caa tcg cgt cga 880 Ala Gly Ala Ile Asp
Leu Lys Ser Leu Ala Ser Thr Gln Ser Arg Arg 95 100 105 cat ttg ttc
cag cgc gca gtt ttt gtg aat ctc acc aat ccc aaa agt 928 His Leu Phe
Gln Arg Ala Val Phe Val Asn Leu Thr Asn Pro Lys Ser 110 115 120 att
gtg ttt ctg gcg gcg cta ttt ccg caa ttc atc atg ccg caa cag 976 Ile
Val Phe Leu Ala Ala Leu Phe Pro Gln Phe Ile Met Pro Gln Gln 125 130
135 140 ccg caa ctg atg cag tat atc gtg ctc ggc gtc acc act att gtg
gtc 1024 Pro Gln Leu Met Gln Tyr Ile Val Leu Gly Val Thr Thr Ile
Val Val 145 150 155 gat att att gtg atg atc ggt tac gcc acc ctt gct
caa cgg att gct 1072 Asp Ile Ile Val Met Ile Gly Tyr Ala Thr Leu
Ala Gln Arg Ile Ala 160 165 170 cta tgg att aaa gga cca aag cag atg
aag gcg ctg aat aag att ttc 1120 Leu Trp Ile Lys Gly Pro Lys Gln
Met Lys Ala Leu Asn Lys Ile Phe 175 180 185 ggc tcg ttg ttt atg ctg
gtg gga gcg ctg tta gca tcg gcg agg cat 1168 Gly Ser Leu Phe Met
Leu Val Gly Ala Leu Leu Ala Ser Ala Arg His 190 195 200 gcg
tgaaaaataa tgtcggatgc ggcgtaaac 1200 Ala 205 2 205 PRT Escherichia
coli 2 Met Thr Leu Glu Trp Trp Phe Ala Tyr Leu Leu Thr Ser Ile Ile
Leu 1 5 10 15 Thr Leu Ser Pro Gly Ser Gly Ala Ile Asn Thr Met Thr
Thr Ser Leu 20 25 30 Asn His Gly Tyr Pro Ala Gly Gly Val Tyr Cys
Trp Ala Ser Asp Arg 35 40 45 Thr Gly Asp Ser Tyr Cys Ala Gly Trp
Arg Gly Val Gly Thr Leu Phe 50 55 60 Ser Arg Ser Val Ile Ala Phe
Glu Val Leu Lys Trp Ala Gly Ala Ala 65 70 75 80 Tyr Leu Ile Trp Leu
Gly Ile Gln Gln Trp Arg Ala Ala Gly Ala Ile 85 90 95 Asp Leu Lys
Ser Leu Ala Ser Thr Gln Ser Arg Arg His Leu Phe Gln 100 105 110 Arg
Ala Val Phe Val Asn Leu Thr Asn Pro Lys Ser Ile Val Phe Leu 115 120
125 Ala Ala Leu Phe Pro Gln Phe Ile Met Pro Gln Gln Pro Gln Leu Met
130 135 140 Gln Tyr Ile Val Leu Gly Val Thr Thr Ile Val Val Asp Ile
Ile Val 145 150 155 160 Met Ile Gly Tyr Ala Thr Leu Ala Gln Arg Ile
Ala Leu Trp Ile Lys 165 170 175 Gly Pro Lys Gln Met Lys Ala Leu Asn
Lys Ile Phe Gly Ser Leu Phe 180 185 190 Met Leu Val Gly Ala Leu Leu
Ala Ser Ala Arg His Ala 195 200 205
* * * * *