U.S. patent application number 11/061298 was filed with the patent office on 2005-09-01 for corynebacterium glutamicum genes encoding phosphoenolpyruvate: sugar phosphotransferase system proteins.
This patent application is currently assigned to BASF AG. Invention is credited to Haberhauer, Gregor, Kroger, Burkhard, Pompejus, Markus, Schroder, Hartwig, Zelder, Oskar.
Application Number | 20050191733 11/061298 |
Document ID | / |
Family ID | 34891344 |
Filed Date | 2005-09-01 |
United States Patent
Application |
20050191733 |
Kind Code |
A1 |
Pompejus, Markus ; et
al. |
September 1, 2005 |
Corynebacterium glutamicum genes encoding phosphoenolpyruvate:
sugar phosphotransferase system proteins
Abstract
Isolated nucleic acid molecules, designated PTS nucleic acid
molecules, which encode novel PTS proteins from Corynebacterium
glutamicum are described. The invention also provides antisense
nucleic acid molecules, recombinant expression vectors containing
PTS nucleic acid molecules, and host cells into which the
expression vectors have been introduced. The invention still
further provides isolated PTS proteins, mutated PTS proteins,
fusion proteins, antigenic peptides and methods for the improvement
of production of a desired compound from C. glutamicum based on
genetic engineering of PTS genes in this organism.
Inventors: |
Pompejus, Markus;
(Freinsheim, DE) ; Kroger, Burkhard;
(Limburgerhof, DE) ; Schroder, Hartwig; (Nussloch,
DE) ; Zelder, Oskar; (Speyer, DE) ;
Haberhauer, Gregor; (Limburgerhof, DE) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP.
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
BASF AG
Ludwigshafen
DE
|
Family ID: |
34891344 |
Appl. No.: |
11/061298 |
Filed: |
February 17, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11061298 |
Feb 17, 2005 |
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09604231 |
Jun 27, 2000 |
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6884614 |
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60142691 |
Jul 1, 1999 |
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60150310 |
Aug 23, 1999 |
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Current U.S.
Class: |
435/106 ;
435/194; 435/252.3; 435/471; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 1/205 20210501;
C12Y 207/01069 20130101; C07K 14/34 20130101; C12R 2001/15
20210501; C12P 1/04 20130101; C12N 9/00 20130101; C12N 9/1223
20130101; C12N 9/1205 20130101; C12P 13/04 20130101; C12P 1/04
20130101; C12R 1/15 20130101; C12P 1/04 20130101; C12R 2001/15
20210501; C12P 1/04 20130101; C12N 1/205 20210501 |
Class at
Publication: |
435/106 ;
435/069.1; 435/194; 435/252.3; 435/471; 536/023.2 |
International
Class: |
C12P 013/04; C07H
021/04; C12P 021/06; C12N 009/12; C12N 015/74; C12N 001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 3, 1999 |
DE |
19942095.5 |
Sep 3, 1999 |
DE |
19942097.1 |
Claims
What is claimed:
1. An isolated nucleic acid molecule from Corynebacterium
glutamicum encoding a phosphoenolpyruvate: sugar phosphotransferase
system protein, or a portion thereof, provided that the nucleic
acid molecule does not consist of any of the F-designated genes set
forth in Table 1.
2. The isolated nucleic acid molecule of claim 1, wherein said
phosphoenolpyruvate: sugar phosphotransferase system protein is
selected from the group consisting of proteins involved in the
transport of glucose, sucrose, mannose, fructose, mannitol,
raffinose, ribulose, ribose, lactose, maltose, sorbose, sorbitol,
xylose, and galactose.
3. An isolated Corynebacterium glutamicum nucleic acid molecule
selected from the group consisting of those sequences set forth in
Appendix A, or a portion thereof, provided that the nucleic acid
molecule does not consist of any of the F-designated genes set
forth in Table 1.
4. An isolated nucleic acid molecule which encodes a polypeptide
sequence selected from the group consisting of those sequences set
forth in Appendix B, provided that the nucleic acid molecule does
not consist of any of the F-designated genes set forth in Table
1.
5. An isolated nucleic acid molecule which encodes a naturally
occurring allelic variant of a polypeptide selected from the group
of amino acid sequences consisting of those sequences set forth in
Appendix B, provided that the nucleic acid molecule does not
consist of any of the F-designated genes set forth in Table 1.
6. An isolated nucleic acid molecule comprising a nucleotide
sequence which is at least 50% homologous to a nucleotide sequence
selected from the group consisting of those sequences set forth in
Appendix A, or a portion thereof, provided that the nucleic acid
molecule does not consist of any of the F-designated genes set
forth in Table 1.
7. An isolated nucleic acid molecule comprising a fragment of at
least 15 nucleotides of a nucleic acid comprising a nucleotide
sequence selected from the group consisting of those sequences set
forth in Appendix A, provided that the nucleic acid molecule does
not consist of any of the F-designated genes set forth in Table
1.
8. An isolated nucleic acid molecule which hybridizes to the
nucleic acid molecule of any one of claims 1-7 under stringent
conditions.
9. An isolated nucleic acid molecule comprising the nucleic acid
molecule of claim 1 or a portion thereof and a nucleotide sequence
encoding a heterologous polypeptide.
10. A vector comprising the nucleic acid molecule of claim 1.
11. The vector of claim 10, which is an expression vector.
12. A host cell transfected with the expression vector of claim
11.
13. The host cell of claim 12, wherein said cell is a
microorganism.
14. The host cell of claim 13, wherein said cell belongs to the
genus Corynebacterium or Brevibacterium.
15. The host cell of claim 12, wherein the expression of said
nucleic acid molecule results in the modulation in production of a
fine chemical from said cell.
16. The host cell of claim 15, wherein said fine chemical is
selected from the group consisting of: organic acids, proteinogenic
amino acids, nonproteinogenic amino acids, purine and pyrimidine
bases, nucleosides, nucleotides, lipids, saturated and unsaturated
fatty acids, diols, carbohydrates, aromatic compounds, vitamins,
cofactors, polyketides, and enzymes.
17. A method of producing a polypeptide comprising culturing the
host cell of claim 12 in an appropriate culture medium to, thereby,
produce the polypeptide.
18. An isolated phosphoenolpyruvate: sugar phosphotransferase
system polypeptide from Corynebacterium glutamicum, or a portion
thereof.
19. The protein of claim 18, wherein said phosphoenolpyruvate:
sugar phosphotransferase system protein is selected from the group
consisting of proteins involved in the transport of glucose,
sucrose, mannose, fructose, mannitol, raffinose, ribulose, ribose,
lactose, maltose, sorbose, and galactose.
20. An isolated polypeptide comprising an amino acid sequence
selected from the group consisting of those sequences set forth in
Appendix B, provided that the amino acid sequence is not encoded by
any of the F-designated genes set forth in Table 1.
21. An isolated polypeptide comprising a naturally occurring
allelic variant of a polypeptide comprising an amino acid sequence
selected from the group consisting of those sequences set forth in
Appendix B, or a portion thereof, provided that the amino acid
sequence is not encoded by any of the F-designated genes set forth
in Table 1.
22. The isolated polypeptide of claim 18, further comprising
heterologous amino acid sequences.
23. An isolated polypeptide which is encoded by a nucleic acid
molecule comprising a nucleotide sequence which is at least 50%
homologous to a nucleic acid selected from the group consisting of
those sequences set forth in Appendix A, provided that the nucleic
acid molecule does not consist of any of the F-designated nucleic
acid molecules set forth in Table 1.
24. An isolated polypeptide comprising an amino acid sequence which
is at least 50% homologous to an amino acid sequence selected from
the group consisting of those sequences set forth in Appendix B,
provided that the amino acid sequence is not encoded by any of the
F-designated genes set forth in Table 1.
25. A method for producing a fine chemical, comprising culturing a
cell containing a vector of claim 12 such that the fine chemical is
produced.
26. The method of claim 25, wherein said method further comprises
the step of recovering the fine chemical from said culture.
27. The method of claim 25, wherein said method further comprises
the step of transfecting said cell with the vector of claim 11 to
result in a cell containing said vector.
28. The method of claim 25, wherein said cell belongs to the genus
Corynebacterium or Brevibacterium.
29. The method of claim 25, wherein said cell is selected from the
group consisting of: Corynebacterium glutamicum, Corynebacterium
herculis, Corynebacterium, lilium, Corynebacterium
acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium
acetophilum, Corynebacterium ammoniagenes, Corynebacterium
fujiokense, Corynebacterium nitrilophilus, Brevibacterium
ammoniagenes, Brevibacterium butanicum, Brevibacterium divaricatum,
Brevibacterium flavum, Brevibacterium healii, Brevibacterium
ketoglutamicum, Brevibacterium ketosoreductum, Brevibacterium
lactofermentum, Brevibacterium linens, Brevibacterium
paraffinolyticum, and those strains set forth in Table 3.
30. The method of claim 25, wherein expression of the nucleic acid
molecule from said vector results in modulation of production of
said fine chemical.
31. The method of claim 25, wherein said fine chemical is selected
from the group consisting of: organic acids, proteinogenic amino
acids, nonproteinogenic amino acids, purine and pyrimidine bases,
nucleosides, nucleotides, lipids, saturated and unsaturated fatty
acids, diols, carbohydrates, aromatic compounds, vitamins,
cofactors, polyketides, and enzymes.
32. The method of claim 25, wherein said fine chemical is an amino
acid.
33. The method of claim 32, wherein said amino acid is drawn from
the group consisting of: lysine, glutamate, glutamine, alanine,
aspartate, glycine, serine, threonine, methionine, cysteine,
valine, leucine, isoleucine, arginine, proline, histidine,
tyrosine, phenylalanine, and tryptophan.
34. A method for producing a fine chemical, comprising culturing a
cell whose genomic DNA has been altered by the inclusion of a
nucleic acid molecule of any one of claims 1-9.
35. A method for diagnosing the presence or activity of
Corynebacterium diphtheriae in a subject, comprising detecting the
presence of one or more of the sequences set forth in Appendix A or
Appendix B in the subject, provided that the sequences are not or
are not encoded by any of the F-designated sequences set forth in
Table 1, thereby diagnosing the presence or activity of
Corynebacterium diphtheriae in the subject.
36. A host cell comprising a nucleic acid molecule selected from
the group consisting of the nucleic acid molecules set forth in
Appendix A, wherein the nucleic acid molecule is disrupted.
37. A host cell comprising a nucleic acid molecule selected from
the group consisting of the nucleic acid molecules set forth in
Appendix A, wherein the nucleic acid molecule comprises one or more
nucleic acid modifications from the sequence set forth in Appendix
A.
38. A host cell comprising a nucleic acid molecule selected from
the group consisting of the nucleic acid molecules set forth in
Appendix A, wherein the regulatory region of the nucleic acid
molecule is modified relative to the wild-type regulatory region of
the molecule.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 60/142,691, filed on Jul. 1, 1999, and also to U.S.
Provisional Patent Application No. 60/150,310, filed on Aug. 23,
1999, incorporated herein in their entirety by this reference. This
application also claims priority to German Patent Application No.
19942095.5, filed on Sep. 3, 1999, and also to German Patent
Application No. 19942097.1, filed on Sep. 3, 1999, incorporated
herein in their entirety by this reference.
BACKGROUND OF THE INVENTION
[0002] Certain products and by-products of naturally-occurring
metabolic processes in cells have utility in a wide array of
industries, including the food, feed, cosmetics, and pharmaceutical
industries. These molecules, collectively termed `fine chemicals`,
include organic acids, both proteinogenic and non-proteinogenic
amino acids, nucleotides and nucleosides, lipids and fatty acids,
diols, carbohydrates, aromatic compounds, vitamins and cofactors,
and enzymes. Their production is most conveniently performed
through large-scale culture of bacteria developed to produce and
secrete large quantities of a particular desired molecule. One
particularly useful organism for this purpose is Corynebacterium
glutamicum, a gram positive, nonpathogenic bacterium. Through
strain selection, a number of mutant strains have been developed
which produce an array of desirable compounds. However, selection
of strains improved for the production of a particular molecule is
a time-consuming and difficult process.
SUMMARY OF THE INVENTION
[0003] 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
phosphoenolpyruvate:sugar phosphotransferase system (PTS)
proteins.
[0004] 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 PTS nucleic acid molecules of the invention,
therefore, can be used to identify microorganisms which can be used
to produce fine chemicals, e.g., by fermentation processes.
Modulation of the expression of the PTS nucleic acids of the
invention, or modification of the sequence of the PTS nucleic acid
molecules of the invention, can be used to modulate the production
of one or more fine chemicals from a microorganism (e.g., to
improve the yield or production of one or more fine chemicals from
a Corynebacterium or Brevibacterium species).
[0005] The PTS nucleic acids of the invention may also be used to
identify an organism as being Corynebacterium glutamicum or a close
relative thereof, or to identify the presence of C. glutamicum or a
relative thereof in a mixed population of microorganisms. The
invention provides the nucleic acid sequences of a number of C.
glutamicum genes; by probing the extracted genomic DNA of a culture
of a unique or mixed population of microorganisms under stringent
conditions with a probe spanning a region of a C. glutamicum gene
which is unique to this organism, one can ascertain whether this
organism is present. Although Corynebacterium glutamicum itself is
nonpathogenic, it is related to species pathogenic in humans, such
as Corynebacterium diphtheriae (the causative agent of diphtheria);
the detection of such organisms is of significant clinical
relevance.
[0006] The PTS nucleic acid molecules of the invention may also
serve as reference points for mapping of the C. glutamicum genome,
or of genomes of related organisms. Similarly, these molecules, or
variants or portions thereof, may serve as markers for genetically
engineered Corynebacterium or Brevibacterium species.
[0007] The PTS proteins encoded by the novel nucleic acid molecules
of the invention are capable of, for example, transporting
high-energy carbon-containing molecules such as glucose into C.
glutamicum, or of participating in intracellular signal
transduction in this microorganism. Given the availability of
cloning vectors for use in Corynebacterium glutamicum, such as
those disclosed in Sinskey et al., U.S. Pat. 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.
[0008] The PTS molecules of the invention may be modified such that
the yield, production, and/or efficiency of production of one or
more fine chemicals is improved. For example, by modifying a PTS
protein involved in the uptake of glucose such that it is optimized
in activity, the quantity of glucose uptake or the rate at which
glucose is translocated into the cell may be increased. The
breakdown of glucose and other sugars within the cell provides
energy that may be used to drive energetically unfavorable
biochemical reactions, such as those involved in the biosynthesis
of fine chemicals. This breakdown also provides intermediate and
precursor molecules necessary for the biosynthesis of certain fine
chemicals, such as amino acids, vitamins and cofactors. By
increasing the amount of intracellular high-energy carbon molecules
through modification of the PTS molecules of the invention, one may
therefore increase both the energy available to perform metabolic
pathways necessary for the production of one or more fine
chemicals, and also the intracellular pools of metabolites
necessary for such production.
[0009] Further, the PTS molecules of the invention may be involved
in one or more intracellular signal transduction pathways which may
affect the yields and/or rate of production of one or more fine
chemical from C. glutamicum. For example, proteins necessary for
the import of one or more sugars from the extracellular medium
(e.g., HPr, Enzyme I, or a member of an Enzyme II complex) are
frequently posttranslationally modified upon the presence of a
sufficient quantity of the sugar in the cell, such that they are no
longer able to import that sugar. While this quantity of sugar at
which the transport system is shut off may be sufficient to sustain
the normal functioning of the cell, it may be limiting for the
overproduction of the desired fine chemical. Thus, it may be
desirable to modify the PTS proteins of the invention such that
they are no longer responsive to such negative regulation, thereby
permitting greater intracellular concentrations of one or more
sugars to be achieved, and, by extension, more efficient production
or greater yields of one or more fine chemicals from organisms
containing such mutant PTS proteins.
[0010] This invention provides novel nucleic acid molecules which
encode proteins, referred to herein as phosphoenolpyruvate:sugar
phosphotransferase system (PTS) proteins, which are capable of, for
example, participating in the import of high-energy carbon
molecules (e.g., glucose, fructose, or sucrose) into C. glutamicum,
and/or of participating in one or more C. glutamicum intracellular
signal transduction pathways. Nucleic acid molecules encoding a PTS
protein are referred to herein as PTS nucleic acid molecules. In a
preferred embodiment, the PTS protein participates in the import of
high-energy carbon molecules (e.g., glucose, fructose, or sucrose)
into C. glutamicum, and also may participate in one or more C.
glutamicum intracellular signal transduction pathways. Examples of
such proteins include those encoded by the genes set forth in Table
1.
[0011] Accordingly, one aspect of the invention pertains to
isolated nucleic acid molecules (e.g., cDNAs, DNAs, or RNAs)
comprising a nucleotide sequence encoding a PTS protein or
biologically active portions thereof, as well as nucleic acid
fragments suitable as primers or hybridization probes for the
detection or amplification of PTS-encoding nucleic acid (e.g., DNA
or mRNA). In particularly preferred embodiments, the isolated
nucleic acid molecule comprises one of the nucleotide sequences set
forth in Appendix A 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 in Appendix A, or a portion thereof. In other preferred
embodiments, the isolated nucleic acid molecule encodes one of the
amino acid sequences set forth in Appendix B. The preferred PTS
proteins of the present invention also preferably possess at least
one of the PTS activities described herein.
[0012] 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 Appendix B, e.g.,
sufficiently homologous to an amino acid sequence of Appendix B
such that the protein or portion thereof maintains a PTS activity.
Preferably, the protein or portion thereof encoded by the nucleic
acid molecule maintains the ability to participate in the import of
high-energy carbon molecules (e.g., glucose, fructose, or sucrose)
into C. glutamicum, and/or to participate in one or more C.
glutamicum intracellular signal transduction pathways. In one
embodiment, the protein encoded by the nucleic acid molecule is at
least about 50%, preferably at least about 60%, and more preferably
at least about 70%, 80%, or 90% and most preferably at least about
95%, 96%, 97%, 98%, or 99% or more homologous to an amino acid
sequence of Appendix B (e.g., an entire amino acid sequence
selected from those sequences set forth in Appendix B). In another
preferred embodiment, the protein is a full length C. glutamicum
protein which is substantially homologous to an entire amino acid
sequence of Appendix B (encoded by an open reading frame shown in
Appendix A).
[0013] In another preferred embodiment, the isolated nucleic acid
molecule is derived from C. glutamicum and encodes a protein (e.g.,
a PTS fusion protein) which includes a biologically active domain
which is at least about 50% or more homologous to one of the amino
acid sequences of Appendix B and is able to participate in the
import of high-energy carbon molecules (e.g., glucose, fructose, or
sucrose) into C. glutamicum, and/or to participate in one or more
C. glutamicum intracellular signal transduction pathways, or
possesses one or more of the activities set forth in Table 1, and
which also includes heterologous nucleic acid sequences encoding a
heterologous polypeptide or regulatory regions.
[0014] 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 Appendix 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 PTS protein, or a biologically
active portion thereof.
[0015] Another aspect of the invention pertains to vectors, e.g.,
recombinant expression vectors, containing the nucleic acid
molecules of the invention, and host cells into which such vectors
have been introduced. In one embodiment, such a host cell is used
to produce a PTS protein by culturing the host cell in a suitable
medium. The PTS protein can be then isolated from the medium or the
host cell.
[0016] Yet another aspect of the invention pertains to a
genetically altered microorganism in which a PTS gene has been
introduced or altered. In one embodiment, the genome of the
microorganism has been altered by the introduction of a nucleic
acid molecule of the invention encoding wild-type or mutated PTS
sequence as a transgene. In another embodiment, an endogenous PTS
gene within the genome of the microorganism has been altered, e.g.,
functionally disrupted, by homologous recombination with an altered
PTS gene. In another embodiment, an endogenous or introduced PTS
gene in a microorganism has been altered by one or more point
mutations, deletions, or inversions, but still encodes a functional
PTS protein. In still another embodiment, one or more of the
regulatory regions (e.g., a promoter, repressor, or inducer) of a
PTS gene in a microorganism has been altered (e.g., by deletion,
truncation, inversion, or point mutation) such that the expression
of the PTS gene is modulated. In a preferred embodiment, the
microorganism belongs to the genus Corynebacterium or
Brevibacterium, with Corynebacterium glutamicum being particularly
preferred. In a preferred embodiment, the microorganism is also
utilized for the production of a desired compound, such as an amino
acid, with lysine being particularly preferred.
[0017] In another aspect, the invention provides a method of
identifying the presence or activity of Cornyebacterium diphtheriae
in a subject. This method includes detection of one or more of the
nucleic acid or amino acid sequences of the invention (e.g., the
sequences set forth in Appendix A or Appendix B) in a subject,
thereby detecting the presence or activity of Corynebacterium
diphtheriae in the subject.
[0018] Still another aspect of the invention pertains to an
isolated PTS protein or a portion, e.g., a biologically active
portion, thereof. In a preferred embodiment, the isolated PTS
protein or portion thereof can participate in the import of
high-energy carbon molecules (e.g., glucose, fructose, or sucrose)
into C. glutamicum, and also may participate in one or more C.
glutamicum intracellular signal transduction pathways. In another
preferred embodiment, the isolated PTS protein or portion thereof
is sufficiently homologous to an amino acid sequence of Appendix B
such that the protein or portion thereof maintains the ability to
participate in the import of high-energy carbon molecules (e.g.,
glucose, fructose, or sucrose) into C. glutamicum, and /or to
participate in one or more C. glutamicum intracellular signal
transduction pathways.
[0019] The invention also provides an isolated preparation of a PTS
protein. In preferred embodiments, the PTS protein comprises an
amino acid sequence of Appendix B. In another preferred embodiment,
the invention pertains to an isolated full length protein which is
substantially homologous to an entire amino acid sequence of
Appendix B (encoded by an open reading frame set forth in Appendix
A). In yet another embodiment, the protein is at least about 50%,
preferably at least about 60%, and more preferably at least about
70%, 80%, or 90%, and most preferably at least about 95%, 96%, 97%,
98%, or 99% or more homologous to an entire amino acid sequence of
Appendix B. In other embodiments, the isolated PTS protein
comprises an amino acid sequence which is at least about 50% or
more homologous to one of the amino acid sequences of Appendix B
and is able to participate in the import of high-energy carbon
molecules (e.g., glucose, fructose, or sucrose) into C. glutamicum,
and/or to participate in one or more C. glutamicum intracellular
signal transduction pathways, or has one or more of the activities
set forth in Table 1.
[0020] Alternatively, the isolated PTS protein can comprise an
amino acid sequence which is encoded by a nucleotide sequence which
hybridizes, e.g., hybridizes under stringent conditions, or is at
least about 50%, preferably at least about 60%, more preferably at
least about 70%, 80%, or 90%, and even more preferably at least
about 95%, 96%, 97%, 98,%, or 99% or more homologous, to a
nucleotide sequence of Appendix B. It is also preferred that the
preferred forms of PTS proteins also have one or more of the PTS
bioactivities described herein.
[0021] The PTS polypeptide, or a biologically active portion
thereof, can be operatively linked to a non-PTS polypeptide to form
a fusion protein. In preferred embodiments, this fusion protein has
an activity which differs from that of the PTS protein alone. In
other preferred embodiments, this fusion protein results in
increased yields, production, and/or efficiency of production of a
desired fine chemical from C. glutamicum. In particularly preferred
embodiments, integration of this fusion protein into a host cell
modulates the production of a desired compound from the cell.
[0022] In another aspect, the invention provides methods for
screening molecules which modulate the activity of a PTS protein,
either by interacting with the protein itself or a substrate or
binding partner of the PTS protein, or by modulating the
transcription or translation of a PTS nucleic acid molecule of the
invention.
[0023] Another aspect of the invention pertains to a method for
producing a fine chemical. This method involves the culturing of a
cell containing a vector directing the expression of a PTS nucleic
acid molecule of the invention, such that a fine chemical is
produced. In a preferred embodiment, this method further includes
the step of obtaining a cell containing such a vector, in which a
cell is transfected with a vector directing the expression of a PTS
nucleic acid. In another preferred embodiment, this method further
includes the step of recovering the fine chemical from the culture.
In a particularly preferred embodiment, the cell is from the genus
Corynebacterium or Brevibacterium, or is selected from those
strains set forth in Table 3.
[0024] Another aspect of the invention pertains to methods for
modulating production of a molecule from a microorganism. Such
methods include contacting the cell with an agent which modulates
PTS protein activity or PTS nucleic acid expression such that a
cell associated activity is altered relative to this same activity
in the absence of the agent. In a preferred embodiment, the cell is
modulated for the uptake of one or more sugars, such that the
yields or rate of production of a desired fine chemical by this
microorganism is improved. The agent which modulates PTS protein
activity can be an agent which stimulates PTS protein activity or
PTS nucleic acid expression. Examples of agents which stimulate PTS
protein activity or PTS nucleic acid expression include small
molecules, active PTS proteins, and nucleic acids encoding PTS
proteins that have been introduced into the cell. Examples of
agents which inhibit PTS activity or expression include small
molecules, and antisense PTS nucleic acid molecules.
[0025] Another aspect of the invention pertains to methods for
modulating yields of a desired compound from a cell, involving the
introduction of a wild-type or mutant PTS gene into a cell, either
maintained on a separate plasmid or integrated into the genome of
the host cell. If integrated into the genome, such integration can
random, or it can take place by homologous recombination such that
the native gene is replaced by the introduced copy, causing the
production of the desired compound from the cell to be modulated.
In a preferred embodiment, said yields are increased. In another
preferred embodiment, said chemical is a fine chemical. In a
particularly preferred embodiment, said fine chemical is an amino
acid. In especially preferred embodiments, said amino acid is
L-lysine.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention provides PTS nucleic acid and protein
molecules which are involved in the uptake of high-energy carbon
molecules (e.g., sucrose, fructose, or glucose) into C. glutamicum,
and may also participate in intracellular signal transduction
pathways in this microorganism. The molecules of the invention may
be utilized in the modulation of production of fine chemicals from
microorganisms. Such modulation may be due to increased
intracellular levels of high-energy molecules needed to produce,
e.g., ATP, GTP and other molecules utilized to drive energetically
unfavorable biochemical reactions in the cell, such as the
biosynthesis of a fine chemical. This modulation of fine chemical
production may also be due to the fact that the breakdown products
of many sugars serve as intermediates or precursors for other
biosynthetic pathways, including those of certain fine chemicals.
Further, PTS proteins are known to participate in certain
intracellular signal transduction pathways which may have
regulatory activity for one or more fine chemical metabolic
pathways; by manipulating these PTS proteins, one may thereby
activate a fine chemical biosynthetic pathways or repress a fine
chemical degradation pathway. Aspects of the invention are further
explicated below.
[0027] I. Fine Chemicals
[0028] 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
Sep. 1-3, 1994 at Penang, Malaysia, AOCS Press, (1995)), enzymes,
polyketides (Cane et al. (1998) Science 282: 63-68), and all other
chemicals described in Gutcho (1983) Chemicals by Fermentation,
Noyes Data Corporation, ISBN: 0818805086 and references therein.
The metabolism and uses of certain of these fine chemicals are
further explicated below.
[0029] A. Amino Acid Metabolism and Uses
[0030] Amino acids comprise the basic structural units of all
proteins, and as such are essential for normal cellular functioning
in all organisms. The term "amino acid" is art-recognized. The
proteinogenic amino acids, of which there are 20 species, serve as
structural units for proteins, in which they are linked by peptide
bonds, while the nonproteinogenic amino acids (hundreds of which
are known) are not normally found in proteins (see Ulmann's
Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97 VCH:
Weinheim (1985)). Amino acids may be in the D- or L-optical
configuration, though L-amino acids are generally the only type
found in naturally-occurring proteins. Biosynthetic and degradative
pathways of each of the 20 proteinogenic amino acids have been well
characterized in both prokaryotic and eukaryotic cells (see, for
example, Stryer, L. Biochemistry, 3.sup.rd edition, pages 578-590
(1988)). The `essential` amino acids (histidine, isoleucine,
leucine, lysine, methionine, phenylalanine, threonine, tryptophan,
and valine), so named because they are generally a nutritional
requirement due to the complexity of their biosyntheses, are
readily converted by simple biosynthetic pathways to the remaining
11 `nonessential` amino acids (alanine, arginine, asparagine,
aspartate, cysteine, glutamate, glutamine, glycine, proline,
serine, and tyrosine). Higher animals do retain the ability to
synthesize some of these amino acids, but the essential amino acids
must be supplied from the diet in order for normal protein
synthesis to occur.
[0031] Aside from their function in protein biosynthesis, these
amino acids are interesting chemicals in their own right, and many
have been found to have various applications in the food, feed,
chemical, cosmetics, agriculture, and pharmaceutical industries.
Lysine is an important amino acid in the nutrition not only of
humans, but also of monogastric animals such as poultry and swine.
Glutamate is most commonly used as a flavor additive (mono-sodium
glutamate, MSG) and is widely used throughout the food industry, as
are aspartate, phenylalanine, glycine, and cysteine. Glycine,
L-methionine and tryptophan are all utilized in the pharmaceutical
industry. Glutamine, valine, leucine, isoleucine, histidine,
arginine, proline, serine and alanine are of use in both the
pharmaceutical and cosmetics industries. Threonine, tryptophan, and
D/L-methionine are common feed additives. (Leuchtenberger, W.
(1996) Amino aids--technical production and use, p. 466-502 in Rehm
et al. (eds.) Biotechnology vol. 6, chapter 14a, VCH: Weinheim).
Additionally, these amino acids have been found to be useful as
precursors for the synthesis of synthetic amino acids and proteins,
such as N-acetylcysteine, S-carboxymethyl-L-cysteine,
(S)-5-hydroxytryptophan, and others described in Ulmann's
Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97, VCH:
Weinheim, 1985.
[0032] 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 .alpha.-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 .beta.-carbon
atom to tetrahydrofolate, in a reaction catalyzed by serine
transhydroxymethylase. Phenylalanine, and tyrosine are synthesized
from the glycolytic and pentose phosphate pathway precursors
erythrose 4-phosphate and phosphoenolpyruvate in a 9-step
biosynthetic pathway that differ only at the final two steps after
synthesis of prephenate. Tryptophan is also produced from these two
initial molecules, but its synthesis is an 11-step pathway.
Tyrosine may also be synthesized from phenylalanine, in a reaction
catalyzed by phenylalanine hydroxylase. Alanine, valine, and
leucine are all biosynthetic products of pyruvate, the final
product of glycolysis. Aspartate is formed from oxaloacetate, an
intermediate of the citric acid cycle. Asparagine, methionine,
threonine, and lysine are each produced by the conversion of
aspartate. Isoleucine is formed from threonine. A complex 9-step
pathway results in the production of histidine from
5-phosphoribosyl-1-pyrophosphate, an activated sugar.
[0033] 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.sup.rd ed. Ch. 21 "Amino Acid
Degradation and the Urea Cycle" p. 495-516 (1988)). Although the
cell is able to convert unwanted amino acids into useful metabolic
intermediates, amino acid production is costly in terms of energy,
precursor molecules, and the enzymes necessary to synthesize them.
Thus it is not surprising that amino acid biosynthesis is regulated
by feedback inhibition, in which the presence of a particular amino
acid serves to slow or entirely stop its own production (for
overview of feedback mechanisms in amino acid biosynthetic
pathways, see Stryer, L. Biochemistry, 3.sup.rd 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.
[0034] B. Vitamin, Cofactor, and Nutraceutical Metabolism and
Uses
[0035] 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).
[0036] The biosynthesis of these molecules in organisms capable of
producing them, such as bacteria, has been largely characterized
(Ullman's Encyclopedia of Industrial Chemistry, "Vitamins" vol.
A27, p. 443-613, VCH: Weinheim, 1996; Michal, G. (1999) Biochemical
Pathways: An Atlas of Biochemistry and Molecular Biology, John
Wiley & Sons; Ong, A. S., Niki, E. & Packer, L. (1995)
"Nutrition, Lipids, Health, and Disease" Proceedings of the
UNESCO/Confederation of Scientific and Technological Associations
in Malaysia, and the Society for Free Radical Research--Asia, held
Sep. 1-3, 1994 at Penang, Malaysia, AOCS Press: Champaign, Ill. X,
374 S).
[0037] Thiamin (vitamin B.sub.1) is produced by the chemical
coupling of pyrimidine and thiazole moieties. Riboflavin (vitamin
B.sub.2) 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 B.sub.6` (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-1-oxobutyl)-.beta.-alanine)
can be produced either by chemical synthesis or by fermentation.
The final steps in pantothenate biosynthesis consist of the
ATP-driven condensation of .beta.-alanine and pantoic acid. The
enzymes responsible for the biosynthesis steps for the conversion
to pantoic acid, to .beta.-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
B.sub.5), pantetheine (and its derivatives) and coenzyme A.
[0038] 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 .alpha.-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.
[0039] Corrinoids (such as the cobalamines and particularly vitamin
B.sub.12) and porphyrines belong to a group of chemicals
characterized by a tetrapyrole ring system. The biosynthesis of
vitamin B.sub.12 is sufficiently complex that it has not yet been
completely characterized, but many of the enzymes and substrates
involved are now known. Nicotinic acid (nicotinate), and
nicotinamide are pyridine derivatives which are also termed
`niacin`. Niacin is the precursor of the important coenzymes NAD
(nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine
dinucleotide phosphate) and their reduced forms.
[0040] 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 B.sub.6, pantothenate,
and biotin. Only Vitamin B.sub.12 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.
[0041] C. Purine, Pyrimidine, Nucleoside and Nucleotide Metabolism
and Uses
[0042] 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).
[0043] Several publications have described the use of these
chemicals for these medical indications, by influencing purine
and/or pyrimidine metabolism (e.g. Christopherson, R. I. and Lyons,
S. D. (1990) "Potent inhibitors of de novo pyrimidine and purine
biosynthesis as chemotherapeutic agents." Med. Res. Reviews 10:
505-548). Studies of enzymes involved in purine and pyrimidine
metabolism have been focused on the development of new drugs which
can be used, for example, as immunosuppressants or
anti-proliferants (Smith, J. L., (1995) "Enzymes in nucleotide
synthesis." Curr. Opin. Struct. Biol. 5: 752-757; (1995) Biochem
Soc. Transact. 23: 877-902). However, purine and pyrimidine bases,
nucleosides and nucleotides have other utilities: as intermediates
in the biosynthesis of several fine chemicals (e.g., thiamine,
S-adenosyl-methionine, folates, or riboflavin), as energy carriers
for the cell (e.g., ATP or GTP), and for chemicals themselves,
commonly used as flavor enhancers (e.g., IMP or GMP) or for several
medicinal applications (see, for example, Kuninaka, A. (1996)
Nucleotides and Related Compounds in Biotechnology vol. 6, Rehm et
al., eds. VCH: Weinheim, p. 561-612). Also, enzymes involved in
purine, pyrimidine, nucleoside, or nucleotide metabolism are
increasingly serving as targets against which chemicals for crop
protection, including fungicides, herbicides and insecticides, are
developed.
[0044] 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.
[0045] D. Trehalose Metabolism and Uses
[0046] Trehalose consists of two glucose molecules, bound in
.alpha., .alpha.-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. Pat. 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.
[0047] II. The Phosphoenolpyruvate:Sugar Phosphotransferase
System
[0048] The ability of cells to grow and divide rapidly in culture
is to a great degree dependent on the extent to which the cells are
able to take up and utilize high energy molecules, such as glucose
and other sugars. Different transporter proteins exist to transport
different carbon sources into the cell. There are transport
proteins for sugars, such as glucose, fructose, mannose, galactose,
ribose, sorbose, ribulose, lactose, maltose, sucrose, or raffinose,
and also transport proteins for starch or cellulose degradation
products. Other transport systems serve to import alcohols (e.g.,
methanol or ethanol), alkanes, fatty acids and organic acids like
acetic acid or lactic acid. In bacteria, sugars may be transported
into the cell across the cellular membrane by a variety of
mechanisms. Aside from the symport of sugars with protons, one of
the most commonly utilized processes for sugar uptake is the
bacterial phosphoenolpyruvate: sugar phosphotransferase system
(PTS). This system not only catalyzes the translocation (with
concomitant phosphorylation) of sugars and hexitols, but it also
regulates cellular metabolism in response to the availability of
carbohydrates. Such PTS systems are ubiquitous in bacteria but do
not occur in archaebacteria or eukaryotes.
[0049] Functionally, the PTS system consists of two cytoplasmic
proteins, Enzyme I and HPr, and a variable number of sugar-specific
integral and peripheral membrane transport complexes (each termed
`Enzyme II` with a sugar-specific subscript, e.g., `Enzyme
II.sup.Glu` for the Enzyme II complex which binds glucose). Enzymes
II specific for mono-, di-, or oligosaccharides, like glucose,
fructose, mannose, galactose, ribose, sorbose, ribulose, lactose,
maltose, sucrose, raffinose, and others are known. Enzyme I
transfers phosphoryl groups from phosphoenolpyruvate (PEP) to the
phosphoryl carrier protein, HPr. HPr then transfers the phosphoryl
groups to the different Enzyme II transport complexes. While the
amino acid sequences of Enzyme I and HPr are quite similar in all
bacteria, the sequences for PTS transporters can be grouped into
structurally unrelated families. Further, the number and homology
between these genes vary from bacteria to bacteria. The E. coli
genome encodes 38 different PTS proteins, 33 of which are subunits
belonging to 22 different transporters. The M. genitalium genome
contains one gene each for Enzyme I and HPr, and only two genes for
PTS transporters. The genomes of T. palladium and C. trachomatis
contain genes for Enzyme I- and HPr-like proteins but no PTS
transporters.
[0050] All PTS transporters consist of three functional units, IIA,
IIB, and IIC, which occur either as protein subunits in a complex
(e.g., IIA.sup.GlcIICB.sup.Glc) or as domains of a single
polypeptide chain (e.g., IICBA.sup.GlcNAc). IIA and IIB
sequentially transfer phosphoryl groups from HPr to the transported
sugars. IIC contains the sugar binding site, and spans the inner
membrane six or eight times. Sugar translocation is coupled to the
transient phosphorylation of the IIB domain. Enzyme I, HPr, and IIA
are phosphorylated at histidine residues, while IIB subunits are
phosphorylated at either cysteine or histidine residues, depending
on the particular transporter involved. Phosphorylation of the
sugar being imported has the advantage of blocking the diffusion of
the sugar back through the cellular membrane to the extracellular
medium, since the charged phosphate group cannot readily traverse
the hydrophobic core of the membrane.
[0051] Some PTS proteins play a role in intracellular signal
transduction in addition to their function in the active transport
of sugars. These subunits regulate their targets either
allosterically, or by phosphorylation. Their regulatory activity
varies with the degree of their phosphorylation (i.e., the ratio of
the non-phosphorylated to the phosphorylated form), which in turn
varies with the ratio of sugar-dependent dephosphorylation and
phosphoenolpyruvate-dependent rephosphorylation. Examples of such
intracellular regulation by PTS proteins in E. coli include the
inhibition of glycerol kinase by dephosphorylated IIA.sup.Glc, and
the activation of adenylate cyclase by the phosphorylated version
of this protein. Also, the HPr and the IIB domains of some
transporters in these microorganisms regulate gene expression by
reversible phosphorylation of transcription antiterminators. In
gram-positive bacteria, the activity of HPr is modulated by
HPr-specific serine kinases and phosphatases. For example, HPr
phosphorylated at serine-46 functions as a co-repressor of the
transcriptional repressor CcpA. Lastly, it has been found that
unphosphorylated Enzyme I inhibits the sensor kinase CheA of the
bacterial chemotaxis machinery, providing a direct link between the
sugar binding and transport systems of the bacterium and those
systems governing movement of the bacterium (Sonenshein, A. L., et
al., eds. Bacillus subtilis and other gram-positive bacteria. ASM:
Washington, D.C.; Neidhardt, F. C., et al., eds. (1996) Escherichia
coli and Salmonella. ASM Press: Washington, D.C.; Lengeler et al.,
(1999). Biology of Prokaryotes. Section II, pp. 68-87, Thieme
Verlag: Stuttgart).
[0052] III. Elements and Methods of the Invention
[0053] The present invention is based, at least in part, on the
discovery of novel molecules, referred to herein as PTS nucleic
acid and protein molecules, which participate in the uptake of
high-energy carbon molecules (e.g., glucose, sucrose, and fructose)
into C. glutamicum, and may also participate in one or more
intracellular signal transduction pathways in these microorganisms.
In one embodiment, the PTS molecules function to import high-energy
carbon molecules into the cell, where the energy produced by their
degradation may be utilized to power less energetically favorable
biochemical reactions, and their degradation products may serve as
intermediates and precursors for a number of other metabolic
pathways. In another embodiment, the PTS molecules may participate
in one or more intracellular signal transduction pathways, wherein
the presence of a modified form of a PTS molecule (e.g., a
phosphorylated PTS protein) may participate in a signal
transduction cascade which regulates one or more cellular
processes. In a preferred embodiment, the activity of the PTS
molecules of the present invention has an impact on the production
of a desired fine chemical by this organism. In a particularly
preferred embodiment, the PTS molecules of the invention are
modulated in activity, such that the yield, production or
efficiency of production of one or more fine chemicals from C.
glutamicum is also modulated.
[0054] The language, "PTS protein" or "PTS polypeptide" includes
proteins which participate in the uptake of one or more high-energy
carbon compounds (e.g., mono-, di, or oligosaccharides, such as
fructose, mannose, sucrose, glucose, raffinose, galactose, ribose,
lactose, maltose, and ribulose) from the extracellular medium to
the interior of the cell. Such PTS proteins may also participate in
one or more intracellular signal transduction pathways, such as,
but not limited to, those governing the uptake of different sugars
into the cell. Examples of PTS proteins include those encoded by
the PTS genes set forth in Table 1 and Appendix A. For general
references pertaining to the PTS system, see: Stryer, L. (1988)
Biochemistry. Chapter 37: "Membrane Transport", W.H. Freeman: New
York, p. 959-961; Darnell, J. et al. (1990) Molecular Cell Biology
Scientific American Books: New York, p. 552-553, and Michal, G.,
ed. (1999) Biochemical Pathways: An Atlas of Biochemistry and
Molecular Biology, Chapter 15 "Special Bacterial Metabolism". The
terms "PTS gene" or "PTS nucleic acid sequence" include nucleic
acid sequences encoding a PTS protein, which consist of a coding
region and also corresponding untranslated 5' and 3' sequence
regions. Examples of PTS genes include those set forth in Table 1.
The terms "production" or "productivity" are art-recognized and
include the concentration of the fermentation product (for example,
the desired fine chemical) formed within a given time and a given
fermentation volume (e.g., kg product per hour per liter). The term
"efficiency of production" includes the time required for a
particular level of production to be achieved (for example, how
long it takes for the cell to attain a particular rate of output of
a fine chemical). The term "yield" or "product/carbon yield" is
art-recognized and includes the efficiency of the conversion of the
carbon source into the product (i.e., fine chemical). This is
generally written as, for example, kg product per kg carbon source.
By increasing the yield or production of the compound, the quantity
of recovered molecules, or of useful recovered molecules of that
compound in a given amount of culture over a given amount of time
is increased. The terms "biosynthesis" or a "biosynthetic pathway"
are art-recognized and include the synthesis of a compound,
preferably an organic compound, by a cell from intermediate
compounds in what may be a multistep and highly regulated process.
The terms "degradation" or a "degradation pathway" are
art-recognized and include the breakdown of a compound, preferably
an organic compound, by a cell to degradation products (generally
speaking, smaller or less complex molecules) in what may be a
multistep and highly regulated process. The language "metabolism"
is art-recognized and includes the totality of the biochemical
reactions that take place in an organism. The metabolism of a
particular compound, then, (e.g., the metabolism of an amino acid
such as glycine) comprises the overall biosynthetic, modification,
and degradation pathways in the cell related to this compound. The
language "transport" or "import" is art-recognized and includes the
facilitated movement of one or more molecules across a cellular
membrane through which the molecule would otherwise be unable to
pass.
[0055] In another embodiment, the PTS molecules of the invention
are capable of modulating the production of a desired molecule,
such as a fine chemical, in a microorganism such as C. glutamicum.
Using recombinant genetic techniques, one or more of the PTS
proteins of the invention may be manipulated such that its function
is modulated. For example, a protein involved in the PTS-mediated
import of glucose may be altered such that it is optimized in
activity, and the PTS system for the importation of glucose may
thus be able to translocate increased amounts of glucose into the
cell. Since glucose molecules are utilized not only for energy to
drive energetically unfavorable biochemical reactions, such as fine
chemical biosyntheses, but also as precursors and intermediates in
a number of fine chemical biosynthetic pathways (e.g., serine is
synthesized from 3-phosphoglycerate). In each case, the overall
yield or rate of production of one of these desired fine chemicals
may be increased, either by increasing the energy available for
such production to occur, or by increasing the availability of
compounds necessary for such production to take place.
[0056] Further, many PTS proteins are known to play key roles in
intracellular signal transduction pathways which regulate cellular
metabolism and sugar uptake in keeping with the availability of
carbon sources. For example, it is known that an increased
intracellular level of fructose 1,6-bisphosphate (a compound
produced during glycolysis) results in the phosphorylation of a
serine residue on HPr which prevents this protein from serving as a
phosphoryl donor in any PTS sugar transport process, thereby
blocking further sugar uptake. By mutagenizing HPr such that this
serine residue cannot be phosphorylated, one may constitutively
activate HPr and thereby increase sugar transport into the cell,
which in turn will ensure greater intracellular energy stores and
intermediate/precursor molecules for the biosynthesis of one or
more desired fine chemicals.
[0057] 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 PTS DNAs and the predicted amino acid sequences of the
C. glutamicum PTS proteins are shown in Appendices A and B,
respectively. Computational analyses were performed which
classified and/or identified these nucleotide sequences as
sequences which encode metabolic pathway proteins.
[0058] The present invention also pertains to proteins which have
an amino acid sequence which is substantially homologous to an
amino acid sequence of Appendix B. 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.
[0059] The PTS protein or a biologically active portion or fragment
thereof of the invention can participate in the transport of
high-energy carbon-containing molecules such as glucose into C.
glutamicum, or can participate in intracellular signal transduction
in this microorganism, or may have one or more of the activities
set forth in Table 1.
[0060] Various aspects of the invention are described in further
detail in the following subsections:
[0061] A. Isolated Nucleic Acid Molecules
[0062] One aspect of the invention pertains to isolated nucleic
acid molecules that encode PTS polypeptides or biologically active
portions thereof, as well as nucleic acid fragments sufficient for
use as hybridization probes or primers for the identification or
amplification of PTS-encoding nucleic acid (e.g., PTS DNA). As used
herein, the term "nucleic acid molecule" is intended to include DNA
molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g.,
mRNA) and analogs of the DNA or RNA generated using nucleotide
analogs. This term also encompasses untranslated sequence located
at both the 3' and 5' ends of the coding region of the gene: at
least about 100 nucleotides of sequence upstream from the 5' end of
the coding region and at least about 20 nucleotides of sequence
downstream from the 3'end of the coding region of the gene. The
nucleic acid molecule can be single-stranded or double-stranded,
but preferably is double-stranded DNA. An "isolated" nucleic acid
molecule is one which is separated from other nucleic acid
molecules which are present in the natural source of the nucleic
acid. Preferably, an "isolated" nucleic acid is free of sequences
which naturally flank the nucleic acid (i.e., sequences located at
the 5' and 3' ends of the nucleic acid) in the genomic DNA of the
organism from which the nucleic acid is derived. For example, in
various embodiments, the isolated PTS nucleic acid molecule can
contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb
of nucleotide sequences which naturally flank the nucleic acid
molecule in genomic DNA of the cell from which the nucleic acid is
derived (e.g, a C. glutamicum cell). Moreover, an "isolated"
nucleic acid molecule, such as a DNA molecule, can be substantially
free of other cellular material, or culture medium when produced by
recombinant techniques, or chemical precursors or other chemicals
when chemically synthesized.
[0063] A nucleic acid molecule of the present invention, e.g., a
nucleic acid molecule having a nucleotide sequence of Appendix A,
or a portion thereof, can be isolated using standard molecular
biology techniques and the sequence information provided herein.
For example, a C. glutamicum PTS DNA can be isolated from a C.
glutamicum library using all or portion of one of the sequences of
Appendix A 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, N.Y., 1989). Moreover, a nucleic acid molecule
encompassing all or a portion of one of the sequences of Appendix A
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
sequences of Appendix A can be isolated by the polymerase chain
reaction using oligonucleotide primers designed based upon this
same sequence of Appendix A). 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 Gibco/BRL,
Bethesda, Md.; or AMV reverse transcriptase, available from
Seikagaku America, Inc., St. Petersburg, Fla.). Synthetic
oligonucleotide primers for polymerase chain reaction amplification
can be designed based upon one of the nucleotide sequences shown in
Appendix A. A nucleic acid of the invention can be amplified using
cDNA or, alternatively, genomic DNA, as a template and appropriate
oligonucleotide primers according to standard PCR amplification
techniques. The nucleic acid so amplified can be cloned into an
appropriate vector and characterized by DNA sequence analysis.
Furthermore, oligonucleotides corresponding to a PTS nucleotide
sequence can be prepared by standard synthetic techniques, e.g.,
using an automated DNA synthesizer.
[0064] In a preferred embodiment, an isolated nucleic acid molecule
of the invention comprises one of the nucleotide sequences shown in
Appendix A. The sequences of Appendix A correspond to the
Corynebacterium glutamicum PTS DNAs of the invention. This DNA
comprises sequences encoding PTS proteins (i.e., the "coding
region", indicated in each sequence in Appendix A), as well as 5'
untranslated sequences and 3' untranslated sequences, also
indicated in Appendix A. Alternatively, the nucleic acid molecule
can comprise only the coding region of any of the sequences in
Appendix A.
[0065] For the purposes of this application, it will be understood
that each of the sequences set forth in Appendix A has an
identifying RXA, RXN, RXS, or RXC number having the designation
"RXA", or "RXN", "RXS", or "RXC" followed by 5 digits (i.e.,
RXA01503, RXN01299, RXS00315, or RXC00953). Each of these sequences
comprises up to three parts: a 5' upstream region, a coding region,
and a downstream region. Each of these three regions is identified
by the same RXA, RXN, RXS, or RXC designation to eliminate
confusion. The recitation "one of the sequences in Appendix A",
then, refers to any of the sequences in Appendix A, which may be
distinguished by their differing RXA, RXN, RXS, or RXC
designations. The coding region of each of these sequences is
translated into a corresponding amino acid sequence, which is set
forth in Appendix B. The sequences of Appendix B are identified by
the same RXA, RXN, RXS, or RXC designations as Appendix A, such
that they can be readily correlated. For example, the amino acid
sequences in Appendix B designated RXA01503, RXN01299, RXS00315,
and RXC00953 are translations of the coding regions of the
nucleotide sequence of nucleic acid molecules RXA01503, RXN01299,
RXS00315, and RXC00953, respectively, in Appendix A. Each of the
RXA, RXN, RXS, and RXC nucleotide and amino acid sequences of the
invention has also been assigned a SEQ ID NO, as indicated in Table
1. For example, as set forth in Table 1, the nucleotide sequence of
RXN01299 is SEQ ID NO: 7, and the corresponding amino acid sequence
is SEQ ID NO:8.
[0066] Several of the genes of the invention are "F-designated
genes". An F-designated gene includes those genes set forth in
Table 1 which have an `F` in front of the RXA, RXN, RXS, or RXC
designation. For example, SEQ ID NO:3, designated, as indicated on
Table 1, as "F RXA00315", is an F-designated gene, as are SEQ ID
NOs: 9, 11, and 13 (designated on Table 1 as "F RXA01299", "F
RXA01883", and "F RXA01889", respectively).
[0067] In one embodiment, the nucleic acid molecules of the present
invention are not intended to include C. glutamicum those compiled
in Table 2. In the case of the dapD gene, a sequence for this gene
was published in Wehrmann, A., et al. (1998) J. Bacteriol. 180(12):
3159-3165. However, the sequence obtained by the inventors of the
present application is significantly longer than the published
version. It is believed that the published version relied on an
incorrect start codon, and thus represents only a fragment of the
actual coding region.
[0068] 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 shown in
Appendix A, or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences shown in Appendix
A is one which is sufficiently complementary to one of the
nucleotide sequences shown in Appendix A such that it can hybridize
to one of the nucleotide sequences shown in Appendix A, thereby
forming a stable duplex.
[0069] In still another preferred embodiment, an isolated nucleic
acid molecule of the invention comprises a nucleotide sequence
which is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,
58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%,
65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and
even more preferably at least about 95%, 96%, 97%, 98%, 99% or more
homologous to a nucleotide sequence shown in Appendix A, 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 shown in Appendix A,
or a portion thereof.
[0070] Moreover, the nucleic acid molecule of the invention can
comprise only a portion of the coding region of one of the
sequences in Appendix A, for example a fragment which can be used
as a probe or primer or a fragment encoding a biologically active
portion of a PTS protein. The nucleotide sequences determined from
the cloning of the PTS genes from C. glutamicum allows for the
generation of probes and primers designed for use in identifying
and/or cloning PTS homologues in other cell types and organisms, as
well as PTS homologues from other Corynebacteria or related
species. The probe/primer typically comprises substantially
purified oligonucleotide. The oligonucleotide typically comprises a
regionrof 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 sequences set forth in Appendix A, an
anti-sense sequence of one of the sequences set forth in Appendix
A, or naturally occurring mutants thereof. Primers based on a
nucleotide sequence of Appendix A can be used in PCR reactions to
clone PTS homologues. Probes based on the PTS nucleotide sequences
can be used to detect transcripts or genomic sequences encoding the
same or homologous proteins. In preferred embodiments, the probe
further comprises a label group attached thereto, e.g the label
group can be a radioisotope, a fluorescent compound, an enzyme, or
an enzyme co-factor. Such probes can be used as a part of a
diagnostic test kit for identifying cells which misexpress a PTS
protein, such as by measuring a level of a PTS-encoding nucleic
acid in a sample of cells e.g., detecting PTS mRNA levels or
determining whether a genomic PTS gene has been mutated or
deleted.
[0071] 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 Appendix B such that the protein or portion
thereof maintains the ability to participate in the transport of
high-energy carbon molecules (such as glucose) into C. glutamicum,
and may also participate in one or more intracellular signal
transduction pathways. As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent (e.g., an amino acid residue which has a similar side
chain as an amino acid residue in one of the sequences of Appendix
B) amino acid residues to an amino acid sequence of Appendix B such
that the protein or portion thereof is capable of transporting
high-energy carbon-containing molecules such as glucose into C.
glutamicum, and may also participate in intracellular signal
transduction in this microorganism. Protein members of such
metabolic pathways, as described herein, function to transport
high-energy carbon-containing molecules such as glucose into C.
glutamicum, and may also participate in intracellular signal
transduction in this microorganism. Examples of such activities are
also described herein. Thus, "the function of a PTS protein"
contributes to the overall functioning and/or regulation of one or
more phosphoenolpyruvate-based sugar transport pathway, and /or
contributes, either directly or indirectly, to the yield,
production, and/or efficiency of production of one or more fine
chemicals. Examples of PTS protein activities are set forth in
Table 1.
[0072] 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 Appendix B.
[0073] Portions of proteins encoded by the PTS nucleic acid
molecules of the invention are preferably biologically active
portions of one of the PTS proteins. As used herein, the term
"biologically active portion of a PTS protein" is intended to
include a portion, e.g., a domain/motif, of a PTS protein that is
capable of transporting high-energy carbon-containing molecules
such as glucose into C. glutamicum, or of participating in
intracellular signal transduction in this microorganism, or has an
activity as set forth in Table 1. To determine whether a PTS
protein or a biologically active portion thereof can participate in
the transportation of high-energy carbon-containing molecules such
as glucose into C. glutamicum, or can participate in intracellular
signal transduction in this microorganism, an assay of enzymatic
activity may be performed. Such assay methods are well known to
those of ordinary skill in the art, as detailed in Example 8 of the
Exemplification.
[0074] Additional nucleic acid fragments encoding biologically
active portions of a PTS protein can be prepared by isolating a
portion of one of the sequences in Appendix B, expressing the
encoded portion of the PTS protein or peptide (e.g., by recombinant
expression in vitro) and assessing the activity of the encoded
portion of the PTS protein or peptide.
[0075] The invention further encompasses nucleic acid molecules
that differ from one of the nucleotide sequences shown in Appendix
A (and portions thereof) due to degeneracy of the genetic code and
thus encode the same PTS protein as that encoded by the nucleotide
sequences shown in Appendix A. 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 Appendix
B. In a still further embodiment, the nucleic acid molecule of the
invention encodes a full length C. glutamicum protein which is
substantially homologous to an amino acid sequence of Appendix B
(encoded by an open reading frame shown in Appendix A).
[0076] It will be understood by one of ordinary skill in the art
that in one embodiment the sequences of the invention are not meant
to include the sequences of the prior art, such as those Genbank
sequences set forth in Tables 2 or 4 which were available prior to
the present invention. In one embodiment, the invention includes
nucleotide and amino acid sequences having a percent identity to a
nucleotide or amino acid sequence of the invention which is greater
than that of a sequence of the prior art (e.g., a Genbank sequence
(or the protein encoded by such a sequence) set forth in Tables 2
or 4). For example, the invention includes a nucleotide sequence
which is greater than and/or at least 44% identical to the
nucleotide sequence designated RXA01503 (SEQ ID NO:5), a nucleotide
sequence which is greater than and/or at least 41% identical to the
nucleotide sequence designated RXA00951 (SEQ ID NO:15), and a
nucleotide sequence which is greater than and/or at least 38%
identical to the nucleotide sequence designated RXA01300 (SEQ ID
NO:21). One of ordinary skill in the art would be able to calculate
the lower threshold of percent identity for any given sequence of
the invention by examining the GAP-calculated percent identity
scores set forth in Table 4 for each of the three top hits for the
given sequence, and by subtracting the highest GAP-calculated
percent identity from 100 percent. One of ordinary skill in the art
will also appreciate that nucleic acid and amino acid sequences
having percent identities greater than the lower threshold so
calculated (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,
58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%,
65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and
even more preferably at least about 95%, 96%, 97%, 98%, 99% or more
identical) are also encompassed by the invention.
[0077] In addition to the C. glutamicum PTS nucleotide sequences
shown in Appendix A, it will be appreciated by those of ordinary
skill in the art that DNA sequence polymorphisms that lead to
changes in the amino acid sequences of PTS proteins may exist
within a population (e.g., the C. glutamicum population). Such
genetic polymorphism in the PTS gene may exist among individuals
within a population due to natural variation. As used herein, the
terms "gene" and "recombinant gene" refer to nucleic acid molecules
comprising an open reading frame encoding a PTS protein, preferably
a C. glutamicum PTS protein. Such natural variations can typically
result in 1-5% variance in the nucleotide sequence of the PTS gene.
Any and all such nucleotide variations and resulting amino acid
polymorphisms in PTS that are the result of natural variation and
that do not alter the functional activity of PTS proteins are
intended to be within the scope of the invention.
[0078] Nucleic acid molecules corresponding to natural variants and
non-C. glutamicum homologues of the C. glutamicum PTS DNA of the
invention can be isolated based on their homology to the C.
glutamicum PTS nucleic acid disclosed herein using the C.
glutamicum DNA, or a portion thereof, as a hybridization probe
according to standard hybridization techniques under stringent
hybridization conditions. Accordingly, in another embodiment, an
isolated nucleic acid molecule of the invention is at least 15
nucleotides in length and hybridizes under stringent conditions to
the nucleic acid molecule comprising a nucleotide sequence of
Appendix A. In other embodiments, the nucleic acid is at least 30,
50, 100, 250 or more nucleotides in length. As used herein, the
term "hybridizes under stringent conditions" is intended to
describe conditions for hybridization and washing under which
nucleotide sequences at least 60% homologous to each other
typically remain hybridized to each other. Preferably, the
conditions are such that sequences at least about 65%, more
preferably at least about 70%, and even more preferably at least
about 75% or more homologous to each other typically remain
hybridized to each other. Such stringent conditions are known to
those of ordinary skill in the art and can be found in Ausubel et
al., Current Protocols in Molecular Biology, John Wiley & Sons,
N.Y. (1989), 6.3.1-6.3.6. A Vreferred, non-limiting example of
stringent hybridization conditions are hybridization in 6.times.
sodium chloride/sodium citrate (SSC) at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
50-65.degree. C. Preferably, an isolated nucleic acid molecule of
the invention that hybridizes under stringent conditions to a
sequence of Appendix A 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
PTS protein.
[0079] In addition to naturally-occurring variants of the PTS
sequence that may exist in the population, one of ordinary skill in
the art will further appreciate that changes can be introduced by
mutation into a nucleotide sequence of Appendix A, thereby leading
to changes in the amino acid sequence of the encoded PTS protein,
without altering the functional ability of the PTS protein. For
example, nucleotide substitutions leading to amino acid
substitutions at "non-essential" amino acid residues can be made in
a sequence of Appendix A. A "non-essential" amino acid residue is a
residue that can be altered from the wild-type sequence of one of
the PTS proteins (Appendix B) without altering the activity of said
PTS protein, whereas an "essential" amino acid residue is required
for PTS protein activity. Other amino acid residues, however,
(e.g., those that are not conserved or only semi-conserved in the
domain having PTS activity) may not be essential for activity and
thus are likely to be amenable to alteration without altering PTS
activity.
[0080] Accordingly, another aspect of the invention pertains to
nucleic acid molecules encoding PTS proteins that contain changes
in amino acid residues that are not essential for PTS activity.
Such PTS proteins differ in amino acid sequence from a sequence
contained in Appendix B yet retain at least one of the PTS
activities described herein. In one embodiment, the isolated
nucleic acid molecule comprises a nucleotide sequence encoding a
protein, wherein the protein comprises an amino acid sequence at
least about 50% homologous to an amino acid sequence of Appendix B
and is capable of transporting high-energy carbon-containing
molecules such as glucose into C. glutamicum, or of participating
in intracellular signal transduction in this microorganism, or has
one or more activities set forth in Table 1. Preferably, the
protein encoded by the nucleic acid molecule is at least about
50-60% homologous to one of the sequences in Appendix B, more
preferably at least about 60-70% homologous to one of the sequences
in Appendix B, even more preferably at least about 70-80%, 80-90%,
90-95% homologous to one of the sequences in Appendix B, and most
preferably at least about 96%, 97%, 98%, or 99% homologous to one
of the sequences in Appendix B.
[0081] To determine the percent homology of two amino acid
sequences (e.g., one of the sequences of Appendix B 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 onie 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 sequences of Appendix B) is occupied by the same amino
acid residue or nucleotide as the corresponding position in the
other sequence (e.g., a mutant form of the sequence selected from
Appendix B), 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.times.100).
[0082] An isolated nucleic acid molecule encoding a PTS protein
homologous to a protein sequence of Appendix B can be created by
introducing one or more nucleotide substitutions, additions or
deletions into a nucleotide sequence of Appendix A 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 sequences of Appendix A by standard techniques,
such as site-directed mutagenesis and PCR-mediated mutagenesis.
Preferably, conservative amino acid substitutions are made at one
or more predicted non-essential amino acid residues. A
"conservative amino acid substitution" is one in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted nonessential amino acid residue in a PTS protein is
preferably replaced with another amino acid residue from the same
side chain family. Alternatively, in another embodiment, mutations
can be introduced randomly along all or part of a PTS coding
sequence, such as by saturation mutagenesis, and the resultant
mutants can be screened for a PTS activity described herein to
identify mutants that retain PTS activity. Following mutagenesis of
one of the sequences of Appendix A, 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).
[0083] In addition to the nucleic acid molecules encoding PTS
proteins described above, another aspect of the invention pertains
to isolated nucleic acid molecules which are antisense thereto. An
"antisense" nucleic acid comprises a nucleotide sequence which is
complementary to a "sense" nucleic acid encoding a protein, e.g.,
complementary to the coding strand of a double-stranded DNA
molecule or complementary to an mRNA sequence. Accordingly, an
antisense nucleic acid can hydrogen bond to a sense nucleic acid.
The antisense nucleic acid can be complementary to an entire PTS
coding strand, or to only a portion thereof. In one embodiment, an
antisense nucleic acid molecule is antisense to a "coding region"
of the coding strand of a nucleotide sequence encoding a PTS
protein. The term "coding region" refers to the region of the
nucleotide sequence comprising codons which are translated into
amino acid residues (e.g., the entire coding region of SEQ ID NO. 5
(RXA01503) comprises nucleotides 1 to 249). In another embodiment,
the antisense nucleic acid molecule is antisense to a "noncoding
region" of the coding strand of a nucleotide sequence encoding PTS.
The term "noncoding region" refers to 5' and 3' sequences which
flank the coding region that are not translated into amino acids
(i.e., also referred to as 5' and 3' untranslated regions).
[0084] Given the coding strand sequences encoding PTS disclosed
herein (e.g., the sequences set forth in Appendix A), antisense
nucleic acids of the invention can be designed according to the
rules of Watson and Crick base pairing. The antisense nucleic acid
molecule can be complementary to the entire coding region of PTS
mRNA, but more preferably is an oligonucleotide which is antisense
to only a portion of the coding or noncoding region of PTS mRNA.
For example, the antisense oligonucleotide can be complementary to
the region surrounding the translation start site of PTS mRNA. An
antisense oligonucleotide can be, for 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-thiouridin- e,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiour- acil,
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).
[0085] The antisense nucleic acid molecules of the invention are
typically administered to a cell or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding a PTS protein to thereby inhibit expression of the
protein, e.g., by inhibiting transcription and/or translation. The
hybridization can be by conventional nucleotide complementarity to
form a stable duplex, or, for example, in the case of an antisense
nucleic acid molecule which binds to DNA duplexes, through specific
interactions in the major groove of the double helix. The antisense
molecule can 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.
[0086] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An .alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .beta.-units, the strands run parallel to each other
(Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The
antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.
15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987)
FEBS Lett. 215:327-330).
[0087] In still another embodiment, an antisense nucleic acid of
the invention is a ribozyme. Ribozymes are catalytic RNA molecules
with ribonuclease activity which are capable of cleaving a
single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can
be used to catalytically cleave PTS mRNA transcripts to thereby
inhibit translation of PTS mRNA. A ribozyme having specificity for
a PTS-encoding nucleic acid can be designed based upon the
nucleotide sequence of a PTS DNA disclosed herein (i.e., SEQ ID
NO:5 (RXA01503 in Appendix A)). For example, a derivative of a
Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide
sequence of the active site is complementary to the nucleotide
sequence to be cleaved in a PTS-encoding mRNA. See, e.g., Cech et
al. U.S. Pat. No. 4,987,071 and Cech et al. U.S. Pat. No.
5,116,742. Alternatively, PTS mRNA can be used to select a
catalytic RNA having a specific ribonuclease activity from a pool
of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993)
Science 261:1411-1418.
[0088] Alternatively, PTS gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory
region of a PTS nucleotide sequence (e.g., a PTS promoter and/or
enhancers) to form triple helical structures that prevent
transcription of a PTS gene in target cells. See generally, Helene,
C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al.
(1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992)
Bioassays 14(12):807-15.
[0089] B. Recombinant Expression Vectors and Host Cells
[0090] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding a
PTS protein (or a portion thereof). As used herein, the term
"vector" refers to a nucleic acid molecule capable of transporting
another nucleic acid to which it has been linked. One type of
vector is a "plasmid", which refers to a circular double stranded
DNA loop into which additional DNA segments can be ligated. Another
type of vector is a viral vector, wherein additional DNA segments
can be ligated into the viral genome. Certain vectors are capable
of autonomous replication in a host cell into which they are
introduced (e.g., bacterial vectors having a bacterial origin of
replication and episomal mammalian vectors). Other vectors (e.g.,
non-episomal mammalian vectors) are integrated into the genome of a
host cell upon introduction into the host cell, and thereby are
replicated along with the host genome. Moreover, certain vectors
are capable of directing the expression of genes to which they are
operatively linked. Such vectors are referred to herein as
"expression vectors". In general, expression vectors of utility in
recombinant DNA techniques are often in the form of plasmids. In
the present specification, "plasmid" and "vector" can be used
interchangeably as the plasmid is the most commonly used form of
vector. However, the invention is intended to include such other
forms of expression vectors, such as viral vectors (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[0091] 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, Calif. (1990). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence in many
types of host cell and those which direct expression of the
nucleotide sequence only in certain host cells. Preferred
regulatory sequences are, for example, promoters such as cos-,
tac-, trp-, tet-, trp-tet-, lpp-, lac-, lpp-lac-, lacI.sup.q-, T7-,
T5-, T3-, gal-, trc-, ara-, SP6-, arny, SPO2, .lambda.-P.sub.R- or
.lambda.P.sub.L, which are used preferably in bacteria. Additional
regulatory sequences are, for example, promoters from yeasts and
fungi, such as ADC1, MF.alpha., AC, P-60, CYC1, GAPDH, TEF, rp28,
ADH, promoters from plants such as CaMV/35S, SSU, OCS, lib4, usp,
STLS1, B33, nos or ubiquitin- or phaseolin-promoters. It is also
possible to use artificial promoters. It will be appreciated by one
of ordinary skill in the art that the design of the expression
vector can depend on such factors as the choice of the host cell to
be transformed, the level of expression of protein desired, etc.
The expression vectors of the invention can be introduced into host
cells to thereby produce proteins or peptides, including fusion
proteins or peptides, encoded by nucleic acids as described herein
(e.g., PTS proteins, mutant forms of PTS proteins, fusion proteins,
etc.).
[0092] The recombinant expression vectors of the invention can be
designed for expression of PTS proteins in prokaryotic or
eukaryotic cells. For example, PTS genes can be expressed in
bacterial cells such as C. glutamicum, insect cells (using
baculovirus expression vectors), yeast and other fungal cells (see
Romanos, M. A. et al. (1992) "Foreign gene expression in yeast: a
review", Yeast 8: 423-488; van den Hondel, C. A. M. J. J. et al.
(1991) "Heterologous gene expression in filamentous fungi" in: More
Gene Manipulations in Fungi, J. W. Bennet & L. L. Lasure, eds.,
p. 396-428: Academic Press: San Diego; and van den Hondel, C. A. M.
J. J. & Punt, P. J. (1991) "Gene transfer systems and vector
development for filamentous fungi, in: Applied Molecular Genetics
of Fungi, Peberdy, J. F. et al., eds., p. 1-28, Cambridge
University Press: Cambridge), algae and multicellular plant cells
(see Schmidt, R. and Willmitzer, L. (1988) High efficiency
Agrobacterium tumefactiens-mediated transformation of Arabidopsis
thaliana leaf and cotyledon explants" Plant Cell Rep.: 583-586), or
mammalian cells. Suitable host cells are discussed further in
Goeddel, Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, Calif. (1990). Alternatively, the
recombinant expression vector can be transcribed and translated in
vitro, for example using T7 promoter regulatory sequences and T7
polymerase.
[0093] Expression of proteins in prokaryotes is most often carried
out with vectors containing constitutive or inducible promoters
directing the expression of either fusion or non-fusion proteins.
Fusion vectors add a number of amino acids to a protein encoded
therein, usually to the amino terminus of the recombinant protein.
Such fusion vectors typically serve three purposes: 1) to increase
expression of recombinant protein; 2) to increase the solubility of
the recombinant protein; and 3) to aid in the purification of the
recombinant protein by acting as a ligand in affinity purification.
Often, in fusion expression vectors, a proteolytic cleavage site is
introduced at the junction of the fusion moiety and the recombinant
protein to enable separation of the recombinant protein from the
fusion moiety subsequent to purification of the fusion protein.
Such enzymes, and their cognate recognition sequences, include
Factor Xa, thrombin and enterokinase.
[0094] 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, Mass.) and pRIT5 (Pharmacia,
Piscataway, N.J.) which fuse glutathione S-transferase (GST),
maltose E binding protein, or protein A, respectively, to the
target recombinant protein. In one embodiment, the coding sequence
of the PTS protein is cloned into a pGEX expression vector to
create a vector encoding a 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 PTS protein unfused to GST
can be recovered by cleavage of the fusion protein with
thrombin.
[0095] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., (1988) Gene 69:301-315) pLG338,
pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236,
pMBL24, pLG200, pUR290, pIN-III113-B1, .lambda.gt11, pBdCl, and pET
11d (Studier et al., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (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 pET11d vector
relies on transcription from a T7 gn10-lac fusion promoter mediated
by a coexpressed viral RNA polymerase (T7 gn1). This viral
polymerase is supplied by host strains BL21 (DE3) or HMS174(DE3)
from a resident .lambda. prophage harboring a T7 gn1 gene under the
transcriptional control of the lacUV 5 promoter. For transformation
of other varieties of bacteria, appropriate vectors may be
selected. For example, the plasmids pIJ101, pIJ364, pIJ702 and
pIJ361 are known to be useful in transforming Streptomyces, while
plasmids pUB110, pC194, or pBD214 are suited for transformation of
Bacillus species. Several plasmids of use in the transfer of
genetic information into Corynebacterium include pHM1519, pBL1,
pSA77, or pAJ667 (Pouwels et al., eds. (1985) Cloning Vectors.
Elsevier: New York IBSN 0 444 904018).
[0096] 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, Calif. (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.
[0097] In another embodiment, the PTS protein expression vector is
a yeast expression vector. Examples of vectors for expression in
yeast S. cerivisae include pYepSec1 (Baldari, et al., (1987) Embo
J. 6:229-234), 2.mu., 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, Calif.). 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).
[0098] Alternatively, the PTS proteins of the invention can be
expressed in insect cells using baculovirus expression vectors.
Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g., Sf 9 cells) include the pAc series
(Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL
series (Lucklow and Summers (1989) Virology 170:31-39).
[0099] In another embodiment, the PTS proteins of the invention may
be expressed in unicellular plant cells (such as algae) or in plant
cells from higher plants (e.g., the spermatophytes, such as crop
plants). Examples of plant expression vectors include those
detailed in: Becker, D., Kemper, E., Schell, J. and Masterson, R.
(1992) "New plant binary vectors with selectable markers located
proximal to the left border", Plant Mol. Biol. 20:1195-1197; and
Bevan, M. W. (1984) "Binary Agrobacterium vectors for plant
transformation", Nuc. Acid. Res. 12: 8711-8721, and include pLGV23,
pGHlac+, pBIN19, pAK2004, and pDH51 (Pouwels et al., eds. (1985)
Cloning Vectors. Elsevier: New York IBSN 0 444 904018).
[0100] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987)
EMBO J 6:187-195). When used in mammalian cells, the expression
vector's control functions are often provided by viral regulatory
elements. For example, commonly used promoters are derived from
polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For
other suitable expression systems for both prokaryotic and
eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E.
F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd,
ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989.
[0101] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al. (1987) Genes
Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton
(1988) Adv. Immunol. 43:235-275), in particular promoters of T cell
receptors (Winoto and Baltimore (1989) EMBO J 8:729-733) and
immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and
Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle (1989) PNAS
86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)
Science 230:912-916), and mammary gland-specific promoters (e.g.,
milk whey promoter; U.S. Pat. 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
.alpha.-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.
3:537-546).
[0102] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. That is, the DNA
molecule is operatively linked to a regulatory sequence in a manner
which allows for expression (by transcription of the DNA molecule)
of an RNA molecule which is antisense to PTS mRNA. Regulatory
sequences operatively linked to a nucleic acid cloned in the
antisense orientation can be chosen which direct the continuous
expression of the antisense RNA molecule in a variety of cell
types, for instance viral promoters and/or enhancers, or regulatory
sequences can be chosen which direct constitutive, tissue specific
or cell type specific expression of antisense RNA. The antisense
expression vector can be in the form of a recombinant plasmid,
phagemid or attenuated virus in which antisense nucleic acids are
produced under the control of a high efficiency regulatory region,
the activity of which can be determined by the cell type into which
the vector is introduced. For a discussion of the regulation of
gene expression using antisense genes see Weintraub, H. et al.,
Antisense RNA as a molecular tool for genetic analysis,
Reviews--Trends in Genetics, Vol. 1(1) 1986.
[0103] Another aspect of the invention pertains to host cells into
which a recombinant expression vector of the invention has been
introduced. The terms "host cell" and "recombinant host cell" are
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0104] A host cell can be any prokaryotic or eukaryotic cell. For
example, a PTS protein can be expressed in bacterial cells such as
C. glutamicum, insect cells, yeast or mammalian cells (such as
Chinese hamster ovary cells (CHO) or COS cells). Other suitable
host cells are known to one of ordinary skill in the art.
Microorganisms related to Corynebacterium glutamicum which may be
conveniently used as host cells for the nucleic acid and protein
molecules of the invention are set forth in Table 3.
[0105] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety-of art-recognized techniques for
introducing foreign nucleic acid (e.g., linear DNA or RNA (e.g., a
linearized vector or a gene construct alone without a vector) or
nucleic acid in the form of a vector (e.g., a plasmid, phage,
phasmid, phagemid, transposon or other DNA) into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (Molecular Cloning: A
Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),
and other laboratory manuals.
[0106] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Preferred selectable markers
include those which confer resistance to drugs, such as G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable
marker can be introduced into a host cell on the same vector as
that encoding a PTS protein or can be introduced on a separate
vector. Cells stably transfected with the introduced nucleic acid
can be identified by drug selection (e.g., cells that have
incorporated the selectable marker gene will survive, while the
other cells die).
[0107] To create a homologous recombinant microorganism, a vector
is prepared which contains at least a portion of a PTS gene into
which a deletion, addition or substitution has been introduced to
thereby alter, e.g., functionally disrupt, the PTS gene.
Preferably, this PTS gene is a Corynebacterium glutamicum PTS gene,
but it can be a homologue from a related bacterium or even from a
mammalian, yeast, or insect source. In a preferred embodiment, the
vector is designed such that, upon homologous recombination, the
endogenous PTS gene is functionally disrupted (i.e., no longer
encodes a functional protein; also referred to as a "knock out"
vector). Alternatively, the vector can be designed such that, upon
homologous recombination, the endogenous PTS gene is mutated or
otherwise altered but still encodes functional protein (e.g., the
upstream regulatory region can be altered to thereby alter the
expression of the endogenous PTS protein). In the homologous
recombination vector, the altered portion of the PTS gene is
flanked at its 5' and 3' ends by additional nucleic acid of the PTS
gene to allow for homologous recombination to occur between the
exogenous PTS gene carried by the vector and an endogenous PTS gene
in a microorganism. The additional flanking PTS nucleic acid is of
sufficient length for successful homologous recombination with the
endogenous gene. Typically, several kilobases of flanking DNA (both
at the 5' and 3' ends) are included in the vector (see e.g.,
Thomas, K. R., and Capecchi, M. R. (1987) Cell 51: 503 for a
description of homologous recombination vectors). The vector is
introduced into a microorganism (e.g., by electroporation) and
cells in which the introduced PTS gene has homologously recombined
with the endogenous PTS gene are selected, using art-known
techniques.
[0108] In another embodiment, recombinant microorganisms can be
produced which contain selected systems which allow for regulated
expression of the introduced gene. For example, inclusion of a PTS
gene on a vector placing it under control of the lac operon permits
expression of the PTS gene only in the presence of IPTG. Such
regulatory systems are well known in the art.
[0109] In another embodiment, an endogenous PTS gene in a host cell
is disrupted (e.g., by homologous recombination or other genetic
means known in the art) such that expression of its protein product
does not occur. In another embodiment, an endogenous or introduced
PTS gene in a host cell has been altered by one or more point
mutations, deletions, or inversions, but still encodes a functional
PTS protein. In still another embodiment, one or more of the
regulatory regions (e.g., a promoter, repressor, or inducer) of a
PTS gene in a microorganism has been altered (e.g., by deletion,
truncation, inversion, or point mutation) such that the expression
of the PTS gene is modulated. One of ordinary skill in the art will
appreciate that host cells containing more than one of the
described PTS gene and protein modifications may be readily
produced using the methods of the invention, and are meant to be
included in the present invention.
[0110] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) a PTS protein. Accordingly, the invention further provides
methods for producing PTS proteins using the host cells of the
invention. In one embodiment, the method comprises culturing the
host cell of invention (into which a recombinant expression vector
encoding a PTS protein has been introduced, or into which genome
has been introduced a gene encoding a wild-type or altered PTS
protein) in a suitable medium until PTS protein is produced. In
another embodiment, the method further comprises isolating PTS
proteins from the medium or the host cell.
[0111] C. Isolated PTS Proteins
[0112] Another aspect of the invention pertains to isolated PTS
proteins, and biologically active portions thereof. An "isolated"
or "purified" protein or biologically active portion thereof is
substantially free of cellular material when produced by
recombinant DNA techniques, or chemical precursors or other
chemicals when chemically synthesized. The language "substantially
free of cellular material" includes preparations of PTS protein in
which the protein is separated from cellular components of the
cells in which it is naturally or recombinantly produced. In one
embodiment, the language "substantially free of cellular material"
includes preparations of PTS protein having less than about 30% (by
dry weight) of non-PTS protein (also referred to herein as a
"contaminating protein"), more preferably less than about 20% of
non-PTS protein, still more preferably less than about 10% of
non-PTS protein, and most preferably less than about 5% non-PTS
protein. When the PTS protein or biologically active portion
thereof is recombinantly produced, it is also preferably
substantially free of culture medium, i.e., culture medium
represents less than about 20%, more preferably less than about
10%, and most preferably less than about 5% of the volume of the
protein preparation. The language "substantially free of chemical
precursors or other chemicals" includes preparations of PTS protein
in which the protein is separated from chemical precursors or other
chemicals which are involved in the synthesis of the protein. In
one embodiment, the language "substantially free of chemical
precursors or other chemicals" includes preparations of PTS protein
having less than about 30% (by dry weight) of chemical precursors
or non-PTS chemicals, more preferably less than about 20% chemical
precursors or non-PTS chemicals, still more preferably less than
about 10% chemical precursors or non-PTS chemicals, and most
preferably less than about 5% chemical precursors or non-PTS
chemicals. In preferred embodiments, isolated proteins or
biologically active portions thereof lack contaminating proteins
from the same organism from which the PTS protein is derived.
Typically, such proteins are produced by recombinant expression of,
for example, a C. glutamicum PTS protein in a microorganism such as
C. glutamicum.
[0113] An isolated PTS protein or a portion thereof of the
invention can participate in the transport of high-energy
carbon-containing molecules such as glucose into C. glutamicum, and
may also participate in intracellular signal transduction in this
microorganism, or has one or more of the activities set forth in
Table 1. In preferred embodiments, the protein or portion thereof
comprises an amino acid sequence which is sufficiently homologous
to an amino acid sequence of Appendix B such that the protein or
portion thereof maintains the ability to transport high-energy
carbon-containing molecules such as glucose into C. glutamicum, or
to participate in intracellular signal transduction in this
microorganism. The portion of the protein is preferably a
biologically active portion as described herein. In another
preferred embodiment, a PTS protein of the invention has an amino
acid sequence shown in Appendix B. In yet another preferred
embodiment, the PTS protein has an amino acid sequence which is
encoded by a nucleotide sequence which hybridizes, e.g., hybridizes
under stringent conditions, to a nucleotide sequence of Appendix A.
In still another preferred embodiment, the PTS protein has an amino
acid sequence which is encoded by a nucleotide sequence that is at
least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or
60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%,
68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more
preferably at least about 95%, 96%, 97%, 98%, 99% or more
homologous to one of the nucleic acid sequences of Appendix A, or a
portion thereof. Ranges and identity values intermediate to the
above-recited values, (e.g., 70-90% identical or 80-95% identical)
are also intended to be encompassed by the present invention. For
example, ranges of identity values using a combination of any of
the above values recited as upper and/or lower limits are intended
to be included. The preferred PTS proteins of the present invention
also preferably possess at least one of the PTS activities
described herein. For example, a preferred PTS protein of the
present invention includes an amino acid sequence encoded by a
nucleotide sequence which hybridizes, e.g., hybridizes under
stringent conditions, to a nucleotide sequence of Appendix A, and
which can participate in the transport of high-energy
carbon-containing molecules such as glucose into C. glutamicum, and
may also participate in intracellular signal transduction in this
microorganism, or which has one or more of the activities set forth
in Table 1.
[0114] In other embodiments, the PTS protein is substantially
homologous to an amino acid sequence of Appendix B and retains the
functional activity of the protein of one of the sequences of
Appendix B yet differs in amino acid sequence due to natural
variation or mutagenesis, as described in detail in subsection I
above. Accordingly, in another embodiment, the PTS protein is a
protein which comprises an amino acid sequence which is at least
about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%,
preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,
69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably
at least about 95%, 96%, 97%, 98%, 99% or more homologous to an
entire amino acid sequence of Appendix B and which has at least one
of the PTS activities described herein. Ranges and identity values
intermediate to the above-recited values, (e.g., 70-90% identical
or 80-95% identical) are also intended to be encompassed by the
present invention. For example, ranges of identity values using a
combination of any of the above values recited as upper and/or
lower limits are intended to be included. In another embodiment,
the invention pertains to a full length C. glutamicum protein which
is substantially homologous to an entire amino acid sequence of
Appendix B.
[0115] Biologically active portions of a PTS protein include
peptides comprising amino acid sequences derived from the amino
acid sequence of a PTS protein, e.g., the an amino acid sequence
shown in Appendix B or the amino acid sequence of a protein
homologous to a PTS protein, which include fewer amino acids than a
full length PTS protein or the full length protein which is
homologous to a PTS protein, and exhibit at least one activity of a
PTS protein. Typically, biologically active portions (peptides,
e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35, 36,
37, 38, 39, 40, 50, 100 or more amino acids in length) comprise a
domain or motif with at least one activity of a PTS protein.
Moreover, other biologically active portions, in which other
regions of the protein are deleted, can be prepared by recombinant
techniques and evaluated for one or more of the activities
described herein. Preferably, the biologically active portions of a
PTS protein include one or more selected domains/motifs or portions
thereof having biological activity.
[0116] PTS proteins are preferably produced by recombinant DNA
techniques. For example, a nucleic acid molecule encoding the
protein is cloned into an expression vector (as described above),
the expression vector is introduced into a host cell (as described
above) and the PTS protein is expressed in the host cell. The PTS
protein can then be isolated from the cells by an appropriate
purification scheme using standard protein purification techniques.
Alternative to recombinant expression, a PTS protein, polypeptide,
or peptide can be synthesized chemically using standard peptide
synthesis techniques. Moreover, native PTS protein can be isolated
from cells (e.g., endothelial cells), for example using an anti-PTS
antibody, which can be produced by standard techniques utilizing a
PTS protein or fragment thereof of this invention.
[0117] The invention also provides PTS chimeric or fusion proteins.
As used herein, a PTS "chimeric protein" or "fusion protein"
comprises a PTS polypeptide operatively linked to a non-PTS
polypeptide. An "PTS polypeptide" refers to a polypeptide having an
amino acid sequence corresponding to PTS, whereas a "non-PTS
polypeptide" refers to a polypeptide having an amino acid sequence
corresponding to a protein which is not substantially homologous to
the PTS protein, e.g., a protein which is different from the PTS
protein and which is derived from the same or a different organism.
Within the fusion protein, the term "operatively linked" is
intended to indicate that the PTS polypeptide and the non-PTS
polypeptide are fused in-frame to each other. The non-PTS
polypeptide can be fused to the N-terminus or C-terminus of the PTS
polypeptide. For example, in one embodiment the fusion protein is a
GST-PTS fusion protein in which the PTS sequences are fused to the
C-terminus of the GST sequences. Such fusion proteins can
facilitate the purification of recombinant PTS proteins. In another
embodiment, the fusion protein is a PTS protein containing a
heterologous signal sequence at its N-terminus. In certain host
cells (e.g., mammalian host cells), expression and/or secretion of
a PTS protein can be increased through use of a heterologous signal
sequence.
[0118] Preferably, a PTS chimeric or fusion protein of the
invention is produced by standard recombinant DNA techniques. For
example, DNA fragments coding for the different polypeptide
sequences are ligated together in-frame in accordance with
conventional techniques, for example by employing blunt-ended or
stagger-ended termini for ligation, restriction enzyme digestion to
provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. In another embodiment, the fusion
gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and reamplified to
generate a chimeric gene sequence (see, for example, Current
Protocols in Molecular Biology, eds. Ausubel et al. John Wiley
& Sons: 1992). Moreover, many expression vectors are
commercially available that already encode a fusion moiety (e.g., a
GST polypeptide). A PTS-encoding nucleic acid can be cloned into
such an expression vector such that the fusion moiety is linked
in-frame to the PTS protein.
[0119] Homologues of the PTS protein can be generated by
mutagenesis, e.g., discrete point mutation or truncation of the PTS
protein. As used herein, the term "homologue" refers to a variant
form of the PTS protein which acts as an agonist or antagonist of
the activity of the PTS protein. An agonist of the PTS protein can
retain substantially the same, or a subset, of the biological
activities of the PTS protein. An antagonist of the PTS protein can
inhibit one or more of the activities of the naturally occurring
form of the PTS protein, by, for example, competitively binding to
a downstream or upstream member of the PTS system which includes
the PTS protein. Thus, the C. glutamicum PTS protein and homologues
thereof of the present invention may modulate the activity of one
or more sugar transport pathways or intracellular signal
transduction pathways in which PTS proteins play a role in this
microorganism.
[0120] In an alternative embodiment, homologues of the PTS protein
can be identified by screening combinatorial libraries of mutants,
e.g., truncation mutants, of the PTS protein for PTS protein
agonist or antagonist activity. In one embodiment, a variegated
library of PTS variants is generated by combinatorial mutagenesis
at the nucleic acid level and is encoded by a variegated gene
library. A variegated library of PTS variants can be produced by,
for example, enzymatically ligating a mixture of synthetic
oligonucleotides into gene sequences such that a degenerate set of
potential PTS sequences is expressible as individual polypeptides,
or alternatively, as a set of larger fusion proteins (e.g., for
phage display) containing the set of PTS sequences therein. There
are a variety of methods which can be used to produce libraries of
potential PTS homologues from a degenerate oligonucleotide
sequence. Chemical synthesis of a degenerate gene sequence can be
performed in an automatic DNA synthesizer, and the synthetic gene
then ligated into an appropriate expression vector. Use of a
degenerate set of genes allows for the provision, in one mixture,
of all of the sequences encoding the desired set of potential PTS
sequences. Methods for synthesizing degenerate oligonucleotides are
known in the art (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3;
Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al.
(1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res.
11:477.
[0121] In addition, libraries of fragments of the PTS protein
coding can be used to generate a variegated population of PTS
fragments for screening and subsequent selection of homologues of a
PTS protein. In one embodiment, a library of coding sequence
fragments can be generated by treating a double stranded PCR
fragment of a PTS coding sequence with a nuclease under conditions
wherein nicking occurs only about once per molecule, denaturing the
double stranded DNA, renaturing the DNA to form double stranded DNA
which can include sense/antisense pairs from different nicked
products, removing single stranded portions from reformed duplexes
by treatment with S I nuclease, and ligating the resulting fragment
library into an expression vector. By this method, an expression
library can be derived which encodes N-terminal, C-terminal and
internal fragments of various sizes of the PTS protein.
[0122] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. Such techniques are adaptable for rapid
screening of the gene libraries generated by the combinatorial
mutagenesis of PTS homologues. The most widely used techniques,
which are amenable to high through-put analysis, for screening
large gene libraries typically include cloning the gene library
into replicable expression vectors, transforming appropriate cells
with the resulting library of vectors, and expressing the
combinatorial genes under conditions in which detection of a
desired activity facilitates isolation of the vector encoding the
gene whose product was detected. Recursive ensemble mutagenesis
(REM), a new technique which enhances the frequency of functional
mutants in the libraries, can be used in combination with the
screening assays to identify PTS homologues (Arkin and Yourvan
(1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein
Engineering 6(3):327-331).
[0123] In another embodiment, cell based assays can be exploited to
analyze a variegated PTS library, using methods well known in the
art.
[0124] D. Uses and Methods of the Invention
[0125] The nucleic acid molecules, proteins, protein homologues,
fusion proteins, primers, vectors, and host cells described herein
can be used in one or more of the following methods: identification
of C. glutamicum and related organisms; mapping of genomes of
organisms related to C. glutamicum; identification and localization
of C. glutamicum sequences of interest; evolutionary studies;
determination of PTS protein regions required for function;
modulation of a PTS protein activity; modulation of the activity of
a PTS pathway; and modulation of cellular production of a desired
compound, such as a fine chemical.
[0126] The PTS nucleic acid molecules of the invention have a
variety of uses. First, they may be used to identify an organism as
being Corynebacterium glutamicum or a close relative thereof. Also,
they may be used to identify the presence of C. glutamicum or a
relative thereof in a mixed population of microorganisms. The
invention provides the nucleic acid sequences of a number of C.
glutamicum genes; by probing the extracted genomic DNA of a culture
of a unique or mixed population of microorganisms 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.
[0127] Although Corynebacterium glutamicum itself is nonpathogenic,
it is related to pathogenic species, such as Corynebacterium
diphtheriae. Corynebacterium diphtheriae is the causative agent of
diphtheria, a rapidly developing, acute, febrile infection which
involves both local and systemic pathology. In this disease, a
local lesion develops in the upper respiratory tract and involves
necrotic injury to epithelial cells; the bacilli secrete toxin
which is disseminated through this lesion to distal susceptible
tissues of the body. Degenerative changes brought about by the
inhibition of protein synthesis in these tissues, which include
heart, muscle, peripheral nerves, adrenals, kidneys, liver and
spleen, result in the systemic pathology of the disease. Diphtheria
continues to have high incidence in many parts of the world,
including Africa, Asia, Eastern Europe and the independent states
of the former Soviet Union. An ongoing epidemic of diphtheria in
the latter two regions has resulted in at least 5,000 deaths since
1990.
[0128] 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 in Appendix A or Appendix B) in a subject,
thereby detecting the presence or activity of Corynebacterium
diphtheriae in the subject. C. glutamicum and C. diphtheriae are
related bacteria, and many of the nucleic acid and protein
molecules in C. glutamicum are homologous to C. diphtheriae nucleic
acid and protein molecules, and can therefore be used to detect C.
diphtheriae in a subject.
[0129] The nucleic acid and protein molecules of the invention may
also serve as markers for specific regions of the genome. This has
utility not only in the mapping of the genome, but also for
functional studies of C. glutamicum proteins. For example, to
identify the region of the genome to which a particular C.
glutamicum DNA-binding protein binds, the C. glutamicum genome
could be digested, and the fragments incubated with the DNA-binding
protein. Those which bind the protein may be additionally probed
with the nucleic acid molecules of the invention, preferably with
readily detectable labels; binding of such a nucleic acid molecule
to the genome fragment enables the localization of the fragment to
the genome map of C. glutamicum, and, when performed multiple times
with different enzymes, facilitates a rapid determination of the
nucleic acid sequence to which the protein binds. Further, the
nucleic acid molecules of the invention may be sufficiently
homologous to the sequences of related species such that these
nucleic acid molecules may serve as markers for the construction of
a genomic map in related bacteria, such as Brevibacterium
lactofermentum.
[0130] The PTS nucleic acid molecules of the invention are also
useful for evolutionary and protein structural studies. The sugar
uptake system in which the molecules of the invention participate
are utilized by a wide variety of bacteria; by comparing the
sequences of the nucleic acid molecules of the present invention to
those encoding similar enzymes from other organisms, the
evolutionary relatedness of the organisms can be assessed.
Similarly, such a comparison permits an assessment of which regions
of the sequence are conserved and which are not, which may aid in
determining those regions of the protein which are essential for
the functioning of the enzyme. This type of determination is of
value for protein engineering studies and may give an indication of
what the protein can tolerate in terms of mutagenesis without
losing function.
[0131] Manipulation of the PTS nucleic acid molecules of the
invention may result in the production of PTS proteins having
functional differences from the wild-type PTS proteins. These
proteins may be improved in efficiency or activity, may be present
in greater numbers in the cell than is usual, or may be decreased
in efficiency or activity.
[0132] The invention provides methods for screening molecules which
modulate the activity of a PTS protein, either by interacting with
the protein itself or a substrate or binding partner of the PTS
protein, or by modulating the transcription or translation of a PTS
nucleic acid molecule of the invention. In such methods, a
microorganism expressing one or more PTS proteins of the invention
is contacted with one or more test compounds, and the effect of
each test compound on the activity or level of expression of the
PTS protein is assessed.
[0133] The PTS molecules of the invention may be modified such that
the yield, production, and/or efficiency of production of one or
more fine chemicals is improved. For example, by modifying a PTS
protein involved in the uptake of glucose such that it is optimized
in activity, the quantity of glucose uptake or the rate at which
glucose is translocated into the cell may be increased. The
breakdown of glucose and other sugars within the cell provides
energy that may be used to drive energetically unfavorable
biochemical reactions, such as those involved in the biosynthesis
of fine chemicals. This breakdown also provides intermediate and
precursor molecules necessary for the biosynthesis of certain fine
chemicals, such as amino acids, vitamins and cofactors. By
increasing the amount of intracellular high-energy carbon molecules
through modification of the PTS molecules of the invention, one may
therefore increase both the energy available to perform metabolic
pathways necessary for the production of one or more fine
chemicals, and also the intracellular pools of metabolites
necessary for such production. Conversely, by decreasing the
importation of a sugar whose breakdown products include a compound
which is used solely in metabolic pathways which compete with
pathways utilized for the production of a desired fine chemical for
enzymes, cofactors, or intermediates, one may downregulate this
pathway and thus perhaps increase production through the desired
biosynthetic pathway.
[0134] Further, the PTS molecules of the invention may be involved
in one or more intracellular signal transduction pathways which may
affect the yields and/or rate of production of one or more fine
chemical from C. glutamicum. For example, proteins necessary for
the import of one or more sugars from the extracellular medium
(e.g., HPr, Enzyme I, or a member of an Enzyme II complex) are
frequently posttranslationally modified upon the presence of a
sufficient quantity of the sugar in the cell, such that they are no
longer able to import that sugar. An example of this occurs in E.
coli, where high intracellular levels of fructose 1,6-bisphosphate
result in the phosphorylation of HPr at serine-46, upon which this
molecule is no longer able to participate in the transport of any
sugar. While this intracellular level of sugar at which the
transport system is shut off may be sufficient to sustain the
normal functioning of the cell, it may be limiting for the
overproduction of the desired fine chemical. Thus, it may be
desirable to modify the PTS proteins of the invention such that
they are no longer responsive to such negative regulation, thereby
permitting greater intracellular concentrations of one or more
sugars to be achieved, and, by extension, more efficient production
or greater yields of one or more fine chemicals from organisms
containing such mutant PTS proteins.
[0135] This aforementioned list of mutagenesis strategies for PTS
proteins to result in increased yields of a desired compound is not
meant to be limiting; variations on these mutagenesis strategies
will be readily apparent to one of ordinary skill in the art. By
these mechanisms, the nucleic acid and protein molecules of the
invention may be utilized to generate C. glutamicum or related
strains of bacteria expressing mutated PTS nucleic acid and protein
molecules such that the yield, production, and/or efficiency of
production of a desired compound is improved. This desired compound
may be any natural product of C. glutamicum, which includes the
final products of biosynthesis pathways and intermediates of
naturally-occurring metabolic pathways, as well as molecules which
do not naturally occur in the metabolism of C. glutamicum, but
which are produced by a C. glutamicum strain of the invention.
[0136] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patent applications, patents, published patent
applications, Tables, Appendices, and the sequence listing cited
throughout this application are hereby incorporated by
reference.
[0137] Exemplification
EXAMPLE 1
Preparation of Total Genomic DNA of Corynebacterium glutamicum ATCC
13032
[0138] A culture of Corynebacterium glutamicum (ATCC 13032) was
grown overnight at 30.degree. C. with vigorous shaking in BHI
medium (Difco). The cells were harvested by centrifugation, the
supernatant was discarded and the cells were resuspended in 5 ml
buffer-I (5% of the original volume of the culture--all indicated
volumes have been calculated for 100 ml of culture volume).
Composition of buffer-I: 140.34 g/l sucrose, 2.46 g/l
MgSO.sub.4.times.7H.sub.2O, 10 ml/l KH.sub.2PO.sub.4 solution (100
g/l, adjusted to pH 6.7 with KOH), 50 ml/l M12 concentrate (10 g/l
(NH.sub.4).sub.2SO.sub.4, 1 g/l NaCl, 2 g/l
MgSO.sub.4.times.7H.sub.2O, 0.2 g/l CaCl.sub.2, 0.5 g/l yeast
extract (Difco), 10 ml/l trace-elements-mix (200 mg/l
FeSO.sub.4.times.H.sub.2O, 10 mg/l ZnSO.sub.4.times.7 H.sub.2O, 3
mg/l MnCl.sub.2.times.4 H.sub.2O, 30 mg/l H.sub.3BO.sub.3 20 mg/l
CoCl.sub.2.times.6 H.sub.2O, 1 mg/l NiCl.sub.2.times.6 H.sub.2O, 3
mg/l Na.sub.2MoO.sub.4.times.2 H.sub.2O, 500 mg/l complexing agent
(EDTA or critic acid), 100 ml/l vitamins-mix (0.2 mg/l biotin, 0.2
mg/l folic acid, 20 mg/l p-amino benzoic acid, 20 mg/l riboflavin,
40 mg/l ca-panthothenate, 140 mg/l nicotinic acid, 40 mg/l
pyridoxole hydrochloride, 200 mg/l 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.degree. 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 NaCl solution (5 M) are added. After adding of
proteinase K to a final concentration of 200 .mu.g/ml, the
suspension is incubated for ca.18 h at 37.degree. C. The DNA was
purified by extraction with phenol,
phenol-chloroform-isoamylalcohol and chloroform-isoamylalcohol
using standard procedures. Then, the DNA was precipitated by adding
1/50 volume of 3 M sodium acetate and 2 volumes of ethanol,
followed by a 30 min incubation at -20.degree. C. and a 30 min
centrifugation at 12,000 rpm in a high speed centrifuge using a
SS34 rotor (Sorvall). The DNA was dissolved in 1 ml TE-buffer
containing 20 .mu.g/ml RNaseA and dialysed at 4.degree. C. against
1000 ml TE-buffer for at least 3 hours. During this time, the
buffer was exchanged 3 times. To aliquots of 0.4 ml of the dialysed
DNA solution, 0.4 ml of 2 M LiCl and 0.8 ml of ethanol are added.
After a 30 min incubation at -20.degree. C., the DNA was collected
by centrifugation (13,000 rpm, Biofuge Fresco, Heraeus, Hanau,
Germany). The DNA pellet was dissolved in TE-buffer. DNA prepared
by this procedure could be used for all purposes, including
southern blotting or construction of genomic libraries.
EXAMPLE 2
Construction of Genomic Libraries in Escherichia coli of
Corynebacterium glutamicum ATCC13032.
[0139] 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. (1 989)
"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.)
[0140] 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
SuperCos1 (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 pSL109 (Lee, H.-S. and A. J. Sinskey (1994) J.
Microbiol. Biotechnol. 4: 256-263).
EXAMPLE 3
DNA Sequencing and Computational Functional Analysis
[0141] Genomic libraries as described in Example 2 were used for
DNA sequencing according to standard methods, in particular by the
chain termination method using ABI377 sequencing machines (see
e.g., Fleischman, R. D. et al. (1995) "Whole-genome Random
Sequencing and Assembly of Haemophilus Influenzae Rd., Science,
269:496-512). Sequencing primers with the following nucleotide
sequences were used:
1 5'- GGAAACAGTATGACCATG-3' or 5'-GTAAAACGACGGCCAGT-3'.
EXAMPLE 4
In vivo Mutagenesis
[0142] 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 the integrity of their genetic information. Typical
mutator strains have mutations in the genes for the DNA repair
system (e.g., mutHLS, mutD, mutT, etc.; for reference, see Rupp, W.
D. (1996) DNA repair mechanisms, in: Escherichia coli and
Salmonella, p. 2277-2294, ASM: Washington.) Such strains are well
known to one of ordinary skill in the art. The use of such strains
is illustrated, for example, in Greener, A. and Callahan, M. (1994)
Strategies 7: 32-34.
EXAMPLE 5
DNA: Transfer Between Escherichia coli and Corynebacterium
glutamicum
[0143] Several Corynebacterium and Brevibacterium species contain
endogenous plasmids (as e.g., pHM1519 or pBL1) which replicate
autonomously (for review see, e.g., Martin, J. F. et al. (1987)
Biotechnology, 5:137-146). Shuttle vectors for Escherichia coli and
Corynebacterium glutamicum can be readily constructed by using
standard vectors for E. coli (Sambrook, J. et al. (1989),
"Molecular Cloning: A Laboratory Manual", Cold Spring Harbor
Laboratory Press or Ausubel, F. M. et al. (1994) "Current Protocols
in Molecular Biology", John Wiley & Sons) to which a origin or
replication for and a suitable marker from Corynebacterium
glutamicum is added. Such origins of replication are preferably
taken from endogenous plasmids isolated from Corynebacterium and
Brevibacterium species. Of particular use as transformation markers
for these species are genes for kanamycin resistance (such as those
derived from the Tn5 or Tn903 transposons) or chloramphenicol
(Winnacker, E. L. (1987) "From Genes to Clones--Introduction to
Gene Technology, VCH, Weinheim). There are numerous examples in the
literature of the construction of a wide variety of shuttle vectors
which replicate in both E. coli and C. glutamicum, and which can be
used for several purposes, including gene over-expression (for
reference, see e.g., Yoshihama, M. et al. (1985) J Bacteriol.
162:591-597, Martin J. F. et al. (1987) Biotechnology, 5:137-146
and Eikmanns, B. J. et al. (1991) Gene, 102:93-98).
[0144] Using standard methods, it is possible to clone a gene of
interest into one of the shuttle vectors described above and to
introduce such a hybrid vectors into strains of Corynebacterium
glutamicum. Transformation of C. glutamicum can be achieved by
protoplast transformation (Kastsumata, R. et al. (1984) J.
Bacteriol. 159306-311), electroporation (Liebl, E. et al. (1989)
FEMS Microbiol. Letters, 53:399-303) and in cases where special
vectors are used, also by conjugation (as described e.g. in Schfer,
A et al. (1990) J. Bacteriol. 172:1663-1666). It is also possible
to transfer the shuttle vectors for C. glutamicum to E. coli by
preparing plasmid DNA from C. glutamicum (using standard methods
well-known in the art) and transforming it into E. coli. This
transformation step can be performed using standard methods, but it
is advantageous to use an Mcr-deficient E. coli strain, such as
NM522 (Gough & Murray (1983) J. Mol. Biol. 166:1-19).
[0145] Genes may be overexpressed in C. glutamicum strains using
plasmids which comprise pCG1 (U.S. Pat. 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).
[0146] 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
[0147] 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.
(1988) Current Protocols in Molecular Biology, Wiley: New York), in
which a primer designed to bind to the gene of interest is labeled
with a detectable tag (usually radioactive or chemiluminescent),
such that when the total RNA of a culture of the organism is
extracted, run on gel, transferred to a stable matrix and incubated
with this probe, the binding and quantity of binding of the probe
indicates the presence and also the quantity of mRNA for this gene.
This information is evidence of the degree of transcription of the
mutant gene. Total cellular RNA can be prepared from
Corynebacterium glutamicum by several methods, all well-known in
the art, such as that described in Bormann, E. R. et al. (1992)
Mol. Microbiol. 6: 317-326.
[0148] 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
[0149] Genetically modified Corynebacteria are cultured in
synthetic or natural growth media. A number of different growth
media for Corynebacteria are both well-known and readily available
(Lieb et al. (1989) Appl. Microbiol. Biotechnol., 32:205-210; von
der Osten et al. (1998) Biotechnology Letters, 11:11-16; Patent DE
4,120,867; Liebl (1992) "The Genus Corynebacterium, in: The
Procaryotes, Volume II, Balows, A. et al., eds. Springer-Verlag).
These media consist of one or more carbon sources, nitrogen
sources, inorganic salts, vitamins and trace elements. Preferred
carbon sources are sugars, such as mono-, di-, or polysaccharides.
For example, glucose, fructose, mannose, galactose, ribose,
sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or
cellulose serve as very good carbon sources. It is also possible to
supply sugar to the media via complex compounds such as molasses or
other by-products from sugar refinement. It can also be
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.sub.4Cl or
(NH.sub.4).sub.2SO.sub.4, NH.sub.4OH, nitrates, urea, amino acids
or complex nitrogen sources like corn steep liquor, soy bean flour,
soy bean protein, yeast extract, meat extract and others.
[0150] Inorganic salt compounds which may be included in the media
include the chloride-, phosphorous- or sulfate-salts of calcium,
magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc,
copper and iron. Chelating compounds can be added to the medium to
keep the metal ions in solution. Particularly useful chelating
compounds include dihydroxyphenols, like catechol or
protocatechuate, or organic acids, such as citric acid. It is
typical for the media to also contain other growth factors, such as
vitamins or growth promoters, examples of which include biotin,
riboflavin, thiamin, folic acid, nicotinic acid, pantothenate and
pyridoxin. Growth factors and salts frequently originate from
complex media components such as yeast extract, molasses, corn
steep liquor and others. The exact composition of the media
compounds depends strongly on the immediate experiment and is
individually decided for each specific case. Information about
media optimization is available in the textbook "Applied Microbiol.
Physiology, A Practical Approach (eds. P. M. Rhodes, P. F.
Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). It is
also possible to select growth media from commercial suppliers,
like standard 1 (Merck) or BHI (grain heart infusion, DIFCO) or
others.
[0151] All medium components are sterilized, either by heat (20
minutes at 1.5 bar and 121.degree. 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.
[0152] Culture conditions are defined separately for each
experiment. The temperature should be in a range between 15.degree.
C. and 45.degree. 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 is also possible to maintain a constant
culture pH through the addition of NaOH or NH.sub.4OH 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.
[0153] The incubation time is usually in a range from several hours
to several days. This time is selected in order to permit the
maximal amount of product to accumulate in the broth. The disclosed
growth experiments can be carried out in a variety of vessels, such
as microtiter plates, glass tubes, glass flasks or glass or metal
fermentors of different sizes. For screening a large number of
clones, the microorganisms should be cultured in microtiter plates,
glass tubes or shake flasks, either with or without baffles.
Preferably 100 ml shake flasks are used, filled with 10% (by
volume) of the required growth medium. The flasks should be shaken
on a rotary shaker (amplitude 25 mm) using a speed-range of 100-300
rpm. Evaporation losses can be diminished by the maintenance of a
humid atmosphere; alternatively, a mathematical correction for
evaporation losses should be performed.
[0154] 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 OD.sub.600 of O0.5-1.5 using cells grown on agar plates, such
as CM plates (10 g/l glucose, 2,5 g/l NaCl, 2 g/l urea, 10 g/l
polypeptone, 5 g/l yeast extract, 5 g/l meat extract, 22 g/l NaCl,
2 g/l urea, 10 g/l polypeptone, 5 g/l yeast extract, 5 g/l meat
extract, 22 g/l agar, pH 6.8 with 2M NaOH) that had been incubated
at 30.degree. 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
[0155] 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 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.sup.rd ed. Academic Press:
New York; Bisswanger, H., (1994) Enzymkinetik, 2.sup.nd ed. VCH:
Weinheim (ISBN 3527300325); Bergmeyer, H. U., Bergmeyer, J.,
Gra.beta.1, M., eds. (1983-1986) Methods of Enzymatic Analysis,
3.sup.rd ed., vol. I-XII, Verlag Chemie: Weinheim; and Ullmann's
Encyclopedia of Industrial Chemistry (1987) vol. A9, "Enzymes".
VCH: Weinheim, p. 352-363.
[0156] 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.
[0157] 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
[0158] 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, Ullman, Encyclopedia of
Industrial Chemistry, vol. A2, p. 89-90 and p. 443-613, VCH:
Weinheim (1985); Fallon, A. et al., (1987) "Applications of HPLC in
Biochemistry" in: Laboratory Techniques in Biochemistry and
Molecular Biology, vol. 17; Rehm et al. (1993) Biotechnology, vol.
3, Chapter III: "Product recovery and purification", page 469-714,
VCH: Weinheim; Belter, P. A. et al. (1988) Bioseparations:
downstream processing for biotechnology, John Wiley and Sons;
Kennedy, J. F. and Cabral, J. M. S. (1992) Recovery processes for
biological materials, John Wiley and Sons; Shaeiwitz, J. A. and
Henry, J. D. (1988) Biochemical separations, in: Ulmann's
Encyclopedia of Industrial Chemistry, vol. B3, Chapter 11, page
1-27, VCH: Weinheim; and Dechow, F. J. (1989) Separation and
purification techniques in biotechnology, Noyes Publications.) In
addition to the measurement of the final product of fermentation,
it is also possible to analyze other components of the metabolic
pathways utilized for the production of the desired compound, such
as intermediates and side-products, to determine the overall
productivity of the organism, yield, and/or efficiency of
production of the compound. Analysis methods include measurements
of nutrient levels in the medium (e.g., sugars, hydrocarbons,
nitrogen sources, phosphate, and other ions), measurements of
biomass composition and growth, analysis of the production of
common metabolites of biosynthetic pathways, and measurement of
gasses produced during fermentation. Standard methods for these
measurements are outlined in Applied Microbial Physiology, A
Practical Approach, P. M. Rhodes and P. F. Stanbury, eds., IRL
Press, p. 103-129; 131-163; and 165-192 (ISBN: 0199635773) and
references cited therein.
EXAMPLE 10
Purification of the Desired Product from C. glutamicum Culture
[0159] Recovery of the desired product from the C. glutamicum cells
or supernatant of the above-described culture can be performed by
various methods well known in the art. If the desired product is
not secreted from the cells, the cells can be harvested from the
culture by low-speed centrifugation, the cells can be lysed by
standard techniques, such as mechanical force or sonication. The
cellular debris is removed by centrifugation, and the supernatant
fraction containing the soluble proteins is retained for further
purification of the desired compound. If the product is secreted
from the C. glutamicum cells, then the cells are removed from the
culture by low-speed centrifugation, and the supernate fraction is
retained for further purification.
[0160] The supernatant fraction from either purification method is
subjected to chromatography with a suitable resin, in which the
desired molecule is either retained on a chromatography resin while
many of the impurities in the sample are not, or where the
impurities are retained by the resin while the sample is not. Such
chromatography steps may be repeated as necessary, using the same
or different chromatography resins. One of ordinary skill in the
art would be well-versed in the selection of appropriate
chromatography resins and in their most efficacious application for
a particular molecule to be purified. The purified product may be
concentrated by filtration or ultrafiltration, and stored at a
temperature at which the stability of the product is maximized.
[0161] 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).
[0162] The identity and purity of the isolated compounds may be
assessed by techniques standard in the art. These include
high-performance liquid chromatography (HPLC), spectroscopic
methods, staining methods, thin layer chromatography, NIRS,
enzymatic assay, or microbiologically. Such analysis methods are
reviewed in: Patek et al. (1994) Appl. Environ. Microbiol. 60:
133-140; Malakhova et al. (1996) Biotekhnologiya 11: 27-32; and
Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's
Encyclopedia of Industrial Chemistry, (1996) vol. A27, VCH:
Weinheim, p. 89-90, p. 521-540, p. 540-547, p. 559-566, 575-581 and
p. 581-587; Michal, G. (1999) Biochemical Pathways: An Atlas of
Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A.
et al. (1987) Applications of HPLC in Biochemistry in: Laboratory
Techniques in Biochemistry and Molecular Biology, vol. 17.
EXAMPLE 11
Analysis of the Gene Sequences of the Invention
[0163] The comparison of sequences and determination of percent
homology between two sequences are art-known techniques, and can be
accomplished using a mathematical algorithm, such as the algorithm
of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA
87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl.
Acad. Sci. USA 90:5873-77. Such an-algorithm is incorporated into
the NBLAST and XBLAST programs (version 2.0) of Altschul, et al.
(1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be
performed with the NBLAST program, score=100, wordlength=12 to
obtain nucleotide sequences homologous to PTS nucleic acid
molecules of the invention. BLAST protein searches can be performed
with the XBLAST program, score=50, wordlength=3 to obtain amino
acid sequences homologous to PTS protein molecules of the
invention. To obtain gapped alignments for comparison purposes,
Gapped BLAST can be utilized as described in Altschul et al.,
(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST
and Gapped BLAST programs, one of ordinary skill in the art will
know how to optimize the parameters of the program (e.g., XBLAST
and NBLAST) for the specific sequence being analyzed.
[0164] 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 PAM120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4
can be used. Additional algorithms for sequence analysis are known
in the art, and include ADVANCE and ADAM. described in Torelli and
Robotti (1994) Comput. Appl. Biosci. 10:3-5; and FASTA, described
in Pearson and Lipman (1988) P.N.A.S. 85:2444-8.
[0165] 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.
[0166] 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) Bioinfornatics: A Practical Guide to the Analysis of
Genes and Proteins. John Wiley and Sons: New York). The gene
sequences of the invention were compared to genes present in
Genbank in a three-step process. In a first step, a BLASTN analysis
(e.g., a local alignment analysis) was performed for each of the
sequences of the invention against the nucleotide sequences present
in Genbank, and the 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
[0167] 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).
[0168] DNA microarrays are solid or flexible supports consisting of
nitrocellulose, nylon, glass, silicone, or other materials. Nucleic
acid molecules may be attached to the surface in an ordered manner.
After appropriate labeling, other nucleic acids or nucleic acid
mixtures can be hybridized to the immobilized nucleic acid
molecules, and the label may be used to monitor and measure the
individual signal intensities of the hybridized molecules at
defined regions. This methodology allows the simultaneous
quantification of the relative or absolute amount of all or
selected nucleic acids in the applied nucleic 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).
[0169] 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).
[0170] 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.
[0171] The nucleic acid molecules of the invention present in a
sample or mixture of nucleotides may be hybridized to the
microarrays. These nucleic acid molecules can be labeled according
to standard methods. In brief, nucleic acid molecules (e.g., mRNA
molecules or DNA molecules) are labeled by the incorporation of
isotopically or fluorescently labeled nucleotides, e.g., during
reverse transcription or DNA synthesis. Hybridization of labeled
nucleic acids to microarrays is described (e.g., in Schena, M. et
al. (1995) supra; Wodicka, L. et al. (1997), supra; and DeSaizieu
A. et al. (1998), supra). The detection and quantification of the
hybridized molecule are tailored to the specific incorporated
label. Radioactive labels can be detected, for example, as
described in Schena, M. et al. (1995) supra) and fluorescent labels
may be detected, for example, by the method of Shalon et al. (1996)
Genome Research 6: 639-645).
[0172] 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)
[0173] 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.
[0174] Protein populations can be analyzed by various well-known
techniques, such as gel electrophoresis. Cellular proteins may be
obtained, for example, by lysis or extraction, and may be separated
from one another using a variety of electrophoretic techniques.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) separates proteins largely on the basis of their
molecular weight. Isoelectric focusing polyacrylamide gel
electrophoresis (IEF-PAGE) separates proteins by their isoelectric
point (which reflects not only the amino acid sequence but also
posttranslational modifications of the protein). Another, more
preferred method of protein analysis is the consecutive combination
of both IEF-PAGE and SDS-PAGE, known as 2-D-gel electrophoresis
(described, for example, in Hermann et al. (1998) Electrophoresis
19: 3217-3221; Fountoulakis et al. (1998) Electrophoresis 19:
1193-1202; Langen et al. (1997) Electrophoresis 18: 1184-1192;
Antelmann et al. (1997) Electrophoresis 18: 1451-1463). Other
separation techniques may also be utilized for protein separation,
such as capillary gel electrophoresis; such techniques are well
known in the art.
[0175] 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., .sup.35S-methionine,
.sup.35S-cysteine, .sup.14C-labelled amino acids, .sup.15N-amino
acids, .sup.15NO.sub.3 or .sup.15NH.sub.4.sup.+ or
.sup.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.
[0176] 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.
[0177] To determine the identity of any given protein, direct
sequencing or other standard techniques may be employed. For
example, N- and/or C-terminal amino acid sequencing (such as Edman
degradation) may be used, as may mass spectrometry (in particular
MALDI or ESI techniques (see, e.g., Langen et al. (1997)
Electrophoresis 18: 1184-1192)). The protein sequences provided
herein can be used for the identification of C. glutamicum proteins
by these techniques.
[0178] The information obtained by these methods can be used to
compare patterns of protein presence, activity, or modification
between different samples from various biological conditions (e.g.,
different organisms, time points of fermentation, media conditions,
or different biotopes, among others). Data obtained from such
experiments alone, or in combination with other techniques, can be
used for various applications, such as to compare the behavior of
various organisms in a given (e.g., metabolic) situation, to
increase the productivity of strains which produce fine chemicals
or to increase the efficiency of the production of fine
chemicals.
EQUIVALENTS
[0179] 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.
Appendix B: Amino Acid Sequences
[0180]
2 > RXA00315 (1-1086, translated) 362 residues YDFGGPVGGL
LFGLVYSPIV ITGLHQSFPP IELELFNQGG SFIFATASMA NIAQGAACLA VFFLAKSEKL
KGLAGASGVS AVLGITEPAI FGVNLRLRWP FFIGIGTAAI GGALIALFNI KAVALGAAGF
LGVVSIDAPD MVMFLVCAVV TFFIAEGAAI AYGLYLVRRN GSIDPDATAA PVPAGTTKAE
AEAPAEFSND STIIQAPLTG EAIALSSVSD AMFASGKLGS GVAIVPTKGQ LVSPVSGKIV
VAEPSGHAFA VRTKAEDGSN VDILMHIGFD TVNLNGTHFN PLKKQGDEVK AGELLCEFDI
DAIKAAGYEV TTPIVVSNYK KTGPVNTYGL GEIEAGANLL NVAKKEAVPA TP >
RXA00951 (1-393, translated) 131 residues IQAILEKAAA PAKQKAPAVA
PAVTPTDAPA ASVQSKTHDK ILTVCGNGLG TSLFLKNTLE QVFDTWGWGP YMTVEATDTI
SAKGKAKEAD LIMTSGEIAR TLGDVGIPVH VINDFTSTDE IDAALRERYD I >
RXA01244 (1-1509, translated) 503 residues LLERSEAAEG PAAEVLKATA
GMVNDRGWRK AVIKGVKGGH PAEYAVVAAT TKFISMFEAA GGLIAERTTD LRDIRDRVIA
ELRGDEEPGL PAVSGQVILF ADDLSPADTA ALDTDLFVGL VTELGGPTSH TAIIARQLNV
PCIVASGAGI KDIKSGEKVL IDGSLGTIDR NADEAEATKL VSESLERAAR IAEWKGPAQT
KDGYRVQLLA NVQDGNSAQQ AAQTEAEGIG LFRTELCFLS ATEEPSVDEQ AAVYSKVLEA
FPESKVVVRS LDAGSDKPVP FASMADEMNP ALGVRGLRIA RGQVDLLTRQ LDAIAKASEE
LGRGDDAPTW VMAPMVATAY EAKWFADMCR ERGLIAGAMI EVPAASLMAD KIMPHLDFVS
IGTNDLTQYT MAADRMSPEL AYLTDPWQPA VLRLIKHTCD EGARFNTPVG VCGEAAADPL
LATVLTGLGV NSLSAASTAL AAVGAKLSEV TLETCKKAAE AALDAEGATE ARDAVRAVID
AAV > RXA01299 (1-441, translated) 147 residues MEIMAAIMAA
GMVPPIALSI ATLLRKKLFT PAEQENGKSS WLLGLAFVSE GAIPFAAADP ERVIPAMMAG
GATTGAISMA LGVGSRAPHG GIFVVWAIEP WWGWLIALAA GTIVSTIVVI ALKQFWPNKA
VAAEVAKQEA QQAAVNA > RXA01300 (1-267, translated) 89 residues
MASKTVTVGS SVGLHARPAS IIAEAAAEYD DEILLTLVGS DDDEETDASS SLMIMALGAE
HGNEVTVTSD NAEAVEKIAA LIAQDLDAE > RXA01503 (1-249, translated)
83 residues MFLAVILAIT AARKFGANVF TSVALAGALL HTQLQAVTVL VDGELQSMTL
VAFQKAGNDV TFLGIPVVLQ LALHVASLMK LSR > RXA01883 (1-480,
translated) 160 residues MNSVNNSSLV RLDVDFGDST TDVINNLATV
IFDAGRASSA DALAKDALDR EAKSGTGVPG QVAIPHCRSE AVSVPTLGFA RLSKGVDFSG
PDGDANLVFL IAAPAGGGKE HLKILSKLAR SLVKKDFIKA LQEATTEQEI VDVVDAVLNP
APKNHRASCS > RXA01889 (1-555, translated) 185 residues
VAITACPTGI AHTYMAADSL TQNAEGRDDV ELVVETQGSS AVTPVDPKII EAADAVIFAT
DVGVKDRERF AGKPVIESGV KRAINEPAKM IDEAIAASKN PNARKVSGSG VAASAETTGE
KLGWGKRTQQ AVMTGVSYMV PFVAAGGLLL ALGFAPGGYD MANGWQAIAT QFSLTNLPGN
TVDVD > RXA01943 (1-405, translated) 135 residues PDPIFAAGKL
GPGIAIQPTG NTVVAPADAT VILVQKSGHA VALRLDSGVE ILVHVGLDTV QLGGEGFTVH
VERRQQVKAG DPLITFDADF IRSKDLPLIT PVVVSNAAKF GEIEGIPADQ ANSSTTVIKV
NGKNE > RXA02191 (1-1239, translated) 413 residues MASKLTTTSQ
HILENLGGPD NITSMTHCAT RLRFQVKDQS IVDQQEIDSD PSVLGVVPQG STGMQVVMGG
SVANYYQEIL KLDGMKHFAD GEATESSSKK EYGGVRGKYS WIDYAFEFLS DTFRPILWAL
LGASLIITLL VLADTFGLQD FRAPMDEQPD TYVFLHSMWR SVFYFLPIMV GATAARKLGA
NEWIGAAIPA ALLTPEFLAL GSAGDTVTVF GLPMVLNDYS GQVFPPLIAA IGLYWVEKGL
KKIIPEAVQM VFVPFFSLLI MIPATAFLLG PFGIGVGNGI SNLLEAINNF SPFILSIVIP
LLYPFLVPLG LHWPLNAIMI QNINTLGYDF IQGPMGAWNF AGFGLVTGVF LLSIKERNKA
MRQVSLGGML AGLLGGISEP SLYGVLLRFK KTYFRLLPGC LAA >RXN01244
TRANSLATE of: rxn01244.seq check: 8583 from: 1 to: 1704
VATVADVNQDTVLKGTGVVGGVRYASAVWITPRPELP- QAGEVVAEENREAEQERFDAAAA
TVSSRLLERSEAAEGPAAEVLKATAGMVNDRGWR- KAVIKGVKGGHPAEYAVVAATTKFIS
MFEAAGGLIAERTTDLRDIRDRVIAELRGDE- EPGLPAVSGQVILFADDLSPADTAALDTD
LFVGLVTELGGPTSHTAIIARQLNVPCI- VASGAGIKDIKSGEKVLIDGSLGTIDRNADEA
EATKLVSESLERAARIAEWKGPAQT- KDGYRVQLLANVQDGNSAQQAAQTEAEGIGLFRTE
LCFLSATEEPSVDEQAAVYSKVLEAFPESKVVVRSLDAGSDKPVPFASMADEMNPALGVR
GLRIARGQVDLLTRQLDAIAKASEELGRGDDAPTWVMAPMVATAYEAKWFADMCRERGLI
AGAMIEVPAASLMADKIMPHLDFVSIGTNDLTQYTMAADRMSPELAYLTDPWQPAVLRLI
KHTCDEGARFNTPVGVCGEAAADPLLATVLTGLGVNSLSAASTALAAVGAKLSEVTLETC
KKAAEAALDAEGATEARDAVRAVIDAAV >RXN01299 TRANSLATE of:
rxn01299.seq check: 4359 from: 1 to: 2064
MNSVNNSSLVRLDVDFGDSTTDVINNLATVIFDAGRASSADALAKDALDREAKSGTGVPG
QVAIPHCRSEAVSVPTLGFARLSKGVDFSGPDGDANLVFLIAAPAGGGKEHLKILSKLAR
SLVKKDFIKALQEATTEQEIVDVVDAVLNPAPKTTEPAAAPAAAAVAESGAASTSVTRIV
AITACPTGIAHTYMAADSLTQNAEGRDDVELVVETQGSSAVTPVDPKIIEAADAVIFATD
VGVKDRERFAGKPVIESGVKRAINEPAKMIDEAIAASKNPNARKVSGSGVAASAETTGEK
LGWGKRIQQAVMTGVSYMVPFVAAGGLLLALGFAFGGYDMANGWQAIATQFSLTNLP- GNT
VDVDGVAMTFERSGFLLYFGAVLFATGQAAMGFIVAALSGYTAYALAGRPGIAP- GFVGGA
ISVTIGAGFIGGLVTGILAGLIALWIGSWKVPRVVQSLMPVVIIPLLTSVV- VGLVMYLLL
GRPLASIMTGLQDWLSSMSGSSAILLGIILGLMMCFDLGGPVNKAAYL- FGTAGLSTGDQA
SMEIMAAIMAAGMVPPIALSIATLLRKKLFTPAEQENGKSSWLLG- LAFVSEGAIPFAAAD
PFRVIPAMMAGGATTGAISMALGVGSRAPHGGIFVVWAIEPW- WGWLIALAAGTIVSTIVV
IALKQFWPNKAVAAEVAKQEAQQAAVNA >RXN01943 TRANSLATE of:
rxn01943.seq check: 1650 from: 1 to: 2049
MASKLTTTSQHILENLGGPDNITSMTHCATRLRFQVKDQSIVDQQEIDSDPSVLGVVPQG
STGMQVVMGGSVANYYQEILKLDGMKHFADGEATESSSKKEYGGVRGKYSWIDYAFEFLS
DTFRPILWALLGASLIITLLVLADTFGLQDFRAPMDEQPDTYVFLHSMWRSVFYFLPIMV
GATAARKLGANEWIGAAIPAALLTPEFLALGSAGDTVTVFGLPMVLNDYSGQVFPPL- IAA
IGLYWVEKGLKKIIPEAVQMVFVPFFSLLIMIPATAFLLGPFGIGVGNGISNLL- EAINNF
SPFILSIVIPLLYPFLVPLGLHWPLNAIMIQNINTLGYDFIQGPMGAWNFA- CFGLVTGVF
LLSIKERNKAMRQVSLGGMLAGLLGGISEPSLYGVLLRFKKTYFRLLP- GCLAGGIVMGIF
DIKAYAFVFTSLLTIPAMDPWLGYTIGIAVAFFVSMFLVLALDYR- SNEERDEARAKVAAD
KQAEEDLKAEANATPAAPVAAAGAGAGAGAGAAAGAATAVAA- KPKLAAGEVVDIVSPLEG
KAIPLSEVPDPIFAAGKLGPGIAIQPTGNTVVAPADATV- ILVQKSGHAVALRLDSGVEIL
VHVGLDTVQLGGEGFTVHVERRQQVKAGDPLITFDA- DFIRSKDLPLITPVVVSNAAKFGE
IEGIPADQANSSTTVIKVNGKNE >RXN03002 TRANSLATE of: rxn03002.seq
check: 5800 from: 1 to: 408
MFVLKDLLKAERIELDRTVTDWREGIRAAGVLLEKTNSIDSAYTDAMTASVEEKGPYIVV
APGFAFAHARPSRAVRETAMSWVRLASPVSFGHSKNDPLNLIVALAAKDATAHTQAMAA- L
AKALGKYRKDLDEAQS >RXS00315 TRANSLATE of: RXS00315.seq check:
1474 from: 1 to: 1404
MAMVFPSLVNGYDVAATMAAGEMPMWSLFGLDVAQAGYQGTVLPVLVVSWILATIEKFLHKRLKGTADF
LITPVLTLLLTGFLTFIAIGPAMRWVGDVLAHGLQGLYDFGGPVGGLLFGLVYSPIVITGL-
HQSFPPIE LELFNQGGSFIFATASMANIAQGAACLAVFFLAKSEKLKGLAGASGVSA-
VLGITEPAIFGVNLRLRWPF FIGIGTAAIGGALIALFNIKAVALGAAGFLGVVSIDA-
PDMVMFLVCAVVTFFIAFGAAIAYGLYLVRRN GSIDPDATAAPVPAGTTKAEAEAPA-
EFSNDSTIIQAPLTGEAIALSSVSDAMFASGKLGSGVAIVPTKG
QLVSPVSGKIVVAFPSGHAFAVRTKAEDGSNVDILMHIGFDTVNLNGTHFNPLKKQGDEVKAGELLCEF
DIDAIKAAGYEVTTPIVVSNYKKTGPVNTYGLGEIEAGANLLNVAKKEAVPATP >RXC00953
TRANSLATE of: RXC00953.seq check: 8687 from: 1 to: 753
MAPPTVGNYIMQSFTQGLQFGVAVAVILFGVRTILGELVPAFQGIAAKVVPGAIPALDAPIV-
FPYAQNA VLIGFLSSFVGGLVGLTVLASWLNPAFGVALILPGLVPHFFTGGAAGVYG-
NATGGRRGAVFGAEANGLL ITFLPAFLLGVLGSFGSENTTFGDADFGWFGIVVGSAA-
KVEGAGGLILLLIIAAVLLGGAMVFQKRVVN GHWDPAPNRERVEKAEADATPTAGAR-
TYPKIAPPAGAPTPPARS >RXC03001 TRANSLATE of: RXC03001 .seq check:
9853 from: 1 to: 453 MDWLTIPLFLVNEILAVPAFLIGIITAVGLGAM-
GRSVGQVIGGAIKATLFELLIGAGATLVTASLEPLG
AMIMGATGMRGVVPTNEAIAGIAQAEYGAQVAWLMILGFAISLVLARFTNLRYVLLNGHHVLLMCTMLT
MVLATGRVDAWIF
[0181]
3TABLE 1 Genes Included in the Invention PHOSPHOENOLPYRUVATE: SUGAR
PHOSPHOTRANSFERASE SYSTEM Nucle- Amino otide Acid SEQ SEQ
Identification ID NO ID NO Code Contig. NT Start NT Stop Function 1
2 RXS00315 PTS SYSTEM, SUCROSE-SPECIFIC IIABC COMPONENT
(EIIABC-SCR) (SUCROSE-PERMEASE IIABC COMPONENT(PHOSPHOTRANSFERASE
ENZYME II, ABC COMPONENT) (EC 2.7.1.69) 3 4 F RXA00315 GR00053 6537
5452 PTS SYSTEM, BETA-GLUCOSIDES-SPECIFIC IIABC COMPONENT
(EIIABC-BGL) (BETA-GLUCOSIDES-PERMEASE IIABC COMPONENT)
(PHOSPHOTRANSFERASE ENZYME II, ABC COMPONENT) (EC 2.7.1.69) 5 6
RXA01503 GR00424 10392 10640 PTS SYSTEM, BETA-GLUCOSIDES-SPECIFIC
IIABC COMPONENT (EIIABC-BGL) (BETA-GLUCOSIDES-PERMEASE IIABC
COMPONENT) (PHOSPHOTRANSFERASE ENZYME II, ABC COMPONENT) (EC
2.7.1.69) 7 8 RXN01299 VV0068 11954 9891 PTS SYSTEM,
FRUCTOSE-SPECIFIC IIBC COMPONENT (EC 2.7.1.69) 9 10 F RXA01299
GR00375 6 446 PTS SYSTEM, FRUCTOSE-SPECIFIC IIBC COMPONENT (EC
2.7.1.69) 11 12 F RXA01883 GR00538 2154 2633 PTS SYSTEM,
FRUCTOSE-SPECIFIC IIBC COMPONENT (EC 2.7.1.69) 13 14 F RXA01889
GR00540 77 631 PTS SYSTEM, FRUCTOSE-SPECIFIC IIBC COMPONENT (EC
2.7.1.69) 15 16 RXA00951 GR00261 564 172 PTS SYSTEM, MANNITOL
(CRYPTIC)-SPECIFIC IIA COMPONENT (EIIA-(C)MTL) (MANNITOL
(CRYPTIC)-PERMEASE IIA COMPONENT) (PHOSPHOTRANSFERASE ENZYME II, A
COMPONENT) (EC 2.7.1.69) 17 18 RXN01244 VV0068 14141 15844
PHOSPHOENOLPYRUVATE-PROTEIN PHOSPHOTRANSFERASE (EC 2.7.3.9) 19 20 F
RXA01244 GR00359 4837 3329 PHOSPHOENOLPYRUVATE-PROTEIN
PHOSPHOTRANSFERASE (EC 2.7.3.9) 21 22 RXA01300 GR00375 637 903
PHOSPHOCARRIER PROTEIN HPR 23 24 RXN03002 VV0236 1437 1844 PTS
SYSTEM, MANNITOL (CRYPTIC)-SPECIFIC IIA COMPONENT (EIIA-(C)MTL)
(MANNITOL (CRYPTIC)-PERMEASE IIA COMPONENT) (PHOSPHOTRANSFERASE
ENZYME II, A COMPONENT) (EC 2.7.1.69) 25 26 RXC00953 VV0260 1834
1082 Membrane Spanning Protein involved in PTS system 27 28
RXC03001 Membrane Spanning Protein involved in PTS system 29 30
RXN01943 VV0120 4326 6374 PTS SYSTEM, GLUCOSE-SPECIFIC IIABC
COMPONENT (EC 2.7.1.69) 31 32 F RXA02191 GR00642 3395 4633
PHOSPHOENOLPYRUVATE SUGAR PHOSPHOTRANSFERASE 33 34 F RXA01943
GR00557 3944 3540 crr gene; phosphotransferase system
glucose-specific enzyme III
[0182]
4TABLE 2 GENES IDENTIFIED FROM GENBANK GenBank .TM. Accession Gene
No. Name Gene Function Reference A09073 ppg Phosphoenol pyruvate
carboxylase Bachmann, B. et al. "DNA fragment coding for
phosphoenolpyruvat corboxylase, recombinant DNA carrying said
fragment, strains carrying the recombinant DNA and method for
producing L-aminino acids using said strains," Patent: EP 0358940-A
3 Mar. 21, 1990 A45579, Threonine dehydratase Moeckel, B. et al.
"Production of L-isoleucine by means of recombinant A45581,
micro-organisms with deregulated threonine dehydratase," Patent: WO
A45583, 9519442-A 5 Jul. 20, 1995 A45585 A45587 AB003132 murC;
Kobayashi, M. et al. "Cloning, sequencing, and characterization of
the ftsZ ftsQ; ftsZ gene from coryneform bacteria," Biochem.
Biophys. Res. Commun., 236(2): 383-388 (1997) AB015023 murC; Wachi,
M. et al. "A murC gene from Coryneform bacteria," Appl. Microbiol.
ftsQ Biotechnol., 51(2): 223-228 (1999) AB018530 dtsR Kimura, E. et
al. "Molecular cloning of a novel gene, dtsR, which rescues the
detergent sensitivity of a mutant derived from Brevibacterium
lactofermentum," Biosci. Biotechnol. Biochem., 60(10): 1565-1570
(1996) AB018531 dtsR1; dtsR2 AB020624 murI D-glutamate racemase
AB023377 tkt transketolase AB024708 gltB; gltD Glutamine
2-oxoglutarate aminotransferase large and small subunits AB025424
acn aconitase AB027714 rep Replication protein AB027715 rep; aad
Replication protein; aminoglycoside adenyltransferase AF005242 argC
N-acetylglutamate-5-semialdehyde dehydrogenase AF005635 glnA
Glutamine synthetase AF030405 hisF cyclase AF030520 argG
Argininosuccinate synthetase AF031518 argF Ornithine
carbamolytransferase AF036932 aroD 3-dehydroquinate dehydratase
AF038548 pyc Pyruvate carboxylase AF038651 dciAE; Dipeptide-binding
protein; adenine Wehmeier, L. et al. "The role of the
Corynebacterium glutamicum rel gene in apt; rel
phosphoribosyltransferase; GTP (p)ppGpp metabolism," Microbiology,
144: 1853-1862 (1998) pyrophosphokinase AF041436 argR Arginine
repressor AF045998 impA Inositol monophosphate phosphatase AF048764
argH Argininosuccinate lyase AF049897 argC;
N-acetylglutamylphosphate reductase; argJ; ornithine
acetyltransferase; N- argB; acetylglutamate kinase; acetylornithine
argD; transminase; ornithine argF; carbamoyltransferase; arginine
repressor; argR; argininosuccinate synthase; argG;
argininosuccinate lyase argH AF050109 inhA Enoyl-acyl carrier
protein reductase AF050166 hisG ATP phosphoribosyltransferase
AF051846 hisA Phosphoribosylformimino-5-amino-1-
phosphoribosyl-4-imidazolecar- boxamide isomerase AF052652 metA
Homoserine O-acetyltransferase Park, S. et al. "Isolation and
analysis of metA, a methionine biosynthetic gene encoding
homoserine acetyltransferase in Corynebacterium glutamicum," Mol.
Cells., 8(3): 286-294 (1998) AF053071 aroB Dehydroquinate
synthetase AF060558 hisH Glutamine amidotransferase AF086704 hisE
Phosphoribosyl-ATP- pyrophosphohydrolase AF114233 aroA
5-enolpyruvylshikimate 3-phosphate synthase AF116184 panD
L-aspartate-alpha-decarboxylase precursor Dusch, N. et al.
"Expression of the Corynebacterium glutamicum panD gene encoding
L-aspartate-alpha-decarboxylase leads to pantothenate
overproduction in Escherichia coli," Appl. Environ. Microbiol.,
65(4)1530-1539 (1999) AF124518 aroD; 3-dehydroquinase; shikimate
aroE dehydrogenase AF124600 aroC; Chorismate synthase; shikimate
kinase; 3- aroK; dehydroquinate synthase; putative aroB;
cytoplasmic peptidase pepQ AF145897 inhA AF145898 inhA AJ001436
ectP Transport of ectoine, glycine betaine, Peter, H. et al.
"Corynebacterium glutamicum is equipped with four secondary proline
carriers for compatible solutes: Identification, sequencing, and
characterization of the proline/ectoine uptake system, ProP, and
the ectoine/proline/glycine betaine carrier, EctP," J. Bacteriol.,
180(22): 6005-6012 (1998) AJ004934 dapD Tetrahydrodipicolinate
succinylase Wehrmann, A. et al. "Different modes of diaminopimelate
synthesis and their (incomplete.sup.i) role in cell wall integrity:
A study with Corynebacterium glutamicum," J. Bacteriol., 180(12):
3159-3165 (1998) AJ007732 ppc; secG;
Phosphoenolpyruvate-carboxylase; ?; high amt; ocd; affinity
ammonium uptake protein; soxA putative
ornithine-cyclodecarboxylase; sarcosine oxidase AJ010319 ftsY,
Involved in cell division; PII protein; Jakoby, M. et al. "Nitrogen
regulation in Corynebacterium glutamicum; glnB, uridylyltransferase
(uridylyl-removing Isolation of genes involved in biochemical
characterization of corresponding glnD; enzmye); signal recognition
particle; low proteins," FEMS Microbiol., 173(2): 303-310 (1999)
srp; amtP affinity ammonium uptake protein AJ132968 cat
Chloramphenicol aceteyl transferase AJ224946 mqo L-malate: quinone
oxidoreductase Molenaar, D. et al. "Biochemical and genetic
characterization of the membrane-associated malate dehydrogenase
(acceptor) from Corynebacterium glutamicum," Eur. J. Biochem.,
254(2): 395-403 (1998) AJ238250 ndh NADH dehydrogenase AJ238703
porA Porin Lichtinger, T. et al. "Biochemical and biophysical
characterization of the cell wall porin of Corynebacterium
glutamicum: The channel is formed by a low molecular mass
polypeptide," Biochemistry, 37(43): 15024-15032 (1998) D17429
Transposable element IS31831 Vertes, A. A. et al. "Isolation and
characterization of IS31831, a transposable element from
Corynebacterium glutamicum," Mol. Microbiol., 11(4): 739-746 (1994)
D84102 odhA 2-oxoglutarate dehydrogenase Usuda, Y. et al.
"Molecular cloning of the Corynebacterium glutamicum
(Brevibacterium lactofermentum AJ12036) odhA gene encoding a novel
type of 2-oxoglutarate dehydrogenase," Microbiology, 142: 3347-3354
(1996) E01358 hdh; hk Homoserine dehydrogenase; homoserine
Katsumata, R. et al. "Production of L-thereonine and L-isoleucine,"
Patent: JP kinase 1987232392-A 1 Oct. 12, 1987 E01359 Upstream of
the start codon of homoserine Katsumata, R. et al. "Production of
L-thereonine and L-isoleucine," Patent: JP kinase gene 1987232392-A
2 Oct. 12, 1987 E01375 Tryptophan operon E01376 trpL; trpE Leader
peptide; anthranilate synthase Matsui, K. et al. "Tryptophan
operon, peptide and protein coded thereby, utilization of
tryptophan operon gene expression and production of tryptophan,"
Patent: JP 1987244382-A 1 Oct. 24, 1987 E01377 Promoter and
operator regions of Matsui, K. et al. "Tryptophan operon, peptide
and protein coded thereby, tryptophan operon utilization of
tryptophan operon gene expression and production of tryptophan,"
Patent: JP 1987244382-A 1 Oct. 24, 1987 E03937 Biotin-synthase
Hatakeyama, K. et al. "DNA fragment containing gene capable of
coding biotin synthetase and its utilization," Patent: JP
1992278088-A 1 Oct. 02, 1992 E04040 Diamino pelargonic acid
aminotransferase Kohama, K. et al. "Gene coding diaminopelargonic
acid aminotransferase and desthiobiotin synthetase and its
utilization," Patent: JP 1992330284-A 1 Nov. 18, 1992 E04041
Desthiobiotinsynthetase Kohama, K. et al. "Gene coding
diaminopelargonic acid aminotransferase and desthiobiotin
synthetase and its utilization," Patent: JP 1992330284-A 1 Nov. 18,
1992 E04307 Flavum aspartase Kurusu, Y. et al. "Gene DNA coding
aspartase and utilization thereof," Patent: JP 1993030977-A 1 Feb.
09, 1993 E04376 Isocitric acid lyase Katsumata, R. et al. "Gene
manifestation controlling DNA," Patent: JP 1993056782-A 3 Mar. 09,
1993 E04377 Isocitric acid lyase N-terminal fragment Kastumata R.et
al. "Gene manifestation controlling DNA," Patient: JP 1993056782-A
3 Mar. 09, 1993 E04484 Prephenate dehydratase Sotouchi, N. et al.
"Production of L-phenylalanine by fermentation," Patent: JP
1993076352-A 2 Mar. 30, 1993 E05108 Aspartokinase Fugono, N. et al.
"Gene DNA coding Aspartokinase and its use," Patent: JP
1993184366-A 1 Jul. 27, 1993 E05112 Dihydro-dipichorinate
synthetase Hatakeyama, K. et al. "Gene DNA coding
dihydrodipicolinic acid synthetase and its use," Patent: JP
1993184371-A 1 Jul. 27, 1993 E05776 Diaminopimelic acid
dehydrogenase Kobayashi, M. et al. "Gene DNA coding Diaminopimelic
acid dehydrogenase and its use," Patent: JP 1993284970-A 1 Nov. 02,
1993 E05779 Threonine synthase Kohama, K. et al. "Gene DNA coding
threonine synthase and its use," Patent: JP 1993284972-A 1 Nov. 02,
1993 E06110 Prephenate dehydratase Kikuchi, T. et al. "Production
of L-phenylalanine by fermentation method," Patent: JP 1993344881-A
1 Dec. 27, 1993 E06111 Mutated Prephenate dehydratase Kikuchi, T.
et al. "Production of L-phenylalanine by fermentation method,"
Patent: JP 1993344881-A 1 Dec. 27, 1993 E06146 Acetohydroxy acid
synthetase Inui, M. et al. "Gene capable of coding Acetohydroxy
acid synthetase and its use," Patent: JP 1993344893-A 1 Dec. 27,
1993 E06825 Aspartokinase Sugimoto, M. et al. "Mutant aspartokinase
gene," patent: JP 1994062866-A 1 Mar. 08, 1994 E06826 Mutated
aspartokinase alpha subunit Sugimoto, M. et al. "Mutant
aspartokinase gene," patent: JP 1994062866-A 1 Mar. 08, 1994 E06827
Mutated aspartokinase alpha subunit Sugimoto, M. et al. "Mutant
aspartokinase gene," patent: JP 1994062866-A 1 Mar. 08, 1994 E07701
secY Honno, N. et al. "Gene DNA participating in integration of
membraneous protein to membrane," Patent: JP 1994169780-A 1 Jun.
21, 1994 E08177 Aspartokinase Sato, Y. et al. "Genetic DNA capable
of coding Aspartokinase released from feedback inhibition and its
utilization," Patent: JP 1994261766-A 1 Sep. 20, 1994 E08178,
Feedback inhibition-released Sato, Y. et al. "Genetic DNA capable
of coding Aspartokinase released from E08179, Aspartokinase
feedback inhibition and its utilization," Patent: JP 1994261766-A 1
Sep. 20, 1994 E08180, E08181, E08182 E08232 Acetohydroxy-acid
isomeroreductase Inui, M. et al. "Gene DNA coding acetohydroxy acid
isomeroreductase," Patent: JP 1994277067-A 1 Oct. 04, 1994 E08234
secE Asai, Y. et al. "Gene DNA coding for translocation machinery
of protein," Patent: JP 1994277073-A 1 Oct. 04, 1994 E08643 FT
aminotransferase and desthiobiotin Hatakeyama, K. et al. "DNA
fragment having promoter function in synthetase promoter region
coryneform bacterium," Patent: JP 1995031476-A 1 Feb. 03, 1995
E08646 Biotin synthetase Hatakeyama, K. et al. "DNA fragment having
promoter function in coryneform bacterium," Patent: JP 1995031476-A
1 Feb. 03, 1995 E08649 Aspartase Kohama, K. et al "DNA fragment
having promoter function in coryneform bacterium," Patent: JP
1995031478-A 1 Feb. 03, 1995 E08900 Dihydrodipicolinate reductase
Madori, M. et al. "DNA fragment containing gene coding
Dihydrodipicolinate acid reductase and utilization thereof,"
Patent: JP 1995075578-A 1 Mar. 20, 1995 E08901 Diaminopimelic acid
decarboxylase Madori, M. et al. "DNA fragment containing gene
coding Diaminopimelic acid decarboxylase and utilization thereof,"
Patent: JP 1995075579-A 1 Mar. 20, 1995 E12594 Serine
hydroxymethyltransferase Hatakeyama, K. et al. "Production of
L-trypophan," Patent: JP 1997028391-A 1 Feb. 04, 1997 E12760,
transposase Moriya, M. et al. "Amplification of gene using
artificial transposon," Patent: E12759, JP 1997070291-A Mar. 18,
1997 E12758 E12764 Arginyl-tRNA synthetase; diaminopimelic Moriya,
M. et al. "Amplification of gene using artificial transposon,"
Patent: acid decarboxylase JP 1997070291-A Mar. 18, 1997 E12767
Dihydrodipicolinic acid synthetase Moriya, M. et al. "Amplification
of gene using artificial transposon," Patent: JP 1997070291-A Mar.
18, 1997 E12770 aspartokinase Moriya, M. et al. "Amplification of
gene using artificial transposon," Patent: JP 1997070291-A Mar. 18,
1997 E12773 Dihydrodipicolinic acid reductase Moriya, M. et al.
"Amplification of gene using artificial transposon," Patent: JP
1997070291-A Mar. 18, 1997 E13655 Glucose-6-phosphate dehydrogenase
Hatakeyama, K. et al. "Glucose-6-phosphate dehydrogenase and DNA
capable of coding the same," Patent: JP 1997224661-A 1 Sep. 02,
1997 L01508 IlvA Threonine dehydratase Moeckel, B. et al.
"Functional and structural analysis of the threonine dehydratase of
Corynebacterium glutamicum," J. Bacteriol., 174: 8065-8072 (1992)
L07603 EC 3-deoxy-D-arabinoheptulosonate-7- Chen, C. et al. "The
cloning and nucleotide sequence of Corynebacterium 4.2.1.15
phosphate synthase glutamicum 3-deoxy-D-arabinoheptulosonate-7-p-
hosphate synthase gene," FEMS Microbiol. Lett., 107: 223-230 (1993)
L09232 IlvB; ilvN; Acetohydroxy acid synthase large subunit;
Keilhauer, C. et al. "Isoleucine synthesis in Corynebacterium
glutamicum: ilvC Acetohydroxy acid synthase small subunit;
molecular analysis of the ilvB-ilvN-ilvC operon," J. Bacteriol.,
175(17): Acetohydroxy acid isomeroreductase 5595-5603 (1993) L18874
PtsM Phosphoenolpyruvate sugar Fouet, A et al. "Bacillus subtilis
sucrose-specific enzyme II of the phosphotransferase
phosphotransferase system: expression in Escherichia coli and
homology to enzymes II from enteric bacteria," PNAS USA, 84(24):
8773-8777 (1987); Lee, J. K. et al. "Nucleotide sequence of the
gene encoding the Corynebacterium glutamicum mannose enzyme II and
analyses of the deduced protein sequence," FEMS Microbiol. Lett.,
119(1-2): 137-145 (1994) L27123 aceB Malate synthase Lee, H-S. et
al. "Molecular characterization of aceB, a gene encoding malate
synthase in Corynebacterium glutamicum," J. Microbiol. Biotechnol.,
4(4): 256-263 (1994) L27126 Pyruvate kinase Jetten, M. S. et al.
"Structural and functional analysis of pyruvate kinase from
Corynebacterium glutamicum," Appl. Environ. Microbiol., 60(7):
2501-2507 (1994) L28760 aceA Isocitrate lyase L35906 dtxr
Diphtheria toxin repressor Oguiza, J. A. et al. "Molecular cloning,
DNA sequence analysis, and characterization of the Corynebacterium
diphtheriae dtxR from Brevibacterium lactofermentum," J.
Bacteriol., 177(2): 465-467 (1995) M13774 Prephenate dehydratase
Follettie, M. T. et al. "Molecular cloning and nucleotide sequence
of the Corynebacterium glutamicum pheA gene," J. Bacteriol., 167:
695-702 (1986) M16175 5S rRNA Park, Y-H. et al. "Phylogenetic
analysis of the coryneform bacteria by 56 rRNA sequences," J.
Bacteriol., 169: 1801-1806 (1987) M16663 trpE Anthranilate
synthase, 5' end Sano, K. et al. "Structure and function of the trp
operon control regions of Brevibacterium lactofermentum, a
glutamic-acid-producing bacterium," Gene, 52: 191-200 (1987) M16664
trpA Tryptophan synthase, 3' end Sano, K. et al. "Structure and
function of the trp operon control regions of Brevibacterium
lactofermentum, a glutamic-acid-producing bacterium," Gene, 52:
191-200 (1987) M25819 Phosphoenolpyruvate carboxylase O'Regan, M.
et al. "Cloning and nucleotide sequence of the Phosphoenolpyruvate
carboxylase-coding gene of Corynebacterium glutamicum ATCC13032,"
Gene, 77(2): 237-251 (1989) M85106 23S rRNA gene insertion sequence
Roller, C. et al. "Gram-positive bacteria with a high DNA G + C
content are characterized by a common insertion within their 23S
rRNA genes," J. Gen. Microbiol., 138: 1167-1175 (1992) M85107, 23S
rRNA gene insertion sequence Roller, C. et al. "Gram-positive
bacteria with a high DNA G + C content are M85108 characterized by
a common insertion within their 23S rRNA genes," J. Gen.
Microbiol., 138: 1167-1175 (1992) M89931 aecD; Beta C-S lyase;
branched-chain amino Rossol, I. et al. "The Corynebacterium
glutamicum aecD gene encodes a C-S brnQ; acid uptake carrier;
hypothetical protein lyase with alpha, beta-elimination activity
that degrades aminoethylcysteine," yhbw yhbw J.
Bacteriol., 174(9): 2968-2977 (1992); Tauch, A. et al. "Isoleucine
uptake in Corynebacterium glutamicum ATCC 13032 is directed by the
brnQ gene product," Arch. Microbiol., 169(4): 303-312 (1998) S59299
trp Leader gene (promoter) Herry, D. M. et al. "Cloning of the trp
gene cluster from a tryptophan- hyperproducing strain of
Corynebacterium glutamicum: identification of a mutation in the trp
leader sequence," Appl. Environ. Microbiol., 59(3): 791-799 (1993)
U11545 trpD Anthranilate phosphoribosyltransferase O'Gara, J. P.
and Dunican, L. K. (1994) Complete nucleotide sequence of the
Corynebacterium glutamicum ATCC 21850 tpD gene." Thesis,
Microbiology Department, University College Galway, Ireland. U13922
cglIM; Putative type II 5-cytosoine Schafer, A. et al. "Cloning and
characterization of a DNA region encoding a cglIR;
methyltransferase; putative type II stress-sensitive restriction
system from Corynebacterium glutamicum ATCC clgIIR restriction
endonuclease; putative type I or 13032 and analysis of its role in
intergeneric conjugation with Escherichia type III restriction
endonuclease coli," J. Bacteriol., 176(23): 7309-7319 (1994);
Schafer, A. et al. "The Corynebacterium glutamicum cglIM gene
encoding a 5-cytosine in an McrBC- deficient Escherichia coli
strain," Gene, 203(2): 95-101 (1997) U14965 recA U31224 ppx Ankri,
S. et al. "Mutations in the Corynebacterium glutamicumproline
biosynthetic pathway: A natural bypass of the proA step," J.
Bacteriol., 178(15): 4412-4419 (1996) U31225 proC L-proline: NADP+
5-oxidoreductase Ankri, S. et al. "Mutations in the Corynebacterium
glutamicumproline biosynthetic pathway: A natural bypass of the
proA step," J. Bacteriol., 178(15): 4412-4419 (1996) U31230 obg;
proB; ?; gamma glutamyl kinase; similar to D- Ankri, S. et al.
"Mutations in the Corynebacterium glutamicumproline unkdh isomer
specific 2-hydroxyacid biosynthetic pathway: A natural bypass of
the proA step," J. Bacteriol., dehydrogenases 178(15): 4412-4419
(1996) U31281 bioB Biotin synthase Serebriiskii, I. G., "Two new
members of the bio B superfamily: Cloning, sequencing and
expression of bio B genes of Methylobacillus flagellatum and
Corynebacterium glutamicum," Gene, 175: 15-22 (1996) U35023 thtR;
Thiosulfate sulfurtransferase; acyl CoA Jager, W. et al. "A
Corynebacterium glutamicum gene encoding a two-domain accBC
carboxylase protein similar to biotin carboxylases and
biotin-carboxyl-carrier proteins," Arch. Microbiol., 166(2); 76-82
(1996) U43535 cmr Multidrug resistance protein Jager, W. et al. "A
Corynebacterium glutamicum gene conferring multidrug resistance in
the heterologous host Escherichia coli," J. Bacteriol., 179(7):
2449-2451 (1997) U43536 clpB Heat shock ATP-binding protein U53587
aphA-3 3'5"-aminoglycoside phosphotransferase U89648
Corynebacterium glutamicum unidentified sequence involved in
histidine biosynthesis, partial sequence X04960 trpA; Tryptophan
operon Matsui, K. et al. "Complete nucleotide and deduced amino
acid sequences of trpB; the Brevibacterium lactofermentum
tryptophan operon," Nucleic Acids Res., trpC; 14(24): 10113-10114
(1986) trpD; trpE; trpG; trpL X07563 lys A DAP decarboxylase (meso-
Yeh, P. et al. "Nucleic sequence of the lysA gene of
Corynebacterium diaminopimelate decarboxylase, glutamicum and
possible mechanisms for modulation of its expression," Mol. EC
4.1.1.20) Gen. Genet., 212(1): 112-119 (1988) X14234 EC
Phosphoenolpyruvate carboxylase Eikmanns, B. J. et al. "The
Phosphoenolpyruvate carboxylase gene of 4.1.1.31 Corynebacterium
glutamicum: Molecular cloning, nucleotide sequence, and
expression," Mol. Gen. Genet., 218(2): 330-339 (1989); Lepiniec, L.
et al. "Sorghum Phosphoenolpyruvate carboxylase gene family:
structure, function and molecular evolution," Plant. Mol. Biol.,
21(3): 487-502 (1993) X17313 fda Fructose-bisphosphate aldolase Von
der Osten, C. H. et al. "Molecular cloning, nucleotide sequence and
fine- structural analysis of the Corynebacterium glutamicum fda
gene: structural comparison of C. glutamicum fructose-1,
6-biphosphate aldolase to class I and class II aldolases," Mol.
Microbiol., X53993 dapA L-2,3-dihydrodipicolinate synthetase (EC
Bonnassie, S. et al. "Nucleic sequence of the dapA gene from
4.2.1.52) Corynebacterium glutamicum," Nucleic Acids Res., 18(21):
6421 (1990) X54223 AttB-related site Cianciotto, N. et al. "DNA
sequence homology between att B-related sites of Corynebacterium
diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum,
and the attP site of lambdacorynephage," FEMS. Microbiol, Lett.,
66: 299-302 (1990) X54740 argS; lysA Arginyl-tRNA synthetase;
Marcel, T. et al. "Nucleotide sequence and organization of the
upstream region Diaminopimelate decarboxylase of the
Corynebacterium glutamicum lysA gene," Mol. Microbiol., 4(11):
1819-1830 (1990) X55994 trpL; trpE Putative leader peptide;
anthranilate Heery, D. M. et al. "Nucleotide sequence of the
Corynebacterium glutamicum synthase component 1 trpE gene," Nucleic
Acids Res., 18(23): 7138 (1990) X56037 thrC Threonine synthase Han,
K. S. et al. "The molecular structure of the Corynebacterium
glutamicum threonine synthase gene," Mol. Microbiol., 4(10):
1693-1702 (1990) X56075 attB- Attachment site Cianciotto, N. et al.
"DNA sequence homology between att B-related sites of related
Corynebacterium diphtheriae, Corynebacterium ulcerans,
Corynebacterium site glutamicum, and the attP site of
lambdacorynephage," FEMS. Microbiol, Lett., 66: 299-302 (1990)
X57226 lysC- Aspartokinase-alpha subunit; Kalinowski, J. et al.
"Genetic and biochemical analysis of the Aspartokinase alpha;
Aspartokinase-beta subunit; aspartate beta from Corynebacterium
glutamicum," Mol. Microbiol., 5(5): 1197-1204 (1991); lysC-beta;
semialdehyde dehydrogenase Kalinowski, J. et al. "Aspartokinase
genes lysC alpha and lysC beta overlap asd and are adjacent to the
aspertate beta-semialdehyde dehydrogenase gene asd in
Corynebacterium glutamicum," Mol. Gen. Genet., 224(3): 317-324
(1990) X59403 gap; pgk; Glyceraldehyde-3-phosphate; Eikmanns, B. J.
"Identification, sequence analysis, and expression of a tpi
phosphoglycerate kinase; triosephosphate Corynebacterium glutamicum
gene cluster encoding the three glycolytic isomerase enzymes
glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate
kinase, and triosephosphate isomeras," J. Bacteriol., 174(19):
6076-6086 (1992) X59404 gdh Glutamate dehydrogenase Bormann, E. R.
et al. "Molecular analysis of the Corynebacterium glutamicum gdh
gene encoding glutamate dehydrogenase," Mol. Microbiol., 6(3):
317-326 (1992) X60312 lysI L-lysine permease Seep-Feldhaus, A. H.
et al. "Molecular analysis of the Corynebacterium glutamicum lysl
gene involved in lysine uptake," Mol. Microbiol., 5(12): 2995-3005
(1991) X66078 cop1 Ps1 protein Joliff, G. et al. "Cloning and
nucleotide sequence of the csp1 gene encoding PS1, one of the two
major secreted proteins of Corynebacterium glutamicum: The deduced
N-terminal region of PS1 is similar to the Mycobacterium antigen 85
complex," Mol. Microbiol., 6(16): 2349-2362 (1992) X66112 glt
Citrate synthase Eikmanns, B. J. et al. "Cloning sequence,
expression and transcriptional analysis of the Corynebacterium
glutamicum gltA gene encoding citrate synthase," Microbiol., 140:
1817-1828 (1994) X67737 dapB Dihydrodipicolinate reductase X69103
csp2 Surface layer protein PS2 Peyret, J. L. et al.
"Characterization of the cspB gene encoding PS2, an ordered
surface-layer protein in Corynebacterium glutamicum," Mol.
Microbiol., 9(1): 97-109 (1993) X69104 IS3 related insertion
element Bonamy, C. et al. "Identification of IS1206, a
Corynebacterium glutamicum IS3-related insertion sequence and
phylogenetic analysis," Mol. Microbiol., 14(3): 571-581 (1994)
X70959 leuA Isopropylmalate synthase Patek, M. et al. "Leucine
synthesis in Corynebacterium glutamicum: enzyme activities,
structure of leuA, and effect of leuA inactivation on lysine
synthesis," Appl. Environ. Microbiol., 60(1): 133-140 (1994) X71489
icd Isocitrate dehydrogenase (NADP+) Eikmanns, B. J. et al.
"Cloning sequence analysis, expression, and inactivation of the
Corynebacterium glutamicum icd gene encoding isocitrate
dehydrogenase and biochemical characterization of the enzyme," J.
Bacteriol., 177(3): 774-782 (1995) X72855 GDHA Glutamate
dehydrogenase (NADP+) X75083, mtrA 5-methyltryptophan resistance
Heery, D. M. et al. "A sequence from a tryptophan-hyperproducing
strain of X70584 Corynebacterium glutamicum encoding resistance to
5-methyltryptophan," Biochem. Biophys. Res. Commun., 201(3):
1255-1262 (1994) X75085 recA Fitzpatrick, R. et al. "Construction
and characterization of recA mutant strains of Corynebacterium
glutamicum and Brevibacterium lactofermentum," Appl. Microbiol.
Biotechnol., 42(4): 575-580 (1994) X75504 aceA; thiX Partial
Isocitrate lyase; ? Reinscheid, D. J. et al. "Characterization of
the isocitrate lyase gene from Corynebacterium glutamicum and
biochemical analysis of the enzyme," J. Bacteriol., 176(12):
3474-3483 (1994) X76875 ATPase beta-subunit Ludwig, W. et al.
"Phylogenetic relationships of bacteria based on comparative
sequence analysis of elongation factor Tu and ATP-synthase
beta-subunit genes," Antonie Van Leeuwenhoek, 64: 285-305 (1993)
X77034 tuf Elongation factor Tu Ludwig, W. et al. "Phylogenetic
relationships of bacteria based on comparative sequence analysis of
elongation factor Tu and ATP-synthase beta-subunit genes," Antonie
Van Leeuwenhoek, 64: 285-305 (1993) X77384 recA Billman-Jacobe, H.
"Nucleotide sequence of a recA gene from Corynebacterium
glutamicum," DNA Seq., 4(6): 403-404 (1994) X78491 aceB Malate
synthase Reinscheid, D. J. et al. "Malate synthase from
Corynebacterium glutamicum pta-ack operon encoding
phosphotransacetylase: sequence analysis," Microbiology, 140:
3099-3108 (1994) X80629 16S rDNA 16S ribosomal RNA Rainey, F. A. et
al. "Phylogenetic analysis of the genera Rhodococcus and Norcardia
and evidence for the evolutionary origin of the genus Norcardia
from within the radiation of Rhodococcus species," Microbiol., 141:
523-528 (1995) X81191 gluA; Glutamate uptake system Kronemeyer, W.
et al. "Structure of the gluABCD cluster encoding the gluB;
glutamate uptake system of Corynebacterium glutamicum," J.
Bacteriol., gluC; 177(5): 1152-1158 (1995) gluD X81379 dapE
Succinyldiaminopimelate desuccinylase Wehrmann, A. et al. "Analysis
of different DNA fragments of Corynebacterium glutamicum
complementing dapE of Escherichia coli," Microbiology, 40: 3349-56
(1994) X82061 16S rDNA 16S ribosomal RNA Ruimy, R. et al.
"Phylogeny of the genus Corynebacterium deduced from analyses of
small-subunit ribosomal DNA sequences," Int. J. Syst. Bacteriol.,
45(4): 740-746 (1995) X82928 asd; lysC Aspartate-semialdehyde
dehydrogenase; ? Serebrijski, I. et al. "Multicopy suppression by
asd gene and osmotic stress- dependent complementation by
heterologous proA in proA mutants," J. Bacteriol., 177(24):
7255-7260 (1995) X82929 proA Gamma-glutamyl phosphate reductase
Serebrijski, I. et al. "Multicopy suppression by asd gene and
osmotic stress- dependent complementation by heterologous proA in
proA mutants," J. Bacteriol., 177(24): 7255-7260 (1995) X84257 16S
rDNA 16S ribosomal RNA Pascual, C. et al. "Phylogenetic analysis of
the genus Corynebacterium based on 16S rRNA gene sequences," Int.
J. Syst. Bacteriol., 45(4): 724-728 (1995) X85965 aroP; Aromatic
amino acid permease; ? Wehrmann, A. et al. "Functional analysis of
sequences adjacent to dapE of dapE Corynebacterium
glutamicumproline reveals the presence of aroP, which encodes the
aromatic amino acid transporter," J. Bacteriol., 177(20): 5991-5993
(1995) X86157 argB; Acetylglutamate kinase; N-acetyl-gamma-
Sakanyan, V. et al. "Genes and enzymes of the acetyl cycle of
arginine argC; glutamyl-phosphate reductase; biosynthesis in
Corynebacterium glutamicum: enzyme evolution in the early argD;
acetylornithine aminotransferase; ornithine steps of the arginine
pathway," Microbiology, 142: 99-108 (1996) argF; argJ
carbamoyltransferase; glutamate N- acetyltransferase X89084 pta;
ackA Phosphate acetyltransferase; acetate kinase Reinscheid, D. J.
et al. "Cloning, sequence analysis, expression and inactivation of
the Corynebacterium glutamicum pta-ack operon encoding
phosphotransacetylase and acetate kinase," Microbiology, 145:
503-513 (1999) X89850 attB Attachment site Le Marrec, C. et al.
"Genetic characterization of site-specific integration functions of
phi AAU2 infecting "Arthrobacter aureus C70," J. Bacteriol.,
178(7): 1996-2004 (1996) X90356 Promoter fragment F1 Patek, M. et
al. "Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90357 Promoter fragment F2 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90358 Promoter fragment F10 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90359 Promoter fragment F13 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90360 Promoter fragment F22 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90361 Promoter fragment F34 Patek, M. et
al."Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90362 Promoter fragment F37 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90363 Promoter fragment F45 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90364 Promoter fragment F64 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90365 Promoter fragment F75 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90366 Promoter fragment PF101 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90367 Promoter fragment PF104 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90368 Promoter fragment PF109 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X93513 amt Ammonium transport system Siewe, R. M.
et al. "Functional and genetic characterization of the (methyl)
ammonium uptake carrier of Corynebacterium glutamicum," J. Biol.
Chem., 271(10): 5398-5403 (1996) X93514 betP Glycine betaine
transport system Peter, H. et al. "Isolation, characterization, and
expression of the Corynebacterium glutamicum betP gene, encoding
the transport system for the compatible solute glycine betaine," J.
Bacteriol., 178(17): 5229-5234 (1996) X95649 orf4 Patek, M. et al.
"Identification and transcriptional analysis of the dapB-ORF2-
dapA-ORF4 operon of Corynebacterium glutamicum, encoding two
enzymes involved in L-lysine synthesis," Biotechnol. Lett., 19:
1113-1117 (1997) X96471 lysE; lysG Lysine exporter protein; Lysine
export Vrljic, M. et
al. "A new type of transporter with a new type of cellular
regulator protein function: L-lysine export from Corynebacterium
glutamicum," Mol. Microbiol., 22(5): 815-826 (1996) X96580 panB;
3-methyl-2-oxobutanoate Sahm, H. et al. "D-pantothenate synthesis
in Corynebacterium glutamicum and panC; hydroxymethyltransferase;
pantoate-beta- use of panBC and genes encoding L-valine synthesis
for D-pantothenate xylB alanine ligase; xylulokinase
overproduction," Appl. Environ. Microbiol., 65(5): 1973-1979 (1999)
X96962 Insertion sequence IS1207 and transposase X99289 Elongation
factor P Ramos, A. et al. "Cloning, sequencing and expression of
the gene encoding elongation factor P in the amino-acid producer
Brevibacterium lactofermentum (Corynebacterium glutamicum ATCC
13869)," Gene, 198: 217-222 (1997) Y00140 thrB Homoserine kinase
Mateos, L. M. et al. "Nucleotide sequence of the homoserine kinase
(thrB) gene of the Brevibacterium lactofermentum," Nucleic Acids
Res., 15(9): 3922 (1987) Y00151 ddh Meso-diaminopimelate
D-dehydrogenase Ishino, S. et al. "Nucleotide sequence of the
meso-diaminopimelate D- (EC 1.4.1.16) dehydrogenase gene from
Corynebacterium glutamicum," Nucleic Acids Res., 15(9): 3917 (1987)
Y00476 thrA Homoserine dehydrogenase Mateos, L. M. et al.
"Nucleotide sequence of the homoserine dehydrogenase (thrA) gene of
the Brevibacterium lactofermentum," Nucleic Acids Res., 15(24):
10598 (1987) Y00546 hom; thrB Homoserine dehydrogenase; homoserine
Peoples, O. P. et al. "Nucleotide sequence and fine structural
analysis of the kinase Corynebacterium glutamicum hom-thrB operon,"
Mol. Microbiol., 2(1): 63-72 (1988) Y08964 murC;
UPD-N-acetylmuramate-alanine ligase; Honrubia, M. P. et al.
"Identification, characterization, and chromosomal ftsQ/divD;
division initiation protein or cell division organization of the
ftsZ gene from Brevibacterium lactofermentum," Mol. Gen. ftsZ
protein; cell division protein Genet., 259(1): 97-104 (1998) Y09163
putP High affinity proline transport system Peter, H. et al.
"Isolation of the putP gene of Corynebacterium glutamicumproline
and characterization of a low-affinity uptake system for compatible
solutes," Arch. Microbiol., 168(2): 143-151 (1997) Y09548 pyc
Pyruvate carboxylase Peters-Wendisch, P. G. et al. "Pyruvate
carboxylase from Corynebacterium glutamicum: characterization,
expression and inactivation of the pyc gene," Microbiology, 144:
915-927 (1998) Y09578 leuB 3-isopropylmalate dehydrogenase Patek,
M. et al. "Analysis of the leuB gene from Corynebacterium
glutamicum," Appl. Microbiol. Biotechnol., 50(1): 42-47 (1998)
Y12472 Attachment site bacteriophage Phi-16 Moreau, S. et al.
"Site-specific integration of corynephage Phi-16: The construction
of an integration vector," Microbiol., 145: 539-548 (1999) Y12537
proP Proline/ectoine uptake system protein Peter, H. et al.
"Corynebacterium glutamicum is equipped with four secondary
carriers for compatible solutes: Identification, sequencing, and
characterization of the proline/ectoine uptake system, ProP, and
the ectoine/proline/glycine betaine carrier, EctP," J. Bacteriol.,
180(22): 6005-6012 (1998) Y13221 glnA Glutamine synthetase I
Jakoby, M. et al. "Isolation of Corynebacterium glutamicum glnA
gene encoding glutamine synthetase I," FEMS Microbiol. Lett.,
154(1): 81-88 (1997) Y16642 lpd Dihydrolipoamide dehydrogenase
Y18059 Attachment site Corynephage 304L Moreau, S. et al. "Analysis
of the integration functions of φ 304L: An integrase module among
corynephages," Virology, 255(1): 150-159 (1999) Z21501 argS; lysA
Arginyl-tRNA synthetase; Oguiza, J. A. et al. "A gene encoding
arginyl-tRNA synthetase is located in the diaminopimelate
decarboxylase (partial) upstream region of the lysA gene in
Brevibacterium lactofermentum: Regulation of argS-IysA cluster
expression by arginine," J. Bacteriol., 175(22): 7356-7362 (1993)
Z21502 dapA; Dihydrodipicolinate synthase; Pisabarro, A. et al. "A
cluster of three genes (dapA, orf2, and dapB) of dapB
dihydrodipicolinate reductase Brevibacterium lactofermentum encodes
dihydrodipicolinate reductase, and a third polypeptide of unknown
function," J. Bacteriol., 175(9): 2743-2749 (1993) Z29563 thrC
Threonine synthase Malumbres, M. et al. "Analysis and expression of
the thrC gene of the encoded threonine synthase," Appl. Environ.
Microbiol., 60(7)2209-2219 (1994) Z46753 16S rDNA Gene for 16S
ribosomal RNA Z49822 sigA SigA sigma factor Oguiza, J. A. et al.
"Multiple sigma factor genes in Brevibacterium lactofermentum:
Characterization of sigA and sigB," J. Bacteriol., 178(2): 550-553
(1996) Z49823 galE; dtxR Catalytic activity UDP-galactose 4-
Oguiza, J. A. et al. "The galE gene encoding the UDP-galactose
4-epimerase of epimerase; diphtheria toxin regulatory
Brevibacterium lactofermentum is coupled transcriptionally to the
dmdR protein gene," Gene, 177: 103-107 (1996) Z49824 orf1; sigB ?;
SigB sigma factor Oguiza, J. A. et al "Multiple sigma factor genes
in Brevibacterium lactofermentum: Characterization of sigA and
sigB," J. Bacteriol., 178(2): 550-553 (1996) Z66534 Transposase
Correia, A. et al. "Cloning and characterization of an IS-like
element present in the genome of Brevibacterium lactofermentum ATCC
13869," Gene, 170(1): 91-94 (1996) .sup.1A sequence for this gene
was published in the indicated reference. However, the sequence
obtained by the inventors of the present application is
significantly longer than the published version. It is believed
that the published version relied on an incorrect start codon, and
thus represents only a fragment of the actual coding region.
[0183]
5TABLE 3 Corynebacterium and Brevibacterium Strains Which May be
Used in the Practice of the Invention Genus species ATCC FERM NRRL
CECT NCIMB CBS NCTC DSMZ Brevibacterium ammoniagenes 21054
Brevibacterium ammoniagenes 19350 Brevibacterium ammoniagenes 19351
Brevibacterium ammoniagenes 19352 Brevibacterium ammoniagenes 19353
Brevibacterium ammoniagenes 19354 Brevibacterium ammoniagenes 19355
Brevibacterium ammoniagenes 19356 Brevibacterium ammoniagenes 21055
Brevibacterium ammoniagenes 21077 Brevibacterium ammoniagenes 21553
Brevibacterium ammoniagenes 21580 Brevibacterium ammoniagenes 39101
Brevibacterium butanicum 21196 Brevibacterium divaricatum 21792
P928 Brevibacterium flavum 21474 Brevibacterium flavum 21129
Brevibacterium flavum 21518 Brevibacterium flavum B11474
Brevibacterium flavum B11472 Brevibacterium flavum 21127
Brevibacterium flavum 21128 Brevibacterium flavum 21427
Brevibacterium flavum 21475 Brevibacterium flavum 21517
Brevibacterium flavum 21528 Brevibacterium flavum 21529
Brevibacterium flavum B11477 Brevibacterium flavum B11478
Brevibacterium flavum 21127 Brevibacterium flavum B11474
Brevibacterium healii 15527 Brevibacterium ketoglutamicum 21004
Brevibacterium ketoglutamicum 21089 Brevibacterium ketosoreductum
21914 Brevibacterium lactofermentum 70 Brevibacterium
lactofermentum 74 Brevibacterium lactofermentum 77 Brevibacterium
lactofermentum 21798 Brevibacterium lactofermentum 21799
Brevibacterium lactofermentum 21800 Brevibacterium lactofermentum
21801 Brevibacterium lactofermentum B11470 Brevibacterium
lactofermentum B11471 Brevibacterium lactofermentum 21086
Brevibacterium lactofermentum 21420 Brevibacterium lactofermentum
21086 Brevibacterium lactofermentum 31269 Brevibacterium linens
9174 Brevibacterium linens 19391 Brevibacterium linens 8377
Brevibacterium paraffinolyticum 11160 Brevibacterium spec. 717.73
Brevibacterium spec. 717.73 Brevibacterium spec. 14604
Brevibacterium spec. 21860 Brevibacterium spec. 21864
Brevibacterium spec. 21865 Brevibacterium spec. 21866
Brevibacterium spec. 19240 Corynebacterium acetoacidophilum 21476
Corynebacterium acetoacidophilum 13870 Corynebacterium
acetoglutamicum B11473 Corynebacterium acetoglutamicum B11475
Corynebacterium acetoglutamicum 15806 Corynebacterium
acetoglutamicum 21491 Corynebacterium acetoglutamicum 31270
Corynebacterium acetophilum B3671 Corynebacterium ammoniagenes 6872
2399 Corynebacterium ammoniagenes 15511 Corynebacterium fujiokense
21496 Corynebacterium glutamicum 14067 Corynebacterium glutamicum
39137 Corynebacterium glutamicum 21254 Corynebacterium glutamicum
21255 Corynebacterium glutamicum 31830 Corynebacterium glutamicum
13032 Corynebacterium glutamicum 14305 Corynebacterium glutamicum
15455 Corynebacterium glutamicum 13058 Corynebacterium glutamicum
13059 Corynebacterium glutamicum 13060 Corynebacterium glutamicum
21492 Corynebacterium glutamicum 21513 Corynebacterium glutamicum
21526 Corynebacterium glutamicum 21543 Corynebacterium glutamicum
13287 Corynebacterium glutamicum 21851 Corynebacterium glutamicum
21253 Corynebacterium glutamicum 21514 Corynebacterium glutamicum
21516 Corynebacterium glutamicum 21299 Corynebacterium glutamicum
21300 Corynebacterium glutamicum 39684 Corynebacterium glutamicum
21488 Corynebacterium glutamicum 21649 Corynebacterium glutamicum
21650 Corynebacterium glutamicum 19223 Corynebacterium glutamicum
13869 Corynebacterium glutamicum 21157 Corynebacterium glutamicum
21158 Corynebacterium glutamicum 21159 Corynebacterium glutamicum
21355 Corynebacterium glutamicum 31808 Corynebacterium glutamicum
21674 Corynebacterium glutamicum 21562 Corynebacterium glutamicum
21563 Corynebacterium glutamicum 21564 Corynebacterium glutamicum
21565 Corynebacterium glutamicum 21566 Corynebacterium glutamicum
21567 Corynebacterium glutamicum 21568 Corynebacterium glutamicum
21569 Corynebacterium glutamicum 21570 Corynebacterium glutamicum
21571 Corynebacterium glutamicum 21572 Corynebacterium glutamicum
21573 Corynebacterium glutamicum 21579 Corynebacterium glutamicum
19049 Corynebacterium glutamicum 19050 Corynebacterium glutamicum
19051 Corynebacterium glutamicum 19052 Corynebacterium glutamicum
19053 Corynebacterium glutamicum 19054 Corynebacterium glutamicum
19055 Corynebacterium glutamicum 19056 Corynebacterium glutamicum
19057 Corynebacterium glutamicum 19058 Corynebacterium glutamicum
19059 Corynebacterium glutamicum 19060 Corynebacterium glutamicum
19185 Corynebacterium glutamicum 13286 Corynebacterium glutamicum
21515 Corynebacterium glutamicum 21527 Corynebacterium glutamicum
21544 Corynebacterium glutamicum 21492 Corynebacterium glutamicum
B8183 Corynebacterium glutamicum B8182 Corynebacterium glutamicum
B12416 Corynebacterium glutamicum B12417 Corynebacterium glutamicum
B12418 Corynebacterium glutamicum B11476 Corynebacterium glutamicum
21608 Corynebacterium lilium P973 Corynebacterium nitrilophilus
21419 11594 Corynebacterium spec. P4445 Corynebacterium spec. P4446
Corynebacterium spec. 31088 Corynebacterium spec. 31089
Corynebacterium spec. 31090 Corynebacterium spec. 31090
Corynebacterium spec. 31090 Corynebacterium spec. 15954 20145
Corynebacterium spec. 21857 Corynebacterium spec. 21862
Corynebacterium spec. 21863 ATCC: American Type Culture Collection,
Rockville, MD, USA FERM: Fermentation Research Institute, Chiba,
Japan NRRL: ARS Culture Collection, Northern Regional Research
Laboratory, Peoria, IL, USA CECT: Coleccion Espanola de Cultivos
Tipo, Valencia, Spain NCIMB: National Collection of Industrial and
Marine Bacteria Ltd., Aberdeen, UK CBS: Centraalbureau voor
Schimmelcultures, Baarn, NL NCTC: National Collection of Type
Cultures, London, UK DSMZ: Deutsche Sammlung von Mikroorganismen
und Zellkulturen, Braunschweig, Germany For reference see Sugawara,
H. et al. (1993) World directory of collections of cultures of
microorganisms: Bacteria, fungi and yeasts (4.sup.th edn), World
federation for culture collections world data center on
microorganisms, Saimata, Japen.
[0184]
6TABLE 4 ALIGNMENT RESULTS length ID # (NT) Genbank Hit Length
Accession Name of Genbank Hit rxa00315 1527 GB_BA1: AB007125 4078
AB007125 Serratia marcescens slaA gene for surface layer protein,
complete cds, Isolate 8000. GB_IN1: CELC47D2 17381 U64861
Caenorhabditis elegans cosmid C47D2. GB_HTG2: AC006732 159453
AC006732 Caenorhabditis elegans clone Y32G9, ***SEQUENCING IN
PROGRESS***, 9 unordered pieces. rxa01503 372 GB_PR3: AC005019
188362 AC005019 Homo sapiens BAC clone GS250A16 from 7p21-p22,
complete sequence. GB_GSS12: AQ390040 680 AQ390040 RPCI11-157C9.TJ
RPCI-11 Homo sapiens genomic clone RPCI-11- 157C9, genomic survey
sequence. GB_GSS5: AQ784231 542 AQ784231 HS_3087_B1_C10_T7C CIT
Approved Human Genomic Sperm Library D Homo sapiens genomic clone
Plate = 3087 Col = 19 Row = F, genomic survey sequence. rxa01299
2187 GB_EST38: AW047296 614 AW047296 UI-M-BH1-amh-e-03-0-UI.s1
NIH_BMAP_M_S2 Mus musculus CDNA clone UI_M-BH1-amh-e-03-0-UI 3',
mRNA sequence. GB_RO: AB004056 1581 AB004056 Rattus norvegicus mRNA
for BarH-class homeodomain transcription factor, complete cds.
GB_RO: AB004056 1581 AB004056 Rattus norvegicus mRNA for BarH-class
homeodomain transcription factor, complete cds. rxa00951 416
GB_BA1: SCJ21 31717 AL109747 Streptomyces coelicolor cosmid J21.
GB_VI: MCU68299 230278 U68299 Mouse cytomegalovirus 1 complete
genomic sequence. GB_VI: U93872 133661 U93872 Kaposi's
sarcoma-associated herpesvirus glycoprotein M, DNA replication
protein, glycoprotein, DNA replication protein, FLICE inhibitory
protein and v-cyclin genes, complete cds, and tegument protein
gene, partial cds. rxa01244 1827 GB_BA1: AFAPHBHI 4501 M69036
Alcaligenes eutrophus protein H (phbH) and protein I (phbI) genes,
complete cds. GB_PR3: HSJ836E13 78055 AL050326 Human DNA sequence
from clone 836E13 on chromosome 20 Contains ESTs, STS and GSSs,
complete sequence. GB_EST24: AI170227 409 AI170227 EST216152
Normalized rat lung, Bento Soares Rattus sp. cDNA clone RLUCF56 3'
end, mRNA sequence. rx01300 390 GB_PR3: HUMDODDA 26764 L39874 Homo
sapiens deoxycytidylate deaminase gene, complete cds. GB_PAT:
I40899 26764 I40899 Sequence 1 from patent US 5622851. GB_PAT:
I40900 1317 I40900 Sequence 2 from patent US 5622851. rxa00953 789
GB_BA1: SCJ21 31717 AL109747 Streptomyces coelicolor cosmid J21.
GB_BA1: BLTRP 7725 X04960 Brevibacterium lactofermentum tryptophan
operon. GB_PAT: E01375 7726 E01375 DNA sequence of tryptophan
operon. rx01943 2172 GB_BA1: CORPTSMA 2656 L18874 Corynebacterium
glutamicum phosphoenolpyruvate sugar phosphotransferase (ptsM)
mRNA, complete cds. GB_BA1: BRLPTSG 3163 L18875 Brevibacterium
lactofermentum phosphoenolpyruvate sugar phosphotransferase (ptsG)
gene, complete cds. GB_BA2: AF045481 2841 AF045481 Corynebacterium
ammoniagenes glucose permease (ptsG) gene, complete cds. % ID #
Source of Genbank Hit homology (GAP) Date of Deposit rxa00315
Serratia marcescens 40,386 26 Mar. 1998 Caenorhabditis elegans
36,207 28 Jul. 1996 Caenorhabditis elegans 36,436 23 Feb. 1999
rxa01503 Homo sapiens 39,722 27 Aug. 1998 Homo sapiens 43,137 21
May 1999 Homo sapiens 37,643 3 Aug. 1999 rxa01299 Mus musculus
41,475 18 Sep. 1999 Rattus norvegicus 41,031 2 Sep. 1998 Rattus
norvegicus 40,717 2 Sep. 1998 rxa00951 Streptomyces coelicolor
34,913 5 Aug. 1999 A3(2) Mouse cytomegalovirus 1 40,097 04 Dec.
1996 Kaposi's sarcoma- 36,029 9 Jul. 1997 associated herpesvirus
rxa01244 Ralstonia eutropha 45,624 26 Apr. 1993 Homo sapiens 37,303
23 Nov. 1999 Rattus sp. 39,098 20 Jan. 1999 rx01300 Homo sapiens
37,644 11 Aug. 1995 Unknown. 37,644 13 May 1997 Unknown. 37,644 13
May 1997 rxa00953 Streptomyces coelicolor 39,398 5 Aug. 1999 A3(2)
Corynebacterium 39,610 10 Feb. 1999 glutamicum Corynebacterium
46,753 29 Sep. 1997 glutamicum rx01943 Corynebacterium 100,000 24
Nov. 1994 glutamicum Brevibacterium 84,963 01 Oct. 1993
lactofermentum Corynebacterium 53,558 29 Jul. 1998 ammoniagenes
Appendix A: DNA Sequences
[0185]
7 >RXA00315 TATGATTTCGGCGGTCCAGTCGGCGGTCTGCTCTTCGGTCTGGT-
CTACTCACCAATCGTC ATCACTGGTCTGCACCAGTCCTTCCCGCCAATTGAGCTGGA-
GCTGTTTAACCAGGGTGGA TCCTTCATCTTCGCAACGGCATCTATGGCTAATATCGC-
CCAGGGTGCGGCATGTTTGGCA GTGTTCTTCCTGGCGAAGAGTGAAAAGCTCAAGGG-
CCTTGCAGGTGCTTCAGGTGTCTCC GCTGTTCTTGGTATTACGGAGCCTGCGATCTT-
CGGTGTGAACCTTCGCCTGCGCTGGCCG TTCTTCATCGGTATCGGTACCGCAGCTAT-
CGGTGGCGCTTTGATTGCACTCTTTAATATC AAGGCAGTTGCGTTGGGCGCTGCAGG-
TTTCTTGGGTGTTGTTTCTATTGATGCTCCAGAT ATGGTCATGTTCTTGGTGTGTGC-
AGTTGTTACCTTCTTCATCGCATTCGGCGCAGCGATT
GCTTATGGCCTTTACTTGGTTCGCCGCAACGGCAGCATTGATCCAGATGCAACCGCTGCT
CCAGTGCCTGCAGGAACGACCAAAGCCGAAGCAGAAGCACCCGCAGAATTTTCAAACGAT
TCCACCATCATCCAGGCACCTTTGACCGGTGAAGCTATTGCACTGAGCAGCGTCAGCGAT
GCCATGTTTGCCAGCGGAAAGCTTGGCTCGGGCGTTGCCATCGTCCCAACCAAGGGGCAG
TTAGTTTCTCCGGTGAGTGGAAAGATTGTGGTGGCATTCCCATCTGGCCATGCTTTCGCA
GTTCGCACCAAGGCTGAGGATGGTTCCAATGTGGATATCTTGATGCACATTGGTTTC- GAC
ACAGTAAACCTCAACGGCACGCACTTTAACCCGCTGAAGAAGCAGGGCGATGAA- GTCAAA
GCAGGGGAGCTGCTGTGTGAATTCGATATTGATGCCATTAAGGCTGCAGGT- TATGAGGTA
ACCACGCCGATTGTTGTTTCGAATTACAAGAAAACCGGACCTGTAAAC- ACTTACGGTTTG
GGCGAAATTGAAGCGGGAGCCAACCTGCTCAACGTCGCAAAGAAA- GAAGCGGTGCCAGCA
ACACCA >RXA00315-downstream TAAGTTGAAACCTTGAGTGTTCG >RXA00951
ATCCAAGCAATCTTAGAGAAGGCAGCAGCGCCGGCGAAGCAGAAGGCTCCTGC- TGTGGCT
CCTGCTGTAACACCCACTGACGCTCCTGCAGCCTCAGTCCAATCCAAAAC- CCACGACAAG
ATCCTCACCGTCTGTGGCAACGGCTTGGGTACCTCCCTCTTCCTCAA- AAACACCCTTGAG
CAAGTTTTCGACACCTGGGGTTGGGGTCCATACATGACGGTGGA- GGCAACCGACACTATC
TCCGCCAAGGGCAAAGCCAAGGAAGCTGATCTCATCATGAC- CTCTGGTGAAATCGCCCGC
ACGTTGGGTGATGTTGGAATCCCGGTTCAGCTGATCAA- TGACTTCACGAGCACCGATGAA
ATCGATGCTGCGCTTCGTGAACGCTACGACATC >RXA00951-downstream
TAACTACTTTAAAAGGACGAAAA >RXA01244-upstream
AGATGTCGATTTCTCGAGGAAGAAGTTAACGCC- GAAGAAAACCGTGAATCAGAGCAGGAG
CGCTTCGACGCCGCTGCAGCCACAGTCTCT- TCTTCGT >RXA01244
TTGCTTGAGCGCTCCGAAGCTGCTGAAGGA- CCAGCAGCTGAGGTGCTTAAAGCTACTGCT
GGCATGGTCAATGACCGTGGCTGGCGT- AAGGCTGTCATCAAGGGTGTCAAGGGTGGTCAC
CCTGCGGAATACGCCGTGGTTGCA- GCAACAACCAAGTTCATCTCCATGTTCGAAGCCGCA
GGCGGCCTGATCGCGGAGCGCACCACAGACTTGCGCGACATCCGCGACCGCGTCATCGCA
GAACTTCGTGGCGATGAAGAGCCAGGTCTGCCAGCTGTTTCCGGACAGGTCATTCTCTTT
GCAGATGACCTCTCCCCAGCAGACACCGCGGCACTAGACACAGATCTCTTTGTGGGACTT
GTCACTGAGCTGGGTGGCCCAACGAGCCACACCGCGATCATCGCACGCCAGCTCAACGTG
CCTTGCATCGTCGCATCCGGCGCCGGCATCAAGGACATCAAGTCCGGCGAAAAGGTGCTT
ATCGACGGCAGCCTCGGCACCATTGACCGCAACGCGGACGAAGCTGAAGCAACCAAG- CTC
GTCTCCGAGTCCCTCGAGCGCGCTGCTCGCATCGCCGAGTGGAAGGGTCCTGCA- CAAACC
AAGGACGGCTACCGCGTTCAGCTCTTGGCCAACGTCCAAGACGGCAACTCT- GCACAGCAG
GCTGCACAGACCGAAGCAGAAGGCATCGGCCTGTTCCGCACCGAACTG- TGCTTCCTTTCC
GCCACCGAAGAGCCAAGCGTTGATGAGCAGGCTGCGGTCTACTCA- AAGGTGCTTGAAGCA
TTCCCAGAGTCCAAGGTCGTTGTCCGCTCCCTCGACGCAGGT- TCTGACAAGCCAGTTCCA
TTCGCATCGATGGCTGATGAGATGAACCCAGCACTGGGT- GTTCGTGGCCTGCGTATCGCA
CGTGGACAGGTTGATCTGCTGACTCGCCAGCTCGAC- GCAATTGCGAAGGCCAGCGAAGAA
CTCGGCCGTGGCGACGACGCCCCAACCTGGGTT- ATGGCTCCAATGGTGGCTACCGCTTAT
GAAGCAAAGTGGTTTGCTGACATGTGCCGT- GAGCGTGGCCTAATCGCCGGCGCCATGATC
GAAGTTCCAGCAGCATCCCTGATGGCA- GACAAGATCATGCCTCACCTGGACTTTGTTTCC
ATCGGTACCAACGACCTGACCCAG- TACACCATGGCAGCGGACCGCATGTCTCCTGAGCTT
GCCTACCTGACCGATCCTTGGCAGCCAGCAGTCCTGCGCCTGATCAAGCACACCTGTGAC
GAAGGTGCTCGCTTTAACACCCCGGTCGGTGTTTGTGGTGAAGCAGCAGCAGACCCACTG
TTGGCAACTGTCCTCACCGGTCTTGGCGTGAACTCCCTGTCCGCAGCATCCACTGCTCTC
GCAGCAGTCGGTGCAAAGCTGTCAGAGGTCACCCTGGAAACCTGTAAGAAGGCAGCAGAA
GCAGCACTTGACGCTGAAGGTGCAACTGAAGCACGCGATGCTGTACGCGCAGTGATCGAC
GCAGCAGTC >RXA01244-downstream TAAACCACTGTTGAGCTAAAAAG
>RXA01299
ATGGAAATCATGGCCGCGATCATGGCAGCTGGCATGGTCCCACCAATCGCGTTGTCCATT
GCTACCCTGCTGCGCAAGAAGCTGTTCACCCCAGCAGAGCAAGAAAACGGCAAGTCTTCC
TGGCTGCTTGGCCTGGCATTCGTCTCCGAAGGTGCCATCCCATTCGCCGCAGCTGACCCA
TTCCGTGTGATCCCAGCAATGATGGCTGGCGGTGCAACCACTGGTGCAATCTCCATGGCA
CTGGGCGTCGGCTCTCGGGCTCCACACGGCGGTATCTTCGTGGTCTGGGCAATCGAACCA
TGGTGGGGCTGGCTCATCGCACTTGCAGCAGGCACCATCGTGTCCACCATCGTTGTC- ATC
GGACTGAAGCAGTTCTGGCCAAACAAGGCCGTCGCTGCAGAAGTCGCGAAGCAA- GAAGCA
CAACAAGCAGGCGTAAACGCA >RXA01299-downstream
TAATCGGACCTTGACCCGATGTC >RXA01300-upstream
GATCGACATTAAATCCCCTCCCTTGGGGGGTTTAACTAACAAAT- CGCTGCGCCCTAATCC
GTTCGGATTAACGGCGTAGCAACACGAAAGGACACTTTCC >RXA01300
ATGGCTTCCAAGACTGTAACCGTCGGTTCCTCCGTTG- GCCTGCACGCACGTCCAGCATCC
ATCATCGCTGAAGCGGCTGCTGAGTACGACGACG- AAATCTTGCTGACCCTGGTTGGCTCC
GATGATGACGAAGAGACCGACGCGTCCTCTT- CCCTCATGATCATGGCGCTGGGCGCAGAG
CACGGCAACGAAGTTACCGTCACCTCCG- ACAACGCTGAAGCTGTTGAGAAGATCGCTGCG
CTTATCGCACAGGACCTTGACGCTG- AG >RXA01300-downstream
TAAACAACGCTCTGCTTGTTAAA >RXA015O3-upstream
GTATCCTCAAAGGCCTTCTAGCTGTTGC- AGCTGCAGCGCACTCGGTGGATACGACATCCA
CGACCTATCAAATTCTTTATGCTGC- AGGCGATGCCTTTTC >RXA01503
ATGTTCTTGGCAGTCATTTTGGCGATTACTGCGGCTCGTAAATTCGGTGCCAATGTCTTT
ACATCAGTCGCACTCGCTGGTGCATTGCTGCACACACAGCTTCAGGCAGTAACCGTGTTG
GTTGACGGTGAACTCCAGTCGATGACTCTGGTGGCTTTCCAAAAGGCTGGTAATGACGTC
ACCTTCCTGGGCATTCCAGTGGTGCTGCAGTTGGCGTTGCATGTAGCGAGTTTGATGAAG
TTGTCGCGA >RXA01503-downstream TAAGAGGAGGGGCGTGTCGGTCT
>RXA01883-upstream
CGACTGCGGCGTCTCTTCCTGGCACTACCATTCCTCGTCCTGACCAACTCGCCACAGCTG
GTGCAACGGTCACCCAAGTCAAAGGATTGAAAGAATCAGC >RXA01883
ATGAATAGCGTAAATAATTCCTCGCTTGTCCGGCTGGATGTCGATTTCGGCGACTCCACC
ACGGATGTCATCAACAACCTTGCCACTGTTATTTTCGACGCTGGCCGAGGTTCCTCCGCC
GACGCCCTTGCCAAAGACGCGCTGGATCGTGAAGCAAAGTCCGGCACCGGCGTTCCTGGT
CAAGTTGCTATCCCCCACTGCCGTTCCGAAGCCGTATCTGTCCCTACCTTGGGCTTT- GCT
CGCCTGAGCAAGGGTGTGGACTTCAGCGGACCTGATGGCGATGCCAACTTGGTG- TTCCTC
ATTGCAGCACCTGCTGGCGGCGGCAAAGAGCACCTGAAGATCCTGTCCAAG- CTTGCTCGC
TCCTTGGTGAAGAAGGATTTCATCAAGGCTCTGCAGGAAGCCACCACC- GAGCAGGAAATC
GTCGACGTTGTCGATGCCGTGCTCAACCCAGCACCAAAAAACCAC- CGAGCCAGCTGCAGC
>RXA01889-upstream
ACCGAGCCAGCTGCAGCTCCGGCTGCGGCGGCCGGTTGTTAAGAGTGGGGCGGCGTCGAC
AAGCGTTACTCGTATC >RXA01889
GTGGCAATCACCGCATGCCCAACCGGTATCGCACACACCTACATGGCTGCGGATTCCCTG
ACGCAAAACGCGGAAGGCCGCGATGATGTGGAACTCGTTGTGGAGACTCAGGGCTCTTCC
GCTGTCACCCCAGTCGATCCGAAGATCATCGAAGCTGCCGACGCCGTCATCTTCGCCACC
GACGTGGGAGTTAAAGACCGCGAGCGTTTCGCTGGCAAGCCAGTCATTGAATCCGGCGTC
AAGCGCGCGATCAATGAGCCAGCCAAGATGATCGACGAGGCCATCGCAGCCTCCAAGAAC
CCAAACGCCCGCAAGGTTTCCGGTTCCGGTGTCGCGGCATCTGCTGAAACCACCGGC- GAG
AAGCTCGGCTGGGGCAAGCGCATCCAGCAGGCAGTCATGACCGGCGTGTCCTAC- ATGGTT
CCATTCGTAGCTGCCGGCGGCCTCCTGTTGGCTCTCGGCTTCGCATTCGGT- GGATACGAC
ATGGCGAACGGCTGGCAAGCAATCGCCACCCAGTTCTCTCTGACCAAC- CTGCCAGGCAAC
ACCGTCGATGTTGAC >RXA01943
CCTGACCCAATCTTTGCAGCAGGCAAGCTTGGACCAGGCATTGCAATCCAACCAACTGGA
AACACCGTTGTTGCTCCAGCAGACGCTACTGTCATCCTTGTCCAGAAATCTGGACACGCA
GTGGCATTGCGCTTAGATAGCGGAGTTGAAATCCTTGTCCACGTTGGATTGGACACCGTG
CAATTGGGCGGCGAAGGCTTCACCGTTCACGTTGAGCGCAGGCAGCAAGTCAAGGCG- GGG
GATCCACTGATCACTTTTGACGCTGACTTCATTCGATCCAAGGATCTACCTTTG- ATCACC
CCAGTTGTGGTGTCTAACGCCGCGAAATTCGGTGAAATTGAAGGTATTCCT- GCAGATCAG
GCAAATTCTTCCACGACTGTGATCAAGGTCAACGGCAAGAACGAG
>RXA01943-downstream TAACCTGGGATCCATGTTGCGCA
>RXA02191-upstream CCGATTCTTTTTCGGCCCAATTCGTAACGGCGAT-
CCTCTTAAGTGGACAAGAAAGTCTCT TGCCCGCGGGAGACAGACCCTACGTTTAGAA-
AGGTTTGAC >RXA02191 ATGGCGTCCAAACTGACGACGACATGGC-
AACATATTCTGGAAAACCTTGGTGGACCAGAC AATATTACTTCGATGACTCACTGTG-
CGACTCGCCTTGGCTTCCAAGTGAAGGATCAATCC
ATTGTTGATCAACAAGAAATTGACTCCGACCCATCAGTTCTTGGCGTAGTACCCCAAGGA
TCCACCGGTATGCAGGTGGTGATGGGTGGATCTGTTGCAAACTATTACCAAGAAATCCTC
AAACTTGATGGAATGAAGCACTTCGCCGACGGTGAAGCTACAGAGAGTTCATCCAAGAAG
GAATACGGCGGAGTCCGTGGCAAGTACTCGTGGATTGACTACGCCTTCGAGTTCTTGTCT
GATACTTTCCGACCAATCCTGTGGGGCCTGCTTGGTGCCTCACTGATTATTACCTTGTTG
GTTCTTGCGGATACTTTCGGTTTGCAAGACTTCCGCGCTCCAATGGATGAGCAGCCT- GAT
ACTTATGTATTCCTGCACTCCATGTGGCGCTCGGTCTTCTACTTCCTGCCAATT- ATGGTT
GGTGCCACCGCAGCTCGAAAGCTCGGCGCAAACGAGTGGATTGGTGCAGCT- ATTCGAGCC
GCAGTTCTTACTCCAGAATTCTTGGCACTGGGTTCTGCCGGCGATACC- GTCACAGTCTTT
GGCCTGCCAATGGTTCTGAATGACTACTCCGGACAGGTATTCCCA- CCGCTGATTGCAGCA
ATTGGTCTGTACTGGGTGGAAAAGGGACTGAAGAAGATCATC- CCTGAAGCAGTCCAAATG
GTGTTCGTCCCATTCTTCTCCCTGCTGATTATGATCCCA- GCGACCGCATTCCTGCTTGGA
CCTTTCGGCATCGGTGTTGGTAACGGAATTTCCAAC- CTGCTTGAAGCGATTAACAACTTC
AGCCCATTTATTCTTTCCATCGTTATCCCATTG- CTCTACCCATTCTTGGTTCCAGTTGGA
TTGCACTGGCCACTAAACGCCATCATGATC- CAGAACATCAACACCCTGGGTTACGACTTC
ATTCAGGGACCAATGGGTGCCTGGAAC- TTCGCCTGCTTCGGCCTGGTCACCGGCGTGTTC
TTGCTCTCCATTAAGGAACGAAAC- AAGGCCATGCGTCAGGTTTCCCTGGGTGGCATGTTG
GCTGGTTTGCTCGGCGGCATTTCCGAGCCTTCCCTCTACGGTGTTCTGCTCCGATTCAAG
AAGACCTACTTCCGCCTCCTGCCGGGTTGTTTGGCAGCA >RXN01244-upstream
GATATGTGTTTGTTTGTCAATATCCAAATGTTTGAATAGTTGCA- CAACTGTTGGTTTTGT
GGTGATCTTGAGGAAATTAACTCAATGATTGTGAGGATGG >RXN01244
GTGGCTACTGTGGCTGATGTGAATCAAGACACTGTAC- TGAAGGGCACCGGCGTTGTCGGT
GGAGTCCGTTATGCAAGCCGGGTGTGGATTACCC- CACGCCCCGAACTACCCCAAGCAGGC
GAAGTCGTCGCCGAAGAAAACCGTGAAGCAG- AGCAGGAGCGTTTCGACGCCGCTGCAGCC
ACAGTCTCTTCTCGTTTGCTTGAGCGCT- CCGAAGCTGCTGAAGGACCAGCAGCTGAGGTG
GTTAAAGCTACTGCTGGCATGGTGA- ATGACCGTGGCTGGCGTAAGGCTGTCATCAAGGGT
GTCAAGGGTGGTCACCCTGCGGAATACGCCGTGGTTGCAGCAACAACCAAGTTCATCTCC
ATGTTCGAAGCCGCAGGCGGCCTGATCGCGGAGCGCACCACAGACTTGCGCGACATCCGC
GACCGCGTCATCGCAGAACTTCGTGGCGATGAAGAGCCAGGTCTGCCAGCTGTTTCCGGA
CAGGTCATTCTCTTTGCAGATGACCTCTCCCCAGCAGACACCGCGGCACTAGACACAGAT
CTCTTTGTGGGACTTGTCACTGAGCTGGGTGGCCCAACGAGCCACACCGCGATCATCGCA
CGCCAGCTCAACGTGCCTTGCATCGTCGCATCCGGCGCCGGCATCAAGGACATCAAG- TCC
GGCGAAAAGGTGCTTATCGACGGCAGCCTCGGCACCATTGACCGCAACGCGGAC- GAAGCT
GAAGCAACCAAGCTCGTCTCCGAGTCCCTCGAGCGCGCTGCTCGCATCGCC- GAGTGGAAG
GGTCCTGCACAAACCAAGGACGGCTACCGCGTTCAGCTGTTGGGCAAC- GTCCAAGACGGC
AACTCTGCACAGCAGGCTGCACAGACCGAAGCAGAAGGCATCGGC- CTGTTCCGCACCGAA
CTGTGCTTCCTTTCCGCCACCGAAGAGCCAAGCGTTGATGAG- CAGGCTGCGGTCTACTCA
AAGGTGCTTGAAGCATTCCCAGAGTCCAAGGTCGTTGTC- CGCTCCCTCGACGCAGGTTCT
GACAAGCCAGTTCCATTCGCATCGATGGCTGATGAG- ATGAACCCAGCACTGGGTGTTCGT
GGCCTGCGTATCGCACGTGGACAGGTTGATCTG- CTGACTCGCCAGCTCGACGCAATTGCG
AAGGCCAGCGAAGAACTCGGCCGTGGCGAC- GACGCCCCAACCTGGGTTATGGCTCCAATG
GTGGCTACCGCTTATGAAGCAAAGTGG- TTTGCTGACATGTGCCGTGAGCGTGGCCTAATC
GCCGGCGCCATGATCGAAGTTCCA- GCAGCATCCCTGATGGCAGACAAGATCATGCCTCAC
CTGGACTTTGTTTCCATCGGTACCAACGACCTGACCCAGTACACCATGGCAGCGGACCGC
ATGTCTCCTGAGCTTGCCTACCTGACCGATCCTTGGCAGCCAGCAGTCCTGCGCCTGATC
AAGCACACCTGTGACGAAGGTGCTCGCTTTAACACCCCGGTCGGTGTTTGTGGTGAAGCA
GCAGCAGACCCACTGTTGGCAACTGTCCTCACCGGTCTTGGCGTGAACTCCCTGTCCGCA
GCATCCACTGCTCTCGCAGCAGTCGGTGCAAAGCTGTCAGAGGTCACCCTGGAAACCTGT
AAGAAGGCAGCAGAAGCAGCACTTGACGCTGAAGGTGCAACTGAAGCACGCGATGCT- GTA
CGCGCAGTGATCGACGCAGCAGTC >RXN01244-downstream
TAAACCACTGTTGAGCTAAAAAG >RXN01299-upstream
CGACTGCGGCGTCTCTTCCTGGCACTACCATTCCTCGTCCTGAC- CAACTCGCCACAGCTG
GTGCAACGGTCACCCAAGTCAAAGGATTGAAAGAATCAGC >RXN01299
ATGAATAGCGTAAATAATTCCTCGCTTGTCCGGCTGG- ATGTCGATTTCGGCGACTCCACC
ACGGATCTCATCAACAACCTTGCCACTGTTATTT- TCGACGCTGGCCGAGCTTCCTCCGCC
GACGCCCTTGCCAAAGACGCGCTGGATCGTG- AAGCAAAGTCCGGCACCGGCGTTCCTGGT
CAAGTTGCTATCCCCCACTGCCGTTCCG- AAGCCGTATCTGTCCCTACCTTGGGCTTTGCT
CGCCTGAGCAAGGGTGTGGACTTCA- GCGGACCTGATGGCGATGCCAACTTGGTGTTCCTC
ATTGCAGCACCTGCTGGCGGCGGCAAAGAGCACCTGAAGATCCTGTCCAAGCTTGCTCGC
TCCTTGGTGAAGAAGGATTTCATCAAGGCTCTGCAGGAAGCCACCACCGAGGAGGAAATC
GTGGACGTTGTCGATGCCGTGCTCAACCCAGCACCAAAAACCACCGAGCCAGCTGCAGCT
CCGGCTGCGGCGGCGGTTGCTGAGAGTGGGGCGGCGTCGACAAGCGTTACTCGTATCGTG
GCAATCACCGCATGCCCAACCGGTATCGCACACACCTACATGGCTGCGGATTCCCTGACG
CAAAACGCGGAAGGCCGCGATGATGTGGAACTCGTTGTGGAGACTCAGGGCTCTTCC- GCT
GTCACCCCAGTCGATCCGAAGATCATCGAAGCTGCCGACGCCGTCATCTTCGCC- ACCGAC
GTGGGAGTTAAAGACCGCGAGCGTTTCGCTGCGAAGCCAGTCATTGAATCC- GGCGTCAAG
CGCGCGATCAATGAGCCAGCCAAGATGATCGACGAGGCCATCGCAGCC- TCCAAGAACCCA
AACGCCCGCAAGGTTTCCGGTTCCGGTGTCGCGGCATCTGCTGAA- ACCACCGGCGAGAAG
CTCGGCTGGGGCAAGCGCATCCAGCAGGCAGTCATGACCGGC- GTGTCCTACATGGTTCCA
TTCGTAGCTGCCGGCGGCCTCCTGTTGGCTCTCGGCTTC- GCATTCGGTGGATACGACATG
GCGAACGGCTGGCAAGCAATCGCCACCCAGTTCTCT- CTGACCAACCTGCCAGGCAACACC
GTCGATGTTGACGGCGTGGCCATGACCTTCGAG- CGTTCAGGCTTCCTGTTGTACTTCGGC
GCAGTCCTGTTCGCCACCGGCCAAGCAGCC- ATGGGCTTCATCGTGGCAGCCCTGTCTGGC
TACACCGCATACGCACTTGCTGGACGC- CCAGGCATCGCGCCGGGCTTCGTCGGTGGCGCC
ATCTCCGTCACCATCCGCGCTGGC- TTCATTGGTGGTCTGGTTACCGGTATCTTGGCTGGT
CTCATTGCCCTGTGGATTGGCTCCTGGAAGGTGCCACGCGTGGTGCAGTCACTGATGCCT
GTGGTCATCATCCCGCTACTTACCTCAGTGGTTGTTGGTCTCGTCATGTACCTCCTGCTG
GGTCGCCCACTCGCATCCATCATGACTGGTTTGCAGGACTGGCTATCGTCAATGTCCGGA
AGCTCCGCCATCTTGCTGGGTATCATCTTGGGCCTCATGATGTGTTTCGACCTCGGCGGA
CCAGTAAACAAGGCAGCCTACCTCTTTGGTACCGCAGGCCTGTCTACCGGCGACCAAGCT
TCCATGGAAATCATGGCCGCGATCATGGCAGCTGGCATGGTCCCACCAATCGCGTTG- TCC
ATTGCTACCCTGCTGCGCAAGAAGCTGTTCACCCCAGCAGAGCAAGAAAACGGC- AAGTCT
TCCTGGCTGCTTGGCCTGGCATTCGTCTCCGAAGGTGCCATCCCATTCGCC- GCAGCTGAC
CCATTCCGTGTGATCCCAGCAATGATGGCTGGCGGTGCAACCACTGGT- GCAATCTCCATG
GCACTGGGCGTCGGCTCTCGGGCTCCACACGGCGGTATCTTCGTG- GTCTGGGCAATCGAA
CCATGGTGGGGCTGGCTCATCGCACTTGCAGCAGGCACCATC- GTGTCCACCATCGTTGTC
ATCGCACTGAAGCAGTTCTGGCCAAACAAGGCCGTCGCT- GCAGAAGTCGCGAAGCAAGAA
GCACAACAAGCAGCTGTAAACGCA >RXN01299-downstream
TAATCGGACCTTGACCCGATGTC >RXN01943-upstream
CCGATTCTTTTTCGGCCCAATTCGTAACGGCGATCCTCTTAAG- TGGACAAGAAAGTCTCT
TGCCCGCGGGAGACAGACCCTACGTTTAGAAAGGTTTGAC >RXN01943
ATGGCGTCCAAACTGACGACGACATCGCAACATATTC- TGGAAAACCTTGGTGGACCAGAC
AATATTACTTCGATGACTCACTGTGCGACTCGCC- TTCGCTTCCAAGTGAAGGATCAATCC
ATTGTTGATCAACAAGAAATTGACTCCGACC- CATCAGTTCTTGGCGTAGTACCCCAAGGA
TCCACCGGTATGCAGGTGGTGATGGGTG- GATCTGTTGCAAACTATTACCAAGAAATCCTC
AAACTTGATGGAATGAACCACTTCG- CCGACGGTGAAGCTACAGAGAGTTCATCCAAGAAG
GAATACGGCGGAGTCCGTGGCAAGTACTCGTGGATTGACTACGCCTTCGAGTTCTTGTCT
GATACTTTCCGACCAATCCTGTGGGCCCTGCTTGGTGCCTCACTGATTATTACCTTGTTG
GTTCTTGCGGATACTTTCGGTTTGCAAGACTTCCGCGCTCCAATGGATGAGCAGCCTGAT
ACTTATGTATTCCTGCACTCCATGTGGCGCTCGGTCTTCTACTTCCTGCCAATTATGGTT
GGTGCCACCGCAGCTCGAAAGCTCGGCGCAAACGAGTGGATTGGTGCAGCTATTCCAGCC
GCACTTCTTACTCCAGAATTCTTGGCACTGGGTTCTGCCGGCGATACCGTCACAGTC- TTT
GGCCTGCCAATGGTTCTGAATGACTACTCCGGACAGGTATTCCCACCGCTGATT- GCAGCA
ATTGGTCTGTACTGGGTGGAAAAGGGACTGAAGAAGATCATCCCTGAAGCA- GTCCAAATG
GTGTTCGTCCCATTCTTCTCCCTGCTGATTATGATCCCAGCGACCGCA- TTCCTGCTTGGA
CCTTTCGGCATCGGTGTTGGTAACGGAATTTCCAACCTGCTTGAA- GCGATTAACAACTTC
AGCCCATTTATTCTTTCCATCGTTATCCCATTGCTCTACCCA- TTCTTGGTTCCACTTGGA
TTGCACTGGCCACTAAACGCCATCATGATCCAGAACATC- AACACCCTGGGTTACGACTTC
ATTCAGGGACCAATGGGTGCCTGGAACTTCGCCTGC- TTCGGCCTGGTCACCGGCGTGTTC
TTGCTCTCCATTAAGGAACGAAACAAGGCCATG- CGTCAGGTTTCCCTGGGTGGCATGTTG
GCTGGTTTGCTCGGCGGCATTTCCGAGCCT- TCCCTCTACGGTGTTCTGCTCCGATTCAAG
AAGACCTACTTCCGCCTCCTGCCGGGT- TGTTTGGCAGGCGGTATCGTGATGGGCATCTTC
GACATCAAGGCGTACGCTTTCGTG- TTCACCTCCTTGCTTACCATCCCAGCAATGGACCCA
TGGTTGGGCTACACCATTGGTATCGCAGTTGCATTCTTCGTTTCCATGTTCCTTGTTCTC
GCACTGGACTACCGTTCCAACGAAGAGCGCGATGAGGCACGTGCAAAGGTTGCTGCTGAC
AAGCAGGCAGAAGAAGATCTGAAGGCAGAAGCTAATGCAACTCCTGCAGCTCCAGTAGCT
GCTGCAGGTGCGGGAGCCGGTGCAGGTGCAGGAGCCGCTGCTGGCGCTGCAACCGCCGTG
GCAGCTAAGCCGAAGCTGGCCGCTGGGGAAGTAGTGGACATTGTTTCCCCACTCGAAGGC
AAGGCAATTCCACTTTCTGAAGTACCTGACCCAATCTTTGCAGCAGGCAAGCTTGGA- CCA
GGCATTGCAATCCAACCAACTGGAAACACCGTTGTTGCTCCAGCAGACGCTACT- GTCATC
CTTGTCCAGAAATCTGGACACGCAGTGGCATTGCGCTTAGATAGCGGAGTT- GAAATCCTT
GTCCACGTTGGATTGGACACCGTGCAATTGGGCGGCGAAGGCTTCACC-
GTTCACGTTGAG CGCAGGCAGCAAGTCAAGGCGGGGGATCCACTGATCACTTTTGAC-
GCTGACTTCATTCGA TCCAAGGATCTACCTTTGATCACCCCAGTTGTGGTGTCTAAC-
GCCGCGAAATTCGGTGAA ATTGAAGGTATTCCTGCAGATCAGGCAAATTCTTCCACG-
ACTGTGATCAAGGTCAACGGC AAGAACGAG >RXN01943-downstream
TAACCTGGGATCCATGTTGCGCA >RXN03002-upstream
GGAACTTCGAGGTGTCTTCGTGGGGCGTACGGAGATCTAGCAAG- TGTGGCTTTATGTTTG
ACCCTATCCGAATCAACATGCAGTGAATTAACATCTACTT >RXN03002
ATGTTTGTACTCAAAGATCTGCTAAAGGCAGAACGCA- TAGAACTCGACCGCACGGTCACC
GATTGGCGTGAAGGCATCCGCGCCGCAGGTGTAC- TCCTAGAAAAGACAAACAGCATTGAT
TCCGCCTACACCGATGCCATGATCGCCAGCG- TGGAAGAAAAAGGCCCCTACATTGTGGTC
GCTCCAGGTTTCGCTTTCGCGCACGCCG- GCCCCAGCAGAGCAGTCCGCGAGACCGCTATG
TCGTGGGTGCGCCTGGCCTCCCCTG- TTTCCTTCGGTCACAGTAAGAATGATCCCCTCAAT
CTCATCGTTGCTCTCGCTGCCAAAGATGCCACCGCACATACCCAAGCGATGGCGGCATTG
GCTAAAGCTTTAGGAAAATACCGAAAGGATCTCGACGAGGCACAAAGT RXS00315 -
upstream CTCATGGCATCTGCGCCGTTCGCGTTCTTGCCAGTGTTGGTTGGTT-
TCACCGCAACCAAGCGTTTCGGC GGCAATGAGTTCCTGGGCGCCGCGTATTGGT RXS00315
ATGGCGATGGTGTTCCCGAGCTTGGTGAACGGCTACGACGTGGC-
CGGCACCATGGCTGCGGGCGAAATG CCAATGTGGTCCCTGTTTGGTTTAGATGTTGC-
CCAAGCCGGTTACCAGGGCACCGTGCTTCCTGTGCTG
GTGGTTTCTTGGATTCTGGCAACGATCGAGAAGTTCCTGCACAAGCGACTCAAGGGCACTGCAGACTTC
CTGATCACTCCAGTGCTGACGTTGCTGCTCACCGGATTCCTTACATTCATCGCCATTGGCC-
CAGCAATG CGCTGGGTGGGCGATGTGCTGGCACACGGTCTACAGGGACTTTATGATT-
TCGGTGGTCCAGTCGGCGGT CTGCTCTTCGGTCTGGTCTACTCACCAATCGTCATCA-
CTGGTCTGCACCAGTCCTTCCCGCCAATTGAG CTGGAGCTGTTTAACCAGGGTGGAT-
CCTTCATCTTCGCAACGGCATCTATGGCTAATATCGCCCAGGGT
GCGGCATGTTTGGCAGTGTTCTTCCTGGCGAAGAGTGAAAAGCTCAAGGGCCTTGCAGGTGCTTCAGGT
GTCTCCGCTGTTCTTGGTATTACGGAGCCTGCGATCTTCGGTGTGAACCTTCGCCTGCGCT-
GGCCGTTC TTCATCGGTATCGGTACCGCAGCTATCGGTGGCGGTTTGATTGCACTCT-
TTAATATCAAGGCAGTTGCG TTGGGCGCTGCAGGTTTCTTGGGTGTTGTTTCTATTG-
ATGCTCCAGATATGGTCATGTTCTTGGTGTGT GCAGTTGTTACCTTCTTCATCGCAT-
TCGGCGCAGCGATTGCTTATGGCCTTTACTTGGTTCGCCGCAAC
GGCAGCATTGATCCAGATGCAACCGCTGCTCCAGTGCCTGCAGGAACGACCAAAGCCGAAGCAGAAGCA
CCCGCAGAATTTTCAAACGATTCCACCATCATCCAGGCACCTTTGACCGGTGAAGCTATTG-
CACTGAGC AGCGTCAGCGATGCCATGTTTGCCAGCGGAAAGCTTGGCTCGGGCGTTG-
CCATCGTCCCAACCAAGGGG CAGTTAGTTTCTCCGGTGAGTGGAAAGATTGTGGTGG-
CATTCCCATCTGGCCATGCTTTCGCAGTTCGC ACCAAGGCTGAGGATGGTTCCAATG-
TGGATATCTTGATGCACATTGGTTTCGACACAGTAAACCTCAAC
GGCACGCACTTTAACCCGCTGAAGAAGCAGGGCGATGAAGTCAAAGCAGGGGAGCTGCTGTGTGAATTC
GATATTGATGCCATTAAGGCTGCAGGTTATGAGGTAACCACGCCGATTGTTGTTTCGAATT-
ACAAGAAA ACCGGACCTGTAAACACTTACGGTTTGGGCGAAATTGAAGCGGGAGCCA-
ACCTGCTCAACGTCGCAAAG AAAGAAGCGGTGCCAGCAACACCA RXS00315 - downstream
TAAGTTGAAACCTTGAGTGTTCG RXC00953 - upstream CTTGCATTCCCCA RXC00953
-
ATGGCGCCACCAACGGTAGGCAACTACATCATGCAGTCCTTCACTCAAGGTCTGCAGTTCGGCGTTGCA
GTTGCCGTGATTCTCTTTGGTGTCCGCACCATTCTTGGTGAACTGGTCCCCGCATTCCAA-
GGTATTGCT GCGAAGGTTGTTCCCGGAGCTATCCCCGCATTGGATGCACCGATCGTG-
TTCCCCTACGCGCAGAACGCC GTTCTCATTGGTTTCTTGTCTTCCTTCGTCGGTGGC-
TTGGTTGGCCTGACTGTTCTTGCATCGTGGCTG AACCCAGCTTTTGGTGTCGCGTTG-
ATTCTGCCTGGTTTGGTCCCCCACTTCTTCACTGGTGGCGCGGCG
GGCGTTTACGGTAATGCCACGGGTGGTCGTCGAGGAGCAGTATTTGGCGCCTTTGCCAACGGTCTTCTG
ATTACCTTCCTCCCTGCTTTCCTGCTTGGTGTGCTTGGTTCCTTCGGGTCAGAGAACACCA-
CTTTCGGT GATGCGGACTTTGGTTGGTTCGGAATCGTTGTTGGTTCTGCAGCCAAGG-
TGGAAGGTGCTGGCGGGCTC ATCTTGTTGCTCATCATCGCAGCGGTTCTTCTGGGTG-
GCGCGATGGTCTTCCAGAAGCGCGTCGTGAAT GGGCACTGGGATCCAGCTCCCAACC-
GTGAGCGCGTGGAGAAGGCGGAAGCTGATGCCACTCCAACGGCT
GGGGCTCGGACCTACCCTAAGATTGCTCCTCCGGCGGGCGCTCCTACCCCACCGGCTGGAAGC
RXC00953 - downstream TAAGATCTCCAAAACCCTGAGAT RXC03001 - upstream
CCCGGTTCACGTGATCAATGACTTCACGAGCACCGATGAAATCG-
ATGCTGCGCTTCGTGAACGCTACGA CATCTAACTACTTTAAAAGGACGAAAATATT RXC03001
- ATGGACTGGTTAACCATTCCTCTTTTCCTCGTTAATGAA-
ATCCTTGCGGTTCCGGCTTTCCTCATCGGT ATCATCACCGCCGTGGGATTGGGTGCC-
ATGGGGCGTTCCGTCGGTCAGGTTATCGGTGGAGCAATCAAA
GCAACGTTGGGCTTTTTGCTCATTGGTGCGGGTGCCACGTTGGTCACTGCCTCCCTGGAGCCACTGGGT
GCGATGATCATGGGTGCCACAGGCATGCGTGGTGTTGTCCCAACGAATGAAGCCATCGCGG-
GAATCGCA CAGGCTGAATACGGCGCGCAGGTGGCGTGGCTGATGATTCTGGGCTTCG-
CCATCTCTTTGGTGTTGGCT GGTTTCACCAACCTGCGTTATGTCTTGCTCAACGGAC-
ACCACGTGCTGTTGATGTGCACCATGCTCACC ATGGTCTTGGCCACCGGAAGAGTTG-
ATGCGTGGATCTTC
[0186]
Sequence CWU 1
1
34 1 1527 DNA Corynebacterium glutamicum CDS (101)..(1504) RXS00315
1 ctcatggcat ctgcgccgtt cgcgttcttg ccagtgttgg ttggtttcac cgcaaccaag
60 cgtttcggcg gcaatgagtt cctgggcgcc gcgtattggt atg gcg atg gtg ttc
115 Met Ala Met Val Phe 1 5 ccg agc ttg gtg aac ggc tac gac gtg gcc
gcc acc atg gct gcg ggc 163 Pro Ser Leu Val Asn Gly Tyr Asp Val Ala
Ala Thr Met Ala Ala Gly 10 15 20 gaa atg cca atg tgg tcc ctg ttt
ggt tta gat gtt gcc caa gcc ggt 211 Glu Met Pro Met Trp Ser Leu Phe
Gly Leu Asp Val Ala Gln Ala Gly 25 30 35 tac cag ggc acc gtg ctt
cct gtg ctg gtg gtt tct tgg att ctg gca 259 Tyr Gln Gly Thr Val Leu
Pro Val Leu Val Val Ser Trp Ile Leu Ala 40 45 50 acg atc gag aag
ttc ctg cac aag cga ctc aag ggc act gca gac ttc 307 Thr Ile Glu Lys
Phe Leu His Lys Arg Leu Lys Gly Thr Ala Asp Phe 55 60 65 ctg atc
act cca gtg ctg acg ttg ctg ctc acc gga ttc ctt aca ttc 355 Leu Ile
Thr Pro Val Leu Thr Leu Leu Leu Thr Gly Phe Leu Thr Phe 70 75 80 85
atc gcc att ggc cca gca atg cgc tgg gtg ggc gat gtg ctg gca cac 403
Ile Ala Ile Gly Pro Ala Met Arg Trp Val Gly Asp Val Leu Ala His 90
95 100 ggt cta cag gga ctt tat gat ttc ggt ggt cca gtc ggc ggt ctg
ctc 451 Gly Leu Gln Gly Leu Tyr Asp Phe Gly Gly Pro Val Gly Gly Leu
Leu 105 110 115 ttc ggt ctg gtc tac tca cca atc gtc atc act ggt ctg
cac cag tcc 499 Phe Gly Leu Val Tyr Ser Pro Ile Val Ile Thr Gly Leu
His Gln Ser 120 125 130 ttc ccg cca att gag ctg gag ctg ttt aac cag
ggt gga tcc ttc atc 547 Phe Pro Pro Ile Glu Leu Glu Leu Phe Asn Gln
Gly Gly Ser Phe Ile 135 140 145 ttc gca acg gca tct atg gct aat atc
gcc cag ggt gcg gca tgt ttg 595 Phe Ala Thr Ala Ser Met Ala Asn Ile
Ala Gln Gly Ala Ala Cys Leu 150 155 160 165 gca gtg ttc ttc ctg gcg
aag agt gaa aag ctc aag ggc ctt gca ggt 643 Ala Val Phe Phe Leu Ala
Lys Ser Glu Lys Leu Lys Gly Leu Ala Gly 170 175 180 gct tca ggt gtc
tcc gct gtt ctt ggt att acg gag cct gcg atc ttc 691 Ala Ser Gly Val
Ser Ala Val Leu Gly Ile Thr Glu Pro Ala Ile Phe 185 190 195 ggt gtg
aac ctt cgc ctg cgc tgg ccg ttc ttc atc ggt atc ggt acc 739 Gly Val
Asn Leu Arg Leu Arg Trp Pro Phe Phe Ile Gly Ile Gly Thr 200 205 210
gca gct atc ggt ggc gct ttg att gca ctc ttt aat atc aag gca gtt 787
Ala Ala Ile Gly Gly Ala Leu Ile Ala Leu Phe Asn Ile Lys Ala Val 215
220 225 gcg ttg ggc gct gca ggt ttc ttg ggt gtt gtt tct att gat gct
cca 835 Ala Leu Gly Ala Ala Gly Phe Leu Gly Val Val Ser Ile Asp Ala
Pro 230 235 240 245 gat atg gtc atg ttc ttg gtg tgt gca gtt gtt acc
ttc ttc atc gca 883 Asp Met Val Met Phe Leu Val Cys Ala Val Val Thr
Phe Phe Ile Ala 250 255 260 ttc ggc gca gcg att gct tat ggc ctt tac
ttg gtt cgc cgc aac ggc 931 Phe Gly Ala Ala Ile Ala Tyr Gly Leu Tyr
Leu Val Arg Arg Asn Gly 265 270 275 agc att gat cca gat gca acc gct
gct cca gtg cct gca gga acg acc 979 Ser Ile Asp Pro Asp Ala Thr Ala
Ala Pro Val Pro Ala Gly Thr Thr 280 285 290 aaa gcc gaa gca gaa gca
ccc gca gaa ttt tca aac gat tcc acc atc 1027 Lys Ala Glu Ala Glu
Ala Pro Ala Glu Phe Ser Asn Asp Ser Thr Ile 295 300 305 atc cag gca
cct ttg acc ggt gaa gct att gca ctg agc agc gtc agc 1075 Ile Gln
Ala Pro Leu Thr Gly Glu Ala Ile Ala Leu Ser Ser Val Ser 310 315 320
325 gat gcc atg ttt gcc agc gga aag ctt ggc tcg ggc gtt gcc atc gtc
1123 Asp Ala Met Phe Ala Ser Gly Lys Leu Gly Ser Gly Val Ala Ile
Val 330 335 340 cca acc aag ggg cag tta gtt tct ccg gtg agt gga aag
att gtg gtg 1171 Pro Thr Lys Gly Gln Leu Val Ser Pro Val Ser Gly
Lys Ile Val Val 345 350 355 gca ttc cca tct ggc cat gct ttc gca gtt
cgc acc aag gct gag gat 1219 Ala Phe Pro Ser Gly His Ala Phe Ala
Val Arg Thr Lys Ala Glu Asp 360 365 370 ggt tcc aat gtg gat atc ttg
atg cac att ggt ttc gac aca gta aac 1267 Gly Ser Asn Val Asp Ile
Leu Met His Ile Gly Phe Asp Thr Val Asn 375 380 385 ctc aac ggc acg
cac ttt aac ccg ctg aag aag cag ggc gat gaa gtc 1315 Leu Asn Gly
Thr His Phe Asn Pro Leu Lys Lys Gln Gly Asp Glu Val 390 395 400 405
aaa gca ggg gag ctg ctg tgt gaa ttc gat att gat gcc att aag gct
1363 Lys Ala Gly Glu Leu Leu Cys Glu Phe Asp Ile Asp Ala Ile Lys
Ala 410 415 420 gca ggt tat gag gta acc acg ccg att gtt gtt tcg aat
tac aag aaa 1411 Ala Gly Tyr Glu Val Thr Thr Pro Ile Val Val Ser
Asn Tyr Lys Lys 425 430 435 acc gga cct gta aac act tac ggt ttg ggc
gaa att gaa gcg gga gcc 1459 Thr Gly Pro Val Asn Thr Tyr Gly Leu
Gly Glu Ile Glu Ala Gly Ala 440 445 450 aac ctg ctc aac gtc gca aag
aaa gaa gcg gtg cca gca aca cca 1504 Asn Leu Leu Asn Val Ala Lys
Lys Glu Ala Val Pro Ala Thr Pro 455 460 465 taagttgaaa ccttgagtgt
tcg 1527 2 468 PRT Corynebacterium glutamicum 2 Met Ala Met Val Phe
Pro Ser Leu Val Asn Gly Tyr Asp Val Ala Ala 1 5 10 15 Thr Met Ala
Ala Gly Glu Met Pro Met Trp Ser Leu Phe Gly Leu Asp 20 25 30 Val
Ala Gln Ala Gly Tyr Gln Gly Thr Val Leu Pro Val Leu Val Val 35 40
45 Ser Trp Ile Leu Ala Thr Ile Glu Lys Phe Leu His Lys Arg Leu Lys
50 55 60 Gly Thr Ala Asp Phe Leu Ile Thr Pro Val Leu Thr Leu Leu
Leu Thr 65 70 75 80 Gly Phe Leu Thr Phe Ile Ala Ile Gly Pro Ala Met
Arg Trp Val Gly 85 90 95 Asp Val Leu Ala His Gly Leu Gln Gly Leu
Tyr Asp Phe Gly Gly Pro 100 105 110 Val Gly Gly Leu Leu Phe Gly Leu
Val Tyr Ser Pro Ile Val Ile Thr 115 120 125 Gly Leu His Gln Ser Phe
Pro Pro Ile Glu Leu Glu Leu Phe Asn Gln 130 135 140 Gly Gly Ser Phe
Ile Phe Ala Thr Ala Ser Met Ala Asn Ile Ala Gln 145 150 155 160 Gly
Ala Ala Cys Leu Ala Val Phe Phe Leu Ala Lys Ser Glu Lys Leu 165 170
175 Lys Gly Leu Ala Gly Ala Ser Gly Val Ser Ala Val Leu Gly Ile Thr
180 185 190 Glu Pro Ala Ile Phe Gly Val Asn Leu Arg Leu Arg Trp Pro
Phe Phe 195 200 205 Ile Gly Ile Gly Thr Ala Ala Ile Gly Gly Ala Leu
Ile Ala Leu Phe 210 215 220 Asn Ile Lys Ala Val Ala Leu Gly Ala Ala
Gly Phe Leu Gly Val Val 225 230 235 240 Ser Ile Asp Ala Pro Asp Met
Val Met Phe Leu Val Cys Ala Val Val 245 250 255 Thr Phe Phe Ile Ala
Phe Gly Ala Ala Ile Ala Tyr Gly Leu Tyr Leu 260 265 270 Val Arg Arg
Asn Gly Ser Ile Asp Pro Asp Ala Thr Ala Ala Pro Val 275 280 285 Pro
Ala Gly Thr Thr Lys Ala Glu Ala Glu Ala Pro Ala Glu Phe Ser 290 295
300 Asn Asp Ser Thr Ile Ile Gln Ala Pro Leu Thr Gly Glu Ala Ile Ala
305 310 315 320 Leu Ser Ser Val Ser Asp Ala Met Phe Ala Ser Gly Lys
Leu Gly Ser 325 330 335 Gly Val Ala Ile Val Pro Thr Lys Gly Gln Leu
Val Ser Pro Val Ser 340 345 350 Gly Lys Ile Val Val Ala Phe Pro Ser
Gly His Ala Phe Ala Val Arg 355 360 365 Thr Lys Ala Glu Asp Gly Ser
Asn Val Asp Ile Leu Met His Ile Gly 370 375 380 Phe Asp Thr Val Asn
Leu Asn Gly Thr His Phe Asn Pro Leu Lys Lys 385 390 395 400 Gln Gly
Asp Glu Val Lys Ala Gly Glu Leu Leu Cys Glu Phe Asp Ile 405 410 415
Asp Ala Ile Lys Ala Ala Gly Tyr Glu Val Thr Thr Pro Ile Val Val 420
425 430 Ser Asn Tyr Lys Lys Thr Gly Pro Val Asn Thr Tyr Gly Leu Gly
Glu 435 440 445 Ile Glu Ala Gly Ala Asn Leu Leu Asn Val Ala Lys Lys
Glu Ala Val 450 455 460 Pro Ala Thr Pro 465 3 1109 DNA
Corynebacterium glutamicum CDS (1)..(1086) FRXA00315 3 tat gat ttc
ggc ggt cca gtc ggc ggt ctg ctc ttc ggt ctg gtc tac 48 Tyr Asp Phe
Gly Gly Pro Val Gly Gly Leu Leu Phe Gly Leu Val Tyr 1 5 10 15 tca
cca atc gtc atc act ggt ctg cac cag tcc ttc ccg cca att gag 96 Ser
Pro Ile Val Ile Thr Gly Leu His Gln Ser Phe Pro Pro Ile Glu 20 25
30 ctg gag ctg ttt aac cag ggt gga tcc ttc atc ttc gca acg gca tct
144 Leu Glu Leu Phe Asn Gln Gly Gly Ser Phe Ile Phe Ala Thr Ala Ser
35 40 45 atg gct aat atc gcc cag ggt gcg gca tgt ttg gca gtg ttc
ttc ctg 192 Met Ala Asn Ile Ala Gln Gly Ala Ala Cys Leu Ala Val Phe
Phe Leu 50 55 60 gcg aag agt gaa aag ctc aag ggc ctt gca ggt gct
tca ggt gtc tcc 240 Ala Lys Ser Glu Lys Leu Lys Gly Leu Ala Gly Ala
Ser Gly Val Ser 65 70 75 80 gct gtt ctt ggt att acg gag cct gcg atc
ttc ggt gtg aac ctt cgc 288 Ala Val Leu Gly Ile Thr Glu Pro Ala Ile
Phe Gly Val Asn Leu Arg 85 90 95 ctg cgc tgg ccg ttc ttc atc ggt
atc ggt acc gca gct atc ggt ggc 336 Leu Arg Trp Pro Phe Phe Ile Gly
Ile Gly Thr Ala Ala Ile Gly Gly 100 105 110 gct ttg att gca ctc ttt
aat atc aag gca gtt gcg ttg ggc gct gca 384 Ala Leu Ile Ala Leu Phe
Asn Ile Lys Ala Val Ala Leu Gly Ala Ala 115 120 125 ggt ttc ttg ggt
gtt gtt tct att gat gct cca gat atg gtc atg ttc 432 Gly Phe Leu Gly
Val Val Ser Ile Asp Ala Pro Asp Met Val Met Phe 130 135 140 ttg gtg
tgt gca gtt gtt acc ttc ttc atc gca ttc ggc gca gcg att 480 Leu Val
Cys Ala Val Val Thr Phe Phe Ile Ala Phe Gly Ala Ala Ile 145 150 155
160 gct tat ggc ctt tac ttg gtt cgc cgc aac ggc agc att gat cca gat
528 Ala Tyr Gly Leu Tyr Leu Val Arg Arg Asn Gly Ser Ile Asp Pro Asp
165 170 175 gca acc gct gct cca gtg cct gca gga acg acc aaa gcc gaa
gca gaa 576 Ala Thr Ala Ala Pro Val Pro Ala Gly Thr Thr Lys Ala Glu
Ala Glu 180 185 190 gca ccc gca gaa ttt tca aac gat tcc acc atc atc
cag gca cct ttg 624 Ala Pro Ala Glu Phe Ser Asn Asp Ser Thr Ile Ile
Gln Ala Pro Leu 195 200 205 acc ggt gaa gct att gca ctg agc agc gtc
agc gat gcc atg ttt gcc 672 Thr Gly Glu Ala Ile Ala Leu Ser Ser Val
Ser Asp Ala Met Phe Ala 210 215 220 agc gga aag ctt ggc tcg ggc gtt
gcc atc gtc cca acc aag ggg cag 720 Ser Gly Lys Leu Gly Ser Gly Val
Ala Ile Val Pro Thr Lys Gly Gln 225 230 235 240 tta gtt tct ccg gtg
agt gga aag att gtg gtg gca ttc cca tct ggc 768 Leu Val Ser Pro Val
Ser Gly Lys Ile Val Val Ala Phe Pro Ser Gly 245 250 255 cat gct ttc
gca gtt cgc acc aag gct gag gat ggt tcc aat gtg gat 816 His Ala Phe
Ala Val Arg Thr Lys Ala Glu Asp Gly Ser Asn Val Asp 260 265 270 atc
ttg atg cac att ggt ttc gac aca gta aac ctc aac ggc acg cac 864 Ile
Leu Met His Ile Gly Phe Asp Thr Val Asn Leu Asn Gly Thr His 275 280
285 ttt aac ccg ctg aag aag cag ggc gat gaa gtc aaa gca ggg gag ctg
912 Phe Asn Pro Leu Lys Lys Gln Gly Asp Glu Val Lys Ala Gly Glu Leu
290 295 300 ctg tgt gaa ttc gat att gat gcc att aag gct gca ggt tat
gag gta 960 Leu Cys Glu Phe Asp Ile Asp Ala Ile Lys Ala Ala Gly Tyr
Glu Val 305 310 315 320 acc acg ccg att gtt gtt tcg aat tac aag aaa
acc gga cct gta aac 1008 Thr Thr Pro Ile Val Val Ser Asn Tyr Lys
Lys Thr Gly Pro Val Asn 325 330 335 act tac ggt ttg ggc gaa att gaa
gcg gga gcc aac ctg ctc aac gtc 1056 Thr Tyr Gly Leu Gly Glu Ile
Glu Ala Gly Ala Asn Leu Leu Asn Val 340 345 350 gca aag aaa gaa gcg
gtg cca gca aca cca taagttgaaa ccttgagtgt 1106 Ala Lys Lys Glu Ala
Val Pro Ala Thr Pro 355 360 tcg 1109 4 362 PRT Corynebacterium
glutamicum 4 Tyr Asp Phe Gly Gly Pro Val Gly Gly Leu Leu Phe Gly
Leu Val Tyr 1 5 10 15 Ser Pro Ile Val Ile Thr Gly Leu His Gln Ser
Phe Pro Pro Ile Glu 20 25 30 Leu Glu Leu Phe Asn Gln Gly Gly Ser
Phe Ile Phe Ala Thr Ala Ser 35 40 45 Met Ala Asn Ile Ala Gln Gly
Ala Ala Cys Leu Ala Val Phe Phe Leu 50 55 60 Ala Lys Ser Glu Lys
Leu Lys Gly Leu Ala Gly Ala Ser Gly Val Ser 65 70 75 80 Ala Val Leu
Gly Ile Thr Glu Pro Ala Ile Phe Gly Val Asn Leu Arg 85 90 95 Leu
Arg Trp Pro Phe Phe Ile Gly Ile Gly Thr Ala Ala Ile Gly Gly 100 105
110 Ala Leu Ile Ala Leu Phe Asn Ile Lys Ala Val Ala Leu Gly Ala Ala
115 120 125 Gly Phe Leu Gly Val Val Ser Ile Asp Ala Pro Asp Met Val
Met Phe 130 135 140 Leu Val Cys Ala Val Val Thr Phe Phe Ile Ala Phe
Gly Ala Ala Ile 145 150 155 160 Ala Tyr Gly Leu Tyr Leu Val Arg Arg
Asn Gly Ser Ile Asp Pro Asp 165 170 175 Ala Thr Ala Ala Pro Val Pro
Ala Gly Thr Thr Lys Ala Glu Ala Glu 180 185 190 Ala Pro Ala Glu Phe
Ser Asn Asp Ser Thr Ile Ile Gln Ala Pro Leu 195 200 205 Thr Gly Glu
Ala Ile Ala Leu Ser Ser Val Ser Asp Ala Met Phe Ala 210 215 220 Ser
Gly Lys Leu Gly Ser Gly Val Ala Ile Val Pro Thr Lys Gly Gln 225 230
235 240 Leu Val Ser Pro Val Ser Gly Lys Ile Val Val Ala Phe Pro Ser
Gly 245 250 255 His Ala Phe Ala Val Arg Thr Lys Ala Glu Asp Gly Ser
Asn Val Asp 260 265 270 Ile Leu Met His Ile Gly Phe Asp Thr Val Asn
Leu Asn Gly Thr His 275 280 285 Phe Asn Pro Leu Lys Lys Gln Gly Asp
Glu Val Lys Ala Gly Glu Leu 290 295 300 Leu Cys Glu Phe Asp Ile Asp
Ala Ile Lys Ala Ala Gly Tyr Glu Val 305 310 315 320 Thr Thr Pro Ile
Val Val Ser Asn Tyr Lys Lys Thr Gly Pro Val Asn 325 330 335 Thr Tyr
Gly Leu Gly Glu Ile Glu Ala Gly Ala Asn Leu Leu Asn Val 340 345 350
Ala Lys Lys Glu Ala Val Pro Ala Thr Pro 355 360 5 372 DNA
Corynebacterium glutamicum CDS (101)..(349) RXA01503 5 gtatcctcaa
aggccttcta gctgttgcag ctgcagcgca ctcggtggat acgacatcca 60
cgacctatca aattctttat gctgcaggcg atgccttttc atg ttc ttg gca gtc 115
Met Phe Leu Ala Val 1 5 att ttg gcg att act gcg gct cgt aaa ttc ggt
gcc aat gtc ttt aca 163 Ile Leu Ala Ile Thr Ala Ala Arg Lys Phe Gly
Ala Asn Val Phe Thr 10 15 20 tca gtc gca ctc gct ggt gca ttg ctg
cac aca cag ctt cag gca gta 211 Ser Val Ala Leu Ala Gly Ala Leu Leu
His Thr Gln Leu Gln Ala Val 25 30 35 acc gtg ttg gtt gac ggt gaa
ctc cag tcg atg act ctg gtg gct ttc 259 Thr Val Leu Val Asp Gly Glu
Leu Gln Ser Met Thr Leu Val Ala Phe 40 45 50 caa aag gct ggt aat
gac gtc acc ttc ctg ggc att cca gtg gtg ctg 307 Gln Lys Ala Gly Asn
Asp Val Thr Phe Leu Gly Ile Pro Val Val Leu 55 60 65 cag ttg gcg
ttg cat gta gcg agt ttg atg aag ttg tcg cga 349 Gln Leu Ala Leu His
Val Ala Ser Leu Met Lys Leu Ser Arg 70 75 80 taagaggagg ggcgtgtcgg
tct 372 6 83 PRT Corynebacterium glutamicum 6 Met Phe Leu Ala Val
Ile Leu Ala Ile Thr Ala Ala Arg Lys Phe Gly 1 5 10 15 Ala Asn Val
Phe Thr Ser
Val Ala Leu Ala Gly Ala Leu Leu His Thr 20 25 30 Gln Leu Gln Ala
Val Thr Val Leu Val Asp Gly Glu Leu Gln Ser Met 35 40 45 Thr Leu
Val Ala Phe Gln Lys Ala Gly Asn Asp Val Thr Phe Leu Gly 50 55 60
Ile Pro Val Val Leu Gln Leu Ala Leu His Val Ala Ser Leu Met Lys 65
70 75 80 Leu Ser Arg 7 2187 DNA Corynebacterium glutamicum CDS
(101)..(2164) RXN01299 7 cgactgcggc gtctcttcct ggcactacca
ttcctcgtcc tgaccaactc gccacagctg 60 gtgcaacggt cacccaagtc
aaaggattga aagaatcagc atg aat agc gta aat 115 Met Asn Ser Val Asn 1
5 aat tcc tcg ctt gtc cgg ctg gat gtc gat ttc ggc gac tcc acc acg
163 Asn Ser Ser Leu Val Arg Leu Asp Val Asp Phe Gly Asp Ser Thr Thr
10 15 20 gat gtc atc aac aac ctt gcc act gtt att ttc gac gct ggc
cga gct 211 Asp Val Ile Asn Asn Leu Ala Thr Val Ile Phe Asp Ala Gly
Arg Ala 25 30 35 tcc tcc gcc gac gcc ctt gcc aaa gac gcg ctg gat
cgt gaa gca aag 259 Ser Ser Ala Asp Ala Leu Ala Lys Asp Ala Leu Asp
Arg Glu Ala Lys 40 45 50 tcc ggc acc ggc gtt cct ggt caa gtt gct
atc ccc cac tgc cgt tcc 307 Ser Gly Thr Gly Val Pro Gly Gln Val Ala
Ile Pro His Cys Arg Ser 55 60 65 gaa gcc gta tct gtc cct acc ttg
ggc ttt gct cgc ctg agc aag ggt 355 Glu Ala Val Ser Val Pro Thr Leu
Gly Phe Ala Arg Leu Ser Lys Gly 70 75 80 85 gtg gac ttc agc gga cct
gat ggc gat gcc aac ttg gtg ttc ctc att 403 Val Asp Phe Ser Gly Pro
Asp Gly Asp Ala Asn Leu Val Phe Leu Ile 90 95 100 gca gca cct gct
ggc ggc ggc aaa gag cac ctg aag atc ctg tcc aag 451 Ala Ala Pro Ala
Gly Gly Gly Lys Glu His Leu Lys Ile Leu Ser Lys 105 110 115 ctt gct
cgc tcc ttg gtg aag aag gat ttc atc aag gct ctg cag gaa 499 Leu Ala
Arg Ser Leu Val Lys Lys Asp Phe Ile Lys Ala Leu Gln Glu 120 125 130
gcc acc acc gag cag gaa atc gtc gac gtt gtc gat gcc gtg ctc aac 547
Ala Thr Thr Glu Gln Glu Ile Val Asp Val Val Asp Ala Val Leu Asn 135
140 145 cca gca cca aaa acc acc gag cca gct gca gct ccg gct gcg gcg
gcg 595 Pro Ala Pro Lys Thr Thr Glu Pro Ala Ala Ala Pro Ala Ala Ala
Ala 150 155 160 165 gtt gct gag agt ggg gcg gcg tcg aca agc gtt act
cgt atc gtg gca 643 Val Ala Glu Ser Gly Ala Ala Ser Thr Ser Val Thr
Arg Ile Val Ala 170 175 180 atc acc gca tgc cca acc ggt atc gca cac
acc tac atg gct gcg gat 691 Ile Thr Ala Cys Pro Thr Gly Ile Ala His
Thr Tyr Met Ala Ala Asp 185 190 195 tcc ctg acg caa aac gcg gaa ggc
cgc gat gat gtg gaa ctc gtt gtg 739 Ser Leu Thr Gln Asn Ala Glu Gly
Arg Asp Asp Val Glu Leu Val Val 200 205 210 gag act cag ggc tct tcc
gct gtc acc cca gtc gat ccg aag atc atc 787 Glu Thr Gln Gly Ser Ser
Ala Val Thr Pro Val Asp Pro Lys Ile Ile 215 220 225 gaa gct gcc gac
gcc gtc atc ttc gcc acc gac gtg gga gtt aaa gac 835 Glu Ala Ala Asp
Ala Val Ile Phe Ala Thr Asp Val Gly Val Lys Asp 230 235 240 245 cgc
gag cgt ttc gct ggc aag cca gtc att gaa tcc ggc gtc aag cgc 883 Arg
Glu Arg Phe Ala Gly Lys Pro Val Ile Glu Ser Gly Val Lys Arg 250 255
260 gcg atc aat gag cca gcc aag atg atc gac gag gcc atc gca gcc tcc
931 Ala Ile Asn Glu Pro Ala Lys Met Ile Asp Glu Ala Ile Ala Ala Ser
265 270 275 aag aac cca aac gcc cgc aag gtt tcc ggt tcc ggt gtc gcg
gca tct 979 Lys Asn Pro Asn Ala Arg Lys Val Ser Gly Ser Gly Val Ala
Ala Ser 280 285 290 gct gaa acc acc ggc gag aag ctc ggc tgg ggc aag
cgc atc cag cag 1027 Ala Glu Thr Thr Gly Glu Lys Leu Gly Trp Gly
Lys Arg Ile Gln Gln 295 300 305 gca gtc atg acc ggc gtg tcc tac atg
gtt cca ttc gta gct gcc ggc 1075 Ala Val Met Thr Gly Val Ser Tyr
Met Val Pro Phe Val Ala Ala Gly 310 315 320 325 ggc ctc ctg ttg gct
ctc ggc ttc gca ttc ggt gga tac gac atg gcg 1123 Gly Leu Leu Leu
Ala Leu Gly Phe Ala Phe Gly Gly Tyr Asp Met Ala 330 335 340 aac ggc
tgg caa gca atc gcc acc cag ttc tct ctg acc aac ctg cca 1171 Asn
Gly Trp Gln Ala Ile Ala Thr Gln Phe Ser Leu Thr Asn Leu Pro 345 350
355 ggc aac acc gtc gat gtt gac ggc gtg gcc atg acc ttc gag cgt tca
1219 Gly Asn Thr Val Asp Val Asp Gly Val Ala Met Thr Phe Glu Arg
Ser 360 365 370 ggc ttc ctg ttg tac ttc ggc gca gtc ctg ttc gcc acc
ggc caa gca 1267 Gly Phe Leu Leu Tyr Phe Gly Ala Val Leu Phe Ala
Thr Gly Gln Ala 375 380 385 gcc atg ggc ttc atc gtg gca gcc ctg tct
ggc tac acc gca tac gca 1315 Ala Met Gly Phe Ile Val Ala Ala Leu
Ser Gly Tyr Thr Ala Tyr Ala 390 395 400 405 ctt gct gga cgc cca ggc
atc gcg ccg ggc ttc gtc ggt ggc gcc atc 1363 Leu Ala Gly Arg Pro
Gly Ile Ala Pro Gly Phe Val Gly Gly Ala Ile 410 415 420 tcc gtc acc
atc ggc gct ggc ttc att ggt ggt ctg gtt acc ggt atc 1411 Ser Val
Thr Ile Gly Ala Gly Phe Ile Gly Gly Leu Val Thr Gly Ile 425 430 435
ttg gct ggt ctc att gcc ctg tgg att ggc tcc tgg aag gtg cca cgc
1459 Leu Ala Gly Leu Ile Ala Leu Trp Ile Gly Ser Trp Lys Val Pro
Arg 440 445 450 gtg gtg cag tca ctg atg cct gtg gtc atc atc ccg cta
ctt acc tca 1507 Val Val Gln Ser Leu Met Pro Val Val Ile Ile Pro
Leu Leu Thr Ser 455 460 465 gtg gtt gtt ggt ctc gtc atg tac ctc ctg
ctg ggt cgc cca ctc gca 1555 Val Val Val Gly Leu Val Met Tyr Leu
Leu Leu Gly Arg Pro Leu Ala 470 475 480 485 tcc atc atg act ggt ttg
cag gac tgg cta tcg tca atg tcc gga agc 1603 Ser Ile Met Thr Gly
Leu Gln Asp Trp Leu Ser Ser Met Ser Gly Ser 490 495 500 tcc gcc atc
ttg ctg ggt atc atc ttg ggc ctc atg atg tgt ttc gac 1651 Ser Ala
Ile Leu Leu Gly Ile Ile Leu Gly Leu Met Met Cys Phe Asp 505 510 515
ctc ggc gga cca gta aac aag gca gcc tac ctc ttt ggt acc gca ggc
1699 Leu Gly Gly Pro Val Asn Lys Ala Ala Tyr Leu Phe Gly Thr Ala
Gly 520 525 530 ctg tct acc ggc gac caa gct tcc atg gaa atc atg gcc
gcg atc atg 1747 Leu Ser Thr Gly Asp Gln Ala Ser Met Glu Ile Met
Ala Ala Ile Met 535 540 545 gca gct ggc atg gtc cca cca atc gcg ttg
tcc att gct acc ctg ctg 1795 Ala Ala Gly Met Val Pro Pro Ile Ala
Leu Ser Ile Ala Thr Leu Leu 550 555 560 565 cgc aag aag ctg ttc acc
cca gca gag caa gaa aac ggc aag tct tcc 1843 Arg Lys Lys Leu Phe
Thr Pro Ala Glu Gln Glu Asn Gly Lys Ser Ser 570 575 580 tgg ctg ctt
ggc ctg gca ttc gtc tcc gaa ggt gcc atc cca ttc gcc 1891 Trp Leu
Leu Gly Leu Ala Phe Val Ser Glu Gly Ala Ile Pro Phe Ala 585 590 595
gca gct gac cca ttc cgt gtg atc cca gca atg atg gct ggc ggt gca
1939 Ala Ala Asp Pro Phe Arg Val Ile Pro Ala Met Met Ala Gly Gly
Ala 600 605 610 acc act ggt gca atc tcc atg gca ctg ggc gtc ggc tct
cgg gct cca 1987 Thr Thr Gly Ala Ile Ser Met Ala Leu Gly Val Gly
Ser Arg Ala Pro 615 620 625 cac ggc ggt atc ttc gtg gtc tgg gca atc
gaa cca tgg tgg ggc tgg 2035 His Gly Gly Ile Phe Val Val Trp Ala
Ile Glu Pro Trp Trp Gly Trp 630 635 640 645 ctc atc gca ctt gca gca
ggc acc atc gtg tcc acc atc gtt gtc atc 2083 Leu Ile Ala Leu Ala
Ala Gly Thr Ile Val Ser Thr Ile Val Val Ile 650 655 660 gca ctg aag
cag ttc tgg cca aac aag gcc gtc gct gca gaa gtc gcg 2131 Ala Leu
Lys Gln Phe Trp Pro Asn Lys Ala Val Ala Ala Glu Val Ala 665 670 675
aag caa gaa gca caa caa gca gct gta aac gca taatcggacc ttgacccgat
2184 Lys Gln Glu Ala Gln Gln Ala Ala Val Asn Ala 680 685 gtc 2187 8
688 PRT Corynebacterium glutamicum 8 Met Asn Ser Val Asn Asn Ser
Ser Leu Val Arg Leu Asp Val Asp Phe 1 5 10 15 Gly Asp Ser Thr Thr
Asp Val Ile Asn Asn Leu Ala Thr Val Ile Phe 20 25 30 Asp Ala Gly
Arg Ala Ser Ser Ala Asp Ala Leu Ala Lys Asp Ala Leu 35 40 45 Asp
Arg Glu Ala Lys Ser Gly Thr Gly Val Pro Gly Gln Val Ala Ile 50 55
60 Pro His Cys Arg Ser Glu Ala Val Ser Val Pro Thr Leu Gly Phe Ala
65 70 75 80 Arg Leu Ser Lys Gly Val Asp Phe Ser Gly Pro Asp Gly Asp
Ala Asn 85 90 95 Leu Val Phe Leu Ile Ala Ala Pro Ala Gly Gly Gly
Lys Glu His Leu 100 105 110 Lys Ile Leu Ser Lys Leu Ala Arg Ser Leu
Val Lys Lys Asp Phe Ile 115 120 125 Lys Ala Leu Gln Glu Ala Thr Thr
Glu Gln Glu Ile Val Asp Val Val 130 135 140 Asp Ala Val Leu Asn Pro
Ala Pro Lys Thr Thr Glu Pro Ala Ala Ala 145 150 155 160 Pro Ala Ala
Ala Ala Val Ala Glu Ser Gly Ala Ala Ser Thr Ser Val 165 170 175 Thr
Arg Ile Val Ala Ile Thr Ala Cys Pro Thr Gly Ile Ala His Thr 180 185
190 Tyr Met Ala Ala Asp Ser Leu Thr Gln Asn Ala Glu Gly Arg Asp Asp
195 200 205 Val Glu Leu Val Val Glu Thr Gln Gly Ser Ser Ala Val Thr
Pro Val 210 215 220 Asp Pro Lys Ile Ile Glu Ala Ala Asp Ala Val Ile
Phe Ala Thr Asp 225 230 235 240 Val Gly Val Lys Asp Arg Glu Arg Phe
Ala Gly Lys Pro Val Ile Glu 245 250 255 Ser Gly Val Lys Arg Ala Ile
Asn Glu Pro Ala Lys Met Ile Asp Glu 260 265 270 Ala Ile Ala Ala Ser
Lys Asn Pro Asn Ala Arg Lys Val Ser Gly Ser 275 280 285 Gly Val Ala
Ala Ser Ala Glu Thr Thr Gly Glu Lys Leu Gly Trp Gly 290 295 300 Lys
Arg Ile Gln Gln Ala Val Met Thr Gly Val Ser Tyr Met Val Pro 305 310
315 320 Phe Val Ala Ala Gly Gly Leu Leu Leu Ala Leu Gly Phe Ala Phe
Gly 325 330 335 Gly Tyr Asp Met Ala Asn Gly Trp Gln Ala Ile Ala Thr
Gln Phe Ser 340 345 350 Leu Thr Asn Leu Pro Gly Asn Thr Val Asp Val
Asp Gly Val Ala Met 355 360 365 Thr Phe Glu Arg Ser Gly Phe Leu Leu
Tyr Phe Gly Ala Val Leu Phe 370 375 380 Ala Thr Gly Gln Ala Ala Met
Gly Phe Ile Val Ala Ala Leu Ser Gly 385 390 395 400 Tyr Thr Ala Tyr
Ala Leu Ala Gly Arg Pro Gly Ile Ala Pro Gly Phe 405 410 415 Val Gly
Gly Ala Ile Ser Val Thr Ile Gly Ala Gly Phe Ile Gly Gly 420 425 430
Leu Val Thr Gly Ile Leu Ala Gly Leu Ile Ala Leu Trp Ile Gly Ser 435
440 445 Trp Lys Val Pro Arg Val Val Gln Ser Leu Met Pro Val Val Ile
Ile 450 455 460 Pro Leu Leu Thr Ser Val Val Val Gly Leu Val Met Tyr
Leu Leu Leu 465 470 475 480 Gly Arg Pro Leu Ala Ser Ile Met Thr Gly
Leu Gln Asp Trp Leu Ser 485 490 495 Ser Met Ser Gly Ser Ser Ala Ile
Leu Leu Gly Ile Ile Leu Gly Leu 500 505 510 Met Met Cys Phe Asp Leu
Gly Gly Pro Val Asn Lys Ala Ala Tyr Leu 515 520 525 Phe Gly Thr Ala
Gly Leu Ser Thr Gly Asp Gln Ala Ser Met Glu Ile 530 535 540 Met Ala
Ala Ile Met Ala Ala Gly Met Val Pro Pro Ile Ala Leu Ser 545 550 555
560 Ile Ala Thr Leu Leu Arg Lys Lys Leu Phe Thr Pro Ala Glu Gln Glu
565 570 575 Asn Gly Lys Ser Ser Trp Leu Leu Gly Leu Ala Phe Val Ser
Glu Gly 580 585 590 Ala Ile Pro Phe Ala Ala Ala Asp Pro Phe Arg Val
Ile Pro Ala Met 595 600 605 Met Ala Gly Gly Ala Thr Thr Gly Ala Ile
Ser Met Ala Leu Gly Val 610 615 620 Gly Ser Arg Ala Pro His Gly Gly
Ile Phe Val Val Trp Ala Ile Glu 625 630 635 640 Pro Trp Trp Gly Trp
Leu Ile Ala Leu Ala Ala Gly Thr Ile Val Ser 645 650 655 Thr Ile Val
Val Ile Ala Leu Lys Gln Phe Trp Pro Asn Lys Ala Val 660 665 670 Ala
Ala Glu Val Ala Lys Gln Glu Ala Gln Gln Ala Ala Val Asn Ala 675 680
685 9 464 DNA Corynebacterium glutamicum CDS (1)..(441) FRXA01299 9
atg gaa atc atg gcc gcg atc atg gca gct ggc atg gtc cca cca atc 48
Met Glu Ile Met Ala Ala Ile Met Ala Ala Gly Met Val Pro Pro Ile 1 5
10 15 gcg ttg tcc att gct acc ctg ctg cgc aag aag ctg ttc acc cca
gca 96 Ala Leu Ser Ile Ala Thr Leu Leu Arg Lys Lys Leu Phe Thr Pro
Ala 20 25 30 gag caa gaa aac ggc aag tct tcc tgg ctg ctt ggc ctg
gca ttc gtc 144 Glu Gln Glu Asn Gly Lys Ser Ser Trp Leu Leu Gly Leu
Ala Phe Val 35 40 45 tcc gaa ggt gcc atc cca ttc gcc gca gct gac
cca ttc cgt gtg atc 192 Ser Glu Gly Ala Ile Pro Phe Ala Ala Ala Asp
Pro Phe Arg Val Ile 50 55 60 cca gca atg atg gct ggc ggt gca acc
act ggt gca atc tcc atg gca 240 Pro Ala Met Met Ala Gly Gly Ala Thr
Thr Gly Ala Ile Ser Met Ala 65 70 75 80 ctg ggc gtc ggc tct cgg gct
cca cac ggc ggt atc ttc gtg gtc tgg 288 Leu Gly Val Gly Ser Arg Ala
Pro His Gly Gly Ile Phe Val Val Trp 85 90 95 gca atc gaa cca tgg
tgg ggc tgg ctc atc gca ctt gca gca ggc acc 336 Ala Ile Glu Pro Trp
Trp Gly Trp Leu Ile Ala Leu Ala Ala Gly Thr 100 105 110 atc gtg tcc
acc atc gtt gtc atc gca ctg aag cag ttc tgg cca aac 384 Ile Val Ser
Thr Ile Val Val Ile Ala Leu Lys Gln Phe Trp Pro Asn 115 120 125 aag
gcc gtc gct gca gaa gtc gcg aag caa gaa gca caa caa gca gct 432 Lys
Ala Val Ala Ala Glu Val Ala Lys Gln Glu Ala Gln Gln Ala Ala 130 135
140 gta aac gca taatcggacc ttgacccgat gtc 464 Val Asn Ala 145 10
147 PRT Corynebacterium glutamicum 10 Met Glu Ile Met Ala Ala Ile
Met Ala Ala Gly Met Val Pro Pro Ile 1 5 10 15 Ala Leu Ser Ile Ala
Thr Leu Leu Arg Lys Lys Leu Phe Thr Pro Ala 20 25 30 Glu Gln Glu
Asn Gly Lys Ser Ser Trp Leu Leu Gly Leu Ala Phe Val 35 40 45 Ser
Glu Gly Ala Ile Pro Phe Ala Ala Ala Asp Pro Phe Arg Val Ile 50 55
60 Pro Ala Met Met Ala Gly Gly Ala Thr Thr Gly Ala Ile Ser Met Ala
65 70 75 80 Leu Gly Val Gly Ser Arg Ala Pro His Gly Gly Ile Phe Val
Val Trp 85 90 95 Ala Ile Glu Pro Trp Trp Gly Trp Leu Ile Ala Leu
Ala Ala Gly Thr 100 105 110 Ile Val Ser Thr Ile Val Val Ile Ala Leu
Lys Gln Phe Trp Pro Asn 115 120 125 Lys Ala Val Ala Ala Glu Val Ala
Lys Gln Glu Ala Gln Gln Ala Ala 130 135 140 Val Asn Ala 145 11 580
DNA Corynebacterium glutamicum CDS (101)..(580) FRXA01883 11
cgactgcggc gtctcttcct ggcactacca ttcctcgtcc tgaccaactc gccacagctg
60 gtgcaacggt cacccaagtc aaaggattga aagaatcagc atg aat agc gta aat
115 Met Asn Ser Val Asn 1 5 aat tcc tcg ctt gtc cgg ctg gat gtc gat
ttc ggc gac tcc acc acg 163 Asn Ser Ser Leu Val Arg Leu Asp Val Asp
Phe Gly Asp Ser Thr Thr 10 15 20 gat gtc atc aac aac ctt gcc act
gtt att ttc gac gct ggc cga gct 211 Asp Val Ile Asn Asn Leu Ala Thr
Val Ile Phe Asp Ala Gly Arg Ala 25 30 35 tcc tcc gcc gac gcc ctt
gcc aaa gac gcg ctg gat cgt gaa gca aag 259 Ser Ser Ala Asp Ala Leu
Ala Lys Asp Ala Leu Asp Arg Glu Ala Lys 40 45 50 tcc ggc acc ggc
gtt cct ggt caa gtt gct atc ccc
cac tgc cgt tcc 307 Ser Gly Thr Gly Val Pro Gly Gln Val Ala Ile Pro
His Cys Arg Ser 55 60 65 gaa gcc gta tct gtc cct acc ttg ggc ttt
gct cgc ctg agc aag ggt 355 Glu Ala Val Ser Val Pro Thr Leu Gly Phe
Ala Arg Leu Ser Lys Gly 70 75 80 85 gtg gac ttc agc gga cct gat ggc
gat gcc aac ttg gtg ttc ctc att 403 Val Asp Phe Ser Gly Pro Asp Gly
Asp Ala Asn Leu Val Phe Leu Ile 90 95 100 gca gca cct gct ggc ggc
ggc aaa gag cac ctg aag atc ctg tcc aag 451 Ala Ala Pro Ala Gly Gly
Gly Lys Glu His Leu Lys Ile Leu Ser Lys 105 110 115 ctt gct cgc tcc
ttg gtg aag aag gat ttc atc aag gct ctg cag gaa 499 Leu Ala Arg Ser
Leu Val Lys Lys Asp Phe Ile Lys Ala Leu Gln Glu 120 125 130 gcc acc
acc gag cag gaa atc gtc gac gtt gtc gat gcc gtg ctc aac 547 Ala Thr
Thr Glu Gln Glu Ile Val Asp Val Val Asp Ala Val Leu Asn 135 140 145
cca gca cca aaa aac cac cga gcc agc tgc agc 580 Pro Ala Pro Lys Asn
His Arg Ala Ser Cys Ser 150 155 160 12 160 PRT Corynebacterium
glutamicum 12 Met Asn Ser Val Asn Asn Ser Ser Leu Val Arg Leu Asp
Val Asp Phe 1 5 10 15 Gly Asp Ser Thr Thr Asp Val Ile Asn Asn Leu
Ala Thr Val Ile Phe 20 25 30 Asp Ala Gly Arg Ala Ser Ser Ala Asp
Ala Leu Ala Lys Asp Ala Leu 35 40 45 Asp Arg Glu Ala Lys Ser Gly
Thr Gly Val Pro Gly Gln Val Ala Ile 50 55 60 Pro His Cys Arg Ser
Glu Ala Val Ser Val Pro Thr Leu Gly Phe Ala 65 70 75 80 Arg Leu Ser
Lys Gly Val Asp Phe Ser Gly Pro Asp Gly Asp Ala Asn 85 90 95 Leu
Val Phe Leu Ile Ala Ala Pro Ala Gly Gly Gly Lys Glu His Leu 100 105
110 Lys Ile Leu Ser Lys Leu Ala Arg Ser Leu Val Lys Lys Asp Phe Ile
115 120 125 Lys Ala Leu Gln Glu Ala Thr Thr Glu Gln Glu Ile Val Asp
Val Val 130 135 140 Asp Ala Val Leu Asn Pro Ala Pro Lys Asn His Arg
Ala Ser Cys Ser 145 150 155 160 13 631 DNA Corynebacterium
glutamicum CDS (77)..(631) FRXA01889 13 accgagccag ctgcagctcc
ggctgcggcg gccggttgtt aagagtgggg cggcgtcgac 60 aagcgttact cgtatc
gtg gca atc acc gca tgc cca acc ggt atc gca cac 112 Val Ala Ile Thr
Ala Cys Pro Thr Gly Ile Ala His 1 5 10 acc tac atg gct gcg gat tcc
ctg acg caa aac gcg gaa ggc cgc gat 160 Thr Tyr Met Ala Ala Asp Ser
Leu Thr Gln Asn Ala Glu Gly Arg Asp 15 20 25 gat gtg gaa ctc gtt
gtg gag act cag ggc tct tcc gct gtc acc cca 208 Asp Val Glu Leu Val
Val Glu Thr Gln Gly Ser Ser Ala Val Thr Pro 30 35 40 gtc gat ccg
aag atc atc gaa gct gcc gac gcc gtc atc ttc gcc acc 256 Val Asp Pro
Lys Ile Ile Glu Ala Ala Asp Ala Val Ile Phe Ala Thr 45 50 55 60 gac
gtg gga gtt aaa gac cgc gag cgt ttc gct ggc aag cca gtc att 304 Asp
Val Gly Val Lys Asp Arg Glu Arg Phe Ala Gly Lys Pro Val Ile 65 70
75 gaa tcc ggc gtc aag cgc gcg atc aat gag cca gcc aag atg atc gac
352 Glu Ser Gly Val Lys Arg Ala Ile Asn Glu Pro Ala Lys Met Ile Asp
80 85 90 gag gcc atc gca gcc tcc aag aac cca aac gcc cgc aag gtt
tcc ggt 400 Glu Ala Ile Ala Ala Ser Lys Asn Pro Asn Ala Arg Lys Val
Ser Gly 95 100 105 tcc ggt gtc gcg gca tct gct gaa acc acc ggc gag
aag ctc ggc tgg 448 Ser Gly Val Ala Ala Ser Ala Glu Thr Thr Gly Glu
Lys Leu Gly Trp 110 115 120 ggc aag cgc atc cag cag gca gtc atg acc
ggc gtg tcc tac atg gtt 496 Gly Lys Arg Ile Gln Gln Ala Val Met Thr
Gly Val Ser Tyr Met Val 125 130 135 140 cca ttc gta gct gcc ggc ggc
ctc ctg ttg gct ctc ggc ttc gca ttc 544 Pro Phe Val Ala Ala Gly Gly
Leu Leu Leu Ala Leu Gly Phe Ala Phe 145 150 155 ggt gga tac gac atg
gcg aac ggc tgg caa gca atc gcc acc cag ttc 592 Gly Gly Tyr Asp Met
Ala Asn Gly Trp Gln Ala Ile Ala Thr Gln Phe 160 165 170 tct ctg acc
aac ctg cca ggc aac acc gtc gat gtt gac 631 Ser Leu Thr Asn Leu Pro
Gly Asn Thr Val Asp Val Asp 175 180 185 14 185 PRT Corynebacterium
glutamicum 14 Val Ala Ile Thr Ala Cys Pro Thr Gly Ile Ala His Thr
Tyr Met Ala 1 5 10 15 Ala Asp Ser Leu Thr Gln Asn Ala Glu Gly Arg
Asp Asp Val Glu Leu 20 25 30 Val Val Glu Thr Gln Gly Ser Ser Ala
Val Thr Pro Val Asp Pro Lys 35 40 45 Ile Ile Glu Ala Ala Asp Ala
Val Ile Phe Ala Thr Asp Val Gly Val 50 55 60 Lys Asp Arg Glu Arg
Phe Ala Gly Lys Pro Val Ile Glu Ser Gly Val 65 70 75 80 Lys Arg Ala
Ile Asn Glu Pro Ala Lys Met Ile Asp Glu Ala Ile Ala 85 90 95 Ala
Ser Lys Asn Pro Asn Ala Arg Lys Val Ser Gly Ser Gly Val Ala 100 105
110 Ala Ser Ala Glu Thr Thr Gly Glu Lys Leu Gly Trp Gly Lys Arg Ile
115 120 125 Gln Gln Ala Val Met Thr Gly Val Ser Tyr Met Val Pro Phe
Val Ala 130 135 140 Ala Gly Gly Leu Leu Leu Ala Leu Gly Phe Ala Phe
Gly Gly Tyr Asp 145 150 155 160 Met Ala Asn Gly Trp Gln Ala Ile Ala
Thr Gln Phe Ser Leu Thr Asn 165 170 175 Leu Pro Gly Asn Thr Val Asp
Val Asp 180 185 15 416 DNA Corynebacterium glutamicum CDS
(1)..(393) RXA00951 15 atc caa gca atc tta gag aag gca gca gcg ccg
gcg aag cag aag gct 48 Ile Gln Ala Ile Leu Glu Lys Ala Ala Ala Pro
Ala Lys Gln Lys Ala 1 5 10 15 cct gct gtg gct cct gct gta aca ccc
act gac gct cct gca gcc tca 96 Pro Ala Val Ala Pro Ala Val Thr Pro
Thr Asp Ala Pro Ala Ala Ser 20 25 30 gtc caa tcc aaa acc cac gac
aag atc ctc acc gtc tgt ggc aac ggc 144 Val Gln Ser Lys Thr His Asp
Lys Ile Leu Thr Val Cys Gly Asn Gly 35 40 45 ttg ggt acc tcc ctc
ttc ctc aaa aac acc ctt gag caa gtt ttc gac 192 Leu Gly Thr Ser Leu
Phe Leu Lys Asn Thr Leu Glu Gln Val Phe Asp 50 55 60 acc tgg ggt
tgg ggt cca tac atg acg gtg gag gca acc gac act atc 240 Thr Trp Gly
Trp Gly Pro Tyr Met Thr Val Glu Ala Thr Asp Thr Ile 65 70 75 80 tcc
gcc aag ggc aaa gcc aag gaa gct gat ctc atc atg acc tct ggt 288 Ser
Ala Lys Gly Lys Ala Lys Glu Ala Asp Leu Ile Met Thr Ser Gly 85 90
95 gaa atc gcc cgc acg ttg ggt gat gtt gga atc ccg gtt cac gtg atc
336 Glu Ile Ala Arg Thr Leu Gly Asp Val Gly Ile Pro Val His Val Ile
100 105 110 aat gac ttc acg agc acc gat gaa atc gat gct gcg ctt cgt
gaa cgc 384 Asn Asp Phe Thr Ser Thr Asp Glu Ile Asp Ala Ala Leu Arg
Glu Arg 115 120 125 tac gac atc taactacttt aaaaggacga aaa 416 Tyr
Asp Ile 130 16 131 PRT Corynebacterium glutamicum 16 Ile Gln Ala
Ile Leu Glu Lys Ala Ala Ala Pro Ala Lys Gln Lys Ala 1 5 10 15 Pro
Ala Val Ala Pro Ala Val Thr Pro Thr Asp Ala Pro Ala Ala Ser 20 25
30 Val Gln Ser Lys Thr His Asp Lys Ile Leu Thr Val Cys Gly Asn Gly
35 40 45 Leu Gly Thr Ser Leu Phe Leu Lys Asn Thr Leu Glu Gln Val
Phe Asp 50 55 60 Thr Trp Gly Trp Gly Pro Tyr Met Thr Val Glu Ala
Thr Asp Thr Ile 65 70 75 80 Ser Ala Lys Gly Lys Ala Lys Glu Ala Asp
Leu Ile Met Thr Ser Gly 85 90 95 Glu Ile Ala Arg Thr Leu Gly Asp
Val Gly Ile Pro Val His Val Ile 100 105 110 Asn Asp Phe Thr Ser Thr
Asp Glu Ile Asp Ala Ala Leu Arg Glu Arg 115 120 125 Tyr Asp Ile 130
17 1827 DNA Corynebacterium glutamicum CDS (101)..(1804) RXN01244
17 gatatgtgtt tgtttgtcaa tatccaaatg tttgaatagt tgcacaactg
ttggttttgt 60 ggtgatcttg aggaaattaa ctcaatgatt gtgaggatgg gtg gct
act gtg gct 115 Val Ala Thr Val Ala 1 5 gat gtg aat caa gac act gta
ctg aag ggc acc ggc gtt gtc ggt gga 163 Asp Val Asn Gln Asp Thr Val
Leu Lys Gly Thr Gly Val Val Gly Gly 10 15 20 gtc cgt tat gca agc
gcg gtg tgg att acc cca cgc ccc gaa cta ccc 211 Val Arg Tyr Ala Ser
Ala Val Trp Ile Thr Pro Arg Pro Glu Leu Pro 25 30 35 caa gca ggc
gaa gtc gtc gcc gaa gaa aac cgt gaa gca gag cag gag 259 Gln Ala Gly
Glu Val Val Ala Glu Glu Asn Arg Glu Ala Glu Gln Glu 40 45 50 cgt
ttc gac gcc gct gca gcc aca gtc tct tct cgt ttg ctt gag cgc 307 Arg
Phe Asp Ala Ala Ala Ala Thr Val Ser Ser Arg Leu Leu Glu Arg 55 60
65 tcc gaa gct gct gaa gga cca gca gct gag gtg ctt aaa gct act gct
355 Ser Glu Ala Ala Glu Gly Pro Ala Ala Glu Val Leu Lys Ala Thr Ala
70 75 80 85 ggc atg gtc aat gac cgt ggc tgg cgt aag gct gtc atc aag
ggt gtc 403 Gly Met Val Asn Asp Arg Gly Trp Arg Lys Ala Val Ile Lys
Gly Val 90 95 100 aag ggt ggt cac cct gcg gaa tac gcc gtg gtt gca
gca aca acc aag 451 Lys Gly Gly His Pro Ala Glu Tyr Ala Val Val Ala
Ala Thr Thr Lys 105 110 115 ttc atc tcc atg ttc gaa gcc gca ggc ggc
ctg atc gcg gag cgc acc 499 Phe Ile Ser Met Phe Glu Ala Ala Gly Gly
Leu Ile Ala Glu Arg Thr 120 125 130 aca gac ttg cgc gac atc cgc gac
cgc gtc atc gca gaa ctt cgt ggc 547 Thr Asp Leu Arg Asp Ile Arg Asp
Arg Val Ile Ala Glu Leu Arg Gly 135 140 145 gat gaa gag cca ggt ctg
cca gct gtt tcc gga cag gtc att ctc ttt 595 Asp Glu Glu Pro Gly Leu
Pro Ala Val Ser Gly Gln Val Ile Leu Phe 150 155 160 165 gca gat gac
ctc tcc cca gca gac acc gcg gca cta gac aca gat ctc 643 Ala Asp Asp
Leu Ser Pro Ala Asp Thr Ala Ala Leu Asp Thr Asp Leu 170 175 180 ttt
gtg gga ctt gtc act gag ctg ggt ggc cca acg agc cac acc gcg 691 Phe
Val Gly Leu Val Thr Glu Leu Gly Gly Pro Thr Ser His Thr Ala 185 190
195 atc atc gca cgc cag ctc aac gtg cct tgc atc gtc gca tcc ggc gcc
739 Ile Ile Ala Arg Gln Leu Asn Val Pro Cys Ile Val Ala Ser Gly Ala
200 205 210 ggc atc aag gac atc aag tcc ggc gaa aag gtg ctt atc gac
ggc agc 787 Gly Ile Lys Asp Ile Lys Ser Gly Glu Lys Val Leu Ile Asp
Gly Ser 215 220 225 ctc ggc acc att gac cgc aac gcg gac gaa gct gaa
gca acc aag ctc 835 Leu Gly Thr Ile Asp Arg Asn Ala Asp Glu Ala Glu
Ala Thr Lys Leu 230 235 240 245 gtc tcc gag tcc ctc gag cgc gct gct
cgc atc gcc gag tgg aag ggt 883 Val Ser Glu Ser Leu Glu Arg Ala Ala
Arg Ile Ala Glu Trp Lys Gly 250 255 260 cct gca caa acc aag gac ggc
tac cgc gtt cag ctg ttg gcc aac gtc 931 Pro Ala Gln Thr Lys Asp Gly
Tyr Arg Val Gln Leu Leu Ala Asn Val 265 270 275 caa gac ggc aac tct
gca cag cag gct gca cag acc gaa gca gaa ggc 979 Gln Asp Gly Asn Ser
Ala Gln Gln Ala Ala Gln Thr Glu Ala Glu Gly 280 285 290 atc ggc ctg
ttc cgc acc gaa ctg tgc ttc ctt tcc gcc acc gaa gag 1027 Ile Gly
Leu Phe Arg Thr Glu Leu Cys Phe Leu Ser Ala Thr Glu Glu 295 300 305
cca agc gtt gat gag cag gct gcg gtc tac tca aag gtg ctt gaa gca
1075 Pro Ser Val Asp Glu Gln Ala Ala Val Tyr Ser Lys Val Leu Glu
Ala 310 315 320 325 ttc cca gag tcc aag gtc gtt gtc cgc tcc ctc gac
gca ggt tct gac 1123 Phe Pro Glu Ser Lys Val Val Val Arg Ser Leu
Asp Ala Gly Ser Asp 330 335 340 aag cca gtt cca ttc gca tcg atg gct
gat gag atg aac cca gca ctg 1171 Lys Pro Val Pro Phe Ala Ser Met
Ala Asp Glu Met Asn Pro Ala Leu 345 350 355 ggt gtt cgt ggc ctg cgt
atc gca cgt gga cag gtt gat ctg ctg act 1219 Gly Val Arg Gly Leu
Arg Ile Ala Arg Gly Gln Val Asp Leu Leu Thr 360 365 370 cgc cag ctc
gac gca att gcg aag gcc agc gaa gaa ctc ggc cgt ggc 1267 Arg Gln
Leu Asp Ala Ile Ala Lys Ala Ser Glu Glu Leu Gly Arg Gly 375 380 385
gac gac gcc cca acc tgg gtt atg gct cca atg gtg gct acc gct tat
1315 Asp Asp Ala Pro Thr Trp Val Met Ala Pro Met Val Ala Thr Ala
Tyr 390 395 400 405 gaa gca aag tgg ttt gct gac atg tgc cgt gag cgt
ggc cta atc gcc 1363 Glu Ala Lys Trp Phe Ala Asp Met Cys Arg Glu
Arg Gly Leu Ile Ala 410 415 420 ggc gcc atg atc gaa gtt cca gca gca
tcc ctg atg gca gac aag atc 1411 Gly Ala Met Ile Glu Val Pro Ala
Ala Ser Leu Met Ala Asp Lys Ile 425 430 435 atg cct cac ctg gac ttt
gtt tcc atc ggt acc aac gac ctg acc cag 1459 Met Pro His Leu Asp
Phe Val Ser Ile Gly Thr Asn Asp Leu Thr Gln 440 445 450 tac acc atg
gca gcg gac cgc atg tct cct gag ctt gcc tac ctg acc 1507 Tyr Thr
Met Ala Ala Asp Arg Met Ser Pro Glu Leu Ala Tyr Leu Thr 455 460 465
gat cct tgg cag cca gca gtc ctg cgc ctg atc aag cac acc tgt gac
1555 Asp Pro Trp Gln Pro Ala Val Leu Arg Leu Ile Lys His Thr Cys
Asp 470 475 480 485 gaa ggt gct cgc ttt aac acc ccg gtc ggt gtt tgt
ggt gaa gca gca 1603 Glu Gly Ala Arg Phe Asn Thr Pro Val Gly Val
Cys Gly Glu Ala Ala 490 495 500 gca gac cca ctg ttg gca act gtc ctc
acc ggt ctt ggc gtg aac tcc 1651 Ala Asp Pro Leu Leu Ala Thr Val
Leu Thr Gly Leu Gly Val Asn Ser 505 510 515 ctg tcc gca gca tcc act
gct ctc gca gca gtc ggt gca aag ctg tca 1699 Leu Ser Ala Ala Ser
Thr Ala Leu Ala Ala Val Gly Ala Lys Leu Ser 520 525 530 gag gtc acc
ctg gaa acc tgt aag aag gca gca gaa gca gca ctt gac 1747 Glu Val
Thr Leu Glu Thr Cys Lys Lys Ala Ala Glu Ala Ala Leu Asp 535 540 545
gct gaa ggt gca act gaa gca cgc gat gct gta cgc gca gtg atc gac
1795 Ala Glu Gly Ala Thr Glu Ala Arg Asp Ala Val Arg Ala Val Ile
Asp 550 555 560 565 gca gca gtc taaaccactg ttgagctaaa aag 1827 Ala
Ala Val 18 568 PRT Corynebacterium glutamicum 18 Val Ala Thr Val
Ala Asp Val Asn Gln Asp Thr Val Leu Lys Gly Thr 1 5 10 15 Gly Val
Val Gly Gly Val Arg Tyr Ala Ser Ala Val Trp Ile Thr Pro 20 25 30
Arg Pro Glu Leu Pro Gln Ala Gly Glu Val Val Ala Glu Glu Asn Arg 35
40 45 Glu Ala Glu Gln Glu Arg Phe Asp Ala Ala Ala Ala Thr Val Ser
Ser 50 55 60 Arg Leu Leu Glu Arg Ser Glu Ala Ala Glu Gly Pro Ala
Ala Glu Val 65 70 75 80 Leu Lys Ala Thr Ala Gly Met Val Asn Asp Arg
Gly Trp Arg Lys Ala 85 90 95 Val Ile Lys Gly Val Lys Gly Gly His
Pro Ala Glu Tyr Ala Val Val 100 105 110 Ala Ala Thr Thr Lys Phe Ile
Ser Met Phe Glu Ala Ala Gly Gly Leu 115 120 125 Ile Ala Glu Arg Thr
Thr Asp Leu Arg Asp Ile Arg Asp Arg Val Ile 130 135 140 Ala Glu Leu
Arg Gly Asp Glu Glu Pro Gly Leu Pro Ala Val Ser Gly 145 150 155 160
Gln Val Ile Leu Phe Ala Asp Asp Leu Ser Pro Ala Asp Thr Ala Ala 165
170 175 Leu Asp Thr Asp Leu Phe Val Gly Leu Val Thr Glu Leu Gly Gly
Pro 180 185 190 Thr Ser His Thr Ala Ile Ile Ala Arg Gln Leu Asn Val
Pro Cys Ile 195 200 205 Val Ala Ser Gly Ala Gly Ile Lys Asp Ile Lys
Ser Gly Glu Lys Val 210 215 220 Leu Ile Asp Gly Ser Leu Gly Thr Ile
Asp Arg Asn Ala Asp Glu Ala 225 230 235 240 Glu Ala Thr Lys Leu Val
Ser Glu Ser Leu Glu Arg Ala Ala Arg Ile 245 250 255 Ala Glu Trp Lys
Gly Pro Ala Gln Thr Lys Asp Gly Tyr Arg Val Gln
260 265 270 Leu Leu Ala Asn Val Gln Asp Gly Asn Ser Ala Gln Gln Ala
Ala Gln 275 280 285 Thr Glu Ala Glu Gly Ile Gly Leu Phe Arg Thr Glu
Leu Cys Phe Leu 290 295 300 Ser Ala Thr Glu Glu Pro Ser Val Asp Glu
Gln Ala Ala Val Tyr Ser 305 310 315 320 Lys Val Leu Glu Ala Phe Pro
Glu Ser Lys Val Val Val Arg Ser Leu 325 330 335 Asp Ala Gly Ser Asp
Lys Pro Val Pro Phe Ala Ser Met Ala Asp Glu 340 345 350 Met Asn Pro
Ala Leu Gly Val Arg Gly Leu Arg Ile Ala Arg Gly Gln 355 360 365 Val
Asp Leu Leu Thr Arg Gln Leu Asp Ala Ile Ala Lys Ala Ser Glu 370 375
380 Glu Leu Gly Arg Gly Asp Asp Ala Pro Thr Trp Val Met Ala Pro Met
385 390 395 400 Val Ala Thr Ala Tyr Glu Ala Lys Trp Phe Ala Asp Met
Cys Arg Glu 405 410 415 Arg Gly Leu Ile Ala Gly Ala Met Ile Glu Val
Pro Ala Ala Ser Leu 420 425 430 Met Ala Asp Lys Ile Met Pro His Leu
Asp Phe Val Ser Ile Gly Thr 435 440 445 Asn Asp Leu Thr Gln Tyr Thr
Met Ala Ala Asp Arg Met Ser Pro Glu 450 455 460 Leu Ala Tyr Leu Thr
Asp Pro Trp Gln Pro Ala Val Leu Arg Leu Ile 465 470 475 480 Lys His
Thr Cys Asp Glu Gly Ala Arg Phe Asn Thr Pro Val Gly Val 485 490 495
Cys Gly Glu Ala Ala Ala Asp Pro Leu Leu Ala Thr Val Leu Thr Gly 500
505 510 Leu Gly Val Asn Ser Leu Ser Ala Ala Ser Thr Ala Leu Ala Ala
Val 515 520 525 Gly Ala Lys Leu Ser Glu Val Thr Leu Glu Thr Cys Lys
Lys Ala Ala 530 535 540 Glu Ala Ala Leu Asp Ala Glu Gly Ala Thr Glu
Ala Arg Asp Ala Val 545 550 555 560 Arg Ala Val Ile Asp Ala Ala Val
565 19 1629 DNA Corynebacterium glutamicum CDS (98)..(1606)
FRXA01244 19 agatgtcgat ttctcgagga agaagttaac gccgaagaaa accgtgaatc
agagcaggag 60 cgcttcgacg ccgctgcagc cacagtctct tcttcgt ttg ctt gag
cgc tcc gaa 115 Leu Leu Glu Arg Ser Glu 1 5 gct gct gaa gga cca gca
gct gag gtg ctt aaa gct act gct ggc atg 163 Ala Ala Glu Gly Pro Ala
Ala Glu Val Leu Lys Ala Thr Ala Gly Met 10 15 20 gtc aat gac cgt
ggc tgg cgt aag gct gtc atc aag ggt gtc aag ggt 211 Val Asn Asp Arg
Gly Trp Arg Lys Ala Val Ile Lys Gly Val Lys Gly 25 30 35 ggt cac
cct gcg gaa tac gcc gtg gtt gca gca aca acc aag ttc atc 259 Gly His
Pro Ala Glu Tyr Ala Val Val Ala Ala Thr Thr Lys Phe Ile 40 45 50
tcc atg ttc gaa gcc gca ggc ggc ctg atc gcg gag cgc acc aca gac 307
Ser Met Phe Glu Ala Ala Gly Gly Leu Ile Ala Glu Arg Thr Thr Asp 55
60 65 70 ttg cgc gac atc cgc gac cgc gtc atc gca gaa ctt cgt ggc
gat gaa 355 Leu Arg Asp Ile Arg Asp Arg Val Ile Ala Glu Leu Arg Gly
Asp Glu 75 80 85 gag cca ggt ctg cca gct gtt tcc gga cag gtc att
ctc ttt gca gat 403 Glu Pro Gly Leu Pro Ala Val Ser Gly Gln Val Ile
Leu Phe Ala Asp 90 95 100 gac ctc tcc cca gca gac acc gcg gca cta
gac aca gat ctc ttt gtg 451 Asp Leu Ser Pro Ala Asp Thr Ala Ala Leu
Asp Thr Asp Leu Phe Val 105 110 115 gga ctt gtc act gag ctg ggt ggc
cca acg agc cac acc gcg atc atc 499 Gly Leu Val Thr Glu Leu Gly Gly
Pro Thr Ser His Thr Ala Ile Ile 120 125 130 gca cgc cag ctc aac gtg
cct tgc atc gtc gca tcc ggc gcc ggc atc 547 Ala Arg Gln Leu Asn Val
Pro Cys Ile Val Ala Ser Gly Ala Gly Ile 135 140 145 150 aag gac atc
aag tcc ggc gaa aag gtg ctt atc gac ggc agc ctc ggc 595 Lys Asp Ile
Lys Ser Gly Glu Lys Val Leu Ile Asp Gly Ser Leu Gly 155 160 165 acc
att gac cgc aac gcg gac gaa gct gaa gca acc aag ctc gtc tcc 643 Thr
Ile Asp Arg Asn Ala Asp Glu Ala Glu Ala Thr Lys Leu Val Ser 170 175
180 gag tcc ctc gag cgc gct gct cgc atc gcc gag tgg aag ggt cct gca
691 Glu Ser Leu Glu Arg Ala Ala Arg Ile Ala Glu Trp Lys Gly Pro Ala
185 190 195 caa acc aag gac ggc tac cgc gtt cag ctg ttg gcc aac gtc
caa gac 739 Gln Thr Lys Asp Gly Tyr Arg Val Gln Leu Leu Ala Asn Val
Gln Asp 200 205 210 ggc aac tct gca cag cag gct gca cag acc gaa gca
gaa ggc atc ggc 787 Gly Asn Ser Ala Gln Gln Ala Ala Gln Thr Glu Ala
Glu Gly Ile Gly 215 220 225 230 ctg ttc cgc acc gaa ctg tgc ttc ctt
tcc gcc acc gaa gag cca agc 835 Leu Phe Arg Thr Glu Leu Cys Phe Leu
Ser Ala Thr Glu Glu Pro Ser 235 240 245 gtt gat gag cag gct gcg gtc
tac tca aag gtg ctt gaa gca ttc cca 883 Val Asp Glu Gln Ala Ala Val
Tyr Ser Lys Val Leu Glu Ala Phe Pro 250 255 260 gag tcc aag gtc gtt
gtc cgc tcc ctc gac gca ggt tct gac aag cca 931 Glu Ser Lys Val Val
Val Arg Ser Leu Asp Ala Gly Ser Asp Lys Pro 265 270 275 gtt cca ttc
gca tcg atg gct gat gag atg aac cca gca ctg ggt gtt 979 Val Pro Phe
Ala Ser Met Ala Asp Glu Met Asn Pro Ala Leu Gly Val 280 285 290 cgt
ggc ctg cgt atc gca cgt gga cag gtt gat ctg ctg act cgc cag 1027
Arg Gly Leu Arg Ile Ala Arg Gly Gln Val Asp Leu Leu Thr Arg Gln 295
300 305 310 ctc gac gca att gcg aag gcc agc gaa gaa ctc ggc cgt ggc
gac gac 1075 Leu Asp Ala Ile Ala Lys Ala Ser Glu Glu Leu Gly Arg
Gly Asp Asp 315 320 325 gcc cca acc tgg gtt atg gct cca atg gtg gct
acc gct tat gaa gca 1123 Ala Pro Thr Trp Val Met Ala Pro Met Val
Ala Thr Ala Tyr Glu Ala 330 335 340 aag tgg ttt gct gac atg tgc cgt
gag cgt ggc cta atc gcc ggc gcc 1171 Lys Trp Phe Ala Asp Met Cys
Arg Glu Arg Gly Leu Ile Ala Gly Ala 345 350 355 atg atc gaa gtt cca
gca gca tcc ctg atg gca gac aag atc atg cct 1219 Met Ile Glu Val
Pro Ala Ala Ser Leu Met Ala Asp Lys Ile Met Pro 360 365 370 cac ctg
gac ttt gtt tcc atc ggt acc aac gac ctg acc cag tac acc 1267 His
Leu Asp Phe Val Ser Ile Gly Thr Asn Asp Leu Thr Gln Tyr Thr 375 380
385 390 atg gca gcg gac cgc atg tct cct gag ctt gcc tac ctg acc gat
cct 1315 Met Ala Ala Asp Arg Met Ser Pro Glu Leu Ala Tyr Leu Thr
Asp Pro 395 400 405 tgg cag cca gca gtc ctg cgc ctg atc aag cac acc
tgt gac gaa ggt 1363 Trp Gln Pro Ala Val Leu Arg Leu Ile Lys His
Thr Cys Asp Glu Gly 410 415 420 gct cgc ttt aac acc ccg gtc ggt gtt
tgt ggt gaa gca gca gca gac 1411 Ala Arg Phe Asn Thr Pro Val Gly
Val Cys Gly Glu Ala Ala Ala Asp 425 430 435 cca ctg ttg gca act gtc
ctc acc ggt ctt ggc gtg aac tcc ctg tcc 1459 Pro Leu Leu Ala Thr
Val Leu Thr Gly Leu Gly Val Asn Ser Leu Ser 440 445 450 gca gca tcc
act gct ctc gca gca gtc ggt gca aag ctg tca gag gtc 1507 Ala Ala
Ser Thr Ala Leu Ala Ala Val Gly Ala Lys Leu Ser Glu Val 455 460 465
470 acc ctg gaa acc tgt aag aag gca gca gaa gca gca ctt gac gct gaa
1555 Thr Leu Glu Thr Cys Lys Lys Ala Ala Glu Ala Ala Leu Asp Ala
Glu 475 480 485 ggt gca act gaa gca cgc gat gct gta cgc gca gtg atc
gac gca gca 1603 Gly Ala Thr Glu Ala Arg Asp Ala Val Arg Ala Val
Ile Asp Ala Ala 490 495 500 gtc taaaccactg ttgagctaaa aag 1629 Val
20 503 PRT Corynebacterium glutamicum 20 Leu Leu Glu Arg Ser Glu
Ala Ala Glu Gly Pro Ala Ala Glu Val Leu 1 5 10 15 Lys Ala Thr Ala
Gly Met Val Asn Asp Arg Gly Trp Arg Lys Ala Val 20 25 30 Ile Lys
Gly Val Lys Gly Gly His Pro Ala Glu Tyr Ala Val Val Ala 35 40 45
Ala Thr Thr Lys Phe Ile Ser Met Phe Glu Ala Ala Gly Gly Leu Ile 50
55 60 Ala Glu Arg Thr Thr Asp Leu Arg Asp Ile Arg Asp Arg Val Ile
Ala 65 70 75 80 Glu Leu Arg Gly Asp Glu Glu Pro Gly Leu Pro Ala Val
Ser Gly Gln 85 90 95 Val Ile Leu Phe Ala Asp Asp Leu Ser Pro Ala
Asp Thr Ala Ala Leu 100 105 110 Asp Thr Asp Leu Phe Val Gly Leu Val
Thr Glu Leu Gly Gly Pro Thr 115 120 125 Ser His Thr Ala Ile Ile Ala
Arg Gln Leu Asn Val Pro Cys Ile Val 130 135 140 Ala Ser Gly Ala Gly
Ile Lys Asp Ile Lys Ser Gly Glu Lys Val Leu 145 150 155 160 Ile Asp
Gly Ser Leu Gly Thr Ile Asp Arg Asn Ala Asp Glu Ala Glu 165 170 175
Ala Thr Lys Leu Val Ser Glu Ser Leu Glu Arg Ala Ala Arg Ile Ala 180
185 190 Glu Trp Lys Gly Pro Ala Gln Thr Lys Asp Gly Tyr Arg Val Gln
Leu 195 200 205 Leu Ala Asn Val Gln Asp Gly Asn Ser Ala Gln Gln Ala
Ala Gln Thr 210 215 220 Glu Ala Glu Gly Ile Gly Leu Phe Arg Thr Glu
Leu Cys Phe Leu Ser 225 230 235 240 Ala Thr Glu Glu Pro Ser Val Asp
Glu Gln Ala Ala Val Tyr Ser Lys 245 250 255 Val Leu Glu Ala Phe Pro
Glu Ser Lys Val Val Val Arg Ser Leu Asp 260 265 270 Ala Gly Ser Asp
Lys Pro Val Pro Phe Ala Ser Met Ala Asp Glu Met 275 280 285 Asn Pro
Ala Leu Gly Val Arg Gly Leu Arg Ile Ala Arg Gly Gln Val 290 295 300
Asp Leu Leu Thr Arg Gln Leu Asp Ala Ile Ala Lys Ala Ser Glu Glu 305
310 315 320 Leu Gly Arg Gly Asp Asp Ala Pro Thr Trp Val Met Ala Pro
Met Val 325 330 335 Ala Thr Ala Tyr Glu Ala Lys Trp Phe Ala Asp Met
Cys Arg Glu Arg 340 345 350 Gly Leu Ile Ala Gly Ala Met Ile Glu Val
Pro Ala Ala Ser Leu Met 355 360 365 Ala Asp Lys Ile Met Pro His Leu
Asp Phe Val Ser Ile Gly Thr Asn 370 375 380 Asp Leu Thr Gln Tyr Thr
Met Ala Ala Asp Arg Met Ser Pro Glu Leu 385 390 395 400 Ala Tyr Leu
Thr Asp Pro Trp Gln Pro Ala Val Leu Arg Leu Ile Lys 405 410 415 His
Thr Cys Asp Glu Gly Ala Arg Phe Asn Thr Pro Val Gly Val Cys 420 425
430 Gly Glu Ala Ala Ala Asp Pro Leu Leu Ala Thr Val Leu Thr Gly Leu
435 440 445 Gly Val Asn Ser Leu Ser Ala Ala Ser Thr Ala Leu Ala Ala
Val Gly 450 455 460 Ala Lys Leu Ser Glu Val Thr Leu Glu Thr Cys Lys
Lys Ala Ala Glu 465 470 475 480 Ala Ala Leu Asp Ala Glu Gly Ala Thr
Glu Ala Arg Asp Ala Val Arg 485 490 495 Ala Val Ile Asp Ala Ala Val
500 21 390 DNA Corynebacterium glutamicum CDS (101)..(367) RXA01300
21 gatcgacatt aaatcccctc ccttgggggg tttaactaac aaatcgctgc
gccctaatcc 60 gttcggatta acggcgtagc aacacgaaag gacactttcc atg gct
tcc aag act 115 Met Ala Ser Lys Thr 1 5 gta acc gtc ggt tcc tcc gtt
ggc ctg cac gca cgt cca gca tcc atc 163 Val Thr Val Gly Ser Ser Val
Gly Leu His Ala Arg Pro Ala Ser Ile 10 15 20 atc gct gaa gcg gct
gct gag tac gac gac gaa atc ttg ctg acc ctg 211 Ile Ala Glu Ala Ala
Ala Glu Tyr Asp Asp Glu Ile Leu Leu Thr Leu 25 30 35 gtt ggc tcc
gat gat gac gaa gag acc gac gcg tcc tct tcc ctc atg 259 Val Gly Ser
Asp Asp Asp Glu Glu Thr Asp Ala Ser Ser Ser Leu Met 40 45 50 atc
atg gcg ctg ggc gca gag cac ggc aac gaa gtt acc gtc acc tcc 307 Ile
Met Ala Leu Gly Ala Glu His Gly Asn Glu Val Thr Val Thr Ser 55 60
65 gac aac gct gaa gct gtt gag aag atc gct gcg ctt atc gca cag gac
355 Asp Asn Ala Glu Ala Val Glu Lys Ile Ala Ala Leu Ile Ala Gln Asp
70 75 80 85 ctt gac gct gag taaacaacgc tctgcttgtt aaa 390 Leu Asp
Ala Glu 22 89 PRT Corynebacterium glutamicum 22 Met Ala Ser Lys Thr
Val Thr Val Gly Ser Ser Val Gly Leu His Ala 1 5 10 15 Arg Pro Ala
Ser Ile Ile Ala Glu Ala Ala Ala Glu Tyr Asp Asp Glu 20 25 30 Ile
Leu Leu Thr Leu Val Gly Ser Asp Asp Asp Glu Glu Thr Asp Ala 35 40
45 Ser Ser Ser Leu Met Ile Met Ala Leu Gly Ala Glu His Gly Asn Glu
50 55 60 Val Thr Val Thr Ser Asp Asn Ala Glu Ala Val Glu Lys Ile
Ala Ala 65 70 75 80 Leu Ile Ala Gln Asp Leu Asp Ala Glu 85 23 508
DNA Corynebacterium glutamicum CDS (101)..(508) RXN03002 23
ggaacttcga ggtgtcttcg tggggcgtac ggagatctag caagtgtggc tttatgtttg
60 accctatccg aatcaacatg cagtgaatta acatctactt atg ttt gta ctc aaa
115 Met Phe Val Leu Lys 1 5 gat ctg cta aag gca gaa cgc ata gaa ctc
gac cgc acg gtc acc gat 163 Asp Leu Leu Lys Ala Glu Arg Ile Glu Leu
Asp Arg Thr Val Thr Asp 10 15 20 tgg cgt gaa ggc atc cgc gcc gca
ggt gta ctc cta gaa aag aca aac 211 Trp Arg Glu Gly Ile Arg Ala Ala
Gly Val Leu Leu Glu Lys Thr Asn 25 30 35 agc att gat tcc gcc tac
acc gat gcc atg atc gcc agc gtg gaa gaa 259 Ser Ile Asp Ser Ala Tyr
Thr Asp Ala Met Ile Ala Ser Val Glu Glu 40 45 50 aaa ggc ccc tac
att gtg gtc gct cca ggt ttc gct ttc gcg cac gcc 307 Lys Gly Pro Tyr
Ile Val Val Ala Pro Gly Phe Ala Phe Ala His Ala 55 60 65 cgc ccc
agc aga gca gtc cgc gag acc gct atg tcg tgg gtg cgc ctg 355 Arg Pro
Ser Arg Ala Val Arg Glu Thr Ala Met Ser Trp Val Arg Leu 70 75 80 85
gcc tcc cct gtt tcc ttc ggt cac agt aag aat gat ccc ctc aat ctc 403
Ala Ser Pro Val Ser Phe Gly His Ser Lys Asn Asp Pro Leu Asn Leu 90
95 100 atc gtt gct ctc gct gcc aaa gat gcc acc gca cat acc caa gcg
atg 451 Ile Val Ala Leu Ala Ala Lys Asp Ala Thr Ala His Thr Gln Ala
Met 105 110 115 gcg gca ttg gct aaa gct tta gga aaa tac cga aag gat
ctc gac gag 499 Ala Ala Leu Ala Lys Ala Leu Gly Lys Tyr Arg Lys Asp
Leu Asp Glu 120 125 130 gca caa agt 508 Ala Gln Ser 135 24 136 PRT
Corynebacterium glutamicum 24 Met Phe Val Leu Lys Asp Leu Leu Lys
Ala Glu Arg Ile Glu Leu Asp 1 5 10 15 Arg Thr Val Thr Asp Trp Arg
Glu Gly Ile Arg Ala Ala Gly Val Leu 20 25 30 Leu Glu Lys Thr Asn
Ser Ile Asp Ser Ala Tyr Thr Asp Ala Met Ile 35 40 45 Ala Ser Val
Glu Glu Lys Gly Pro Tyr Ile Val Val Ala Pro Gly Phe 50 55 60 Ala
Phe Ala His Ala Arg Pro Ser Arg Ala Val Arg Glu Thr Ala Met 65 70
75 80 Ser Trp Val Arg Leu Ala Ser Pro Val Ser Phe Gly His Ser Lys
Asn 85 90 95 Asp Pro Leu Asn Leu Ile Val Ala Leu Ala Ala Lys Asp
Ala Thr Ala 100 105 110 His Thr Gln Ala Met Ala Ala Leu Ala Lys Ala
Leu Gly Lys Tyr Arg 115 120 125 Lys Asp Leu Asp Glu Ala Gln Ser 130
135 25 789 DNA Corynebacterium glutamicum CDS (14)..(766) RXC00953
25 cttgcattcc cca atg gcg cca cca acg gta ggc aac tac atc atg cag
tcc 52 Met Ala Pro Pro Thr Val Gly Asn Tyr Ile Met Gln Ser 1 5 10
ttc act caa ggt ctg cag ttc ggc gtt gca gtt gcc gtg att ctc ttt 100
Phe Thr Gln Gly Leu Gln Phe Gly Val Ala Val Ala Val Ile Leu Phe 15
20 25 ggt gtc cgc acc att ctt ggt gaa ctg gtc ccc gca ttc caa ggt
att 148 Gly Val Arg Thr Ile Leu Gly Glu Leu Val Pro Ala Phe Gln Gly
Ile 30 35 40 45 gct gcg aag gtt gtt ccc gga gct atc ccc gca ttg gat
gca ccg atc 196 Ala Ala Lys Val Val
Pro Gly Ala Ile Pro Ala Leu Asp Ala Pro Ile 50 55 60 gtg ttc ccc
tac gcg cag aac gcc gtt ctc att ggt ttc ttg tct tcc 244 Val Phe Pro
Tyr Ala Gln Asn Ala Val Leu Ile Gly Phe Leu Ser Ser 65 70 75 ttc
gtc ggt ggc ttg gtt ggc ctg act gtt ctt gca tcg tgg ctg aac 292 Phe
Val Gly Gly Leu Val Gly Leu Thr Val Leu Ala Ser Trp Leu Asn 80 85
90 cca gct ttt ggt gtc gcg ttg att ctg cct ggt ttg gtc ccc cac ttc
340 Pro Ala Phe Gly Val Ala Leu Ile Leu Pro Gly Leu Val Pro His Phe
95 100 105 ttc act ggt ggc gcg gcg ggc gtt tac ggt aat gcc acg ggt
ggt cgt 388 Phe Thr Gly Gly Ala Ala Gly Val Tyr Gly Asn Ala Thr Gly
Gly Arg 110 115 120 125 cga gga gca gta ttt ggc gcc ttt gcc aac ggt
ctt ctg att acc ttc 436 Arg Gly Ala Val Phe Gly Ala Phe Ala Asn Gly
Leu Leu Ile Thr Phe 130 135 140 ctc cct gct ttc ctg ctt ggt gtg ctt
ggt tcc ttc ggg tca gag aac 484 Leu Pro Ala Phe Leu Leu Gly Val Leu
Gly Ser Phe Gly Ser Glu Asn 145 150 155 acc act ttc ggt gat gcg gac
ttt ggt tgg ttc gga atc gtt gtt ggt 532 Thr Thr Phe Gly Asp Ala Asp
Phe Gly Trp Phe Gly Ile Val Val Gly 160 165 170 tct gca gcc aag gtg
gaa ggt gct ggc ggg ctc atc ttg ttg ctc atc 580 Ser Ala Ala Lys Val
Glu Gly Ala Gly Gly Leu Ile Leu Leu Leu Ile 175 180 185 atc gca gcg
gtt ctt ctg ggt ggc gcg atg gtc ttc cag aag cgc gtc 628 Ile Ala Ala
Val Leu Leu Gly Gly Ala Met Val Phe Gln Lys Arg Val 190 195 200 205
gtg aat ggg cac tgg gat cca gct ccc aac cgt gag cgc gtg gag aag 676
Val Asn Gly His Trp Asp Pro Ala Pro Asn Arg Glu Arg Val Glu Lys 210
215 220 gcg gaa gct gat gcc act cca acg gct ggg gct cgg acc tac cct
aag 724 Ala Glu Ala Asp Ala Thr Pro Thr Ala Gly Ala Arg Thr Tyr Pro
Lys 225 230 235 att gct cct ccg gcg ggc gct cct acc cca ccg gct cga
agc 766 Ile Ala Pro Pro Ala Gly Ala Pro Thr Pro Pro Ala Arg Ser 240
245 250 taagatctcc aaaaccctga gat 789 26 251 PRT Corynebacterium
glutamicum 26 Met Ala Pro Pro Thr Val Gly Asn Tyr Ile Met Gln Ser
Phe Thr Gln 1 5 10 15 Gly Leu Gln Phe Gly Val Ala Val Ala Val Ile
Leu Phe Gly Val Arg 20 25 30 Thr Ile Leu Gly Glu Leu Val Pro Ala
Phe Gln Gly Ile Ala Ala Lys 35 40 45 Val Val Pro Gly Ala Ile Pro
Ala Leu Asp Ala Pro Ile Val Phe Pro 50 55 60 Tyr Ala Gln Asn Ala
Val Leu Ile Gly Phe Leu Ser Ser Phe Val Gly 65 70 75 80 Gly Leu Val
Gly Leu Thr Val Leu Ala Ser Trp Leu Asn Pro Ala Phe 85 90 95 Gly
Val Ala Leu Ile Leu Pro Gly Leu Val Pro His Phe Phe Thr Gly 100 105
110 Gly Ala Ala Gly Val Tyr Gly Asn Ala Thr Gly Gly Arg Arg Gly Ala
115 120 125 Val Phe Gly Ala Phe Ala Asn Gly Leu Leu Ile Thr Phe Leu
Pro Ala 130 135 140 Phe Leu Leu Gly Val Leu Gly Ser Phe Gly Ser Glu
Asn Thr Thr Phe 145 150 155 160 Gly Asp Ala Asp Phe Gly Trp Phe Gly
Ile Val Val Gly Ser Ala Ala 165 170 175 Lys Val Glu Gly Ala Gly Gly
Leu Ile Leu Leu Leu Ile Ile Ala Ala 180 185 190 Val Leu Leu Gly Gly
Ala Met Val Phe Gln Lys Arg Val Val Asn Gly 195 200 205 His Trp Asp
Pro Ala Pro Asn Arg Glu Arg Val Glu Lys Ala Glu Ala 210 215 220 Asp
Ala Thr Pro Thr Ala Gly Ala Arg Thr Tyr Pro Lys Ile Ala Pro 225 230
235 240 Pro Ala Gly Ala Pro Thr Pro Pro Ala Arg Ser 245 250 27 553
DNA Corynebacterium glutamicum CDS (101)..(553) RXC03001 27
cccggttcac gtgatcaatg acttcacgag caccgatgaa atcgatgctg cgcttcgtga
60 acgctacgac atctaactac tttaaaagga cgaaaatatt atg gac tgg tta acc
115 Met Asp Trp Leu Thr 1 5 att cct ctt ttc ctc gtt aat gaa atc ctt
gcg gtt ccg gct ttc ctc 163 Ile Pro Leu Phe Leu Val Asn Glu Ile Leu
Ala Val Pro Ala Phe Leu 10 15 20 atc ggt atc atc acc gcc gtg gga
ttg ggt gcc atg ggg cgt tcc gtc 211 Ile Gly Ile Ile Thr Ala Val Gly
Leu Gly Ala Met Gly Arg Ser Val 25 30 35 ggt cag gtt atc ggt gga
gca atc aaa gca acg ttg ggc ttt ttg ctc 259 Gly Gln Val Ile Gly Gly
Ala Ile Lys Ala Thr Leu Gly Phe Leu Leu 40 45 50 att ggt gcg ggt
gcc acg ttg gtc act gcc tcc ctg gag cca ctg ggt 307 Ile Gly Ala Gly
Ala Thr Leu Val Thr Ala Ser Leu Glu Pro Leu Gly 55 60 65 gcg atg
atc atg ggt gcc aca ggc atg cgt ggt gtt gtc cca acg aat 355 Ala Met
Ile Met Gly Ala Thr Gly Met Arg Gly Val Val Pro Thr Asn 70 75 80 85
gaa gcc atc gcc gga atc gca cag gct gaa tac ggc gcg cag gtg gcg 403
Glu Ala Ile Ala Gly Ile Ala Gln Ala Glu Tyr Gly Ala Gln Val Ala 90
95 100 tgg ctg atg att ctg ggc ttc gcc atc tct ttg gtg ttg gct cgt
ttc 451 Trp Leu Met Ile Leu Gly Phe Ala Ile Ser Leu Val Leu Ala Arg
Phe 105 110 115 acc aac ctg cgt tat gtc ttg ctc aac gga cac cac gtg
ctg ttg atg 499 Thr Asn Leu Arg Tyr Val Leu Leu Asn Gly His His Val
Leu Leu Met 120 125 130 tgc acc atg ctc acc atg gtc ttg gcc acc gga
aga gtt gat gcg tgg 547 Cys Thr Met Leu Thr Met Val Leu Ala Thr Gly
Arg Val Asp Ala Trp 135 140 145 atc ttc 553 Ile Phe 150 28 151 PRT
Corynebacterium glutamicum 28 Met Asp Trp Leu Thr Ile Pro Leu Phe
Leu Val Asn Glu Ile Leu Ala 1 5 10 15 Val Pro Ala Phe Leu Ile Gly
Ile Ile Thr Ala Val Gly Leu Gly Ala 20 25 30 Met Gly Arg Ser Val
Gly Gln Val Ile Gly Gly Ala Ile Lys Ala Thr 35 40 45 Leu Gly Phe
Leu Leu Ile Gly Ala Gly Ala Thr Leu Val Thr Ala Ser 50 55 60 Leu
Glu Pro Leu Gly Ala Met Ile Met Gly Ala Thr Gly Met Arg Gly 65 70
75 80 Val Val Pro Thr Asn Glu Ala Ile Ala Gly Ile Ala Gln Ala Glu
Tyr 85 90 95 Gly Ala Gln Val Ala Trp Leu Met Ile Leu Gly Phe Ala
Ile Ser Leu 100 105 110 Val Leu Ala Arg Phe Thr Asn Leu Arg Tyr Val
Leu Leu Asn Gly His 115 120 125 His Val Leu Leu Met Cys Thr Met Leu
Thr Met Val Leu Ala Thr Gly 130 135 140 Arg Val Asp Ala Trp Ile Phe
145 150 29 2172 DNA Corynebacterium glutamicum CDS (101)..(2149)
RXN01943 29 ccgattcttt ttcggcccaa ttcgtaacgg cgatcctctt aagtggacaa
gaaagtctct 60 tgcccgcggg agacagaccc tacgtttaga aaggtttgac atg gcg
tcc aaa ctg 115 Met Ala Ser Lys Leu 1 5 acg acg aca tcg caa cat att
ctg gaa aac ctt ggt gga cca gac aat 163 Thr Thr Thr Ser Gln His Ile
Leu Glu Asn Leu Gly Gly Pro Asp Asn 10 15 20 att act tcg atg act
cac tgt gcg act cgc ctt cgc ttc caa gtg aag 211 Ile Thr Ser Met Thr
His Cys Ala Thr Arg Leu Arg Phe Gln Val Lys 25 30 35 gat caa tcc
att gtt gat caa caa gaa att gac tcc gac cca tca gtt 259 Asp Gln Ser
Ile Val Asp Gln Gln Glu Ile Asp Ser Asp Pro Ser Val 40 45 50 ctt
ggc gta gta ccc caa gga tcc acc ggt atg cag gtg gtg atg ggt 307 Leu
Gly Val Val Pro Gln Gly Ser Thr Gly Met Gln Val Val Met Gly 55 60
65 gga tct gtt gca aac tat tac caa gaa atc ctc aaa ctt gat gga atg
355 Gly Ser Val Ala Asn Tyr Tyr Gln Glu Ile Leu Lys Leu Asp Gly Met
70 75 80 85 aag cac ttc gcc gac ggt gaa gct aca gag agt tca tcc aag
aag gaa 403 Lys His Phe Ala Asp Gly Glu Ala Thr Glu Ser Ser Ser Lys
Lys Glu 90 95 100 tac ggc gga gtc cgt ggc aag tac tcg tgg att gac
tac gcc ttc gag 451 Tyr Gly Gly Val Arg Gly Lys Tyr Ser Trp Ile Asp
Tyr Ala Phe Glu 105 110 115 ttc ttg tct gat act ttc cga cca atc ctg
tgg gcc ctg ctt ggt gcc 499 Phe Leu Ser Asp Thr Phe Arg Pro Ile Leu
Trp Ala Leu Leu Gly Ala 120 125 130 tca ctg att att acc ttg ttg gtt
ctt gcg gat act ttc ggt ttg caa 547 Ser Leu Ile Ile Thr Leu Leu Val
Leu Ala Asp Thr Phe Gly Leu Gln 135 140 145 gac ttc cgc gct cca atg
gat gag cag cct gat act tat gta ttc ctg 595 Asp Phe Arg Ala Pro Met
Asp Glu Gln Pro Asp Thr Tyr Val Phe Leu 150 155 160 165 cac tcc atg
tgg cgc tcg gtc ttc tac ttc ctg cca att atg gtt ggt 643 His Ser Met
Trp Arg Ser Val Phe Tyr Phe Leu Pro Ile Met Val Gly 170 175 180 gcc
acc gca gct cga aag ctc ggc gca aac gag tgg att ggt gca gct 691 Ala
Thr Ala Ala Arg Lys Leu Gly Ala Asn Glu Trp Ile Gly Ala Ala 185 190
195 att cca gcc gca ctt ctt act cca gaa ttc ttg gca ctg ggt tct gcc
739 Ile Pro Ala Ala Leu Leu Thr Pro Glu Phe Leu Ala Leu Gly Ser Ala
200 205 210 ggc gat acc gtc aca gtc ttt ggc ctg cca atg gtt ctg aat
gac tac 787 Gly Asp Thr Val Thr Val Phe Gly Leu Pro Met Val Leu Asn
Asp Tyr 215 220 225 tcc gga cag gta ttc cca ccg ctg att gca gca att
ggt ctg tac tgg 835 Ser Gly Gln Val Phe Pro Pro Leu Ile Ala Ala Ile
Gly Leu Tyr Trp 230 235 240 245 gtg gaa aag gga ctg aag aag atc atc
cct gaa gca gtc caa atg gtg 883 Val Glu Lys Gly Leu Lys Lys Ile Ile
Pro Glu Ala Val Gln Met Val 250 255 260 ttc gtc cca ttc ttc tcc ctg
ctg att atg atc cca gcg acc gca ttc 931 Phe Val Pro Phe Phe Ser Leu
Leu Ile Met Ile Pro Ala Thr Ala Phe 265 270 275 ctg ctt gga cct ttc
ggc atc ggt gtt ggt aac gga att tcc aac ctg 979 Leu Leu Gly Pro Phe
Gly Ile Gly Val Gly Asn Gly Ile Ser Asn Leu 280 285 290 ctt gaa gcg
att aac aac ttc agc cca ttt att ctt tcc atc gtt atc 1027 Leu Glu
Ala Ile Asn Asn Phe Ser Pro Phe Ile Leu Ser Ile Val Ile 295 300 305
cca ttg ctc tac cca ttc ttg gtt cca ctt gga ttg cac tgg cca cta
1075 Pro Leu Leu Tyr Pro Phe Leu Val Pro Leu Gly Leu His Trp Pro
Leu 310 315 320 325 aac gcc atc atg atc cag aac atc aac acc ctg ggt
tac gac ttc att 1123 Asn Ala Ile Met Ile Gln Asn Ile Asn Thr Leu
Gly Tyr Asp Phe Ile 330 335 340 cag gga cca atg ggt gcc tgg aac ttc
gcc tgc ttc ggc ctg gtc acc 1171 Gln Gly Pro Met Gly Ala Trp Asn
Phe Ala Cys Phe Gly Leu Val Thr 345 350 355 ggc gtg ttc ttg ctc tcc
att aag gaa cga aac aag gcc atg cgt cag 1219 Gly Val Phe Leu Leu
Ser Ile Lys Glu Arg Asn Lys Ala Met Arg Gln 360 365 370 gtt tcc ctg
ggt ggc atg ttg gct ggt ttg ctc ggc ggc att tcc gag 1267 Val Ser
Leu Gly Gly Met Leu Ala Gly Leu Leu Gly Gly Ile Ser Glu 375 380 385
cct tcc ctc tac ggt gtt ctg ctc cga ttc aag aag acc tac ttc cgc
1315 Pro Ser Leu Tyr Gly Val Leu Leu Arg Phe Lys Lys Thr Tyr Phe
Arg 390 395 400 405 ctc ctg ccg ggt tgt ttg gca ggc ggt atc gtg atg
ggc atc ttc gac 1363 Leu Leu Pro Gly Cys Leu Ala Gly Gly Ile Val
Met Gly Ile Phe Asp 410 415 420 atc aag gcg tac gct ttc gtg ttc acc
tcc ttg ctt acc atc cca gca 1411 Ile Lys Ala Tyr Ala Phe Val Phe
Thr Ser Leu Leu Thr Ile Pro Ala 425 430 435 atg gac cca tgg ttg ggc
tac acc att ggt atc gca gtt gca ttc ttc 1459 Met Asp Pro Trp Leu
Gly Tyr Thr Ile Gly Ile Ala Val Ala Phe Phe 440 445 450 gtt tcc atg
ttc ctt gtt ctc gca ctg gac tac cgt tcc aac gaa gag 1507 Val Ser
Met Phe Leu Val Leu Ala Leu Asp Tyr Arg Ser Asn Glu Glu 455 460 465
cgc gat gag gca cgt gca aag gtt gct gct gac aag cag gca gaa gaa
1555 Arg Asp Glu Ala Arg Ala Lys Val Ala Ala Asp Lys Gln Ala Glu
Glu 470 475 480 485 gat ctg aag gca gaa gct aat gca act cct gca gct
cca gta gct gct 1603 Asp Leu Lys Ala Glu Ala Asn Ala Thr Pro Ala
Ala Pro Val Ala Ala 490 495 500 gca ggt gcg gga gcc ggt gca ggt gca
gga gcc gct gct ggc gct gca 1651 Ala Gly Ala Gly Ala Gly Ala Gly
Ala Gly Ala Ala Ala Gly Ala Ala 505 510 515 acc gcc gtg gca gct aag
ccg aag ctg gcc gct ggg gaa gta gtg gac 1699 Thr Ala Val Ala Ala
Lys Pro Lys Leu Ala Ala Gly Glu Val Val Asp 520 525 530 att gtt tcc
cca ctc gaa ggc aag gca att cca ctt tct gaa gta cct 1747 Ile Val
Ser Pro Leu Glu Gly Lys Ala Ile Pro Leu Ser Glu Val Pro 535 540 545
gac cca atc ttt gca gca ggc aag ctt gga cca ggc att gca atc caa
1795 Asp Pro Ile Phe Ala Ala Gly Lys Leu Gly Pro Gly Ile Ala Ile
Gln 550 555 560 565 cca act gga aac acc gtt gtt gct cca gca gac gct
act gtc atc ctt 1843 Pro Thr Gly Asn Thr Val Val Ala Pro Ala Asp
Ala Thr Val Ile Leu 570 575 580 gtc cag aaa tct gga cac gca gtg gca
ttg cgc tta gat agc gga gtt 1891 Val Gln Lys Ser Gly His Ala Val
Ala Leu Arg Leu Asp Ser Gly Val 585 590 595 gaa atc ctt gtc cac gtt
gga ttg gac acc gtg caa ttg ggc ggc gaa 1939 Glu Ile Leu Val His
Val Gly Leu Asp Thr Val Gln Leu Gly Gly Glu 600 605 610 ggc ttc acc
gtt cac gtt gag cgc agg cag caa gtc aag gcg ggg gat 1987 Gly Phe
Thr Val His Val Glu Arg Arg Gln Gln Val Lys Ala Gly Asp 615 620 625
cca ctg atc act ttt gac gct gac ttc att cga tcc aag gat cta cct
2035 Pro Leu Ile Thr Phe Asp Ala Asp Phe Ile Arg Ser Lys Asp Leu
Pro 630 635 640 645 ttg atc acc cca gtt gtg gtg tct aac gcc gcg aaa
ttc ggt gaa att 2083 Leu Ile Thr Pro Val Val Val Ser Asn Ala Ala
Lys Phe Gly Glu Ile 650 655 660 gaa ggt att cct gca gat cag gca aat
tct tcc acg act gtg atc aag 2131 Glu Gly Ile Pro Ala Asp Gln Ala
Asn Ser Ser Thr Thr Val Ile Lys 665 670 675 gtc aac ggc aag aac gag
taacctggga tccatgttgc gca 2172 Val Asn Gly Lys Asn Glu 680 30 683
PRT Corynebacterium glutamicum 30 Met Ala Ser Lys Leu Thr Thr Thr
Ser Gln His Ile Leu Glu Asn Leu 1 5 10 15 Gly Gly Pro Asp Asn Ile
Thr Ser Met Thr His Cys Ala Thr Arg Leu 20 25 30 Arg Phe Gln Val
Lys Asp Gln Ser Ile Val Asp Gln Gln Glu Ile Asp 35 40 45 Ser Asp
Pro Ser Val Leu Gly Val Val Pro Gln Gly Ser Thr Gly Met 50 55 60
Gln Val Val Met Gly Gly Ser Val Ala Asn Tyr Tyr Gln Glu Ile Leu 65
70 75 80 Lys Leu Asp Gly Met Lys His Phe Ala Asp Gly Glu Ala Thr
Glu Ser 85 90 95 Ser Ser Lys Lys Glu Tyr Gly Gly Val Arg Gly Lys
Tyr Ser Trp Ile 100 105 110 Asp Tyr Ala Phe Glu Phe Leu Ser Asp Thr
Phe Arg Pro Ile Leu Trp 115 120 125 Ala Leu Leu Gly Ala Ser Leu Ile
Ile Thr Leu Leu Val Leu Ala Asp 130 135 140 Thr Phe Gly Leu Gln Asp
Phe Arg Ala Pro Met Asp Glu Gln Pro Asp 145 150 155 160 Thr Tyr Val
Phe Leu His Ser Met Trp Arg Ser Val Phe Tyr Phe Leu 165 170 175 Pro
Ile Met Val Gly Ala Thr Ala Ala Arg Lys Leu Gly Ala Asn Glu 180 185
190 Trp Ile Gly Ala Ala Ile Pro Ala Ala Leu Leu Thr Pro Glu Phe Leu
195 200 205 Ala Leu Gly Ser Ala Gly Asp Thr Val Thr Val Phe Gly Leu
Pro Met 210 215 220 Val Leu Asn Asp Tyr Ser Gly Gln Val Phe Pro Pro
Leu Ile Ala Ala 225 230 235 240 Ile Gly Leu Tyr Trp Val Glu Lys Gly
Leu Lys Lys Ile Ile Pro Glu 245 250 255 Ala Val Gln Met Val Phe Val
Pro Phe Phe Ser Leu Leu Ile Met Ile 260
265 270 Pro Ala Thr Ala Phe Leu Leu Gly Pro Phe Gly Ile Gly Val Gly
Asn 275 280 285 Gly Ile Ser Asn Leu Leu Glu Ala Ile Asn Asn Phe Ser
Pro Phe Ile 290 295 300 Leu Ser Ile Val Ile Pro Leu Leu Tyr Pro Phe
Leu Val Pro Leu Gly 305 310 315 320 Leu His Trp Pro Leu Asn Ala Ile
Met Ile Gln Asn Ile Asn Thr Leu 325 330 335 Gly Tyr Asp Phe Ile Gln
Gly Pro Met Gly Ala Trp Asn Phe Ala Cys 340 345 350 Phe Gly Leu Val
Thr Gly Val Phe Leu Leu Ser Ile Lys Glu Arg Asn 355 360 365 Lys Ala
Met Arg Gln Val Ser Leu Gly Gly Met Leu Ala Gly Leu Leu 370 375 380
Gly Gly Ile Ser Glu Pro Ser Leu Tyr Gly Val Leu Leu Arg Phe Lys 385
390 395 400 Lys Thr Tyr Phe Arg Leu Leu Pro Gly Cys Leu Ala Gly Gly
Ile Val 405 410 415 Met Gly Ile Phe Asp Ile Lys Ala Tyr Ala Phe Val
Phe Thr Ser Leu 420 425 430 Leu Thr Ile Pro Ala Met Asp Pro Trp Leu
Gly Tyr Thr Ile Gly Ile 435 440 445 Ala Val Ala Phe Phe Val Ser Met
Phe Leu Val Leu Ala Leu Asp Tyr 450 455 460 Arg Ser Asn Glu Glu Arg
Asp Glu Ala Arg Ala Lys Val Ala Ala Asp 465 470 475 480 Lys Gln Ala
Glu Glu Asp Leu Lys Ala Glu Ala Asn Ala Thr Pro Ala 485 490 495 Ala
Pro Val Ala Ala Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala 500 505
510 Ala Ala Gly Ala Ala Thr Ala Val Ala Ala Lys Pro Lys Leu Ala Ala
515 520 525 Gly Glu Val Val Asp Ile Val Ser Pro Leu Glu Gly Lys Ala
Ile Pro 530 535 540 Leu Ser Glu Val Pro Asp Pro Ile Phe Ala Ala Gly
Lys Leu Gly Pro 545 550 555 560 Gly Ile Ala Ile Gln Pro Thr Gly Asn
Thr Val Val Ala Pro Ala Asp 565 570 575 Ala Thr Val Ile Leu Val Gln
Lys Ser Gly His Ala Val Ala Leu Arg 580 585 590 Leu Asp Ser Gly Val
Glu Ile Leu Val His Val Gly Leu Asp Thr Val 595 600 605 Gln Leu Gly
Gly Glu Gly Phe Thr Val His Val Glu Arg Arg Gln Gln 610 615 620 Val
Lys Ala Gly Asp Pro Leu Ile Thr Phe Asp Ala Asp Phe Ile Arg 625 630
635 640 Ser Lys Asp Leu Pro Leu Ile Thr Pro Val Val Val Ser Asn Ala
Ala 645 650 655 Lys Phe Gly Glu Ile Glu Gly Ile Pro Ala Asp Gln Ala
Asn Ser Ser 660 665 670 Thr Thr Val Ile Lys Val Asn Gly Lys Asn Glu
675 680 31 1339 DNA Corynebacterium glutamicum CDS (101)..(1339)
FRXA02191 31 ccgattcttt ttcggcccaa ttcgtaacgg cgatcctctt aagtggacaa
gaaagtctct 60 tgcccgcggg agacagaccc tacgtttaga aaggtttgac atg gcg
tcc aaa ctg 115 Met Ala Ser Lys Leu 1 5 acg acg aca tcg caa cat att
ctg gaa aac ctt ggt gga cca gac aat 163 Thr Thr Thr Ser Gln His Ile
Leu Glu Asn Leu Gly Gly Pro Asp Asn 10 15 20 att act tcg atg act
cac tgt gcg act cgc ctt cgc ttc caa gtg aag 211 Ile Thr Ser Met Thr
His Cys Ala Thr Arg Leu Arg Phe Gln Val Lys 25 30 35 gat caa tcc
att gtt gat caa caa gaa att gac tcc gac cca tca gtt 259 Asp Gln Ser
Ile Val Asp Gln Gln Glu Ile Asp Ser Asp Pro Ser Val 40 45 50 ctt
ggc gta gta ccc caa gga tcc acc ggt atg cag gtg gtg atg ggt 307 Leu
Gly Val Val Pro Gln Gly Ser Thr Gly Met Gln Val Val Met Gly 55 60
65 gga tct gtt gca aac tat tac caa gaa atc ctc aaa ctt gat gga atg
355 Gly Ser Val Ala Asn Tyr Tyr Gln Glu Ile Leu Lys Leu Asp Gly Met
70 75 80 85 aag cac ttc gcc gac ggt gaa gct aca gag agt tca tcc aag
aag gaa 403 Lys His Phe Ala Asp Gly Glu Ala Thr Glu Ser Ser Ser Lys
Lys Glu 90 95 100 tac ggc gga gtc cgt ggc aag tac tcg tgg att gac
tac gcc ttc gag 451 Tyr Gly Gly Val Arg Gly Lys Tyr Ser Trp Ile Asp
Tyr Ala Phe Glu 105 110 115 ttc ttg tct gat act ttc cga cca atc ctg
tgg gcc ctg ctt ggt gcc 499 Phe Leu Ser Asp Thr Phe Arg Pro Ile Leu
Trp Ala Leu Leu Gly Ala 120 125 130 tca ctg att att acc ttg ttg gtt
ctt gcg gat act ttc ggt ttg caa 547 Ser Leu Ile Ile Thr Leu Leu Val
Leu Ala Asp Thr Phe Gly Leu Gln 135 140 145 gac ttc cgc gct cca atg
gat gag cag cct gat act tat gta ttc ctg 595 Asp Phe Arg Ala Pro Met
Asp Glu Gln Pro Asp Thr Tyr Val Phe Leu 150 155 160 165 cac tcc atg
tgg cgc tcg gtc ttc tac ttc ctg cca att atg gtt ggt 643 His Ser Met
Trp Arg Ser Val Phe Tyr Phe Leu Pro Ile Met Val Gly 170 175 180 gcc
acc gca gct cga aag ctc ggc gca aac gag tgg att ggt gca gct 691 Ala
Thr Ala Ala Arg Lys Leu Gly Ala Asn Glu Trp Ile Gly Ala Ala 185 190
195 att cca gcc gca ctt ctt act cca gaa ttc ttg gca ctg ggt tct gcc
739 Ile Pro Ala Ala Leu Leu Thr Pro Glu Phe Leu Ala Leu Gly Ser Ala
200 205 210 ggc gat acc gtc aca gtc ttt ggc ctg cca atg gtt ctg aat
gac tac 787 Gly Asp Thr Val Thr Val Phe Gly Leu Pro Met Val Leu Asn
Asp Tyr 215 220 225 tcc gga cag gta ttc cca ccg ctg att gca gca att
ggt ctg tac tgg 835 Ser Gly Gln Val Phe Pro Pro Leu Ile Ala Ala Ile
Gly Leu Tyr Trp 230 235 240 245 gtg gaa aag gga ctg aag aag atc atc
cct gaa gca gtc caa atg gtg 883 Val Glu Lys Gly Leu Lys Lys Ile Ile
Pro Glu Ala Val Gln Met Val 250 255 260 ttc gtc cca ttc ttc tcc ctg
ctg att atg atc cca gcg acc gca ttc 931 Phe Val Pro Phe Phe Ser Leu
Leu Ile Met Ile Pro Ala Thr Ala Phe 265 270 275 ctg ctt gga cct ttc
ggc atc ggt gtt ggt aac gga att tcc aac ctg 979 Leu Leu Gly Pro Phe
Gly Ile Gly Val Gly Asn Gly Ile Ser Asn Leu 280 285 290 ctt gaa gcg
att aac aac ttc agc cca ttt att ctt tcc atc gtt atc 1027 Leu Glu
Ala Ile Asn Asn Phe Ser Pro Phe Ile Leu Ser Ile Val Ile 295 300 305
cca ttg ctc tac cca ttc ttg gtt cca ctt gga ttg cac tgg cca cta
1075 Pro Leu Leu Tyr Pro Phe Leu Val Pro Leu Gly Leu His Trp Pro
Leu 310 315 320 325 aac gcc atc atg atc cag aac atc aac acc ctg ggt
tac gac ttc att 1123 Asn Ala Ile Met Ile Gln Asn Ile Asn Thr Leu
Gly Tyr Asp Phe Ile 330 335 340 cag gga cca atg ggt gcc tgg aac ttc
gcc tgc ttc ggc ctg gtc acc 1171 Gln Gly Pro Met Gly Ala Trp Asn
Phe Ala Cys Phe Gly Leu Val Thr 345 350 355 ggc gtg ttc ttg ctc tcc
att aag gaa cga aac aag gcc atg cgt cag 1219 Gly Val Phe Leu Leu
Ser Ile Lys Glu Arg Asn Lys Ala Met Arg Gln 360 365 370 gtt tcc ctg
ggt ggc atg ttg gct ggt ttg ctc ggc ggc att tcc gag 1267 Val Ser
Leu Gly Gly Met Leu Ala Gly Leu Leu Gly Gly Ile Ser Glu 375 380 385
cct tcc ctc tac ggt gtt ctg ctc cga ttc aag aag acc tac ttc cgc
1315 Pro Ser Leu Tyr Gly Val Leu Leu Arg Phe Lys Lys Thr Tyr Phe
Arg 390 395 400 405 ctc ctg ccg ggt tgt ttg gca gca 1339 Leu Leu
Pro Gly Cys Leu Ala Ala 410 32 413 PRT Corynebacterium glutamicum
32 Met Ala Ser Lys Leu Thr Thr Thr Ser Gln His Ile Leu Glu Asn Leu
1 5 10 15 Gly Gly Pro Asp Asn Ile Thr Ser Met Thr His Cys Ala Thr
Arg Leu 20 25 30 Arg Phe Gln Val Lys Asp Gln Ser Ile Val Asp Gln
Gln Glu Ile Asp 35 40 45 Ser Asp Pro Ser Val Leu Gly Val Val Pro
Gln Gly Ser Thr Gly Met 50 55 60 Gln Val Val Met Gly Gly Ser Val
Ala Asn Tyr Tyr Gln Glu Ile Leu 65 70 75 80 Lys Leu Asp Gly Met Lys
His Phe Ala Asp Gly Glu Ala Thr Glu Ser 85 90 95 Ser Ser Lys Lys
Glu Tyr Gly Gly Val Arg Gly Lys Tyr Ser Trp Ile 100 105 110 Asp Tyr
Ala Phe Glu Phe Leu Ser Asp Thr Phe Arg Pro Ile Leu Trp 115 120 125
Ala Leu Leu Gly Ala Ser Leu Ile Ile Thr Leu Leu Val Leu Ala Asp 130
135 140 Thr Phe Gly Leu Gln Asp Phe Arg Ala Pro Met Asp Glu Gln Pro
Asp 145 150 155 160 Thr Tyr Val Phe Leu His Ser Met Trp Arg Ser Val
Phe Tyr Phe Leu 165 170 175 Pro Ile Met Val Gly Ala Thr Ala Ala Arg
Lys Leu Gly Ala Asn Glu 180 185 190 Trp Ile Gly Ala Ala Ile Pro Ala
Ala Leu Leu Thr Pro Glu Phe Leu 195 200 205 Ala Leu Gly Ser Ala Gly
Asp Thr Val Thr Val Phe Gly Leu Pro Met 210 215 220 Val Leu Asn Asp
Tyr Ser Gly Gln Val Phe Pro Pro Leu Ile Ala Ala 225 230 235 240 Ile
Gly Leu Tyr Trp Val Glu Lys Gly Leu Lys Lys Ile Ile Pro Glu 245 250
255 Ala Val Gln Met Val Phe Val Pro Phe Phe Ser Leu Leu Ile Met Ile
260 265 270 Pro Ala Thr Ala Phe Leu Leu Gly Pro Phe Gly Ile Gly Val
Gly Asn 275 280 285 Gly Ile Ser Asn Leu Leu Glu Ala Ile Asn Asn Phe
Ser Pro Phe Ile 290 295 300 Leu Ser Ile Val Ile Pro Leu Leu Tyr Pro
Phe Leu Val Pro Leu Gly 305 310 315 320 Leu His Trp Pro Leu Asn Ala
Ile Met Ile Gln Asn Ile Asn Thr Leu 325 330 335 Gly Tyr Asp Phe Ile
Gln Gly Pro Met Gly Ala Trp Asn Phe Ala Cys 340 345 350 Phe Gly Leu
Val Thr Gly Val Phe Leu Leu Ser Ile Lys Glu Arg Asn 355 360 365 Lys
Ala Met Arg Gln Val Ser Leu Gly Gly Met Leu Ala Gly Leu Leu 370 375
380 Gly Gly Ile Ser Glu Pro Ser Leu Tyr Gly Val Leu Leu Arg Phe Lys
385 390 395 400 Lys Thr Tyr Phe Arg Leu Leu Pro Gly Cys Leu Ala Ala
405 410 33 428 DNA Corynebacterium glutamicum CDS (1)..(405)
FRXA01943 33 cct gac cca atc ttt gca gca ggc aag ctt gga cca ggc
att gca atc 48 Pro Asp Pro Ile Phe Ala Ala Gly Lys Leu Gly Pro Gly
Ile Ala Ile 1 5 10 15 caa cca act gga aac acc gtt gtt gct cca gca
gac gct act gtc atc 96 Gln Pro Thr Gly Asn Thr Val Val Ala Pro Ala
Asp Ala Thr Val Ile 20 25 30 ctt gtc cag aaa tct gga cac gca gtg
gca ttg cgc tta gat agc gga 144 Leu Val Gln Lys Ser Gly His Ala Val
Ala Leu Arg Leu Asp Ser Gly 35 40 45 gtt gaa atc ctt gtc cac gtt
gga ttg gac acc gtg caa ttg ggc ggc 192 Val Glu Ile Leu Val His Val
Gly Leu Asp Thr Val Gln Leu Gly Gly 50 55 60 gaa ggc ttc acc gtt
cac gtt gag cgc agg cag caa gtc aag gcg ggg 240 Glu Gly Phe Thr Val
His Val Glu Arg Arg Gln Gln Val Lys Ala Gly 65 70 75 80 gat cca ctg
atc act ttt gac gct gac ttc att cga tcc aag gat cta 288 Asp Pro Leu
Ile Thr Phe Asp Ala Asp Phe Ile Arg Ser Lys Asp Leu 85 90 95 cct
ttg atc acc cca gtt gtg gtg tct aac gcc gcg aaa ttc ggt gaa 336 Pro
Leu Ile Thr Pro Val Val Val Ser Asn Ala Ala Lys Phe Gly Glu 100 105
110 att gaa ggt att cct gca gat cag gca aat tct tcc acg act gtg atc
384 Ile Glu Gly Ile Pro Ala Asp Gln Ala Asn Ser Ser Thr Thr Val Ile
115 120 125 aag gtc aac ggc aag aac gag taacctggga tccatgttgc gca
428 Lys Val Asn Gly Lys Asn Glu 130 135 34 135 PRT Corynebacterium
glutamicum 34 Pro Asp Pro Ile Phe Ala Ala Gly Lys Leu Gly Pro Gly
Ile Ala Ile 1 5 10 15 Gln Pro Thr Gly Asn Thr Val Val Ala Pro Ala
Asp Ala Thr Val Ile 20 25 30 Leu Val Gln Lys Ser Gly His Ala Val
Ala Leu Arg Leu Asp Ser Gly 35 40 45 Val Glu Ile Leu Val His Val
Gly Leu Asp Thr Val Gln Leu Gly Gly 50 55 60 Glu Gly Phe Thr Val
His Val Glu Arg Arg Gln Gln Val Lys Ala Gly 65 70 75 80 Asp Pro Leu
Ile Thr Phe Asp Ala Asp Phe Ile Arg Ser Lys Asp Leu 85 90 95 Pro
Leu Ile Thr Pro Val Val Val Ser Asn Ala Ala Lys Phe Gly Glu 100 105
110 Ile Glu Gly Ile Pro Ala Asp Gln Ala Asn Ser Ser Thr Thr Val Ile
115 120 125 Lys Val Asn Gly Lys Asn Glu 130 135
* * * * *
References