Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Coryneform Bacteria which Produce Chemical Compounds I
Prior Art
Chemical compounds, which means, in particular, L-amino
acids, vitamins, nucleosides and nucleotides and D-amino
acids, are used in human medicine, in the~pharmaceuticals
industry, in cosmetics, in the foodstuffs industry and in
animal nutrition.
Numerous of these compounds are prepared by fermentation
from strains of coryneform bacteria, in particular
Corynebacterium glutamicum. Because of their great
importance, work is constantly being undertaken to improve
the preparation processes. Improvements to the process can
relate to fermentation measures, such as, for example,
stirring and supply of oxygen, or the composition of the
nutrient media, such as, for example, the sugar
concentration during the fermentation, or the working up to
the product form by, for example, ion exchange
chromatography, or the intrinsic output properties of the
microorganism itself.
Methods of mutagenesis, selection and mutant selection are
used to improve the output properties of these
microorganisms. Strains which are resistant to
antimetabolites or are auxotrophic for metabolites of
regulatory importance and which produce the particular
compounds are obtained in this manner.
Methods of the recombinant DNA technique have also been
employed for some years for improving the strain of
Corynebacterium strains, by amplifying individual
biosynthesis genes and investigating the effect on
production.
A common method comprises amplification of certain
biosynthesis genes in the particular microorganism by means
of episomally replicating plasmids. This procedure has the
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disadvantage that during the fermentation, which in
industrial processes is in general associated with numerous
generations, the plasmids are lost spontaneously
(segregational instability).
Another method comprises duplicating certain biosynthesis
genes by means of plasmids which do not replicate in the
particular microorganism. In this method, the plasmid,
including the cloned biosynthesis gene, is integrated into
the chromosomal biosynthesis gene of the microorganism
(Reinscheid et al., Applied and Environmental Microbiology
60(1), 126-132 (1994); Jetten et al., Applied Microbiology
and Biotechnology 43(1):76-82 (1995)). A disadvantage of
this method is that the nucleotide sequences of the plasmid
and of the antibiotic resistance gene necessary for the
selection remain in the microorganism. This is a
disadvantage, for example, for the disposal and utilization
of the biomass. Moreover, the expert expects such strains
to be unstable as a result of disintegration by "Campbell
type cross over" in a corresponding number of generations
such as are usual in industrial fermentations.
Object of the Invention
The inventors had the object of providing new measures for
improved fermentative preparation chemical compounds using
coryneform bacteria.
Summary of the Invention
Coryneform bacteria which produce chemical compounds,
characterised in that these have, in addition to at least
one copy, present at the natural site (locus), of an open
reading frame (ORF), gene or allele which codes for the
synthesis of a protein or an RNA, a second, optionally
third or fourth copy of the open reading frame (ORF), gene
or allele in question at a second, optionally third or
fourth site in a form integrated into the chromosome, no
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nucleotide sequence which is capable of/enables episomal
replication or transposition in microorganisms and no
nucleotide sequences) which imparts) resistance to
antibiotics being present at the second, optionally third
or fourth site, and the second, optionally third or fourth
site not relating to open reading frames (ORF), genes or
alleles which are essential for the growth of the bacteria
and the production of the desired compound.
The invention also provides processes for the preparation
of one or more chemical compounds, in which the following
steps are carried out:
a) fermentation of coryneform bacteria,
a1) which have, in addition to at least one copy,
present at the natural site (locus), of an open
reading frame (ORF), gene or allele which codes
for the synthesis of a protein or an RNA, a
second, optionally third or fourth copy of this
open reading frame (ORF), gene or allele at a
second, optionally third.orr fourth site_in a .form
integrated into the chromosome, no nucleotide
sequence which is capable of/enables episomal
replication or transposition in microorganisms
and no nucleotide sequences) which imparts)
resistance to antibiotics being present at the
second, optionally third or fourth site, and the
second, optionally third or fourth site not
relating to open reading frames (ORF), genes or
alleles which are essential for the growth of the
bacteria and the production of the desired
compound, and
a2) in which the intracellular activity of the
corresponding protein is increased, in particular
the nucleotide sequence which codes for this
protein is over-expressed,
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b) concentration of the chemical compounds) in the
fermentation broth andlor in the cells of the
bacteria,
c) isolation of the chemical compound(s), optionally
d) with constituents from the fermentation broth
and/or the biomass to the extent of > (greater
than) 0 to 100 wt.~.
The invention also provides processes for the preparation
of one or more chemical compounds, which comprise the
following steps:
a) fermentation of coryneform bacteria, in particular of
the genus Corynebacterium, which have, in addition to
the copy of an open reading frame (ORF), gene or
allele present at the natural site (locus), in each
case a second, optionally third or fourth copy of the
open reading frame (ORF), gene or allele in question
at in each case a second, optionally third or fourth
site in integrated form, no nucleotide sequence which
is capable of/enables episomal replication in
microorganisms, no nucleotide sequence which is
capable of/enables transposition and no nucleotide
sequence which imparts resistance to antibiotics
being present at the particular second, optionally
third or fourth site,
under conditions which allow expression of the said
open reading frames (ORF), genes or alleles
b) concentration of the chemical compounds) in the
fermentation broth and/or in the cells of the
bacteria,
c) isolation of the chemical compound(s), optionally
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d) with constituents from the fermentation broth and/or
the biomass to the extent of > (greater than) 0 to
100.
Detailed Description of the Invention
5 Chemical compounds are to be understood, in particular, as
meaning amino acids, vitamins, nucleosides and nucleotides.
The biosynthesis pathways of these compounds are known and
are available in the prior art.
Amino acids mean, preferably, L-amino acids, in particular
the proteinogenic L-amino acids, chosen from the group
consisting of L-aspartic acid, L-asparagine, L-threonine,
L-serine, L-glutamic acid, L-glutamine, glycine, L-alanine,
L-cysteine, L-valine, L-methionine, L-isoleucine, L-
leucine, L-tyrosine, L-phenylalanine, L-histidine, L-
lysine, L-tryptophan, L-proline and L-arginine and salts
thereof, in particular L-lysine, L-methionine and L-
threonine. L-Lysine is very particularly preferred.
Proteinogenic amino acids are understood-as meaning the
amino acids which occur in natural proteins, that is to say
in proteins of microorganisms, plants, animals and humans.
Vitamins mean, in particular, vitamin B1 (thiamine),
vitamin B2 (riboflavin), vitamin B5 (pantothenic acid),
vitamin B6 (pyridoxines), vitamin B12 (cyanocobalamin),
nicotinic acid/nicotinamide, vitamin M (folic acid) and
vitamin E (tocopherol) and salts thereof, pantothenic acid
being preferred.
Nucleosides and nucleotides mean, inter alia, S-adenosyl-
methionine, inosine-5'-monophosphoric acid and guanosine-
5'-monophosphoric acid and salts thereof.
The coryneform bacteria are, in particular, those of the
genus Corynebacterium. Of the genus Corynebacterium, the
species Corynebacterium glutamicum, Corynebacterium
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ammoniagenes and Corynebacterium thermoaminogenes are
preferred. Information on the taxonomic classification of
strains of this group of bacteria is to be found, inter
alia, in Kampfer and Kroppenstedt (Canadian Journal of
Microbiology 42, 989-1005 (1996)) and in US-A-5,250,434.
Suitable strains of the species Corynebacterium glutamicum
(C. glutamicum) are, in particular, the known wild-type
strains
Corynebacterium glutamicum ATCC13032
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium lilium ATCC15990
Corynebacterium melassecola ATCC17965
Corynebacterium herculis ATCC13868
Arthrobacter sp. ATCC243
Brevibacterium chang-fua ATCC14017
Brevibacterium flavum ATCC14067
Brevibacterium lactofermentum ATCC13869
Brevibacterium divaricatum ATCC14020
Brevibacterium taipei ATCC13744 and
Microbacterium ammoniaphilum ATCC21645
and mutants or strains, such as are known from the prior
art, produced therefrom which produce chemical compounds.
Suitable strains of the species Corynebacterium
ammoniagenes (C. ammoniagenes) are, in particular, the
known wild-type strains
Brevibacterium ammoniagenes ATCC6871
Brevibacterium ammoniagenes ATCC15137 and
Corynebacterium sp. ATCC21084
and mutants or strains, such as are known from the prior
art, produced therefrom which produce chemical compounds.
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Suitable strains of the species Corynebacterium
thermoaminogenes (C. thermoaminogenes) are, in particular,
the known wild-type strains
Corynebacterium thermoaminogenes FERM BP-1539
Corynebacterium thermoaminogenes FERM BP-1540
Corynebacterium thermoaminogenes FERM BP-1541 and
Corynebacterium thermoaminogenes FERM BP-1542
and mutants or strains, such as are known from the prior
art, produced therefrom which produce chemical compounds.
Strains with the designation "ATCC" can be obtained from
the American Type Culture Collection (Manassas, VA, USA).
Strains with the designation "FERM" can be obtained from
the National Institute of Advanced Industrial Science and
Technology (AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba
Ibaraki, Japan). The strains of Corynebacterium
thermoaminogenes mentioned (FERM BP-1539, FERM BP-1540,
FERM BP-1541 and FERM BP-1542) are described in US-A
5,250,434.
Open reading frame (ORF) describes a section of a
nucleotide sequence which codes or can code for a protein
or polypeptide or ribonucleic acid to which no function can
be assigned according to the prior art.
After assignment of a function to the nucleotide sequence
section in question, it is in general referred to as a
gene.
Alleles are in general understood as meaning alternative
forms of a given gene. The forms are distinguished by
differences in the nucleotide sequence.
In the context of the present invention, endogenous, that
is to say species-characteristic, open reading frames,
genes or alleles are preferably used. These are understood
as meaning the open reading frames, genes or alleles or
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nucleotide sequences thereof present in the population of a
species, such as, for example, Corynebacterium glutamicum.
"A copy of an open reading frame (ORF), a gene or allele
present at the natural site (locus)" in the context of this
invention is understood as meaning the position or
situation of the ORF or gene or allele in relation to the
adjacent ORFs or genes or alleles such as exists in the
corresponding wild-type or corresponding parent organism or
starting organism.
Thus, for example, the natural site of the lysC gene or of
an lysCFBR allele, which codes for a "feed back" resistant
aspartate kinase from Corynebacterium glutamicum is the
lysC site or lysC locus or lysC gene site with the directly
adjacent genes or open reading frames orfX and leuA on one
flank and the asd gene on the other flank.
"Feed back" resistant aspartate kinase is understood as
meaning aspartate kinases which, compared with the wild-
type form, have a lower sensitivity to inhibition by
mixtures of lysine and threonine or mixtures of AEC
(aminoethylcysteine) and threonine or lysine by itself or
AEC by itself. Strains which produce L-lysine typically
contain such "feed back" resistant or desensitized
aspartate kinases.
The nucleotide sequence of the chromosome of
Corynebacterium glutamicum is known and can be found in
Patent Application EP-A-1108790 and Access Number
(Accession No.) AX114121 of the nucleotide sequence
databank of the European Molecular Biologies Laboratories
(EMBL, Heidelberg, Germany and Cambridge, UK). The
nucleotide sequences of orfX, the leuA gene and the asd
gene have the Access Numbers AX120364 (orfX), AX123517
(leuA) and AX123519 (asd).
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Further databanks, such as, for example, that of the
National Center for Biotechnology Information (NCBI,
Bethesda, Nm, USA) or that of the Swiss Institute of
Bioinformatics (Swissprot, Geneva, Switzerland) or that of
the Protein Information Resource Database (PIR, Washington,
DC, USA) can also be used.
"In each case a second, optionally third or fourth site" is
understood as meaning a site which differs from the
"natural site". It is also called a "target site" or
"target sequence" in the following. It can also be called
an "integration site" or "transformation site". This
second, optionally third or fourth site, or the nucleotide
sequence present at the corresponding sites, is preferably
in the chromosome and is in general not essential for
growth and for production of the desired chemical
compounds.
To produce the coryneform bacteria according to the
invention, the nucleotide sequence of the desired ORF, gene
or allele, optionally including expression and/or
regulation signals, is isolated and provided with
nucleotide sequences of the target site at the ends, these
are then transferred into the desired coryneform bacterium,
preferably with the aid of vectors which do not replicate
or replicate to only a limited extent in coryneform
bacteria, and those bacteria in which the desired ORF, gene
or allele is incorporated at the target site are isolated,
no nucleotide sequence which is capable of/enables episomal
replication in microorganisms, no nucleotide sequence which
is capable of/enables transposition and no nucleotide
sequence which imparts resistance to antibiotics remaining
at the target site.
The invention accordingly also provides a process for the
production of coryneform bacteria which produce one or more
chemical compounds, which comprises
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a) isolating the nucleotide sequence of at least one
desired ORF, gene or allele, optionally including the
expression and/or regulation signals,
b) providing the 5' and the 3' end of the ORF, gene or
5 allele with nucleotide sequences of the target site,
c) preferably incorporating the nucleotide sequence of
the desired ORF, gene or allele provided with
nucleotide sequences of the target site into a vector
which does not replicate or replicates to only a
10 limited extent in coryneform bacteria,
d) transferring the nucleotide sequence according to b)
or c) into coryneform bacteria, and
e) isolating coryneform bacteria in which the nucleotide
sequence according to a) is incorporated at the
target site, no nucleotide sequence which is capable
of/enables episomal replication in microorganisms, no
nucleotide sequence which is capable of/enables
transposition and no nucleotide sequence which
imparts resistance to antibiotics remaining at the
target site.
Preferably, also, no residues of sequences of the vectors
used or species-foreign DNA, such as, for example,
restriction cleavage sites, remain at the target site. A
maximum of 24, preferably a maximum of 12, particularly
preferably a maximum of 6 nucleotides of such DNA upstream
or downstream of the ORF, gene or allele incorporated
optionally remain at the target site.
By the measures according to the invention, the
productivity of the coryneform bacteria or of the
fermentative processes for the preparation of chemical
compounds is improved in respect of one or more of the
features chosen from the group consisting of concentration
(chemical compound formed, based on the unit volume), yield
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(chemical compound formed, based on the source of carbon
consumed) and product formation rate (chemical compound
formed, based on the time) by at least 0.5 - 1.0~ or at
least 1.0 to 1.5~ or at least 1.5~- 2.0o.
Instructions on conventional genetic engineering methods,
such as, for example, isolation of chromosomal DNA, plasmid
DNA, handling of restriction enzymes etc., are found in
Sambrook et al. (Molecular Cloning - A Laboratory Manual
(1989) Cold Spring Harbor Laboratory Press). Instructions
on transformation and conjugation in coryneform bacteria
are found, inter alia, in Thierbach et al. (Applied
Microbiology and Biotechnology 29, 356-362 (1988)), in
Schafer et a1. (Journal of Bacteriology 172, 1663-1666
(1990) and Gene 145, 69-73 (1994)) and in Schwarzer and
Piihler (Bio/Technology 9, 84-87 (1991)).
Vectors which replicate to only a limited extent are
understood as meaning plasmid vectors which, as a function
of the conditions under which the host or carrier is
cultured, replicate or do not replicate. Thus, a
temperature-sensitive plasmid for coryneform bacteria which
can replicate only at temperatures below 31°-C has been
described by Nakamura et al. (US-A-6,303,383).
The invention furthermore provides coryneform bacteria, in
particular of the genus Corynebacterium, which produce L
lysine, characterized in that these have, in addition to at
least one of the copy of an open reading frame (ORF), gene
or allele of lysine production present at the natural site
(locus), in each case a second, optionally third or fourth
copy of the open reading frame (ORF), gene or allele in
question at in each case a second, optionally third or
fourth site in integrated form, no nucleotide sequence
which is capable of/enables episomal replication in
microorganisms, no nucleotide sequence.which is capable
of/enables transposition and no nucleotide sequence which
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imparts resistance to antibiotics being present at the
particular second, optionally third or fourth site.
The invention also furthermore provides a process for the
preparation of L-lysine, which comprises the following
steps:
a) fermentation of coryneform bacteria, in particular
Corynebacterium glutamicum, characterized in that
these have, in addition to at least one of the copy
of an open reading frame (ORF), gene or allele of
lysine production present at the natural site
(locus), in each case a second, optionally third or
fourth copy of the open reading frame (ORF), gene or
allele in question at in each case a second,
optionally third or fourth site in integrated form,
no nucleotide sequence which is capable of/enables
episomal replication in microorganisms, no nucleotide
sequence which is capable of/enables transposition
and no nucleotide sequence which imparts resistance
to antibiotics being present at the particular
second, optionally third or fourth site,
under conditions which allow expression of the said
open reading frames (ORF), genes or alleles,
b) concentration of the L-lysine in the fermentation
broth,
c) isolation of the L-lysine from the fermentation
broth, optionally
d) with constituents from the fermentation broth and/or
the biomass to the extent of > (greater than) 0 to
100.
A "copy of an open reading frame (ORF), gene or allele of
lysine production" is to be understood as meaning all the,
preferably endogenous, open reading frames, genes or
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alleles of which enhancement/over-expression can have the
effect of improving lysine production. Enhancement is
understood as meaning an increase in the intracellular
concentration or activity of the particular gene product,
protein or enzyme.
These include, inter alia, the following open reading
frames, genes or alleles: accBC, accDA, cstA, cysD, cysE,
cysH, cysK, cysN, cysQ, dapA, dapB, dapC, dapD, dapE, dapF,
ddh, dps, eno, gap, gap2, gdh, gnd, lysC, lysCF$R, lysE,
msiK, opcA, oxyR, ppc, ppcFBR, pgk, pknA, pknB, pknD, pknG,
ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE,
sigH, sigh, tal, thyA, tkt, tpi, zwal, zwf and zwf A213T.
These are summarized and explained in Table 1.
These include, in particular, the lysCFBR alleles which code
for a "feed back" resistant aspartate kinase. Various
lysCFBR alleles are summarized and explained in Table 2.
The following lysCFBR alleles are preferred: lysC A279T
(replacement of alanine at position 279 of the aspartate
kinase protein coded, according to SEQ ID NO: 2, by
threonine), lysC A279V (replacement of alanine at position
279 of the aspartate kinase protein coded, according to SEQ
ID NO: 2, by valine), lysC S301F (replacement of serine at
position 301 of the aspartate kinase protein coded,
according to SEQ ID NO: 2, by phenylalanine), lysC T308I
(replacement of threonine at position 308 of the aspartate
kinase protein coded, according to SEQ ID N0: 2, by
isoleucine), lysC S301Y (replacement of serine at position
308 of the aspartate kinase protein coded, according to SEQ
ID NO: 2, by tyrosine), lysC G345D (replacement of glycine
at position 345 of the aspartate kinase protein coded,
according to SEQ ID N0: 2, by aspartic acid), lysC R320G
(replacement of arginine at position 320 of the aspartate
kinase protein coded, according to SEQ ID N0: 2, by
glycine), lysC T311I (replacement of threonine at position
311 of.the aspartate kinase protein coded, according to SEQ
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ID N0: 2, by isoleucine), lysC S381F (replacement of serine
at position 381 of the aspartate kinase protein coded,
according to SEQ ID N0: 2, by phenylalanine).
The lysCF$R allele lysC T311I (replacement of threonine at
position 311 of the aspartate kinase protein coded,
according to SEQ ID N0: 2, by isoleucine), the nucleotide
sequence of which is shown as SEQ ID N0:3, is particularly
preferred; the amino acid sequence of the aspartate kinase
protein coded is shown as SEQ ID N0:4.
The second, optionally third or fourth copy of the open
reading frame (ORF), gene or allele of lysine production in
question can be integrated at in each case a second,
optionally third or fourth site. The following open reading
frames, genes or nucleotide sequences, inter alia, can be
used for this: aecD, ccpAl, ccpA2, citA, citB, citE, fda,
gluA, gluB, gluC, gluD, luxR, luxS, lysRl, lysR2, lysR3,
menE, mqo, pck, pgi, poxB and zwa2, in particular the genes
aecD, gluA, gluB, gluC, gluD and pck. These are summarized
and explained in Table 3.
The sites mentioned include, of course, not only the coding
regions of the open reading frames or genes mentioned, but
also the regions or nucleotide sequences lying upstream
which are responsible for expression and regulation, such
as, for example, ribosome binding sites, promoters, binding
sites for regulatory proteins, binding sites for regulatory
ribonucleic acids and attenuators. These regions in general
lie in a range of 1-800, 1-600, 1-400, 1-200, 1-100 or 1-50
nucleotides upstream of the coding region. In the same way,
regions lying downstream, such as, for example,
transcription terminators, are also included. These regions
in general lie in a range of 1-400, 1-200, 1-100, ~.-50 or
1-25 nucleotides downstream of the coding region.
Intergenic regions in the chromosome, that is to say
nucleotide sequences without a coding function, can
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furthermore be used. Finally, prophages or defective phages
contained in the chromosome can be used for this.
A prophage is understood as meaning a bacteriophage, in
particular the genome thereof, where this is replicated
5 together with the genome of the host and the formation of
infectious particles does not take place. A defective phage
is understood as meaning a prophage, in particular the
genome thereof, which, as a result of various mutations,
has lost the ability to form so-called infectious
10 particles. Defective phages are also called cryptic.
Prophages and defective phages are often present in
integrated form in the chromosome of their host. Further
details exist in the prior art, for example in the textbook
by Edward A. Birge (Bacterial and Bacteriophage Genetics,
15 3rd ed., Springer-Verlag, New York, USA, 1994) or in the
textbook by S. Klaus et al. (Bakterienviren, Gustav Fischer
Verlag, Jena, Germany, 1992).
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Table 1
Open reading frames, genes and alleles of lysine production
Name Description of the coded enzymeReference Access
or
protein Number
accBC Acyl-CoA Carboxylase Jager et al. U35023
EC 6.3.4.14 Archives of
(acyl-CoA carboxylase) ' Microbiology
(1996) 166:76-
82
EP1108790; AX123524
W00100805 AX066441
accDA Acetyl-CoA Carboxylase EP1055725
EC 6.4.1.2 EP1108790 AX121013
(acetyl-CoA carboxylase) W00100805 AX066443
cstA Carbon Starvation Protein A EP1108790 AX120811
(carbon starvation protein A) W00100804 AX066109
cysD Sulfate Adenylyltransferase EP1108790 AX123177
sub-unit II
EC 2.7.7.4
(sulfate adenylyltransferase
small
chain )
cysE Serine Acetyltransferase EP1108790 AX122902
EC 2.3.1.30 W00100843 AX063961
(serine acetyltransferase)
cysH 3'-Phosphoadenyl Sulfate ReductaseEP1108790 AX123178
EC 1.8.99.4 W00100842 AX066001
(3'-phosphoadenosine 5'-
phosphosulfate reductase)
cysK. Cysteine Synthase EP1108790 AX122901
EC 4.2.99.8 W00100843 AX063963
(cysteine synthase)
cysN Sulfate Adenylyltransferase EP1108790 AX123176
sub-
unit I AX127152
EC 2.7.7.4
(sulfate adenylyltransferase)
cysQ Transport Protein CysQ EP1108790 AX127145
(transporter cysQ) W00100805 AX066423
dapA Dihydrodipicolinate Synthase Bonnassie et X53993
EC 4.2.1.52 al. Nucleic
(dihydrodipicolinate synthase) Acids Research
18:6421 (1990)
Pisabarro et
al., Journal
of
Bacteriology X21502
175:2743-
2749(1993)
EP1108790
W00100805
EP0435132
EP1067192 AX123560
EP1067193 AX063773
dapB Dihydrodipicolinate Reductase EP1108790 AX127149
EC 1.3.1.26 W00100843 AX063753
(dihydrodipicolinate reductase)EP1067192 AX137723
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EP1067193 AX137602
Pisabarro X67737
et
al., Journal 221502
of
Bacteriology
175:2743-
2749(1993)
JP1998215883 E16749
JP1997322774 E14520
JP1997070291 E12773
JP1995075578 E08900
dapC N-Succinyl Aminoketopimelate EP1108790 AX127146
Transaminase W00100843 AX064219
EC 2.6.1.17 EP1136559
(N-succinyl diaminopimelate
transaminase)
dapD Tetrahydrodipicolinate SuccinylaseEP1108790 AX127146
EC 2.3.1.117 W00100843 AX063757
(tetrahydrodipicolinate Wehrmann et AJ004934
al.
succinylase) Journal of
Bacteriology
180:3159-
3165(1998)
dapE N-Succinyl Diaminopimelate EP1108790 AX127146
Desuccinylase W00100843 AX063749
EC 3.5.1.18 Wehrmann et X81379
al.
(N-succinyl diaminopimelate Microbiology
desuccinylase) 140:3349-3356
(1994)
dapF Diaminopimelate Epimerase EP1108790 AX127149
EC 5.1.1.7 W00100843 AX063719
(diaminopimelate epimerase) EP1085094 AX137620
ddh Diaminopimelate Dehydrogenase EP1108790 AX127152
EC 1.4.1.16 W00100843 AX063759
(diaminopimelate dehydrogenase)Ishino et Y00151
al.,
Nucleic Acids
Research
15:3917-
3917(1987)
JP1997322774 E14511
JP1993284970 E05776
Kim et al., D87976
Journal of
Microbiology
and
Biotechnology
5:250-256(1995)
dps DNA Protection Protein EP1108790 AX127153
(protection during starvation
protein)
eno Enolase EP1108790 AX127146
EC 4.2.1.11 W00100844 AX064945
(enolase) EP1090998 AX136862
Hermann et
al.,
Electrophoresis
19:3217-3221
(1998)
gap Glyceraldehyde 3-Phosphate EP1108790 AX127148
Dehydrogenase W00100844 AX064941
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EC 1.2.1.12 Eikmanns et X59403
(glyceraldehyde 3-phosphate al., Journal
of
dehydrogenase) ~ Bacteriology
174:6076-
6086(1992)
gap2 Glyceraldehyde 3-Phosphate EP1108790 AX127146
Dehydrogenase W00100844 AX064939
EC 1.2.1.12
(glyceraldehyde 3-phosphate
dehydrogenase 2)
gdh Glutamate Dehydrogenase EP1108790 AX127150
EC 1.4.1.4 W00100844 AX063811
(glutamate dehydrogenase) Boermann et X59404
al., Molecular
Microbiology
6:317-326
(1992) .
Guyonvarch X72855
et
al., NCBI
gnd 6-Phosphogluconate DehydrogenaseEP1108790 AX127147
EC 1.1.1.44 AX121689
(6-phosphogluconate dehydrogenase)W00100844 . AX065125
lysC Aspartate Kinase EP1108790 AX120365
EC 2.7.2.4 ~ W00100844 AX063743
(aspartate kinase) Italinowski X57226
et
al., Molecular
Microbiology
5:119.7-204
(1991)
lysC~R Aspartate Kinase feedback resistantsee Table 2
( fbr)
EC 2.7.2.4
(aspartate kinase fbr)
lysE Lysine Exporter EP1108790 AX123539
(lysine exporter protein) W00100843 AX123539
Vrljic et al.,X96471
Molecular
Microbiology
22:815-826
(1996)
msiK Sugar Importer EP1108790 AX120892
(multiple sugar import protein)
opcA Glucose 6-phosphate DehydrogenaseW00104325 AX076272
(subunit of glucose 6-phosphate
dehydrogenase)
oxyR Transcription Regulator EP1108790 AX122198
(transcriptional regulator) AX127149
ppcF$R Phosphoenol Pyruvate CarboxylaseEP0723011
feedback resistant W00100852
EC 4.1.1.31
(phosphoenol pyruvate carboxylase
feedback resistant)
ppc Phosphoenol Pyruvate CarboxylaseEP1108790 AX127148
EC 4.1.1.31 AX123554
(phosphoenol pyruvate carboxylase)O'Reagan et M25819
al., Gene
77(2):237-
251(1989)
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pgk Phosphoglycerate Kinase EP1108790 AX121838
EC 2.7.2.3 AX127148
(phosphoglycerate kinase) W00100844 AX064943
Eikmanns, X59403
Journal of
Bacteriology
174:6076-6086
(1992)
pknA Protein Kinase A EP1108790 AX120131
(protein kinase A) AX120085
pknB Protein Kinase B EP1108790 AX120130
(protein kinase B) AX120085
pknD Protein Kinase D EP1108790 AX127150
(protein kinase D) AX122469
AX122468
pknG Protein Kinase G EP1108790 AX127152
(protein kinase G) AX123109
ppsA Phosphoenol Pyruvate Synthase EP1108790 AX127144
EC 2.7.9.2 AX120700
(phosphoenol pyruvate synthase) AX122469
ptsH Phosphotransferase System ProteinEP1108790 AX122210
H
EC 2.7'.1.69 AX127149
(phosphotransferase system W00100844 AX069154
component H)
ptsI Phosphotransferase System EnzymeEP1108790 AX122206
I
EC 2.7.3.9 AX127149
(phosphotransferase system
enzyme I)
ptsM Glukose-specific Phosphotransferase.Lee et al., L18874
System Enzyme II FEMS
EC 2.7.1.69 Microbiology
(glucose phosphotransferase Letters 119
system
enzyme II) (1-2):137-145
(1994)
pyc Pyruvate Carboxylase W09918228 A97276
EC 6.4.1.1 Peters-WendischY09548
(pyruvate carboxylase) et al.,
Microbiology
144:915-927
(1998)
pyc Pyruvate Carboxylase EP1108790
P458S EC 6.4.1.1
(pyruvate carboxylase)
amino acid exchange P458S
sigC Sigma Factor C EP1108790 AX120368
EC 2.7.7.6 AX120085
(extracytoplasmic function
alternative sigma factor C)
sigD RNA Polymerase Sigma Factor EP1108790 AX120753
D
EC 2.7.7.6 AX127144
(RNA polymerase sigma factor)
sigE Sigma Factor E EP1108790 AX127146
EC 2.7.7.6 AX121325
(extracytoplasmic function
alternative sigma factor E)
sigH Sigma Factor H EP1108790 AX127145
EC 2.7.7.6 AX120939
(sigma factor SigH)
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sigh Sigma Factor M EP1108790 AX123500
EC 2.7.7.6 AX127145
(sigma factor Sigh)
tal Transaldolase W00104325 AX076272
EC 2.2.1.2
(transaldolase)
thyA Thymidylate Synthase EP1108790 AX121026
EC 2.1.1.45 AX127145
(thymidylate synthase)
tkt Transketolase Ikeda et al., AB023377
EC 2.2.1.1 NCBI
(transketolase)
tpi Triose Phosphate Isomerase Eikmanns, X59403
EC 5.3.1.1 Journal of
(triose phosphate isomerase) Bacteriology
174:6076-6086
(1992)
zwal Cell Growth Factor 1 EP1111062 AX133781
(growth factor 1)
zwf Glucose 6-phosphate 1-DehydrogenaseEP1108790 AX127148
EC 1.1.1.49 AX121827
(glucose 6-phosphate 1- W00104325 AX076272
dehydrogenase)
zwf Glucose 6-phosphate 1-DehydrogenaseEP1108790
A213T EC 1.1.1.49
(glucose 6-phosphate 1-
dehydrogenase)
amino acid exchange A213T
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Table 2
lysCF$R alleles which code for feed back resistant aspartate
kinases
Name of the Further Reference Access Number
allele information
lysCFaa-E05108 JP 1993184366-A E05108
(sequence 1)
lysCFax-E06825 lysC A279T JP 1994062866-A E06825
(sequence 1)
lysCFBa-E06826 lysC A279T JP 1994062866-A E06826
(sequence 2)
lysCFax-E06827 JP 1994062866-A E06827
(sequence 3)
lysCFax-E08177 JP 1994261766-A E08177
(sequence 1)
lysCFax-E08178 lysC A279T JP 1994261766-A E08178
(sequence 2)
lysCFax-E08179 lysC A279V JP 1994261766-A E08179
(sequence 3)
lysCFaR-E08180 lysC 5301F JP 1994261766-A E08180
(sequence 4)
lysCFaR-E08181 lysC T308I JP 1994261766-A E08181
(sequence .5)
lysC~-E08182 JP 1994261766-A E08182
(sequence 6)
lysCFaR-E12770 JP 1997070291-A E12770
(sequence 13)
lysCFSa-E14514 JP 1997322774-A E14514
(sequence 9)
lysCFaR-E16352 JP 1998165180-A E16352
(sequence 3)
lysCFax-E16745 JP 1998215883-A E16745
(sequence 3)
lysCF'BR-E16746 JP 1998215883-A E16746
(sequence 4)
lysC~R-174588 US 5688671-A I74588
(sequence 1)
lysCFaR-174589 lysC A279T US 5688671-A I74589
(sequence 2)
lysCFHR_I74590 US 5688671-A ~ 174590
(sequence 7)
lysCFaR-174591 lysC A279T US 5688671-A I74591
(sequence 8)
lysCFaR_I74592 US 5688671-A I74592
(sequence 9)
lysCFaR-174593 lysC A279T US 5688671-A I74593
(sequence 10)
lysCFaR-174594 US 5688671-A I74594
(sequence 11)
lysCFax-174595 lysC A279T US 5688671-A I74595
(sequence 12)
lysCFaR-174596 US 5688671-A 174596
(sequence 13)
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lysCFHR-174597 lysC A279T US 5688671-A I74597
(sequence 14)
lysCFSR-X57226 lysC S301Y EP0387527 X57226
Kalinowski et al.,
Molecular and
General Genetics
224:317-324 (1990)
lysCFHR-L16848 lysC G345D Follettie and L16848
Sinskey
NCBI Nucleotide
Database (1990)
lysCFBR-L27125 lysC R320G Jetten et al., L27125
lysC G345D Applied Microbiology
Biotechnology 43:76-
82 (1995)
lysCFBR lysC T311I W00063388
(sequence 17)
lysCF$R lysC S301F US3732144
lysCF$R lysC S381F
lysC~R JP6261766
(sequence 1)
lysC~R lysC A279T JP6261766
(sequence 2)
lysCF$R lysC A279V JP6261766
(sequence 3)
__ lysC S301F JP6261766
lysC~R
(sequence 4)
lysC~R lysC T308I JP6261766
(sequence 5)
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Table 3
Target sites for integration of open reading frames, genes
and alleles of lysine production
Gene Description of the Ref erence Access
coded
name enzyme or protein Number
aecD beta C-S Lyase Rossol et al., JournalM89931
EC 2.6.1.1 of Bacteriology
(beta C-S lyase) 174(9):2968-77 (1992)
ccpAl Catabolite Control W00100844 AX065267
Protein EP1108790 AX127147
(catabolite control
protein A1)
ccpA2 Catabolite Control W00100844 AX065267
Protein EP1108790 AX121594
(catabolite control
protein A2)
citA Sensor Kinase CitA EP1108790 AX120161
(sensor kinase CitA)
_
cit8 Transcription RegulatorEP1108790 AX120163
CltB
(tranSCriptiOn regulator
citB)
citE Citrate Lyase W00100844 AX065421
EC 4.1.3.6 EP1108790 AX127146
(citrate lyase)
fda Fructose Bisphosphate von der Osten et al.,X17323
Aldolase Molecular Microbiology
EC 4.1.2.13 3(11):2625-37 (1989)
(fructose 1,6-
bisphosphate aldolase)
gluA Glutamate Transport Kronemeyer et al., X8119
ATP- 1
binding Protein Journal of Bacteriology
(glutamate transport 177(5):1152-8 (1995)
ATP-binding protein)
gluB Glutamate-binding Kronemeyer et al., X81191
Protein Journal of Bacteriology
(glutamate-binding 177(5):1152-8 (1995)
protein)
gluC Glutamate Transport Kronemeyer et al., X81191
Permease Journal of Bacteriology
(glutamate transport 177(5):1152-8 (1995)
system permease)
gluD Glutamate Transport Kronemeyer et al., X81191
Permease Journal of Bacteriology
(glutamate transport 177(5):1152-8 (1995)
system permease)
luxR Transcription RegulatorW00100842 AX065953
LuxR EP1108790 AX123320
(transcription regulator
LuxR)
luxS Histidine Kinase LuxS EP1108790 AX123323
(histidine kinase LuxS) AX127145
lysR1 Transcription RegulatorEP1108790 AX064673
LysR1 AX127144
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(transcription regulator
LysR1)
lysR2 Transcription ActivatorEP1108790 AX123312
LysR2
(transcription regulator
LysR2)
lysR3 Transcription RegulatorW00100842 AX065957
LysR3 EP1108790 AX127150
(transcription regulator
LysR3)
menE 0-Succinylbenzoic AcidW00100843 AX064599
CoA Lipase EP1108790 AX064193
EC 6.2.1.26 AX127144
(0-succinylbenzoate
CoA
lipase)
mqo Malate-Quinone Molenaar et al., Eur.AJ224946
Oxidoreductase Journal of Biochemistry
(malate-quinone- 1;254(2):395-403 (1998)
oxidoreductase)
pck Phosphoenol Pyruvate W00100844 AJ269506
Carboxykinase Ax065053
(phosphoenol pyruvate
carboxykinase)
pgi Glucose 6-phosphate EP1087015 AX136015
Isomerase EP1108790 AX127146
EC 5.3.1.9
(glucose 6-phosphate
isomerase)
poxB Pyruvate Oxidase ' W00100844 AX064959
EC 1.2.3.3 EP1096013 AX137665
(pyruvate oxidase)
zwa2 Cell Growth Factor EP1106693 AX113822
2
(growth factor 2) EP1108790 AX127146
The invention accordingly also provides a process for the
production of coryneform bacteria which produce L-lysine,
which comprises
a) isolating the nucleotide sequence of at least one
desired ORF, gene or allele of lysine production,
optionally including the expression and/or regulation
signals,
b) providing the 5' and the 3' end of the ORF, gene or
allele of lysine production with nucleotide sequences
of the target site,
c) preferably incorporating the nucleotide sequence of
the desired ORF, gene or allele provided with
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nucleotide sequences of the target site into a vector
which does not replicate or replicates to only a
limited extent in coryneform bacteria,
d) transferring the nucleotide sequence according to b)
5 or c) into coryneform bacteria, and
e) isolating coryneform bacteria in which the nucleotide
sequence according to a) is incorporated at the target
site, no nucleotide sequence which is capable
of/enables episomal replication in microorganisms, no
10 nucleotide sequence which is capable of/enables
transposition and no nucleotide sequence which imparts
resistance to antibiotics remaining at the target
site.
The invention furthermore provides coryneform bacteria, in
15 particular of the genus Corynebacterium, which produce L-
methionine and/or L-threonine, characterized in that these
have, in addition to at least one of the copy of an open
reading frame (ORF), gene or allele of methionine
production or threonine production present at the natural
20 site (locus), in each case a second, optionally third or
fourth copy of the open reading frame (ORF), gene or allele
in question at in each case a second, optionally third or
fourth site in integrated form, no nucleotide sequence
which is capable of/enables episomal replication in
25 microorganisms, no nucleotide sequence which is capable
of/enables transposition and no nucleotide sequence which
imparts resistance to antibiotics being present at the
particular second, optionally third or fourth site.
The invention also furthermore provides a process for the
preparation of L-methionine and/or L-threonine, which
comprises the following steps:
a) fermentation of coryneform bacteria, in particular
Corynebacterium glutamicum, characterized in that
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these have, in addition to at least one of the copy of
an open reading frame (ORF), gene or allele of
methionine production or threonine production present
at the natural site (locus), in each case a second,
optionally third or fourth copy of the open reading
frame (ORF), gene or allele in question at in each
case a second, optionally third or fourth site in
integrated form, no nucleotide sequence which is
capable of/enables episomal replication in
microorganisms, no nucleotide sequence which is
capable of/enables transposition and no nucleotide
sequence which imparts resistance to antibiotics being
present at the particular second, optionally third or
fourth site,
under conditions which allow expression of the said
open reading frames (ORF), genes or alleles,
b) concentration of the L-methionine and/or L-threonine
in the fermentation broth,
c)~ isolation of the L-methionine and/or L-threonine from
the fermentation broth, optionally
d) with constituents from the fermentation broth and/or
the biomass to the extent of > (greater than) 0 to
100.
A "copy of an open reading frame (ORF), gene or allele of
methionine production" is to be understood as meaning all
the, preferably endogenous, open reading frames, genes or
alleles of which enhancement/over-expression can have the
effect of improving methionine production.
These include, inter alia, the following open reading
frames, genes or alleles: accBC, accDA, aecD, cstA, cysD,
cysE, cysH, cysI~, cysN, cysQ, dps, eno, fda, gap, gap2,
gdh, gnd, glyA, hom, homFBR, lysC, lysCF$R, metA, metB, metE,
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metes, metY, msiK, opcA, oxyR, ppc, ppcFBR, pgk, pknA, pknB,
pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC,
sigD, sigE, sigH, sigh, tal, thyA, tkt, tpi, zwal, zwf and
zwf A213T, These are summarized and explained in Table 4.
These include, in particular, the lysCFBR alleles which code
for a "feed back" resistant aspartate kinase (see Table 2)
and the hom~BR alleles which code for a "feed back"
resistant homoserine dehydrogenase.
The second, optionally third or fourth copy of the opera.
reading frame (ORF), gene or allele of methionine
production in question can be integrated at in each case a
second, optionally third or fourth site. The following open
reading frames, genes or nucleotide sequences, inter alia,
can be used for this: brnE, brnF, brnQ, ccpAl, ccpA2, citA,
citB, citE, ddh, gluA, gluB, gluC, gluD, luxR, luxS, lysRl,
lysR2, lysR3, menE, metD, metK, pck, pgi, poxB and zwa2.
These are summarized, and explained in Table 5.
The sites mentioned include, of course, not only the coding
regions of the open reading frames or genes mentioned, but
also the regions or nucleotide sequences lying upstream
which are responsible for expression and regulation, such
as, for example, ribosome binding sites, promoters, binding
sites for regulatory proteins, binding sites for regulatory
ribonucleic acids and attenuators. These regions in general
lie in a range of 1-800, 1-600, 1-400, 1-200, 1-100 or 1-50
nucleotides upstream of the coding region. In the same way,
regions lying downstream, such as, for example,
transcription terminators, are also included. These regions
in general lie in a range of 1-400, 1-200, 1-100, 1-50 or
1-25 nucleotides downstream of the coding region.
Intergenic regions in the chromosome, that is to say
nucleotide sequences without a coding function, can
furthermore be used. Finally, prophages or defective phages
contained in the chromosome can be used for this.
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Table 4
Open reading frames, genes and alleles of methionine
production
Name Description of the coded enzymeReference Access
or
protein Number
AccBC Acyl-CoA Carboxylase Jager et al. U35023
EC 6.3.4.14 Archives of
(acyl-CoA carboxylase) , Microbiology
(1996) 166:76-82
EP1108790; AX123524
W00100805 AX066441
AccDA Acetyl-CoA Carboxylase EP1055725
EC 6.4.1.2 EP1108790 AX121013
(acetyl-CoA carboxylase) W00100805 AX066443
AecD Cystathionine beta-Lyase Rossol et al., M89931
EC 4.4.1.8 Journal of
(cystathionine beta-lyase) Bacteriology
174:2968-2977
(1992)
CstA Carbon Starvation Protein A EP2108790 AX220811
(carbon starvation protein A) W00100804 AX066109
CysD Sulfate Adenylyltransferase EP1108790 AX123177
sub-unit II
EC 2.7.7.4
(sulfate adenylyltransferase
small
chain)
CysE Serine Acetyltransferase EP1108790 AX122902
EC 2.3.1.30 W00100843 AX063961
(serine acetyltransferase)
CysH 3'-Phosphoadenyl Sulfate ReductaseEP1108790 AX123178
EC 1.8.99.4 W00200842 AX066001
t3'-phosphoadenosine 5'-
phosphosulfate reductase)
CysK Cysteine Synthase EP1108790 AX122901
EC 4.2.99.8 W00100843 AX063963
(cysteine synthase)
CysN Sulfate Adenylyltransferase EP1108790 AK123176
sub-
unit I AX127152
EC 2.7.7.4
tsulfate adenylyltransferase)~
CysQ Transport protein CysQ EP1108790 AX127145
(transporter cysQ) W00100805 AX066423
Dps DNA Protection Protein EP1108790 AX127153
(protection during starvation
protein)
Eno Enolase EP1108790 AX127146
EC 4.2.1.11 W00100844 AX064945
(enolase) EP1090998 AX136862
Hermann et al.,
Electrophoresis
19:3217-3221
(1998)
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Fda Fructose Bisphosphate Aldolase van der Osten X17313
et
EC 4.1.2.13 al., Molecular
(fructose bisphosphate aldolase)Microbiology
3:1625-1637
(1989)
Gap Glyceraldehyde 3-Phosphate EP1108790 AX127148
Dehydrogenase W00100844 AX064941
EC 1.2.1.12 Eikmanns et X59403
al.,
(glyceraldehyde 3-phosphate Journal of
dehydrogenase) Bacteriology
174:6076-
6086(1992)
gap2 Glyceraldehyde 3-Phosphate EP1108790 AX127146
Dehydrogenase W00100844 AX064939
EC 1.2.1.12
(glyceraldehyde 3-phosphate
dehydrogenase 2)
Gdh Glutamate Dehydrogenase EP1108790 AX127150
EC 1.4.1.4 W00100844 AX063811
(glutamate dehydrogenase) Boermann et X59404
al.,
Molecular
Microbiology
6:317-326
(1992);
Guyonvarch et X72855
al., NCBI
GlyA Glycine/Serine EP1108790 AX127146
Hydroxymethyltransferase AX121194
EC 2.1.2.1
(glycine/serine
hydroxymethyltransferase)
Gnd 6-Phosphogluconate DehydrogenaseEP1108790 AX127147
EC 1.1.1.44 ~ AX121689
(6-phosphogluconate dehydrogenase)W00100844 AX065125
Hom Homoserine Dehydrogenase Peoples et al.,Y00546
EC 1.1.1.3 Molecular
(homoserine dehydrogenase) Microbiology
2:63-72 (1988)
homFBRHomoserine Dehydrogenase feedbackReinscheid et
resistant (fbr) al., Journal
of
EC 1.1.1.3 Bacteriology
(homoserine dehydrogenase fbr) 173:3228-30
(1991)
LysC Aspartate Kinase EP1108790 AX120365
EC 2.7.2.4 W00100844 AX063743
(aspartate kinase) Kalinowski et X57226
al., Molecular
Microbiology
5:1197-204
( 1991 )
lysC~RAspartate Kinase feedback resistantsee Table 2
( fbr )
EC 2.7.2.4
(aspartate kinase fbr)
MetA Homoserine Acetyltransferase Park et al., AF052652
EC 2.3.1.31 Molecular Cells
(homoserine acetyltransferase) 8:286-94 (1998)
MetB Cystathionine y-Lyase Hwang et al., AF126953
I
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EC 4.4.1.1 Molecular Cells
(cystathionine gamma-synthase)9:300-308 (1999)
MetE Homocysteine MethyltransferaseEP1108790 AX127146
EC 2.1.1.14 ~ AX121345
(homocysteine methyltransferase)
Metes Homocysteine MethyltransferaseEP1108790 AX127148
(Vitamin B12-dependent) AX121747
EC 2.1.1.14
(homocysteine methyltransferase)
MetY Acetylhomoserine SulfhydrolaseEP1108790 AX120810
(acetylhomoserine sulfhydrolase) AX127145
MsiK Sugar Importer EP1108790 AX120892
(multiple sugar import protein)
OpcA Glucose 6-phosphate DehydrogenaseW00104325 AX076272
(subunit of glucose 6-phosphate
dehydrogenase)
OxyR Transcription Regulator EP1108790 AX122198
(transcriptional regulator) AX127149
ppc~R Phosphoenol Pyruvate CarboxylaseEP0723011
feedback resistent W00100852
EC 4.1.1.31
(phosphoenol pyruvate carboxylase
feedback resistant)
Ppc Phosphoenol Pyruvate CarboxylaseEP1108790 AX127148
EC 4.1.1.31 AX123554
(phosphoenol pyruvate carboxylase)O'Reagan et M25819
al.,
Gene 77(2):237-
251(1989)
Pgk Phosphoglycerate Kinase EP1108790 AX121838
EC 2.7.2.3 AX127148
(phosphoglycerate kinase) W00100844 AX064943
Eikmanns, X59403
Journal of
Bacteriology
174:6076-6086
(1992 )
PknA Protein k~inase A EP1108790 AX120131
(protein kinase A) AX120085
PknB Protein Kinase B EP1108790 AX120130
(protein kinase B) AX120085
PknD Protein Kinase D EP1108790 AX127150
(protein Kinase D) AX122469
AX122468
PknG Protein Kinase G EP1108790 AX127152
(protein kinase G) AX123109
PpsA Phosphoenol Pyruvate Synthase EP1108790 AX127144
EC 2.7.9.2 AX120700
(phosphoenol pyruvate synthase) AX122469
PtsH Phosphotransferase System ProteinEP1108790 AX122210
H
EC 2.7.1.69 AX127149
(phosphotransferase system W00100844 AX069154
component H)
PtsI Phosphotransferase System EnzymeEP1108790 AX122206
I
EC 2.7.3.9 AX127149
(phosphotransferase system
enzyme I)
PtsM Glucose-specific PhosphotransferaseLee et al., L18874
FEMS
System Enzyme II Microbiology
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EC 2.7.1.69 Letters 119
(glucose phosphotransferase (1-2):137-145
system
enzyme II) (1994)
Pyc Pyruvate Carboxylase W09918228 A97276
EC 6.4.1.1 Peters-WendischY09548
(pyruvate carboxylase) et al.,
Microbiology
144:915-927
(1998)
Pyc Pyruvate Carboxylase EP1108790
P458s EC 6.4.1.1
(pyruvate carboxylase)
amino acid exchange P458S
SigC Sigma Factor C EP1108790 AX120368
EC 2.7.7.6 AX120085
(extracytoplasmic function
alternative sigma factor C)
SigD RNA Polymerase Sigma Factor EP1108790 AX120753
D
EC 2.7.7.6 AX127144
(RNA polymerase sigma factor)
SigE Sigma Factor E EP1108790 AX127146
EC 2.7.7.6 AX121325
(extracytoplasmic function
alternative sigma factor E)
SigH Sigma Factor H EP1108790 AX127145
EC 2.7.7.6 AX120939
(sigma factor SigH)
Sigh Sigma Factor M EP1108790 AX123500
EC 2.7.7.6 AX127153
(sigma factor Sigh)
Tal Transaldolase W00104325 AX076272
EC 2.2.1.2
(transaldolase)
ThyA Thymidylate Synthase EP1108790 AX121026
EC 2.1.1.45 AX127145
(thymidylate synthase)
Tkt Transketolase Ikeda et al., AB023377
EC 2.2.1.1 NCBI
(transktolase)
Tpi Triose Phosphate Isomerase Eikmanns, X59403
EC 5.3.1.1 Journal of
(triose phosphate isomerase) Bacteriology
174:6076-6086
(1992)
zwal Cell Growth Factor 1 EP1111062 AX133781
(growth factor 1)
Zwf Glucose 6-phosphate 1-DehydrogenaseEP1108790 AX127148
EC 1.1.1.49 AX121827
(glucose 6-phosphate 1- W00104325 AX076272
dehydrogenase)
Zwf Glucose 6-phosphate 1-DehydrogenaseEP1108790
A213T EC 1.1.1.49
(glucose 6-phosphate 1-
dehydrogenase)
amino acid exchange A213T
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Table 5
Target sites for integration of open reading frames, genes
and alleles of methionine production
Gene Description of the Reference Access
name coded enzyme or protein Number
BrnE Transporter of EP1096010 AX137709
branched-chain amino AX137714
acids
(branched-chain amino
acid transporter)
BrnF Transporter of EP1096010 AX137709
branched-chain amino AX137714
acids
(branched-chain amino
acid transporter)
BrnQ Carrier protein of Tauch et al., ArchivesM89931
branched-chain amino of Microbiology AX066841
acids 169(4):303-12 (1998) AX127150
(branched-chain aminoW00100805
acid transport systemEP1108790
carrier protein)
ccpA1 Catabolite Control W00100844 AX065267
Protein EP1108790 AX127147
(catabolite control
protein A1)
ccpA2 Catabolite Control W00100844 AX065267
Protein EP1108790 AX121594
(catabolite control
protein A2)
citA Sensor Kinase CitA EP1108790 AX120161
(sensor kinase CitA)
citB Transcription RegulatorEP1108790 AX120163
CitB
(transcription
regulator CitB)
citE Citrate Lyase W00100844 AX065421
EC 4.1.3.6 EP1108790 AX127146
(citrate lyase)
ddh Diaminopimelate Ishino et al., Nucleic507384
Dehydrogenase Acids Research 15: AX127152
3917
EC 1.4.1.16 (1987)
(diaminopimelate EP1108790
dehydrogenase)
gluA Glutamate Transport Kronemeyer et al., X81191
ATP-binding Protein Journal of Bacteriology
(glutamate transport 177(5):1152-8 (1995)
ATP-binding protein)
gluB Glutamate-binding Kronemeyer et al., X81191
Protein Journal of Bacteriology
(glutamate-binding 177(5):1152-8 (1995)
protein)
gluC Glutamate Transport Kronemeyer et al., X81191
Permease Journal of Bacteriology
(glutamate transport 177(5):1152-8 (1995)
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system permease)
gluD Glutamate Transport Kronemeyer et al., X81191
Permease Journal of Bacteriology
(glutamate transport177(5):1152-8 (1995)
system permease)
luxR Transcription RegulatorW00100842 AX065953
LuxR EP1108790 AX123320
(transcription
regulator LuxR)
luxS Histidine Kinase EP1108790 AX123323
LuxS
(histidine kinase AX127145
LuxS)
lysR1 Transcription RegulatorEP1108790 AX064673
LysR1 AX127144
(transcription
regulator LysR1)
lysR2 Transcription ActivatorEP1108790 AX123312
LysR2
(transcription
regulator LysR2)
lysR3 Transcription RegulatorW00100842 AX065957
LysR3 EP1108790 AX127150
(transcription
regulator LysR3)
menE 0-Succinylbenzoic W00100843 AX064599
Acid
CoA Lipase EP1108790 AX064193
EC 6.2.1.26 AX127144
(0-succinylbenzoate
CoA
lipase)
metD Transcription RegulatorEP1108790 AX123327
MetD AX127153
(transcription
regulator MetD)
metK Methionine Adenosyl W00100843 AX063959
Transferase EP1108790 AX127148
EC 2.5.1.6
(S-adenosylmethionine
synthetase)
pck Phosphoenol PyruvateW00100844 AJ269506
Carboxykinase AX065053
(phosphoenol pyruvate
carboxykinase)
pgi Glucose 6-Phosphate EP1087015 AX136015
Isomerase EP1108790 AX127146
EC 5.3.1.9
(glucose-6-phosphate
isomerase)
poxB Pyruvate Oxidase W00100844 AX064959
EC 1.2.3.3 EP1096013 AX137665
(pyruvate oxidase)
zwa2 Cell Growth Factor EP1106693 AX113822
2
(growth factor 2) EP1108790 AX127146
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A "copy of an open reading frame (ORF), gene or allele of
threonine production" is to be understood as meaning all
the open reading frames, genes or alleles of which
enhancement/over-expression can have the effect of
improving threonine production.
These include, inter alia, the following open reading
frames, genes or alleles: accBC, accDA, cstA, cysD, cysE,
cysH, cysI, cysN, cysQ, dps, eno, fda, gap, gap2, gdh, gnd,
hom, homFBR, lysC, lysCFBR, msiK, opcA, oxyR, ppc, ppcFBR,
pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc,
pyc P458S, sigC, sigD, sigE, sigH, sigh, tal, thyA, tkt,
tpi, thrB, thrC, thrE, zwal, zwf and zwf A213T. These are
summarized and explained in Table 6. These include, in
particular, the lysCFBR alleles which code for a "feed back"
resistant aspartate kinase (See Table 2) and the homF$R
alleles which code for a "feed back" resistant homoserine
dehydrogenase.
The second, optionally third or fourth copy of the open
reading frame (0RF), gene or allele of threonine production
in question can be integrated at in each case a second,
optionally third or fourth site. The following open reading
frames, genes or nucleotide sequences, inter alia, can be
used for this: ccpAl, ccpA2, citA, citB, citE, ddh, gluA,
gluB, gluC, gluD, glyA, ilvA, ilvBN, ilvC, ilvD, luxR,
luxS, lysRl, lysR2, lysR3, mdh, menE, metA, metD, pck,
poxB, sigB and zwa2. These are summarized and explained in
Table 7.
The sites mentioned include, of course, not only the coding
regions of the open reading frames or genes mentioned, but
also the regions or nucleotide sequences lying upstream
which are responsible for expression and regulation, such
as, for example, ribosome binding sites, promoters, binding
sites for regulatory proteins, binding sites for regulatory
ribonucleic acids and attenuators. These regions in general
lie in a range of ~.-800, 1-600, 1-400, 1-200, 1-100 or 1-'50
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nucleotides upstream of the coding region. In the same way,
regions lying downstream, such as, for example,.
transcription terminators, are also included. These regions
in general lie in a range of 1-400, 1-200, 1-100, 1-50 or
5 1-25 nucleotides downstream of the coding region.
Intergenic regions in the chromosome, that is to say
nucleotide sequences without a coding function, can
furthermore be used. Finally, prophages or defective phages
contained in the chromosome can be used for this.
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Table 6
Open reading frames, genes and alleles of threonine production
Name Description of the coded enzymeReference Access
or
protein Number
accBC Acyl-CoA Carboxylase Jager et al. U35023
EC 6.3.4.14 Archives of
(acyl-CoA carboxylase) Microbiology
166:76-82 (1996)
EP1108790 AX123524
W00100805 AX066441
accDA Acetyl-CoA Carboxylase EP1055725
EC 6.4.1.2 EP1108790 AX121013
(acetyl-CoA carboxylase) W00100805 AX066443
cstA Carbon Starvation Protein A EP1108790 AX120812
(carbon starvation protein A) W00100804 AX066109
cysD Sulfate Aderiylyltransferase EP1108790 AX123177
sub-unit II
EC 2.7.7.4
(sulfate adenylyltransferase
small
chain)
cysE Serine Acetyltransferase EP1108790 AX122902
EC 2.3.1.30 W00100843 AX063961
(serine acetyltransferase)
cysH 3'-Phosphoadenyl Sulfate ReductaseEP1108790 AX123178
EC 1.8.99.4 WO0100842 AX066001
(3'-phosphoadenosine 5'-
phosphosulfate reductase)
cysK Cysteine Synthase EP1108790 AX122901
EC 4.2.99.8 W00100843 AX063963
(cysteine synthase)
cysN Sulfate Adenylyltransferase EP1108790 AX123176
sub-
unit I AX127152
EC 2.7.7.4
(sulfate adenylyltransferase)
cysQ Transport protein CysQ EP1108790 AX127145
(transporter cysQ) W00100805 AX066423
dps DNA Protection Protein EP1108790 AX127153
(protection during starvation
protein)
eno Enolase EP1108790 AX127146
EC 4.2.2.11 W00100844 AX064945
(enolase) EP1090998 AX136862
Hermann et al.,
Electrophoresis
19:3217-3221
(1998)
fda Fructose Bisphosphate Aldolase van der Osten X17313
et
EC 4.1.2.13 al., Molecular
(fructose bisphosphate aldolase)Microbiology
3:1625-1637
(1989)
gap Glyceraldehyde 3-Phosphate EP11fl8790 AX127148
Dehydrogenase W00100844 AX064942
EC 1.2.1.12 Eikmanns et X59403
al.,
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(glyceraldehyde 3-phosphate Journal of
dehydrogenase) Bacteriology
174:6076-
6086(1992)
gap2 Glyceraldehyde 3-Phosphate EP1108790 AX127146
Dehydrogenase W00100844 AX064939
EC 1.2.1.12
(glyceraldehyde 3-phosphate
dehydrogenase 2)
gdh Glutamate Dehydrogenase EP1108790 AX127150
EC 1.4.1.4 W00100844 AX063811
(glutamate dehydrogenase) Boermann et X59404
al.,
Molecular
Microbiology
6:317-326
(1992);
Guyonvarch et X72855
al., NCBI
gnd 6-Phosphogluconate DehydrogenaseEP1108790 AX127147
EC 1.1.1.44 AX121689
(6-phosphogluconate dehydrogenase)W00100844 AX065125
hom Homoserine Dehydrogenase Peoples et al.,Y00546
EC 1.1.1.3 Molecular
(homoserine dehydrogenase) Microbiology
2:63-72 (1988)
hom~R Homoserine Dehydrogenase feedbackReinscheid et
resistant (fbr) al., Journal
of
EC 1.1.1.3 Bacteriology
(homoserine dehydrogenase fbr)173:3228-30
(1991)
lysC Aspartate Kinase EP1108790 AX120365
EC 2.7.2.4 W00100844 AX063743
(aspartate kinase) Kalinowski et X57226
al., Molecular
Microbiology
5:1197-204
(1991)
lysC~R Aspartate Kinase feedback resistentsee Table 2
( fbr)
EC 2.7.2.4
(aspartate kinase fbr)
msiK Sugar Importer EP1108790 AX120892
(multiple sugar import protein)
opcA Glucose 6-Phosphate DehydrogenaseW00104325 AX076272
(subunit of glucose 6-phosphate
dehydrogenase)
oxyR Transcription Regulator EP1108790 AX122198
(transcriptional regulator) AX127149
ppc~R Phosphoenol Pyruvate CarboxylaseEP0723011
feedback resistent W00100852
EC 4.1.1.31
(phosphoenol pyruvate carboxylase
feedback resistant)
ppc Phosphoenol Pyruvate CarboxylaseEP1108790 AX127148
EC 4.1.1.31 AX123554
(phosphoenol pyruvate carboxylase)0'Reagan et M25819
al.,
Gene 77(2):237-
251(1989)
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pgk Phosphoglycerate Kinase EP1108790 AX121838
EC 2.7.2.3 AX127148
(phosphoglycerate kinase) W00100844 AX064943
Eikmanns, X59403
Journal of
Bacteriology
174:6076-6086
(1992)
pknA Protein Kinase A EP1108790 AX120131
(protein kinase A) AX120085
pknB Protein Kinase B EP1108790 AX120130
(protein kinase B) AX120085
pknD Protein Kinase D EP1108790 'AX127150
(protein kinase D) AX122469
AX122468
pknG Protein Kinase G EP1108790 AX127152
(protein kinase G) AX123109
ppsA Phosphoenol Pyruvate Synthase EP1108790 AX127144
EC 2.7.9'.2 ' AX120700
(phosphoenol pyruvate synthase) AX122469
ptsH Phosphotransferase System ProteinEP1108790 AX122210
H
EC 2.7.1.69 AX127149
(phosphotransferase system WOU100844 AX069154
component H)
ptsI Phosphotransferase System EnzymeEP1108790 AX122206
I
EC 2.7.3.9 ~ AX127149
(phosphotransferase system
enzyme I)
ptsM Glukose-specific PhosphotransferaseLee, et al., L18874
FEMS
System Enzyme II Microbiology
EC 2.7.1.69 Letters 119
(glucose phosphotransferase-system(1-2):137-145
enzyme II) (1994)
pyc Pyruvate Carboxylase W09918228 A97276
EC 6.4.1.1 Peters-WendischY09548
(pyruvate carboxylase) et al.,
Microbiology
144:915-927
(1998)
pyc Pyruvate Carboxylase EP1108790
P458S EC 6.4.1.1
(pyruvate carboxylase)
amino acid exchange P458S
sigC Sigma Factor C EP1108790 AX120368
EC 2.7.7.6 AX120085
(extracytoplasmic function
alternative sigma factor C)
sigD RNA Polymerase Sigma Factor EP1108790 AX120753
D
EC 2.7.7.6 AX127144
(RNA polymerase sigma factor)
sigE Sigma Factor E EP1108790 AX127146
EC 2.7.7.6 AX121325
(extracytoplasmic function
alternative sigma factor E)
sigH Sigma Factor H EP1108790 AX127145
EC 2.7.7.6 A'X120939
(sigma factor SigH)
sigh Sigma Factor M EP1108790 AX123500
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EC 2.7.7.6 AX127153
(sigma factor Sigh)
tal Transaldolase W00104325 AX076272
EC 2.2.1.2
(transaldolase)
thrB Homoserine Kinase Peoples et al.,Y00546
EC 2.7.1.39 Molecular
(homoserine kinase) Microbiology
2:63-72 (1988)
thrC Threonine Synthase Han et al., X56037
EC 4.2.99.2 Molecular
(threonine synthase) Microbiology
4:1693-1702
(1990)
thrE Threonine Exporter EP1085091 AX137526
(threonine export carrier)
thyA Thymidylate Synthase EP1108790 AX121026
EC 2.1.1.45 AX127145
(thymidylate synthase)
tkt Transketolase Ikeda et al., AB023377
EC 2.2.1.1 NCBI
(transketolase)
tpi Triose phosphate Isomerase Eikmanns, X59403
EC 5.3.1.1 Journal of
(triose phosphate isomerase) Bacteriology
174:6076-6086
(1992)
zwal Cel1 Growth Factor 1 EP1111062 AX133781
(growth factor 1)
zwf Glucose 6-Phosphate 1-DehydrogenaseEP1108790 AX127148
EC 1.1.1.49 AX121827
(glucose 6-phosphate 1- W00104325 AX076272
dehydrogenase)
zwf Glucose 6-Phosphate 1-DehydrogenaseEP1108790
A213T EC 1.1.1.49
(glucose 6-phosphate 1-
dehydrogenase)
amino acid exchange A213T
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Table 7
Target sites for integration of open reading frames, genes
and alleles of threonine production
Gene Description of the codedReference Access
name enzyme or protein Number
ccpA1 Catabolite Control W00100844 AX065267
Protein EP1108790 AX127147
(catabolite control
protein A1)
ccpA2 Catabolite Control W00100844 AX065267
Protein EP1108790 AX121594
(catabolite control
protein A2)
citA Sensor Kinase CitA EP1108790 AX120161
(sensor kinase CitA)
citB Transcription RegulatorEP1108790 AX120163
CitB
(transcription regulator
CitB)
citE Citrate Lyase W00100844 AX065421
EC 4.1.3.6 EP1108790 AX127146
(citrate lyase)
ddh Diaminopimelate Ishino et al., NucleicS07384
Dehydrogenase Acids Research 15: AX127152
3917
EC 1.4.1.16 . (1987)
(diaminopimelate EP1108790
dehydrogenase)
gluA Glutamate Transport Kronemeyer et al., X81191
ATP-
binding Protein Journal of Bacteriology
(glutamate transport 177(5):1152-8 (1995)
ATP-
binding protein)
gluB Glutamate-binding ProteinKronemeyer et al., X81191
(glutamate-binding Journal of Bacteriology
protein) 177(5):1152-8 (1995)
gluC Glutamate Transport Kronemeyer et al., X81191
Permease Journal of Bacteriology
(glutamate transport 177(5):1152-8 (1995)
system permease)
,gluD Glutamate Transport Kronemeyer et al., X82191
Permease Journal of Bacteriology
(glutamate transport 177(5):1152-8 (1995)
system permease)
glyA Glycine WO0100843 AX063861
Hydroxymethyltransferase AF327063
EC 2.1.2.1
(glycine
hydroxymethyltransferase)
ilvA Threonine Dehydratase Mockel et al., JournalA47044
EC 4.2.1.16 of Bacteriology 174 L01508
(threonine dehydratase)(24), 8065-8072 (1992)AX127150
EP1108790
ilvBN Acetolactate Synthase Keilhauer et al., L09232
EC 4.1.3.18 Journal of Bacteriology
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(acetolactate synthase)175(17):5595-603 (1993)
EP1108790 AX127147
ilvC Reductoisomerase Keilhauer et al., C48648
EC 1.1.1.86 Journal of BacteriologyAX127147
(ketol-acid 175(17):5595-603 (1993)
reductoisomerase) EP1108790
ilvD Dihydroxy-acid EP1006189 AX136925
Dehydratase
EC 4.2.1.9
(dihydroxy-acid
dehydratase)
luxR Transcription RegulatorW00100842 AX065953
LuxR EP1108790 AX123320
(transcription regulator
LuxR)
luxS Histidine Kinase LuxS EP1108790 AX123323
(histidine kinase LuxS) AX127153
lysR1 Transcription RegulatorEP1108790 AX064673
LysR1 AX127144
(transcription regulator
LysR1)
lysR2 .Transcription ActivatorEP1108790 AX123312
LysR2
(transcription regulator
LysR2)
lysR3 Transcription RegulatorW00100842 AX065957
LysR3 EP1108790 AX127150
(transcription regulator
LysR3)
mdh Malate Dehydrogenase W00100844 AX064895
EC 1.1.1.37
(malate dehydrogenase)
menE O-Succinylbenzoic AcidW00100843 AX064599
CoA Ligase EP1108790 AX064193
EC 6.2.1.26 AX127144
(O-succinylbenzoate
CoA
ligase)
metA Homoserine 0- Park et al., MolecularAX063895
Acetyltransferase Cells 30;8(3):286-94 AX127145
EC 2.3.1.31 (1998)
(homoserine 0- W00100843
acetyltransferase) EP1108790
metD Transcription RegulatorEP1108790 AX123327
MetD AX127153
(transcription regulator
MetD)
pck Phosphoenol Pyruvate W00100844 AJ269506
Carboxykinase AX065053
(phosphoenol pyruvate
carboxykinase)
poxB Pyruvate Oxidase W00100844 AX064959
EC 1.2.3.3 EP1096013 AX137665
(pyruvate oxidase)
sigB RNA Polymerase EP1108790 AX127149
Transcription Factor
(RNA polymerase
transcription factor)
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zwa2 Cell Growth Factor 2 EP1106693 AX113822
(growth factor 2) EP1108790 AX127146
The invention accordingly also provides a process for the
production of coryneform bacteria which produce L-
methionine and/or L-threonine, which comprises
a) isolating the nucleotide sequence of at least one
desired ORF, gene or allele of methionine production
or threonine production, optionally including the
expression and/or regulation signals,
b) providing the 5' and the 3' end of the ORF, gene or
20 allele with nucleotide sequences of the target site,
c) preferably incorporating the nucleotide sequence of
the desired ORF, gene or allele provided with
nucleotide sequences of the target site into a vector
which does not replicate or replicates to only a
limited extent in coryneform bacteria,
d) transferring the nucleotide sequence according to b)
or c) into coryneform bacteria, and
e) isolating coryneform bacteria in which the nucleotide
sequence according to a) is incorporated at the target
site, no nucleotide sequence which is capable.
of/enables episomal replication in microorganisms, no
nucleotide sequence which is capable oflenables
transposition and no nucleotide sequence which imparts
resistance to antibiotics remaining at the target
site.
The invention furthermore provides coryneform bacteria, in
particular of the genus Corynebacterium, which produce L-
valine, wherein these have, in addition to at least one of
the copy of an open reading frame (ORF), gene or allele of
valine production present at the natural.. site (locus), in
each case a second, optionally third. or fourth copy of the
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open reading frame (ORF), gene or allele in question at in
each case a second, optionally third or fourth site in
integrated form, no nucleotide sequence which is capable
of/enables episomal replication in microorganisms, no
nucleotide sequence which is capable of/enables
transposition and no nucleotide sequence which imparts
resistance to antibiotics being present at the particular
second, optionally third or fourth site.
The invention also furthermore provides a process for the
preparation of L-valine, which comprises the following
steps:
a) fermentation of coryneform bacteria, in particular
Corynebacterium glutamicum, characterized in that
these have, in addition to at least one of the copy of
an open reading frame (ORF), gene or allele of valine
production present at the natural site (locus), in
each case a second, optionally third or fourth copy of
the open reading frame (ORF), gene or allele in
question at in each case a second, optionally third or
fourth site in integrated form, no nucleotide sequence
which is capable of/enables episomal replication in
microorganisms, no nucleotide sequence which is
capable of/enables transposition and no nucleotide
sequence which imparts resistance to antibiotics being
present at the particular second, optionally third or
fourth site,
under conditions which allow expression of the said
open reading frames (ORF), genes or alleles,
b) concentration of the L-valine in the fermentation
broth,
c) isolation of the L-valine from the fermentation broth,
optionally
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d) with constituents from the fermentation broth and/or
the biomass to the extent of > (greater than) 0 to
100.
A "copy of an open reading frame (ORF), gene or allele of
valine production" is to be understood as meaning all the
open reading frames, genes or alleles of which
enhancement/over-expression can have the effect of
improving valine production.
These include, inter alia, the following open reading
frames, genes or alleles: brnE, brnF, brnEF, cstA, cysD,
dps, eno, fda, gap, gap2, gdh, ilvB, ilvN, ilvBN, ilvC,
ilvD, ilvE msiK, pgk, ptsH, ptsl, ptsM, sigC, sigD, sigE,
sigH, sigh, tpi, zwal. These are summarized and explained
in Table 8. These include in particular the acetolactate
synthase which codes for a valine-resistant.
The second, optionally third or fourth copy of the open
reading frame (ORF), gene or allele of threonine production
in question can be integrated at in each case a second,
optionally third or fourth site. The following open reading
frames, genes or nucleotide sequences, inter alia, can be
used for this: aecD, ccpAl, ccpA2, citA, citB, citE, ddh,
gluA, gluB, gluC, gluD, glyA, ilvA, luxR, lysRl, lysR2,
lysR3, pang, panC, poxB and zwa2. These are summarized and
explained in Table 9.
The sites mentioned include, of course, not only the coding
regions of the open reading frames or genes mentioned, but
also the regions or nucleotide sequences lying upstream
which are responsible for expression and regulation, such
as, for example, ribosome binding sites, promoters, binding
sites for regulatory proteins, binding sites for regulatory
ribonucleic acids and attenuators. These regions in general
lie in a range of 1-800, 1-600, 1-400, 1-200, 1-100 or 1-50
nucleotides upstream of the coding region. In the same way,
regions lying downstream, such as, for example,
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transcription terminators, are also included. These regions
in general lie in a range of 1-400, 1-200, 1-100, 1-50 or
1-25 nucleotides downstream of the coding region.
Intergenic regions in the chromosome, that is to say
5 nucleotide sequences without a coding function, can
furthermore be used. Finally prophages or defective phages
contained in the chromosome can be used for this.
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46
Table 8
Open reading frames, genes and alleles of valine production
Name Description of the coded enzymeReference Access
or
protein Number
brnEF Export of branched-chain amino EP1096010
acids
(branched chain amino acid export)Kennerknecht AF454053
et
al., NCBI
cstA Carbon Starvation Protein A EP1108790 AX120811
(carbon starvation protein A) W00100804 AX066109
dps DNA Protection Protein EP1108790 AX127153
(protection during starvation
protein)
eno Enolase EP1108790 AX127146
EC 4.2.1.12 W00100844 AX064945
(enolase) EP1090998 AX136862
Hermann et al.,
Electrophoresis
19:3217-3221
(1998)
fda Fructose Bisphosphate Aldolase van der Osten X17313
et
EC 4.1.2.13 al., Molecular
(fructose bisphosphate aldolase)Microbiology
3:1625-1637
(1989)
gap Glyceraldehyde 3-Phosphate EP1108790 AX127148
Dehydrogenase W00100844 AX064941
EC 1.2.1.22 Eikmanns et X59403
al.,
(glyceraldehyde 3-phosphate Journal of
dehydrogenase) Bacteriology
174:6076-
6086(1992)
gap2 Glyceraldehyde 3-Phosphate EP1108790 AX127146
Dehydrogenase W00100844 AX064939
EC 1.2.1.12
(glyceraldehyde 3-phosphate
dehydrogenase 2)
gdh Glutamate Dehydrogenase EP1108790 AX127150
EC 1.4.1.4 W00100844 AX063811
(glutamate dehydrogenase) Boermann et X59404
al.,
Molecular
Microbiology
6:317-326
(1992);
Guyonvarch et X72855
al., NCBI
ilvBN Acetolactate Synthase Keilhauer et L09232
EC 4.1.3.18 al., Journal
of
(aeetolactate synthase) Bacteriology
175(17):5595-603
(1993)
EP1108790 AX127147
ilvC Isomeroreductase Keilhauer et C48648
EC 1.1,1.86 al., Journal AX127147
of
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47
(acetohydroxy acid Bacteriology
isomeroreductase) 175(17):5595-603
(1993)
EP1108790
ilvD Dihydroxy-acid Dehydratase EP1006189 AX136925
EC 4.2.1.9
(dihydroxy acid dehydratase)
ilvE Transaminase B EP1108790 AX127150
EC 2.6.1.42 AX122498
(transaminase B)
msiK Sugar Importer EP1108790 AX120892
(multiple sugar import protein)
pgk Phosphoglycerate Kinase EP1108790 AX121838
EC 2.7.2.3 AX127148
(phosphoglycerate kinase) W00100844 AX064943
Eikmanns, X59403
Journal of
Bacteriology
174:6076-6086
(1992)
ptsH Phosphotransferase System ProteinEP1108790 AX122210
H
EC 2.7.1.69 AX127149
(phosphotransferase system W00100844 AX069154
component H)
ptsI Phosphotransferase System EnzymeEP1108790 AX122206
I
EC 2.7.3.9 AX127149
(phosphotransferase system
enzyme I )
ptsM Glucose-specific PhosphotransferaseLee et al., L18874
FEMS
System Enzyme II Microbiology
EC 2.7.1.69 ' Letters 119
(glucose phosphotransferase-system(1-2):137-145
enzyme II) (1994)
sigC Sigma Factor C EP1108790 AX120368
EC 2.7.7.6 AX120085
(extracytoplasmic function
alternative sigma factor C)
sigD RNA Polymerase Sigma Factor EP1108790 AX120753
D
EC 2.7.7.6 AX127144
(RNA polymerase sigma factor)
sigE Sigma Factor E EP1108790 AX127146
EC 2.7.7.6 AX121325
(extracytoplasmic function
alternative sigma factor E)
sigH Sigma Factor H EP1108790 AX127145
EC 2.7.7.6 AX120939
(sigma factor SigH)
sigh Sigma Factor M EP1108790 AX123500
EC 2.7.7.6 AX127153
(sigma factor Sigh)
tpi Triose Phosphate Isomerase Eikmanns, X59403
EC 5.3.1.1 Journal of
(triose phosphate isomerase) Bacteriology
174:6076-6086
(1992)
zwal Cell Growth Factor 1 EP1111062 AX133781
(growth factor 1)
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Table 9
Target sites for integration of open reading frames, genes
and alleles of valine production
Gene Description of the codedReference Access
name enzyme or protein Number
aecD beta C-S Lyase Rossol et al., JournalM89931
EC 2.6.1.1 of Bacteriology
(beta C-S lyase) 174(9):2968-77 (1992)
ccpA1 Catabolite Control W00100844 AX065267
Protein EP1108790 AX127147
(catabolite control
protein A1)
ccpA2 Catabolite Control W00100844 AX065267
Protein EP1108790 AX121594
(catabolite control
protein A2)
citA Sensor Kinase CitA EP1108790 AX120161
(sensor kinase CitA)
citB Transcription RegulatorEP1108790 AX120163
CitB
(transcription regulator
CitB)
citE Citrate Lyase Wo0100844 AX065421
EC 4.1.3.6 EP1108790 AX227146
(citrate lyase)
ddh Diaminopimelate Ishino et al., Nucleic507384
Dehydrogenase Acids Research 15: AX227152
3927
EC 1.4.1.16 (1987)
(diaminopimelate EP1108790
dehydrogenase)
gluA Glutamate Transport Kronemeyer et al., X81191
ATP-
binding Protein Journal of Bacteriology
(glutamate transport 177(5):1152-8 (1995)
ATP-
binding protein)
gluB Glutamate-binding ProteinKronemeyer et al., X81191
(glutamate-binding Journal of Bacteriology
protein) 177(5):1152-8 (1995)
gluC Glutamate Transport Kronemeyer et al., X81191
Permease Journal of Bacteriology
(glutamate transport 177(5):1152-8 (1995)
system permease)
gluD Glutamate Transport Kronemeyer et al., X81292
Permease Journal of Bacteriology
(glutamate transport 177(5):1152-8 (2995)
system permease)
glyA Glycine W00100843 AX063861
Hydroxymethyltransferase AF327063
EC 2.1.2,2
(glycine
hydroxyme thyl t-rans
f erase )
ilvA Threonine Dehydratase Mockel et al., JournalA47044
EC 4.2.1.16 of Bacteriology 174 La1508
(threonine dehydratase)(24), 8065-8072 (1992)AX127150
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EP1108790
luxR Transcription RegulatorW00100842 AX065953
LuxR EP1108790 AX123320
(transcription regulator
LuxR)
lysR1 Transcription RegulatorEP1108790 AX064673
LysR1 AX127144
(transcription regulator
LysR1)
lysR2 Transcription ActivatorEP1108790 AX123312
LysR2
(transcription regulator
LysR2)
lysR3 Transcription RegulatorW00100842 AX065957
LysR3 EP1108790 AX127150
(transcription regulator
LysR3 )
pang Ketopantoate US6177264 X96580
Hydroxymethyltransferase
EC 2. 1. 2. 11
(ketopantoate
hydroxymethyltransferase)
panC Pantothenate SynthetaseUS6177264 X96580
EC 6.3.2.1
(pantothenate synthetase)
poxB Pyruvate Oxidase WOOi00844 AX064959
EC 1.2.3.3 EP1096013 AX137665
(pyruvate oxidase)
zwa2 Cell Growth Factor EP1106693 AX113822
2
(growth factor 2) EP1108790 AX127146
The invention accordingly also provides a process for the
production of coryneform bacteria which produce L-valine,
which comprises
a) isolating the nucleotide sequence of at least one
desired ORF, gene or allele of valine production,.
optionally including the expression andlor regulation
signals,
b) providing the 5' and the 3' end of the ORF, gene or
1p allele with nucleotide sequences of the target site,
c) preferably incorporating the nucleotide sequence of
the desired ORF, gene or allele provided with
nucleotide sequences of the target site into a vector
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which does not replicate or replicates to only a
limited extent in coryneform bacteria,
d) transferring the nucleotide sequence according to b)
or c) into coryneform bacteria, and
5 e) isolating coryneform bacteria in which the nucleotide
sequence according to a) is incorporated at the target
site, no nucleotide sequence which is capable
of/enables episomal replication in microorganisms, no
nucleotide sequence which is capable of/enables
10 transposition and no nucleotide sequence which imparts
resistance to antibiotics remaining at the target
site.
During work on the present invention, it was possible to
incorporate a second copy of an lysCFBR allele into the gluB
15 gene of Corynebacterium glutamicum such that no nucleotide
sequence which is capable oflenables episomal replication
in microorganisms, no nucleotide sequence which is capable
of/enables transposition and no nucleotide sequence which
imparts resistance to antibiotics remained at the gluB gene
20 site. This strain, which is called DSM13994g1u::lysC,
carries the lysCFBR allele lysC T311I at its natural lysC
site and a second copy of the lysCF$R allele lysC T311I at a
second site (target site), namely the gluB gene. A plasmid
with the aid of which the incorporation of the lysCFBR
25 allele into the gluB gene can be achieved is shown in
Figure 1. It carries the name pKl8mobsacBglul 1.
During work on the present invention, it was furthermore
possible to incorporate a copy of an lysCFBR allele into the
target site of the gluB gene of Corynebacterium glutamicum
30 such that no nucleotide sequence which is capable
of/enables episomal'replication in microorganisms, no
nucleotide sequence which is capable of/enables
transposition and no nucleotide sequence which imparts
resistance to antibiotics remained at the gluB gene site.
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This strain, which is called DSM12866g1u::lysC, carries the
wild-type form of the lysC gene at its natural lysC site
and a second copy of the lysC gene in the form of the
lysCFBR allele lysC T311I at a second site (target site),
namely the gluB gene. It has been deposited under number
DSM15039 at the Deutsche Sammlung fur Mikroorganismen and
Zellkulturen (German Collection of Microorganisms and Cell
Cultures). A plasmid with the aid of which the
incorporation of the lys~CFBR allele into the gluB gene can
be achieved is shown in Figure 1. It carries the name
pKl8mobsacBglul 1.
During work on the present invention, it was furthermore
possible to incorporate a copy of an lysCFBR allele into the
target site of the aecD gene of Corynebacterium glutamicum
such that no nucleotide sequence which is capable
of/enables episomal replication in microorganisms, no
nucleotide sequence which is capable of/enables
transposition and no nucleotide sequence which imparts
resistance to antibiotics remained at the aecD gene site.
This strain, which is called DSM12866aecD::lysC, carries
the wild-type form of the lysC gene at its natural lysC
site and a second copy of the lysC gene in the form of the
lysCFBR allele lysC T311I at a second site (target site),
namely the aecD gene. A plasmid with the aid of which the
incorporation of the lysCFBR allele into the aecD gene can
be achieved is shown in Figure 2. It carries the name
pKl8mobsacBaecD1 1.
During work on the present invention, it was furthermore
possible to incorporate a copy of an lySCF~R allele into the
target site of the pck gene of Corynebacterium glutamicum
such that no nucleotide sequence which is capable
of/enables episomal replication in microorganisms, no
nucleotide sequence which is capable of/enables
transposition and no nucleotide sequence which imparts
resistance to antibiotics remained at the pck gene site.
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This strain, which is called DSM12866pck::lysC, carries the
wild-type form of the lysC gene at its natural l.ysC site
and a second copy of the lysC gene in the form of the
lysCF$~ allele lysC T311I at a second site (target site),
namely the pck gene. A plasmid with the aid of which the
incorporation into the pck gene can be achieved is shown in
Figure 3. It carries the name pKl8mobsacBpckl 1.
During work on the present invention, it was furthermore
possible to incorporate a copy of the ddh gene into the
target site of the gluB gene of Corynebacterium glutamicum
such that no nucleotide sequence which is capable
of/enables episomal replication in microorganisms, no
nucleotide sequence which is capable of/enables
transposition and no nucleotide sequence which imparts
resistance to antibiotics remained at the gluB gene site.
This strain, which is called DSM12866g1u::ddh, carries a
copy of the ddh gene at its natural ddh site and a second
copy of the ddh gene at a second site (target site), namely
the gluB gene. A plasmid with the aid of which the
incorporation of the ddh gene into the gluB gene can be
achieved is shown in Figure 4. It carries the name
pKl8mobsacBgluB2 1.
During work on the present invention, it was furthermore
possible to incorporate a copy of the dapA gene into the
target site of the aecD gene of Corynebacterium glutamicum
such that no nucleotide sequence which is capable
of/enables episomal replication in microorganisms, no
nucleotide sequence which is capable of/enables
transposition and no nucleotide sequence which imparts
resistance to antibiotics remained at the aecD gene site.
This strain, which is called DSM12866aecD::dapA, carries a
copy of the dapA gene at its natural dapA site and a second
copy of the dapA gene at a second site (target site),
namely the aecD gene. A plasmid with the aid of which the
incorporation of the dapA gene into the aecD gene can be
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53
achieved is shown in Figure 5. It carries the name
pKl8mobsacBaecD2 1.
During work on the present invention, it was furthermore
possible to incorporate a copy of a pyc allele into the
target site of the pck gene of Corynebacterium glutamicum
such that no nucleotide sequence which is capable
of/enables episomal replication in microorganisms, no
nucleotide sequence which is capable of/enables
transposition and no nucleotide sequence which imparts
resistance to antibiotics remained at the pck gene site.
This strain, which is called DSM12866pck::pyc, carries a
copy of the wild-type form of the pyc gene at its natural
pyc site and a second copy of the pyc gene in the form of
the pyc allele pyc P458S at a second site (target site),
namely the pck gene. A plasmid with the aid of which the
incorporation of the pyc allele into the pck gene can be
achieved is shown in Figure 6. It carries the name
pKl8mobsacBpckl_3.
The coryneform bacteria produced according to the invention
can be cultured continuously or discontinuously in the
batch process (batch culture) or in the fed batch (feed
process) or repeated fed batch process (repetitive feed
process) for the purpose of production of chemical
compounds. A summary of known culture methods is described
in the textbook by Chmiel (Biopro~esstechnik 1. Einfiihrung
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 meet the requirements of
the particular strains in a suitable manner. Descriptions
of culture media for various microorganisms are contained
in the handbook "Manual of Methods for General
Bacteriology" of the American Society for Bacteriology
(Washington D.C., USA, 1981).
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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 or lactic 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 sulfate, ammonium chloride, ammonium
phosphate, ammonium carbonate and ammonium nitrate, can be
used as the source of nitrogen. The sources of nitrogen can
be used individually or as a mixture.
Phosphoric acid, potassium dihydrogen phosphate or
dipotassium hydrogen phosphate or the corresponding sodium-
containing salts can be used as the source of phosphorus.
The culture medium must furthermore comprise salts of
metals, such as e. g. magnesium sulfate or iron sulfate,
which are necessary for growth. Finally, essential growth
substances, such as amino acids and vitamins, can be
employed in addition to the above-mentioned substances.
Suitable precursors can moreover be added to the culture
medium. The starting substances mentioned can be added to
the culture in the form of a single batch, or can be fed in
during the culture in a suitable manner.
Basic compounds, such as sodium hydroxide, potassium
hydroxide, ammonia or aqueous ammonia, or acid compounds.
such as phosphoric acid or sulfuric acid, can be employed
in a suitable manner to control the pH of the culture.
An.tifoams, such as e.g. fatty acid polyglycol esters, can
be employed to control the development of foam. Suitable
substances having a selective action, such as e.g.
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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
5 culture is usually 20~C to 45~C, and preferably 25pC to
40°-C. Culturing is continued until a maximum of the desired
chemical compound has formed. This target is usually
reached within 10 hours to 160 hours.
It has been found that the coryneform bacteria according to
10 the invention, in particular the coryneform bacteria which
produce L-lysine, have an unexpectedly high stability. They
were stable for at least 10-20, 20-30, 30-40, 40-50,
preferably at least 50-60, 60-70, 70-80 and 80-90
generations or cell division cycles.
15 The following microorganisms have been deposited:
The strain Corynebacterium glutamicum DSM12866g1u::lysC was
deposited in the form of a pure culture on 5th June 2002
under number DSM15039 at the Deutsche Sammlung fu.r
Mikroorganismen and Zellkulturen (DSMZ = German Collection
20 of Microorganisms and Cell Cultures, Braunschweig, Germany)
in accordance with the Budapest Treaty.
The plasmid pKl8mobsacBglul 1 was deposited in the form of
a pure culture of the strain E. coli
DH5amcr/pKl8mobsacBglul 1 (_
25 DHSalphamcr/pKl.BmobsacBglul_1) on 20th April 2001 under
number DSM14243 at the Deutsche Sammlung fur
Mikroorganismen and Zellkulturen (DSMZ, Braunschweig,
Germany) in accordance with the Budapest Treaty.
The plasmid pKl8mobsacBaecD1_1 was deposited in the form of
30 a pure culture of the strain E. coli
DHSamcr/pKl8mobsacBaecD1_1 (_
DHSalphamcr/pKl8mobsacBaecD1 1) on 5th June 2002 under
number DSM15040 at the Deutsche Sammlung fur
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Mikroorganismen and Zellkulturen (DSMZ, Braunschweig,
Germany) in accordance with the Budapest Treaty.
Example 1
Incorporation of a second copy of the lysCFBR allele into
the chromosome of the strain DSM13994 and of the strain
DSM12866
The Corynebacterium glutamicum strain DSM13994 was produced
by multiple, non-directed mutagenesis, selection and mutant
selection from C. glutamicum ATCC13032. The strain is
resistant to the lysine analogue S-(2-aminoethyl)-L-
cysteine and has a feed back-resistant aspartate kinase
which is insensitive to inhibition by a mixture of lysine
and threonine (in each case 25 mM). The nucleotide sequence
of the lysCF$R allele of this strain is shown as SEQ ID
N0:3. It is also called lysC T311I in the following. The
amino acid sequence of the aspartate kinase protein coded
is shown as SEQ ID N0:4. A pure culture of this strain was
deposited on 16th January 2001 at the Deutsche Sammlung fur
Mikroorganismen and Zellkulturen (DSMZ = German Collection
of Microorganisms and Cell Cultures, Braunschweig, Germany)
in accordance with the Budapest Treaty.
The strain DSM12866 was produced from C. glutamicum
ATCC13032 by non-directed mutagenesis and selection of the
mutants with the best L-lysine accumulation. It is
methionine-sensitive. Growth on minimal medium comprising
L-methionine can be re-established by addition of
threonine. This strain has the wild-type form of the lysC
gene shown as SEQ ID N0:1. The corresponding amino acid
sequence of the wild-type aspartate kinase protein is shown
as SEQ ID N0:2. A pure culture of this strain was deposited
on 10th June 1999 at the Deutsche Sammlung fur
Mikroorganismen and Zellkulturen (DSMZ, Braunschweig,
Germany) in accordance with the Budapest Treaty.
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1.1 Isolation and sequencing of the DNA of the lysC
allele of strain DSM13994
From the strain DSM13994, chromosomal DNA is isolated by
the conventional methods (Eikmanns et al., Microbiology
140: 1817 - 1828 (1994)). With the aid of the polymerase
chain reaction, a DNA section which carries the lysC gene
or allele is amplified. On the basis of the sequence of the
lysC gene known for C. glutamicum (Kalinowski et al.,
Molecular Microbiology, 5 (5), 1197 - 1204 (1991);
Accession Number X57226), the following primer
oligonucleotides were chosen for the PCR:
lysClbeg (SEQ ID No: 5):
5~ TA(G GAT CC)T CCG GTG TCT GAC CAC GGT G 3~
lysC2end: (SEQ ID NO: 6):
5~ AC(G GAT CC)G CTG GGA AAT TGC GCT CTT CC 3~
The primers shown are synthesized by MWG Biotech and the
PCR reaction is carried out by the standard PCR method of
Innis et al. (PCR Protocols. A Guide to Methods and
Applications, 1990, Academic Press). The primers allow
amplification of a DNA section of approx. 1.7 kb in length,
which carries the lysC gene or allele. The primers moreover
contain the sequence for a cleavage site of the restriction
endonuclease BamHI, which is marked by parentheses in the
nucleotide sequence shown above.
The amplified DNA fragment of approx. 1.7 kb in length
which carries the lysC allele of the strain DSM13994 is
identified by electrophoresis in a 0.8~ agarose gel,
isolated from the gel and purified by conventional methods
(QIAquick Gel Extraction Kit, Qiagen, Hilden).
Ligation of the fragment is then carried out by means of
the Topo TA Cloning Kit (Invitrogen, Leek, The Netherlands,
Cat. Number K4600-01) in the vector pCRII-TOPO. The
ligation batch is transformed in the E. coli strain TOP10
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58
(Invitrogen, Leek, The Netherlands). Selection of plasmid-
carrying cells is made by plating out the transformation
batch on kanamycin (50 mg/1)-containing LB agar with X-Gal
(5-bromo-4-chloro-3-indolyl ~i-D-galactopyranoside,
64 mg/1) .
The plasmid obtained is checked by means of restriction
cleavage, after isolation of the DNA, and identified in
agarose gel. The resulting plasmid is called pCRIITOPOlysC.
The nucleotide sequence of the amplified DNA fragment or
PCR product is determined by the dideoxy chain termination
method of Sanger et al. (Proceedings of the National
Academy of Sciences USA, 74:5463-5467 (1977)) using the
"ABI Prism 377" sequencing apparatus of PE Applied
Biosystems (Weiterstadt, Germany). The sequence of the
coding region of the PCR product is shown in SEQ ID No:3.
The amino acid sequence of the associated aspartate kinase
protein is shown in SEQ ID N0:4.
The base thymine is found at position 932 of the nucleotide
sequence of the coding region of the lysCFBR allele of
strain DSM13994 (SEQ ID N0:3). The base cytosine is found
at the corresponding position of the wild-type gene (SEQ ID
N0:1) .
The amino acid isoleucine is found at position 311 of the
amino acid sequence of the aspartate kinase protein of
strain DSM13994 (SEQ ID No:4). The amino acid threonine is
found at the corresponding position of the wild-type
protein (SEQ ID No:2).
The lysC allele, which contains the base thymine at
position 932 of the coding region and accordingly codes for
an aspartate kinase protein which contains the amino acid
isoleucine at position 311 of the amino acid sequence, is
called the lysCFBR allele or lysC T311I.in the following.
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The plasmid pCRIITOPOlysC, which carries the lysCFBR allele
lysC T311I, was deposited in the form of a pure culture of
the strain E. coli TOP 10/pCRIITOPOlysC under number
DSM14242 on 20th April 2001 at the Deutsche Sammlung fur
Mikroorganismen and Zellkulturen (DSMZ, Braunschweig,
Germany) in accordance with the Budapest Treaty.
1.2 Construction of the replacement vector
pKl8mobsacBglul_1
The Cor'ynebacterium glutamicum strain ATCC13032 is used as
the donor for the chromosomal DNA. From the strain
ATCC13032, chromosomal DNA is isolated using the
conventional methods (Eikmanns et al., Microbiology 140:
1817 - 1828 (1994)). With the aid of the polymerase chain
reaction, a DNA fragment which carries the gluB gene and
surrounding regions is amplified. On the basis of the
sequence of the gluABCD gene cluster known for C.
glutamicum (Kronemeyer et al., Journal of Bacteriology,
177: 1152 - 1158 (1995)) (Accession Number X81191), the
following primer oligonucleotides are chosen for the PCR:
gluBgll (SEQ ID N0: 7):
5~ TA(A GAT CT)G TGT TGG ACG TCA TGG CAA G 3~
gluBgl2 (SEQ ID N0: 8):
5~ AC(A GAT CT)T GAA GCC AAG TAC GGC CAA G 3~
The primers shown are synthesized by MTnIG Biotech and the
PCR reaction is carried out by the standard PCR method of
Innis et al. (PCR Protocols. A Guide to Methods and
Applications, 1990, Academic Press). The primers allow
amplification of a DNA fragment of approx 1.7 kb in size,
which carries the gluB gene and surrounding regions. The
surrounding regions are a sequence section approx. 0.33 kb
in length upstream of the gluB gene, which represents the
3' end of the gluA gene, and a sequence section approx.
0.44 kb in length downstream of the gluB gene, which
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represents the 5' end of the gluC gene. The primers
moreover contain the sequence for the cleavage site of the
restriction endonuclease BgIII, which is marked by
parentheses in the nucleotide sequence shown above.
5 The amplified DNA fragment of approx. 1.7 kb in length
which carries the gluB gene and surrounding regions is
identified by means of electrophoresis in a 0.8~ agarose
gel and isolated from the gel and purified by conventional
methods (QIAquick Gel Extraction Kit, Qiagen, Hilden).
10 Ligation of the fragment is then carried out by means of
the TOPO TA Cloning Kit (Invitrogen, Leek, The Netherlands,
Cat. Number K4600-O1) in the vector pCRII-TOPO. The
ligation batch is transformed in the E. coli strain TOP10
(Invitrogen, Leek, The Netherlands). Selection of plasmid-
15 carrying cells is made by plating out the transformation
batch on kanamycin (50 mg/1)-containing LB agar with .X-Gal
(5-bromo-4-chloro-3-indolyl ~i-D-galactopyranoside,
64 mg/1).
The plasmid obtained is checked by means of restriction
20 cleavage, after isolation of the DNA, and identified in
agarose gel. The resulting plasmid is called pCRII-TOPOglu.
The plasmid pCRII-TOPOglu is cleaved with the restriction
enzyme BglII (Amersham-Pharmacia, Freiburg, Germany) and
after separation in an agarose gel (0.8~) with the aid of
25 the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany)
the gluB fragment of approx. 1.7 kb is isolated from the
agarose gel and employed for ligation with the mobilizable
cloning vector pKl8mobsacB described by Schafer et a1.
(Gene 14: 69-73 (1994)), This is cleaved beforehand with
30 the restriction enzyme BamHI and dephosphoryl.ated with
alkaline phosphatase (Alkaline Phosphatase, Boehringer
Mannheim), mixed with the gluB fragment of approx. 1.7 kb,
and the mixture is treated with T4 DNA Lipase (Amersham-
Pharmacia, Freiburg, Germany).
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The E. coli strain DH5a, (Grant et al.; Proceedings of the
National Academy of Sciences USA, 87 (1990) 4645-4649) is
then transformed with the ligation batch (Hanahan, In. DNA
Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold
Spring Harbor, New York, 1989). Selection of plasmid-
carrying cells~is made by plating out the transformation
batch on LB agar (Sambrook et al., Molecular Cloning: A
Laboratory Manual. 2nd Ed., Cold Spring Harbor, New York,
1989), which is supplemented with 50 mg/1 kanamycin.
Plasmid DNA is isolated from a transformant with the aid of
the QIAprep Spin Miniprep Kit from Qiagen and checked by
restriction cleavage and subsequent agarose gel
electrophoresis. The plasmid is called pKl8mobsacBglul.
Plasmid DNA was isolated from the strain DSM14242 (see
Example 1.1), which carries the plasmid pCRIITOPOlysC, and
cleaved with the restriction enzyme BamHI (Amersham-
Pharmacia, Freiburg, Germany), and after separation in an
agarose gel (0.8~) with the aid of the QIAquick Gel
Extraction Kit (Qiagen, Hilden, Germany) the lysCF$R-
containing DNA fragment of approx. 1.7 kb in length was
isolated from the agarose gel and employed for ligation
with the vector pKl8mobsacBglul described above. This is
cleaved beforehand with the restriction enzyme BamHI,
dephosphorylated with alkaline phosphatase (Alkaline
Phosphatase, Boehringer Mannheim, Germany), mixed with the
lysCF~R fragment of approx. 1.7 kb and the mixture is
treated with T4 DNA Ligase (Amersham-Pharmacia, Freiburg,
Germany).
The E. coli strain DH5amcr (Life Technologies GmbH,
Karlsruhe, Germany) is then transformed with the ligation
batch (Hanahan, In: DNA Cloning. A Practical Approach. Vol.
1, ILR-Press, Cold Spring Harbor, New York, 1989).
Selection of plasmid-carrying cells is made by plating out
the transformation batch on LB agar (Sambrook et al.,
Molecular Cloning: A Laboratflry Manual. 2nd Ed., Cold
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Spring Harbor, New York, 1989), which was supplemented with
50 mg/1 kanamycin.
Plasmid DNA is isolated from a transformant with the aid of
the QIAprep Spin Miniprep Kit from Qiagen and checked by
restriction cleavage and subsequent agarose gel
electrophoresis. The plasmid is called pKlBmobsacBglul 1. A
map of the plasmid is shown in Figure 1.
The plasmid pKl8mobsacBglul 1 was deposited in the form of
a pure culture of the strain E. coli
DHSamcr/pKl8mobsacBglul_1 (_
DH5alphamcr/pKl8mobsacBglul 1) under number DSM14243 on
20.04.2001 at the Deutsche Sammlung fur Mikroorganismen and
zellkulturen (DSMZ, Braunschweig, Germany) in accordance
with the Budapest Treaty.
1.3 Incorporation of a second copy of the lysCF$R allele
lysC T311I into the chromosome (target site: gluB
gene) of the strain DSM13994 by means of the
replacement vector pKl8mobsacBglul_1
The vector pKl8mobsacBglul 1 described in Example 1.2 is
transferred by the protocol of Schafer et al. (Journal of
Microbiology 172: 1663-1666 (1990)) into the C. glutamicum
strain DSM13994 by conjugation. The vector cannot replicate
independently in DSM13994 and is retained in the cell only
if it has integrated into the chromosome. Selection of
clones or transconjugants with integrated pKl8mobsacBglul 1
is made by plating out the conjugation batch on LB agar
(Sambrook et al., Molecular Cloning: A Laboratory Manual.
2nd Ed., Cold Spring Harbor, New York, 1989), which is
supplemented with 15 mg/1 kanamycin and 50 mg/1 nalidixic
acid. Kanamycin-resistant transconjugants are plated out on
LB agar plates with 25 mg/1 kanamycin and incubated for 48
hours at 33°C.
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For selection of mutants in which excision of the plasmid
has taken place as a consequence of a second recombination
event, the clones are cultured for 20 hours in LB liquid
medium and then plated out on LB agar with 10~ sucrose and
incubated for 48 hours.
The.plasmid pKl8mobsacBglul_1, like the starting plasmid
pKl8mobsacB, contains, in addition to the kanamycin
resistance gene, a copy of the sacB gene which codes for
levan sucrase from Bacillus subtilis. The expression which
can be induced by sucrose leads to the formation of levan
sucrase, which catalyses the synthesis of the product"
levan, which is toxic to C. glutamicum. Only those clones
in which the integrated pKlBmobsacBglul_1 has excised as
the consequence of a second recombination event therefore
grow on LB agar. Depending on the position of the second
recombination event, after the excision the second copy of
the lysCFBR allele manifests itself in the chromosome at the
gluB locus, or the original gluB locus of the host remains.
Approximately 40 to 50 colonies are tested for the
phenotype "growth in the presence of sucrose" and "non-
growth in the presence of kanamycin". Approximately 20
colonies which show the phenotype "growth in the presence
of sucrose" and "non-growth in the presence of kanamycin"
are investigated with the aid of the polymerase chain
reaction. A DNA fragment which carries the gluB gene and
surrounding regions is amplified here from the chromosomal
DNA of the colonies. The same primer oligonucleotides as
are described in Example 1.2 for the construction of the
integration plasmid are chosen for the PCR.
gluBgll (SEQ ID N0: 7):
5~ TA(A GAT CT)G TGT TGG ACG TCA TGG CAA G 3~
gluBgl2 (SEQ ID N0: 8):
5' AC(A GAT CT)T GAA GCC AAG TAC GGC CAA G 3~
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The primers allow amplification of a DNA fragment approx.
1.7 kb in size in control clones with the original gluB
locus. In clones with a second copy of the lysCFBR allele in
the chromosome at the gluB locus, DNA fragments with a size
of approx. 3.4 kb are amplified.
The amplified DNA fragments are identified by means of
electrophoresis in a 0.8o agarose gel.
A clone which, in addition to the copy present at the lysC
locus, has a second copy of the lysCF~ allele lysC T311I at
the gluB locus in the chromosome was identified in this
manner. This clone was called strain DSM13994g1u::lysC.
1.4 Incorporation of a second copy of the lysC gene in
the form of the lysCF$R allele lysC T311I into the
chromosome (target site: gluB gene) of the strain
DSM12866 by means of the replacement vector
pKl8mobsacBglul 1
As described in Example 1.3, the plasmid pKl8mobsacBglul 1
is transferred into the C. glutamicum strain DSM12866 by
conjugation. A clone which, in addition to the copy of the
wild-type gene present at the lysC locus, has a second copy
of the lysC gene in the form of the lysCFBR allele lysC
T311I at the gluB locus in the chromosome was identified in
the manner described in 1.3. This clone was called strain
DSM12866g1u::lysC.
The Corynebacterium glutamicum strain according to the
invention which carries a second copy of an lySCF$R allele
in the gluB gene was deposited in the form of a pure
culture of the strain Corynebacterium glutamicum
DSM12866g1u::lysC on 5th June 2002 under number DSM15039 at
the Deutsche Sammlung fur Mikroorganismen and Zellkulturen
(DSMZ, Braunschweig, Germany) in accordance with the
Budapest Treaty.
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1.5 'Construction of the replacement vector
pKl8mobsacBpckl_1
The Corynebacterium glutamicum strain ATCC13032 is used as
the donor for the chromosomal DNA. From the strain
5 ATCC13032, chromosomal DNA is isolated using the
conventional methods (Eikmanns et al., Microbiology 140:
1817 - 1828 (1994)). With the aid of the polymerase chain
reaction, a DNA fragment which carries the pck gene and
surrounding regions is amplified. On the basis of the
10 sequence of the pck gene known for C. glutamicum (EP1094111
and Riedel et al., Journal of Molecular and Microbiological
Biotechnology 3:573-583 (2001)) (Accession Number
AJ269506), the following primer oligonucleotides are chosen
for the PCR:
15 pck_beg (SEQ ID N0: 9):
5~ TA(A GAT~CT) G CCG GCA TGA CTT CAG TTT 3~
pck_end (SEQ ID N0: 10):
5~ AC(A GAT CT) G GTG GGA GCC TTT CTT GTT ATT3
The primers shown are synthesized by MWG Biotech and the
20 PCR reaction is carried out by the standard PCR method of
Innis et al. (PCR Protocols. A Guide to Methods and
Applications, 1990, Academic Press). The primers allow
amplification of a DNA fragment of approx.2.9 kb in size,
which carries the pck gene and adjacent regions. The
25 primers moreover contain the sequence for the cleavage site
of the restriction endonuclease BglII, which is marked by
parentheses in the nucleotide sequence shown above.
The amplified DNA fragment of approx. 2.9 kb in length
which carries the pck gene and surrounding regions is
30 identified by means of electrophoresis in a 0.8o agarose
gel and isolated from the gel and purified by conventional
methods (QIAquick Gel Extraction Kit, Qiagen, Hilden).
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Ligation of the fragment is then carried out by means of
the TOPO TA Cloning Kit (Invitrogen, Leek, The Netherlands,
Cat. Number K4600-01) in the vector pCRII-TOPO. The
ligation batch is transformed in the E. coli strain TOP10
(Invitrogen, Leek, The Netherlands). Selection of plasmid-
carrying cells is made by plating out the transformation
batch on kanamycin (50 mg/1)-containing LB agar with X-Gal
(64 mg/1).
The plasmid obtained is checked by means of restriction
cleavage, after isolation of the DNA, and identified in
agarose gel. The resulting plasmid is called pCRII-TOPOpck.
The plasmid pCRII-TOPOpck is cleaved with the restriction
enzyme BglII (Amersham-Pharmacia, Freiburg, Germany) and
after separation in an agarose gel (0.8~) with the aid of
the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany)
the pck fragment of approx. 2.9 kb is isolated from the
agarose gel and employed for ligation with the mobilizable
cloning vector pKlBmobsacB described by Schafer et al.
(Gene 14: 69-73 (1994)). This is cleaved beforehand with
the restriction enzyme BamHI and dephosphorylated with
alkaline phosphatase (Alkaline Phosphatase, Boehringer
Mannheim), mixed with the pck fragment of approx. 2.9 kb,
and the mixture is treated with T4 DNA Ligase (Amersham-
Pharmacia, Freiburg, Germany).
The E. coli Strain DHSo~ (Grant et al.~ Proceedings of the
National Academy of Sciences USA, 87 (1990) 4645-4649) is
then transformed with the ligation batch (Hanahan, In. DNA
Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold
Spring Harbor, New York, 1989) Selection of plasmid-
carrying cells is made by plating out the transformation
batch on LB agar (Sambrook et al., Molecular Cloning: A
Laboratory Manual. 2nd Ed., Cold Spring Harbor, New York,
1989), which is supplemented with 50 mg/1 kanamycin.
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Plasmid DNA is isolated from a transformant with the aid of
the QIAprep Spin Miniprep Kit from Qiagen and checked by
restriction cleavage and subsequent agarose gel
electrophoresis. The plasmid is called pKl8mobsacBpckl.
Plasmid DNA was isolated from the strain DSM14242 (see
Example 1.1), which carries the plasmid pCRIITOP0IysC, and
cleaved with the restriction enzyme BamHI (Amersham-
Pharmacia, Freiburg, Germany), and after separation in an
agarose gel (0.8o) with the aid of the QIAquick Gel
Extraction Kit (Qiagen, Hilden, Germany) the lysCF$R-
containing DNA fragment approx. 1.7 kb long was isolated
from the agarose gel and employed for ligation with the
vector pKl8mobsacBpckl described above. This is cleaved
beforehand with the restriction enzyme BamHI,
15~ dephosphorylated with alkaline phosphatase (Alkaline
Phosphatase, Boehringer Mannheim, Germany), mixed with the
lysCFBR fragment of approx. 1.7 kb and the mixture is
treated with T4 DNA Lipase (Amersham-Pharmacia, Freiburg,
Germany).
The E. coli strain DH5amcr (Life Technologies GmbH,
Karlsruhe, Germany) is then transformed with the ligation
batch (Hanahan, In: DNA Cloning. A Practical Approach. Vol.
1, ILR-Press, Cold Spring Harbor, New York, 1989).
Selection of plasmid-carrying cells is made by plating out
the transformation batch on LB agar (Sambrook et al.,
Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold
Spring Harbor, New York, 1989), which was supplemented with
50 mg/1 kanamycin.
Plasmid DNA is isolated from a transformant with the aid of
the QIAprep Spin Miniprep Kit from Qiagen and checked by
restriction cleavage and subsequent agarose gel
electrophoresis. The plasmid is called pKl8mobdsacBpckl 1.
A map of the plasmid is shown in Figure 3.
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1.6 Incorporation of a second copy of the lysC gene in
the form of the lysCF$R allele lysC T311I into the
chromosome (target site: pck gene) of the strain
DSM12866 by means of the replacement vector
pKl8mobsacBpckl_1
As described in Example 1.3, the plasmid pKl8mobsacBpckl_1.
described in Example 1.5 is transferred into the C.
glutamicum strain DSM12866 by conjugation. Selection is
made for targeted recombination events in the chromosome of
C. glutamicum DSM12866 as described in Example 1.3.
Depending on the position of the second recombination
event, after the excision the second copy of the lysCFBR
allele manifests itself in the chromosome at the pck locus,
or the original pck locus of the host remains.
Approximately 40 to 50 colonies are tested for the
phenotype "growth in the presence of sucrose" and "non-
growth in the presence of kanamycin". Approximately 20
colonies which show the phenotype "growth in the presence
of sucrose" and "non-growth in the presence of kanamycin"
are investigated with the aid of the polymerase chain
reaction. A DNA fragment which carries the pck gene and
surrounding regions is amplified here from the chromosomal
DNA of the colonies. The same primer oligonucleotides as
are described in Example 1.5 for the construction of the
integration plasmid are chosen for the PCR.
pck beg (SEQ ID N0: 9):
5~ TA(A GAT CT) G CCG GCA TGA CTT CAG TTT 3~
pck_end (SEQ ID NO: 10):
5~ AC(A GAT CT) G GTG GGA GCC TTT CTT GTT ATT3
The primers allow amplification of a DNA fragment approx.
2.9 kb in size in control clones with the original pck
locus. In clones with a second copy of the lysCFBR allele in
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the chromosome at the pck locus, DNA fragments with a size
of approx. 4.6 kb are amplified.
The amplified DNA fragments are identified by means of
electrophoresis in a 0.8~ agarose gel.
A clone which, in addition to the copy of the wild-type
gene present at the lysC locus, has a second copy of the
lysC gene in the form of the lysCF$R allele lysC T311I at
the pck locus in the chromosome was identified in this
manner. This clone was called strain DSM12866pck::lysC.
1.7 Construction of the replacement vector
pKlBmobsacBaecD1 1
The Corynebacterium glutamicum strain ATCC13032 is used as
the donor for the chromosomal DNA. From the strain
ATCC13032, chromosomal DNA is isolated using the
conventional methods (Eikmanns et al., Microbiology 140:
1817 - 1828 (1994)). With the aid of the polymerase chain
reaction, a DNA fragment which carries the aecD gene and
surrounding regions is amplified. On the basis of the
sequence of the aecD gene known for C. glutamicum (Rossol
et al., Journal of Bacteriology 174:2968-2977 (1992))
(Accession Number M89931), the following primer
oligonucleotides are chosen for the PCR:
aecD beg (SEQ ID NO: 11):
5~ GAA CTT ACG CCA AGC TGT TC 3~
aecD_end (SEQ ID NO: 12):
5~ AGC ACC ACA ATC AAC GTG AG 3~
The primers shown are synthesized by MWG Biotech and the
PCR reaction is carried out by the standard PCR method of
Innis et a1. (PCR Protocols. A Guide to Methods and
Applications, 1990, Academic Press). The primers allow
amplification of a DNA fragment of approx 2.1 kb in size,
which carries the aecD gene and adjacent regions.
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The amplified DNA fragment of approx. 2.1 kb in length is
identified by means of electrophoresis in a 0.8~ agarose
gel and isolated from the gel and purified by conventional
methods (QIAquick Gel Extraction Kit, Qiagen, Hilden).
5 The DNA fragment purified is cleaved with the restriction
enzyme BamHI and EcoRV (Amersham Pharmacia, Freiburg,
Germany). The ligation of the fragment in the vector pUCl8
then takes place (Norrander et al., Gene 26:101-106
(1983)). This is cleaved beforehand with the restriction
10 enzymes BglII and SmaI, dephosphorylated, mixed with the
aecD-carrying fragment of approx. 1.5 kb, and the mixture
is treated with T4 DNA Lipase (Amersham-Pharmacia,
Freiburg, Germany). The ligation batch is transformed in
the E. coli strain TOP10 (Invitrogen, Leek, The
15 Netherlands). Selection of plasmid-carrying cells is made
by plating out the transformation batch on kanamycin
(50 mg/1)-containing LB agar with X-Gal (64 mg/1).
The plasmid obtained is checked by means of restriction
cleavage, after isolation of the DNA, and identified in
20 agarose gel. The resulting plasmid is called pUCl8aecD.
Plasmid DNA was isolated from the strain DSM14242 (see
Example 1.1) which carries the plasmid pCRIiTOPOlysC and
cleaved with the restriction enzyme BamHI (Amersham-
Pharmacia, Freiburg, Germany) and then treated with Klenow
25 polymerase. After separation in an agarose gel (0.8~) with
the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden,
Germany) the lysCF$R-containing DNA fragment approx. 1.7 kb
in length is isolated from the agarose gel and employed for
ligation with the vector pUCl8aecD described above. This is
30 cleaved beforehand with the restriction enzyme StuI,
dephosphorylated with alkaline phosphatase (Alkaline
Phosphatase, Boehringer Mannheim, Germany), mixed with the
lysCFBR fragment of approx. 1.7 kb and the mixture is
treated with T4 DNA Lipase (Amersham-Pharmacia, Freiburg,
35 Germany).
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The E. coli strain DH5amcr (Life Technologies GmbH,
Karlsruhe, Germany) is then transformed with the ligation
batch (Hanahan, In: DNA Cloning. A Practical Approach. Vol.
1, ILR-Press, Cold Spring Harbor, New York, 1989).
Selection of plasmid-carrying cells is made by plating out
the transformation batch on LB agar (Sambrook et al.,
Molecular Cloning: A Laboratory Manual. 2na Ed., Cold
Spring Harbor, New York, 1989), which was supplemented with
50 mg/1 kanamycin.
Plasmid DNA is isolated from a transformant with the aid of
the QIAprep Spin Miniprep Kit from Qiagen and checked by
restriction cleavage and subsequent agarose gel
electrophoresis. The plasmid is called pUCl8aecDl.
The plasmid pUCl8aecD1 is cleaved with the restriction
enzyme Kpnl and then treated with Klenow polymerase. The
plasmid is then cleaved with the restriction enzyme Sall
(Amersham-Pharmacia, Freiburg, Germany) and after
separation in an agarose gel (0.8~) with the aid of the
QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) the
fragment of approx. 3.2 kb which carries aecD and lysC is
isolated from the agarose gel and employed for ligation
with the mobilizable cloning vector pKl8mobsacB described
by Schafex et al. (Gene 14: &9-73 (1994)). This is cleaved
beforehand with the restriction enzymes Smal and Sall and
dephosphorylated with alkaline phosphatase (Alkaline
Phosphatase, Boehringer Mannheim), mixed with the fragment
of approx. 3.2 kb which carries aecD and lysC, and the
mixture is treated with T4 DNA Ligase (Amersham-Pharmacia,
Freiburg, Germany).
The E. coli strain DHSa (Grant et al.; Proceedings of the
National Academy of Sciences USA, 87 (1990) 4645-4649) is
then transformed with the ligation batch (Hanahan, In. DNA
Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold
Spring Harbor, New York, 1989). Selection of plasmid-
carrying cells is made by plating out the transformation
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batch on LB agar (Sambrook et al., Molecular Cloning: A
Laboratory Manual. 2nd Ed., Cold Spring Harbor, New York,
1989), which is supplemented with 50 mg/1 kanamycin.
Plasmid DNA is isolated from a transformant with the aid of
the QIAprep Spin Miniprep Kit from Qiagen and checked by
restriction cleavage and subsequent agarose gel
electrophoresis. The plasmid is called pKl8mobsacBaecD1_1.
A map of the plasmid is shown in Figure 2.
The plasmid pKl8mobsacBaecD1 1 was deposited in the form of
a pure culture of the strain E. coli
DHSamcr/pKl8mobsacBaecD1_1 (_
DHSalphamcr/pKl8mobsacBaecD1 1) on 5th June 2002 under
number DSM15040 at the Deutsche Sammlung fur
Mikroorganismen and Zellkulturen (DSMZ, Braunschweig,
Germany) in accordance with the Budapest Treaty.
1.8 Incorporation of a second copy of the lysC gene as
the lysCFBR allele into the chromosome (target site:
aecD gene) of the strain DSM12866 by means of the
replacement vector pKl8mobsacBaecD1_1
As described in Example 1.3, the plasmid pKl8mobsacBaecD1 1
described in Example 1.4 is transferred into the C.
glutamicum strain DSM12866 by conjugation. Selection is
made for targeted recombination events in the chromosome of
C, glutamicum DSM12866 as described in Example 1.3.
Depending on the position of the second recombination
event, after the excision the second copy of the lysCFBR
allele manifests itself in the chromosome at the aecD
locus, or the original aecD locus of the host remains.
Approximately 40 to 50 colonies are tested for the
phenotype "growth in the presence of sucrose" and "non-
growth in the presence of kanamycin". Approximately 20
colonies which show the phenotype "growth in the presence
of sucrose" and "non-growth in the presence of kanamycin"
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are investigated with the aid of the polymerase chain
reaction. A DNA fragment which carries the aecD gene and
surrounding regions is amplified here from the chromosomal
DNA of the colonies. The same primer oligonucleotides as
are described in Example 1.7 for the construction of the
integration plasmid are chosen for the PCR.
aecD beg (SEQ ID N0: 11):
5~ GAA CTT ACG CCA AGC TGT TC 3~
aecD_end (SEQ ID N0: 12):
5~ AGC ACC ACA ATC AAC GTG AG 3~
The primers allow amplification of a DNA fragment approx.
2.1 kb in size in control clones with the original aecD
locus. In clones with a second copy of the IySCFBR allele in
the chromosome at the aecD locus, DNA fragments with a size
of approx. 3..8 kb are amplified.
The amplified DNA fragments are identified by means of
electrophoresis in a 0.8o agarose gel.
A clone which, in addition to the copy of the wild-type
gene present at the lysC locus, has a second copy of the
lysC gene in the form of the lysCFBR allele lysC T311I at
the aecD locus in the chromosome was identified in this
manner. This clone was called strain DSM12866aecD::lysC.
Example 2
Incorporation of a second copy of the ddh gene into the
chromosome (target site: gluB gene) of the strain DSM12866
2.1 Construction of the replacement vector
pKl8mobsacBglu2 1
The Corynebacterium glutamicum strain ATCC13032 is used as
the donor for the chromosomal DNA. From the strain
ATCC13032, chromosomal DNA is isolated using the
conventional methods (Eikmanns et al., Microbiology 140:
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1817 - 1828 (1994)). With the aid of the polymerase chain
reaction, a DNA fragment which carries the gluB gene and
surrounding regions is amplified. On the basis of the
sequence of the gluABCD gene cluster known for C.
glutamicum (Kronemeyer et al., Journal of Bacteriology,
177: 1152 - 1158 (1995); EP1108790) (Accession Number
X81191 and AX127149), the following primer oligonucleotides
are chosen for the PCR:
gluA_beg (SEQ ID NO: 13):
5~ CAC GGT TGC TCA TTG TAT CC 3~
gluD end (SEQ ID NO: 14):
5~ CGA GGC GAA TCA GAC TTC TT 3~
The primers shown are synthesized by MWG Biotech and the
PCR reaction is carried out by the standard PCR method of
25 Innis et al. (PCR Protocols. A Guide to Methods and
Applications, 1990, Academic Press). The primers allow
amplification of a DNA fragment of approx 4.4 kb in size,
which carries the gluB gene and surrounding regions.
The amplified DNA fragment is identified by means of
electrophoresis in a 0.8~ agarose gel and isolated from the
gel and purified by conventional methods (QIAquick Gel
Extraction Kit, Qiagen, Hilden).
Ligation of the fragment is then carried out by means of
the TOPO TA Cloning Kit (Invitrogen, Leek, The Netherlands,
Cat. Number K4600-01) in the vector pCRII-TOPO. The
ligation batch is transformed in the E. eoli strain TOP10
(Invitrogen, Leek, The Netherlands). Selection of plasmid-
carrying cells is made by plating out the transformation
batch on kanamycin (50 mg/1)-containing LB agar with X-Gal
(64 mg/1) .
The plasmid obtained is checked by means of restriction
cleavage, after isolation of the DNA, and identified in
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agarose gel. The resulting plasmid is called pCRII-
TOPOglu2.
The plasmid pCP.II-TOPOglu2 is cleaved with the restriction
enzymes EcoRI and Sall (Amersham-Pharmacia, Freiburg,
5 Germany) and after separation in an agarose gel (0.80) with
the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden,
Germany) the gluB fragment of approx. 3.7 kb is isolated
from the agarose gel and employed for ligation with the
mobilizable cloning vector pKl8mobsacB described by Schafer
10 et al. (Gene 14, 69-73 (1994)). This is cleaved beforehand
with the restriction enzymes EcoRI and SalI and
dephosphorylated with alkaline phosphatase (Alkaline
Phosphatase, Boehringer Mannheim), mixed with the gluB
fragment of approx. 3.7 kb, and the mixture is treated with
15 T4 DNA Ligase (Amersham-Pharmacia, Freiburg, Germany).
The E. coli Strain DH5a, (Grant et al.; Proceedings of the
National Academy of Sciences USA, 87 (1990) 4645-4649) is
then transformed with the ligation batch (Hanahan, In. DNA
Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold
20 Spring Harbor, New York, 1989). Selection of plasmid-
carrying cells is made by plating out the transformation
batch on LB agar (Sambrook et al., Molecular Cloning: A
Laboratory Manual. 2nd Ed., Cold Spring Harbor, New York,
1989), which is supplemented with 50 mg/1 kanamycin.
25 Plasmid DNA is isolated from a transformant with the aid of
the QIAprep Spin Miniprep Kit from Qiagen and checked by
restriction cleavage and subsequent agarose gel
electrophoresis. The plasmid is called pKl8mobsacBglu2.
As described in Example 2.1, a DNA fragment which carries
30 the ddh gene and surrounding regions is also amplified with
the aid of the polymerase chain reaction. On the basis of
the sequence of the ddh gene cluster known for C.
glutamicum (Ishino et al., Nucleic Acids Research 15,
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3917(1987)) (Accession Number Y00151), the following primer
oligonucleotides are chosen for the PCR:
ddh beg ( SEQ ID NO : 15 )
5' CTG AAT CAA AGG CGG ACA TG 3'
ddh_end (SEQ ID NO: 16):
5' TCG AGC TAA ATT AGA CGT CG 3'
The primers shown are synthesized by MWG Biotech and the
PCR reaction is carried out by the standard PCR method of
Innis et al. (PCR Protocols. A Guide to Methods and
Applications, 1990, Academic Press). The primers allow
amplification of a DNA fragment of approx 1.6 kb in size,
which carries the ddh gene.
The amplified DNA fragment of approx. 1.6 kb in length,
which the ddh gene, is identified by means of
electrophoresis in a 0.8o agarose gel and isolated from the
gel and purified by conventional methods (QIAquick Gel
Extraction Kit, Qiageri, Hilden).
After purification, the fragment carrying the ddh gene is
employed for ligation in the vector pKl8mobsacBglu~
described. This is partly cleaved beforehand with the
restriction enzyme BamHI. By treatment of the vector with a
Klenow polymerise (Amersham-Pharmacia, Freiburg, Germany),
the overhangs of the cleaved ends are completed to blunt
ends, the vector is then mixed with the DNA fragment of
approx. 1.6 kb which carries the ddh gene and the mixture
is treated with T4 DNA lipase (Amersham-Pharmacia,
Freiburg, Germany). By using Vent Polymerise (New England
Biolabs, Frankfurt, Germany) for the PCR reaction, a ddh-
carrying DNA fragment which has blunt ends and is suitable
for ligation in the pretreated vector pKl8mobsacBglu2 is
generated.
The E. coli strain DH5amcr (Life Technologies GmbH,
Karlsruhe, Germany) is then transformed with the ligation
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batch (Hanahan, In: DNA Cloning. A Practical Approach. Vol.
1, ILR-Press, Cold Spring Harbor, New York, 1989).
Selection of plasmid-carrying cells is made by plating out
the transformation batch on LB agar (Sambrook et al.~,
Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold
Spring Harbor, New York, 1989), which was supplemented with
50 mg/1 kanamycin. '
Plasmid DNA is isolated from a transformant with the aid of
the QIAprep Spin Miniprep Kit from Qiagen and checked by
restriction cleavage and subsequent agarose gel
electrophoresis. The plasmid is called pKl8mobsacBglu2 1. A
map of the plasmid is shown in Figure 4.
2.2 Incorporation of a second copy of the ddh gene into
the chromosome (target site: gluB gene) of the strain
DSM12866 by means of the replacement vector
pKlBmobsacBglu2 1
As described ir? Example 1.3, the plasmid pKl8mobsacBglu2_1
described in Example 2.1 is transferred into the C.
glutamicum strain DSM12866 by conjugation. Selection is
made for targeted recombination events in the chromosome of
C. glutamicum DSM12866 as described in Example 1.3.
Depending on the position of the second recombination
event, after the excision the second copy of the ddh gene
manifests itself in the chromosome at the gluB locus, or
the original gluB locus of the host remains.
Approximately 40 to 50 colonies are tested for the
phenotype "growth in the presence of sucrose" and "non-
growth in the presence of kanamycin". Approximately 20
colonies which show the phenotype "growth in the presence
of sucrose" and "non-growth in the presence of kanamycin"
are investigav~ed with the aid of the polymerase chain
reaction. A DPdA fragment which carries the glu region
described is amplified here from the chromosomal DNA of the
colonies. The same primer oligonucheotides as are described
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in Example 2.1 for the construction of the replacement
plasmid are chosen for the PCR.
gluA beg ( SEQ TD NO : 13 )
5~ CAC GGT TGC TCA TTG TAT CC 3~
gluD end (SEQ ID N0: 14):
5~ CGA GGC GAA TCA GAC TTC TT 3~
The primers allow amplification of a DNA fragment approx.
4.4 kb in sire in control clones with the original glu
locus. In clonE~s with a second copy of the ddh gene in the
chromosome at the gluB locus, DNA fragments with a size of
approx. 6 kb are amplified.
The amplified DNA fragments are identified by means of
electrophoresis in a 0.8o agarose gel.
A clone which, in addition to the copy present at the ddh
locus, has a sncon.d copy of the ddh gene at the gluB locus
in the chromosome was identified in this manner. This clone
was called strain DSM12866g1u::ddh.
Example 3
Incorporation of a second copy of the dapA gene into the
chromosome (target site: aecD gene) of the strain DSM12866
3.1 Construction of the replacement vector
pKl8mobsacBaecD2_1
The Corynebacterium glutamicum strain ATCC13032 is used as
the donor for the chromosomal DNA. From the strain
ATCC13032, chromosomal DNA is isolated using the
conventional methods (Eikmanns et al., Microbiology 140:
1817 - 1828 (1994)). With the aid of the polymerase chain
reaction, a DNA fragment which carries the aecD gene and
surrounding regions is amplified. On the basis of the
sequence of the aecD gene known for C. glutamicum (Rossol
et al., Journal of Bacteriology 174:2968-2977 (1992))
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(Accession Number M89931), the following primer
oligonucleotides are chosen for the PCR:
aecD beg (SEQ TD NO: 11):
5~ GAA CTT ACG CCA AGC TGT TC 3~
aecD_end (SEA ID NO: 12):
5~ AGC ACC ACA ATC AAC GTG AG 3~
The primers shown are synthesized by MWG Biotech and the
PCR reaction is carried out by the standard PCR method of
Innis et al. (pCR Protocols. A Guide to Methods and
Applications, 1990, Academic Press). The primers allow
amplification of a DNA fragment of approx 2.1 kb in size,
which carries the aecD gene and adjacent regions.
The amplified DNA fragment of approx. 2.1 kb in length is
identified by means of electrophoresis in a 0.8~ agarose
gel and isolated from the gel and purified by conventional
methods (QIAquvck Gel Extraction Kit, Qiagen, Hilden).
The DNA fragment purified is cleaved with the restriction
enzyme BglII and EcoRV.(Amersham Pharmacia, Freiburg,
Germany). The ligation of the fragment in the vector pUCl8
then takes place (Norrander et al., Gene 26:101-106
(1983)). This is cleaved beforehand with the restriction
enzymes BamHI and Smal and dephosphorylated, mixed with the
aecD-carrying fragment of approx. 1.5 kb, and the mixture
is treated with T4 DNA Ligase (Amersham-Pharmacia,
Freiburg, Germany). The ligation batch is transformed in
the E. coli strain TOP10 (Invitrogen, Leek, The
Netherlands). Selection of plasmid-carrying cells is made
by plating out the transformation batch on kanamycin
(50 mg/1)-containing LB agar with X-Gal (64 mg/1).
The plasmid obtained is checked by means of restriction
cleavage, after isolation of the DNA, and identified in
agarose gel. The resulting plasmid is called pUCl8aecD.
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With the aid of the polymerase chain reaction, a further
DNA fragment which carries the dapA gene and surrounding
regions is amplified. On the basis of the sequence of the
dapA gene kno~,rur~ for C. glutamicum (Bonassi et al., Nucleic
5 Acids Research 18:6421 (1990)) (Accession Number X53993 and
AX127149), the following primer oligonucleotides are chosen
for the PCR:
dapA beg (SEQ ID N0: 17):
5~ CGA GCC AGT GAA CAT GCA GA 3~
10 dapA end (SEQ ID NO: 18):
5~ CTT GAG CAC CTT GCG CAG CA 3~
The primers shown are synthesized by MWG Biotech and the
PCR reaction is carried out by the standard PCR method of
Innis et al. (PCR Protocols. A Guide to Methods and
15 Applications, 1990, Academic Press). The primers allow
amplification of a DNA fragment of approx. 1.4 kb~in size,
which carries the dapA gene and adjacent regions.
The amplified DNA fragment of approx. 1.4 kb in length is
identified by means of electrophoresis in a 0.8~ agarose
20 gel and isolated from the gel and purified by conventional
methods (QIAquick Gel Extraction Kit, Qiagen, Hilden).
After purification, the dapA-containing DNA fragment
approx. 1.4 kb in length is employed for ligation with the
vector pUCl8aecD described above. This is cleaved
25 beforehand with the restriction enzyme StuI, mixed with the
DNA fragment of approx. 1.4 kb, and the mixture is treated
with T4 DNA Lipase (Amersham-pharmacia, Freiburg, Germany).
The E. coli strain DH5amcr (Life Technologies GmbH,
Karlsruhe, Germany) is then transformed with the ligation
30 batch (Hanahan, In: DNA Cloning. A Practical Approach. Vol.
1, ILR-Press, Cold Spring Harbor, New York, 1989).
Selection of plasmid-carrying cells is made by plating out
the transformation batch on LB agar (Sambrook et al.,
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Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold
Spring Harbor, New York, 1989), which was supplemented with
50 mg/1 kanamycin.
Plasmid DNA is isolated from a transformant with the aid of
the QIAprep Spin Miniprep Kit from Qiagen and checked by
restriction cleavage and subsequent agarose gel
electrophoresis. The plasmid is called pUCl8aecD2.
The plasmid pUClBaecD2 is cleaved with the restriction
enzyme Sall arid partly with EcoRI (Amersham-Pharmacies,
Freiburg, Germany) and of ter separation in an agarose gel
(0.8~) with the aid of the QIAquick Gel Extraction Kit
(Qiagen, Hilden, Germany) the fragment of approx. ~.7 kb
which carries aecD and dapA is isolated from the agarose
gel and employed for ligation with the mobilizable cloning
vector pKlBmobsacB described by Schafer et al. (Gene 14:
69-73 (1994).). This is cleaved beforehand with the
restriction enzymes EcoRI and with SalI and.
dephosphorylated with alkaline phosphatase (Alkaline
Phosphatase, Boehringer Mannheim), mixed with the fragment
of approx. 2.7 kb which carries aecD and dapA, and the
mixture is treated with T4 DNA Lipase (Amersham-Pharmacies,
Freiburg, Germany).
The E. coli strain DHSa (Grant et al.; Proceedings of the
National Acaderly of Sciences USA, 87 (1990) 4f45-4649) is
then transformed with the ligation batch (Hanahan, In. DNA
Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold
Spring Harbor, New York, 1989). Selection of plasmid-
carrying cells is made by plating out the transformation
batch on LB agar (Sambrook et al., Molecular Cloning: A
Laboratory Manual. 2nd Ed., Cold Spring Harbor, New York,
1989), which is supplemented with 50 mg/1 kanamycin.
Plasmid DNA is isolated from a transformant with the aid of
the QIAprep.Spin Miniprep Kit from Qiagen and checked by
restriction cleavage and subsequent agarose gel
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electrophoresis. The plasmid is called pKl8mobsacBaecD2_1.
A map of the plasmid is shown in Figure 5.
3.2 Incorpnwation of a second copy of the dapA gene into
the chromosome (target site: aecD gene) of the strain
DSM12866 by means of the replacement vector
pKl8mobsacBaecD2 1
As described in Example 1.3, the plasmid pKl8mobsacBaecD2 1
described in Example 3.1 is transferred into the C.
glutamicum strain DSM12866 by conjugation. Selection is
made for targeted recombination events in the chromosome of
C. glutamicum DSM12866 as described in Example 1.3.
Depending on the position of the second recombination
event, after the excision the second copy of the dapA gene
manifests itself in the chromosome at the aecD locus, or
the original aeeD locus of the host remains.
Approximately 40 to 50 colonies are tested for the
phenotype "growth in the presence of sucrose" and "non-
growth in the presence of kanamycin". Approximately 20
colonies which show the phenotype "growth in the presence
of sucrose" and "non-growth in the presence of kanamycin"
are investigated with the aid of the polymerase chain
reaction. A DNA fragment which carries the aecD gene and
surrounding regions is amplified here from the chromosomal
DNA of the colonies. The same primer oligonucleotides as
are described in. Example 3.1 for the construction of the
integration plasmid are chosen for the PCR.
aecD beg (SEQ ID N0: 11):
5~ GAA CTT ACG CCA AGC TGT TC 3~
aecD end (SEQ ID N0: 12):
5~ AGC ACC ACA ATC AAC GTG AG 3~
The primers allow amplification of a DNA fragment approx.
2.1 kb in size in control clones with the original aecD
locus. In clones ~~Vlth a second copy of the dapA gene in the
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chromosome at the aecD locus, DNA fragments with a size of
approx. 3.6 kb are amplified.
The amplified DNA fragments are identified by means of
electrophoresis in a 0.8~ agarose gel.
A clone which, in addition to the copy present at the dapA
locus, has a second copy of the dapA gene at the aecD locus
in the chromosome was identified in this manner. This clone
was called strain DSM12866aecD::dapA.
Example 4
Incorporation of a second copy of the pyc gene in the form
of the pyc allele pycP458S into the chromosome (target
site: pck gene) of the strain DSM12866
4.1 Construction of the replacement vector
pKl8mobsacBpckl_3
The replacement vector pKlBmobsacBpckl described in
Example 1.5 is used as the base vector for insertion of the
pyc allele.
As described in Example 2.1, a DNA fragment which carries
the pyc gene and surrounding regions is also amplified with
the aid of the polymerase chain reaction. On the basis of
the sequence of the pyc gene cluster known for C.
glutamicum (Peters-Wendisch et al., Journal of Microbiology
144: 915-927 (1998)) (Accession Number Y09548), the
following prirner oligonucleotides are chosen for the PCR:
pyc beg (SEQ ID NO: 19):
5~ TC(A CGC GT)C TTG AAG TCG TGC AGG TCA G 3~
pyc_end (SEQ ID NO: 20):
5~ TC(A CGC GT)C GCC TCC TCC ATG AGG AAG A 3~
The primers shown are synthesized by MWG Biotech and the
PCR reaction is carried out by the standard PCR method of
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Innis et al. (PCR Protocols. A Guide to Methods and
Applications, 1990, Academic Press). The primers allow
amplification of a DNA fragment of approx 3.6 kb in size,
which carriea +.:he pyc gene. The primers moreover contain
the sequence for the cleavage site of the restriction
endonuclease MluI, which is marked by parentheses in the
nucleotide sequence shown above. .
The amplified DIVA fragment of approx. 3.6 kb in length,
which carries the pyc gene, is cleaved with the restriction
endonuclease MluI, identified by means of electrophoresis
in a 0.8~ agarose gel and isolated from the gel and
purified by conventional methods (QIAquick Gel Extraction
Kit, Qiagen, riilden).
After purification, the fragment carrying the pyc gene is
employed for ligation in the vector pKl8mobsacBpckl
described. This is cleaved beforehand with the restriction
enzyme BssHII, dephosphorylated with alkaline phosphatase
(Alkaline Phosphatase, Boehringer Mannheim, Germany), mixed
with the DNA fragment of approx. 3.6 kb which carries the
pyc gene, and the mixture is treated with T4 DNA Lipase
(Amersham-Pharmacia, Freiburg, Germany).
The E. coli strain DH5amcr (Life Technologies GmbH,
Karlsruhe, Germany) is then transformed with the ligation
batch (Hanaha~~; In: DNA Cloning. A Practical Approach. Vol.
1, ILR-Press, Cold Spring Harbor, New York, 1989).
Selection of plasmid-carrying cells is made by plating out
the transformation batch on LB agar (Sambrook et al.,
Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold
Spring Harbor, New York, 1989), which was supplemented with
50 mg/1 kanamycin.
Plasmid DNA is isolated from a transformant with the aid of
the QIAprep Spin Niiniprep Kit from Qiagen and checked by
restriction cleavage and subsequent agarose gel
electrophoresis. The plasmid -is called pKl8mobsacBpckl 2.
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4.2 Construction of the pyc allele pyc P458S by means of
site-specific mutagenesis of the wild-type pyc gene
The site-direr_ted mutagenesis is carried out with the
QuikChange Site-Directed Mutagenesis Kit (Stratagene, La
5 Jolla, USA). EP-A-1108790 describes a point mutation in the
pyc gene for C. glutamicum which allows improved L-lysine
production. On the basis of the point mutation in the
nucleotide sequence of cytosine to thymine in the pyc gene
at position i3i2, replacement in the amino acid sequence
10 derived therefrom of proline for serine at position 458
results. The allele is called pyc P458S. To generate the
mutation described, the following primer oligonucleotides
are chosen for the linear amplification:
P458S-1 (SEQ ID NO: 21):
15 5° GGATTCATTGCCGATCAC (TCG) CACCTCCTTCAGGCTCCA 3'
P458S-2 (SEQ ZD NO: 22):
5'GTGGAGGAAGTCCGAGGT (CGA) GTGATCGGCAATGAATCC 3'
The primers shown are synthesized by MWG Biotech. The codon
for serine, which is to replace the proline at position
20 458, is marked by parentheses in the nucleotide sequence
shown above. The plasmid pKl8mobsacBpckl 2 described in
Example 4.1 is employed with the two primers, which are
each complemeW~ary to a strand of the plasmid, for linear
amplification by means of Pfu Turbo DNA polymerase. By this
25 lengthening of the primers, a mutated plasmid with broken
circular strands is formed. The product of the linear
amplification is treated with Dpnl - this endonuclease
cleaves the methylated and half-methylated template DNA
specifically. The newly synthesized broken, mutated vector
30 DNA is transformed in the E. coli strain XL1 Blue (Bullock,
Fernandez and Short, BioTechniques (5) 376-379 (1987)).
After the transformation, the XL1 Blue cells repair the
breaks in the mutated plasmids. Selection of the
transformants was carried out on LB medium with kanamycin
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50 mg/1. The plasmid obtained is checked by means of
restriction cleavage, after isolation of the DNA, and
identified in agarose gel. The DNA sequence of the mutated
DNA fragment ~~~ checked by sequencing. The sequence of the
PCR product coincides with the sequence described Ohnishi
et al. (2002). The resulting plasmid is called
pKl8mobsacBpckl_3. A map of the plasmid is shown in
Figure 6.
4.3 Incorporation of a second copy of the pyc gene in the
form of the pyc allele pycP458S into the chromosome
(target site pck gene) of the strain DSM12866 by
means of the replacement vector pkl8mobsacBpckl 3
The plasmid pK~8mobsacBpcki._3 described in Example 4.2 is
transferred as described in Example 1.3 into the C.
glutamicum strain DSM12866 by conjugation. Selection is
made for targeted recombination events in the chromosome of
C. glutamicum DSM12866 as described in Example 1.3.
Depending on the position of the second recombination
event, of ter the excision the second copy of the pyc allele
manifests itself in the chromosome at the pck locus, or the
original pck locus of the host remains.
Approximately 40 to 50 colonies are tested for the
phenotype "growth in the presence of sucrose" and "non-
growth in the presence of kanamycin". Approximately 20
colonies which show the phenotype "growth in the presence
of sucrose" and "non-growth in the presence of kanamycin"
are investigated with the aid of the polymerase chain
reaction. A DNA fragment which carries the pck gene and
surrounding regions is amplified here from the chromosomal
DNA of the colonies. The same primer.oligonucleotides as
are described ~.n Example 1.5 for the construction of the
replacement plasmid are chosen for the PCR.
pck_beg (SEQ ID N0: 9):
5~ TA(A GA~1 C'i) G CCG GCA TGA CTT CAG TTT 3~
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pck end (SEQ ID NO: 10):
5~ AC(A GAT CT} G GTG GGA GCC TTT CTT GTT ATT3
The primers ,z1 _'_ow amplification of a DNA fragment approx.
2.9 kb in size in control clones with the original pck
locus. In clones with a second copy of the pyc allele in
the chromosome at the pck locus, DNA fragments with a size
of approx. 6.5 kb are amplified.
The amplified DNA fragments are identified by means of
electrophoresis in a 0.8o agarose gel.
A clone which, in addition to the copy of the wild-type
gene present at the pyc locus, has a second copy of the pyc
gene in the form of the pyc allele pycP458S at the pck
locus in the chromosome was identified in this manner. This
clone was called strain DSM12866pck::pyc.
Example 5
Preparation of Lysine
The C. glutamicum strains DSM13994g1u::lysC,
DSM12866g1u::lysC, DSM12866pck::lysC, DSM12866aecD::lysC,
DSM12866g1u::ddh, DSM12866aecD::dapA and DSM12866pck::pyc
obtained in Example 1, 2, 3 and 4 are cultured in a
nutrient mediurz suitable for the production of lysine and
the lysine content in the culture supernatant was
determined.
For this, the cultures are first incubated on a brain-heart
agar plate (Merck, Darmstadt, Germany) for 24 hours at
33qC. Starting from this agar plate culture, a preculture
is seeded (10 mi medium in a 100 ml conical flask). The
medium MM is used as the medium for the preculture. The
preculture is incubated for 24 hours at 33°-C at 240 rpm on
a shaking machine. A main culture is seeded from this
preculture such that the initial OD (660 nm) of the main
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culture is 0.1 OD. The Medium MM is also used for the main
culture.
Medium MM
CSL 5 g/1
MOPS 20 g/1
Glucose (autoc7_aved separately) 50 g/1
Salts:
(NH4) 2504 25 g/1
KH2P04 0.1 g/1
MgS04 * ~ H~0 1.0 g/1
CaCl2 * 2 H20 10 mg/ 1
FeS04 * 7 HBO 10 mg/1
MnS04 * H20 5.0 mg/1
Biotin (sterile-filtered) 0.3 mg/1
Thiamine * HC1 (sterile-filtered) 0.2 mg/1
25 g/1
CaC03
The CSL (corn steep liquor), MOPS
(morpholinopropanesulfonic acid) and the salt solution are
brought to pH 7 with aqueous ammonia and autoclaved. The
sterile subs trate and vitamin solutions, as well as the
CaC03 autoclaved in the dry state, are then added.
Culturing is carried out in a 10 ml volume in a 100 ml
conical flask with baffles. Culturing is carried out at
33sC and 80o atmospheric humidity.
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After 48 hours, the OD is determined at a measurement
wavelength of 660 nm with a Biomek 1000 (Beckmann
Instruments GmbH, Munich). The amount of lysine formed is
determined winch an amino acid analyzer from Eppendorf-
BioTronik (Hamburg, Germany) by ion exchange chromatography
and post-column derivation with ninhydrin detection.
The result of the experiment is shown in Table 10.
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~a'hl r~ 1 fl
Strain OD Lysine HC1
(660 nm) gll
DSM13994 12.0 19.1
DSM13994g1u::lysC 9.9 20.0
DSM12866 12.5 14.9
DSM15039 11.4 16.2
DSM12866pck::lysC 12.6 16.5
DSM12866aecD::lysC ~ 12.0 15.9
DSM12866g1u::ddh ~ 11.0 15.5
DSM12856aecD::dapA ~ 11.1 16.2
DSM12866pck::pyc = 10.9 16.9
i
Brief Description of the Figures:
5 The base pair numbers stated are approximate values
obtained in the context of reproducibility of measurements.
Figure 1: I~iap of the plasmid pKl8mobsacBglul 1.
The abbreviations and designations used have the following
meaning:
KanR: Kanamycin resistance gene
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HindIII: Cleavage site of the restriction enzyme
HindIII
BamHI: Cleavage site of the restriction enzyme
BamHI
lysC: lySCFBR allele, lysC T311I
'gluA: 3' terminal fragment of the gluA gene
gluB': 5' terminal fragment of the gluB gene
'gluB: 3' terminal fragment of the gluB gene
gluC'- 5' terminal fragment of the gluC gene
sacB: sacB gene
RP4mob: mob region with the replication origin
for
the transfer (oriT)
oriV: Replication origin V
Figure 2: Map of the plasmid pKl8mobsacBaecD1
1.
The abbreviations following
and designations
used have
the
meaning:
KanR: Kanamycin resistance gene
SalI: Cleavage site of the restriction enzyme SalI
lysC: lysCFBR allele, lysC T311I
aecD': 5' terminal fragment of the aecD gene
'aecD: 3' terminal fragment of the aecD.gene
sacB: sacB gene
RP4mob: mob region with the replication origin for
the transfer (oriT)
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oriV: Replication origin V
Figure 3: Map of the plasmid pKl8mobsacBpckl-1.
The abbreviations following
and designations
used have
the
meaning:
KanR: Kanamycin resistance gene
BamHI: Cleavage site of the restriction enzyme
BamHI
lysC: lysCFBR allele, lysC T311I
pck': 5' terminal fragment of the pck gene
'pck: 3' terminal fragment of the pck gene
sacB: sacB gene
RP4mob: mob region with the replication origin for
the transfer (oriT)
oriV: Replication origin V
Figure 4: Map of the plasmid pKl8mobsacBgluB2 .
1
The abbreviations following
and designations
used have
the
meaning:
KanR: Kanamycin resistance gene
SalI Cleavage site of the restriction enzyme SalI
EcoRI Cleavage site of the restriction enzyme
ECORI
BamHI: Cleavage site of the restriction enzyme
BamHI
ddh: ddh gene
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gluA gluA gene
gluB': 5' terminal fragment of the gluB gene
'gluB: 3' terminal fragment of the gluB gene
gluC gluC gene
gluD': 5' terminal fragment of the gluD gene
sacB: sacB gene
RP4mob: mob region with the replication origin for
the transfer (oriT)
oriV: Replication origin V
Figure Map of the plasmid pKl8mobsacBaecD2_1.
5:
The abbreviations and designations following
used have the
meaning:
KanR: I~anamycin resistance gene
EcoRI Cl.eavag~ site of the restriction enzyme
ECORI
Sall: Cleavage site of the restriction enzyme SalI
dapA: dapA gene
aecD': 5' terminal fragment of the aecD gene
'aecD: 3' terminal fragment of the aecD gene
sacB: ~sacB gene
RP4mob: mob region with the replication origin for
the transfer (oriT)
oriV: Replication origin V
Figure 6: Map of the plasmid pKl8mobsacBpckl 3.
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94
The abbreviations and designations used have the following
meaning:
KanR: Kanamycin resistance gene
pyc: pyc allele, pyc P458S
pck': 5' terminal fragment of the pck gene
'pck: 3' terminal fragment of the pck gene
sacB: sacB gene
RP4mob: mob region with the replication origin for
the transfer (oriT)
oriV: Replication origin V
CA 02455878 2004-02-04
WO 03/040373 PCT/EP02/08464
BUDAPEST TREATY ON THE INTERNATIONAL Deutsche Sammlung von
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Mikroorganismen undo
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,.,
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issued pursuant to Rule 7.1 by the
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identified at the bottom of this page
I. ll~ENTIFICATION OF THE MICROORGANISM
Identification reference given Accession number given by the
by the DEPOSITOR:
DSIVI12866g1u::lysC INTERNATIONAL DEPOSITARY AUTHORITY:
DSM 15039
II. SCIENTIFIC DESCRIPTION AND/OR
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The microorganism identified under
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llI. RECEIPT AND ACCEPTANCE
This International Depositary
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IV. RECEIPT OF REQUEST FOR CONVERSION
The microorganism identified under
I above was received by this
International Depositary Authority
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and a request to convert the original
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by it on (date of receipt of
request
for conversion).
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON Signatures) of persons) having
the power to represent the
MIKROORGANISMEN UND ZELLKULTUREN International Depositary Authonty
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Address: Mascheroder Weg 1b
D-38124 Braunschweig
r
Date: 2002-06-06
Where Rule 6.4 (d) applies, such date is the date on which the staNS of
international depositary authority was acquired,
Form DSMZ-BP/4 (sole-page) 12/2001
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96
BUDAPEST TREATY ON THE INTERNATIONAL Deutsche Sammlung von
RECOGNITION OF TIIE DEPOSIT OF MICROORGANISMS Mikroorganismen and
FOR THE PURPOSES OF PATENT PROCEDURE Zellkulturen GmbH
INTERNATIONAL FORM
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Kantstr. 2
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VIABILITY STATEMENT
issued pursuant to Rule 10.2 by the
INTERNATIONAL DEPOSTfARY AUTHORITY
identified at the bottom of this page
I. DEPOSTfOR II. IDENTIFICATION OF THE MICROORGANISM
Name: Degussa AG Accession number given by the
ICantstr. 2 INTERNATIONAL DEPOSITARY AUTHORITY:
Address: 33790 Halle (Westf.)
DSM 15039
Date of the deposit or the transfer:
2002-06-OS
III. VIABILITY STATEMENT
The viability of the microorganism
identified under R above was tested
on 2~~2-~6-05
On that date, the said microorganism
was
( R)' viable
( )' no longer viable
IV. CONDTfIONS UNDER WHICH THE
VIABILITY TEST HAS BEEN PERFORMED
V. INTERNATIONAL DEPOS1TARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON Signatures) of persons) having
the power to represent the
MIKROORGANISMEN UND ZELLICULI'URENInternational Depositary Authority
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Date: 2002-06-06
Indicate the date of original deposit or, where a new deposit or a hansfer has
been made, the most recent relevant date (date of the new deposit or date
of the transfer).
' In the cases referred to in Rule 10.2(a) (ii) and (iii), refer to the most
recent viability test.
Mark with a cross the applicable box.
Fill in if the information has been requested and if the results of the test
were negative.
Fonn DSMZ-BP/9 (sole page) 12/2001
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97
BUDAPEST TREATY ON THE INTERNATIONAL Deutsche Sammlung von
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Mikroorganismen and
FOR THE PURPOSES OF PATENT PROCEDURE Zellkulturen GmbH
Y ~.
INTERNATIONAL FORM
Degussa AG
Kantstr. 2
33790 Halle (Westf.) RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
issued pursuant to Rule 7.1 by the
INTERNATIONAL DEPOSITARY AUTHORITY
identified at the bottom of this page
I. E)EIdTIFICATION OF THE MICROORGANISM
Identification reference given Accession number given by the
by the DEPOSITOR:
DHSalphamcr/pKl8mobsacBaecD1_1 ~TE~ATIONAL DEPOS1TARY AUTHORITY:
DSM 15040
E. SCIENTIFIC DESCRIPTION AND/OR
PROPOSED TA3CONOMIC DESIGNATION
The microorganism identified under
I. above was accompanied by:
g ) a scientific description
a proposed taxonomic designation
(Mark with a cross when; applicable).
III. RECEIPT AND ACCEPTANCE
This International Depository
Authority accepts the microorganism
identified under I, above, which
was received by it on 2002-06-OS
(Date of the original deposit).
IV. RECEIPT OF REQUEST FOR CONVERSION
The microorganism identified under
1 above was received by this
International Depository Authority
on (date of original deposit)
and a request to convert the original
deposit to a deposit under the
Budapest Treaty was received
by it on (date ofreceipt of request
for conversion).
V. INTERNATIONAL DEPOS1TARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON Signatures) of persons) having
the power to represent the
MIKROORGANISMEN UND 2ELLKULTUREN International Depository Authonty
GmbH or of authorized official(s);
Address: Mascheroder Weg 1b
D-38124 Braunschweig
Date: 2002-06-06
Where Rule 6.4 (d) applies, such date is the date on which the status of
international depository authority was acquired,
Fonn DSMZ-$P/4 (sole page) 12/2001
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98
BUDAPEST TREATY ON THE INTERNATIONAL Deutsche Sammlung von
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Mikroorganismen and
FOR THE PURPOSES OF PATENT PROCEDURE Zellkulturen GmbH
INTERNATIONAL FORM
Degussa AG
Kantstr. 2
33790 Halle t,Westf.)
VIABILITY STATEMENT
issued pursuant to Rule 10.2 by the
INTERNATIONAL DEPOS1TARY AUTHORITY
identified at the bottom of this page
I. DEPOSTfOR II. IDENTIFICATION OF THE MICROORGANISM
Name: Degussa AG
Accession number given by the
Kantstr. 2 INTERNATIONAL DEPOSITARY A>ffFIORITY:
Address: 33790 Halle (Westf.)
DSM 15040
Date of the deposit or the transfer:
2002-06-OS
III. VIABILITY STATEMENT
The viability of the microorganism
identified under II above was
tested on 2002-06-OS
On that date, the said microorganism
was
(X)' viable
( )' no longer viable
IV. CONDITIONS UNDER WHICH THE
VIABILITY TEST HAS BEEN PERFORMED'
V. INTERNATIONAL DEPOSiTARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON Signatures) of persons) having
the power to represent the
MIKROORGANISMEN UND ZELLKULTUREN International Depositary Authority
GmbH or of authorized offtciai(s):
Address: Mascheroder Weg 1b
D-38124 Braunschweig
Date: 2002-06-06
Indicate the date of original deposit o5 where a new deposit or a transfer has
been made, the most recent relevant date (date of the new deposit or date
of the transfer).
In the cases referred to in Rule 10.2(a) (ii) and (iii), refer to the most
recent viability test.
Mark with a cross the applicable box.
Fill in if the information has been requested and if the results of the test
were negative.
Form DSMZ-BP/9 (sole page) 12/2001
CA 02455878 2004-02-04
WO 03/040373 PCT/EP02/08464
99
BUDAPEST TREATY ON TFIE 1NTERNATiONAL
fECO(ihIITION OF THE DEPOSIT OF MICROORGAMSMS
FOR THE PURPOSES OF PATENT PROCEDURE
INTERNATIONAL FORM
Degussa AG
Kantstr. 2
33790 Aalle/Kiznsebeck
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
issued pursuant to Rule 7.1 by the
INTERNATIONAL DEPOSITARY AIJTHORTfSC
identified at the bottom of this page
I. IDENTIFICATION OF THE MICROORGANISM
I
Identification reference given Accession number given by the
by the DEPOSITOR:
INTERNATIONAL DEP05ITARY AUTHORITY:
DHSalphamcr/
pKlBmobsacBglul 1 DSM 14243
II. SCIENTIFIC DESCRIPTION AND/OR
PROPOSED TAXONOMIC DESIGNATION
The microorganism identified under
I, above was accompanied by:
(X ) a scientific description
(X ) a proposed taxonomic designation
(Mark with a cross where applicable).
III. RECEB~T AND ACCEPTANCE
This International Depository Authority
accepts the microorganism identified
under I. above, which was received
by it on 2 0 O 1-0 4 - 2 0
(Date of the original deposit)'.
IV. RECEIPT OF REQUEST FOR CONVERSION
The microorganism identified under
I above wes received by this International
Depository Authority on (date
of original deposit}
and a request to convert the original
deposit to a deposit under the
Budapest Treaty was received by
it on (date of receipt of request
for conversion).
V. B~ITERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON Signatures) of persons) having
the power to represent the
MIKROORGANISMEN UND ZELLIt;ULTURENIatemational Depository Authority
GmbH or of authorized official(s):
Address: Mascheroder Weg 1b
D-38124 Braunschwei
g
Date: 2001-04-26
' Where Rule 6.4 (d) applies, such date is the date on which the status of
international depository authority was acquired.
Form DSMZ-Bpl4 (sole page) OI96
CA 02455878 2004-02-04
WO 03/040373 PCT/EP02/08464
100
BUDAPEST TREATY ON THE INTERNATIONAL
COGNITION OF THE DEP05IT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE
INTERNATIONAL FORM
Degussa AG
Kantstr. 2
33790 Halle/Kunsebeck
VIABILITY STATEMENT
issued pursuant to Rule 10.2 by the
INTERNATIONAL DEPOSITARY AUTHORITY
identified at the bottom of this page
I. DEPOSITOR II. IDENTIFICATION OF THE MICROORGANISM
Name: Degas sa AG Accession number given by the
Kantstr. 2 INTERNATIONAL DEPOSITARY AUTHORITY:
Aaaress: 33?90 Halle/Kunsebeck DSM 14243
Date of the deposit or the transfer':
2001-04-20
III. VIABIL1TY STATEMENT
The viability of the microorganism
identified under II above was tested
on 2 ~ O 1- ~ 4 - 2 ~ 1 .
On that date, the said microorganism
was
(X)' viable
( )' no longer viable
IV. CONDITIONS UNDER WHICH TfIE
VIABIISTY TEST HAS BEEN PERFORMED
V. INTERNATIONAL DEPOSTfARY AUTHORITY
Name: DSMZ DEUTSCHE SAMMLUNG VON Signatures) of persons) having
MIKROORGANISMEN UND ZELLKULTUREN the power to represent the
GmbH International Depositary Authority
or of authorized official(s):
'
Address: Mascheroder Weg 1b /~
~
~
D-38124 Braunschweig ~
_.~s~~ I
Date: 2001-04-26
Indicate the date of original deposit or, where a new deposit or a transfer
has been made, the most recent relevant date (date of the new deposit or
date of the transfer).
= In the cases referred to in Rule 1 D.2(a) (ii) and (iii), refer to the most
recent viability test.
' Mark with a cross the applicable box.
° Fill in if the information has been requested and if the results of
the test were negative.
Form DSMZ-BP19 (sale page) 0196
CA 02455878 2004-02-04
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1/11
SEøLIENCE LISTING
<110> Degussa AG
<120> Coryneform bacteria which produce chemical
compounds I
<130> 010301 BT
<160> 22
<170> PatentIn version 3.1
<210> 1
<211> 1263
<212> DNA
<213> Corynebacterium glutamicum
<220>
<221> CDS
<222> (1)..(1263)
<223> lysC wild-type gene
<400>
1
gtg gccctg gtcgtacagaaa tatggcggttcctcgctt gagagtgcg 48
Met AlaLeu ValValGlnLys TyrGlyGlySerSerLeu GluSerAla
1 5 10 15
gaa cgcatt agaaacgtcget gaacggatcgttgccacc aagaagget 96
Glu ArgIle ArgAsnValAla GluArgIleValAlaThr LysLysAla
20 25 30
3 gga aatgat gtcgtggttgtc tgctccgcaatgggagac accacggat 144
5
Gly AsnAsp ValValValVal CysSerAlaMetGlyAsp ThrThrAsp
35 40 45
gaa cttcta gaacttgcagcg gcagtgaatcccgttccg ccagetcgt 192
4 Glu LeuLeu GluLeuAlaAla AlaValAsnProValPro ProAlaArg
0
50 55 60
gaa atggat atgctcctgact getggtgagcgtatttct aacgetctc 240
Glu MetAsp MetLeuLeuThr AlaGlyGluArgIleSer AsnAlaLeu
45 65 70 75 80
gtc gccatg getattgagtcc cttggcgcagaagcccaa tctttcacg 288
Val AlaMet AlaIleGluSer LeuGlyAlaGluAlaGln SerPheThr
85 90 95
50
ggc tctcag getggtgtgctc accaccgagcgccacgga aacgcacgc 336
G1y SerGln AlaGlyValLeu ThrThrGluArgHisGly AsnAlaArg
100 105 110
55 att gttgat gtcactccaggt cgtgtgcgtgaagcactc gatgagggc 384
Ile ValAsp ValThrProGly ArgValArgGluAlaLeu AspG1uGly
115 120 125
aag atctgc attgttgetggt ttccagggtgttaataaa gaaacccgc 432
Lys I1eCys IleValAlaGly PheGlnGlyValAsnLys GluThrArg
13D 135 140
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gat gtcaccacg ttgggtcgtggtggttct gacaccact gcagttgcg 480
Asp Va1ThrThr LeuGlyArgGlyGlySer AspThrThr AlaValAla
145 150 155 160
ttg gcagetget ttgaacgct~gatgtgtgt gagatttac tcggacgtt 528
Leu AlaAlaAla LeuAsnAlaAspValCys GluIleTyr SerAspVal
165 170 175
gac ggtgtgtat accgetgacccgcgcatc gttcctaat gcacagaag 576
1 Asp GlyValTyr ThrAlaAspProArgIle ValProAsn AlaGlnLys
0
180 185 190
ctg gaaaagctc agcttcgaagaaatgctg gaacttget getgttggc 624
Leu GluLysLeu SerPheGluGluMetLeu GluLeuAla AlaValGly
195 200 205
tcc aagattttg gtgctgcgcagtgttgaa tacgetcgt gcattcaat 672
Ser LysIleLeu ValLeuArgSerValGlu TyrAlaArg AlaPheAsn
210 215 220
gtg ccactt cgcgtacgctcgtcttat agtaatgatccc ggcactttg 720
Val ProLeu ArgValArgSerSerTyr SerAsnAspPro GlyThrLeu
225 230 235 240
2 att gccggc tctatggaggatattcct gtggaagaagca gtccttacc 768
5
Ile AlaGly SerMetGluAspI1ePro ValGluGluAla ValLeuThr
245 250 255
ggt gtcgca accgacaagtccgaagcc aaagtaaccgtt ctgggtatt 816
3 Gly ValAla ThrAspLysSerG1uAla LysValThrVal LeuGlyIle
0
260 265 270
tcc gataag ccaggcgaggetgcgaag gttttccgtgcg ttggetgat 864
Ser AspLys ProGlyG1uAlaAlaLys ValPheArgAla LeuAlaAsp
275 280 285
gca gaaatc aacattgacatggttctg cagaacgtctct tctgtagaa 912
Ala GluIle AsnIleAspMetValLeu GlnAsnValSer SerValGlu
290 295 300
40 ,
gac ggcacc accgacatcaccttcacc tgccctcgttcc gacggccgc 960
Asp GlyThr ThrAspIleThrPheThr CysProArgSer AspGlyArg
305 310 315 320
45 cgc gcgatg gagatcttgaagaagctt caggttcagggc aactggacc 1008
Arg AlaMet GluIleLeuLysLysLeu GlnValG1nGly AsnTrpThr
325 330 335
aat gtgctt tacgacgaccaggtcggc aaagtctccctc gtgggtget 1056
50 Asn Va1Leu TyrAspAspGlnValGly LysValSerLeu ValG1yAla
340 345 350
ggc atgaag tctcacccaggtgttacc gcagagttcatg gaagetctg 1104
Gly MetLys SerHisProGlyValThr AlaGluPheMet GluAlaLeu
55 355 360 365
cgc gatgtc aacgtgaacatcgaattg atttccacctct gagattcgt 1152
Arg AspVal AsnValAsnIleGluLeu IleSerThrSer GluIleArg
370 375 380
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att tcc gtg ctg atc cgt gaa gat gat ctg gat get get gca cgt gca 1200
Ile Ser Va1 Leu Ile Arg Glu Asp Asp Leu Asp Ala Ala Ala Arg Ala
385 390 395 400
ttg cat gag cag ttc cag ctg ggc ggc gaa gac gaa gcc gtc gtt tat 1248
Leu His Glu Gln Phe Gln Leu Gly Gly Glu Asp G1u Ala Val Val Tyr
405 410 415
gca ggc acc gga cgc 1263
1 0 Ala Gly Thr G1y Arg
420
<210> 2
<211> 421
15 <212> PRT
<213> Corynebacterium glutamicum
<400> 2
2 0 Met Ala Leu Val Val Gln Lys Tyr Gly Gly Ser Ser Leu Glu Ser Ala
1 5 10 15
Glu Arg Ile Arg Asn Val Ala Glu Arg Ile Va1 Ala Thr Lys Lys Ala
20 25 30
~5
Gly Asn Asp Val Val Val Val Cys Ser A1a Met Gly Asp Thr Thr Asp
35 40 45
Glu Leu Leu Glu Leu Ala Ala Ala Val Asn Pro Va1 Pro Pro A1a Arg
30 50 55 60
Glu Met Asp Met Leu Leu Thr Ala Gly Glu Arg Ile Ser Asn A1a Leu
65 70 75 80
3 5 Val A1a Met Ala Ile Glu Ser Leu Gly Ala Glu Ala Gln Ser Phe Thr
85 90 95
Gly Ser Gln Ala Gly Val Leu Thr Thr Glu Arg His Gly Asn Ala Arg
100 105 110
Ile Val Asp Val Thr Pro Gly Arg Val Arg Glu Ala Leu Asp Glu Gly
115 120 125
Lys Ile Cys Ile Val Ala Gly Phe Gln Gly Val Asn Lys Glu Thr Arg
130 135 140
Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala
145 150 155 160
5 0 Leu Ala Ala Ala Leu Asn Ala Asp Val Cys Glu Ile Tyr Ser Asp Val
165 170 175
Asp Gly Val Tyr Thr Ala Asp Pro Arg .Ile Val Pro Asn Ala Gln Lys
180 185 190
Leu Glu Lys Leu Ser Phe Glu G1u Met Leu Glu Leu Ala Ala Val Gly
195 200 205
Ser Lys Ile Leu Val Leu Arg Ser Val Glu Tyr Ala Arg Ala Phe Asn
$0 210 215 220
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Val Pro Leu Arg Val Arg Ser Ser Tyr Ser Asn Asp Pro G1y Thr Leu
225 230 235 240
Ile Ala Gly Ser Met G1u Asp Ile Pro Val Glu Glu Ala Val Leu Thr
245 250 255
Gly Val Ala Thr Asp Lys Ser Glu Ala Lys Val Thr Val Leu Gly I1e
260 265 270
1 Ser AspLysProGly Glu'AlaAlaLysVal PheArgAlaLeuAlaAsp
0
275 280 285
Ala GluIleAsnIle AspMetValLeuGln AsnVa1SerSerValGlu
290 295 300
Asp GlyThrThrAsp IleThrPheThrCys ProArgSerAspGlyArg
305 310 315 320
Arg AlaMetG1uIle LeuLysLysLeuGln ValGlnGlyAsnTrpThr
325 330 335
Asn ValLeuTyrAsp AspGlnValG1yLys ValSerLeuValGlyAla
340 345 350
2 Gly MetLysSerHis ProGlyValThrAla GluPheMetGluA1aLeu
5
355 360 365
Arg AspValAsnVal AsnIleGluLeuIle SerThrSerGluIleArg
370 375 380
30
Ile SerValLeuIle ArgGluAspAspLeu AspAlaAlaA1aArgAla
385 390 395 400
Leu HisGluGlnPhe GlnLeuGlyGlyGlu AspGluAlaValValTyr
35 405 410 415
Ala GlyThrGlyArg
420
40 <210> 3
<211> 1263
<212> DNA
<213> Corynebacterium
glutamicum
45 <220>
<221> CDS
<222> (1)..(1263)
<223> lysC-fbrallele T311I
lysC
5fl <400> 3
gtg gcc ctg gta cagaaatatggc ggttcctcgcttgagagt gcg 48
gtc
Met A1a Leu Val GlnLysTyrGly G1ySerSerLeuGluSer A1a
Val
1 5 10 15
5 gaa cgc att aac gtcgetgaacgg atcgttgccaCCaagaag get 96
5 aga
Glu Arg 21e Asn ValAlaGluArg IleValAlaThrLysLys Ala
Arg
25 30
gga aat gat gtg gttgtctgctcc gcaatgggagacaccacg gat 1.44
gtc
6 Gly Asn Asp Val Va1ValCysSer AlaMetGlyAspThrThr Asp
0 Val
35 40 45
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gaa ctt cta gaa ctt gca gcg gca gtg aat ccc gtt ccg c.ca get cgt 192
Glu Leu Leu Glu Leu Ala Ala A1a Val Asn Pro Val Pro Pro Ala Arg
50 55 60
gaa atg gat atg ctc ctg act get ggt gag cgt att tct aac get ctc 240
Glu Met Asp Met Leu Leu Thr Ala Gly Glu Arg Ile Ser Asn Ala Leu
65 70 75 80
gtc gcc atg get att gag tcc .ctt ggc gca gaa gcc caa tct ttc acg 288
Val Ala Met Ala Ile Glu Ser Leu Gly Ala Glu Ala Gln Ser Phe Thr
85 90 95
ggc tct cag get ggt gtg ctc acc acc gag cgc cac gga aac gca cgc 336
Gly Ser Gln Ala Gly Val Leu Thr Thr Glu Arg His Gly Asn Ala Arg
100 105 110
att gtt gat gtc act cca ggt cgt gtg cgt gaa gca ctc gat gag ggc 384
Ile Val Asp Val Thr Pro Gly Arg Val Arg Glu Ala Leu Asp Glu Gly
115 120 125
aag atc tgc att gtt get ggt ttc cag ggt gtt aat aaa gaa acc cgc 432
Lys Ile Cys Ile Val Ala Gly Phe G1n Gly Val Asn Lys Glu Thr Arg
130 135 140
2 5 gat gtc acc acg ttg ggt cgt ggt ggt tct gac acc act gca gtt gcg 480
Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala
145 150 155 160
ttg gca get get ttg aac get gat gtg tgt gag att tac tcg gac gtt 528
3 0 Leu Ala A1a Ala Leu Asn Ala Asp Val Cys Glu Ile Tyr Ser Asp Val
165 170 175
gac ggt gtg tat acc get gac ccg cgc atc gtt cct aat gca cag aag 576
Asp Gly Va1 Tyr Thr Ala Asp Pro Arg Ile Val Pro Asn A1a Gln Lys
3 5 180 185 190
ctg gaa aag ctc agc ttc gaa gaa atg ctg gaa ctt get get gtt ggc 624
Leu Glu Lys Leu Ser Phe Glu Glu Met Leu Glu Leu Ala A1a Val Gly
195 200 205
tcc aag att ttg gtg ctg cgc agt gtt gaa tac get cgt gca ttc aat 672
Ser Lys Ile Leu Val Leu Arg Ser Val Glu Tyr Ala Arg Ala Phe Asn
210 215 220
gtg cca ctt cgc gta cgc tcg tct tat agt aat gat ccc ggc act ttg 720
Val Pro Leu Arg Val Arg Ser Ser Tyr Ser Asn Asp Pro Gly Thr Leu
225 230 235 240
att gcc ggc tct atg gag gat att cct gtg gaa gaa gca gtc ctt acc 768
5 0 Ile Ala Gly Ser Met Glu Asp Ile Pro Val Glu Glu Ala Val Leu Thr
245 250 255
ggt gtc gca acc gac aag tcc gaa gcc aaa gta acc gtt ctg ggt att 816
Gly Val Ala Thr Asp Lys Ser Glu Ala Lys Va1 Thr Val Leu G1y Ile
2g0 265 270
tcc gat aag cca ggc gag get gcg aag gtt ttc cgt gcg ttg get gat 864
Ser Asp Lys Pro Gly G1u Ala Ala Lys Val Phe Arg Ala Leu A1a Asp
275 28D 285
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gca gaaatcaac attgacatggtt ctgcagaacgtctcttct gtagaa 912
Ala GluIleAsn I1eAspMetVal LeuGlnAsnValSerSer ValGlu
290 295 300
gac ggcaccacc gacatcatcttc acctgccctcgttccgac ggccgc 960
Asp GlyThrThr AspIleIlePhe ThrCysProArgSerAsp GlyArg
305 310 315 320
cgc gcgatggag atcttgaagaag cttcaggttcagggcaac tggacc 1008
1,.0Arg AlaMetGlu IleLeuLysLys LeuGlnValGlnGlyAsn TrpThr
325 330 335
aat gtgctttac gacgaccaggtc ggcaaagtctccctcgtg ggtget 1056
Asn ValLeuTyr AspAspGlnVal GlyLysValSerLeuVal GlyAla
340 345 350
ggc atgaagtct cacccaggtgtt accgcagagttcatggaa getctg 1104
Gly MetLysSer HisProGlyVal ThrAlaGluPheMetGlu AlaLeu
355 360 365
cgc gatgtcaac gtgaacatcgaa ttgatttccacctctgag attcgt 1152
Arg AspValAsn ValAsnIleGlu LeuIleSerThrSerGlu I1eArg
370 375 380
2 att tccgtgctg atccgtgaagat gatctggatgetgetgca cgtgca 1200
5
Ile SerValLeu IleArgGluAsp AspLeuAspAlaAlaAla ArgAla
385 390 395 400
ttg catgagcag ttccagctgggc ggcgaagacgaagccgtc gtttat 1248
3flLeu HisGluGln PheG1nLeuGly GlyGluAspGluA1aVal ValTyr
405. 410 415
gca ggcaccgga cgc 1263
Ala GlyThrGly Arg
3 420
5
<210> 4
<211> 421
<212> PRT
<213> Corynebacterium
glutamicum
<400> 4
Met Ala Leu Val Lys Tyr Gly Gly Ser GluSer
Val Gln Ser Leu Ala
45 1 5 10 15
Glu Arg Ile Asn Ala G1u Arg Ile Ala LysLys
Arg Val Val Thr Ala
20 25 30
5 Gly Asn Asp Val Val Cys Ser Ala Gly ThrThr
~ Val Val Met Asp Asp
35 40 45
Glu Leu Leu Leu Ala Ala Val Asn Val ProAla
Glu Ala Pro Pro Arg
50 55 60
55
Glu Met Asp Leu Thr Ala Gly Glu Ile AsnAla
Met Leu Arg Ser Leu
65 70 75 80
Val Ala Met Ile Ser Leu Gly Ala Ala SerPhe
Ala Glu Glu Gln Thr
85 90 95
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Gly Ser Gln Ala Gly Val Leu Thr Thr Glu Arg His Gly Asn Ala Arg
100 105 110
Ile Val Asp Val Thr Pro Gly Arg Val Arg Glu Ala Leu Asp Glu Gly
115 120 125
Lys Ile Cys Ile Val Ala Gly Phe Gln Gly Val Asn Lys Glu Thr Arg
130 135 140
1 0 Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala
145 150 155 160
Leu Ala Ala Ala Leu Asn Ala Asp Va1 Cys Glu Ile T'yr Ser Asp Val
165 170 175
Asp Gly Val Tyr Thr Ala Asp Pro Arg Ile Val Pro Asn Ala Gln Lys
180 185 190
Leu Glu Lys Leu Ser Phe Glu Glu Met Leu Glu Leu Ala Ala Val Gly
195 200 205
Ser Lys Ile Leu Val Leu Arg Ser Va1 Glu 'I'yr Ala Arg Ala the Asn
210 215 220
~ 5 Val Pro Leu Arg Val Arg Ser Ser Tyr Ser Asn Asp Pro Gly Thr Leu
225 230 235 240
Ile Ala Gly Ser Met Glu Asp Ile Pro Val Glu Glu A1a Val Leu Thr
245 250 255
Gly Val Ala Thr Asp Lys Ser Glu Ala Lys Val Thr Val Leu Gly Ile
260 265 270
Ser Asp Lys Pro Gly Glu Ala Ala Lys Val Phe Arg A1a Leu Ala Asp
275 280 285
Ala G1u Ile Asn Ile Asp Met Val Leu Gln Asn Val Ser Ser Val Glu
290 295 300
4 0 Asp G1y Thr Thr Asp Ile Ile Phe Thr Cys Pro Arg Ser Asp Gly Arg
305 310 315 320
Arg Ala Met Glu Ile Leu Lys Lys Leu G1n Val Gln Gly Asn Trp Thr
325 330 335
Asn Val Leu Tyr Asp Asp Gln Val Gly Lys Val Ser Leu Val Gly Ala
340 345 350
Gly Met Lys Ser His Pro Gly Val Thr Ala Glu Phe Met Glu Ala Leu
355 360 365
Arg Asp Val Asn Val Asn I1e Glu Leu Ile Ser Thr Ser Glu Ile Arg
370 375 380
5 5 Ile Ser Val Leu Ile Arg Glu Asp Asp Leu Asp Ala Ala Ala Arg Ala
385 390 395 ' 400
Leu His Glu Gln Phe Gln Leu Gly Gly Glu Asp Glu A1a Val Val Tyr
405 410 415
b0
Ala Gly Thr Gly Arg
420
CA 02455878 2004-02-04
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<210> 5
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<221> misc
feature
_
<222> (1). (28)
1 <223> Primer lysClbeg
0
<400> 5
taggatcctc cggtgtctga ccacggtg 28
<210> 6
<211> 29
<212> DNA
<213> Artificial sequence
<220>
<221> misc
feature
_
<222> (1). (29)
<223> Primer lysC2end
<~oo>
acggatccgc tgggaaattg cgctcttcc 29
<210> 7
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<221> misc
feature
3 _
5 <222> (1). (28)
<223> Primer gluBgll
<400> 7
taagatctgt gttggacgtc atggcaag 28
<210> 8
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<221> misc
feature
_
<222> (1). (28)
<223> Primer gluBgl2
<400> 8
acagatcttg aagccaagta cggccaag 28
<210> 9
<211> 27
<212 > DNA
<213> Artificial sequence
<220>
$fl<221> misc feature
<222> (1). (27)
<223> Primer pck beg
CA 02455878 2004-02-04
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9/11
<400> 9
taagatctgc cggcatgact tcagttt 27
<210> 10
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<221> misc
feature
_
<222> (1). {30)
<223> Primer pck_end
<400> so
acagatctgg tgggagcctt tcttgttatt 30
<210> 11
<211> 20
<212> DNA
<213> Corynebacterium glutamicum
<220>
<221> misc
feature
2 _
5 <222> (1). (20)
<223> Primer aecD beg
<400> 11
gaacttacgc caagctgttc 20
<210> 12
<211> 20
<212> DNA
<213> Corynebacterium glutamicum
<220>
<221> misc
feature
_
<222> (1). (20)
<223> Primer aecD_end
<400> 12
agcaccacaa tCaacgtgag 20
<210> 13
<211> 20
<212> DNA
<213> Corynebacterium glutamicum
<220>
<221> misc
feature
_
<222> (1). (20)
<223> Primer gluA_beg
<400> 13
cacggttgct cattgtatcc 20
<210> 14
<211> 20
<212> DNA
<213> Corynebacterium glutamicum
CA 02455878 2004-02-04
WO 03/040373 PCT/EP02/08464
10/11
<220>
<221> misc_feature
<222> (1)..(20)
<223> Primer gluD_end
<400> 14
cgaggcgaat cagacttctt 2p
<210> 15
<211> 20
<212> DNA
<213> Corynebacterium glutamicum
<220>
1 <221> misc_feature
5
<222> (1)..(20)
<223> Primer ddh_beg
<400> 15
2 ctgaatcaaa ggcggacatg 20
0
<210> 16
<211> 20
<212> DNA
2 <2l3> Corynebacterium glutamicum
5
<220>
<221> misc feature
<222> (1)..(20)
3 <223> Primer ddh end
0
<400> 16
tcgagctaaa ttagacgtcg 20
3 <210> 17
5
<211> 20
<212> DNA
<213> Corynebacterium glutamicum
40 <220>
<221> misc
feature
_
<222> (1). (20)
<223> Primer dapA beg
45 <400> 17
cgagccagtg aacatgcaga 20
<210> 18
<211> 20
50 <212> DNA
<213> Corynebacterium glutamicum
<220>
<221> misc
feature
5 _
5 <222> (1). (20)
<223> Primer dapA_end
<400> 18
cttgagcacc ttgcgcagca 20
60
CA 02455878 2004-02-04
WO 03/040373 PCT/EP02/08464
11/11
<210> 19
<211> 28
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (1). (28)
<223> Primer pyc beg
<400> 19
tcacgcgtct tgaagtcgtg caggtcag 28
<210> 20
<211> 28
<212> DNA
<213> Artificial sequence
<220>
2 0 <221> misc_feature
<222> (1). (28)
<223> Primer pyc_end
<400> 20
~ 5 tcacgcgtcg cctcctccat gaggaaga 28
<210> 21
<211> 39
<212> DNA
3 0 <213> Corynebacterium glutamicum
<220>
<221> misc_feature
<222> (1). (39)
3 5 <223> Primer P458S-1
<400> 21
ggattcattg ccgatcactc gcacctcctt caggctcca 39
40 <210> 22
<211> 39
<212> DNA
<213> Corynebacterium glutamicum
45 <220>
<221> misc_feature
<222> (1)..(39)
<223> Primer P458S-2
<400> 22
gtggaggaag tccgaggtcg agtgatcggc aatgaatcc 39
