U.S. patent application number 09/813932 was filed with the patent office on 2002-06-27 for novel nucleotide sequences coding for the glbo gene.
Invention is credited to Marx, Achim, Mockel, Bettina, Pfefferle, Walter.
Application Number | 20020081673 09/813932 |
Document ID | / |
Family ID | 27079456 |
Filed Date | 2002-06-27 |
United States Patent
Application |
20020081673 |
Kind Code |
A1 |
Mockel, Bettina ; et
al. |
June 27, 2002 |
Novel nucleotide sequences coding for the glbO gene
Abstract
Isolated polynucleotides containing at least one nucleic acid
sequence selected from the group a) polynucleotides which is at
least 70% identical to a polynucleotide, b) b) polynucleotide which
codes for a polypeptide which contains an amino acid sequence which
is at least 70% identical to an amino acid sequence o, c)
polynucleotide which is complementary to the polynucleotides of a)
or b), and d) polynucleotide containing at least 15 successive
nucleotides of the polynucleotide sequence of a), b) or c), and a
method for the fermentative production of L-amino acids with
amplification of the glb0 gene which codes for the hemoglobin-like
protein and the use of the above polynucleotides as a primer or
hybridization probe.
Inventors: |
Mockel, Bettina;
(Dusseldorf, DE) ; Marx, Achim; (Bielefeld,
DE) ; Pfefferle, Walter; (Halle, DE) |
Correspondence
Address: |
SMITH, GAMBRELL & RUSSELL, LLP
1850 M STREET, N.W., SUITE 800
WASHINGTON
DC
20036
US
|
Family ID: |
27079456 |
Appl. No.: |
09/813932 |
Filed: |
March 22, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09813932 |
Mar 22, 2001 |
|
|
|
09585642 |
Jun 2, 2000 |
|
|
|
Current U.S.
Class: |
435/115 ;
435/183; 435/252.3; 435/320.1; 536/23.2 |
Current CPC
Class: |
C12P 13/08 20130101;
C07K 14/805 20130101; C07K 14/34 20130101; C12P 13/04 20130101 |
Class at
Publication: |
435/115 ;
435/183; 435/252.3; 536/23.2; 435/320.1 |
International
Class: |
C12P 013/04; C12P
013/08; C07H 021/04; C12N 009/00; C12N 001/21; C12N 015/74 |
Claims
What is claimed is:
1. An isolated polynucleotide from coryneform bacteria containing
one of the following polynucleotide sequences: a) a polynucleotide
which is at least 70% identical to a polynucleotide which codes for
a polypeptide containing the amino acid sequence of SEQ ID NO: 2,
b) a polynucleotide which codes for a polypeptide which contains an
amino acid sequence which is at least 70% identical 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 containing at least 15 consecutive nucleotides of
the polynucleotide sequence of a), b) or c).
2. The polynucleotide of claim 1, wherein the polynucleotide is a
recombinant DNA replicable in coryneform bacteria.
3. The polynucleotide of claim 1, wherein the polynucleotide is an
RNA.
4. The polynucleotide of claim 2, wherein the replicable DNA
comprises: (i) the nucleotide sequence shown in SEQ ID NO: 1, or
(ii) at least one sequence which matches the sequence of (i) within
the degeneration range of the genetic code, or (iii) at least one
sequence which hybridizes with the complementary sequence to
sequence (i) or (ii), or (iv) functionally neutral sense mutations
in (i).
5. The polynucleotide of claim 2 and having a sequence which codes
for a polypeptide which comprises the amino acid sequence as shown
in SEQ ID NO: 2.
6. A vector containing the polynucleotide sequence of claim 1.
7. A coryneform bacterium containing the vector as claimed in claim
6.
8. A method for the fermentative production of L-amino acids,
comprising: a) fermenting L-amino acid producing coryneform
bacteria in which at least the gene which codes for the
hemoglobin-like protein is amplified.
9. The method of claim 8, wherein bacteria are used in which
further genes of the biosynthetic pathway of the desired L-amino
acid are additionally amplified.
10. The method of claim 8, wherein bacteria are used in which the
metabolic pathways which reduce the formation of the L-amino acid
are at least partially suppressed.
11. The method of claim 8, wherein bacteria transformed with a
plasmid vector are used and the plasmid vector bears the nucleotide
sequence of the gene which codes for the hemoglobin-like
protein.
12. The method of claim 8, wherein coryneform bacteria are used
which produce L-lysine.
13. The method claim 9, wherein one or more genes selected from the
group consisting of the dapA gene which codes for dihydropicolinate
synthase,the pyc gene which codes for pyruvate carboxylase,the tpi
gene which codes for triosephosphate isomerase, the gap gene which
codes for glyceraldehyde-3-phosphate dehydrogenase,the pgk gene
which codes for 3-phosphoglycerate kinase, and the lysE gene which
codes for lysine export is/are simultaneously amplified.
14. The method of claim 10, wherein bacteria are fermented for the
production of L-lysine in which one or more of the genes selected
from the group consisting of the pck gene which codes for
phosphoenolpyruvate carboxykinase and the pgi gene which codes for
glucose-6-phosphate isomerase is/are simultaneously attenuated.
15. The method of claim 8, wherein microorganisms of the genus
Corynebacterium glutamicum are used.
16. The method of claim 8 further comprising: b. accumulating
L-amino acid in the medium or in the cells of the bacteria.
17. The method of claim 16 further comprising: c. isolating the
L-amino acid.
18. A primer which comprises a polynucleotide sequence of claim 1,
wherein the primer is used for the production of DNA of genes which
code for the gene product of Glb0 by the polymerase chain
reaction.
19. A hybridization probe comprising a polynucleotide sequence of
claim 1.
Description
[0001] The invention provides nucleotide sequences coding for the
glb0 gene and a process for the fermentative production of L-amino
acids, in particular L-lysine, using coryneform bacteria in which
the glb0 gene is amplified. All references cited herein are
expressly incorporated by reference. Incorporation by reference is
also designated by the term "I.B.R." following any citation.
PRIOR ART
[0002] L-amino acids, in particular L-lysine, are used in human
medicine and in the pharmaceuticals industry, but especially in
animal nutrition.
[0003] It is known that L-amino acids are produced by fermentation
of strains of coryneform bacteria, in particular Corynebacterium
glutamicum. Due to their great significance, efforts are constantly
being made to improve the production process. Improvements to the
process may relate to measures concerning fermentation technology,
for example stirring and oxygen supply, or to the composition of
the nutrient media, such as for example sugar concentration during
fermentation, or to working up to yield the product by, for
example, ion exchange chromatography, or to the intrinsic
performance characteristics of the microorganism itself.
[0004] The performance characteristics of these microorganisms are
improved using methods of mutagenesis, selection and mutant
selection. In this manner, strains are obtained which are resistant
to antimetabolites, such as for example the lysine analogue
S-2-aminoethyl)cysteine, or are auxotrophic for regulatorily
significant metabolites and produce L-lysine.
[0005] For some years, methods of recombinant DNA technology have
also been used to improve strains of Corynebacterium which produce
L-amino acids by amplifying individual biosynthesis genes for
L-amino acids and investigating the effect on L-amino acid
production.
[0006] Review articles on this subject may be found inter alia in
Kinoshita ("Glutamic Acid Bacteria", in: Biology of Industrial
Microorganisms, Demain and Solomon (Eds.) I.B.R., Benjamin
Cummings, London, UK, 1985, 115-142), Hilliger (BioTec 2, 40-44
(1991)) I.B.R., Eggeling (Amino Acids 6:261-272 (1994)) I.B.R.,
Jetten and Sinskey (Critical Reviews in Biotechnology 15, 73-103
(1995)) and Sahm et al. (Annuals of the New York Academy of Science
782, 25-39 (1996)) I.B.R.
OBJECT OF THE INVENTION
[0007] An object of the invention is to provide new measures for
improved fermentative preparation of L-amino acids, in particular
L-lysine. Amino acids, in particular L-lysine, are used in human
medicine, in the pharmaceuticals industry and, in particular, in
animal nutrition. Therefore, there is a general interest in
providing new improved processes for the preparation of amino
acids, in particular L-lysine. When L-lysine or lysine are
mentioned in the following, not only the base but also the salts,
such as e. g. lysine monohydrochloride or lysine sulfate, are also
meant by this.
SUMMARY OF THE INVENTION
[0008] The new DNA sequence of C. glutamicum which codes for the
glb0 gene and which as a constituent of the present invention is
SEQ ID NO 1 and related sequences. The amino acid sequence of the
corresponding gene product of the glb0 gene has furthermore been
derived from the present DNA sequence. The resulting amino acid
sequence of the glb0 gene product is SEQ ID NO 2 and related
sequences.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] The present invention will be further understood with
reference to the drawing offered here for illustration only and not
in limitation of this invention.
[0010] FIG. 1 is a map of the plasmid pEC-K18mob2.
[0011] FIG. 2 is a map of the plasmid pEC-K18mob2glb0exp.
DETAILED DESCRIPTION OF THE INVENTION
[0012] The invention provides an isolated polynucleotide from
coryneform bacteria containing a polynucleotide sequence selected
from the group consisting of:
[0013] a) polynucleotide which is at least 70% identical to a
polynucleotide which codes for a polypeptide containing the amino
acid sequence of SEQ ID NO: 2,
[0014] b) polynucleotide which codes for a polypeptide which
contains an amino acid sequence which is at least 70% identical to
the amino acid sequence of SEQ ID NO: 2,
[0015] c) polynucleotide which is complementary to the
polynucleotides of a) or b), and
[0016] d) polynucleotide containing at least 15 successive
nucleotides of the polynucleotide sequence of a), b) or c).
[0017] The invention also provides a polynucleotide which is a
preferably recombinant DNA replicable in coryneform bacteria.
[0018] The invention also provides a polynucleotide which is an
RNA.
[0019] The invention also provides a polynucleotide as described
above, wherein it preferably comprises a replicable DNA
containing:
[0020] (i) the nucleotide sequence shown in SEQ ID NO: 1, or
[0021] (ii) at least one sequence which matches the sequence (i)
within the degeneration range of the genetic code, or
[0022] (iii) at least one sequence which hybridizes with the
complementary sequence to sequence (i) or (ii) and optionally
[0023] (iv) functionally neutral sense mutations in (i).
[0024] The relative degree of substitution or mutation in the
polynucleotide or amino acid sequence to produce a desired
percentage of sequence identity can be established or determined by
well-known methods of sequence analysis. These methods are
disclosed and demonstrated in Bishop, et al. "DNA & Protein
Sequence Analysis (A Practical Approach"), Oxford Univ. Press, Inc.
(1997) I.B.R. and by Steinberg, Michael "Protein Structure
Prediction" (A Practical Approach), Oxford Univ. Press, Inc. (1997)
I.B.R. Hybridization of complementary sequences can occur at
varying degrees of stringency. Sambrook et al.: Molecular Cloning,
A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989)
I.B.R.
[0025] Hybridization of complementary sequences can occur at
varying degrees of stringency. Sambrook et al.: Molecular Cloning,
A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989)
I.B.R. 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)I.B.R. and in
Liebl et al. (International Journal of Systematic Bacteriology
(1991) 41: 255-260) I.B.R.
[0026] Comprehensive descriptions can be found in known textbooks
of genetics and molecular biology, such as e. g. that by Hagemann
("Allgemeine Genetik" [General Genetics], Gustav Fischer Verlag,
Stuttgart, 1986) I.B.R.
[0027] Possible mutations are transitions, transversions,
insertions and deletions. Depending on the effect of the amino acid
exchange on the enzyme activity, missense mutations or nonsense
mutations are referred to. Insertions or deletions of at least one
base pair in a gene lead to frame shift mutations, as a consequence
of which incorrect amino acids are incorporated or translation is
interrupted prematurely. Deletions of several codons typically lead
to a complete loss of the enzyme activity.
[0028] Instructions on generation of such mutations are prior art
and can be found in known textbooks of genetics and molecular
biology, such as e. g. the textbook by Knippers ("Molekulare
Genetik" [Molecular Genetics], 6th edition, Georg Thieme Verlag,
Stuttgart, Germany, 1995) I.B.R., that by Winnacker ("Gene und
Klone" [Genes and Clones], VCH Verlagsgesellschaft, Weinheim,
Germany, 1990) I.B.R. or that by Hagemann ("Allgemeine Genetik"
[General Genetics], Gustav Fischer Verlag, Stuttgart, 1986)
I.B.R.
[0029] The invention also provides a vector containing one of the
stated polynucleotides and coryneform bacteria acting as host cell
which contain the vector or in which the glb0 gene is
amplified.
[0030] The invention also provides polynucleotides which
substantially consist of a polynucleotide sequence, which are
obtainable by screening by means of hybridization of a suitable
gene library, which contains the complete gene having the
polynucleotide sequence according to SEQ ID NO: 1, with a probe
which contains the sequence of the stated polynucleotide according
to SEQ ID NO: 1, or a fragment thereof, and isolation of the stated
DNA sequence.
[0031] Polynucleotide sequences according to the invention are
suitable as hybridization probes for RNA, cDNA and DNA in order to
isolate full length cDNA which code for the hemoglobin-like protein
(glb0) and to isolate such cDNA or genes, the sequence of which
exhibits a high level of similarity with that of the gene for the
hemoglobin-like protein.
[0032] Polynucleotide sequences according to the invention are
furthermore suitable as primers for the production of DNA of genes
which code for the hemoglobin-like protein by the polymerase chain
reaction (PCR).
[0033] Such oligonucleotides acting as probes or primers contain at
least 30, preferably at least 20, very particularly preferably at
least 15 successive nucleotides. Oligonucleotides having a length
of at least 40 or 50 nucleotides are also suitable.
[0034] "Isolated" means separated from its natural environment.
[0035] "Polynucleotide" generally relates to polyribonucleotides
and polydeoxyribonucleotides, wherein the RNA or DNA may be
unmodified or modified.
[0036] "Polypeptides" are taken to mean peptides or proteins which
contain two or more amino acids connected by peptide bonds.
[0037] The polypeptides according to the invention include a
polypeptide according to SEQ ID NO: 2, in particular those having
the biological activity of the hemoglobin-like protein and also
those which are at least 70% identical to the polypeptide according
to SEQ ID NO: 2 and which preferably exhibit at least 80% and in
particular at least 90% to 95% identity to the polypeptide
according to SEQ ID NO: 2 and exhibit the stated activity.
[0038] The invention furthermore relates to a process for the
fermentative production of L-amino acids, in particular L-lysine,
using coryneform bacteria, which in particular already produce an
L-amino acid and in which the nucleotide sequences which code for
the glb0 gene are amplified, in particular overexpressed.
[0039] In this connection, the term "amplification" describes the
increase in the intracellular activity of one or more enzymes in a
microorganism, which enzymes are coded by the corresponding DNA,
for example by increasing the copy number of the gene or genes, by
using a strong promoter or a gene which codes for a corresponding
enzyme having elevated activity and optionally by combining these
measures.
[0040] The microorganisms, provided by the present invention, may
produce L-amino acids, in particular L-lysine, from glucose,
sucrose, lactose, fructose, maltose, molasses, starch, cellulose or
from glycerol and ethanol. The microorganisms may comprise
representatives of the coryneform bacteria in particular of the
genus Corynebacterium. Within the genus Corynebacterium, the
species Corynebacterium glutamicum may in particular be mentioned,
which is known in specialist circles for its ability to produce
L-amino acids.
[0041] Suitable strains of the genus Corynebacterium, in particular
of the species Corynebacterium glutamicum, are for example the
known wild type strains
[0042] Corynebacterium glutamicum ATCC13032
[0043] Corynebacterium acetoglutamicum ATCC15806
[0044] Corynebacterium acetoacidophilum ATCC13870
[0045] Corynebacterium thermoaminogenes FERM BP-1539
[0046] Corynebacterium melassecola ATCC17965
[0047] Brevibacterium flavum ATCC14067
[0048] Brevibacterium lactofermentum ATCC13869 and
[0049] Brevibacterium divaricatum ATCC14020
[0050] and L-lysine producing mutants or strains produced
therefrom, such as for example
[0051] Corynebacterium glutamicum FERM-P 1709
[0052] Brevibacterium flavum FERM-P 1708
[0053] Brevibacterium lactofermentum FERM-P 1712
[0054] Corynebacterium glutamicum FERM-P 6463
[0055] Corynebacterium glutamicum FERM-P 6464 and
[0056] Corynebacterium glutamicum DSM5715.
[0057] The inventors have succeeded in isolating the novel glb0
gene which codes for the hemoglobin-like protein from C.
glutamicum.
[0058] The glb0 gene or also other genes from C. glutamicum are
isolated by initially constructing a gene library of this
microorganism in E. coli. The construction of gene libraries is
described in generally known textbooks and manuals. Examples which
may be mentioned are the textbook by Winnacker, Gene und Klone,
Eine Elnruhrung in die Gentechnologie (Verlag Chemie, Weinheim,
Germany, 1990) I.B.R. or the manual by Sambrook et al., Molecular
Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press,
1989) I.B.R. One very well known gene library is that of E. coli
K-12 strain W3110, which was constructed by Kohara et al. (Cell 50,
495-508 (1987)) I.B.R. in .lambda.-vectors. Bathe et al. (Molecular
and General Genetics, 252:255-265, 1996) I.B.R. describe a gene
library of C. glutamicum ATCC13032, which was constructed using the
cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the
National Academy of Sciences USA, 84:2160-2164) I.B.R. in E. coli
K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research
16:1563-1575) I.B.R. Bormann et al. (Molecular Microbiology 6(3),
317-326, 1992)) I.B.R. also describe a gene library of C.
glutamicum ATCC 13032, using cosmid pHC79 (Hohn and Collins, Gene
11, 291-298 (1980)) I.B.R. A gene library of C. glutamicum in E.
coli may also be produced using plasmids such as pBR322 (Bolivar,
Life Sciences, 25, 807-818 (1979)) I.B.R. or pUC9 (Vieira et al.,
1982, Gene, 19:259-268) I.B.R. Suitable hosts are in particular
those E. coli strains with restriction and recombination defects.
One example of such a strain 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) I.B.R. The long DNA fragments
cloned with the assistance of cosmids may then in turn be
sub-cloned in usual vectors suitable for sequencing and then be
sequenced, as described, for example, in Sanger et al. (Proceedings
of the National Academy of Sciences of the United States of
America, 74:5463-5467, 1977) I.B.R.
[0059] The novel DNA sequence from C. glutamicum which codes for
the glb0 gene and, as SEQ ID NO: 1, is provided by the present
invention, was obtained in this manner. The amino acid sequence of
the corresponding protein was furthermore deduced from the above
DNA sequence using the methods described above. SEQ ID NO: 2 shows
the resultant amino acid sequence of the product of the glb0
gene.
[0060] Coding DNA sequences arising from SEQ ID NO: 1 due to the
degeneracy of the genetic code are also provided by the invention.
Conservative substitutions of amino acids in proteins, for example
the substitution of glycine for alanine or of aspartic acid for
glutamic acid, are known in specialist circles as "sense
mutations", which result in no fundamental change in activity of
the protein, i.e. they are functionally neutral. It is furthermore
known that changes to the N and/or C terminus of a protein do not
substantially impair or may even stabilize the function thereof.
The person skilled in the art will find information in this
connection inter alia in Ben-Bassat et al. (Journal of Bacteriology
169:751-757 (1987)) I.B.R., in O'Regan et al. (Gene 77:237-251
(1989)) I.B.R., in Sahin-Toth et al. (Protein Sciences 3:240-247
(1994)) I.B.R., in Hochuli et al. (Bio/Technology 6:1321-1325
(1988)) I.B.R. and in known textbooks of genetics and molecular
biology. Amino acid sequences arising in a corresponding manner
from SEQ ID NO: 2 and DNA sequences which code for these amino acid
sequences are also provided by the invention.
[0061] 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)) I.B.R., in O'Regan et al. (Gene
77:237-251 (1989)) I.B.R., in Sahin-Toth et al. (Protein Sciences
3:240-247 (1994)) I.B.R., in Hochuli et al. (Bio/Technology
6:1321-1325 (1988)) I.B.R. 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.
[0062] DNA sequences which hybridize with SEQ ID NO: 1 or parts of
SEQ ID NO: 1 are similarly provided by the invention. Finally, DNA
sequences produced by the polymerase chain reaction (PCR) using
primers obtained from SEQ ID NO: 1 are also provided by the
invention. Such oligonucleotides typically have a length of at
least 15 nucleotides.
[0063] 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) I.B.R. and in
Liebl et al. (International Journal of Systematic Bacteriology
(1991) 41: 255-260) I.B.R. 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:
Oligonukleotide synthesis: a practical approach (IRL Press, Oxford,
UK, 1984) I.B.R. and in Newton and Graham: PCR (Spektrum
Akademischer Verlag, Heidelberg, Germany, 1994) I.B.R.
[0064] The inventors discovered that coryneform bacteria produce
L-amino acids, in particular L-lysine, in an improved manner once
the glb0 has been overexpressed.
[0065] Overexpression may be achieved by increasing the copy number
of the corresponding genes or by mutating the promoter and
regulation region or the ribosome-binding site located upstream
from the structural gene. Expression cassettes incorporated
upstream from the structural gene act in the same manner. It is
additionally possible to increase expression during fermentative
L-amino acid production by means of inducible promoters. Expression
is also improved by measures to extend the lifetime of the mRNA.
Enzyme activity is moreover amplified by preventing degradation of
the enzyme protein. The genes or gene constructs may either be
present in plasmids in a variable copy number or be integrated in
the chromosome and amplified. Alternatively, overexpression of the
genes concerned may also be achieved by modifying the composition
of the nutrient media and culture conditions.
[0066] The person skilled in the art will find guidance in this
connection inter alia in Martin et al. (Bio/Technology 5, 137-146
(1987)) I.B.R., in Guerrero et al. (Gene 138, 35-41 (1994)) I.B.R.,
Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)) I.B.R., in
Eikmanns et al. (Gene 102, 93-98 (1991)) I.B.R., in European patent
EPS 0 472 869, in U.S. Pat. No. 4,601,893 I.B.R., in Schwarzer and
Puhler (Bio/Technology 9, 84-87 (1991) [sic] I.B.R., in Fitzpatrick
et al. (Applied Microbiology and Biotechnology 42, 575-580 (1994)
I.B.R. in Reinscheid et al. (Applied and Environmental Microbiology
60, 126-132 (1994)), in LaBarre et al. (Journal of Bacteriology
175, 1001-1007 (1993)) I.B.R., in patent application WO 96/15246
I.B.R., in Malumbres et al. (Gene 134, 15-24 (1993)) I.B.R., in
Japanese published patent application JP-A-10-229891 I.B.R., in
Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195
(1998)) I.B.R., in Makrides (Microbiological Reviews 60:512-538
(1996)) I.B.R. and in known textbooks of genetics and molecular
biology.
[0067] The expert can find additional information on this e.g. in
the patent application WO 96/15246 I.B.R., in Boyd and Murphy
(Journal of Bacteriology 170: 5949 (1988)) I.B.R., in Voskuil and
Chambliss (Nucleic Acids Research 26: 3548 (1998) I.B.R., in Jensen
and Hammer (Biotechnology and Bioengineering 58: 191 (1998))
I.B.R., in Ptek et al. (Microbiology 142: 1297 (1996)) I.B.R. and
in known textbooks of genetics and molecular biology, such as e. g.
the textbook by Knippers ("Molekulare Genetik" [Molecular
Genetics], 6th edition, Georg Thieme Verlag, Stuttgart, Germany,
1995) I.B.R. or that by Winnacker ("Gene und Klone" [Genes and
Clones], VCH Verlagsgesellschaft, Weinheim, Germany, 1990)
I.B.R.
[0068] By way of example, the glb0 gene according to the invention
was overexpressed with the assistance of plasmids.
[0069] Suitable plasmids are those which are replicated in
coryneform bacteria. Numerous known plasmid vectors, such as for
example pZ1 (Menkel et al., Applied and Environmental Microbiology,
64: 549-554(1989)) I.B.R., pEKEx1 (Eikmanns et al., Gene 102:93-98
(1991)) I.B.R. or pHS2-1 (Sonnen et al., Gene 107:69-74 (1991))
I.B.R. are based on the cryptic plasmids pHM1519, pBL1 or pGA1.
Other plasmid vectors, such as for example those based on pCG4
(U.S. Pat. No. 4,489,160) I.B.R., or pNG2 (Serwold-Davis et al.,
FEMS Microbiology Letters 66, 119-124 (1990)) I.B.R., or pAG1 (U.S.
Pat. No. 5,158,891) I.B.R. may be used in the same manner.
[0070] Further suitable plasmid vectors are those with the
assistance of which gene amplification may be performed by
integration into the chromosome, as has for example been described
by Reinscheid et al. (Applied and Environmental Microbiology 60,
126-132 (1994)) I.B.R. for the duplication or amplification of the
hom-thrB operon. In this method, the complete gene is cloned into a
plasmid vector which can replicate in a host (typically E. coli),
but not in C. glutamicum. Vectors which may be considered are, for
example, pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983))
I.B.R., pK18mob or pK19mob (Schfer et al., Gene 145, 69-73 (1994))
I.B.R., pGEM-T (Promega corporation, Madison, Wis., USA),
pCR2.1-TOPO (Shuman (1994) I.B.R. Journal of Biological Chemistry
269:32678-84 I.B.R.; U.S. Pat. No. 5,487,993) I.B.R., pCR.RTM.Blunt
(Invitrogen, Groningen, Netherlands; Bernard et al., Journal of
Molecular Biology, 234: 534-541 (1993)) I.B.R. or pEM1 (Schrumpf et
al, 1991, Journal of Bacteriology 173:4510-4516) I.B.R. 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 conjugation method is described, for example,
in Schfer et al. (Applied and Environmental Microbiology 60,
756-759 (1994)) I.B.R. Transformation methods are described, for
example, in Thierbach et al. (Applied Microbiology and
Biotechnology 29, 356-362 (1988)) I.B.R., Dunican and Shivnan
(Bio/Technology 7, 1067-1070 (1989)) I.B.R. and Tauch et al. (FEMS
Microbiological Letters 123, 343-347 (1994)) I.B.R. After
homologous recombination by means of "crossing over", the resultant
strain contains at least two copies of the gene in question.
[0071] The invention accordingly also provides a process for the
fermentative production of L-amino acids, in particular L-lysine,
in which a strain transformed with a plasmid vector is used and the
plasmid vector bears the nucleotide sequence of the gene which
codes for the hemoglobin-like protein.
[0072] It may additionally be advantageous for the production of
L-amino acids, in particular L-lysine, to amplify not only the glb0
gene, but also further genes of the biosynthetic pathway of the
desired L-amino acid, such that one or more enzymes of the
particular biosynthetic pathway, of glycolysis, of anaplerotic
metabolism or of amino acid export is/are overexpressed.
[0073] For the production of L-lysine, for example, it is thus
possible simultaneously to overexpress one or more genes selected
from the group
[0074] the dapA gene which codes for dihydropicolinate synthase
(EP-B 0 197 335) I.B.R.,
[0075] the gap gene which codes for glyceraldehyde-3-phosphate
dehydrogenase (Eikmanns (1992), Journal of Bacteriology
174:6076-6086) I.B.R.,
[0076] the tpi gene which codes for triosephosphate isomerase
(Eikmanns (1992), Journal of Bacteriology 174:6076-6086)
I.B.R.,
[0077] the pgk gene which codes for 3-phosphoglycerate kinase
(Eikmanns (1992), Journal of Bacteriology 174:6076-6086)
I.B.R.,
[0078] the pyc gene which codes for pyruvate carboxylase (Eikmanns
(1992), Journal of Bacteriology 174:6076-6086) I.B.R. and
[0079] the lysE gene which codes for lysine export (DE-A-195 48
222) I.B.R.
[0080] It may furthermore be advantageous for the production of
L-amino acids, in particular L-lysine, apart from the glb0 gene,
simultaneously to attenuate
[0081] the pck gene which codes for phosphoenolpyruvate
carboxykinase (DE 199 50 409.1, DSM 13047) I.B.R. and/or
[0082] the pgi gene which codes for glucose-6-phosphate isomerase
(U.S. Ser. No. 09/396,478, DSM 12969) I.B.R.
[0083] the poxB gene which codes for pyruvate oxidase (DE:
19951975.79) I.B.R.
[0084] It may furthermore be advantageous for the production of
L-amino acids, in particular L-lysine, in addition to
overexpressing the glb0 gene, to suppress unwanted secondary
reactions (Nakayama: "Breeding of Amino Acid Producing
Micro-organisms", in: Overproduction of Microbial Products,
Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982)
I.B.R.
[0085] For the purposes of amino acid production, in particular of
L-lysine, the microorganisms produced according to the invention
may be cultured continuously or discontinuously using the batch
process or the fed batch process or repeated fed batch process. A
summary of known culture methods is given in the textbook by Chmiel
(Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik
(Gustav Fischer Verlag, Stuttgart, 1991)) I.B.R. or in the textbook
by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg
Verlag, Braunschweig/Wiesbaden, 1994)) I.B.R.
[0086] The culture medium to be used must adequately satisfy the
requirements of the particular strains. Culture media for various
microorganisms are described in "Manual of Methods for General
Bacteriology" from the American Society for Bacteriology
(Washington D.C., USA, 1981) I.B.R. Carbon sources which may be
used are sugars and carbohydrates, such as glucose, sucrose,
lactose, fructose, maltose, molasses, starch and cellulose for
example, oils and fats, such as soya oil, sunflower oil, peanut oil
and coconut oil for example, fatty acids, such as palmitic acid,
stearic acid and linoleic acid for example, alcohols, such as
glycerol and ethanol for example, and organic acids, such as acetic
acid for example. These substances may be used individually or as a
mixture. Nitrogen sources which may be used comprise organic
compounds containing nitrogen, such as peptones, yeast extract,
meat extract, malt extract, corn steep liquor, soya 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. Phosphorus sources which may be used are phosphoric acid,
potassium dihydrogen phosphate or dipotassium hydrogen phosphate or
the corresponding salts containing sodium. The culture medium has
additionally to contain salts of metals, such as magnesium sulfate
or iron sulfate for example, which are necessary for growth.
Finally, essential growth-promoting substances such as amino acids
and vitamins may also be used in addition to the above-stated
substances. Suitable precursors may furthermore be added to the
culture medium. The stated feed substances may be added to the
culture as a single batch or be fed appropriately during
cultivation.
[0087] Basic compounds, such as sodium hydroxide, potassium
hydroxide, ammonia or ammonia water, or acidic compounds, such as
phosphoric acid or sulfuric acid, are used appropriately to control
the pH of the culture. Foaming may be controlled by using
antifoaming agents such as fatty acid polyglycol esters for
example. Plasmid stability may be maintained by the addition to the
medium of suitable selectively acting substances, for example
antibiotics. Oxygen or oxygen-containing gas mixtures, such as air
for example, are introduced into the culture in order to maintain
aerobic conditions. The temperature of the culture is normally from
20.degree. C. to 45.degree. C. and preferably from 25.degree. C. to
40.degree. C. The culture is continued until the maximum quantity
of L-lysine has formed. This objective is normally achieved within
10 hours to 160 hours.
[0088] The invention accordingly provides a process for the
fermentative production of L-amino acids, in particular L-lysine,
in which the following steps are performed:
[0089] a) fermentation of the L-amino acid producing coryneform
bacteria in which at least the glb0 gene which codes for the
hemoglobin-like protein is amplified, in particular
overexpressed.
[0090] b) accumulation of the L-amino acid in the medium or in the
cells of the bacteria and
[0091] c) isolation of the L-amino acid.
[0092] Analysis of L-lysine may be performed by anion exchange
chromatography with subsequent ninhydrin derivatization, as
described in Spackman et al. (Analytical Chemistry, 30, (1958),
1190) I.B.R.
[0093] The purpose of the process according to the invention is the
fermentative production of L-amino acids, in particular
L-lysine.
EXAMPLES
[0094] The present invention is illustrated in greater detail by
the following practical examples.
Example 1
[0095] Production of a genomic cosmid gene library from
Corynebacterium glutamicum ATCC13032
[0096] Chromosomal DNA from Corynebacterium glutamicum ATCC 13032
was isolated as described in Tauch et al., (1995, Plasmid
33:168-179) I.B.R. and partially cleaved with the restriction
enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, product
description Sau3AI, code no. 27-0913-02) I.B.R. The DNA fragments
were dephosphorylated with shrimp alkaline phosphatase (Roche
Molecular Biochemicals, Mannheim, Germany, product description SAP,
code no. 1758250) I.B.R. The DNA of cosmid vector SuperCos1 (Wahl
et al. (1987) Proceedings of the National Academy of Sciences USA
84:2160-2164) I.B.R., purchased 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)
I.B.R. and also dephosphorylated with shrimp alkaline phosphatase.
The cosmid DNA was then cleaved with the restriction enzyme BamHI
(Amersham Pharmacia, Freiburg, Germany, product description BamHI,
code no. 27-0868-04) I.B.R. Cosmid DNA treated in this manner was
mixed with the treated ATCC 13032 DNA and the batch was treated
with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, product
description T4 DNA Ligase, code no. 27-0870-04) I.B.R. The ligation
mixture was then packed in phages using Gigapack II XL Packing
Extracts (Stratagene, La Jolla, USA, product description Gigapack
II XL Packing Extract, code no. 200217). E. coli strain NM554
(Raleigh et al. 1988, Nucleic Acid Research 16:1563-1575) I.B.R.
was infected by suspending the cells in 10 mM MgSO.sub.4 and mixing
them with an aliquot of the phage suspension. The cosmid library
was infected and titred as described in Sambrook et al. (1989,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor) I.B.R.,
the cells being plated out on LB agar (Lennox, 1955, Virology,
1:190) I.B.R. with 100 .mu.g/ml of ampicillin. After overnight
incubation at 37.degree. C., individual recombinant clones were
selected.
Example 2
[0097] Isolation and sequencing of the glb0 gene.
[0098] Cosmid DNA from an individual colony was isolated in
accordance with the manufacturer's instructions using the Qiaprep
Spin Miniprep Kit (product no. 27106, Qiagen, Hilden, Germany) and
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).
Once separated by gel electrophoresis, the cosmid fragments of a
size of 1500 to 2000 bp were isolated using the QiaExII Gel
Extraction Kit (product no. 20021, Qiagen, Hilden, Germany). The
DNA of the sequencing vector pZero-1 purchased 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). Ligation of the cosmid
fragments into the sequencing vector pZero-1 was performed as
described by Sambrook et al. (1989, Molecular Cloning: A laboratory
Manual, Cold Spring Harbor) I.B.R., the DNA mixture being incubated
overnight with T4 ligase (Pharmacia Biotech, Freiburg, Germany).
This ligation mixture was then electroporated into the E. coli
strain DH5aMCR (Grant, 1990, Proceedings of the National Academy of
Sciences U.S.A., 87:4645-4649) I.B.R. (Tauch et al. 1994, FEMS
Microbiol Letters, 123:343-7) I.B.R. and plated out onto LB agar
(Lennox, 1955, Virology, 1:190) I.B.R. with 50 .mu.g/ml of Zeocin.
Plasmids of the recombinant clones were prepared using the Biorobot
9600 (product no. 900200, Qiagen, Hilden, Germany, Germany).
Sequencing was performed using the dideoxy chain termination method
according to Sanger et al. (1977, Proceedings of the National
Academy of Sciences U.S.A., 74:5463-5467) I.B.R. as modified by
Zimmermann et al. (1990, Nucleic Acids Research, 18:1067) I.B.R.
The "RR dRhodamin Terminator Cycle Sequencing Kit" from PE Applied
Biosystems (product no. 403044, Weiterstadt, Germany) was used.
Separation by gel electrophoresis and analysis of the sequencing
reaction was performed in a "Rotiphorese NF"
acrylamide/bisacrylamide gel (29:1) (product no. A124.1, Roth,
Karlsruhe, Germany) using the "ABI Prism 377" sequencer from PE
Applied Biosystems (Weiterstadt, Germany).
[0099] The resultant raw sequence data were then processed using
the Staden software package (1986, Nucleic Acids Research,
14:217-231) I.B.R., version 97-0. The individual sequences of the
pZero 1 derivatives were assembled into a cohesive contig.
Computer-aided coding range analysis was performed using XNIP
software (Staden, 1986, Nucleic Acids Research, 14:217-231) I.B.R.
Further analysis was performed using the "BLAST search programs"
(Altschul et al., 1997, Nucleic Acids Research, 25:3389-3402)
I.B.R., against the non-redundant database of the "National Center
for Biotechnology Information" (NCBI, Bethesda, Md., USA).
[0100] The resultant nucleotide sequence is stated in SEQ ID NO: 1.
Analysis of the nucleotide sequence revealed an open reading frame
of 393 base pairs, which was designated the glb0 gene. The glb0
gene codes for a protein of 131 amino acids.
Sequence CWU 1
1
2 1 840 DNA Corynebacterium glutamicum CDS (186)..(578) 1
atggtggttt taccggcgcc atttggacca agtacggccc agtgcgaccc ttgcggaatt
60 gagagggaga tatcgtcgag aagcaatttc tcgccgcgcc gcacggtgac
gccggccatg 120 ttgagtgcta gttcgcgcat gggtaacacc ttactggcgt
aaggccaggg cttagactgg 180 taccc atg aca acc tca gaa aat ttt tat gat
tct gtg ggc ggc gag gaa 230 Met Thr Thr Ser Glu Asn Phe Tyr Asp Ser
Val Gly Gly Glu Glu 1 5 10 15 acg ttt tcc ctc atc gtc cac cgt ttt
tat gaa cag gtc ccc aac gac 278 Thr Phe Ser Leu Ile Val His Arg Phe
Tyr Glu Gln Val Pro Asn Asp 20 25 30 gat att tta ggc ccg atg tat
ccg ccg gat gat ttt gag ggc gcc gag 326 Asp Ile Leu Gly Pro Met Tyr
Pro Pro Asp Asp Phe Glu Gly Ala Glu 35 40 45 cag cgt cta aag atg
ttc ctc agc cag tac tgg ggc ggc ccg aag gat 374 Gln Arg Leu Lys Met
Phe Leu Ser Gln Tyr Trp Gly Gly Pro Lys Asp 50 55 60 tat cag gag
cag cgt gga cac cct cgt ctg cgc atg cgt cac gtc aat 422 Tyr Gln Glu
Gln Arg Gly His Pro Arg Leu Arg Met Arg His Val Asn 65 70 75 tac
ccc atc ggc gtc acc gca gcg gag cgt tgg ctg cag ctc atg tcc 470 Tyr
Pro Ile Gly Val Thr Ala Ala Glu Arg Trp Leu Gln Leu Met Ser 80 85
90 95 aat gca ctc gac ggc gtg gat ttg acc gcg gag cag cgt gaa gcg
att 518 Asn Ala Leu Asp Gly Val Asp Leu Thr Ala Glu Gln Arg Glu Ala
Ile 100 105 110 tgg gag cat atg gtg cgc gcg gcc gat atg ctg atc aat
tcc aac ccc 566 Trp Glu His Met Val Arg Ala Ala Asp Met Leu Ile Asn
Ser Asn Pro 115 120 125 gat ccg cac gct taacttctgc caaaaagtcg
ttttgaccat aagctaagcg 618 Asp Pro His Ala 130 attgtgaatc gaattgcaga
aatcgcacgc agtttcggcg tgctgggctt cagcgctttc 678 ggcggcccca
ccgcgcacct cggatatttc cgcacggaat tcgtggagcg gcggcgctgg 738
ctggatgatc gccaatattc cgagatcgta gcgctcagcc aactacttcc cggacctgga
798 tcgtcgcagg tcggtatgat gctgggctac caccgcgccg gt 840 2 131 PRT
Corynebacterium glutamicum 2 Met Thr Thr Ser Glu Asn Phe Tyr Asp
Ser Val Gly Gly Glu Glu Thr 1 5 10 15 Phe Ser Leu Ile Val His Arg
Phe Tyr Glu Gln Val Pro Asn Asp Asp 20 25 30 Ile Leu Gly Pro Met
Tyr Pro Pro Asp Asp Phe Glu Gly Ala Glu Gln 35 40 45 Arg Leu Lys
Met Phe Leu Ser Gln Tyr Trp Gly Gly Pro Lys Asp Tyr 50 55 60 Gln
Glu Gln Arg Gly His Pro Arg Leu Arg Met Arg His Val Asn Tyr 65 70
75 80 Pro Ile Gly Val Thr Ala Ala Glu Arg Trp Leu Gln Leu Met Ser
Asn 85 90 95 Ala Leu Asp Gly Val Asp Leu Thr Ala Glu Gln Arg Glu
Ala Ile Trp 100 105 110 Glu His Met Val Arg Ala Ala Asp Met Leu Ile
Asn Ser Asn Pro Asp 115 120 125 Pro His Ala 130
* * * * *