Note: Descriptions are shown in the official language in which they were submitted.
CA 02590625 2007-06-18
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CA 02590625 2007-06-18
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CORYNEBACTERIUM GLUTAMICUM GENES ENCODING
PHOSPHOENOLPYRUVATE: SUGAR PHOSPHOTRANSFERASE SYSTEM
PROTEINS
This is a divisional application of Canadian Patent Application Serial No.
2,377,378. In some aspects, this divisional application relates to an isolated
nucleic acid
from Corynebacterium glutamicum encoding a phosphoenolpyruvate:sugar
phosphotransferase system protein as set forth in SEQ ID NO:7, as well as a
vector, a
host, a polypeptide as set forth in SEQ ID NO: 8, a method for producing fine
chemicals,
and a method for diagnosing using said nucleic acid, described in greater
detail herein.
However, it should be understood that the expression "the invention" and the
like
encompass the subject matter of both the parent and this divisional
application.
Back ro~ und of the Invention
Certain products and by-products of naturally-occurring metabolic processes in
cells have utility in a wide array of industries, including the food, feed,
cosmetics, and
pharmaceutical industries. These molecules, collectively termed 'fine
chemicals',
include organic acids, both proteinogenic and non-proteinogenic amino acids,
nucleotides and nucleosides, lipids and fatty acids, diols, carbohydrates,
aromatic
compounds, vitamins and cofactors, and enzymes. Their production is most
conveniently performed through large-scale culture of bacteria developed to
produce
and secrete large quantities of a particular desired molecule. One
particularly useful
organism for this purpose is Corynebacterium glutamicum, a gram positive,
nonpathogenic bacterium. Through strain selection, a number of mutant strains
have
been developed which produce an array of desirable eompounds_ However,
selection of
strains improved for the production of a particular molecule is a time-
consuming and
difficult process.
Summary of the Invention
The invention provides novel bacterial nucleic acid molecules which have a
variety of uses. These uses include the identification of microorganisms which
can be
used to produce fine chemicals, the modulation of fine chemical production in
C.
CA 02590625 2007-06-18
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glutamicum or related bacteria, the typing or identification of C. glutamicum
or related
bacteria, as reference points for mapping the C. glutamicum genome, and as
markers for
transformation. These novel nucleic acid molecules encode proteins, referred
to herein
as phosphoenolpyruvate:sugar phosphotransferase system (PTS) proteins.
C. glutamicum is a grain positive, aerobic bacterium which is commonly used in
industry for the large-scale production of a variety of fine chemicals, and
also for the
degradation of hydrocarbons (such as in petroleum spills) and for the
oxidation of
terpenoids. The PTS nucleic acid molecules of the invention, therefore, can be
used to
identify microorganisms which can be used to produce fine chemicals, e.g., by
fermentation processes. Modulation of the expression of the PTS nucleic acids
of the
invention, or modification of the sequence of the PTS nucleic acid molecules
of the
invention, can be used to modulate the production of one or more fine
chemicals from a
microorganism (e.g., to improve the yield or production of one or more fine
chemicals
from a Corynebacterium or Brevibacterium species).
'1'he PTS nucleic acids of the invention may also be used to identify an
organism
as being Corynebacterium glutamicum or a close relative thereof, or to
identify the
presence of C. glutamicum or a relative thereof in a mixed population of
microorganisms. The invention provides the nucleic acid sequences of a number
of C.
glutamicum genes; by probing the extracted genomic DNA of a culture of a
unique or
mixed population of microorganisms under stringent conditions with a probe
spanning a
region of a C. glutamicum gene which is unique to this organism, one can
ascertain
whether this organism is present. Although Corynebacterium glutamicum itself
is
nonpathogenic, it is related to species pathogenic in humans, such as
Corynebacterium
diphtheriae (the causative agent of diphtheria); the detection of such
organisms is of
significant clinical relevance.
The PTS nucleic acid molecules of the invention may also serve as reference
points for mapping of the C. glutamicum genome, or of genomes of related
organisms.
Similarly, these molecules, or variants or portions thereof, may serve as
markers for
genetically engineered Corynebacterium or Brevibacterium species.
The PTS proteins encoded by the novel nucleic acid molecules of the invention
are capable of, for example, transporting high-energy carbon-containing
molecules such
as glucose into C. glutamicum, or of participating in intracellular signal
transduction in
CA 02590625 2007-06-18
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this microorganism. Given the availability of cloning vectors for use in
Corynebacterium glutamicum, such as those disclosed in Sinskey et al., U.S.
Patent No.
4,649,119, and techniques for genetic manipulation of C. glutamicum and the
related
Brevibacterium species (e.g., lactofermentum) (Yoshihama et al, J. Bacteriol.
162: 591-
597 (1985); Katsumata et at., J. Bacteriol. 159: 306-31 l(1984); and
Santamaria et al., J.
Gen. Microbiol. 130: 2237-2246 (1984)), the nucleic acid molecules of the
invention
may be utilized in the genetic engineering of this organism to make it a
better or more
efficient producer of one or more fine chemicals.
The PTS molecules of the invention may be modified such that the yield,
production, and/or efficiency of production of one or more fine chemicals is
improved.
For example, by modifying a PTS protein involved in the uptake of glucose such
that it
is optimized in activity, the quantity of glucose uptake or the rate at which
glucose is
translocated into the cell may be increased. The breakdown of glucose and
other sugars
within the cell provides energy that may be used to drive energetically
unfavorable
biochemical reactions, such as those involved in the biosynthesis of fine
chemicals.
This breakdown also provides intermediate and precursor molecules necessary
for the
biosynthesis of certain fine chemicals, such as amino acids, vitamins and
cofactors. By
increasing the amount of intracellular high-energy carbon molecules through
modification of the PTS molecules of the invention, one may therefore increase
both the
energy available to perform metabolic pathways necessary for the production of
one or
more fine chemicals, and also the intracellular pools of metabolites necessary
for such
production.
Further, the PTS molecules of the invention may be involved in one or more
intracellular signal transduction pathways which may affect the yields and/or
rate of
production of one or more fine chemical from C. glutamicum. For example,
proteins
necessary for the import of one or more sugars from the extracellular medium
(e.g., HPr,
Enzyme I, or a member of an Enzyme II complex) are frequently
posttranslationally
modified upon the presence of a sufficient quantity of the sugar in the cell,
such that
they are no longer able to import that sugar. While this quantity of sugar at
which the
transport system is shut off may be sufficient to sustain the normal
functioning of the
cell, it may be limiting for the overproduction of the desired fine chemical.
Thus, it may
be desirable to modify the PTS proteins of the invention such that they are no
longer
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responsive to such negative regulation, thereby permitting greater
intracellular
concentrations of one or more sugars to be achieved, and, by extension, more
efficient
production or greater yields of one or more fine chemicals from organisms
containing
such mutant PTS proteins.
This invention provides novel nucleic acid molecules which encode proteins,
referred to herein as phosphoenolpyruvate:sugar phosphotransferase system
(PTS)
proteins, which are capable of, for example, participating in the import of
high-energy
carbon molecules (e.g., glucose, fructose, or sucrose) into C. glutamicum,
and/or of
participating in one or more C. glutamicum intracellular signal transduction
pathways.
Nucleic acid molecules encoding a PTS protein are referred to herein as PTS
nucleic
acid molecules. In a preferred embodiment, the PTS protein participates in the
import of
high-energy carbon molecules (e.g., glucose, fructose, or sucrose) into C.
glulamicum,
and also may participate in one or more C. glutamicum intracellular signal
transduction
pathways. Examples of such proteins include those encoded by the genes set
forth in
Table 1.
Accordingly, one aspect of the invention pertains to isolated nucleic acid
molecules (e.g., cDNAs, DNAs, or RNAs) comprising a nucleotide sequence
encoding a
PTS protein or biologically active portions thereof, as well as nucleic acid
fragments
suitable as primers or hybridization probes for the detection or amplification
of PTS-
encoding nucleic acid (e.g., DNA or mRNA). In particularly preferred
embodiments,
the isolated nucleic acid molecule comprises one of the nucleotide sequences
set forth in
as the odd-numbered SEQ ID NOs in the Sequence Listing (e.g., SEQ ID NO: 1,
SEQ ID
NO:3, SEQ ID NO:5, SEQ ID NO:7....) or the coding region or a complement
thereof
of one of these nucleotide sequences. In other particularly preferred
embodiments, the
isolated nucleic acid molecule of the invention comprises a nucleotide
sequence which
hybridizes to or is at least about 50%, preferably at least about 60%, more
preferably at
least about 70%, 80% or 90%, and even more preferably at least about 95%, 96%,
97%,
98%, 99% or more homologous to a nucleotide sequence set forth, as an odd-
numbered
SEQ ID NO in the Sequence Listing (e.g., SEQ ID NO: 1, SEQ ID NO:3, SEQ ID
NO:5,
SEQ ID NO:7....), or a portion thereof. In other preferred embodiments, the
isolated
nucleic acid molecule encodes one of the amino acid sequences set forth in as
an even-
numbered SEQ ID NO in the Sequence Listing (e.g., SEQ ID NO:2, SEQ ID NO:4,
SEQ
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ID NO:6, SEQ ID NO:8....). The preferred PTS proteins of the present invention
also
preferably possess at least one of the PTS activities described herein.
In another embodiment, the isolated nucleic acid molecule encodes a protein or
portion thereof wherein the protein or portion thereof includes an amino acid
sequence
which is sufficiently homologous to an amino acid sequence of the invention
(e.g., a
sequence having an even-numbered SEQ ID NO: in the Sequence Listing), e.g.,
sufficiently homologous to an amino acid sequence of the invention such that
the protein
or portion thereof maintains a PTS activity. Preferably, the protein or
portion thereof
encoded by the nucleic acid molecule maintains the ability to participate in
the import of
high-energy carbon molecules (e.g., glucose, fructose, or sucrose) into C.
glutamicum,
andlor to participate in one or more C. glutamicum intracellular signal
transduction
pathways. In one embodiment, the protein encoded by the nucleic acid molecule
is at
least about 50%, preferably at least about 60%, and more preferably at least
about 70%,
80%, or 90% and most preferably at least about 95%, 96%, 97%, 98%, or 99% or
more
homologous to an amino acid sequence of the invention (e.g., an entire amino
acid
sequence selected from those having an even-numbered SEQ ID NO in the Sequence
Listing). In another preferred embodiment, the protein is a full length C.
glutamicum
protein which is substantially homologous to an entire amino acid sequence of
the
invention (encoded by an open reading frame shown in the corresponding odd-
numbered
SEQ ID NOs in the Sequence Listing (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:5, SEQ ID NO:7....).
In another preferred embodiment, the isolated nucleic acid molecule is derived
from C. glutamicum and encodes a protein (e.g., a PTS fusion protein) which
includes a
biologically active domain which is at least about 50% or more homologous to
one of
the amino acid sequences of the invention (e.g., a sequence of one of the even-
numbered
SEQ ID NOs in the Sequence Listing) and is able to participate in the import
of high-
energy carbon molecules (e.g., glucose, fructose, or sucrose) into C.
glutamicum, and/or
to participate in one or more C. glutamicum intracellular signal transduction
pathways,
or possesses one or more of the activities set forth in Table 1, and which
also includes
heterologous nucleic acid sequences encoding a heterologous polypeptide or
regulatory
regions.
CA 02590625 2007-06-18
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In another embodiment, the isolated nucleic acid molecule is at least 15
nucleotides in length and hybridizes under stringent conditions to a nucleic
acid
molecule comprising a nucleotide sequence of the invention (e.g., a sequence
of an odd-
numbered SEQ ID NO in the Sequence Listing). Preferably, the isolated nucleic
acid
molecule corresponds to a naturally-occun ing nucleic acid molecule. More
preferably,
the isolated nucleic acid encodes a naturally-occurring C. glutamicum PTS
protein, or a
biologically active portion thereof.
Another aspect of the invention pertains to vectors, e.g., recombinant
expression
vectors, containing the nucleic acid molecules of the invention, and host
cells into which
such vectors have been introduced. In one embodiment, such a host cell is used
to
produce a PTS protein by culturing the host cell in a suitable medium. The PTS
protein
can be then isolated from the medium or the host cell.
Yet another aspect of the invention pertains to a genetically altered
microorganism in which a PTS gene has been introduced or altered. In one
embodiment, the genome of the microorganism has been altered by the
introduction of a
nucleic acid molecule of the invention encoding wild-type or mutated PTS
sequence as a
transgene. In another embodiment, an endogenous PTS gene within the genome of
the
microorganism has been altered, e.g., functionally disrupted, by homologous
recombination with an altered PTS gene. In another embodiment, an endogenous
or
introduced PTS gene in a microorganism has been altered by one or more point
mutations, deletions, or inversions, but still encodes a functional PTS
protein. In still
another embodiment, one or more of the regulatory regions (e.g., a promoter,
repressor,
or inducer) of a PTS gene in a microorganism has been altered (e.g., by
deletion,
truncation, inversion, or point mutation) such that the expression of the PTS
gene is
modulated. In a preferred embodiment, the microorganism belongs to the genus
Corynebacterium or Brevibacterium, with Corynebacterium glutamicum being
particularly preferred. In a preferred embodiment, the microorganism is also
utilized for
the production of a desired compound, such as an amino acid, with lysine being
particularly preferred.
In another aspect, the invention provides a method of identifying the presence
or
activity of Cornyebacterium diphtheriae in a subject. This method includes
detection of
one or more of the nucleic acid or amino acid sequences of the invention
(e.g., the
CA 02590625 2007-06-18
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sequences set forth in the Sequence Listing as SEQ ID NOs I through 34)) in a
subject,
thereby detecting the presence or activity of Corynebacterium diphtheriae in
the subject.
Still another aspect of the invention pertains to an isolated PTS protein or a
portion, e.g., a biologically active portion, thereof. In a preferred
embodiment, the
isolated PTS protein or portion thereof can participate in the import of high-
energy
carbon molecules (e.g., glucose, fructose, or sucrose) into C. glutamicum, and
also may
participate in one or more C. glutamicum intracellular signal transduction
pathways. In
another preferred embodiment, the isolated PTS protein or portion thereof is
sufficiently
homologous to an amino acid sequence of the invention (e.g., a sequence of an
even-
numbered SEQ ID NO: in the Sequence Listing) such that the protein or portion
thereof
maintains the ability to participate in the import of high-energy carbon
molecules (e.g.,
glucose, fructose, or sucrose) into C. glutamicum, and /or to participate in
one or more
C. glutamicum intracellular signal transduction pathways.
The invention also provides an isolated preparation of a PTS protein. In
preferred embodiments, the PTS protein comprises an amino acid sequence of the
invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence
Listing).
In another preferred embodiment, the invention pertains to an isolated full
length protein
which is substantially homologous to an entire amino acid sequence of the
invention
(e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing)
(encoded
by an open reading frame set in a corresponding odd-numbered SEQ ID NO: of the
Sequence Listing). In yet another embodiment, the protein is at least about
50%,
preferably at least about 60%, and more preferably at least about 70%, 80%, or
90%,
and most preferably at least about 95%, 96%, 97%, 98%, or 99% or more
homologous
to an entire amino acid sequence of the invention (e.g., a sequence of an even-
numbered
SEQ ID NO: of the Sequence Listing). In other embodiments, the isolated PTS
protein
comprises an amino acid sequence which is at least about 50% or more
homologous to
one of the amino acid sequences of the invention (e.g., a sequence of an even-
numbered
SEQ ID NO: of the Sequence Listing) and is able to participate in the import
of high-
energy carbon molecules (e.g., glucose, fructose, or sucrose) into C.
glutamicum, and/or
to participate in one or more C. glutamicum intracellular signal transduction
pathways,
or has one or more of the activities set forth in Table 1.
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Alternatively, the isolated PTS protein can comprise an amino acid sequence
which is encoded by a nucleotide sequence which hybridizes, e.g., hybridizes
under
stringent conditions, or is at least about 50%, preferably at least about 60%,
more
preferably at least about 70%, 80%, or 90%, and even more preferably at least
about
95%, 96%, 97%, 98,%, or 99% or more homologous, to a nucleotide sequence of
one of
the even-numbered SEQ ID NOs set forth in the Sequence Listing. It is also
preferred
that the preferred forms of PTS proteins also have one or more of the PTS
bioactivities
described herein.
The PTS polypeptide, or a biologically active portion thereof, can be
operatively
linked to a non-PTS polypeptide to form a fusion protein. In preferred
embodiments,
this fusion protein has an activity which differs from that of the PTS protein
alone. In
other preferred embodiments, this fusion protein results in increased yields,
production,
and/or efficiency of production of a desired fine chemical from C. glutamicum.
In
particularly preferred embodiments, integration of this fusion protein into a
host cell
modulates the production of a desired compound from the cell.
In another aspect, the invention provides methods for screening molecules
which
modulate the activity of a PTS protein, either by interacting with the protein
itself or a
substrate or binding partner of the PTS protein, or by modulating the
transcription or
translation of a PTS nucleic acid molecule of the invention.
Another aspect of the invention pertains to a method for producing a fine
chemical. This method involves the culturing of a cell containing a vector
directing the
expression of a PTS nucleic acid molecule of the invention, such that a fine
chemical is
produced. In a preferred embodiment, this method further includes the step of
obtaining
a cell containing such a vector, in which a cell is transfected with a vector
directing the
expression of a PTS nucleic acid. In another preferred embodiment, this method
further
includes the step of recovering the fine chemical from the culture. In a
particularly
preferred embodiment, the cell is from the genus Corynebacterium or
Brevibacterium,
or is selected from those strains set forth in Table 3.
Another aspect of the invention pertains to methods for modulating production
of
a molecule from a microorganism. Such methods include contacting the cell with
an
agent which modulates PTS protein activity or PTS nucleic acid expression such
that a
cell associated activity is altered relative to this same activity in the
absence of the
CA 02590625 2007-06-18
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agent. In a preferred embodiment, the cell is modulated for the uptake of one
or more
sugars, such that the yields or rate of production of a desired fine chemical
by this
microorganism is improved. The agent which modulates PTS protein activity can
be an
agent which stimulates PTS protein activity or PTS nucleic acid expression.
Examples
of agents which stimulate PTS protein activity or PTS nucleic acid expression
include
small molecules, active PTS proteins, and nucleic acids encoding PTS proteins
that have
been introduced into the cell. Examples of agents which inhibit PTS activity
or
expression include small molecules, and antisense PTS nucleic acid molecules.
Another aspect of the invention pertains to methods for modulating yields of a
desired compound from a cell, involving the introduction of a wild-type or
mutant PTS
gene into a cell, either maintained on a separate plasmid or integrated into
the genome of
the host cell. If integrated into the genome, such integration can random, or
it can take
place by homologous recombination such that the native gene is replaced by the
introduced copy, causing the production of the desired compound from the cell
to be
modulated. In a preferred embodiment, said yields are increased. In another
preferred
embodiment, said chemical is a fine chemical. In a particularly preferred
embodiment,
said fine chemical is an amino acid. In especially prefened embodiments, said
amino
acid is L-lysine.
Detailed Description of the Invention
The present invention provides PTS nucleic acid and protein molecules which
are involved in the uptake of high-energy carbon molecules (e.g., sucrose,
fructose, or
glucose) into C. glutamicum, and may also participate in intracellular signal
transduction
pathways in this microorganism. The molecules of the invention may be utilized
in the
modulation of production of fine chemicals from microorganisms. Such
modulation may
be due to increased intracellular levels of high-energy molecules needed to
produce,
e.g., ATP, GTP and other molecules utilized to drive energetically unfavorable
biochemical reactions in the cell, such as the biosynthesis of a fine
chemical. This
modulation of fine chemical production may also be due to the fact that the
breakdown
products of many sugars serve as intermediates or precursors for other
biosynthetic
pathways, including those of certain fine chemicals. Further, PTS proteins are
known to
participate in certain intracellular signal transduction pathways which may
have
CA 02590625 2007-06-18
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regulatory activity for one or more fine chemical metabolic pathways; by
manipulating
these PTS proteins, one may thereby activate a fine chemical biosynthetic
pathways or
repress a fine chemical degradation pathway. Aspects of the invention are
further
explicated below.
I. Fine Chemicals
The term 'fine chemical' is art-recognized and includes molecules produced by
an organism which have applications in various industries, such as, but not
limited to,
the pharmaceutical, agriculture, and cosmetics industries. Such compounds
include
organic acids, such as tartaric acid, itaconic acid, and diaminopimelic acid,
both
proteinogenic and non-proteinogenic amino acids, purine and pyrimidine bases,
nucleosides, and nucleotides (as described e.g. in Kuninaka, A. (1996)
Nucleotides and
related compounds, p. 561-612, in Biotechnology vol. 6, Rehm et al., eds. VCH:
Weinheim, and references contained therein), lipids, both saturated and
unsaturated fatty
acids (e.g., arachidonic acid), diols (e.g., propane diol, and butane diol),
carbohydrates
(e.g., hyaluronic acid and trehalose), aromatic compounds (e.g., aromatic
amines,
vanillin, and indigo), vitamins and cofactors (as described in Ullmann's
Encyclopedia of
Industrial Chemistry, vol. A27, "Vitamins", p. 443-613 (1996) VCH: Weinheim
and
references therein; and Ong, A.S., Niki, E. & Packer, L. (1995) "Nutrition,
Lipids,
Health, and Disease" Proceedings of the UNESCO/Confederation of Scientific and
Technological Associations in Malaysia, and the Society for Free Radical
Research -
Asia, held Sept. 1-3, 1994 at Penang, Malaysia, AOCS Press, (1995)), enzymes,
polyketides (Cane et al. (1998) Science 282: 63-68), and all other chemicals
described in
Gutcho (1983) Chemicals by Fermentation, Noyes Data Corporation, ISBN:
0818805086 and references therein. The metabolism and uses of certain of these
fine
chemicals are further explicated below.
A. Amino Acid Metabolism and Uses
Amino acids comprise the basic structural units of all proteins, and as such
are
essential for normal cellular functioning in all organisms. The term "amino
acid" is art-
recognized. The proteinogenic amino acids, of which there are 20 species,
serve as
structural units for proteins, in which they are linked by peptide bonds,
while the
CA 02590625 2007-06-18
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nonproteinogenic amino acids (hundreds of which are known) are not normally
found in
proteins (see Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97
VCH:
Weinheim (1985)). Amino acids may be in the D- or L- optical configuration,
though L-
amino acids are generally the only type found in naturally-occurring proteins.
Biosynthetic and degradative pathways of each of the 20 proteinogenic amino
acids
have been well characterized in both prokaryotic and eukaryotic cells (see,
for example,
Stryer, L. Biochemistry, 3d edition, pages 578-590 (1988)). The 'essential'
amino acids
(histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine,
tryptophan,
and valine), so named because they are generally a nutritional requirement due
to the
complexity of their biosyntheses, are readily converted by simple biosynthetic
pathways
to the remaining 11 'nonessential' amino acids (alanine, arginine, asparagine,
aspartate,
cysteine, glutamate, glutamine, glycine, proline, serine, and tyrosine).
Higher animals
do retain the ability to synthesize some of these amino acids, but the
essential amino
acids must be supplied from the diet in order for normal protein synthesis to
occur.
Aside from their function in protein biosynthesis, these amino acids are
interesting chemicals in their own right, and many have been found to have
various
applications in the food, feed, chemical, cosmetics, agriculture, and
pharmaceutical
industries. Lysine is an important amino acid in the nutrition not only of
humans, but
also of monogastric animals such as poultry and swine. Glutamate is most
commonly
used as a flavor additive (mono-sodium glutamate, MSG) and is widely used
throughout
the food industry, as are aspartate, phenylalanine, glycine, and cysteine.
Glycine, L-
methionine and tryptophan are all utilized in the pharmaceutical industry.
Glutamine,
valine, leucine, isoleucine, histidine, arginine, proline, serine and alanine
are of use in
both the pharmaceutical and cosmetics industries. Threonine, tryptophan, and
D/ L-
methionine are common feed additives. (Leuchtenberger, W. (1996) Amino aids -
technical production and use, p. 466-502 in Rehm et al. (eds.) Biotechnology
vol. 6,
chapter 14a, VCH: Weinheim). Additionally, these amino acids have been found
to be
useful as precursors for the synthesis of synthetic amino acids and proteins,
such as N-
acetylcysteine, S-carboxymethyl-L-cysteine, (S)-5-hydroxytryptophan, and
others
described in Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97,
VCH:
Weinheim, 1985.
CA 02590625 2007-06-18
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The biosynthesis of these natural amino acids in organisms capable of
producing them, such as bacteria, has been well characterized (for review of
bacterial
amino acid biosynthesis and regulation thereof, see Umbarger, H.E.(1978) Ann.
Rev.
Biochem. 47: 533-606). Glutamate is synthesized by the reductive amination of
a-
ketoglutarate, an intermediate in the citric acid cycle. Glutamine, proline,
and arginine
are each subsequently produced from glutamate. The biosynthesis of serine is a
three-
step process beginning with 3-phosphoglycerate (an intermediate in
glycolysis), and
resulting in this amino acid after oxidation, transamination, and hydrolysis
steps. Both
cysteine and glycine are produced from serine; the former by the condensation
of
homocysteine with serine, and the latter by the transferal of the side-chain 0-
carbon
atom to tetrahydrofolate, in a reaction catalyzed by serine
transhydroxymethylase.
Phenylalanine, and tyrosine are synthesized from the glycolytic and pentose
phosphate
pathway precursors erythrose 4-phosphate and phosphoenolpyruvate in a 9-step
biosynthetic pathway that differ only at the final two steps after synthesis
of prephenate.
Tryptophan is also produced from these two initial molecules, but its
synthesis is an 11-
step pathway. Tyrosine may also be synthesized from phenylalanine, in a
reaction
catalyzed by phenylalanine hydroxylase. Alanine, valine, and leucine are all
biosynthetic products of pyruvate, the final product of glycolysis. Aspartate
is formed
from oxaloacetate, an intermediate of the citric acid cycle. Asparagine,
methionine,
threonine, and lysine are each produced by the conversion of aspartate.
Isoleucine is
formed from threonine. A complex 9-step pathway results in the production of
histidine
from 5-phosphoribosyl-l-pyrophosphate, an activated sugar.
Amino acids in excess of the protein synthesis needs of the cell cannot be
stored,
and are instead degraded to provide intermediates for the major metabolic
pathways of
the cell (for review see Stryer, L. Biochemistry 3d ed. Ch. 21 "Amino Acid
Degradation
and the Urea Cycle" p. 495-516 (1988)). Although the cell is able to convert
unwanted
amino acids into useful metabolic intermediates, amino acid production is
costly in
terms of energy, precursor molecules, and the enzymes necessary to synthesize
them.
Thus it is not surprising that amino acid biosynthesis is regulated by
feedback inhibition,
in which the presence of a particular amino acid serves to slow or entirely
stop its own
production (for overview of feedback mechanisms in amino acid biosynthetic
pathways,
see Stryer, L. Biochemistry, 3'd ed. Ch. 24: "Biosynthesis of Amino Acids and
Heme" p.
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575-600 (1988)). Thus, the output of any particular amino acid is limited by
the amount
of that amino acid present in the cell.
B. Vitamin, Cofactor, and Nutraceutical Metabolism and Uses
Vitamins, cofactors, and nutraceuticals comprise another group of molecules
which the higher animals have lost the ability to synthesize and so must
ingest, although
they are readily synthesized by other organisms, such as bacteria. These
molecules are
either bioactive substances themselves, or are precursors of biologically
active
substances which may serve as electron carriers or intermediates in a variety
of
metabolic pathways. Aside from their nutritive value, these compounds also
have
significant industrial value as coloring agents, antioxidants, and catalysts
or other
processing aids. (For an overview of the structure, activity, and industrial
applications
of these compounds, see, for example, Ullman's Encyclopedia of Industrial
Chemistry,
"Vitamins" vol. A27, p. 443-613, VCH: Weinheim, 1996.) The term "vitamin" is
art-
recognized, and includes nutrients which are required by an organism for
normal
functioning, but which that organism cannot synthesize by itself. The group of
vitamins
may encompass cofactors and nutraceutical compounds. The language "cofactor"
includes nonproteinaceous compounds required for a nonmal enzymatic activity
to
occur. Such compounds may be organic or inorganic; the cofactor molecules of
the
invention are preferably organic. The term "nutraceutical" includes dietary
supplements
having health benefits in plants and animals, particularly humans. Examples of
such
molecules are vitamins, antioxidants, and also certain lipids (e.g.,
polyunsaturated fatty
acids).
The biosynthesis of these molecules in organisms capable of producing them,
such as bacteria, has been largely characterized (Ullman's Encyclopedia of
Industrial
Chemistry, "Vitamins" vol. A27, p. 443-613, VCH: Weinheim, 1996; Michal, G.
(1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John
Wiley
& Sons; Ong, A.S., Niki, E. & Packer, L. (1995) "Nutrition, Lipids, Health,
and
Disease" Proceedings of the UNESCO/Confederation of Scientific and
Technological
Associations in Malaysia, and the Society for Free Radical Research - Asia,
held Sept.
1-3, 1994 at Penang, Malaysia, AOCS Press: Champaign, IL X, 374 S).
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Thiamin (vitamin B1) is produced by the chemical coupling of pyrimidine and
thiazole moieties. Riboflavin (vitamin BZ) is synthesized from guanosine-5'-
triphosphate
(GTP) and ribose-5'-phosphate. Riboflavin, in tunn, is utilized for the
synthesis of flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The family of
compounds collectively termed 'vitamin B6' (e.g., pyridoxine, pyridoxamine,
pyridoxa-
5'-phosphate, and the commercially used pyridoxin hydrochloride) are all
derivatives of
the common structural unit, 5-hydroxy-6-methylpyridine. Pantothenate
(pantothenic
acid, (R)-(+)-N-(2,4-dihydroxy-3,3-dimethyl-l-oxobutyl)-o-alanine) can be
produced
either by chemical synthesis or by fermentation. The final steps in
pantothenate
biosynthesis consist of the ATP-driven condensation of 0-alanine and pantoic
acid. The
enzymes responsible for the biosynthesis steps for the conversion to pantoic
acid, to (3-
alanine and for the condensation to panthotenic acid are known. The
metabolically
active form of pantothenate is Coenzyme A, for which the biosynthesis proceeds
in 5
enzymatic steps. Pantothenate, pyridoxal-5'-phosphate, cysteine and ATP are
the
precursors of Coenzyme A. These enzymes not only catalyze the formation of
panthothante, but also the production of (R)-pantoic acid, (R)-pantolacton,
(R)-
panthenol (provitamin B5), pantetheine (and its derivatives) and coenzyme A.
Biotin biosynthesis from the precursor molecule pimeloyl-CoA in
microorganisms has been studied in detail and several of the genes involved
have been
identified. Many of the corresponding proteins have been found to also be
involved in
Fe-cluster synthesis and are members of the nifS class of proteins. Lipoic
acid is
derived from octanoic acid, and serves as a coenzyme in energy metabolism,
where it
becomes part of the pyruvate dehydrogenase complex and the a-ketoglutarate
dehydrogenase complex. The folates are a group of substances which are all
derivatives
of folic acid, which is turn is derived from L-glutamic acid, p-amino-benzoic
acid and 6-
methylpterin. The biosynthesis of folic acid and its derivatives, starting
from the
metabolism intermediates guanosine-5'-triphosphate (GTP), L-glutamic acid and
p-
amino-benzoic acid has been studied in detail in certain microorganisms.
Corrinoids (such as the cobalamines and particularly vitamin B12) and
porphyrines belong to a group of chemicals characterized by a tetrapyrole ring
system.
The biosynthesis of vitamin B12 is sufficiently complex that it has not yet
been
completely characterized, but many of the enzymes and substrates involved are
now
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known. Nicotinic acid (nicotinate), and nicotinamide are pyridine derivatives
which are
also termed 'niacin'. Niacin is the precursor of the important coenzymes NAD
(nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine
dinucleotide
phosphate) and their reduced forms.
The large-scale production of these compounds has largely relied on cell-free
chemical syntheses, though some of these chemicals have also been produced by
large-
scale culture of microorganisms, such as riboflavin, Vitamin B6, pantothenate,
and
biotin. Only Vitamin B12 is produced solely by fermentation, due to the
complexity of
its synthesis. In vitro methodologies require significant inputs of materials
and time,
often at great cost.
C. Purine, Pyrimidine, Nucleoside and Nucleotide Metabolism and Uses
Purine and pyrimidine metabolism genes and their corresponding proteins are
important targets for the therapy of tumor diseases and viral infections. The
language
"purine" or "pyrimidine" includes the nitrogenous bases which are constituents
of
nucleic acids, co-enzymes, and nucleotides. The term "nucleotide" includes the
basic
structural units of nucleic acid molecules, which are comprised of a
nitrogenous base, a
pentose sugar (in the case of RNA, the sugar is ribose; in the case of DNA,
the sugar is
D-deoxyribose), and phosphoric acid. The language "nucleoside" includes
molecules
which serve as precursors to nucleotides, but which are lacking the phosphoric
acid
moiety that nucleotides possess. By inhibiting the biosynthesis of these
molecules, or
their mobilization to form nucleic acid molecules, it is possible to inhibit
RNA and DNA
synthesis; by inhibiting this activity in a fashion targeted to cancerous
cells, the ability
of tumor cells to divide and replicate may be inhibited. Additionally, there
are
nucleotides which do not form nucleic acid molecules, but rather serve as
energy stores
(i.e., AMP) or as coenzymes (i.e., FAD and NAD).
Several publications have described the use of these chemicals for these
medical
indications, by influencing purine and/or pyrimidine metabolism (e.g.
Christopherson,
R.I. and Lyons, S.D. (1990) "Potent inhibitors of de novo pyrimidine and
purine
biosynthesis as chemotherapeutic agents." Med. Res. Reviews 10: 505-548).
Studies of
enzymes involved in purine and pyrimidine metabolism have been focused on the
development of new drugs which can be used, for example, as immunosuppressants
or
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anti-proliferants (Smith, J.L., (1995) "Enzymes in nucleotide synthesis."
Curr. Opin.
Struct. Biol. 5: 752-757; (1995) Biochem Soc. Transact. 23: 877-902). However,
purine
and pyrimidine bases, nucleosides and nucleotides have other utilities: as
intermediates
in the biosynthesis of several fine chemicals (e.g., thiamine, S-adenosyl-
methionine,
folates, or riboflavin), as energy carriers for the cell (e.g., ATP or GTP),
and for
chemicals themselves, commonly used as flavor enhancers (e.g., IMP or GMP) or
for
several medicinal applications (see, for example, Kuninaka, A. (1996)
Nucleotides and
Related Compounds in Biotechnology vol. 6, Rehm et al., eds. VCH: Weinheim, p.
561-
612). Also, enzymes involved in purine, pyrimidine, nucleoside, or nucleotide
metabolism are increasingly serving as targets against which chemicals for
crop
protection, including fungicides, herbicides and insecticides, are developed.
The metabolism of these compounds in bacteria has been characterized (for
reviews see, for example, Zalkin, H. and Dixon, J.E. (1992) "de novo purine
nucleotide
biosynthesis", in: Progress in Nucleic Acid Research and Molecular Biology,
vol. 42,
Academic Press:, p. 259-287; and Michal, G. (1999) "Nucleotides and
Nucleosides",
Chapter 8 in: Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology,
Wiley: New York). Purine metabolism has been the subject of intensive
research, and is
essential to the normal functioning of the cell. Impaired purine metabolism in
higher
animals can cause severe disease, such as gout. Purine nucleotides are
synthesized from
ribose-5-phosphate, in a series of steps through the intermediate compound
inosine-5'-
phosphate (IMP), resulting in the production of guanosine-5'-monophosphate
(GMP) or
adenosine-5'-monophosphate (AMP), from which the triphosphate forms utilized
as
nucleotides are readily formed. These compounds are also utilized as energy
stores, so
their degradation provides energy for many different biochemical processes in
the cell.
Pyrimidine biosynthesis proceeds by the formation of uridine-5'-monophosphate
(UMP)
from ribose-5-phosphate. UMP, in turn, is converted to cytidine-5'-
triphosphate (CTP).
The deoxy- forms of all of these nucleotides are produced in a one step
reduction
reaction from the diphosphate ribose form of the nucleotide to the diphosphate
deoxyribose form of the nucleotide. Upon phosphorylation, these molecules are
able to
participate in DNA synthesis.
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D. Trehalose Metabolism and Uses
Trehalose consists of two glucose molecules, bound in a, a-1,1 linkage. It is
commonly used in the food industry as a sweetener, an additive for dried or
frozen
foods, and in beverages. However, it also has applications in the
pharmaceutical,
cosmetics and biotechnology industries (see, for example, Nishimoto et al.,
(1998) U.S.
Patent No. 5,759,610; Singer, M.A. and Lindquist, S. (1998) Trends Biotech.
16: 460-
467; Paiva, C.L.A. and Panek, A.D. (1996) Biotech. Ann. Rev. 2: 293-314; and
Shiosaka, M. (1997) J. Japan 172: 97-102). Trehalose is produced by enzymes
from
many microorganisms and is naturally released into the surrounding medium,
from
which it can be collected using methods known in the art.
II. The Phosphoenolpyruvate: Sugar Phosphotransferase System
The ability of cells to grow and divide rapidly in culture is to a great
degree
dependent on the extent to which the cells are able to take up and utilize
high energy
molecules, such as glucose and other sugars. Different transporter proteins
exist to
transport different carbon sources into the cell. There are transport proteins
for sugars,
such as glucose, fructose, mannose, galactose, ribose, sorbose, ribulose,
lactose, maltose,
sucrose, or raffinose, and also transport proteins for starch or cellulose
degradation
products. Other transport systems serve to import alcohols (e.g., methanol or
ethanol),
alkanes, fatty acids and organic acids like acetic acid or lactic acid. In
bacteria, sugars
may be transported into the cell across the cellular membrane by a variety of
mechanisms. Aside from the symport of sugars with protons, one of the most
commonly utilized processes for sugar uptake is the bacterial
phosphoenolpyruvate:
sugar phosphotransferase system (PTS). This system not only catalyzes the
translocation (with concomitant phosphorylation) of sugars and hexitols, but
it also
regulates cellular metabolism in response to the availability of
carbohydrates. Such PTS
systems are ubiquitous in bacteria but do not occur in archaebacteria or
eukaryotes.
Functionally, the PTS system consists of two cytoplasmic proteins, Enzyme I
and HPr, and a variable number of sugar-specific integral and peripheral
membrane
transport complexes (each termed 'Enzyme 11' with a sugar-specific subscript,
e.g.,
'Enzyme IIG'Oi for the Enzyme II complex which binds glucose). Enzymes II
specific
for mono-, di-, or oligosaccharides, like glucose, fructose, mannose,
galactose, ribose,
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sorbose, ribulose, lactose, maltose, sucrose, raffinose, and others are known.
Enzyme I
transfers phosphoryl groups from phosphoenolpyruvate (PEP) to the phosphoryl
carrier
protein, HPr. HPr then transfers the phosphoryl groups to the different Enzyme
II
transport complexes. While the amino acid sequences of Enzyme I and HPr are
quite
similar in all bacteria, the sequences for PTS transporters can be grouped
into
structurally unrelated families. Further, the number and homology between
these genes
vary from bacteria to bacteria. The E. colf genome encodes 38 different PTS
proteins,
33 of which are subunits belonging to 22 different transporters. The M.
genitalium
genome contains one gene each for Enzyme I and HPr, and only two genes for PTS
transporters. The genomes of T. palladium and C. trachomatis contain genes for
Enzyme I- and HPr-like proteins but no PTS transporters.
All PTS transporters consist of three functional units, IIA, IIB, and IIC,
which
occur either as protein subunits in a complex (e.g., IIAGI'IICBC;") or as
domains of a
single polypeptide chain (e.g., IICBAci'NA') IIA and IIB sequentially transfer
phosphoryl groups from HPr to the transported sugars. IIC contains the sugar
binding
site, and spans the inner membrane six or eight times. Sugar translocation is
coupled to
the transient phosphorylation of the IIB domain. Enzyme I, HPr, and IIA are
phosphorylated at histidine residues, while IIB subunits are phosphorylated at
either
cysteine or histidine residues, depending on the particular transporter
involved.
Phosphorylation of the sugar being imported has the advantage of blocking the
diffusion
of the sugar back through the cellular membrane to the extracellular medium,
since the
charged phosphate group cannot readily traverse the hydrophobic core of the
membrane.
Some PTS proteins play a role in intracellular signal transduction in addition
to
their function in the active transport of sugars. These subunits regulate
their targets
either allosterically, or by phosphorylation. Their regulatory activity varies
with the
degree of their phosphorylation (i.e., the ratio of the non-phosphorylated to
the
phosphorylated form), which in turn varies with the ratio of sugar-dependent
dephosphorylation and phosphoenolpyruvate-dependent rephosphorylation.
Examples
of such intracellular regulation by PTS proteins in E. coli include the
inhibition of
glycerol kinase by dephosphorylated IIAGIO, and the activation of adenylate
cyclase by
the phosphorylated version of this protein. Also, the HPr and the IIB domains
of some
transporters in these microorganisms regulate gene expression by reversible
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phosphorylation of transcription antiterminators. In gram-positive bacteria,
the activity
of HPr is modulated by HPr-specific serine kinases and phosphatases. For
example, HPr
phosphorylated at serine-46 functions as a co-repressor of the transcriptional
repressor
CcpA. Lastly, it has been found that unphosphorylated Enzyme I inhibits the
sensor
kinase CheA of the bacterial chemotaxis machinery, providing a direct link
between the
sugar binding and transport systems of the bacterium and those systems
governing
movement of the bacterium (Sonenshein, A. L., et al., eds. Bacillus subtilis
and other
gram-positive bacteria. ASM: Washington, D.C.; Neidhardt, F.C., et al., eds.
(1996)
Escherichia coli and Salmonella. ASM Press: Washington, D.C.; Lengeler et al.,
(1999).
Biology of Prokaryotes. Section II, pp. 68-87, Thieme Verlag; Stuttgart).
III. Elements and Methods of the Invention
The present invention is based, at least in part, on the discovery of novel
molecules, referred to herein as PTS nucleic acid and protein molecules, which
participate in the uptake of high-energy carbon molecules (e.g., glucose,
sucrose, and
fructose) into C. glutamicum, and may also participate in one or more
intracellular signal
transduction pathways in these microorganisms. In one embodiment, the PTS
molecules
function to import high-energy carbon molecules into the cell, where the
energy
produced by their degradation may be utilized to power less energetically
favorable
biochemical reactions, and their degradation products may serve as
intermediates and
precursors for a number of other metabolic pathways. In another embodiment,
the PTS
molecules may participate in one or more intracellular signal transduction
pathways,
wherein the presence of a modified form of a PTS molecule (e.g., a
phosphorylated PTS
protein) may participate in a signal transduction cascade which regulates one
or more
cellular processes. In a preferred embodiment, the activity of the PTS
molecules of the
present invention has an impact on the production of a desired fine chemical
by this
organism. In a particularly preferred embodiment, the PTS molecules of the
invention
are modulated in activity, such that the yield, production or efficiency of
production of
one or more fine chemicals from C. glulamicum is also modulated.
The language, "PTS protein" or "PTS polypeptide" includes proteins which
participate in the uptake of one or more high-energy carbon compounds (e.g.,
mono-, di,
or oligosaccharides, such as fructose, mannose, sucrose, glucose, raffinose,
galactose,
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ribose, lactose, maltose, and ribulose) from the extracellular medium to the
interior of
the cell. Such PTS proteins may also participate in one or more intracellular
signal
transduction pathways, such as, but not limited to, those goveming the uptake
of
different sugars into the cell. Examples of PTS proteins include those encoded
by the
PTS genes set forth in Table 1 and by the odd-numbered SEQ ID NOs. For general
references pertaining to the PTS system, see: Stryer, L. (1988) Biochemistry.
Chapter
37: "Membrane Transport", W.H. Freeman: New York, p. 959-961; Darnell, J. et
al.
(1990) Molecular Cell Biology Scientific American Books: New York, p. 552-553,
and
Michal, G., ed. (1999) Biochemical Pathways: An Atlas of Biochemistry and
Molecular
Biology, Chapter 15 "Special Bacterial Metabolism". The terms "PTS gene" or
"PTS
nucleic acid sequence" include nucleic acid sequences encoding a PTS protein,
which
consist of a coding region and also corresponding untranslated 5' and 3'
sequence
regions. Examples of PTS genes include those set forth in Table 1. The terms
"production" or "productivity" are art-recognized and include the
concentration of the
fermentation product (for example, the desired fine chemical) formed within a
given
time and a given fermentation volume (e.g., kg product per hour per liter),
The term
"efficiency of production" includes the time required for a particular level
of production
to be achieved (for example, how long it takes for the cell to attain a
particular rate of
output of a fine chemical). The term "yield" or "product/carbon yield" is art-
recognized
and includes the efficiency of the conversion of the carbon source into the
product (i.e.,
fine chemical). This is generally written as, for example, kg product per kg
carbon
source. By increasing the yield or production of the compound, the quantity of
recovered molecules, or of useful recovered molecules of that compound in a
given
amount of culture over a given amount of time is increased. The terms
"biosynthesis" or
a "biosynthetic pathway" are art-recognized and include the synthesis of a
compound,
preferably an organic compound, by a cell from intermediate compounds in what
may
be a multistep and highly regulated process. The terms "degradation" or a
"degradation
pathway" are art-recognized and include the breakdown of a compound,
preferably an
organic compound, by a cell to degradation products (generally speaking,
smaller or less
complex molecules) in what may be a multistep and highly regulated process.
The
language "metabolism" is art-recognized and includes the totality of the
biochemical
reactions that take place in an organism. The metabolism of a particular
compound,
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then, (e.g., the metabolism of an amino acid such as glycine) comprises the
overall
biosynthetic, modification, and degradation pathways in the cell related to
this
compound. The language "transport" or "import" is art-recognized and includes
the
facilitated movement of one or more molecules across a cellular membrane
through
which the molecule would otherwise be unable to pass.
In another embodiment, the PTS molecules of the invention are capable of
modulating the production of a desired molecule, such as a fine chemical, in a
microorganism such as C. glutamicum. Using recombinant genetic techniques, one
or
more of the PTS proteins of the invention may be manipulated such that its
function is
modulated. For example, a protein involved in the PTS-mediated import of
glucose may
be altered such that it is optimized in activity, and the PTS system for the
importation of
glucose may thus be able to translocate increased amounts of glucose into the
cell.
Since glucose molecules are utilized not only for energy to drive
energetically
unfavorable biochemical reactions, such as fine chemical biosyntheses, but
also as
precursors and intermediates in a number of fine chemical biosynthetic
pathways (e.g.,
serine is synthesized from 3-phosphoglycerate). In each case, the overall
yield or rate of
production of one of these desired fine chemicals may be increased, either by
increasing
the energy available for such production to occur, or by increasing the
availability of
compounds necessary for such production to take place.
Further, many PTS proteins are known to play key roles in intracellular signal
transduction pathways which regulate cellular metabolism and sugar uptake in
keeping
with the availability of carbon sources. For example, it is known that an
increased
intracellular level of fructose 1,6-bisphosphate (a compound produced during
glycolysis) results in the phosphorylation of a serine residue on HPr which
prevents this
protein from serving as a phosphoryl donor in any PTS sugar transport process,
thereby
blocking further sugar uptake. By mutagenizing HPr such that this serine
residue cannot
be phosphorylated, one may constitutively activate HPr and thereby increase
sugar
transport into the cell, which in turn will ensure greater intracellular
energy stores and
intermediate/precursor molecules for the biosynthesis of one or more desired
fine
chemicals.
The isolated nucleic acid sequences of the invention are contained within the
genome of a Corynebacterium glutamicum strain available through the American
Type
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Culture Collection, given designation ATCC 13032. The nucleotide sequence of
the
isolated C. glutamicum PTS DNAs and the predicted amino acid sequences of the
C.
glutamicum PTS proteins are shown in the Sequence Listing as odd-numbered SEQ
ID
NOs and even-numbered SEQ ID NOs, respectively.
Computational analyses were performed which classified and/or identified these
nucleotide sequences as sequences which encode metabolic pathway proteins.
The present invention also pertains to proteins which have an amino acid
sequence which is substantially homologous to an amino acid sequence of the
invention
(e.g., the sequence of an even-numbered SEQ ID NO of the Sequence Listing). As
used
herein, a protein which has an amino acid sequence which is substantially
homologous
to a selected amino acid sequence is least about 50% homologous to the
selected amino
acid sequence, e.g., the entire selected amino acid sequence. A protein which
has an
amino acid sequence which is substantially homologous to a selected amino acid
sequence can also be least about 50-60%, preferably at least about 60-70%, and
more
preferably at least about 70-80%, 80-90%, or 90-95%, and most preferably at
least about
96%, 97%, 98%, 99% or more homologous to the selected amino acid sequence.
The PTS protein or a biologically active portion or fragment thereof of the
invention can participate in the transport of high-energy carbon-containing
molecules
such as glucose into C. glutamicum, or can participate in intracellular signal
transduction
in this microorganism, or may have one or more of the activities set forth in
Table 1.
Various aspects of the invention are described in further detail in the
following
subsections:
A. Isolated Nucleic Acid Molecules
One aspect of the invention pertains to isolated nucleic acid molecules that
encode PTS polypeptides or biologically active portions thereof, as well as
nucleic acid
fragments sufficient for use as hybridization probes or primers for the
identification or
amplification of PTS-encoding nucleic acid (e.g., PTS DNA). As used herein,
the term
"nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or
genomic
DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated
using nucleotide analogs. This term also encompasses untranslated sequence
located at
both the 3' and 5' ends of the coding region of the gene: at least about 100
nucleotides
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of sequence upstream from the 5' end of the coding region and at least about
20
nucleotides of sequence downstream from the 3'end of the coding region of the
gene.
The nucleic acid molecule can be single-stranded or double-stranded, but
preferably is
double-stranded DNA. An "isolated" nucleic acid molecule is one which is
separated
from other nucleic acid molecules which are present in the natural source of
the nucleic
acid. Preferably, an "isolated" nucleic acid is free of sequences which
naturally flank
the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic
acid) in the
genomic DNA of the organism from which the nucleic acid is derived. For
example, in
various embodiments, the isolated PTS nucleic acid molecule can contain less
than
about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences
which
naturally flank the nucleic acid molecule in genomic DNA of the cell from
which the
nucleic acid is derived (e.g, a C. glutamicum cell). Moreover, an "isolated"
nucleic acid
molecule, such as a DNA molecule, can be substantially free of other cellular
material,
or culture medium when produced by recombinant techniques, or chemical
precursors or
other chemicals when chemically synthesized.
A nucleic acid molecule of the present invention, e.g., a nucleic acid
molecule
having a nucleotide sequence of an odd-numbered SEQ ID NO of the Sequence
Listing,
or a portion thereof, can be isolated using standard molecular biology
techniques and the
sequence information provided herein_ For example, a C. glulamicum PTS DNA can
be
isolated from a C. glutamicum library using all or portion of one of the odd-
numbered
SEQ ID NO sequences of the Sequence Listing as a hybridization probe and
standard
hybridization techniques (e.g., as described in Sambrook, J., Fritsh, E. F.,
and Maniatis,
T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY,
1989).
Moreover, a nucleic acid molecule encompassing all or a portion of one of the
nucleic
acid sequences of the invention (e.g., an odd-numbered SEQ ID NO of the
Sequence
Listing) can be isolated by the polymerase chain reaction using
oligonucleotide primers
designed based upon this sequence (e.g., a nucleic acid molecule encompassing
all or a
portion of one of the nucleic acid sequences of the invention (e.g., an odd-
numbered
SEQ ID NO of the Sequence Listing) can be isolated by the polymerase chain
reaction
using oligonucleotide primers designed based upon this same sequence). For
example,
mRNA can be isolated from normal endothelial cells (e.g., by the guanidinium-
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thiocyanate extraction procedure of Chirgwin et al. (1979) Biochemistry 18:
5294-5299)
and DNA can be prepared using reverse transcriptase (e.g., Moloney MLV reverse
transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse
transcriptase,
available from Seikagaku America, Inc., St. Petersburg, FL). Synthetic
oligonucleotide
primers for polymerase chain reaction amplification can be designed based upon
one of
the nucleotide sequences shown in the Sequence Listing. A nucleic acid of the
invention
can be amplified using cDNA or, alternatively, genomic DNA, as a template and
appropriate oligonucleotide primers according to standard PCR amplification
techniques. The nucleic acid so amplified can be cloned into an appropriate
vector and
characterized by DNA sequence analysis. Furthermore, oligonucleotides
corresponding
to a PTS nucleotide sequence can be prepared by standard synthetic techniques,
e.g.,
using an automated DNA synthesizer.
In a preferred embodiment, an isolated nucleic acid molecule of the invention
comprises one of the nucleotide sequences shown in the Sequence Listing. The
nucleic
acid sequences of the invention, as set forth in the Sequence Listing,
correspond to the
Corynebacterium glutamicum PTS DNAs of the invention. This DNA comprises
sequences encoding PTS proteins (i.e., the "coding region", indicated in each
odd-
numbered SEQ ID NO: sequence in the Sequence Listing), as well as 5'
untranslated
sequences and 3' untranslated sequences, also indicated in each odd-numbered
SEQ ID
NO: in the Sequence Listing. Alternatively, the nucleic acid molecule can
comprise
only the coding region of any of the nucleic acid sequences of the Sequence
Listing.
For the purposes of this application, it will be understood that each of the
nucleic
acid and amino acid sequences set forth in the Sequence Listing has an
identifying RXA,
RXN, RXS, or RXC number having the designation "RXA", "RXN", "RXS", or "RXC"
followed by 5 digits (i.e., RXA01503, RXNO1299, RXS00315, or RXC00953). Each
of
the nucleic acid sequences comprises up to three parts: a 5' upstream region,
a coding
region, and a downstream region. Each of these three regions is identified by
the same
RXA, RXN, RXS, or RXC designation to eliminate confusion. The recitation "one
of
the odd-numbered sequences of the Sequence Listing" then, refers to any of the
nucleic
acid sequences in the Sequence Listing, which may be also be , distinguished
by their
differing RXA, RXN, RXS, or RXC designations. The coding region of each of
these
sequences is translated into a corresponding amino acid sequence, which is
also set forth
CA 02590625 2007-06-18
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in the Sequence Listing, as an even-numbered SEQ ID NO: immediately following
the
corresponding nucleic acid sequence . For example, the coding region for
RXA02229 is
set forth in SEQ ID NO: 1, while the amino acid sequence which it encodes is
set forth as
SEQ ID NO:2. The sequences of the nucleic acid molecules of the invention are
identified by the same RXA, RXN, RXS, or RXC designations as the amino acid
molecules which they encode, such that they can be readily correlated. For
example, the
amino acid sequences designated RXA01503, RXN01299, RXS00315, and RXC00953
are translations of the coding regions of the nucleotide sequence of nucleic
acid
molecules RXA01503, RXN01299, RXS00315, and RXC00953, respectively. The
correspondence between the RXA, RXN, RXS, and RXC nucleotide and amino acid
sequences of the invention and their assigned SEQ ID NOs, is set forth in
Table 1. For
example, as set forth in Table 1, the nucleotide sequence of RXN01299 is SEQ
ID NO:
7, and the corresponding amino acid sequence is SEQ ID NO:8.
Several of the genes of the invention are "F-designated genes". An F-
designated
gene includes those genes set forth in Table 1 which have an 'F' in front of
the RXA,
RXN, RXS, or RXC designation. For example, SEQ ID NO:3, designated, as
indicated
on Table 1, as "F RXA00315", is an F-designated gene, as are SEQ ID NOs: 9,
11, and
13 (designated on Table 1 as "F RXA01299", "F RXA01883", and "F RXA01889",
respectively).
In one embodiment, the nucleic acid molecules of the present invention are not
intended to include C. glutamicum those compiled in Table 2. In the case of
the dapD
gene, a sequence for this gene was published in Wehrmann, A., et al. (1998) J.
Bacteriol. 180(12): 3159-3165. However, the sequence obtained by the inventors
of the
present application is significantly longer than the published version. It is
believed that
the published version relied on an incorrect start codon, and thus represents
only a
fragment of the actual coding region.
In another preferred embodiment, an isolated nueleic acid molecule of the
invention comprises a nucleic acid molecule which is a complement of one of
the
nucleotide sequences of the invention (e.g., a sequence of an odd-numbered SEQ
ID
NO: of the Sequence Listing), or a portion thereof. A nucleic acid molecule
which is
complementary to one of the nucleotide sequences of the invention is one which
is
sufficiently complementary to one of the nucleotide sequences shown in the
Sequence
CA 02590625 2007-06-18
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Listing (e.g., the sequence of an odd-numbered SEQ ID NO:) such that it can
hybridize
to one of the nucleotide sequences of the invention, thereby fonning a stable
duplex.
In still another preferred embodiment, an isolated nucleic acid molecule of
the
invention comprises a nucleotide sequence which is at least about 50%, 51%,
52%, 53%,
54 /a, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%,
63%,
64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%,
73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at
least
about 95%, 96%, 97%, 98%, 99% or more homologous to a nucleotide sequence of
the
invention (e.g., a sequence of an odd-numbered SEQ ID NO: of the Sequence
Listing),
or a portion thereof. Ranges and identity values intermediate to the above-
recited
ranges, (e.g., 70-90% identical or 80-95% identical) are also intended to be
encompassed by the present invention. For example, ranges of identity values
using a
combination of any of the above values recited as upper and/or lower limits
are intended
to be included. In an additional preferred embodiment, an isolated nucleic
acid
molecule of the invention comprises a nucleotide sequence which hybridizes,
e.g.,
hybridizes under stringent conditions, to one of the nucleotide sequences of
the
invention (e.g., a sequence of an odd-numbered SEQ ID NO: of the Sequence
Listing),
or a portion thereof.
Moreover, the nucleic acid molecule of the invention can comprise only a
portion of the coding region of the sequence of one of the odd-numbered SEQ ID
NOs
of the Sequence Listing, for example a fragment which can be used as a probe
or primer
or a fragment encoding a biologically active portion of a PTS protein. The
nucleotide
sequences determined from the cloning of the PTS genes from C. glutamicum
allows for
the generation of probes and primers designed for use in identifying and/or
cloning PTS
homologues in other cell types and organisms, as well as PTS homologues from
other
Corynebacteria or related species. The probe/primer typically comprises
substantially
purified oligonucleotide. The oligonucleotide typically comprises a region of
nucleotide
sequence that hybridizes under stringent conditions to at least about 12,
preferably about
25, more preferably about 40, 50 or 75 consecutive nucleotides of a sense
strand of one
of the nucleotide sequences of the invention (e.g., a sequence of one of the
odd-
numbered SEQ ID NOs of the Sequence Listing, an anti-sense sequence of one of
these
CA 02590625 2007-06-18
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sequences, or naturally occurring mutants thereof. Primers based on a
nucleotide
sequence of the invention can be used in PCR reactions to clone PTS
homologues.
Probes based on the PTS nucleotide sequences can be used to detect transcripts
or
genomic sequences encoding the same or homologous proteins. In preferred
embodiments, the probe further comprises a label group attached thereto, e.g.
the label
group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-
factor. Such probes can be used as a part of a diagnostic test kit for
identifying cells
which misexpress a PTS protein, such as by measuring a level of a PTS-encoding
nucleic acid in a sample of cells e.g., detecting PTS mRNA levels or
determining
whether a genomic PTS gene has been mutated or deleted.
In one embodiment, the nucleic acid molecule of the invention encodes a
protein
or portion thereof which includes an amino acid sequence which is sufficiently
homologous to an amino acid sequence of the invention (e.g., a sequence of an
even-
numbered SEQ ID NO of the Sequence Listing), such that the protein or portion
thereof
maintains the ability to participate in the transport of high-energy carbon
molecules
(such as glucose) into C. glutamicum, and may also participate in one or more
intracellular signal transduction pathways. As used herein, the language
"sufficiently
homologous" refers to proteins or portions thereof which have amino acid
sequences
which include a minimum number of identical or equivalent (e.g., an amino acid
residue
which has a similar side chain as an amino acid residue in a sequence of one
of the even-
numbered SEQ ID NOs of the Sequence Listing) amino acid residues to an amino
acid
sequence of the invention such that the protein or portion thereof is capable
of
transporting high-energy carbon-containing molecules such as glucose into C.
glutamicum, and may also participate in intracellular signal transduction in
this
microorganism. Protein members of such metabolic pathways, as described
herein,
function to transport high-energy carbon-containing molecules such as glucose
into C.
glutamicum, and may also participate in intracellular signal transduction in
this
microorganism. Examples of such activities are also described herein. Thus,
"the
function of a PTS protein" contributes to the overall functioning and/or
regulation of one
or more phosphoenolpyruvate-based sugar transport pathway, and /or
contributes, either
directly or indirectly, to the yield, production, and/or efficiency of
production of one or
more fine chemicals. Examples of PTS protein activities are set forth in
T'able 1.
CA 02590625 2007-06-18
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In another embodiment, the protein is at least about 50-60%, preferably at
least
about 60-70%, and more preferably at least about 70-80%, 80-90%, 90-95%, and
most
preferably at least about 96%, 97%, 98%, 99% or more homologous to an entire
amino
acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID
NO: of
the Sequence Listing).
Portions of proteins encoded by the PTS nucleic acid molecules of the
invention
are preferably biologically active portions of one of the PTS proteins. As
used herein,
the term "biologically active portion of a PTS protein" is intended to include
a portion,
e.g., a domain/motif, of a PTS protein that is capable of transporting high-
energy
carbon-containing molecules such as glucose into C. glutamicum, or of
participating in
intracellular signal transduction in this microorganism, or has an activity as
set forth in
Table 1. To determine whether a PTS protein or a biologically active portion
thereof
can participate in the transportation of high-energy carbon-containing
molecules such as
glucose into C. glutamicum, or can participate in intracellular signal
transduction in this
microorganism, an assay of enzymatic activity may be performed. Such assay
methods
are well known to those of ordinary skill in the art, as detailed in Example 8
of the
Exemplification.
Additional nucleic acid fragments encoding biologically active portions of a
PTS protein can be prepared by isolating a portion of one of the amino acid
sequences of
the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence
Listing), expressing the encoded portion of the PTS protein or peptide (e.g.,
by
recombinant expression in vitro) and assessing the activity of the encoded
portion of the
PTS protein or peptide.
The invention further encompasses nucleic acid molecules that differ from one
of
the nucleotide sequences of the invention (e.g., a sequence of an odd-numbered
SEQ ID
NO: of the Sequence Listing) (and portions thereof) due to degeneracy of the
genetic
code and thus encode the same PTS protein as that encoded by the nucleotide
sequences
of the invention. In another embodiment, an isolated nucleic acid molecule of
the
invention has a nucleotide sequence encoding a protein having an amino acid
sequence
shown in the Sequence Listing (e.g., an even-numbered SEQ ID NO:). In a still
further
embodiment, the nucleic acid molecule of the invention encodes a full length
C.
CA 02590625 2007-06-18
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glutamicum protein which is substantially homologous to an amino acid sequence
of the
invention (encoded by an open reading frame shown in an odd-numbered SEQ ID
NO:
of the Sequence Listing).
It will be understood by one of ordinary skill in the art that in one
embodiment
the sequences of the invention are not meant to include the sequences of the
prior art,
such as those Genbank sequences set forth in Tables 2 or 4 which were
available prior to
the present invention. In one embodiment, the invention includes nucleotide
and amino
acid sequences having a percent identity to a nucleotide or amino acid
sequence of the
invention which is greater than that of a sequence of the prior art (e.g., a
Genbank
sequence (or the protein encoded by such a sequence) set forth in Tables 2 or
4). For
example, the invention includes a nucleotide sequence which is greater than
and/or at
least 44% identical to the nucleotide sequence designated RXA01503 (SEQ ID
NO:5), a
nucleotide sequence which is greater than and/or at least 41% identical to the
nucleotide
sequence designated RXA00951 (SEQ ID NO: 15), and a nucleotide sequence which
is
greater than and/or at least 38% identical to the nucleotide sequence
designated
RXA01300 (SEQ ID NO:21). One of ordinary skill in the art would be able to
calculate
the lower threshold of percent identity for any given sequence of the
invention by
examining the GAP-calculated percent identity scores set forth in Table 4 for
each of the
three top hits for the given sequence, and by subtracting the highest GAP-
calculated
percent identity from 100 percent. One of ordinary skill in the art will also
appreciate
that nucleic acid and amino acid sequences having percent identities greater
than the
lower threshold so calculated (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%,
56%,
57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%,
67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or
90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%,
97%,
98%, 99% or more identical) are also encompassed by the invention.
In addition to the C. glutamicum PTS nucleotide sequences set forth in the
Sequence
Listing as odd-numbered SEQ ID NOs, it will be appreciated by those of
ordinary skill
in the art that DNA sequence polymorphisms that lead to changes in the amino
acid
sequences of PTS proteins may exist within a population (e.g., the C.
glutamicum
population). Such genetic polymorphism in the PTS gene may exist among
individuals
CA 02590625 2007-06-18
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within a population due to natural variation. As used herein, the terms "gene"
and
"recombinant gene" refer to nucleic acid molecules comprising an open reading
frame
encoding a PTS protein, preferably a C. glutamicum PTS protein. Such natural
variations can typically result in 1-5% variance in the nucleotide sequence of
the PTS
gene. Any and all such nucleotide variations and resulting amino acid
polymorphisms
in PTS that are the result of natural variation and that do not alter the
functional activity
of PTS proteins are intended to be within the scope of the invention.
Nucleic acid molecules corresponding to natural variants and non-C. glutamicum
homologues of the C. glulamicum PTS DNA of the invention can be isolated based
on
their homology to the C. glutamicum PTS nucleic acid disclosed herein using
the C.
glutamicum DNA, or a portion thereof, as a hybridization probe according to
standard
hybridization techniques under stringent hybridization conditions.
Accordingly, in
another embodiment, an isolated nucleic acid molecule of the invention is at
least 15
nucleotides in length and hybridizes under stringent conditions to the nucleic
acid
molecule comprising a nucleotide sequence of an odd-numbered SEQ ID NO: of the
Sequence Listing. In other embodiments, the nucleic acid is at least 30, 50,
100, 250 or
more nucleotides in length. As used herein, the term "hybridizes under
stringent
conditions" is intended to describe conditions for hybridization and washing
under
which nucleotide sequences at least 60% homologous to each other typically
remain
hybridized to each other. Preferably, the conditions are such that sequences
at least
about 65%, more preferably at least about 70%, and even more preferably at
least about
75% or more homologous to each other typically remain hybridized to each
other. Such
stringent conditions are known to those of ordinary skill in the art and can
be found in
Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons,
N.Y.
(1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringent
hybridization
conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at
about 45 C,
followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65 C. Preferably,
an
isolated nucleic acid molecule of the invention that hybridizes under
stringent conditions
to a nucleotide sequence of the invention corresponds to a naturally-occurring
nucleic
acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule
refers to
an RNA or DNA molecule having a nucleotide sequence that occurs in nature
(e.g.,
CA 02590625 2007-06-18
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encodes a natural protein). In one embodiment, the nucleic acid encodes a
natural C.
glutamicum PTS protein.
In addition to naturally-occuning variants of the PTS sequence that may exist
in
the population, one of ordinary skill in the art will further appreciate that
changes can be
introduced by mutation into a nucleotide sequence of the invention, thereby
leading to
changes in the amino acid sequence of the encoded PTS protein, without
altering the
functional ability of the PTS protein. For example, nucleotide substitutions
leading to
amino acid substitutions at "non-essential" amino acid residues can be made in
a
nucleotide sequence of the invention. A "non-essential" amino acid residue is
a residue
that can be altered from the wild-type sequence of one of the PTS proteins
(e.g., an
even-numbered SEQ ID NO: of the Sequence Listing ) without altering the
activity of
said PTS protein, whereas an "essential" amino acid residue is required for
PTS protein
activity. Other amino acid residues, however, (e.g., those that are not
conserved or only
semi-conserved in the domain having PTS activity) may not be essential for
activity and
thus are likely to be amenable to alteration without altering PTS activity.
Accordingly, another aspect of the invention pertains to nucleic acid
molecules
encoding PTS proteins that contain changes in amino acid residues that are not
essential
for PTS activity. Such PTS proteins differ in amino acid sequence from a
sequence of
an even-numbered SEQ ID NO: of the Sequence Listing yet retain at least one of
the
PTS activities described herein. In one embodiment, the isolated nucleic acid
molecule
comprises a nucleotide sequence encoding a protein, wherein the protein
comprises an
amino acid sequence at least about 50% homologous to an amino acid sequence of
the
invention and is capable of transporting high-energy carbon-containing
molecules such
as glucose into C. glutamicum, or of participating in intracellular signal
transduction in
this microorganism, or has one or more activities set forth in Table 1.
Preferably, the
protein encoded by the nucleic acid molecule is at least about 50-60%
homologous to
the amino acid sequence of one of the odd-numbered SEQ ID NOs of the Sequence
Listing, more preferably at least about 60-70% homologous to one of these
sequences,
even more preferably at least about 70-80%, 80-90%, 90-95% homologous to one
of
these sequences, and most preferably at least about 96%, 97%, 98%, or 99%
homologous to one of the amino acid sequences of the invention.
CA 02590625 2007-06-18
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To determine the percent homology of two amino acid sequences (e.g., one of
the amino acid sequences of the invention and a mutant form thereof) or of two
nucleic
acids, the sequences are aligned for optimal comparison purposes (e.g., gaps
can be
introduced in the sequence of one protein or nucleic acid for optimal
alignment with the
other protein or nucleic acid). The amino acid residues or nucleotides at
corresponding
amino acid positions or nucleotide positions are then compared. When a
position in one
sequence (e.g., one of the amino acid sequences of the invention) is occupied
by the
same amino acid residue or nucleotide as the corresponding position in the
other
sequence (e.g., a mutant form of the amino acid sequence), then the molecules
are
homologous at that position (i.e., as used herein amino acid or nucleic acid
"homology"
is equivalent to amino acid or nucleic acid "identity"). The percent homology
between
the two sequences is a function of the number of identical positions shared by
the
sequences (i.e., % homology = # of identical positions/total # of positions x
100).
An isolated nucleic acid molecule encoding a PTS protein homologous to a
protein sequence of the invention (e.g., a sequence of an even-numbered SEQ ID
NO: of
the Sequence Listing) can be created by introducing one or more nucleotide
substitutions, additions or deletions into a nucleotide sequence of the
invention such that
one or more amino acid substitutions, additions or deletions are introduced
into the
encoded protein. Mutations can be introduced into one of the nucleotide
sequences of
the invention by standard techniques, such as site-directed mutagenesis and
PCR-
mediated mutagenesis. Preferably, conservative amino acid substitutions are
made at
one or more predicted non-essential amino acid residues. A "conservative amino
acid
substitution" is one in which the amino acid residue is replaced with an amino
acid
residue having a similar side chain. Families of amino acid residues having
similar side
chains have been defined in the art. These families include amino acids with
basic side
chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic
acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine,
serine,
threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,
leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched
side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains (e.g.,
tyrosine,
phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino
acid residue
in a PTS protein is preferably replaced with another amino acid residue from
the same
CA 02590625 2007-06-18
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side chain family. Altematively, in another embodiment, mutations can be
introduced
randomly along all or part of a PTS coding sequence, such as by saturation
mutagenesis,
and the resultant mutants can be screened for a PTS activity described herein
to identify
mutants that retain PTS activity. Following mutagenesis of one of the
nucleotide
sequence of one of the odd-numbered SEQ ID NOs of the Sequence Listing, the
encoded protein can be expressed recombinantly and the activity of the protein
can be
determined using, for example, assays described herein (see Example 8 of the
Exemplification).
In addition to the nucleic acid molecules encoding PTS proteins described
above,
another aspect of the invention pertains to isolated nucleic acid molecules
which are
antisense thereto. An "antisense" nucleic acid comprises a nucleotide sequence
which is
complementary to a "sense" nucleic acid encoding a protein, e.g.,
complementary to the
coding strand of a double-stranded DNA molecule or complementary to an mRNA
sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense
nucleic
acid. The antisense nucleic acid can be complementary to an entire PTS coding
strand,
or to only a portion thereof. In one embodiment, an antisense nucleic acid
molecule is
antisense to a "coding region" of the coding strand of a nucleotide sequence
encoding a
PTS protein. The term "coding region" refers to the region of the nucleotide
sequence
comprising codons which are translated into amino acid residues (e.g., the
entire coding
region of SEQ ID NO. 5(RXA01503) comprises nucleotides 1 to 249). In another
embodiment, the antisense nucleic acid molecule is antisense to a "noncoding
region" of
the coding strand of a nucleotide sequence encoding PTS. The term "noncoding
region"
refers to 5' and 3' sequences which flank the coding region that are not
translated into
amino acids (i.e., also referred to as 5' and 3' untranslated regions).
Given the coding strand sequences encoding PTS disclosed herein (e.g., the
sequences set forth as odd-numbered SEQ ID NOs in the Sequence Listing),
antisense
nucleic acids of the invention can be designed according to the rules of
Watson and
Crick base pairing. The antisense nucleic acid molecule can be complementary
to the
entire coding region of PTS mRNA, but more preferably is an oligonucleotide
which is
antisense to only a portion of the coding or noncoding region of PTS mRNA. For
example, the antisense oligonucleotide can be complementary to the region
surrounding
the translation start site of PTS mRNA. An antisense oligonucleotide can be,
for
CA 02590625 2007-06-18
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example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
An
antisense nucleic acid of the invention can be constructed using chemical
synthesis and
enzymatic ligation reactions using procedures known in the art. For example,
an
antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically
synthesized
using naturally occurring nucleotides or variously modified nucleotides
designed to
increase the biological stability of the molecules or to increase the physical
stability of
the duplex formed between the antisense and sense nucleic acids, e.g.,
phosphorothioate
derivatives and acridine substituted nucleotides can be used. Examples of
modified
nucleotides which can be used to generate the antisense nucleic acid include 5-
fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xanthine, 4-
acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-
thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-
galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-
methylinosine,
2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-
methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-
methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-
methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-
isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-
thiocytosine, 5-
methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-
oxyacetic acid
methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-
N-2-
carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the
antisense
nucleic acid can be produced biologically using an expression vector into
which a
nucleic acid has been subcloned in an antisense orientation (i.e., RNA
transcribed from
the inserted nucleic acid will be of an antisense orientation to a target
nucleic acid of
interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically
administered
to a cell or generated in situ such that they hybridize with or bind to
cellular mRNA
and/or genomic DNA encoding a PTS protein to thereby inhibit expression of the
protein, e.g., by inhibiting transcription and/or translation. The
hybridization can be by
conventional nucleotide complementarity to form a stable duplex, or, for
example, in the
case of an antisense nucleic acid molecule which binds to DNA duplexes,
through
specific interactions in the major groove of the double helix. The antisense
molecule can
CA 02590625 2007-06-18
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be modified such that it specifically binds to a receptor or an antigen
expressed on a
selected cell surface, e.g., by linking the antisense nucleic acid molecule to
a peptide or
an antibody which binds to a cell surface receptor or antigen. The antisense
nucleic acid
molecule can also be delivered to cells using the vectors described herein. To
achieve
sufficient intracellular concentrations of the antisense molecules, vector
constructs in
which the antisense nucleic acid molecule is placed under the control of a
strong
prokaryotic, viral, or eukaryotic promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the
invention
is an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule
forms
specific double-stranded hybrids with complementary RNA in which, contrary to
the
usual (3-units, the strands run parallel to each other (Gaultier et al. (1987)
Nucleic Acids.
Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-
o-
methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or
a
chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
In still another embodiment, an antisense nucleic acid of the invention is a
ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity
which are
capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which
they
have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to
catalytically cleave PTS mRNA transcripts to thereby inhibit translation of
PTS mRNA.
A ribozyme having specificity for a PTS-encoding nucleic acid can be designed
based
upon the nucleotide sequence of a PTS DNA disclosed herein (i.e., SEQ ID NO:5
(RXA01503)). For example, a derivative of a Tetrahymena L-19 IVS RNA can be
constructed in which the nucleotide sequence of the active site is
complementary to the
nucleotide sequence to be cleaved in a PTS-encoding mRNA. See, e.g., Cech et
al. U.S.
Patent No. 4,987,071 and Cech et al. U.S. Patent No. 5,116,742. Alternatively,
PTS
mRNA can be used to select a catalytic RNA having a specific ribonuclease
activity
from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993)
Science
261:1411-1418.
Alternatively, PTS gene expression can be inhibited by targeting nucleotide
sequences complementary to the regulatory region of a PTS nucleotide sequence
(e.g., a
PTS promoter and/or enhancers) to form triple helical structures that prevent
CA 02590625 2007-06-18
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transcription of a PTS gene in target cells. See generally, Helene, C. (1991)
Anticancer
Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N. Y. Acad. Sci. 660:27-
36; and
Maher, L.J. (1992) Bioassays 14(12):807-15.
B. Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression
vectors, containing a nucleic acid encoding a PTS protein (or a portion
thereof). As
used herein, the term "vector" refers to a nucleic acid molecule capable of
transporting
another nucleic acid to which it has been linked. One type of vector is a
"plasmid",
which refers to a circular double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector, wherein
additional
DNA segments can be ligated into the viral genome. Certain vectors are capable
of
autonomous replication in a host cell into which they are introduced (e.g.,
bacterial
vectors having a bacterial origin of replication and episomal mammalian
vectors). Other
vectors (e.g., non-episomal mammalian vectors) are integrated into the genome
of a host
cell upon introduction into the host cell, and thereby are replicated along
with the host
genome. Moreover, certain vectors are capable of directing the expression of
genes to
which they are operatively linked. Such vectors are referred to herein as
"expression
vectors". In general, expression vectors of utility in recombinant DNA
techniques are
often in the form of plasmids. In the present specification, "plasmid" and
"vector" can
be used interchangeably as the plasmid is the most commonly used form of
vector.
However, the invention is intended to include such other forms of expression
vectors,
such as viral vectors (e.g., replication defective retroviruses, adenoviruses
and adeno-
associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of
the invention in a form suitable for expression of the nucleic acid in a host
cell, which
means that the recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for expression,
which is
operatively linked to the nucleic acid sequence to be expressed. Within a
recombinant
expression vector, "operably linked" is intended to mean that the nucleotide
sequence of
interest is linked to the regulatory sequence(s) in a manner which allows for
expression
of the nucleotide sequence (e.g., in an in vitro transcription/translation
system or in a
CA 02590625 2007-06-18
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host cell when the vector is introduced into the host cell). The term
"regulatory
sequence" is intended to include promoters, enhancers and other expression
control
elements (e.g., polyadenylation signals). Such regulatory sequences are
described, for
example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, CA (1990). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence in many types of host
cell and
those which direct expression of the nucleotide sequence only in certain host
cells.
Preferred regulatory sequences are, for example, promoters such as cos-, tac-,
trp-, tet-,
trp-tet-, lpp-, lac-, lpp-lac-, lacIq-, T7-, T5-, T3-, gal-, trc-, ara-, SP6-,
amy, SPO2, X-PR-
or k PL, which are used preferably in bacteria. Additional regulatory
sequences are, for
example, promoters from yeasts and fungi, such as ADC 1, MFa, AC, P-60, CYC 1,
GAPDH, TEF, rp28, ADH, promoters from plants such as CaMV/35S, SSU, OCS, lib4,
usp, STLSI, B33, nos or ubiquitin- or phaseolin-promoters. It is also possible
to use
artificial promoters. It will be appreciated by one of ordinary skill in the
art that the
design of the expression vector can depend on such factors as the choice of
the host cell
to be transformed, the level of expression of protein desired, etc. The
expression vectors
of the invention can be introduced into host cells to thereby produce proteins
or
peptides, including fusion proteins or peptides, encoded by nucleic acids as
described
herein (e.g., PTS proteins, mutant forms of PTS proteins, fusion proteins,
etc.).
The recombinant expression vectors of the invention can be designed for
expression of PTS proteins in prokaryotic or eukaryotic cells. For example,
PTS genes
can be expressed in bacterial cells such as C. glutamicum, insect cells (using
baculovirus
expression vectors), yeast and other fungal cells (see Romanos, M.A. et al.
(1992)
"Foreign gene expression in yeast: a review", Yeast 8: 423-488; van den
Hondel,
C.A.M.J.J. et al. (1991) "Heterologous gene expression in filamentous fungi"
in: More
Gene Manipulations in Fungi, J.W. Bennet & L.L. Lasure, eds., p. 396-428:
Academic
Press: San Diego; and van den Hondel, C.A.M.J.J. & Punt, P.J. (1991) "Gene
transfer
systems and vector development for filamentous fungi, in: Applied Molecular
Genetics
of Fungi, Peberdy, J.F. et al., eds., p. 1-28, Cambridge University Press:
Cambridge),
algae and multicellular plant cells (see Schmidt, R. and Willmitzer, L. (1988)
High
efficiency Agrobacterium tumefactiens -mediated transformation of Arabidopsis
thaliana leaf and cotyledon explants" Plant Cell Rep.: 583-586), or mammalian
cells.
CA 02590625 2007-06-18
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Suitable host cells are discussed further in Goeddel, Gene Expression
Technology:
Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
Alternatively, the
recombinant expression vector can be transcribed and translated in vitro, for
example
using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out with vectors
containing constitutive or inducible promoters directing the expression of
either fusion
or non-fusion proteins. Fusion vectors add a number of amino acids to a
protein
encoded therein, usually to the amino terminus of the recombinant protein.
Such fusion
vectors typically serve three purposes: 1) to increase expression of
recombinant protein;
2) to increase the solubility of the recombinant protein; and 3) to aid in the
purification
of the recombinant protein by acting as a ligand in affinity purification.
Often, in fusion
expression vectors, a proteolytic cleavage site is introduced at the junction
of the fusion
moiety and the recombinant protein to enable separation of the recombinant
protein
from the fusion moiety subsequent to purification of the fusion protein. Such
enzymes,
and their cognate recognition sequences, include Factor Xa, thrombin and
enterokinase.
Typical fusion expression vectors include pGEX (Pharmacia Biotech Ine; Smith,
D.B. and Johnson, K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs,
Beverly,
MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase
(GST), maltose E binding protein, or protein A, respectively, to the target
recombinant
protein. In one embodiment, the coding sequence of the PTS protein is cloned
into a
pGEX expression vector to create a vector encoding a fi-sion protein
comprising, from
the N-terminus to the C-terminus, GST-thrombin cleavage site-X protein. The
fusion
protein can be purified by affinity chromatography using glutathione-agarose
resin.
Recombinant PTS protein unfused to GST can be recovered by cleavage of the
fusion
protein with thrombin.
Examples of suitable inducible non-fusion E. coli expression vectors include
pTrc (Amann et al., (1988) Gene 69:301-315) pLG338, pACYC184, pBR322, pUC18,
pUC 19, pKC30, pRep4, pHS 1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-
III 113-B 1, ),gt 11, pBdCl, and pET I 1 d (Studier et al., Gene Expression
Technology:
Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89
; and
Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: New York IBSN 0 444
904018).
Target gene expression from the pTrc vector relies on host RNA polymerase
CA 02590625 2007-06-18
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transcription from a hybrid trp-lac fusion promoter. Target gene expression
from the
pET l ld vector relies on transcription from a T7 gn10-lac fusion promoter
mediated by
a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied
by
host strains BL21(DE3) or HMS 174(DE3) from a resident a, prophage harboring a
T7
gnl gene under the transcriptional control of the lacUV 5 promoter. For
transformation
of other varieties of bacteria, appropriate vectors may be selected. For
example, the
plasmids pIJ101, pIJ364, pIJ702 and pIJ361 are known to be useful in
transforming
Streptomyces, while plasmids pUB 110, pC 194, or pBD214 are suited for
transformation
of Bacillus species. Several plasmids of use in the transfer of genetic
information into
Corynebacterium include pHM1519, pBL1, pSA77, or pAJ667 (Pouwels et al., eds.
(1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018).
One strategy to maximize recombinant protein expression is to express the
protein in a host bacteria with an impaired capacity to proteolytically cleave
the
recombinant protein (Gottesman, S., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Another
strategy is to alter the nucleic acid sequence of the nucleic acid to be
inserted into an
expression vector so that the individual codons for each amino acid are those
preferentially utilized in the bacterium chosen for expression, such as C.
glutamicum
(Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of
nucleic acid
sequences of the invention can be carried out by standard DNA synthesis
techniques.
In another embodiment, the PTS protein expression vector is a yeast expression
vector. Examples of vectors for expression in yeast S. cerivisae include
pYepSec 1
(Baldari, et al., (1987) Embo J. 6:229-234), 2 , pAG-1, Yep6, Yep13,
pEMBLYe23,
pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al.,
(1987)
Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors
and
methods for the construction of vectors appropriate for use in other fungi,
such as the
filamentous fungi, include those detailed in: van den Hondel, C.A.M.J.J. &
Punt, P.J.
(1991) "Gene transfer systems and vector development for filamentous fungi,
in:
Applied Molecular Genetics of Fungi, J.F. Peberdy, et al., eds., p. 1-28,
Cambridge
University Press: Cambridge, and Pouwels et al., eds. (1985) Cloning Vectors.
Elsevier:
New York (IBSN 0 444 904018).
CA 02590625 2007-06-18
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Alternatively, the PTS proteins of the invention can be expressed in insect
cells
using baculovirus expression vectors. Baculovirus vectors available for
expression of
proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series
(Smith et al.
(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers
(1989)
Virology 170:31-39).
In another embodiment, the PTS proteins of the invention may be expressed in
unicellular plant cells (such as algae) or in plant cells from higher plants
(e.g., the
spermatophytes, such as crop plants). Examples of plant expression vectors
include
those detailed in: Becker, D., Kemper, E., Schell, J. and Masterson, R. (1992)
"New
plant binary vectors with selectable markers located proximal to the left
border", Plant
Mol. Biol. 20: 1195-1197; and Bevan, M.W. (1984) "Binary Agrobacterium vectors
for
plant transformation", Nucl. Acid. Res. 12: 8711-8721, and include pLGV23,
pGHlac+,
pBIN19, pAK2004, and pDH51 (Pouwels et al., eds. (1985) Cloning Vectors.
Elsevier:
New York IBSN 0 444 904018).
In yet another embodiment, a nucleic acid of the invention is expressed in
mammalian cells using a mammalian expression vector. Examples of mammalian
expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC
(Kaufman et al. (1987) EMBOJ. 6:187-195). When used in mammalian cells, the
expression vector's control functions are often provided by viral regulatory
elements.
For example, commonly used promoters are derived from polyoma, Adenovirus 2,
cytomegalovirus and Simian Virus 40. For other suitable expression systems for
both
prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,
Fritsh, E. F.,
and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
1989.
In another embodiment, the recombinant mammalian expression vector is
capable of directing expression of the nucleic acid preferentially in a
particular cell type
(e.g., tissue-specific regulatory elements are used to express the nucleic
acid). Tissue-
specific regulatory elements are known in the art. Non-limiting examples of
suitable
tissue-specific promoters include the albumin promoter (liver-specific;
Pinkert et al_
(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton
(1988)
Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto
and
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Baltimore (1989) EMBOJ. 8:729-733) and immunoglobulins (Banerji et al. (1983)
Cell
33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific
promoters
(e.g., the neurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477),
pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and
mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316
and
European Application Publication No. 264,166). Developmentally-regulated
promoters
are also encompassed, for example the murine hox promoters (Kessel and Gruss
(1990)
Science 249:374-379) and the a-fetoprotein promoter (Campes and Tilghman
(1989)
Genes Dev. 3:537-546).
The invention further provides a recombinant expression vector comprising a
DNA molecule of the invention cloned into the expression vector in an
antisense
orientation. That is, the DNA molecule is operatively linked to a regulatory
sequence in
a manner which allows for expression (by transcription of the DNA molecule) of
an
RNA molecule which is antisense to PTS mRNA. Regulatory sequences operatively
linked to a nucleic acid cloned in the antisense orientation can be chosen
which direct
the continuous expression of the antisense RNA molecule in a variety of cell
types, for
instance viral promoters and/or enhancers, or regulatory sequences can be
chosen which
direct constitutive, tissue specific or cell type specific expression of
antisense RNA.
The antisense expression vector can be in the form of a recombinant plasmid,
phagemid
or attenuated virus in which antisense nucleic acids are produced under the
control of a
high efficiency regulatory region, the activity of which can be determined by
the cell
type into which the vector is introduced. For a discussion of the regulation
of gene
expression using antisense genes see Weintraub, H. et al., Antisense RNA as a
molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1)
1986.
Another aspect of the invention pertains to host cells into which a
recombinant
expression vector of the invention has been introduced. The terms "host cell"
and
"recombinant host cell" are used interchangeably herein. It is understood that
such
terms refer not only to the particular subject cell but to the progeny or
potential progeny
of such a cell. Because certain modifications may occur in succeeding
generations due
to either mutation or environmental influences, such progeny may not, in fact,
be
identical to the parent cell, but are still included within the scope of the
term as used
herein.
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A host cell can be any prokaryotic or eukaryotic cell. For example, a PTS
protein can be expressed in bacterial cells such as C. glutamicum, insect
cells, yeast or
mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
Other
suitable host cells are known to one of ordinary skill in the art.
Microorganisms related
to Corynebacterium glutamicum which may be conveniently used as host cells for
the
nucleic acid and protein molecules of the invention are set forth in Table 3.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional transformation or transfection techniques. As used herein, the
terms
"transformation" and "transfection" are intended to refer to a variety of art-
recognized
techniques for introducing foreign nucleic acid (e.g., linear DNA or RNA
(e.g., a
linearized vector or a gene construct alone without a vector) or nucleic acid
in the form
of a vector (e.g., a plasmid, phage, phasmid, phagemid, transposon or other
DNA) into a
host cell, including calcium phosphate or calcium chloride co-precipitation,
DEAE-
dextran-mediated transfection, lipofection, or electroporation. Suitable
methods for
transforming or transfecting host cells can be found in Sambrook, et al.
(Molecular
Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold
Spring
Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory
manuals.
For stable transfection of mammalian cells, it is known that, depending upon
the
expression vector and transfection technique used, only a small fraction of
cells may
integrate the foreign DNA into their genome. In order to identify and select
these
integrants, a gene that encodes a selectable marker (e.g., resistance to
antibiotics) is
generally introduced into the host cells along with the gene of interest.
Preferred
selectable markers include those which confer resistance to drugs, such as
G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be
introduced into a host cell on the same vector as that encoding a PTS protein
or can be
introduced on a separate vector. Cells stably transfected with the introduced
nucleic
acid can be identified by drug selection (e.g., cells that have incorporated
the selectable
marker gene will survive, while the other cells die).
To create a homologous recombinant microorganism, a vector is prepared which
contains at least a portion of a PTS gene into which a deletion, addition or
substitution
has been introduced to thereby alter, e.g., functionally disrupt, the PTS
gene.
Preferably, this PTS gene is a Corynebacterium glutamicum PTS gene, but it can
be a
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homologue from a related bacterium or even from a mammalian, yeast, or insect
source.
In a preferred embodiment, the vector is designed such that, upon homologous
recombination, the endogenous PTS gene is functionally disrupted (i.e., no
longer
encodes a functional protein; also referred to as a "knock out" vector).
Alternatively,
the vector can be designed such that, upon homologous recombination, the
endogenous
PTS gene is mutated or otherwise altered but still encodes functional protein
(e.g., the
upstream regulatory region can be altered to thereby alter the expression of
the
endogenous PTS protein). In the homologous recombination vector, the altered
portion
of the PTS gene is flanked at its 5' and 3' ends by additional nucleic acid of
the PTS
gene to allow for homologous recombination to occur between the exogenous PTS
gene
carried by the vector and an endogenous PTS gene in a microorganism. The
additional
flanking PTS nucleic acid is of sufficient length for successful homologous
recombination with the endogenous gene. Typically, several kilobases of
flanking DNA
(both at the 5' and 3' ends) are included in the vector (see e.g., Thomas,
K.R., and
Capecchi, M.R. (1987) Cell 51: 503 for a description of homologous
recombination
vectors). The vector is introduced into a microorganism (e.g., by
electroporation) and
cells in which the introduced PTS gene has homologously recombined with the
endogenous PTS gene are selected, using art-known techniques.
In another embodiment, recombinant microorganisms can be produced which
contain selected systems which allow for regulated expression of the
introduced gene.
For example, inclusion of a PTS gene on a vector placing it under control of
the lac
operon pennits expression of the PTS gene only in the presence of IPTG. Such
regulatory systems are well known in the art.
In another embodiment, an endogenous PTS gene in a host cell is disrupted
(e.g.,
by homologous recombination or other genetic means known in the art) such that
expression of its protein product does not occur. In another embodiment, an
endogenous
or introduced PTS gene in a host cell has been altered by one or more point
mutations,
deletions, or inversions, but still encodes a functional PTS protein. In still
another
embodiment, one or more of the regulatory regions (e.g., a promoter,
repressor, or
inducer) of a PTS gene in a microorganism has been altered (e.g., by deletion,
truncation, inversion, or point mutation) such that the expression of the PTS
gene is
modulated. One of ordinary skill in the art will appreciate that host cells
containing
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more than one of the described PTS gene and protein modifications may be
readily
produced using the methods of the invention, and are meant to be included in
the present
invention.
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in
culture, can be used to produce (i.e., express) a PTS protein. Accordingly,
the invention
further provides methods for producing PTS proteins using the host cells of
the
invention. In one embodiment, the method comprises culturing the host cell of
invention (into which a recombinant expression vector encoding a PTS protein
has been
introduced, or into which genome has been introduced a gene encoding a wild-
type or
altered PTS protein) in a suitable medium until PTS protein is produced. In
another
embodiment, the method further comprises isolating PTS proteins from the
medium or
the host cell.
C. Isolated PTS Proteins
Another aspect of the invention pertains to isolated PTS proteins, and
biologically active portions thereof. An "isolated" or "purified" protein or
biologically
active portion thereof is substantially free of cellular material when
produced by
recombinant DNA techniques, or chemical precursors or other chemicals when
chemically synthesized. The language "substantially free of cellular material"
includes
preparations of PTS protein in which the protein is separated from cellular
components
of the cells in which it is naturally or recombinantly produced. In one
embodiment, the
language "substantially free of cellular material" includes preparations of
PTS protein
having less than about 30% (by dry weight) of non-PTS protein (also referred
to herein
as a "contaminating protein"), more preferably less than about 20% of non-PTS
protein,
still more preferably less than about 10% of non-PTS protein, and most
preferably less
than about 5% non-PTS protein. When the PTS protein or biologically active
portion
thereof is recombinantly produced, it is also preferably substantially free of
culture
medium, i.e., culture medium represents less than about 20%, more preferably
less than
about 10%, and most preferably less than about 5% of the volume of the protein
preparation. The language "substantially free of chemical precursors or other
chemicals" includes preparations of PTS protein in which the protein is
separated from
chemical precursors or other chemicals which are involved in the synthesis of
the
CA 02590625 2007-06-18
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protein. In one embodiment, the language "substantially free of chemical
precursors or
other chemicals" includes preparations of PTS protein having less than about
30% (by
dry weight) of chemical precursors or non-PTS chemicals, more preferably less
than
about 20% chemical precursors or non-PTS chemicals, still more preferably less
than
about 10% chemical precursors or non-PTS chemicals, and most preferably less
than
about 5% chemical precursors or non-PTS chemicals. In preferred embodiments,
isolated proteins or biologically active portions thereof lack contaminating
proteins from
the same organism from which the PTS protein is derived. Typically, such
proteins are
produced by recombinant expression of, for example, a C. glutamicum PTS
protein in a
microorganism such as C. glutamicum.
An isolated PTS protein or a portion thereof of the invention can participate
in
the transport of high-energy carbon-containing molecules such as glucose into
C.
glutamicum, and may also participate in intracellular signal transduction in
this
microorganism, or has one or more of the activities set forth in Table 1. In
preferred
embodiments, the protein or portion thereof comprises an amino acid sequence
which is
sufficiently homologous to an amino acid sequence of the invention (e.g., a
sequence of
an even-numbered SEQ ID NO: of the Sequence Listing) such that the protein or
portion
thereof maintains the ability to transport high-energy carbon-containing
molecules such
as glucose into C. glutamicum, or to participate in intracellular signal
transduction in this
microorganism. The portion of the protein is preferably a biologically active
portion as
described herein. In another preferred embodiment, a PTS protein of the
invention has
an amino acid sequence set forth as an even-numbered SEQ ID NO: of the
Sequence
Listing. In yet another preferred embodiment, the PTS protein has an amino
acid
sequence which is encoded by a nucleotide sequence which hybridizes, e.g.,
hybridizes
under stringent conditions, to a nucleotide sequence of the invention (e.g., a
sequence of
an odd-numbered SEQ ID NO: of the Sequence Listing). In still another
preferred
embodiment, the PTS protein has an amino acid sequence which is encoded by a
nucleotide sequence that is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%,
57%,
58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%,
68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%,
77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%,
or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%,
98%,
CA 02590625 2007-06-18
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99% or more homologous to one of the nucleic acid sequences of the invention,
or a
portion thereof. Ranges and identity values intermediate to the above-recited
values,
(e.g., 70-90% identical or 80-95% identical) are also intended to be
encompassed by the
present invention. For example, ranges of identity values using a combination
of any of
the above values recited as upper and/or lower limits are intended to be
included. The
preferred PTS proteins of the present invention also preferably possess at
least one of
the PTS activities described herein. For example, a preferred PTS protein of
the present
invention includes an amino acid sequence encoded by a nucleotide sequence
which
hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide
sequence of the
invention, and which can participate in the transport of high-energy carbon-
containing
molecules such as glucose into C. glutamicum, and may also participate in
intracellular
signal transduction in this microorganism, or which has one or more of the
activities set
forth in Table 1.
In other embodiments, the PTS protein is substantially homologous to an amino
acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID
NO: of
the Sequence Listing) and retains the functional activity of the protein of
one of the
amino acid sequences of the invention yet differs in amino acid sequence due
to natural
variation or mutagenesis, as described in detail in subsection I above.
Accordingly, in
another embodiment, the PTS protein is a protein which comprises an amino acid
sequence which is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,
59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,
69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or
91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%,
98%,
99% or more homologous to an entire amino acid sequence of the invention and
which
has at least one of the PTS activities described herein. Ranges and identity
values
intermediate to the above-recited values, (e.g., 70-90% identical or 80-95%
identical)
are also intended to be encompassed by the present invention. For example,
ranges of
identity values using a combination of any of the above values recited as
upper and/or
lower limits are intended to be included. In another embodiment, the invention
pertains
to a full length C. glutamicum protein which is substantially homologous to an
entire
amino acid sequence of the invention.
CA 02590625 2007-06-18
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Biologically active portions of a PTS protein include peptides comprising
amino
acid sequences derived from the amino acid sequence of a PTS protein, e.g., an
amino
acid sequence of an even-numbered SEQ ID NO: of the Sequence Listing or the
amino
acid sequence of a protein homologous to a PTS protein, which include fewer
amino
acids than a full length PTS protein or the full length protein which is
homologous to a
PTS protein, and exhibit at least one activity of a PTS protein. Typically,
biologically
active portions (peptides, e.g., peptides which are, for example, 5, 10, 15,
20, 30, 35, 36,
37, 38, 39, 40, 50, 100 or more amino acids in length) comprise a domain or
motif with
at least one activity of a PTS protein. Moreover, other biologically active
portions, in
which other regions of the protein are deleted, can be prepared by recombinant
techniques and evaluated for one or more of the activities described herein.
Preferably,
the biologically active portions of a PTS protein include one or more selected
domains/motifs or portions thereof having biological activity.
PTS proteins are preferably produced by recombinant DNA techniques. For
example, a nucleic acid molecule encoding the protein is cloned into an
expression
vector (as described above), the expression vector is introduced into a host
cell (as
described above) and the PTS protein is expressed in the host cell. The PTS
protein can
then be isolated from the cells by an appropriate purification scheme using
standard
protein purification techniques. Alternative to recombinant expression, a PTS
protein,
polypeptide, or peptide can be synthesized chemically using standard peptide
synthesis
techniques. Moreover, native PTS protein can be isolated from cells (e.g.,
endothelial
cells), for example using an anti-PTS antibody, which can be produced by
standard
techniques utilizing a PTS protein or fragment thereof of this invention.
The invention also provides PTS chimeric or fusion proteins. As used herein, a
PTS "chimeric protein" or "fusion protein" comprises a PTS polypeptide
operatively
linked to a non-PTS polypeptide. An "PTS polypeptide" refers to a polypeptide
having
an amino acid sequence corresponding to PTS, whereas a "non-PTS polypeptide"
refers
to a polypeptide having an amino acid sequence corresponding to a protein
which is not
substantially homologous to the PTS protein, e.g., a protein which is
different from the
PTS protein and which is derived from the same or a different organism. Within
the
fusion protein, the term "operatively linked" is intended to indicate that the
PTS
polypeptide and the non-PTS polypeptide are fused in-frame to each other. The
non-
CA 02590625 2007-06-18
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PTS polypeptide can be fused to the N-terminus or C-terminus of the PTS
polypeptide.
For example, in one embodiment the fusion protein is a GST-PTS fusion protein
in
which the PTS sequences are fused to the C-terminus of the GST sequences. Such
fusion proteins can facilitate the purification of recombinant PTS proteins.
In another
embodiment, the fusion protein is a PTS protein containing a heterologous
signal
sequence at its N-terminus. In certain host cells (e.g., mammalian host
cells), expression
and/or secretion of a PTS protein can be increased through use of a
heterologous signal
sequence.
Preferably, a PTS chimeric or fusion protein of the invention is produced by
standard recombinant DNA techniques. For example, DNA fragments coding for the
different polypeptide sequences are ligated together in-frame in accordance
with
conventional techniques, for example by employing blunt-ended or stagger-ended
termini for ligation, restriction enzyme digestion to provide for appropriate
termini,
filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to
avoid
undesirable joining, and enzymatic ligation. In another embodiment, the fusion
gene
can be synthesized by conventional techniques including automated DNA
synthesizers.
Alternatively, PCR amplification of gene fragments can be carried out using
anchor
primers which give rise to complementary overhangs between two consecutive
gene
fragments which can subsequently be annealed and reamplified to generate a
chimeric
gene sequence (see, for example, Current Protocols in Molecular Biology, eds.
Ausubel
et al. John Wiley & Sons: 1992). Moreover, many expression vectors are
commercially
available that already encode a fusion moiety (e.g., a GST polypeptide). A PTS-
encoding nucleic acid can be cloned into such an expression vector such that
the fusion
moiety is linked in-frame to the PTS protein.
Homologues of the PTS protein can be generated by mutagenesis, e.g., discrete
point mutation or truncation of the PTS protein. As used herein, the term
"homologue"
refers to a variant form of the PTS protein which acts as an agonist or
antagonist of the
activity of the PTS protein. An agonist of the PTS protein can retain
substantially the
same, or a subset, of the biological activities of the PTS protein. An
antagonist of the
PTS protein can inhibit one or more of the activities of the naturally occun-
ing form of
the PTS protein, by, for example, competitively binding to a downstream or
upstream
member of the PTS system which includes the PTS protein. Thus, the C.
glutamicum
CA 02590625 2007-06-18
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PTS protein and homologues thereof of the present invention may modulate the
activity
of one or more sugar transport pathways or intracellular signal transduction
pathways in
which PTS proteins play a role in this microorganism.
In an altecnative embodiment, homologues of the PTS protein can be identified
by screening combinatorial libraries of mutants, e.g., truncation mutants, of
the PTS
protein for PTS protein agonist or antagonist activity. In one embodiment, a
variegated
library of PTS variants is generated by combinatorial mutagenesis at the
nucleic acid
level and is encoded by a variegated gene library. A variegated library of PTS
variants
can be produced by, for example, enzymatically ligating a mixture of synthetic
oligonucleotides into gene sequences such that a degenerate set of potential
PTS
sequences is expressible as individual polypeptides, or alternatively, as a
set of larger
fusion proteins (e.g., for phage display) containing the set of PTS sequences
therein.
There are a variety of inethods which can be used to produce libraries of
potential PTS
homologues from a degenerate oligonucleotide sequence. Chemical synthesis of a
degenerate gene sequence can be performed in an automatic DNA synthesizer, and
the
synthetic gene then ligated into an appropriate expression vector. Use of a
degenerate
set of genes allows for the provision, in one mixture, of all of the sequences
encoding
the desired set of potential PTS sequences. Methods for synthesizing
degenerate
oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983)
Tetrahedron 39:3;
Itakura et a!_ (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984)
Science
198:1056; Ike et a!. (1983) Nucleic Acid Res. 11:477.
In addition, libraries of fragments of the PTS protein coding can be used to
generate a variegated population of PTS fragments for screening and subsequent
selection of homologues of a PTS protein. In one embodiment, a library of
coding
sequence fragments can be generated by treating a double stranded PCR fragment
of a
PTS coding sequence with a nuclease under conditions wherein nicking occurs
only
about once per molecule, denaturing the double stranded DNA, renaturing the
DNA to
form double stranded DNA which can include sense/antisense pairs from
different
nicked products, removing single stranded portions from reformed duplexes by
treatment with SI nuclease, and ligating the resulting fragment library into
an expression
vector. By this method, an expression library can be derived which encodes N-
terminal,
C-terminal and internal fragments of various sizes of the PTS protein.
CA 02590625 2007-06-18
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Several techniques are known in the art for screening gene products of
combinatorial libraries made by point mutations or truncation, and for
screening cDNA
libraries for gene products having a selected property. Such techniques are
adaptable for
rapid screening of the gene libraries generated by the combinatorial
mutagenesis of PTS
homologues. The most widely used techniques, which are amenable to high
through-put
analysis, for screening large gene libraries typically include cloning the
gene library into
replicable expression vectors, transforming appropriate cells with the
resulting library of
vectors, and expressing the combinatorial genes under conditions in which
detection of a
desired activity facilitates isolation of the vector encoding the gene whose
product was
detected. Recursive ensemble mutagenesis (REM), a new technique which enhances
the
frequency of functional mutants in the libraries, can be used in combination
with the
screening assays to identify PTS homologues (Arkin and Yourvan (1992) PNAS
89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-33 1).
In another embodiment, cell based assays can be exploited to analyze a
variegated PTS library, using methods well known in the art.
D. Uses and Methods of the Invention
The nucleic acid molecules, proteins, protein homologues, fusion proteins,
primers, vectors, and host cells described herein can be used in one or more
of the
following methods: identification of C. glutamicum and related organisms;
mapping of
genomes of organisms related to C. glutamicum; identification and localization
of C.
glutamicum sequences of interest; evolutionary studies; determination of PTS
protein
regions required for function; modulation of a PTS protein activity;
modulation of the
activity of a PTS pathway; and modulation of cellular production of a desired
compound, such as a fine chemical.
The PTS nucleic acid molecules of the invention have a variety of uses. First,
they may be used to identify an organism as being Corynebacterium glutamicum
or a
close relative thereof. Also, they may be used to identify the presence of C.
glutamicum
or a relative thereof in a mixed population of microorganisms. The invention
provides
the nucleic acid sequences of a number of C. glutamicum genes; by probing the
extracted genomic DNA of a culture of a unique or mixed population of
microorganisms
CA 02590625 2007-06-18
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under stringent conditions with a probe spanning a region of a C. glutamicum
gene
which is unique to this organism, one can ascertain whether this organism is
present.
Although Corynebacterium glutamicum itself is nonpathogenic, it is related to
pathogenic species, such as Corynebacterium diphtheriae. Corynebacterium
diphtheriae is the causative agent of diphtheria, a rapidly developing, acute,
febrile
infection which involves both local and systemic pathology. In this disease, a
local
lesion develops in the upper respiratory tract and involves necrotic injury to
epithelial
cells; the bacilli secrete toxin which is disseminated through this lesion to
distal
susceptible tissues of the body. Degenerative changes brought about by the
inhibition of
protein synthesis in these tissues, which include heart, muscle, peripheral
nerves,
adrenals, kidneys, liver and spleen, result in the systemic pathology of the
disease.
Diphtheria continues to have high incidence in many parts of the world,
including
Africa, Asia, Eastern Europe and the independent states of the former Soviet
Union. An
ongoing epidemic of diphtheria in the latter two regions has resulted in at
least 5,000
deaths since 1990.
In one embodiment, the invention provides a method of identifying the presence
or activity of Cornyebacterium diphtheriae in a subject. This method includes
detection
of one or more of the nucleic acid or amino acid sequences of the invention
(e.g., the
sequences set forth as odd-numbered or even-numbered SEQ ID NOs, respectively,
in
the Sequence Listing) in a subject, thereby detecting the presence or activity
of
Corynebacterium diphtheriae in the subject. C. glutamicum and C. diphtheriae
are
related bacteria, and many of the nucleic acid and protein molecules in C.
glutamicum
are homologous to C. diphtheriae nucleic acid and protein molecules, and can
therefore
be used to detect C. diphtheriae in a subject.
The nucleic acid and protein molecules of the invention may also serve as
markers for specific regions of the genome. This has utility not only in the
mapping of
the genome, but also for functional studies of C. glutamicum proteins. For
example, to
identify the region of the genome to which a particular C. glutamicum DNA-
binding
protein binds, the C. glutamicum genome could be digested, and the fragments
incubated
with the DNA-binding protein. Those which bind the protein may be additionally
probed
with the nucleic acid molecules of the invention, preferably with readily
detectable
labels; binding of such a nucleic acid molecule to the genome fragment enables
the
CA 02590625 2007-06-18
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localization of the fragment to the genome map of C. glutamicum, and, when
performed
multiple times with different enzymes, facilitates a rapid determination of
the nucleic
acid sequence to which the protein binds. Further, the nucleic acid molecules
of the
invention may be sufficiently homologous to the sequences of related species
such that
these nucleic acid molecules may serve as markers for the construction of a
genomic
map in related bacteria, such as Brevibacterium lactofermentum.
The PTS nucleic acid molecules of the invention are also useful for
evolutionary
and protein structural studies. The sugar uptake system in which the molecules
of the
invention participate are utilized by a wide variety of bacteria; by comparing
the
sequences of the nucleic acid molecules of the present invention to those
encoding
similar enzymes from other organisms, the evolutionary relatedness of the
organisms
can be assessed. Similarly, such a comparison permits an assessment of which
regions
of the sequence are conserved and which are not, which may aid in determining
those
regions of the protein which are essential for the functioning of the enzyme.
This type
of determination is of value for protein engineering studies and may give an
indication
of what the protein can tolerate in terms of mutagenesis without losing
function.
Manipulation of the PTS nucleic acid molecules of the invention may result in
the production of PTS proteins having functional differences from the wild-
type PTS
proteins. These proteins may be improved in efficiency or activity, may be
present in
greater numbers in the cell than is usual, or may be decreased in efficiency
or activity.
The invention provides methods for screening molecules which modulate the
activity of a PTS protein, either by interacting with the protein itself or a
substrate or
binding partner of the PTS protein, or by modulating the transcription or
translation of a
PTS nucleic acid molecule of the invention. In such methods, a microorganism
expressing one or more PTS proteins of the invention is contacted with one or
more test
compounds, and the effect of each test compound on the activity or level of
expression
of the PTS protein is assessed.
The PTS molecules of the invention may be modified such that the yield,
production, and/or efficiency of production of one or more fine chemicals is
improved.
For example, by modifying a PTS protein involved in the uptake of glucose such
that it
is optimized in activity, the quantity of glucose uptake or the rate at which
glucose is
translocated into the cell may be increased. The breakdown of glucose and
other sugars
CA 02590625 2007-06-18
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within the cell provides energy that may be used to drive energetically
unfavorable
biochemical reactions, such as those involved in the biosynthesis of fine
chemicals.
This breakdown also provides intermediate and precursor molecules necessary
for the
biosynthesis of certain fine chemicals, such as amino acids, vitamins and
cofactors. By
increasing the amount of intracellular high-energy carbon molecules through
modification of the PTS molecules of the invention, one may therefore increase
both the
energy available to perform metabolic pathways necessary for the production of
one or
more fine chemicals, and also the intracellular pools of metabolites necessary
for such
production. Conversely, by decreasing the importation of a sugar whose
breakdown
products include a compound which is used solely in metabolic pathways which
compete with pathways utilized for the production of a desired fine chemical
for
enzymes, cofactors, or intermediates, one may downregulate this pathway and
thus
perhaps increase production through the desired biosynthetic pathway.
Further, the PTS molecules of the invention may be involved in one or more
intracellular signal transduction pathways which may affect the yields and/or
rate of
production of one or more fine chemical from C. glutamicum. For example,
proteins
necessary for the import of one or more sugars from the extracellular medium
(e.g., HPr,
Enzyme I, or a member of an Enzyme II complex) are frequently
posttranslationally
modified upon the presence of a sufficient quantity of the sugar in the cell,
such that
they are no longer able to import that sugar. An example of this occurs in E.
coli, where
high intracellular levels of fructose 1,6-bisphosphate result in the
phosphorylation of
HPr at serine-46, upon which this molecule is no longer able to participate in
the
transport of any sugar. While this intracellular level of sugar at which the
transport
system is shut off may be sufficient to sustain the normal functioning of the
cell, it may
be limiting for the overproduction of the desired fine chemical. Thus, it may
be
desirable to modify the PTS proteins of the invention such that they are no
longer
responsive to such negative regulation, thereby permitting greater
intracellular
concentrations of one or more sugars to be achieved, and, by extension, more
efficient
production or greater yields of one or more fine chemicals from organisms
containing
such mutant PTS proteins.
This aforementioned list of mutagenesis strategies for PTS proteins to result
in
increased yields of a desired compound is not meant to be limiting; variations
on these
CA 02590625 2007-06-18
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mutagenesis strategies will be readily apparent to one of ordinary skill in
the art. By
these mechanisms, the nucleic acid and protein molecules of the invention may
be
utilized to generate C. glutamicum or related strains of bacteria expressing
mutated PTS
nucleic acid and protein molecules such that the yield, production, and/or
efficiency of
production of a desired compound is improved. This desired compound may be any
natural product of C. glutamicum, which includes the final products of
biosynthesis
pathways and intermediates of naturally-occurring metabolic pathways, as well
as
molecules which do not naturally occur in the metabolism of C. glutamicum, but
which
are produced by a C. glutamicum strain of the invention.
This invention is further illustrated by the following examples which should
not
be construed as limiting. The contents of all references, patent applications,
patents,
published patent applications, Tables, and the Sequence Listing cited
throughout this
application are hereby incorporated by reference.
TABLE 1: Genes Included in the Invention
PHOSPHOENOLPYRUVATE: SUGAR PHOSPHOTRANSFERASE SYSTEM
Nucteotide Amino Acid ldentification Contig. NT Start NT Stop Function
SEQ ID NO SEQ ID NO Code
1 2 RXS00315 PTS SYSTEM, SUCROSE-SPECIFIC IIABC COMPONENT (EIIABC-SCR)
(SUCROSE- PERMEASE
IIABC COMPONENT(PHOSPHOTRANSFERASE ENZYME II, ABC COMPONENT) (EC 2.7.1.69)
3 4 F RXA00315 GR00053 6537 5452 PTS SYSTEM, BETA-GLUCOSIDES-SPECIFIC IIABC
COMPONENT (EI{ABC-BGL) (BETA-
GLUCOSIDES- PERMEASE IIABC COMPONENT) (PHOSPHOTRANSFERASE ENZYME II, ABC
COMPONENT) (EC 2.71.69)
6 RXA01503 GR00424 10392 10640 PTS SYSTEM, BETA-GLUCOSIDES-SPECIFIC IIABC
COMPONENT (EIIABC-BGL) (BETA-
GLUCOSIDES- PERMEASE IIABC COMPONENT) (PHOSPHOTRANSFERASE ENZYME Il, ABC
COMPONENT) (EC 2.7.1.69)
7 8 RXN01299 W0068 11954 9891 PTS SYSTEM, FRUCTOSE-SPECIFIC IIBC COMPONENT (EC
2.7.1.69)
9 10 F RXA01299 GR00375 6 446 PTS SYSTEM, FRUCTOSE-SPECIFIC IIBC COMPONENT (EC
2.7.1.69) t"
11 12 F RXA01883 GR00538 2154 2633 PTS SYSTEM, FRUCTOSE-SPECIFIC IIBC
COMPONENT (EC 2.7.1.69) 0)
N
13 14 F RXA01889 GR00540 77 631 PTS SYSTEM, FRUCTOSE-SPECIFIC IIBC COMPONENT
(EC 2.7.1.69) tn
16 RXA00951 GR00261 564 172 PTS SYSTEM, MANNITOL (CRYPTIC) -SPECIFIC IIA
COMPONENT (EIIA-(C)MTL) (MANNITOL
(CRYPTIC)- PERMEASE IIA COMPONENT) (PHOSPHOTRANSFERASE ENZYME II, A cn o
COMPONENT) (EC 2.7.1.69) v' -.3
17 18 RXN01244 W0068 14141 15844 PHOSPHOENOLPYRUVATE-PROTEIN
PHOSPHOTRANSFERASE (EC 2.7.3.9) o
19 20 F RXA01244 GR00359 4837 3329 PHOSPHOENOLPYRUVATE-PROTEIN
PHOSPHOTRANSFERASE (EC 2.7.3.9) 0)
21 22 RXA01300 GR00375 637 903 PHOSPHOCARRIER PROTEIN HPR
ao
23 24 RXN03002 W0236 1437 1844 PTS SYSTEM, MANNITOL (CRYPTIC) -SPECIFIC IIA
COMPONENT (EIIA-(C)MTL) (MANNITOL
(CRYPTIC)-PERMEASE IIA COMPONENT) (PHOSPHOTRANSFERASE ENZYME II, A
COMPONENT) (EC 2.7.1.69)
26 RXC00953 W0260 1834 1082 Membrane Spanning Protein involved in PTS system
27 28 RXC03001 Membrane Spanning Protein involved in PTS system
29 30 RXN01943 W0120 4326 6374 PTS SYSTEM, GLUCOSE-SPECIFIC IIABC COMPONENT
(EC 2.7.1.69)
31 32 F RXA02191 GR00642 3395 4633 PHOSPHOENOLPYRUVATE SUGAR
PHOSPHOTRANSFERASE
33 34 F RXA01943 GR00557 3944 3540 crr gene; phosphotransferase system glucose-
specific enzyme III
TABLE 2 - Excluded Genes
GenBankTM' Gene Name Gene Function Reference
Accession No.
A09073 ppg Phosphoenol pyruvate carboxylase Bachmann, B. et at. "DNA fragment
coding for phosphoenolpyruvat
corboxylase, recombinant DNA carrying said fragment, strains carrying the
recombinant DNA and method for producing L-aminino acids using said
strains," Patent: EP 0358940-A 3 03/21/90
A45579, Threonine dehydratase Moeckel, B. et al. "Production of L-isoleucine
by means of recombinant
A45581, micro-organisms with deregulated threonine dehydratase," Patent: WO
A45583, - 9519442-A 5 07/20/95
A45585
A45587
AB003132 murC; ftsQ; ftsZ Kobayashi, M. et al. "Cloning, sequencing, and
characterization of the ftsZ
gene from coryneform bacteria," Biochem. Biophys. Res. Commun.,
236(2):383-388 (1997) r v
AB015023 murC; ftsQ Wachi, M. et al. "A murC gene from Coryneform bacteria,"
Appl. Microbiol. Ln
Biolechnol., 51(2):223-228(1999)
AB018530 dtsR Kimura, E. et al. "Molecular cloning of a novel gene, dtsR,
which rescues the Lõ
detergent sensitivity of a mutant derived from Brevibacterium
laclofermentum," Biosci. Biolechnol. Biochem., 60(10):1565-1570 (1996)
AB018531 dtsR]; dtsR2 -.3
AB020624 murl D-glutamate racemase a,
AB023377 tkt transketolase '
r
AB024708 gltB; gltD Glutamine 2-oxoglutarate aminotransferase D
large and small subunits
AB025424 acn aconitase
AB027714 rep Replication protein
AB027715 rep; aad Replication protein; aminoglycoside
adenyltransferase
AF005242 argC N-acetylglutamate-5-semialdehyde
dehydrogenase
AF005635 glnA Glutamine synthetase
AF030405 hisF cyclase
AF030520 argG Argininosuccinate synthetase
AF031518 argF Ornithine carbamolytransferase
AF036932 aroD 3-dehydroquinate dehydratase
AF038548 pyc Pyruvate carboxylase
Table 2 (continued)
AF038651 dciAE; apt; rel Dipeptide-binding protein; adenine Wehmeier, L. et
al. "The role of the Corynebacterium glutamicum rel gene in
phosphoribosyltransferase; GTP (p)ppGpp metabolism," Microbiology, 144:1853-
1862 (1998)
pyrophosphokinase
AF041436 argR Arginine repressor
AF045998 impA Inositol monophosphate phosphatase
AF048764 argH Argininosuccinate lyase
AF049897 argC; argJ; argB; N-acetylglutamylphosphate reductase;
argD; argF; argR; ornithine acetyltransferase; N-
argG; argH acetylglutamate kinase; acetylornithine
transminase; ornithine
carbamoyltransferase; arginine repressor;
argininosuccinate synthase;
argininosuccinatelyase
AF050109 inhA Enoyl-acyl carrier protein reductase
AF050166 hisG ATP phosphoribosyltransferase Lõ
AF051846 hisA Phosphoribosylformimino-5-amino- 1-
1O
phosphoribosyl-4-imidazolecarboxamide
isomerase o+
AF052652 metA Homoserine 0-acetyltransferase Park, S. et al. "Isolation and
analysis of metA, a methionine biosynthetic gene o
encoding homoserine acetyltransferase in Corynebacterium glutamicum," Mol.
Cells., 8(3):286-294 (1998)
AF053071 aroB Dehydroquinate synthetase rn
AF060558 hisH Glutamine amidotransferase
eo
AF086704 hisE Phosphoribosyl-ATP-
pyrophosphohydrolase
AF114233 aroA 5-enolpyruvyishikimate 3-phosphate
synthase
AF116184 panD L-aspartate-alpha-decarboxylase precursor Dusch, N. et al.
"Expression of the Corynebacterium glutamicum panD gene
encoding L-aspartate-alpha-decarboxy lase leads to pantothenate
overproduction in Escherichia coli," Appl. Environ. Microbiol., 65(4)1530-
1539(1999)
AF124518 aroD; aroE 3-dehydroquinase; shikimate
dehydrogenase
AF124600 aroC; aroK; aroB; Chorismate synthase; shikimate kinase; 3-
pepQ dehydroquinate synthase; putative
cytoplasmic peptidase
AF145897 inhA
AF145898 inhA
Table 2 (continued)
AJ001436 ectP Transport of ectoine, glycine betaine, Peter, H. et al.
"Corynebacterium glutamicum is equipped with four secondary
proline carriers for compatible solutes: Identification, sequencing, and
characterization
of the proline/ectoine uptake system, ProP, and the ectoine/proline/glycine
betaine carrier, EctP," J. Bacteriol., 180(22):6005-6012 (1998)
AJ004934 dapD Tetrahydrodipicolinate succinylase Wehrmann, A. et al.
"Different modes of diaminopimelate synthesis and their
(incomplete') role in cell wall integrity: A study with Corynebacterium
glutamicum," J.
Bacteriol., 180(12):3159-3165 (1998)
AJ007732 ppc; secG; amt; ocd; Phosphoenolpyruvate-carboxylase; ?; high
soxA affinity ammonium uptake protein; putative
ornithine-cyclodecarboxylase;sarcosine
oxidase
AJ010319 ftsY, glnB, glnD; srp; Involved in cell division; Pll protein;
Jakoby, M. et al. "Nitrogen regulation in Corynebacterium glutamicum;
amtP uridylyltransferase (uridylyl-removing Isolation of genes involved in
biochemical characterization of corresponding
enzmye); signal recognition particle; low proteins," FEMS Microbiol.,
173(2):303-310 (1999)
affinity ammonium uptake protein Ln
AJ132968 cat Chloramphenicol aceteyl transferase
AJ224946 mqo L-malate: quinone oxidoreductase Molenaar, D. et al. "Biochemical
and genetic characterization of the Lõ
membrane-associated malate dehydrogenase (acceptor) from Corynebacterium
glutamicum," Eur. J. Biochem., 254(2):395-403 (1998)
AJ238250 ndh NADH dehydrogenase -.3
I
AJ238703 porA Porin Lichtinger, T. et al. "Biochemical and biophysical
characterization of the cell
wall porin of Corynebacterium glutamicum: The channel is formed by a low
molecular mass polypeptide," Biochemistry, 37(43):15024-15032 (1998) 00
D17429 Transposable element IS31831 Vertes et al."Isolation and
characterization of IS31831, a transposable element
from Corynebacterium glutamicum," Mol. Microbiol., 11(4):739-746 (1994)
D84102 odhA 2-oxoglutarate dehydrogenase Usuda, Y. et al. "Molecular cloning
of the Corynebacterium glutamicum
(Brevibacterium lactofermentum AJ 12036) odhA gene encoding a novel type
of 2-oxoglutarate dehydrogenase," Microbiology, 142:3347-3354 (1996)
E01358 hdh; hk Homoserine dehydrogenase; homoserine Katsumata, R. et al.
"Production of L-thereonine and L-isoleucine," Patent: JP
kinase 1987232392-A 1 10/12/87
E01359 Upstream of the start codon of homoserine Katsumata, R. et al.
"Production of L-thereonine and L-isoleucine," Patent: JP
kinase gene 1987232392-A 2 10/12/87
E01375 Tryptophan operon
E01376 trpL; trpE Leader peptide; anthranilate synthase Matsui, K. et al.
"Tryptophan operon, peptide and protein coded thereby,
utilization of tryptophan operon gene expression and production of
tryptophan," Patent: JP 1987244382-A 1 10/24/87
Table 2 continued
E01377 Promoter and operator regions of Matsui, K. et al. "Tryptophan operon,
peptide and protein coded thereby,
tryptophan operon utilization of tryptophan operon gene expression and
production of
tryptophan," Patent: JP 1987244382-A 1 10/24/87
E03937 Biotin-synthase Hatakeyama, K. et al. "DNA fragment containing gene
capable of coding
biotin synthetase and its utilization," Patent: JP 1992278088-A 1 10/02/92
E04040 Diamino pelargonic acid aminotransferase Kohama, K. et al. "Gene coding
diaminopelargonic acid aminotransferase and
desthiobiotin synthetase and its utilization," Patent: JP 1992330284-A 1
11/18/92
E04041 Desthiobiotinsynthetase Kohama, K. et al. "Gene coding
diaminopelargonic acid aminotransferase and
desthiobiotin synthetase and its utilization," Patent: JP 1992330284-A 1
I 1 / 18/92
E04307 Flavum aspartase Kurusu, Y. et al. "Gene DNA coding aspartase and
utilization thereof," Patent:
JP 1993030977-A l 02/09/93
E04376 Isocitric acid lyase Katsumata, R. et al. "Gene manifestation
controlling DNA," Patent: JP
1993056782-A 3 03/09/93 Lõ
E04377 Isocitric acid lyase N-terminal fragment Katsumata, R. et al. "Gene
manifestation controlling DNA," Patent: JP
1993056782-A 3 03/09/93
E04484 Prephenate dehydratase Sotouchi, N. et al. "Production of L-
phenylalanine by fetmentation," Patent: JP "'
1993076352-A 2 03/30/93 0
E05108 Aspartokinase Fugono, N. et al. "Gene DNA coding Aspartokinase and its
use," Patent: JP 1~
1993184366-A 1 07/27/93
E05112 Dihydro-dipichorinate synthetase Hatakeyama, K. et al. "Gene DNA coding
dihydrodipicolinic acid synthetase
and its use," Patent: JP 1993184371-A 1 07/27/93
ao
E05776 Diaminopirnelic acid dehydrogenase Kobayashi, M. et al. "Gene DNA
coding Diaminopimelic acid dehydrogenase
and its use," Patent: JP 1993284970-A 1 11/02/93
E05779 Threonine synthase Kohama, K. et al. "Gene DNA coding threonine
synthase and its use," Patent:
JP 1993284972-A I 11/02/93
E061 10 Prephenate dehydratase Kikuchi, T. et al. "Production of L-
phenylalanine by fermentation method,"
Patent: JP 1993344881-A 1 12/27/93
E06111 Mutated Prephenate dehydratase Kikuchi, T. et al. "Production of L-
phenylalanine by fermentation method,"
Patent: JP 1993344881-A I 12/27/93
E06146 Acetohydroxy acid synthetase Inui, M. et al. "Gene capable of coding
Acetohydroxy acid synthetase and its
use," Patent: JP 1993344893-A 1 12/27/93
E06825 Aspartokinase Sugimoto, M. et al. "Mutant aspartokinase gene," patent:
JP 1994062866-A I
03/08/94
E06826 Mutated aspartokinase alpha subunit Sugimoto, M. et al. "Mutant
aspartokinase gene," patent: JP 1994062866-A 1
03/08/94
Table 2 (continued)
E06827 Mutated aspartokinase alpha subunit Sugimoto, M. et al. "Mutant
aspartokinase gene," patent: JP 1994062866-A I
03/08/94
E07701 secY Honno, N. et al. "Gene DNA participating in integration of
membraneous
protein to membrane," Patent: JP 1994169780-A 1 06/21/94
E08177 Aspartokinase Sato, Y. et al. "Genetic DNA capable of coding
Aspartokinase released from
feedback inhibition and its utilization," Patent: JP 1994261766-A 1 09/20/94
E08178, Feedback inhibition-released Aspartokinase Sato, Y. et al. "Genetic
DNA capable of coding Aspartokinase released from
E08179, feedback inhibition and its utilization," Patent: JP 1 99426 1 766-A 1
09/20/94
E08180,
E08181,
E08182
E08232 Acetohydroxy-acid isomeroreductase Inui, M. et al. "Gene DNA coding
acetohydroxy acid isomeroreductase,"
Patent: JP 1994277067-A 1 10/04/94
E08234 secE Asai, Y. et al. "Gene DNA coding for translocation machinery of
protein,"
Patent: JP 1994277073-A 1 10/04/94 "'
E08643 FT aminotransferase and desthiobiotin Hatakeyama, K. et al. "DNA
fragment having promoter function in
synthetase promoter region coryneform bacterium," Patent: JP 1995031476-A 1
02/03/95 <,,
E08646 Biotin synthetase Hatakeyama, K. et al. "DNA fragment having promoter
function in
coryneform bacterium," Patent: JP 1995031476-A 1 02/03/95
E08649 Aspartase Kohama, K. et al "DNA fragment having promoter function in
coryneform - 1 .3
bacterium," Patent: JP 1995031478-A 1 02/03/95
~ rn
E08900 Dihydrodipicolinate reductase Madori, M. et al. "DNA fragment
containing gene coding Dihydrodipicolinate
acid reductase and utilization thereof," Patent: JP 1995075578-A 1 03/20/95 co
E08901 Diaminopimelic acid decarboxylase Madori, M. et al. "DNA fragment
containing gene coding Diaminopimelic acid
decarboxylase and utilization thereof," Patent: JP 1995075579-A 1 03/20/95
E12594 Serine hydroxymethyltransferase Hatakeyama, K. et a1. "Production of L-
trypophan," Patent: JP 1997028391-A
1 02/04/97
E12760, transposase Moriya, M. et aI. "Amplification of gene using artificial
transposon," Patent:
E12759, JP 1997070291-A 03/18/97
E12758
E12764 Arginyl-tRNA synthetase; diaminopimelic Moriya, M. et al.
"Amplification of gene using artificial transposon," Patent:
acid decarboxylase JP 1997070291-A 03/18/97
E 12767 Dihydrodipicolinic acid synthetase Moriya, M. et al. "Amplification of
gene using artificial transposon," Patent:
JP 1997070291-A 03/18/97
E12770 aspartokinase Moriya, M. et al. "Amplification of gene using artificial
transposon," Patent:
JP 1997070291-A 03/18/97
E 12773 Dihydrodipicolinic acid reductase Moriya, M. et al. "Amplification of
gene using artificial transposon," Patent:
JP 1997070291-A 03/18/97
Table 2 (continued)
E13655 Glucose-6-phosphate dehydrogenase Hatakeyama, K. et al. "Glucose-6-
phosphate dehydrogenase and DNA capable
of coding the same," Patent: JP 1997224661-A 1 09/02/97
L01508 IIvA Threonine dehydratase Moeckel, B. et al. "Functional and
structural analysis of the threonine
dehydratase of Corynebacterium glutamicum," J. Bacteriol., 174:8065-8072
(1992)
L07603 EC 4.2.1.15 3-deoxy-D-arabinoheptulosonate-7- Chen, C. et al. "The
cloning and nucleotide sequence of Corynebacterium
phosphate synthase glutamicum 3-deoxy-D-arabinoheptulosonate-7-phosphate
synthase gene,"
FEMS Microbiol. Lett., 107:223-230 (1993)
L09232 IIvB; ilvN; ilvC Acetohydroxy acid synthase large subunit; Keilhauer,
C. et al. "Isoleucine synthesis in Corynebacterium glutamicum:
Acetohydroxy acid synthase small subunit; molecular analysis of the ilvB-ilvN-
ilvC operon," J. Bacteriol., 175(17):5595-
Acetohydroxy acid isomeroreductase 5603 (1993)
L 18874 PtsM Phosphoenolpyruvate sugar Fouet, A et al. "Bacillus subtilis
sucrose-specific enzyme II of the
phosphotransferase phosphotransferase system: expression in Escherichia coli
and homology to
enzymes lI from enteric bacteria," PNAS USA, 84(24):8773-8777 (1987); Lee, o
J.K. et al. "Nucleotide sequence of the gene encoding the Corynebacterium ci,
glutamicum mannose enzyme 11 and analyses of the deduced protein
sequence," FEMS Microbiol. Lett., 119(1-2):137- t45 (1994)
L27123 aceB Malate synthase Lee, H-S. et al. "Molecular characterization of
aceB, a gene encoding malate "'
synthase in Corynebacterium glutamicum," J. Microbiol. Biotechnol., o
4(4):256-263 (1994)
L27126 Pyruvate kinase Jetten, M. S. et al. "Structural and functional
analysis of pyruvate kinase from ~ 'o
Corynebacterium glutamicum," Appl. Environ. Microbiol., 60(7):2501-2507
(1994) 00
L28760 aceA Isocitrate lyase
L35906 dtxr Diphtheria toxin repressor Oguiza, J.A. et al. "Molecular cloning,
DNA sequence analysis, and
characterization of the Corynebacterium diphtheriae dtxR from Brevibacterium
lactofermentum," J. Bacteriol., 177(2):465-467 (1995)
M 13774 Prephenate dehydratase Follettie, M.T. et al. "Molecular cloning and
nucteotide sequence of the
Corynebacterium glutamicum pheA gene," J. Bacteriol., 167:695-702 (1986)
M 16175 5S rRNA Park, Y-H. et al. "Phylogenetic analysis of the coryneform
bacteria by 56
rRNA sequences," J. Bacteriol., 169:1801-1806 (1987)
M 16663 trpE Anthranilate synthase, 5' end Sano, K. et al. "Structure and
function of the trp operon control regions of
Brevibacterium lactofermentum, a glutamic-acid-producing bacterium," Gene,
52:191-200 (1987)
M 16664 trpA Tryptophan synthase, 3'end Sano, K. et al. "Structure and
function of the trp operon control regions of
Brevibacterium lactofermentum, a glutamic-acid-producing bacterium," Gene,
52:191-200 (1987)
Table 2 continued
M25819 Phosphoenolpyruvate carboxylase O'Regan, M. et al. "Cloning and
nucleotide sequence of the
Phosphoenolpyruvate carboxylase-coding gene of Corynebacterium
glutamicum ATCC13032," Gene, 77(2):237-251 (1989)
M85106 23S rRNA gene insertion sequence Roller, C. et al. "Gram-positive
bacteria with a high DNA G+C content are
characterized by a common insertion within their 23S rRNA genes," J. Gen.
Microbiol., 138:1167-1175 (1992)
M85107, 23S rRNA gene insertion sequence Roller, C. et al. "Gram-positive
bacteria with a high DNA G+C content are
M85108 characterized by a common insertion within their 23S rRNA genes," J.
Gen.
Microbiol., 138:1167-1 175 (1992)
M89931 aecD; bmQ; yhbw Beta C-S lyase; branched-chain amino acid Rossol, I. et
al. "The Corynebacterium glutamicum aecD gene encodes a C-S
uptake carrier; hypothetical protein yhbw lyase with alpha, beta-elimination
activity that degrades aminoethylcysteine,"
J. Bacteriol., 174(9):2968-2977 (1992); Tauch, A. et al. "Isoleucine uptake in
Corynebacterium glutamicum ATCC 13032 is directed by the binQ gene
product," Arch. Microbiol., 169(4):303-312 (1998)
S59299 trp Leader gene (promoter) Herry, D.M. et al. "Cloning of the trp gene
cluster from a tryptophan- ci,
hyperproducing strain of Corynebacterium glutamicum: identification of a o
mutation in the trp leader sequence," Appl. Environ. Microbiol., 59(3):791-799
nO1i
(1993) L"
U11545 trpD Anthranilate phosphoribosyltransferase O'Gara, J.P. and Dunican,
L.K. (1994) Complete nucleotide sequence of the
Corynebacterium glutamicum ATCC 21850 tpD gene." Thesis, Microbiology
Department, University College Galway, Ireland. N o
U 13922 cgI1M; cgI1R; c1gIIR Putative type 11 5-cytosoine Schafer, A. et al.
"Cloning and characterization of a DNA region encoding a
methyltransferase; putative type lI stress-sensitive restriction system from
Corynebacterium glutamicum ATCC co
restriction endonuclease; putative type I or 13032 and analysis of its role in
intergeneric conjugation with Escherichia
type I lI restriction endonuclease coli," J. Bacteriol., 176(23):7309-7319
(1994); Schafer, A. et al. "The
Corynebacterium glutamicum cg1IM gene encoding a 5-cytosine in an McrBC-
deficient Escherichia coli strain," Gene, 203(2):95-101 (1997)
U14965 recA
U31224 ppx Ankri, S. et al. "Mutations in the Corynebacterium
glutamicumproline
biosynthetic pathway: A natural bypass of the proA step," J. Bacteriol.,
178(15):4412-4419 (1996)
U31225 proC L-proline: NADP+ 5-oxidoreductase Ankri, S. et al. "Mutations in
the Corynebacterium glutamicumproline
biosynthetic pathway: A natural bypass of the proA step," J. Bacteriol.,
178(15):4412-4419 (1996)
U31230 obg; proB; unkdh ?;gamma glutamyl kinase;similar to D- Ankri, S. et al.
"Mutations in the Corynebacterium glutamicumproline
isomer specific 2-hydroxyacid biosynthetic pathway: A natural bypass of the
proA step," J. Bacteriol.,
dehydrogenases 178(15):4412-4419(1996)
Table 2 continued
U3 t281 bioB Biotin synthase Serebriiskii, I.G., "Two new members of the bio B
superfamily: Cloning,
sequencing and expression of bio B genes of Methylobacillus flagellatum and
Corynebacterium glutamicum," Gene, 175:15-22 (1996)
U35023 thtR; accBC Thiosulfate sulfurtransferase; acyl CoA Jager, W. et al. "A
Corynebacterium glutamicum gene encoding a two-domain
carboxylase protein similar to biotin carboxylases and biotin-carboxyl-carrier
proteins,"
Arch. Microbiol., 166(2);76-82 (1996)
U43535 cmr Multidrug resistance protein Jager, W. et al. "A Corynebacterium
glutamicum gene conferring multidrug
resistance in the heterologous host Escherichia coli," J. Bacteriol.,
179(7):2449-2451 (1997)
U43536 clpB Heat shock ATP-binding protein
U53587 aphA-3 3'5"-aminoglycoside phosphotransferase
U89648 Corynebacterium glutamicum unidentified
sequence involved in histidine biosynthesis,
partial sequence
X04960 trpA; trpB; trpC; trpD; Tryptophan operon Matsui, K. et al. "Complete
nucteotide and deduced amino acid sequences of Lõ
trpE; trpG; trpL the Brevibacterium lactofermentum tryptophan operon," Nucleic
Acids Res., o
14(24):101 l 3-10114 (1986)
X07563 lys A DAP decarboxylase (meso-diaminopimelate Yeh, P. et al. "Nucleic
sequence of the lysA gene of Corynebacterium "'
decarboxylase, EC 4.1.1.20) glutamicum and possible mechanisms for modulation
of its expression," Mol.
Gen. Genet., 212(1):112-119 (1988) ~ =
-.3
X14234 EC 4.1.1.31 Phosphoenolpyruvate carboxylase Eikmanns, B.J. et al. "The
Phosphoenolpyruvate carboxylase gene of
Corynebacterium glutamicum: Molecular cloning, nucleotide sequence, and
expression," Mol. Gen. Genet., 218(2):330-339 (1989); Lepiniec, L. et al.
eo
"Sorghum Phosphoenolpyruvate carboxylase gene family: structure, function
and molecular evolution," Plant. Mol. Biol., 21 (3):487-502 (1993)
X17313 fda Fructose-bisphosphate aldolase Von der Osten, C.H. et al.
"Molecular cloning, nucleotide sequence and fine-
structural analysis of the Corynebacterium glutamicum fda gene: structural
comparison of C. glutamicum fructose-1, 6-biphosphate aldolase to class I and
class 11 aldolases," Mol. Microbiol.,
X53993 dapA L-2, 3-dihydrodipicolinate synthetase (EC Bonnassie, S. et al.
"Nucleic sequence of the dapA gene from
4.2.1.52) Corynebacterium glutam icum," Nucleic Acids Res., 18(21):6421 (1990)
X54223 AttB-related site Cianciotto, N. et al. "DNA sequence homology between
att B-related sites of
Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium
glutamicum , and the attP site of lambdacorynephage," FEMS. Mrcrobiol,
Lett., 66:299-302 (1990)
X54740 argS; lysA Arginyl-tRNA synthetase; Diaminopimelate Marcel, T. et al.
"Nucleotide sequence and organization of the upstream region
decarboxylase of the Corynebacterium glutamicum lysA gene," Mol. Microbiol.,
4(I 1):1819-
1830(1990)
Table 2 (continued)
X55994 trpL; trpE Putative leader peptide; anthranilate Heery, D.M. et al.
"Nucleotide sequence of the Corynebacterium glutamicum
synthase component I trpE gene," Nucleic Acids Res., 18(23):7138 (1990)
X56037 thrC Threonine synthase Han, K.S. et al. "The molecular structure of
the Corynebacterium glutamicum
threonine synthase gene," Mol. Microbiol., 4(10):1693-1702 (1990)
X56075 attB-related site Attachment site Cianciotto, N. et al. "DNA sequence
homology between att B-related sites of
Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium
glutamicum , and the attP site of lambdacorynephage," FEMS. Microbiol,
Lett., 66:299-302 (1990)
X57226 lysC-alpha; lysC-beta; Aspartokinase-alpha subunit; Kalinowski, J. et
al. "Genetic and biochemical analysis of the Aspartokinase
asd Aspartokinase-beta subunit; aspartate beta from Corynebacterium
glutamicum," Mol. Microbiol., 5(5):1197-1204 (1991);
semialdehyde dehydrogenase Kalinowski, J. et al. "Aspartokinase genes lysC
alpha and lysC beta overlap
and are adjacent to the aspertate beta-semialdehyde dehydrogenase gene asd in
Corynebacterium glutamicum," Mol. Gen. Genet., 224(3):317-324 (1990)
X59403 gap;pgk; tpi Glyceraldehyde-3-phosphate; Eikmanns, B.J.
"Identification, sequence analysis, and expression of a
phosphoglycerate kinase; triosephosphate Corynebacterium glutamicum gene
cluster encoding the three glycolytic c.i,
isomerase enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate
kinase, and triosephosphate isomeras," J. Bacteriol., 174(19):6076-6086
(1992) L'
X59404 gdh Glutamate dehydrogenase Bon:nann, E.R. et al. "Molecular analysis
of the Corynebacterium glutamicum
gdh gene encoding glutamate dehydrogenase," Mol. Microbiol., 6(3):317-326 ON
(1992)
X60312 lysl L-lysine permease Seep-Feldhaus, A.H. et al. "Molecular analysis
of the Corynebacterium ~ O1
glutamicum lysl gene involved in lysine uptake," Mol. Microbiol., 5(12):2995-
00
3005(1991) X66078 copl Psl protein Joliff, G. et al. "Cloning and nucleotide
sequence of the cspl gene encoding
PSI, one of the two major secreted proteins of Corynebacterium glutamicum:
The deduced N-terminal region of PSI is similar to the Mycobacterium antigen
85 complex," Mol. Microbiol., 6(16):2349-2362 (1992)
X66112 glt Citrate synthase Eikmanns, B.J. et al. "Cloning sequence,
expression and transcriptional
analysis of the Corynebacterium glutamicum gItA gene encoding citrate
synthase," Microbiol., 140:1817-1828 (1994)
X67737 dapB Dihydrodipicolinate reductase
X69103 csp2 Surface layer protein PS2 Peyret, J.L. et al. "Characterization of
the cspB gene encoding PS2, an ordered
surface-layer protein in Corynebacterium glutamicum," Mol. Microbiol.,
9(1):97-109 (1993)
X69104 IS3 related insertion element Bonamy, C. et al. "Identification of
IS1206, a Corynebacterium glutamicum
IS3-related insertion sequence and phylogenetic analysis," Mol. Microbiol.,
14(3):571-581 (1994)
Table 2 (continued)
X70959 leuA Isopropylmalate synthase Patek, M. et al. "Leucine synthesis in
Corynebacterium glutamicum: enzyme
activities, structure of leuA, and effect of leuA inactivation on lysine
synthesis," Appl. Environ. Microbiol., 60(1):133-140 (1994)
X71489 icd lsocitrate dehydrogenase (NADP+) Eikmanns, B.J. et al. "Cloning
sequence analysis, expression, and inactivation
of the Corynebacterium glutamicum icd gene encoding isocitrate
dehydrogenase and biochemical characterization of the enzyme," J. Bacteriol.,
177(3):774-782(1995)
X72855 GDHA Glutamate dehydrogenase (NADP+)
X75083, mtrA 5-methyltryptophan resistance Heery, D.M. et al. "A sequence from
a tryptophan-hyperproducing strain of
X70584 Corynebacterium glutamicum encoding resistance to 5-methyltryptophan,"
Biochem. Biophys. Res. Commun., 201(3):1255-1262 (1994)
X75085 recA Fitzpatrick, R. et al. "Construction and characterization of recA
mutant strains
of Corynebacterium glutamicum and Brevibacterium lactofermentum," Appl.
Microbio/. Biotechnol., 42(4):575-580 (1994)
X75504 aceA; thiX Partial lsocitrate lyase; ? Reinscheid, D.J. et al.
"Characterization of the isocitrate lyase gene from <,,
Corynebacterium glutamicum and biochemical analysis of the enzyme," J. o
Bacteriol., 176(12):3474-3483 (1994)
X76875 ATPase beta-subunit Ludwig, W. et al. "Phylogenetic relationships of
bacteria based on comparative "'
sequence analysis of elongation factor Tu and ATP-synthase beta-subunit
genes," Antonie Van Leeuwenhoek, 64:285-305 (1993)
oN -.3
X77034 tuf Elongation factor Tu Ludwig, W. et al. "Phylogenetic relationships
of bacteria based on comparative
sequence analysis of elongation factor Tu and ATP-synthase beta-subunit
genes," Antonie Van Leeuwenhoek, 64:285-305 (1993) co
X77384 recA Billman-Jacobe, H. "Nucleotide sequence of a recA gene from
Corynebacterium glutamicum," DNA Seq., 4(6):403-404 (1994)
X78491 aceB Malate synthase Reinscheid, D.J. et al. "Malate synthase from
Corynebacterium glutamicum
pta-ack operon encoding phosphotransacetylase: sequence analysis,"
Microbiology, 140:3099-3108 (1994)
X80629 16S rDNA 16S ribosomal RNA Rainey, F.A. et al. "Phylogenetic analysis
of the genera Rhodococcus and
Norcardia and evidence for the evolutionary origin of the genus Norcardia
from within the radiation of Rhodococcus species," MicrobioL, 141:523-528
(1995)
X81191 gluA; gluB; gluC; Glutamate uptake system Kronemeyer, W. et al.
"Structure of the gIuABCD cluster encoding the
gluD glutamate uptake system of Corynebacterium glutamicum," J. Bacteriol.,
177(5):1152-1158(1995)
X81379 dapE Succinyldiaminopimelate desuccinylase Wehrmann, A. et al.
"Analysis of different DNA fragments of
Corynebacterium glutamicum complementing dapE of Escherichia coli,"
Microbiology, 40:3349-56 (1994)
Table 2 continued
X82061 16S rDNA 16S ribosomal RNA Ruimy, R. et al. "Phylogeny of the genus
Corynebacterium deduced from
analyses of small-subunit ribosomal DNA sequences," Int. J. Syst. Bacteriol.,
45(4):740-746 (1995)
X82928 asd; lysC Aspartate-semialdehyde dehydrogenase; ? Serebrijski, I. et
al. "Multicopy suppression by asd gene and osmotic stress-
dependent complementation by heterologous proA in proA mutants," J.
Bacteriol., 177(24):7255-7260 (1995)
X82929 proA Gamma-glutamyl phosphate reductase Serebrijski, I. et al.
"Multicopy suppression by asd gene and osmotic stress-
dependent complementation by heterologous proA in proA mutants," J.
Bacteriol., 177(24):7255-7260 (1995)
X84257 16S rDNA 16S ribosomal RNA Pascual, C. et al. "Phylogenetic analysis of
the genus Corynebacterium based
on 16S rRNA gene sequences," /nt. J. Syst. Bacteriol., 45(4):724-728 (1995)
X85965 aroP; dapE Aromatic amino acid permease; ? Wehrmann et al. "Functional
analysis of sequences adjacent to dapE of C.
glutamicum proline reveals the presence of aroP, which encodes the aromatic
amino acid transporter," J. Bacteriol., l77(20):5991-5993 (1995) o
X86157 argB; argC; argD; Acetylglutamate kinase; N-acetyl-gamma- Sakanyan, V.
et al. "Genes and enzymes of the acetyl cycle of arginine cn
argF; argJ glutatnyl-phosphate reductase; biosynthesis in Corynebacterium
glutamicum: enzyme evolution in the early o
acetylornithine aminotransferase; omithine steps of the arginine pathway,"
Microbiology, 142:99-108 (1996) nO1i
carbamoyltransferase; glutamate N- "'
acetyltransferase
X89084 pta; ackA Phosphate acetyltransferase; acetate kinase Reinscheid, D.J.
et al. "Cloning, sequence analysis, expression and inactivation ~
of the Corynebacterium glutamicum pta-ack operon encoding
phosphotransacety lase and acetate kinase," Microbiology, 145:503-513 (1999) ~
O1
X89850 attB Attachment site Le Marrec, C. et al. "Genetic characterization of
site-specific integration co
functions of phi AAU2 infecting "Arthrobacter aureus C70," J. Bacteriol.,
178(7):1996-2004 (1996)
X90356 Promoter fragment F 1 Patek, M. et al. "Promoters from Corynebacterium
glutamicum: cloning,
molecular analysis and search for a consensus motif," Microbiology,
142:1297-1309 (1996)
X90357 Promoter fragment F2 Patek, M. et al. "Promoters from Corynebacterium
glutamicum: cloning,
molecular analysis and search for a consensus motif," Microbiology,
142:1297-1309 (1996)
X90358 Promoter fragment F10 Patek, M. et al. "Promoters from Corynebacterium
glutamicum: cloning,
molecular analysis and search for a consensus motif," Microbiology,
142:1297-1309 (1996)
X90359 Promoter fragment F 13 Patek, M. et al. "Promoters from Corynebacterium
glutamicum: cloning,
molecular analysis and search for a consensus motif," Microbiology,
142:1297-1309(1996)
Table 2 (continued)
X90360 Promoter fragment F22 Patek, M. et al. "Promoters from Corynebacterium
glutamicum: cloning,
molecular analysis and search for a consensus motif," Microbiology,
142:1297-1309 (1996)
X90361 Promoter fragment F34 Patek, M. et al. "Promoters from Corynebacterium
glutamicum: cloning,
molecular analysis and search for a consensus motif," Microbiology,
142:1297-1309 (1996)
X90362 Promoter fragment F37 Patek, M. et al. "Promoters from C. glutamicum:
cloning, molecular analysis
and search for a consensus motif," Microbiology, 142:1297-1309 (1996)
X90363 Promoter fragment F45 Patek, M. et al. "Promoters from Corynebacterium
glutamicum: cloning,
molecular analysis and search for a consensus motif," Microbiology,
142:1297-1309 (1996)
X90364 Promoter fragment F64 Patek, M. et al. "Promoters from Corynebacterium
glutamicum: cloning,
molecular analysis and search for a consensus motif," Microbiology,
142:1297-1309(1996)
X90365 Promoter fragment F75 Patek, M. et al. "Promoters from Corynebacterium
glutamicum: cloning, Ln
molecular analysis and search for a consensus motif," Microbiology, o
142:1297-1309(1996) nO1i
X90366 Promoter fragment PF101 Patek, M. et al. "Promoters from
Corynebacterium glutamicum: cloning, "'
molecular analysis and search for a consensus motif," Microbiology, o
142:1297-1309(1996)
X90367 Promoter fragment PF104 Patek, M. et al. "Promoters from
Corynebacterium glutamicum: cloning,
molecular analysis and search for a consensus motif," Microbiology, O1
142:1297-1309 (1996)
00
X90368 Promoter fragment PF109 Patek, M. et al. "Promoters from
Corynebacterium glutamicum: cloning,
molecular analysis and search for a consensus motif," Microbiology,
142:1297-1309 (1996)
X93513 amt Ammonium transport system Siewe, R.M. et al. "Functional and
genetic characterization of the (methyl)
ammonium uptake carrier of Corynebacterium glutamicum," J. Biol. Chem.,
271(10):5398-5403(1996)
X93514 betP Glycine betaine transport system Peter, H. et al. "Isolation,
characterization, and expression of the
Corynebacterium glutamicum betP gene, encoding the transport system for the
compatible solute glycine betaine," J. Bacteriol., 178(17):5229-5234 (1996)
X95649 orf4 Patek, M. et al. "Identification and transcriptional analysis of
the dapB-ORF2-
dapA-ORF4 operon of Corynebacterium glutamicum, encoding two enzymes
involved in L-lysine synthesis," Biotechnol. Le1t., 19:1113-1117 (1997)
X96471 IysE; IysG Lysine exporter protein; Lysine export Vrljic, M. et al. "A
new type of transporter with a new type of cellular
regulator protein function: L-lysine export from Corynebacterium glutamicum,"
Mol.
Microbiol., 22(5):815-826 (1996)
Table 2 (continued)
X96580 panB; panC; xylB 3-methyl-2-oxobutanoate Sahm, H. et al. "D-
pantothenate synthesis in Corynebacterium glutamicum and
hydroxymethyltransferase; pantoate-beta- use of panBC and genes encoding L-
valine synthesis for D-pantothenate
alanine ligase; xylulokinase overproduction," Appl. Environ. Microbiol.,
65(5):1973-1979 (1999)
X96962 Insertion sequence 1S1207 and transposase
X99289 Elongation factor P Ramos, A. et al. "Cloning, sequencing and
expression of the gene encoding
elongation factor P in the amino-acid producer Brevibacterium lactofermentum
(Corynebacterium glutamicum ATCC 13869)," Gene, 198:217-222 (1997)
Y00140 thrB Homoserine kinase Mateos, L.M. et al. "Nucleotide sequence of the
homoserine kinase (thrB) gene
of the Brevibacterium lactofermentum," Nucleic Acids Res., 15(9):3922 (1987)
Y00151 ddh Meso-diaminopimelate D-dehydrogenase Ishino, S. et al. "Nucleotide
sequence of the meso-diaminopimelate D-
(EC 1.4.1.16) dehydrogenase gene from Corynebacterium glutarnicum," Nucleic
Acids Res., r v
15(9):3917 (1987) Ln
Y00476 thrA Homoserine dehydrogenase Mateos, L.M. et al. "Nucleotide sequence
of the homoserine dehydrogenase
(thrA) gene of the Brevibacterium lactofermentum," Nucleic Acids Res., Lõ
15(24):10598 (1987)
Y00546 hom; thrB Homoserine dehydrogenase; homoserine Peoples, O.P. et al.
"Nucleotide sequence and fine structural analysis of the
kinase Corynebacterium glutamicum hom-thrB operon," Mol. Microbiol., 2(1):63-
72 ~ : ~
(1988)
rn
Y08964 murC; ftsQ/divD; ftsZ UPD-N-acetylmuramate-alanine ligase; Honrubia,
M.P. et al. "Identification, characterization, and chromosomal
division initiation protein or cell division organization of the ftsZ gene
from Brevibacterium lactofermentum," Mol. Gen. 00
protein; cell division protein Genet.. 259(l):97-104 (1998)
Y09163 putP High affinity proline transport system Peter, H. et al. "Isolation
of the putP gene of Corynebacterium
glutamicumproline and characterization of a low-affinity uptake system for
compatible solutes," Arch. Microbiol., 168(2):143-151 (1997)
Y09548 pyc Pyruvate carboxylase Peters- Wendisch, P.G. et al. "Pyruvate
carboxylase from Corynebacterium
glutamicum: characterization, expression and inactivation of the pyc gene,"
Microbiology, 144:915-927 (1998)
Y09578 leuB 3-isopropylmalate dehydrogenase Patek, M. et al. "Analysis of the
leuB gene from Corynebacterium
glutamicum," Appl. Microbiol. Biolechnol., 50(1):42-47 (1998)
Y12472 Attachment site bacteriophage Phi-16 Moreau, S. et al. "Site-specific
integration of corynephage Phi-16: The
construction of an integration vector," Microbiol., 145:539-548 (1999)
Y12537 proP Proline/ectoine uptake system protein Peter, H. et al.
"Corynebacterium glutamicum is equipped with four secondarycarriers for
compatible solutes: Identification, sequencing, and characterization
of the proline/ectoine uptake system, ProP, and the ectoine/proline/glycine
betaine carrier, EctP," J. Bacteriol., 180(22):6005-6012 (1998)
Table 2 (continued)
Y 13221 glnA Glutamine synthetase I Jakoby, M. et al. "Isolation of
Corynebacterium glutamicum glnA gene
encoding glutamine synthetase I," FEMS Microbiol. Lett., 154(l):81-88 (1997)
Y 16642 lpd Dihydrolipoamide dehydrogenase
Y 18059 Attachment site Corynephage 304L Moreau, S. et al. "Analysis of the
integration functions of φ304L: An
integrase module among corynephages," Virology, 255(1):150-159 (1999)
Z21501 argS; lysA Arginyl-tRNA synthetase; diaminopimelate Oguiza, J.A. et al.
"A gene encoding arginyl-tRNA synthetase is located in the
decarboxylase (partial) upstream region of the lysA gene in Brevibacterium
lactofermentum:
Regulation of argS-lysA cluster expression by arginine," J.
Bacterrol.,175(22):7356-7362 (1993)
Z21502 dapA; dapB Dihydrodipicolinate synthase; Pisabarro, A. et al. "A
cluster of three genes (dapA, orf2, and dapB) of
dihydrodipicolinate reductase Brevibacterium lactofermentum encodes
dihydrodipicolinate reductase, and a
third polypeptide of unknown function," J. Bacteriol., 175(9):2743-2749
(1993)
Z29563 thrC Threonine synthase Malumbres, M. et al. "Analysis and expression
of the thrC gene of the encoded
threonine synthase," Appl. Environ. Microbiol., 60(7)2209-2219 (1994) L"
to
Z46753 16S rDNA Gene for 16S ribosomal RNA
rn
Z49822 sigA SigA sigma factor Oguiza, J.A. et al "Multiple sigma factor genes
in Brevibacterium N
cn
lactofermentum: Characterization of sigA and sigB," J. Bacteriol., 178(2):550-
553 (1996)
Z49823 galE; dtxR Catalytic activity UDP-galactose 4- Oguiza, J.A. et al "The
galE gene encoding the UDP-galactose 4-epimerase of CN -.3
epimerase; diphtheria toxin regulatory Brevibacterium lactofermentum is
coupled transcriptionally to the dmdR o
protein gene," Gene, 177:103-107 (1996) O1
Z49824 orfl ; sigB ?; SigB sigma factor Oguiza, J.A. et al "Multiple sigma
factor genes in Brevibacterium o~o
lactofermentum: Characterization of sigA and sigB," J. Bacteriol., 178(2):550-
553 (1996)
Z66534 Transposase Correia, A. et al. "Cloning and characterization of an IS-
like element present in
the genome of Brevibacterium lactofermentum ATCC 13869," Gene,
170(1):91-94 (1996)
A sequence for this gene was published in the indicated reference. However,
the sequence obtained by the inventors of the present application is
significantly longer than
the published version. It is believed that the published version relied on an
incorrect start codon, and thus represents only a fragment of the actual
coding region.
CA 02590625 2007-06-18
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TABLE 3: Corynebacterium and Brevibacterium Strains Which May be Used in
the Practice of the Invention
Genus s i =TC } ; ;F~RM ~N ~ ~E = G11VIB' ~BS~ N C~ DSMZ
_. .. , ,. . . . - .._ ~. _ . _ . ..._.
Brevibacterium ammoniagenes 21054
Brevibacterium ammoniagenes 19350
Brevibacterium ammoniagenes 19351
Brevibacterium ammoniagenes 19352
Brevibacterium ammoniagenes 19353
Brevibacterium ammoniagenes 19354
Brevibacterium ammoniagenes 19355
Brevibacterium ammoniagenes 19356
Brevibacterium ammoniagenes 21055
Brevibacterium ammoniagenes 21077
Brevibacterium ammoniagenes 21553
Brevibacterium ammoniagenes 21580
Brevibacterium arnmoniagenes 39101
Brevibacterium butanicum 21196
Brevibacterium divaricatum 21792 P928
Brevibacterium flavum 21474
Brevibacterium flavum 21129
Brevibacterium flavum 21518
Brevibacterium flavum B11474
Brevibacterium flavum B 11472
Brevibacterium flavum 21127
Brevibacterium flavum 21128
Brevibacterium flavum 21427
Brevibacterium flavum 21475
Brevibacterium flavum 21517
Brevibacterium flavum 21528
Brevibacterium flavum 21529
Brevibacterium flavum B11477
Brevibacterium flavum B11478
Brevibacterium flavum 21127
Brevibacterium flavum B11474
Brevibacterium healii 15527
Brevibacterium ketoglutamicum 21004
Brevibacterium ketoglutamicum 21089
Brevibacterium ketosoreductum 21914
Brevibacterium lactofetmentum 70
Brevibacterium lactofermentum 74
Brevibacterium lactofermentum 77
Brevibacterium lactofermentum 21798
Brevibacterium lactofermentum 21799
Brevibacterium lactofermentum 21800
Brevibacterium lactofermentum 21801
Brevibacterium lactofermentum B11470
Brevibacterium lacto ermentum B11471
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Brevibacterium lactofermentum 21086
Brevibacterium Iactofermentum 21420
Brevibacterium lactofermenturn 21086
Brevibacterium lactofermentum 31269
Brevibacterium linens 9174
Brevibacterium linens 19391
Brevibacterium linens 8377
Brevibacterium paraffinolyticum 11160
Brevibacterium spec. 717.73
Brevibacterium spec. 717.73
Brevibacterium spec. 14604
Brevibacterium spec. 21860
Brevibacterium spec. 21864
Brevibacterium spec. 21865
Brevibacterium spec. 21866
Brevibacterium spec. 19240
Corynebacterium acetoacidophilum 21476
Corynebacterium acetoacidophilum 13870
Corynebacterium acetoglutamicum B 11473
Corynebacterium acetoglutamicum B 11475
Corynebacterium acetoglutamicum 15806
Corynebacterium acetoglutamicum 21491
Corynebacterium acetoglutamicum 31270
Corynebacterium acetophilum B3671
Corynebacterium ammoniagenes 6872 2399
Corynebacterium ammoniagenes 15511
Corynebacterium fujiokense 21496
Corynebacterium glutamicum 14067
Corynebacterium glutamicum 39137
Corynebacterium glutamicum 21254
Corynebacterium glutamicum 21255
Corynebacterium glutamicum 31830
Corynebacterium glutamicum 13032
Corynebacterium glutamicum 14305
Corynebacterium glutamicum 15455
Corynebacterium glutamicum 13058
Corynebacterium glutarnicum 13059
Corynebacterium glutamicum 13060
Corynebacterium glutamicum 21492
Corynebacterium glutamicum 21513
Corynebacterium glutamicum 21526
Corynebacterium glutamicum 21543
Corynebacterium glutaznicum 13287
Corynebacterium glutamicum 21851
Corynebacterium glutamicum 21253
Corynebacterium glutamicum 21514
Corynebacterium glutamicum 21516
Corynebacterium glutamicum 21299
Corynebacterium glutamicum 21300
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Corynebacterium glutamicum 39684
Corynebacterium glutamicum 21488
Corynebacterium glutamicum 21649
Corynebacterium glutamicum 21650
Corynebacterium glutamicum 19223
Corynebacterium glutamicum 13869
Corynebacterium glutamicum 21157
Corynebacterium glutamicum 21158
Corynebacterium glutamicum 21159
Corynebacterium glutamicum 21355
Corynebacterium glutamicum 31808
Corynebacterium glutamicum 21674
Corynebacterium glutamicum 21562
Corynebacterium glutamicum 21563
Corynebacterium glutamicum 21564
Corynebacterium glutamicum 21565
Corynebacterium glutamicum 21566
Corynebacterium glutamicum 21567
Corynebacterium glutamicum 21568
Corynebacterium glutamicum 21569
Corynebacterium glutamicum 21570
Corynebacterium glutamicum 21571
Corynebacterium glutamicum 21572
Corynebacterium glutamicum 21573
Corynebacterium glutamicum 21579
Corynebacterium glutamicum 19049
Corynebacterium glutamicum 19050
Corynebacterium glutamicum 19051
Corynebacterium glutamicum 19052
Corynebacterium glutamicum 19053
Corynebacterium glutamicum 19054
Corynebacterium glutamicum 19055
Corynebacterium glutamicum 19056
Corynebacterium glutamicum 19057
Corynebacterium glutamicum 19058
Corynebacterium glutamicum 19059
Corynebacterium glutamicum 19060
Corynebacterium glutamicum 19185
Corynebacterium glutamicum 13286
Corynebacterium glutamicum 21515
Corynebacterium glutamicum 21527
Corynebacterium glutamicum 21544
Corynebacterium glutamicum 21492
Corynebacterium glutamicum B8183
Corynebacterium glutamicum B8182
Corynebacterium glutamicum B12416
Corynebacterium glutamicum B12417
Corynebacterium glutamicum B12418
Corynebacterium glutamicum B11476
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Corynebacterium glutamicum 21608
Corynebacterium lilium P973
Corynebacterium nitrilophilus 21419 11594
Corynebacterium spec. P4445
Corynebacterium spec. P4446
Corynebacterium spec. 31088
Corynebacterium spec. 31089
Corynebacterium spec. 31090
Corynebacterium spec. 31090
Corynebacterium spec. 31090
Corynebacterium spec. 15954 20145
Corynebacterium spec. 21857
Corynebacterium spec. 21862
Corynebacterium spec. 21863
ATCC: American Type Culture Collection, Rockville, MD, USA
FERM: Fermentation Research Institute, Chiba, Japan
NRRL: ARS Culture Collection, Northern Regional Research Laboratory, Peoria,
IL, USA
CECT: Coleccion Espanola de Cultivos Tipo, Valencia, Spain
NCIMB: National Collection of Industrial and Marine Bacteria Ltd., Aberdeen,
UK
CBS: Centraalbureau voor Schimmelcultures, Baarn, NL
NCTC: National Collection of Type Cultures, London, UK
DSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig,
Germany
For reference see Sugawara, H. et al. (1993) World directory of collections of
cultures of
microorganisms: Bacteria, fungi and yeasts (4'h edn), World federation for
culture collections world
data center on microorganisms, Saimata, Japen.
TABLE 4: ALIGNMENT RESULTS
ID# lenath Genbank Hit Len h Accession Name of Genbank Hit Source of Genbank
Hit 9_ Date of
NT homoloav Deposit
G~ APy
rxa00315 1527 GB BA1:AB007125 4078 A6007125 Serratia marcescens slaA gene for
surface layer protein, complete Serratia marcescens 40,386 26-MAR-1998
cds, isolate 8000.
GB_IN1:CELC47D2 17381 U64861 Caenorhabditis elegans cosmid C47D2.
Caenorhabditis elegans 36,207 28-Jul-96
GB_HTG2:AC006732 159453 AC006732 Caenorhabditis elegans clone Y32G9, "'
SEQUENCING IN Caenorhabditis elegans 36,436 23-Feb-99
PROGRESS =",9 unordered pieces.
rxa01503 372 GB PR3:AC005019 188362 AC005019 Homo sapiens SAC clone GS250A16
from 7p21-p22, complete Homo sapiens 39,722 27-Aug-98
GB_GSS12:AQ390040 680 AQ390040 RPCI11-157C9.TJ RPCI-11 Homo sapiens genomic
clone RPCI- Homo sapiens 43,137 21-MAY-1999
11-157C9,genomic survey sequence.
GB_GSS5:AQ784231 542 AQ784231 HS_3087_B1_C10_T7C CIT Approved Human Genomic
Sperm Homo sapiens 37,643 3-Aug-99
Library D Homo sapiens genomic clone Plate=3087 Col=19
Row=F, genomic survey sequence.
rxa01299 2187 GB_EST38:AW047296 614 AW047296 UI-M-BH1-amh-e-03-0-UI.s1
NIH_BMAP_M_S2 Mus musculus Mus musculus 41,475 18-Sep-99 Ln
cDNA clone UI-M-BH1-amh-e-03-0-UI 3', mRNA sequence.
GB_RO:AB004056 1581 AB004056 Rattus norvegicus mRNA for BarH-class homeodomain
Rattus norvegicus 41,031 2-Sep-98 N
transcription factor, complete cds. L"
GB_RO:AS004056 1581 AB004056 Rattus norvegicus mRNA for BarH-class homeodomain
Rattus norvegicus 40,717 2-Sep-98
transcription factor, complete cds. 0
nca00951 416 GB_BAI:SCJ21 31717 AL109747 Streptomyces coelicolor cosmid J21.
Streptomyces coelicolor 34,913 5-Aug-99 .3
A3(2) 46
0)
GS_VI:MCU68299 230278 U68299 Mouse cytomegalovirus 1 complete genomic
sequence. Mouse cytomegalovirus 1 40,097 04-DEC-1996 N
~
00
GB_VI:U93872 133661 U93872 Kaposi's sarcoma-associated herpesvirus
glycoprotein M, DNA Kaposi's sarcoma- 36,029 9-Jul-97
replication protein, glycoprotein, DNA replication protein, FLICE associated
herpesvirus
inhibitory protein and v-cyclin genes, complete cds, and tegument
rxa01244 1827 GB BA1:AFAPHBHI 4501 M69036 Alcaligenes eutrophus protein H
(phbH) and protein I(phbl) Ralstonia eutropha 45,624 26-Apr-93
genes, complete cds.
GB_PR3:HSJ836E13 78055 AL050326 Human DNA sequence from clone 836E13 on
chromosome 20 Homo sapiens 37,303 23-Nov-99
Contains ESTs, STS and GSSs, complete sequence.
GB_EST24:AI170227 409 A1170227 EST216152 Normalized rat lung, Bento Soares
Rattus sp. cDNA Rattus sp. 39,098 20-Jan-99
clone RLUCF56 3' end, mRNA sequence.
nca01300 390 GB_PR3:HUMDODDA 26764 L39874 Homo sapiens deoxycytidylate
deaminase gene, complete cds. Homo sapiens 37,644 11-Aug-95
GB_PAT:140899 26764 140899 Sequence 1 from patent US 5622851. Unknown. 37,644
13-MAY-1997
GB_PAT:140900 1317 140900 Sequence 2 from patent US 5622851. Unknown. 37.644
13-MAY-1997
rxa00953 789 GB_BAI:SCJ21 31717 AL109747 Streptomyces coelicolor cosmid J21.
Streptomyces coelicolor 39,398 5-Aug-99
A3(2)
GB_BA1:BLTRP 7725 X04960 Brevibacterium lactofermentum tryptophan operon.
Corynebacterium 39,610 10-Feb-99
glutamicum
GB_PAT:E01375 7726 E01375 DNA sequence of tryptophan operon. Corynebacterium
46,753 29-Sep-97
glutamicum
Table 4 (continued)
nca01943 2172 GB_BA1:CORPTSMA 2656 L18874 Corynebacterium glutamicum
phosphoenolpyruvate sugar Corynebacterium 100,000 24-Nov-94
phosphotransferase (ptsM) mRNA, complete cds. glutamicum
GB BA1:BRLPTSG 3163 L18875 Brevibacterium lactofermentum phosphoenolpyruvate
sugar Brevibacterium 84,963 01-OCT-1993
phosphotransferase (ptsG) gene, complete cds. lactofermentum
GB_BA2:AF045481 2841 AF045481 Corynebacterium ammoniagenes glucose permease
(ptsG) gene, Corynebacterium 53,558 29-Jul-98
complete cds. ammoniagenes
O
U9
O
0)
U9
O
O
i . J
~ I
0)
F-'
00
CA 02590625 2007-06-18
- 76 -
Exemplification
Example 1: Preparation of total genomic DNA of Corynebacterium glutamicum
ATCC 13032
A culture of Corynebacterium glutamicum (ATCC 13032) was grown overnight
at 30 C with vigorous shaking in BHI medium (Difco). The cells were harvested
by
centrifugation, the supernatant was discarded and the cells were resuspended
in 5 ml
buffer-I (5% of the original volume of the culture - all indicated volumes
have been
calculated for 100 ml of culture volume). Composition of buffer-I: 140.34 g/I
sucrose,
2.46 g/I MgSO4 x 7Hz0, 10 ml/1 KHZPO, solution (100 g/l, adjusted to pH 6.7
with
KOH), 50 ml/1 M12 concentrate (10 g/l (NH,)ZSO,, I g/l NaCI, 2 g/l MgSO4 x
7HZ0,
0.2 g/1 CaClZ, 0.5 g/t yeast extract (Difco), 10 ml/I trace-elements-mix (200
mg/i FeSOq
x HZO, 10 mg/1 ZnSO4 x 7 HZO, 3 mg/1 MnClz x 4 HzO, 30 mg/1 H3BO3 20 mg/1
CoC1z x
6 H20, 1 mg/1 NiCIZ x 6 H2O, 3 mg/1 NaZMoO4 x 2 H2O, 500 mg/1 complexing agent
(EDTA or critic acid), 100 ml/1 vitamins-mix (0.2 mg/1 biotin, 0.2 mg/1 folic
acid, 20
mg/1 p-amino benzoic acid, 20 mg/1 riboflavin, 40 mg/1 ca-panthothenate, 140
mg/1
nicotinic acid, 40 mg/1 pyridoxole hydrochloride, 200 mg/1 myo-inositol).
Lysozyme
was added to the suspension to a final concentration of 2.5 mg/mi. After an
approximately 4 h incubation at 37 C, the cell wall was degraded and the
resulting
protoplasts are harvested by centrifugation. The pellet was washed once with 5
ml
buffer-I and once with 5 ml TE-buffer (10 mM Tris-HCI,1 mM EDTA, pI-I 8). The
pellet was resuspended in 4 ml TE-buffer and 0.5 ml SDS solution (10%) and 0.5
ml
NaCI solution (5 M) areadded. After adding of proteinase K to a final
concentration of
200 g/ml, the suspension is incubated for ca.18 h at 37 C. The DNA was
purified by
extraction with phenol, phenol-chloroform-isoamylalcohol and chloroform-
isoamylalcohol using standard procedures. Then, the DNA was precipitated by
adding
1/50 volume of 3 M sodium acetate and 2 volumes of ethanol, followed by a 30
min
incubation at -20 C and a 30 min centrifugation at 12,000 rpm in a high speed
centrifuge
using a SS34 rotor (Sorvall). The DNA was dissolved in 1 ml TE-buffer
containing 20
g/ml RNaseA and dialysed at 4 C against 1000 ml TE-buffer for at least 3
hours.
During this time, the buffer was exchanged 3 times. To aliquots of 0.4 ml of
the
dialysed DNA solution, 0.4 ml of 2 M LiCI and 0.8 ml of ethanol are added.
After a 30
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min incubation at -20 C, the DNA was collected by centrifugation (13,000 rpm,
Biofuge
Fresco, Heraeus, Hanau, Germany). The DNA pellet was dissolved in TE-buffer.
DNA
prepared by this procedure could be used for all purposes, including southern
blotting or
construction of genomic libraries.
Example 2: Construction of genomic libraries in Escherichia coli of
Corynebacterium
glutamicum ATCC13032.
Using DNA prepared as described in Example 1, cosmid and plasmid libraries
were
constructed according to known and well established methods (see e.g.,
Sambrook, J. et al.
(1989) "Molecular Cloning : A Laboratory Manual", Cold Spring Harbor
Laboratory Press,
or Ausubel, F.M. et al. (1994) "Current Protocols in Molecular Biology", John
Wiley &
Sons.)
Any plasmid or cosmid could be used. Of particular use were the plasmids
pBR322
(Sutcliffe, J.G. (1979) Proc. Natl. Acad. Sci. USA, 75:3737-3741); pACYC177
(Change &
Cohen (1978) J. Bacteriol 134:1141-1156), plasmids of the pBS series (pBSSK+,
pBSSK- and
others; Stratagene, LaJolla, USA), or cosmids as SuperCosl (Stratagene,
LaJolla, USA) or
Lorist6 (Gibson, T.J., Rosenthal A. and Waterson, R.H. (1987) Gene 53:283-286.
Gene libraries
specifically for use in C. glutamicum may be constructed using plasmid pSL 109
(Lee, H.-S. and
A. J. Sinskey (1994) J. Microbiol. Biotechnol. 4: 256-263).
Example 3: DNA Sequencing and Computational Functional Analysis
Genomic libraries as described in Example 2 were used for DNA sequencing
according to standard methods, in particular by the chain termination method
using
AB1377 sequencing machines (see e.g., Fleischman, R.D. et al. (1995) "Whole-
genome
Random Sequencing and Assembly of Haemophilus Influenzae Rd., Science, 269:496-
512). Sequencing primers with the following nucleotide sequences were used: 5'-
GGAAACAGTATGACCATG-3' or 5'-GTAAAACGACGGCCAGT-3'.
Example 4: In vivo Mutagenesis
In vivo mutagenesis of Corynebacterium glutamicum can be performed by passage
of
plasmid (or other vector) DNA through E. coli or other microorganisms (e.g.
Bacillus spp. or
yeasts such as Saccharomyces cerevisiae) which are impaired in their
capabilities to maintain
CA 02590625 2007-06-18
-78-
the integrity of their genetic information. Typical mutator strains have
mutations in the genes
for the DNA repair system (e.g., mutHLS, mutD, mutT, etc.; for reference, see
Rupp, W.D.
(1996) DNA repair mechanisms, in: Escherichia coli and Salmonella, p. 2277-
2294, ASM:
Washington.) Such strains are well known to one of ordinary skill in the art.
The use of such
strains is illustrated, for example, in Greener, A. and Callahan, M. (1994)
Strategies 7: 32-34.
Example 5: DNA Transfer Between Escherichia coli and Corynebacterium
glutamicum
Several Corynebacterium and Brevibacterium species contain endogenous
plasmids (as e.g., pHM1519 or pBLI) which replicate autonomously (for review
see, e.g.,
Martin, J.F. et al. (1987) Biotechnology, 5:137-146). Shuttle vectors for
Escherichia coli
and Corynebacterium glutamicum can be readily construeted by using standard
vectors for
E. coli (Sambrook, J. el al. (1989), "Molecular Cloning: A Laboratory Manual",
Cold
Spring Harbor Laboratory Press or Ausubel, F.M. et al. (1994) "Current
Protocols in
Molecular Biology", John Wiley & Sons) to which a origin or replication for
and a
suitable marker from Corynebacterium glutamicum is added. Such origins of
replication
are preferably taken from endogenous plasmids isolated from Corynebacterium
and
Brevibacterium species. Of particular use as transformation markers for these
species are
genes for kanamycin resistance (such as those derived from the Tn5 or Tn903
transposons) or chloramphenicol (Winnacker, E.L. (1987) "From Genes to Clones -
Introduction to Gene Technology, VCH, Weinheim). There are numerous examples
in the
literature of the construction of a wide variety of shuttle vectors which
replicate in both E.
coli and C. glutamicum, and which can be used for several purposes, including
gene over-
expression (for reference, see e.g., Yoshihama, M. et al. (1985) J. Bacteriol.
162:591-597,
Martin J.F. et al. (1987) Biotechnology, 5:137-146 and Eikmanns, B.J. et al.
(1991) Gene,
102:93-98).
Using standard methods, it is possible to clone a gene of interest into one of
the
shuttle vectors described above and to introduce such a hybrid vectors into
strains of
Corynebacterium glutamicum. Transformation of C. glutamicum can be achieved by
protoplast transformation (Kastsumata, R. et al. (1984) J. Bacteriol. 1 59306-
3 1 1),
electroporation (Liebl, E. et al. (1989) FEMSMicrobiol. Letters, 53:399-303)
and in cases
where special vectors are used, also by conjugation (as described e.g. in
Schafer, A et al.
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(1990) J. Bacteriol. 172:1663-1666). It is also possible to transfer the
shuttle vectors for
C. glutamicum to E. coli by preparing plasmid DNA from C. glutamicum (using
standard
methods well-known in the art) and transforming it into E. coli. This
transformation step
can be performed using standard methods, but it is advantageous to use an Mcr-
deficient
E. coli strain, such as NM522 (Gough & Murray (1983) J. Mol. Biol. 166:1-19).
Genes may be overexpressed in C. glutamicum strains using plasmids which
comprise pCGI (U.S. Patent No. 4,617,267) or fragments thereof, and optionally
the
gene for kanamycin resistance from TN903 (Grindley, N.D. and Joyce, C.M.
(1980)
Proc. Natl. Acad. Sci. USA 77(12): 7176-7180). In addition, genes may be
overexpressed in C. glutamicum strains using plasmid pSL 109 (Lee, H.-S. and
A. J.
Sinskey (1994)J. Microbiol. Biotechnol. 4: 256-263).
Aside from the use of replicative plasmids, gene overexpression can also be
achieved by integration into the genome. Genomic integration in C. glutamicum
or other
Corynebacterium or Brevibacterium species may be accomplished by well-known
methods, such as homologous recombination with genomic region(s), restriction
endonuclease mediated integration (REMI) (see, e.g., DE Patent 19823834), or
through
the use of transposons. It is also possible to modulate the activity of a gene
of interest by
modifying the regulatory regions (e.g., a promoter, a repressor, and/or an
enhancer) by
sequence modification, insertion, or deletion using site-directed methods
(such as
homologous recombination) or methods based on random events (such as
transposon
mutagenesis or REMI). Nucleic acid sequences which function as transcriptional
terminators may also be inserted 3' to the coding region of one or more genes
of the
invention; such terminators are well-known in the art and are described, for
example, in
Winnacker, E.L. (1987) From Genes to Clones - Introduction to Gene Technology.
VCH:
Weinheim.
Example 6: Assessment of the Expression of the Mutant Protein
Observations of the activity of a mutated protein in a transformed host cell
rely on
the fact that the mutant protein is expressed in a similar fashion and in a
similar quantity
to that of the wild-type protein. A useful method to ascertain the level of
transcription of
the mutant gene (an indicator of the amount of mRNA available for translation
to the gene
product) is to perform a Northern blot (for reference see, for example,
Ausubel et al.
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(1988) Current Protocols in Molecular Biology, Wiley: New York), in which a
primer
designed to bind to the gene of interest is labeled with a detectable tag
(usually radioactive
or chemiluminescent), such that when the total RNA of a culture of the
organism is
extracted, run on gel, transferred to a stable matrix and incubated with this
probe, the
binding and quantity of binding of the probe indicates the presence and also
the quantity
of mRNA for this gene. This information is evidence of the degree of
transcription of the
mutant gene. Total cellular RNA can be prepared from Corynebacterium
glutamicum by
several methods, all well-known in the art, such as that described in Bormann,
E.R. et al.
(1992) Mol. Microbiol. 6: 317-326.
To assess the presence or relative quantity of protein translated from this
mRNA,
standard techniques, such as a Westem blot, may be employed (see, for example,
Ausubel
et al. (1988) Current Protocols in Molecular Biology, Wiley: New York). In
this process,
total cellular proteins are extracted, separated by gel electrophoresis,
transferred to a
matrix such as nitrocellulose, and incubated with a probe, such as an
antibody, which
specifically binds to the desired protein. This probe is generally tagged with
a
chemiluminescent or colorimetric label which may be readily detected. The
presence and
quantity of label observed indicates the presence and quantity of the desired
mutant
protein present in the cell.
Example 7: Growth of Genetically Modified Corynebacterium glutamicum - Media
and Culture Conditions
Genetically modified Corynebacteria are cultured in synthetic or natural
growth
media. A number of different growth media for Corynebacteria are both well-
known and
readily available (Lieb et al. (1989) Appl. Microbiol. Biotechnol., 32:205-
210; von der
Osten et al. (1998) Biotechnology Letters, 11:11-16; Patent DE 4,120,867;
Liebl (1992)
"The Genus Corynebacterium, in: The Procaryotes, Volume II, Balows, A. et al.,
eds.
Springer-Verlag). These media consist of one or more carbon sources, nitrogen
sources,
inorganic salts, vitamins and trace elements. Preferred carbon sources are
sugars, such as
mono-, di-, or polysaccharides. For example, glucose, fructose, mannose,
galactose,
ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or
cellulose serve as
very good carbon sources. It is also possible to supply sugar to the media via
complex
compounds such as molasses or other by-products from sugar refinement. It can
also be
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advantageous to supply mixtures of different carbon sources. Other possible
carbon
sources are alcohols and organic acids, such as methanol, ethanol, acetic acid
or lactic
acid. Nitrogen sources are usually organic or inorganic nitrogen compounds, or
materials
which contain these compounds. Exemplary nitrogen sources include ammonia gas
or
ammonia salts, such as NH4CI or (NI-I4)ZSO,, NH4OH, nitrates, urea, amino
acids or
complex nitrogen sources like corn steep liquor, soy bean flour, soy bean
protein, yeast
extract, meat extract and others.
Inorganic salt compounds which may be included in the media include the
chloride-, phosphorous- or sulfate- salts of calcium, magnesium, sodium,
cobalt,
molybdenum, potassium, manganese, zinc, copper and iron. Chelating compounds
can be
added to the medium to keep the metal ions in solution. Particularly useful
chelating
compounds include dihydroxyphenols, like catechol or protocatechuate, or
organic acids,
such as citric acid. It is typical for the media to also contain other growth
factors, such as
vitamins or growth promoters, examples of which include biotin, riboflavin,
thiamin, folic
acid, nicotinic acid, pantothenate and pyridoxin. Growth factors and salts
frequently
originate from complex media components such as yeast extract, molasses, corn
steep
liquor and others. The exact composition of the media compounds depends
strongly on
the immediate experiment and is individually decided for each specific case.
Information
about media optimization is available in the textbook "Applied Microbiol.
Physiology, A
Practical Approach (eds. P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-
73, ISBN 0
19 963577 3). It is also possible to select growth media from commercial
suppliers, like
standard 1(Merck) or BHI (grain heart infusion, DIFCO) or others.
All medium components are sterilized, either by heat (20 minutes at 1.5 bar
and
121 C) or by sterile filtration. The components can either be sterilized
together or, if
necessary, separately. All media components can be present at the beginning of
growth,
or they can optionally be added continuously or batchwise.
Culture conditions are defined separately for each experiment. The temperature
should be in a range between 15 C and 45 C. The temperature can be kept
constant or can
be altered during the experiment. The pH of the medium should be in the range
of 5 to
8.5, preferably around 7.0, and can be maintained by the addition of buffers
to the media.
An exemplary buffer for this purpose is a potassium phosphate buffer.
Synthetic buffers
such as MOPS, HEPES, ACES and others can altematively or simultaneously be
used. It
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is also possible to maintain a constant culture pH through the addition of
NaOH or
NH4OH during growth. If complex medium components such as yeast extract are
utilized,
the necessity for additional buffers may be reduced, due to the fact that many
complex
compounds have high buffer capacities. If a fermentor is utilized for
culturing the micro-
organisms, the pH can also be controlled using gaseous ammonia.
The incubation time is usually in a range from several hours to several days.
This
time is selected in order to permit the maximal amount of product to
accumulate in the
broth. The disclosed growth experiments can be carried out in a variety of
vessels, such as
microtiter plates, glass tubes, glass flasks or glass or metal fermentors of
different sizes.
For screening a large number of clones, the microorganisms should be cultured
in
microtiter plates, glass tubes or shake flasks, either with or without
baffles. Preferably
100 ml shake flasks are used, filled with 10% (by volume) of the required
growth
medium. The flasks should be shaken on a rotary shaker (amplitude 25 mm) using
a
speed-range of 100 - 300 rpm. Evaporation losses can be diminished by the
maintenance
of a humid atmosphere; alternatively, a mathematical correction for
evaporation losses
should be performed.
If genetically modified clones are tested, an unmodified control clone or a
control
clone containing the basic plasmid without any insert should also be tested.
The medium
is inoculated to an OD600 of 0.5 - 1.5 using cells grown on agar plates, such
as CM plates
(10 g/1 glucose, 2,5 g/1 NaCI, 2 g/1 urea, 10 g/1 polypeptone, 5 g/l yeast
extract, 5 g/l meat
extract, 22 g/l NaCI, 2 g/l urea, 10 g/I polypeptone, 5 g/1 yeast extract, 5
g/l meat extract,
22 g/l agar, pH 6.8 with 2M NaOH) that had been incubated at 30 C. Inoculation
of the
media is accomplished by either introduction of a saline suspension of C.
glutamicum cells
from CM plates or addition of a liquid preculture of this bacterium.
Example 8 - In vitro Analysis of the Function of Mutant Proteins
The determination of activities and kinetic parameters of enzymes is well
established in the art. Experiments to determine the activity of any given
altered
enzyme must be tailored to the specific activity of the wild-type enzyme,
which is well
within the ability of one of ordinary skill in the art. Overviews about
enzymes in
general, as well as specific details concerning structure, kinetics,
principles, methods,
applications and examples for the determination of many enzyme activities may
be
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found, for example, in the following references: Dixon, M., and Webb, E.C.,
(1979)
Enzymes. Longmans: London; Fersht, (1985) Enzyme Structure and Mechanism.
Freeman: New York; Walsh, (1979) Enzymatic Reaction Mechanisms. Freeman: San
Francisco; Price, N.C., Stevens, L. (1982) Fundamentals of Enzymology. Oxford
Univ.
Press: Oxford; Boyer, P.D., ed. (1983) The Enzymes, 3d ed. Academic Press: New
York; Bisswanger, H., (1994) Enzymkinetik, 2"d ed. VCH: Weinheim (ISBN
3527300325); Bergmeyer, H.U., Bergmeyer, J., Graf31, M., eds. (1983-1986)
Methods of
Enzymatic Analysis, 3'd ed., vol. I-XII, Verlag Chemie: Weinheim; and
Ullmann's
Encyclopedia of Industrial Chemistry (1987) vol. A9, "Enzymes". VCH: Weinheim,
p.
352-363.
The activity of proteins which bind to DNA can be measured by several well-
established methods, such as DNA band-shift assays (also called gel
retardation assays).
The effect of such proteins on the expression of other molecules can be
measured using
reporter gene assays (such as that described in Kolmar, H. et al. (1995) EMBO
J. 14:
3895-3904 and references cited therein). Reporter gene test systems are well
known and
established for applications in both pro- and eukaryotic cells, using enzymes
such as
beta-galactosidase, green fluorescent protein, and several others.
The determination of activity of membrane-transport proteins can be performed
according to techniques such as those described in Gennis, R.B. (1989) "Pores,
Channels and Transporters", in Biomembranes, Molecular Structure and Function,
Springer: Heidelberg, p. 85-137; 199-234; and 270-322.
Example 9: Analysis of Impact of Mutant Protein on the Production of the
Desired
Product
The effect of the genetic modification in C. glutamicum on production of a
desired compound (such as an amino acid) can be assessed by growing the
modified
microorganism under suitable conditions (such as those described above) and
analyzing
the medium and/or the cellular component for increased production of the
desired
product (i.e., an amino acid). Such analysis techniques are well known to one
of
ordinary skill in the art, and include spectroscopy, thin layer
chromatography, staining
methods of various kinds, enzymatic and microbiological methods, and
analytical
chromatography such as high performance liquid chromatography (see, for
example,
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Ullman, Encyclopedia of Industrial Chemistry, vol. A2, p. 89-90 and p. 443-
613, VCH:
Weinheim (1985); Fallon, A. et al., (1987) "Applications of HPLC in
Biochemistry" in:
Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17; Rehm et
al.
(1993) Biotechnology, vol. 3, Chapter III: "Product recovery and
purification", page
469-714, VCH: Weinheim; Belter, P.A. et al. (1988) Bioseparations: downstream
processing for biotechnology, John Wiley and Sons; Kennedy, J.F. and Cabral,
J.M.S.
(1992) Recovery processes for biological materials, John Wiley and Sons;
Shaeiwitz,
J.A. and Henry, J.D. (1988) Biochemical separations, in: Ulmann's Encyclopedia
of
Industrial Chemistry, vol. B3, Chapter 11, page 1-27, VCH: Weinheim; and
Dechow,
F.J. (1989) Separation and purification techniques in biotechnology, Noyes
Publications.)
In addition to the measurement of the final product of fermentation, it is
also
possible to analyze other components of the metabolic pathways utilized for
the
production of the desired compound, such as intermediates and side-products,
to
determine the overall productivity of the organism, yield, and/or efficiency
of
production of the compound. Analysis methods include measurements of nutrient
levels
in the medium (e.g., sugars, hydrocarbons, nitrogen sources, phosphate, and
other ions),
measurements of biomass composition and growth, analysis of the production of
common metabolites of biosynthetic pathways, and measurement of gasses
produced
during fermentation. Standard methods for these measurements are outlined in
Applied
Microbial Physiology, A Practical Approach, P.M. Rhodes and P.F. Stanbury,
eds., IRL
Press, p. 103-129; 131-163; and 165-192 (ISBN: 0199635773) and references
cited
therein.
Example 10: Purification of the Desired Product from C. glutamicum Culture
Recovery of the desired product from the C. glutamicum cells or supernatant of
the above-described culture can be performed by various methods well known in
the art.
If the desired product is not secreted from the cells, the cells can be
harvested from the
culture by low-speed centrifugation, the cells can be lysed by standard
techniques, such
as mechanical force or sonication. The cellular debris is removed by
centrifugation, and
the supernatant fraction containing the soluble proteins is retained for
further
purification of the desired compound. If the product is secreted from the C.
glutamicum
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cells, then the cells are removed from the culture by low-speed
centrifugation, and the
supernate fraction is retained for further purification.
The supernatant fraction from either purification method is subjected to
chromatography with a suitable resin, in which the desired molecule is either
retained on
a chromatography resin while many of the impurities in the sample are not, or
where the
impurities are retained by the resin while the sample is not. Such
chromatography steps
may be repeated as necessary, using the same or different chromatography
resins. One
of ordinary skill in the art would be well-versed in the selection of
appropriate
chromatography resins and in their most efficacious application for a
particular molecule
to be purified. The purified product may be concentrated by filtration or
ultrafiltration,
and stored at a temperature at which the stability of the product is
maximized.
There are a wide array of purification methods known to the art and the
preceding method of purification is not meant to be limiting. Such
purification
techniques are described, for example, in Bailey, J.E. & Ollis, D.F.
Biochemical
Engineering Fundamentals, McGraw-Hill: New York (1986).
The identity and purity of the isolated compounds may be assessed by
techniques
standard in the art. These include high-performance liquid chromatography
(HPLC),
spectroscopic methods, staining methods, thin layer chromatography, NIRS,
enzymatic
assay, or microbiologically. Such analysis methods are reviewed in: Patek et
al. (1994)
Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya
11: 27-
32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's
Encyclopedia
of Industrial Chemistry, (1996) vol. A27, VCH: Weinheim, p. 89-90, p. 521-540,
p. 540-
547, p. 559-566, 575-581 and p. 581-587; Michal, G. (1999) Biochemical
Pathways: An
Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A.
et al.
(1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in
Biochemistry and Molecular Biology, vol. 17.
Example 11: Analysis of the Gene Sequences of the Invention
The comparison of sequences and determination of percent homology between
two sequences are art-known techniques, and can be accomplished using a
mathematical
algorithm, such as the algorithm of Karlin and Altschul (1990) Proc. Natl.
Acad. Sci.
USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.
Sci. USA
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90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST
programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
BLAST
nucleotide searches can be performed with the NBLAST program, score = 100,
wordlength = 12 to obtain nucleotide sequences homologous to PTS nucleic acid
molecules of the invention. BLAST protein searches can be performed with the
XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences
homologous to PTS protein molecules of the invention. To obtain gapped
alignments
for comparison purposes, Gapped BLAST can be utilized as described in Altschul
et al.,
(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped
BLAST programs, one of ordinary skill in the art will know how to optimize the
parameters of the program (e.g., XBLAST and NBLAST) for the specific sequence
being analyzed.
Another example of a mathematical algorithm utilized for the comparison of
sequences is the algorithm of Meyers and Milier ((1988) Comput. Appl. Biosci.
4: 11-
17). Such an algorithm is incorporated into the ALIGN program (version 2.0)
which is
part of the GCG sequence alignment software package. When utilizing the ALIGN
program for comparing amino acid sequences, a PAM120 weight residue table, a
gap
length penalty of 12, and a gap penalty of 4 can be used. Additional
algorithms for
sequence analysis are known in the art, and include ADVANCE and ADAM.
described
in Torelli and Robotti (1994) Comput. Appl. Biosci. 10:3-5; and FASTA,
described in
Pearson and Lipman (1988) P.N.A.S. 85:2444-8.
The percent homology between two amino acid sequences can also be
accomplished using the GAP program in the GCG software package (available at
http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a
gap
weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4. The percent
homology
between two nucleic acid sequences can be accomplished using the GAP program
in the
GCG software package, using standard parameters, such as a gap weight of 50
and a
length weight of 3.
A comparative analysis of the gene sequences of the invention with those
present
in Genbank has been performed.using techniques known in the art (see, e.g.,
Bexevanis
and Ouellette, eds. (1998) Bioinformatics: A Practical Guide to the Analysis
of Genes
and Proteins. John Wiley and Sons: New York). The gene sequences of the
invention
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were compared to genes present in Genbank in a three-step process. In a first
step, a
BLASTN analysis (e.g., a local alignment analysis) was performed for each of
the
sequences of the invention against the nucleotide sequences present in
Genbank, and the
top 500 hits were retained for further analysis. A subsequent FASTA search
(e.g., a
combined local and global alignment analysis, in which limited regions of the
sequences
are aligned) was performed on these 500 hits. Each gene sequence of the
invention was
subsequently globally aligned to each of the top three FASTA hits, using the
GAP
program in the GCG software package (using standard parameters). In order to
obtain
correct results, the length of the sequences extracted from Genbank were
adjusted to the
length of the query sequences by methods well-known in the art. The results of
this
analysis are set forth in Table 4. The resulting data is identical to that
which would have
been obtained had a GAP (global) analysis alone been performed on each of the
genes of
the invention in comparison with each of the references in Genbank, but
required
significantly reduced computational time as compared to such a database-wide
GAP
(global) analysis. Sequences of the invention for which no alignments above
the cutoff
values were obtained are indicated on Table 4 by the absence of alignment
information.
It will further be understood by one of ordinary skill in the art that the GAP
alignment
homology percentages set forth in Table 4 under the heading "% homology (GAP)"
are
listed in the European numerical fonnat, wherein a',' represents a decimal
point. For
example, a value of "40,345" in this colurnn represents "40.345 /a".
Example 12: Construction and Operation of DNA Microarrays
The sequences of the invention may additionally be used in the construction
and
application of DNA microarrays (the design, methodology, and uses of DNA
arrays are
well known in the art, and are described, for example, in Schena, M. et al.
(1995)
Science 270: 467-470; Wodicka, L. et al. (1997) Nature Biotechnology 15: 1359-
1367;
DeSaizieu, A. et al. (1998) Nature Biotechnology 16: 45-48; and DeRisi, J.L.
et al.
(1997) Science 278: 680-686).
DNA microarrays are solid or flexible supports consisting of nitrocellulose,
nylon, glass, silicone, or other materials. Nucleic acid molecules may be
attached to the
surface in an ordered manner. After appropriate labeling, other nucleic acids
or nucleic
acid mixtures can be hybridized to the immobilized nucleic acid molecules, and
the label
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may be used to monitor and measure the individual signal intensities of the
hybridized
molecules at defined regions. This methodology allows the
simultaneousquantification
of the relative or absolute amount of all or selected nucleic acids in the
applied nucleic
acid sample or mixture. DNA microarrays, therefore, permit an analysis of the
expression of multiple (as many as 6800 or more) nucleic acids in parallel
(see, e.g.,
Schena, M. (1996) BioEssays 18(5): 427-431).
The sequences of the invention may be used to design oligonucleotide primers
which are able to amplify defined regions of one or more C. glutamicum genes
by a
nucleic acid amplification reaction such as the polymerase chain reaction. The
choice
and design of the 5' or 3' oligonucleotide primers or of appropriate linkers
allows the
covalent attachment of the resulting PCR products to the surface of a support
medium
described above (and.also described, for example, Schena, M. et al. (1995)
Science 270:
467-470).
Nucleic acid microarrays may also be constructed by in situ oligonucleotide
synthesis as described by Wodicka, L. et al. (1997) Nature Biotechnology 15:
1359-
1367. By photolithographic methods, precisely defined regions of the matrix
are
exposed to light. Protective groups which are photolabile are thereby
activated and
undergo nucleotide addition, whereas regions that are masked from light do not
undergo
any modification. Subsequent cycles of protection and light activation permit
the
synthesis of different oligonucleotides at defined positions. Small, defined
regions of
the genes of the invention may be synthesized on microarrays by solid phase
oligonucleotide synthesis.
The nucleic acid molecules of the invention present in a sample or mixture of
nucleotides may be hybridized to the microarrays. These nucleic acid molecules
can be
labeled according to standard methods. In brief, nucleic acid molecules (e.g.,
mRNA
molecules or DNA molecules) are labeled by the incorporation of isotopically
or
fluorescently labeled nucleotides, e.g., during reverse transcription or DNA
synthesis.
Hybridization of labeled nucleic acids to microarrays is described (e.g., in
Schena, M. et
al. (1995) supra; Wodicka, L. et al. (1997), supra; and DeSaizieu A. et al.
(1998),
supra). The detection and quantification of the hybridized molecule are
tailored to the
specific incorporated label. Radioactive labels can be detected, for example,
as
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described in Schena, M. et al. (1995) supra) and fluorescent labels may be
detected, for
example, by the method of Shalon et al. (1996) Genome Research 6: 639-645).
The application of the sequences of the invention to DNA microarray
technology, as described above, permits comparative analyses of different
strains of C.
glutamicum or other Corynebacteria. For example, studies of inter-strain
variations
based on individual transcript profiles and the identification of genes that
are important
for specific and/or desired strain properties such as pathogenicity,
productivity and
stress tolerance are facilitated by nucleic acid array methodologies. Also,
comparisons
of the profile of expression of genes of the invention during the course of a
fermentation
reaction are possible using nucleic acid array technology.
Example 13: Analysis of the Dynamics of Cellular Protein Populations
(Proteomics)
The genes, compositions, and methods of the invention may be applied to study
the interactions and dynamics of populations of proteins, termed 'proteomics'.
Protein
populations of interest include, but are not limited to, the total protein
population of C.
glutamicum (e.g., in comparison with the protein populations of other
organisms), those
proteins which are active under specific environmental or metabolic conditions
(e.g.,
during fermentation, at high or low temperature, or at high or low pH), or
those proteins
which are active during specific phases of growth and development.
Protein populations can be analyzed by various well-known techniques, such as
gel electrophoresis. Cellular proteins may be obtained, for example, by lysis
or
extraction, and may be separated from one another using a variety of
electrophoretic
techniques. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE)
separates proteins largely on the basis of their molecular weight. Isoelectric
focusing
polyacrylamide gel electrophoresis (IEF-PAGE) separates proteins by their
isoelectric
point (which reflects not only the amino acid sequence but also
posttranslational
modifications of the protein). Another, more preferred method of protein
analysis is the
consecutive combination of both IEF-PAGE and SDS-PAGE, known as 2-D-gel
electrophoresis (described, for example, in Hermann et al. (1998)
Electrophoresis 19:
3217-3221; Fountoulakis et al. (1998) Electrophoresis 19: 1193-1202; Langen et
al.
(1997) Electrophoresis 18: 1184-1192; Antelmann et al. (1997) Electrophoresis
18:
CA 02590625 2007-06-18
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1451-1463). Other separation techniques may also be utilized for protein
separation,
such as capillary gel electrophoresis; such techniques are well known in the
art.
Proteins separated by these methodologies can be visualized by standard
techniques, such as by staining or labeling. Suitable stains are known in the
art, and
include Coomassie Brilliant Blue, silver stain, or fluorescent dyes such as
Sypro Ruby
(Molecular Probes). The inclusion of radioactively labeled amino acids or
other protein
precursors (e.g., 35S-methionine, 35S-cysteine, 14C-labelled amino acids, 15N-
amino
acids, "NO3 or "NH4+ or 13C-labelled amino acids) in the medium of C.
glutamicum
permits the labeling of proteins from these cells prior to their separation.
Similarly,
fluorescent labels may be employed. These labeled proteins can be extracted,
isolated
and separated according to the previously described techniques.
Proteins visualized by these techniques can be further analyzed by measuring
the
amount of dye or label used. The amount of a given protein can be determined
quantitatively using, for exarnple, optical methods and can be compared to the
amount
of other proteins in the same gel or in other gels. Comparisons of proteins on
gels can
be made, for example, by optical comparison, by spectroscopy, by image
scanning and
analysis of gels, or through the use of photographic films and. screens. Such
techniques
are well-known in the art.
To determine the identity of any given protein, direct sequencing or other
standard techniques may be employed. For example, N- and/or C-terminal amino
acid
sequencing (such as Edman degradation) may be used, as may mass spectrometry
(in
particular MALDI or ESI techniques (see, e.g., Langen el al. (1997)
Electrophoresis 18:
1184-1192)). The protein sequences provided herein can be used for the
identification
of C. glutamicum proteins by these techniques.
The information obtained by these methods can be used to compare patterns of
protein presence, activity, or modification between different samples from
various
biological conditions (e.g., different organisms, time points of fermentation,
media
conditions, or different biotopes, among others). Data obtained from such
experiments
alone, or in combination with other techniques, can be used for various
applications,
such as to compare the behavior of various organisms in a given (e.g.,
metabolic)
situation, to increase the productivity of strains which produce fine
chemicals or to
increase the efficiency of the production of fine chemicals.
CA 02590625 2007-06-18
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Equivalents
Those of ordinary skill in the art will recognize, or will be able to
ascertain using
no more than routine experimentation, many equivalents to the specific
embodiments of
the invention described herein. Such equivalents are intended to be
encompassed by the
following claims.
CA 02590625 2007-06-18
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