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CA 02587112 2007-05-01
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CORYNEBACTERIUM GL UTAMlCUM GENES ENCODING STRESS,
RESISTANCE AND TOLERANCE PROTEINS
This application is a divisional of Canadian Patent Application 2,380,870
filed on June 23rd, 2000 as PCT/IB00/00922.
A first divisional application relates to subject matter associated with
SEQ IDNOS: 5 and 6.
A second divisional application relates to subject matter associated with
SEQ ID NOS: 79 and 80.
Background 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 Corynebac[erium glutamicum, a gram positive,
nonpathogenic bacterium. Through strain selection, a riumber of mutant strains
have
been developed which produce an array of desirable compounds. However,
selection of
strains improved for the production of a particular molecule is a time-
consuming and
difficult process.
CA 02587112 2007-05-01
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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.
glulamicum 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 stress, resistance and tolerance (SRT) proteins.
C. glutamicum is a gram 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 SRT 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 SRT nucleic acids
of the
invention, or modification of the sequence of the SRT 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).
The SRT 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
dfphtheriae (the causative agent of diphtheria); the detection of such
organisms is of
significant clinical relevance.
The SRT 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.
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Similarly, these molecules, or variants or portions thereof, may serve as
markers for
genetically engineered Corynebacterium or Brevibacterium species.
The SRT proteins encoded by the novel nucleic acid molecules of the invention
are capable of, for example, pennitting C. glutamicum to survive in a setting
which is
either chemically or environmentally hazardous to this microorganism. Given
the
availability of cloning vectors for use in Corynebacteriurn 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 al.,
J. Bacteriol. 159: 306-311 (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, through the ability of these proteins to permit growth
and
multiplication of C. glutamicum (and also continuous production of one or more
fine
chemicals) under circumstances which would normally impede growth of the
organism,
such as those conditions frequently encountered during large-scale
fermentative growth.
For example, by overexpressing or engineering a heat-shock induced protease
molecule
such that it is optimized in activity, one may increase the ability of the
bacterium to
degrade incorrectly folded proteins when the bacterium is challenged with high
temperatures. By having fewer misfolded (and possibly misregulated or
nonfunctional)
proteins to interfere with normal reaction mechanisms in the cell, the cell is
increased in
its ability to function normally in such a culture, which should in tum
provide increased
viability. This overall increase in number of cells having greater viability
and activity in
the culture should also result in an increase in yield, production, and/or
efficiency of
production of one or more desired fine chemicals, due at least to the
relatively greater
number of cells producing these chemicals in the culture.
This invention provides novel SRT nucleic acid molecules which encode SRT
proteins which are capable of, for example, permitting C. glutamicum to
survive in a
setting which is either chemically or environmentally hazardous to this
microorganism.
Nucleic acid molecules encoding an SRT protein are referred to herein as SRT
nucleic
acid molecules. In a preferred embodiment, the SRT protein participates in
metabolic
pathways permitting C. glutamicum to survive in a setting which is either
chemically or
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environmentally hazardous to this microorganism. Examples of such proteins
include
those encoded by the genes set forth in Table I.
Accordingly, one aspect of the invention pertains to isolated nucleic acid
molecules (e.g., cDNAs, DNAs, or RNAs) comprising a nucleotide sequence
encoding
an SRT protein or biologically active portions thereof, as well as nucleic
acid fragments
suitable as primers or hybridization probes for the detection or amplification
of SRT-
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 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 as an
even-
numbered SEQ ID NO in the Sequence Listing (e.g., SEQ ID NO:2, SEQ ID NO:4,
SEQ
ID NO:6, SEQ ID NO:8....).. The preferred SRT proteins of the present
invention also
preferably possess at least one of the SRT 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 an SRT activity. Preferably, the protein or
portion thereof
encoded by the nucleic acid molecule maintains the ability to increase the
survival of C.
glutamicum in a setting which is either chemically or environmentally
hazardous to this
microorganism. 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
CA 02587112 2007-05-01
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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 the corresponding odd-
numbered
SEQ ID NOs in the Sequence Listing (e.g., SEQ ID NO:I, 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., an SRT 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 has the ability to increase the
survival of C.
glutamicum in a setting which is either chemically or environmentally
hazardous to this
microorganism, 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.
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
rnolecule corresponds to a naturally-occurring nucleic acid molecule. More
preferably,
the isolated nucleic acid encodes a naturally-occurring C. glutamicum SRT
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- an SRT protein by culturing the host cell in a suitable medium. The
SRT
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 an SRT 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 SRT
sequence as
CA 02587112 2007-05-01
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a transgene. In another embodiment, an endogenous SRT gene within the genome
of the
microorganism has been altered, e.g., functionally disrupted, by homologous
recombination with an altered SRT gene. In another embodiment, an endogenous
or
introduced SRT gene in a microorganism has been altered by one or more point
mutations, deletions, or inversions, but still encodes a functional SRT
protein. In still
another embodiment, one or more of the regulatory regions (e.g., a promoter,
repressor,
or inducer) of a SRT gene in a microorganism has been altered (e.g., by
deletion,
truncation, inversion, or point mutation) such that the expression of the SRT
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
sequences set forth in the Sequence Listing as SEQ ID NOs I through 304)) 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 SRT protein or a
portion, e.g., a biologically active portion, thereof. In a preferred
embodiment, the
isolated SRT protein or portion thereof possesses the ability to increase the
survival of
C. glutamicum in a setting which is either chemically or environmentally
hazardous to
this microorganism. In another preferred embodiment, the isolated SRT 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 increase the survival
of C.
glutamicum in a setting which is either chemically or environmentally
hazardous to this
microorganism.
The invention also provides an isolated preparation of an SRT protein. In
preferred embodiments, the SRT 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
CA 02587112 2007-05-01
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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 forth 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 SRT
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 improve the survival rate
of C.
glufamicum in a setting which is either chemically or environmentally
hazardous to this
microorganism, or has one or more of the activities set forth in Table 1.
Alternatively, the isolated SRT 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 homologousto 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 fonms of SRT proteins also have one or more of the SRT
bioactivities
described herein.
The SRT polypeptide, or a biologically active portion thereof, can be
operatively
linked to a non-SRT polypeptide to form a fusion protein. In preferred
embodiments,
this fusion protein has an activity which differs from that of the SRT 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 an SRT protein, either by interacting with the
protein itself or a
substrate or binding partner of the SRT protein, or by modulating the
transcription or
translation of an SRT nucleic acid molecule of the invention.
CA 02587112 2007-05-01
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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 an SRT 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 an SRT 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 SRT protein activity or SRT nucleic acid expression such
that a
cell associated activity is altered relative to this same activity in the
absence of the
agent. In a preferred embodiment, the cell is modulated in resistance to one
or more
toxic chemicals or in resistance to one or more environmental stresses, such
that the
yields or rate of production of a desired fine chemical by this microorganism
is
improved. The agent which modulates SRT protein activity can be an agent which
=
stimulates SRT protein activity or SRT nucleic acid expression. Examples of
agents
which stimulate SRT protein activity or SRT nucleic acid expression include
small
molecules, active SRT proteins, and nucleic acids encoding SRT proteins that
have been
introduced into the cell. Examples of agents which inhibit SRT activity or
expression
include small molecules, and antisense SRT 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 SRT
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 preferred embodiments, said
amino
acid is L-lysine.
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Detailed Description of the Invention
The present invention provides SRT nucleic acid and protein molecules which
are involved in the survival of C. glutamicum upon exposure of this
microorganism to
chemical or environmental hazards. The molecules of the invention may be
utilized in
the modulation of production of fine chemicals from microorganisms, since
these SRT
proteins provide a means for continued growth and multiplication of C.
glutamicum in
the presence of toxic chemicals or hazardous environmental conditions, such as
may be
encountered during large-scale fermentative growth. By increasing the growth
rate or at
least maintaining normal growth in the face of poor, if not toxic, conditions,
one may
increase the yield, production, and/or efficiency of production of one or more
fine
chemicals from such a culture, at least due to the relatively greater number
of cells
producing the fine chemical in the culture. Aspects of the invention are
further
explicated below.
1. 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
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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
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, 3'd 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
CA 02587112 2007-05-01
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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 a!. (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.
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 I 1-
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
CA 02587112 2007-05-01
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the cell (for review see Stryer, L. Biochemistry 3rd 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
ten-ns 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, 3rd ed. Ch. 24: "Biosynthesis of Amino Acids and
Heme" p.
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 normal 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).
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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).
Thiamin (vitamin Bi) is produced by the chemical coupling of pyrimidine and
thiazole moieties. Riboflavin (vitamin B2) is synthesized from guanosine-5'-
triphosphate
(GTP) and ribose-5'-phosphate. Riboflavin, in turn, 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 a-alanine and pantoic
acid. The
enzymes responsible for the biosynthesis steps for the conversion to pantoic
acid, to 0-
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
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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
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 Bi2 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
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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
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.
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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.
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. Resistance to Damage from Chemicals, Environmental Stress, and Antibiotics
Production of fine chemicals is typically performed by large-scale culture of
bacteria developed to produce and secrete large quantities of these molecules.
However,
this type of large-scale fermentation results in the subjection of the
microorganisms to
stresses of various kinds. These stresses include environmental stress and
chemical
stress.
A. Resistance to Environmental Stress
Examples of environmental stresses typically encountered in large-scale
fermentative culture include mechanical stress, heat stress, stress due to
limited oxygen,
stress due to oxygen radicals, pH stress, and osmotic stress. The stirring
mechanism
used in most large-scale fermentors to ensure aeration of the culture produces
heat, thus
increasing the temperature of the culture. Increases in temperature induce the
well-
characterized heat shock response, in which a set of proteins are expressed
which not
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only aid in the survival of the bacterium in the face of high temperatures,
but also
increase survival in response to a number of other environmental stresses (see
Neidhardt, F.C., et al., eds. (1996) E. coli and Salmonella. ASM Press:
Washington,
D.C., p. 1382-1399; Wosten, M. M. (1998) FEMS Microbiology Reviews 22(3): 127-
50;
Bahl, H. et al. (1995) FEMS Microbiology Reviews 17(3): 341-348; Zimmerman,
J.L.,
Cohill, P.R. (1991) New Biologist 3(7): 641-650; Samali, A., and Orrenius, S.
(1998)
Cell. Stress Chaperones 3(4): 228-236, and references contained therein from
each of
these citations). Regulation of the heat shock response in bacteria is
facilitated by
specific sigma factors and other cellular regulators of gene expression
(Hecker, M.,
Volker, U (1998). Molecular Microbiology 29(5): 1129-1136). One of the largest
problems that the cell encounters when exposed to high temperature is that
protein
folding is impaired; nascent proteins have sufficient kinetic energy in high
temperature
circumstances that it is difficult for the growing polypeptide chain to remain
in a stable
conformation long enough to fold properly. Thus, two of the key types of
proteins
expressed during the heat shock response consist of chaperones (proteins which
assist in
the folding or unfolding of other proteins - see, e.g., Fink, A.L. (1999)
Physiol. Rev.
79(2): 425-449), and proteases, which can destroy any improperly folded
proteins.
Examples of chaperones expressed during the heat shock response include GroEL
and
DNAK; proteases known to be expressed during this cellular reaction to heat
shock
include Lon, FtsH, and C1pB.
Other environmental stresses besides heat may also provoke a stress response.
Though the fermentor stirring process is meant to introduce oxygen into the
culture,
oxygen may remain in limited supply, particularly when the culture is advanced
in
growth and the oxygen needs of the culture are thereby increased; an
insufficient supply
of oxygen is another stress for the microorganism. Cells in fermentor cultures
are also
subjected to a number of osmotic stresses, particularly when nutrients are
added to the
culture, resulting in a high extracellular and low intracellular concentration
of these
molecules. Further, the large quantities of the desired molecules produced by
these
organisms in culture may contribute to osmotic stress of the bacteria. Lastly,
aerobic
metabolism such as that used by C. glutamicum results in carbon dioxide as a
waste
product; secretion of this molecule may acidify the culture medium due to
conversion of
this molecule to carboxylic acid. Thus, bacteria in culture are also
frequently subjected
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to acidic pH stress. The converse may also be true - when high levels of basic
waste
molecules such as ammonium are present in the culture medium, the bacteria in
culture
may be subjected to basic pH stress as well.
To combat such environmental stresses, bacteria have elegant gene systems
which are expressed upon exposure to one or more stresses, such as the
aforementioned
heat shock system. Genes expressed in response to osmotic stress, for example,
encode
proteins capable of transporting or synthesizing compatible solutes such that
osmotic
intake or export of a particular molecule is slowed to manageable levels.
Other examples
of stress-induced bacterial proteins are those involved in trehalose
biosynthesis, those
encoding enzymes involved in ppGpp metabolism, those involved in signal
transduction,
particularly those encoding two-component systems which are sensitive to
osmotic
pressure, and those encoding transcription factors which are responsive to a
variety of
stress factors (e.g., RssB analogues and/or sigma factors). Many other such
genes and
their protein products are known in the art.
B. Resistance to Chemical Stress
Aside from environmental stresses, cells may also experience a number of
chemical stresses. These may fall into two categories. The first are natural
waste
products of metabolism and other cellular processes which are secreted by the
cell to the
surrounding medium. The second are chemicals present in the extracellular
medium
which do not originate from the cell. Generally, when cells excrete toxic
waste products
from the concentrated intracellular cytoplasm into the relatively much more
dilute
extracellular medium, these products dissipate such that extracellular levels
of the
possibly toxic compound are quite low. However, in large-scale fermentative
culture of
the bacterium, this may not be the case: so many bacteria are grown in a
relatively small
environment and at such a high metabolic rate that waste products may
accumulate in
the medium to nearly toxic levels. Examples of such wastes are carbon dioxide,
metal
ions, and reactive oxygen species such as hydrogen peroxide. These compounds
may
interfere with the activity or structure of cell surface molecules, or may re-
enter the cell,
where they can seriously damage proteins and nucleic acids alike. Certain
other
chemicals hazardous to the normal functioning of cells may be naturally found
in the
extracellular medium. For example, metal ions such as mercury, cadmium, nickel
or
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copper are frequently found in water sources, and may form tight complexes
with
cellular enzymes which prevent the nonnal functioning of these proteins.
C. Resistance to Antibiotics
Bacteriocidal proteins or antibiotics, may also be found in the extracellular
milieu, either through the intervention of the researcher, or as a natural
product from
another organism, utilized to gain a competitive advantage. Microorganisms
have
several art-known mechanisms to protect themselves against antimicrobial
chemicals.
Degradation, modification, and export of compounds toxic to the cell are
common
methods by which microorganisms eliminate or detoxify antibiotics. Cytoplasmic
'efflux-pumps' are known in several prokaryotes and show similarities to the
so-called
'multidrug resistance' proteins from higher eukaryotes (Neyfakh, A. A. , et
al. (1991)
Proc. Natl. Acad. Sci. USA 88: 4781-4785). Examples of such proteins include
emrAB
from E. coli (Lomovskaya, O. and K. Lewis (1992) Proc. Natl. Acad. Sci. USA
89:
8938-8942), ImrB from B. subtilis (Kumano, M. et al. (1997) Microbiology 143:
2775-
2782), smr from S. aureus (Grinius, L.G. et al. (1992) Plasmid 27: 119-129) or
crnr
from C. glutamicum (Kaidoh, K. et al. (1997) Micro. Drug Resist. 3: 345-350).
C.
glutamicum itself is non-pathogenic, in contrast to several other members of
the genus
Corynebacterium , such as C. diphtheriae or C. pseudotuberculosis. Several
pathogenic
Corynebacteria are known to have multiple resistances against a variety of
antibiotics,
such as C. jeikeium and C. urealyticum (Soriano, F. et al. (1995) Antimicrob.
Agents
Chemother. 39: 208-214).
Lincosamides are recognized as effective antibiotics against Corynebacterium
species (Soriano, F. et al. (1995) Antimicrob. Agents Chemother. 39: 208-214).
An
unexpected result of the present invention was the identification of a gene
encoding a
lincosamide-resistance protein (in particular, a lincomycin-resistance
protein). The
LMRB protein from C. glutamicum shows 40% homology to the product of the 1mrB
gene from B. subtilis (see Genbank accession no. AL009126), as calculated
using
version 1.7 of the program CLUSTALW (Thompson, J.D., Higgins, D.G., Gibson, T.
J.
(1994) Nucl. Acids Res. 22: 4673-4680) using standard parameters (PAIRWISE
ALIGNMENT PARAMETERS: slow/accurate alignments: Gap Open Penalty = 10.00,
Gap Extension Penalty = 0.10, Protein weight matrix = BLOSUM 30, DNA weight
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matrix = IUB, Fast/Approximate alignments: Gap penalty = 3, K-tuple (word)
size = l,
No. of top diagonals = 5, Window size = 5, Toggle Slow/Fast pairwise
alignments =
slow. Multiple alignment parameters: Gap Opening Penalty = 10.00, Gap
Extension
Penalty = 0.05, Delay divergent sequences = 40%, DNA transitions weight =
0.50,
Protein weight matrix = BLOSUM series, DNA weight matrix = IUB, Use negative
matrix = OFF).
Environmental stress, chemical stress, and antibiotic or other antimicrobial
stress may influence the behavior of the microorganisms during fermentor
culture, and
may have an impact on the production of the desired compound from these
organisms.
For example, osmotic stress of a microorganism may cause inappropriate or
inappropriately rapid uptake of one or more compounds which can ultimately
lead to
cellular damage or death due to osmotic shock. Similarly, chemicals present in
the
culture, either exogenously added (e.g., antimicrobial compounds intended to
eliminate
unwanted microbes) or generated by the bacteria themselves (e.g., waste
compounds
such as heavy metals or oxygen radicals, or even antimicrobial compounds) may
result
in inhibition of fine chemical production or even death of the organism. The
genes of
the invention encode C. glutamicum proteins which act to prevent cell damage
or death,
by specifically counteracting the source or effect of the environmental or
chemical
stress.
111. 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 SRT nucleic acid and protein molecules, which
increase
the ability of C. glutamicum to survive in chemically or environmentally
hazardous
settings. In one embodiment, the SRT molecules function to confer resistance
to one or
more environmental or chemical stresses to C. glutamicum. In a preferred
embodiment,
the activity of the SRT 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 SRT molecules of the invention are modulated in activity, such
that the
yield, production, and/or efficiency of production of one or more fine
chemicals from C.
glutamicum is also modulated.
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The language, "SRT protein" or "SRT polypeptide" includes proteins which
participate in the resistance of C. gluramicum to one or more environmental or
chemical
stresses. Examples of SRT proteins include those encoded by the SRT genes set
forth in
Table I and by the odd-numbered SEQ ID NOs. The terms "SRT gene" or "SRT
nucleic acid sequence" include nucleic acid sequences encoding an SRT protein,
which
consist of a coding region and also corresponding untranslated 5' and 3'
sequence
regions. Examples of SRT 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 tenm
"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,
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 tenns "resistance" and "tolerance" are art-known and include the
ability
of a cell to not be affected by exposure to a chemical or an environment which
would
otherwise be detrimental to the normal functioning of these organisms. The
terms
"stress" or "hazard" include factors which are detrimental to the normal
functioning of
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cells such as C. glulamicum. Examples of stresses include "chemical stress",
in which a
cell is exposed to one or more chemicals which are detrimental to the cell,
and
"environmental stress" where a cell is exposed to an environmental condition
outside of
those to which it is adapted. Chemical stresses may be either natural
metabolic waste
products such as, but not limited to reactive oxygen species or carbon
dioxide, or
chemicals otherwise present in the environment, including, but not limited to
heavy
metal ions or bacteriocidal proteins such as antibiotics. Environrnental
stresses may be,
but are not limited to temperatures outside of the normal range, suboptimal
oxygen
availability, osmotic pressures, or extremes of pH, for example.
In another embodiment, the SRT 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 SRT proteins of the invention may be manipulated such that its
function is
modulated. The alteration of activity of stress response, resistance or
tolerance genes
such that the cell is increased in tolerance to one or more stresses may
improve the
ability of that cell to grow and multiply in the relatively stressful
conditions of large-
scale fermentor culture. For example, by overexpressing or engineering a heat-
shock
induced chaperone molecule such that it is optimized in activity, one may
increase the
ability of the bacterium to correctly fold proteins in the face of nonoptimal
temperature
conditions. By having fewer misfolded (and possibly misregulated or
nonfunctional)
proteins, the cell is increased in its ability to function normally in such a
culture, which
should in tum provide increased viability. This overall increase in number of
cells
having greater viability and activity in the culture should also result in an
increase in the
yield, production, andlor efficiency of production of one or more desired fine
chemicals,
due at least to the relatively greater number of cells producing these
chemicals in the
culture.
The isolated nucleic acid sequences of the invention are contained within the
genome of a Corynebacterium glutamicum strain available through the American
Type
Culture Collection, given designation ATCC 13032. The nucleotide sequence of
the
isolated C. glulamicum SRT DNAs and the predicted amino acid sequences of the
C.
glutamicum SRT proteins are shown the Sequence Listing as odd-numbered SEQ ID
NOs and even-numbered SEQ ID NOs, respectively.,.
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Computational analyses were performed which classified and/or identified these
nucleotide sequences as sequences which encode chemical and environmental
stress,
resistance, and tolerance 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.
Ranges and identity values intermediate to the above-recited values, (e.g.,
75%-80%
identical, 85-87% identical, 91-92% 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 andlor lower limits are intended to
be included.
The SRT proteins or biologically active portions or fragments thereof of the
invention can confer resistance or tolerance to one or more chemical or
environmental
stresses, 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 SRT 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 SRT-encoding nucleic acid (e.g., SRT 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 SRT 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. glutamicum SRT 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:) 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-
thiocyanate
CA 02587112 2007-05-01
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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 AMY 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 an SRT 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 SRT DNAs of the invention. This DNA comprises
sequences encoding SRT 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, or RXS number having the designation "RXA", "RXN", or "RXS" followed by 5
digits (i.e., RXA01524, RXN00493, or RXS01027). 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, or RXS
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, or
RXS designations. The coding region of each of these sequences is translated
into a
corresponding amino acid sequence, which is also et forth in the Sequence
Listing, as an
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even-numbered SEQ ID NO: immediately following the corresponding nucleic acid
sequence. For example, the coding region for RXA01524 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, or RXS designations as the amino acid molecules which they encode,
such
that they can be readily correlated. For example, the amino acid sequence
designated
RXA01524 is a translation of the coding region of the nucleotide sequence of
nucleic
acid molecule RXA01524, the amino acid sequence designated RXN00034 is a
translation of the coding region of the nucleotide sequence of nucleic acid
molecule
RXN00034, and the amino acid sequence in designated RXS00568 is a translation
of the
coding region of the nucleotide sequence of nucleic acid molecule RXS00568.
The
correspondence between the RXA, RXN, and RXS nucleotide and amino acid
sequences
of the invention and their assigned SEQ ID NOs is set forth in Table 1.
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, or RXS designation. For example, SEQ ID NO:7, designated, as indicated on
Table 1, as "F RXA00498", is an F-designated gene, as are SEQ ID NOs: 25, 33,
and 37
(designated on Table 1 as "F RXA01345", "F RXA02543", and "F RXA02282",
respectively).
In one embodiment, the nucleic acid molecules of the present invention are not
intended to include 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 nucleic 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
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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 forming 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%, 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,, 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 an SRT protein. The
nucleotide
sequences determined from the cloning of the SRT genes from C. glutamicum
allows for
the generation of probes and primers designed for use in identifying and/or
cloning SRT
homologues in other cell types and organisms, as well as SRT homologues from
other
Corynebacteria or related species. The probelprimer 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 sequences, or naturally occurring mutants thereof. Primers based on a
nucleotide
sequence of the invention can be used in PCR reactions to clone SRT
homologues.
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Probes based on the SRT 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 an SRT protein, such as by measuring a level of an SRT-
encoding
nucleic acid in a sample of cells, e.g., detecting SRT mRNA levels or
determining
whether a genomic SRT 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 confer resistance or tolerance of C. glutamicum to
one or more
chemical or environmental stresses. 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
participating in the resistance of C. glutamicum to one or more chemical or
environmental stresses. Protein members of such metabolic pathways, as
described
herein, function to increase the resistance or tolerance of C. glutamicum to
one or more
environmental or chernical hazards or stresses. Examples of such activities
are also
described herein. Thus, "the function of an SRT protein" contributes to the
overall
resistance of C. glutamicum to elements of its surroundings which may impede
its
nonmal growth or functioning, and/or contributes, either directly or
indirectly, to the
yield, production, andlor efficiency of production of one or more fine
chemicals.
Examples of SRT protein activities are set forth in Table 1.
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
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the Sequence Listing). Ranges and identity values intermediate to the above-
recited
values, (e.g., 75%-80% identical, 85-87% identical, or 91-92% 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.
Portions of proteins encoded by the SRT nucleic acid molecules of the
invention
are preferably biologically active portions of one of the SRT proteins. As
used herein,
the term "biologically active portion of an SRT protein" is intended to
include a portion,
e.g., a domain/motif, of an SRT protein that is capable of imparting
resistance or
tolerance to one or more environmental or chemical stresses or hazards, or has
an
activity as set forth in Table I. To detennine whether an SRT protein or a
biologically
active portion thereof can increase the resistance or tolerance of C.
glutamicum to one or
more chemical or environmental stresses or hazards, 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 an
SRT 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 SRT protein or peptide (e.g.,
by
recombinant expression in vitro) and assessing the activity of the encoded
portion of the
SRT 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 SRT 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
fuither
embodiment, the nucleic acid molecule of the invention encodes a full length
C.
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).
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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 39% identical to the nucleotide sequence designated RXA00084 (SEQ ID
NO: 189), a nucleotide sequence which is greater than and/or at least 56%
identical to the
nucleotide sequence designated RXA00605 (SEQ ID NO: 11), and a nucleotide
sequence
which is greater than and/or at least 50% identical to the nucleotide sequence
designated
RXA00886 (SEQ ID NO:39). 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 SRT nucleotide sequences set forth in the
Sequence Listing as odd-numbered SEQ ID NOs, it will be appreciated by one of
ordinary skill in the art that DNA sequence polymorphisms that lead to changes
in the
amino acid sequences of SRT proteins may exist within a population (e.g., the
C.
glutamicum population). Such genetic polymorphism in the SRT gene may exist
among
individuals 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 an SRT protein, preferably a C. gluramicum SRT protein.
Such
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natural variations can typically result in 1-5% variance in the nucleotide
sequence of the
SRT gene. Any and all such nucleotide variations and resulting amino acid
polymorphisms in SRT that are the result of natural variation and that do not
alter the
functional activity of SRT 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. glutamicum SRT DNA of the invention can be isolated based
on
their homology to the C. glutamicum SRT 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 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-occutring
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.,
encodes a natural protein). In one embodiment, the nucleic acid encodes a
natural C.
glutamicum SRT protein.
In addition to naturally-occurring variants of the SRT sequence that may exist
in
the population, one of ordinary skill in the art will further appreciate that
changes can be
CA 02587112 2007-05-01
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introduced by mutation into a nucleotide sequence of the invention, thereby
leading to
changes in the amino acid sequence of the encoded SRT protein, without
altering the
functional ability of the SRT 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 SRT proteins
(e.g., an
even-numbered SEQ ID NO: of the Sequence Listing) without altering the
activity of
said SRT protein, whereas an "essential" amino acid residue is required for
SRT protein
activity. Other amino acid residues, however, (e.g., those that are not
conserved or only
semi-conserved in the domain having SRT activity) may not be essential for
activity and
thus are likely to be amenable to alteration without altering SRT activity.
Accordingly, another aspect of the invention pertains to nucleic acid
molecules
encoding SRT proteins that contain changes in amino acid residues that are not
essential
for SRT activity. Such SRT 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
SRT 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 increasing the resistance or tolerance of C.
glutamicum to
one or more environmental or chemical stresses, or has one or more of the
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 in, and most preferably at least about
96%, 97%,
98%, or 99% homologous to one of the amino acid sequences of the invention.
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
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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 an SRT 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 an SRT protein is preferably replaced with another amino acid residue from
the same
side chain family. Alternatively, in another embodiment, mutations can be
introduced
randomly along all or part of an SRT coding sequence, such as by saturation
mutagenesis, and the resultant mutants can be screened for an SRT activity
described
herein to identify mutants that retain SRT activity. Following mutagenesis of
one 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
CA 02587112 2007-05-01
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be determined using, for example, assays described herein (see Example 8 of
the
Exemplification).
In addition to the nucleic acid molecules encoding SRT 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 SRT 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 an SRT 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_: 120
(R.XA00600) comprises nucleotides I to 1098). In another embodiment, the
antisense
nucleic acid molecule is antisense to a "noncoding region" of the coding
strand of a
nucleotide sequence encoding SRT. 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 SRT 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 SRT mRNA, but more preferably is an oligonucleotide
which is
antisense to only a portion of the coding or noncoding region of SRT mRNA. For
example, the antisense oligonucleotide can be complementary to the region
surrounding
the translation start site of SRT mRNA. An antisense oligonucleotide can be,
for
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
CA 02587112 2007-05-01
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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, I-
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-
isopentenyladeni.ne,
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 silu such that they hybridize with or bind to
cellular mRNA
and/or genomic DNA encoding an SRT 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
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
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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-
methyiribonucleotide (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 SRT mRNA transcripts to thereby inhibit translation of
SRT mRNA.
A ribozyme having specificity for an SRT-encoding nucleic acid can be designed
based
upon the nucleotide sequence of an SRT cDNA disclosed herein (i.e., SEQ ID
NO:119
(RXA00600)). 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 an SRT-encoding mRNA. See, e.g., Cech el
al.
U.S. Patent No. 4,987,071 and Cech et al. U.S. Patent No. 5,116,742.
Alternatively,
SRT 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, SRT gene expression can be inhibited by targeting nucleotide
sequences complementary to the regulatory region of an SRT nucleotide sequence
(e.g.,
an SRT promoter and/or enhancers) to form triple helical structures that
prevent
transcription of an SRT 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.
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B. Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression
vectors, containing a nucleic acid encoding an SRT 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 fonns 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 fonn 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
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,
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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-, Ipp-, lac-, Ipp-lac-, IacIq-, T7-, T5-, T3-, gal-, trc-, ara-, SP6-,
amy, SP02, ?,-PR-
or a. 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/355, SSU, OCS, lib4,
usp, STLS l, 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., SRT proteins, mutant forms of SRT proteins, fusion proteins,
etc.).
The recombinant expression vectors of the invention can be designed for
expression of SRT proteins in prokaryotic or eukaryotic cells. For example,
SRT genes
can be expressed in bacterial cells such as C. glulamicum, 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 lumefaciens -mediated transformation of Arabidopsis
ihaliana leaf and cotyledon explants" Plant Cell Rep.: 583-586), or mammalian
cells.
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.
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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 tenninus 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 Inc; 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 SRT protein is cloned
into a
pGEX expression vector to create a vector encoding a fusion 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 SRT 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 69:301-3 l 5) pLG338, pACYC 184, pBR322, pUC
18,
pUC19, pKC30, pRep4, pHSI, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-
III113-B1, kgt11, pBdCl, and pET l ld (Studier et al., Gene Expression
Technology:
Methods in Enzymotogy 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
transcription from a hybrid trp-lac fusion promoter. Target gene expression
from the
pET 11 d vector relies on transcription from a T7 gn 10-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 74 prophage harboring a
T7
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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 pUB110, 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 el 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 SRT protein expression vector is a yeast expression
vector. Examples of vectors for expression in yeast S. cerevisiae include
pYepSecl
(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).
Altecnatively, the SRT 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).
CA 02587112 2007-05-01
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In another embodiment, the SRT 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) EMBO J. 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
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
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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 SRT 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.
A host cell can be any prokaryotic or eukaryotic cell. For example, an SRT
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 those of ordinary skill in the art.
Microorganisms related
CA 02587112 2007-05-01
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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 fotrn
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 a!.
(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 an SRT 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 an SRT gene into which a deletion, addition or
substitution
has been introduced to thereby alter, e.g., functionally disrupt, the SRT
gene.
Preferably, this SRT gene is a Corynebacterium glutamicum SRT gene, but it can
be a
homologue from a related bacterium or even from a mannnalian, yeast, or insect
source.
In a preferred embodiment, the vector is designed such that, upon homologous ,
recombination, the endogenous SRT gene is functionally disrupted (i.e., no
longer
encodes a functional protein; also referred to as a "knock out" vector).
Alternatively,
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the vector can be designed such that, upon homologous recombination, the
endogenous
SRT 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 SRT protein). In the homologous recombination vector, the altered
portion
of the SRT gene is flanked at its 5' and 3' ends by additional nucleic acid of
the SRT
gene to allow for homologous recombination to occur between the exogenous SRT
gene
carried by the vector and an endogenous SRT gene in a microorganism. The
additional
flanking SRT 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 SRT gene has homologously recombined with the
endogenous SRT 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 an SRT gene on a vector placing it under control of
the lac
operon permits expression of the SRT gene only in the presence of IPTG. Such
regulatory systems are well known in the art.
In another embodiment, an endogenous SRT 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 SRT gene in a host cell has been altered by one or more point
mutations,
deletions, or inversions, but still encodes a functional SRT protein. In still
another
embodiment, one or more of the regulatory regions (e. g. , a promoter,
repressor, or
inducer) of an SRT gene in a microorganism has been altered (e.g., by
deletion,
truncation, inversion, or point mutation) such that the expression of the SRT
gene is
modulated. One of ordinary skill in the art will appreciate that host cells
containing
more than one of the described SRT gene and protein modifications may be
readily
produced using the methods of the invention, and are meant to be included in
the present
invention.
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A host cell of the invention, such as a prokaryotic or eukaryotic host cell in
culture, can be used to produce (i.e., express) an SRT protein. Accordingly,
the
invention further provides methods for producing SRT 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 an SRT protein
has
been introduced, or into which genome has been introduced a gene encoding a
wild-type
or altered SRT protein) in a suitable medium until SRT protein is produced. In
another
embodiment, the method further comprises isolating SRT proteins from the
medium or
the host cell.
C. Isolated SRT Proteins
Another aspect of the invention pertains to isolated SRT 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 SRT 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
SRT protein
having less than about 30% (by dry weight) of non-SRT protein (also referred
to herein
as a "contaminating protein"), more preferably less than about 20% of non-SRT
protein,
still more preferably less than about 10% of non-SRT protein, and most
preferably less
than about 5% non-SRT protein. When the SRT 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 SRT protein in which the protein is
separated from
chemical precursors or other chemicals which are involved in the synthesis of
the
protein. In one embodiment, the language "substantially free of chemical
precursors or
other chemicals" includes preparations of SRT protein having less than about
30% (by
dry weight) of chemical precursors or non-SRT chemicals, more preferably less
than
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about 20% chemical precursors or non-SRT chemicals, still more preferably less
than
about 10% chemical precursors or non-SRT chemicals, and most preferably less
than
about 5% chemical precursors or non-SRT chemicals. In preferred embodiments,
isolated proteins or biologically active portions thereof lack contaminating
proteins from
the same organism from which the SRT protein is derived. Typically, such
proteins are
produced by recombinant expression of, for example, a C. glutamicum SRT
protein in a
microorganism such as C. glutamicum.
An isolated SRT protein or a portion thereof of the invention can contribute
to
the resistance or tolerance of C. glutamicum to one or more chemical or
environmental
stresses or hazards, 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 mediate the resistance or tolerance of C.
glutamicum to
one or more chemical or environmental stresses or hazards. The portion of the
protein is
preferably a biologically active portion as described herein. In another
preferred
embodiment, an SRT 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 SRT 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 SRT
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%, 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
CA 02587112 2007-05-01
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and/or lower limits are intended to be included. The prefeffed SRT proteins of
the
present invention also preferably possess at least one of the SRT activities
described
herein. For example, a preferred SRT 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
increase the resistance or tolerance of C. glutamicum to one or more
environmental or
chemical stresses, or which has one or more of the activities set forth in
Table 1.
In other embodiments, the SRT 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 SRT 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 SRT 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 .
Biologically active portions of an SRT protein include peptides comprising
amino acid sequences derived from the amino acid sequence of an SRT 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 an SRT protein, which include
fewer
amino acids than a full length SRT protein or the full length protein which is
homologous to an SRT protein, and exhibit at least one activity of an SRT
protein.
CA 02587112 2007-05-01
- 48 -
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 an SRT 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 an SRT
protein include
one or more selected domains/motifs or portions thereof having biological
activity.
SRT 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 SRT protein is expressed in the host cell. The SRT
protein can
then be isolated from the cells by an appropriate purification scheme using
standard
protein purification techniques. Alternative to recombinant expression, an SRT
protein,
polypeptide, or peptide can be synthesized chemically using standard peptide
synthesis
techniques. Moreover, native SRT protein can be isolated from cells (e.g.,
endothelial
cells), for example using an anti-SRT antibody, which can be produced by
standard
techniques utilizing an SRT protein or fragment thereof of this invention.
The invention also provides SRT chimeric or fusion proteins. As used herein,
an
SRT "chimeric protein" or "fusion protein" comprises an SRT polypeptide
operatively
linked to a non-SRT polypeptide. An "SRT polypeptide" refers to a polypeptide
having
an amino acid sequence corresponding to SRT, whereas a "non-SRT polypeptide"
refers
to a polypeptide having an amino acid sequence corresponding to a protein
which is not
substantially homologous to the SRT protein, e.g., a protein which is
different from the
SRT 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
SRT
polypeptide and the non-SRT polypeptide are fused in-frame to each other. The
non-
SRT polypeptide can be fused to the N-terminus or C-terminus of the SRT
polypeptide.
For example, in one embodiment the fusion protein is a GST-SRT fusion protein
in
which the SRT sequences are fused to the C-terminus of the GST sequences. Such
fusion proteins can facilitate the purification of recombinant SRT proteins.
In another
embodiment, the fusion protein is an SRT protein containing a heterologous
signal
sequence at its N-terminus. In certain host cells (e.g., mammalian host
cells), expression
CA 02587112 2007-05-01
-49-
and/or secretion of an SRT protein can be increased through use of a
heterologous signal
sequence.
Preferably, an SRT 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). An
SRT-
encoding nucleic acid can be cloned into such an expression vector such that
the fusion
moiety is linked in-frame to the SRT protein.
Homologues of the SRT protein can be generated by mutagenesis, e.g., discrete
point mutation or truncation of the SRT protein. As used herein, the term
"homologue"
refers to a variant form of the SRT protein which acts as an agonist or
antagonist of the
activity of the SRT protein. An agonist of the SRT protein can retain
substantially the
same, or a subset, of the biological activities of the SRT protein. An
antagonist of the
SRT protein can inhibit one or more of the activities of the naturally
occurring form of
the SRT protein, by, for example, competitively binding to a downstream or
upstream
member of the SRT system which includes the SRT protein. Thus, the C.
glutamicum
SRT protein and homologues thereof of the present invention may increase the
tolerance
or resistance of C. glutamicum to one or more chemical or environmental
stresses.
In an alternative embodiment, homologues of the SRT protein can be identified
by screening combinatorial libraries of mutants, e.g., truncation mutants, of
the SRT
protein for SRT protein agonist or antagonist activity. In one embodiment, a
variegated
library of SRT variants is generated by combinatorial mutagenesis at the
nucleic acid
CA 02587112 2007-05-01
-50-
level and is encoded by a variegated gene library. A variegated library of SRT
variants
can be produced by, for example, enzymatically ligating a mixture of synthetic
oligonucleotides into gene sequences such that a degenerate set of potential
SRT
sequences is expressible as individual polypeptides, or alternatively, as a
set of larger
fusion proteins (e.g., for phage display) containing the set of SRT sequences
therein.
There are a variety of methods which can be used to produce libraries of
potential SRT
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 SRT sequences. Methods for synthesizing
degenerate
oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983)
Tetrahedron 39:3;
Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura ec al. (1984)
Science
198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.
In addition, libraries of fragments of the SRT protein coding can be used to
generate a variegated population of SRT fragments for screening and subsequent
selection of homologues of an SRT protein. In one embodiment, a library of
coding
sequence fragments can be generated by treating a double stranded PCR fragment
of an
SRT 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 intemal fragments of various sizes of the SRT protein.
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 SRT
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
CA 02587112 2007-05-01
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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 SRT 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 SRT 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 SRT
protein
regions required for function; modulation of an SRT protein activity;
modulation of the
activity of an SRT pathway; and modulation of cellular production of a desired
compound, such as a fine chemical.
The SRT 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
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
CA 02587112 2007-05-01
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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
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 lactojermentum.
The SRT nucleic acid molecules of the invention are also useful for
evolutionary
and protein structural studies. The resistance processes in which the
molecules of the
invention participate are utilized by a wide variety of cells; by comparing
the sequences
CA 02587112 2007-05-01
.
-53-
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 penmits 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.
The genes of the invention, e.g., the gene encoding LMRB (SEQ ID NO:1) or
other gene of the invention encoding a chemical or environmental resistance or
tolerance
protein (e.g_, resistance against one or more antibiotics), may be used as
genetic markers
for the genetic transformation of (e.g., the transfer of additional genes into
or disruption
of preexisting genes of) organisms such as C. glutamicum or other bacterial
species_
Use of these nucleic acid molecules permits efficient selection of organisms
which have
incorporated a given transgene cassette (e.g., a plasmid, phage, phasmid,
phagemid,
transposon, or other nucleic acid element), based on a trait which permits the
survival of
the organism in an otherwise hostile or toxic environment (e.g., in the
presence of an
antimicrobial compound). By employing one or more of the genes of the
invention as
genetic markers, the speed and ease with which organisms having desirable
transformed
traits (e.g., modulated fine chemical production) are engineered and isolated
are
improved. While it is advantageous to use the genes of the invention for
selection of
transformed C. glutamicum and related bacteria, it is possible, as described
herein, to use
homologs (e.g., homologs from other organisms), allelic variants or fragments
of the
gene retaining desired activity. Furthermore, 5' and 3' regulatory elements of
the genes
of the invention may be modified as described herein (e.g., by nucleotide
substitution,
insertion, deletion, or replacement with a more desirable genetic element) to
modulate
the transcription of the gene. For example, an LMRB variant in which the
nucleotide
sequence in the region from -1 to -200 5' to the start codon has been altered
to
modulate (preferably increase) the transcription and/or translation of LMRB
may be
employed, as can constructs in which a gene of the invention (e.g., the LMRB
gene
(SEQ ID NO:1)) is functionally coupled to one or more regulatory signals
(e.g., inducer
or repressor binding sequences) which can be used for modulating gene
expression.
CA 02587112 2007-05-01
-54-
Similarly, more than one copy of a gene (functional or inactivated) of the
invention may
be employed.
An additional application of the genes of the invention (e.g., the gene
encoding
LMRB (SEQ ID NO:I) or other drug- or antibiotic-resistance gene) is in the
discovery
of new antibiotics which are active against Corynebacteria and/or other
bacteria. For
example, a gene of the invention may be expressed (or overexpressed) in a
suitable host
to generate an organism with increased resistance to one or more drugs or
antibiotics (in
the case of LMRB, lincosamides in particular, especially lincomycin). This
type of
resistant host can subsequently be used to screen for chemicals with
bacteriostatic and/or
bacteriocidal activity, such as novel antibiotic compounds. It is possible, in
particular,
to use the genes of the invention (e.g., the LMRB gene) to identify new
antibiotics
which are active against those microorganisms which are already resistant to
standard
antibiotic compounds.
The invention provides methods for screening molecules which modulate the
activity of an SRT protein, either by interacting with the protein itself or a
substrate or
binding partner of the SRT protein, or by modulating the transcription or
translation of,T
SRT nucleic acid molecule of the invention. In such methods, a microorganism
expressing one or more SRT 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 SRT protein is assessed.
Manipulation of the SRT nucleic acid molecules of the invention may result in
the production of SRT proteins having functional differences from the wild-
type SRT
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 goal of such manipulations is to increase the viability and activity of
the cell when
the cell is exposed to the environmental and chemical stresses and hazards
which
frequently accompany large-scale fermentative culture. Thus, by increasing the
activity
or copy number of a heat-shock-regulated protease, one may increase the
ability of the
cell to destroy incorrectly folded proteins, which may otherwise interfere
with normal
cellular functioning (for example, by continuing to bind substrates or
cofactors although
the protein lacks the activity to act on these molecules appropriately). The
same is true
for the overexpression or optimization of activity of one or more chaperone
molecules
CA 02587112 2007-05-01
-55-
induced by heat or cold shock. These proteins aid in the correct folding of
nascent
polypeptide chains, and thus their increased activity or presence should
increase the
percentage of correctly folded proteins in the cell, which in turn should
increase the
overall metabolic efficiency and viability of the cells in culture. The
overexpression or
optimization of the transporter molecules activated by osmotic shock should
result in an
increased ability on the part of the cell to maintain intracellular
homeostasis, thereby
increasing the viability of these cells in culture. Similarly, the
overproduction or
increase in activity by mutagenesis of proteins involved in the development of
cellular
resistance to chemical stresses of various kinds (either by transport of the
offending
chemical out of the cell or by modification of the chemical to a less
hazardous
substance) should increase the fitness of the organism in the environment
containing the
hazardous substance (i.e., large-scale fermentative culture), and thereby may
permit
relatively larger numbers of cells to survive in such a culture. The net
effect of all of
these mutagenesis strategies is to increase the quantity of fi ne-chemical-
producing
compounds in the culture, thereby increasing the yield, production, and/or
efficiency of
production of one or more desired fine chemicals from the culture.
This aforementioned list of mutagenesis strategies for SRT proteins to result
in
increased yields of a desired compound is not meant to be limiting; variations
on these
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 SRT
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.
CA 02587112 2007-05-01
56
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CA 02587112 2007-05-01
-74-
TABLE 3: Corynebacterium and Brevibacterium Strains Which May be Used in
the Practice of the Invention
i.'_tC;
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 ammoniagenes 39101
Brevibacterium butanicum 21196
Brevibacterium divaricatum 21792 P928
Brevibacterium flavum 21474
Brevibacterium flavum 21129
Brevibacterium flavum 21518
Brevibacterium flavum B 11474
Brevibacterium flavum B11472
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 B 11478
Brevibacterium flavum 21127
Brevibacterium flavum B 11474
Brevibacterium healii 15527
Brevibacterium ketoglutamicum 21004
Brevibacterium ketoglutamicum 21089
Brevibacterium ketosoreductum 21914
Brevibacterium lactofermentum 70
Brevibacterium lactofermentum 74
Brevibacterium lactofermentum 77
Brevibacterium Iactofermentum 21798
Brevibacterium lactofermentum 21799
Brevibacterium lactofermentum 21800
Brevibacterium lactofermentum 21801
Brevibacterium lactofermentum B 11470
Brevibacterium lactofermentum B 11471
CA 02587112 2007-05-01
-75-
A.. c;.. w-s :. c cr ; c~M$ >9s"~ Kcr nS1V~:
Brevibacterium lactofermentum 21086
Brevibacterium lactofermentum 21420
Brevibacterium lactofermentum 21086
Brevibacterium lactofermentum 31269
Brevibacterium linens 9174
Brevibacterium linens 19391
Brevibacterium linens 8377
Brevibacterium paraftinolyticum 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 aceloglutamicum 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 glutamicum 13059
Corynebacterium glutamicum 13060
Corynebacterium glutamicum 21492
Corynebacterium glutamicum 21513
Corynebacterium glutamicum 21526
Corynebacterium glutamicum 21543
Corynebacterium glutamicum 13287
Corynebacterium glutamicum 21851
Corynebacterium glutamicum 21253
Corynebacterium glutamicum 21514
Corynebacterium glutamicum 21516
Corynebacterium glutamicum 21299
CA 02587112 2007-05-01
-76-
.
,; .:: . .,.. ~
.x
~..=..
. ~.,: . . .; . ...._.: . n .. . __. ._~
Corynebacterium glutamicum 21300
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
CA 02587112 2007-05-01
-77-
- =-
,fr
G CT. .
Corynebacterium glutamicum B12418
Corynebacterium glutamicum B11476
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, Baam, NL
NCTC: National Collection of Type Cultures, London, UK
DSMZ: Deutsche Sammiung 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 (4w edn), World federation for
culture collections world
data center on microorganisms, Saimata, Japen.
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CA 02587112 2007-05-01
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CA 02587112 2007-05-01
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Exemplification
Example 1: Preparation of total genomic DNA of Corynebacterium glulamicum
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 mi of culture volume). Composition of buffer-I: 140.34 g/1
sucrose,
2.46 g/l MgSO4 x 7H2O, 10 ml/1 KH2PO4 solution (100 g/l, adjusted to pH 6.7
with
KOH), 50 mUl M12 concentrate (10 g/1(NH.)ZSO,, 1 g/l NaCI, 2 g/t MgSO. x 7H,0,
0.2 gll CaCI=, 0.5 g/1 yeast extract (Difco), 10 ml/I trace-elements-mix (200
mg/I FeSO,
x HjO, 10 mg/i ZnSO, x 7 H2O, 3 mg/i MnCl2 x 4 H2O, 30 mg/I H3BO3 20 mg/1
CoCl2 x
6 H2O, 1 mg/i NiCI2 x 6 H2O, 3 mg/i Na2MoO4x 2 H2O, 500 mg/1 complexing agent
(EDTA or critic acid), 100 mI/) vitamins-mix (0.2 mg/i biotin, 0.2 mg/1 folic
acid, 20
mg/i p-amino benzoic acid, 20 mg/1 riboflavin, 40 mg/i ca-panthothenate, 140
mg/1
nicotinic acid, 40 mg/i pyridoxole hydrochloride, 200 mg/1 myo-inositol).
Lysozyme
was added to the suspension to a final concentration of 2.5 mg/ml. 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, I mM EDTA, pH 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) are added. 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-isoamylalcohot 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 LiCl and 0.8 ml of ethanol are added.
After a 30
CA 02587112 2007-05-01
-87-
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
Corynebaclerium
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); pACYCl77
(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. glulamicum 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' (SEQ ID No:305) or
5'-GTAAAACGACGGCCAGT-3' (SEQ ID No:306).
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
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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 those 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 constructed by using standard
vectors for
E. coli (Sambrook, J. et al. (1989), "Molecular Cloning: A Laboratory Manual",
Cold
Spring Harbor Laboratory Press or Ausubel, F.M. et a!. (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: l 37-146 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 vector into
strains of
Corynebacterium glutamicum. Transformation of C. glutamicum can be achieved by
protoplast transformation (Kastsumata, R. et al. (1984) J. Bacteriol. 159306-
311),
electroporation (Liebl, E. et al. (1989) FEMS Microbiol. 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. glulamicum
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) BiotechnologyLetters, 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 NH.CI or (NH.)2SOõ 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 mnge 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 alternatively or simultaneously be
used. It
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is also possible to maintain a constant culture pH through the addition of
NaOH or
NH.OH 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 mi 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/I NaCI, 2 g/l urea, 10 g/l polypeptone, 5 g/1 yeast
ektract, 5 g/1 meat
extract, 22 g/l NaCI, 2 g/l urea, 10 g/l polypeptone, 5 g/i yeast extract, 5
g/t meat extract,
22 g/1 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 conceming 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, 3rd ed. Academic Press:
New
York; Bisswanger, H., (1994) Enzymkinetik, 2"d ed. VCH: Weinheim (ISBN
3527300325); Bergmeyer, H.U., Bergmeyer, J., GraBl, M., eds. (1983-1986)
Methods of
Enzymatic Analysis, 3rd 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
a!.
(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 yield, production, 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 cluomatography 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: Cloning of a Corynebacterium glutamicum Gene Involved in
Lincomycin Resistance Using a Reporter Gene Approach
A. ldentification of the Gene Encoding the LMRB Protein
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Plasmid pSL 130 was constructed by ligation of the aceB promoter region
(paceB) of C. glutamicum (Kim, H.J. et al. (1997) J. Microbiol. Biotechnol. 7:
287-292)
into the polylinker of the lac operon fusion vector pRS415, which lacks a
promoter
(Simon, R.W. et al. (1987) Gene 53: 85-96). Plasmid pSL 145 was constructed by
ligating the resulting paceB-lac region into the E. coli cloning vector
pACYC184. E.
coli DH5aF' was transformed with pSL 145 and the resulting strain was used as
a host
for screening of a genomic C. glutamicum library (in pSL 109).
Transfonnants were screened by growth on agar medium containing 5-bromo-4-
chloro-3-indolyl-beta-D-glalactopyranoside (X-Gal). A white colony, containing
DNA
influencing lacZ expression, was selected for further analysis. This clone was
found to
contain a 4 kB fragment from the gene library. Subclones were constructed in
pSL 109
and a subclone which retained the white phenotype on X-Gal plates was
identified. This
subclone was found to contain a 2.6 kB BamHl-Xhol fragment (plasmid pSL149-5).
The fragment was sequenced and identified as a membrane protein-encoding gene
(LMRB gene).
The 1442 nucleotides of the coding sequence of the LMRB gene encode a
polypeptide of 481 amino acid residues with a high percentage of hydrophobic
amino
acids. A Genbank search determined that the LMRB protein is 40% identical to
the
protein product of the ImrB gene from Bacillus subtilis (Genbank Accession
AL009126,
TREMBL Accession P94422), as determined using a CLUSTAL W analysis (using
standard parameters).
The LMRN protein contains a sequence pattern: 158-A-P-A-L-G-P-T-L-S-G-167
(SEQ ID NO:301), which resembles the known multi-drug-resistance-protein
consensus
motif G-X-X-X-G-P-X-X-G-G (SEQ ID NO:302) (Paulsen, I.T., and Skurray, R.A.
(1993) Gene 124: 1-11). Therefore, the LMRB protein was classified as a drug
resistance protein.
B. In vivo Analysis of lmrB Function
The lmrB gene was overexpressed in C. glutamicum ASO19E12 (Kim, H.J. et al.
(1997) J. Microbiol. Biotechnol. 7: 287-292) using the plasmid pSL 149-5,
described
above.
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Disruption of the LMRB gene was accomplished by use of the vector pSLl8-
lmrB. This vector was constructed as follows: an internal fragment of the LMRB
gene
was amplified by PCR under standard conditions using primers 5'-
CTCCAGGATTGCTCCGAAGG-3' (SEQ ID NO:303) and 5'-
CACAGTGGTTGACCACTGGC-3' (SEQ ID NO:304). The resulting PCR product
was treated with T7 DNA polymerase and T7 polynucleotide kinase, and was
cloned
into the SmaI site of plasmid pSL18 (Kim, Y.H. and H.-S. Lee (1996) J.
Microbiol.
Biotechnol. 6: 315-320). The disruption of the LMRB gene in C. glutamicum
ASO 19E 12 was performed by conjugation, as previously described (Schwarzer
and
Puhler (1991) Bio/Technology 9:84-87).
C. glutamicum cells transformed with pSL149-5 displayed similar resistances as
untransformed cells against erythromycin, penicillin G, tetracycline,
chloramphenicol,
spectinomycin, nalidixic acid, gentamycin, streptomycin, ethidium bromide,
carbonyl
cyanide m-chlorophenylhydrazone (CCCP), and sodium dodecyl sulfate.
Significant
differences were observed, however, in the resistance of transformed and
untransformed
cells to lincomycin.
LMRB-overexpressing C. glutamicum cells were found to be able to grow in the
presence of 20 g/ml lincomycin. In contrast, cells which do not overexpress
LMRB (or
cells carrying a LMRB disruption) were not able to grow on agar media
containing 5
g/ml lincomycin. This effect was clearly visible in liquid culture. LMRB
overexpression led to a 9-fold increased resistance (compared to wild-type)
against
lincomycin and LMRB disruption resulted in a decreased resistance (28% of wild-
type)
to this antibiotic.
Example 12: 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
90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST
programs (version 2.0) of Altschul, et a!. (1990) J. Mol. Biol. 215:403-10.
BLAST
nucleotide searches can be performed with the NBLAST program, score = 100,
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wordlength = 12 to obtain nucleotide sequences homologous to SRT 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 SRT 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 Miller ((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 PAM 120 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
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
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top 500 hits were retained for further analysis. A subsequent FASTA search
(e.g., a
combined local and global aligmnent 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 format, wherein a',' represents a decimal
point. For
example, a value of "40,345" in this column represents "40.345%".
Example 13: 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 a!.
(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
may be used to monitor and measure the individual signal intensities of the
hybridized
molecules at defined regions. This methodology allows the simultaneous
quantification
of the relative or absolute amount of all or selected nucleic acids in the
applied nucleic
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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
described in Schena, M. et al. (1995) supra) and fluorescent labels may be
detected, for
example, by the method of Shalon et a!. (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.
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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 14: 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:
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
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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,'SN03 or'SNH4+ or 13C-labelled amino acids) in the medium of C.
glutamicurn
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 example, 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 et 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 pattems 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.
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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.
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