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CORYIYEBACTERIUM GLUTAMICUM GENES ENCODING PROTEINS
INVOLVED IN MEMBRANE SYNTHESIS AND MEMBRANE TRANSPORT
Related Applications
This application claims priority to prior filed U.S. Provisional Patent
Application
Serial No. 60/141031, filed June 25, 1999. This application also claims
priority to
German Patent Application No. 19931454.3, filed July 8, 1999, German Patent
Application No. 19931478.0, filed July 8, 1999, German Patent Application No.
19931563.9, filed July 8, 1999, German Patent Application No. 19932122.1,
filed July
9, 1999, German Patent Application No. 19932124.8, filed 99709, German Patent
Application No. 19932125.6, filed July 9, 1999, German Patent Application No.
19932128.0, filed July 9, 1999, German Patent Application No. 19932180.9,
filed July
9, 1999, German Patent Application No. 19932182.5, filed July 9, 1999, German
Patent
Application No. 19932190.6, filed July 9, 1999, German Patent Application No.
19932191.4, filed July 9, 1999, German Patent Application No. 19932209.0,
filed July
9, 1999, German Patent Application No. 19932212.0, filed July 9, 1999, German
Patent
Application No. 19932227.9, filed July 9, 1999, German Patent Application No.
19932228.7, filed July 9, 1999, German Patent Application No. 19932229.5,
filed
99070, German Patent Application No. 19932230.9, filed July 9, 1999, German
Patent
Application No. 19932927.3, filed July 14, 1999, German Patent Application No.
19933005.0, filed July 14, 1999, German Patent Application No. 19933006.9,
filed July
14, 1999, German Patent Application No. 19940764.9, filed August 27, 1999,
German
Patent Application No. 19940765.7, filed August 27, 1999, German Patent
Application
No. 19940766.5, filed August 27, 1999, German Patent Application No.
19940830.0,
filed August 27, 1999, German Patent Application No. 19940831.9, filed August
27,
1999, German Patent Application No. 19940832.7, filed August 27, 1999, German
Patent Application No. 19940833.5, filed August 27, 1999, German Patent
Application
No. 19941378.9 filed August 31, 1999, German Patent Application No.
19941379.7,
filed August 31, 1999, German Patent Application No. 19941395.9, filed August
31,
1999, German Patent Application No. 19942077.7, filed September 3, 1999,
German
Patent Application No. 19942078.5, filed September 3, 1999, German Patent
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Application No. 19942079.3, filed September 3, 1999, and German Patent
Application
No. 19942088.2, filed September 3, 1999. The entire contents of all of the
above
referenced applications are hereby expressly incorporated herein by this
reference.
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 the large-scale culture of bacteria developed
to produce
and secrete large quantities of one or more desired molecules. One
particularly useful
organism for this purpose is Corynebacterium glutamicum, a gram positive,
nonpathogenic bacterium. Through strain selection, a number of mutant strains
have
been developed which produce an array of desirable compounds. However,
selection of
strains improved for the production of a particular molecule is a time-
consuming and
difficult process.
Summary of the Invention
The invention provides novel bacterial nucleic acid molecules which have a
variety of uses. These uses include the identification of microorganisms which
can be
used to produce fine chemicals, the modulation of fine chemical production in
C.
glutamicum or related bacteria, the typing or identification of C. glutamicum
or related
bacteria, as reference points for mapping the C. glutamicum genome, and as
markers for
transformation. These novel nucleic acid molecules encode proteins, referred
to herein
as membrane construction and membrane transport (MCT) 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 MCT nucleic acid molecules of the invention, therefore, can be
used to
identify microorganisms which can be used to produce fine chemicals, e.g., by
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fermentation processes. Modulation of the expression of the MCT nucleic acids
of the
invention, or modification of the sequence of the MCT 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 MCT nucleic acids of the invention may also be used to identify an
organism as being Corynebacterium glutamicum or a close relative thereof, or
to
identify the presence of C. glutamicum or a relative thereof in a mixed
population of
microorganisms. The invention provides the nucleic acid sequences of a number
of C.
glutamicum genes; by probing the extracted genomic DNA of a culture of a
unique or
mixed population of microorganisms under stringent conditions with a probe
spanning a
region of a C. glutamicum gene which is unique to this organism, one can
ascertain
whether this organism is present. Although Corynebacterium glutamicum itself
is
nonpathogenic, it is related to species pathogenic in humans, such as
Corynebacterium
diphtheriae (the causative agent of diphtheria); the detection of such
organisms is of
significant clinical relevance.
The MCT nucleic acid molecules of the invention may also serve as reference
points for mapping of the C. glutamicum genome, or of genomes of related
organisms.
Similarly, these molecules, or variants or portions thereof, may serve as
markers for
genetically engineered Corynebacterium or Brevibacterium species.
The MCT proteins encoded by-the novel nucleic acid molecules of the invention
are capable of, for example, performing a function involved in the metabolism
(e.g., the
biosynthesis or degradation) of compounds necessary for membrane biosynthesis,
or of
assisting in the transmembrane transport of one or more compounds either into
or out of
the cell. Given the availability of cloning vectors for use in Corynebacterium
glutamicum, such as those disclosed in Sinskey et al., U.S. Patent No.
4,649,119, and
techniques for genetic manipulation of C. glutamicum and the related
Brevibacterium
species (e.g., lactofermentum) (Yoshihama et al, J. Bacteriol. 162: 591-597
(1985);
Katsumata et 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. This improved production or efficiency
of
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production of a fine chemical may be due to a direct effect of manipulation of
a gene of
the invention, or it may be due to an indirect effect of such manipulation.
There are a number of mechanisms by which the alteration of an MCT protein of
the invention may directly affect the yield, production, and/or efficiency of
production
of a fine chemical from a C. glutamicum strain incorporating such an altered
protein.
Those MCT proteins involved in the export of fine chemical molecules from the
cell
may be increased in number or activity such that greater quantities of these
compounds
are secreted to the extracellular medium, from which they are more readily
recovered.
Similarly, those MCT proteins involved in the import of nutrients necessary
for the
biosynthesis of one or more fine chemicals (e.g., phosphate, sulfate, nitrogen
compounds, etc.) may be increased in number or activity such that these
precursors,
cofactors, or intermediate compounds are increased in concentration within the
cell.
Further, fatty acids and lipids themselves are desirable fine chemicals; by
optimizing the
activity or increasing the number of one or more MCT proteins of the invention
which
participate in the biosynthesis of these compounds, or by impairing the
activity of one or
more MCT proteins which are involved in the degradation of these compounds, it
may
be possible to increase the yield, production, and/or efficiency of production
of fatty
acid and lipid molecules from C. glutamicum.
The mutagenesis of one or more MCT genes of the invention may also result in
MCT proteins having altered activities which indirectly impact the production
of one or
more desired fine chemicals from C .glutamicum. For example, MCT proteins of
the
invention involved in the export of waste products may be increased in number
or
activity such that the normal metabolic wastes of the cell (possibly increased
in quantity
due to the overproduction of the desired fine chemical) are efficiently
exported before
they are able to damage nucleotides and proteins within the cell (which would
decrease
the viability of the cell) or to interfere with fine chemical biosynthetic
pathways (which
would decrease the yield, production, or efficiency of production of the
desired fine
chemical). Further, the relatively large intracellular quantities of the
desired fine
chemical may in itself be toxic to the cell, so by increasing the activity or
number of
transporters able to export this compound from the cell, one may increase the
viability of
the cell in culture, in turn leading to a greater number of cells in the
culture producing
the desired fine chemical. The MCT proteins of the invention may also be
manipulated
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such that the relative amounts of different lipid and fatty acid molecules are
produced.
This may have a profound effect on the lipid composition of the membrane of
the cell.
Since each type of lipid has different physical properties, an alteration in
the lipid
composition of a membrane may significantly alter membrane fluidity. Changes
in
membrane fluidity can impact the transport of molecules across the membrane,
as well
as the integrity of the cell, both of which have a profound effect on the
production of
fine chemicals from C. glutamicum in large-scale fermentative culture.
The invention provides novel nucleic acid molecules which encode proteins,
referred to herein as MCT proteins, which are capable of, for example,
participating in
the metabolism of compounds necessary for the construction of cellular
membranes in
C. glutamicum, or in the transport of molecules across these membranes.
Nucleic acid
molecules encoding an MCT protein are referred to herein as MCT nucleic acid
molecules. In a preferred embodiment, the MCT protein participates in the
metabolism
of compounds necessary for the construction of cellular membranes in C.
glutamicum,
or in the transport of molecules across these membranes. Examples of such
proteins
include those encoded by the genes set forth in Table 1.
Accordingly, one aspect of the invention pertains to isolated nucleic acid
molecules (e.g., cDNAs, DNAs, or RNAs) comprising a nucleotide sequence
encoding
an MCT protein or biologically active portions thereof, as well as nucleic
acid fragments
suitable as primers or hybridization probes for the detection or amplification
of MCT-
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
N0:3, SEQ ID NO:S, SEQ ID N0:7....), forth as the odd-numbered SEQ ID NOs in
the
Sequence Listing (e.g., SEQ ID NO:1, SEQ ID N0:3, SEQ ID NO:S, SEQ ID
N0:7....),
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 N0:3, SEQ ID NO:S, SEQ ID N0:7....), or a portion
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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 N0:2, SEQ ID N0:4, SEQ ID N0:6, SEQ ID N0:8...). The
preferred MCT proteins of the present invention also preferably possess at
least one of
the MCT 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 MCT activity. Preferably; the protein or
portion thereof
encoded by the nucleic acid molecule maintains. the ability to participate in
the
metabolism of compounds necessary for the construction of cellular membranes
in C.
glutamicum, or in the transport of molecules across these membranes. In one
embodiment, the protein encoded by the nucleic acid molecule is at least about
50%,
preferably at least about 60%, and more preferably at least about 70%, 80%, or
90% and
most preferably at least about 95%, 96%, 97%, 98%, or 99% or more homologous
to an
amino acid sequence of the invention (e.g., an entire amino acid sequence
selected from
those having an even-numbered SEQ ID NO in the Sequence Listing). In another
preferred embodiment, the protein is a full length C. glutamicum protein which
is
substantially homologous to an entire amino acid sequence of the invention
(encoded by
an open reading frame shown in the corresponding odd-numbered SEQ ID NOs in
the
Sequence Listing (e.g., SEQ ID NO:1, SEQ ID N0:3, SEQ ID NO:S, SEQ ID
N0:7....).
In another preferred embodiment, the isolated nucleic acid molecule is derived
from C. glutamicum and encodes a protein (e.g., an MCT fusion protein) which
includes
a biologically active domain which is at least about 50% or more homologous to
one of
the amino acid sequences of the invention (e.g., a sequence of one of the even-
numbered
SEQ ID NOs in the Sequence Listing) and is able to participate in the
metabolism of
compounds necessary for the construction of cellular membranes in C.
glutamicum, or in
the transport of molecules across these membranes, or has one or more of the
activities
set forth in Table l, and which also includes heterologous nucleic acid
sequences
encoding a heterologous polypeptide or regulatory regions.
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In another embodiment, the isolated nucleic acid molecule is at least 1 S
nucleotides in length and hybridizes under stringent conditions to a nucleic
acid
molecule comprising a nucleotide sequence of the invention (e.g., a sequence
of an odd-
numbered SEQ ID NO in the Sequence Listing). Preferably, the isolated nucleic
acid
molecule corresponds to a naturally-occurring nucleic acid molecule. More
preferably,
the isolated nucleic acid encodes a naturally-occurring C. glutamicum MCT
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 MCT protein by culturing the host cell in a suitable medium. The
MCT
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 MCT gene has been introduced or altered. In one
embodiment, the genome of the microorganism has been altered by introduction
of a
nucleic acid molecule of the invention encoding wild-type or mutated MCT
sequence as
a transgene. In another embodiment, an endogenous MCT gene within the genome
of
the microorganism has been altered, e.g., functionally disrupted, by
homologous
recombination with an altered MCT gene. In another embodiment, an endogenous
or
introduced MCT gene in a microorganism has been altered by one or more point
mutations, deletions, or inversions, but still encodes a functional MCT
protein. In still
another embodiment, one or more of the regulatory regions (e.g., a promoter,
repressor,
or inducer) of an MCT gene in a microorganism has been altered (e.g., by
deletion,
truncation, inversion, or point mutation) such that the expression of the MCT
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
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sequences set forth in the Sequence Listing as SEQ ID NOs 1 through 676) 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 MCT protein or a
portion, e.g., a biologically active portion, thereof. In a preferred
embodiment, the
isolated MCT protein or portion thereof can participate in the metabolism of
compounds
necessary for the construction of cellular membranes in C. glutamicum, or in
the
transport of molecules across these membranes. In another preferred
embodiment, the
isolated MCT protein or portion thereof is sufficiently homologous to an amino
acid
sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: in
the
Sequence Listing) such that the protein or portion thereof maintains the
ability to
participate in the metabolism of compounds necessary for the construction of
cellular
membranes in C. glutamicum, or in the transport of molecules across these
membranes.
The invention also provides an isolated preparation of an MCT protein. In
preferred embodiments, the MCT protein comprises an amino acid sequence of the
invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence
Listing).
In another preferred embodiment, the invention pertains to an isolated full
length protein
which is substantially homologous to an entire amino acid sequence of the
invention
(e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing)
(encoded
by an open reading frame set forth in a corresponding odd-numbered SEQ ID NO:
of the
Sequence Listing A). 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 MCT
protein
comprises an amino acid sequence which is at least about 50% or more
homologous to
one of the amino acid sequences of the invention (e.g., a sequence of an even-
numbered
SEQ ID NO: of the Sequence Listing) and is able to participate in the
metabolism of
compounds necessary for the construction of cellular membranes in C.
glutamicum, or in
the transport of molecules across these membranes, or has one or more of the
activities
set forth in Table 1.
Alternatively, the isolated MCT protein can comprise an amino acid sequence
which is encoded by a nucleotide sequence which hybridizes, e.g., hybridizes
under
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stringent conditions, or is at least about 50%, preferably at least about 60%,
more
preferably at least about 70%, 80%, or 90%, and even more preferably at least
about
95%, 96%, 97%, 98,%, or 99% or more homologous, to a nucleotide sequence of
one of
the even-numbered SEQ ID NOs set forth in the Sequence Listing. It is also
preferred
that the preferred forms of MCT proteins also have one or more of the MCT
bioactivities described herein.
The MCT polypeptide, or a biologically active portion thereof, can be
operatively linked to a non-MCT polypeptide to form a fusion protein. In
preferred
embodiments, this fusion protein has an activity which differs from that of
the MCT
protein alone. In other preferred embodiments, this fusion protein participate
in the
metabolism of compounds necessary for the construction of cellular membranes
in C.
glutamicum, or in the transport of molecules across these membranes. In
particularly
preferred embodiments, integration of this fusion protein into a host cell
modulates
production of a desired compound from the cell.
In another aspect, the invention provides methods for screening molecules
which
modulate the activity of an MCT protein, either by interacting with the
protein itself or a
substrate or binding partner of the MCT protein, or by modulating the
transcription or
translation of an MCT nucleic acid molecule of the invention.
Another aspect of the invention pertains to a method for producing a fine
chemical. This method involves the culturing of a cell containing a vector
directing the
expression of an MCT 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 MCT 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 MCT protein activity or MCT 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 for one or more C.
glutamicum
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metabolic pathways for cell membrane components or is modulated for the
transport of
compounds across such membranes, such that the yields or rate of production of
a
desired fine chemical by this microorganism is improved. The agent which
modulates
MCT protein activity can be an agent which stimulates MCT protein activity or
MCT
nucleic acid expression. Examples of agents which stimulate MCT protein
activity or
MCT nucleic acid expression include small molecules, active MCT proteins, and
nucleic
acids encoding MCT proteins that have been introduced into the cell. Examples
of
agents which inhibit MCT activity or expression include small molecules and
antisense
MCT 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 MCT
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 be random,
or it can
take place by homologous recombination such that the native gene is replaced
by the
1 S 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.
Detailed Description of the Invention
The present invention provides MCT nucleic acid and protein molecules which
are involved in the metabolism of cellular membrane components in C.
glutamicum or
in the transport of compounds across such membranes. The molecules of the
invention
may be utilized in the modulation of production of fine chemicals from
microorganisms,
such as C. glutamicum, either directly (e.g., where overexpression or
optimization of a
fatty acid biosynthesis protein has a direct impact on the yield, production,
and/or
efficiency of production of the fatty acid from modified C glutamicum), or may
have an
indirect impact which nonetheless results in an increase of yield, production,
and/or
efficiency of production of the desired compound (e.g., where modulation of
the
metabolism of cell membrane components results in alterations in the yield,
production,
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and/or efficiency of production or the composition of the cell membrane, which
in turn
may impact the production of one or more fine chemicals). Aspects of the
invention are
further explicated below.
I. Fine Chemicals
The term 'fine chemical' is art-recognized and includes molecules produced by
an organism which have applications in various industries, such as, but not
limited to,
the pharmaceutical, agriculture, and cosmetics industries. Such compounds
include
organic acids, such as tartaric acid, itaconic acid, and diaminopimelic acid,
both
proteinogenic and non-proteinogenic amino acids, purine and pyrimidine bases,
nucleosides, and nucleotides (as described e.g. in Kuninaka, A. (1996)
Nucleotides and
related compounds, p. 561-612, in Biotechnology vol. 6, Rehm et al., eds. VCH:
Weinheim, and references contained therein), lipids, both saturated and
unsaturated fatty
acids (e.g., arachidonic acid), diols (e.g., propane diol, and butane diol),
carbohydrates
(e.g., hyaluronic acid and trehalose), aromatic compounds (e.g., aromatic
amines,
vanillin, and indigo), vitamins and cofactors (as described in Ullmann's
Encyclopedia of
Industrial Chemistry, vol. A27, "Vitamins", p. 443-613 (1996) VCH: Weinheim
and
references therein; and Ong, A.S., Niki, E. & Packer, L. (1995) "Nutrition,
Lipids,
Health, and Disease" Proceedings of the UNESCO/Confederation of Scientific and
Technological Associations in Malaysia, and the Society for Free Radical
Research -
Asia, held Sept. 1-3, 1994 at Penang, Malaysia, AOCS Press, (1995)), enzymes,
polyketides (Cane et al. (1998) Science 282: 63-68), and all other chemicals
described in
Gutcho (1983) Chemicals by Fermentation, Noyes Data Corporation, ISBN:
0818805086 and references therein. The metabolism and uses of certain of these
fine
chemicals are further explicated below.
A. Amino Acid Metabolism and Uses
Amino acids comprise the basic structural units of all proteins, and as such
are
essential for normal cellular functioning in all organisms. The term "amino
acid" is art-
recognized. The proteinogenic amino acids, of which there are 20 species,
serve as
structural units for proteins, in which they are linked by peptide bonds,
while the
nonproteinogenic amino acids (hundreds of which are known) are not normally
found in
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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
S 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
both the pharmaceutical and cosmetics industries. Threonine, tryptophan, and
D/ L-
methionine are common feed additives. (Leuchtenberger, W. (1996) Amino aids -
technical production and use, p. 466-502 in Rehm et al. (eds.) Biotechnology
vol. 6,
chapter 14a, VCH: Weinheim). Additionally, these amino acids have been found
to be
useful as precursors for the synthesis of synthetic amino acids and proteins,
such as N-
acetylcysteine, S-carboxymethyl-L-cysteine, (S)-5-hydroxytryptophan, and
others
described in Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97,
VCH:
Weinheim, 1985.
The biosynthesis of these natural amino acids in organisms capable of
producing them, such as bacteria, has been well characterized (for review of
bacterial
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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-
s 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
(3-carbon
atom to tetrahydrofolate, in a reaction catalyzed by serine
transhydroxymethylase.
Phenylalanine, and tyrosine are synthesized from the glycolytic and pentose
phosphate
pathway precursors erythrose 4-phosphate and phosphoenolpyruvate in a 9-step
biosynthetic pathway that differ only at the final two steps after synthesis
of prephenate.
Tryptophan is also produced from these two initial molecules, but its
synthesis is an 11-
step pathway. Tyrosine may also be synthesized from phenylalanine, in a
reaction
catalyzed by phenylalanine hydroxylase. Alanine, valine, and leucine are all
biosynthetic products of pyruvate, the final product of glycolysis. Aspartate
is formed
from oxaloacetate, an intermediate of the citric acid cycle. Asparagine,
methionine,
threonine, and lysine are each produced by the conversion of aspartate.
Isoleucine is
formed from threonine. A complex 9-step pathway results in the production of
histidine
from 5-phosphoribosyl-1-pyrophosphate, an activated sugar.
Amino acids in excess of the protein synthesis needs of the cell cannot be
stored,
and are instead degraded to provide intermediates for the major metabolic
pathways of
the cell (for review see Stryer, L. Biochemistry 3'd ed. Ch. 21 "Amino Acid
Degradation
and the Urea Cycle" p. 495-516 (1988)). Although the cell is able to convert
unwanted
amino acids into useful metabolic intermediates, amino acid production is
costly in
terms of energy, precursor molecules, and the enzymes necessary to synthesize
them.
Thus it is not surprising that amino acid biosynthesis is regulated by
feedback inhibition,
in which the presence of a particular amino acid serves to slow or entirely
stop its own
production (for overview of feedback mechanisms in amino acid biosynthetic
pathways,
see Stryer, L. Biochemistry, 3'd ed. Ch. 24: "Biosynthesis of Amino Acids and
Heme" p.
575-600 (1988)). Thus, the output of any particular amino acid is limited by
the amount
of that amino acid present in the cell.
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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).
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 B~) 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
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compounds collectively termed 'vitamin B6' (e.g., pyridoxine, pyridoxamine,
pyridoxa-
S'-phosphate, and the commercially used pyridoxin hydrochloride) are all
derivatives of
the common structural unit, S-hydroxy-6-methylpyridine. Pantothenate
(pantothenic
acid, (R)-(+)-N-(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)-(3-alanine) can be
produced
either by chemical synthesis or by fermentation. The final steps in
pantothenate
biosynthesis consist of the ATP-driven condensation of ~i-alanine and pantoic
acid. The
enzymes responsible for the biosynthesis steps for the conversion to pantoic
acid, to (3-
alanine and for the condensation to panthotenic acid are known. The
metabolically
active form of pantothenate is Coenzyme A, for which the biosynthesis proceeds
in 5
enzymatic steps. Pantothenate, pyridoxal-5'-phosphate, cysteine and ATP are
the
precursors of Coenzyme A. These enzymes not only catalyze the formation of
panthothante, but also the production of (R)-pantoic acid, (R)-pantolacton,
(R)-
panthenol (provitamin BS), pantetheine (and its derivatives) and coenzyme A.
Biotin biosynthesis from the precursor molecule pimeloyl-CoA in
microorganisms has been studied in detail and several of the genes involved
have been
identified. Many of the corresponding proteins have been found to also be
involved in
Fe-cluster synthesis and are members of the nifs class of proteins. Lipoic
acid is
derived from octanoic acid, and serves as a coenzyme in energy metabolism,
where it
becomes part of the pyruvate dehydrogenase complex and the a-ketoglutarate
dehydrogenase complex. The folates are a group of substances which are all
derivatives
of folic acid, which is turn is derived from L-glutamic acid, p-amino-benzoic
acid and 6-
methylpterin. The biosynthesis of folic acid and its derivatives, starting
from the
metabolism intermediates guanosine-5'-triphosphate (GTP), L-glutamic acid and
p-
amino-benzoic acid has been studied in detail in certain microorganisms.
Corrinoids (such as the cobalamines and particularly vitamin BIZ) and
porphyrines belong to a group of chemicals characterized by a tetrapyrole ring
system.
The biosynthesis of vitamin BlZ 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.
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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 B,2 is produced solely by fermentation, due to the
complexity of
its synthesis. In vitro methodologies require significant inputs of materials
and time,
often at great cost.
C. Purine, Pyrimidine, Nucleoside and Nucleotide Metabolism and Uses
Purine and pyrimidine metabolism genes and their corresponding proteins are
important targets for the therapy of tumor diseases and viral infections. The
language
"purine" or "pyrimidine" includes the nitrogenous bases which are constituents
of
nucleic acids, co-enzymes, and nucleotides. The term "nucleotide" includes the
basic
structural units of nucleic acid molecules, which are comprised of a
nitrogenous base, a
pentose sugar (in the case of RNA, the sugar is ribose; in the case of DNA,
the sugar is
D-deoxyribose), and phosphoric acid. The language "nucleoside" includes
molecules
which serve as precursors to nucleotides, but which are lacking the phosphoric
acid
moiety that nucleotides possess. By inhibiting the biosynthesis of these
molecules, or
their mobilization to form nucleic acid molecules, it is possible to inhibit
RNA and DNA
synthesis; by inhibiting this activity in a fashion targeted to cancerous
cells, the ability
of tumor cells to divide and replicate may be inhibited. Additionally, there
are
nucleotides which do not form nucleic acid molecules, but rather serve as
energy stores
(i.e., AMP) or as coenzymes (i.e., FAD and NAD).
Several publications have described the use of these chemicals for these
medical
indications, by influencing purine and/or pyrimidine metabolism (e.g.
Christopherson,
R.I. and Lyons, S.D. (1990) "Potent inhibitors of de novo pyrimidine and
purine
biosynthesis as chemotherapeutic agents." Med. Res. Reviews 10: 505-548).
Studies of
enzymes involved in purine and pyrimidine metabolism have been focused on the
development of new drugs which can be used, for example, as immunosuppressants
or
anti-proliferants (Smith, J.L., (1995) "Enzymes in nucleotide synthesis."
Curr. Opin.
Struct. Biol. S: 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,
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folates, or riboflavin), as energy carriers for the cell (e.g., ATP or GTP),
and for
chemicals themselves, commonly used as flavor enhancers (e.g., IMP or GMP) or
for
several medicinal applications (see, for example, Kuninaka, A. ( 1996)
Nucleotides and
Related Compounds in Biotechnology vol. 6, Rehm et al., eds. VCH: Weinheim, p.
561-
612). Also, enzymes involved in purine, pyrimidine, nucleoside, or nucleotide
metabolism are increasingly serving as targets against which chemicals for
crop
protection, including fungicides, herbicides and insecticides, are developed.
The metabolism of these compounds in bacteria has been characterized (for
reviews see, for example, Zalkin, H. and Dixon, J.E. (1992) "de novo purine
nucleotide
biosynthesis", in: Progress in Nucleic Acid Research and Molecular Biology,
vol. 42,
Academic Press:, p. 259-287; and Michal, G. (1999) "Nucleotides and
Nucleosides",
Chapter 8 in: Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology,
Wiley: New York). Purine metabolism has been the subject of intensive
research, and is
essential to the normal functioning of the cell. Impaired purine metabolism in
higher
animals can cause severe disease, such as gout. Purine nucleotides are
synthesized from
ribose-5-phosphate, in a series of steps through the intermediate compound
inosine-5'-
phosphate (IMP), resulting in the production of guanosine-5'-monophosphate
(GMP) or
adenosine-5'-monophosphate (AMP), from which the triphosphate forms utilized
as
nucleotides are readily formed. These compounds are also utilized as energy
stores, so
their degradation provides energy for many different biochemical processes in
the cell.
Pyrimidine biosynthesis proceeds by the formation of uridine-5'-monophosphate
(UMP)
from ribose-5-phosphate. UMP, in turn, is converted to cytidine-5'-
triphosphate (CTP).
The deoxy- forms of all of these nucleotides are produced in a one step
reduction
reaction from the diphosphate ribose form of the nucleotide to the diphosphate
deoxyribose form of the nucleotide. Upon phosphorylation, these molecules are
able to
participate in DNA synthesis.
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.
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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
S which it can be collected using methods known in the art.
II. Membrane Biosynthesis and Transmembrane Transport
Cellular membranes serve a variety of functions in a cell. First and foremost,
a
membrane differentiates the contents of a cell from the surrounding
environment, thus
giving integrity to the cell. Membranes may also serve as barriers to the
influx of
hazardous or unwanted compounds, and also to the efflux of desired compounds.
Cellular membranes are by nature impervious to the unfacilitated diffusion of
hydrophilic compounds such as proteins, water molecules and ions due to their
structure:
a bilayer of lipid molecules in which the polar head groups face outwards
(towards the
exterior and interior of the cell, respectively) and the nonpolar tails face
inwards at the
center of the bilayer, forming a hydrophobic core (for a general review of
membrane
structure and function, see Gennis, R.B. (1989) Biomembranes, Molecular
Structure and
Function, Springer: Heidelberg). This barrier enables cells to maintain a
relatively
higher concentration of desired compounds and a relatively lower concentration
of
undesired compounds than are contained within the surrounding medium, since
the
diffusion of these compounds is effectively blocked by the membrane.
However, the membrane also presents an effective barrier to the import of
desired
compounds and the export of waste molecules. To overcome this difficulty,
cellular
membranes incorporate many kinds of transporter proteins which are able to
facilitate
the transmembrane transport of different kinds of compounds. There are two
general
classes of these transport proteins: pores or channels and transporters. The
former are
integral membrane proteins, sometimes complexes of proteins, which form a
regulated
hole through the membrane. This regulation, or 'gating' is generally specific
to the
molecules to be transported by the pore or channel, rendering these
transmembrane
constructs selectively permeable to a specific class of substrates; for
example, a
potassium channel is constructed such that only ions having a like charge and
size to that
of potassium may pass through. Channel and pore proteins tend to have discrete
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hydrophobic and hydrophilic domains, such that the hydrophobic face of the
protein
may associate with the interior of the membrane while the hydrophilic face
lines the
interior of the channel, thus providing a sheltered hydrophilic environment
through
which the selected hydrophilic molecule may pass. Many such pores/channels are
known in the art, including those for potassium, calcium, sodium, and chloride
ions.
This pore and channel-mediated system of facilitated diffusion is limited to
very
small molecules, such as ions, because pores or channels large enough to
permit the
passage of whole proteins by facilitated diffusion would be unable to prevent
the
passage of smaller hydrophilic molecules as well. Transport of molecules by
this process
is sometimes termed 'facilitated diffusion' since the driving force of a
concentration
gradient is required for the transport to occur. Permeases also permit
facilitated
diffusion of larger molecules, such as glucose or other sugars, into the cell
when the
concentration of these molecules on one side of the membrane is greater than
that on the
other (also called 'uniport'). In contrast to pores or channels, these
integral membrane
1 S proteins (often having between 6-14 membrane-spanning a-helices) do not
form open
channels through the membrane, but rather bind to the target molecule at the
surface of
the membrane and then undergo a conformational shift such that the target
molecule is
released on the opposite side of the membrane.
However, cells frequently require the import or export of molecules against
the
existing concentration gradient ('active transport'), a situation in which
facilitated
diffusion cannot occur. There are two general mechanisms used by cells for
such
membrane transport: symport or antiport, and energy-coupled transport such as
that
mediated by the ABC transporters. Symport and antiport systems couple the
movement
of two different molecules across the membrane (via permeases having two
separate
binding sites for the two different molecules); in symport, both molecules are
transported in the same direction, while in antiport, one molecule is imported
while the
other is exported. This is possible energetically because one of the two
molecules
moves in accordance with a concentration gradient, and this energetically
favorable
event is permitted only upon concomitant movement of a desired compound
against the
prevailing concentration gradient. Single molecules may be transported across
the
membrane against the concentration gradient in an energy-driven process, such
as that
utilized by the ABC transporters. In this system, the transport protein
located in the
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membrane has an ATP-binding cassette; upon binding of the target molecule, the
ATP is
converted to ADP + Pi, and the resulting release of energy is used to drive
the
movement of the target molecule to the opposite face of the membrane,
facilitated by the
transporter. For more detailed descriptions of all of these transport systems,
see:
Bamberg, E. et al., (1993) "Charge transport of ion pumps on lipid bilayer
membranes",
Q. Rev. Biophys. 26: 1-25; Findlay, J.B.C. (1991) "Structure and function in
membrane
transport systems", Curr. Opin. Struct. Biol. 1:804-810; Higgins, C.F. (1992)
"ABC
transporters from microorganisms to man", Ann. Rev. Cell Biol. 8: 67-113;
Gennis, R.B.
(1989) "Pores, Channels and Transporters", in: Biomembranes, Molecular
Structure and
Function, Springer: Heidelberg, p. 270-322; and Nikaido, H. and Saier, H.
(1992)
"Transport proteins in bacteria: common themes in their design", Science 258:
936-942,
and references contained within each of these references.
The synthesis of membranes is a well-characterized process involving a number
of components, the most important of which are lipid molecules. Lipid
synthesis may
be divided into two parts: the synthesis of fatty acids and their attachment
to sn-
glycerol-3-phosphate, and the addition or modification of a polar head group.
Typical
lipids utilized in bacterial membranes include phospholipids, glycolipids,
sphingolipids,
and phosphoglycerides. Fatty acid synthesis begins with the conversion of
acetyl CoA
either to malonyl CoA by acetyl CoA carboxylase, or to acetyl-ACP by
acetyltransacylase. Following a condensation reaction, these two product
molecules
together form acetoacetyl-ACP, which is converted by a series of condensation,
reduction and dehydration reactions to yield a saturated fatty acid molecule
having a
desired chain length. The production of unsaturated fatty acids from such
molecules is
catalyzed by specific desaturases either aerobically, with the help of
molecular oxygen,
or anaerobically (for reference on fatty acid synthesis, see F.C. Neidhardt et
al. (1996)
E. coli and Salmonella. ASM Press: Washington, D.C., p. 612-636 and references
contained therein; Lengeler et al. (eds) (1999) Biology of Procaryotes.
Thieme:
Stuttgart, New York, and references contained therein; and Magnuson, K. et
al., (1993)
Microbiological Reviews 57: 522-542, and references contained therein). The
cyclopropane fatty acids (CFA) are synthesized by a specific CFA-synthase
using SAM
as a cosubstrate. Branched chain fatty acids are synthesized from branched
chain amino
acids that are deaminated to yield branched chain 2-oxo-acids (see Lengeler et
al., eds.
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(1999) Biology of Procaryotes. Thieme: Stuttgart, New York, and references
contained
therein). Another essential step in lipid synthesis is the transfer of fatty
acids onto the
polar head groups by, for example, glycerol-phosphate-acyltransferases. The
combination of various precursor molecules and biosynthetic enzymes results in
the
production of different fatty acid molecules, which has a profound effect on
the
composition of the membrane.
III. Elements and Methods of the Invention
The present invention is based, at least in part, on the discovery of novel
molecules, referred to herein as MCT nucleic acid and protein molecules, which
control
the production of cellular membranes in C. glutamicum and govern the movement
of
molecules across such membranes. In one embodiment, the MCT molecules
participate
in the metabolism of compounds necessary for the construction of cellular
membranes in
C. glutamicum, or in the transport of molecules across these membranes. In a
preferred
embodiment, the activity of the MCT molecules of the present invention to
regulate
membrane component production and membrane transport has an impact on the
production of a desired fine chemical by this organism. In a particularly
preferred
embodiment, the MCT molecules of the invention are modulated in activity, such
that
the C. glutamicum metabolic pathways which the MCT proteins of the invention
regulate are modulated in yield, production, and/or efficiency of production
and the
transport of compounds through the membranes is altered in efficiency, which
either
directly or indirectly modulates the yield, production, and/or efficiency of
production of
a desired fine chemical by C. glutamicum.
The language, "MCT protein" or "MCT polypeptide" includes proteins which
participate in the metabolism of compounds necessary for the construction of
cellular
membranes in C. glutamicum, or in the transport of molecules across these
membranes.
Examples of MCT proteins include those encoded by the MCT genes set forth in
Table 1
and by the odd-numbered SEQ ID NOs. The terms "MCT gene" or "MCT nucleic acid
sequence" include nucleic acid sequences encoding an MCT protein, which
consist of a
coding region and also corresponding untranslated 5' and 3' sequence regions.
Examples of MCT genes include those set forth in Table 1. The terms
"production" or
"productivity" are art-recognized and include the concentration of the
fermentation
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product (for example, the desired fine chemical) formed within a given time
and a given
fermentation volume (e.g., kg product per hour per liter). The term
"efficiency of
production" includes the time required for a particular level of production to
be achieved
(for example, how long it takes for the cell to attain a particular rate of
output of a fine
chemical). The term "yield" or "product/carbon yield" is art-recognized and
includes
the efficiency of the conversion of the carbon source into the product (i. e.,
fine
chemical). This is generally written as, for example, kg product per kg carbon
source.
By increasing the yield or production of the compound, the quantity of
recovered
molecules, or of useful recovered molecules of that compound in a given amount
of
culture over a given amount of time is increased. The terms "biosynthesis" or
a
"biosynthetic pathway" are art-recognized and include,the synthesis of a
compound,
preferably an organic compound, by a cell from intermediate compounds in what
may
be a multistep and highly regulated process. The terms "degradation" or a
"degradation
pathway" are art-recognized and include the breakdown of a compound,
preferably an
organic compound, by a cell to degradation products (generally speaking,
smaller or less
complex molecules) in what may be a multistep and highly regulated process.
The
language "metabolism" is art-recognized and includes the totality of the
biochemical
reactions that take place in an organism. The metabolism of a particular
compound,
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.
In another embodiment, the MCT 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. There are a number of mechanisms by which
the alteration of an MCT protein of the invention may directly affect the
yield,
production, and/or efficiency of production of a fine chemical from a C
glutamicum
strain incorporating such an altered protein. Those MCT proteins involved in
the export
of fine chemical molecules from the cell may be increased in number or
activity such
that greater quantities of these compounds are secreted to the extracellular
medium,
from which they are more readily recovered. Similarly, those MCT proteins
involved in
the import of nutrients necessary for the biosynthesis of one or more fine
chemicals
(e.g., phosphate, sulfate, nitrogen compounds, etc.) may be increased in
number or
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activity such that these precursor , cofactor, or intermediate compounds are
increased in
concentration within the cell. Further, fatty acids and lipids themselves are
desirable fine
chemicals; by optimizing the activity or increasing the number of one or more
MCT
proteins of the invention which participate in the biosynthesis of these
compounds, or by
impairing the activity of one or more MCT proteins which are involved in the
degradation of these compounds, it may be possible to increase the yield,
production,
and/or efficiency of production of fatty acid and lipid molecules from C.
glutamicum.
The mutagenesis of one or more MCT genes of the invention may also result in
MCT proteins having altered activities which indirectly impact the production
of one or
more desired fine chemicals from C .glutamicum. For example, MCT proteins of
the
invention involved in the export of waste products may be increased in number
or
activity such that the normal metabolic wastes of the cell (possibly increased
in quantity
due to the overproduction of the desired fine chemical) are efficiently
exported before
they are able to damage nucleotides and proteins within the cell (which would
decrease
the viability of the cell) or to interfere with fine chemical biosynthetic
pathways (which
would decrease the yield, production, or efficiency of production of the
desired fine
chemical). Further, the relatively large intracellular quantities of the
desired fine
chemical may in itself be toxic to the cell, so by increasing the activity or
number of
transporters able to export this compound from the cell, one may increase the
viability of
the cell in culture, in turn leading to a greater number of cells in the
culture producing
the desired fine chemical. The MCT proteins of the invention may also be
manipulated
such that the relative amounts of different lipid and fatty acid molecules are
produced.
This may have a profound effect on the lipid composition of the membrane of
the cell.
Since each type of lipid has different physical properties, an alteration in
the lipid
composition of a membrane may significantly alter membrane fluidity. Changes
in
membrane fluidity can impact the transport of molecules across the membrane,
as well
as the integrity of the cell, both of which have a profound effect on the
production of
fine chemicals from C. glutamicum in large-scale fermentative 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. glutamicum MCT DNAs and the predicted amino acid sequences of the
C.
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glutamicum MCT proteins are shown in the Sequence Listing as odd-numbered SEQ
ID
NOs and even-numbered SEQ ID NOs, respectively. Computational analyses were
performed which classified and/or identified these nucleotide sequences as
sequences
which encode proteins involved in the metabolism of cellular membrane
components or
proteins involved in the transport of compounds across such membranes.
The present invention also pertains to proteins which have an amino acid
sequence which is substantially homologous to an amino acid sequence of the
invention
(e.g., the sequence of an even-numbered SEQ ID NO of the Sequence Listing)..
As used
herein, a protein which has an amino acid sequence which is substantially
homologous
to a selected amino acid sequence is least about 50% homologous to the
selected amino
acid sequence, e.g., the entire selected amino acid sequence. A protein which
has an
amino acid sequence which is substantially homologous to a selected amino acid
sequence can also be least about 50-60%, preferably at least about 60-70%, and
more
preferably at least about 70-80%, 80-90%, or 90-95%, and most preferably at
least about
96%, 97%, 98%, 99% or more homologous to the selected amino acid sequence.
The MCT protein or a biologically active portion or fragment thereof of the
invention can participate in the metabolism of compounds necessary for the
'construction
of cellular membranes in C. glutamicum, or in the transport of molecules
across these
membranes, or 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 MCT 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 MCT-encoding nucleic acid (e.g., MCT 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 of sequence upstream from the 5' end of the coding region and at
least about
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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 MCT 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 MCT 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 polymerise 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 polymerise 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
extraction procedure of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and
DNA
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can be prepared using reverse transcriptase (e.g., Moloney MLV reverse
transcriptase,
available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase,
available
from Seikagaku America, Inc., St. Petersburg, FL). Synthetic oligonucleotide
primers
for polymerase chain reaction amplification can be designed based upon one of
the
nucleotide sequences shown in the Sequence Listing. A nucleic acid of the
invention
can be amplified using cDNA or, alternatively, genomic DNA, as a template and
appropriate oligonucleotide primers according to standard PCR amplification
techniques. The nucleic acid so amplified can be cloned into an appropriate
vector and
characterized by DNA sequence analysis. Furthermore, oligonucleotides
corresponding
to an MCT 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 MCT DNAs of the invention. This DNA comprises
sequences encoding MCT proteins (i.e., the "coding region", indicated in each
odd-
numbered SEQ ID NO: in the Sequence Listing), as well as 5' untranslated
sequences
and 3' untranslated sequences, also indicated in each odd-numbered SEQ ID NO:
in the
Sequence Listing. Alternatively, the nucleic acid molecule can comprise only
the
coding region of any of the nucleic acid sequences of the Sequence Listing.
For the purposes of this application, it will be understood that each of the
nucleic
acid and amino acid sequences set forth in the Sequence Listing has an
identifying RXA,
RXN, RXS, or RXC number having the designation "RXA", "RXN", "RXS" or "RXC"
followed by 5 digits (i.e., RXA02099, RXN03097, RXS00148, or RXC01748). Each
of
the nucleic acid sequences comprises up to three parts: a 5' upstream region,
a coding
region, and a downstream region. Each of these three regions is identified by
the same
RXA, RXN, RXS, or RXC designation to eliminate confusion. The recitation "one
of
the odd-numbered sequences in of the Sequence Listing", then, refers to any of
the
nucleic acid sequences in the Sequence Listing, which may also be
distinguished by
their differing RXA, RXN, RXS, or RXC designations. The coding region of each
of
these sequences is translated into a corresponding amino acid sequence, which
is also set
forth in the Sequence Listing, as an even-numbered SEQ ID NO: immediately
following
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the corresponding nucleic acid sequence. For example, the coding region for
RXA03097 is set forth in SEQ ID NO:1, while the amino acid sequence which it
encodes is set forth as SEQ ID N0:2. The sequences of the nucleic acid
molecules of
the invention are identified by the same RXA, RXN, RXS, or RXC designations as
the
amino acid molecules which they encode, such that they can be readily
correlated. For
example, the amino acid sequences designated RXA02099, RXN03097, RXS00148, and
RXC01748 are translations of the coding region of the nucleotide sequences of
nucleic
acid molecules RXA02099, RXN03097, RXS00148, and RXC01748, respectively. The
correspondence between the RXA, RXN, RXS, and RXC nucleotide and amino acid
sequences of the invention and their assigned SEQ ID NOs is set forth in Table
1. For
example, as set forth in Table 1, the nucleotide sequence of RXA00104 is SEQ
ID
NO:S, and the amino acid sequence of RXA00104 is SEQ ID N0:6.
Several of the genes of the invention are "F-designated genes". An F-
designated
gene includes those genes set forth in Table 1 which have an 'F' in front of
the RXA,
RXN, RXS, or RXC designation. For example, SEQ ID NO:1 l, designated, as
indicated
on Table 1, as "F RXA02581 ", is an F-designated gene, as are SEQ ID NOs: 31,
33, and
43 (designated on Table 1 as "F RXA02487", "F RXA02490", and "F RXA02809",
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 shown in of the invention is
one
which is sufficiently complementary to one of the nucleotide sequences shown
in the
Sequence Listing (e.g., the sequence of an odd-numbered SEQ ID NO:)such that
it can
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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%, SS%, 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 MCT protein. The
nucleotide
sequences determined from the cloning of the MCT genes from C. glutamicum
allows
for the generation of probes and primers designed for use in identifying
and/or cloning
MCT homologues in other cell types and organisms, as well as MCT homologues
from
other Corynebacteria or related species. The probe/primer typically comprises
substantially purified oligonucleotide. The oligonucleotide typically
comprises a region
of nucleotide sequence that hybridizes under stringent conditions to at least
about 12,
preferably about 25, more preferably about 40, 50 or 75 consecutive
nucleotides of a
sense strand of one of the nucleotide sequences of the invention (e.g., a
sequence of one
of the odd-numbered SEQ ID NOs of the Sequence Listing), an anti-sense
sequence of
one of these sequences, or naturally occurring mutants thereof. Primers based
on a
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nucleotide sequence of the invention can be used in PCR reactions to clone MCT
homologues. Probes based on the MCT 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 MCT protein, such as by measuring a level of an MCT-
encoding
nucleic acid in a sample of cells, e.g., detecting MCT mRNA levels or
determining
whether a genomic MCT gene has been mutated or deleted.
In one embodiment, the nucleic acid molecule of the invention encodes a
protein
or portion thereof which includes an amino acid sequence which is sufficiently
homologous to an amino acid sequence of the invention (e.g., a sequence of an
even-
numbered SEQ ID NO of the Sequence Listing) such that the protein or portion
thereof
maintains the ability to participate in the metabolism of compounds necessary
for the
construction of cellular membranes in C. glutamicum, or in the transport of
molecules
across these membranes. 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 able to participate
in the
metabolism of compounds necessary for the construction of cellular membranes
in C
glutamicum, or in the transport of molecules across these membranes. Protein
members
of such membrane component metabolic pathways or membrane transport systems,
as
described herein, may play a role in the production and secretion of one or
more fine
chemicals. Examples of such activities are also described herein. Thus, "the
function of
an MCT protein" contributes either directly or indirectly to the yield,
production, and/or
efficiency of production of one or more fine chemicals. Examples of MCT
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
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acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID
NO: of
the Sequence Listing).
Portions of proteins encoded by the MCT nucleic acid molecules of the
invention
are preferably biologically active portions of one of the MCT proteins. As
used herein,
the term "biologically active portion of an MCT protein" is intended to
include a
portion, e.g., a domain/motif, of an MCT protein that participates in the
metabolism of
compounds necessary for the construction of cellular membranes in C
glutamicum, or in
the transport of molecules across these membranes, or has an activity as set
forth in
Table 1. To determine whether an MCT protein or a biologically active portion
thereof
can participate in the metabolism of compounds necessary for the construction
of
cellular membranes in C. glutamicum, or in the transport of molecules across
these
membranes, 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
MCT 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 MCT protein or peptide (e.g.,
by
recombinant expression in vitro) and assessing the activity of the encoded
portion of the
MCT protein or peptide.
The invention further encompasses nucleic acid molecules that differ from one
of
the nucleotide sequences shown 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 MCT protein as that encoded by the
nucleotide
sequences of the invention. In another embodiment, an isolated nucleic acid
molecule of
the invention has a nucleotide sequence encoding a protein having an amino
acid
sequence shown in the Sequence Listing (e.g., an even-numbered SEQ ID NO:). In
a
still further embodiment, the nucleic acid molecule of the invention encodes a
full length
C. 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 38% identical to the nucleotide sequence designated RXA01420 (SEQ ID
N0:7), a
nucleotide sequence which is greater than and/or at least 43% identical to the
nucleotide
sequence designated RXA00104 (SEQ ID NO:S), and a nucleotide sequence which is
greater than and/or at least 45% identical to the nucleotide sequence
designated
RXA02173 (SEQ ID N0:25). 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 MCT 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 MCT proteins may exist within a population (e.g., the
C.
glutamicum population). Such genetic polymorphism in the MCT 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 MCT protein, preferably a C. glutamicum MCT
protein.
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Such natural variations can typically result in 1-5% variance in the
nucleotide sequence
of the MCT gene. Any and all such nucleotide variations and resulting amino
acid
polymorphisms in MCT that are the result of natural variation and that do not
alter the
functional activity of MCT 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 MCT DNA of the invention can be isolated based
on
their homology to the C. glutamicum MCT nucleic acid disclosed herein using
the C.
glutamicum DNA, or a portion thereof, as a hybridization probe according to
standard
hybridization techniques under stringent hybridization conditions.
Accordingly, in
another embodiment, an isolated nucleic acid molecule of the invention is at
least 15
nucleotides in length and hybridizes under stringent conditions to the nucleic
acid
molecule comprising a nucleotide sequence of an odd-numbered SEQ ID NO: of the
Sequence Listing. In other embodiments, the nucleic acid is at least 30, 50,
100, 250 or
more nucleotides in length. As used herein, the term "hybridizes under
stringent
conditions" is intended to describe conditions for hybridization and washing
under
which nucleotide sequences at least 60% homologous to each other typically
remain
hybridized to each other. Preferably, the conditions are such that sequences
at least
about 65%, more preferably at least about 70%, and even more preferably at
least about
75% or more homologous to each other typically remain hybridized to each
other. Such
stringent conditions are known to those of ordinary skill in the art and can
be found in
Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-
6.3.6.
A preferred, non-limiting example of stringent hybridization conditions are
hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C,
followed by
one or more washes in 0.2 X SSC, 0.1 % SDS at 50-65°C. Preferably, an
isolated
nucleic acid molecule of the invention that hybridizes under stringent
conditions to a
nucleotide sequence of the invention corresponds to a naturally-occurring
nucleic acid
molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers
to an
RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g.,
encodes a natural protein). In one embodiment, the nucleic acid encodes a
natural C.
glutamicum MCT protein.
In addition to naturally-occurring variants of the MCT sequence that may exist
in
the population, one of ordinary skill in the art will further appreciate that
changes can be
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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 MCT protein, without
altering the
functional ability of the MCT 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 MCT proteins
(e.g., an
even-numbered SEQ ID NO: of the Sequence Listing) without altering the
activity of
said MCT protein, whereas an "essential" amino acid residue is required for
MCT
protein activity. Other amino acid residues, however, (e.g., those that are
not conserved
or only semi-conserved in the domain having MCT activity) may not be essential
for
activity and thus are likely to be amenable to alteration without altering MCT
activity.
Accordingly, another aspect of the invention pertains to nucleic acid
molecules
encoding MCT proteins that contain changes in amino acid residues that are not
essential for MCT activity. Such MCT 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 MCT 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 SO% homologous to an amino
acid
sequence of the invention and is capable of participate in the metabolism of
compounds
necessary for the construction of cellular membranes in C. glutamicum, or in
the
transport of molecules across these membranes, or has one or more activities
set forth in
Table 1. Preferably, the protein encoded by the nucleic acid molecule is at
least about
50-60% homologous to the amino acid sequence of one of the odd-numbered SEQ ID
NOs of the Sequence Listing , more preferably at least about 60-70% homologous
to one
of these sequences, even more preferably at least about 70-80%, 80-90%, 90-95%
homologous to one of these sequences, and most preferably at least about 96%,
97%,
98%, or 99% homologous to one of the amino acid sequences of the invention..
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
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amino acid positions or nucleotide positions are then compared. When a
position in one
sequence (e.g., one of the amino acid sequences of the invention) is occupied
by the
same amino acid residue or nucleotide as the corresponding position in the
other
sequence (e.g., a mutant form of the amino acid sequence), then the molecules
are
homologous at that position (i.e., as used herein amino acid or nucleic acid
"homology"
is equivalent to amino acid or nucleic acid "identity"). The percent homology
between
the two sequences is a function of the number of identical positions shared by
the
sequences (i.e., % homology = # of identical positions/total # of positions x
100).
An isolated nucleic acid molecule encoding an MCT 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 MCT 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 MCT coding sequence, such as by saturation
mutagenesis, and the resultant mutants can be screened for an MCT activity
described
herein to identify mutants that retain MCT activity. Following mutagenesis of
the
nucleotide sequence of one of the odd-numbered SEQ ID NOs of the Sequence
Listing,
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the encoded protein can be expressed recombinantly and the activity of the
protein can
be determined using, for example, assays described herein (see Example 8 of
the
Exemplification).
In addition to the nucleic acid molecules encoding MCT 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 cDNA 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 MCT 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 MCT 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:S
(RXA00104) comprises nucleotides 1 to 756). In another embodiment, the
antisense
nucleic acid molecule is antisense to a "noncoding region" of the coding
strand of a
nucleotide sequence encoding MCT. 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 MCT 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 MCT mRNA, but more preferably is an oligonucleotide
which is
antisense to only a portion of the coding or noncoding region of MCT mRNA. For
example, the antisense oligonucleotide can be complementary to the region
surrounding
the translation start site of MCT 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
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using naturally occurring nucleotides or variously modified nucleotides
designed to
increase the biological stability of the molecules or to increase the physical
stability of
the duplex formed between the antisense and sense nucleic acids, e.g.,
phosphorothioate
derivatives and acridine substituted nucleotides can be used. Examples of
modified
nucleotides which can be used to generate the antisense nucleic acid include 5-
fluorouracil, S-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xanthine, 4-
acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-
thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-
galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-
methylinosine,
2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-
methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-
methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-
methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-
isopentenyladenine,
uracil-S-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-
thiocytosine, 5-
methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-
oxyacetic acid
methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-
N-2-
carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the
antisense
nucleic acid can be produced biologically using an expression vector into
which a
nucleic acid has been subcloned in an antisense orientation (i.e., RNA
transcribed from
the inserted nucleic acid will be of an antisense orientation to a target
nucleic acid of
interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically
administered
to a cell or generated in situ such that they hybridize with or bind to
cellular mRNA
and/or genomic DNA encoding an MCT 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
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sufficient intracellular concentrations of the antisense molecules, vector
constructs in
which the antisense nucleic acid molecule is placed under the control of a
strong
prokaryotic, viral, or eukaryotic promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the
invention
is an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule
forms
specific double-stranded hybrids with complementary RNA in which, contrary to
the
usual (3-units, the strands run parallel to each other (Gaultier et al. (
1987) Nucleic Acids.
Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-
0-
methylribonucleotide (moue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a
chimeric RNA-DNA analogue (moue 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 MCT mRNA transcripts to thereby inhibit translation of
MCT
mRNA. A ribozyme having specificity for an MCT-encoding nucleic acid can be
designed based upon the nucleotide sequence of an MCT DNA disclosed herein
(i.e.,
SEQ ID NO. 5 (RXA00104). 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 MCT-encoding
mRNA.
See, e.g., Cech et al. U.S. Patent No. 4,987,071 and Cech et al. U.S. Patent
No.
5,116,742. Alternatively, MCT 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, MCT gene expression can be inhibited by targeting nucleotide
sequences complementary to the regulatory region of an MCT nucleotide sequence
(e.g.,
an MCT promoter and/or enhancers) to form triple helical structures that
prevent
transcription of an MCT 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 MCT protein (or a portion
thereof). As
used herein, the term "vector" refers to a nucleic acid molecule capable of
transporting
another nucleic acid to which it has been linked. One type of vector is a
"plasmid",
which refers to a circular double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector, wherein
additional
DNA segments can be ligated into the viral genome. Certain vectors are capable
of
autonomous replication in a host cell into which they are introduced (e.g.,
bacterial
vectors having a bacterial origin of replication and episomal mammalian
vectors). Other
vectors (e.g., non-episomal mammalian vectors) are integrated into the genome
of a host
cell upon introduction into the host cell, and thereby are replicated along
with the host
genome. Moreover, certain vectors are capable of directing the expression of
genes to
which they are operatively linked. Such vectors are referred to herein as
"expression
vectors". In general, expression vectors of utility in recombinant DNA
techniques are
often in the form of plasmids. In the present specification, "plasmid" and
"vector" can
be used interchangeably as the plasmid is the most commonly used form of
vector.
However, the invention is intended to include such other forms of expression
vectors,
such as viral vectors (e.g., replication defective retroviruses, adenoviruses
and adeno-
associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of
the invention in a form suitable for,expression of the nucleic acid in a host
cell, which
means that the recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for expression,
which is
operatively linked to the nucleic acid sequence to be expressed. Within a
recombinant
expression vector, "operably linked" is intended to mean that the nucleotide
sequence of
interest is linked to the regulatory sequences) 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-, lpp-, lac-, lpp-lac-, lacIq-, T7-, TS-, T3-, gal-, trc-, ara-, SP6-,
arny, SP02, ~.-PR-
or ~, P~, which are used preferably in bacteria. Additional regulatory
sequences are, for
example, promoters from yeasts and fungi, such as ADC1, MFa, AC, P-60, CYCI,
GAPDH, TEF, rp28, ADH, promoters from plants such as CaMV/355, SSU, OCS, lib4,
usp, STLS1, 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., MCT proteins, mutant forms of MCT proteins, fusion proteins,
etc.).
The recombinant expression vectors of the invention can be designed for
expression of MCT proteins in prokaryotic or eukaryotic cells. For example,
MCT
genes can be expressed in bacterial cells such as C. glutamicum, insect cells
(using
baculovirus expression vectors), yeast and other fungal cells (see Romanos,
M.A. et al.
( 1992) "Foreign gene expression in yeast: a review", Yeast 8: 423-488; van
den Hondel,
C.A.M.J.J. et al. (1991) "Heterologous gene expression in filamentous fungi"
in: More
Gene Manipulations in Fungi, J.W. Bennet & L.L. Lasure, eds., p. 396-428:
Academic
Press: San Diego; and van den Hondel, C.A.M.J.J. & Punt, P.J. (1991) "Gene
transfer
systems and vector development for filamentous fungi, in: Applied Molecular
Genetics
of Fungi, Peberdy, J.F. et al., eds., p. 1-28, Cambridge University Press:
Cambridge),
algae and multicellular plant cells (see Schmidt, R. and Willmitzer, L. (1988)
High
efficiency Agrobacterium tumefaciens -mediated transformation of Arabidopsis
thaliana 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 terminus of the recombinant protein but
also to the
C-terminus or fused within suitable regions in the proteins. 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 pRITS (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 MCT 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 MCT protein unfused to GST can be recovered by cleavage of the
fusion
protein with thrombin.
Examples of suitable inducible non-fusion E. coli expression vectors include
pTrc (Amann et al., (1988) Gene 69:301-315) pLG338, pACYC184, pBR322, pUCl8,
pUCl9, pKC30, pRep4, pHSI, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-
III113-B1, ~,gtl l, pBdCl, and pET l 1d (Studier et al., Gene Expression
Technology:
Methods in Enzymolo~ 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 l 1d vector relies on transcription from a T7 gnl0-lac fusion promoter
mediated by
a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied
by
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host strains BL21 (DE3) or HMS 174(DE3) from a resident ~, prophage harboring
a T7
gnl gene under the transcriptional control of the lacUV 5 promoter. For
transformation
of other varieties of bacteria, appropriate vectors may be selected. For
example, the
plasmids pIJ101, pIJ364, pIJ702 and pIJ361 are known to be useful in
transforming
Streptomyces, while plasmids pUB 110, pC 194, or pBD214 are suited for
transformation
of Bacillus species. Several plasmids of use in the transfer of genetic
information into
Corynebacterium include pHM1519, pBLI, pSA77, or pAJ667 (Pouwels et al., eds.
(1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018).
One strategy to maximize recombinant protein expression is to express the
protein in a host bacteria with an impaired capacity to proteolytically cleave
the
recombinant protein (Gottesman, S., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Another
strategy is to alter the nucleic acid sequence of the nucleic acid to be
inserted into an
expression vector so that the individual codons for each amino acid are those
preferentially utilized in the bacterium chosen for expression, such as C.
glutamicum
(Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of
nucleic acid
sequences of the invention can be carried out by standard DNA synthesis
techniques.
In another embodiment, the MCT protein expression vector is a yeast expression
vector. Examples of vectors for expression in yeast S. cerevisiae include
pYepSec 1
(Baldari, et al., (1987) Embo J. 6:229-234), 2 p, pAG-1, Yep6, Yepl3,
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).
Alternatively, the MCT 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.
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(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers
(1989)
Virology 170:31-39).
In another embodiment, the MCT 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+,
pBINl9, 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) EMBO J. 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),
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pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and
mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316
and
European Application Publication No. 264,166). Developmentally-regulated
promoters
are also encompassed, for example the murine hox promoters (Kessel and Gruss
(1990)
S 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 MCT 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 MCT
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
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suitable host cells are known to one of ordinary skill in the art.
Microorganisms related
to Corynebacterium glutamicum which may be conveniently used as host cells for
the
nucleic acid and protein molecules of the invention are set forth in Table 3.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional transformation or transfection techniques. As used herein, the
terms
"transformation" and "transfection", "conjugation" and "transduction" are
intended to
refer to a variety of art-recognized techniques for introducing foreign
nucleic acid (e.g.,
linear DNA or RNA (e.g., a linearized vector or a gene construct alone without
a vector)
or nucleic acid in the form of a vector (e.g., a plasmid, phage, phasmid,
phagemid,
transposon or other DNA) into a host cell, including calcium phosphate or
calcium
chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection,
natural
competence, chemical-mediated transfer, or electroporation. Suitable methods
for
transforming or transfecting host cells can be found in Sambrook, et al.
(Molecular
Cloning: A Laboratory Manual. 2nd, ed , Cold Spring Harbor Laboratory, Cold
Spring
Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory
manuals.
For stable transfection of mammalian cells, it is known that, depending upon
the
expression vector and transfection technique used, only a small fraction of
cells may
integrate the foreign DNA into their genome. In order to identify and select
these
integrants, a gene that encodes a selectable marker (e.g., resistance to
antibiotics) is
generally introduced into the host cells along with the gene of interest.
Preferred
selectable markers include those which confer resistance to drugs, such as
6418,
hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be
introduced into a host cell on the same vector as that encoding an MCT protein
or can be
introduced on a separate vector. Cells stably transfected with the introduced
nucleic
acid can be identified by, for example, 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 MCT gene into which a deletion, addition or
substitution
has been introduced to thereby alter, e.g., functionally disrupt, the MCT
gene.
Preferably, this MCT gene is a Corynebacterium glutamicum MCT gene, but it can
be a
homologue from a related bacterium or even from a mammalian, yeast, or insect
source.
In a preferred embodiment, the vector is designed such that, upon homologous
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recombination, the endogenous MCT gene is functionally disrupted (i.e., no
longer
encodes a functional protein; also referred to as a "knock out" vector).
Alternatively,
the vector can be designed such that, upon homologous recombination, the
endogenous
MCT 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 MCT protein). In the homologous recombination vector, the altered
portion
of the MCT gene is flanked at its 5' and 3' ends by additional nucleic acid of
the MCT
gene to allow for homologous recombination to occur between the exogenous MCT
gene carried by the vector and an endogenous MCT gene in a microorganism. The
additional flanking MCT 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
1 S cells in which the introduced MCT gene has homologously recombined with
the
endogenous MCT 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 MCT gene on a vector placing it under control of
the lac
operon permits expression of the MCT gene only in the presence of IPTG. Such
regulatory systems are well known in the art.
In another embodiment, an endogenous MCT 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 MCT gene in a host cell has been altered by one or more point
mutations,
deletions, or inversions, but still encodes a functional MCT protein. In still
another
embodiment, one or more of the regulatory regions (e.g., a promoter,
repressor, or
inducer) of an MCT gene in a microorganism has been altered (e.g., by
deletion,
truncation, inversion, or point mutation) such that the expression of the MCT
gene is
modulated. One of ordinary skill in the art will appreciate that host cells
containing
more than one of the described MCT gene and protein modifications may be
readily
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produced using the methods of the invention, and are meant to be included in
the present
invention.
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in
culture, can be used to produce (i.e., express) an MCT protein. Accordingly,
the
invention further provides methods for producing MCT 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 MCT protein
has
been introduced, or into which genome has been introduced a gene encoding a
wild-type
or altered MCT protein) in a suitable medium until MCT protein is produced. In
another
embodiment, the method further comprises isolating MCT proteins from the
medium or
the host cell.
C. Isolated MCT Proteins
Another aspect of the invention pertains to isolated MCT 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 MCT 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
MCT protein
having less than about 30% (by dry weight) of non-MCT protein (also referred
to herein
as a "contaminating protein"), more preferably less than about 20% of non-MCT
protein, still more preferably less than about 10% of non-MCT protein, and
most
preferably less than about 5% non-MCT protein. When the MCT 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 MCT 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
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other chemicals" includes preparations of MCT protein having less than about
30% (by
dry weight) of chemical precursors or non-MCT chemicals, more preferably less
than
about 20% chemical precursors or non-MCT chemicals, still more preferably less
than
about 10% chemical precursors or non-MCT chemicals, and most preferably less
than
about 5% chemical precursors or non-MCT chemicals. In preferred embodiments,
isolated proteins or biologically active portions thereof lack contaminating
proteins from
the same organism from which the MCT protein is derived. Typically, such
proteins are
produced by recombinant expression of, for example, a C. glutamicum MCT
protein in a
microorganism such as C. glutamicum.
An isolated MCT protein or a portion thereof of the invention can participate
in
the metabolism of compounds necessary for the construction of cellular
membranes in
C. glutamicum, or in the transport of molecules across these membranes, 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 participate in the metabolism of compounds necessary for the
construction of
cellular membranes in C. glutamicum, or in the transport of molecules across
these
membranes. The portion of the protein is preferably a biologically active
portion as
described herein. In another preferred embodiment, an MCT 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 MCT 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 MCT 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
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portion thereof. Ranges and identity values intermediate to the above-recited
values,
(e.g., 70-90% identical or 80-95% identical) are also intended to be
encompassed by the
present invention. For example, ranges of identity values using a combination
of any of
the above values recited as upper and/or lower limits are intended to be
included. The
S preferred MCT proteins of the present invention also preferably possess at
least one of
the MCT activities described herein. For example, a preferred MCT protein of
the
present invention includes an amino acid sequence encoded by a nucleotide
sequence
which hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide
sequence of
the invention, and which can participate in the metabolism of compounds
necessary for
the construction of cellular membranes in C. glutamicum, or in the transport
of
molecules across these membranes, or which has one or more of the activities
set forth
in Table 1.
In other embodiments, the MCT 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 MCT protein is a protein which comprises an amino acid
sequence which is at least about SO%,.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 MCT 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.
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Biologically active portions of an MCT protein include peptides comprising
amino acid sequences derived from the amino acid sequence of an MCT protein,
e.g.,
the 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 MCT protein, which
include
fewer amino acids than a full length MCT protein or the full length protein
which is
homologous to an MCT protein, and exhibit at least one activity of an MCT
protein.
Typically, biologically active portions (peptides, e.g., peptides which are,
for example,
5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in
length) comprise
a domain or motif with at least one activity of an MCT 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 MCT
protein include
one or more selected domains/motifs or portions thereof having biological
activity.
MCT proteins are preferably produced by recombinant DNA techniques. For
1 S 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 MCT protein is expressed in the host cell. The MCT
protein
can then be isolated from the cells by an appropriate purification scheme
using standard
protein purification techniques. Alternative to recombinant expression, an MCT
protein,
polypeptide, or peptide can be synthesized chemically using standard peptide
synthesis
techniques. Moreover, native MCT protein can be isolated from cells (e.g.,
endothelial
cells), for example using an anti-MCT antibody, which can be produced by
standard
techniques utilizing an MCT protein or fragment thereof of this invention.
The invention also provides MCT chimeric or fusion proteins. As used herein,
an MCT "chimeric protein" or "fusion protein" comprises an MCT polypeptide
operatively linked to a non-MCT polypeptide. An "MCT polypeptide" refers to a
polypeptide having an amino acid sequence corresponding to an MCT protein,
whereas
a "non-MCT polypeptide" refers to a polypeptide having an amino acid sequence
corresponding to a protein which is not substantially homologous to the MCT
protein,
e.g., a protein which is different from the MCT 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 MCT polypeptide and the non-MCT polypeptide are
fused
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in-frame to each other. The non-MCT polypeptide can be fused to the N-terminus
or C-
terminus of the MCT polypeptide. For example, in one embodiment the fusion
protein
is a GST-MCT fusion protein in which the MCT sequences are fused to the C-
terminus
of the GST sequences. Such fusion proteins can facilitate the purification of
recombinant MCT proteins. In another embodiment, the fusion protein is an MCT
protein containing a heterologous signal sequence at its N-terminus. In
certain host cells
(e.g., mammalian host cells), expression and/or secretion of an MCT protein
can be
increased through use of a heterologous signal sequence.
Preferably, an MCT 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
MCT-
encoding nucleic acid can be cloned into such an expression vector such that
the fusion
moiety is linked in-frame to the MCT protein.
Homologues of the MCT protein can be generated by mutagenesis, e.g., discrete
point mutation or truncation of the MCT protein. As used herein, the term
"homologue"
refers to a variant form of the MCT protein which acts as an agonist or
antagonist of the
activity of the MCT protein. An agonist of the MCT protein can retain
substantially the
same, or a subset, of the biological activities of the MCT protein. An
antagonist of the
MCT protein can inhibit one or more of the activities of the naturally
occurring form of
the MCT protein, by, for example, competitively binding to a downstream or
upstream
member of the cell membrane component metabolic cascade which includes the MCT
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protein, or by binding to an MCT protein which mediates transport of compounds
across
such membranes, thereby preventing translocation from taking place.
In an alternative embodiment, homologues of the MCT protein can be identified
by screening combinatorial libraries of mutants, e.g., truncation mutants, of
the MCT
protein for MCT protein agonist or antagonist activity. In one embodiment, a
variegated
library of MCT variants is generated by combinatorial mutagenesis at the
nucleic acid
level and is encoded by a variegated gene library. A variegated library of MCT
variants
can be produced by, for example, enzymatically ligating a mixture of synthetic
oligonucleotides into gene sequences such that a degenerate set of potential
MCT
sequences is expressible as individual polypeptides, or alternatively, as a
set of larger
fusion proteins (e.g., for phage display) containing the set of MCT sequences
therein.
There are a variety of methods which can be used to produce libraries of
potential MCT
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 MCT 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 et al. (1984)
Science
198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.
In addition, libraries of fragments of the MCT protein coding can be used to
generate a variegated population of MCT fragments for screening and subsequent
selection of homologues of an MCT protein. In one embodiment, a library of
coding
sequence fragments can be generated by treating a double stranded PCR fragment
of an
MCT 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 S 1 nuclease, and ligating the resulting fragment library into
an expression
vector. By this method, an expression library can be derived which encodes N-
terminal,
C-terminal and internal fragments of various sizes of the MCT protein.
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Several techniques are known in the art for screening gene products of
combinatorial libraries made by point mutations or truncation, and for
screening cDNA
libraries for gene products having a selected property. Such techniques are
adaptable for
rapid screening of the gene libraries generated by the combinatorial
mutagenesis of
S MCT homologues. The most widely used techniques, which are amenable to high
through-put analysis, for screening large gene libraries typically include
cloning the
gene library into replicable expression vectors, transforming appropriate
cells with the
resulting library of vectors, and expressing the combinatorial genes under
conditions in
which detection of a desired activity facilitates isolation of the vector
encoding the gene
whose product was detected. Recursive ensemble mutagenesis (REM), a new
technique
which enhances the frequency of functional mutants in the libraries, can be
used in
combination with the screening assays to identify MCT homologues (Arkin and
Yourvan (1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein Engineering
6(3):327-331).
In another embodiment, cell based assays can be exploited to analyze a
variegated MCT 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 MCT
protein
regions required for function; modulation of an MCT protein activity;
modulation of the
metabolism of one or more cell membrane components; modulation of the
transmembrane transport of one or more compounds; and modulation of cellular
production of a desired compound, such as a fine chemical.
The MCT 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
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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
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
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localization of the fragment to the genome map of C. glutamicum, and, when
performed
multiple times with different enzymes, facilitates a rapid determination of
the nucleic
acid sequence to which the protein binds. Further, the nucleic acid molecules
of the
invention may be sufficiently homologous to the sequences of related species
such that
these nucleic acid molecules may serve as markers for the construction of a
genomic
map in related bacteria, such as Brevibacterium lactofermentum.
The MCT nucleic acid molecules of the invention are also useful for
evolutionary and protein structural studies. The metabolic and transport
processes in
which the molecules of the invention participate are utilized by a wide
variety of
prokaryotic and eukaryotic cells; by comparing the sequences of the nucleic
acid
molecules of the present invention to those encoding similar enzymes from
other
organisms, the evolutionary relatedness of the organisms can be assessed.
Similarly,
such a comparison permits an assessment of which regions of the sequence are
conserved and which are not, which may aid in determining those regions of the
protein
which are essential for the functioning of the enzyme. This type of
determination is of
value for protein engineering studies and may give an indication of what the
protein can
tolerate in terms of mutagenesis without losing function.
Manipulation of the MCT nucleic acid molecules of the invention may result in
the production of MCT proteins having functional differences from the wild-
type MCT
proteins. These proteins may be improved in efficiency or activity, may be
present in
greater numbers in the cell than is usual, or may be decreased in efficiency
or activity.
The invention provides methods for screening molecules which modulate the
activity of an MCT protein, either by interacting with the protein itself or a
substrate or
binding partner of the MCT protein, or by modulating the transcription or
translation of
an MCT nucleic acid molecule of the invention. In such methods, a
microorganism
expressing one or more MCT 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 MCT protein is assessed.
There are a number of mechanisms by which the alteration of an MCT protein of
the invention may directly affect the yield, production, and/or efficiency of
production
of a fine chemical from a C. glutamicum strain incorporating such an altered
protein.
Recovery of fine chemical compounds from large-scale cultures of C. glutamicum
is
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significantly improved if C. glutamicum secretes the desired compounds, since
such
compounds may be readily purified from the culture medium (as opposed to
extracted
from the mass of C. glutamicum cells). By either increasing the number or the
activity
of transporter molecules which export fine chemicals from the cell, it may be
possible to
increase the amount of the produced fine chemical which is present in the
extracellular
medium, thus permitting greater ease of harvesting and purification.
Conversely, in
order to efficiently overproduce one or more fine chemicals, increased amounts
of the
cofactors, precursor molecules, and intermediate compounds for the appropriate
biosynthetic pathways are required. Therefore, by increasing the number and/or
activity
of transporter proteins involved in the import of nutrients, such as carbon
sources (i. e. ,
sugars), nitrogen sources (i.e., amino acids, ammonium salts), phosphate, and
sulfur, it
may be possible to improve the production of a fine chemical, due to the
removal of any
nutrient supply limitations on the biosynthetic process. Further, fatty acids
and lipids
are themselves desirable. fine chemicals, so by optimizing the activity or
increasing the
number of one or more MCT proteins of the invention which participate in the
biosynthesis of these compounds, or by impairing the activity of one or more
MCT
proteins which are involved in the degradation of these compounds, it may be
possible
to increase the yield, production, and/or efficiency of production of fatty
acid and lipid
molecules from C. glutamicum.
The engineering of one or more MCT genes of the invention may also result in
MCT proteins having altered activities which indirectly impact the production
of one or
more desired fme chemicals from C .glutamicum. For example, the normal
biochemical
processes of metabolism result in the production of a variety of waste
products (e.g.,
hydrogen peroxide and other reactive oxygen species) which may actively
interfere with
these same metabolic processes (for example, peroxynitrite is known to nitrate
tyrosine
side chains, thereby inactivating some enzymes having tyrosine in the active
site
(Groves, J.T. (1999) Curr. Opin. Chem. Biol. 3(2): 226-235). While these waste
products are typically excreted, the C. glutamicum strains utilized for large-
scale
fermentative production are optimized for the overproduction of one or more
fine
chemicals, and thus may produce more waste products than is typical for a wild-
type C
glutamicum. By optimizing the activity of one or more MCT proteins of the
invention
which are involved in the export of waste molecules, it may be possible to
improve the
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viability of the cell and to maintain efficient metabolic activity. Also, the
presence of
high intracellular levels of the desired fine chemical may actually be toxic
to the cell, so
by increasing the ability of the cell to secrete these compounds, one may
improve the
viability of the cell.
Further, the MCT proteins of the invention may be manipulated such that the
relative amounts of various lipid and fatty acid molecules produced are
altered. This
may have a profound effect on the lipid composition of the membrane of the
cell. Since
each type of lipid has different physical properties, an alteration in the
lipid composition
of a membrane may significantly alter membrane fluidity. Changes in membrane
fluidity
can impact the transport of molecules across the membrane, which, as
previously
explicated, may modify the export of waste products or the produced fine
chemical or
the import of necessary nutrients. Such membrane fluidity changes may also
profoundly
affect the integrity of the cell; cells with relatively weaker membranes are
more
vulnerable in the large-scale fermentor environment to mechanical stresses
which may
damage or kill the cell. By manipulating MCT proteins involved in the
production of
fatty acids and lipids for membrane construction such that the resulting
membrane has a
membrane composition more amenable to the environmental conditions extant in
the
cultures utilized to produce fine chemicals, a greater proportion of the C.
glutamicum
cells should survive and multiply. Greater numbers of C. glutamicum cells in a
culture
should translate into greater yields, production, or efficiency of production
of the fine
chemical from the culture.
The aforementioned mutagenesis strategies for MCT proteins to result in
increased yields of a fine chemical from C. glutamicum are not meant to be
limiting;
variations on these strategies will be readily apparent to one of ordinary
skill in the art.
Using such strategies, and incorporating the mechanisms disclosed herein, the
nucleic
acid and protein molecules of the invention may be utilized to generate C.
glutamicum
or related strains of bacteria expressing mutated MCT 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
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not naturally occur in the metabolism of C. glutamicum, but which are produced
by a C.
glutamicum strain of the invention.
This invention is further illustrated by the following examples which should
not
be construed as limiting. The contents of all references, patent applications,
patents,
S published patent applications, Tables, and the sequence listing cited
throughout this
application are hereby incorporated by reference.
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CA 02380863 2001-12-20
WO 01/00805 PCT/IB00/00926
-81 -
TABLE 3: Corynebacterium and Brevibacterium Strains Which May be Used in
the Practice of the Invention
Genus : _ s ~ cies ATCC~ FERM~--NRRI::CELT NCIMB. -N.CTCDSIGIZ
.CBS
Brevibacteriumammoniagenes21054
Brevibacteriumammoniagenes19350
Brevibacteriumammoniagenes19351
Brevibacteriumammoniagenes19352
Brevibacteriumammoniagenes19353
Brevibacteriumammoniagenes19354
Brevibacteriumammoniagenes19355
Brevibacteriumammoniagenes19356
Brevibacteriumammoniagenes21055
Brevibacteriumammoniagenes21077
Brevibacteriumammoniagenes21553
Brevibacteriumammoniagenes21580
Brevibacteriumammoniagenes39101
Brevibacteriumbutanicum 21196
Brevibacteriumdivaricatum 21792 P928
Brevibacteriumflavum 21474
Brevibacteriumflavum 21129
Brevibacteriumflavum 21518
Brevibacteriumflavum B
11474
Brevibacteriumflavum B11472-
Brevibacteriumflavum 21127
Brevibacteriumflavum 21128
Brevibacteriumflavum 21427
Brevibacteriumflavum 21475
Brevibacteriumflavum 21517
Brevibacteriumflavum 21528
Brevibacteriumflavum 21529
Brevibacteriumflavum B11477
Brevibacteriumflavum B11478
Brevibacteriumflavum 21127
Brevibacteriumflavum B
11474
Brevibacteriumhealii 15527
Brevibacteriumketoglutamicum21004
Brevibacteriumketoglutamicum21089
Brevibacteriumketosoreductum21914
Brevibacteriumlactofermentum 70
Brevibacteriumlactofermentum 74
Brevibacteriumlactofermentum 77
Brevibacteriumlactofermentum21798
Brevibacteriumlactofermentum21799
Brevibacteriumlactofermentum21800
Brevibacteriumlactofermentum21801
Brevibacteriumlactofermentum B
11470
BrevibacteriumlactofermentumI I B
I I 11471
CA 02380863 2001-12-20
WO 01/00805 PCT/IB00/00926
-82-
Genus s'~ eles- ~ ~ATCC.FERMNRRI:.CELT;;NCIMB: CBS NCTG~
w.'yA,'~ .~~a8.. ;'..H ~ ~- k~ ry4...,Y.-~'w:- H,.LA''~SMZ=
!: ~, .' u. : SKe,J, ..,u~ ",:Y" uJ'~.,,.p",S
~ . yi'
,
,...
Brevibacteriumlactofermentum21086
Brevibacteriumlactofermentum21420
Brevibacteriumlactofermentum21086
Brevibacteriumlactofermentum31269
Brevibacteriumlinens 9174
Brevibacteriumlinens 19391
Brevibacteriumlinens 8377
Brevibacteriumparaffinolyticum 11160
Brevibacteriumspec. 717.73
Brevibacteriumspec. 717.73
Brevibacteriumspec. 14604
Brevibacteriumspec. 21860
Brevibacteriumspec. 21864
Brevibacteriumspec. 21865
Brevibacteriumspec. 21866
Brevibacteriumspec. 19240
Corynebacteriumacetoacidophilum21476
Corynebacteriumacetoacidophilum13870
Corynebacteriumacetoglutamicum B11473
Corynebacteriumacetoglutamicum B11475
Corynebacteriumacetoglutamicum15806
Corynebacteriumacetoglutamicum21491
Corynebacteriumacetoglutamicum31270
Corynebacteriumacetophilum B3671
Corynebacteriumammoniagenes6872 2399
Corynebacteriumammoniagenes15511
Corynebacteriumfujiokense 21496
Corynebacteriumglutamicum 14067
Corynebacteriumglutamicum 39137
Corynebacteriumglutamicum 21254
Corynebacteriumglutamicum 21255
Corynebacteriumglutamicum 31830
Corynebacteriumglutamicum 13032
Corynebacteriumglutamicum 14305
Corynebacteriumglutamicum 15455
Corynebacteriumglutamicum 13058
Corynebacteriumglutamicum 13059
Corynebacteriumglutamicum 13060
Corynebacteriumglutamicum 21492
Corynebacteriumglutamicum 21513
Corynebacteriumglutamicum 21526
Corynebacteriumglutamicum 21543
Corynebacteriumglutamicum 13287
Corynebacteriumglutamicum 21851
Corynebacteriumglutamicum 21253
Corynebacteriumglutamicum 21514
Corynebacteriumglutamicum 21516
ICorynebacteriumglutamicum 21299
I
CA 02380863 2001-12-20
WO 01/00805 PCT/IB00/00926
-83-
Genus ~. species ' rITCCFERM NRRL CECT''NCI1VTB: CBS:NCTCDSMZ
, ,
Corynebacteriumglutamicum 21300
Corynebacteriumglutamicum 39684
Corynebacteriumglutamicum 21488
Corynebacteriumglutamicum 21649
Corynebacteriumglutamicum 21650
Corynebacteriumglutamicum 19223
Corynebacteriumglutamicum 13869
Corynebacteriumglutamicum 21157
Corynebacteriumglutamicum 21158
Corynebacteriumglutamicum 21159
Corynebacteriumglutamicum 21355
Corynebacteriumglutamicum 31808
Corynebacteriumglutamicum 21674
Corynebacteriumglutamicum 21562
Corynebacteriumglutamicum 21563
Corynebacteriumglutamicum 21564
Corynebacteriumglutamicum 21565
Corynebacteriumglutamicum 21566
Corynebacteriumglutamicum 21567
Corynebacteriumglutamicum 21568
Corynebacteriumglutamicum 21569
Corynebacteriumglutamicum 21570
Corynebacteriumglutamicum 21571
Corynebacteriumglutamicum 21572
Corynebacteriumglutamicum 21573
Corynebacteriumglutamicum 21579
Corynebacteriumglutamicum 19049
Corynebacteriumglutamicum 19050
Corynebacteriumglutamicum 19051
Corynebacteriumglutamicum 19052
Corynebacteriumglutamicum 19053
Corynebacteriumglutamicum 19054
Corynebacteriumglutamicum 19055
Corynebacteriumglutamicum 19056
Corynebacteriumglutamicum 19057
Corynebacteriumglutamicum 19058
Corynebacteriumglutamicum 19059
Corynebacteriumglutamicum 19060
Corynebacteriumglutamicum 19185
Corynebacteriumglutamicum 13286
Corynebacteriumglutamicum 21515
Corynebacteriumglutamicum 21527
Corynebacteriumglutamicum 21544
Corynebacteriumglutamicum 21492
Corynebacteriumglutamicum B8183
Corynebacteriumglutamicum B8182
Corynebacteriumglutamicum B
12416
Corynebacteriumglutamicum B12417
CA 02380863 2001-12-20
WO 01/00805 PCT/IB00/00926
-84-
Genus s- ecies ~ ATGC FERM '~NRRLCELT-NC_IIVIB., NCTCDSMZ
' -~ -~m = ".._~,__..x~.~.:~-:CBS
.:F . ~...w. _.~_
.
Corynebacteriumglutamicum B12418
Corynebacteriumglutamicum B11476
Corynebacteriumglutamicum 21608
Corynebacteriumlilium P973
Corynebacteriumnitrilophilus21419 11594
Corynebacteriumspec. P4445
Corynebacteriumspec. P4446
Corynebacteriumspec. 31088
Corynebacteriumspec. 31089
Corynebacteriumspec. 31090
Corynebacteriumspec. 31090
Corynebacteriumspec. 31090
Corynebacteriumspec. 15954 20145
Corynebacteriumspec. 21857
Corynebacteriumspec. 21862
Corynebacteriumspec. 21863
ATCC: American Type Culture Collection, Rockville, MD, USA
FERM: Fermentation Research Institute, Chiba, Japan
NRRL: ARS Culture Collection, Northern Regional Research Laboratory, Peoria,
IL, USA
CECT: Coleccion Espanola de Cultivos Tipo, Valencia, Spain
NCIMB: National Collection of Industrial and Marine Bacteria Ltd., Aberdeen,
UK
CBS: Centraalbureau voor Schimmelcultures, Baarn, NL
NCTC: National Collection of Type Cultures, London, UK
DSMZ: Deutsche Sammlung von Mikroorganismen and Zellkulturen, Braunschweig,
Germany
For reference see Sugawara, H. et al. (1993) World directory of collections of
cultures of
microorganisms: Bacteria, fungi and yeasts (4'~' edn), World federation for
culture collections world
data center on microorganisms, Saimata, Japen.
CA 023808632001-12-20
WO 1/00805 85 PCT/IB00/00926
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CA 02380863 2001-12-20
WO 01/00805 PCT/IB00/00926
- 107 -
Exemplification
Example 1: Preparation of total genomic DNA of Corynebacterium glutamicum
ATCC 13032
S A culture of Corynebacterium glutamicum (ATCC 13032) was grown overnight
at 30°C with vigorous shaking in BHI medium (Difco). The cells were
harvested by
centrifugation, the supernatant was discarded and the cells were resuspended
in 5 ml
buffer-I (5% of the original volume of the culture - all indicated volumes
have been
calculated for 100 ml of culture volume). Composition of buffer-I: 140.34 g/1
sucrose,
2.46 g/1 MgS04 x 7H20, 10 m1/1 KHZPOQ solution (100 g/1, adjusted to pH 6.7
with
KOH), 50 m1/1 M12 concentrate (10 g/1 (NHQ)ZS04, 1 g/1 NaCI, 2 g/1 MgS04 x
7H20,
0.2 g/1 CaClz, 0.5 g/1 yeast extract (Difco), 10 m1/1 trace-elements-mix (200
mg/1 FeS04
x HZO, 10 mg/1 ZnS04 x 7 HZO, 3 mg/1 MnCl2 x 4 HzO, 30 mg/1 H,BO, 20 mg/1
CoCIZ x
6 H20, 1 mg/1 NiCl2 x 6 H20, 3 mg/1 NazMo04 x 2 H,O, 500 mg/1 complexing agent
(EDTA or critic acid), 100 m1/1 vitamins-mix (0.2 mg/1 biotin, 0.2 mg/1 folic
acid, 20
mg/1 p-amino benzoic acid, 20 mg/1 riboflavin, 40 mg/1 ca-panthothenate, 140
mg/1
nicotinic acid, 40 mg/1 pyridoxole hydrochloride, 200 mg/1 myo-inositol).
Lysozyme
was added to the suspension to a final concentration of 2.5 mg/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, l 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.l8 h at 37°C. The DNA was
purified by
extraction with phenol, phenol-chloroform-isoamylalcohol and chloroform-
isoamylalcohol using standard procedures. Then, the DNA was precipitated by
adding
1/50 volume of 3 M sodium acetate and 2 volumes of ethanol, followed by a 30
min
incubation at -20°C and a 30 min centrifugation at 12,000 rpm in a high
speed centrifuge
using a SS34 rotor (Sorvall). The DNA was dissolved in 1 ml TE-buffer
containing 20
~g/ml RNaseA and dialysed at 4°C against 1000 ml TE-buffer for at least
3 hours.
During this time, the buffer was exchanged 3 times. To aliquots of 0.4 ml of
the
dialysed DNA solution, 0.4 ml of 2 M LiCI and 0.8 ml of ethanol are added.
After a 30
CA 02380863 2001-12-20
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min incubation at -20°C, the DNA was collected by centrifugation
(13,000 rpm, Biofuge
Fresco, Heraeus, Hanau, Germany). The DNA pellet was dissolved in TE-buffer.
DNA
prepared by this procedure could be used for all purposes, including southern
blotting or
construction of genomic libraries.
Example 2: Construction of genomic libraries in Escherichia coli of
Corynebacterium
glutamicum ATCC13032.
Using DNA prepared as described in Example 1, cosmid and plasmid libraries
were
constructed according to known and well established methods (see e. g.,
Sambrook, J. et al.
(1989) "Molecular Cloning : A Laboratory Manual", Cold Spring Harbor
Laboratory Press,
or Ausubel, F.M. et al. (1994) "Current Protocols in Molecular Biology", John
Wiley &
Sons.)
Any plasmid or cosmid could be used. Of particular use were the plasmids
pBR322
(Sutcliffe, J.G. (1979) Proc. Natl. Acad. Sci. USA, 75:3737-3741); pACYC177
(Change &
Cohen (1978) J. Bacteriol 134:1141-1156), plasmids of the pBS series (pBSSK+,
pBSSK- and
others; Stratagene, LaJolla, USA), or cosmids as SuperCosl (Stratagene,
LaJolla, USA) or
Lorist6 (Gibson, T.J., Rosenthal A. and Waterson, R.H. (1987) Gene 53:283-286.
Gene libraries
specifically for use in C. glutamicum may be constructed using plasmid pSL 109
(Lee, H.-S. and
A. J. Sinskey (1994) J. Microbiol. Biotechnol. 4: 256-263).
Example 3: DNA Sequencing and Computational Functional Analysis
Genomic libraries as described in Example 2 were used for DNA sequencing
according to standard methods, in particular by the chain termination method
using
ABI377 sequencing machines (see e.g., Fleischman, R.D. et al. (1995) "Whole-
genome
Random Sequencing and Assembly of Haemophilus Influenzae Rd., Science, 269:496-
512). Sequencing primers with the following nucleotide sequences were used: 5'-
GGAAACAGTATGACCATG-3' or S'-GTAAAACGACGGCCAGT-3'.
Example 4: In vivo Mutagenesis
In vivo mutagenesis of Corynebacterium glutamicum can be performed by passage
of
plasmid (or other vector) DNA through E. coli or other microorganisms (e.g.
Bacillus spp. or
yeasts such as Saccharomyces cerevisiae) which are impaired in their
capabilities to maintain
CA 02380863 2001-12-20
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- 109 -
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 al. (1994) "Current
Protocols in
Molecular Biology", John Wiley & Sons) to which a origin or replication for
and a
suitable marker from Corynebacterium glutamicum is added. Such origins of
replication
are preferably taken from endogenous plasmids isolated from Corynebacterium
and
Brevibacterium species. Of particular use as transformation markers for these
species are
genes for kanamycin resistance (such as those derived from the Tn5 or Tn903
transposons) or chloramphenicol (Winnacker, E.L. (1987) "From Genes to Clones -
Introduction to Gene Technology, VCH, Weinheim). There are numerous examples
in the
literature of the construction of a wide variety of shuttle vectors which
replicate in both E.
coli and C. glutamicum, and which can be used for several purposes, including
gene over-
expression (for reference, see e.g., Yoshihama, M. et al. (1985) J. Bacteriol.
162:591-597,
Martin J.F. et al. (1987) Biotechnology, 5:137-146 and Eikmanns, B.J. et al.
(1991) Gene,
102:93-98).
Using standard methods, it is possible to clone a gene of interest into one of
the shuttle
vectors described above and to introduce such a hybrid vectors into strains of
Corynebacterium glutamicum. Transformation of C. glutamicum can be achieved by
protoplast transformation (Kastsumata, R. et al. (1984) J. Bacteriol. 159306-
311),
electroporation (Liebl, E. et al. (1989) FEMSMicrobiol. Letters, 53:399-303)
and in
cases where special vectors are used, also by conjugation (as described e.g.
in Schafer,
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A et al. (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 pSL109 (Lee, H.-S. and A.
J.
Sinskey (1994) J. Microbiol. Biotechnol. 4: 256-263).
Aside from the use of replicative plasmids, gene overexpression can also be
achieved by integration into the genome. Genomic integration in C. glutamicum
or other
1 S 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 mIRNA available for translation
to the gene
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product) is to perform a Northern blot (for reference see, for example,
Ausubel et al.
( 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 Western blot, may be employed (see, for
example, Ausubel
et al. (1988) Current Protocols in Molecular Biology, .Whey: New York). In
this process,
total cellular proteins are extracted, separated by gel electrophoresis,
transferred to a
matrix such as nitrocellulose, and incubated with a probe, such as an
antibody, which
specifically binds to the desired protein. This probe is generally tagged with
a
chemiluminescent or colorimetric label which may be readily detected. The
presence and
quantity of label observed indicates the presence and quantity of the desired
mutant
protein present in the cell.
Example 7: Growth of Genetically Modified Corynebacterium glutamicum - Media
and Culture Conditions
Genetically modified Corynebacteria are cultured in synthetic or natural
growth
media. A number of different growth media for Corynebacteria are both well-
known and
readily available (Lieb et al. (1989) Appl. Microbiol. Biotechnol., 32:205-
210; von der
Osten et al. (1998) Biotechnology Letters, 11:11-16; Patent DE 4,120,867;
Liebl (1992)
"The Genus Corynebacterium, in: The Procaryotes, Volume II, Balows, A. et al.,
eds.
Springer-Verlag). These media consist of one or more carbon sources, nitrogen
sources,
inorganic salts, vitamins and trace elements. Preferred carbon sources are
sugars, such as
mono-, di-, or polysaccharides. For example, glucose, fructose, mannose,
galactose,
ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or
cellulose serve as
very good carbon sources. It is also possible to supply sugar to the media via
complex
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compounds such as molasses or other by-products from sugar refinement. It can
also be
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 NH4Cl or (NHQ)zS04, NH40H, nitrates, urea, amino acids
or
complex nitrogen sources like corn steep liquor, soy bean flour, soy bean
protein, yeast
extract, meat extract and others.
Inorganic salt compounds which may be included in the media include the
chloride-, phosphorous- or sulfate- salts of calcium, magnesium, sodium,
cobalt,
molybdenum, potassium, manganese, zinc, copper and iron. Chelating compounds
can be
added to the medium to keep the metal ions in solution. Particularly useful
chelating
compounds include dihydroxyphenols, like catechol or protocatechuate, or
organic acids,
such as citric acid. It is typical for the media to also contain other growth
factors, such as
vitamins or growth promoters, examples of which include biotin, riboflavin,
thiamin, folic
acid, nicotinic acid, pantothenate and pyridoxin. Growth factors and salts
frequently
originate from complex media components such as yeast extract, molasses, corn
steep
liquor and others. The exact composition of the media compounds depends
strongly on
the immediate experiment and is individually decided for each specific case.
Information
about media optimization is available in the textbook "Applied Microbiol.
Physiology, A
Practical Approach (eds. P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-
73, ISBN 0
19 963577 3). It is also possible to select growth media from commercial
suppliers, like
standard 1 (Merck) or BHI (grain heart infusion, DIFCO) or others.
All medium components are sterilized, either by heat (20 minutes at 1.5 bar
and
121 °C) or by sterile filtration. The components can either be
sterilized together or, if
necessary, separately. All media components can be present at the beginning of
growth,
or they can optionally be added continuously or batchwise.
Culture conditions are defined separately for each experiment. The temperature
should be in a range between 15°C and 45°C. The temperature can
be kept constant or can
be altered during the experiment. The pH of the medium should be in the range
of 5 to
8.5, preferably around 7.0, and can be maintained by the addition of buffers
to the media.
An exemplary buffer for this purpose is a potassium phosphate buffer.
Synthetic buffers
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such as MOPS, HEPES, ACES and others can alternatively or simultaneously be
used. It
is also possible to maintain a constant culture pH through the addition of
NaOH or
NHQOH during growth. If complex medium components such as yeast extract are
utilized,
the necessity for additional buffers may be reduced, due to the fact that many
complex
compounds have high buffer capacities. If a fermentor is utilized for
culturing the micro-
organisms, the pH can also be controlled using gaseous ammonia.
The incubation time is usually in a range from several hours to several days.
This
time is selected in order to permit the maximal amount of product to
accumulate in the
broth. The disclosed growth experiments can be carried out in a variety of
vessels, such as
microtiter plates, glass tubes, glass flasks or glass or metal fermentors of
different sizes.
For screening a large number of clones, the microorganisms should be cultured
in
microtiter plates, glass tubes or shake flasks, either with or without
baffles. Preferably
100 ml shake flasks are used, filled with 10% (by volume) of the required
growth
medium. The flasks should be shaken on a rotary shaker (amplitude 25 mm) using
a
speed-range of 100 - 300 rpm. Evaporation losses can be diminished by the
maintenance
of a humid atmosphere; alternatively, a mathematical correction for
evaporation losses
should be performed.
If genetically modified clones are tested, an unmodified control clone or a
control
clone containing the basic plasmid without any insert should also be tested.
The medium
is inoculated to an OD6oo of O.5 - 1.5 using cells grown on agar plates, such
as CM plates
(10 g/1 glucose, 2,5 g/1 NaCI, 2 g/1 urea, 10 g/1 polypeptone, 5 g/1 yeast
extract, 5 g/1 meat
extract, 22 g/1 NaCI, 2 g/1 urea, 10 g/1 polypeptone, 5 g/1 yeast extract, 5
g/1 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 concerning structure, kinetics,
principles, methods,
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applications and examples for the determination of many enzyme activities may
be
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, 3'd ed. Academic Press:
New
York; Bisswanger, H., (1994) Enzymkinetik, 2"d ed. VCH: Weinheim (ISBN
3527300325); Bergmeyer, H.U., Bergmeyer, J., Gra131, M., eds. (1983-1986)
Methods of
Enzymatic Analysis, 3'd ed., vol. I-XII, Verlag Chemie: Weinheim; and
Ullmann's
Encyclopedia of Industrial Chemistry (1987) vol. A9, "Enzymes". VCH: Weinheim,
p.
352-363.
The activity of proteins which bind to DNA can be measured by several well-
established methods, such as DNA band-shift assays (also called gel
retardation assays).
The effect of such proteins on the expression of other molecules can be
measured using
reporter gene assays (such as that described in Kolmar, H. et al. (1995) EMBO
J. 14:
3895-3904 and references cited therein). Reporter gene test systems are well
known and
established for applications in both pro- and eukaryotic cells, using enzymes
such as
beta-galactosidase, green fluorescent protein, and several others.
The determination of activity of membrane-transport proteins can be performed
according to techniques such as those described in Gennis, R.B. (1989) "Pores,
Channels and Transporters", in Biomembranes, Molecular Structure and Function,
Springer: Heidelberg, p. 85-137; 199-234; and 270-322.
Example 9: Analysis of Impact of Mutant Protein on the Production of the
Desired
Product
The effect of the genetic modification in C. glutamicum on production of a
desired compound (such as an amino acid) can be assessed by growing the
modified
microorganism under suitable conditions (such as those described above) and
analyzing
the medium and/or the cellular component for increased production of the
desired
product (i.e., an amino acid). Such analysis techniques are well known to one
of
ordinary skill in the art, and include spectroscopy, thin layer
chromatography, staining
methods of various kinds, enzymatic and microbiological methods, and
analytical
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chromatography such as high performance liquid chromatography (see, for
example,
Ullman, Encyclopedia of Industrial Chemistry, vol. A2, p. 89-90 and p. 443-
613, VCH:
Weinheim (1985); Fallon, A. et al., (1987) "Applications of HPLC in
Biochemistry" in:
Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17; Rehm et
al.
(1993) Biotechnology, vol. 3, Chapter III: "Product recovery and
purification", page
469-714, VCH: Weinheim; Belter, P.A. et al. (1988) Bioseparations: downstream
processing for biotechnology, John Wiley and Sons; Kennedy, J.F. and Cabral,
J.M.S.
(1992) Recovery processes for biological materials, John Wiley and Sons;
Shaeiwitz,
J.A. and Henry, J.D. (1988) Biochemical separations, in: Ulmann's Encyclopedia
of
Industrial Chemistry, vol. B3, Chapter 11, page 1-27, VCH: Weinheim; and
Dechow,
F.J. (1989) Separation and purification techniques in biotechnology, Noyes
Publications.)
In addition to the measurement of the final product of fermentation, it is
also
possible to analyze other components of the metabolic pathways utilized for
the
1 S production of the desired compound, such as intermediates and side-
products, to ,
determine the overall efficiency of production of the compound. Analysis
methods
include measurements of nutrient levels in the medium (e.g., sugars,
hydrocarbons,
nitrogen sources, phosphate, and other ions), measurements of biomass
composition and
growth, analysis of the production of common metabolites of biosynthetic
pathways, and
measurement of gasses produced during fermentation. Standard methods for these
measurements are outlined in Applied Microbial Physiology, A Practical
Approach,
P.M. Rhodes and P.F. Stanbury, eds., IRL Press, p. 103-129; 131-163; and 165-
192
(ISBN: 0199635773) and references cited therein.
Example 10: Purification of the Desired Product from C. glutamicum Culture
Recovery of the desired product from the C. glutamicum cells or supernatant of
the above-described culture can be performed by various methods well known in
the art.
If the desired product is not secreted from the cells, the cells can be
harvested from the
culture by low-speed centrifugation, the cells can be lysed by standard
techniques, such
as mechanical force or sonication. The cellular debris is removed by
centrifugation, and
the supernatant fraction containing the soluble proteins is retained for
further
purification of the desired compound. If the product is secreted from the C
glutamicum
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cells, then the cells are removed from the culture by low-speed
centrifugation, and the
supernate fraction is retained for further purification.
The supernatant fraction from either purification method is subjected to
chromatography with a suitable resin, in which the desired molecule is either
retained on
a chromatography resin while many of the impurities in the sample are not, or
where the
impurities are retained by the resin while the sample is not. Such
chromatography steps
may be repeated as necessary, using the same or different chromatography
resins. One
of ordinary skill in the art would be well-versed in the selection of
appropriate
chromatography resins and in their most efficacious application for a
particular molecule
to be purified. The purified product may be concentrated by filtration or
ultrafiltration,
and stored at a temperature at which the stability of the product is
maximized.
There are a wide array of purification methods known to the art and the
preceding method of purification is not meant to be limiting. Such
purification
techniques are described, for example, in Bailey, J.E. & Ollis, D.F.
Biochemical
Engineering Fundamentals, McGraw-Hill: New York (1986).
The identity and purity of the isolated compounds may be assessed by
techniques
standard in the art. These include high-performance liquid chromatography
(HPLC),
spectroscopic methods, staining methods, thin layer chromatography, NIRS,
enzymatic
assay, or microbiologically. Such analysis methods are reviewed in: Patek et
al. (1994)
Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya
11: 27-
32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's
Encyclopedia
of Industrial Chemistry, (1996) vol. A27, VCH: Weinheim, p. 89-90, p. 521-540,
p. 540-
547, p. 559-566, 575-581 and p. 581-587; Michal, G. (1999) Biochemical
Pathways: An
Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A.
et al.
(1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in
Biochemistry and Molecular Biology, vol. 17.
Example 11: Analysis of the Gene Sequences of the Invention
The comparison of sequences and determination of percent homology between
two sequences are art-known techniques, and can be accomplished using a
mathematical
algorithm, such as the algorithm of Karlin and Altschul (1990) Proc. Natl.
Acad. Sci.
USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.
Sci. USA
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90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST
programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
BLAST
nucleotide searches can be performed with the NBLAST program, score = 100,
wordlength = 12 to obtain nucleotide sequences homologous to MCT 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 MCT 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.NA.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 SO
and a
length weight of 3.
A comparative analysis of the gene sequences of the invention with those
present
in Genbank has been performed using techniques known in the art (see, e.g.,
Bexevanis
and Ouellette, eds. (1998) Bioinformatics: A Practical Guide to the Analysis
of Genes
and Proteins. John Wiley and Sons: New York). The gene sequences of the
invention
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were compared to genes present in Genbank in a three-step process. In a first
step, a
BLASTN analysis (e.g., a local alignment analysis) was performed for each of
the
sequences of the invention against the nucleotide sequences present in
Genbank, and the
top 500 hits were retained for further analysis. A subsequent FASTA search
(e.g., a
combined local and global alignment analysis, in which limited regions of the
sequences
are aligned) was performed on these 500 hits. Each gene sequence of the
invention was
subsequently globally aligned to each of the top three FASTA hits, using the
GAP
program in the GCG software package (using standard parameters). In order to
obtain
correct results, the length of the sequences extracted from Genbank were
adjusted to the
length of the query sequences by methods well-known in the art. The results of
this
analysis are set forth in Table 4. The resulting data is identical to that
which would have
been obtained had a GAP (global) analysis alone been performed on each of the
genes of
the invention in comparison with each of the references in Genbank, but
required
significantly reduced computational time as compared to such a database-wide
GAP
(global) analysis. Sequences of the invention for which no alignments above
the cutoff
values were obtained are indicated on Table 4 by the absence of alignment
information.
It will further be understood by one of ordinary skill in the art that the GAP
alignment
homology percentages set forth in Table 4 under the heading "% homology (GAP)"
are
listed in the European numerical format, wherein a ',' represents a decimal
point. For
example, a value of "40,345" in this column represents "40.345%".
Example 12: Construction and Operation of DNA Microarrays
The sequences of the invention may additionally be used in the construction
and
application of DNA microarrays (the design, methodology, and uses of DNA
arrays are
well known in the art, and are described, for example, in Schena, M. et al.
(1995)
Science 270: 467-470; Wodicka, L. et al. (1997) Nature Biotechnology 15: 1359-
1367;
DeSaizieu, A. et al. (1998) Nature Biotechnology 16: 45-48; and DeRisi, J.L.
et al.
(1997) Science 278: 680-686).
DNA microarrays are solid or flexible supports consisting of nitrocellulose,
nylon, glass, silicone, or other materials. Nucleic acid molecules may be
attached to the
surface in an ordered manner. After appropriate labeling, other nucleic acids
or nucleic
acid mixtures can be hybridized to the immobilized nucleic acid molecules, and
the label
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may be used to monitor and measure the individual signal intensities of the
hybridized
molecules at defined regions. This methodology allows the simultaneous
quantification
of the relative or absolute amount of all or selected nucleic acids in the
applied nucleic
acid sample or mixture. DNA microarrays, therefore, permit an analysis of the
expression of multiple (as many as 6800 or more) nucleic acids in parallel
(see, e.g.,
Schena, M. (1996) BioEssays 18(5): 427-431).
The sequences of the invention may be used to design oligonucleotide primers
which are able to amplify defined regions of one or more C. glutamicum genes
by a
nucleic acid amplification reaction such as the polymerise chain reaction. The
choice
and design of the S' or 3' oligonucleotide primers or of appropriate linkers
allows the
covalent attachment of the resulting PCR products to the surface of a support
medium
described above (and also described, for example, Schena, M. et al. (1995)
Science 270:
467-470).
Nucleic acid microarrays may also be constructed by in situ oligonucleotide
synthesis as described by Wodicka, L. et al. (1997) Nature Biotechnology 15:
1359-
1367. By photolithographic methods, precisely defined regions of the matrix
are
exposed to light. Protective groups which are photolabile are thereby
activated and
undergo nucleotide addition, whereas regions that are masked from light do not
undergo
any modification. Subsequent cycles of protection and light activation permit
the
synthesis of different oligonucleotides at defined positions. Small, defined
regions of
the genes of the invention may be synthesized on microarrays by solid phase
oligonucleotide synthesis.
The nucleic acid molecules of the invention present in a sample or mixture of
nucleotides may be hybridized to the microarrays. These nucleic acid molecules
can be
labeled according to standard methods. In brief, nucleic acid molecules (e.g.,
mRNA
molecules or DNA molecules) are labeled by the incorporation of isotopically
or
fluorescently labeled nucleotides, e.g., during reverse transcription or DNA
synthesis.
Hybridization of labeled nucleic acids to microarrays is described (e.g., in
Schena, M. et
al. (1995) supra; Wodicka, L. et al. (1997), supra; and DeSaizieu A. et al.
(1998),
supra). The detection and quantification of the hybridized molecule are
tailored to the
specific incorporated label. Radioactive labels can be detected, for example,
as
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described in Schena, M. et al. (1995) supra) and fluorescent labels may be
detected, for
example, by the method of Shalon et al. (1996) Genome Research 6: 639-645).
The application of the sequences of the invention to DNA microarray
technology, as described above, permits comparative analyses of different
strains of C.
glutamicum or other Corynebacteria. For example, studies of inter-strain
variations
based on individual transcript profiles and the identification of genes that
are important
for specific and/or desired strain properties such as pathogenicity,
productivity and
stress tolerance are facilitated by nucleic acid array methodologies. Also,
comparisons
of the profile of expression of genes of the invention during the course of a
fermentation
reaction are possible using nucleic acid array technology.
Example 13: Analysis of the Dynamics of Cellular Protein Populations
(Proteomics)
The genes, compositions, and methods of the invention may be applied to study
the interactions and dynamics of populations of proteins, termed 'proteomics'.
Protein
populations of interest include, but are not limited to, the total protein
population of C.
glutamicum (e.g., in comparison with the protein populations of other
organisms), those
proteins which are active under specific environmental or metabolic conditions
(e.g.,
during fermentation, at high or low temperature, or at high or low pH), or
those proteins
which are active during specific phases of growth and development.
Protein populations can be analyzed by various well-known techniques, such as
gel electrophoresis. Cellular proteins may be obtained, for example, by lysis
or
extraction, and may be separated from one another using a variety of
electrophoretic
techniques. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE)
separates proteins largely on the basis of their molecular weight. Isoelectric
focusing
polyacrylamide gel electrophoresis (IEF-PAGE) separates proteins by their
isoelectric
point (which reflects not only the amino acid sequence but also
posttranslational
modifications of the protein). Another, more preferred method of protein
analysis is the
consecutive combination of both IEF-PAGE and SDS-PAGE, known as 2-D-gel
electrophoresis (described, for example, in Hermann et al. (1998)
Electrophoresis 19:
3217-3221; Fountoulakis et al. (1998) Electrophoresis 19: 1193-1202; Langen et
al.
(1997) Electrophoresis 18: 1184-1192; Antelmann et al. (1997) Electrophoresis
18:
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1451-1463). Other separation techniques may also be utilized for protein
separation,
such as capillary gel electrophoresis; such techniques are well known in the
art.
Proteins separated by these methodologies can be visualized by standard
techniques, such as by staining or labeling. Suitable stains are known in the
art, and
include Coomassie Brilliant Blue, silver stain, or fluorescent dyes such as
Sypro Ruby
(Molecular Probes). The inclusion of radioactively labeled amino acids or
other protein
precursors (e.g., 35S-methionine, 35S-cysteine,'4C-labelled amino acids,'SN-
amino
acids, ~SN03 or 15NH4+ or ~3C-labelled amino acids) in the medium of C.
glutamicum
permits the labeling of proteins from these cells prior to their separation.
Similarly,
fluorescent labels may be employed. These labeled proteins can be extracted,
isolated
and separated according to the previously described techniques.
Proteins visualized by these techniques can be further analyzed by measuring
the
amount of dye or label used. The amount of a given protein can be determined
quantitatively using, for 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 patterns of
protein presence, activity, or modification between different samples from
various
biological conditions (e.g., different organisms, time points of fermentation,
media
conditions, or different biotopes, among others). Data obtained from such
experiments
alone, or in combination with other techniques, can be used for various
applications,
such as to compare the behavior of various organisms in a given (e.g.,
metabolic)
situation, to increase the productivity of strains which produce fine
chemicals or to
increase the efficiency of the production of fine chemicals.
CA 02380863 2001-12-20
WO 01/00805 PCT/IB00/00926
- 122 -
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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