Note: Descriptions are shown in the official language in which they were submitted.
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Novel nucleotide sequences coding for the pfkA gene
The present invention provides nucleotide sequences coding
for the pfkA gene and a process for the fermentative
production of L-amino acids, in particular L-lysine, using
coryneform bacteria in which the pfkA 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 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 of the
product by, for example, ion exchange chromatography, or to
the intrinsic performance characteristics of the
microorganism itself.
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-amino acids, such as for example L-lysine.
For some years, methods of recombinant DNA technology have
likewise been used to improve strains of Corynebacterium
which produce amino acids by amplifying individual
biosynthesis genes and investigating the effect on amino
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2
acid production. Review articles on this subject may 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) ) .
15
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Object of the invention
The inventors set themselves the object of providing novel
measures for the improved fermentative production 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 accordingly
general interest in providing novel improved processes for
the production of amino acids, in particular L-lysine.
Any subsequent mention of L-lysine or lysine should be
taken to mean not only the base, but also salts, such as
for example lysine monohydrochloride or lysine sulfate.
The invention provides an isolated polynucleotide from
coryneform bacteria containing a polynucleotide sequence
selected from the group
a) polynucleotide which is at least 70a identical to a
polynucleotide which codes for a polypeptide containing
the amino acid sequence of SEQ ID no. 2,
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,
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) .
The present invention also provides the polynucleotide
according to claim 1, wherein it preferably comprises
replicable DNA containing:
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(i) the nucleotide sequence shown in SEQ ID no. 1, or
(ii) at least one sequence which matches the sequence
(i) within the degeneration range of the genetic
code, or
(iii) at least one sequence which hybridises with the
complementary sequence to sequence (i) or (ii)
and optionally
(iv) functionally neutral sense mutations in (i).
The present invention also provides
a polynucleotide according to claim 4, containing the
nucleotide sequence as shown in SEQ ID no. 1,
a polynucleotide according to claim 6 which codes for a
polypeptide which contains the amino acid sequence as
shown in SEQ ID no. 2,
a vector containing the polynucleotide according to claim
1, in particular a shuttle vector or plasmid vector
and coryneform bacteria acting as host cell which contain
the vector.
The present invention also provides polynucleotides which
substantially consist of a polynucleotide sequence, which
are obtainable by screening by means of hybridisation 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.
Polynucleotide sequences according to the invention are
suitable as hybridisation probes for RNA, cDNA and DNA in
order to isolate full length cDNA which code for
phosphofructokinase and to isolate such cDNA or genes, the
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sequence of which exhibits a high level of similarity with
that of the phosphofructokinase gene.
Polynucleotide sequences according to the invention are
furthermore suitable as primers for the production of DNA
5 of genes which code for phosphofructokinase by the
polymerase chain reaction (PCR).
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 90 or 50
nucleotides are also suitable.
"Isolated" means separated from its natural environment.
"Polynucleotide" generally relates to polyribonucleotides
and polydeoxyribonucleotides, wherein the RNA or DNA may be
unmodified or modified.
"Polypeptides" are taken to mean peptides or proteins which
contain two or more amino acids connected by peptide bonds.
The polypeptides according to the invention include a
polypeptide according to SEQ ID no. 2, in particular those
having the biological activity of phosphofructokinase and
also those, which are at least 70$, preferably at least
80~, identical to the polypeptide according to SEQ ID no. 2
and in particular are 90~ to 95$ identical to the
polypeptide according to SEQ ID no. 2 and exhibit the
stated activity.
The invention furthermore relates to a process for the
fermentative production 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 pfkA gene are amplified, in
particular overexpressed.
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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.
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.
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 produced
therefrom, such as for example
Corynebacterium glutamicum FERM-P 1709
Brevibacterium flavum FERM-P 1708
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Brevibacterium lactofermentum FERM-P 1712
Corynebacterium glutamicum FERM-P 6463
Corynebacterium glutamicum FERM-P 6464 and
Corynebacterium glutamicum DSM5715.
The inventors succeeded in isolating the novel pfkA gene,
which codes for the enzyme phosphofructokinase (EC
2.7.1.11), from C. glutamicum.
The pfkA 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 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). 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)) in ~-vectors. Bathe et al.
(Molecular and General Genetics, 252:255-265, 1996)
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) in 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)) also
describe a gene library of C. glutamicum ATCC 13032, using
cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)). 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)) or pUC9 (Vieira et al., 1982,
Gene, 19:259-268). Suitable hosts are in particular those
E. coli strains with restriction and recombination defects.
One example of such a strain is the strain DHSamcr, which
has been described by Grant et al. (Proceedings of the
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8
National Academy of Sciences USA, 87 (1990) 4695-4649). 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).
The novel DNA sequence from C. glutamicum which codes for
the pfkA 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 pfkA gene.
Coding DNA sequences arising from the degeneracy of the
genetic code are also provided by the present invention.
DNA sequences which hybridise with SEQ ID no. 1 or parts of
SEQ ID no. 1 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 stabilise 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)), 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
arising in a corresponding manner from SEQ ID no. 2 are
also provided by the present invention.
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Similarly, DNA sequences which hybridise with SEQ ID no. 1
or portions of SEQ ID no. 1 are also provided by the
present invention. Finally, DNA sequences produced by the
polymerase chain reaction (PCR) using primers obtained from
SEQ ID no. 1 are also provided by the present invention.
Such oligonucleotides typically have a length of at least
nucleotides.
The person skilled in the art may find instructions for
identifying DNA sequences by means of hybridisation inter
10 alia in the manual "The DIG System Users Guide for Filter
Hybridization" from Boehringer Mannheim GmbH (Mannheim,
Germany, 1993) and in Liebl et al. (International Journal
of Systematic Bacteriology (1991) 41: 255-260). The person
skilled in the art may find instructions for amplifying DNA
15 sequences using the polymerase chain reaction (PCR) inter
alia in the manual by Gait, Oligonucleotide synthesis: a
practical approach (IRL Press, Oxford, UK, 1984) and in
Newton & Graham, PCR (Spektrum Akademischer Verlag,
Heidelberg, Germany, 1994).
The inventors discovered that coryneform bacteria produce
L-amino acids, in particular L-lysine, in an improved
manner once the pfkA has been overexpressed.
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-lysine 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
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amplified. Alternatively, overexpression of the genes
concerned may also be achieved by modifying the composition
of the nutrient media and culture conditions.
The person skilled in the art will find guidance in this
5 connection inter alia in Martin et al. (Bio/Technology 5,
137-146 (1987)), in Guerrero et al. (Gene 138, 35-41
(1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430
(1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in
European patent EP 0 472 869, in US patent 4,601,893, in
10 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 published patent application JP-A-10-229891, in
Jensen and Hammer (Biotechnology and Bioengineering 58,
191-195 (1998)), in Makrides (Microbiological Reviews
60:512-538 (1996)) and in known textbooks of genetics and
molecular biology.
By way of example, the pfkA gene according to the invention
was overexpressed with the assistance of plasmids.
Suitable plasmids are those which are replicated in
coryneform bacteria. Numerous known plasmid vectors, such
as for example pZl (Menkel et al., Applied and
Environmental Microbiology (1989) 64: 549-554), pEKEx1
(Eikmanns et al., Gene 102:93-98 (1991)) or pHS2-1 (Sonnen
et al., Gene 107:69-74 (1991)) are based on the cryptic
plasmids pHM1519, pBLl or pGAl. Other plasmid vectors, such
as for example those based on pCG9 (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) may be used in
the same manner.
Further suitable plasmid vectors are those with the
assistance of which gene amplification may be performed by
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integration into the chromosome, as has for example been
described by Reinscheid et al. (Applied and Environmental
Microbiology 60, 126-132 (1994)) 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)), pKlBmob or pKl9mob (Schafer et al., Gene 145, 69-
73 (1994)), pGEM-T (Promega corporation, Madison, WI, USA),
pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry
269:32678-84; US-A 5,487,993), pCR~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 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 Schafer et al. (Applied and
Environmental Microbiology 60, 756-759 (1994)).
Transformation methods 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 "crossing over", the resultant
strain contains at least two copies of the gene in
question.
It may additionally be advantageous for the production of
amino acids, in particular L-lysine, to amplify or
overexpress not only the pfkA gene, but also one or more
enzymes of the particular biosynthetic pathway, of
glycolysis, of anaplerotic metabolism, of the citric acid
cycle or of amino acid export.
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For the production of L-lysine, for example, it is thus
possible simultaneously to overexpress one or more genes
selected from the group
~ the dapA gene which codes for dihydropicolinate synthase
(EP-B 0 197 335), or
~ the gap gene which codes for glyceraldehyde-3-phosphate
dehydrogenase (Eikmanns (1992), Journal of Bacteriology
174:6076-6086), or
~ the tpi gene which codes for triosephosphate isomerase
(Eikmanns (1992), Journal of Bacteriology 174:6076-6086),
or
~ the pgk gene which codes for 3-phosphoglycerate kinase
(Eikmanns (1992), Journal of Bacteriology 174:6076-6086),
or
~ the pyc gene which codes for pyruvate carboxylase
(Eikmanns (1992), Journal of Bacteriology 174:6076-6086),
or
~ the lysE gene which codes for lysine export
(DE-A-195 48 222).
It may furthermore be advantageous for the production of
amino acids, in particular L-lysine, in addition to
amplifying the pfkA gene, simultaneously to attenuate
~ the pck gene which codes for phosphoenolpyruvate
carboxykinase (DE 199 50 409.1, DSM 13047) and/or
~ the pgi gene which codes for glucose 6-phosphate
isomerase (US 09/396,478, DSM 12969).
It may furthermore be advantageous for the production of
amino acids, in particular L-lysine, in addition to
overexpressing the pfkA gene, to suppress unwanted
secondary reactions (Nakayama: "Breeding of Amino Acid
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Producing Micro-organisms", in: Overproduction of Microbial
Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press,
London, UK, 1982).
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)) or in the textbook by Storhas (Bioreaktoren and
periphere Einrichtungen (Vieweg Verlag,
Braunschweig/Wiesbaden, 1994)).
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). 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,
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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
culturing.
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°C to 45°C and preferably from 25°C to
40°C. The culture is continued until the maximum quantity
of lysine has formed. This aim is normally achieved within
10 to 160 hours.
Analysis of L-lysine may be performed by anion exchange
chromatography with subsequent ninhydrin derivatisation, as
described in Spackman et al. (Analytical Chemistry, 30,
(1958), 1190).
The purpose of the process according to the invention is
the fermentative production of amino acids, in particular
L-lysine.
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Examples
The present invention is illustrated in greater detail by
the following practical examples.
5 Example 1
Production of a genomic cosmid gene library from
Corynebacterium glutamicum ATCC13032
Chromosomal DNA from Corynebacterium glutamicum ATCC 13032
was isolated as described in Tauch et al., (1995, Plasmid
10 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,
15 Germany, product description SAP, code no. 1758250). The
DNA of cosmid vector SuperCosl (Wahl et al. (1987)
Proceedings of the National Academy of Sciences USA
84:2160-2164), purchased from Stratagene (La Jolla, USA,
product description SuperCosl 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 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). 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). 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 Res. 16:1563-1575) was infected by
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16
suspending the cells in 10 mM MgSOq 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), the cells being plated out on LB agar (Lennox,
1955, Virology, 1:190) with 100ug/ml of ampicillin. After
overnight incubation at 37°C, individual recombinant clones
were selected.
Example 2
Isolation and sequencing of the pfkA gene
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 by 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-
Ol) was cleaved with the restriction enzyme BamHI (Amersham
Pharmacia, Freiburg, Germany, product description BamHI,
product no. 27-0868-09). 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), the DNA mixture
being incubated overnight with T4 ligase (Pharmacia
Biotech, Freiburg, Germany). This ligation mixture was then
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17
electroporated into the E. coli strain DHSaMCR (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 ug/ml of Zeocin. Plasmids of
the recombinant clones were prepared using the Biorobot
9600 (product no. 900200, Qiagen, Hilden, 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) 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.
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).
The resultant raw sequence data were then processed using
the Staden software package (1986, Nucleic Acids Research,
14:217-231), 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). Further analysis was performed using the
"BLAST search programs" (Altschul et al., 1997, Nucleic
Acids Research, 25:3389-3402), against the non-redundant
database of the "National Center for Biotechnology
Information" (NCBI, Bethesda, MD, USA).
The resultant nucleotide sequence is stated in SEQ ID no.
1. Analysis of the nucleotide sequence revealed an open
reading frame of 1029 base pairs, which was designated the
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18
pfkA gene. The pfkA gene codes for a protein of 343 amino
acids.
Example 3
Production of a plasmid for overexpressing pfkA in
Corynebacterium glutamicum
3.1. Cloning of pfkA in the pCR2-Blunt vector
Chromosomal DNA from Corynebacterium glutamicum ATCC 13032
was isolated as described in Tauch et al., (1995, Plasmid
33:168-179). On the basis of the sequence of the pfkA gene
for C. glutamicum known from Example 2, the following
oligonucleotides were selected for the polymerase chain
reaction:
pf kA-exp
5'-AAC TGC AGC TCT GGC GAT TA-3'
pf k-ex2
5'-AAC TAT CCA AAC ATT GCC TG-3'
The stated primers were synthesised by the company MWG
Biotech (Ebersberg, Germany) and the PCR reaction performed
in accordance with the standard PCR method of Innis et al.
(PCR protocols. A guide to methods and applications,1990,
Academic Press) using Pwo polymerase from Roche Diagnostics
GmbH (Mannheim, Germany). A DNA fragment of approx. 1160 by
in size, which bears the pfkA gene, was isolated with the
assistance of the polymerase chain reaction.
The amplified DNA fragment was ligated into the vector pCR2
Blunt Vector (Bernard et al., (1983) Journal of Molecular
Biology. 234:534-541) using the Zero Blunt PCR Cloning Kit
from Invitrogen Corporation (Carlsbad, CA, USA; catalogue
number K2700-20). The E. coli strain ToplOF (Grand et al.
(1990) Proceedings of the National Academy of Sciences,
USA. 87:4645-4649) was transformed with the ligation batch.
CA 02324485 2000-11-21
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19
Plasmid-bearing cells were selected by plating the
transformation batch out onto LB agar (Sambrook et al.,
Molecular cloning: a laboratory manual. 2nd Ed. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.) which
had been supplemented with 50 mg/1 of kanamycin. Plasmid
DNA was isolated from a transformant using the QIAprep Spin
Miniprep Kit from Qiagen and verified by restriction with
the restriction enzyme EcoRI and subsequent agarose gel
electrophoresis (0.8%). The plasmid was named pCRBl-
pfkAexpl.
3.2. Production of the shuttle vector pEC-Tl8mob2
The E. coli-C. glutamicum shuttle vector was constructed in
accordance with the prior art. The vector contains the
replication region rep of plasmid pGAl, including the
replication effector per (US-A- 5,175,108; Nesvera et al.,
Journal of Bacteriology 179, 1525-1532 (1997)). the tetA(Z)
gene, which imparts tetracycline resistance, of plasmid
pAGl (US-A- 5,158,891; gene library entry at the National
Center for Biotechnology Information (NCBI, Bethesda, MD,
USA) with the accession number AF121000), the replication
origin oriV of plasmid pMBl (Sutcliffe, Cold Spring Harbor
Symposium on Quantitative Biology 43, 77-90 (1979)), the
lacZa gene fragment including the lac promoter and a
multiple cloning site (mcs) (Norrander et al. Gene 26, 101-
106 (1983)) and the mob region of plasmid RP4 (Simon et
al.,(1983) Bio/Technology 1:784-791). The constructed
vector was then transformed into E. coli strain DH5a
(Hanahan, in: DNA cloning. A practical approach. Vol. I.
IRL-Press, Oxford, Washington DC, USA). Plasmid-bearing
cells were selected by plating the transformation batch out
onto LB agar (Sambrook et al., Molecular cloning: a
laboratory manual. 2"d Ed. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.) which had been
supplemented with 5 mg/1 of tetracycline. Plasmid DNA was
CA 02324485 2000-11-21
990169 BT / A1
isolated from a transformant using the QIAprep Spin
Miniprep Kit from Qiagen and verified by restriction with
the restriction enzymes EcoRI and HindIII and subsequent
agarose gel electrophoresis (0.8%). The plasmid was named
5 pEC-Tl8mob2 and is shown in Figure 1.
3.3. Cloning of pfkA into shuttle vector pEC-Tl8mob2
The vector used was the E. coli-C. glutamicum shuttle
vector pEC-Tl8mob2 described in Example 3.2. DNA from this
10 plasmid was completely cleaved with the restriction enzyme
EcoRI and then dephosphorylated with shrimp alkaline
phosphatase (Roche Molecular Biochemicals, Mannheim,
Germany, product description SAP, product no. 1758250).
The pfkA gene was isolated from the plasmid pCRBl-pfkAexpl
15 described in Example 3.1. by complete cleavage with the
enzyme EcoRI. The approx. 1160 by pfkA fragment was
isolated from the agarose gel using the QiaExII Gel
Extraction Kit (product no. 20021, Qiagen, Hilden,
Germany).
20 The pfkA fragment obtained in this manner was mixed with
the prepared pEC-Tl8mob2 vector and the batch was treated
with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany,
product description T4 DNA Ligase, code no. 27-0870-04).
The ligation batch was then transformed into E. coli strain
DHSa (Hanahan, in: DNA cloning. A practical approach. Vol.
I. IRL-Press, Oxford, Washington DC, USA). Plasmid-bearing
cells were selected by plating the transformation batch out
onto LB agar (Lennox, 1955, Virology, 1:190) with 5 mg/1 of
tetracycline. After overnight incubation at 37°C,
individual recombinant clones were selected. Plasmid DNA
was isolated from a transformant in accordance with the
manufacturer's instructions using the Qiaprep Spin Miniprep
Kit (product no. 27106, Qiagen, Hilden, Germany) and
CA 02324485 2000-11-21
990169 BT / A1
. 21
cleaved with the restriction enzyme EcoRI in order to check
the plasmid by subsequent agarose gel electrophoresis. The
resultant plasmid was named pT-pfkAexp. It is shown in
Figure 2.
Example 4:
Transformation of strain DSM5715 with plasmid pT-pfkAexp
Strain DSM5715 (EP-B-0 435 132) was then transformed with
plasmid pT-pfkAexp using the electroporation method
described by Liebl et al. (FEMS Microbiology Letters,
53:299-303 (1989)). Transformant selection proceeded on
LBHIS agar consisting of 18.5 g/1 of brain-heart infusion
bouillon, 0.5 M sorbitol, 5 g/1 of Bacto tryptone, 2.5 g/1
of Bacto yeast extract, 5 g/1 of NaCl and 18 g/1 of Bacto
agar, which had been supplemented with 5 mg/1 of
tetracycline. Incubation was performed for 2 days at 33°C.
Plasmid DNA was isolated from a transformant using the
conventional methods (Peters-Wendisch et al., 1998,
Microbiology, 144, 915 - 927), cut with the restriction
endonuclease EcoRI in order to check the plasmid by
subsequent agarose gel electrophoresis. The resultant
strain was named DSM5715/pT-pfkAexp.
Example 5
Production of lysine
The C. glutamicum strain DSM5715/pT-pfkAexp obtained in
Example 4 was cultured in a nutrient medium suitable for
the production of lysine and the lysine content of the
culture supernatant was determined.
CA 02324485 2000-11-21
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22
To this end, the strain was initially incubated for 24
hours at 33°C on an agar plate with the appropriate
antibiotic (brain/heart agar with tetracycline (5 mg/1)).
Starting from this agar plate culture, a preculture was
inoculated (10 ml of medium in a 100 ml Erlenmeyer flask).
The medium used for the preculture was complete medium
CgIII (2.5 g/1 of NaCl, 10 g/1 of Bacto peptone, 10 g/1 of
Bacto yeast extract, 20 g/1 of glucose, pH 7.4).
Tetracycline (5 mg/1) was added to this medium. The
preculture was incubated for 16 hours at 33°C on a shaker
at 240 rpm. A main culture was inoculated from this
preculture, such that the initial OD (660 nm) of the main
culture was 0.1. Medium MM was used for the main culture.
CA 02324485 2000-11-21
990169 BT / Al
~ 23
Medium MM
CSL (Corn Steep Liquor) 5
g/1
MOPS (Morpholinopropanesulfonic 20 g/1
acid)
Glucose (separately autoclaved) 50 g/1
(NHq) ZS04 25 g/1
KHzP04 0.1 g/1
MgSOq * 7 H20 1.0 g/1
CaCl2 * 2 HZO 10 mg/1
FeS09 * 7 HZO 10 mg/1
MnS04 * Hz0 5 mg
. /
0 1
Biotin (sterile-filtered) 0.3 mg/1
Thiamine * HC1 (sterile-filtered) 0.2 mg/1
L-leucine (sterile-filtered) 0.1 g/1
CaC03 25 g/1
CSL, MOPS and the salt solution were adjusted to pH 7 with
ammonia water and autoclaved. The sterile substrate and
vitamin solutions, together with the dry-autoclaved CaC03
are then added.
Culturing is performed in a volume of 10 ml in a 100 ml
Erlenmeyer flask with flow spoilers. Kanamycin (25 mg/1)
was added. Culturing was performed at 33°C and 80~
atmospheric humidity.
CA 02324485 2000-11-21
990169 BT / A1
24
After 24 hours, the OD was determined at a measurement
wavelength of 660 nm using a Biomek 1000 (Beckmann
Instruments GmbH, Munich). The quantity of lysine formed
was determined using an amino acid analyser from Eppendorf-
BioTronik (Hamburg, Germany) by ion exchange chromatography
and post-column derivatisation with ninhydrin detection.
Table 1 shows the result of the test.
Table 1
Strain OD(660) Lysine HC1
g/1
DSM5715/pT-pfkAexp 14.6 10.1
DSM5715 15.2 8.1
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The following Figures are attached:
Figure l: Map of plasmid pT-pfkAexp
Figure 2: Map of plasmid pT-pfkAexp
The abbreviations and names are defined as follows.
5 Tet: Resistance gene for tetracycline
oriV: Plasmid-code replication origin of
E. coli
RP4mob: mob region for plasmid mobilisation
rep: Plasmid-coded replication origin from
10 C. glutamicum plasmid pGAl
per: Gene for controlling copy number from pGAl
lacZ-alpha: lacZa gene fragment (N terminus) of
the ~-galactosidase gene
'lacZa': 5' end of the lacZa gene fragment
15 lacZ-alpha': 3' end of the lacZa gene fragment
pfkA: pfkA gene from C. glutamicum ATCC13032
BamHI: Restriction site of the restriction enzyme
BamHI
EcoRI: Restriction site of the restriction enzyme
20 EcoRI
HindIII: Restriction site of the restriction enzyme
HindIII
KpnI: Restriction site of the restriction enzyme
KpnI
25 PstI: Restriction site of the restriction enzyme
PstI
PvuI: Restriction site of the restriction enzyme
PvuI
SalI: Restriction site of the restriction enzyme
SalI
SacI: Restriction site of the restriction enzyme
SacI
SmaI: Restriction site of the restriction enzyme
SmaI
CA 02324485 2000-11-21
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26
SphI: Restriction site of the restriction enzyme
SphI
XbaI: Restriction site of the restriction enzyme
XbaI
XhoI: Restriction site of the restriction enzyme
XhoI
CA 02324485 2001-O1-30
27
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Degussa-Hiils Aktiengesellschaft
(B) STREET: Weissfrauenstrasse 9
(C) CITY: Frankfurt am Main
(D) COUNTRY: Germany
(E) POSTAL CODE (ZIP): D-60287
(ii) TITLE OF INVENTION: NOVEL NUCLEOTIDE SEQUENCES CODING
FOR THE PFKA GENE
(iii) NUMBER OF SEQUENCES: 2
(iv) CORRESPONDENCE ADDRESS:
(A) NAME: Marks & Clerk
(B) STREET: 280 Slater Street, Suite 1800
(C) CITY: Ottawa
(D) STATE: Ontario
(E) COUNTRY: Canada
(F) POSTAL CODE (ZIP): K1P 1C2
(v) COMPUTER-READABLE FORM:
(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM PC
(C) OPERATING SYSTEM: MS DOS
(D) SOFTWARE: PatentIn Ver. 2.1
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,324,985
(B) FILING DATE: 2000-11-21
(C) CLASSIFICATION: Unknown
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 199 56 133.8
(B) FILING DATE: 1999-11-23
(C) CLASSIFICATION: Unknown
(viii) PATENT AGENT INFORMATION:
(A) NAME: Richard J. Mitchell
(B) REGISTRATION NUMBER:
(C) REFERENCE/DOCKET NUMBER: 10055-2
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 613-236-9561
(B) TELEFAX: 613-230-8821
(2) INFORMATION FOR SEQ ID NO.: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1279
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
CA 02324485 2001-O1-30
28
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Corynebacterium glutamicum
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: (143)..(1171)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 1:
GTCGATTTGT TAATGAAACT AGATGGTCAG 60
GCAGCTCTGG AGACAGTTTT
CGATTAAATA
TTGGCCTGTC TTGTTCCGGC GCGGGTGTTG 120
AACCCCTGTG
ATTCTCTTAT
TTTTGGGTGA
TGATGGGTTT T 172
AATATGGAAG CTC
AC ACG
ATG TCA
CGA GGC
ATT GGC
GCT
AC
Me t r u r
Arg Le Thr Gly
Ile Se Gly
Ala
Th
1 5 10
GACTGC CCCGGACTAAAC GCCGTCATC CGAGGAATC GTCCGCACA GCC 220
AspCys ProGlyLeuAsn AlaValIle ArgGlyIle ValArgThr Ala
15 20 25
AGCAAT GAATTTGGCTCC ACCGTCGTT GGTTATCAA GACGGTTGG GAA 268
SerAsn GluPheGlySer ThrValVal GlyTyrGln AspGlyTrp Glu
30 35 40
GGACTG TTAGGCGATCGT CGCGTACAG CTGTATGAC GATGAAGAT ATT 316
GlyLeu LeuGlyAspArg ArgValGln LeuTyrAsp AspGluAsp Ile
45 50 55
GACCGA ATCCTCCTTCGA GGCGGCACC ATTTTGGGC ACTGGTCGC CTC 364
AspArg IleLeuLeuArg GlyGlyThr IleLeuGly ThrGlyArg Leu
60 65 70
CATCCG GACAAGTTTAAG GCCGGAATT GATCAGATT AAGGCCAAC TTA 412
HisPro AspLysPheLys AlaGlyIle AspGlnIle LysAlaAsn Leu
75 80 85 90
GAAGAC GCCGGCATCGAT GCCCTTATC CCAATCGGT GGCGAAGGA ACC 460
GluAsp AlaGlyIleAsp AlaLeuIle ProIleGly GlyGluGly Thr
95 100 105
CTGAAG GGTGCCAAGTGG CTGTCTGAT AACGGTATC CCTGTTGTC GGT 508
LeuLys GlyAlaLysTrp LeuSerAsp AsnGlyIle ProValVal Gly
110 115 120
GTCCCA AAGACCATTGAC AATGACGTG AATGGCACT GACTTCACC TTC 556
ValPro LysThrIleAsp AsnAspVal AsnGlyThr AspPheThr Phe
125 130 135
GGTTTC GATACTGCTGTG GCAGTGGCT ACCGACGCT GTTGACCGC CTG 604
GlyPhe AspThrAlaVal AlaValAla ThrAspAla ValAspArg Leu
140 145 150
CACACC ACCGCTGAATCT CACAACCGT GTGATGATC GTGGAGGTC ATG 652
HisThr ThrAlaGluSer HisAsnArg ValMetIle ValGluVal Met
155 160 165 170
GGCCGC CACGTGGGTTGG ATTGCTCTG CACGCAGGT ATGGCCGGC GGT 700
GlyArg HisValGlyTrp IleAlaLeu HisAlaGly MetAlaGly Gly
175 180 185
CA 02324485 2001-O1-30
29
GCT CAC TAC ACC GTT ATT CCA GAA GTA CCT TTC GAT ATT GCA GAG ATC 748
Ala His Tyr Thr Val Ile Pro Glu Val Pro Phe Asp Ile Ala Glu Ile
190 195 200
TGCAAG GCGATGGAA CGTCGCTTC CAGATGGGC GAGAAGTAC GGCATT 796
CysLys AlaMetGlu ArgArgPhe GlnMetGly GluLysTyr GlyIle
205 210 215
ATCGTC GTTGCGGAA GGTGCGTTG CCACGCGAA GGCACCATG GAGCTT 849
IleVal ValAlaGlu GlyAlaLeu ProArgGlu GlyThrMet GluLeu
220 225 230
CGTGAA GGCCACATT GACCAGTTC GGTCACAAG ACCTTCACG GGAATT 892
ArgGlu GlyHisIle AspGlnPhe GlyHisLys ThrPheThr GlyIle
235 240 245 250
GGACAG CAGATCGCT GATGAGATC CACGTGCGC CTCGGCCAC GATGTT 940
GlyGln GlnIleAla AspGluIle HisValArg LeuGlyHis AspVal
255 260 265
CGTACG ACCGTTCTT GGCCACATT CAACGTGGT GGAACCCCA ACTGCT 988
ArgThr ThrValLeu GlyHisIle GlnArgGly GlyThrPro ThrAla
270 275 280
TTCGAC CGTGTTCTG GCCACTCGT TATGGTGTT CGTGCAGCT CGTGCG 1036
PheAsp ArgValLeu AlaThrArg TyrGlyVal ArgAlaAla ArgAla
285 290 295
TGCCAT GAGGGAAGC TTTGACAAG GTTGTTGCT TTGAAGGGT GAGAGC 1089
CysHis GluGlySer PheAspLys ValValAla LeuLysGly GluSer
300 305 310
ATTGAG ATGATCACC TTTGAAGAA GCAGTCGGA ACCTTGAAG GAAGTT 1132
IleGlu MetIleThr PheGluGlu AlaValGly ThrLeuLys GluVal
315 320 325 330
CCATTC GAACGCTGG GTTACTGCC CAGGCAATG TTTGGATAGTTTTTCG 1181
ProPhe GluArgTrp ValThrAla GlnAlaMet PheGly
335 340
GGCTTTTATC AACAGCCAAT AACAGCTCTT TCGCCCATTG AGGTGGAGGG GCTGTTTTTT 1241
CATGCCGTAA GGAAAGTGCA AGTAAGTGAA ATC 1274
(2) INFORMATION FOR SEQ ID NO.: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 343
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: polypeptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Corynebacterium glutamicum
CA 02324485 2001-O1-30
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 2:
Met Arg Ile Ala Thr Leu Thr Ser Gly Gly Asp Cys Pro Gly Leu Asn
1 5 10 15
Ala Val Ile Arg Gly Ile Val Arg Thr Ala Ser Asn Glu Phe Gly Ser
20 25 30
Thr Val Val Gly Tyr Gln Asp Gly Trp Glu Gly Leu Leu Gly Asp Arg
40 45
Arg Val Gln Leu Tyr Asp Asp Glu Asp Ile Asp Arg Ile Leu Leu Arg
50 55 60
Gly Gly Thr Ile Leu Gly Thr Gly Arg Leu His Pro Asp Lys Phe Lys
65 70 75 80
Ala Gly Ile Asp Gln Ile Lys Ala Asn Leu Glu Asp Ala Gly Ile Asp
85 90 95
Ala Leu Ile Pro Ile Gly Gly Glu Gly Thr Leu Lys Gly Ala Lys Trp
100 105 110
Leu Ser Asp Asn Gly Ile Pro Val Val Gly Val Pro Lys Thr Ile Asp
115 120 125
Asn Asp Val Asn Gly Thr Asp Phe Thr Phe Gly Phe Asp Thr Ala Val
130 135 140
Ala Val Ala Thr Asp Ala Val Asp Arg Leu His Thr Thr Ala Glu Ser
145 150 155 160
His Asn Arg Val Met Ile Val Glu Val Met Gly Arg His Val Gly Trp
165 170 175
Ile Ala Leu His Ala Gly Met Ala Gly Gly Ala His Tyr Thr Val Ile
180 185 190
Pro Glu Val Pro Phe Asp Ile Ala Glu Ile Cys Lys Ala Met Glu Arg
195 200 205
Arg Phe Gln Met Gly Glu Lys Tyr Gly Ile Ile Val Val Ala Glu Gly
210 215 220
Ala Leu Pro Arg Glu Gly Thr Met Glu Leu Arg Glu Gly His Ile Asp
225 230 235 240
Gln Phe Gly His Lys Thr Phe Thr Gly Ile Gly Gln Gln Ile Ala Asp
245 250 255
Glu Ile His Val Arg Leu Gly His Asp Val Arg Thr Thr Val Leu Gly
260 265 270
His Ile Gln Arg Gly Gly Thr Pro Thr Ala Phe Asp Arg Val Leu Ala
275 280 285
Thr Arg Tyr Gly Val Arg Ala Ala Arg Ala Cys His Glu Gly Ser Phe
290 295 300
CA 02324485 2001-O1-30
31
Asp Lys Val Val Ala Leu Lys Gly Glu Ser Ile Glu Met Ile Thr Phe
305 310 315 320
Glu Glu Ala Val Gly Thr Leu Lys Glu Val Pro Phe Glu Arg Trp Val
325 330 335
Thr Ala Gln Ala Met Phe Gly
340
