U.S. patent application number 11/137504 was filed with the patent office on 2005-10-06 for methods of making l-amino acids in coryneform bacteria using the sige gene.
This patent application is currently assigned to Degussa AG. Invention is credited to Binder, Michael, Farwick, Mike, Hermann, Thoams, Mockel, Bettina, Pfefferle, Walter.
Application Number | 20050221450 11/137504 |
Document ID | / |
Family ID | 27437871 |
Filed Date | 2005-10-06 |
United States Patent
Application |
20050221450 |
Kind Code |
A1 |
Mockel, Bettina ; et
al. |
October 6, 2005 |
Methods of making L-amino acids in coryneform bacteria using the
sigE gene
Abstract
The present invention relates to an isolated polynucleotide from
Corynebacterium glutamicum comprising a polynucleotide sequence
chosen from the group consisting of (a) a polynucleotide which is
identical to the extent of at least 70% to a polynucleotide which
codes for a polypeptide which comprises the amino acid sequence of
SEQ ID NO: 2; (b) a polynucleotide which codes for a polypeptide
which comprises an amino acid sequence which is identical to the
extent of at least 70% to the amino acid sequence of SEQ ID NO: 2;
(c) a polynucleotide which is complementary to the polynucleotides
of (a) or (b); and (d) a polynucleotide comprising at least 15
successive nucleotides of the polynucleotide sequence of (a), (b),
or (c), and a process for the fermentative preparation of L-amino
acids using coryneform bacteria in which at least the sigE gene is
present in enhanced form, and the use of polynucleotides which
comprise the sequence according to the invention as hybridization
probes.
Inventors: |
Mockel, Bettina;
(Dusseldorf, DE) ; Hermann, Thoams; (Bielefeld,
DE) ; Farwick, Mike; (Bielefeld, DE) ; Binder,
Michael; (Steinhagen, DE) ; Pfefferle, Walter;
(Halle, DE) |
Correspondence
Address: |
PILLSBURY WINTHROP SHAW PITTMAN, LLP
P.O. BOX 10500
MCLEAN
VA
22102
US
|
Assignee: |
Degussa AG
|
Family ID: |
27437871 |
Appl. No.: |
11/137504 |
Filed: |
May 26, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11137504 |
May 26, 2005 |
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09935757 |
Aug 24, 2001 |
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6913908 |
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60295009 |
Jun 4, 2001 |
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Current U.S.
Class: |
435/106 ;
435/193; 435/252.3; 435/471; 536/23.2 |
Current CPC
Class: |
C12N 1/205 20210501;
C12R 2001/15 20210501; C07K 14/34 20130101; C12P 13/08
20130101 |
Class at
Publication: |
435/106 ;
435/193; 435/252.3; 435/471; 536/023.2 |
International
Class: |
C12P 013/04; C07H
021/04; C12N 009/10; C12N 009/12; C12N 015/74 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 2, 2000 |
DE |
100 43 336.7 |
May 31, 2001 |
DE |
101 26 422.4 |
Claims
1-20. (canceled)
21. An isolated nucleic acid comprising a nucleotide sequence
selected from the group consisting of: (a) a nucleotide sequence as
set forth in SEQ ID NO: 1; (b) a nucleotide sequence encoding the
polypeptide as set forth in SEQ ID NO: 2; and (c) a nucleotide
sequence fully complementary to (a) or (b).
22. A vector comprising the nucleic acid of claim 21.
23. A bacterium comprising the vector of claim 22, wherein said
bacterium is an E. coli or coryneform bacterium.
24. A recombinant Corynebacterium glutamicum comprising an
overexpressed polynucleotide encoding a polypeptide having the
amino acid sequence of SEQ ID NO: 2, wherein overexpression is
achieved by increasing the copy number of said polynucleotide or by
operably linking promoter to said polynucleotide.
25. An isolated nucleic acid that encodes a polypeptide comprising
an amino acid sequence that is at least 95% identical to the amino
acid sequence of SEQ ID NO: 2, wherein said polypeptide has a
biological activity of a sigma factor E protein.
26. A vector comprising the nucleic acid of claim 25.
27. A host cell comprising the vector of claim 26.
28. An isolated polynucleotide primer or probe of a nucleic acid
fragment, wherein said fragment consists of at least 30 consecutive
nucleotides from SEQ ID NO: 1 or the full complement of said
fragment.
29. An isolated primer or probe consisting of a nucleic acid
fragment, wherein said fragment consists of at least 40 consecutive
nucleotides from SEQ ID NO: 1 or the full complement of said
fragment.
30. The shuttle vector pEC-T 18mob2sigEexp having (a) a 1930 bp
nucleic acid fragment of SEQ ID NO: 4 which harbors the C.
glutamicum gene; and (b) a restriction map as set forth in FIG.
2.
31. The shuttle vector of claim 30, wherein the vector has been
deposited in Corynebacterium glutamicum strain
DSM5715/pEC-T18mob2sigEexp under accession no. DSM 14229.
32. An isolated polynucleotide comprising nucleotides 236 to 907 of
SEQ ID NO: 1.
Description
FIELD OF THE INVENTION
[0001] The invention provides nucleotide sequences from coryneform
bacteria which code for the sigE gene and a process for the
fermentative preparation of amino acids using bacteria in which the
sigE gene is enhanced.
PRIOR ART
[0002] L-Amino acids are used in human medicine and in the
pharmaceuticals industry, in the foodstuffs industry and especially
in animal nutrition.
[0003] It is known that amino acids are prepared by fermentation
from strains of coryneform bacteria, in particular Corynebacterium
glutamicum. Because of their great importance, work is constantly
being undertaken to improve the preparation processes. Improvements
to the process can relate to fermentation measures, such as, for
example, stirring and supply of oxygen, or the composition of the
nutrient media, such as, for example, the sugar concentration
during the fermentation, or the working up to the product form by,
for example, ion exchange chromatography, or the intrinsic output
properties of the microorganism itself.
[0004] Methods of mutagenesis, selection and mutant selection are
used to improve the output properties of these microorganisms.
Strains which are resistant to antimetabolites or are auxotrophic
for metabolites of regulatory importance and produce amino acids
are obtained in this manner.
[0005] Methods of the recombinant DNA technique have also been
employed for some years for improving the strain of Corynebacterium
strains which produce L-amino acid, by amplifying individual amino
acid biosynthesis genes and investigating the effect on the amino
acid production.
OBJECT OF THE INVENTION
[0006] The inventors had the object of providing new measures for
improved fermentative preparation of amino acids.
SUMMARY OF THE INVENTION
[0007] Where L-amino acids or amino acids are mentioned in the
following, this means one or more amino acids, including their
salts, chosen from the group consisting of L-asparagine,
L-threonine, L-serine, L-glutamate, L-glycine, L-alanine,
L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine,
L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan
and L-arginine. Lysine is particularly preferred.
[0008] The invention provides an isolated polynucleotide from
coryneform bacteria, comprising a polynucleotide sequence which
codes for the sigE gene, chosen from the group consisting of
[0009] a) polynucleotide which is identical to the extent of at
least 70% to a polynucleotide which codes for a polypeptide which
comprises the amino acid sequence of SEQ ID No. 2,
[0010] b) polynucleotide which codes for a polypeptide which
comprises an amino acid sequence which is identical to the extent
of at least 70% to the amino acid sequence of SEQ ID No. 2,
[0011] c) polynucleotide which is complementary to the
polynucleotides of a) or b), and
[0012] d) polynucleotide comprising at least 15 successive
nucleotides of the polynucleotide sequence of a), b) or c),
[0013] the polypeptide preferably having the activity of sigma
factor E.
[0014] The invention also provides the above-mentioned
polynucleotide, this preferably being a DNA which is capable of
replication, comprising:
[0015] (i) the nucleotide sequences shown in SEQ ID No. 1, SEQ ID
NO. 3 or 4, or
[0016] (ii) at least one sequence which corresponds to sequence (i)
within the range of the degeneration of the genetic code, or
[0017] (iii) at least one sequence which hybridizes with the
sequence complementary to sequence (i) or (ii), and optionally
[0018] (iv) sense mutations of neutral function in (i).
[0019] The invention also provides
[0020] a polynucleotide, in particular DNA, which is capable of
replication and comprises the nucleotide sequence as shown in SEQ
ID No. 1;
[0021] a polynucleotide which codes for a polypeptide which
comprises the amino acid sequence as shown in SEQ ID No. 2;
[0022] a vector containing the polynucleotide according to the
invention, in particular a shuttle vector or plasmid vector,
and
[0023] coryneform bacteria which contain the vector or in which the
sigE gene is enhanced.
[0024] The invention also provides polynucleotides which
substantially comprise a polynucleotide sequence, which are
obtainable by screening by means of hybridization of a
corresponding gene library of a coryneform bacterium, which
comprises the complete gene or parts thereof, with a probe which
comprises the sequence of the polynucleotide according to the
invention according to SEQ ID No. 1 or a fragment thereof, and
isolation of the polynucleotide sequence mentioned.
DETAILED DESCRIPTION OF THE INVENTION
[0025] Polynucleotides which comprise the sequences according to
the invention are suitable as hybridization probes for RNA, cDNA
and DNA, in order to isolate, in the full length, nucleic acids or
polynucleotides or genes which code for sigma factor E or to
isolate those nucleic acids or polynucleotides or genes which have
a high similarity of sequence with that of the sigE gene.
[0026] Polynucleotides which comprise the sequences according to
the invention are furthermore suitable as primers with the aid of
which DNA of genes which code for sigma factor E can be prepared by
the polymerase chain reaction (PCR).
[0027] Such oligonucleotides which serve as probes or primers
comprise at least 30, preferably at least 20, very particularly
preferably at least 15 successive nucleotides. Oligonucleotides
which have a length of at least 40 or 50 nucleotides are also
suitable.
[0028] "Isolated" means separated out of its natural
environment.
[0029] "Polynucleotide" in general relates to polyribonucleotides
and polydeoxyribonucleotides, it being possible for these to be
non-modified RNA or DNA or modified RNA or DNA.
[0030] The polynucleotides according to the invention include a
polynucleotide according to SEQ ID No. 1 or a fragment prepared
therefrom and also those which are at least 70%, preferably at
least 80% and in particular at least 90% to 95% identical to the
polynucleotide according to SEQ ID No. 1 or a fragment prepared
therefrom.
[0031] "Polypeptides" are understood as meaning peptides or
proteins which comprise two or more amino acids bonded via peptide
bonds.
[0032] The polypeptides according to the invention include a
polypeptide according to SEQ ID No. 2, in particular those with the
biological activity of sigma factor E, and also those which are at
least 70%, preferably at least 80% and in particular at least 90%
to 95% identical to the polypeptide according to SEQ ID No. 2 and
have the activity mentioned.
[0033] The invention furthermore relates to a process for the
fermentative preparation of amino acids chosen from the group
consisting of L-asparagine, L-threonine, L-serine, L-glutamate,
L-glycine, L-alanine, L-cysteine, L-valine, L-methionine,
L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine,
L-lysine, L-tryptophan and L-arginine using coryneform bacteria
which in particular already produce amino acids and in which the
nucleotide sequences which code for the sigE gene are enhanced, in
particular over-expressed.
[0034] The term "enhancement" in this connection describes the
increase in the intracellular activity of one or more enzymes in a
microorganism which are coded by the corresponding DNA, for example
by increasing the number of copies of the gene or allele or of the
genes or alleles, using a potent promoter or using a gene or allele
which codes for a corresponding enzyme having a high activity, and
optionally combining these measures.
[0035] The microorganisms which the present invention provides can
produce L-amino acids from glucose, sucrose, lactose, fructose,
maltose, molasses, starch, cellulose or from glycerol and ethanol.
They can be representatives of coryneform bacteria, in particular
of the genus Corynebacterium. Of the genus Corynebacterium, there
may be mentioned in particular the species Corynebacterium
glutamicum, which is known among experts for its ability to produce
L-amino acids.
[0036] Suitable strains of the genus Corynebacterium, in particular
of the species Corynebacterium glutamicum (C. glutamicum), are in
particular the known wild-type strains
[0037] Corynebacterium glutamicum ATCC13032
[0038] Corynebacterium acetoglutamicum ATCC15806
[0039] Corynebacterium acetoacidophilum ATCC13870
[0040] Corynebacterium thermoaminogenes FERM BP-1539
[0041] Corynebacterium melassecola ATCC17965.
[0042] Brevibacterium flavum ATCC14067
[0043] Brevibacterium lactofermentum ATCC13869 and
[0044] Brevibacterium divaricatum ATCC14020
[0045] and L-amino acid-producing mutants or strains prepared
therefrom.
[0046] The new sigE gene from C. glutamicum which codes for the
enzyme sigma factor E has been isolated.
[0047] To isolate the sigE gene or also other genes of C.
glutamicum, a gene library of this microorganism is first set up in
Escherichia coli (E. coli). The setting up of gene libraries is
described in generally known textbooks and handbooks. The textbook
by Winnacker: Gene und Klone, Eine Einfuhrung in die Gentechnologie
(Verlag Chemie, Weinheim, Germany, 1990), or the handbook by
Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold
Spring Harbor Laboratory Press, 1989) may be mentioned as an
example. A well-known gene library is that of the E. coli K-12
strain W3110 set up in .lambda. vectors by Kohara et al. (Cell 50,
495-508 (1987)). Bathe et al. (Molecular and General Genetics,
252:255-265, 1996) describe a gene library of C. glutamicum
ATCC13032, which was set up with the aid of the cosmid vector
SuperCos I (Wahl et al., 1987, Proceedings of the National Academy
of Sciences USA, 84:2160-2164) in the E. coli K-12 strain NM554
(Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575).
[0048] Bormann et al. (Molecular Microbiology 6 (3), 317-326)
(1992)) in turn describe a gene library of C. glutamicum ATCC13032
using the cosmid pHC79 (Hohn and Collins, Gene 11, 291-298
(1980)).
[0049] To prepare a gene library of C. glutamicum in E. coli it is
also possible to use plasmids such as pBR322 (Bolivar, Life
Sciences, 25, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene,
19:259-268). Suitable hosts are, in particular, those E. coli
strains which are restriction- and recombination-defective. An
example of these is the strain DH5.alpha.mcr, which has been
described by Grant et al. (Proceedings of the National Academy of
Sciences USA, 87 (1990) 4645-4649). The long DNA fragments cloned
with the aid of cosmids can in turn be subcloned in the usual
vectors suitable for sequencing and then sequenced, as is described
e.g. by Sanger et al. (Proceedings of the National Academy of
Sciences of the United States of America, 74:5463-5467, 1977).
[0050] The resulting DNA sequences can then be investigated with
known algorithms or sequence analysis programs, such as e.g. that
of Staden (Nucleic Acids Research 14, 217-232 (1986)), that of
Marck (Nucleic Acids Research 16, 1829-1836 (1988)) or the GCG
program of Butler (Methods of Biochemical Analysis 39, 74-97
(1998)).
[0051] The new DNA sequence of C. glutamicum which codes for the
sigE gene and which, as SEQ ID No. 1, is a constituent of the
present invention has been found. The amino acid sequence of the
corresponding protein has furthermore been derived from the present
DNA sequence by the methods described above. The resulting amino
acid sequence of the sigE gene product is shown in SEQ ID No.
2.
[0052] Coding DNA sequences which result from SEQ ID No. 1 by the
degeneracy of the genetic code are also a constituent of the
invention. In the same way, DNA sequences which hybridize with SEQ
ID No. 1 or parts of SEQ ID No. 1 are a constituent of the
invention. Conservative amino acid exchanges, such as e.g. exchange
of glycine for alanine or of aspartic acid for glutamic acid in
proteins, are furthermore known among experts as "sense mutations"
which do not lead to a fundamental change in the activity of the
protein, i.e. are of neutral function. It is furthermore known that
changes on the N and/or C terminus of a protein cannot
substantially impair or can even stabilize the function thereof.
Information in this context can be found by the expert, inter alia,
in Ben-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)),
in O'Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth et al.
(Protein Sciences 3:240-247 (1994)), in Hochuli et al.
(Bio/Technology 6:1321-1325 (1988)) and in known textbooks of
genetics and molecular biology. Amino acid sequences which result
in a corresponding manner from SEQ ID No. 2 are also a constituent
of the invention.
[0053] In the same way, DNA sequences which hybridize with SEQ ID
No. 1 or parts of SEQ ID No. 1 are a constituent of the invention.
Finally, DNA sequences which are prepared by the polymerase chain
reaction (PCR) using primers which result from SEQ ID No. 1 are a
constituent of the invention. Such oligonucleotides typically have
a length of at least 15 nucleotides.
[0054] Instructions for identifying DNA sequences by means of
hybridization can be found by the expert, inter alia, in the
handbook "The DIG System Users Guide for Filter Hybridization" from
Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et
al. (International Journal of Systematic Bacteriology (1991) 41:
255-260). The hybridization takes place under stringent conditions,
that is to say only hybrids in which the probe and target sequence,
i.e. the polynucleotides treated with the probe, are at least 70%
identical are formed. It is known that the stringency of the
hybridization, including the washing steps, is influenced or
determined by varying the buffer composition, the temperature and
the salt concentration. The hybridization reaction is preferably
carried out under a relatively low stringency compared with the
washing steps (Hybaid Hybridisation Guide, Hybaid Limited,
Teddington, UK, 1996).
[0055] A 5.times.SSC buffer at a temperature of approx.
50-68.degree. C., for example, can be employed for the
hybridization reaction. Probes can also hybridize here with
polynucleotides which are less than 70% identical to the sequence
of the probe. Such hybrids are less stable and are removed by
washing under stringent conditions. This can be achieved, for
example, by lowering the salt concentration to 2.times.SSC and
optionally subsequently 0.5.times.SSC (The DIG System User's Guide
for Filter Hybridisation, Boehringer Mannheim, Mannheim, Germany,
1995) a temperature of approx. 50-68.degree. C. being established.
It is optionally possible to lower the salt concentration to
0.1.times.SSC. Polynucleotide fragments which are, for example, at
least 70% or at least 80% or at least 90% to 95% identical to the
sequence of the probe employed can be isolated by increasing the
hybridization temperature stepwise from 50 to 68.degree. C. in
steps of approx. 1-2.degree. C. Further instructions on
hybridization are obtainable on the market in the form of so-called
kits (e.g. DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim,
Germany, Catalogue No. 1603558).
[0056] Instructions for amplification of DNA sequences with the aid
of the polymerase chain reaction (PCR) can be found by the expert,
inter alia, in the handbook by Gait: Oligonucleotide Synthesis: A
Practical Approach (IRL Press, Oxford, UK, 1984) and in Newton and
Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany,
1994).
[0057] In the work on the present invention, it has been found that
coryneform bacteria produce amino acids in an improved manner after
over-expression of the sigE gene.
[0058] To achieve an over-expression, the number of copies of the
corresponding genes can be increased, or the promoter and
regulation region or the ribosome binding site upstream of the
structural gene can be mutated. Expression cassettes which are
incorporated upstream of the structural gene act in the same way.
By inducible promoters, it is additionally possible to increase the
expression in the course of fermentative amino acid production. The
expression is likewise improved by measures to prolong the life of
the m-RNA. Furthermore, the enzyme activity is also increased by
preventing the degradation of the enzyme protein. The genes or gene
constructs can either be present in plasmids with a varying number
of copies, or can be integrated and amplified in the chromosome.
Alternatively, an over-expression of the genes in question can
furthermore be achieved by changing the composition of the media
and the culture procedure.
[0059] Instructions in this context can be found by the expert,
inter alia, in Martin et al. (Bio/Technology 5, 137-146 (1987)), in
Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga
(Bio/Technology 6, 428-430 (1988)), in Eikmanns et al. (Gene 102,
93-98 (1991)), in EP 0 472 869, in U.S. Pat. No. 4,601,893, in
Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991), in Reinscheid
et al. (Applied and Environmental Microbiology 60, 126-132 (1994)),
in LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)),
in WO 96/15246, in Malumbres et al. (Gene 134, 15-24 (1993)), in
JP-A-10-229891, in Jensen and Hammer (Biotechnology and
Bioengineering 58, 191-195 (1998)), in Makrides (Microbiological
Reviews 60:512-538 (1996)) and in known textbooks of genetics and
molecular biology.
[0060] By way of example, for enhancement the sigE gene according
to the invention was over-expressed with the aid of episomal
plasmids. Suitable plasmids are those which are replicated in
coryneform bacteria. Numerous known plasmid vectors, such as e.g.
pZ1 (Menkel et al., Applied and Environmental Microbiology (1989)
64: 549-554), pEKEx1 (Eikmanns et al., Gene 102:93-98 (1991)) or
pHS2-1 (Sonnen et al., Gene 107:69-74 (1991)) are based on the
cryptic plasmids pHM1519, pBL1 or pGA1. Other plasmid vectors, such
as e.g. those based on pCG4 (U.S. Pat. No. 4,489,160), or pNG2
(Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124
(1990)), or pAG1 (U.S. Pat. No. 5,158,891), can be used in the same
manner.
[0061] Plasmid vectors which are furthermore suitable are also
those with the aid of which the process of gene amplification by
integration into the chromosome can be used, as has been described,
for example, by Reinscheid et al. (Applied and Environmental
Microbiology 60, 126-132 (1994)) for duplication or amplification
of the hom-thrB operon. In this method, the complete gene is cloned
in a plasmid vector which can replicate in a host (typically E.
coli), but not in C. glutamicum. Possible vectors are, for example,
pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)), pK18mob
or pK19mob (Schfer et al., Gene 145, 69-73 (1994)), pGEM-T (Promega
corporation, Madison, Wis., USA), pCR2.1-TOPO (Shuman (1994).
Journal of Biological Chemistry 269:32678-84; U.S. Pat. No.
5,487,993), pCR.RTM.Blunt (Invitrogen, Groningen, Holland; Bernard
et al., Journal of Molecular Biology, 234: 534-541 (1993)), pEM1
(Schrumpf et al, 1991, Journal of Bacteriology 173:4510-4516) or
pBGS8 (Spratt et al., 1986, Gene 41: 337-342). The plasmid vector
which contains the gene to be amplified is then transferred into
the desired strain of C. glutamicum by conjugation or
transformation. The method of conjugation is described, for
example, by Schfer et al. (Applied and Environmental Microbiology
60, 756-759 (1994)). Methods for transformation are described, for
example, by Thierbach et al. (Applied Microbiology and
Biotechnology 29, 356-362 (1988)), Dunican and Shivnan
(Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS
Microbiological Letters 123, 343-347 (1994)). After homologous
recombination by means of a "cross over" event, the resulting
strain contains at least two copies of the gene in question.
[0062] In addition, it may be advantageous for the production of
L-amino acids to enhance, in particular over-express one or more
enzymes of the particular biosynthesis pathway, of glycolysis, of
anaplerosis, of the citric acid cycle, of the pentose phosphate
cycle, of amino acid export and optionally regulatory proteins, in
addition to the sigE gene.
[0063] Thus, for example, for the preparation of L-amino acids, in
addition to enhancement of the sigE gene, one or more genes chosen
from the group consisting of
[0064] the dapA gene which codes for dihydrodipicolinate synthase
(EP-B 0 197 335),
[0065] the gap gene which codes for glyceraldehyde 3-phosphate
dehydrogenase (Eikmanns (1992), Journal of Bacteriology
174:6076-6086),
[0066] the tpi gene which codes for triose phosphate isomerase
(Eikmanns (1992), Journal of Bacteriology 174:6076-6086),
[0067] the pgk gene which codes for 3-phosphoglycerate kinase
(Eikmanns (1992), Journal of Bacteriology 174:6076-6086),
[0068] the zwf gene which codes for glucose 6-phosphate
dehydrogenase (JP-A-09224661),
[0069] the pyc gene which codes for pyruvate carboxylase (DE-A-198
31 609),
[0070] the mqo gene which codes for malate-quinone oxidoreductase
(Molenaar et al., European Journal of Biochemistry 254, 395-403
(1998)),
[0071] the lysC gene which codes for a feed-back resistant
aspartate kinase (Accession No. P26512; EP-A-0699759),
[0072] the lysE gene which codes for lysine export (DE-A-195 48
222),
[0073] the hom gene which codes for homoserine dehydrogenase (EP-A
0131171),
[0074] the ilvA gene which codes for threonine dehydratase (Mockel
et al., Journal of Bacteriology (1992) 8065-8072)) or the ilvA(Fbr)
allele which codes for a "feed back resistant" threonine
dehydratase (Mockel et al., (1994) Molecular Microbiology 13:
833-842),
[0075] the ilvBN gene which codes for acetohydroxy-acid synthase
(EP-B 0356739),
[0076] the ilvD gene which codes for dihydroxy-acid dehydratase
(Sahm and Eggeling (1999) Applied and Environmental Microbiology
65: 1973-1979),
[0077] the zwa1 gene which codes for the Zwa1 protein (DE:
19959328.0, DSM 13115)
[0078] can be enhanced, in particular over-expressed.
[0079] It may furthermore be advantageous for the production of
L-amino acids, in addition to the enhancement of the sigE gene, for
one or more of the genes chosen from the group consisting of:
[0080] the pck gene which codes for phosphoenol pyruvate
carboxykinase (DE 199 50 409.1; DSM 13047),
[0081] the pgi gene which codes for glucose 6-phosphate isomerase
(U.S. Ser. No. 09/396,478; DSM 12969),
[0082] the poxB gene which codes for pyruvate oxidase (DE: 1995
1975.7; DSM 13114),
[0083] the zwa2 gene which codes for the Zwa2 protein (DE:
19959327.2, DSM 13113)
[0084] to be attenuated, in particular for the expression thereof
to be reduced.
[0085] In addition to over-expression of the sigE gene it may
furthermore be advantageous for the production of amino acids to
eliminate undesirable side reactions (Nakayama: "Breeding of Amino
Acid Producing Micro-organisms", in: Overproduction of Microbial
Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London,
UK, 1982).
[0086] The invention also provides the microorganisms prepared
according to the invention, and these can be cultured continuously
or discontinuously in the batch process (batch culture) or in the
fed batch (feed process) or repeated fed batch process (repetitive
feed process) for the purpose of production of amino acids. A
summary of known culture methods is described in the textbook by
Chmiel (Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik
(Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by
Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag,
Braunschweig/Wiesbaden, 1994)).
[0087] The culture medium to be used must meet the requirements of
the particular strains in a suitable manner. Descriptions of
culture media for various microorganisms are contained in the
handbook "Manual of Methods for General Bacteriology" of the
American Society for Bacteriology (Washington D.C., USA, 1981).
[0088] Sugars and carbohydrates, such as e.g. glucose, sucrose,
lactose, fructose, maltose, molasses, starch and cellulose, oils
and fats, such as e.g. soya oil, sunflower oil, groundnut oil and
coconut fat, fatty acids, such as e.g. palmitic acid, stearic acid
and linoleic acid, alcohols, such as e.g. glycerol and ethanol, and
organic acids, such as e.g. acetic acid, can be used as the source
of carbon. These substances can be used individually or as a
mixture.
[0089] Organic nitrogen-containing compounds, such as peptones,
yeast extract, meat extract, malt extract, corn steep liquor, soya
bean flour and urea, or inorganic compounds, such as ammonium
sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate
and ammonium nitrate, can be used as the source of nitrogen. The
sources of nitrogen can be used individually or as a mixture.
[0090] Phosphoric acid, potassium dihydrogen phosphate or
dipotassium hydrogen phosphate or the corresponding
sodium-containing salts can be used as the source of phosphorus.
The culture medium must furthermore comprise salts of metals, such
as e.g. magnesium sulfate or iron sulfate, which are necessary for
growth. Finally, essential growth substances, such as amino acids
and vitamins, can be employed in addition to the above-mentioned
substances. Suitable precursors can moreover be added to the
culture medium. The starting substances mentioned can be added to
the culture in the form of a single batch, or can be fed in during
the culture in a suitable manner.
[0091] Basic compounds, such as sodium hydroxide, potassium
hydroxide, ammonia or aqueous ammonia, or acid compounds, such as
phosphoric acid or sulfuric acid, can be employed in a suitable
manner to control the pH of the culture. Antifoams, such as e.g.
fatty acid polyglycol esters, can be employed to control the
development of foam. Suitable substances having a selective action,
such as e.g. antibiotics, can be added to the medium to maintain
the stability of plasmids. To maintain aerobic conditions, oxygen
or oxygen-containing gas mixtures, such as e.g. air, are introduced
into the culture. The temperature of the culture is usually
20.degree. C. to 45.degree. C., and preferably 25.degree. C. to
40.degree. C. Culturing is continued until a maximum of the desired
product has formed. This target is usually reached within 10 hours
to 160 hours.
[0092] Methods for the determination of L-amino acids are known
from the prior art. The analysis can thus be carried out, for
example, as described by Spackman et al. (Analytical Chemistry, 30,
(1958), 1190) by ion exchange chromatography with subsequent
ninhydrin derivation, or it can be carried out by reversed phase
HPLC, for example as described by Lindroth et al. (Analytical
Chemistry (1979) 51: 1167-1174).
[0093] The process according to the invention is used for
fermentative preparation of amino acids.
[0094] The following microorganism was deposited as a pure culture
on 11th Apr. 2001 at the Deutsche Sammlung fur Mikroorganismen und
Zellkulturen (DSMZ=German Collection of Microorganisms and Cell
Cultures, Braunschweig, Germany) in accordance with the Budapest
Treaty:
[0095] Corynebacterium glutamicum DSM5715/pEC-T18mob2sigEexp as DSM
14229.
[0096] The present invention is explained in more detail in the
following with the aid of embodiment examples.
[0097] The isolation of plasmid DNA from Escherichia coli and all
techniques of restriction, Klenow and alkaline phosphatase
treatment were carried out by the method of Sambrook et al.
(Molecular Cloning. A Laboratory Manual (1989) Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., USA). Methods for
transformation of Escherichia coli are also described in this
handbook.
[0098] The composition of the usual nutrient media, such as LB or
TY medium, can also be found in the handbook by Sambrook et al.
EXAMPLE 1
[0099] Preparation of a Genomic Cosmid Gene Library from
Corynebacterium glutamicum ATCC 13032
[0100] Chromosomal DNA from Corynebacterium glutamicum ATCC 13032
was isolated as described by Tauch et al. (1995, Plasmid
33:168-179) and partly cleaved with the restriction enzyme Sau3AI
(Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI,
Code no. 27-0913-02). The DNA fragments were dephosphorylated with
shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim,
Germany, Product Description SAP, Code no. 1758250). The DNA of the
cosmid vector SuperCos1 (Wahl et al. (1987) Proceedings of the
National Academy of Sciences USA 84:2160-2164), obtained from
Stratagene (La Jolla, USA, Product Description SuperCos1 Cosmid
Vector Kit, Code no. 251301) was cleaved with the restriction
enzyme XbaI (Amersham Pharmacia, Freiburg, Germany, Product
Description XbaI, Code no. 27-0948-02) and likewise
dephosphorylated with shrimp alkaline phosphatase.
[0101] The cosmid DNA was then cleaved with the restriction enzyme
BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description
BamHI, Code no. 27-0868-04). The cosmid DNA treated in this manner
was mixed with the treated ATCC13032 DNA and the batch was treated
with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product
Description T4-DNA-Ligase, Code no. 27-0870-04). The ligation
mixture was then packed in phages with the aid of Gigapack II XL
Packing Extract (Stratagene, La Jolla, USA, Product Description
Gigapack II XL Packing Extract, Code no. 200217).
[0102] For infection of the E. coli strain NM554 (Raleigh et al.
1988, Nucleic Acid Research 16:1563-1575) the cells were taken up
in 10 mM MgSO.sub.4 and mixed with an aliquot of the phage
suspension. The infection and titering of the cosmid library were
carried out as described by Sambrook et al. (1989, Molecular
Cloning: A laboratory Manual, Cold Spring Harbor), the cells being
plated out on LB agar (Lennox, 1955, Virology, 1:190) with 100 mg/l
ampicillin. After incubation overnight at 37.degree. C.,
recombinant individual clones were selected.
EXAMPLE 2
[0103] Isolation and Sequencing of the sigE Gene
[0104] The cosmid DNA of an individual colony was isolated with the
Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden,
Germany) in accordance with the manufacturer's instructions and
partly cleaved with the restriction enzyme Sau3AI (Amersham
Pharmacia, Freiburg, Germany, Product Description Sau3AI, Product
No. 27-0913-02). The DNA fragments were dephosphorylated with
shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim,
Germany, Product Description SAP, Product No. 1758250). After
separation by gel electrophoresis, the cosmid fragments in the size
range of 1500 to 2000 bp were isolated with the QiaExII Gel
Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany).
[0105] The DNA of the sequencing vector pZero-1, obtained from
Invitrogen (Groningen, Holland, Product Description Zero Background
Cloning Kit, Product No. K2500-01), was cleaved with the
restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany,
Product Description BamHI, Product No. 27-0868-04). The ligation of
the cosmid fragments in the sequencing vector pZero-1 was carried
out as described by Sambrook et al. (1989, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor), the DNA mixture being
incubated overnight with T4 ligase (Pharmacia Biotech, Freiburg,
Germany). This ligation mixture was then electroporated (Tauch et
al. 1994, FEMS Microbiol Letters, 123:343-7) into the E. coli
strain DH5.alpha.MCR (Grant, 1990, Proceedings of the National
Academy of Sciences U.S.A., 87:4645-4649) and plated out on LB agar
(Lennox, 1955, Virology, 1:190) with 50 mg/l zeocin.
[0106] The plasmid preparation of the recombinant clones was
carried out with the Biorobot 9600 (Product No. 900200, Qiagen,
Hilden, Germany). The sequencing was carried out by the dideoxy
chain termination method of Sanger et al. (1977, Proceedings of the
National Academy of Sciences U.S.A., 74:5463-5467) with
modifications according to Zimmermann et al. (1990, Nucleic Acids
Research, 18:1067). The "RR dRhodamin Terminator Cycle Sequencing
Kit" from PE Applied Biosystems (Product No. 403044, Weiterstadt,
Germany) was used. The separation by gel electrophoresis and
analysis of the sequencing reaction were carried out in a
"Rotiphoresis NF Acrylamide/Bisacrylamide" Gel (29:1) (Product No.
A124.1, Roth, Karlsruhe, Germany) with the "ABI Prism 377"
sequencer from PE Applied Biosystems (Weiterstadt, Germany).
[0107] The raw sequence data obtained were then processed using the
Staden program package (1986, Nucleic Acids Research, 14:217-231)
version 97-0. The individual sequences of the pZero1 derivatives
were assembled to a continuous contig. The computer-assisted coding
region analysis was prepared with the XNIP program (Staden, 1986,
Nucleic Acids Research, 14:217-231).
[0108] The resulting nucleotide sequence is shown in SEQ ID No. 1.
Analysis of the nucleotide sequence showed an open reading frame of
651 base pairs, which was called the sigE gene. The sigE gene codes
for a protein of 216 amino acids (SEQ ID NO. 2).
[0109] The DNA sections lying upstream and downstream of SEQ ID NO.
1, which are shown in SEQ ID NO. 3 and SEQ ID NO. 4, were
identified in the same manner. The sigE gene region extended by SEQ
ID NO. 3 and SEQ ID NO. 4 is shown in SEQ ID NO. 5.
EXAMPLE 3
[0110] Preparation of a Shuttle Vector pEC-T18mob2sigEexp for
Enhancement of the sigE Gene in C. glutamicum
[0111] 3.1. Cloning of the sigE Gene
[0112] From the strain ATCC 13032, chromosomal DNA was isolated by
the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)).
On the basis of the sequence of the sigE gene known for C.
glutamicum from example 2, the following oligonucleotides were
chosen for the polymerase chain reaction (see SEQ ID No. 7 and SEQ
ID No. 8).
1 sigE1: 5'TAG TCA CCA CGG TTA AGC CT 3' sigE2: 5'GCC TTG GTT CTT
ACG AAC TG 3'
[0113] The primers shown were synthesized by ARK Scientific GmbH
Biosystems (Darmstadt, Germany) and the PCR reaction was carried
out by the standard PCR method of Innis et al. (PCR protocols. A
guide to methods and applications, 1990, Academic Press) with
Taq-Polymerase from Qiagen (Hilden, Germany). With the aid of the
polymerase chain reaction, the primers allow amplification of a DNA
fragment approx. 2.03 kb in size, which carries the sigE gene.
[0114] The amplified DNA fragment of approx. 2.03 kb in size which
carries the sigE gene was ligated with the TOPO TA Cloning.RTM. Kit
from Invitrogen Corporation (Carlsbad, Calif., USA) in the vector
pCR.RTM.2.1TOPO (Bernard et al., Journal of Molecular Biology, 234:
534-541 (1993)). The E. coli strain Top10 (Grant et al.,
Proceedings of the National Academy of Sciences USA, 87 (1990)
4645-4649) was then transformed with the ligation batch in
accordance with the instructions of the manufacturer of the kit
(Invitrogen Corporation, Carlsbad, Calif., USA). Selection of
plasmid-carrying cells was carried out by plating out the
transformation batch on LB Agar (Sambrook et al., Molecular
cloning: a laboratory manual. 2.sup.nd Ed. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989), which had been
supplemented with 50 mg/l kanamycin. Plasmid DNA was isolated from
a transformant with the aid of the QIAprep Spin Miniprep Kit from
Qiagen (Hilden, Germany) and checked by treatment with the
restriction enzyme SphI and EcoRI with subsequent agarose gel
electrophoresis (0.8%). The DNA sequence of the amplified DNA
fragment was checked by sequencing. The plasmid was called
pCR2.1sigEexp. The strain was called E. coli
Top10/pCR2.1sigEexp.
[0115] 3.2. Preparation of the E. coli-C. glutamicum Shuttle Vector
pEC-T18mob2
[0116] The E. coli-C. glutamicum shuttle vector was constructed
according to the prior art. The vector contains the replication
region reg of the plasmid pGA1 including the replication effector
per (U.S. Pat. No. 5,175,108; Nesvera et al., Journal of
Bacteriology 179, 1525-1532 (1997)), the tetracycline
resistance-imparting tetA(Z) gene of the plasmid pAG1 (U.S. Pat.
No. 5,158,891; gene library entry at the National Center for
Biotechnology Information (NCBI, Bethesda, Md., USA) with the
accession number AF121000), the replication region oriV of the
plasmid pMB1 (Sutcliffe, Cold Spring Harbor Symposium on
Quantitative Biology 43, 77-90 (1979)), the lacZ.alpha. gene
fragment including the lac promoter and a multiple cloning site
(mcs) (Norrander, J. M. et al. Gene 26, 101-106 (1983)) and the mob
region of the plasmid RP4 (Simon et al., (1983) Bio/Technology
1:784-791). The vector constructed was transformed in the E. coli
strain DH5.alpha. (Hanahan, In: DNA cloning. A Practical Approach.
Vol. I. IRL-Press, Oxford, Washington D.C., USA).
[0117] Selection for plasmid-carrying cells was made by plating out
the transformation batch on LB agar (Sambrook et al., Molecular
cloning: A Laboratory Manual. 2.sup.nd Ed. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.), which had been
supplemented with 5 mg/l tetracycline. Plasmid DNA was isolated
from a transformant with the aid of the QIAprep Spin Miniprep Kit
from Qiagen and checked by restriction with the restriction enzymes
EcoRI and HindIII and subsequent agarose gel electrophoresis
(0.8%). The plasmid was called pEC-T18mob2 and is shown in FIG.
1.
[0118] 3.3. Cloning of sigE in the E. coli-C. glutamicum Shuttle
Vector pEC-T18mob2
[0119] The E. coli-C. glutamicum shuttle vector pEC-T18mob2
described in example 3.2 was used as the vector. DNA of this
plasmid was cleaved completely with the restriction enzymes BamHI
and SalI and then dephosphorylated with shrimp alkaline phosphatase
(Roche Diagnostics GmbH, Mannheim, Germany, Product Description
SAP, Product No. 1758250).
[0120] The sigE gene was isolated from the plasmid pCR2.1sigEexp
described in example 3.1. by complete cleavage with the enzymes
BamHI and SalI. The sigE fragment 1930 bp in size was isolated from
the agarose gel with the QiaExII Gel Extraction Kit (Product No.
20021, Qiagen, Hilden, Germany).
[0121] The sigE fragment obtained in this manner was mixed with the
prepared vector pEC-T18mob2 and the batch was treated with T4 DNA
ligase (Amersham Pharmacia, Freiburg, Germany, Product Description
T4-DNA-Ligase, Code no. 27-0870-04). The ligation batch was
transformed in the E. coli strain DH5.alpha.MCR (Hanahan, In: DNA
cloning. A Practical Approach. Vol. I. IRL-Press, Oxford,
Washington D.C., USA). Selection of plasmid-carrying cells was made
by plating out the transformation batch on LB agar (Lennox, 1955,
Virology, 1:190) with 5 mg/l tetracycline. After incubation
overnight at 37.degree. C., recombinant individual clones were
selected. Plasmid DNA was isolated from a transformant with the
Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden,
Germany) in accordance with the manufacturer's instructions and
cleaved with the restriction enzymes BamHI and SalI to check the
plasmid by subsequent agarose gel electrophoresis. The plasmid
obtained was called pEC-T18mob2sigEexp. It is shown in FIG. 2.
EXAMPLE 4
[0122] Transformation of the Strain DSM5715 with the Plasmid
pEC-T18mob2sigEexp
[0123] The strain DSM5715 was transformed with the plasmid
pEC-T18mob2sigEexp using the electroporation method described by
Liebl et al., (FEMS Microbiology Letters, 53:299-303 (1989)).
Selection of the transformants took place on LBHIS agar comprising
18.5 g/l brain-heart infusion broth, 0.5 M sorbitol, 5 g/l
Bacto-tryptone, 2.5 g/l Bacto-yeast extract, 5 g/l NaCl and 18 g/l
Bacto-agar, which had been supplemented with 5 mg/l tetracycline.
Incubation was carried out for 2 days at 33.degree. C.
[0124] Plasmid DNA was isolated from a transformant by conventional
methods (Peters-Wendisch et al., 1998, Microbiology, 144, 915-927),
cleaved with the restriction endonucleases BamHI and SalI, and the
plasmid was checked by subsequent agarose gel electrophoresis. The
strain obtained was called DSM5715/pEC-T18mob2sigEexp.
EXAMPLE 5
[0125] Preparation of Lysine
[0126] The C. glutamicum strain DSM5715/pEC-T18mob2sigEexp obtained
in example 4 was cultured in a nutrient medium suitable for the
production of lysine and the lysine content in the culture
supernatant was determined.
[0127] For this, the strain was first incubated on an agar plate
with the corresponding antibiotic (brain-heart agar with
tetracycline (5 mg/l)) for 24 hours at 33.degree. C. Starting from
this agar plate culture, a pre-culture was seeded (10 ml medium in
a 100 ml conical flask). The complete medium CgIII was used as the
medium for the pre-culture.
Medium Cg III
[0128]
2 NaCl 2.5 g/l Bacto-Peptone 10 g/l Bacto-Yeast extract 10 g/l
Glucose (autoclaved separately) 2% (w/v)
[0129] The pH was brought to pH 7.4
[0130] Tetracycline (5 mg/l) was added to this. The pre-culture was
incubated for 16 hours at 33.degree. C. at 240 rpm on a shaking
machine. A main culture was seeded from this pre-culture such that
the initial OD (660 nm) of the main culture was 0.05. Medium MM was
used for the main culture.
Medium MM
[0131]
3 CSL (corn steep liquor) 5 g/l MOPS (morpholinopropanesulfonic
acid) 20 g/l Glucose (autoclaved separately) 50 g/l
(NH.sub.4).sub.2SO.sub.4 25 g/l KH.sub.2PO.sub.4 0.1 g/l MgSO.sub.4
* 7 H.sub.2O 1.0 g/l CaCl.sub.2 * 2 H.sub.2O 10 mg/l FeSO.sub.4 * 7
H.sub.2O 10 mg/l MnSO.sub.4 * H.sub.2O 5.0 mg/l Biotin
(sterile-filtered) 0.3 mg/l Thiamine * HCl (sterile-filtered) 0.2
mg/l L-Leucine (sterile-filtered) 0.1 g/l CaCO.sub.3 25 g/l
[0132] The CSL, MOPS and the salt solution were brought to pH 7
with aqueous ammonia and autoclaved. The sterile substrate and
vitamin solutions were then added, as well as the CaCO.sub.3
autoclaved in the dry state.
[0133] Culturing is carried out in a 10 ml volume in a 100 ml
conical flask with baffles. Tetracycline (5 mg/l) was added.
Culturing was carried out at 33.degree. C. and 80% atmospheric
humidity.
[0134] After 48 hours, the OD was determined at a measurement
wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH,
Munich). The amount of lysine formed was determined with an amino
acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion
exchange chromatography and post-column derivation with ninhydrin
detection.
[0135] The result of the experiment is shown in Table 1.
4 TABLE 1 OD Lysine HCl Strain (660 nm) g/l DSM5715/pEC-T18mob2
12.2 13.14 DSM5715/pEC- 13.07 14.09 T18mob2sigEexp
BRIEF DESCRIPTION OF THE FIGURES
[0136] FIG. 1: Map of the plasmid pEC-T18mob2.
[0137] FIG. 2: Map of the plasmid pEC-T18mob2sigEexp.
[0138] The abbreviations and designations used have the following
meaning.
[0139] per: Gene for controlling the number of copies from PGA1
[0140] oriV: ColE1-similar origin from pMB1
[0141] rep: Plasmid-coded replication region from C. glutamicum
plasmid pGA1
[0142] RP4mob: RP4 mobilization site
[0143] lacZ-alpha: lacZ gene fragment from E. coli
[0144] Tet: Resistance gene for tetracycline
[0145] sigE: sigE gene of C. glutamicum
[0146] BamHI: Cleavage site of the restriction enzyme BamHI
[0147] SalI: Cleavage site of the restriction enzyme SalI
[0148] sigE: sigE gene of C. glutamicum
[0149] BamHI: Cleavage site of the restriction enzyme BamHI
[0150] SalI: Cleavage site of the restriction enzyme SalI
Sequence CWU 1
1
8 1 1330 DNA Corynebacterium glutamicum CDS (302)..(949) sigE gene
1 accagtggag ccgttgccat tggtggtggc agccaaagtg gttagcagct ggccagtcat
60 ttcatccggg gcggggagac cgaactcggc ggcgtcttca cgagcgcgcg
ctacagcagc 120 gtcggtttca gtagtggact cgacataagt gcgaagatac
tcgaaggcgt tactcacgcg 180 ttatagtcta gagcgagcag gcgagatgtg
aagtacctac acgcattaag tgcaaatgaa 240 ttcacaattg ccagaagatg
cacaggatgt aatctagatt tcccaagttc agtggggcaa 300 a atg act tat atg
aaa aag aag tcc cga gat gac gca ccc gtc gta atc 349 Met Thr Tyr Met
Lys Lys Lys Ser Arg Asp Asp Ala Pro Val Val Ile 1 5 10 15 gaa acc
gtt caa gca gaa cat gct gaa gaa ctc acg ggc act gca gca 397 Glu Thr
Val Gln Ala Glu His Ala Glu Glu Leu Thr Gly Thr Ala Ala 20 25 30
ttc gat gct gga cag gca gac atg cca aca tgg ggc gag cta gtc gca 445
Phe Asp Ala Gly Gln Ala Asp Met Pro Thr Trp Gly Glu Leu Val Ala 35
40 45 gaa cat gca gat agc gtt tac cgc ctc gcg tac cgt ctt tcc ggc
aac 493 Glu His Ala Asp Ser Val Tyr Arg Leu Ala Tyr Arg Leu Ser Gly
Asn 50 55 60 cag cac gat gct gaa gac ctg acc caa gaa aca ttc atg
cgt gtc ttc 541 Gln His Asp Ala Glu Asp Leu Thr Gln Glu Thr Phe Met
Arg Val Phe 65 70 75 80 cgc tcg ttg aag agc tac cag cca ggc acc ttt
gag ggc tgg ctg cac 589 Arg Ser Leu Lys Ser Tyr Gln Pro Gly Thr Phe
Glu Gly Trp Leu His 85 90 95 cgc atc acc acc aac ttg ttc ctt gat
atg gtt cgc cac cgc ggc aag 637 Arg Ile Thr Thr Asn Leu Phe Leu Asp
Met Val Arg His Arg Gly Lys 100 105 110 atc cgc atg gag gcg ctg cct
gaa gat tat gag cgc gtt ccg ggc aat 685 Ile Arg Met Glu Ala Leu Pro
Glu Asp Tyr Glu Arg Val Pro Gly Asn 115 120 125 gac atc acc cca gag
cag gca tac acc gaa gct aac ctt gac cca gct 733 Asp Ile Thr Pro Glu
Gln Ala Tyr Thr Glu Ala Asn Leu Asp Pro Ala 130 135 140 ctg cag gca
gcc ctc gat gag ttg agc cca gac ttc cgc gtg gca gtg 781 Leu Gln Ala
Ala Leu Asp Glu Leu Ser Pro Asp Phe Arg Val Ala Val 145 150 155 160
atc ctc tgt gat gtt gtt ggt atg agc tat gac gaa atc gca gag acc 829
Ile Leu Cys Asp Val Val Gly Met Ser Tyr Asp Glu Ile Ala Glu Thr 165
170 175 ctc gga gtg aaa atg ggt acc gtg cgt tcc cgt att cac cgt gga
cgc 877 Leu Gly Val Lys Met Gly Thr Val Arg Ser Arg Ile His Arg Gly
Arg 180 185 190 agc cag ctt cgt gca agt ttg gaa gct gca gca atg acc
agc gag gaa 925 Ser Gln Leu Arg Ala Ser Leu Glu Ala Ala Ala Met Thr
Ser Glu Glu 195 200 205 gtt tct ttg ttg gtt cca acc cac taaagttggt
gtgttttctg acacgacaaa 979 Val Ser Leu Leu Val Pro Thr His 210 215
cgcaaatgtc gtgtcatttt tgcagctcag tgcattattt tggggttcgt ggtgcggaca
1039 gggaacttat cacaggcgac atccgttttg agtagtaggt atcttggata
agaagttacc 1099 cacatccttg aaagtcgaga cacaggaggt catcggaaga
tatgttcaat tccgacacca 1159 ccgcgaatct ccaagctaaa agtcgagatc
gtgcaggatc taaagcaaag cgcagcaggc 1219 caagttttga ttcagtagcg
cgggatgttt tggatgttcg aacaaaaaca gcacaagtta 1279 aaaacaaggc
taaagagttt tcctctgttg atcacctttc agcagacgcc g 1330 2 216 PRT
Corynebacterium glutamicum 2 Met Thr Tyr Met Lys Lys Lys Ser Arg
Asp Asp Ala Pro Val Val Ile 1 5 10 15 Glu Thr Val Gln Ala Glu His
Ala Glu Glu Leu Thr Gly Thr Ala Ala 20 25 30 Phe Asp Ala Gly Gln
Ala Asp Met Pro Thr Trp Gly Glu Leu Val Ala 35 40 45 Glu His Ala
Asp Ser Val Tyr Arg Leu Ala Tyr Arg Leu Ser Gly Asn 50 55 60 Gln
His Asp Ala Glu Asp Leu Thr Gln Glu Thr Phe Met Arg Val Phe 65 70
75 80 Arg Ser Leu Lys Ser Tyr Gln Pro Gly Thr Phe Glu Gly Trp Leu
His 85 90 95 Arg Ile Thr Thr Asn Leu Phe Leu Asp Met Val Arg His
Arg Gly Lys 100 105 110 Ile Arg Met Glu Ala Leu Pro Glu Asp Tyr Glu
Arg Val Pro Gly Asn 115 120 125 Asp Ile Thr Pro Glu Gln Ala Tyr Thr
Glu Ala Asn Leu Asp Pro Ala 130 135 140 Leu Gln Ala Ala Leu Asp Glu
Leu Ser Pro Asp Phe Arg Val Ala Val 145 150 155 160 Ile Leu Cys Asp
Val Val Gly Met Ser Tyr Asp Glu Ile Ala Glu Thr 165 170 175 Leu Gly
Val Lys Met Gly Thr Val Arg Ser Arg Ile His Arg Gly Arg 180 185 190
Ser Gln Leu Arg Ala Ser Leu Glu Ala Ala Ala Met Thr Ser Glu Glu 195
200 205 Val Ser Leu Leu Val Pro Thr His 210 215 3 457 DNA
Corynebacterium glutamicum upstream region 3 tagtcaccac ggttaagcct
gcaccaaggg gcaggcgagc aacgtgtgcg ccttcaatgg 60 aacgaatata
ttcatcggcg tcacgtgctg cttgggtgtc acgatccttg cgggtttgat 120
ccgcaatggt gccgtcaagg agcgcatcgg cgagcaccag cgcaccgcct cgtcgaagaa
180 gcggccaggc ggcgtcgaca agcgccttta aatccatggg ggagacttgg
ccgaagacaa 240 gctgatagct gtcgttggca aggcgactca tcacgtcgag
cgggcgcgag agcaagaagc 300 gtacgcggct gggggaatag ccggcctcgc
ggaagagtgc tttggcctgg cgctgatgct 360 ctgattcagg atcaatgcag
gtcagtgtgg tgttatcggc cagtccgttc aggatataca 420 gacccaccaa
cccggcagcc ggggtaatcg cgatggc 457 4 299 DNA Corynebacterium
glutamicum downstream region 4 cagccatgtt tgtagacaat gaactgtccc
gtggcgccat gcatcgcgcc aggctgcaca 60 ttgtgcactg cgctgaatgt
agggaagaga ttaaccgtca gcgggaaacc gttgattatc 120 tccgctcaga
gtgcaaaaac gaagaagtgt ccgccccaat ggacctcaaa gcacggcttg 180
ccagcctcgc cactgagtgc atgcctggcc ctggcgcaga gaatttagca atgcagcgcc
240 cagagtcttt tgtggctaaa gttgagtccg tagtgcgcgc agttcgtaag
aaccaaggc 299 5 2086 DNA Corynebacterium glutamicum CDS
(759)..(1406) sigE 5 tagtcaccac ggttaagcct gcaccaaggg gcaggcgagc
aacgtgtgcg ccttcaatgg 60 aacgaatata ttcatcggcg tcacgtgctg
cttgggtgtc acgatccttg cgggtttgat 120 ccgcaatggt gccgtcaagg
agcgcatcgg cgagcaccag cgcaccgcct cgtcgaagaa 180 gcggccaggc
ggcgtcgaca agcgccttta aatccatggg ggagacttgg ccgaagacaa 240
gctgatagct gtcgttggca aggcgactca tcacgtcgag cgggcgcgag agcaagaagc
300 gtacgcggct gggggaatag ccggcctcgc ggaagagtgc tttggcctgg
cgctgatgct 360 ctgattcagg atcaatgcag gtcagtgtgg tgttatcggc
cagtccgttc aggatataca 420 gacccaccaa cccggcagcc ggggtaatcg
cgatggcacc agtggagccg ttgccattgg 480 tggtggcagc caaagtggtt
agcagctggc cagtcatttc atccggggcg gggagaccga 540 actcggcggc
gtcttcacga gcgcgcgcta cagcagcgtc ggtttcagta gtggactcga 600
cataagtgcg aagatactcg aaggcgttac tcacgcgtta tagtctagag cgagcaggcg
660 agatgtgaag tacctacacg cattaagtgc aaatgaattc acaattgcca
gaagatgcac 720 aggatgtaat ctagatttcc caagttcagt ggggcaaa atg act
tat atg aaa aag 776 Met Thr Tyr Met Lys Lys 1 5 aag tcc cga gat gac
gca ccc gtc gta atc gaa acc gtt caa gca gaa 824 Lys Ser Arg Asp Asp
Ala Pro Val Val Ile Glu Thr Val Gln Ala Glu 10 15 20 cat gct gaa
gaa ctc acg ggc act gca gca ttc gat gct gga cag gca 872 His Ala Glu
Glu Leu Thr Gly Thr Ala Ala Phe Asp Ala Gly Gln Ala 25 30 35 gac
atg cca aca tgg ggc gag cta gtc gca gaa cat gca gat agc gtt 920 Asp
Met Pro Thr Trp Gly Glu Leu Val Ala Glu His Ala Asp Ser Val 40 45
50 tac cgc ctc gcg tac cgt ctt tcc ggc aac cag cac gat gct gaa gac
968 Tyr Arg Leu Ala Tyr Arg Leu Ser Gly Asn Gln His Asp Ala Glu Asp
55 60 65 70 ctg acc caa gaa aca ttc atg cgt gtc ttc cgc tcg ttg aag
agc tac 1016 Leu Thr Gln Glu Thr Phe Met Arg Val Phe Arg Ser Leu
Lys Ser Tyr 75 80 85 cag cca ggc acc ttt gag ggc tgg ctg cac cgc
atc acc acc aac ttg 1064 Gln Pro Gly Thr Phe Glu Gly Trp Leu His
Arg Ile Thr Thr Asn Leu 90 95 100 ttc ctt gat atg gtt cgc cac cgc
ggc aag atc cgc atg gag gcg ctg 1112 Phe Leu Asp Met Val Arg His
Arg Gly Lys Ile Arg Met Glu Ala Leu 105 110 115 cct gaa gat tat gag
cgc gtt ccg ggc aat gac atc acc cca gag cag 1160 Pro Glu Asp Tyr
Glu Arg Val Pro Gly Asn Asp Ile Thr Pro Glu Gln 120 125 130 gca tac
acc gaa gct aac ctt gac cca gct ctg cag gca gcc ctc gat 1208 Ala
Tyr Thr Glu Ala Asn Leu Asp Pro Ala Leu Gln Ala Ala Leu Asp 135 140
145 150 gag ttg agc cca gac ttc cgc gtg gca gtg atc ctc tgt gat gtt
gtt 1256 Glu Leu Ser Pro Asp Phe Arg Val Ala Val Ile Leu Cys Asp
Val Val 155 160 165 ggt atg agc tat gac gaa atc gca gag acc ctc gga
gtg aaa atg ggt 1304 Gly Met Ser Tyr Asp Glu Ile Ala Glu Thr Leu
Gly Val Lys Met Gly 170 175 180 acc gtg cgt tcc cgt att cac cgt gga
cgc agc cag ctt cgt gca agt 1352 Thr Val Arg Ser Arg Ile His Arg
Gly Arg Ser Gln Leu Arg Ala Ser 185 190 195 ttg gaa gct gca gca atg
acc agc gag gaa gtt tct ttg ttg gtt cca 1400 Leu Glu Ala Ala Ala
Met Thr Ser Glu Glu Val Ser Leu Leu Val Pro 200 205 210 acc cac
taaagttggt gtgttttctg acacgacaaa cgcaaatgtc gtgtcatttt 1456 Thr His
215 tgcagctcag tgcattattt tggggttcgt ggtgcggaca gggaacttat
cacaggcgac 1516 atccgttttg agtagtaggt atcttggata agaagttacc
cacatccttg aaagtcgaga 1576 cacaggaggt catcggaaga tatgttcaat
tccgacacca ccgcgaatct ccaagctaaa 1636 agtcgagatc gtgcaggatc
taaagcaaag cgcagcaggc caagttttga ttcagtagcg 1696 cgggatgttt
tggatgttcg aacaaaaaca gcacaagtta aaaacaaggc taaagagttt 1756
tcctctgttg atcacctttc agcagacgcc gcagccatgt ttgtagacaa tgaactgtcc
1816 cgtggcgcca tgcatcgcgc caggctgcac attgtgcact gcgctgaatg
tagggaagag 1876 attaaccgtc agcgggaaac cgttgattat ctccgctcag
agtgcaaaaa cgaagaagtg 1936 tccgccccaa tggacctcaa agcacggctt
gccagcctcg ccactgagtg catgcctggc 1996 cctggcgcag agaatttagc
aatgcagcgc ccagagtctt ttgtggctaa agttgagtcc 2056 gtagtgcgcg
cagttcgtaa gaaccaaggc 2086 6 216 PRT Corynebacterium glutamicum 6
Met Thr Tyr Met Lys Lys Lys Ser Arg Asp Asp Ala Pro Val Val Ile 1 5
10 15 Glu Thr Val Gln Ala Glu His Ala Glu Glu Leu Thr Gly Thr Ala
Ala 20 25 30 Phe Asp Ala Gly Gln Ala Asp Met Pro Thr Trp Gly Glu
Leu Val Ala 35 40 45 Glu His Ala Asp Ser Val Tyr Arg Leu Ala Tyr
Arg Leu Ser Gly Asn 50 55 60 Gln His Asp Ala Glu Asp Leu Thr Gln
Glu Thr Phe Met Arg Val Phe 65 70 75 80 Arg Ser Leu Lys Ser Tyr Gln
Pro Gly Thr Phe Glu Gly Trp Leu His 85 90 95 Arg Ile Thr Thr Asn
Leu Phe Leu Asp Met Val Arg His Arg Gly Lys 100 105 110 Ile Arg Met
Glu Ala Leu Pro Glu Asp Tyr Glu Arg Val Pro Gly Asn 115 120 125 Asp
Ile Thr Pro Glu Gln Ala Tyr Thr Glu Ala Asn Leu Asp Pro Ala 130 135
140 Leu Gln Ala Ala Leu Asp Glu Leu Ser Pro Asp Phe Arg Val Ala Val
145 150 155 160 Ile Leu Cys Asp Val Val Gly Met Ser Tyr Asp Glu Ile
Ala Glu Thr 165 170 175 Leu Gly Val Lys Met Gly Thr Val Arg Ser Arg
Ile His Arg Gly Arg 180 185 190 Ser Gln Leu Arg Ala Ser Leu Glu Ala
Ala Ala Met Thr Ser Glu Glu 195 200 205 Val Ser Leu Leu Val Pro Thr
His 210 215 7 20 DNA Corynebacterium glutamicum Primer sigE1 7
tagtcaccac ggttaagcct 20 8 20 DNA Corynebacterium glutamicum Primer
sigE2 8 gccttggttc ttacgaactg 20
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