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CORYNEBACTERIUM GLUTAMICUM GENES ENCODING PROTEINS
INVOLVED IN CARBON METABOLISM AND ENERGY PRODUCTION
This application is a divisional application of Canadian Application Serial
No. 2,383,875 filed June 23, 2000.
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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
phanmaceutical 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 sugar metabolism and oxidative phosphorylation (SMP) 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 SMP nucleic acid molecules of the invention, therefore, can be
used to
identify microorganisms which can be used to produce fine chemicals, e.g., by
fermentation processes. Modulation of the expression of the SMP nucleic acids
of the
invention, or modification of the sequence of the SMP nucleic acid molecules
of the
invention, can be used to modulate the production of one or more fine
chemicals from a
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microorganism (e.g., to improve the yield or production of one or more fine
chemicals
from a Corynebacterium or Brevibacterium species).
The SMP 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 detectiori of such
organisms is of
significant clinical relevance.
The SMP 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 SMP ptoteins encoded by the novel nucleic acid molecules of the invention
are capable of, for example, performing a function involved in the metabolism
of carbon
compounds such as sugars or in the generation of energy molecules by processes
such as
oxidative phosphorylation in Corynebacterium glutamicum. 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 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.
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There are a number of mechanisms by which the alteration of an SMP protein of
the invention may directly affect the yield, production, and/or efficiency of
production
of a fine chenucal from a C. glutamicum strain incorporating such an altered
protein.
The degradation of high-energy carbon molecules such as sugars, and the
conversion of
compounds such as NADH and FADH2 to compounds containing high energy phosphate
bonds via oxidative phosphorylation results in a number of compounds which
themselves may be desirable fine chemicals, such as pyruvate, ATP, NADH, and a
number of intermediate sugar compounds. Further, the energy molecules (such as
ATP)
and the reducing equivalents (such as NADH or NADPH) produced by these
metabolic
pathways are utilized in the cell to drive reactions which would otherwise be
energetically unfavorable. Such unfavorable reactions include many
biosynthetic
pathways for fine chemicals. By improving the ability of the cell to utilize a
particular
sugar (e.g., by manipulating the genes encoding enzymes involved in the
degradation
and conversion of that sugar into energy for the cell), one may increase the
amount of
energy available to permit unfavorable, yet desired metabolic reactions (e.g.,
the
biosynthesis of a desired fine chemical) to occur.
The mutagenesis of one or more SMP genes of the invention may also result in
SMP proteins having altered activities which indirectly impact the production
of one or
more desired fine chemicals from C. glutamicum. For example, by increasing the
efficiency of utilization of one or more sugars (such that the conversion of
the sugar to
useful energy molecules is improved), or by increasing the efficiency of
conversion of
reducing equivalents to useful energy molecules (e.g., by improving the
efficiency of
oxidative phosphorylation, or the activity of the ATP synthase), one can
increase the
amount of these high-energy compounds available to the cell to drive normally
unfavorable metabolic processes. These processes include the construction of
cell walls,
transcription, translation, and the biosynthesis of compounds necessary for
growth and
division of the cells (e.g., nucleotides, amino acids, vitamins, lipids, etc.)
(Lengeler et al.
(1999) Biology of Prokaryotes, Thieme Verlag: Stuttgart, p. 88-109; 913-918;
875-899).
By improving the growth and multiplication of these engineered cells, it is
possible to
increase both the viability of the cells in large-scale culture, and also to
improve their
rate of division, such that a relatively larger number of cells can survive in
fermentor
culture. The yield, production, or efficiency of production may be increased,
at least
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due to the presence of a greater number of viable cells, each producing the
desired fine
chemical. Also, many of the degradation products produced during sugar
metabolism
are utilized by the cell as precursors or intermediates in the production of
other desirable
products, such as fine chemicals. So, by increasing the ability of the cell to
metabolize
sugars, the number of these degradation products available to the cell for
other processes
should also be increased.
The invention provides novel nucleic acid molecules which encode proteins,
referred to herein as SMP proteins, which are capable of, for example,
performing a
function involved in the metabolism of carbon compounds such as sugars and the
generation of energy molecules by processes such as oxidative phosphorylation
in
Corynebacterium glutamicum. Nucleic acid molecules encoding an SMP protein are
referred to herein as SMP nucleic acid molecules. In a preferred embodiment,
the SMP
protein participates in the conversion of carbon molecules and degradation
products
thereof to energy which is utilized by the cell for metabolic processes.
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 SMP protein or biologically active portions thereof, as well as nucleic
acid fragments
suitable as primers or hybridization probes for the detection or amplification
of SMP-
encoding nucleic acid (e.g., DNA or mRNA). In particularly preferred
embodiments,
the isolated nucleic acid molecule comprises one of the nucleotide sequences
set forth as
the odd-numbered SEQ ID NOs in the Sequence Listing (e.g., SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:5, SEQ ID NO:7....), or the coding region or a complement
thereof
of one of these nucleotide sequences. In other particularly preferred
embodiments, the
isolated nucleic acid molecule of the invention comprises a nucleotide
sequence which
hybridizes to or is at least about 50%, preferably at least about 60%, more
preferably at
least about 70%, 80% or 90%, and even more preferably at least about 95%, 96%,
97%,
98%, 99% or more homologous to a nucleotide sequence set forth as an odd-
numbered
SEQ ID NO in the Sequence Listing (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:5,
SEQ ID NO:7....), or a portion thereof. In other preferred embodiments, the
isolated
nucleic acid molecule encodes one of the amino acid sequences set forth as an
even-
numbered SEQ ID NO in the Sequence Listing (e.g., SEQ ID NO:2, SEQ ID NO:4,
SEQ
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ID NO:6, SEQ ID NO:8....).. The preferred SMP proteins of the present
invention also
preferably possess at least one of the SMP 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 SMP activity. Preferably, the protein or
portion thereof
encoded by the nucleic acid molecule maintains the ability to perform a
function
involved in the metabolism of carbon compounds such as sugars or the
generation of
energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in
Corynebacterium glutamicum. 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 those having an even-numbered SEQ ID NO in
the
Sequence Listing). In another preferred embodiment, the protein is a full
length C.
glutamicum protein which is substantially homologous to an entire amino acid
sequence
of the invention (encoded by an open reading frame shown in the corresponding
odd-
numbered SEQ ID NOs in the Sequence Listing (e.g., SEQ ID NO:1, SEQ ID NO:3,
SEQ ID NO:5, SEQ ID NO:7....).
In another preferred embodiment, the isolated nucleic acid molecule is derived
from C. glutamicum and encodes a protein (e.g., an SMP 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 perform a function involved
in the
metabolism of carbon compounds such as sugars or the generation of energy
molecules
(e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium
glutamicum, or has one or more of the activities set forth in Table 1, and
which also
includes heterologous nucleic acid sequences encoding a heterologous
polypeptide or
regulatory regions.
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In another embodiment, the isolated nucleic acid molecule is at least 15
nucleotides in length and hybridizes under stringent conditions to a nucleic
acid
molecule comprising a nucleotide sequence of the invention (e.g., a sequence
of an odd-
numbered SEQ ID NO in the Sequence Listing) A. 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 SMP
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 SMP protein by culturing the host cell in a suitable medium. The
SMP
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 SMP 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 SMP
sequence as
a transgene. In another embodiment, an endogenous SMP gene within the genome
of
the microorganism has been altered, e.g., functionally disrupted, by
homologous
recombination with an altered SMP gene. In another embodiment, an endogenous
or
introduced SMP gene in a microorganism has been altered by one or more point
mutations, deletions, or inversions, but still encodes a functional SMP
protein. In still
another embodiment, one or more of the regulatory regions (e.g., a promoter,
repressor,
or inducer) of an SMP gene in a microorganism has been altered (e.g., by
deletion,
truncation, inversion, or point mutation) such that the expression of the SMP
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 I through 782) 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 SMP protein or a
portion, e.g., a biologically active portion, thereof. In a preferred
embodiment, the
isolated SMP protein or portion thereof is capable of performing a function
involved in
the metabolism of carbon compounds such as sugars or in the generation of
energy
molecules (e.g., ATP) by processes such as oxidative phosphorylation in
Corynebacterium glutamicum. In another preferred embodiment, the isolated SMP
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 perfonm a
function
involved in the metabolism of.carbon compounds such as sugars or in the
generation of
energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in
Corynebacterium glutamicum.
The invention also provides an isolated preparation of an SMP protein. In
preferred embodiments, the SMP 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). 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 SMP
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 perform a function involved
in the
metabolism of carbon compounds such as sugars or in the generation of energy
molecules (e.g., ATP) by processes such as oxidative phosphorylation in
Corynebacterium glutamicum, or has one or more of the activities set forth in
Table 1.
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Altematively, the isolated SMP protein can comprise an amino acid sequence
which is encoded by a nucleotide sequence which hybridizes, e.g., hybridizes
under
stringent conditions, or is at least about 50%, preferably at least about 60%,
more
preferably at least about 70%, 80%, or 90%, and even more preferably at least
about
95%, 96%, 97%, 98,%, or 99% or more homologous to a nucleotide sequence of one
of
the even-numbered SEQ ID NOs set forth in the Sequence Listing. It is also
preferred
that the preferred fonns of SMP proteins also have one or more of the SMP
bioactivities
described herein.
The SMP polypeptide, or a biologically active portion thereof, can be
operatively
linked to a non-SMP polypeptide to form a fusion protein. In preferred
embodiments,
this fusion protein has an activity which differs from that of the SMP protein
alone. In
other preferred embodiments, this fusion protein performs a function involved
in the
metabolism of carbon compounds such as sugars or in the generation of energy
molecules (e.g., ATP) by processes such as oxidative phosphorylation in
Corynebacterium glutamicum. 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 SMP protein, either by interacting with the
protein itself or a
substrate or binding partner of the SMP protein, or by modulating the
transcription or
translation of an SMP 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 SMP 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 SMP 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
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agent which modulates SMP protein activity or SMP nucleic acid expression such
that a
celi 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
carbon metabolism pathways or for the production of energy through processes
such as
oxidative phosphorylation, such that the yields or rate of production of a
desired fine
chemical by this microorganism is improved. The agent which modulates SMP
protein
activity can be an agent which stimulates SMP protein activity or SMP nucleic
acid
expression. Examples of agents which stimulate SMP protein activity or SMP
nucleic
acid expression include small molecules, active SMP proteins, and nucleic
acids
encoding SMP proteins that have been introduced into the cell. Examples of
agents
which inhibit SMP activity or expression include small molecules and antisense
SMP
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 SMP
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
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 SMP nucleic acid and protein molecules which
are involved in the metabolism of carbon compounds such as sugars and the
generation
of energy molecules by processes such as oxidative phosphorylation in
Corynebacterium glutamicum. 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
glycolytic
pathway protein has a direct impact on the yield, production, and/or
efficiency of
production of, e.g., pyruvate from modified C. glutamicum), or may have an
indirect
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impact which nonetheless results in an increase of yield, production, and/or
efficiency of
production of the desired compound (e.g., where modulation of proteins
involved in
oxidative phosphorylation results in alterations in the amount of energy
available to
perform necessary metabolic processes and other cellular functions, such as
nucleic acid
and protein biosynthesis and transcription/translation). Aspects of the
invention are
further explicated below.
1. Fine Chemicals
The term 'fine chemical' is art-recognized and includes molecules produced by
an organism which have applications in various industries, such as, but not
limited to,
the pharmaceutical, agriculture, and cosmetics industries. Such compounds
include
organic acids, such as tartaric acid, itaconic acid, and diaminopimelic acid,
both
proteinogenic and non-proteinogenic amino acids, purine and pyrimidine bases,
nucleosides, and nucleotides (as described e.g. in Kuninaka, A. (1996)
Nucleotides and
related compounds, p. 561-612, in Biotechnology vol. 6, Rehm et al., eds. VCH:
Weinheim, and references contained therein), lipids, both saturated and
unsaturated fatty
acids (e.g., arachidonic acid), diols (e.g., propane diol, and butane diol),
carbohydrates
(e.g., hyaluronic acid and trehalose), aromatic compounds (e.g., aromatic
amines,
vanillin, and indigo), vitamins and cofactors (as described in Ullmann's
Encyclopedia of
Industrial Chemistry, vol. A27, "Vitamins", p. 443-613 (1996) VCH: Weinheim
and
references therein; and Ong, A.S., Niki, E. & Packer, L. (1995) "Nutrition,
Lipids,
Health, and Disease" Proceedings of the UNESCO/Confederation of Scientific and
Technological Associations in Malaysia, and the Society for Free Radical
Research -
Asia, held Sept. 1-3, 1994 at Penang, Malaysia, AOCS Press, (1995)), enzymes,
polyketides (Cane el 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-
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recognized. The proteinogenic amino acids, of which there are 20 species,
serve as
structural units for proteins, in which they are linked by peptide bonds,
while the
nonproteinogenic amino acids (hundreds of which are known) are not normally
found in
proteins (see Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97
VCH:
Weinheim (1985)). Amino acids may be in the D- or L- optical configuration,
though L-
amino acids are generally the only type found in naturally-occurring proteins.
Biosynthetic and degradative pathways of each of the 20 proteinogenic amino
acids
have been well characterized in both prokaryotic and eukaryotic cells (see,
for example,
Stryer, L. Biochemistry, 3d edition, pages 578-590 (1988)). The 'essential'
amino acids
(histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine,
tryptophan,
and valine), so named because they are generally a nutritional requirement due
to the
complexity of their biosyntheses, are readily converted by simple biosynthetic
pathways
to the remaining 11 'nonessential' amino acids (alanine, arginine, asparagine,
aspartate,
cysteine, glutamate, glutamine, glycine, proline, serine, and tyrosine).
Higher animals
do retain the ability to synthesize some of these amino acids, but the
essential amino
acids must be supplied from the diet in order for nonnal 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
conunonly
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
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described in Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97,
VCH:
Weinheim, 1985.
The biosynthesis of these natural amino acids in organisms capable of
producing them, such as bacteria, has been well characterized (for review of
bacterial
amino acid biosynthesis and regulation thereof, see Umbarger, H.E.(1978) Ann.
Rev.
Biochem. 47: 533-606). Glutamate is synthesized by the reductive amination of
a-
ketoglutarate, an intermediate in the citric acid cycle. Glutamine, proline,
and arginine
are each subsequently produced from glutamate. The biosynthesis of serine is a
three-
step process beginning with 3-phosphoglycerate (an intermediate in
glycolysis), and
resulting in this amino acid after oxidation, transamination, and hydrolysis
steps. Both
cysteine and glycine are produced from serine; the former by the condensation
of
homocysteine with serine, and the latter by the transferal of the side-chain 0-
carbon
atom to tetrahydrofolate, in a reaction catalyzed by serine
transhydroxymethylase.
Phenylalanine, and tyrosine are synthesized from the glycolytic and pentose
phosphate
pathway precursors erythrose 4-phosphate and phosphoenolpyruvate in a 9-step
biosynthetic pathway that differ only at the final two steps after synthesis
of prephenate.
Tryptophan is also produced from these two initial molecules, but its
synthesis is an 11-
step pathway. Tyrosine may also be synthesized from phenylalanine, in a
reaction
catalyzed by phenylalanine hydroxylase. Alanine, valine, and leucine are all
biosynthetic products of pyruvate, the final product of glycolysis. Aspartate
is formed
from oxaloacetate, an intermediate of the citric acid cycle. Asparagine,
methionine,
threonine, and lysine are each produced by the conversion of aspartate.
Isoleucine is
formed from threonine. A complex 9-step pathway results in the production of
histidine
from 5-phosphoribosyl-l-pyrophosphate, an activated sugar.
Amino acids in excess of the protein synthesis needs of the cell cannot be
stored,
and are instead degraded to provide intermediates for the major metabolic
pathways of
the cell (for review see Stryer, L. Biochemistry 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
tenms 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
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production (for overview of feedback mechanisms in amino acid biosynthetic
pathways,
see Stryer, L. Biochemistry, 3rd ed. Ch. 24: "Biosynthesis of Amino Acids and
Heme" p.
575-600 (1988)). Thus, the output of any particular amino acid is limited by
the amount
of that amino acid present in the cell.
B. Vitamin, Cofactor, and Nutraceutical Metabolism and Uses
Vitamins, cofactors, and nutraceuticals comprise another group of molecules
which the higher animals have lost the ability to synthesize and so must
ingest, although
they are readily synthesized by other organisms such as bacteria. These
molecules are
either bioactive substances themselves, or are precursors of biologically
active
substances which may serve as electron carriers or intermediates in a variety
of
metabolic pathways. Aside from their nutritive value, these compounds also
have
significant industrial value as coloring agents, antioxidants, and catalysts
or other
processing aids. (For an overview of the structure, activity, and industrial
applications
of these compounds, see, for example, Ullman's Encyclopedia of Industrial
Chemistry,
"Vitamins" vol. A27, p. 443-613, VCH: Weinheim, 1996.) The term "vitamin" is
art-
recognized, and includes nutrients which are required by an organism for
normal
functioning, but which that organism cannot synthesize by itself. The group of
vitamins
may encompass cofactors and nutraceutical compounds. The language "cofactor"
includes nonproteinaceous compounds required for a normal enzymatic activity
to
occur. Such compounds may be organic or inorganic; the cofactor molecules of
the
invention are preferably organic. The term "nutraceutical" includes dietary
supplements
having health benefits in plants and animals, particularly humans. Examples of
such
molecules are vitamins, antioxidants, and also certain lipids (e.g.,
polyunsaturated fatty
acids).
The biosynthesis of these molecules in organisms capable of producing them,
such as bacteria, has been largely characterized (Uliman'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
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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 i) is produced by the chemical coupling of pyrimidine and
thiazole moieties. Riboflavin (vitamin BZ) is synthesized from guanosine-5'-
triphosphate
(GTP) and ribose-5'-phosphate. Riboflavin, in turn, is utilized for the
synthesis of flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The family of
compounds collectively termed 'vitamin B6' (e.g., pyridoxine, pyridoxamine,
pyridoxa-
5'-phosphate, and the commercially used pyridoxin hydrochloride) are all
derivatives of
the common structural unit, 5-hydroxy-6-methylpyridine. Pantothenate
(pantothenic
acid, (R)-(+)-N-(2,4-dihydroxy-3,3-dimethyl-l-oxobutyl)-p-alanine) can be
produced
either by chemical synthesis or by fermentation. The final steps in
pantothenate
biosynthesis consist of the ATP-driven condensation of 0-alanine and pantoic
acid. The
enzymes responsible for the biosynthesis steps for the conversion to pantoic
acid, to 0-
alanine and for the condensation to panthotenic acid are known. The
metabolically
active form of pantothenate is Coenzyme A, for which the biosynthesis proceeds
in 5
enzymatic steps. Pantothenate, pyridoxal-5'-phosphate, cysteine and ATP are
the
precursors of Coenzyme A. These enzymes not only catalyze the formation of
panthothante, but also the production of (R)-pantoic acid, (R)-pantolacton,
(R)-
panthenol (provitamin B5), pantetheine (and its derivatives) and coenzyme A.
Biotin biosynthesis from the precursor molecule pimeloyl-CoA in
microorganisms has been studied in detail and several of the genes involved
have been
identified. Many of the corresponding proteins have been found to also be
involved in
Fe-cluster synthesis and are members of the nifS class of proteins. Lipoic
acid is
derived from octanoic acid, and serves as a coenzyme in energy metabolism,
where it
becomes part of the pyruvate dehydrogenase complex and the a-ketoglutarate
dehydrogenase complex. The folates are a group of substances which are all
derivatives
of folic acid, which is turn is derived from L-glutamic acid, p-amino-benzoic
acid and 6-
methylpterin. The biosynthesis of folic acid and its derivatives, starting
from the
metabolism intermediates guanosine-5'-triphosphate (GTP), L-glutamic acid and
p-
amino-benzoic acid has been studied in detail in certain microorganisms.
Corrinoids (such as the cobalamines and particularly vitamin B12) and
porphyrines belong to a group of chemicals characterized by a tetrapyrole ring
system.
t
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The biosynthesis of vitamin B12 is sufficiently complex that it has not yet
been
completely characterized, but many of the enzymes and substrates involved are
now
known. Nicotinic acid (nicotinate), and nicotinamide are pyridine derivatives
which are
also tenmed 'niacin'. Niacin is the precursor of the important coenzymes NAD
5(nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine
dinucleotide
phosphate) and their reduced forms.
The large-scale production of these compounds has largely relied on cell-free
chemical syntheses, though some of these chemicals have also been produced by
large-
scale culture of microorganisms, such as riboflavin, Vitamin B6, pantothenate,
and
biotin. Only Vitamin BiZ 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
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enzymes involved in purine and pyrimidine metabolism have been focused on the
development of new drugs which can be used, for example, as immunosuppressants
or
anti-proliferants (Smith, J.L., (1995) "Enzymes in nucleotide synthesis."
Curr. Opin.
Struct. Biol. 5: 752-757; (1995) Biochem Soc. Transact. 23: 877-902). However,
purine
and pyrimidine bases, nucleosides and nucleotides have other utilities: as
intermediates
in the biosynthesis of several fine chemicals (e.g., thiamine, S-adenosyl-
methionine,
folates, or riboflavin), as energy carriers for the cell (e.g., ATP or GTP),
and for
chemicals themselves, commonly used as flavor enhancers (e.g., IMP or GMP) or
for
several medicinal applications (see, for example, Kuninaka, A. (1996)
Nucleotides and
Related Compounds in Biotechnology vol. 6, Rehm et al., eds. VCH: Weinheim, p.
561-
612). Also, enzymes involved in purine, pyrimidine, nucleoside, or nucleotide
metabolism are increasingly serving as targets against which chemicals for
crop
protection, including fungicides, herbicides and insecticides, are developed.
The metabolism of these compounds in bacteria has been characterized (for
reviews see, for example, Zalkin, H. and Dixon, J.E. (1992) "de novo purine
nucleotide
biosynthesis", in: Progress in Nucleic Acid Research and Molecular Biology,
vol. 42,
Academic Press:, p. 259-287; and Michal, G. (1999) "Nucleotides and
Nucleosides",
Chapter 8 in: Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology,
Wiley: New York). Purine metabolism has been the subject of intensive
research, and is
essential to the normal functioning of the cell. Impaired purine metabolism in
higher
animals can cause severe disease, such as gout. Purine nucleotides are
synthesized from
ribose-5-phosphate, in a series of steps through the intermediate compound
inosine-5'-
phosphate (IMP), resulting in the production of guanosine-5'-monophosphate
(GMP) or
adenosine-5'-monophosphate (AMP), from which the triphosphate forms utilized
as
nucleotides are readily formed. These compounds are also utilized as energy
stores, so
their degradation provides energy for many different biochemical processes in
the cell.
Pyrimidine biosynthesis proceeds by the formation of uridine-5'-monophosphate
(UMP)
from ribose-5-phosphate. UMP, in turn, is converted to cytidine-5'-
triphosphate (CTP).
The deoxy- forms of all of these nucleotides are produced in a one step
reduction
reaction from the diphosphate ribose form of the nucleotide to the diphosphate
deoxyribose form of the nucleotide. Upon phosphorylation, these molecules are
able to
participate in DNA synthesis.
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D. Trehalose Metabolism and Uses
Trehalose consists of two glucose molecules, bound in a, a-1,1 linkage. It is
commonly used in the food industry as a sweetener, an additive for dried or
frozen
foods, and in beverages. However, it also has applications in the
pharmaceutical,
cosmetics and biotechnology industries (see, for example, Nishimoto et al.,
(1998) U.S.
Patent No. 5,759,610; Singer, M.A. and Lindquist, S. (1998) Trends Biotech.
16: 460-
467; Paiva, C.L.A. and Panek, A.D. (1996) Biotech. Ann. Rev. 2: 293-314; and
Shiosaka, M. (1997) J. Japan 172: 97-102). Trehalose is produced by enzymes
from
many microorganisms and is naturally released into the surrounding medium,
from
which it can be collected using methods known in the art.
II. Sugar and Carbon Molecule Utilization and Oxidative Phosphorylation
Carbon is a critically important element for the formation of all organic
compounds, and thus is a nutritional requirement not only for the growth and
division of
C. glutamicum, but also for the overproduction of fine chemicals from this
microorganism. Sugars, such as mono-, di-, or polysaccharides, are
particularly good
carbon sources, and thus standard growth media typically contain one or more
of:
glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose,
maltose,
sucrose, raffinose, starch, or cellulose (Ullmann's Encyclopedia of Industrial
Chemistry
(1987) vol. A9, "Enzymes", VCH: Weinheim). Alternatively, more complex forms
of
sugar may be utilized in the media, such as molasses, or other by-products of
sugar
refinement. Other compounds aside from the sugars may be used as alternate
carbon
sources, including alcohols (e.g., ethanol or methanol), alkanes, sugar
alcohols, fatty
acids, and organic acids (e.g., acetic acid or lactic acid). For a review of
carbon sources
and their utilization by microorganisms in culture, see: Ullman's Encyclopedia
of
Industrial Chemistry (1987) vol. A9, "Enzymes", VCH: Weinheim; Stoppok, E. and
Buchholz, K. (1996) "Sugar-based raw materials for fennentation applications"
in
Biotechnology (Rehm, H.J. et al., eds.) vol. 6, VCH: Weinheim, p. 5-29; Rehm,
H.J.
(1980) Industrielle Mikrobiologie, Springer: Berlin; Bartholomew, W.H., and
Reiman,
H.B. (1979). Economics of Fermentation Processes, in: Peppler, H.J. and
Perlman, D.,
eds. Microbial Technology 2"d ed., vol. 2, chapter 18, Academic Press: New
York; and
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Kockova-Kratachvilova, A. (1981) Characteristics of Industrial Microorganisms,
in:
Rehm, H.J. and Reed, G., eds. Handbook of Biotechnology, vol. 1, chapter 1,
Verlag
Chemie: Weinheim.
After uptake, these energy-rich carbon molecules must be processed such that
they are able to be degraded by one of the major sugar metabolic pathways.
Such
pathways lead directly to useful degradation products, such as ribose-5-
phosphate and
phosphoenolpyruvate, which may be subsequently converted to pyruvate. Three of
the
most important pathways in bacteria for sugar metabolism include the Embden-
Meyerhoff-Pamas (EMP) pathway (also known as the glycolytic or fructose
bisphosphate pathway), the hexosemonophosphate (HMP) pathway (also known as
the
pentose shunt or pentose phosphate pathway), and the Entner-Doudoroff (ED)
pathway
(for review, see Michal, G. (1999) Biochemical Pathways: An Atlas of
Biochemistry
and Molecular Biology, Wiley: New York, and Stryer, L. (1988) Biochemistry,
Chapters
13-19, Freeman: New York, and references therein).
The EMP pathway converts hexose molecules to pyruvate, and in the process
produces 2 molecules of ATP and 2 molecules of NADH. Starting with glucose-l-
phosphate (which may be either directly taken up from the medium, or
altematively may
be generated from glycogen, starch, or cellulose), the glucose molecule is
isomerized to
fructose-6-phosphate, is phosphorylated, and split into two 3-carbon molecules
of
glyceraldehyde-3-phosphate. After dehydrogenation, phosphorylation, and
successive
rearrangements, pyruvate results.
The HMP pathway converts glucose to reducing equivalents, such as NADPH,
and produces pentose and tetrose compounds which are necessary as
intenmediates and
precursors in a number of other metabolic pathways. In the HMP pathway,
glucose-6-
phosphate is converted to ribulose-5-phosphate by two successive dehydrogenase
reactions (which also release two NADPH molecules), and a carboxylation step.
Ribulose-5-phosphate may also be converted to xyulose-5-phosphate and ribose-5-
phosphate; the former can undergo a series of biochemical steps to glucose-6-
phosphate,
which may enter the EMP pathway, while the latter is commonly utilized as an
intermediate in other biosynthetic pathways within the cell.
The ED pathway begins with the compound glucose or gluconate, which is
subsequently phosphorylated and dehydrated to form 2-dehydro-3-deoxy-6-P-
gluconate.
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Glucuronate and galacturonate may also be converted to 2-dehydro-3-deoxy-6-P-
gluconate through more complex biochemical pathways. This product molecule is
subsequently cleaved into glyceraldehyde-3-P and pyruvate; glyceraldehyde-3-P
may
itself also be converted to pyruvate.
The EMP and HMP pathways share many features, including intermediates and
enzymes. The EMP pathway provides the greatest amount of ATP, but it does not
produce ribose-5-phosphate, an important precursor for, e.g., nucleic acid
biosynthesis,
nor does it produce erythrose-4-phosphate, which is important for amino acid
biosynthesis. Microorganisms that are capable of using only the EMP pathway
for
glucose utilization are thus not able to grow on simple media with glucose as
the sole
carbon source. They are referred to as fastidious organisms, and their growth
requires
inputs of complex organic compounds, such as those found in yeast extract.
In contrast, the HMP pathway produces all of the precursors necessary for both
nucleic acid and amino acid biosynthesis, yet yields only half the amount of
ATP energy
that the EMP pathway does. The HMP pathway also produces NADPH, which may be
used for redox reactions in biosynthetic pathways. The HMP pathway does not
directly
produce pyruvate, however, and thus these microorganisms must also possess
this
portion of the EMP pathway. It is therefore not surprising that a number of
microorganisms, particularly the facultative anerobes, have evolved such that
they
possess both of these pathways.
The ED pathway has thus far has only been found in bacteria. Although this
pathway is linked partly to the HMP pathway in the reverse direction for
precursor
formation, the ED pathway directly forms pyruvate by the aldolase cleavage of
3-
ketodeoxy-6-phosphogluconate. The ED pathway can exist on its own and is
utilized by
the majority of strictly aerobic microorganisms. The net result is similar to
that of the
HMP pathway, although one mole of ATP can be formed only if the carbon atoms
are
converted into pyruvate, instead of into precursor molecules.
The pyruvate molecules produced through any of these pathways can be readily
converted into energy via the Krebs cycle (also known as the citric acid
cycle, the citrate
cycle, or the tricarboxylic acid cycle (TCA cycle)). In this process, pyruvate
is first
decarboxylated, resulting in the production of one molecule of NADH, I
molecule of
acetyl-CoA, and I molecule of CO2. The acetyl group of acetyl CoA then reacts
with
CA 02593287 2007-07-27
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the 4 carbon unit, oxaolacetate, leading to the formation of citric acid, a 6
carbon
organic acid. Dehydration and two additional COZ molecules are released.
Ultimately,
oxaloacetate is regenerated and can serve again as an acetyl acceptor, thus
completing
the cycle. The electrons released during the oxidation of intermediates in the
TCA cycle
are transferred to NAD+ to yield NADH.
During respiration, the electrons from NADH are transferred to molecular
oxygen or other terminal electron acceptors. This process is catalyzed by the
respiratory
chain, an electron transport system containing both integral membrane proteins
and
membrane associated proteins. This system serves two basic functions: first,
to accept
electrons from an electron donor and to transfer them to an electron acceptor,
and
second, to conserve some of the energy released during electron transfer by
the synthesis
of ATP. Several types of oxidation-reduction enzymes and electron transport
proteins
are known to be involved in such processes, including the NADH dehydrogenases,
flavin-containing electron carriers, iron sulfur proteins, and cytochromes.
The NADH
dehydrogenases are located at the cytoplasmic surface of the plasma membrane,
and
transfer hydrogen atoms from NADH to flavoproteins, in turn accepting
electrons from
NADH. The flavoproteins are a group of electron carriers possessing a flavin
prosthetic
group which is alternately reduced and oxidized as it accepts and transfers
electrons.
Three flavins are known to participate in these reactions: riboflavin, flavin-
adenine
dinucleotide (FAD) and flavin-mononucleotide (FMN). Iron sulfur proteins
contain a
cluster of iron and sulfur atoms which are not bonded to a heme group, but
which still
are able to participate in dehydration and rehydration reactions. Succinate
dehydrogenase and aconitase are exemplary iron-sulfur proteins; their iron-
sulfur
complexes serve to accept and transfer electrons as part of the overall
electron-transport
chain. The cytochromes are proteins containing an iron porphyrin ring (heme).
There
are a number of different classes of cytochromes, differing in their reduction
potentials.
Functionally, these cytochromes form pathways in which electrons may be
transferred to
other cytochromes having increasingly more positive reduction potentials. A
further
class of non-protein electron carriers is known: the lipid-soluble quinones
(e.g.,
coenzyme Q). These molecules also serve as hydrogen atom acceptors and
electron
donors.
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The action of the respiratory chain generates a proton gradient across the
cell
membrane, resulting in proton motive force. This force is utilized by the cell
to
synthesize ATP, via the membrane-spanning enzyme, ATP synthase. This enzyme is
a
multiprotein complex in which the transport of H+ molecules through the
membrane
results in the physicaI rotation of the intracellular subunits and concomitant
phosphorylation of ADP to form ATP (for review, see Fillingame, R.H. and
Divall, S.
(1999) Novartis Found. Symp. 221: 218-229, 229-234).
Non-hexose carbon substrates may also serve as carbon and energy sources for
cells. Such substrates may first be converted to hexose sugars in the
gluconeogenesis
pathway, where glucose is first synthesized by the cell and then is degraded
to produce
energy. The starting material for this reaction is phosphoenolpyruvate (PEP),
which is
one of the key intermediates in the glycolytic pathway. PEP may be formed from
substrates other than sugars, such as acetic acid, or by decarboxylation of
oxaloacetate
(itself an intermediate in the TCA cycle). By reversing the glycolytic pathway
(utilizing
a cascade of enzymes different than those of the original glycolysis pathway),
glucose-6-
phosphate may be formed. The conversion of pyruvate to glucose requires the
utilization of 6 high energy phosphate bonds, whereas glycolysis only produces
2 ATP
in the conversion of glucose to pyruvate. However, the complete oxidation of
glucose
(glycolysis, conversion of pyruvate into acetyl CoA, citric acid cycle, and
oxidative
phosphorylation) yields between 36-38 ATP, so the net loss of high energy
phosphate
bonds experienced during gluconeogenesis is offset by the overall greater gain
in such
high-energy molecules produced by the oxidation of glucose.
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 SMP nucleic acid and protein molecules, which
participate in the conversion of sugars to useful degradation products and
energy (e.g.,
ATP) in C. glutamicum or which may participate in the production of useful
energy-rich
molecules (e.g., ATP) by other processes, such as oxidative phosphorylation.
In one
embodiment, the SMP molecules participate in the metabolism of carbon
compounds
such as sugars or the generation of energy molecules (e.g., ATP) by processes
such as
oxidative phosphorylation in Corynebacterium glutamicum. In a preferred
embodiment,
CA 02593287 2007-07-27
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the activity of the SMP molecules of the present invention to contribute to
carbon
metabolism or energy production in C. glutamicum has an impact on the
production of a
desired fine chemical by this organism. In a particularly prefen-ed
embodiment, the SMP
molecules of the invention are modulated in activity, such that the C.
glutamicum
metabolic and energetic pathways in which the SMP proteins of the invention
participate
are modulated in yield, production, and/or efficiency of production, which
either directly
or indirectly modulates the yield, production, and/or efficiency of production
of a
desired fine chemical by C. glutamicum.
The language, "SMP protein" or "SMP polypeptide" includes proteins which are
capable of performing a function involved in the metabolism of carbon
compounds such
as sugars and the generation of energy molecules by processes such as
oxidative
phosphorylation in Corynebacterium glutamicum. Examples of SMP proteins
include
those encoded by the SMP genes set forth in Table 1 and by the odd-numbered
SEQ ID
NOs. The tenns "SMP gene" or "SMP nucleic acid sequence" include nucleic acid
sequences encoding an SMP protein, which consist of a coding region and also
corresponding untranslated 5' and 3' sequence regions. Examples of SMP genes
include
those set forth in Table 1. The tenns "production" or "productivity" are art-
recognized
and include the concentration of the fermentation product (for example, the
desired fine
chemical) formed within a given time and a given fermentation volume (e.g., kg
product
per hour per liter). The term "efficiency of production" includes the time
required for a.
particular level of production to be achieved (for example, how long it takes
for the cell
to attain a particular rate of output of a fine chemical). The term "yield" or
"product/carbon yield" is art-recognized and includes the efficiency of the
conversion of
the carbon source into the product (f.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
intenmediate
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
CA 02593287 2007-07-27
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products (generally speaking, smaller or less complex molecules) in what may
be a
multistep and highly regulated process. The term "degradation product" is art-
recognized and includes breakdown products of a compound. Such products may
themselves have utility as precursor (starting point) or intermediate
molecules necessary
for the biosynthesis of other compounds by the cell. 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 SMP 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 SMP 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. The degradation of high-energy
carbon
molecules such as sugars, and the conversion of compounds such as NADH and
FADH2
to more useful forms via oxidative phosphorylation results in a number of
compounds
which themselves may be desirable fine chemicals, such as pyruvate, ATP, NADH,
and
a number of intermediate sugar compounds. Further, the energy molecules (such
as
ATP) and the reducing equivalents (such as NADH or NADPH) produced by these
metabolic pathways are utilized in the cell to drive reactions which would
otherwise be
energetically unfavorable. Such unfavorable reactions include many
biosynthetic
pathways for fine chemicals. By improving the ability of the cell to utilize a
particular
sugar (e.g., by manipulating the genes encoding enzymes involved in the
degradation
and conversion of that sugar into energy for the cell), one may increase the
amount of
energy available to pemiit unfavorable, yet desired metabolic reactions (e.g.,
the
biosynthesis of a desired fine chemical) to occur.
The mutagenesis of one or more SMP genes of the invention may also result in
SMP proteins having altered activities which indirectly impact the production
of one or
more desired fine chemicals from C. glutamicum. For example, by increasing the
efficiency of utilization of one or more sugars (such that the conversion of
the sugar to
useful energy molecules is improved), or by increasing the efficiency of
conversion of
CA 02593287 2007-07-27
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reducing equivalents to useful energy molecules (e.g., by improving the
efficiency of
oxidative phosphorylation, or the activity of the ATP synthase), one can
increase the
amount of these high-energy compounds available to the cell to drive normally
unfavorable metabolic processes. These processes include the construction of
cell walls,
transcription, translation, and the biosynthesis of compounds necessary for
growth and
division of the cells (e.g., nucleotides, amino acids, vitamins, lipids, etc.)
(Lengeler et al.
(1999) Biology of Prokaryotes, Thieme Verlag: Stuttgart, p. 88-109; 913-918;
875-899).
By improving the growth and multiplication of these engineered cells, it is
possible to
increase both the viability of the cells in large-scale culture, and also to
improve their
rate of division, such that a relatively larger number of cells can survive in
fennentor
culture. The yield, production, or efficiency of production may be increased,
at least
due to the presence of a greater number of viable cells, each producing the
desired fine
chemical. Further, a number of the degradation and intermediate compounds
produced
during sugar metabolism are necessary precursors and intermediates for other
biosynthetic pathways throughout the cell. For example, many amino acids are
synthesized directly from compounds normally resulting from glycolysis or the
TCA
cycle (e.g_, serine is synthesized from 3-phosphoglycerate, an intermediate in
glycolysis). Thus, by increasing the efficiency of conversion of sugars to
useful energy
molecules, it is also possible to increase the amount of useful degradation
products as
well.
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 SMP DNAs and the predicted amino acid sequences of the
C.
glutamicum SMP 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 having a function involved in the metabolism
of
carbon compounds such as sugars or in the generation of energy molecules by
processes
such as oxidative phosphorylation in Corynebacterium glutamicum.
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
CA 02593287 2007-07-27
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(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.
An SMP protein or a biologically active portion or fragment thereof of the
invention can participate in the metabolism of carbon compounds such as sugars
or in
the generation of energy molecules (e.g., ATP) by processes such as oxidative
phosphorylation in Corynebacterium gtutamrcum, or can 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 SMP 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 SMP-encoding nucleic acid (e.g., SMP 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
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
CA 02593287 2007-07-27
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genomic DNA of the organism from which the nucleic acid is derived. For
example, in
various embodiments, the isolated SMP 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 SMP DNA can
be
isolated from a C. glutamicum library using all or portion of one of the odd-
numbered
SEQ ID NO sequences of the Sequence Listing as a hybridization probe and
standard
hybridization techniques (e.g., as described in Sambrook, J., Fritsh, E. F.,
and Maniatis,
T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY,
1989).
Moreover, a nucleic acid molecule encompassing all or a portion of one of the
nucleic
acid sequences of the invention (e.g., an odd-numbered SEQ ID NO:) can be
isolated by
the polymerase chain reaction using oligonucleotide primers designed based
upon this
sequence (e.g., a nucleic acid molecule encompassing all or a portion of one
of the
nucleic acid sequences of the invention (e.g., an odd-numbered SEQ ID NO of
the
Sequence Listing) can be isolated by the polymerase chain reaction using
oligonucleotide primers designed based upon this same sequence). For example,
mRNA
can be isolated from normal endothelial cells (e.g., by the guanidinium-
thiocyanate
extraction procedure of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and
DNA
can be prepared using reverse transcriptase (e.g., Moloney MLV reverse
transcriptase,
available from GibcoBRL, 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
CA 02593287 2007-07-27
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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 SMP 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 gtutamicum SMP DNAs of the invention. This DNA comprises
sequences encoding SMP proteins (i.e., the "coding region", indicated in each
odd-
numbered SEQ ID NO: sequence in the Sequence Listing), as well as 5'
untranslated
sequences and 3' untranslated sequences, also indicated in each odd-numbered
SEQ ID
NO: in the Sequence Listing.. Alternatively, the nucleic acid molecule can
comprise
only the coding region of any of the sequences in nucleic acid sequences of
the
Sequence Listing.
For the purposes of this application, it will be understood that each of the
nucleic
acid and amino acid sequences set forth in the Sequence Listing has an
identifying RXA,
RXN, or RXS number having the designation "RXA," "RXN," or "RXS" followed by 5
digits (i.e., RXA01626, RXN00043, or RXS0735). Each of the nucleic acid
sequences
comprises up to three parts: a 5' upstream region, a coding region, and a
downstream
region. Each of these three regions is identified by the same RXA, RXN, or RXS
designation to eliminate confusion. The recitation "one of the odd-numbered
sequences
of the Sequence Listing", then, refers to any of the nucleic acid sequences in
the
Sequence Listing, which may also be distinguished by their differing RXA, RXN,
or
RXS designations. The coding region of each of these sequences is translated
into a
corresponding amino acid sequence, which is also set forth in the Sequence
Listing, as
an even-numbered SEQ ID NO: immediately following the corresponding nucleic
acid
sequence. For example, the coding region for RXA02735 is set forth in SEQ ID
NO:1,
while the amino acid sequence which it encodes is set forth as SEQ ID NO:2.
The
sequences of the nucleic acid molecules of the invention are identified by the
same
RXA, RXN, or RXS designations as the amino acid molecules which they encode,
such
that they can be readily correlated. For example, the amino acid sequence
designated
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RXA00042 is a translation of the coding region of the nucleotide sequence of
nucleic
acid molecule RXA00042, and the amino acid sequence designated RXN00043 is a
translation of the coding region of the nucleotide sequence of nucleic acid
molecule
RXN00043. The correspondence between the RXA, RXN and RXS nucleotide and
amino acid sequences of the invention and their assigned SEQ ID NOs is set
forth in
Table 1.
Several of the genes of the invention are "F-designated genes". An F-
designated
gene includes those genes set forth in Table 1 which have an 'F' in front of
the
RXAdesignation. For example, SEQ ID NO:11, designated, as indicated on Table
1, as
"F RXA01312", is an F-designated gene, as are SEQ ID NOs: 29, 33, and 39
(designated on Table I as "F RXA02803", "F RXA02854", and "F RXA01365",
respectively).
In one embodiment, the nucleic acid molecules of the present invention are not
intended to include those compiled in Table 2. In the case of the dapD gene, a
sequence
for this gene was published in Wehrmann, A., et al. (1998) J. Bacteriol.
180(12): 3159-
3165. However, the sequence obtained by the inventors of the present
application is
significantly longer than the published version. It is believed that the
published version
relied on an incorrect start codon, and thus represents only a fragment of the
actual
coding region.
In another preferred embodiment, an isolated nucleic acid molecule of the
invention comprises a nucleic acid molecule which is a complement of one of
the
nucleotide sequences of the invention (e.g., a sequence of an odd-numbered SEQ
ID
NO: of the Sequence Listing), or a portion thereof. A nucleic acid molecule
which is
complementary to one of the nucleotide sequences of the invention is one which
is
sufficiently complementary to one of the nucleotide sequences shown in the
Sequence
Listing (e.g., the sequence of an odd-numbered SEQ ID NO:) such that it can
hybridize
to one of the nucleotide sequences of the invention, thereby forming a stable
duplex.
In still another preferred embodiment, an isolated nucleic acid molecule of
the
invention comprises a nucleotide sequence which is at least about 50'/0, 51%,
52%, 53%,
54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62010,
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%,
CA 02593287 2007-07-27
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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 SMP protein. The
nucleotide
sequences determined from the cloning of the SMP genes from C. glutamicum
allows
for the generation of probes and primers designed for use in identifying
and/or cloning
SMP homologues in other cell types and organisms, as well as SMP 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
nucleotide sequence of the invention can be used in PCR reactions to clone SMP
homologues. Probes based on the SMP 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 SMP protein, such as by measuring a level of an SMP-
encoding
CA 02593287 2007-07-27
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nucleic acid in a sample of cells, e.g., detecting SMP mRNA levels or
determining
whether a genomic SMP 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 perform a function involved in the metabolism of
carbon
compounds such as sugars or in the generation of energy molecules (e.g., ATP)
by
processes such as oxidative phosphorylation in Corynebacterium glutamicum. 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 perfonn a function involved in the
metabolism of
carbon compounds such as sugars or in the generation of energy molecules
(e.g., ATP)
by processes such as oxidative phosphorylation in Corynebacterium glutamicum.
Protein members of such sugar metabolic pathways or energy producing 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 SMP protein" contributes either directly or indirectly to the yield,
production, and/or
efficiency of production of one or more fine chemicals. Examples of SMP
protein
activities are set forth in Table 1.
In another embodiment, the protein is at least about 50-60%, preferably at
least
about 60-70%, and more preferably at least about 70-80%, 80-90%, 90-95%, and
most
preferably at least about 96%, 97%, 98%, 99% or more homologous to an entire
amino
acid sequence of the invention(e.g., a sequence of an even-numbered SEQ ID NO:
of the
Sequence Listing).
Portions of proteins encoded by the SMP nucleic acid molecules of the
invention
are preferably biologically active portions of one of the SMP proteins. As
used herein,
the term "biologically active portion of an SMP protein" is intended to
include a portion,
e.g., a domain/motif, of an SMP protein that participates in the metabolism of
carbon
CA 02593287 2007-07-27
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compounds such as sugars, or in energy-generating pathways in C glutamicum, or
has
an activity as set forth in Table 1. To determine whether an SMP protein or a
biologically active portion thereof can participate in the metabolism of
carbon
compounds or in the production of energy-rich molecules in C. glutamicum, an
assay of
enzymatic activity may be performed. Such assay methods are well known to
those of
ordinary slcili in the art, as detailed in Example 8 of the Exemplification.
Additional nucleic acid fragments encoding biologically active portions of an
SMP 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 SMP protein or peptide (e.g.,
by
recombinant expression in vitro) and assessing the activity of the encoded
portion of the
SMP protein or peptide.
The invention further encompasses nucleic acid molecules that differ from one
of
the nucleotide sequences of the invention (e.g., a sequence of an odd-numbered
SEQ ID
NO: of the Sequence Listing) (and portions thereof) due to degeneracy of the
genetic
code and thus encode the same SMP 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 of the
invention
(encoded by an open reading frame shown in an odd-numbered SEQ ID NO: of the
Sequence Listing).
It will be understood by one of ordinary skill in the art that in one
embodiment
the sequences of the invention are not meant to include the sequences of the
prior art,
such as those Genbank sequences set forth in Tables 2 or 4 which were
available prior to
the present invention. In one embodiment, the invention includes nucleotide
and amino
acid sequences having a percent identity to a nucleotide or amino acid
sequence of the
invention which is greater than that of a sequence of the prior art (e.g., a
Genbank
sequence (or the protein encoded by such a sequence) set forth in Tables 2 or
4). For
example, the invention includes a nucleotide sequence which is greater than
and/or at
least 58% identical to the nucleotide sequence designated RXA00014 (SEQ ID
NO:41),
CA 02593287 2007-07-27
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a nucleotide sequence which is greater than and/or at least % identical to the
nucleotide
sequence designated RXA00195 (SEQ ID NO:399), and a nucleotide sequence which
is
greater than and/or at least 42% identical to the nucleotide sequence
designated
RXA00196 (SEQ ID NO:401). 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 SMP nucleotide sequences set forth in the
Sequence Listing as odd-numbered SEQ ID NOs, it will be appreciated by those
of
ordinary skill in the art that DNA sequence polymorphisms that lead to changes
in the
amino acid sequences of SMP proteins may exist within a population (e.g., the
C.
glutamicum population). Such genetic polymorphism in the SMP 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 SMP protein, preferably a C. glutamicum SMP protein.
Such
natural variations can typically result in 1-5% variance in the nucleotide
sequence of the
SMP gene. Any and all such nucleotide variations and resulting amino acid
polymorphisms in SMP that are the result of natural variation and that do not
alter the
functional activity of SMP 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 SMP DNA of the invention can be isolated based
on
their homology to the C. glutamicum SMP 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
CA 02593287 2007-07-27
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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 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 SMP protein.
In addition to naturally-occurring variants of the SMP sequence that may exist
in
the population, one of ordinary skill in the art will further appreciate that
changes can be
introduced by mutation into a nucleotide sequence of the invention, thereby
leading to
changes in the amino acid sequence of the encoded SMP protein, without
altering the
functional ability of the SMP 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 SMP proteins
(e.g., an
even-numbered SEQ ID NO: of the Sequence Listing) without altering the
activity of
said SMP protein, whereas an "essential" amino acid residue is required for
SMP protein
activity. Other amino acid residues, however, (e.g., those that are not
conserved or only
CA 02593287 2007-07-27
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semi-conserved in the domain having SMP activity) may not be essential for
activity and
thus are likely to be amenable to alteration without altering SMP activity.
Accordingly, another aspect of the invention pertains to nucleic acid
molecules
encoding SMP proteins that contain changes in amino acid residues that are not
essential
for SMP activity. Such SMP 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
SMP activities described herein. In one embodiment, the isolated nucleic acid
molecule
comprises a nucleotide sequence encoding a protein, wherein the protein
comprises an
amino acid sequence at least about 50% homologous to an amino acid sequence of
the
invention and is capable of participate in the metabolism of carbon compounds
such as
sugars, or in the biosynthesis of high-energy compounds in C. glutamicum, 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
amino acid positions or nucleotide positions are then compared. When a
position in one
sequence (e.g., one of the amino acid sequences the invention) is occupied by
the same
amino acid residue or nucleotide as the corresponding position in the other
sequence
(e.g., a mutant fon=n 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).
CA 02593287 2007-07-27
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An isolated nucleic acid molecule encoding an SMP 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., al.anine, 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 SMP protein is preferably replaced with another amino acid residue from
the same
side chain family. Altematively, in another embodiment, mutations can be
introduced
randomly along all or part of an SMP coding sequence, such as by saturation
mutagenesis, and the resultant mutants can be screened for an SMP activity
described
herein to identify mutants that retain SMP activity. Following mutagenesis of
the
nucleotide sequence of one of the odd-numbered SEQ ID NOs of the Sequence
Listing,
the encoded protein can be expressed recombinantly and the activity of the
protein can
be determined using, for example, assays described herein (see Example 8 of
the
Exemplification).
In addition to the nucleic acid molecules encoding SMP proteins described
above, another aspect of the invention pertains to isolated nucleic acid
molecules which
are antisense thereto. An "antisense" nucleic acid comprises a nucleotide
sequence
which is complementary to a "sense" nucleic acid encoding a protein, e.g.,
complementary to the coding strand of a double-stranded DNA molecule or
CA 02593287 2007-07-27
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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 SMP 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 SMP 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 NO.
3(RXA01626)
comprises nucleotides I to 345). In another embodiment, the antisense nucleic
acid
molecule is antisense to a"noncoding region" of the coding strand of a
nucleotide
sequence encoding SMP. The term "noncoding region" refers to 5' and 3'
sequences
which flank the coding region that are not transtated into amino acids (i.e.,
also referred
to as 5' and 3' untranslated regions).
Given the coding strand sequences encoding SMP 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 SMP mRNA, but more preferably is an oligonucleotide
which is
antisense to only a portion of the coding or noncoding region of SMP mRNA. For
example, the antisense oligonucleotide can be complementary to the region
surrounding
the translation start site of SMP mRNA. An antisense oligonucleotide can be,
for
example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
An
antisense nucleic acid of the invention can be constructed using chemical
synthesis and
enzymatic ligation reactions using procedures known in the art. For example,
an
antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically
synthesized
using naturally occurring nucleotides or variously modified nucleotides
designed to
increase the biological stability of the molecules or to increase the physical
stability of
the duplex formed between the antisense and sense nucleic acids, e.g.,
phosphorothioate
derivatives and acridine substituted nucleotides can be used. Examples of
modified
nucleotides which can be used to generate the antisense nucleic acid include 5-
fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xanthine, 4-
acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-
thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-
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galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-
methylinosine,
2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-
methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-
methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-
methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-
isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-
thiocytosine, 5-
methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-
oxyacetic acid
methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-
N-2-
carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the
antisense
nucleic acid can be produced biologically using an expression vector into
which a
nucleic acid has been subcloned in an antisense orientation (i.e., RNA
transcribed from
the inserted nucleic acid will be of an antisense orientation to a target
nucleic acid of
interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically
administered
to a cell or generated in situ such that they hybridize with or bind to
cellular mRNA
and/or genomic DNA encoding an SMP 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 fonn a stable duplex, or, for
example, in the
case of an antisense nucleic acid molecule which binds to DNA duplexes,
through
specific interactions in the major groove of the double helix. The antisense
molecule can
be modified such that it specifically binds to a receptor or an antigen
expressed on a
selected cell surface, e.g., by linking the antisense nucleic acid molecule to
a peptide or
an antibody which binds to a cell surface receptor or antigen. The antisense
nucleic acid
molecule can also be delivered to cells using the vectors described herein. To
achieve
sufficient intracellular concentrations of the antisense molecules, vector
constructs in
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 nucteic acid molecule
forms
specific double-stranded hybrids with complementary RNA in which, contrary to
the
usual ~-units, the strands run parallel to each other (Gaultier e1 al. (1987)
Nucleic Acids.
Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-
o-
CA 02593287 2007-07-27
39
methylribonucleotide (Inoue et aL (1987) Nucleic Acids Res. 15:6131-6148) or a
chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
In still another embodiment, an antisense nucleic acid of the invention is a
ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity
which are
capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which
they
have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to
catalytically cleave SMP mRNA transcripts to thereby inhibit translation of
SMP
mRNA. A ribozyme having specificity for an SMP-encoding nucleic acid can be
designed based upon the nucleotide sequence of an SMP cDNA disclosed herein
(i.e.,
SEQ ID NO. 3(RXA01626)). For example, a derivative of a Teirahymena 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 SMP-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, SMP 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.
Altematively, SMP gene expression can be inhibited by targeting nucleotide
sequences complementary to the regulatory region of an SMP nucleotide sequence
(e.g.,
an SMP promoter and/or enhancers) to form triple helical structures that
prevent
transcription of an SMP gene in target cells. See generally, Helene, C. (1991)
Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N. Y. Acad.
Sci. 660:27-
36; and Maher, L.J. (1992) Bioassays 14(12):807-15.
B. Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression
vectors, containing a nucleic acid encoding an SMP 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
CA 02593287 2007-07-27
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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 fonn.n of plasmids. In the present specification, "plasmid" and
"vector" can
be used interchangeably as the plasmid is the most commonly used form of
vector.
However, the invention is intended to include such other forms of expression
vectors,
such as viral vectors (e.g., replication defective retroviruses, adenoviruses
and adeno-
associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of
the invention in a form suitable for expression of the nucleic acid in a host
cell, which
means that the recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for expression,
which is
operatively linked to the nucleic acid sequence to be expressed. Within a
recombinant
expression vector, "operably linked" is intended to mean that the nucleotide
sequence of
interest is linked to the regulatory sequence(s) in a manner which allows for
expression
of the nucleotide sequence (e.g., in an in vitro transcription/translation
system or in a
host cell when the vector is introduced into the host cell). The term
"regulatory
sequence" is intended to include promoters, enhancers and other expression
control
elements (e.g., polyadenylation signals). Such regulatory sequences are
described, for
example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, CA (1990). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence in many types of host
cell and
those which direct expression of the nucleotide sequence only in certain host
cells.
Preferred regulatory sequences are, for example, promoters such as cos-, tac-,
trp-, tet-,
trp-tet-, lpp-, lac-, Ipp-lac-, IacIq-, T7-, T5-, T3-, gal-, trc-, ara-, SP6-,
amy, SPO2, X-PR-
or X PL, which are used preferably in bacteria. Additional regulatory
sequences are, for
example, promoters from yeasts and fungi, such as ADC 1, MFa, AC, P-60, CYC1,
GAPDH, TEF, rp28, ADH, promoters from plants such as CaMV/35S, SSU, OCS, lib4,
CA 02593287 2007-07-27
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usp, STLS 1, B33, nos or ubiquitin- or phaseolin-promoters. It is also
possible to use
artificial promoters. It will be appreciated by those 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., SMP proteins, mutant forms of SMP proteins, fusion proteins,
etc.).
The recombinant expression vectors of the invention can be designed for
expression of SMP proteins in prokaryotic or eukaryotic cells. For example,
SMP 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.
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
CA 02593287 2007-07-27
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expression vectors, a proteolytic cleavage site is introduced at the junction
of the fusion
moiety and the recombinant protein to enable separation of the recombinant
protein
from the fusion moiety subsequent to purification of the fusion protein. Such
enzymes,
and their cognate recognition sequences, include Factor Xa, thrombin and
enterokinase.
Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith,
D.B. and Johnson, K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs,
Beverly,
MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase
(GST), maltose E binding protein, or protein A, respectively, to the target
recombinant
protein. In one embodiment, the coding sequence of the SMP 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 SMP protein unfused to GST can be recovered by cleavage of the
fusion
protein with thrombin.
Examples of suitable inducible non-fusion E. coli expression vectors include
pTrc (Amann et al., (1988) Gene 69:301-315), pLG338, pACYC184, pBR322, pUC18,
pUC 19, pKC30, pRep4, pHS 1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-
I1I113-B1, kgt11, pBdCl, and pET I id (Studier et al., Gene Expression
Technology:
Methods in Enzymology 185, Academic Press, San Diego, Califonva (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 I ld vector relies on transcription from a T7 gn10-lac fusion promoter
mediated by
a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied
by
host strains BL21(DE3) or HMS174(DE3) from a resident k prophage harboring a
T7
gn 1 gene under the transcriptional control of the lacUV 5 promoter. For
transformation
of other varieties of bacteria, appropriate vectors may be selected. For
example, the
plasmids pIJ101, pIJ364, pIJ702 and pIJ361 are known to be useful in
transforming
Streptomyces, while plasmids pUB 110, pC 194, or pBD214 are suited for
transformation
of Bacillus species. Several plasmids of use in the transfer of genetic
information into
Corynebacterium include pHM1519, pBL1, pSA77, or pAJ667 (Pouwels et al., eds.
(1985) Cloning Vectors. Elsevier: New York IBSN 0 444 90401 8)_
CA 02593287 2007-07-27
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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, Califomia (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 SMP protein expression vector is a yeast expression
vector. Examples of vectors for expression in yeast S. cerevisiae include
pYepSecl
(Baldari, et al., (1987) EmboJ. 6:229-234), 2 , pAG-1, Yep6, Yep13,
pEMBLYe23,
pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al.,
(1987)
Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors
and
methods for the construction of vectors appropriate for use in other fungi,
such as the
filamentous fungi, include those detailed in: van den Hondel, C.A.M.J.J. &
Punt, P.J.
(1991) "Gene transfer systems and vector development for filamentous fungi,
in:
Applied Molecular Genetics of Fungi, J.F. Peberdy, et al., eds., p. 1-28,
Cambridge
University Press: Cambridge, and Pouwels et al., eds. (1985) Cloning Vectors.
Elsevier:
New York (IBSN 0 444 904018).
Alternatively, the SMP proteins of the invention can be expressed in insect
cells
using baculovirus expression vectors. Baculovirus vectors available for
expression of
proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series
(Smith et al.
(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers
(1989)
Virology 170:31-39).
In another embodiment, the SMP 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+,
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pBIN19, pAK2004, and pDH51 (Pouwels et al., eds. (1985) Cloning Vectors.
Elsevier:
New York IBSN 0 444 904018). .
In yet another embodiment, a nucleic acid of the invention is expressed in
mammalian cells using a mammalian expression vector. Examples of mammalian
expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC
(Kaufman et al. (1987) EMBOJ. 6:187-195). When used in mammalian cells, the
expression vector's control functions are often provided by viral regulatory
elements.
For example, commonly used promoters are derived from polyoma, Adenovirus 2,
cytomegalovirus and Simian Virus 40. For other suitable expression systems for
both
prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,
Fritsh, E. F.,
and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
1989.
In another embodiment, the recombinant mammalian expression vector is
capable of directing expression of the nucleic acid preferentially in a
particular cell type
(e.g., tissue-specific regulatory elements are used to express the nucleic
acid). Tissue-
specific regulatory elements are known in the art. Non-limiting examples of
suitable
tissue-specific promoters include the albumin promoter (liver-specific;
Pinkert et al.
(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton
(1988)
Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto
and
Baltimore (1989) EMBOJ. 8:729-733) and immunoglobulins (Banerji et al, (1983)
Cell
33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific
promoters
(e.g., the neurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477),
pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and
mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316
and
European Application Publication No. 264,166). Developmentally-regulated
promoters
are also encompassed, for example the murine hox promoters (Kessel and Gruss
(1990)
Science 249:374-379) and the a-fetoprotein promoter (Campes and Tilghman
(1989)
Genes Dev. 3:537-546).
The invention further provides a recombinant expression vector comprising a
DNA molecule of the invention cloned into the expression vector in an
antisense
orientation. That is, the DNA molecule is operatively linked to a regulatory
sequence in
CA 02593287 2007-07-27
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a manner which allows for expression (by transcription of the DNA molecule) of
an
RNA molecule which is antisense to SMP 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 deterrnined 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. l(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
tenns 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 SMP
protein can be expressed in bacterial cells such as C. glutamicum, insect
cells, yeast or
mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
Other
suitable host cells are known to one of ordinary skill in the art.
Microorganisms related
to Corynebacterium glutamicum which may be conveniently used as host cells for
the
nucleic acid and protein molecules of the invention are set forth in Table 3.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional transformation or transfection techniques. As used herein, the
terms
"transformation" and "transfection", "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,
CA 02593287 2007-07-27
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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 G41
8,
hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be
introduced into a host cell on the same vector as that encoding an SMP 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 SMP gene into which a deletion, addition or
substitution
has been introduced to thereby alter, e.g., functionally disrupt, the SMP
gene.
Preferably, this SMP gene is a Corynebacterium glutamicum SMP 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
recombination, the endogenous SMP gene is functionally disrupted (I.e., no
longer
encodes a functional protein; also referred to as a "knock out" vector).
Altematively,
the vector can be designed such that, upon homologous recombination, the
endogenous
SMP 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 SMP protein). In the homologous recombination vector, the altered
portion
of the SMP gene is flanked at its 5' and 3' ends by additional nucleic acid of
the SMP
gene to allow for homologous recombination to occur between the exogenous SMP
gene
carried by the vector and an endogenous SMP gene in a microorganism. The
additional
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flanking SMP nucleic acid is of sufficient length for successful homologous
recombination with the endogenous gene. Typically, several kilobases of
flanking DNA
(both at the 5' and 3' ends) are included in the vector (see e.g., Thomas,
K.R., and
Capecchi, M.R. (1987) Cell 51: 503 for a description of homologous
recombination
vectors). The vector is introduced into a microorganism (e.g., by
electroporation) and
cells in which the introduced SMP gene has homologously recombined with the
endogenous SMP 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 SMP gene on a vector placing it under control of
the lac
operon permits expression of the SMP gene only in the presence of IPTG. Such
regulatory systems are well known in the art.
In another embodiment, an endogenous SMP 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 SMP gene in a host cell has been altered by one or more point
mutations,
deletions, or inversions, but still encodes a functional SMP protein. In still
another
embodiment, one or more of the regulatory regions (e.g., a promoter,
repressor, or
inducer) of an SMP gene in a microorganism has been altered (e.g., by
deletion,
truncation, inversion, or point mutation) such that the expression of the SMP
gene is
modulated. One of ordinary skill in the art will appreciate that host cells
containing
more than one of the described SMP gene and protein modifications may be
readily
produced using the methods of the invention, and are meant to be included in
the present
invention.
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in
culture, can be used to produce (i.e., express) an SMP protein. Accordingly,
the
invention further provides methods for producing SMP 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 SMP protein
has
been introduced, or into which genome has been introduced a gene encoding a
wild-type
or altered SMP protein) in a suitable medium until SMP protein is produced. In
another
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embodiment, the method further comprises isolating SMP proteins from the
medium or
the host cell.
C. Isolated SMP Proteins
Another aspect of the invention pertains to isolated SMP 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 SMP 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
SMP protein
having less than about 30% (by dry weight) of non-SMP protein (also referred
to herein
as a"contaminating protein"), more preferably less than about 20% of non-SMP
protein,
still more preferably less than about 10% of non-SMP protein, and most
preferably less
than about 5% non-SMP protein. When the SMP 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 SMP protein in which the protein is
separated from
chemical precursors or other chemicals which are involved in the synthesis of
the
protein. In one embodiment, the language "substantially free of chemical
precursors or
other chemicals" includes preparations of SMP protein having less than about
30% (by
dry weight) of chemical precursors or non-SMP chemicals, more preferably less
than
about 20% chemical precursors or non-SMP chemicals, still more preferably less
than
about 10% chemical precursors or non-SMP chemicals, and most preferably less
than
about 5% chemical precursors or non-SMP chemicals. In preferred embodiments,
isolated proteins or biologically active portions thereof lack contaminating
proteins from
the same organism from which the SMP protein is derived. Typically, such
proteins are
produced by recombinant expression of, for example, a C. glutamicum SMP
protein in a
microorganism such as C. glutamicum.
CA 02593287 2007-07-27
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An isolated SMP protein or a portion thereof of the invention can participate
in
the metabolism of carbon compounds such as sugars, or in the production of
energy
compounds (e.g., by oxidative phosphorylation) utilized to drive unfavorable
metabolic
pathways, or has one or more of the activities set forth in Table 1. In
preferred
embodiments, the protein or portion thereof comprises an amino acid sequence
which is
sufficiently homologous to an amino acid sequence of the invention (e.g., a
sequence of
an even-numbered SEQ ID NO: of the Sequence Listing) such that the protein or
portion
thereof maintains the ability to perform a function involved in the metabolism
of carbon
compounds such as sugars or in the generation of energy molecules by processes
such as
oxidative phosphorylation in Corynebacterium glutamicum. The portion of the
protein
is preferably a biologically active portion as described herein. In another
preferred
embodiment, an SMP 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 SMP 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 SMP
protein has an
amino acid sequence which is encoded by a nucleotide sequence that is at least
about
50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least
about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at
least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even
more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to
one
of the nucleic acid sequences of the invention, or a portion thereof. Ranges
and identity
values intermediate to the above-recited values, (e.g., 70-90% identical or 80-
95%
identical) are also intended to be encompassed by the present invention. For
example,
ranges of identity values using a combination of any of the above values
recited as upper
and/or lower limits are intended to be included. The preferred SMP proteins of
the
present invention also preferably possess at least one of the SMP activities
described
herein. For example, a preferred SMP 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
CA 02593287 2007-07-27
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which can perform a function involved in the metabolism of carbon compounds
such as
sugars or in the generation of energy molecules (e.g., ATP) by processes such
as
oxidative phosphorylation in Corynebacterium glutamicum, or which has one or
more of
the activities set forth in Table 1.
In other embodiments, the SMP protein is substantially homologous to an amino
acid sequence of 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 SMP protein is a protein which comprises an amino acid
sequence which is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,
59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,
69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or
91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%,
98%,
99% or more homologous to an entire amino acid sequence of the invention and
which
has at least one of the SMP 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. glutamrcum protein which is substantially homologous to an
entire
amino acid sequenoe of the invention.
Biologically active portions of an SMP protein include peptides comprising
amino acid sequences derived from the amino acid sequence of an SMP protein,
e.g., an
amino acid sequence of an even-numbered SEQ ID NO: of the Sequence Listing or
the
amino acid sequence of a protein homologous to an SMP protein, which include
fewer
amino acids than a full length SMP protein or the full length protein which is
homologous to an SMP protein, and exhibit at least one activity of an SMP
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 SMP protein. Moreover,
other
CA 02593287 2007-07-27
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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 SMP
protein include
one or more selected domains/motifs or portions thereof having biological
activity.
SMP proteins are preferably produced by recombinant DNA techniques. For
example, a nucleic acid molecule encoding the protein is cloned into an
expression
vector (as described above), the expression vector is introduced into a host
cell (as
described above) and the SMP protein is expressed in the host cell. The SMP
protein
can then be isolated from the cells by an appropriate purification scheme
using standard
protein purification techniques. Altemative to recombinant expression, an SMP
protein,
polypeptide, or peptide can be synthesized chemically using standard peptide
synthesis
techniques. Moreover, native SMP protein can be isolated from cells (e.g.,
endothelial
cells), for example using an anti-SMP antibody, which can be produced by
standard
techniques utilizing an SMP protein or fragment thereof of this invention.
The invention also provides SMP chimeric or fusion proteins. As used herein,
an
SMP "chimeric protein" or "fusion protein" comprises an SMP polypeptide
operatively
linked to a non-SMP polypeptide. An "SMP polypeptide" refers to a polypeptide
having
an amino acid sequence corresponding to an SMP protein, whereas a "non-SMP
polypeptide" refers to a polypeptide having an amino acid sequence
corresponding to a
protein which is not substantially homologous to the SMP protein, e.g., a
protein which
is different from the SMP 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 SMP polypeptide and the non-SMP polypeptide are fused in-
frame to
each other. The non-SMP polypeptide can be fused to the N-terminus or C-
terminus of
the SMP polypeptide. For example, in one embodiment the fusion protein is a
GST-
SMP fusion protein in which the SMP sequences are fused to the C-terminus of
the GST
sequences. Such fusion proteins can facilitate the purification of recombinant
SMP
proteins. In another embodiment, the fusion protein is an SMP 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 SMP protein can be increased
through use
of a heterologous signal sequence.
CA 02593287 2007-07-27
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Preferably, an SMP 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,
Ausubel et
al., eds. John Wiley & Sons: 1992). Moreover, many expression vectors are
commercially available that already encode a fusion moiety (e.g., a GST
polypeptide).
An SMP-encoding nucleic acid can be cloned into such an expression vector such
that
the fusion moiety is linked in-frame to the SMP protein.
Homologues of the SMP protein can be generated by mutagenesis, e.g., discrete
point mutation or truncation of the SMP protein. As used herein, the term
"homologue"
refers to a variant form of the SMP protein which acts as an agonist or
antagonist of the
activity of the SMP protein. An agonist of the SMP protein can retain
substantially the
same, or a subset, of the biological activities of the SMP protein. An
antagonist of the
SMP protein can inhibit one or more of the activities of the naturally
occurring form of
the SMP protein, by, for example, competitively binding to a downstream or
upstream
member of the sugar molecule metabolic cascade or the energy-producing pathway
which includes the SMP protein.
In an alternative embodiment, homologues of the SMP protein can be identified
by screening combinatorial libraries of mutants, e.g., truncation mutants, of
the SMP
protein for SMP protein agonist or antagonist activity. In one embodiment, a
variegated
library of SMP variants is generated by combinatorial mutagenesis at the
nucleic acid
level and is encoded by a variegated gene library. A variegated library of SMP
variants
can be produced by, for example, enzymatically ligating a mixture of synthetic
oligonucleotides into gene sequences such that a degenerate set of potential
SMP
CA 02593287 2007-07-27
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sequences is expressible as individual polypeptides, or alternatively, as a
set of larger
fusion proteins (e.g., for phage display) containing the set of SMP sequences
therein.
There are a variety of methods which can be used to produce libraries of
potential SMP
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 SMP 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 SMP protein coding can be used to
generate a variegated population of SMP fragments for screening and subsequent
selection of homologues of an SMP protein. In one embodiment, a library of
coding
sequence fragments can be generated by treating a double stranded PCR fragment
of an
SMP 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 SMP protein.
Several techniques are known in the art for screening gene products of
combinatorial libraries made by point mutations or truncation, and for
screening cDNA
libraries for gene products having a selected property. Such techniques are
adaptable for
rapid screening of the gene libraries generated by the combinatorial
mutagenesis of SMP
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
CA 02593287 2007-07-27
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frequency of functional mutants in the libraries, can be used in combination
with the
screening assays to identify SMP 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 SMP 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 SMP
protein
regions required for function; modulation of an SMP protein activity;
modulation of the
metabolism of one or more sugars; modulation of high-energy molecule
production in a
cell (i.e., ATP, NADPH); and modulation of cellular production of a desired
compound,
such as a fine chemical.
The SMP nucleic acid molecules of the invention have a variety of uses. First,
they may be used to identify an organism as being Corynebacterium glutamicum
or a
close relative thereof. Also, they may be used to identify the presence of C.
glutamicum
or a relative thereof in a mixed population of microorganisms. The invention
provides
the nucleic acid sequences of a number of C. glutamicum genes; by probing the
extracted genomic DNA of a culture of a unique or mixed population of
microorganisms
under stringent conditions with a probe spanning a region of a C. glutamicum
gene
which is unique to this organism, one can ascertain whether this organism is
present.
Although Corynebacterium glutamicum itself is nonpathogenic, it is related to
pathogenic species, such as Corynebacterium diphtheriae. Corynebacterium
diphtheriae
is the causative agent of diphtheria, a rapidly developing, acute, febrile
infection which
involves both local and systemic pathology. In this disease, a local lesion
develops in
the upper respiratory tract and involves necrotic injury to epithelial cells;
the bacilli
secrete toxin which is disseminated through this lesion to distal susceptible
tissues of the
body. Degenerative changes brought about by the inhibition of protein
synthesis in
these tissues, which include heart, muscle, peripheral nerves, adrenals,
kidneys, liver and
CA 02593287 2007-07-27
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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 diphiheriae 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. glutarnicum genome could be digested, and the fragments
incubated
with the DNA-binding protein. Those which bind the protein may be additionally
probed
with the nucleic acid molecules of the invention, preferably with readily
detectable
labels; binding of such a nucleic acid molecule to the genome fragment enables
the
localization of the fragment to the genome map of C. glutamicum, and, when
performed
multiple times with different enzymes, facilitates a rapid determination of
the nucleic
acid sequence to which the protein binds. Further, the nucleic acid molecules
of the
invention may be sufficiently homologous to the sequences of related species
such that
these nucleic acid molecules may serve as markers for the construction of a
genomic
map in related bacteria, such as Brevibacterium lactofermentum.
The SMP nucleic acid molecules of the invention are also useful for
evolutionary
and protein structural studies. The metabolic and energy-releasing 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
CA 02593287 2007-07-27
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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 SMP nucleic acid molecules of the invention may result in
the production of SMP proteins having functional differences from the wild-
type SMP
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 SMP protein, either by interacting with the protein itself or a
substrate or
binding partner of the SMP protein, or by modulating the transcription or
translation of
an SMP nucleic acid molecule of the invention. In such methods, a
microorganism
expressing one or more SMP 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 SMP protein is assessed.
There are a number of mechanisms by which the alteration of an SMP 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.
The degradation of high-energy carbon molecules such as sugars, and the
conversion of
compounds such as NADH and FADH2 to more useful fon;ns via oxidative
phosphorylation results in a number of compounds which themselves may be
desirable
fine chemicals, such as pyruvate, ATP, NADH, and a number of intermediate
sugar
compounds. Further, the energy molecules (such as ATP) and the reducing
equivalents
(such as NADH or NADPH) produced by these metabolic pathways are utilized in
the
cell to drive reactions which would otherwise be energetically unfavorable.
Such
unfavorable reactions include many biosynthetic pathways for fine chemicals.
By
improving the ability of the cell to utilize a particular sugar (e,g., by
manipulating the
genes encoding enzymes involved in the degradation and conversion of that
sugar into
energy for the cell), one may increase the amount of energy available to
permit
CA 02593287 2007-07-27
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unfavorable, yet desired metabolic reactions (e.g., the biosynthesis of a
desired fine
chemical) to occur.
Further, modulation of one or more pathways involved in sugar utilization
permits optimization of the conversion of the energy contained within the
sugar
molecule to the production of one or more desired fine chemicals. For example,
by
reducing the activity of enzymes involved in, for example, gluconeogenesis,
more ATP
is available to drive desired biochemical reactions (such as fine chemical
biosyntheses)
in the cell. Also, the overall production of energy molecules from sugars may
be
modulated to ensure that the cell maximizes its energy production from each
sugar
molecule. Inefficient sugar utilization can lead to excess COz production and
excess
energy, which may result in futile metabolic cycles. By improving the
metabolism of
sugar molecules, the cell should be able to function more efficiently, with a
need for
fewer carbon molecules. This should result in an improved fine chemical
product: sugar
molecule ratio (improved carbon yield), and permits a decrease in the amount
of sugars
that must be added to the medium in large-scale fermentor culture of such
engineered C.
glutamicum.
The mutagenesis of one or more SMP genes of the invention may also result in
SMP proteins having altered activities which indirectly impact the production
of one or
more desired fine chemicals from C. glutamicum. For example, by increasing the
efficiency of utilization of one or more sugars (such that the conversion of
the sugar to
useful energy molecules is improved), or by increasing the efficiency of
conversion of
reducing equivalents to useful energy molecules (e.g., by improving the
efficiency of
oxidative phosphorylation, or the activity of the ATP synthase), one can
increase the
amount of these high-energy compounds available to the cell to drive normally
unfavorable metabolic processes. These processes include the construction of
cell walls,
transcription, translation, and the biosynthesis of compounds necessary for
growth and
division of the cells (e.g., nucleotides, amino acids, vitamins, lipids, etc.)
(Lengeler et al.
(1999) Biology of Prokaryotes, Thieme Verlag: Stuttgart, p. 88-109; 913-918;
875-899).
By improving the growth and multiplication of these engineered cells, it is
possible to
increase both the viability of the cells in large-scale culture, and also to
improve their
rate of division, such that a relatively larger number of cells can survive in
fermentor
culture. The yield, production, or efficiency of production may be increased,
at least
CA 02593287 2007-07-27
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due to the presence of a greater number of viable cells, each producing the
desired fine
chemical.
Further, many of the degradation products produced during sugar metabolism are
themselves utilized by the cell as precursors or intermediates for the
production of a
number of other useful compounds, some of which are fine chemicals. For
example,
pyruvate is converted into the amino acid alanine, and ribose-5-phosphate is
an integral
part of, for example, nucleotide molecules. The amount and efficiency of sugar
metabolism, then, has a profound effect on the availability of these
degradation products
in the cell. By increasing the ability of the cell to process sugars, either
in terms of
efficiency of existing pathways (e.g., by engineering enzymes involved in
these
pathways such that they are optimized in activity), or by increasing the
availability of
the enzymes involved in such pathways (e.g., by increasing the number of these
enzymes present in the cell), it is possible to also increase the availability
of these
degradation products in the cell, which should in turn increase the production
of many
different other desirable compounds in the cell (e.g., fine chemicals).
The aforementioned mutagenesis strategies for SMP 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 SMP 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 product produced by C. glutamicum,
which includes the final products of biosynthesis pathways and intermediates
of
naturally-occurring metabolic pathways, as well as molecules which do not
naturally
occur in the metabolism of C. glutamicum, but which are produced by a C.
glutamicum
strain of the invention.
This invention is further illustrated by the following examples which should
not
be construed as limiting.
CA 02593287 2007-07-27
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CA 02593287 2007-07-27
-86-
TABLE 3: Corynebacterium and Brevibacterium Strains Which May be Used in
the Practice of the Invention
Brevibacterium ammoniagenes 2i054
Brevibacterium ammoniagenes 19350
Brevibacterium ammoniagenes 19351
Brevibacterium ammoniagenes 19352
Brevibacterium ammoniagenes 19353
Brevibacterium ammoniagenes 19354
Brevibacterium ammoniagenes 19355
Brevibacterium ammoniagenes 19356
Brevibacterium ammoniagenes 21055
Brevibacterium ammoniagenes 21077
Brevibacterium ammoniagenes 21553
Brevibacterium ammoniagenes 21580
Brevibacterium ammoniagenes 39101
Brevibacterium butanicum 21196
Brevibacterium divaricatum 21792 P928
Brevibacterium flavum 21474
Brevibacterium flavum 21129
Brevibacterium flavum 21518
Brevibacterium flavum B 11474
Brevibacterium flavum B 11472
Brevibacterium flavum 21127
Brevibacterium flavum 21128
Brevibacterium flavum 21427
Brevibacterium flavum 21475
Brevibacterium flavum 21517
Brevibacterium flavum 21528
Brevibacterium flavum 21529
Brevibacterium flavum B11477
Brevibacterium flavum B 11478
Brevi acterium flavum 21127
Brevibacterium flavum B 11474
Brevibacterium heatii 15527
Brevibacterium ketoglutamicum 21004
Brevibacterium ketoglutamicum 21089
Brevibacterium ketosoreductum 21914
Brevtbacterium actofermentum 70
Brevibacterium actofermentum 74
Brevibacterium actofermentum 77
Brevibacterium actofertnentum 21798
Brevibacterium actoferrnentum 21799
Brevibacterium actofermentum 21800
Brevt cterium acto ermentum 21801
Brevibacterium lactofennentum B11470
Brevibacterium lactofermentum B 11471
CA 02593287 2007-07-27
-87-
(~u3 FERM ' NR1ti:. CEGT = C11MB CW;: NCTG DSm
Brevibacterium lactofetmentum 21086
Brevibacterium lactofermentum 21420
Brevibacterium actofermentum 21086
Brevibacterium iactofermentum 312 9
Brevibacterium nens 9174
Bravibacterium nens 19391
Brevibacterium nens 8377
Brevibacterium paraffinolyticum 11160
Brevibacterium spec. 717.73
Brevibacterium spec. 717.73
Brevibacterium spec. 14604
Brevibacterium spec. 21860
Brevibacterium spec. 21864
Brevibacterium spec. 21865
Brevibacterium spec. 21866
Bnevibacterium spec. 19240
Corynebacterium acetoacidophilum 21476
Corynebacterium acetoacidophilum 13870
Corynebacterium acetoglutamicum BI1473
Corynebacterium acetoglutamicum B11475
Corynebacterium acetoglutamicum 15806
Corynebacterium acetoglutamicum 21491
Corynebacterium acetoglutamicum 31270
Corynebacterium acetophilum B3671
Corynebacterium ammontagenes 6872 2399
Corynebacterium ammoniagenes 15511
Corynebacterium fi-jiokense 21496
Corynebacterium glutamicum 14067
Corynebacterium glutamicum 39137
Corynebacterium glutamicum 21254
Corynebacterium glutamicum 21255
Corynebacterium glutamicum 31830
Corynebacterium glutamicum 13032
Corynebacterium glutamicum 14305
Corynebacterium glutamicum 15455
Corynebactertum glutamicum 13058
Corynebacterium glutamicum 13059
Corynebacterium glutamicum 13060
Corynebacterium glutamicum 21492
Corynebacterium glutamicum 21513
Corynebacterium g utamicum 21526
Corynebacterium glutamicum 21543
Corynebacterium glutamicum 13287
Corynebacterium glutamicum 21851
Corynebacterium glutamicum 21253
Corynebacterium glutamicum 21514
Corynebacterium glutamicum 21516
Corynebacterium glutamicum 21299
CA 02593287 2007-07-27
-88-
.. : _. .. __.
:.; . , .. .:.,
usss.-, s es.:: _;;:~; x!.=. .'. ,=.' _ 4TN
'
Corynebacterium glutamicum 21300
Corynebacterium glutamicum 39684
Corynebacterium glutamicum 21488
Corynebactertum glutamicum 21649
Corynebacterium glutamicum 21650
Corynebacterium glutamicum 19223
Corynebacterium glutamieum 13869
Corynebacterium glutamicum 21157
Corynebacterium glutamicum 21158
Corynebacterium glutamicum 21159
Corynebacterium glutamicum 21355
Corynebacterium glutamicum 31808
Corynebacterium glutamicum 21674
Corynebacterium glutamicum 21562
Corynebacterium glutamicum 21563
Corynebacterium glutamicum 21564
Corynebacterium glutamicum 21565
Corynebacterium glutamicum 21566
Corynebacterium glutamicum 21567
Corynebacterium glutamicum 21568
Corynebacterium glutamicum 21569
Corynebacterium glutamicum 21570
Corynebacterium glutamicum 21571
Corynebacterium glutamicum 21572
Corynebacterium glutamicum 21573
Corynebacterium glutamicum 21579
Corynebacterium glutamicum 19049
Corynebacterium glutamicum 19050
Corynebacterium glutamicum 19051
Corynebacterium glutam icum 19052
Corynebacterium glutamicum 19053
Corynebacterium glutamicum 19054
Corynebacterium glutamicum 19055
Corynebacterium glutamicum 19056
Corynebacterium glutamicum 19057
Corynebacterium glutamicum 19058
Corynebacterium glutamicum 19059
Corynebacterium glutamicum 19060
Corynebacterium glutamicum 19185
Coryne acterium glutamicum 13286
Corynebacterium glutamicum 21515
Corynebacterium glutamicum 21527
Corynebacterium glutamicum 21544
Corynebacterium glutamicum 21492
Corynebacterium glutamicum B8183
Corynebacterium glutamicum B8182
Corynebacterium glutamicum B124I6
Corynebacterium glutamicum B12417
CA 02593287 2007-07-27
-89-
. .......... ._, ._. .
' P2~
Corynebacterium glutamicum B12418
Corynebacterium glutamicum B11476
Corynebacterium glutamicum 21608
Corynebacterium lilium P973
Corynebacterium nitrilophilus 21419 11594
Corynebacterium spec. P4445
Corynebacterium spec. P4446
Corynebacterium spec. 31088
Corynebacterlum spec. 31089
Corynebacterium spec. 31090
Corynebacterium spec. 31090
Corynebacterium spec. 31090
Corynebacterium spec. 15954 20145
Corynebacterium spec. 21857
Corynebacterium spec. 21862
Corynebacterium spec. 21863
ATCC: American Type Culture Collection, Rockville, MD, USA
FERM: Fermentation Research Institute, Chiba, Japan
NRRL: ARS Culture Collection, Northern Regional Research Laboratory, Peoria,
IL, USA
CECT: Coleccion Espanola de Cultivos Tipo, Valencia, Spain
NCIMB: National Collection of Industrial and Marine Bacteria Ltd., Aberdeen,
UK
CBS: Centraalbureau voor Schimmelcultures, Baam, NL
NCTC: National Collection of Type Cultures, London, UK
DSMZ: Deutsche Sammiung von Mikroorganismen und Zellkulturen, Braunschweig,
Gennany
For reference see Sugawara, H. et al. (1993) World directory of collections of
cultures of
microorganisms: Bacteria, fungi and yeasts (41 edn), World federation for
culture collections world
data center on microorganisms, Saimata, Japen.
CA 02593287 2007-07-27
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CA 02593287 2007-07-27
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CA 02593287 2007-07-27
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CA 02593287 2007-07-27
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CA 02593287 2007-07-27
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CA 02593287 2007-07-27
- 118-
Exempliiication
Example 1: Preparation of total genomic DNA of Corynebacteriumglutamicum
ATCC 13032
A culture of Corynebacterium glutamicum (ATCC 13032) was grown overnight
at 30 C with vigorous shaking in BHI medium (Difco). The cells were harvested
by
centrifugation, the supematant 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/l
sucrose,
2.46 g/l MgSO4 x 7H2O, 10 ml/I. KH2PO4 solution (100 g/l, adjusted to pH 6.7
with
KOH), 50 ml/I. M12 concentrate (10 g/1(NH.)2S0., I g/1 NaCI, 2 g/l MgSO, x
7H2O,
0.2 g/l CaCI2, 0.5 g/1 yeast extract (Difco), 10 ml/1 trace-elements-mix (200
mg/l FeSO4
x H2O, 10 mg/1 ZnSO4 x 7 H2O, 3 mg/1 MnCI2 x 4 H2O, 30 mg/1 H,BO3 20 mg/1
CoC12 x
6 H2O, 1 mg/l NiCl2 x 6 HZO, 3 mg/1 Na2MoO4 x 2 H1O, 500 mg/I complexing agent
(EDTA or critic acid), 100 ml/I vitamins-mix (0.2 mg/I biotin, 0.2 mg/1 folic
acid, 20
mg/1 p-amino benzoic acid, 20 mg/1 riboflavin, 40 mg/I 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-HCl,1 mM EDTA, pH 8). The
pellet was resuspended in 4 ml TE-buffer and 0.5 ml SDS solution (10%) and 0.5
ml
NaCI solution (5 M) are added. After adding of proteinase K to a final
concentration of
200 g/ml, the suspension is incubated for ca.18 h at 37 C. The DNA was
purified by
extraction with phenol, phenol-chloroform-isoamylalcohol and chloroform-
isoamytalcohol 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 I 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 02593287 2007-07-27
- 119-
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 southem
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. el al. (1995) "Whole-
genome
Random Sequencing and Assembly of Haemophilus Influenzae Rd., Science, 269:496-
512). Sequencing primers with the following nucleotide sequences were used: 5'-
GGAAACAGTATGACCATG-3' (SEQ ID NO:783) or 5'-GTAAAACGACGGCCAGT-3' (SEQ ID
NO:784).
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 02593287 2007-07-27
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the integrity of their genetic information. Typical mutator strains have
mutations in the genes
for the DNA repair system (e.g., mutHLS, mutD, mutT, etc.; for reference, see
Rupp, W.D.
(1996) DNA repair mechanisms, in: Escherichia coli and Salmonella, p. 2277-
2294, ASM:
Washington.) Such strains are well known to those of ordinary skill in the
art. The use of such
strains is illustrated, for example, in Greener, A. and Callahan, M. (1994)
Strategies 7: 32-34.
Example 5: DNA Transfer Between Escherichia coli and Corynebacteriunt
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 Corynebacierium glulamicum 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. glutamieum, and which can be used for several purposes, including
gene over-
expression (for reference, see e.g., Yoshihama, M. el 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. Transfonmation of C. glutamicum can be achieved by
protoplast transformation (Kastsumata, R. et al. (1984) J. Bacteriol. 159306-
311),
electroporation (Liebl, E. et al. (1989) FEMS Microbiol. Letters, 53:399-303)
and in cases
where special vectors are used, also by conjugation (as described e.g. in
Schtifer, A et al.
CA 02593287 2007-07-27
- 121 -
(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
Corynebacterium or Brevibacterium species may be accomplished by well-known
methods, such as homologous recombination with genomic region(s), restriction
endonuclease mediated integration (REMI) (see, e.g., DE Patent 19823834), or
through
the use of transposons. It is also possible to modulate the activity of a gene
of interest by
modifying the regulatory regions (e.g., a promoter, a repressor, and/or an
enhancer) by
sequence modification, insertion, or deletion using site-directed methods
(such as
homologous recombination) or methods based on random events (such as
transposon
mutagenesis or REMI). Nucleic acid sequences which function as transcriptional
terminators may also be inserted 3' to the coding region of one or more genes
of the
invention; such terminators are well-known in the art and are described, for
example, in
Winnacker, E.L. (1987) From Genes to Clones - Introduction to Gene Technology.
VCH:
Weinheim.
Example 6: Assessment of the Expression of the Mutant Protein
Observations of the activity of a mutated protein in a transformed host cell
rely on
the fact that the mutant protein is expressed in a similar fashion and in a
similar quantity
to that of the wild-type protein. A useful method to ascertain the level of
transcription of
the mutant gene (an indicator of the amount of mRNA available for translation
to the gene
product) is to perform a Northern blot (for reference see, for example,
Ausubel et al.
CA 02593287 2007-07-27
- 122 -
(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. Microbrol. 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, Wiley: New York). In
this process,
total cellular proteins are extracted, separated by gel electrophoresis,
transferred to a
matrix such as nitrocellulose, and incubated with a probe, such as an
antibody, which
specifically binds to the desired protein. This probe is generally tagged with
a
chemiluminescent or colorimetric label which may be readily detected. The
presence and
quantity of label observed indicates the presence and quantity of the desired
mutant
protein present in the cell.
Example 7: Growth of Genetically Modified Corynebacterium glutamicum - Media
and Culture Conditions
Genetically modified Corynebacteria are cultured in synthetic or natural
growth
media. A number of different growth media for Corynebacteria are both well-
known and
readily available (Lieb et a!. (1989) Appl. Microbiol. Biotechnol., 32:205-2
10; von der
Osten et al. (1998) Biotechnology Let[ers, 11:11-16; Patent DE 4,120,867;
Liebl (1992)
"The Genus Corynebacterium, in: The Procaryotes, Volume 11, Balows, A. et al.,
eds.
Springer-Verlag). These media consist of one or more carbon sources, nitrogen
sources,
inorganic salts, vitamins and trace elements. Preferred carbon sources are
sugars, such as
mono-, di-, or polysaccharides. For example, glucose, fructose, mannose,
galactose,
ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or
cellulose serve as
very good carbon sources. It is also possible to supply sugar to the media via
complex
compounds such as molasses or other by-products from sugar refinement. It can
also be
CA 02593287 2007-07-27
- 123 -
advantageous to supply mixtures of different carbon sources. Other possible
carbon
sources are alcohols and organic acids, such as methanol, ethanol, acetic acid
or lactic
acid. Nitrogen sources are usually organic or inorganic nitrogen compounds, or
materials
which contain these compounds. Exemplary nitrogen sources include ammonia gas
or
ammonia salts, such as NH.CI or (NH.)2SO4, NH4OH, nitrates, urea, amino acids
or
complex nitrogen sources like com 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 l(Merck) or BHI (grain heart infusion, DIFCO) or others.
All medium components are sterilized, either by heat (20 minutes at 1.5 bar
and
121 C) or by sterile filtration. The components can either be sterilized
together or, if
necessary, separately. All media components can be present at the beginning of
growth,
or they can optionally be added continuously or batchwise.
Culture conditions are defined separately for each experiment. The temperature
should be in a range between 15'C and 45'C. The temperature can be kept
constant or can
be altered during the experiment. The pH of the medium should be in the range
of 5 to
8.5, preferably around 7.0, and can be maintained by the addition of buffers
to the media.
An exemplary buffer for this purpose is a potassium phosphate buffer.
Synthetic buffers
such as MOPS, HEPES, ACES and others can alternatively or simultaneously be
used. It
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is also possible to maintain a constant culture pH through the addition of
NaOH or
NH,OH during growth. If complex medium components such as yeast extract are
utilized,
the necessity for additional buffers may be reduced, due to the fact that many
complex
compounds have high buffer capacities. If a fermentor is utilized for
culturing the micro-
organisms, the pH can also be controlled using gaseous ammonia.
The incubation time is usually in a range from several hours to several days.
This
time is selected in order to permit the maximal amount of product to
accumulate in the
broth. The disclosed growth experiments can be carried out in a variety of
vessels, such as
microtiter plates, glass tubes, glass flasks or glass or metal fermentors of
different sizes.
For screening a large number of clones, the microorganisms should be cultured
in
microtiter plates, glass tubes or shake flasks, either with or without
baffles. Preferably
100 mi shake flasks are used, filled with 10% (by volume) of the required
growth
medium. The flasks should be shaken on a rotary shaker (amplitude 25 mm) using
a
speed-range of 100 - 300 rpm. Evaporation losses can be diminished by the
maintenance
of a humid atmosphere; alternatively, a mathematical correction for
evaporation losses
should be performed.
If genetically modified clones are tested, an unmodified control clone or a
control
clone containing the basic plasmid without any insert should also be tested.
The medium
is inoculated to an OD6oo of 0.5 - 1.5 using cells grown on agar plates, such
as CM plates
(10 g/1 glucose, 2,5 g/l NaCI, 2 g/1 urea, 10 g/l polypeptone, 5 g/1 yeast
extract, 5 g/l meat
extract, 22 g/l NaCI, 2 g/l urea, 10 g/l polypeptone, 5 g/1 yeast extract, 5
g/l meat extract,
22 g/l agar, pH 6.8 with 2M NaOH) that had been incubated at 30 C. Inoculation
of the
media is accomplished by either introduction of a saline suspension of C.
glutamicum cells
from CM plates or addition of a liquid preculture of this bacterium.
Example 8- In vitro Analysis of the Function of Mutant Proteins
The determination of activities and kinetic parameters of enzymes is well
established in the art. Experiments to determine the activity of any given
altered
enzyme must be tailored to the specific activity of the wild-type enzyme,
which is well
within the ability of one of ordinary skill in the art. Overviews about
enzymes in
general, as well as specific details concerning structure, kinetics,
principles, methods,
applications and examples for the determination of many enzyme activities may
be
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found, for example, in the following references: Dixon, M., and Webb, E.C.,
(1979)
Enzymes. Longmans: London; Fersht, (1985) Enzyme Structure and Mechanism.
Freeman: New York; Walsh, (1979) Enzymatic Reaction Mechanisms. Freeman: San
Francisco; Price, N.C., Stevens, L. (1982) Fundamentals of Enzymology. Oxford
Univ.
Press: Oxford; Boyer, P.D., ed. (1983) The Enzymes, 3'd ed. Academic Press:
New
York; Bisswanger, H., (1994) Enzymkinetik, 2"d ed. VCH: Weinheim (ISBN
3527300325); Bergmeyer, H.U., Bergmeyer, J., Grafil, M., eds. (1983-1986)
Methods of
Enzymatic Analysis, 3rd ed., vol. I-XII, Verlag Chemie: Weinheim; and
Ullmann's
Encyclopedia of Industrial Chemistry (1987) vol. A9, "Enzymes". VCH: Weinheim,
p.
352-363.
The activity of proteins which bind to DNA can be measured by several well-
established methods, such as DNA band-shift assays (also called gel
retardation assays).
The effect of such proteins on the expression of other molecules can be
measured using
reporter gene assays (such as that described in Kolmar, H. et al. (1995) EMBO
J. 14:
3895-3904 and references cited therein). Reporter gene test systems are well
known and
established for applications in both pro- and eukaryotic cells, using enzymes
such as
beta-galactosidase, green fluorescent protein, and several others.
The determination of activity of membrane-transport proteins can be performed
according to techniques such as those described in Gennis, R.B. (1989) "Pores,
Channels and Transporters", in Biomembranes, Molecular Structure and Function,
Springer: Heidelberg, p. 85-137; 199-234; and 270-322.
Example 9: Analysis of Impact of Mutant Protein on the Production of the
Desired
Product
The effect of the genetic modification in C. glutamicum on production of a
desired compound (such as an amino acid) can be assessed by growing the
modified
microorganism under suitable conditions (such as those described above) and
analyzing
the medium and/or the cellular component for increased production of the
desired
product (i.e., an amino acid). Such analysis techniques are well known to one
of
ordinary skill in the art, and include spectroscopy, thin layer
chromatography, staining
methods of various kinds, enzymatic and microbiological methods, and
analytical
chromatography such as high performance liquid chromatography (see, for
example,
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Ullman, Encyclopedia of Industrial Chemistry, vol. A2, p. 89-90 and p. 443-
613, VCH:
Weinheim (1985); Fallon, A. et al., (1987) "Applications of HPLC in
Biochemistry" in:
Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17; Rehm et
al.
(1993) Biotechnology, vol. 3, Chapter III: "Product recovery and
purification", page
469-714, VCH: Weinheim; Belter, P.A. et al. (1988) Bioseparations: downstream
processing for biotechnology, John Wiley and Sons; Kennedy, J.F. and Cabral,
J.M.S.
(1992) Recovery processes for biological materials, John Wiley and Sons;
Shaeiwitz,
J.A. and Henry, J.D. (1988) Biochemical separations, in: Ulmann's Encyclopedia
of
Industrial Chemistry, vol. B3, Chapter 11, page 1-27, VCH: Weinheim; and
Dechow,
F.J. (1989) Separation and purification techniques in biotechnology, Noyes
Publications.)
In addition to the measurement of the final product of fermentation, it
is.also
possible to analyze other components of the metabolic pathways utilized for
the
production of the desired compound, such as intermediates and side-products,
to
determine the overall 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 supematant 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
supemate fraction is retained for further purification.
The supematant 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.
el 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. Nall. 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 SMP 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 SMP 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. 13iosci. 10:3-5; and FASTA,
described in
Pearson and Lipman (1988) P.N.A.S. 85:2444-8.
The percent homology between two amino acid sequences can also be
accomplished using the GAP program in the GCG software package (available at
http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a
gap
weight of 12, 10, 8, 6, or 4 and a length weight of 2; 3, or 4. The percent
homology
between two nucleic acid sequences can be accomplished using the GAP program
in the
GCG software package, using standard parameters, such as a gap weight of 50
and a
length weight of 3.
A comparative analysis of the gene sequences of the invention with those
present
in Genbank has been performed using techniques known in the art (see, e.g.,
Bexevanis
and Ouellette, eds. (1998) Bioinformatics: A Practical Guide to the Analysis
of Genes
and Proteins. John Wiley and Sons: New York). The gene sequences of the
invention
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were compared to genes present in Genbank in a three-step process. In a first
step, a
BLASTN analysis (e.g., a local alignment analysis) was performed for each of
the
sequences of the invention against the nucleotide sequences present in
Genbank, and the
top 500 hits were retained for further analysis. A subsequent FASTA search
(e.g., a
combined local and global alignment analysis, in which limited regions of the
sequences
are aligned) was performed on these 500 hits. Each gene sequence of the
invention was
subsequently globally aligned to each of the top three FASTA hits, using the
GAP
program in the GCG software package (using standard parameters). In order to
obtain
correct results, the length of the sequences extracted from Genbank were
adjusted to the
length of the query sequences by methods well-known in the art. The results of
this
analysis are set forth in Table 4. The resulting data is identical to that
which would have
been obtained had a GAP (global) analysis alone been performed on each of the
genes of
the invention in comparison with each of the references in Genbank, but
required
significantly reduced computational time as compared to such a database-wide
GAP
(global) analysis. Sequences of the invention for which no alignments above
the cutoff
values were obtained are indicated on Table 4 by the absence of alignment
information.
It will further be understood by one of ordinary skill in the art that the GAP
alignment
homology percentages set forth in Table 4 under the heading "% homology (GAP)"
are
listed in the European numerical 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 polymerase chain reaction. The
choice
and design of the 5' or 3' oligonucleotide primers or of appropriate linkers
allows the
covalent attachment of the resulting PCR products to the surface of a support
medium
described above (and also described, for example, Schena, M. et al. (1995)
Science 270:
467-470).
Nucleic acid microarrays may also be constructed by in situ oligonucleotide
synthesis as described by Wodicka, L. et al. (1997) Nature Biotechnology 15:
1359-
1367. By photolithographic methods, precisely defined regions of the matrix
are
exposed to light. Protective groups which are photolabile are thereby
activated and
undergo nucleotide addition, whereas regions that are masked from light do not
undergo
any modification. Subsequent cycles of protection and light activation permit
the
synthesis of different oligonucleotides at defined positions. Small, defined
regions of
the genes of the invention may be synthesized on microarrays by solid phase
oligonucleotide synthesis.
The nucleic acid molecules of the invention present in a sample or mixture of
nucleotides may be hybridized to the microarrays. These nucleic acid molecules
can be
labeled according to standard methods. In brief, nucleic acid molecules (e.g.,
mRNA
molecules or DNA molecules) are labeled by the incorporation of isotopically
or
fluorescently labeled nucleotides, e.g., during reverse transcription or DNA
synthesis.
Hybridization of labeled nucleic acids to microarrays is described (e.g., in
Schena, M. et
al. (1995) supra; Wodicka, L. et al. (1997), supra; and DeSaizieu A. et al.
(1998),
supra). The detection and quantification of the hybridized molecule are
tailored to the
specific incorporated label. Radioactive labels can be detected, for example,
as
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described in Schena, M. et al. (1995) supra) and fluorescent labels may be
detected, for
example, by the method of Shalon et al. (1996) Genome Research 6: 639-645).
The application of the sequences of the invention to DNA microarray
technology, as described above, permits comparative analyses of different
strains of C.
glutamicum or other Corynebacteria. For example, studies of inter-strain
variations
based on individual transcript profiles and the identification of genes that
are important
for specific and/or desired strain properties such as pathogenicity,
productivity and
stress tolerance are facilitated by nucleic acid array methodologies. Also,
comparisons
of the profile of expression of genes of the invention during the course of a
fermentation
reaction are possible using nucleic acid array technology.
Example 13: Analysis of the Dynamics of Cellular Protein Populations
(Proteomics)
The genes, compositions, and methods of the invention may be applied to study
the interactions and dynamics of populations of proteins, termed 'proteomics'.
Protein
populations of interest include, but are not limited to, the total protein
population of C.
glutamicum (e.g., in comparison with the protein populations of other
organisms), those
proteins which are active under specific environmental or metabolic conditions
(e.g.,
during fermentation, at high or low temperature, or at high or low pH), or
those proteins
which are active during specific phases of growth and development.
Protein populations can be analyzed by various well-known techniques, such as
gel electrophoresis. Cellular proteins may be obtained, for example, by lysis
or
extraction, and may be separated from one another using a variety of
electrophoretic
techniques. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE)
separates proteins largely on the basis of their molecular weight. Isoelectric
focusing
polyacrylamide gel electrophoresis (IEF-PAGE) separates proteins by their
isoelectric
point (which reflects not only the amino acid sequence but also
posttranslational
modifications of the protein). Another, more preferred method of protein
analysis is the
consecutive combination of both IEF-PAGE and SDS-PAGE, known as 2-D-gel
electrophoresis (described, for example, in Hermann et al. (1998)
Electrophoresis 19:
3217-3221; Fountoulakis et al. (1998) Electrophoresis 19: 1193-1202; Langen et
al.
(1997) Electrophoresis 18: 1184-1192; Antelmann et al. (1997) Electrophoresis
18 :
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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, 35 S-cysteine, "C-labelled amino acids, 15N-
amino
acids, 15NO3 or 'SNH4+ or 13C-labelled amino acids) in the medium of C.
glutamicum
permits the labeling of proteins from these cells prior to their separation.
Similarly,
fluorescent labels may be employed. These labeled proteins can be extracted,
isolated
and separated according to the previously described techniques.
Proteins visualized by these techniques can be further analyzed by measuring
the
amount of dye or label used. The amount of a given protein can be determined
quantitatively using, for 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)
Electraphoresis 18:
1184-1192)). The protein sequences provided herein can be used for the
identification
of C. glulamicum proteins by these techniques.
The information obtained by these methods can be used to compare pattems of
protein presence, activity, or modification between different samples from
various
biological conditions (e.g., different organisms, time points of fermentation,
media
conditions, or different biotopes, among others). Data obtained from such
experiments
alone, or in combination with other techniques, can be used for various
applications,
such as to compare the behavior of various organisms in a given (e.g.,
metabolic)
situation, to increase the productivity of strains which produce fine
chemicals or to
increase the efficiency of the production of fine chemicals.
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Equivalents
Those of ordinary skill in the art will recognize, or will be able to
ascertain using
no more than routine experimentation, many equivalents to the specific
embodiments of
the invention described herein. Such equivalents are intended to be
encompassed by the
following claims.
