U.S. patent application number 11/091889 was filed with the patent office on 2006-01-19 for dna coding for a protein which imparts l-homoserine resistance to escherichia coli bacterium, and a method for producing l-amino acids.
Invention is credited to Vladimir Venyamiovich Aleshin, Alla Valentinovna Belareva, Vitaly Arkadievich Livshits, Irina Lvovna Tokmakova, Natalia Pavlovna Zakataeva.
Application Number | 20060014258 11/091889 |
Document ID | / |
Family ID | 20211138 |
Filed Date | 2006-01-19 |
United States Patent
Application |
20060014258 |
Kind Code |
A1 |
Livshits; Vitaly Arkadievich ;
et al. |
January 19, 2006 |
DNA coding for a protein which imparts L-homoserine resistance to
Escherichia coli bacterium, and a method for producing L-amino
acids
Abstract
A bacterium which has an ability to produce an amino acid and in
which a novel rhtB gene encodes 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 cause
accumulation of the amino acid in the medium, and the amino acid is
recovered from the medium.
Inventors: |
Livshits; Vitaly Arkadievich;
(Moscow, RU) ; Zakataeva; Natalia Pavlovna;
(Moscow, RU) ; Aleshin; Vladimir Venyamiovich;
(Kaluga region, RU) ; Belareva; Alla Valentinovna;
(Moscow, RU) ; Tokmakova; Irina Lvovna; (Moscow
region, RU) |
Correspondence
Address: |
CERMAK & KENEALY LLP;ACS LLC
515 EAST BRADDOCK ROAD
SUITE B
ALEXANDRIA
VA
22314
US
|
Family ID: |
20211138 |
Appl. No.: |
11/091889 |
Filed: |
March 29, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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|
09847392 |
May 3, 2001 |
6887691 |
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11091889 |
Mar 29, 2005 |
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09396357 |
Sep 15, 1999 |
6303348 |
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09847392 |
May 3, 2001 |
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Current U.S.
Class: |
435/106 ;
435/115; 435/252.33 |
Current CPC
Class: |
C12P 13/06 20130101;
C07K 14/245 20130101; C12P 13/08 20130101; C12P 13/04 20130101 |
Class at
Publication: |
435/106 ;
435/115; 435/252.33 |
International
Class: |
C12P 13/04 20060101
C12P013/04; C12P 13/08 20060101 C12P013/08; C12N 1/21 20060101
C12N001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 13, 1998 |
RU |
98118425 |
Claims
1. A method for producing an amino acid selected from the group
consisting of L-threonine, L-homoserine, L-alanine, L-isoleucine,
and L-valine comprising: A) cultivating a bacterium which has an
ability to produce an amino acid in a medium, and b) recovering
said amino acid from the medium, wherein said bacterium belongs to
the genus Escherichia, and wherein L-homoserine resistance of said
bacterium is enhanced by amplifying a copy number of a DNA in said
bacterium, wherein said DNA is able to hybridize under stringent
conditions to nucleotides 557 to 1171 of SEQ ID NO: 1, wherein said
DNA is not less than 70% homologous to nucleotides 557 to 1171 of
SEQ ID NO: 1, and wherein said DNA encodes a protein which has an
activity of imparting L-homoserine resistance to a bacterium having
the protein.
2. The method according to claim 1, wherein said amino acid is at
least one selected from the group consisting of L-alanine,
L-isoleucine, and L-valine.
3. The method according to claim 1, wherein said DNA is carried on
a multi-copy vector.
4. The method according to claim 1, wherein said DNA is carried on
a transposon.
5. A method for producing an amino acid selected from the group
consisting of L-threonine, L-homoserine, L-alanine, L-isoleucine,
and L-valine comprising: A) cultivating a Escherichia bacterium
which has been transformed with a mutant DNA in a medium, wherein
said Escherichia bacterium has an ability to produce the amino
acid, and B) recovering the amino acid from the medium, wherein
said mutant DNA is obtainable by mutating a DNA comprising
nucleotides 557 to 1171 of SEQ ID NO: 1 and selecting a mutant DNA
which, when transferred into a bacterium increases the homoserine
resistance of the bacterium as compared to the bacterium prior to
receiving said mutant DNA, and wherein said mutant DNA is not less
than 70% homologous to nucleotides 557 to 1171 of SEQ ID NO:1.
6. The method according to claim 5, wherein said amino acid is at
least one selected from the group consisting of L-alanine,
L-isoleucine, and L-valine.
7. The method according to claim 5, wherein said DNA is carried on
a multicopy vector.
8. The method according to claim 5, wherein said DNA is carried on
a transposon.
9. The method according to claim 5, wherein said DNA is derived
from a bacterium belonging to the genus Escherichia.
10. The method according to claim 9, wherein said amino acid is at
least one selected from the group consisting of L-alanine,
L-isoleucine, and L-valine.
11. The method according to claim 9, wherein said DNA is carried on
a multicopy vector.
12. The method according to claim 9, wherein said DNA is carried on
a transposon.
13. A method for producing an amino acid selected from the group
consisting of L-threonine, L-homoserine, L-alanine, L-isoleucine,
and L-valine comprising: A) cultivating a bacterium which has an
ability to produce the amino acid in a medium, and B) recovering
the amino acid from the medium, wherein said bacterium belongs to
the genus Escherichia, wherein L-homoserine resistance of said
bacterium is enhanced by amplifying a copy number of a DNA in said
bacterium, wherein said DNA is derived from a bacterium belonging
to the genus Escherichia, wherein said DNA is able to hybridize
under stringent conditions to nucleotides 557 to 1171 of SEQ ID NO:
1, wherein said DNA is not less than 70% homologous to nucleotides
557 to 1171 of SEQ ID NO: 1, and wherein said DNA encodes a protein
which has an activity of imparting L-homoserine resistance to a
bacterium having the protein.
14. The method according to claim 13, wherein said amino acid is at
least one selected from the group consisting of L-alanine,
L-isoleucine, and L-valine.
15. The method according to claim 13, wherein said DNA is carried
on a multicopy vector.
16. The method according to claim 13, wherein said DNA is carried
on a transposon.
Description
[0001] This application claims benefit as a continuation under 35
U.S.C. .sctn.120 to U.S. patent application Ser. No. 09/847,392,
which is a divisional of 09/396,357, now U.S. Pat. No.
6,303,348.
BACKGROUND OF THE INVENTION
[0002] 1. Technical Field
[0003] The present invention relates to a method for producing an
amino acid, and especially a method for producing L-homoserine,
L-alanine, L-isoleucine, L-valine, or L-threonine using a bacterium
belonging to the genus Escherichia.
[0004] 2. Background Art
[0005] The present inventors obtained, with respect to E. coli
K-12, a mutant having a mutation thrR (hereinafter, "rhtA23") that
results 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 the rhtA23 mutation, an improved threonine-producing strain (SU
patent No. 974817), and homoserine- and glutamic acid-producing
strains (Astaurova et al., Appl. Boch. And Microbiol., 27, 556-561
(1991)) were obtained.
[0006] Furthermore, the present inventors have reported that the
rhtA gene exists at 18 min on the E. coli chromosome, and is
identical to the ORF1 between pexB and ompX genes. DNA expressing a
protein encoded by the ORF1 has been designated the rhtA (rht:
resistance to homoserine and threonine) gene. The rhtA gene
includes a 5'-noncoding region, which includes a SD sequence, an
ORF1, and a terminator. Also, the present inventors have found that
a wild-type rhtA gene imparts resistance to threonine and
homoserine if cloned in a multi-copy state, and that enhancement of
expression of the rhtA gene improves amino acid productivity of a
bacterium belonging to the genus Escherichia which has 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. Aug.
24-29, 1997, abstract No. 457).
[0007] The present inventors have found during the cloning of the
rhtA gene that at least two distinct genes which impart homoserine
resistance in a multi-copy state exist in E. coli. One is the rhtA
gene, and the other has not been previously reported.
SUMMARY OF THE INVENTION
[0008] An object of the present invention is to provide a novel
gene which imparts 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.
[0009] The inventors have found that a region at 86 min on the E.
coli chromosome, when cloned into a multi-copy vector, is able to
impart resistance to L-homoserine to E. coli, and that when the
region is amplified, the amino acid productivity of the E. coli is
improved, similar to the rhtA gene. On the basis of these findings,
the present invention has been completed.
[0010] It is an object of the present invention to provide a DNA
coding for a protein selected from the group consisting of: [0011]
(A) a protein comprising an amino acid sequence shown in SEQ ID NO:
2; and [0012] (B) a protein comprising an amino acid sequence which
includes deletion, substitution, insertion, or addition of one or
several amino acids in the amino acid sequence shown in SEQ ID NO:
2, and wherein said protein has an activity of making a bacterium
having the protein L-homoserine-resistant,
[0013] It is an object of the present invention to provide the DNA
as described above which is a DNA selected from the group
consisting of: [0014] (a) a DNA comprising a nucleotide sequence
corresponding to numbers 557 to 1171 shown in SEQ ID NO: 1; and
[0015] (b) a DNA which is able to hybridize with the nucleotide
sequence of numbers 557 to 1171 shown in SEQ ID NO: 1 under
stringent conditions, and wherein said DNA codes for a protein
having the activity of making the bacterium having the protein
L-homoserine-resistant.
[0016] It is an object of the present invention to provide a
bacterium belonging to the genus Escherichia, wherein L-homoserine
resistance of the bacterium is enhanced by amplifying a copy number
of the above-described DNA in the bacterium.
[0017] It is a further object of the present invention to provide
the bacterium as described above wherein the above-described DNA is
carried on a multicopy vector in the bacterium,
[0018] It is an object of the present invention to provide the
bacterium as described above wherein the above-described DNA is
carried on a transposon in the bacterium.
[0019] It is an object of the present invention to provide a method
for producing an amino acid, comprising cultivating the bacterium
as described above which has an ability to produce the amino acid,
in a culture medium to produce and cause accumulatation of the
amino acid in the medium, and recovering the amino acid from the
medium.
[0020] It is an object of the present invention to provide the
method as described above, 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 shows the cloning, identification, and inactivation
of the rhtB gene.
[0022] FIG. 2 shows the amino acid sequence of the RhtB
protein.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0023] The DNA of the present invention may be referred to as the
"rhtB gene." A protein encoded by the rhtB gene may be referred to
as the "RhtB protein." An activity of the RhtB protein which
imparts resistance to L-homoserine to a bacterium, i.e. an activity
of making a bacterium having the RhtB protein
L-homoserine-resistant, may be referred to as the "Rh activity." A
structural gene encoding the RhtB protein within the rhtB gene may
be referred to as the "rhtB structural gene". The phrase "enhancing
the Rh activity" means imparting resistance to homoserine to a
bacterium or enhancing the resistance by increasing the number of
RhtB protein molecules, increasing a specific activity of the RhtB
protein, or desensitizing negative regulation of the expression or
the activity of the RhtB protein, or the like. The phrase "DNA
coding for a protein" means a double-stranded DNA in which one
strand codes for the protein. "L-homoserine resistance" means that
a bacterium is able to grow on a minimal medium containing
L-homoserine at a concentration at which a wild-type strain thereof
cannot grow, usually 10 mg/ml. A bacterium having an "ability to
produce an amino acid" means that the bacterium produces and is
able to cause accumulation of larger amounts of an amino acid in a
medium than a wild-type strain thereof.
[0024] According to the present invention, resistance to a high
concentration of homoserine can be imparted to a bacterium
belonging to the genus Escherichia. A bacterium belonging to the
genus Escherichia, which has an increased resistance to homoserine
and an ability to cause accumulation of an amino acid, especially,
L-homoserine, L-alanine, L-isoleucine, L-valine, or L-threonine in
a medium with a high yield is disclosed.
[0025] The present invention will be explained in detail below.
[0026] <1> DNA of the Present Invention
[0027] The DNA of the present invention encodes a protein which has
Rh activity and an amino acid sequence of SEQ ID NO: 2.
Specifically, the DNA of the present invention may be exemplified
by a DNA comprising a nucleotide sequence of the numbers 557 to
1171 in SEQ ID NO: 1.
[0028] The DNA of the present invention includes a DNA fragment
encoding the RhtB protein which imparts resistance to homoserine to
the bacterium Escherichia coli. The DNA of the present invention
includes a DNA fragment which includes the regulatory elements of
the rhtB gene and the structural part of rhtB gene, and which has
the nucleotide sequence shown in SEQ ID NO: 1.
[0029] The nucleotide sequence shown in SEQ ID NO: 1 corresponds to
a part of a sequence complementary 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 ORF (open reading frame) located at 86 min on the E. coli
chromosome, and 5'-flanking and 3'-flanking regions thereof, but
the function of f138 is unknown. The f138, which contains only 160
nucleotides in the 5'-flanking region, cannot impart resistance to
homoserine. No termination codon is present between the 62160 and
61959 of M87049 (upstream to the ORF f138). Hence, the coding
region is 201 bp longer. Thus, the RhtB protein and the rhtB gene
are novel.
[0030] 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
using mini-Mu d5005 phagemid (Groisman, E. A., et al., J.
Bacteriol., 168, 357-364 (1986)), and isolating plasmid DNAs from
colonies grown 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 an oligonucleotide(s)
which has a sequence corresponding to the region near the portion
at 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 an upstream region from
nucleotide number 557 and a downstream region from nucleotide
number 1171 in SEQ ID NO: 1 as the primers for PCR, the entire
coding region can be amplified.
[0031] Synthesis of the oligonucleotides can be performed by an
ordinary method such as the 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). Furthermore, PCR can be performed 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.
[0032] The DNA coding for the RhtB protein of the present invention
may code for a RhtB protein which includes deletion, substitution,
insertion, or addition of one or several amino acids at one or a
plurality of positions, provided the Rh activity of RhtB protein
encoded thereby is not disrupted. The DNA, which codes for
substantially the same protein as the RhtB protein 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 is
deleted, substituted, inserted, or added. DNA modified as described
above may be obtained by conventionally known mutation treatments.
These treatments includes treating DNA coding for the RhtB protein
in vitro, for example, with hydroxylamine, and 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.
[0033] The DNA which codes for substantially the same protein as
the RhtB protein can be obtained by expressing a DNA which has been
subjected to in vitro mutation treatment as described above in a
multi-copy vector in an appropriate cell, investigating the
resistance to homoserine, and selecting the DNA which imparts an
increase in 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 numbers 557 to 1171 shown in SEQ ID NO: 1 under
stringent conditions, and which codes for a protein having Rh
activity, from a bacterium belonging to the genus Escherichia which
has been subjected to a mutation treatment, or a spontaneous mutant
or variant of a bacterium belonging to the genus Escherichia. The
term "stringent conditions" referred to herein are conditions under
which a so-called specific hybrid is formed, and a 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 those under which DNAs having high
homology, for example, DNAs having homology of not less than 70% to
each other are able to hybridize, and DNAs having homology lower
than the above to each other are not able to hybridize.
[0034] <2> Bacterium Belonging to the Genus Escherichia of
the Present Invention
[0035] The bacterium belonging the genus Escherichia of the present
invention is a bacterium belonging to the genus Escherichia having
enhanced Rh activity. A bacterium belonging to the genus
Escherichia is exemplified by Escherichia coli. The Rh activity can
be enhanced by, for example, amplifying the copy number of the rhtB
structural gene in a cell, or transforming a bacterium belonging to
the genus Escherichia with a recombinant DNA which includes the
rhtB structural gene encoding the RhtB protein ligated to a
promoter sequence which functions efficiently in a bacterium
belonging to the genus Escherichia. The Rh activity can be also
enhanced by substituting 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.
[0036] The amplification of the copy number of the rhtB structural
gene in a cell can be performed by introducing a multi-copy vector
which carries the rhtB structural gene into 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 bacterium belonging to the
genus Escherichia.
[0037] Examples of a multicopy vector include plasmid vectors such
as pBR322, pMW118, pUC19, or the like, and phage vectors such as
.lamda.1059, .lamda.BF101, M13 mp9, or the like. The transposon is
exemplified by Mu, Tn10, Tn5, or the like.
[0038] The introduction of a DNA into a bacterium belonging to the
genus Escherichia can be performed, for example, by the 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 the permeability of DNA (Mandel, M. and Higa,
A., J. Mol. Biol., 53, 159 (1970)), and the like.
[0039] If the Rh activity is enhanced in an amino acid-producing
bacterium belonging to the genus Escherichia as described above,
the amount of amino acid produced can be increased. As the
bacterium belonging to the genus Escherichia which is to have the
Rh. activity enhanced, strains which have abilities to produce
desired amino acids are used. Alternatively, 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.
[0040] L-Threonine-Producing Bacteria
[0041] 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)).
[0042] (2) L-Homoserine-Producing Bacteria
[0043] 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)) and is Leu.sup.+ revertant.
[0044] 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 useful for the production of amino acids by fermentation.
[0045] The new strains have been deposited in accordance with the
Budapest Treaty in the Russian National Collection of Industrial
Microorganisms (VKPM) on Oct. 6, 1998. The strain E. coli
NZ10/pAL4, pRhtB was deposited and given accession number VKPM
B-7658; the strain E. coli MG442/pRhtB was deposited and given
accession number of VKPM B-7659; and the strain E. coli
MG442/pVIC40,pRhtB was deposited and given accession number of VKPM
B-7660.
[0046] The strain E. coli NZ10/pAL4, pRhtB (VKPM B-7658) exhibits
the following culture, morphological, and biochemical features.
[0047] Cytomorphology: Gram-negative weakly-motile rods having
rounded ends. Longitudinal size: 1.5 to 2 .mu.m.
[0048] Culture Features:
[0049] 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.
[0050] 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.
[0051] 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 greyish-white in color, semitransparent, slightly convex, with
a lustrous surface.
[0052] Growth in a beef-extract broth--After 24-hour growth at
37.degree. C., exhibits strong uniform cloudiness, has a
characteristic odor.
[0053] Physiological and Biochemical Features:
[0054] Grows upon thrust inoculation in a beef-extract agar.
Exhibits good growth throughout the inoculated area. The
microorganism proves to be a facultative anaerobe.
[0055] It does not liquefy gelatin.
[0056] Features good growth on milk, accompanied by milk
coagulation.
[0057] Does not produce indole.
[0058] Temperature conditions--Grows on beef-extract broth at
20-42.degree. C., an optimum temperature being within 33-37.degree.
C.
[0059] pH value of culture medium--Grows on liquid media having the
pH value from 6 to 8, an optimum value being 7.2.
[0060] Carbon sources--Exhibits good growth on glucose, fructose,
lactose, mannose, galactose, xylose, glycerol, and mannitol to
produce an acid and gas.
[0061] Nitrogen sources--Assimilates nitrogen in the form of
ammonium, nitric acid salts, as well as from some organic
compounds.
[0062] Resistant to ampicillin, kanamycin and L-homoserine.
[0063] L-Threonine is used as a growth factor.
[0064] Content of plasmids--The cells contain the multi-copy hybrid
plasmid pAL4 which ensures resistance to ampicillin and carries the
thrA gene from the threonine operon, which codes for aspartate
kinase-homoserine dehydrogenase I and is responsible for increased
homoserine biosynthesis. Besides, the cells contain a multi-copy
hybrid plasmid pRhtB which ensures resistance to kanamycin and
carries the rhtB gene which confers resistance to homoserine (10
mg/l).
[0065] The strain E. coli MG442/pRhtB (VKPM B-7659) has the same
culture, morphological, and biochemical features as the strain
NZ10/pAL4, pRhtB, except that 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 multi-copy hybrid plasmid pRhtB which ensures resistance to
kanamycin and carryies the rhtB gene which confers resistance to
homoserine (10 mg/l).
[0066] The strain E. coli MG442/pVIC40,pRhtB (VKPM B-7660) has the
same culture, 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 the multi-copy
hybrid plasmid pVIC40 which ensures resistance to streptomycin and
carryies the genes of the threonine operon. Besides, they contain
multi-copy hybrid plasmid pRhtB which ensures resistance to
kanamycin and carryies the rhtB gene which confers resistance to
homoserine (10 mg/l).
[0067] <3> Method for Producing an Amino Acid
[0068] An amino acid can be efficiently produced by cultivating the
bacterium having enhanced Rh activity 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 causing
accumulation of 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.
[0069] In the method of present invention, the cultivation of the
bacterium belonging to the genus Escherichia, and the collection
and purification of an amino acid from the liquid medium may be
performed in a manner similar to conventional methods for producing
an amino acid by fermentation using a bacterium. The cultivation
medium may be either synthetic or natural, so long as the medium
includes a carbon and nitrogen source and minerals and, if
necessary, nutrients which the chosen bacterium requires for growth
in appropriate amounts. The carbon source may include various
carbohydrates such as glucose and sucrose, and various organic
acids. Depending on the assimilatory ability of the chosen
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 can be used. As minerals,
monopotassium phosphate, magnesium sulfate, sodium chloride,
ferrous sulfate, manganese sulfate, and calcium carbonate can be
used.
[0070] The cultivation is preferably performed under aerobic
conditions such as shaking, aeration, and stirring. The temperature
of the 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.
[0071] Recovering the amino acid after cultivation can be performed
by removing solids such as cells from the medium by centrifugation
or membrane filtration, and then collecting and purifying the
target amino acid by ion exchange, concentration, and crystalline
fraction methods, and the like.
EXAMPLES
[0072] The present invention will be more concretely explained
below with reference to the following non-limiting Examples. In the
Examples, an amino acid is of L-configuration unless otherwise
noted.
Example 1
Obtaining the rhtB DNA Fragment
[0073] Cloning the rhtB Gene into Mini-Mu Phagemid
[0074] 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 the
inserted fragments were analyzed by restriction mapping. It
appeared that two types of inserts belonging to different
chromosome regions had been cloned from the donor. Thus, at least
two different genes are present in multi-copy form and impart
resistance to homoserine exist in E. coli. One of the two types 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. Aug.
24-29, 1997). The other type of insert is a fragment having a
minimum length which imparts resistance to homoserine of 0.8 kb
(FIG. 1).
[0075] (2) Identification of rhtB Gene
[0076] 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 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), but the function is unknown. The
f138 having only 160 nucleotides in the 5'-flanking region was not
able to impart resistance to homoserine. No termination codon is
present upstream the ORF f138 between 62160 and 61959 nucleotides
of M87049. Furthermore, one ATG following a predicted ribosome
binding site is present in the sequence. The larger ORF (nucleotide
numbers 62160 to 61546) is designated as the 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
[0077] Strain NZ10 of E. coli was transformed with the plasmid pAL4
to obtain the strain NZ10/pAL4. This plasmid was constructed by
inserting a thrA gene, which encodes aspartokinase-homoserine
dehydrogenase I, into a pBR322 vector. 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)).
[0078] The rhtB gene was inserted into plasmid pUK21, which is a
known plasmid pUC19 having a kanamycin resistance gene substituted
for an ampicillin resistance gene (Vieira, J. and Messing, J.,
Gene, 100, 189-194 (1991)), to obtain pRhtB.
[0079] The strain NZ10/pAL4 was transformed with pUK21 or pRhtB to
obtain strains NZ 10/pAL4, pUK21, and NZ 10/pAL4, 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 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 cultivation,
the amount of homoserine which accumulates in the medium, and the
absorbance at 560 nm of the medium were determined by known
methods.
[0081] Fermentation medium composition (g/L) TABLE-US-00001 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)
[0082] The results are shown in Table 1. The strain NZ10/pAL4,
pRhtB was able to cause accumulation of a larger amount of
homoserine than the strains NZ 10/pAL4 and NZ 10/pAL4, pUK21, which
do not have an enhanced rhtB gene. TABLE-US-00002 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 Using a
RhtB-Transformed Strain
[0083] E. coli strain MG442 is a known strain (Gusyatiner, et al.,
1978, Genetika (in Russian), 14:947-956).
[0084] The strain MG442 was transformed with the plasmids pUK21 and
pRhtB to obtain strains MG442/pUK21 and MG442/pRhtB.
[0085] 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, which
contains 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 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.
[0086] The results are shown in Table 2. The strain MG442/pRhtB was
able to cause accumulation of larger amounts of alanine, valine,
and isoleucine than the strain MG442/pUK21, which does not have an
enhanced rhtB gene. TABLE-US-00003 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
[0087] The strain MG442 (Example 3) was transformed with the 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.
[0088] The strain MG442/pVIC40 was transformed with pUK21 or pRhtB
to obtain strains MG442/pVIC40, pUK21 and MG442/pVIC40, pRhtB.
[0089] 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, which contains 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
cultivation, the accumulated amount of threonine in the medium, and
an absorbance at 560 nm of the medium were determined by known
methods.
[0090] The results are shown in Table 3. The strain MG442/pVIC40,
pRhtB was able to cause accumulation of larger amounts of threonine
than the strains MG442/pVIC40 and MG442/pVIC40,pUK21, which do not
have an enhanced rhtB gene. TABLE-US-00004 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
[0091] 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 an AsuII-BsrBI fragment containing a cat (Cm.sup.R)
gene from the 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 accomplished by
phage P1 transduction and Southern hybridization (Southern, E. M.,
J. Mol. Biol., 98, 503-517, 1975).
[0092] 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 live 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 was examined after a 40-46 h cultivation.
The results are shown in Table 4. TABLE-US-00005 TABLE 4 MIC
(.mu.g/ml) Substrate N99 (rhtB.sup.+) N99/pRhtB N99 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
[0093] It follows from Table 4 that multiple copies of rhtB
imparted to 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
cells' sensitivity to homoserine and AHVA. These results in
conjunction with the data on homology of the RhtB protein to the
LysE lysine efflux transporter of Corynebacterium glutamicum
(Vrljic et al., Mol. Microbiol., 22, 815-826, 1996) indicate the
presence of functional analogues for the rhtB gene product. The
presumed efflux transporter, RhtB, has specificity to several
substrates (amino acids), or may show non-specific effects as a
result of amplification.
[0094] 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. The aforementioned documents, as well as the foreign
priority document, RU98118425, and the parent application Ser. Nos.
09/847,392 and 09/396,357 which is now U.S. Pat. No. 6,303,348, are
incorporated by reference herein in their entirety.
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
* * * * *