U.S. patent application number 15/233258 was filed with the patent office on 2016-11-24 for method for producing an l-amino acid using a bacterium of the family enterobacteriaceae having overexpressed the yajl gene.
This patent application is currently assigned to AJINOMOTO CO., INC.. The applicant listed for this patent is AJINOMOTO CO., INC.. Invention is credited to Vera Georgievna Doroshenko, Natalia Sergeevna Eremina, Valery Vasilievich Samsonov, Natalia Viktorovna Stoynova.
Application Number | 20160340681 15/233258 |
Document ID | / |
Family ID | 52633554 |
Filed Date | 2016-11-24 |
United States Patent
Application |
20160340681 |
Kind Code |
A1 |
Samsonov; Valery Vasilievich ;
et al. |
November 24, 2016 |
Method for Producing an L-Amino Acid Using a Bacterium of the
Family Enterobacteriaceae Having Overexpressed the yajL Gene
Abstract
The present invention provides a method for producing L-amino
acids or salts thereof by fermentation using a bacterium of the
family Enterobacteriaceae, particularly a bacterium belonging to
the genus Escherichia, which has been modified to overexpress the
yajL gene.
Inventors: |
Samsonov; Valery Vasilievich;
(Moscow region, RU) ; Eremina; Natalia Sergeevna;
(Moscow, RU) ; Stoynova; Natalia Viktorovna;
(Moscow, RU) ; Doroshenko; Vera Georgievna;
(Moscow, RU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AJINOMOTO CO., INC. |
Tokyo |
|
JP |
|
|
Assignee: |
AJINOMOTO CO., INC.
Tokyo
JP
|
Family ID: |
52633554 |
Appl. No.: |
15/233258 |
Filed: |
August 10, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/JP2015/054508 |
Feb 12, 2015 |
|
|
|
15233258 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 13/12 20130101;
C12P 13/222 20130101; C12P 13/227 20130101; C12P 13/04 20130101;
C12P 13/08 20130101; C12P 13/24 20130101; C12P 13/14 20130101; C12N
15/70 20130101; C12P 13/06 20130101; C12P 13/10 20130101 |
International
Class: |
C12N 15/70 20060101
C12N015/70; C12P 13/22 20060101 C12P013/22; C12P 13/24 20060101
C12P013/24; C12P 13/12 20060101 C12P013/12; C12P 13/14 20060101
C12P013/14; C12P 13/06 20060101 C12P013/06; C12P 13/08 20060101
C12P013/08; C12P 13/10 20060101 C12P013/10 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 14, 2014 |
RU |
2014105547 |
Claims
1. A method for producing an L-amino acid comprising: (i)
cultivating an L-amino acid-producing bacterium of the family
Enterobacteriaceae in a culture medium to produce said L-amino acid
in the bacterium or the culture medium, or both; and, (ii)
collecting said L-amino acid from the bacterium or the culture
medium, or both, wherein said bacterium has been modified to
overexpress the yajL gene.
2. The method according to claim 1, wherein said bacterium belongs
to the genus Escherichia.
3. The method according to claim 2, wherein said bacterium is
Escherichia coli.
4. The method according to claim 1, wherein said yajL gene is
overexpressed by increasing a copy number of the yajL gene or
modifying an expression control sequence of the yajL gene so that
the expression of said gene is enhanced as compared to a
non-modified bacterium.
5. The method according to claim 1, wherein said L-amino acid is
selected from the group consisting of L-phenylalanine,
L-tryptophan, L-tyrosine, L-alanine, L-arginine, L-asparagine,
L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid,
L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine,
L-lysine, L-methionine, L-ornithine, L-proline, L-serine,
L-threonine, and L-valine.
6. The method according to claim 5, wherein said L-amino acid is
selected from the group consisting of L-alanine, L-isoleucine,
L-leucine, L-valine, and L-phenylalanine.
7. The method according to claim 5, wherein said L-amino acid is
L-phenylalanine.
8. The method according to claim 5, wherein said L-amino acid is
L-valine.
Description
[0001] This application is a Continuation of, and claims priority
under 35 U.S.C. .sctn.120 to, International Application No.
PCT/JP2015/054508, filed Feb. 12, 2015, and claims priority
therethrough under 35 U.S.C. .sctn.119 to Russian Patent
Application No. 2014105547, filed Feb. 14, 2014, the entireties of
which are incorporated by reference herein. Also, the Sequence
Listing filed electronically herewith is hereby incorporated by
reference (File name: 2016-08-10T_US-529_Seq_List; File size: 12
KB; Date recorded: Aug. 10, 2016).
BACKGROUND OF THE INVENTION
[0002] Field of the Invention
[0003] The present invention relates to the microbiological
industry, and specifically to a method for producing L-amino acids
by fermentation of a bacterium of the family Enterobacteriaceae
which has been modified to overexpress the yajL gene.
[0004] Brief Description of the Related Art
[0005] Conventionally, L-amino acids are industrially produced by
fermentation methods utilizing strains of microorganisms obtained
from natural sources, or mutants thereof. Typically, the
microorganisms are modified to enhance production yields of L-amino
acids.
[0006] Many techniques to enhance L-amino acids production yields
have been reported, including transformation of microorganisms with
recombinant DNA (see, for example, U.S. Pat. No. 4,278,765 A) and
alteration of regulatory regions such as a promoter, leader
sequence, and/or attenuator, or others known to the person skilled
in the art (see, for example, US20060216796 A1 and WO9615246 A1).
Other techniques for enhancing production yields include increasing
the activities of enzymes involved in amino acid biosynthesis
and/or desensitizing the target enzymes to the feedback inhibition
by the resulting L-amino acid (see, for example, WO9516042 A1,
EP0685555 A1 or U.S. Pat. Nos. 4,346,170 A, 5,661,012 A, and
6,040,160 A).
[0007] Another method for enhancing L-amino acid production yields
is to attenuate expression of a gene or several genes which are
involved in degradation of the target L-amino acid, genes which
divert the precursors of the target L-amino acid from the L-amino
acid biosynthetic pathway, genes involved in the redistribution of
the carbon, nitrogen, and phosphate fluxes, and genes encoding
toxins, etc.
[0008] The yajL gene encodes the YajL protein which belongs to the
PfpI/Hsp31/DJ-1 superfamily that includes chaperones, peptidases,
and the protein DJ-1 (Messaoudi N. et al., Global stress response
in a prokaryotic model of DJ-1-associated Parkinsonism, J.
Bacteriol., 2013, 195(6):1167-1178). The YajL is the closest
prokaryotic homolog of DJ-1. For example, the Escherichia coli (E.
coli) YajL protein has 40% sequence identity and a similar
three-dimensional structure with human DJ-1, an oncogene and
neuroprotective protein whose loss-of-function mutants are
associated with certain types of familial, autosomal recessive
Parkinsonism (Wilson M. A. et al., The atomic resolution crystal
structure of the YajL (ThiJ) protein from Escherichia coli: a dose
prokaryotic homologue of the Parkinsonism-associated protein DJ-1,
J. Mol. Biol., 2005, 353(4678-691). The high homology and
similarity of crystal structures of YajL and DJ-1 suggest that the
proteins have similar function. It was found recently that YajL
protects bacteria against oxidative stress and
oxidative-stress-induced protein aggregation possibly through its
chaperone function and control of gene expression (Kthiri F. et
al., Protein aggregation in a mutant deficient in YajL, the
bacterial homolog of the Parkinsonism-associated protein DJ-1, J.
Biol. Chem., 2010, 285:10328-10336). In E. coli, YajL functions as
a covalent chaperone that, upon oxidative stress, forms mixed
disulfides with chaperones, proteases, ribosomal proteins,
catalases, peroxidases, and FeS proteins (Messaoudi N. et al., J.
Bacteriol., 2013, 195(6):1167-1178).
[0009] Until now, no data has been reported demonstrating the
effect from overexpression of the yajL gene on L-amino acid
production by the modified bacterial strains of the family
Enterobacteriaceae.
SUMMARY OF THE INVENTION
[0010] An aspect of the present invention is to provide a bacterium
belonging to the family Enterobacteriaceae, which can belong to the
genus Escherichia and, more specifically, to the species
Escherichia coli (E. coli), which has been modified to overexpress
the yajL gene.
[0011] Another aspect of the present invention is to provide a
method for producing an L-amino acid such as L-alanine, L-arginine,
L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic
acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine,
L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline,
L-serine, L-threonine, L-tryptophan, L-tyrosine or L-valine using a
bacterium of the family Enterobacteriaceae as described
hereinafter.
[0012] These aims were achieved by the unexpected finding that
overexpression of the yajL gene in a bacterium belonging to the
family Enterobacteriaceae, which can belong to the genus
Escherichia and, more specifically, to the species E. coli, confers
on the microorganism higher productivity of L-amino acids such as,
in particular, but not limited to, L-amino acids of the pyruvate
family such as L-valine, and aromatic L-amino acids such as
L-phenylalanine.
[0013] An aspect of the present invention is to provide a method
for producing an L-amino acid comprising:
[0014] (i) cultivating an L-amino acid-producing bacterium of the
family Enterobacteriaceae, in a culture medium to produce said
L-amino acid in the bacterium or the culture medium, or both;
and,
[0015] (ii) collecting said L-amino acid from the bacterium or the
culture medium, or both,
[0016] wherein the bacterium has been modified to overexpress the
yajL gene.
[0017] It is a further aspect of the present invention to provide
the method as described above, wherein the bacterium belongs to the
genus Escherichia.
[0018] It is a further aspect of the present invention to provide
the method as described above, wherein the bacterium is Escherichia
coli.
[0019] It is a further aspect of the present invention to provide
the method as described above, wherein the yajL gene is
overexpressed by increasing a copy number of the yajL gene or
modifying an expression control sequence of the yajL gene so that
the expression of the gene is enhanced as compared to a
non-modified bacterium.
[0020] It is a further aspect of the present invention to provide
the method as described above, wherein the L-amino acid is selected
from the group consisting of L-phenylalanine, L-tryptophan,
L-tyrosine, L-alanine, L-arginine, L-asparagine, L-aspartic acid,
L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine,
L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,
L-ornithine, L-proline, L-serine, L-threonine, and L-valine.
[0021] It is a further aspect of the present invention to provide
the method as described above, wherein the L-amino acid is selected
from the group consisting of L-alanine, L-isoleucine, L-leucine,
L-valine and L-phenylalanine.
[0022] It is a further aspect of the present invention to provide
the method as described above, wherein the L-amino acid is
L-phenylalanine.
[0023] It is a further aspect of the present invention to provide
the method as described above, wherein the L-amino acid is
L-valine.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0024] The present invention is described in detail below.
[0025] 1. Bacterium
[0026] The phrase "an L-amino acid-producing bacterium" can mean a
bacterium of the family Enterobacteriaceae which has an ability to
produce, excrete or secrete, and/or cause accumulation of an
L-amino acid in a culture medium or the bacterial cells when the
bacterium is cultured in the medium.
[0027] The phrase "an L-amino acid-producing bacterium" can also
mean a bacterium which is able to produce, excrete or secrete,
and/or cause accumulation of an L-amino acid in a culture medium in
an amount larger than a wild-type or parental strain, such as E.
coli K-12, and can mean that the microorganism is able to cause
accumulation in a medium of an amount not less than 0.5 g/L or not
less than 1.0 g/L of the target L-amino acid.
[0028] The phrase "L-amino acid-producing ability" can mean the
ability of the bacterium to produce, excrete or secrete, and/or
cause accumulation of the L-amino acid in a medium or the bacterial
cells to such a level that the L-amino acid can be collected from
the medium or the bacterial cells, when the bacterium is cultured
in the medium.
[0029] The phrase "L-amino acid" can mean L-alanine, L-arginine,
L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic
acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine,
L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline,
L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.
[0030] The phrase "aromatic L-amino acid" includes, for example,
L-phenylalanine, L-tryptophan, and L-tyrosine. As L-histidine has
an aromatic moiety such as an imidazole ring, the phrase "aromatic
L-amino acid" can also include, besides the aforementioned aromatic
L-amino acids, L-histidine.
[0031] The phrase "non-aromatic L-amino acid" includes, for
example, L-alanine, L-arginine, L-asparagine, L-aspartic acid,
L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine,
L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine,
L-proline, L-serine, L-threonine, and L-valine. As the biosynthetic
pathway of aromatic amino acids such as L-phenylalanine,
L-tryptophan, or L-tyrosine is different from the biosynthetic
pathway of L-histidine, the phrase "non-aromatic L-amino acid" can
also include, besides the aforementioned non-aromatic L-amino
acids, L-histidine.
[0032] An L-amino acid can belong to one or more L-amino acid
families. As an example, the L-amino acid can belong to the
glutamate family including L-arginine, L-glutamic acid,
L-glutamine, and L-proline; the serine family including L-cysteine,
glycine, and L-serine; the aspartate family including L-asparagine,
L-aspartic acid, L-isoleucine, L-lysine, L-methionine, and
L-threonine; the pyruvate family including L-alanine, L-isoleucine,
L-valine, and L-leucine; and the aromatic family including
L-phenylalanine, L-tryptophan, and L-tyrosine. As some L-amino
acids can be the intermediate amino acids in a biosynthetic pathway
of a particular L-amino acid, the aforementioned families of amino
acids may also include other L-amino acids, for example,
non-proteinogenic L-amino acids. For example, L-citrulline and
L-ornithine are amino acids from the arginine biosynthetic pathway.
Therefore, the glutamate family may include L-arginine,
L-citrulline, L-glutamic acid, L-glutamine, L-ornithine, and
L-proline
[0033] L-Arginine, L-cysteine, L-glutamic acid, L-histidine,
L-isoleucine, L-lysine, L-ornithine, L-phenylalanine, L-proline,
L-threonine, L-tryptophan, and L-valine are particular examples.
The pyruvate family amino acids such as L-alanine, L-isoleucine,
L-valine, and L-leucine are preferable examples. The aromatic amino
acids such as L-phenylalanine, L-tryptophan, and L-tyrosine are yet
preferable examples. L-valine and L-phenylalanine are more
preferable examples.
[0034] The phrase "L-amino acid" includes not only an L-amino acid
in a free form, but may also include a salt or a hydrate of the
L-amino acid, or an adduct formed by the L-amino acid and another
organic or inorganic compound.
[0035] The bacteria belonging to the family Enterobacteriaceae can
be from the genera Enterobacter, Envinia, Escherichia, Klebsiella,
Morganella, Pantoea, Photorhabdus, Providencia, Salmonella,
Yersinia, and so forth, and can have the ability to produce an
L-amino acid. Specifically, those classified into the family
Enterobacteriaceae according to the taxonomy used in the NCBI
(National Center for Biotechnology Information) database
(ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=543) can be used.
Examples of strains from the family Enterobacteriaceae which can be
modified include a bacterium of the genus Escherichia, Enterobacter
or Pantoea.
[0036] Strains of Escherichia bacterium which can be modified to
obtain Escherichia bacteria in accordance with the presently
disclosed subject matter are not particularly limited, and
specifically, those described in the work of Neidhardt et al. can
be used (Bachmann, B. J., Derivations and genotypes of some mutant
derivatives of E. coli K-12, p. 2460-2488. In F. C. Neidhardt et
al. (ed.), E. coli and Salmonella: cellular and molecular biology,
2.sup.nd ed. ASM Press, Washington, D.C., 1996). The species E.
coli is a particular example. Specific examples of E. coli include
E. coli W3110 (ATCC 27325), E. coli MG1655 (ATCC 47076), and so
forth, which are derived from the prototype wild-type strain, E.
coli K-12 strain. These strains are available from, for example,
the American Type Culture Collection (P.O. Box 1549, Manassas, Va.
20108, United States of America). That is, registration numbers are
given to each of the strains, and the strains can be ordered by
using these registration numbers (refer to atcc.org). The
registration numbers of the strains are listed in the catalogue of
the American Type Culture Collection.
[0037] Examples of the Enterobacter bacteria include Enterobacter
agglomerans, Enterobacter aerogenes, and so forth. Examples of the
Pantoea bacteria include Pantoea ananatis, and so forth. Some
strains of Enterobacter agglomerans were recently reclassified into
Pantoea agglomerans, Pantoea ananatis or Pantoea stewartii on the
basis of nucleotide sequence analysis of 16S rRNA, etc. A bacterium
belonging to any of the genus Enterobacter or Pantoea may be used
so long as it is a bacterium classified into the family
Enterobacteriaceae. When a Pantoea ananatis strain is bred by
genetic engineering techniques, Pantoea ananatis AJ13355 strain
(FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ13601 strain (FERM
BP-7207) and derivatives thereof can be used. These strains were
identified as Enterobacter agglomerans when they were isolated, and
deposited as Enterobacter agglomerans. However, they were recently
re-classified as Pantoea ananatis on the basis of nucleotide
sequencing of 16S rRNA and so forth as described above.
[0038] L-Amino Acid-Producing Bacteria
[0039] A bacterium belonging to the family Enterobacteriaceae and
modified to overexpress the yajL gene, which is able to produce
either an aromatic or a non-aromatic L-amino acid, can be used.
[0040] The bacterium may inherently have the L-amino acid-producing
ability or may be modified to have an L-amino acid-producing
ability by using a mutation method or DNA recombination techniques.
The bacterium can be obtained by overexpressing the yajL gene in a
bacterium which inherently has the ability to produce an L-amino
acid. Alternatively, the bacterium can be obtained by imparting the
ability to produce an L-amino acid to a bacterium already having
the yajL gene overexpressed.
[0041] The bacterium can produced an L-amino acid either alone or
as a mixture of two or more kinds of L-amino acids.
[0042] L-Arginine-Producing Bacteria
[0043] Examples of parental strains which can be used to derive
L-arginine-producing bacteria include, but are not limited to,
strains belonging to the genus Escherichia such as E. coli strain
237 (VKPM B-7925) (U.S. Patent Application 2002/058315 A1) and its
derivative strains harboring mutant N-acetylglutamate synthase
(RU2215783), E. coli strain 382 (VKPM B-7926) (EP1170358 A1), an
arginine-producing strain into which argA gene encoding
N-acetylglutamate synthetase is introduced therein (EP1170361 A1),
and the like.
[0044] Examples of parental strains which can be used to derive
L-arginine-producing bacteria also include strains in which
expression of one or more genes encoding an L-arginine biosynthetic
enzyme are enhanced. Examples of such genes include genes encoding
N-acetyl-.gamma.-glutamylphosphate reductase (argC), ornithine
acetyltransferase (argJ), N-acetylglutamate kinase (argB),
N-acetylornithine aminotransferase (argD), ornithine
carbamoyltransferase (argF), argininosuccinate synthase (argG),
argininosuccinate lyase (argH), and carbamoyl phosphate synthetase
(carAB).
[0045] L-Citrulline-Producing Bacteria
[0046] Examples of parental strains which can be used to derive
L-citrulline-producing bacteria include, but are not limited to,
strains belonging to the genus Escherichia such as E. coli mutant
N-acetylglutamate synthase strains 237/pMADS11, 237/pMADS12, and
237/pMADS13 (Russian patent No. 2215783, European patent No.
1170361 B1, U.S. Pat. No. 6,790,647 B2), E. coli strains 333 (VKPM
B-8084) and 374 (VKPM B-8086), both harboring mutant
feedback-resistant carbamoyl phosphate synthetase (Russian Patent
RU2264459 C2), strains E. coli, in which .alpha.-ketoglutarate
synthase activity is increased, and ferredoxin NADP.sup.+
reductase, pyruvate synthase or .alpha.-ketoglutarate dehydrogenase
activities are additionally modified (EP 2133417 A1), and strain P.
ananantis NA1sucAsdhA, in which succinate dehydrogenase and
.alpha.-ketoglutarate dehydrogenase activities are decreased (US
Patent Application No 2009286290), and the like.
[0047] As L-citrulline is an intermediate of L-arginine
biosynthetic pathway, examples of parent strains, which can be used
to derive L-citrulline-producing bacteria, include strains in which
expression of one or more genes encoding an L-arginine biosynthetic
enzyme is enhanced. Examples of such genes include, but are not
limited to, genes encoding N-acetylglutamate synthase (argA),
N-acetylglutamate kinase (argB), N-acetylglutamyl phosphate
reductase (argC), acetylornithine transaminase (argD),
acetylornithine deacetylase (argE), ornithine carbamoyltransferase
(argF/I), and carbamoyl phosphate synthetase (carAB), or
combinations thereof.
[0048] L-citrulline-producing bacterium can be also easily obtained
from any L-arginine-producing bacterium, for example E. coli 382
stain (VKPM B-7926), by inactivation of argininosuccinate synthase
encoded by argG gene.
[0049] L-Cysteine-Producing Bacteria
[0050] Examples of parental strains which can be used to derive
L-cysteine-producing bacteria include, but are not limited to,
strains belonging to the genus Escherichia such as E. coli JM15
which is transformed with different cysE alleles encoding
feedback-resistant serine acetyltransferases (U.S. Pat. No.
6,218,168, Russian patent No. 2279477), E. coli W3110 having
overexpressed genes which encode proteins suitable for secreting
substances toxic for cells (U.S. Pat. No. 5,972,663), E. coli
strains having lowered cysteine desulfohydrase activity (JP11155571
A2), E. coli W3110 with increased activity of a positive
transcriptional regulator for cysteine regulon encoded by the cysB
gene (WO0127307 A1), and the like.
[0051] L-Glutamic Acid-Producing Bacteria
[0052] Examples of parental strains which can be used to derive
L-glutamic acid-producing bacteria include, but are not limited to
strains belonging to the genus Escherichia such as E. coli
VL334thrC.sup.+ (EP 1172433). The E. coli VL334 (VKPM B-1641) is an
L-isoleucine and L-threonine auxotrophic strain having mutations in
thrC and ilvA genes (U.S. Pat. No. 4,278,765). A wild-type allele
of the thrC gene was transferred by the method of general
transduction using a bacteriophage P1 grown on the wild-type E.
coli strain K12 (VKPM B-7) cells. As a result, an L-isoleucine
auxotrophic strain VL334thrC.sup.+ (VKPM B-8961), which is able to
produce L-glutamic acid, was obtained.
[0053] Examples of parental strains which can be used to derive the
L-glutamic acid-producing bacteria include, but are not limited to
strains in which expression of one or more genes encoding an
L-glutamic acid biosynthetic enzyme are enhanced. Examples of such
genes include genes encoding glutamate dehydrogenase (gdhA),
glutamine synthetase (glnA), glutamate synthetase (gltAB),
isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB),
citrate synthase (gltA), phosphoenolpyruvate carboxylase (ppc),
pyruvate carboxylase (pyc), pyruvate dehydrogenase (aceEF, lpdA),
pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA),
enolase (eno), phosphoglyceromutase (pgmA, pgmI), phosphoglycerate
kinase (pgk), glyceraldehyde-3-phophate dehydrogenase (gapA),
triose phosphate isomerase (tpiA), fructose bisphosphate aldolase
(fbp), phosphofructokinase (pfkA, pfkB), and glucose phosphate
isomerase (pgi).
[0054] Examples of strains modified so that expression of the
citrate synthetase gene, the phosphoenolpyruvate carboxylase gene,
and/or the glutamate dehydrogenase gene is/are enhanced include
those disclosed in EP1078989 A2, EP955368 A2, and EP952221 A2.
[0055] Examples of parental strains which can be used to derive the
L-glutamic acid-producing bacteria also include strains having
decreased or eliminated activity of an enzyme that catalyzes
synthesis of a compound other than L-glutamic acid by branching off
from an L-glutamic acid biosynthesis pathway. Examples of such
enzymes include isocitrate lyase (aceA), .alpha.-ketoglutarate
dehydrogenase (sucA), phosphotransacetylase (pta), acetate kinase
(ack), acetohydroxy acid synthase (ilvG), acetolactate synthase
(ilvI), formate acetyltransferase (pfl), lactate dehydrogenase
(ldh), and glutamate decarboxylase (gadAB). Bacteria belonging to
the genus Escherichia deficient in the .alpha.-ketoglutarate
dehydrogenase activity or having a reduced .alpha.-ketoglutarate
dehydrogenase activity and methods for obtaining them are described
in U.S. Pat. Nos. 5,378,616 and 5,573,945. Specifically, these
strains include the following:
[0056] E. coli W3110sucA::Km.sup.R
[0057] E. coli AJ12624 (FERM BP-3853)
[0058] E. coli AJ12628 (FERM BP-3854)
[0059] E. coli AJ12949 (FERM BP-4881)
[0060] E. coli W3110sucA::Km.sup.R is a strain obtained by
disrupting the .alpha.-ketoglutarate dehydrogenase gene
(hereinafter referred to as "sucA gene") of E. coli W3110. This
strain is completely deficient in .alpha.-ketoglutarate
dehydrogenase.
[0061] Other examples of L-glutamic acid-producing bacterium
include those which belong to the genus Escherichia and have
resistance to an aspartic acid antimetabolite. These strains can
also be deficient in the .alpha.-ketoglutarate dehydrogenase
activity and include, for example, E. coli AJ13199 (FERM BP-5807)
(U.S. Pat. No. 5,908,768), FFRM P-12379, which additionally has a
low L-glutamic acid decomposing ability (U.S. Pat. No. 5,393,671),
AJ13138 (FERM BP-5565) (U.S. Pat. No. 6,110,714), and the like.
[0062] Examples of L-glutamic acid-producing bacteria include
mutant strains belonging to the genus Pantoea which are deficient
in the .alpha.-ketoglutarate dehydrogenase activity or have a
decreased .alpha.-ketoglutarate dehydrogenase activity, and can be
obtained as described above. Such strains include Pantoea ananatis
AJ13356. (U.S. Pat. No. 6,331,419). Pantoea ananatis AJ13356 was
deposited at the National Institute of Bioscience and
Human-Technology, Agency of Industrial Science and Technology,
Ministry of International Trade and Industry (currently,
Incorporated Administrative Agency, National Institute of
Technology and Evaluation, International Patent Organism Depositary
(NITE-IPOD), #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken
292-0818, JAPAN) on Feb. 19, 1998 under an accession number of FERM
P-16645. It was then converted to an international deposit under
the provisions of Budapest Treaty on Jan. 11, 1999 and received an
accession number of FERM BP-6615. Pantoea ananatis AJ13356 is
deficient in .alpha.-ketoglutarate dehydrogenase activity as a
result of disruption of the .alpha.KGDH-E1 subunit gene (sucA). The
above strain was identified as Enterobacter agglomerans when it was
isolated and deposited as the Enterobacter agglomerans AJ13356.
However, it was recently re-classified as Pantoea ananatis on the
basis of nucleotide sequencing of 16S rRNA and so forth. Although
AJ13356 was deposited at the aforementioned depository as
Enterobacter agglomerans, for the purposes of this specification,
they are described as Pantoea ananatis.
[0063] L-Histidine-Producing Bacteria
[0064] Examples of parental strains which can be used to derive
L-histidine-producing bacteria include, but are not limited to,
strains belonging to the genus Escherichia such as E. coli strain
24 (VKPM B-5945, RU2003677), E. coli strain 80 (VKPM B-7270,
RU2119536), E. coli NRRL B-12116-B12121 (U.S. Pat. No. 4,388,405),
E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (U.S. Pat.
No. 6,344,347), E. coli H-9341 (FERM BP-6674) (EP1085087), E. coli
AI80/pFM201 (U.S. Pat. No. 6,258,554), and the like.
[0065] Examples of parental strains which can be used to derive
L-histidine-producing bacteria also include strains in which
expression of one or more genes encoding an L-histidine
biosynthetic enzyme are enhanced. Examples of such genes include
genes encoding ATP phosphoribosyltransferase (hisG),
phosphoribosyl-AMP cyclohydrolase (hisI), phosphoribosyl-AMP
cyclohydrolase/phosphoribosyl-ATP pyrophosphatase (hisIE),
phosphoribosylformimino-5-aminoimidazole carboxamide ribotide
isomerase (hisA), amidotransferase (hisH), histidinol phosphate
aminotransferase (hisC), histidinol phosphatase (hisB), histidinol
dehydrogenase (hisD), and so forth.
[0066] It is known that the L-histidine biosynthetic enzymes
encoded by hisG and hisBHAFI are inhibited by L-histidine, and
therefore an L-histidine-producing ability can also be efficiently
enhanced by introducing a mutation conferring resistance to the
feedback inhibition into ATP phosphoribosyltransferase (Russian
Patent Nos. 2003677 and 2119536).
[0067] Specific examples of strains having an L-histidine-producing
ability include E. coli FERM-P 5038 and 5048 which have been
transformed with a vector carrying a DNA encoding an
L-histidine-biosynthetic enzyme (JP 56-005099 A), E. coli strains
transformed with rht, a gene for an amino acid-export (EP1016710
A), E. coli 80 strain imparted with sulfaguanidine,
DL-1,2,4-triazole-3-alanine, and streptomycin-resistance (VKPM
B-7270, RU2119536), and so forth.
[0068] L-Isoleucine-Producing Bacteria
[0069] Examples of parental strains which can be used to derive
L-isoleucine-producing bacteria include, but are not limited to,
mutants having resistance to 6-dimethylaminopurine (JP 5-304969 A),
mutants having resistance to an isoleucine analogue such as
thiaisoleucine and isoleucine hydroxamate, and mutants additionally
having resistance to DL-ethionine and/or arginine hydroxamate (JP
5-130882 A). In addition, recombinant strains transformed with
genes encoding proteins involved in L-isoleucine biosynthesis, such
as threonine deaminase and acetohydroxate synthase, can also be
used as parental strains (JP 2-458 A, EP0356739 A1, and U.S. Pat.
No. 5,998,178).
[0070] L-Leucine-Producing Bacteria
[0071] Examples of parental strains which can be used to derive
L-leucine-producing bacteria include, but are not limited to,
strains belonging to the genus Escherichia such as E. coli strains
resistant to leucine (for example, the strain 57 (VKPM B-7386, U.S.
Pat. No. 6,124,121)) or leucine analogs including
.beta.-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine,
5,5,5-trifluoroleucine (JP 62-34397 B and JP 8-70879 A); E. coli
strains obtained by the gene engineering method described in
WO96/06926; E. coli H-9068 (JP 8-70879 A), and the like.
[0072] The bacterium can be improved by enhancing the expression of
one or more genes involved in L-leucine biosynthesis. Examples
include genes of the leuABCD operon, which can be represented by a
mutant leuA gene encoding isopropylmalate synthase freed from
feedback inhibition by L-leucine (U.S. Pat. No. 6,403,342). In
addition, the bacterium can be improved by enhancing the expression
of one or more genes encoding proteins which excrete L-amino acid
from the bacterial cell. Examples of such genes include the b2682
and b2683 genes (ygaZH genes) (EP1239041 A2).
[0073] L-Lysine-Producing Bacteria
[0074] Examples of L-lysine-producing bacteria belonging to the
family Escherichia include mutants having resistance to an L-lysine
analogue. The L-lysine analogue inhibits growth of bacteria
belonging to the genus Escherichia, but this inhibition is fully or
partially desensitized when L-lysine is present in the medium.
Examples of the L-lysine analogue include, but are not limited to,
oxalysine, lysine hydroxamate, S-(2-aminoethyl)-L-cysteine (AEC),
.gamma.-methyllysine, .alpha.-chlorocaprolactam, and so forth.
Mutants having resistance to these lysine analogues can be obtained
by subjecting bacteria belonging to the genus Escherichia to a
conventional artificial mutagenesis treatment. Specific examples of
bacterial strains useful for producing L-lysine include E. coli
AJ11442 (FERM BP-1543, NRRL B-12185; see U.S. Pat. No. 4,346,170)
and E. coli VL611. In these microorganisms, feedback inhibition of
aspartokinase by L-lysine is desensitized.
[0075] Examples of parental strains which can be used to derive
L-lysine-producing bacteria also include strains in which
expression of one or more genes encoding an L-lysine biosynthetic
enzyme are enhanced. Examples of such genes include, but are not
limited to, genes encoding dihydrodipicolinate synthase (dapA),
aspartokinase (lysC), dihydrodipicolinate reductase (dapB),
diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase
(ddh) (U.S. Pat. No. 6,040,160), phosphoenolpyrvate carboxylase
(ppc), aspartate semialdehyde dehydrogenease (asd), and aspartase
(aspA) (EP1253195 A1). In addition, the parental strains may have
an increased level of expression of the gene involved in energy
efficiency (cyo) (EP1170376 A1), the gene encoding nicotinamide
nucleotide transhydrogenase (pntAB) (U.S. Pat. No. 5,830,716 A),
the ybjE gene (WO2005/073390), or combinations thereof.
[0076] L-Amino acid-producing bacteria may have reduced or no
activity of an enzyme that catalyzes a reaction which causes a
branching off from the L-amino acid biosynthesis pathway and
results in the production of another compound. Also, the bacteria
may have reduced or no activity of an enzyme that negatively acts
on L-amino acid synthesis or accumulation. Examples of such enzymes
involved in L-lysine production include homoserine dehydrogenase,
lysine decarboxylase (cadA, ldcC), malic enzyme, and so forth, and
strains in which activities of these enzymes are decreased or
deleted are disclosed in WO95/23864, WO96/17930, WO2005/010175, and
so forth.
[0077] Expression of both the cadA and ldcC genes encoding lysine
decarboxylase can be decreased in order to decrease or delete the
lysine decarboxylase activity. Expression of the both genes can be
decreased by, for example, the method described in
WO2006/078039.
[0078] Examples of L-lysine-producing bacteria can include the E.
coli WC196.DELTA.cadA.DELTA.ldcC/pCABD2 strain (WO2006/078039). The
strain was constructed by introducing the plasmid pCABD2 containing
lysine biosynthesis genes (U.S. Pat. No. 6,040,160) into the WC196
strain having disrupted cadA and ldcC genes which encode lysine
decarboxylase.
[0079] The WC196 strain was bred from the W3110 strain, which was
derived from E. coli K-12 by replacing the wild-type lysC gene on
the chromosome of the W3110 strain with a mutant lysC gene encoding
a mutant aspartokinase III in which threonine at position 352 was
replaced with isoleucine, resulting in desensitization of the
feedback inhibition by L-lysine (U.S. Pat. No. 5,661,012), and
conferring AEC resistance to the resulting strain (U.S. Pat. No.
5,827,698). The WC196 strain was designated E. coli AJ13069,
deposited at the National Institute of Bioscience and
Human-Technology, Agency of Industrial Science and Technology
(currently NITE-IPOD, #120, 2-5-8 Kazusakamatari, Kisarazu-shi,
Chiba-ken 292-0818, JAPAN) on Dec. 6, 1994, and assigned an
accession number of FERM P-14690. Then, it was converted to an
international deposit under the provisions of the Budapest Treaty
on Sep. 29, 1995, and assigned an accession number of FERM BP-5252
(U.S. Pat. No. 5,827,698).
[0080] The WC196.DELTA.cadA.DELTA.ldcC strain itself is also an
exemplary L-lysine-producing bacterium. The
WC196.DELTA.cadA.DELTA.ldcC was designated AJ110692 and deposited
at the National Institute of Bioscience and Human-Technology,
Agency of Industrial Science and Technology (currently (NITE-IPOD),
#120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken 292-0818,
JAPAN) on Oct. 7, 2008 as an international deposit under an
accession number of FERM BP-11027.
[0081] L-Methionine-Producing Bacteria
[0082] Examples of L-methionine-producing bacteria and parent
strains which can be used to derive L-methionine-producing bacteria
include, but are not limited to, Escherichia bacteria strains such
as strains AJ11539 (NRRL B-12399), AJ11540 (NRRL B-12400), AJ11541
(NRRL B-12401), AJ 11542 (NRRL B-12402) (patent GB2075055); strains
218 (VKPM B-8125) (patent RU2209248) and 73 (VKPM B-8126) (patent
RU2215782) resistant to norleucine, the L-methionine analog, or the
like. The strain E. coli 73 has been deposited in the Russian
National Collection of Industrial Microorganisms (VKPM) FGUP GosNII
Genetika (1 Dorozhny proezd, 1 Moscow 117545, Russian Federation)
on May 14, 2001 under accession number VKPM B-8126, and was
converted to an international deposit under the Budapest Treaty on
Feb. 1, 2002. Furthermore, a methionine repressor-deficient strain
and recombinant strains transformed with genes encoding proteins
involved in L-methionine biosynthesis such as homoserine
transsuccinylase and cystathionine .gamma.-synthase (JP 2000-139471
A) can also be used as parent strains.
[0083] L-Ornithine-Producing Bacteria
[0084] L-ornithine-producing bacterium can be easily obtained from
any L-arginine-producing bacterium, for example E. coli 382 stain
(VKPM B-7926), by inactivation of ornithine carbamoyltransferase
encoded by both argF and argI genes. Methods for inactivation of
ornithine carbamoyltransferase are described herein.
[0085] L-Phenylalanine-Producing Bacteria
[0086] Examples of parental strains which can be used to derive
L-phenylalanine-producing bacteria include, but are limited to
strains belonging to the genus Escherichia such as E. coli AJ12739
(tyrA::Tn10, tyrR) (VKPM B-8197), E. coli HW1089 (ATCC 55371)
harboring the mutant pheA34 gene (U.S. Pat. No. 5,354,672), E. coli
MWEC101-b (KR8903681), E. coli NRRL B-12141, NRRL B-12145, NRRL
B-12146 and NRRL B-12147 (U.S. Pat. No. 4,407,952). Also, E. coli
K-12 [W3110 (tyrA)/pPHAB (FERM BP-3566), E. coli K-12 [W3110
(tyrA)/pPHAD] (FERM BP-12659), E. coli K-12 [W3110 (tyrA)/pPHATerm]
(FERM BP-12662), and E. coli K-12 [W3110 (tyrA)/pBR-aroG4, pACMAB]
named as AJ 12604 (FERM BP-3579) may be used as a parental strain
(EP488424 B1). Furthermore, L-phenylalanine-producing bacteria
belonging to the genus Escherichia with an enhanced activity of the
protein encoded by the yedA gene or the yddG gene may also be used
(U.S. patent applications 2003/0148473 A1 and 2003/0157667 A1).
[0087] L-Proline-Producing Bacteria
[0088] Examples of parental strains which can be used to derive
L-proline-producing bacteria include, but are not limited to,
strains belonging to the genus Escherichia such as E. coli 702ilvA
(VKPM B-8012) which is deficient in the ilvA gene and is able to
produce L-proline (EP1172433 A1). The bacterium can be improved by
enhancing the expression of one or more genes involved in L-proline
biosynthesis. Examples of genes which can be used in
L-proline-producing bacteria include the proB gene encoding
glutamate kinase with desensitized feedback inhibition by L-proline
(DE3127361 A1). In addition, the bacterium can be improved by
enhancing the expression of one or more genes encoding proteins
excreting L-amino acid from bacterial cell. Such genes are
exemplified by b2682 and b2683 genes (ygaZH genes) (EP1239041
A2).
[0089] Examples of bacteria belonging to the genus Escherichia,
which have an activity to produce L-proline include the following
E. coli strains: NRRL B-12403 and NRRL B-12404 (GB Patent 2075056),
VKPM B-8012 (Russian patent application No. 2000124295), plasmid
mutants described in DE3127361 A1, plasmid mutants described by
Bloom F. R. et al. in "The 15.sup.th Miami winter symposium", 1983,
p. 34, and the like.
[0090] L-Threonine-Producing Bacteria
[0091] Examples of parental strains which can be used to derive
L-threonine-producing bacteria include, but are not limited to,
strains belonging to the genus Escherichia such as E. coli
TDH-6/pVIC40 (VKPM B-3996) (U.S. Pat. No. 5,175,107, U.S. Pat. No.
5,705,371), E. coli 472T23/pYN7 (ATCC 98081) (U.S. Pat. No.
5,631,157), E. coli NRRL-21593 (U.S. Pat. No. 5,939,307), E. coli
FERM BP-3756 (U.S. Pat. No. 5,474,918), E. coli FERM BP-3519 and
FERM BP-3520 (U.S. Pat. No. 5,376,538), E. coli MG442 (Gusyatiner
M. et al., Genetika (Russian), 1978, 14:947-956), E. coli VL643 and
VL2055 (EP1149911 A2), and the like.
[0092] The strain TDH-6 is deficient in the thrC gene, as well as
being sucrose-assimilative, and the ilvA gene has a leaky mutation.
This strain also has a mutation in the rhtA gene, which imparts
resistance to high concentrations of threonine or homoserine. The
strain B-3996 contains the plasmid pVIC40 which was obtained by
inserting a thrA*BC operon which includes a mutant thrA gene into a
RSF1010-derived vector. This mutant thrA gene encodes aspartokinase
homoserine dehydrogenase I which has substantially desensitized
feedback inhibition by threonine. The strain B-3996 was deposited
on Nov. 19, 1987 in the All-Union Scientific Center of Antibiotics
(Russian Federation, 117105 Moscow, Nagatinskaya Street 3-A) under
the accession number RIA 1867. The strain was also deposited in the
Russian National Collection of Industrial Microorganisms (VKPM)
FGUP GosNII Genetika (1 Dorozhny proezd, 1 Moscow 117545, Russian
Federation) on Apr. 7, 1987 under the accession number VKPM
B-3996.
[0093] E. coli VKPM B-5318 (EP0593792 A1) may also be used as a
parental strain for deriving L-threonine-producing bacteria. The
strain B-5318 is prototrophic with regard to isoleucine; and a
temperature-sensitive lambda-phage C1 repressor and PR promoter
replace the regulatory region of the threonine operon in plasmid
pVIC40. The strain VKPM B-5318 was deposited in the Russian
National Collection of Industrial Microorganisms (VKPM) FGUP GosNII
Genetika (1 Dorozhny proezd, 1 Moscow 117545, Russian Federation)
on May 3, 1990 under the accession number of VKPM B-5318.
[0094] The bacterium can be additionally modified to enhance
expression of one or more of the following genes: [0095] the mutant
thrA gene which encodes aspartokinase homoserine dehydrogenase I
resistant to feedback inhibition by threonine; [0096] the thrB gene
which encodes homoserine kinase; [0097] the thrC gene which encodes
threonine synthase; [0098] the rhtA gene which encodes a putative
transmembrane protein of the threonine and homoserine efflux
system; [0099] the asd gene which encodes
aspartate-.beta.-semialdehyde dehydrogenase; and [0100] the aspC
gene which encodes aspartate aminotransferase (aspartate
transaminase);
[0101] The thrA gene which encodes aspartokinase I and homoserine
dehydrogenase I of E. coli has been elucidated (KEGG, Kyoto
Encyclopedia of Genes and Genomes, entry No. b0002; GenBank
accession No. NC_000913.2; nucleotide positions: 337 to 2,799; Gene
ID: 945803). The thrA gene is located between the thrL and thrB
genes on the chromosome of E. coli K-12.
[0102] The thrB gene which encodes homoserine kinase of E. coli has
been elucidated (KEGG entry No. b0003; GenBank accession No.
NC_000913.2; nucleotide positions: 2,801 to 3,733; Gene ID:
947498). The thrB gene is located between the thrA and thrC genes
on the chromosome of E. coli K-12.
[0103] The thrC gene which encodes threonine synthase of E. coli
has been elucidated (KEGG entry No. b0004; GenBank accession No.
NC_000913.2; nucleotide positions: 3,734 to 5,020; Gene ID:
945198). The thrC gene is located between the thrB and yaaX genes
on the chromosome of E. coli K-12. All three genes function as a
single threonine operon thrABC. To enhance expression of the
threonine operon, the attenuator region which affects the
transcription is desirably removed from the operon (WO2005049808
A1, WO2003097839 A1).
[0104] The mutant thrA gene which encodes aspartokinase I and
homoserine dehydrogenase I resistant to feedback inhibition by
L-threonine, as well as, the thrB and thrC genes can be obtained as
one operon from the well-known plasmid pVIC40 which is present in
the L-threonine-producing E. coli strain VKPM B-3996. Plasmid
pVIC40 is described in detail in U.S. Pat. No. 5,705,371.
[0105] The rhtA gene which encodes a protein of the threonine and
homoserine efflux system (an inner membrane transporter) of E. coli
has been elucidated (KEGG entry No. b0813; GenBank accession No.
NC_000913.2; nucleotide positions: 848,433 to 849,320, complement;
Gene ID: 947045). The rhtA gene is located between the dps and ompX
genes on the chromosome of E. coli K-12 close to the glnHPQ operon,
which encodes components of the glutamine transport system. The
rhtA gene is identical to the ybiF gene (KEGG entry No. B0813).
[0106] The asd gene which encodes aspartate-.beta.-semialdehyde
dehydrogenase of E. coli has been elucidated (KEGG entry No. b3433;
GenBank accession No. NC_000913.2; nucleotide positions: 3,571,798
to 3,572,901, complement; Gene ID: 947939). The asd gene is located
between the glgB and gntU gene on the same strand (yhgN gene on the
opposite strand) on the chromosome of E. coli K-12.
[0107] Also, the aspC gene which encodes aspartate aminotransferase
of E. coli has been elucidated (KEGG entry No. b0928; GenBank
accession No. NC_000913.2; nucleotide positions: 983,742 to
984,932, complement; Gene ID: 945553). The aspC gene is located
between the ycbL gene on the opposite strand and the ompF gene on
the same strand on the chromosome of E. coli K-12.
[0108] L-Tryptophan-Producing Bacteria
[0109] Examples of parental strains which can be used to derive the
L-tryptophan-producing bacteria include, but are not limited to,
strains belonging to the genus Escherichia such as E. coli
JP4735/pMU3028 (DSM10122) and JP6015/pMU91 (DSM10123) deficient in
the tryptophanyl-tRNA synthetase encoded by mutant trpS gene (U.S.
Pat. No. 5,756,345), E. coli SV164 (pGH5) having a serA allele
encoding phosphoglycerate dehydrogenase free from feedback
inhibition by serine and a trpE allele encoding anthranilate
synthase free from feedback inhibition by tryptophan (U.S. Pat. No.
6,180,373), E. coli AGX17 (pGX44) (NRRL B-12263) and
AGX6(pGX50)aroP (NRRL B-12264) deficient in the enzyme
tryptophanase (U.S. Pat. No. 4,371,614), E. coli AGX17/pGX50,
pACKG4-pps in which a phosphoenolpyruvate-producing ability is
enhanced (WO9708333, U.S. Pat. No. 6,319,696), and the like may be
used. L-tryptophan-producing bacteria belonging to the genus
Escherichia with an enhanced activity of the identified protein
encoded by and the yedA gene or the yddG gene may also be used
(U.S. patent applications 2003/0148473 A1 and 2003/0157667 A1).
[0110] Examples of parental strains which can be used to derive the
L-tryptophan-producing bacteria also include strains in which one
or more activities of the enzymes selected from anthranilate
synthase, phosphoglycerate dehydrogenase, and tryptophan synthase
are enhanced. The anthranilate synthase and phosphoglycerate
dehydrogenase are both subject to feedback inhibition by
L-tryptophan and L-serine, so that a mutation desensitizing the
feedback inhibition may be introduced into these enzymes. Specific
examples of strains having such a mutation include an E. coli SV164
which harbors desensitized anthranilate synthase and a transformant
strain obtained by introducing into the E. coli SV164 the plasmid
pGH5 (WO 94/08031), which contains a mutant serA gene encoding
feedback-desensitized phosphoglycerate dehydrogenase.
[0111] Examples of parental strains which can be used to derive the
L-tryptophan-producing bacteria also include strains into which the
tryptophan operon which contains a gene encoding desensitized
anthranilate synthase has been introduced (JP 57-71397 A, JP
62-244382 A, U.S. Pat. No. 4,371,614). Moreover,
L-tryptophan-producing ability may be imparted by enhancing
expression of a gene which encodes tryptophan synthase, among
tryptophan operons (trpBA). The tryptophan synthase consists of
.alpha. and .beta. subunits which are encoded by the trpA and trpB
genes, respectively. In addition, L-tryptophan-producing ability
may be improved by enhancing expression of the isocitrate
lyase-malate synthase operon (WO2005/103275).
[0112] L-Valine-Producing Bacteria
[0113] Examples of parental strains which can be used to derive
L-valine-producing bacteria include, but are not limited to,
strains which have been modified to overexpress the ilvGMEDA operon
(U.S. Pat. No. 5,998,178). It is desirable to remove the region of
the ilvGMEDA operon which is required for attenuation so that
expression of the operon is not attenuated by the L-valine that is
produced. Furthermore, the ilvA gene in the operon is desirably
disrupted so that threonine deaminase activity is decreased.
[0114] Examples of parental strains for deriving L-valine-producing
bacteria also include mutants having a mutation of aminoacyl-tRNA
synthetase (U.S. Pat. No. 5,658,766). For example, E. coli VL1970,
which has a mutation in the ileS gene encoding isoleucine tRNA
synthetase, can be used. E. coli VL1970 was deposited in the
Russian National Collection of Industrial Microorganisms (VKPM)
FGUP GosNII Genetika (1 Dorozhny proezd, 1 Moscow 117545, Russian
Federation) on Jun. 24, 1988 under the accession number VKPM
B-4411.
[0115] Furthermore, mutants requiring lipoic acid for growth and/or
lacking H.sup.+-ATPase can also be used as parental strains
(WO96/06926).
[0116] Parent strains for deriving L-valine-producing bacteria also
include E. coli H81 strain (VKPM B-8066; see, for example,
EP1942183 B1), NRRL B-12287 and NRRL B-12288 (U.S. Pat. No.
4,391,907), VKPM B-4411 (U.S. Pat. No. 5,658,766), VKPM B-7707
(European patent application EP1016710 A2) or the like.
[0117] The bacterium of the present invention belonging to the
family Enterobacteriaceae has been modified to overexpress the yajL
gene.
[0118] The phrase "a bacterium modified to overexpress the yajL
gene" can mean that the bacterium has been modified in such a way
that in the modified bacterium the total enzymatic activity of the
corresponding gene protein product such as YajL is increased as
compared with, or the expression level of the yajL gene is higher
than that level in, a non-modified strain, for example, a wild-type
or parental strain as described above and hereinafter. Examples of
a non-modified strain serving as a reference for the above
comparison can include a wild-type strain of a microorganism
belonging to the genus Escherichia such as the E. coli MG1655
strain (ATCC 47076), W3110 strain (ATCC 27325), and so forth.
[0119] The phrase "the yajL gene is overexpressed" can mean that
the total enzymatic activity of the corresponding gene protein
product such as the YajL protein is increased by, for example,
introducing and/or increasing the copy number of the yajL gene in
bacterial genome, or increasing the activity per molecule (may be
referred to as a specific activity) of the protein encoded by said
gene, as compared with a non-modified strain. The bacterium can be
modified so that the activity of the YajL protein per cell is
increased to 150% or more, 200% or more, 300% or more, of the
activity of a non-modified strain. The chaperone activity of YajL
can be used to determine a specific enzymatic activity of the
protein per mg. For example, citrate synthase can be used as a
control protein to measure refolding activity of YajL under
reducing or oxidizing conditions (Kthiri F. et al., J. Biol. Chem.,
2010, 285:10328-10336). The protein concentration can be determined
by the Bradford protein assay (Bradford M. M., Anal. Biochem.,
1976, 72:248-254) using bovine serum albumin as a standard.
[0120] The phrase "the yajL gene is overexpressed" can also mean
that the expression level of the yajL gene is higher than that
level in a non-modified strain. Therefore, the phrase "the yajL
gene is overexpressed" is equivalent to the phrase "expression of
the yajL gene is enhanced".
[0121] Methods which can be used to enhance expression of the yajL
gene include, but are not limited to, increasing the yajL gene copy
number in bacterial genome (in the chromosome and/or in the
autonomously replicable plasmid) and/or introducing the yajL gene
into a vector that is able to increase the copy number and/or the
expression level of the yajL gene in a bacterium of the family
Enterobacteriaceae according to genetic engineering methods known
to the one skilled in the art.
[0122] Examples of the vectors include, but are not limited to,
broad-host-range plasmids such as pMW118/119, pBR322, pUC19, and
the like. Multiple copies of the yajL gene can also be introduced
into the chromosomal DNA of a bacterium by, for example, homologous
recombination, Mu-driven integration, or the like. Homologous
recombination can be carried out using sequence with multiple
copies in the chromosomal DNA. Sequences with multiple copies in
the chromosomal DNA include, but are not limited to, repetitive DNA
or inverted repeats present at the end of a transposable element.
In addition, it is possible to incorporate the yajL gene into a
transposon and allow it to be transferred to introduce multiple
copies of the yajL gene into the chromosomal DNA. By using
Mu-driven integration, more than 3 copies of the gene can be
introduced into the chromosomal DNA during a single act (Akhverdyan
V. Z. et al., Biotechnol. (Russian), 2007, 3:3-20).
[0123] Enhancing of the yajL gene expression can also be achieved
by increasing the expression level of the yajL gene by modification
of adjacent regulatory regions of the yajL gene or introducing
native and/or modified foreign regulatory regions. Regulatory
regions or sequences can be exemplified by promoters, enhancers,
attenuators and termination signals, anti-termination signals,
ribosome-binding sites (RBS) and other expression control elements
(e.g., regions to which repressors or inducers bind and/or binding
sites for transcriptional and translational regulatory proteins,
for example, in the transcribed mRNA). Such regulatory regions are
described, for example, in Sambrook J., Fritsch E. F. and Maniatis
T., "Molecular Cloning: A Laboratory Manual", 2.sup.nd ed., Cold
Spring Harbor Laboratory Press (1989). Modifications of regions
controlling gene(s) expression can be combined with increasing the
copy number of the modified gene(s) in bacterial genome using the
known methods (see, for example, Akhverdyan V. Z. et al., Appl.
Microbiol. Biotechnol., 2011, 91:857-871; Tyo K. E. J. et al.,
Nature Biotechnol., 2009, 27:760-765).
[0124] The exemplary promoters enhancing the yajL gene expression
can be the potent promoters that are stronger than the native yajL
promoter. For example, the lac promoter, the trp promoter, the trc
promoter, the tac promoter, the PR or the PL promoters of lambda
phage are all known to be potent promoters. Potent promoters
providing a high level of gene expression in a bacterium belonging
to the family Enterobacteriaceae can be used. Alternatively, the
effect of a promoter can be enhanced by, for example, introducing a
mutation into the promoter region of the yajL gene to obtain a
stronger promoter function, thus resulting in the increased
transcription level of the yajL gene located downstream of the
promoter. Furthermore, it is known that substitution of several
nucleotides in the Shine-Dalgarno (SD) sequence, and/or in the
spacer between the SD sequence and the start codon, and/or a
sequence immediately upstream and/or downstream from the start
codon in the ribosome-binding site greatly affects the translation
efficiency of mRNA. For example, a 20-fold range in the expression
levels was found, depending on the nature of the three nucleotides
preceding the start codon (Gold L. et al., Annu. Rev. Microbiol.,
1981, 35:365-403; Hui A. et al., EMBO J., 1984, 3:623-629).
[0125] The copy number, presence or absence of the gene can be
measured, for example, by restricting the chromosomal DNA followed
by Southern blotting using a probe based on the gene sequence,
fluorescence in situ hybridization (FISH), and the like. The level
of gene expression can be determined by measuring the amount of
mRNA transcribed from the gene using various well-known methods,
including Northern blotting, quantitative RT-PCR, and the like. The
amount of the protein encoded by the gene can be measured by known
methods including SDS-PAGE followed by immunoblotting assay
(Western blotting analysis), or mass spectrometry analysis of the
protein samples, and the like.
[0126] Methods for manipulation with recombinant molecules of DNA
and molecular cloning such as preparation of plasmid DNA,
digestion, ligation and transformation of DNA, selection of an
oligonucleotide as a primer, incorporation of mutations, and the
like may be ordinary methods well-known to the person skilled in
the art. These methods are described, for example, in Sambrook J.,
Fritsch E. F. and Maniatis T., "Molecular Cloning: A Laboratory
Manual", 2.sup.nd ed., Cold Spring Harbor Laboratory Press (1989)
or Green M. R. and Sambrook J. R., "Molecular Cloning: A Laboratory
Manual", 4.sup.th ed., Cold Spring Harbor Laboratory Press (2012);
Bernard R. Glick, Jack J. Pasternak and Cheryl L. Patten,
"Molecular Biotechnology: principles and applications of
recombinant DNA", 4.sup.th ed., Washington, D.C., ASM Press
(2009).
[0127] The yajL gene encodes the oxidative-stress-resistance
chaperone YajL (KEGG, Kyoto Encyclopedia of Genes and Genomes,
entry No. b0424; Protein Knowledgebase, UniProtKB/Swiss-Prot,
accession No. Q46948). The yajL gene (GenBank accession No.
NC_000913.2; nucleotide positions: 442275 to 442865, complement;
Gene ID: 945066) is located between the panE gene on the same
strand and the thiI gene on the opposite strand on the chromosome
of E. coli strain K-12. The nucleotide sequence of the yajL gene
and the amino acid sequence of the YajL protein encoded by the yajL
gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. In
some microorganisms, the YajL protein has been mistakenly described
as ThiJ involved in thiamine metabolism (EcoGene: EG13272;
ecogene.org/old/geneinfo.php?eg_id=EG13272). This phenotype is now
attributed to the adjacent thiI gene (T. Begley, personal
communication cited in Mueller E. G. et al., Identification of a
gene involved in the generation of 4-thiouridine in tRNA, Nucleic
Acids Res., 1998, 26(11):2606-2610).
[0128] Since there may be some differences in DNA sequences between
the genera, species or strains of the family Enterobacteriaceae,
the yajL gene is not limited to the gene shown in SEQ ID NO: 1, but
may include genes which are variant nucleotide sequences of or
homologous to SEQ ID NO: 1, and which encode variants of the YajL
protein.
[0129] The phrase "a variant protein" can mean a protein which has
one or several changes in the sequence compared with SEQ ID NO: 2,
whether they are substitutions, deletions, insertions, and/or
additions of one or several amino acid residues, but still
maintains an activity or function similar to that of the YajL
protein, or the three-dimensional structure of the YajL protein is
not significantly changed relative to the wild-type or non-modified
protein. The number of changes in the variant protein depends on
the position in the three-dimensional structure of the protein or
the type of amino acid residues. It can be, but is not strictly
limited to, 1 to 30, in another example 1 to 15, in another example
1 to 10, and in another example 1 to 5, in SEQ ID NO: 2. This is
because some amino acids have high homology to one another so that
the activity or function is not affected by such a change, or the
three-dimensional structure of YajL is not significantly changed
relative to the wild-type or non-modified protein. Therefore, the
protein variants encoded by the yajL gene may have a homology,
defined as the parameter "identity" when using the computer program
BLAST, of not less than 70%, not less than 80%, not less than 90%,
not less than 95%, not less than 98%, or not less than 99% with
respect to the entire amino acid sequence shown in SEQ ID NO: 2, as
long as the activity or function of the YajL protein is maintained,
or the three-dimensional structure of YajL is not significantly
changed relative to the wild-type or non-modified protein.
[0130] The exemplary substitution, deletion, insertion, and/or
addition of one or several amino acid residues can be a
conservative mutation(s). The representative conservative mutation
is a conservative substitution. The conservative substitution can
be, but is not limited to, a substitution, wherein substitution
takes place mutually among Phe, Trp and Tyr, if the substitution
site is an aromatic amino acid; among Ala, Leu, Ile and Val, if the
substitution site is a hydrophobic amino acid; between Glu, Asp,
Gln, Asn, Ser, His and Thr, if the substitution site is a
hydrophilic amino acid; between Gln and Asn, if the substitution
site is a polar amino acid; among Lys, Arg and His, if the
substitution site is a basic amino acid; between Asp and Glu, if
the substitution site is an acidic amino acid; and between Ser and
Thr, if the substitution site is an amino acid having hydroxyl
group. Examples of conservative substitutions include substitution
of Ser or Thr for Ala, substitution of Gln, His or Lys for Arg,
substitution of Glu, Gln, Lys, His or Asp for Asn, substitution
Asn, Glu or Gln for Asp, substitution of Ser or Ala for Cys,
substitution Asn, Glu, Lys, His, Asp or Arg for Gln, substitution
Asn, Gln, Lys or Asp for Glu, substitution of Pro for Gly,
substitution Asn, Lys, Gln, Arg or Tyr for His, substitution of
Leu, Met, Val or Phe for Ile, substitution of Ile, Met, Val or Phe
for Leu, substitution Asn, Glu, Gln, His or Arg for Lys,
substitution of Ile, Leu, Val or Phe for Met, substitution of Trp,
Tyr, Met, Ile or Leu for Phe, substitution of Thr or Ala for Ser,
substitution of Ser or Ala for Thr, substitution of Phe or Tyr for
Trp, substitution of His, Phe or Trp for Tyr, and substitution of
Met, Ile or Leu for Val.
[0131] The exemplary substitution, deletion, insertion, and/or
addition of one or several amino acid residues can also be a
non-conservative mutation(s) provided that the mutation(s) is/are
compensated by one or more secondary mutation(s) in the different
position(s) of amino acids sequence so that the activity or
function of the variant protein is maintained and similar to that
of the YajL protein, or the three-dimensional structure of YajL is
not significantly changed relative to the wild-type or non-modified
protein.
[0132] To evaluate the degree of protein or DNA homology, several
calculation methods can be used, such as BLAST search, FASTA search
and ClustalW method. The BLAST (Basic Local Alignment Search Tool,
ncbi.nlm.nih.gov/BLAST/) search is the heuristic search algorithm
employed by the programs blastp, blastn, blastx, megablast,
tblastn, and tblastx; these programs ascribe significance to their
findings using the statistical methods of Samuel K. and Altschul S.
F. ("Methods for assessing the statistical significance of
molecular sequence features by using general scoring schemes" Proc.
Natl. Acad. Sci. USA, 1990, 87:2264-2268; "Applications and
statistics for multiple high-scoring segments in molecular
sequences". Proc. Natl. Acad. Sci. USA, 1993, 90:5873-5877). The
computer program BLAST calculates three parameters: score, identity
and similarity. The FASTA search method is described by Pearson W.
R. ("Rapid and sensitive sequence comparison with FASTP and FASTA",
Methods Enzymol., 1990, 183:63-98). The ClustalW method is
described by Thompson J. D. et al. ("CLUSTAL W: improving the
sensitivity of progressive multiple sequence alignment through
sequence weighting, position-specific gap penalties and weight
matrix choice", Nucleic Acids Res., 1994, 22:4673-4680).
[0133] Moreover, the yajL gene can be a variant nucleotide
sequence. The phrase "a variant nucleotide sequence" can mean a
nucleotide sequence which encodes "a variant protein" using any
synonymous amino acid codons according to the standard genetic code
table (see, e.g., Lewin B., "Genes VIII", 2004, Pearson Education,
Inc., Upper Saddle River, N.J. 07458). Therefore, the yajL gene can
be a variant nucleotide sequence due to degeneracy of genetic
code.
[0134] The phrase "a variant nucleotide sequence" can also mean,
but is not limited to, a nucleotide sequence which hybridizes under
stringent conditions with the nucleotide sequence complementary to
the sequence shown in SEQ ID NO: 1, or a probe which can be
prepared from the nucleotide sequence under stringent conditions
provided that it encodes active or functional protein. "Stringent
conditions" include those under which a specific hybrid, for
example, a hybrid having homology, defined as the parameter
"identity" when using the computer program BLAST, of not less than
60%, not less than 70%, not less than 80%, not less than 90%, not
less than 95%, not less than 96%, not less than 97%, not less than
98%, or not less than 99% is formed, and a non-specific hybrid, for
example, a hybrid having homology lower than the above is not
formed. For example, stringent conditions can be exemplified by
washing one time or more, or in another example, two or three
times, at a salt concentration of 1.times.SSC (standard sodium
citrate or standard sodium chloride), 0.1% SDS (sodium dodecyl
sulphate), or in another example, 0.1.times.SSC, 0.1% SDS at
60.degree. C. or 65.degree. C. Duration of washing depends on the
type of membrane used for blotting and, as a rule, should be what
is recommended by the manufacturer. For example, the recommended
duration of washing for the Amersham Hybond.TM.-N+ positively
charged nylon membrane (GE Healthcare) under stringent conditions
is 15 minutes. The washing step can be performed 2 to 3 times. As
the probe, a part of the sequence complementary to the sequences
shown in SEQ ID NO: 1 may also be used. Such a probe can be
produced by PCR using oligonucleotides as primers prepared on the
basis of the sequence shown in SEQ ID NO: 1 and a DNA fragment
containing the nucleotide sequence as a template. The length of the
probe is recommended to be >50 bp; it can be suitably selected
depending on the hybridization conditions, and is usually 100 bp to
1 kbp. For example, when a DNA fragment having a length of about
300 bp is used as the probe, the washing conditions after
hybridization can be exemplified by 2.times.SSC, 0.1% SDS at
50.degree. C., 60.degree. C. or 65.degree. C.
[0135] As the gene encoding the YajL protein of the species E. coli
has already been elucidated (see above), the yajL gene encoding the
YajL protein, and the variant nucleotide sequences encoding variant
proteins of YajL protein can be obtained by PCR (polymerase chain
reaction; refer to White T. J. et al., The polymerase chain
reaction, Trends Genet., 1989, 5:185-189) from a bacterium
belonging to the family Enterobacteriaceae utilizing primers
prepared based on the nucleotide sequence of the yajL gene; or the
site-directed mutagenesis method by treating a DNA containing the
wild-type yajL gene in vitro, for example, with hydroxylamine, or a
method for treating a microorganism, for example, a bacterium
belonging to the family Enterobacteriaceae harboring the wild-type
yajL gene with ultraviolet (UV) irradiation or a mutating agent
such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and nitrous acid
usually used for the such treatment; or chemically synthesized as
full-length gene structure. Genes encoding the YajL protein or its
variant proteins of other microorganisms of the family
Enterobacteriaceae can be obtained in a similar manner.
[0136] The phrase "a wild-type protein" can mean a native protein
naturally produced by a wild-type or parent bacterial strain of the
family Enterobacteriaceae, for example, by the wild-type E. coli
MG1655 strain. A wild-type protein can be encoded by the "wild-type
gene", or the "non-modified gene" naturally occurring in genome of
a wild-type bacterium.
[0137] The bacterium as described herein can be obtained by
modifying a bacterium inherently having an ability to produce an
L-amino acid to overexpress the yajL gene, for example, by
introducing the aforementioned DNAs into the bacterium.
Alternatively, the bacterium as described herein can be obtained by
imparting the ability to produce an L-amino acid to a bacterium
already modified to overexpress the yajL gene.
[0138] The bacterium can have, in addition to the properties
already mentioned, other specific properties such as various
nutrient requirements, drug resistance, drug sensitivity, and drug
dependence, without departing from the scope of the present
invention.
[0139] 2. Method
[0140] A method of the present invention includes the method for
producing an L-amino acid such as L-alanine, L-arginine,
L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic
acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine,
L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline,
L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, or a
mixture thereof. The method for producing an L-amino acid can
include the steps of cultivating the bacterium in a culture medium
to allow the L-amino acid to be produced, excreted, and/or
accumulated in the culture medium or in the bacterial cells, and
collecting the L-amino acid from the culture medium and/or the
bacterial cells. The L-amino acid can be produced in a salt or a
hydrate form thereof, or a combination thereof. For example,
sodium, potassium, ammonium, and the like salts of the L-amino acid
can be produced by the method. The L-amino acid can be produced in
an adduct form thereof with, for example, another organic or
inorganic compound. Specifically, a monochlorhydrate salt of an
L-amino acid can be produced by the method such as monochlorhydrate
salt of L-lysine.
[0141] The cultivation of the bacterium, and collection and
purification of L-amino acid from the medium and the like may be
performed in a manner similar to conventional fermentation methods
wherein L-amino acid is produced using a microorganism. The culture
medium for production of the L-amino acid can be either a synthetic
or natural medium such as a typical medium that contains a carbon
source, a nitrogen source, a sulphur source, inorganic ions, and
other organic and inorganic components as required. As the carbon
source, saccharides such as glucose, sucrose, lactose, galactose,
fructose, arabinose, maltose, xylose, trehalose, ribose, and
hydrolyzates of starches; alcohols such as ethanol, glycerol,
mannitol, and sorbitol; organic acids such as gluconic acid,
fumaric acid, citric acid, malic acid, and succinic acid; fatty
acid, and the like can be used. As the nitrogen source, inorganic
ammonium salts such as ammonium sulfate, ammonium chloride, and
ammonium phosphate; organic nitrogen such as of soy bean
hydrolyzates; ammonia gas; aqueous ammonia; and the like can be
used. The sulphur source can include ammonium sulphate, magnesium
sulphate, ferrous sulphate, manganese sulphate, and the like.
Vitamins such as vitamin B1, required substances, for example,
organic nutrients such as nucleic acids such as adenine and RNA, or
yeast extract, and the like may be present in appropriate, even if
trace, amounts. Other than these, small amounts of calcium
phosphate, iron ions, manganese ions, and the like may be added, if
necessary.
[0142] Cultivation can be performed under aerobic conditions for 16
to 72 h, or for 32 to 68 h; the culture temperature during
cultivation can be controlled within 30 to 45.degree. C., or within
30 to 37.degree. C.; and the pH can be adjusted between 5 and 8, or
between 6 and 7.5. The pH can be adjusted by using an inorganic or
organic acidic or alkaline substance, as well as ammonia gas.
[0143] After cultivation, solids such as cells and cell debris can
be removed from the liquid medium by centrifugation or membrane
filtration, and then the target L-amino acid can be recovered from
the fermentation liquor by any combination of conventional
techniques such as concentration, ion-exchange chromatography, and
crystallization.
EXAMPLES
[0144] The present invention will be more precisely explained below
with reference to the following non-limiting Examples.
Example 1
Construction of the E. coli L-Valine-Producing Strain Modified to
Overexpress the yajL Gene
1.1 Construction of the E. coli MG1655 Strain Having Modified a
Regulatory Region of yajL
[0145] Expression of the yajL gene in E. coli was changed using the
method developed by Datsenko K. A. and Wanner B. L. called
".lamda.Red/ET-mediated integration" (Datsenko K. A. and Wanner B.
L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645). According
to this procedure, the PCR primers P1 (SEQ ID NO: 3) and P2 (SEQ ID
NO: 4), which are homologous to both regions adjacent to the yajL
gene and the gene conferring chloramphenicol resistance (Cm.sup.R)
and the regions adjacent to the promoter in the template
chromosome, were constructed. The chromosome of the
chloramphenicol-resistant E. coli strain MG1655 which contains a
tac-like promoter having the structure of the -35 region as TGGCAA
(from 5'-end to 3'-end) upstream lacZ (refer to Table 1, clone 7 in
Katashkina J. L. et al., Tuning the expression level of a gene
located on a bacterial chromosome, Mol. Biol. (Mosk., in Russian),
2005, 39(5):719-726) was used as the template in PCR reaction.
Conditions for PCR were as follows: denaturation for 3 min at
95.degree. C.; profile for 35 cycles: 1 min at 95.degree. C., 1 min
at 58.degree. C., 1 min at 72.degree. C.; final elongation for 5
min at 72.degree. C. The obtained DNA-fragment 1 (1,768 bp) (SEQ ID
NO: 5) was purified in an agarose gel and used for electroporation
of the strain E. coli MG1655 (ATCC 47076) containing the plasmid
pKD46 with a temperature-sensitive replication origin. The plasmid
pKD46 (Datsenko K. A. and Wanner B. L., Proc. Natl. Acad. Sci. USA,
2000, 97(12):6640-6645) includes a 2,154 nt (31088-33241)
DNA-fragment of phage .lamda. (GenBank accession No. J02459) and
contains genes of the .lamda.Red homologous recombination system
(.gamma., .beta., exo genes) under the control of
arabinose-inducible P.sub.araB promoter. The plasmid pKD46 is
necessary to integrate the DNA-fragment into the chromosome of
strain E. coli MG1655.
[0146] Electrocompetent cells were prepared as follows: E. coli
MG1655 cells were grown overnight at 30.degree. C. in LB-medium
(Sambrook, J. and Russell, D. W. "Molecular Cloning: A Laboratory
Manual", 3.sup.rd ed., Cold Spring Harbor Laboratory Press (2001))
containing ampicillin (100 mg/L), and the culture was diluted 100
times with 5 mL of SOB-medium (Sambrook J., Fritsch E. F. and
Maniatis T., "Molecular Cloning: A Laboratory Manual", 2.sup.nd
ed., Cold Spring Harbor Laboratory Press (1989)) containing
ampicillin (100 mg/L) and L-arabinose (1 mM). The obtained culture
was grown with aeration (250 rpm) at 30.degree. C. to an OD.sub.600
of .about.0.6 and then made electrocompetent by concentrating
100-fold and washing three times with ice-cold deionized H.sub.2O.
Electroporation was performed using 70 .mu.L of cells and
.about.100 ng of the DNA-fragment 1. Then, cells were incubated
with 1 mL of SOC-medium (Sambrook J., Fritsch E. F. and Maniatis
T., "Molecular Cloning: A Laboratory Manual", 2.sup.nd ed., Cold
Spring Harbor Laboratory Press (1989)) at 37.degree. C. for 2.5 h,
placed onto the plates containing LB-medium (Sambrook, J. and
Russell, D. W. "Molecular Cloning: A Laboratory Manual", 3.sup.rd
ed., Cold Spring Harbor Laboratory Press (2001)), agar (1.5%) and
chloramphenicol (20 mg/L), and grown at 37.degree. C. to select
Cm.sup.R-recombinants. To eliminate the pKD46 plasmid, 1 passage on
L-agar with chloramphenicol (20 mg/L) at 42.degree. C. were
performed, and the obtained colonies were tested for sensitivity to
ampicillin. Thus the strain E. coli MG1655 P.sub.tac7-yajL was
obtained.
1.2 Verification of a Modification of the Regulatory Region of
yajL
[0147] Mutants containing the replacement of a promoter region of
the yajL gene marked with Cm.sup.R-gene (cat) were verified by PCR
using locus-specific primers P3 (SEQ ID NO: 6) and P4 (SEQ ID NO:
7). Conditions for PCR were as follows: denaturation for 3 min at
94.degree. C.; profile for 30 cycles: 30 sec at 94.degree. C., 30
sec at 58.degree. C., 2 min at 72.degree. C.; final elongation for
6 min at 72.degree. C. DNA-fragment 2, obtained in the reaction
with the chromosomal DNA from the parent strain E. coli MG1655 as
the template, was 966 bp in length (SEQ ID NO: 8). DNA-fragment 3,
obtained in the reaction with the chromosomal DNA from the mutant
strain E. coli MG1655 P.sub.tac7-yajL as a template, was 1,820 bp
in length (SEQ ID NO: 9).
1.3. Construction of the E. coli L-Valine-Producing Strain
[0148] The yajL gene under control of P.sub.tac7 promoter was
introduced into valine-producing E. coli strain H81 (Russian patent
No. 2355763, EP1942183 B1) by P1-transduction (Miller J. H.
"Experiments in molecular genetics", Cold Spring Harbor Laboratory,
Cold Spring Harbor (1972)). The E. coli MG1655 P.sub.tac7-yajL
strain (see Example 1.1) was used as a donor. The strain H81 was
deposited in the Russian National Collection of Industrial
Microorganisms (VKPM) FGUP GosNII Genetika (1 Dorozhny proezd, 1
Moscow 117545, Russian Federation) on Jan. 30, 2001 under accession
number VKPM B-8066 and then converted to a deposit under the
Budapest Treaty on Feb. 1, 2002. The E. coli H81 mutants harboring
the P.sub.tac7-yajL cassette were selected on the plates containing
LB-medium, agar (1.5%) and chloramphenicol (20 mg/L). Thus the
strain E. coli H81 P.sub.tac7-yajL was obtained. Replacement of the
promoter region of the yajL gene was verified by PCR as described
in Section 1.2 of Example 1.
Example 2
Production of L-Valine by the E. coli H81 P.sub.tac7-yajL
Strain
[0149] The modified E. coli H81 P.sub.tac7-yajL and the control E.
coli H81 strains were each cultivated at 32.degree. C. for 18 hours
in LB-medium (also referred to as lysogenic broth or Luria-Bertani
medium as described in Sambrook, J. and Russell, D. W. "Molecular
Cloning: A Laboratory Manual", 3.sup.rd ed., Cold Spring Harbor
Laboratory Press (2001)). Then, 0.2 mL of the obtained culture was
inoculated into 2 mL of a fermentation medium in 20.times.200-mm
test tubes and cultivated at 32.degree. C. for 66 hours on a rotary
shaker at 250 rpm to an OD.sub.550 of .about.29 until glucose
consumption.
[0150] The composition of the fermentation medium (g/L) was as
follows:
TABLE-US-00001 Glucose 60.0 (NH.sub.4).sub.2SO.sub.4 15.0
KH.sub.2PO.sub.4 1.5 MgSO.sub.4.cndot.7H.sub.2O 1.0 Thiamine-HCl
0.1 CaCO.sub.3 25.0 LB-medium 10% (v/v)
[0151] The fermentation medium was sterilized at 116.degree. C. for
30 min, except that glucose and CaCO.sub.3 were sterilized
separately as follows: glucose at 110.degree. C. for 30 min and
CaCO.sub.3 at 116.degree. C. for 30 min. The pH was adjusted to 7.0
by KOH solution.
[0152] After cultivation, accumulated L-valine was measured using
thin-layer chromatography (TLC). TLC plates (10.times.20 cm) were
coated with 0.11-mm layers of Sorbfil.TM. silica gel containing
non-fluorescent indicator (Sorbpolymer.TM., Krasnodar, Russian
Federation). Samples were applied onto the plates with the Camag
Linomat.TM. 5 sample applicator. The Sorbfil plates were developed
with a mobile phase consisting of propan-2-ol:ethylacetate:25%
aqueous ammonia:water (16:16:5:10, v/v). A solution of ninhydrin
(2%, w/v) in acetone was used as the visualizing reagent. After
development, plates were dried and scanned with the Camag TLC
Scanner 3.TM. in absorbance mode with detection at 520 nm using
winCATS.TM. software (version 1.4.2).
[0153] The results of 3 independent test-tube fermentations are
shown in Table 1. As it can be seen from the Table 1, the modified
E. coli H81 P.sub.tac7-yajL strain was able to accumulate a higher
amount of L-valine as compared with the parent E. coli H81
strain.
TABLE-US-00002 TABLE 1 Production of L-valine. Strain Val, g/L E.
coli H81 (control) 9.7 E. coli H81 P.sub.tac7-yajL 10.7
Example 3
Construction of the E. coli L-Phenylalanine-Producing Strain
Modified to Overexpress the yajL Gene
[0154] The P.sub.tac7-yajL fragment was introduced into E. coli
DV269 (TyrA-LAA) strain, also known as E. coli MG1655
htrE:(P.sub.L-yddG)[MUDaroG4-pheA.sup.fbr-aroL], as described in
Section 1.3 of Example 1. The E. coli MG1655 P.sub.tac7-yajL strain
(see Example 1.1) was used as a donor. Construction of the E. coli
DV269 (TyrA-LAA) strain is described in Doroshenko V. G. et al.,
Construction of an L-phenylalanine-producing tyrosine-prototrophic
Escherichia coli strain using tyrA ssrA-like tagged alleles,
Biotechnol. Lett., 2010, 35:1117-1121. The E. coli DV269 (TyrA-LAA)
mutants harboring the P.sub.tac7-yajL cassette were selected on the
plates containing LB-medium, agar (1.5%) and chloramphenicol (20
mg/L). Thus the strain E. coli DV269 (TyrA-LAA) P.sub.tac7-yajL was
obtained.
Example 4
Production of L-Phenylalanine by the E. coli DV269 (TyrA-LAA)
P.sub.tac7-yajL Strain
[0155] The freshly grown cells of L-phenylalanine-producing E. coli
DV269 (TyrA-LAA) P.sub.tac7-yajL and control E. coli DV269
(TyrA-LAA) strains were taken from L-agar plates in an amount of
10.sup.8 CFU/mL (colony-forming unit, CFU), inoculated into 3 mL of
LB-medium in 15.times.150-mm test tubes and cultivated at
34.degree. C. for 3 hours on a rotary shaker at 250 rpm. Then, 0.2
mL of the obtained culture was inoculated into 2 mL of a
fermentation medium in 20.times.200-mm test tubes and cultivated at
34.degree. C. for 30 hours on a rotary shaker at 240 rpm to an
OD.sub.540 of .about.6.5-7 until glucose consumption.
[0156] The composition of the fermentation medium (g/L) is as
follows:
TABLE-US-00003 Glucose 50.0 (NH.sub.4).sub.2SO.sub.4 0.6
K.sub.2HPO.sub.4.cndot.3H.sub.2O 0.6 FeSO.sub.4.cndot.7H.sub.2O
0.01 MnSO.sub.4.cndot.5H.sub.2O 0.01 Yeast extract 2.0 CaCO.sub.3
20.0
[0157] Glucose was sterilized separately. CaCO.sub.3 was dry-heat
sterilized at 180.degree. C. for 2 h.
[0158] After cultivation, accumulated L-phenylalanine was measured
using thin-layer chromatography (TLC). TLC plates (10.times.20 cm)
were coated with 0.11-mm layers of Sorbfil silica gel containing
non-fluorescent indicator (Sorbpolymer, Krasnodar, Russian
Federation). Samples were applied onto the plates with the Camag
Linomat 5 sample applicator. The Sorbfil plates were developed with
a mobile phase consisting of propan-2-ol:ethylacetate 25% aqueous
ammonia:water (16:16:3:5, v/v). A solution of ninhydrin (1%, w/v)
in acetone was used as the visualizing reagent. After development,
plates were dried and scanned with the Camag TLC Scanner 3 in
absorbance mode with detection at 520 nm using winCATS software
(version 1.4.2).
[0159] The results of 10 independent test-tube fermentations are
shown in Table 2. As it can be seen from the Table 2, the modified
E. coli DV269 (TyrA-LAA) P.sub.tac7-yajL strain was able to
accumulate a higher amount of L-phenylalanine as compared with the
parent E. coli DV269 (TyrA-LAA) strain.
TABLE-US-00004 TABLE 2 Production of L-phenylalanine. Strain Phe,
g/L E. coli DV269 (TyrA-LAA) (control) 3.8 E. coli DV269 (TyrA-LAA)
P.sub.tac7-yajL 4.1
Example 5
Production of L-Arginine by E. coli 382 P.sub.tac7-yajL Strain
[0160] To test the effect of overexpression of the yajL gene on
L-arginine production, the DNA-fragments from the chromosome of the
above-described E. coli MG1655 P.sub.tac7-yajL strain are
transferred to the arginine-producing E. coli strain 382 by
P1-transduction (Miller, J. H. (1972), Experiments in Molecular
Genetics, Cold Spring Harbor Lab. Press, Plainview, N.Y.) to obtain
the strain 382 P.sub.tac7-yajL. The strain 382 was deposited in the
Russian National Collection of Industrial Microorganisms (VKPM)
FGUP GosNII Genetika (1 Dorozhny proezd, 1 Moscow 117545, Russian
Federation) on Apr. 10, 2000 under the accession number VKPM B-7926
and then converted to a deposit under the Budapest Treaty on May
18, 2001.
[0161] E. coli strains 382 and 382 P.sub.tac7-yajL are separately
cultivated with shaking (220 rpm) at 37.degree. C. for 18 h in 3 mL
of nutrient broth, and 0.3 mL of the obtained cultures are
inoculated into 2 mL of a fermentation medium in 20.times.200-mm
test tubes and cultivated at 32.degree. C. for 48 h on a rotary
shaker (220 rpm).
[0162] After the cultivation, the amount of L-arginine which
accumulates in the medium is determined by paper chromatography
using a mobile phase consisting of butan-1-ol:acetic
acid:water=4:1:1 (v/v). A solution of ninhydrin (2%) in acetone is
used as a visualizing reagent. A spot containing L-arginine is cut
out, L-arginine is eluted with 0.5% water solution of CdCl.sub.2,
and the amount of L-arginine is estimated spectrophotometrically at
540 nm.
[0163] The composition of the fermentation medium (g/L) is as
follows:
TABLE-US-00005 Glucose 48.0 (NH.sub.4).sub.2SO4 35.0
KH.sub.2PO.sub.4 2.0 MgSO.sub.4.cndot.7H.sub.2O 1.0 Thiamine-HCl
0.0002 Yeast extract 1.0 L-isoleucine 0.1 CaCO.sub.3 5.0
[0164] Glucose and magnesium sulfate are sterilized separately.
CaCO.sub.3 is dry-heat sterilized at 180.degree. C. for 2 h. The pH
is adjusted to 7.0.
Example 6
Production of L-Cysteine by E. coli JM15(ydeD)P.sub.tac7-yajL
[0165] To test the effect of overexpression of the yajL gene on
L-cysteine production, the DNA fragments from the chromosome of the
above-described E. coli MG1655 P.sub.tac7-yajL strain are
transferred to the L-cysteine-producing E. coli strain JM15(ydeD)
by P1-transduction to obtain the strain
JM15(ydeD)P.sub.tac7-yajL.
[0166] E. coli JM15(ydeD) is a derivative of E. coli JM15 (U.S.
Pat. No. 6,218,168), which is transformed with DNA having the ydeD
gene encoding a membrane protein, and is not involved in a
biosynthetic pathway of any L-amino acid (U.S. Pat. No.
5,972,663).
[0167] Fermentation conditions and procedure for evaluation of
L-cysteine production were described in detail in Example 6 of U.S.
Pat. No. 6,218,168.
Example 7
Production of L-Glutamic Acid by E. coli VL334thrC.sup.+
P.sub.tac7-yajL
[0168] To test the effect of overexpression of the yajL gene on
L-glutamic acid production, the DNA fragments from the chromosome
of the above-described E. coli MG1655 P.sub.tac7-yajL strain are
transferred to the E. coli L-glutamate-producing strain
VL334thrC.sup.+ (EP1172433 A1) by P1-transduction to obtain the
strain VL334thrC.sup.+ P.sub.tac7-yajL. The strain VL334thrC.sup.+
was deposited in the Russian National Collection of Industrial
Microorganisms (VKPM) FGUP GosNII Genetika (1 Dorozhny proezd, 1
Moscow 117545, Russian Federation) on Dec. 6, 2004 under the
accession number VKPM B-8961 and then converted to a deposit under
the Budapest Treaty on Dec. 8, 2004.
[0169] E. coli strains VL334thrC.sup.+ and VL334thrC.sup.+
P.sub.tac7-yajL are separately cultivated for 18-24 h at 37.degree.
C. on L-agar plates. Then, one loop of the cells is transferred
into test tubes containing 2 mL of fermentation medium. Cultivation
is carried out at 30.degree. C. for 3 days with shaking.
[0170] After the cultivation, the amount of L-glutamic acid which
accumulates in the medium is determined by paper chromatography
using a mobile phase consisting of butan-1-ol:acetic
acid:water=4:1:1 (v/v) with subsequent staining by ninhydrin (1%
solution in acetone), elution of the compounds in 50% ethanol with
0.5% CdCl.sub.2 and further estimation of L-glutamic acid at 540
nm.
[0171] The composition of the fermentation medium (g/L) is as
follows:
TABLE-US-00006 Glucose 60.0 (NH.sub.4).sub.2SO.sub.4 25.0
KH.sub.2PO.sub.4 2.0 MgSO.sub.4.cndot.7H.sub.2O 1.0 Thiamine-HCl
0.1 L-isoleucine 0.07 CaCO.sub.3 25.0
[0172] Glucose and CaCO.sub.3 are sterilized separately. The pH is
adjusted to 7.2.
Example 8
Production of L-Leucine by E. coli 57 P.sub.tac7-yajL
[0173] To test the effect of overexpression of the yajL gene on
L-leucine production, the DNA fragments from the chromosome of the
above-described E. coli MG1655 P.sub.tac7-yajL strain are
transferred to the E. coli L-leucine-producing strain 57 (VKPM
B-7386, U.S. Pat. No. 6,124,121) by P1-transduction to obtain the
strain 57 P.sub.tac7-yajL. The strain 57 was deposited in the
Russian National Collection of Industrial Microorganisms (VKPM)
FGUP GosNII Genetika (1 Dorozhny proezd, 1 Moscow 117545, Russian
Federation) on May 19, 1997 under the accession number VKPM
B-7386.
[0174] E. coli strains 57 and 57 P.sub.tac7-yajL are separately
cultivated for 18-24 h at 37.degree. C. on L-agar plates. To obtain
a seed culture, the strains are grown on a rotary shaker (250 rpm)
at 32.degree. C. for 18 h in 20.times.200-mm test tubes containing
2 mL of L-broth (Sambrook, J. and Russell, D. W. (2001) "Molecular
Cloning: A Laboratory Manual", 3.sup.rd ed., Cold Spring Harbor
Laboratory Press) supplemented with sucrose (4%). Then, the
fermentation medium is inoculated with 0.2 mL of seed material
(10%). The fermentation is performed in 2 mL of a minimal
fermentation medium in 20.times.200-mm test tubes. Cells are grown
for 48-72 h at 32.degree. C. with shaking at 250 rpm. The amount of
L-leucine which accumulates in the medium is determined by paper
chromatography using a mobile phase consisting of butan-1-ol:acetic
acid:water=4:1:1 (v/v).
[0175] The composition of the fermentation medium (g/L) is as
follows:
TABLE-US-00007 Glucose 60.0 (NH.sub.4).sub.2SO.sub.4 25.0
K.sub.2HPO.sub.4 2.0 MgSO.sub.4.cndot.7H.sub.2O 1.0 Thiamine-HCl
0.01 CaCO.sub.3 25.0
[0176] Glucose is sterilized separately. CaCO.sub.3 is dry-heat
sterilized at 180.degree. C. for 2 h. The pH is adjusted to
7.2.
Example 9
Production of L-Lysine by E. coli AJ11442 P.sub.tac7-yajL
[0177] To test the effect of overexpression of the yajL gene on
L-lysine production, the DNA fragments from the chromosome of the
above-described E. coli MG1655 P.sub.tac7-yajL strain are
transferred to the L-lysine-producing E. coli strain AJ11442 by
P1-transduction to obtain the AJ11442 P.sub.tac7-yajL strain. The
strain AJ11442 was deposited in National Institute of Bioscience
and Human Technology of Agency of Industrial Science and Technology
(currently Incorporated Administrative Agency, National Institute
of Technology and Evaluation, International Patent Organism
Depositary (NITE-IPOD, #120, 2-5-8 Kazusakamatari, Kisarazu-shi,
Chiba-ken 292-0818, JAPAN) on May 1, 1981 under a deposition number
of FERM P-5084. Then it was converted to an international deposit
under the provisions of the Budapest Treaty on Oct. 29, 1987 and
received an accession number of FERM BP-1543. The pCABD2 plasmid
includes the dapA gene encoding dihydrodipicolinate synthase having
a mutation which desensitizes feedback inhibition by L-lysine, the
lysC gene encoding aspartokinase III having a mutation which
desensitizes feedback inhibition by L-lysine, the dapB gene
encoding dihydrodipicolinate reductase, and the ddh gene encoding
diaminopimelate dehydrogenase (U.S. Pat. No. 6,040,160).
[0178] E. coli strains AJ11442 and AJ11442 P.sub.tac7-yajL are
separately cultivated in L-medium containing streptomycin (20 mg/L)
at 37.degree. C., and 0.3 mL of the obtained culture is inoculated
into 20 mL of the fermentation medium containing the required drugs
in a 500-mL flask. The cultivation is carried out at 37.degree. C.
for 16 h by using a reciprocal shaker at the agitation speed of 115
rpm. After the cultivation, the amounts of L-lysine and residual
glucose in the medium are determined by a known method
(Biotech-analyzer.TM. AS210, Sakura Seiki Co.). Then, the yield of
L-lysine is calculated relative to consumed glucose for each of the
strains.
[0179] The composition of the fermentation medium (g/L) is as
follows:
TABLE-US-00008 Glucose 40.0 (NH.sub.4).sub.2SO.sub.4 24.0
K.sub.2HPO.sub.4 1.0 MgSO.sub.4.cndot.7H.sub.2O 1.0
FeSO.sub.4.cndot.7H.sub.2O 0.01 MnSO.sub.4.cndot.5H.sub.2O 0.01
Yeast extract 2.0
[0180] The pH is adjusted to 7.0 by KOH and the medium is
autoclaved at 115.degree. C. for 10 min. Glucose and magnesium
sulfate are sterilized separately. CaCO.sub.3 is dry-heat
sterilized at 180.degree. C. for 2 h and added to the medium for a
final concentration of 30 g/L.
Example 10
Production of L-Proline by E. coli 702ilvA P.sub.tac7-yajL
[0181] To test the effect of overexpression of the yajL gene on
L-proline production, the DNA fragments from the chromosome of the
above-described E. coli MG1655 P.sub.tac7-yajL strain are
transferred to the proline-producing E. coli strain 702ilvA by
P1-transduction to obtain the strain 702ilvA P.sub.tac7-yajL. The
strain 702ilvA was deposited in the Russian National Collection of
Industrial Microorganisms (VKPM) FGUP GosNII Genetika (1 Dorozhny
proezd, 1 Moscow 117545, Russian Federation) on Jul. 18, 2000 under
the accession number VKPM B-8012 and then converted to a deposit
under the Budapest Treaty on May 18, 2001.
[0182] E. coli strains 702ilvA and 702ilvA P.sub.tac7-yajL are
separately cultivated for 18-24 h at 37.degree. C. on L-agar
plates. Then, these strains are cultivated under the same
conditions as in Example 7 (Production of L-glutamic acid).
Example 11
Production of L-Threonine by E. coli B-3996 P.sub.tac7-yajL
[0183] To test the effect of overexpression of the yajL gene on
L-threonine production, the DNA fragments from the chromosome of
the above-described E. coli MG1655 P.sub.tac7-yajL strain are
transferred to the L-threonine-producing E. coli strain VKPM B-3996
by P1-transduction to obtain the strain B-3996 P.sub.tac7-yajL. The
strain B-3996 was deposited on Nov. 19, 1987 in the All-Union
Scientific Center of Antibiotics (Russian Federation, 117105
Moscow, Nagatinskaya Street, 3-A) under the accession number RIA
1867. The strain was also deposited in the Russian National
Collection of Industrial Microorganisms (VKPM) FGUP GosNII Genetika
(1 Dorozhny proezd, 1 Moscow 117545, Russian Federation) on Apr. 7,
1987 under the accession number VKPM B-3996.
[0184] E. coli strains B-3996 and B-3996 P.sub.tac7-yajL are
separately cultivated for 18-24 h at 37.degree. C. on L-agar
plates. To obtain a seed culture, the strains are grown on a rotary
shaker (250 rpm) at 32.degree. C. for 18 h in 20.times.200-mm test
tubes containing 2 mL of L-broth (Sambrook, J. and Russell, D. W.
(2001) "Molecular Cloning: A Laboratory Manual", 3.sup.rd ed., Cold
Spring Harbor Laboratory Press) supplemented with glucose (4%).
Then, the fermentation medium is inoculated with 0.2 mL (10%) of
seed material. The fermentation is performed in 2 mL of minimal
medium in 20.times.200-mm test tubes. Cells are grown for 65 h at
32.degree. C. with shaking at 250 rpm.
[0185] After cultivation, the amount of L-threonine which
accumulates in the medium is determined by paper chromatography
using a mobile phase consisting of butan-1-ol:acetic
acid:water=4:1:1 (v/v). A solution of ninhydrin (2%) in acetone is
used as a visualizing reagent. A spot containing L-threonine is cut
out, L-threonine is eluted with 0.5% water solution of CdCl.sub.2,
and the amount of L-threonine is estimated spectrophotometrically
at 540 nm.
[0186] The composition of the fermentation medium (g/L) is as
follows:
TABLE-US-00009 Glucose 80.0 (NH.sub.4).sub.2SO.sub.4 22.0 NaCl 0.8
KH.sub.2PO.sub.4 2.0 MgSO.sub.4.cndot.7H.sub.2O 0.8
FeSO.sub.4.cndot.7H.sub.2O 0.02 MnSO.sub.4.cndot.5H.sub.2O 0.02
Thiamine-HCl 0.0002 Yeast extract 1.0 CaCO.sub.3 30.0
[0187] Glucose and magnesium sulfate are sterilized separately.
CaCO.sub.3 is sterilized by dry-heat at 180.degree. C. for 2 h. The
pH is adjusted to 7.0. The antibiotic is introduced into the medium
after sterilization.
Example 12
Production of L-Tryptophan by E. coli
SV164(pGH5)P.sub.tac7-yajL
[0188] To test the effect of overexpression of the yajL gene on
L-tryptophan production, the DNA fragments from the chromosome of
the above-described E. coli MG1655 P.sub.tac7-yajL strain are
transferred to the L-tryptophan-producing E. coli strain
SV164(pGH5) by P1-transduction to obtain the strain
SV164(pGH5)P.sub.tac7-yajL. The strain SV164 has the trpE allele
encoding anthranilate synthase free from feedback inhibition by
tryptophan. The plasmid pGH5 harbors a mutant serA gene encoding
phosphoglycerate dehydrogenase free from feedback inhibition by
serine. The strain SV164(pGH5) was described in detail in U.S. Pat.
No. 6,180,373 or EP0662143 B1.
[0189] E. coli strains SV164(pGH5) and SV164(pGH5)P.sub.tac7-yajL
are separately cultivated with shaking at 37.degree. C. for 18 h in
3 mL of nutrient broth supplemented with tetracycline (20 mg/L,
marker of pGH5 plasmid). The obtained cultures (0.3 mL each) are
inoculated into 3 mL of a fermentation medium containing
tetracycline (20 mg/L) in 20.times.200-mm test tubes, and
cultivated at 37.degree. C. for 48 h with a rotary shaker at 250
rpm. After cultivation, the amount of L-tryptophan which
accumulates in the medium is determined by TLC. The 10.times.15-cm
TLC plates coated with 0.11-mm layers of Sorbfil silica gel
containing non-fluorescent indicator (Stock Company Sorbpolymer,
Krasnodar, Russian Federation) are used. The Sorbfil plates are
developed with a mobile phase consisting of
propan-2-ol:ethylacetate:25% aqueous ammonia:water=40:40:7:16
(v/v). A solution of ninhydrin (2%) in acetone is used as a
visualizing reagent. The fermentation medium components are listed
in Table 3, but should be sterilized in separate groups (A, B, C,
D, E, F, and H), as shown, to avoid adverse interactions during
sterilization.
TABLE-US-00010 TABLE 3 Final concentration, Solutions Component g/L
A KH.sub.2PO.sub.4 1.5 NaCl 0.5 (NH.sub.4).sub.2SO.sub.4 1.5
L-Methionine 0.05 L-Phenylalanine 0.1 L-Tyrosine 0.1 Mameno* (as
the amount of nitrogen) 0.07 B Glucose 40.0
MgSO.sub.4.cndot.7H.sub.2O 0.3 C CaCl.sub.2 0.011 D
FeSO.sub.4.cndot.7H.sub.2O 0.075 Sodium citrate 1.0 E
Na.sub.2MoO.sub.4.cndot.2H.sub.2O 0.00015 H.sub.3BO.sub.3 0.0025
CoCl.sub.2.cndot.6H.sub.2O 0.00007 CuSO.sub.4.cndot.5H.sub.2O
0.00025 MnCl.sub.2.cndot.4H.sub.2O 0.0016
ZnSO.sub.4.cndot.7H.sub.2O 0.0003 F Thiamine-HCl 0.005 G CaCO.sub.3
30.0 H Pyridoxine 0.03 The pH of solution A is adjusted to 7.1 with
NH.sub.4OH. *Mameno .TM. is the soybean meal hydrolysate (Ajinomoto
Co, Inc.).
Example 13
Production of L-Citrulline by E. coli 382.DELTA.argG
P.sub.tac7-yajL
[0190] To test the effect of overexpression of the yajL gene on
L-citrulline production, the DNA fragments from the chromosome of
the above-described E. coli MG1655 P.sub.tac7-yajL strain are
transferred to the L-citrulline producing E. coli strain
382.DELTA.argG by P1-transduction to obtain the strain
382.DELTA.argG P.sub.tac7-yajL. The strain 382.DELTA.argG is
obtained by deletion of argG gene on the chromosome of 382 strain
(VKPM B-7926) by the method initially developed by Datsenko K. A.
and Wanner B. L. called ".lamda.Red/ET-mediated integration"
(Datsenko K. A. and Wanner B. L., Proc. Natl. Acad. Sci. USA, 2000,
97(12):6640-6645). According to this procedure, the PCR primers
homologous to both the region adjacent to the argG gene and the
gene which confers antibiotic resistance in the template plasmid
are constructed. The plasmid pMW118-.lamda.attL-cat-.lamda.aaR (WO
05/010175) is used as the template in the PCR reaction.
[0191] E. coli strains 382.DELTA.argG and 382.DELTA.argG
P.sub.tac7-yajL are separately cultivated with shaking at
37.degree. C. for 18 h in 3 mL of nutrient broth, and 0.3 mL of the
obtained cultures are inoculated into 2 mL of a fermentation medium
in 20.times.200-mm test tubes and cultivated at 32.degree. C. for
48 h on a rotary shaker.
[0192] After the cultivation, the amount of L-citrulline which
accumulates in the medium is determined by paper chromatography
using a mobile phase consisting of butan-1-ol:acetic
acid:water=4:1:1 (v/v). A solution of ninhydrin (2%) in acetone is
used as a visualizing reagent. A spot containing citrulline is cut
out, L-citrulline is eluted with 0.5% water solution of CdCl.sub.2,
and the amount of L-citrulline is estimated spectrophotometrically
at 540 nm.
[0193] The composition of the fermentation medium (g/L) is as
follows:
TABLE-US-00011 Glucose 48.0 (NH.sub.4).sub.2SO.sub.4 35.0
KH.sub.2PO.sub.4 2.0 MgSO.sub.4.cndot.7H.sub.2O 1.0 Thiamine-HCl
0.0002 Yeast extract 1.0 L-isoleucine 0.1 L-arginine 0.1 CaCO.sub.3
5.0
[0194] Glucose and magnesium sulfate are sterilized separately.
CaCO.sub.3 is dry-heat sterilized at 180.degree. C. for 2 h. The pH
is adjusted to 7.0.
Example 14
Production of L-Ornithine by E. coli 382.DELTA.argF.DELTA.argI
P.sub.tac7-yajL
[0195] To test the effect of overexpression of the yajL gene on
L-ornithine production, the DNA fragments from the chromosome of
the above-described E. coli MG1655 P.sub.tac7-yajL strain are
transferred to the L-ornithine producing E. coli strain
382.DELTA.argF.DELTA.argI by P1-transduction to obtain the strain
382.DELTA.argF.DELTA.argI P.sub.tac7-yajL. The strain
382.DELTA.argF.DELTA.argI is obtained by consecutive deletion of
argF and argI genes on the chromosome of 382 strain (VKPM B-7926)
by the method initially developed by Datsenko K. A. and Wanner B.
L. called ".lamda.Red/ET-mediated integration" (Datsenko K. A. and
Wanner B. L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645).
According to this procedure, two pairs of PCR primers homologous to
both the region adjacent to the argF or argI gene and the gene
which confers antibiotic resistance in the template plasmid are
constructed. The plasmid pMW118-.lamda.attL-cat-.lamda.attR (WO
05/010175) is used as the template in the PCR reaction.
[0196] E. coli strains 382.DELTA.argF.DELTA.argI and
382.DELTA.argF.DELTA.argI P.sub.tac7-yajL are separately cultivated
with shaking at 37.degree. C. for 18 h in 3 mL of nutrient broth,
and 0.3 mL of the obtained cultures are inoculated into 2 mL of a
fermentation medium in 20.times.200-mm test tubes and cultivated at
32.degree. C. for 48 h on a rotary shaker.
[0197] After the cultivation, the amount of ornithine which
accumulates in the medium is determined by paper chromatography
using a mobile phase consisting of butan-1-ol:acetic
acid:water=4:1:1 (v/v). A solution of ninhydrin (2%) in acetone is
used as a visualizing reagent. A spot containing ornithine is cut
out, ornithine is eluted with 0.5% water solution of CdCl.sub.2,
and the amount of ornithine is estimated spectrophotometrically at
540 nm.
[0198] The composition of the fermentation medium (g/L) is as
follows:
TABLE-US-00012 Glucose 48.0 (NH.sub.4).sub.2SO.sub.4 35.0
KH.sub.2PO.sub.4 2.0 MgSO.sub.4.cndot.7H.sub.2O 1.0 Thiamine-HCl
0.0002 Yeast extract 1.0 L-isoleucine 0.1 L-arginine 0.1 CaCO.sub.3
5.0
[0199] Glucose and magnesium sulfate are sterilized separately.
CaCO.sub.3 is dry-heat sterilized at 180.degree. C. for 2 h. The pH
is adjusted to 7.0.
[0200] 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. All the cited references herein are incorporated by
reference as a part of this application.
Sequence CWU 1
1
91591DNAEscherichia coliCDS(1)..(591) 1atg agc gca tcg gca ctg gtt
tgc ctc gcc cct ggt agt gaa gag act 48Met Ser Ala Ser Ala Leu Val
Cys Leu Ala Pro Gly Ser Glu Glu Thr 1 5 10 15 gaa gcc gtc acc act
atc gat ctg ctg gtt cgc ggc ggt atc aaa gtc 96Glu Ala Val Thr Thr
Ile Asp Leu Leu Val Arg Gly Gly Ile Lys Val 20 25 30 acc act gcc
agc gtc gcc agc gat ggt aac ctg gcg att acc tgc tcg 144Thr Thr Ala
Ser Val Ala Ser Asp Gly Asn Leu Ala Ile Thr Cys Ser 35 40 45 cgc
ggc gtg aag ctg ctg gcg gat gcg ccg ctg gtc gaa gtg gct gat 192Arg
Gly Val Lys Leu Leu Ala Asp Ala Pro Leu Val Glu Val Ala Asp 50 55
60 ggc gaa tat gac gtg atc gtg ctg cct ggt ggc att aaa ggc gcg gag
240Gly Glu Tyr Asp Val Ile Val Leu Pro Gly Gly Ile Lys Gly Ala Glu
65 70 75 80 tgt ttt cgc gat agc act ctg ctg gtt gaa acc gtt aaa cag
ttc cac 288Cys Phe Arg Asp Ser Thr Leu Leu Val Glu Thr Val Lys Gln
Phe His 85 90 95 cgt tcc ggg cgt atc gtc gcg gct att tgc gcc gcg
cca gcc acc gtg 336Arg Ser Gly Arg Ile Val Ala Ala Ile Cys Ala Ala
Pro Ala Thr Val 100 105 110 ctg gtg ccg cac gat atc ttc ccg att ggt
aat atg acc ggc ttc ccg 384Leu Val Pro His Asp Ile Phe Pro Ile Gly
Asn Met Thr Gly Phe Pro 115 120 125 acg ctg aaa gac aaa att ccc gcc
gaa caa tgg ctg gac aag cgc gtc 432Thr Leu Lys Asp Lys Ile Pro Ala
Glu Gln Trp Leu Asp Lys Arg Val 130 135 140 gtc tgg gat gca cgg gta
aaa ttg ctg acc agc cag ggg ccg ggt aca 480Val Trp Asp Ala Arg Val
Lys Leu Leu Thr Ser Gln Gly Pro Gly Thr 145 150 155 160 gct atc gac
ttt ggt ctg aaa att atc gac ctg ttg gtt ggg cgt gaa 528Ala Ile Asp
Phe Gly Leu Lys Ile Ile Asp Leu Leu Val Gly Arg Glu 165 170 175 aaa
gcc cat gaa gtg gca tca caa ctg gtg atg gcg gca ggg att tat 576Lys
Ala His Glu Val Ala Ser Gln Leu Val Met Ala Ala Gly Ile Tyr 180 185
190 aat tat tac gag tag 591Asn Tyr Tyr Glu 195 2196PRTEscherichia
coli 2Met Ser Ala Ser Ala Leu Val Cys Leu Ala Pro Gly Ser Glu Glu
Thr 1 5 10 15 Glu Ala Val Thr Thr Ile Asp Leu Leu Val Arg Gly Gly
Ile Lys Val 20 25 30 Thr Thr Ala Ser Val Ala Ser Asp Gly Asn Leu
Ala Ile Thr Cys Ser 35 40 45 Arg Gly Val Lys Leu Leu Ala Asp Ala
Pro Leu Val Glu Val Ala Asp 50 55 60 Gly Glu Tyr Asp Val Ile Val
Leu Pro Gly Gly Ile Lys Gly Ala Glu 65 70 75 80 Cys Phe Arg Asp Ser
Thr Leu Leu Val Glu Thr Val Lys Gln Phe His 85 90 95 Arg Ser Gly
Arg Ile Val Ala Ala Ile Cys Ala Ala Pro Ala Thr Val 100 105 110 Leu
Val Pro His Asp Ile Phe Pro Ile Gly Asn Met Thr Gly Phe Pro 115 120
125 Thr Leu Lys Asp Lys Ile Pro Ala Glu Gln Trp Leu Asp Lys Arg Val
130 135 140 Val Trp Asp Ala Arg Val Lys Leu Leu Thr Ser Gln Gly Pro
Gly Thr 145 150 155 160 Ala Ile Asp Phe Gly Leu Lys Ile Ile Asp Leu
Leu Val Gly Arg Glu 165 170 175 Lys Ala His Glu Val Ala Ser Gln Leu
Val Met Ala Ala Gly Ile Tyr 180 185 190 Asn Tyr Tyr Glu 195
362DNAArtificial SequencePrimer P1 3tcatattcac tctcctttct
ttttaccatt tcaaacgctc acaattccac acattatacg 60ag 62464DNAArtificial
SequencePrimer P2 4ttgaacaccc ggagtggttg cgggtgagga ggaacacgct
caagttagta taaaaaagct 60gaac 6451768DNAArtificial
SequenceDNA-fragment 1 5ttgaacaccc ggagtggttg cgggtgagga ggaacacgct
caagttagta taaaaaagct 60gaacgagaaa cgtaaaatga tataaatatc aatatattaa
attagatttt gcataaaaaa 120cagactacat aatactgtaa aacacaacat
atgcagtcac tatgaatcaa ctacttagat 180ggtattagtg acctgtaaca
gactgcagtg gtcgaaaaaa aaagcccgca ctgtcaggtg 240cgggcttttt
tctgtgttaa gcttcgacga atttctgcca ttcatccgct tattatcact
300tattcaggcg tagcaccagg cgtttaaggg caccaataac tgccttaaaa
aaattacgcc 360ccgccctgcc actcatcgca gtactgttgt aattcattaa
gcattctgcc gacatggaag 420ccatcacaga cggcatgatg aacctgaatc
gccagcggca tcagcacctt gtcgccttgc 480gtataatatt tgcccatggt
gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt 540aaatcaaaac
tggtgaaact cacccaggga ttggctgaga cgaaaaacat attctcaata
600aaccctttag ggaaataggc caggttttca ccgtaacacg ccacatcttg
cgaatatatg 660tgtagaaact gccggaaatc gtcgtggtat tcactccaga
gcgatgaaaa cgtttcagtt 720tgctcatgga aaacggtgta acaagggtga
acactatccc atatcaccag ctcaccgtct 780ttcattgcca tacggaattc
cggatgagca ttcatcaggc gggcaagaat gtgaataaag 840gccggataaa
acttgtgctt atttttcttt acggtcttta aaaaggccgt aatatccagc
900tgaacggtct ggttataggt acattgagca actgactgaa atgcctcaaa
atgttcttta 960cgatgccatt gggatatatc aacggtggta tatccagtga
tttttttctc cattttagct 1020tccttagctc ctgaaaatct cggatccggc
caagctagct tggctctagc tagagcgccc 1080ggttgacgct gctagtgtta
cctagcgatt tgtatcttac tgcatgttac ttcatgttgt 1140caatacctgt
ttttcgtgcg acttatcagg ctgtctactt atccggagat ccacaggacg
1200ggtgtggtcg ccatgatcgc gtagtcgata gtggctccaa gtagcgaagc
gagcaggact 1260gggcggcggc caaagcggtc ggacagtgct ccgagaacgg
gtgcgcatag aaattgcatc 1320aacgcatata gcgctagcag cacgccatag
tgactggcga tgctgtcgga atggacgata 1380tcccgcaaga ggcccggcag
taccggcata accaagccta tgcctacagc atccagggtg 1440acggtgccga
ggatgacgat gagcgcattg ttagatttca tacacggtgc ctgactgcgt
1500tagcaattta actgtgataa actaccgcat taaagcttat cgatgataag
ctgtcaaaca 1560tgagaattcg aaatcaaata atgattttat tttgactgat
agtgacctgt tcgttgcaac 1620aaattgataa gcaatgcttt tttataatgc
caacttagta taaaaaagca ggcttcaaga 1680tctccctgtg gcaaattaat
catcggctcg tataatgtgt ggaattgtga gcgtttgaaa 1740tggtaaaaag
aaaggagagt gaatatga 1768620DNAArtificial SequencePrimer P3
6agtcgcaaca gcatgcacaa 20720DNAArtificial SequencePrimer P4
7accagtgccg atgcgctcat 208966DNAArtificial SequenceDNA-fragment 2
8accagtgccg atgcgctcat attcactctc ctttcttttt accatttcaa acaggcgggt
60gttttccggt acggcaatcc catgcgcgcg ggcgcggcgt aagagaaaac cattgatata
120gtcgatttca gtgtggcgca gcgcgcggat atcctgcaac atcgacgaga
tattttccgc 180tgtggcatca atcacctgca tcacgtaatc acgcaaatct
tctgctgaag tatgatgccc 240ttcgcgttcg atcaccgccg cgacttcttc
gcatatctgc ataatttctt gcggatgatg 300acgtaattca ccgttcgggc
aattccagat ggcagtcagt ggattaatca cgcagttgac 360tgccagcttg
cgccacagct cggcgcgaat attgttatgc caggcaacgt caggcaacac
420ggtttgcaaa atatccgcca gataactgta atccccgtcc tgttgccgtg
ccgggccaat 480atgcgtgata ccgtttgcca catgaataat gacattgccg
tcgcggcggg ctgcatgggt 540ggtggtgccc atcagtaatg gctgctgaat
gttttgcaac tcttcgatgg tgcccatgcc 600gttgtgaatt aacagtattg
gcgtagttac aggcagtgtg gacgcgaggc ttttgacggc 660atcggaaacc
tgccatgctt tcagcgtcac caggagcaga tcgctggtgg cgagaaaatc
720gggatcgttg gcggtcagcg attcgttaaa tatcgaacca tctgtctcaa
ccagattcac 780gctacaataa ggttgcggta cgcgcagcca gccctgaact
tcatgaccct gtttgcaaag 840tgctgtaagc cataattgcc ctaaggcacc
gcatcccaat acggtaattt tcattgttcc 900tcctcacccg caaccactcc
gggtgttcaa taaggctatc ccttaattgt gcatgctgtt 960gcgact
96691820DNAArtificial SequenceDNA-fragment 3 9agtcgcaaca gcatgcacaa
ttaagggata gccttattga acacccggag tggttgcggg 60tgaggaggaa cacgctcaag
ttagtataaa aaagctgaac gagaaacgta aaatgatata 120aatatcaata
tattaaatta gattttgcat aaaaaacaga ctacataata ctgtaaaaca
180caacatatgc agtcactatg aatcaactac ttagatggta ttagtgacct
gtaacagact 240gcagtggtcg aaaaaaaaag cccgcactgt caggtgcggg
cttttttctg tgttaagctt 300cgacgaattt ctgccattca tccgcttatt
atcacttatt caggcgtagc accaggcgtt 360taagggcacc aataactgcc
ttaaaaaaat tacgccccgc cctgccactc atcgcagtac 420tgttgtaatt
cattaagcat tctgccgaca tggaagccat cacagacggc atgatgaacc
480tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat aatatttgcc
catggtgaaa 540acgggggcga agaagttgtc catattggcc acgtttaaat
caaaactggt gaaactcacc 600cagggattgg ctgagacgaa aaacatattc
tcaataaacc ctttagggaa ataggccagg 660ttttcaccgt aacacgccac
atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg 720tggtattcac
tccagagcga tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa
780gggtgaacac tatcccatat caccagctca ccgtctttca ttgccatacg
gaattccgga 840tgagcattca tcaggcgggc aagaatgtga ataaaggccg
gataaaactt gtgcttattt 900ttctttacgg tctttaaaaa ggccgtaata
tccagctgaa cggtctggtt ataggtacat 960tgagcaactg actgaaatgc
ctcaaaatgt tctttacgat gccattggga tatatcaacg 1020gtggtatatc
cagtgatttt tttctccatt ttagcttcct tagctcctga aaatctcgga
1080tccggccaag ctagcttggc tctagctaga gcgcccggtt gacgctgcta
gtgttaccta 1140gcgatttgta tcttactgca tgttacttca tgttgtcaat
acctgttttt cgtgcgactt 1200atcaggctgt ctacttatcc ggagatccac
aggacgggtg tggtcgccat gatcgcgtag 1260tcgatagtgg ctccaagtag
cgaagcgagc aggactgggc ggcggccaaa gcggtcggac 1320agtgctccga
gaacgggtgc gcatagaaat tgcatcaacg catatagcgc tagcagcacg
1380ccatagtgac tggcgatgct gtcggaatgg acgatatccc gcaagaggcc
cggcagtacc 1440ggcataacca agcctatgcc tacagcatcc agggtgacgg
tgccgaggat gacgatgagc 1500gcattgttag atttcataca cggtgcctga
ctgcgttagc aatttaactg tgataaacta 1560ccgcattaaa gcttatcgat
gataagctgt caaacatgag aattcgaaat caaataatga 1620ttttattttg
actgatagtg acctgttcgt tgcaacaaat tgataagcaa tgctttttta
1680taatgccaac ttagtataaa aaagcaggct tcaagatctc cctgtggcaa
attaatcatc 1740ggctcgtata atgtgtggaa ttgtgagcgt ttgaaatggt
aaaaagaaag gagagtgaat 1800atgagcgcat cggcactggt 1820
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