Note: Descriptions are shown in the official language in which they were submitted.
CA 02319716 2000-10-04
990152 BT
1
New nucleotide sequences which code for the eno gene
The invention provides nucleotide sequences which code for
the eno gene and processes for the fermentative preparation
of amino acids, in particular L-lysine, using coryneform
bacteria in which the eno gene is amplified.
Prior art
Amino acids, in particular L-lysine, are used in human
medicine and in the pharmaceuticals industry, but in
particular in animal nutrition.
It is known that amino acids are prepared by fermentation
of strains of coryneform bacteria, in particular
Corynebacterium glutamicum. Because of its great
importance, work is constantly being undertaken to improve
the preparation processes. Improvements to the processes
can relate to fermentation measures, such as e.g. stirring
and supply of oxygen, or the composition of the nutrient
media, such as e.g. the sugar concentration during the
fermentation, or the working up to the product form by e.g.
ion exchange chromatography, or the intrinsic output
properties of the microorganism itself.
Methods of mutagenesis, selection and mutant selection are
used to improve the output properties of these -
microorganisms. Strains which are resistant to
antimetabolites, such as e.g. the lysine analogue S-(2-
aminoethyl)-cysteine, or are auxotrophic for amino acids of
regulatory importance and produce L-lysine are obtained in
this manner.
CA 02319716 2000-10-04
.,
990152 BT
2
Methods of the recombinant DNA technique have also been
employed for some years for improving the strain of
Corynebacterium strains which produce amino acids, by
amplifying individual amino acid biosynthesis genes and
investigating the effect on the amino acid production.
Review articles in this context are to be found, inter
alia, in Kinoshita ("Glutamic Acid Bacteria", in: Biology
of Industrial Microorganisms, Demain and Solomon (Eds.),
Benjamin Cummings, London, UK, 1985, 115-142), Hilliger
(BioTec 2, 40-44 (1991)), Eggeling (Amino Acids 6:261-272
(1994)), 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)).
990152 BT
CA 02319716 2000-10-04
3
Object of the invention
The inventors had the object of providing new measures for
improved fermentative preparation of amino acids, in
particular L-lysine.
Description of the invention
Amino acids, in particular L-Lysine, are used in human
medicine, in the pharmaceuticals industry and in particular
in animal nutrition. There is therefore 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.
The invention provides an isolated polynucleotide from
coryneform bacteria, comprising a polynucleotide sequence
chosen from the group consisting of
a) polynucleotide which is identical to the extent of at
least 70 % to a polynucleotide which codes for a
polypeptide which comprises the amino acid sequence.of
SEQ ID No. 2,
b) polynucleotide which codes for a polypeptide which
comprises an amino acid sequence which is identical to
the extent of at least 70o to the amino acid sequence of
SEQ ID No. 2,
' ~ 990152 BT
CA 02319716 2000-10-04
4
c) polynucleotide which is complementary to the
polynucleotides of a) or b) and
d) polynucleotide comprising at least 15 successive bases
of the polynucleotide sequence of a), b) or c).
The invention also provides the polynucleotide according to
claim 1, this preferably being a DNA which is capable of
replication, comprising:
(i) the nucleotide sequence shown in SEQ ID no. 1, or
(ii) at least one sequence which corresponds to
sequence (i) within the range of the degeneration
of the genetic code, or
(iii) at least one sequence which hybridizes with the
sequence complementary to sequence (i) or (ii),
and optionally
(iv) sense mutations of neutral function in (i).
The invention also provides
a polynucleotide according to claim 4, comprising the
nucleotide sequence as shown in SEQ ID no. 1,
a polynucleotide according to claim 6, which codes for a
polypeptide which comprises the amino acid sequence as
shown in SEQ ID No. 2,
a vector containing the polypeptide according to claim 1,
in particular a shuttle vector or plasmid vector
CA 02319716 2000-10-04
990152 BT
S
and coryneform bacteria serving as the host cell, which
contain the vector.
The invention also provides polynucleot:ides which
substantially comprise a polynucleotide sequence, which are
obtainable by screening by means of hybridization of a
corresponding gene library, which contains the complete
gene with the polynucleotide sequence corresponding to SEQ
ID no. 1, with a probe which contains the sequence of the
polynucleotide mentioned, according to SEQ ID no. 1, or a
fragment thereof, and isolation of the DNA sequence
mentioned.
Polynucleotide sequences according to the invention are
suitable as hybridization probes for RNA, cDNA and DNA, in
order to isolate, in the full length, cDNA which code for
enolase and to isolate those cDNA or genes which have a
high similarity of sequence with that of the enolase gene.
Polynucleotide sequences according to the invention are
furthermore suitable as primers for the preparation of DNA
of genes which code for enolase by the polymerase chain
reaction (PCR).
Such oligonucleotides which serve as probes or primers
comprise at least 30, preferably at least 20, especially
preferably at least 15 successive bases. Oligonucleotides
which have a length of at least 40 or 50 base pairs are
also suitable.
"Isolated" means separated out of its natural environment.
CA 02319716 2000-10-04
990152 BT
6
"Polynucleotide" in general relates to polyribonucleotides
and polydeoxyribonucleotides, it being possible for these
to be non-modified RNA or DNA or modified RNA or DNA.
"Polypeptides" is understood as meaning peptides or
proteins which comprise two or more amino acids bonded via
peptide bonds.
The polypeptides according to the invention include a
polypeptide according to SEQ ID No. 2, in particular those
with the biological activity of enolase, and also those
which are identical to the extent of at least 70 % to the
polypeptide according to SEQ ID No. 2, and preferably are
identical to the extent of 80o and in particular to the
extent of at least 90 % to 95 % to the polypeptide
according to SEQ ID no. 2, and have the activity mentioned.
The invention also provides a process for the fermentative
preparation of amino acids, in particular L-lysine, using
coryneform bacteria which in particular already produce an
amino acid, and in which the nucleotide sequences which
code for the eno gene are amplified, in particular over-
expressed.
The term "amplification" in this connection describes the
increase in the intracellular activity of one or more
enzymes in a microorganism which are coded by the
corresponding DNA, for example by increasing the number of
copies of the gene or genes, using a potent promoter or
using a gene which codes for a corresponding enzyme having
a high activity, and optionally combining these measures.
CA 02319716 2000-10-04
990152 BT
7
The microorganisms which the present invention provides can
prepare L-amino acids, in particular L-lysine, from
glucose, sucrose, lactose, fructose, maltose, molasses,
starch, cellulose or from glycerol and ethanol. They can be
S representatives of coryneform bacteria, in particular of
the genus Corynebacterium. Of the genus Corynebacterium,
there may be mentioned in particular the species
Corynebacterium glutamicum, which is known among
specialists for its ability to produce L-amino acids.
Suitable strains of the genus Corynebacterium, in
particular of the species Corynebacterium glutamicum, are,
for example, the known wild-type strains
Corynebacterium glutamicum ATCC13032
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium thermoaminogenes FERM BP-1539
Corynebacterium melassecola ATCC17965
Brevibacterium flavum ATCC14067
Brevibacterium lactofermentum ATCC13869 and
Brevibacterium divaricatum ATCC14020
and L-lysine-producing mutants or strains prepared
therefrom, such as, for example
Corynebacterium glutamicum FERM-P 170
Brevibacterium flavum FERM-P 1708
Brevibacterium lactofermentum. FERM-P 1712
Corynebacterium glutamicum FERM-P 6463
Corynebacterium glutamicum FERM-P 6464 and
Corynebacterium glutamicum DSMS71S.
CA 02319716 2000-10-04
990152 BT
8
The inventors have succeeded in isolating the eno gene of
C. glutamicum which codes for the enzyme enolase (EC
4.2.1.11).
y
To isolate the eno gene or also other genes of C.
glutamicum, a gene library of this microorganism is first
set up in E. coli. The setting up of gene libraries is
described in generally known textbooks and handbooks. The
textbook by Winnacker: Gene and Klone, Eine Einfuhrung in
die Gentechnologie [Genes and Clones, An Introduction to
l0 Genetic Engineering] (Verlag Chemie, Weinheim, Germany,
1990) or the handbook by Sambrook et al.: Molecular
Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory
Press, 1989) may be mentioned as an example. A well-known
gene library is that of the E. coli K-12 strain W3110 set
up in ~ vectors by Kohara et al. (Cell 50, 495 - 508
(1987)). Bathe et al. (Molecular and General Genetics,
252:255-265, 1996) describe a gene library of C. glutamicum
ATCC13032, which was set up with the aid of the cosmid
vector SuperCos I (Wahl et al., 1987, Proceedings of the
National Academy of Sciences USA, 84:2160-2164) in the
E.coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic
Acids Research 16:1563-1575). Bormann et al. (Molecular
Microbiology 6(3), 317-326)) in turn describe a gene
library of C. glutamicum ATCC13032 using the cosmid pHC79
(Hohn and Collins, Gene 11, 291-298 (1980)). To prepare a
gene library of C. glutamicum in E. coli it is also
possible to use plasmids such as pBR322 (Bolivar, Life
Sciences, 25, 807-818 (1979)) or pUC9 (Viera et al., 1982,
Gene, 19:259-268). Suitable hosts are, in particular, those
E. coli strains which are restriction- and recombination-
CA 02319716 2000-10-04
990152 BT
9
defective. An example of these is the strain DHSamcr, which
has been described by Grant et al. (Proceedings of the
National Academy of Sciences USA, 87 (1990) 4645-4649). The
long DNA fragments cloned with the aid of cosmids can then
in turn be subcloned and subsequently sequenced in the
usual vectors which are suitable for sequencing, such as is
described e.g. by Sanger et al. (Proceedings of the
National Academy of Sciences of the United States of
America, 74:5463-5467, 1977).
The new DNA sequence of C. glutamicum which codes for the
eno gene and which is a constituent of the present
invention as SEQ ID NO l.was obtained in this manner. The
amino acid sequence of the corresponding protein has
furthermore been derived from the present DNA sequence by
the methods described above. The resulting amino acid
sequence of the eno gene product is shown in SEQ ID NO 2.
Coding DNA sequences which result from SEQ ID NO 1 by the
degeneracy of the genetic code are also a constituent of
the invention. In the same way, DNA sequences which -
hybridize with SEQ ID NO 1 or parts of SEQ ID NO 1 are a
constituent of the invention. Conservative amino acid
exchanges, such as e.g. exchange of glycine for alanine or
of aspartic acid for glutamic acid in proteins, are
furthermore known among experts as "sense mutations" which
do not lead to a fundamental change in the activity of the
protein, i.e. are of neutral function. It is furthermore
known that changes on the N and/or C terminus of a protein
cannot substantially impair or can even stabilize the
function thereof. Information in this context can be found
by the expert, inter alia, in Ben-Bass<~t et al. (Journal of
CA 02319716 2000-10-04
990152 BT
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
5 molecular biology. Amino acid sequences which result in a
corresponding manner from SEQ ID NO 2 are also a
constituent of the invention.
In the same way, DNA sequences which hybridize with SEQ ID
NO 1 or parts of SEQ ID NO 1 are a constituent of the
10 invention. Finally, DNA sequences which are prepared by the
polymerase chain reaction (PCR) using primers which result
from SEQ ID NO 1 are a constituent of the invention. Such
oligonucleotides typically have a length of at least 15
base pairs.
Instructions for identifying DNA sequences by means of
hybridization can be found by the expert, inter alia, in
the handbook "The DIG System Users Guide for Filter
Hybridization" from Boehringer Mannheim GmbH (Mannheim,
Germany, 1993) and in Liebl et al. (International Journal
of Systematic Bacteriology (1991) 41: 255-260).
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 [sic] synthesis: a practical approach (IRL
Press, Oxford, UK, 1984) and in Newton and Graham: PCR
(Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).
The inventors have found that coryneform bacteria produce
amino acids, in particular L-lysine in an improved manner
after over-expression of the eno gene.
CA 02319716 2000-10-04
990152 BT
11
To achieve an over-expression, the number of copies of the
corresponding genes can be increased, or the promoter and
regulation region or the ribosome binding site upstream of
the structural gene can be mutated. Expression cassettes
which are incorporated upstream of the structural gene act
in the same way. By inducible promoters, it is additionally
possible to increase the expression in the course of
fermentative L-lysine production. The expression is
likewise improved by measures to prolong the life of the m-
l0 RNA. Furthermore, the enzyme activity is also increased by
preventing the degradation of the enzyme protein. The genes
or gene constructions can either be present in plasmids
with a varying number of-copies, or can be integrated and
amplified in the chromosome. Alternatively, an over-
expression of the genes in question can furthermore be
achieved by changing the composition of the media and the
culture procedure.
Instructions in this context can be found by the expert,
inter alia, in Martin et al. (Bio/Technology 5, 137-146
(1987)), in Guerrero et al. (Gene 138, 35-41 (1994))r
Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)),
in Eikmanns et al. (Gene 102, 93-98 (1991)), in European
Patent Specification EPS 0 472 869, in US Patent 4,601,893,
in Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991), in
Reinscheid et al. (Applied and Environmental Microbiology
60, 126-132 (1994)), in LaBarre et al. (Journal of
Bacteriology 175, 1001-1007 (1993)), in Patent Application
WO 96/15246, in Malumbres et al. (Gene 134, 15 - 24
(1993)), in Japanese Laid-Open Specification JP-A-10-
229891, in Jensen and Hammer (Biotechnology and
CA 02319716 2000-10-04
990152 BT
12
Bioengineering 58, 191-195 (1998)), in Makrides
(Microbiological Reviews 60:512-538 (1996)) and in known
textbooks of genetics and molecular biology.
By way of example, the eno gene according to the invention
was over-expressed with the aid of plasmids. Suitable
plasmids are those which are replicated in coryneform
bacteria. Numerous known plasmid vectors, such as e.g. pZl
(Menkel et al., Applied and Environmental Microbiology
(1989) 64: 549-554), pEKExl (Eikmanns et al., Gene 102:
93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107:69-74
(1991)) are based on the cryptic plasmids pHM1519, pBLl or
pGAl. Other plasmid vectors, such as e. g. those based on
pCG4~(US-A 4,489,160), or pNG2 (Serwold-Davis et al., FEMS
Microbiology Letters 66, 119-124 (1990)), or pAGl (US-A
5,158,891) can be used in the same manner.
In addition, it may be advantageous for the production of
amino acids, in particular L-lysine, to over-express one or
more enzymes of the particular biosynthesis route, of
glycolysis, of anaplerosis, of the citric acid cycle or of
amino acid export, in addition to the eno gene.
Thus, for example, for the preparation of L-lysine
~ at the same time the dapA gene which codes for
dihydrodipicolinate synthase (EP-B 0 197 335),-or
~ at the same time the gap gene which codes for
glyceraldehyde 3-phosphate dehydrogenase (Eikmanns
(1992). Journal of Bacteriology 174:6076-6086), or
CA 02319716 2000-10-04
990152 BT
13
~ at the same time the tpi gene which codes for triose
phosphate isomerase (Eikmanns (1992). Journal of
Bacteriology 174:6076-6086), or
~ at the same time the pgk gene which codes for 3-
phosphoglycerate kinase (Eikmanns (1992). Journal of
Bacteriology 174:6076-6086), or
~ at the same time the pyc gene which codes for pyruvate
carboxylase (Eikmanns (1992). Journal. of Bacteriology
174:6076-6086), or
~ at the same time the lysE gene which codes for lysine
export (DE-A-195 48 222)
can be over-expressed.
In addition to over-expression of the eno gene it may
furthermore be advantageous, for the production of amino
acids, in particular L-lysine, to eliminate undesirable
side reactions (Nakayama: "Breeding of Amino Acid Producing
Micro-organisms", in: Overproduction of Microbial Products,
Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London,
UK, 1982). ,
The microorganisms prepared according to the invention can
be cultured continuously or discontinuously in t-he batch
process (batch culture) or in the fed batch (feed process)
or repeated fed batch process (repetitive feed process) for
the purpose of production of amino acids, in particular L-
lysine. A summary of known culture methods are [sic]
described in the textbook by Chmiel (Bioprozesstechnik 1.
CA 02319716 2000-10-04
990152 BT
14
Einfuhrung in die Bioverfahrenstechnik [Bioprocess
Technology 1. Introduction to Bioprocess Technology (Gustav
Fischer Verlag, Stuttgart, 1991)) or in the textbook by
Storhas (Bioreaktoren and periphere Einrichtungen
S [Bioreactors and Peripheral Equipment] (Vieweg Verlag,
Braunschweig/Wiesbaden, 1994)).
The culture medium to be used must meet the requirements of
the particular strains in a suitable manner. Descriptions
of culture media for various microorganisms are contained
in the handbook "Manual of Methods for General
Bacteriology" of the American Society for Bacteriology
(Washington D.C., USA, 181). Sugars and carbohydrates,
such as e.g. glucose, sucrose, lactose, fructose, maltose,
molasses, starch and cellulose, oils and fats, such as
e. g. Soya oil, sunflower oil, groundnut oil and coconut
fat, fatty acids, such as e. g. palmitic acid, stearic acid
and linoleic acid, alcohols, such as e. g. glycerol and
ethanol, and organic acids, such as e. g. acetic acid, can
be used as the source of carbon. These substances can be
used individually or as a mixture. 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
sulphate, ammonium chloride, ammonium phosphate, ammonium
carbonate and ammonium nitrate, can be used as the source
of nitrogen. The sources of nitrogen can be used
individually or as a mixture. Phosphoric acid, potassium
dihydrogen phosphate or dipotassium hydrogen phosphate or
the corresponding sodium-containing salts can be used as
the source of phosphorus. The culture medium must
CA 02319716 2000-10-04
990152 BT
furthermore comprise salts of metals, such as e. g.
magnesium sulfate or iron sulfate, which are necessary for
growth. Finally, essential growth substances, such as amino
acids and vitamins, can be employed in addition to the
5 abovementioned substances. Suitable precursors can moreover
be added to the culture medium. The starting substances
mentioned can be added to the culture in the form of a
single batch, or can be fed in during the culture in a
suitable manner.
10 Basic compounds, such as sodium hydroxide, potassium
hydroxide, ammonia or aqueous ammonia, or acid compounds,
such as phosphoric acid or sulfuric acid, can be employed
in a suitable manner to control the pH. Antifoams, such as
e.g. fatty acid polyglycol esters, can be employed to
15 control the development of foam. Suitable substances having
a selective action, e.g. antibiotics, can be added to the
medium to maintain the stability of plasmids. To maintain
aerobic conditions, oxygen or oxygen-containing gas
mixtures, such as e.g, air, are introduced into the
culture. The temperature of the culture is usually 20°C to
45°C, and preferably 25°C to 40°C. Culturing is continued
until a maximum of lysine has formed. This target is
usually reached within 10 hours to 160 hours.
The analysis of L-lysine takes place can be carried out
[sic) by anion exchange chromatography with subsequent
ninhydrin derivatization, as described by Spackman et al.
(Analytical Chemistry, 30, (1958), 1190).
CA 02319716 2000-10-04
990152 BT
16
The process according to the invention is used for the
fermentative preparation of amino acids, in particular
L-lysine.
CA 02319716 2000-10-04
990152 BT
17
Examples
The present invention is explained in more detail in the
following with the aid of embodiment examples.
Example 1
Preparation of a genomic cosmid gene library from
Corynebacterium glutamicum ATCC 13032
Chromosomal DNA from Corynebacterium glutamicum ATCC 13032
was isolated as described by Tauch et al. (1995, Plasmid
33:168-179) and partly cleaved with the restriction enzyme
Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product
Description Sau3AI, Code no. 27-0913-02). The DNA fragments
were dephosphorylated with shrimp alkaline phosphatase
(Roche Molecular Biochemicals, Mannheim, Germany, Product
Description SAP, Code no. 1758250). The DNA of the cosmid
vector SuperCosl (Wahl et al. (1987) Proceedings of the
National Academy of Sciences USA 84:2160-2164), obtained
from the company Stratagene (La Jolla, USA, Product
Description SuperCosl Cosmid Vektor 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. The cosmid DNA was then
cleaved with the restriction enzyme BamHI (Amersham
Pharmacia, Freiburg, Germany, Product Description BamHI,
Code no. 27-0868-04). The cosmid DNA treated in this manner
was mixed with the treated ATCC13032 DNA and the batch was
treated with T4 DNA lipase (Amersham Pharmacia, Freiburg,
Germany, Product Description T4-DNA-Lipase, Code no.27-
CA 02319716 2000-10-04
990152 BT
18
0870-04). The ligation mixture was then packed in phages
with the aid of Gigapack II XL Packing Extracts
(Stratagene, La Jolla, USA, Product Description Gigapack II
XL Packing Extract, Code no. 200217). F'or infection of the
E. coli strain NM554 (Raleigh et al. 1988, Nucleic Acid
Research 16:1563-1575) the cells were taken up in 10 mM
MgS04 and mixed with an aliquot of the phage suspension.
The infection and titering of the cosmid library were
carried out as described by Sambrook et al. (1989,
Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor), the cells being plated out on LB agar (Lennox,
1955, Virology, 1:190) with 100 ~g/ml ampicillin. After
incubation overnight at 37°C, recombinant individual clones
were selected.
Example 2
Isolation and sequencing of the eno gene
The cosmid DNA of an individual colony was isolated with
the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen,
Hilden, Germany) in accordance with the manufacturer's
instructions and partly cleaved with the restriction enzyme
Sau3AI (Amersham Pharmacies, 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 separation by gel electrophoresis, the cosmid
fragments in the size range of 1500 to 2000 by were
isolated with the QiaExII Gel Extraction Kit (Product No.
20021, Qiagen, Hilden, Germany). The DNA of the sequencing
CA 02319716 2000-10-04
990152 BT
19
vector pZero-1, obtained from the company Invitrogen
(Groningen, The Netherlands, Product Description Zero
Background Cloning Kit, Product No. K2500-O1) was cleaved
with the restriction enzyme BamHI (Amersham Pharmacia,
Freiburg, Germany, Product Description BamHI, Product No.
27-0868-04). The ligation of the cosmid fragments in the
sequencing vector pZero-1 was carried out as described by
Sambrook et al. (1989, Molecular Cloning: A laboratory
Manual, Cold Spring Harbor), the DNA mixture being
incubated overnight with T4 ligase (Pharmacia Biotech,
Freiburg, Germany). This ligation mixture was then
electroporated (Tauch et al. 1994, FEMS Microbiol Letters,
123:343-7) into the E. coli strain DHSaMCR (Grant, 1990,
Proceedings of the National Academy of Sciences U.S.A.,
87:4645-4649) and plated out on LB agar (Lennox, 1955,
Virology, 1:190) with 50 ~g/ml zeocin. The plasmid
preparation of the recombinant clones was carried out with
Biorobot 9600 (Product No. 900200, Qiagen, Hilden,
Germany). The sequencing was carried out by the dideoxy
chain-stopping method of Sanger et al. (1977, Proceedings
of the National Academy of Sciences U.S.A., 74:5463-567)
with modifications according to Zimmermann et al. (1990,
Nucleic Acids Research, 18:1067). The "RR dRhodamin
Terminator Cycle Sequencing Kit" from PE Applied
Biosystems(Product No. 403044, Weiterstadt, Germany) was
used. The separation by gel electrophoresis and analysis of
the sequencing reaction were carried outs in a "Rotiphoresis
NF Acrylamide/Bisacrylamide" Gel (29:1) (Product No.
A124.1, Roth, Karlsruhe, Germany) with t:he "ABI Prism 377"
sequencer from PE Applied Biosystems (Weiterstadt,
Germany).
CA 02319716 2000-10-04
990152 BT
The raw sequence data obtained were then processed using
the Staden program package (1986, Nucleic Acids Research,
14:217-231) version 97-0. The individual sequences of the
pZerol derivatives were assembled to a continuous contig.
5 The computer-assisted coding region analysis [sic] were
prepared with the XNIP program (Staden, 1986, Nucleic Acids
Research, 14:217-231). Further analyses were carried out
with the "BLAST search program" (Altschul et al., 1997,
Nucleic Acids Research, 25:3389-3402), against the non-
10 redundant databank of the "National Center for
Biotechnology Information" (NCBI, Bethesda, MD, USA).
The nucleotide sequence obtained is shown in SEQ ID NO 1.
Analysis of the nucleotide sequence showed an open reading
frame of 1275 base pairs, which was called the eno gene.
15 The eno gene codes for a protein of 425 amino acids.
CA 02319716 2000-11-28
21
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Degussa-Hiils Aktiengesellschaft
(B) CITY: Frankfurt: am Main
(C) COUNTRY: Germany
(D) POSTAL CODE (ZI_f): DE-60287
(ii) TITLE OF INVENTION: NEW NUCLEOTIDE SEQUENCES WHICH
CODE FOR THE ENO GENE
(iii) NUMBER OF SEQUENCES: 2
(iv) CORRESPONDENCE ADDRESS:
(A) NAME: Marks & Clerk
(B) STREET: 280 Slat:er Street, Suite 1800
(C) CITY: Ottawa
(D) STATE: Ontario
(E) COUNTRY: Canada
(F) POSTAL CODE (Z:CP): K1P 1C2
(v) COMPUTER-READABLE FORM:
(A) MEDIUM TYPE: D_L:~kette
(B) COMPUTER: IBM I?C
(C) OPERATING SYSTEM: MS DOS
(D) SOFTWARE: Patent:In Ver. 2.1
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,319,716
(B) FILING DATE: 2000-10-04
(C) CLASSIFICATION: Unknown
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 199 47 791.4
(B) FILING DATE: 1999-10-05
(C) CLASSIFICATION: Unknown
(viii) PATENT AGENT INFORhIATION:
(A) NAME: Richard .J. Mitchell
(B) REGISTRATION NI7MBER:
(C) REFERENCE/DOCKET NUMBER: 99472-3
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613) 236-9561
(B) TELEFAX: (613) 230-8821
(2) INFORMATION FOR SEQ ID NO.: 7.:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1578
(B) TYPE: nucleic.~ acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Corynebacterium glutamicum
CA 02319716 2000-11-28
22
(ix)
FEATURE:
(A) CDS
NAME/KEY:
(B) (151)..(1425)
LOCATION:
(xi ) SEQUENCE SEQID O.: l:
DESCRIPTION: N
GGCTGGGGAT TCAACTG ATTGCCTC AT GAAACAAGA 60
ATGGGTAGTT C
TTCGCCACTA
A'CT
TTCGTGCAAC GAAGACATTT GATCACGT GA TAATTCTAG120
AATTGGGTGT A
AGACGTGATT
TTAGCTCCCA GCTGAA CAC GTA 174
AGTTGGCATA ATC TTC
GGAGGCCACA ATG
G'PG
Va31AlaGlu IleMetHis ValPhe
1 5
GCTCGC GAA CTC GACTCCCGC GG'L'AACCCA ACCGTCGAG GCAGAG 222
ATT
AlaArg Glu Leu AspSerArg GlyAsnPro ThrValGlu AlaGlu
Ile
15 20
GTTTTC CTG GAC GGTTCCCAC GG'PGTCGCA GGTGTTCCA TCCGGC 270
GAT
ValPhe Leu Asp GlySerHis GlyValAla GlyValPro SerGly
Asp
25 30 35 40
GCATCC ACC GTC CACGAGGCT CA'PGAGCTG CGTGACGGT GGCGAT 318
GGC
AlaSer Thr Val HisGluAla HisGluLeu ArgAspGly GlyAsp
Gly
45 50 55
CGCTAC CTG AAG GGCGT'PTTG AAGGCAGTT GAAAACGTC AACGAA 366
GGC
ArgTyr Leu Lys GlyVa.1Leu LysAlaVal GluAsnVal AsnGlu
Gly
60 6.5 70
GAAATC GGC GAG CTCGC'rGGC CTAGAGGCT GACGATCAG CGCCTC 414
GAC
GluIle Gly Glu LeuAlaGly LeuGluAla AspAspGln ArgLeu
Asp
75 80 85
ATCGAC GAA ATG ATCAAGCTT GA'rGGCACC GCCAACAAG TCCCGC 962
GCA
IleAsp Glu Met IleLysLeu AspGlyThr AlaAsnLys SerArg
Ala
90 95 100
CTGGGT GCA GCA ATCCTTGGT GT'rTCCATG GCTGTTGCA AAGGCT 510
AAC
LeuGly Ala Ala IleLeuGly ValSerMet AlaValAla LysAla
Asn
105 110 115 120
GCTGCT GAT GCA GGCCTCCCA CTGTTCCGC TACATCGGT GGACCA 558
TCC
AlaAla Asp Ala GlyLeuPro LeuPheArg TyrIleGly GlyPro
Ser
125 130 135
AACGCA CAC CTT CCAGTTCCA ATGATGAAC ATCATCAAC GGTGGC 606
GTT
AsnAla His Leu ProVa1Pro MetMetAsn I:leIleAsn GlyGly
Val
140 145 150
GCTCAC GCT TCC GGTGTTGAC GTTCAGGAA TTCATGATC GCTCCA 654
GAC
AlaHis Ala Ser GlyVa.LA.spValGlnGlu PheMetIle AlaPro
Asp
155 160 165
ATCGGT GCA ACC TTCTCTGAG GCTCTCCGC AACGGCGCG GAGGTC 702
GAG
IleGly Ala Thr PheSerGlu AlaLeuArg AsnGlyAla GluVal
Glu
170 175 180
CA 02319716 2000-11-28
23
TACCACGCA CTGAAGTCCGTC ATCAAGGAA GGCCTG TCCACCGGA 750
AAG
TyrHisAla LeuLysSerVal :I:LeLysoGlu LysGlyLeu SerThrGly
185 190 195 200
CTTGGCGAT GAGGGCGGCTTC GCTCCTTCC GTCGGCTCC ACCCGTGAG 798
LeuGlyAsp GluGlyGlyPheeAlaProSer ValGlySer ThrArgGlu
205 210 215
GCTCTTGAC CTTATCGTTGAG (~CAATCGAG AAGGCTGGC TTCACCCCA 846
AlaLeuAsp LeuIleValGlu RlaIleGlu LysAlaGly PheThrPro
220 225 230
GGCAAGGACATC GCTCTTGCT (.'CGGAC:GTT GCTTCCTCT GAGTTCTTC 894
GlyLysAspIle AlaLeuAla LeuAspVal AlaSerSer GluPhePhe
235 240 295
AAGGACGGCACC TACCACTTC GRAGGTGGC CAGCACTCC GCAGCTGAG 942
LysAspGlyThr TyrHisPhe GluGlyGly GlnHisSer AlaAlaGlu
250 255 260
ATGGCAAACGTT TACGCTGAG (~TCGTTGAC GCGTACCCA ATCGTCTCC 990
MetAlaAsnVal TyrAlaGlu LeuVal.Rsp AlaTyrPro IleValSer
265 270 275 280
ATCGAGGACCCA CTGCAGGAA GATGACTGG GAGGGTTAC ACCAACCTC 1038
IleGluAspPro LeuGlnGlu AspAspTrp GluGlyTyr ThrAsnLeu
285 290 295
ACCGCAACCATC GGCGACAAG (iTTCAGATC GTTGGCGAC GACTTCTTC 1086
ThrAlaThrIle GlyAspLys ValGlr~Ile ValGlyAsp AspPhePhe
300 305 310
GTC ACC AAC CCT GAG CGC CTG ARG GAG GGC ATC GCT AAG AAG GCT GCC 1134
Val Thr Asn Pro Glu Arg Leu Lys Glu Gly Ile Ala Lys Lys Ala Ala
315 320 325
AAC TCC ATC CTG GTT AAG GTG RAC CAG ATC GGT ACC CTC ACC GAG ACC 1182
Asn Ser Ile Leu Val Lys Val. Asn Gln Ile Gly Thr Leu Thr Glu Thr
330 335 340
TTCGACGCTGTC GACATGGCT CACCGCGCA GGCTACACC TCCATGATG 1230
PheAspAlaVal AspMetAla l3isArgAla GlyTyrThr SerMetMet
395 350 355 360
TCCCACCGTTCC GGTGAGACC GAGGAC:ACC ACCATTGCT GACCTCGCA 1278
SerHisArgSer GlyGluThr GluAs~>Thr ThrIleAla AspLeuAla
365 370 375
GTTGCACTCAAC TGTGGCCAG RTCAAGACT GGTGCTCCA GCACGTTCC 1326
ValAlaLeuAsn CysGlyGln :LleLysThr GlyAlaPro AlaArgSer
380 385 390
GACCGTGTCGCA AAGTACAAC:CRGCTTCTC CGCATCGAG CAGCTGCTT 1374
AspArgValAla LysTyrAsn (~lnLeuLeu ArgIleGlu GlnLeuLeu
395 400 905
GGCGACGCCGGC GTCTACGCA GGTCGC:AGC GCATTCCCA CGCTTTCAG 1922
GlyAspAlaGly ValTyrAla (~lyArgSer AlaPhePro ArgPheGln
910 415 920
CA 02319716 2000-11-28
24
GGC TAAATAAAAG CGCTTTTCGA CGCCCGGTAA CCTCAAGGTT GCCGGGCGTC 1975
Gly
425
GTTGCCTTAC TACTGTTACT GGTGTGACTA TGATCGAGGA TTATGGCAAA GCAGAAGAAA 1535
ACTCATAAAG GCCTTGTTCC TGTCTCAAGC AGGGAACGTG CTT 1578
(2) INFORMATION FOR SEQ ID NO.: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 425
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: polypeptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Corynebacterium glutamicum
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 2:
Val Ala Glu Ile Met His Val Phe Ala Arg Glu Ile Leu Asp Ser Arg
1 5 10 15
Gly Asn Pro Thr Val Glu Ala Glu Val Phe Leu Asp Asp Gly Ser His
20 25 30
Gly Val Ala Gly Val Pro Ser Gly Ala Ser Thr Gly Val His Glu Ala
35 40 45
His Glu Leu Arg Asp Gly Gly Asp Arg Tyr Leu Gly Lys Gly Val Leu
50 55 60
Lys Ala Val Glu Asn Val Asn Glu Glu Ile Gly Asp Glu Leu Ala Gly
65 70 75 80
Leu Glu Ala Asp Asp Gln Arg heu Ile Asp Glu Ala Met Ile Lys Leu
85 90 95
Asp Gly Thr Ala Asn Lys Ser Arg Leu Gly Ala Asn Ala Ile Leu Gly
100 105 110
Val Ser Met Ala Val Ala Lys Ala Ala Ala Asp Ser Ala Gly Leu Pro
115 1.20 125
Leu Phe Arg Tyr Ile Gly Gly Pro Asn Ala His Val Leu Pro Val Pro
130 135 140
Met Met Asn Ile Ile Asn Gly Gly Ala His Ala Asp Ser Gly Val Asp
145 150 155 160
Val Gln Glu Phe Met Ile Al.a f'ro Ile Gly Ala Glu Thr Phe Ser Glu
165 170 175
Ala Leu Arg Asn Gly Ala Glu Val Tyr His Ala Leu Lys Ser Val Ile
180 185 190
CA 02319716 2000-11-28
Lys Glu Lys Gly Leu Ser Thr Gl.y Leu Gly Asp Glu Gly Gly Phe Ala
195 200 205
Pro Ser Val Gly Ser Thr Arg Glu Ala Leu Asp Leu Ile Val Glu Ala
210 215 220
Ile Glu Lys Ala Gly Phe Thr E'ro Gly Lys Asp Ile Ala Leu Ala Leu
225 230 235 240
Asp Val Ala Ser Ser Glu Phe E'he Lys Asp Gly Thr Tyr His Phe Glu
245 250 255
Gly Gly Gln His Ser Ala Ala Gl.u Met Ala Asn Val Tyr Ala Glu Leu
260 265 270
Val Asp Ala Tyr Pro Ile Val :>er Ile Glu Asp Pro Leu Gln Glu Asp
275 280 285
Asp Trp Glu Gly Tyr Thr Asn Leu Thr Ala Thr Ile Gly Asp Lys Val
290 295 300
Gln Ile Val Gly Asp Asp Phe Phe Val Thr Asn Pro Glu Arg Leu Lys
305 310 315 320
Glu Gly Ile Ala Lys Lys Ala F~l.a Asn Ser Ile Leu Val Lys Val Asn
325 330 335
Gln Ile Gly Thr Leu Thr Glu Thr Phe Asp Ala Val Asp Met Ala His
390 395 350
Arg Ala Gly Tyr Thr Ser Met Met Ser His Arg Ser Gly Glu Thr Glu
355 ..60 365
Asp Thr Thr Ile Ala Asp Leu Al.a Val Ala Leu Asn Cys Gly Gln Ile
370 375 380
Lys Thr Gly Ala Pro Ala Arg ~le~r Asp Arg Val Ala Lys Tyr Asn Gln
385 390 395 400
Leu Leu Arg Ile Glu Gln Leu Leu Gly Asp Ala Gly Val Tyr Ala Gly
405 410 415
Arg Ser Ala Phe Pro Arg Phe Gl.n Gly
420 425
