U.S. patent application number 10/872565 was filed with the patent office on 2005-04-28 for nucleotide sequences coding for the lysri gene.
This patent application is currently assigned to Degussa AG. Invention is credited to Farwick, Mike, Hermann, Thomas, Kreutzer, Caroline, Moeckel, Bettina, Pfefferle, Walter.
Application Number | 20050089976 10/872565 |
Document ID | / |
Family ID | 7651972 |
Filed Date | 2005-04-28 |
United States Patent
Application |
20050089976 |
Kind Code |
A1 |
Moeckel, Bettina ; et
al. |
April 28, 2005 |
Nucleotide sequences coding for the lysRI gene
Abstract
The present invention relates to polynucleotides corresponding
to the lysR1 gene and which encode a LysR1 transcriptional
regulator, methods of producing L-amino acids, and methods of
screening for polynucleotides which encode proteins having LysR3
transcriptional regulator activity.
Inventors: |
Moeckel, Bettina;
(Duesseldorf, DE) ; Farwick, Mike; (Bielefeld,
DE) ; Hermann, Thomas; (Bielefeld, DE) ;
Kreutzer, Caroline; (Melle, DE) ; Pfefferle,
Walter; (Halle, DE) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
Degussa AG
Duesseldorf
DE
|
Family ID: |
7651972 |
Appl. No.: |
10/872565 |
Filed: |
June 22, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10872565 |
Jun 22, 2004 |
|
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09903770 |
Jul 13, 2001 |
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Current U.S.
Class: |
435/115 ;
435/252.3 |
Current CPC
Class: |
C12P 13/08 20130101;
C07K 14/34 20130101 |
Class at
Publication: |
435/115 ;
435/252.3 |
International
Class: |
C12P 013/08; C12P
021/04; C12N 001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 10, 2000 |
DE |
100 39 044.7 |
Claims
1-22. (canceled)
23. A process for producing L-amino acids comprising culturing a
recombinant bacterial cell in a medium suitable for producing
L-amino acids, and collecting the L-amino acids produced wherein
said bacterial cell comprises an attenuated lysR1 gene.
24. The process of claim 23, wherein said recombinant bacterial
cell is a Coryneform bacterium or Brevibacterim.
25. The process of claim 24, wherein said recombinant bacterial
cell is selected from the group consisting of Coryneform
glutamicum, Corynebacterium acetoglutamicum, Corynebacterium
acetoacidophilum, Corynebacterium melassecola, Corynebacterium
thermoaminogenes, Brevibacteriumflavum, Brevibacterium
lactofermentum, Brevibacterium divaricatum.
26. The process of claim 23, wherein said lysR1 gene prior to being
attenuated comprises the polynucleotide sequence of SEQ ID NO: 1 or
a polynucleotide which hybridizes under stringent conditions to SEQ
ID NO: 1 or the full-complement of SEQ ID NO: 1 wherein the
stringent conditions comprise washing in 0.5.times.SSC at a
temperature of 68.degree. C., and wherein the polynucleotide
encodes a protein that inhibits lysine production in a bacterial
cell.
27. The process of claim 23, wherein said L-amino acid is
L-lysine.
28. The process of claim 23, wherein said L-amino acid is
L-valine.
29. The process of claim 23, wherein said recombinant bacterial
cell further comprises at least one gene whose expression is
enhanced, wherein said gene is selected from the group consisting
of dapA, eno, zwf, pyc, and lysE.
30. The process of claim 23, wherein said recombinant bacterial
cell further comprises at least one gene whose expression is
attenuated, wherein said gene is selected from the group consisting
of pck, pgi, and poxB.
31. A process for screening for polynucleotides which encode a
protein having LysR1 transcriptional regulatory activity comprising
hybridizing the isolated polynucleotide which encodes a protein
comprising the amino acid sequence of SEQ ID NO:2 to the
polynucleotide to be screened; expressing the polynucleotide to
produce a protein; and detecting the presence or absence of LysR1
transcriptional regulatory activity in said protein.
32. A process for screening for polynucleotides which encode a
protein having LysR1 transcriptional regulatory activity comprising
hybridizing isolated polynucleotide comprising SEQ ID NO: 1 to the
polynucleotide to be screened; expressing the polynucleotide to
produce a protein; and detecting the presence or absence of LysR1
transcriptional regulatory activity in said protein.
33. A method for detecting a nucleic acid with at least 70%
homology to an isolated polynucleotide which encodes a protein
comprising SEQ ID NO:2, comprising contacting a nucleic acid sample
with a probe or primer comprising at least 15 consecutive
nucleotides of said isolated polvnucleotide, or at least 15
consecutive nucleotides of the complement thereof.
34. A method for producing a nucleic acid with at least 70%
homology to an isolated polynucleotide which encodes a protein
comprising SEQ ID NO:2, comprising contacting a nucleic acid sample
with a primer comprising at least 15 consecutive nucleotides of the
isolated polynucleotide, or at least 15 consecutive nucleotides of
the complement thereof.
35. A method for detecting a nucleic acid with at least 70%
homology to isolated polynucleotide comprising SEQ ID NO: 1,
comprising contacting a nucleic acid sample with a probe or primer
comprising at least 15 consecutive nucleotides of the nucleotide
sequence of said isolated polynucleotide, or at least 15
consecutive nucleotides of the complement thereof.
36. A method for producing a nucleic acid with at least 70%
homology to comprising contacting a nucleic acid sample with a
primer comprising at least 15 consecutive nucleotides of the
isolated polynucleotide, or at least 15 consecutive nucleotides of
the complement thereof.
37-38. (canceled)
39. An isolated polypeptide comprising the amino acid sequence of
SEQ ID NO:2.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application claims priority to German
Application No. DE 100 39 044.7 filed Aug. 10, 2000, the entire
contents of which are incorporated herein by refeference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention provides nucleotide sequences from Coryneform
bacteria which code for the lysR1 gene and a process for the
fermentative preparation of amino acids, in particular L-lysine by
attenuation of the lysR1 gene. The lysR1 gene codes for the LysR1
protein, which is a transcription regulator of the LysR family.
[0004] 2. Discussion of the Background
[0005] L-amino acids, in particular L-lysine, are used in human
medicine and in the pharmaceutical industry, in the foodstuffs
industry, and most particularly in animal nutrition.
[0006] It is known that amino acids can be produced by fermentation
of strains of Coryneform bacteria, in particular Corynebacterium
glutamicum. On account of the great importance of these amino acids
constant efforts are being made to improve the production
processes. Improvements in production processes may involve
fermentation technology measures, such as for example stirring and
provision 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 performance properties of the
microorganism itself.
[0007] Methods involving mutagenesis, selection and mutant
selection are used to improve the performance properties of these
microorganisms. In this way strains are obtained that are resistant
to antimetabolites or are auxotrophic for regulatorily significant
metabolites and that produce amino acids.
[0008] Methods of recombinant DNA have also been employed for
improving strains of Corynebacterium strains which produce L-amino
acids.
[0009] However, there remains a critical need for improved methods
of producing L-amino acids and thus for the provision of strains of
bacteria producing higher amounts of L-amino acids. On a commercial
or industrial scale even small improvements in the yield of L-amino
acids, or the efficiency of their production, are economically
significant. Prior to the present invention, it was not recognized
that attenuation of lysR1 gene encoding the a LysR1 transcriptional
regulation protein would improve L-amino acid yields.
SUMMARY OF THE INVENTION
[0010] One object of the present invention, is providing a new
process adjuvant for improving the fermentative production of
L-amino acids, particularly L-lysine and L-glutamate. Such process
adjuvants include enhanced bacteria, preferably enhanced Coryneform
bacteria which express attenuated amounts of LysR1 transcriptional
regulator which is encoded by the lysR1 gene.
[0011] Thus, another object of the present invention is providing
such an bacterium, which expresses an attenuated amount of LysR1
transcriptional regulator or gene products of the lysR1 gene.
[0012] Another object of the present invention is providing a
bacterium, preferably a Coryneform bacterium, which expresses a
polypeptide that has an attenuated LysR1 transcriptional regulator
activity.
[0013] Another object of the invention is to provide a nucleotide
sequence encoding a polypeptide which has LysR1 transcriptional
regulator sequence. One embodiment of such a sequence is the
nucleotide sequence of SEQ ID NO: 1.
[0014] A further object of the invention is a method of making
LysR3 transcriptional regulator or an isolated polypeptide having a
LysR1 transcriptional regulator activity, as well as use of such
isolated polypeptides in the production of amino acids. One
embodiment of such a polypeptide is the polypeptide having the
amino acid sequence of SEQ ID NO: 2.
[0015] Other objects of the invention include methods of detecting
nucleic acid sequences homologous to SEQ ID NO: 1, particularly
nucleic acid sequences encoding polypeptides that have LysR1
transcriptional regulator activity, and methods of making nucleic
acids encoding such polypeptides.
[0016] The above objects highlight certain aspects of the
invention. Additional objects, aspects and embodiments of the
invention are found in the following detailed description of the
invention.
BRIEF DESCRIPTION OF THE DRAWING
[0017] FIG. 1: Map of the plasmid pCR2.1lysR1 int.
DETAILED DESCRIPTION OF THE INVENTION
[0018] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art of molecular biology. Although methods
and materials similar or equivalent to those described herein can
be used in the practice or testing of the present invention,
suitable methods and materials are described herein. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In addition, the materials, methods, and examples are illustrative
only and are not intended to be limiting.
[0019] Reference is made to standard textbooks of molecular biology
that contain definitions and methods and means for carrying out
basic techniques, encompassed by the present invention. See, for
example, Maniatis et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory, New York (1982) and Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, New York (1989) and the various references cited
therein.
[0020] As used herein, L-amino acids or amino acids are understood
to mean and amino acid or its salt. Preferably, the amnio acids are
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. L-Lysine is
particularly preferred.
[0021] As used herein L-lysine or lysine include not only the bases
but also the salts, such as e.g. lysine monohydrochloride or lysine
sulfate.
[0022] The invention provides an isolated polynucleotide of
Coryneform bacteria containing a polynucleotide sequence coding for
the lysR1 gene, selected from the group comprising
[0023] a) polynucleotide that is at least 70% identical to a
polynucleotide coding for a polypeptide that contains the amino
acid sequence of SEQ ID No. 2,
[0024] b) polynucleotide coding for a polypeptide that contains an
amino acid sequence that is at least 70% identical to the amino
acid sequence of SEQ ID No. 2,
[0025] c) polynucleotide that is complementary to the
polynucleotides of a) or b), and
[0026] d) polynucleotide containing at least 15 successive
nucleotides of the polynucleotide sequence of a), b) or c),
[0027] the polypeptide preferably having the activity of the
transcription regulator lysR1.
[0028] The invention also provides the aforementioned
polynucleotide, which is preferably a replicable DNA
containing:
[0029] (i) the nucleotide sequence shown in SEQ ID No.1, or
[0030] (ii) at least one sequence that corresponds to the sequence
(i) within the region of degeneration of the genetic code, or
[0031] (iii) at least one sequence that hybridises with the
sequences that are complementary to the sequences (i) or (ii), and
optionally
[0032] (iv) functionally neutral sense mutations in (i).
[0033] The invention furthermore provides:
[0034] a replicable DNA containing the nucleotide sequence as
illustrated in SEQ ID No.1;
[0035] a polynucleotide coding for a polypeptide that contains the
amino acid sequence as is illustrated in SEQ ID No. 2;
[0036] a vector containing the polynucleotide d) according to the
invention, in particular pCR2.1lysR1int inserted into E. Coli DSM
13616 and filed at DSMZ, Brunswick, (Germany);
[0037] and Coryneform bacteria that in the lysR1 gene contain an
insertion or deletion, in particular by using the vector
pCR2.1lysR1int.
[0038] The invention thus provides polynucleotides consisting
substantially of a polynucleotide sequence, that are obtainable by
screening by hybridising a corresponding gene library that contains
the complete gene with the polynucleotide sequence corresponding to
SEQ ID No.1, with a probe that contains the sequence of the
aforementioned polynucleotide according to SEQ ID No. 1 or a
fragment thereof, and isolating the aforementioned DNA
sequence.
[0039] Polynucleotide sequences according to the invention are
suitable as hybridisation probes for RNA, cDNA and DNA, in order to
isolate nucleic acids, polynucleotides or genes in their full
length that code for lysR1 protein, or to isolate such nucleic
acids or polynucleotides or genes that have a high degree of
similarity to the sequence of the lysR1 gene.
[0040] Polynucleotide sequences according to the invention are
further suitable as primers with the polymerase chain reaction
(PCR), DNA of genes can be produced that code for lysR1
protein.
[0041] Such oligonucleotides serving as probes or primers contain
at least 30, preferably at least 20, and most particularly
preferably at least 15 successive nucleotides. Also suitable are
oligonucleotides having a length of at least 40 or 50
nucleotides.
[0042] "Isolated" denotes separated from its natural
environment.
[0043] "Polynucleotide" refers in general to polyribonucleotides
and polydeoxyribonucleotides, which may either be unmodified RNA or
DNA or modified RNA or DNA.
[0044] The term "polypeptides" denotes peptides or proteins that
contain two or more amino acids bound via peptide bonds.
[0045] The polypeptides according to the invention include a
polypeptide according to SEQ ID No. 2, in particular those peptides
having the biological activity of lysR1 protein, and also those
polypeptides that are at least 70%, preferably at least 80% and
particularly preferably at least 90% to 95% identical to the
polypeptide according to SEQ ID No. 2 and have the aforementioned
activity.
[0046] The present invention furthermore relates to a process for
the enzymatic production of amino acids, in particular L-lysine,
using Coryneform bacteria that in particular already produce amino
acids and in which the nucleotide sequences coding for the lysR1
gene are attenuated, in particular are switched off or are
expressed at a low level.
[0047] The term "attenuation" used in this context denotes the
reduction or switching off of the intracellular activity of one or
more enzymes (proteins) in a microorganism that are coded by the
corresponding DNA, by for example using a weak promoter or using a
gene or allele that codes for a corresponding gene having a low
activity or that inactivates the corresponding gene or enzyme
(protein), and optionally combining these measures.
[0048] The microorganisms that are the subject of the present
invention may produce amino acids, in particular L-lysine, from
glucose, sucrose, lactose, fructose, maltose, molasses, starch,
cellulose or from glycerol and ethanol. These microorganisms may be
representatives of Coryneform bacteria, in particular of the genus
Corynebacterium. In the genus Corynebacterium the species
Corynebacterium glutamicum should in particular be mentioned, which
is known to those skilled in the art for its ability to produce
L-amino acids.
[0049] Suitable strains of the genus Corynebacterium, in particular
of the species Corynebacterium glutamicum (C. glutamicum), are in
particular the known wild type strains
[0050] Corynebacterium glutamicum ATCC13032
[0051] Corynebacterium acetoglutamicum ATCC15806
[0052] Corynebacterium acetoacidophilum ATCC13870
[0053] Corynebacterium melassecola ATCC17965
[0054] Corynebacterium thermoaminogenes FERM BP-1539
[0055] Brevibacterium flavum ATCC14067
[0056] Brevibacterium lactofermentum ATCC13869 and
[0057] Brevibacterium divaricatum ATCC14020
[0058] or mutants or strains formed therefrom that produce L-amino
acids, such as for example the strains producing L-lysine.
[0059] Corynebacterium glutamicum FERM-P 1709
[0060] Brevibacterium flavum FERM-P 1708
[0061] Brevibacterium lactofermentum FERM-P 1712
[0062] Corynebacterium glutamicum FERM-P 6463
[0063] Corynebacterium glutamicum FERM-P 6464
[0064] Corynebacterium glutamicum DM58-1
[0065] Corynebacterium glutamicum DG52-5
[0066] Corynebacterium glutamicum DSM 5714 and
[0067] Corynebacterium glutamicum DSM 12866
[0068] Preferably, a bacterial strain with attenuated expression of
a lysR1 gene that encodes a polypeptide with LysR1 transcriptional
regulation activity will improve amino acid yield at least 1%.
[0069] The inventors have successfully isolated the new lysR1 gene
from C. glutamicum coding for lysR1 protein, which is a
transcription regulator of the lysR family.
[0070] In order to isolate the lysR1 gene or also other genes from
C. glutamicum, a gene library of this microorganism is first of all
introduced into Escherichia coli (E. coli). The introduction of
gene libraries is described in generally known textbooks and
manuals. As an example there may be mentioned the textbook by
Winnacker: Gene and Klone, Eine Einfuhrung in die Gentechnologie
(Verlag Chemie, Weinheim, Germany, 1990), or the manual by Sambrook
et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor
Laboratory Press, 1989). A very well-known gene library is that of
the E. coli K-12 strain W3110, which was introduced by Kohara et
al. (Cell 50, 495-508 (1987)) into X-vectors. Bathe et al.
(Molecular and General Genetics, 252:255-265, 1996) describe a gene
library of C. glutamicum ATCC13032, which was introduced by means
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). Bormann et al. (Molecular Microbiology 6(3), 317-326
(1992)) again describe a gene library of C. glutamicum ATCC13032
using the cosmid pHC79 (Hohn and Collins, 1980, Gene 11,
291-298).
[0071] In order to produce a gene library of C. glutamicum in E.
coli plasmids such as pBR322 (Bolivar, 1979, Life Sciences, 25,
807-818) or pUC9 (Vieira et al., 1982, Gene, 19:259-268) may also
be employed. Suitable hosts are in particular those E. coli strains
that are restriction and recombinant defective, for example the
strain DH5.alpha., (Jeffrey H. Miller: "A Short Course in Bacterial
Genetics, A Laboratory Manual and Handbook for Escherichia coli and
Related Bacteria", Cold Spring Harbor Laboratory Press, 1992)
[0072] The long DNA fragments cloned with the help of cosmids or
other .lambda.-vectors may then in turn be subcloned into
conventional vectors suitable for DNA sequencing.
[0073] Methods of DNA sequencing are described in, inter alia,
Sanger et al. (Proceedings of the National Academy of Sciences of
the United States of America USA, 74:5463-5467, 1977).
[0074] The DNA sequences obtained may then be investigated with
known algorithms or sequence analysis programs, such as for example
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)).
[0075] The new DNA sequence of C. glutamicum coding for the lysR1
gene was obtained in this way, and as SEQ ID No. 1 is part of the
present invention. The amino acid sequence of the corresponding
protein was also derived from the existing DNA sequence using the
aforedescribed methods. The resulting amino acid sequence of the
lysR1 gene product is shown in SEQ ID No. 2.
[0076] Coding DNA sequences that are obtained from SEQ ID No. 1 as
a result of the degenerability of the genetic code are also covered
by the invention. Similarly, DNA sequences that hybridise with SEQ
ID No. 1 or parts of SEQ ID No. 1 are also covered by the
invention. Furthermore, in this specialist field conservative amino
acid replacements, such as for example the replacement of glycine
by alanine or of aspartic acid by glutamic acid in proteins, are
known as sense mutations, which do not lead to any fundamental
change in the activity of the protein, i.e. are functionally
neutral. Furthermore, it is known that changes at the N-terminus
and/or C-terminus of a protein do not significantly impair or may
even stabilise its function. Those skilled in the art can find
details of this in, inter alia, 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 that are obtained in a corresponding manner
from SEQ ID No. 2 are likewise covered by the invention.
[0077] Finally, DNA sequences that are produced by the polymerase
chain reaction (PCR) using primers resulting from SEQ ID No. 1, are
also covered by the invention. Such oligonucleotides typically have
a length of at least 15 nucleotides.
[0078] The person skilled in the art can find details of the
identification of DNA sequences by means of hybridisation in, inter
alia, the textbook "The DIG System User's Guide for Filter
Hybridization" published by Boehringer Mannheim GmbH (Mannheim,
Germany, 1993) and in Liebl et al. (International Journal of
Systematic Bacteriology 41: 255-260 (1991)). The person skilled in
the art can obtain details of the amplification of DNA sequences by
means of the polymerase chain reaction (PCR) in, inter alia, the
handbook by Gait: Oligonucleotide Synthesis: A Practical Approach
(IRL Press, Oxford, UK, 1984) and the Newton and Graham: PCR
(Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).
[0079] In the course of work carried out on the present invention
it was found that Coryneform bacteria after attenuation of the
lysR1 gene produce amino acids, in particular L-lysine, in an
improved manner.
[0080] In order to achieve an attenuation, either the expression of
the lysR1 gene or the catalytic properties of the enzyme protein
may be reduced or switched off. Optionally both measures may be
combined.
[0081] The reduction of the gene expression may be achieved by
suitable culture conditions or by genetic alteration (mutation) of
the signal structures of the gene expression. Signal structures of
the gene expression are for example repressor genes, activator
genes, operators, promoters, attenuators, ribosome binding sites,
the start codon and terminators. The person skilled in the art can
obtain further information on this in for example patent
application WO 96/15246, in Boyd and Murphy (Journal of
Bacteriology 170: 5949 (1988)), in Voskuil and Chambliss (Nucleic
Acids Research 26: 3548 (1998), in Jensen and Hammer (Biotechnology
and Bioengineering 58: 191 (1998)), in Ptek et al. (Microbiology
142: 1297 (1996)), Vasicova et al. (Journal of Bacteriology 181:
6188 (1999)) and in known textbooks of genetics and molecular
biology, such as for example the textbook by Knippers ("Molekulare
Genetik", 6.sup.th Edition, Georg Thieme Verlag, Stuttgart,
Germany, 1995) or the textbook by Winnacker ("Gene and Klone", VCH
Verlagsgesellschaft, Weinheim, Germany, 1990).
[0082] Mutations that lead to an alteration or reduction of the
catalytic properties of enzyme proteins are known in the prior art;
as examples there may be mentioned the work of Qiu and Goodman
(Journal of Biological Chemistry 272: 8611-8617 (1997)), Sugimoto
et al. (Bioscience Biotechnology and Biochemistry 61: 1760-1762
(1997)) and Mockel ("Die Threonindehydratase aus Corynebacterium
glutamicum: Aufhebung der allosterischen Regulation and Struktur
des Enzyms", and reports published by the Julich Research Centre,
Jul-2906, ISSN09442952, Julich, Germany, 1994). Overviews may be
obtained from known textbooks on genetics and molecular biology,
for example that of Hagemann ("Allgemeine Genetik", Gustav Fischer
Verlag, Stuttgart, 1986).
[0083] Mutations in the present context include transitions,
transversions, insertions and deletions. Depending on the effect of
the amino acid replacement on the enzyme activity, one talks either
of missense mutations or nonsense mutations. Insertions or
deletions of at least one base pair (bp) in a gene lead to frame
shift mutations, following which false amino acids are incorporated
or the translation terminates prematurely. Deletions of several
codons typically lead to a complete cessation of enzyme activity.
Details of the production of such mutations are part of the prior
art and may be obtained from known textbooks on genetics and
molecular biology, such as for example the textbook by Knippers
("Molekulare Genetik", 6.sup.th Edition, Georg Thieme Verlag,
Stuttgart, Germany, 1995), the textbook by Winnacker ("Gene and
Klone", VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or the
textbook by Hagemann ("Allgemeine Genetik", Gustav Fischer Verlag,
Stuttgart, 1986).
[0084] A conventional method of mutating genes of C. glutamicum is
the method of gene disruption and gene replacement described by
Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991)).
[0085] In the method of gene disruption a central part of the
coding region of the gene in question is cloned into a plasmid
vector that can replicate in a host (typically E. coli), but not in
C. glutamicum. Suitable 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)), pK18mobsacB or pK19mobsacB (Jger
et al., Journal of Bacteriology 174: 5462-65 (1992)), 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, Netherlands;
Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993))
or pEM1 (Schrumpf et al, 1991, Journal of Bacteriology
173:4510-4516). The plasmid vector that contains the central part
of the coding region of the gene is then converted by conjugation
or transformation into the desired strain of C. glutamicum. The
method of conjugation is described for example by Schafer et al.
(Applied and Environmental Microbiology 60, 756-759 (1994)).
Methods of transformation are described for example in 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 coding
region of the relevant gene is disrupted by the vector sequence and
two incomplete alleles are obtained, missing respectively the 3'-
and 5'-end. This method has been used for example by Fitzpatrick et
al. (Applied Microbiology and Biotechnology 42, 575-580 (1994)) to
switch off the recA gene of C. glutamicum.
[0086] FIG. 1 shows for example the plasmid vector pCR2.1lysR1int,
by means of which the lysRl gene can be disrupted or switched
off.
[0087] In the gene replacement method a mutation, such as for
example a deletion, insertion or base replacement, is produced in
vitro in the gene that is of interest. The resultant allele is in
turn cloned into a non-replicative vector for C. glutamicum, and
this is then converted by transformation or conjugation into the
desired host of C. glutamicum. After homologous recombination by
means of a first cross-over event effecting integration, and an
appropriate second cross-over event effecting an excision, the
incorporation of the mutation or allele in the target gene or in
the target sequence is achieved. This method has been used for
example by Peters-Wendisch et al. (Microbiology 144, 915-927
(1998)) to switch off the pyc gene of C. glutamicum by a
deletion.
[0088] A deletion, insertion or a base replacement can be
incorporated into the lysRl gene in this way.
[0089] In addition, it may be advantageous for the production of
L-amino acids, in particular L-lysine, in addition to the
attenuation of the lysRl gene, also to enhance, in particular
overexpress, one or more enzymes of the respective biosynthesis
pathway, glycolysis, anapleurosis, pentose phosphate cycle, or
amino acid export.
[0090] Thus for example, for the production of L-lysine one or more
of the genes selected from the following group may simultaneously
be enhanced, in particular overexpressed
[0091] the gene dapA coding for dihydrodipicolinate synthase (EP-B
0 197 335),
[0092] the gene eno coding for enolase (DE: 19947791.4),
[0093] the gene zwf coding for the zwf gene product
(JP-A-09224661)
[0094] the gene pyc coding for pyruvate carboxylase
(Peters-Wendisch et al.(Microbiology 144, 915-927 (1998))
[0095] the gene lysE coding for lysine export (DE-A-195 48
222).
[0096] Also, it may be advantageous for the production of amino
acids, especially L-lysine, besides attenuating the lysR1 gene, at
the same time to attenuate one or more of the genes selected from
the group
[0097] the gene pck coding for phosphoenol pyruvate carboxykinase
(DE 199 50 409.1, DSM 13047),
[0098] the gene pgi coding for glucose-6-phosphate isomerase (U.S.
Ser. No. 09/396,478, DSM 12969),
[0099] the gene poxB coding for pyruvate oxidase (DE:1995 1975.7,
DSM 13114)
[0100] Moreover, it may be advantageous for the production of amino
acids, in particular L-lysine, in addition to attenuating the lysRl
gene also to switch off undesirable secondary reactions (Nakayama:
"Breeding of Amino Acid Producing Microorganisms", in:
Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek
(eds.), Academic Press, London, UK, 1982).
[0101] The microorganisms produced according to the invention are
likewise covered by the invention and for the purposes of producing
L-amino acids, in particular L-lysine, may be cultivated
continuously or batchwise in a batch process, or in a feed batch
process or repeated batch process. A summary of known cultivation
methods is described in the textbook by Chmiel (Bioprozesstechnik
1. Einfuhrung in die Bioverfahrens-technik (Gustav Fischer Verlag,
Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und
periphere Einrichtungen (Vieweg Verlag, Brunswick/Wiesbaden,
1994)).
[0102] The culture medium to be used must satisfy in an appropriate
manner the requirements of the respective strains. Descriptions of
culture media for various microorganisms are given in the handbook
"Manual of Methods for General Bacteriology" of the American
Society for Bacteriology (Washington D.C., USA, 1981). As carbon
source there may be used sugars and carbohydrates such as for
example glucose, sucrose, lactose, fructose, maltose, molasses,
starch and cellulose, oils and fats such as for example soya oil,
sunflower oil, groundnut oil and coconut oil, fatty acids such as
for example palmitic acid, stearic acid and linoleic acid, alcohols
such as for example glycerol and ethanol, and organic acids such as
for example acetic acid. These substances may be used individually
or as a mixture.
[0103] As nitrogen source there may be used 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. The
nitrogen sources may be used individually or as a mixture.
[0104] As phosphorus source there may be used phosphoric acid,
potassium dihydrogen phosphate or dipotassium hydrogen phosphate,
or the corresponding sodium-containing salts. The culture medium
must furthermore contain salts of metals, such as for example
magnesium sulfate or iron sulfate, that are necessary for growth.
Finally, essential growth promoters such as amino acids and
vitamins may in addition be added to the aforementioned substances.
Suitable precursors may moreover be added to the culture medium.
The aforementioned starting substances may be added to the culture
in the form of a single batch, or metered in in an appropriate
manner during the cultivation procedure.
[0105] In order to control the pH of the culture basic compounds
such as sodium hydroxide, potassium hydroxide, ammonia or ammonia
water, or acidic compounds such as phosphoric acid or sulfuric acid
may be added in an appropriate manner. In order to control foam
formation anti-foaming agents such as for example fatty acid
polyglycol esters may be used. In order to maintain the stability
of plasmids selectively acting substances, such as for example
antibiotics, may be added to the medium. In order to maintain
aerobic conditions, oxygen or oxygen-containing gas mixtures, such
as for example air, are pumped into the culture. The temperature of
the culture is normally 20.degree. C. to 45.degree. C. and
preferably 25.degree. C. to 40.degree. C. The cultivation is
continued until a maximum amount of the desired product has been
formed. This target is normally reached within 10 hours to 160
hours.
[0106] Methods for determining L-amino acids are known from the
prior art. The analysis may for example be carried out as described
by Spackman et al. (Analytical Chemistry, 30, (1958), 1190) by
anion exchange chromatography followed by ninhydrin derivatisation,
or it may be carried out by reversed phase HPLC, as described by
Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174).
[0107] The following microorganism has been filed according to the
Budapest Convention at the German Collection for Microorganisms and
Cell Cultures (DSMZ, Brunswick, Germany).
[0108] Escherichia coli strain E. coli TOP10F/pCR2.1lysR1int as DSM
13616.
[0109] The process according to the invention serves for the
enzymatic production of amino acids, in particular L-lysine.
[0110] The present invention is illustrated in more detail
hereinafter with the aid of examples of implementation.
[0111] The isolation of plasmid DNA from Escherichia coli as well
as all techniques for the restriction, Klenow and alkaline
phosphatase treatment were carried out according to Sambrook et al.
(Molecular Cloning. A Laboratory Manual, 1989, Cold Spring Harbour
Laboratory Press, Cold Spring Harbor, N.Y., USA). Methods for the
transformation of Escherichia coli are likewise described in this
handbook. The compositions of conventional nutrient media such as
LB medium or TY medium may also be obtained from the handbook by
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York (1989).
EXAMPLE 1
[0112] Production of a Genomic Cosmid Gene Library from C.
glutamicum ATCC 13032
[0113] Chromosomal DNA from C. glutamicum ATCC 13032 was isolated
as described by Tauch et al., (1995, Plasmid 33:168-179) and
partially 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 Molecular Biochemicals, 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.
[0114] 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 way was
mixed with the treated ATCC13032-DNA, and the batch was then
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 into phages using Gigapack II XL
Packing Extracts (Stratagene, La Jolla, USA, Product Description
Gigapack II XL Packing Extract, Code no. 200217).
[0115] In order to infect the E. coli strain NM554 (Raleigh et al.
1988, Nucleic Acid Res. 16:1563-1575) the cells were taken up in 10
mM MgSO.sub.4 and mixed with an aliquot of the phage suspension.
Infection and titration 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)+100 .mu.g/ml ampicillin. After
incubation overnight at 37.degree. C. recombinant individual clones
were selected.
EXAMPLE 2
[0116] Isolation and Sequencing of the Gene lysR1
[0117] The cosmid DNA of an individual colony was isolated with the
Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden,
Germany) according to the manufacturer's instructions and then
partially 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 Molecular Biochemicals,
Mannheim, Germany, Product Description SAP, Product No. 1758250).
After gel electrophoresis separation the cosmid fragments were
isolated in the size range from 1500 to 2000 bp using the QiaExII
Gel Extraction Kit (Product No. 20021, Qiagen, Hilden,
Germany).
[0118] The DNA of the sequencing vector pZero-1 obtained from
Invitrogen (Groningen, Netherlands, 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 into 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 having been
incubated overnight with T4 ligase (Pharmacia Biotech, Freiburg,
Germany). This ligation mixture was then electroporated into the E.
coli strain DH5 MCR (Grant, 1990, Proceedings of the National
Academy of Sciences, U.S.A., 87:4645-4649) (Tauch et al. 1994, FEMS
Microbiol. Letters, 123:343-7) and plated out onto LB-agar (Lennox,
1955, Virology, 1:190) with 50 .mu.g/l zeocin.
[0119] The plasmid preparation of the recombinant clone was carried
out with the Biorobot 9600 (Product No. 900200, Qiagen, Hilden,
Germany). The sequencing was performed according to the dideoxy
chain termination method of Sanger et al. (1977, Proceedings of the
National Academies of Sciences, U.S.A., 74:5463-5467) as modified
by 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
gel electrophoresis separation 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) using the "ABI Prism 377" sequencing device
from PE Applied Biosystems (Weiterstadt, Germany).
[0120] The raw sequence data thus obtained were then processed
using the Staden Program Package (1986, Nucleic Acids Research,
14:217-231) Version 97-0. The individual sequences of the pzerol
derivates were assembled to form a coherent contig. The
computer-assisted coding region analysis was prepared using the
XNIP program (Staden, 1986, Nucleic Acids Research, 14:217-231).
Further analyses were carried out with the "BLAST search programs"
(Altschul et al., 1997, Nucleic Acids Research, 25:33893402)
against the non-redundant database of the National Center for
Biotechnology Information (NCBI, Bethesda, Md., USA).
[0121] The nucleotide sequence thus obtained is represented in SEQ
ID No. 1. Analysis of the nucleotide sequence revealed an open
reading frame of 912 base pairs, which was termed the lysR1 gene.
The lysRl gene codes for a polypeptide of 304 amino acids.
EXAMPLE 3
[0122] Production of an Integration Vector for the Integration
Mutagenesis of the lysR1 Gene
[0123] Chromosomal DNA was isolated from the strain ATCC 13032 by
the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)).
On account of the sequence of the lysR1 gene known from Example 2
for C. glutamicum, the following oligonucleotides were selected for
the polymerase chain reaction:
1 lysRlintA: 5'TTC CAA TCC CTG CTG TTC AC 3' (SEQ ID NO;4)
lysRlintB: 5'GTG ACC TTT GAA ACC AGC GA 3' (SEQ ID NO:5)
[0124] The represented primers were synthesised by MWG Biotech
(Ebersberg, Germany) and the PCR reaction was carried out according
to the standard PCR method of Innis et al. (PCR protocols. A guide
to methods and applications, 1990, Academic Press) using Pwo
polymerase from Boehringer. By means of the polymerase chain
reaction a 383 bp long internal fragment of the lysR1 gene was
isolated, which is shown in SEQ ID No. 3.
[0125] The amplified DNA fragment was ligated into the vector pCR2.
1-TOPO (Mead at al. (1991) Bio/Technology 9:657-663) using the TOPO
TA Cloning Kit from Invitrogen Corporation (Carlsbad, Calif., USA;
Cat. No. K4500-01).
[0126] The E. coli strain TOP10F was then transformed with the
ligation batch (Hanahan, In: DNA cloning. A practical approach.
Vol. I. IRL-Press, Oxford, Washington D.C., USA, 1985).
Plasmid-carrying cells were selected by plating out the
transformation batch onto 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) that had been
supplemented with 25 mg/l of kanamycin. Plasmid DNA was isolated
from a transformant using the QIAprep Spin Miniprep Kit from Qiagen
and was checked by restriction with the restriction enzyme EcoRI
followed by agarose gel electrophoresis (0.8%). The plasmid was
named pCR2.1lysR1int.
EXAMPLE 4
[0127] Integration Mutagenesis of the lysR1 Gene in the Lysine
Producer DSM 5715
[0128] The vector pCR2. 1lysR1int mentioned in Example 3 was
electroporated into Corynebacterium glutamicum DSM 5715 according
to the electroporation method of Tauch et. al.(FEMS Microbiological
Letters, 123:343-347 (1994)). The strain DSM 5715 is an
AEC-resistant lysine producer. The vector pCR2.1lysR1int cannot
replicate independently in DSM 5715 and thus only remains in the
cell if it has integrated into the chromosome of DSM 5715. The
selection of clones with pCR2.1lysR1int integrated into the
chromosome was made by plating out the electroporation batch onto
LB agar (Sambrook et al., Molecular cloning: a laboratory manual.
2.sup.nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.) that had been supplemented with 15 mg/l of
kanamycin.
[0129] In order to demonstrate the integration the lysR1int
fragment was labelled using the Dig Hybridisation Kit from
Boehringer according to the method described in "The DIG System
User's Guide for Filter Hybridization" published by Boehringer
Mannheim GmbH (Mannheim, Germany, 1993). Chromosomal DNA of a
potential integrant was isolated according to the method of
Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) and was in
each case cleaved with the restriction enzymes SalI, SacI and
HindIII. The resultant fragments were separated by means of agarose
gel electrophoresis and hybridised at 68.degree. C. using the Dig
Hybridisation Kit from Boehringer. The plasmid pCR2.1lysR1int
mentioned in Example 3 had inserted itself into the chromosome of
DSM 5715 within the chromosomal lysR1 gene. The strain was
designated DSM 5715::pCR2.1lysR1int.
EXAMPLE 5
[0130] Production of Lysine
[0131] The C. glutamicum strain DSM 5715: pCR2.1lysR1int obtained
in Example 4 was cultivated in a nutrient medium suitable for the
production of lysine and the lysine content in the culture
supernatant was determined.
[0132] For this purpose the strain was first of all incubated for
24 hours at 33.degree. C. on an agar plate with the corresponding
antibiotic (brain-heart agar with kanamycin (25 mg/1). Starting
from this agar plate culture a preculture was inoculated (10 ml of
medium in a 100 ml Erlenmeyer flask). The full medium CgIII was
used as medium for the preculture.
2 Medium Cg III NaCl 2.5 g/l Bacto-Peptone 10 g/l Bacto-Yeast
Extract 10 g/l Glucose (autoclaved separately) 2% (w/v)
[0133] The pH value was adjusted to pH 7.4
[0134] Kanamycin (25 mg/l) was added to this preculture. The
preculture was then incubated for 24 hours at 33.degree. C. at 240
rpm on a shaker table. From this preculture a main culture was
inoculated so that the initial OD (660 nm) of the main culture was
0.1 OD. The medium MM was used for the main culture.
3 Medium MM CSL (Corn Steep Liquor) 5 g/l MOPS 20 g/l Glucose
(autoclaved separately) 50 g/l Salts: (NH.sub.4).sub.2SO.sub.4) 25
g/l KH.sub.2PO.sub.4 0.1 g/l MgSO.sub.4.7H.sub.2O 1.0 g/l
CaCl.sub.2.2H.sub.2O 10 mg/l FeSO.sub.4.7H.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 Leucine (sterile filtered)
0.1 g/l CaCO.sub.3 25 g/l
[0135] CSL, MOPS and the salt solution are adjusted with ammonia
water to pH 7 and autoclaved. The sterile substrate and vitamin
solutions as well as the dry autoclaved CaCO.sub.3 are then
added.
[0136] Cultivation is carried out in a 10 ml volume in a 100 ml
Erlenmeyer flask equipped with baffles. Kanamycin was added (25
mg/l). The cultivation was carried out at 33.degree. C. and 80%
atmospheric humidity.
[0137] After 72 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 by ion exchange
chromatography and post-column derivatisation with ninhydrin
detection using an amino acid analyser from Eppendorf-BioTronik
(Hamburg, Germany).
[0138] The results of the experiment are shown in Table 1.
4 TABLE 1 Lysine-HCl Strain OD (660) g/l DSM 5715 7.5 13.01 DSM
5715:: pCR2.1lysR1int 7.7 15.64
[0139] The acronyms and abbreviations used have the following
meanings.
[0140] KmR: Kanamycin resistance gene
[0141] EcoRI: Cleavage site of the restriction enzyme EcoRI
[0142] lysR1int: Internal fragment of the lysR1 gene
[0143] ColEl ori: Replication origin of the plasmid ColE1
[0144] Obviously, numerous modifications and variations on the
present invention are possible in light of the above teachings. It
is therefore to be understood that within the scope of the appended
claims, the invention may be practiced otherwise than as
specifically described herein.
Sequence CWU 1
1
5 1 1311 DNA Corynebacterium glutamicum CDS (201)..(1109) 1
acagcccagg ggccgttgag ggggaaaagc tgcgttccaa tggcagcacc aaattgcagg
60 gatagggcgg aacccatcac catcaacact gcagcggact gtttattcat
gcccttgatt 120 attgccaaag aaacctttaa ggactagatc gaaaaacagc
caactatagt taagtaatac 180 tgaacaattt tggaggtgtc gtg ctc aat ctc aac
cgc tta cac atc ctg cag 233 Val Leu Asn Leu Asn Arg Leu His Ile Leu
Gln 1 5 10 gaa ttc cac cgc ctg gga acg att aca gca gtg gcg gaa tcc
atg aac 281 Glu Phe His Arg Leu Gly Thr Ile Thr Ala Val Ala Glu Ser
Met Asn 15 20 25 tac agc cgc tct gcc atc tcc caa caa atg gcg ctg
ctg gaa aaa gaa 329 Tyr Ser Arg Ser Ala Ile Ser Gln Gln Met Ala Leu
Leu Glu Lys Glu 30 35 40 att ggt gtg aaa ctc ttt gaa aaa agc ggc
cga aac ctc tac ttc aca 377 Ile Gly Val Lys Leu Phe Glu Lys Ser Gly
Arg Asn Leu Tyr Phe Thr 45 50 55 gaa caa ggc gaa gtg ttg gcc tca
gaa aca cat gcg atc atg gca gca 425 Glu Gln Gly Glu Val Leu Ala Ser
Glu Thr His Ala Ile Met Ala Ala 60 65 70 75 gtc gac cat gcc cgc gca
gcc gtt cta gat tcg ctg tct gaa gtg tcc 473 Val Asp His Ala Arg Ala
Ala Val Leu Asp Ser Leu Ser Glu Val Ser 80 85 90 gga acg ctg aaa
gtc acc tcc ttc caa tcc ctg ctg ttc acc ctt gcc 521 Gly Thr Leu Lys
Val Thr Ser Phe Gln Ser Leu Leu Phe Thr Leu Ala 95 100 105 ccg aaa
gcc atc gcg cgc ctg acc gag aaa tac cca cac ctg caa gta 569 Pro Lys
Ala Ile Ala Arg Leu Thr Glu Lys Tyr Pro His Leu Gln Val 110 115 120
gaa atc tcc caa cta gaa gtc acc gca gcg ctc gaa gaa ctc cgc gcc 617
Glu Ile Ser Gln Leu Glu Val Thr Ala Ala Leu Glu Glu Leu Arg Ala 125
130 135 cgc cgc gtc gac gtc gca ctc ggc gag gaa tac ccc gtg gaa gtc
ccc 665 Arg Arg Val Asp Val Ala Leu Gly Glu Glu Tyr Pro Val Glu Val
Pro 140 145 150 155 ctt gtt gag gcc agc att cac cgc gaa gtc ctc ttc
gaa gac ccc atg 713 Leu Val Glu Ala Ser Ile His Arg Glu Val Leu Phe
Glu Asp Pro Met 160 165 170 ctg ctc gtc acc cca gca agc ggc cca tac
tct ggc ctc acc ctg cca 761 Leu Leu Val Thr Pro Ala Ser Gly Pro Tyr
Ser Gly Leu Thr Leu Pro 175 180 185 gaa ctc cgc gac atc ccc atc gcc
atc gat cca ccc gac ctt ccc gcg 809 Glu Leu Arg Asp Ile Pro Ile Ala
Ile Asp Pro Pro Asp Leu Pro Ala 190 195 200 ggc gaa tgg gtc cat agg
ctc tgc cgg cgc gcc ggg ttt gag ccc cgc 857 Gly Glu Trp Val His Arg
Leu Cys Arg Arg Ala Gly Phe Glu Pro Arg 205 210 215 gtg acc ttt gaa
acc agc gat ccc atg ctc caa gca cac ctc gtg cgt 905 Val Thr Phe Glu
Thr Ser Asp Pro Met Leu Gln Ala His Leu Val Arg 220 225 230 235 agc
ggc ttg gcc gtg aca ttt tcc ccc aca ctg ctc acc ccg atg ctg 953 Ser
Gly Leu Ala Val Thr Phe Ser Pro Thr Leu Leu Thr Pro Met Leu 240 245
250 gaa agc gtg cac atc cag ccg ctg ccc ggc aac ccc acg cgc acg ctc
1001 Glu Ser Val His Ile Gln Pro Leu Pro Gly Asn Pro Thr Arg Thr
Leu 255 260 265 tac acc gcg gtc agg gaa ggg cgc cag ggg cat cca gcc
att aaa gct 1049 Tyr Thr Ala Val Arg Glu Gly Arg Gln Gly His Pro
Ala Ile Lys Ala 270 275 280 ttt cga cga gcc ctc gcc cat gtg gcc aaa
gaa tct tat ttg gag gct 1097 Phe Arg Arg Ala Leu Ala His Val Ala
Lys Glu Ser Tyr Leu Glu Ala 285 290 295 cgt cta gta gag tgagttcttg
tgagccttca gacaaatcat cgcccagtac 1149 Arg Leu Val Glu 300
tcgtcgttga cttcggcgca cagtacgcgc agctgatcgc acgtcgtgtg cgtgaggccg
1209 gcatctactc cgaagtcatc ccgcacaccg ccaccgcaga cgatgtgcgc
gctaaaaatg 1269 cagcagccct cgtcctttcc ggtggcccat cctccgtgta tg 1311
2 303 PRT Corynebacterium glutamicum 2 Val Leu Asn Leu Asn Arg Leu
His Ile Leu Gln Glu Phe His Arg Leu 1 5 10 15 Gly Thr Ile Thr Ala
Val Ala Glu Ser Met Asn Tyr Ser Arg Ser Ala 20 25 30 Ile Ser Gln
Gln Met Ala Leu Leu Glu Lys Glu Ile Gly Val Lys Leu 35 40 45 Phe
Glu Lys Ser Gly Arg Asn Leu Tyr Phe Thr Glu Gln Gly Glu Val 50 55
60 Leu Ala Ser Glu Thr His Ala Ile Met Ala Ala Val Asp His Ala Arg
65 70 75 80 Ala Ala Val Leu Asp Ser Leu Ser Glu Val Ser Gly Thr Leu
Lys Val 85 90 95 Thr Ser Phe Gln Ser Leu Leu Phe Thr Leu Ala Pro
Lys Ala Ile Ala 100 105 110 Arg Leu Thr Glu Lys Tyr Pro His Leu Gln
Val Glu Ile Ser Gln Leu 115 120 125 Glu Val Thr Ala Ala Leu Glu Glu
Leu Arg Ala Arg Arg Val Asp Val 130 135 140 Ala Leu Gly Glu Glu Tyr
Pro Val Glu Val Pro Leu Val Glu Ala Ser 145 150 155 160 Ile His Arg
Glu Val Leu Phe Glu Asp Pro Met Leu Leu Val Thr Pro 165 170 175 Ala
Ser Gly Pro Tyr Ser Gly Leu Thr Leu Pro Glu Leu Arg Asp Ile 180 185
190 Pro Ile Ala Ile Asp Pro Pro Asp Leu Pro Ala Gly Glu Trp Val His
195 200 205 Arg Leu Cys Arg Arg Ala Gly Phe Glu Pro Arg Val Thr Phe
Glu Thr 210 215 220 Ser Asp Pro Met Leu Gln Ala His Leu Val Arg Ser
Gly Leu Ala Val 225 230 235 240 Thr Phe Ser Pro Thr Leu Leu Thr Pro
Met Leu Glu Ser Val His Ile 245 250 255 Gln Pro Leu Pro Gly Asn Pro
Thr Arg Thr Leu Tyr Thr Ala Val Arg 260 265 270 Glu Gly Arg Gln Gly
His Pro Ala Ile Lys Ala Phe Arg Arg Ala Leu 275 280 285 Ala His Val
Ala Lys Glu Ser Tyr Leu Glu Ala Arg Leu Val Glu 290 295 300 3 383
DNA Corynebacterium glutamicum 3 ttccaatccc tgctgttcac ccttgccccg
aaagccatcg cgcgcctgac cgagaaatac 60 ccacacctgc aagtagaaat
ctcccaacta gaagtcaccg cagcgctcga agaactccgc 120 gcccgccgcg
tcgacgtcgc actcggcgag gaataccccg tggaagtccc ccttgttgag 180
gccagcattc accgcgaagt cctcttcgaa gaccccatgc tgctcgtcac cccagcaagc
240 ggcccatact ctggcctcac cctgccagaa ctccgcgaca tccccatcgc
catcgatcca 300 cccgaccttc ccgcgggcga atgggtccat aggctctgcc
ggcgcgccgg gtttgagccc 360 cgcgtgacct ttgaaaccag cga 383 4 20 DNA
Artificial Sequence synthetic DNA 4 ttccaatccc tgctgttcac 20 5 20
DNA Artificial Sequence synthetic DNA 5 gtgacctttg aaaccagcga
20
* * * * *