U.S. patent application number 10/454437 was filed with the patent office on 2005-12-15 for corynebacterium glutamicum genes encoding proteins involved in homeostasis and adaptation.
Invention is credited to Haberhauer, Gregor, Kroger, Burkhard, Pompejus, Markus, Schroder, Hartwig, Zelder, Oskar.
Application Number | 20050277115 10/454437 |
Document ID | / |
Family ID | 33492654 |
Filed Date | 2005-12-15 |
United States Patent
Application |
20050277115 |
Kind Code |
A1 |
Pompejus, Markus ; et
al. |
December 15, 2005 |
Corynebacterium glutamicum genes encoding proteins involved in
homeostasis and adaptation
Abstract
Isolated nucleic acid molecules, designated HA nucleic acid
molecules, which encode novel HA proteins from Corynebacterium
glutamicum are described. The invention also provides antisense
nucleic acid molecules, recombinant expression vectors containing
HA nucleic acid molecules, and host cells into which the expression
vectors have been introduced. The invention still further provides
isolated HA proteins, mutated HA proteins, fusion proteins,
antigenic peptides and methods for the improvement of production of
a desired compound from C. glutamicum based on genetic engineering
of HA 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
|
Family ID: |
33492654 |
Appl. No.: |
10/454437 |
Filed: |
June 4, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10454437 |
Jun 4, 2003 |
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09602777 |
Jun 23, 2000 |
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6831165 |
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60141031 |
Jun 25, 1999 |
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Current U.S.
Class: |
435/134 |
Current CPC
Class: |
C12N 9/00 20130101; C07K
14/34 20130101; C12N 9/18 20130101; C12P 13/04 20130101; C12Q 1/689
20130101; C07K 2319/00 20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 8, 1999 |
DE |
19931636.8 |
Jul 9, 1999 |
DE |
19932125.6 |
Jul 9, 1999 |
DE |
19932126.4 |
Jul 9, 1999 |
DE |
19932127.2 |
Jul 9, 1999 |
DE |
19932128.0 |
Jul 9, 1999 |
DE |
19932129.9 |
Jul 9, 1999 |
DE |
19932226.0 |
Jul 14, 1999 |
DE |
19932920.6 |
Jul 14, 1999 |
DE |
19932922.2 |
Jul 14, 1999 |
DE |
19932924.9 |
Jul 14, 1999 |
DE |
19932928.1 |
Jul 14, 1999 |
DE |
19932930.3 |
Jul 14, 1999 |
DE |
19932933.8 |
Jul 14, 1999 |
DE |
19932935.4 |
Jul 14, 1999 |
DE |
19932973.7 |
Jul 14, 1999 |
DE |
19933002.6 |
Jul 14, 1999 |
DE |
19933003.4 |
Jul 14, 1999 |
DE |
19933005.0 |
Jul 14, 1999 |
DE |
19933006.9 |
Aug 31, 1999 |
DE |
19941378.9 |
Aug 31, 1999 |
DE |
19941379.7 |
Aug 31, 1999 |
DE |
19941390.8 |
Aug 31, 1999 |
DE |
19941391.6 |
Sep 3, 1999 |
DE |
19942088.2 |
Claims
What is claimed:
1. An isolated nucleic acid molecule from Corynebacterium
glutamicum encoding an HA 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
nucleic acid molecule encodes an HA protein involved in the
production of a fine chemical.
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. Tile 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
and 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 HA polypeptide from Corynebacterium glutamicum, or
a portion thereof.
19. The polypeptide of claim 18, wherein said polypeptide is
involved in the production of a fine chemical production.
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 kelosoreductum, 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 and
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 1-7.
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 is a continuation application of U.S.
patent application Ser. No. 09/602,777, filed on Jun. 23, 2000
(allowed), which claims priority to prior filed U.S. Provisional
Patent Application Ser. No. 60/141,031, filed Jun. 25, 1999. This
application also claims priority to prior filed German Patent
Application No. 19931636.8, filed Jul. 8, 1999, German Patent
Application No. 19932125.6, filed Jul. 9, 1999, German Patent
Application No. 19932126.4, filed Jul. 9, 1999, German Patent
Application No. 19932127.2, filed Jul. 9, 1999, German Patent
Application No. 19932128.0, filed Jul. 9, 1999, German Patent
Application No. 19932129.9, filed Jul. 9, 1999, German Patent
Application No. 19932226.0, filed Jul. 9, 1999, German Patent
Application No. 19932920.6, filed Jul. 14, 1999, German Patent
Application No. 19932922.2, filed Jul. 14, 1999, German Patent
Application No. 19932924.9, filed Jul. 14, 1999, German Patent
Application No. 19932928.1, filed Jul. 14, 1999, German Patent
Application No. 19932930.3, filed Jul. 14, 1999, German Patent
Application No. 19932933.8, filed Jul. 14, 1999, German Patent
Application No. 19932935.4, filed Jul. 14, 1999, German Patent
Application No. 19932973.7, filed Jul. 14, 1999, German Patent
Application No. 19933002.6, filed Jul. 14, 1999, German Patent
Application No. 19933003.4, filed Jul. 14, 1999, German Patent
Application No. 19933005.0, filed Jul. 14, 1999, German Patent
Application No. 19933006.9, filed Jul. 14, 1999, German Patent
Application No. 19941378.9, filed Aug. 31, 1999, German Patent
Application No. 19941379.7, filed Aug. 31, 1999, German Patent
Application No. 19941390.8, filed Aug. 31, 1999, German Patent
Application No. 19941391.6, filed Aug. 31, 1999, and German Patent
Application No. 19942088.2, filed Sep. 3, 1999. The entire contents
of all of the aforementioned applications are hereby expressly
incorporated herein by this reference.
REFERENCE TO COMPACT DISK APPENDIX
[0002] The Sequence Listing is being filed concurrently herewith on
two (2) compact discs in lieu of paper. Each compact disc submitted
is formatted from an IBM-PC and is compatible with MS-Windows. Each
disc contains one (1) file named SeqList.txt, each file contains
1,488 kb. These files were created on May 20, 2003. The material on
the compact discs, namely the Sequence Listing, are expressly
incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] Certain products and by-products of naturally-occurring
metabolic processes in cells have utility in a wide array of
industries, including the food, feed, cosmetics, and pharmaceutical
industries. These molecules, collectively termed `fine chemicals`,
include organic acids, both proteinogenic and non-proteinogenic
amino acids, nucleotides and nucleosides, lipids and fatty acids,
diols, carbohydrates, aromatic compounds, vitamins and cofactors,
and enzymes. Their production is most conveniently performed
through the large-scale culture of bacteria developed to produce
and secrete large quantities of one or more desired molecules. One
particularly useful organism for this purpose is Corynebacterium
glutamicum, a gram positive, nonpathogenic bacterium. Through
strain selection, a number of mutant strains have been developed
which produce an array of desirable compounds. However, selection
of strains improved for the production of a particular molecule is
a time-consuming and difficult process.
SUMMARY OF THE INVENTION
[0004] 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
homeostasis and adaptation (HA) proteins.
[0005] 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 HA 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 HA nucleic acids of the
invention, or modification of the sequence of the HA 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).
[0006] The HA 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.
[0007] The HA 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.
[0008] e.g. e.g. The HA proteins encoded by the novel nucleic acid
molecules of the invention are capable of, for example, performing
a function involved in the maintenance of homeostasis in C.
glutamicum, or in the ability of this microorganism to adapt to
different environmental conditions. 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. Bacieriol. 162: 591-597 (1985); Katsumata et al, J.
Bacteriol. 159: 306-311 (1984); and Santamaria et al., J. Gen.
Microbiol. 130: 2237-2246 (1984)), the nucleic acid molecules of
the invention may be utilized in the genetic engineering of this
organism to make it a better or more efficient producer of one or
more fine chemicals. This improved production or efficiency of
production of a fine chemical may be due to a direct effect of
manipulation of a gene of the invention, or it may be due to an
indirect effect of such manipulation.
[0009] There are a number of mechanisms by which the alteration of
an HA protein of the invention may directly affect the yield,
production, and/or efficiency of production of a fine chemical from
a C. glutamicum strain incorporating such an altered protein. For
example, by engineering enzymes which modify or degrade aromatic or
aliphatic compounds such that these enzymes are increased or
decreased in activity or number, it may be possible to modulate the
production of one or more fine chemicals which are the modification
or degradation products of these compounds. Similarly, enzymes
involved in the metabolism of inorganic compounds provide key
molecules (e.g. phosphorous, sulfur, and nitrogen molecules) for
the biosynthesis of such fine chemicals as amino acids, vitamins,
and nucleic acids. By altering the activity or number of these
enzymes in C. glutamicum, it may be possible to increase the
conversion of these inorganic compounds (or to use alternate
inorganic compounds) to thus permit improved rates of incorporation
of inorganic atoms into these fine chemicals. Genetic engineering
of C. glutamicum enzymes involved in general cellular processes may
also directly improve fine chemical production, since many of these
enzymes directly modify fine chemicals (e.g., amino acids) or the
enzymes which are involved in fine chemical synthesis or secretion.
Modulation of the activity or number of cellular proteases may also
have a direct effect on fine chemical production, since many
proteases may degrade fine chemicals or enzymes involved in fine
chemical production or breakdown.
[0010] Further, the aforementioned enzymes which participate in
aromatic/aliphatic compound modification or degradation, general
biocatalysis, inorganic compound metabolism or proteolysis are each
themselves fine chemicals, desirable for their activity in various
in vitro industrial applications. By altering the number of copies
of the gene for one or more of these enzymes in C. glutamicum it
may be possible to increase the number of these proteins produced
by the cell, thereby increasing the potential yield or efficiency
of production of these proteins from large-scale C. glutamicum or
related bacterial cultures.
[0011] The alteration of an HA protein of the invention may also
indirectly affect the yield, production, and/or efficiency of
production of a fine chemical from a C. glutamicum strain
incorporating such an altered protein. For example, by modulating
the activity and/or number of those proteins involved in the
construction or rearrangement of the cell wall, it may be possible
to modify the structure of the cell wall itself such that the cell
is able to better withstand the mechanical and other stresses
present during large-scale fermentative culture. Also, large-scale
growth of C. glutamicum requires significant cell wall production.
Modulation of the activity or number of cell wall biosynthetic or
degradative enzymes may allow more rapid rates of cell wall
biosynthesis, which in turn may permit increased growth rates of
this microorganism in culture and thereby increase the number of
cells producing the desired fine chemical.
[0012] By modifying the HA enzymes of the invention, one may also
indirectly impact the yield, production, or efficiency of
production of one or more fine chemicals from C. glutamicum. For
example, many of the general enzymes in C. glutamicum may have a
significant impact on global cellular processes (e.g., regulatory
processes) which in turn have a significant effect on fine chemical
metabolism. Similarly, proteases, enzymes which modify or degrade
possibly toxic aromatic or aliphatic compounds, and enzymes which
promote the metabolism of inorganic compounds all serve to increase
the viability of C. glutamicum. The proteases aid in the selective
removal of misfolded or misregulated proteins, such as those that
might occur under the relatively stressful environmental conditions
encountered during large-scale fermentor culture. By altering these
proteins, it may be possible to further enhance this activity and
to improve the viability of C. glutamicum in culture. The
aromatic/aliphatic modification or degradation proteins not only
serve to detoxify these waste compounds (which may be encountered
as impurities in culture medium or as waste products from cells
themselves), but also to permit the cells to utilize alternate
carbon sources if the optimal carbon source is limiting in the
culture. By increasing their number and/or activity, the survival
of C. glutamicum cells in culture may be enhanced. The inorganic
metabolism proteins of the invention supply the cell with inorganic
molecules required for all protein and nucleotide (among others)
synthesis, and thus are critical for the overall viability of the
cell. An increase in the number of viable cells producing one or
more desired fine chemicals in large-scale culture should result in
a concomitant increase in the yield, production, and/or efficiency
of production of the fine chemical in the culture.
[0013] The invention provides novel nucleic acid molecules which
encode proteins, referred to herein as HA proteins, which are
capable of, for example, performing a function involved in the
maintenance of homeostasis in C. glutamicum, or of participating in
the ability of this microorganism to adapt to different
environmental conditions. Nucleic acid molecules encoding an HA
protein are referred to herein as HA nucleic acid molecules. In a
preferred embodiment, an HA protein participates in C. glutamicum
cell wall biosynthesis or rearrangements, metabolism of inorganic
compounds, modification or degradation of aromatic or aliphatic
compounds, or possesses a C. glutamicum enzymatic or proteolytic
activity. Examples of such proteins include those encoded by the
genes set forth in Table 1.
[0014] Accordingly, one aspect of the invention pertains to
isolated nucleic acid molecules (e.g., cDNAs, DNAs, or RNAs)
comprising a nucleotide sequence encoding an HA protein or
biologically active portions thereof, as well as nucleic acid
fragments suitable as primers or hybridization probes for the
detection or amplification of HA- encoding nucleic acids (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 HA
proteins of the present invention also preferably possess at least
one of the HA activities described herein.
[0015] 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 an HA activity.
Preferably, the protein or portion thereof encoded by the nucleic
acid molecule maintains the ability to participate in the
maintenance of homeostasis in C. glutamicum, or to perform a
function involved in the adaptation of this microorganism to
different environmental conditions. 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).
[0016] In another preferred embodiment, the isolated nucleic acid
molecule is derived from C. glutamicum and encodes a protein (e.g.,
an HA 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
repair or recombination of DNA, in the transposition of genetic
material, in gene expression (i.e., the processes of transcription
or translation), in protein folding, or in protein secretion in
Corynebacterium glutamicum, or has one or more of the activities
set forth in Table 1, and which also includes heterologous nucleic
acid sequences encoding a heterologous polypeptide or regulatory
regions.
[0017] 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 HA protein, or a biologically
active portion thereof.
[0018] Another aspect of the invention pertains to vectors, e.g.,
recombinant expression vectors, containing the nucleic acid
molecules of the invention, and host cells into which such vectors
have been introduced. In one embodiment, such a host cell is used
to produce an HA protein by culturing the host cell in a suitable
medium. The HA protein can be then isolated from the medium or the
host cell.
[0019] Yet another aspect of the invention pertains to a
genetically altered microorganism in which an HA gene has been
introduced or altered. In one embodiment, the genome of the
microorganism has been altered by introduction of a nucleic acid
molecule of the invention encoding wild-type or mutated HA sequence
as a transgene. In another embodiment, an endogenous HA gene within
the genome of the microorganism has been altered, e.g.,
functionally disrupted, by homologous recombination with an altered
HA gene. In another embodiment, an endogenous or introduced HA gene
in a microorganism has been altered by one or more point mutations,
deletions, or inversions, but still encodes a functional HA
protein. In still another embodiment, one or more of the regulatory
regions (e.g., a promoter, repressor, or inducer) of an HA gene in
a microorganism has been altered (e.g., by deletion, truncation,
inversion, or point mutation) such that the expression of the HA
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.
[0020] 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.
[0021] Still another aspect of the invention pertains to an
isolated HA protein or a portion, e.g., a biologically active
portion, thereof. In a preferred embodiment, the isolated HA
protein or portion thereof can participate in the maintenance of
homeostasis in C. glutamicum, or can perform a function involved in
the adaptation of this microorganism to different environmental
conditions. In another preferred embodiment, the isolated HA
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 maintenance of
homeostasis in C. glutamicum, or to perform a function involved in
the adaptation of this microorganism to different environmental
conditions.
[0022] The invention also provides an isolated preparation of an HA
protein. In preferred embodiments, the HA 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 HA 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 maintenance of homeostasis in C.
glutamicum, or to perform a function involved in the adaptation of
this microorganism to different environmental conditions, or has
one or more of the activities set forth in Table 1.
[0023] Alternatively, the isolated HA 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 HA proteins also have one or more of the HA
bioactivities described herein.
[0024] The HA polypeptide, or a biologically active portion
thereof, can be operatively linked to a non-HA polypeptide to form
a fusion protein. In preferred embodiments, this fusion protein has
an activity which differs from that of the HA protein alone. In
other preferred embodiments, this fusion protein participates in
the maintenance of homeostasis in C. glutamicum, or performs a
function involved in the adaptation of this microorganism to
different environmental conditions. In particularly preferred
embodiments, integration of this fusion protein into a host cell
modulates production of a desired compound from the cell.
[0025] In another aspect, the invention provides methods for
screening molecules which modulate the activity of an HA protein,
either by interacting with the protein itself or a substrate or
binding partner of the HA protein, or by modulating the
transcription or translation of an HA nucleic acid molecule of the
invention.
[0026] Another aspect of the invention pertains to a method for
producing a fine chemical. This method involves the culturing of a
cell containing a vector directing the expression of an HA nucleic
acid molecule of the invention, such that a fine chemical is
produced. In a preferred embodiment, this method further includes
the step of obtaining a cell containing such a vector, in which a
cell is transfected with a vector directing the expression of an HA
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.
[0027] 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
HA protein activity or HA nucleic acid expression such that a cell
associated activity is altered relative to this same activity in
the absence of the agent. In a preferred embodiment, the cell is
modulated for one or more C. glutamicum processes involved in cell
wall biosynthesis or rearrangements, metabolism of inorganic
compounds, modification or degradation of aromatic or aliphatic
compounds, or enzymatic or proteolytic activities. The agent which
modulates' HA protein activity can be an agent which stimulates HA
protein activity or HA nucleic acid expression. Examples of agents
which stimulate HA protein activity or HA nucleic acid expression
include small molecules, active HA proteins, and nucleic acids
encoding HA proteins that have been introduced into the cell.
Examples of agents which inhibit HA activity or expression include
small molecules and antisense HA nucleic acid molecules.
[0028] 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 HA gene into a cell, either
maintained on a separate plasmid or integrated into the genome of
the host cell. If integrated into the genome, such integration can
be random, or it can take place by homologous recombination such
that the native gene is replaced by the introduced copy, causing
the production of the desired compound from the cell to be
modulated. In a preferred embodiment, said yields are increased. In
another preferred embodiment, said chemical is a fine chemical. In
a particularly preferred embodiment, said fine chemical is an amino
acid. In especially preferred embodiments, said amino acid is
L-lysine.
DETAILED DESCRIPTION OF THE INVENTION
[0029] The present invention provides HA nucleic acid and protein
molecules which are involved in C. glutamicum cell wall
biosynthesis or rearrangements, metabolism of inorganic compounds,
modification or degradation of aromatic or aliphatic compounds, or
that have a C. glutamicum enzymatic or proteolytic activity. The
molecules of the invention may be utilized in the modulation of
production of fine chemicals from microorganisms, such as C.
glutamicum, either directly (e.g., where overexpression or
optimization of activity of a protein involved in the production of
a fine chemical (e.g., an enzyme) has a direct impact on the yield,
production, and/or efficiency of production of a fine chemical from
the modified C. glutamicum), or an indirect impact which
nonetheless results in an increase of yield, production, and/or
efficiency of production of the desired compound (e.g., where
modulation of the activity or number of copies of a C. glutamicum
aromatic or aliphatic modification or degradation protein results
in an increase in the viability of C. glutamicum cells, which in
turn permits increased production in a large-scale culture
setting). Aspects of the invention are further explicated
below.
[0030] 1. Fine Chemicals
[0031] 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.
[0032] A. Amino Acid Metabolism and Uses
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] B. Vitamin, Cofactor, and Nutraceutical Metabolism and
Uses
[0038] 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).
[0039] 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, IL X,
374 S).
[0040] 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 Bs),
pantetheine (and its derivatives) and coenzyme A.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] C. Purine, Pyrimidine, Nucleoside and Nucleotide Metabolism
and Uses
[0045] 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).
[0046] 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-mcthionine, 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.
[0047] 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.
[0048] D. Trehalose Metabolism and Uses
[0049] Trehalose consists of two glucose molecules, bound in a,
a-1,1 linkage. It is commonly used in the food industry as a
sweetener, an additive for dried or frozen foods, and in beverages.
However, it also has applications in the pharmaceutical, cosmetics
and biotechnology industries (see, for example, Nishimoto et al.,
(1998) U.S. 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.
[0050] II. Maintenance of Homeostasis in C. glutamicum and
Environmental Adaptation
[0051] The metabolic and other biochemical processes by which cells
function are sensitive to environmental conditions such as
temperature, pressure, solute concentration, and availability of
oxygen. When one or more such environmental condition is perturbed
or altered in a fashion that is incompatible with the normal
functioning of these cellular processes, the cell must act to
maintain an intracellular environment which will permit them to
occur despite the hostile extracellular environment. Gram positive
bacterial cells, such as C. glutamicum cells, have a number of
mechanisms by which internal homeostasis may be maintained despite
unfavorable extracellular conditions. These include a cell wall,
proteins which are able to degrade possibly toxic aromatic and
aliphatic compounds, mechanisms of proteolysis whereby misfolded or
misregulated proteins may be rapidly destroyed, and catalysts which
permit intracellular reactions to occur which would not normally
take place under the conditions optimal for bacterial growth.
[0052] Aside from merely surviving in a hostile environment,
bacterial cells (e.g. C. glutamicum cells) are also frequently able
to adapt such that they are able to take advantage of such
conditions. For example, cells in an environment lacking desired
carbon sources may be able to adapt to growth on a less-suitable
carbon source. Also, cells may be able to utilize less desirable
inorganic compounds when the commonly utilized ones are
unavailable. C. glutamicum cells possess a number of genes which
permit them to adapt to utilize inorganic and organic molecules
which they would normally not encounter under optimal growth
conditions as nutrients and precursors for metabolism. Aspects of
cellular processes involved in homeostasis and adaptation are
further explicated below.
[0053] A. Modification and Degradation of Aromatic and Aliphatic
Compounds
[0054] Bacterial cells are routinely exposed to a variety of
aromatic and aliphatic compounds in nature. Aromatic compounds are
organic molecules having a cyclic ring structure, while aliphatic
compounds are organic molecules having open chain structures rather
than ring structures. Such compounds may arise as by-products of
industrial processes (e.g., benzene or toluene), but may also be
produced by certain microorganisms (e.g., alcohols). Many of these
compounds are toxic to cells, particularly the aromatic compounds,
which are highly reactive due to the high-energy ring structure.
Thus, certain bacteria have developed mechanisms by which they are
able to modify or degrade these compounds such that they are no
longer hazardous to the cell. Cells may possess enzymes that are
able to, for example, hydroxylate, isomerize, or methylate aromatic
or aliphatic compounds such that they are either rendered less
toxic, or such that the modified form is able to be processed by
standard cellular waste and degradation pathways. Also, cells may
possess enzymes which are able to specifically degrade one or more
such potentially hazardous substance, thereby protecting the cell.
Principles and examples of these types of modification and
degradation processes in bacteria are described in several
publications, e.g., Sahm, H. (1999) "Procaryotes in Industrial
Production" in Lengeler, J. W. et al., eds. Biology of the
Procaryotes, Thieme Verlag: Stuttgart; and Schlegel, H. G. (1992)
Allgemeine Mikrobiologie, Thieme: Stuttgart).
[0055] Aside from simply inactivating hazardous aromatic or
aliphatic compounds, many bacteria have evolved to be able to
utilize these compounds as carbon sources for continued metabolism
when the preferred carbon sources of the cell are not available.
For example, Pseudomonas strains able to utilize toluene, benzene,
and 1,10-dichlorodecane as carbon sources are known (Chang, B. V.
et al. (1997) Chemosphere 35(12): 2807-2815; Wischnak, C. et al.
(1998) Appl. Environ. Microbiol. 64(9): 3507-3511; Churchill, S. A.
et al. (1999) Appl. Environ. Microbiol. 65(2): 549-552). There are
similar examples from many other bacterial species which are known
in the art.
[0056] The ability of certain bacteria to modify or degrade
aromatic and aliphatic compounds has begun to be exploited.
Petroleum is a complex mixture of chemicals which includes
aliphatic molecules and aromatic compounds. By applying bacteria
having the ability to degrade or modify these toxic compounds to an
oil spill, for example, it is possible to eliminate much of the
environmental damage with high efficiency and low cost (see, for
example, Smith, M. R. (1990) "The biodegradation of aromatic
hydrocarbons by bacteria" Biodegradation 1(2-3): 191-206; and
Suyama, T. et al. (1998) "Bacterial isolates degrading aliphatic
polycarbonates," FEMS Microbiol. Lett. 161(2): 255-261).
[0057] B. Metabolism of Inorganic Compounds
[0058] Cells (e.g., bacterial cells) contain large quantities of
different molecules, such as water, inorganic ions, and organic
substances (e.g., proteins, sugars, and other macromolecules). The
bulk of the mass of a typical cell consists of only 4 types of
atoms: carbon, oxygen, hydrogen, and nitrogen. Although they
represent a smaller percentage of the content of a cell, inorganic
substances are equally as important to the proper functioning of
the cell. Such molecules include phosphorous, sulfur, calcium,
magnesium, iron, zinc, manganese, copper, molybdenum, tungsten, and
cobalt. Many of these compounds arc critical for the construction
of important molecules, such as nucleotides (phosphorous) and amino
acids (nitrogen and sulfur). Others of these inorganic ions serve
as cofactors for enzymic reactions or contribute to osmotic
pressure. All such molecules must be taken up by the bacterium from
the surrounding environment.
[0059] For each of these inorganic compounds it is desirable for
the bacterium to take up the form which can be most readily used by
the standard metabolic machinery of the cell. However, the
bacterium may encounter environments in which these preferred forms
are not readily available. In order to survive under these
circumstances, it is important for bacteria to have additional
biochemical mechanisms which are able to convert less metabolically
active but readily available forms of these inorganic compounds to
ones which may be used in cellular metabolism. Bacteria frequently
possess a number of genes encoding enzymes for this purpose, which
are not expressed unless the desired inorganic species are not
available. Thus, these genes for the metabolism of various
inorganic compounds serve as another tool which bacteria may use to
adapt to suboptimal environmental conditions.
[0060] After carbon, the most important element in the cell is
nitrogen. A typical bacterial cell contains between 12-15%
nitrogen. It is a constituent of amino acids and nucleotides, as
well as many other important molecules in the cell. Further,
nitrogen may serve as a substitute for oxygen as a terminal
electron acceptor in energy metabolism. Good sources of nitrogen
include many organic and inorganic compounds, such ammonia gas or
ammonia salts (e.g., NH.sub.4Cl, (NH.sub.4).sub.2SO.sub.4, or
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, etc. Ammonia nitrogen is fixed by the
action of particular enzymes: glutamate dehydrogenase, glutamine
synthase, and glutamine-2-oxoglutarate aminotransferase. The
transfer of amino-nitrogen from one organic molecule to another is
accomplished by the aminotransferases, a class of enzymes which
transfer one amino group from an alpha-amino acid to an alpha-keto
acid. Nitrate may be reduced via nitrate reductase, nitrite
reductase, and further redox enzymes until it is converted to
molecular nitrogen or ammonia, which may be readily utilized by the
cell in standard metabolic pathways.
[0061] Phosphorous is typically found intracellularly in both
organic and inorganic forms, and may be taken up by the cell in
either of these forms as well, though most microorganisms
preferentially take up inorganic phosphate. The conversion of
organic phosphate to a form which the cell can utilize requires the
action of phosphatases (e.g., phytases, which hydrolyze
phyate-yielding phosphate and inositol derivatives). Phosphate is a
key element in the synthesis of nucleic acids, and also has a
significant role in cellular energy metabolism (e.g., in the
synthesis of ATP, ADP, and AMP).
[0062] Sulfur is a requirement for the synthesis of amino acids
(e.g., methionine and cysteine), vitamins (e.g., thiamine, biotin,
and lipoic acid) and iron sulfur proteins. Bacteria obtain sulfur
primarily from inorganic sulfate, though thiosulfate, sulfite, and
sulfide are also commonly utilized. Under conditions where these
compounds may not be readily available, many bacteria express genes
which enable them to utilize sulfonate compounds such as
2-aminosulfonate (taurine) (Kertesz, M. A. (1993) "Proteins induced
by sulfate limitation in Escherichia coli, Pseudomonas putido, or
Staphylococcus aureus." J. Bacteriol. 175: 1187-1190).
[0063] Other inorganic atoms, e.g., metal or calcium ions, are also
critical for the viability of cells. Iron, for example, plays a key
role in redox reactions and is a cofactor of iron-sulfur proteins,
heme proteins, and cytochromes. The uptake of iron into bacterial
cells may be accomplished by the action of siderophores, chelating
agents which bind extracellular iron ions and translocate them to
the interior of the cell. For reference on the metabolism of iron
and other inorganic compounds, see: Lengeler et al. (1999) Biology
of Prokaryotes, Thieme Verlag: Stuttgart; Neidhardt, F. C. et al.,
eds. Escherichia coli and Salmonella. ASM Press: Washington, D.C.;
Sonenshein, A. L. et al., eds. (199?) Bacillus subtilis and Other
Gram-Positive Bacteria, ASM Press: Washington, D.C.; Voet, D. and
Voet, J. G. (1992) Biochemie, VCH: Weinheim; Brock, T. D. and
Madigan, M. T. (1991) Biology of Microorgansisms, 6.sup.th ed.
Prentice Hall: Englewood Cliffs, p. 267-269; Rhodes, P. M. and
Stanbury, P. F. Applied Microbial Physiology--A Practical Approach,
Oxford Univ. Press: Oxford.
[0064] C. Enzymes and Proteolysis
[0065] The intracellular conditions for which bacteria such as C.
glutamicum are optimized are frequently not conditions under which
many biochemical reactions would normally take place. In order to
make such reactions proceed under physiological conditions, cells
utilize enzymes. Enzymes are proteinaceous biological catalysts,
spatially orienting reacting molecules or providing a specialized
environment such that the energy barrier to a biochemical reaction
is lowered. Different enzymes catalyze different reactions, and
each enzyme may be the subject of transcriptional, translational,
or posttranslational regulation such that the reaction will only
take place under appropriate conditions and at specified times.
Enzymes may contribute to the degradation (e.g., the proteases),
synthesis (e.g. the synthases), or modification (e.g., transferases
or isomerases) of compounds, all of which enable the production of
necessary compounds within the cell. This, in turn, contributes to
the maintenance of cellular homeostasis.
[0066] However, the fact that enzymes are optimized for activity
under the physiological conditions at which the bacterium is most
viable means that when environmental conditions are perturbed,
there is a significant possibility that enzyme activity will also
be perturbed. For example, changes in temperature may result in
aberrantly folded proteins, and the same is true for changes of
pH--protein folding is largely dependent on electrostatic and
hydrophobic interactions of amino acids within the polypeptide
chain, so any alteration to the charges on individual amino acids
(as might be brought about by a change in cellular pH) may have a
profound effect on the ability of the protein to correctly fold.
Changes in temperature effectively change the amount of kinetic
energy that the polypeptide molecule possesses, which affects the
ability of the polypeptide to settle into a correctly folded,
energetically stable configuration. Misfolded proteins may be
harmful to the cell for two reasons. First, the aberrantly folded
protein may have a similarly aberrant activity, or no activity
whatsoever. Second, misfolded proteins may lack the conformational
regions necessary for proper regulation by other cellular systems
and thus may continue to be active but in an uncontrolled
fashion.
[0067] The cell has a mechanism by which misfolded enzymes and
regulatory proteins may be rapidly destroyed before any damage
occurs to the cell: proteolysis. Proteins such as those of the
la/lon family and those of the Clp family specifically recognize
and degrade misfolded proteins (see, e.g., Sherman, M. Y.,
Goldberg, A. L. (1999) EXS 77: 57-78 and references therein and
Porankiewicz J. (1999) Molec. Microbiol. 32(3): 449-58, and
references therein; Neidhardt, F. C., et al. (1996) E. coli and
Salmonella, ASM Press: Washington, D.C. and references therein; and
Pritchard, G. G., and Coolbear, T. (1993) FEMS Microbiol. Rev.
12(1-3): 179-206 and references therein). These enzymes bind to
misfolded or unfolded proteins and degrade them in an ATP-dependent
manner. Proteolysis thus serves as an important mechanism employed
by the cell to prevent damage to normal cellular functions upon
environmental changes, and it further permits cells to survive
under conditions and in environments which would otherwise be toxic
due to misregulated and/or aberrant enzyme or regulatory
activity.
[0068] Proteolysis also has important functions in the cell under
optimal environmental conditions. Within normal metabolic
processes, proteases aid in the hydrolysis of peptide bonds, in the
catabolism of complex molecules to provide necessary degradation
products, and in protein modification. Secreted proteases play an
important role in the catabolism of external nutrients even prior
to the entry of these compounds into the cell. Further, proteolytic
activity itself may serve regulatory functions; sporulation in B.
subtilis and cell cycle progression in Caulobacter spp. are known
to be regulated by key proteolytic events in each of these species
(Gottesman, S. (1999) Curr. Opin. Microbiol. 2(2): 142-147). Thus,
proteolytic processes are key for cellular survival under both
suboptimal and optimal environmental conditions, and contribute to
the overall maintenance of homeostasis in cells.
[0069] D. Cell Wall Production and Rearrangements
[0070] While the biochemical machinery of the cell may be able to
readily adapt to different and possibly unfavorable environments,
cells still require a general mechanism by which they may be
protected from the environment. For many bacteria, the cell wall
affords such protection, and also plays roles in adhesion, cell
growth and division, and transport of desired solutes and waste
materials.
[0071] In order to function, cells require intracellular
concentrations of metabolites and other molecules that are
substantially higher than those of the surrounding media. Since
these metabolites are largely prevented from leaving the cell due
to the presence of the hydrophobic membrane, the tendency of the
system is for water molecules to enter the cell from the external
medium such that the interior concentrations of solutes match the
exterior concentrations. Water molecules are readily able to cross
the cellular membrane, and this membrane is not able to withstand
the resulting swelling and pressure, which may lead to osmotic
lysis of the cell. The rigidity of the cell wall greatly improves
the ability of the cell to tolerate these pressures, and offers a
further barrier to the unwanted diffusion of these metabolites and
desired solutes from the cell. Similarly, the cell wall also serves
to prevent unwanted material from entering the cell.
[0072] The cell wall also participates in a number of other
cellular processes, such as adhesion and cell growth and division.
Due to the fact that the cell wall completely surrounds the cell,
any interaction of the cell with its surroundings must be mediated
by the cell wall. Thus, the cell wall must participate in any
adherence of the cell to other cells and to desired surfaces.
Further, the cell cannot grow or divide without concomitant changes
in the cell wall. Since the protection that the wall affords
requires its presence during growth, morphogenesis and
multiplication, one of the key steps in cell division is cell wall
synthesis within the cell such that a new cell divides from the
old. Thus, frequently cell wall biosynthesis is regulated in tandem
with cell growth and cell division (see, e.g. Sonenshein, A. L. et
al, eds. (1993) Bacillus subtilis and Other Gram-Positive Bacteria,
ASM: Washington, D.C.).
[0073] The structure of the cell wall varies between gram-positive
and gram-negative bacteria. However, in both types, the fundamental
structural unit of the wall remains similar: an overlapping lattice
of two polysaccharides, N-acetyl glucosamine (NAG) and N- acetyl
muramic acid (NAM) which are cross-linked by amino acids (most
commonly L- alanine, D-glutamate, diaminopimelic acid, and
D-alanine), termed `peptidoglycan`. The processes involved in the
synthesis of the cell wall are known (see, e.g., Michal, G., ed.
(1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, Wiley: New York).
[0074] In gram-negative bacteria, the inner cellular membrane is
coated by a single-layered peptidoglycan (approximately 10 nm
thick), termed the murein-sacculus. This peptidoglycan structure is
very rigid, and its structure determines the shape of the organism.
The outer surface of the murein-sacculus is covered with an outer
membrane, containing porins and other membrane proteins,
phospholipids, and lipopolysaccharides. To maintain a tight
association with the outer membrane, the gram-negative cell wall
also has interspersed lipid molecules which serve to anchor it to
the surrounding membrane.
[0075] In gram-positive bacteria, such as Corynebacterium
glutamicum, the cytoplasmic membrane is covered by a multi-layered
peptidoglycan, which ranges from 20-80 nm in thickness (see, e.g.,
Lengeler et al. (1999) Biology of Prokaryotes Thieme Verlag:
Stuttgart, p. 913-918, p. 875-899, and p. 88-109 and references
therein). The gram-positive cell wall also contains teichoic acid,
a polymer of glycerol or ribitol linked through phosphate groups.
Teichoic acid is also able to associate with amino acids, and forms
covalent bonds with muramic acid. Also present in the cell wall may
be lipoteichoic acids and teichuronic acids. If present, cellular
surface structures such as flagella or capsules will be anchored in
this layer as well.
[0076] III. Elements and Methods of the Invention
[0077] The present invention is based, at least in part, on the
discovery of novel molecules, referred to herein as HA nucleic acid
and protein molecules, which participate in the maintenance of
homeostasis in C. glutamicum, or which perform a function involved
in the adaptation of this microorganism to different environmental
conditions. In one embodiment, the HA molecules participate in C.
glutamicum cell wall biosynthesis or rearrangements, in the
metabolism of inorganic compounds, in the modification or
degradation of aromatic or aliphatic compounds, or have an
enzymatic or proteolytic activity. In a preferred embodiment, the
activity of the HA molecules of the present invention with regard
to C. glutamicum cell wall biosynthesis or rearrangements,
metabolism of inorganic compounds, modification or degradation of
aromatic or aliphatic compounds, or enzymatic or proteolytic
activity has an impact on the production of a desired fine chemical
by this organism. In a particularly preferred embodiment, the HA
molecules of the invention are modulated in activity, such that the
C. glutamicum cellular processes in which the HA molecules
participate (e.g., C. glutamicum cell wall biosynthesis or
rearrangements, metabolism of inorganic compounds, modification or
degradation of aromatic or aliphatic compounds, or enzymatic or
proteolytic activity) are also altered in activity, resulting
either directly or indirectly in a modulation of the yield,
production, and/or efficiency of production of a desired fine
chemical by C. glutamicum.
[0078] The language, "HA protein" or "HA polypeptide" includes
proteins which participate in a number of cellular processes
related to C. glutamicum homeostasis or the ability of C.
glutamicum cells to adapt to unfavorable environmental conditions.
For example, an HA protein may be involved in C. glutamicum cell
wall biosynthesis or rearrangements, in the metabolism of inorganic
compounds in C. glutamicum, in the modification or degradation of
aromatic or aliphatic compounds in C. glutamicum, or have a C.
glutamicum enzymatic or proteolytic activity. Examples of HA
proteins include those encoded by the HA genes set forth in Table I
and Appendix A. The terms "HA gene" or "HA nucleic acid sequence"
include nucleic acid sequences encoding an HA protein, which
consist of a coding region and also corresponding untranslated 5'
and 3' sequence regions. Examples of HA 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 term "homeostasis" is art-recognized
and includes all of the mechanisms utilized by a cell to maintain a
constant intracellular environment despite the prevailing
extracellular environmental conditions. A non-limiting example of
such processes is the utilization of a cell wall to prevent osmotic
lysis due to high intracellular solute concentrations. The term
"adaptation" or "adaptation to an environmental condition" is
art-recognized and includes mechanisms utilized by the cell to
render the cell able to survive under nonpreferred environmental
conditions (generally speaking, those environmental conditions in
which one or more favored nutrients are absent, or in which an
environmental condition such as temperature, pH, osmolarity, oxygen
percentage and the like fall outside of the optimal survival range
of the cell). Many cells, including C. glutamicum cells, possess
genes encoding proteins which are expressed under such
environmental conditions and which permit continued growth in such
suboptimal conditions.
[0079] In another embodiment, the HA molecules of the invention are
capable of modulating the production of a desired molecule, such as
a fine chemical, in a microorganism such as C. glutamicum. There
are a number of mechanisms by which the alteration of an HA protein
of the invention may directly affect the yield, production, and/or
efficiency of production of a fine chemical from a C. glutamicum
strain incorporating such an altered protein. For example, by
engineering enzymes which modify or degrade aromatic or aliphatic
compounds such that these enzymes are increased or decreased in
activity or number, it may be possible to modulate the production
of one or more fine chemicals which are the modification or
degradation products of these compounds. Similarly, enzymes
involved in the metabolism of inorganic compounds provide key
molecules (e.g. phosphorous, sulfur, and nitrogen molecules) for
the biosynthesis of such fine chemicals as amino acids, vitamins,
and nucleic acids. By altering the activity or number of these
enzymes in C. glutamicum, it may be possible to increase the
conversion of these inorganic compounds (or to use alternate
inorganic compounds) to thus permit improved rates of incorporation
of inorganic atoms into these fine chemicals. Genetic engineering
of C. glutamicum enzymes involved in general cellular processes may
also directly improve fine chemical production, since many of these
enzymes directly modify fine chemicals (e.g., amino acids) or the
enzymes which are involved in fine chemical synthesis or secretion.
Modulation of the activity or number of cellular proteases may also
have a direct effect on fine chemical production, since many
proteases may degrade fine chemicals or enzymes involved in fine
chemical production or breakdown.
[0080] Further, the aforementioned enzymes which participate in
aromatic/aliphatic compound modification or degradation, general
biocatalysis, inorganic compound metabolism or proteolysis are each
themselves fine chemicals, desirable for their activity in various
in vitro industrial applications. By altering the number of copies
of the gene for one or more of these enzymes in C. glutamicum it
may be possible to increase the number of these proteins produced
by the cell, thereby increasing the potential yield or efficiency
of production of these proteins from large-scale C. glutamicum or
related bacterial cultures.
[0081] The alteration of an HA protein of the invention may also
indirectly affect the yield, production, and/or efficiency of
production of a fine chemical from a C. glutamicum strain
incorporating such an altered protein. For example, by modulating
the activity and/or number of those proteins involved in the
construction or rearrangement of the cell wall, it may be possible
to modify the structure of the cell wall itself such that the cell
is able to better withstand the mechanical and other stresses
present during large-scale fermentative culture. Also, large-scale
growth of C. glutamicum requires significant cell wall production.
Modulation of the activity or number of cell wall biosynthetic or
degradative enzymes may allow more rapid rates of cell wall
biosynthesis, which in turn may permit increased growth rates of
this microorganism in culture and thereby increase the number of
cells producing the desired fine chemical.
[0082] By modifying the HA enzymes of the invention, one may also
indirectly impact the yield, production, or efficiency of
production of one or more fine chemicals from C. glutamicum. For
example, many of the general enzymes in C. glutamicum may have a
significant impact on global cellular processes (e.g., regulatory
processes) which in turn have a significant effect on fine chemical
metabolism. Similarly, proteases, enzymes which modify or degrade
possibly toxic aromatic or aliphatic compounds, and enzymes which
promote the metabolism of inorganic compounds all serve to increase
the viability of C. glutamicum. The proteases aid in the selective
removal of misfolded or misregulated proteins, such as those that
might occur under the relatively stressful environmental conditions
encountered during large-scale fermentor culture. By altering these
proteins, it may be possible to further enhance this activity and
to improve the viability of C. glutamicum in culture. The
aromatic/aliphatic modification or degradation proteins not only
serve to detoxify these waste compounds (which may be encountered
as impurities in culture medium or as waste products from cells
themselves), but also to permit the cells to utilize alternate
carbon sources if the optimal carbon source is limiting-in the
culture. By increasing their number and/or activity, the survival
of C. glutamicum cells in culture may be enhanced. The inorganic
metabolism proteins of the invention supply the cell with inorganic
molecules required for all protein and nucleotide (among others)
synthesis, and thus are critical for the overall viability of the
cell. An increase in the number of viable cells producing one or
more desired fine chemicals in large-scale culture should result in
a concomitant increase in the yield, production, and/or efficiency
of production of the fine chemical in the culture.
[0083] 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 HA DNAs and the predicted amino acid sequences of the C.
glutamicum HA 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 proteins that participate in C. glutamicum
cell wall biosynthesis or rearrangements, metabolism of inorganic
compounds, modification or degradation of aromatic or aliphatic
compounds, or that have a C. glutamicum enzymatic or proteolytic
activity.
[0084] 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.
[0085] The HA protein or a biologically active portion or fragment
thereof of the invention can participate in the maintenance of
homeostasis in C. glutamicum, or can perform a function involved in
the adaptation of this microorganism to different environmental
conditions, or have one or more of the activities set forth in
Table 1.
[0086] Various aspects of the invention are described in further
detail in the following subsections.
[0087] A. Isolated Nucleic Acid Molecules
[0088] One aspect of the invention pertains to isolated nucleic
acid molecules that encode HA 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 HA-encoding nucleic acid (e.g., HA 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 HA nucleic acid molecule can
contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 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.
[0089] 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 HA 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 an HA nucleotide
sequence can be prepared by standard synthetic techniques, e.g.,
using an automated DNA synthesizer.
[0090] 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 HA DNAs of the invention. This DNA
comprises sequences encoding HA 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.
[0091] 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," "RXN," "RXS, or "RXC" followed by 5 digits (i.e., RXA02458,
RXN00249, RXS00153, or RXC00963). 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 RXA02458, RXN00249, RXS00153, and RXC00963
are translations of the coding regions of the nucleotide sequences
of nucleic acid molecules RXA02458, RXN00249, RXS00153, and
RXC00963, 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.
[0092] Several of the genes of the invention are "F-designated
genes". An F-designated gene includes those genes set forth in
Table I which have an `F` in front of the RXA, RXN, RXS, or RXC
designation. For example, SEQ ID NO:5, designated, as indicated on
Table 1, as "F RXA00249", is an F-designated gene, as are SEQ ID
NOs: 11, 15, and 33 (designated on Table 1 as "F RXA02264", "F
RXA02274", and "F RXA00675", respectively).
[0093] In one embodiment, the nucleic acid molecules of the present
invention are not intended to include those compiled in Table 2. In
the case of the dapD gene, a sequence for this gene was published
in Wehrmann, A., et al. (1998) J. Bacteriol. 180(12): 3159-3165.
However, the sequence obtained by the inventors of the present
application is significantly longer than the published version. It
is believed that the published version relied on an incorrect start
codon, and thus represents only a fragment of the actual coding
region.
[0094] 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.
[0095] 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%, 990% 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.
[0096] 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 an HA protein. The nucleotide sequences determined from
the cloning of the HA genes from C. glutamicum allows for the
generation of probes and primers designed for use in identifying
and/or cloning HA homologues in other cell types and organisms, as
well as HA homologues from other Corynebacteria or related species.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, preferably about 25, more preferably about
40, 50 or 75 consecutive nucleotides of a sense strand of one of
the 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 HA homologues.
Probes based on the HA nucleotide sequences can be used to detect
transcripts or genomic sequences encoding the same or homologous
proteins. In preferred embodiments, the probe further comprises a
label group attached thereto, e.g. the label group can be a
radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-factor. Such probes can be used as a part of a diagnostic test
kit for identifying cells which misexpress an HA protein, such as
by measuring a level of an HA-encoding nucleic acid in a sample of
cells, e.g., detecting HA mRNA levels or determining whether a
genomic HA gene has been mutated or deleted.
[0097] 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 maintenance of
homeostasis in C. glutamicum, or to perform a function involved in
the adaptation of this microorganism to different environmental
conditions. 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 able to participate in the
maintenance of homeostasis in C. glutamicum, or to perform a
function involved in the adaptation of this microorganism to
different environmental conditions. Proteins involved in C.
glutamicum cell wall biosynthesis or rearrangements, metabolism of
inorganic compounds, modification or degradation of aromatic or
aliphatic compounds, or that have a C. glutamicum enzymatic or
proteolytic activity, as described herein, may play a role in the
production and secretion of one or more fine chemicals. Examples of
such activities are also described herein. Thus, "the function of
an HA protein" contributes either directly or indirectly to the
yield, production, and/or efficiency of production of one or more
fine chemicals. Examples of HA protein activities are set forth in
Table 1.
[0098] 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.
[0099] Portions of proteins encoded by the HA nucleic acid
molecules of the invention are preferably biologically active
portions of one of the HA proteins. As used herein, the term
"biologically active portion of an HA protein" is intended to
include a portion, e.g. a domain/motif, of an HA protein that can
participate in the maintenance of homeostasis in C. glutamicum, or
that can perform a function involved in the adaptation of this
microorganism to different environmental conditions, or has an
activity as set forth in Table 1. To determine whether an HA
protein or a biologically active portion thereof can participate in
C. glutamicum cell wall biosynthesis or rearrangements, metabolism
of inorganic compounds, modification or degradation of aromatic or
aliphatic compounds, or has a C. glutamicum enzymatic or
proteolytic activity, 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.
[0100] Additional nucleic acid fragments encoding biologically
active portions of an HA protein can be prepared by isolating a
portion of one of the sequences in Appendix B, expressing the
encoded portion of the HA protein or peptide (e.g., by recombinant
expression in vitro) and assessing the activity of the encoded
portion of the HA protein or peptide.
[0101] 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 HA 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).
[0102] It will be understood by one of ordinary skill in the art
that in one embodiment the sequences of the invention are not meant
to include the sequences of the prior art, such as those Genbank
sequences set forth in Tables 2 or 4 which were available prior to
the present invention. In one embodiment, the invention includes
nucleotide and amino acid sequences having a percent identity to a
nucleotide or amino acid sequence of the invention which is greater
than that of a sequence of the prior art (e.g., a Genbank sequence
(or the protein encoded by such a sequence) set forth in Tables 2
or 4). For example, the invention includes a nucleotide sequence
which is greater than and/or at least 39% identical to the
nucleotide sequence designated RXA00471 (SEQ ID NO:293), a
nucleotide sequence which is greater than and/or at least 41%
identical to the nucleotide sequence designated RXA00500 (SEQ ID
NO:143), and a nucleotide sequence which is greater than and/or at
least 35% identical to the nucleotide sequence designated
RXA00502(SEQ ID NO:147). 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%, 990% or more identical) are also encompassed by the
invention.
[0103] In addition to the C. glutamicum HA 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 HA proteins may exist within
a population (e.g., the C. glutamicum population). Such genetic
polymorphism in the HA gene may exist among individuals within a
population due to natural variation. As used herein, the terms
"gene" and "recombinant gene" refer to nucleic acid molecules
comprising an open reading frame encoding an HA protein, preferably
a C. glutamicum HA protein. Such natural variations can typically
result in 1-5% variance in the nucleotide sequence of the HA gene.
Any and all such nucleotide variations and resulting amino acid
polymorphisms in HA that are the result of natural variation and
that do not alter the functional activity of HA proteins are
intended to be within the scope of the invention.
[0104] Nucleic acid molecules corresponding to natural variants and
non-C. glutamicum homologues of the C. glutamicum HA DNA of the
invention can be isolated based on their homology to the C.
glutamicum HA 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 Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting
example of stringent hybridization conditions are hybridization in
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
HA protein.
[0105] In addition to naturally-occurring variants of the HA
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 HA protein,
without altering the functional ability of the HA 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 HA proteins (Appendix B) without altering the activity of said
HA protein, whereas an "essential" amino acid residue is required
for HA protein activity. Other amino acid residues, however, (e.g.,
those that are not conserved or only semi-conserved in the domain
having HA activity) may not be essential for activity and thus are
likely to be amenable to alteration without altering HA
activity.
[0106] Accordingly, another aspect of the invention pertains to
nucleic acid molecules encoding HA proteins that contain changes in
amino acid residues that are not essential for HA activity. Such HA
proteins differ in amino acid sequence from a sequence contained in
Appendix B yet retain at least one of the HA 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 participating in the maintenance of homeostasis in C.
glutamicum, or of performing a function involved in the adaptation
of this microorganism to different environmental conditions, or has
one or more of the activities set forth in Table 1. Preferably, the
protein encoded by the nucleic acid molecule is at least about
50-60% homologous to 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.
[0107] 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 one protein or nucleic acid for optimal alignment
with the other protein or nucleic acid). The amino acid residues or
nucleotides at corresponding amino acid positions or nucleotide
positions are then compared. When a position in one sequence (e.g.,
one of the 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).
[0108] An isolated nucleic acid molecule encoding an HA 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 an HA protein is
preferably replaced with another amino acid residue from the same
side chain family. Alternatively, in another embodiment, mutations
can be introduced randomly along all or part of an HA coding
sequence, such as by saturation mutagenesis, and the resultant
mutants can be screened for an HA activity described herein to
identify mutants that retain HA 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).
[0109] In addition to the nucleic acid molecules encoding HA
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 HA
coding strand, or to only a portion thereof. In one embodiment, an
antisense nucleic acid molecule is antisense to a "coding region"
of the coding strand of a nucleotide sequence encoding an HA
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. 3
(RXN00249) comprises nucleotides 1 to 957). In another embodiment,
the antisense nucleic acid molecule is antisense to a "noncoding
region" of the coding strand of a nucleotide sequence encoding HA.
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).
[0110] Given the coding strand sequences encoding HA 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 HA
mRNA, but more preferably is an oligonucleotide which is antisense
to only a portion of the coding or noncoding region of HA mRNA. For
example, the antisense oligonucleotide can be complementary to the
region surrounding the translation start site of HA 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 1,2-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).
[0111] The antisense nucleic acid molecules of the invention are
typically administered to a cell or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding an HA 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.
[0112] 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).
[0113] 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 HA mRNA transcripts to thereby
inhibit translation of HA mRNA. A ribozyme having specificity for
an HA-encoding nucleic acid can be designed based upon the
nucleotide sequence of an HA DNA molecule disclosed herein (i.e.,
SEQ ID NO. 3 (RXN00249) 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 an HA-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, HA 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.
[0114] Alternatively, HA gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory
region of an HA nucleotide sequence (e.g., an HA promoter and/or
enhancers) to form triple helical structures that prevent
transcription of an HA 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.
[0115] B. Recombinant Expression Vectors and Host Cells
[0116] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding
an HA 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.
[0117] 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 are 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-, lacl.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
those of ordinary skill in the art that the design of the
expression vector can depend on such factors as the choice of the
host cell to be transformed, the level of expression of protein
desired, etc. The expression vectors of the invention can be
introduced into host cells to thereby produce proteins or peptides,
including fusion proteins or peptides, encoded by nucleic acids as
described herein (e.g., HA proteins, mutant forms of HA proteins,
fusion proteins, etc.).
[0118] The recombinant expression vectors of the invention can be
designed for expression of HA proteins in prokaryotic or eukaryotic
cells. For example, HA genes can be expressed in bacterial cells
such as C. glutamicum, insect cells (using baculovirus expression
vectors), yeast and other fungal cells (see Romanos, M. A. et al.
(1992) "Foreign gene expression in yeast: a review"., Yeast 8:
423-488; van den Hondel, C. A. M. J. J. et al. (1991) "Heterologous
gene expression in filamentous fungi" in: More Gene Manipulations
in Fungi, J. W. Bennet & L. L. Lasure, eds. p. 396-428:
Academic Press: San Diego; and van den Hondel, C. A. M. J. J. &
Punt, P. J. (1991) "Gene transfer systems and vector development
for filamentous fungi, in: Applied Molecular Genetics of Fungi,
Peberdy, J. F. et al., eds., p. 1-28, Cambridge University Press:
Cambridge), algae and multicellular plant cells (see Schmidt, R.
and Willmitzer, L. (1988) High efficiency Agrobacterium
tumefaciens-mediated transformation of Arabidopsis thaliana leaf
and cotyledon explants" Plant Cell Rep.: 583-586), or mammalian
cells. Suitable host cells are discussed further in Goeddel, Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). Alternatively, the recombinant expression
vector can be transcribed and translated in vitro, for example
using T7 promoter regulatory sequences and T7 polymerase.
[0119] Expression of proteins in prokaryotes is most often carried
out with vectors containing constitutive or inducible promoters
directing the expression of either fusion or non-fusion proteins.
Fusion vectors add a number of amino acids to a protein encoded
therein, usually to the amino terminus of the recombinant protein
but also to the C-terminus or fused within suitable regions in the
proteins. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, in fusion expression vectors, a
proteolytic cleavage site is introduced at the junction of the
fusion moiety and the recombinant protein to enable separation of
the recombinant protein from the fusion moiety subsequent to
purification of the fusion protein. Such enzymes, and their cognate
recognition sequences, include Factor Xa, thrombin and
enterokinase.
[0120] 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 fise 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 HA 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
fussion protein can be purified by affinity chromatography using
glutathione-agarose resin. Recombinant HA protein unfused to GST
can be recovered by cleavage of the fusion protein with
thrombin.
[0121] 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, California (1990) 60-89,
nd Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: New York
IBSN 0 444 904018). Target gene expression from the pTrc vector
relies on host RNA polymerase transcription from a hybrid trp-lac
fusion promoter. Target gene expression from the pET 11d 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 BL2 I (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, pU364, 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).
[0122] 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.
[0123] In another embodiment, the HA protein expression vector is a
yeast expression vector. Examples of vectors for expression in
yeast S. cerevisiae 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).
[0124] Alternatively, the HA 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).
[0125] In another embodiment, the HA proteins of the invention may
be expressed in unicellular plant cells (such as algae) or in plant
cells from higher plants (e.g., the spermatophytes, such as crop
plants). Examples of plant expression vectors include those
detailed in: Becker, D., Kemper, E., Schell, J. and Masterson, R.
(1992) "New plant binary vectors with selectable markers located
proximal to the left border", Plant Mol. Biol. 20: 1195-1197; and
Bevan, M. W. (1984) "Binary Agrobacterium vectors for plant
transformation", Nucl. Acid Res. 12: 8711-8721, and include pLGV23,
pGHlac+, pBIN19, pAK2004, and pDH51 (Pouwels et al., eds. (1985)
Cloning Vectors. Elsevier: New York IBSN 0 444 904018).
[0126] 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.
[0127] 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).
[0128] 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 HA 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).
[0129] 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.
[0130] A host cell can be any prokaryotic or eukaryotic cell. For
example, an HA protein can be expressed in bacterial cells such as
C. glutamicum, insect cells, yeast or mammalian cells (such as
Chinese hamster ovary cells (CHO) or COS cells). Other suitable
host cells are known to those of ordinary skill in the art.
Microorganisms related 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.
[0131] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection",
"conjugation" and "transduction" are intended to refer to a variety
of art-recognized techniques for introducing foreign nucleic acid
(e.g., linear DNA or RNA (e.g., a linearized vector or a gene
construct alone without a vector) or nucleic acid in the form of a
vector (e.g., a plasmid, phage, phasmid, phagemid, transposon or
other DNA) into a host cell, including calcium phosphate or calcium
chloride co-precipitation, DEAE-dextran-mediated transfection,
lipofection, natural competence, chemical-mediated transfer, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (Molecular Cloning: A
Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),
and other laboratory manuals.
[0132] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Preferred selectable markers
include those which confer resistance to drugs, such as G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable
marker can be introduced into a host cell on the same vector as
that encoding an HA protein or can be introduced on a separate
vector. Cells stably transfected with the introduced nucleic acid
can be identified by, for example, drug selection (e.g., cells that
have incorporated the selectable marker gene will survive, while
the other cells die).
[0133] To create a homologous recombinant microorganism, a vector
is prepared which contains at least a portion of an HA gene into
which a deletion, addition or substitution has been introduced to
thereby alter, e.g., functionally disrupt, the HA gene. Preferably,
this HA gene is a Corynebacterium glutamicum HA 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
HA 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 HA 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 HA protein). In the homologous
recombination vector, the altered portion of the HA gene is flanked
at its 5' and 3' ends by additional nucleic acid of the HA gene to
allow for homologous recombination to occur between the exogenous
HA gene carried by the vector and an endogenous HA gene in a
microorganism. The additional flanking HA 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 HA gene has homologously recombined
with the endogenous HA gene are selected, using art-known
techniques.
[0134] In another embodiment, recombinant microorganisms can be
produced which contain selected systems which allow for regulated
expression of the introduced gene. For example, inclusion of an HA
gene on a vector placing it under control of the lac operon permits
expression of the HA gene only in the presence of IPTG. Such
regulatory systems are well known in the art.
[0135] In another embodiment, an endogenous HA 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
HA gene in a host cell has been altered by one or more point
mutations, deletions, or inversions, but still encodes a functional
HA protein. In still another embodiment, one or more of the
regulatory regions (e.g., a promoter, repressor, or inducer) of an
HA gene in a microorganism has been altered (e.g., by deletion,
truncation, inversion, or point mutation) such that the expression
of the HA gene is modulated. One of ordinary skill in the art will
appreciate that host cells containing more than one of the
described HA gene and protein modifications may be readily produced
using the methods of the invention, and are meant to be included in
the present invention.
[0136] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) an HA protein. Accordingly, the invention further provides
methods for producing HA proteins using the host cells of the
invention. In one embodiment, the method comprises culturing the
host cell of invention (into which a recombinant expression vector
encoding an HA protein has been introduced, or into which genome
has been introduced a gene encoding a wild-type or altered HA
protein) in a suitable medium until HA protein is produced. In
another embodiment, the method further comprises isolating HA
proteins from the medium or the host cell.
[0137] C. Isolated HA Proteins
[0138] Another aspect of the invention pertains to isolated HA
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 HA 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 HA protein having less than about 30% (by
dry weight) of non-HA protein (also referred to herein as a
"contaminating protein"), more preferably less than about 20% of
non-HA protein, still more preferably less than about 10% of non-HA
protein, and most preferably less than about 5% non-HA protein.
When the HA 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 HA 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 HA protein having less than
about 30% (by dry weight) of chemical precursors or non-HA
chemicals, more preferably less than about 20% chemical precursors
or non-HA chemicals, still more preferably less than about 10%
chemical precursors or non-HA chemicals, and most preferably less
than about 5% chemical precursors or non-HA chemicals. In preferred
embodiments, isolated proteins or biologically active portions
thereof lack contaminating proteins from the same organism from
which the HA protein is derived. Typically, such proteins are
produced by recombinant expression of, for example, a C. glutamicum
HA protein in a microorganism such as C. glutamicum.
[0139] An isolated HA protein or a portion thereof of the invention
can participate in the repair or recombination of DNA, in the
transposition of genetic material, in gene expression (i.e., the
processes of transcription or translation), in protein folding, or
in protein secretion in Corynebacterium glutamicum, 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 participate in the maintenance of homeostasis in C.
glutamicum, or to perform a function involved in the adaptation of
this microorganism to different environmental conditions. The
portion of the protein is preferably a biologically active portion
as described herein. In another preferred embodiment, an HA protein
of the invention has an amino acid sequence shown in Appendix B. In
yet another preferred embodiment, the HA 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 HA 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 HA
proteins of the present invention also preferably possess at least
one of the HA activities described herein. For example, a preferred
HA 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 maintenance of homeostasis in C.
glutamicum, or can perform a function involved in the adaptation of
this microorganism to different environmental conditions, or which
has one or more of the activities set forth in Table 1.
[0140] In other embodiments, the HA 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 HA 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 HA 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.
[0141] Biologically active portions of an HA protein include
peptides comprising amino acid sequences derived from the amino
acid sequence of an HA protein, e.g., the an amino acid sequence
shown in Appendix B or the amino acid sequence of a protein
homologous to an HA protein, which include fewer amino acids than a
full length HA protein or the full length protein which is
homologous to an HA protein, and exhibit at least one activity of
an HA protein. Typically, biologically active portions (peptides,
e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35, 36,
37, 38, 39, 40, 50, 100 or more amino acids in length) comprise a
domain or motif with at least one activity of an HA protein.
Moreover, other biologically active portions, in which other
regions of the protein are deleted, can be prepared by recombinant
techniques and evaluated for one or more of the activities
described herein. Preferably, the biologically active portions of
an HA protein include one or more selected domains/motifs or
portions thereof having biological activity.
[0142] HA 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 HA protein is expressed in the host cell. The HA
protein can then be isolated from the cells by an appropriate
purification scheme using standard protein purification techniques.
Alternative to recombinant expression, an HA protein, polypeptide,
or peptide can be synthesized chemically using standard peptide
synthesis techniques. Moreover, native HA protein can be isolated
from cells (e.g., endothelial cells), for example using an anti-HA
antibody, which can be produced by standard techniques utilizing an
HA protein or fragment thereof of this invention.
[0143] The invention also provides HA chimeric or fusion proteins.
As used herein, an HA "chimeric protein" or "fusion protein"
comprises an HA polypeptide operatively linked to a non-HA
polypeptide. An "HA polypeptide" refers to a polypeptide having an
amino acid sequence corresponding to an HA protein, whereas a
"non-HA polypeptide" refers to a polypeptide having an amino acid
sequence corresponding to a protein which is not substantially
homologous to the HA protein, e.g., a protein which is different
from the HA 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 HA
polypeptide and the non-HA polypeptide are fused in-frame to each
other. The non-HA polypeptide can be fused to the N-terminus or
C-terminus of the HA polypeptide. For example, in one embodiment
the fusion protein is a GST-HA fusion protein in which the HA
sequences are fused to the C-terminus of the GST sequences. Such
fusion proteins can facilitate the purification of recombinant HA
proteins. In another embodiment, the fusion protein is an HA
protein containing a heterologous signal sequence at its N-
terminus. In certain host cells (e.g., mammalian host cells),
expression and/or secretion of an HA protein can be increased
through use of a heterologous signal sequence.
[0144] Preferably, an HA chimeric or fusion protein of the
invention is produced by standard recombinant DNA techniques. For
example, DNA fragments coding for the different polypeptide
sequences are ligated together in-frame in accordance with
conventional techniques, for example by employing blunt-ended or
stagger-ended termini for ligation, restriction enzyme digestion to
provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. In another embodiment, the fusion
gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and reamplified to
generate a chimeric gene sequence (see, for example, Current
Protocols in Molecular Biology, eds. Ausubel et al. John Wiley
& Sons: 1992). Moreover, many expression vectors are
commercially available that already encode a fusion moiety (e.g., a
GST polypeptide). An HA- encoding nucleic acid can be cloned into
such an expression vector such that the fusion moiety is linked
in-frame to the HA protein.
[0145] Homologues of the HA protein can be generated by
mutagenesis, e.g., discrete point mutation or truncation of the HA
protein. As used herein, the term "homologue" refers to a variant
form of the HA protein which acts as an agonist or antagonist of
the activity of the HA protein. An agonist of the HA protein can
retain substantially the same, or a subset, of the biological
activities of the HA protein. An antagonist of the HA protein can
inhibit one or more of the activities of the naturally occurring
form of the HA protein, by, for example, competitively binding to a
downstream or upstream member of a biochemical cascade which
includes the HA protein, by binding to a target molecule with which
the HA protein interacts, such that no functional interaction is
possible, or by binding directly to the HA protein and inhibiting
its normal activity.
[0146] In an alternative embodiment, homologues of the HA protein
can be identified by screening combinatorial libraries of mutants,
e.g., truncation mutants, of the HA protein for HA protein agonist
or antagonist activity. In one embodiment, a variegated library of
HA variants is generated by combinatorial mutagenesis at the
nucleic acid level and is encoded by a variegated gene library. A
variegated library of HA variants can be produced by, for example,
enzymatically ligating a mixture of synthetic oligonucleotides into
gene sequences such that a degenerate set of potential HA sequences
is expressible as individual polypeptides, or alternatively, as a
set of larger fusion proteins (e.g., for phage display) containing
the set of HA sequences therein. There are a variety of methods
which can be used to produce libraries of potential HA 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 HA 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.
[0147] In addition, libraries of fragments of the HA protein coding
can be used to generate a variegated population of HA fragments for
screening and subsequent selection of homologues of an HA protein.
In one embodiment, a library of coding sequence fragments can be
generated by treating a double stranded PCR fragment of an HA
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 S1 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 HA protein.
[0148] 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 HA 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 HA homologues (Arkin and Yourvan
(1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein
Engineering 6(3):327-331).
[0149] In another embodiment, cell based assays can be exploited to
analyze a variegated HA library, using methods well known in the
art.
[0150] D. Uses and Methods of the Invention
[0151] 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 HA protein regions required for function;
modulation of an HA protein activity, modulation of the metabolism
of one or more inorganic compounds; modulation of the modification
or degradation of one or more aromatic or aliphatic compounds;
modulation of cell wall synthesis or rearrangements; modulation of
enzyme activity or proteolysis; and modulation of cellular
production of a desired compound, such as a fine chemical.
[0152] The HA nucleic acid molecules of the invention have a
variety of uses. First, they may be used to identify an organism as
being Corynebacterium glutamicum or a close relative thereof. Also,
they may be used to identify the presence of C. glutamicum or a
relative thereof in a mixed population of microorganisms. The
invention provides the nucleic acid sequences of a number of C.
glutamicum genes; by probing the extracted genomic DNA of a culture
of a unique or mixed population of microorganisms under stringent
conditions with a probe spanning a region of a C. glutamicum gene
which is unique to this organism, one can ascertain whether this
organism is present. Although Corynebacterium glutamicum itself is
nonpathogenic, it is related to pathogenic species, such as
Corynebacterium diphtheriae. Corynebacterium diphtheriae is the
causative agent of diphtheria, a rapidly developing, acute, febrile
infection which involves both local and systemic pathology. In this
disease, a local lesion develops in the upper respiratory tract and
involves necrotic injury to epithelial cells; the bacilli secrete
toxin which is disseminated through this lesion to distal
susceptible tissues of the body. Degenerative changes brought about
by the inhibition of protein synthesis in these tissues, which
include heart, muscle, peripheral nerves, adrenals, kidneys, liver
and 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.
[0153] 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.
[0154] 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.
[0155] The HA nucleic acid molecules of the invention are also
useful for evolutionary and protein structural studies. The
processes involved in adaptation and the maintenance of homeostasis
in which the molecules of the invention participate are utilized by
a wide variety of species; 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.
[0156] Manipulation of the HA nucleic acid molecules of the
invention may result in the production of HA proteins having
functional differences from the wild-type HA 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.
[0157] The invention provides methods for screening molecules which
modulate the activity of an HA protein, either by interacting with
the protein itself or a substrate or binding partner of the HA
protein, or by modulating the transcription or translation of an HA
nucleic acid molecule of the invention. In such methods, a
microorganism expressing one or more HA 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 HA
protein is assessed.
[0158] The modulation of activity or number of HA proteins involved
in cell wall biosynthesis or rearrangements may impact the
production, yield, and/or efficiency of production of one or more
fine chemicals from C. glutamicum cells. For example, by altering
the activity of these proteins, it may be possible to modulate the
structure or thickness of the cell wall. The cell wall serves in
large measure as a protective device against osmotic lysis and
external sources of injury; by modifying the cell wall it may be
possible to increase the ability of C. glutamicum to withstand the
mechanical and shear force stresses encountered by this
microorganism during large-scale fermentor culture. Further, each
C. glutamicum cell is surrounded by a thick cell wall, and thus, a
significant portion of the biomass present in large scale culture
consists of cell wall. By increasing the rate at which the cell
wall is synthesized or by activating cell wall synthesis (through
genetic engineering of the HA cell wall proteins of the invention)
it may be possible to improve the growth rate of the microorganism.
Similarly, by decreasing the activity or number of proteins
involved in the degradation of cell wall or by decreasing the
repression of cell wall biosynthesis, an overall increase in cell
wall production may be achieved. An increase in the number of
viable C. glutamicum cells (as may be accomplished by any of the
foregoing described protein alterations) should result in increased
numbers of cells producing the desired fine chemical in large-scale
fermentor culture, which should permit increased yields or
efficiency of production of these compounds from the culture.
[0159] The modulation of activity or number of C. glutamicum HA
proteins that participate in the modification or degradation of
aromatic or aliphatic compounds may also have direct or indirect
impacts on the production of one or more fine chemicals from these
cells. Certain aromatic or aliphatic modification or degradation
products are desirable fine chemicals (e.g., organic acids or
modified aromatic and aliphatic compounds); thus, by modifying the
enzymes which perform these modifications (e.g., hydroxylation,
methylation, or isomerization) or degradation reactions, it may be
possible to increase the yields of these desired compounds.
Similarly, by decreasing the activity or number of proteins
involved in pathways which further degrade the modified or
breakdown products of the aforementioned reactions it may be
possible to improve the yields of these fine chemicals from C.
glutamicum cells in culture.
[0160] These aromatic and aliphatic modification and degradative
enzymes are themselves fine chemicals. In purified form, these
enzymes may be used to degrade aromatic and aliphatic compounds
(e.g., toxic chemicals such as petroleum products), either for the
bioremediation of polluted sites, for the engineered decomposition
of wastes, or for the large-scale and economically feasible
production of desired modified aromatic or aliphatic compounds or
their breakdown products, some of which may be conveniently used as
carbon or energy sources for other fine chemical-producing
compounds in culture (see, e.g., Faber, K. (1995)
Biotransformations in Organic Chemistry, Springer: Berlin and
references therein; and Roberts, S. M., ed. (1992-1996) Preparative
Biotransformations, Wiley: Chichester, and references therein). By
genetically altering these proteins such that their regulation by
other cellular mechanisms is lessened or abolished, it may be
possible to increase the overall number or activity of these
proteins, thereby improving not only the yield of these fine
chemicals but also the activity of these harvested proteins.
[0161] The modification of these aromatic and aliphatic modifying
and degradation enzymes may also have an indirect effect on the
production of one or more fine chemical. Many aromatic and
aliphatic compounds (such as those that may be encountered as
impurities in culture media or as waste products from cellular
metabolism) are toxic to cells; by modifying and/or degrading these
compounds such that they may be readily removed or destroyed,
cellular viability should be increased. Further, these enzymes may
modify or degrade these compounds in such a manner that the
resulting products may enter the normal carbon metabolism pathways
of the cell, thus rendering the cell able to use these compounds as
alternate carbon or energy sources. In large-scale culture
situations, when there may be limiting amounts of optimal carbon
sources, these enzymes provide a method by which cells may continue
to grow and divide using aromatic or aliphatic compounds as
nutrients. In either case, the resulting increase in the number of
C. glutamicum cells in the culture producing the desired fine
chemical should in turn result in increased yields or efficiency of
production of the fine chemical(s).
[0162] Modifications in activity or number of HA proteins involved
in the metabolism of inorganic compounds may also directly or
indirectly affect the production of one or more fine chemicals from
C. glutamicum or related bacterial cultures. For example, many
desirable fine chemicals, such as nucleic acids, amino acids,
cofactors and vitamins (e.g., thiamine, biotin, and lipoic acid)
cannot be synthesized without inorganic molecules such as
phosphorous, nitrate, sulfate, and iron. The inorganic metabolism
proteins of the invention permit the cell to obtain these molecules
from a variety of inorganic compounds and to divert them into
various fine chemical biosynthetic pathways. Therefore, by
increasing the activity or number of enzymes involved in the
metabolism of these inorganic compounds, it may be possible to
increase the supply of these possibly limiting inorganic molecules,
thereby directly increasing the production or efficiency of
production of various fine chemicals from C. glutamicum cells
containing such altered proteins. Modification of the activity or
number of inorganic metabolism enzymes of the invention may also
render C. glutamicum able to better utilize limited inorganic
compound supplies, or to utilize nonoptimal inorganic compounds to
synthesize amino acids, vitamins, cofactors, or nucleic acids, all
of which are necessary for continued growth and replication of the
cell. By improving the viability of these cells in large-scale
culture, the number of C. glutamicum cells producing one or more
fine chemicals in the culture may also be increased, in turn
increasing the yields or efficiency of production of one or more
fine chemicals.
[0163] C. glutamicum enzymes for general processes are themselves
desirable fine chemicals. The specific properties of enzymes (i.e.,
regio- and stereospecificity, among others) make them useful
catalysts for chemical reactions in vitro. Either whole C.
glutamicum cells may be incubated with an appropriate substrate
such that the desired product is produced by enzymes in the cell,
or the desired enzymes may be overproduced and purified from C.
glutamicum cultures (or those of a related bacterium) and
subsequently utilized in in vitro reactions in an industrial
setting (either in solution or immobilized on a suitable immobile
phase). In either situation, the enzyme can either be a natural C.
glutamicum protein, or it may be mutagenized to have an altered
activity; typical industrial uses for such enzymes include as
catalysts in the chemical industry (e.g. for synthetic organic
chemistry) as food additives, as feed components, for fruit
processing, for leather preparation, in detergents, in analysis and
medicine, and in the textile industry (see, e.g., Yamada, H. (1993)
"Microbial reactions for the production of useful organic
compounds," Chimica 47: 5-10; Roberts, S. M. (1998) Preparative
biotransformations: the employment of enzymes and whole-cells in
synthetic chemistry," J. Chem. Soc. Perkin Trans. 1: 157-169; Zaks,
A. and Dodds, D. R. (1997) "Application of biocatalysis and
biotransformations to the synthesis of pharmaceuticals," DDT2:
513-531; Roberts, S. M. and Williamson, N. M. (1997) "The use of
enzymes for the preparation of biologically active natural products
and analogues in optically active form," Curr. Organ. Chemistry
1:1-20; Faber, K. (1995) Biotransformations in Organic Chemistry,
Springer: Berlin; Roberts, S. M., ed. (1992-96) Preparative
Biotransformations, Wiley: Chichester; Cheetham, P. S. J. (1995)
"The applications of enzymes in industry" in: Handbook of Enzyme
Biotechnology, 3.sup.rd ed., Wiseman, A., ed., Elis: Horwood, p.
419-552; and Ullmann's Encyclopedia of Industrial Chemistry (1987),
vol. A9, Enzymes, p. 390-457). Thus, by increasing the activity or
number of these enzymes, it may be possible to also increase the
ability of the cell to convert supplied substrates to desired
products, or to overproduce these enzymes for increased yields in
large-scale culture. Further, by mutagenizing these proteins it may
be possible to remove feedback inhibition or other repressive
cellular regulatory controls such that greater numbers of these
enzymes may be produced and activated by the cell, thereby leading
to greater yields, production, or efficiency of production of these
fine chemical proteins from large-scale cultures. Further,
manipulation of these enzymes may alter the activity of one or more
C. glutamicum metabolic pathways, such as those for the
biosynthesis or secretion of one or more fine chemicals.
[0164] Mutagenesis of the proteolytic enzymes of the invention such
that they are altered in activity or number may also directly or
indirectly affect the yield, production, and/or efficiency of
production of one or more fine chemicals from C. glutamicum. For
example, by increasing the activity or number of these proteins, it
may be possible to increase the ability of the bacterium to survive
in large-scale culture, due to an increased ability of the cell to
rapidly degrade proteins misfolded in response to the high
temperatures, nonoptimal pH, and other stresses encountered during
fermentor culture. Increased numbers of cells in these cultures may
result in increased yields or efficiency of production of one or
more desired fine chemicals, due to the relatively larger number of
cells producing these compounds in the culture. Also, C. glutamicum
cells possess multiple cell-surface proteases which serve to break
down external nutrients into molecules which may be more readily
incorporated by the cells as carbon/energy sources or nutrients of
other kinds. An increase in activity or number of these enzymes may
improve this turnover and increase the levels of available
nutrients, thereby improving cell growth or production. Thus,
modifications of the proteases of the invention may indirectly
impact C. glutamicum fine chemical production.
[0165] A more direct impact on fine chemical production in response
to the modification of one or more of the proteases of the
invention may occur when these proteases are involved in the
production or degradation of a desired fine chemical. By decreasing
the activity of a protease which degrades a fine chemical or a
protein involved in the synthesis of a fine chemical it may be
possible to increase the levels of that fine chemical (due to the
decreased degradation or increased synthesis of the compound).
Similarly, by increasing the activity of a protease which degrades
a compound to result in a fine chemical or a protein involved in
the degradation of a fine chemical, a similar result should be
achieved: increased levels of the desired fine chemical from C.
glutamicum cells containing these engineered proteins.
[0166] The aforementioned mutagenesis strategies for HA proteins to
result in increased yields of a fine chemical from C. glutamicum
are not meant to be limiting; variations on these strategies will
be readily apparent to one of ordinary skill in the art. Using such
strategies, and incorporating the mechanisms disclosed herein, the
nucleic acid and protein molecules of the invention may be utilized
to generate C. glutamicum or related strains of bacteria expressing
mutated HA nucleic acid and protein molecules such that the yield,
production, and/or efficiency of production of a desired compound
is improved. This desired compound may be any product produced by
C. glutamicum, which includes the final products of biosynthesis
pathways and intermediates of naturally-occurring metabolic
pathways, as well as molecules which do not naturally occur in the
metabolism of C. glutamicum, but which are produced by a C.
glutamicum strain of the invention.
[0167] 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.
[0168] Exemplification
EXAMPLE 1
Preparation of Total Genomic DNA of Corynebacterium glutamicum ATCC
13032
[0169] 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-1: 140.34 g/l sucrose, 2.46 g/l
MgSO.sub.4x7H.sub.2O, 10 mill KH.sub.2PO.sub.4 solution (100 g/l,
adjusted to pH 6.7 with KOH), 50 ml/l M12 concentrate (10 .mu.l
(NH.sub.4).sub.2SO.sub.4, 1 g/l NaCl, 2 g/l MgSO.sub.4x7H.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.4xH.sub.2O, 10 mg/l
ZnSO.sub.4x7H.sub.2O, 3 mg/l MnCl.sub.2x4H.sub.2O, 30 mg/l
H.sub.3BO.sub.3 20 mg/l CoCl.sub.2x6H.sub.2O, 1 mg/l
NiCl.sub.2x6H.sub.2O, 3 mg/l Na.sub.2MoO.sub.4x2H.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
{fraction (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 Enomic Libraries in Escherichia coli of
Corynebacterium glutamicum ATCC13032
[0170] Using DNA prepared as described in Example 1, cosmid and
plasmid libraries were constructed according to known and well
established methods (see e.g., Sambrook, J. et al (1989) "Molecular
Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press,
or Ausubel, F. M. et al. (1994) "Current Protocols in Molecular
Biology", John Wiley & Sons.)
[0171] Any plasmid or cosmid could be used. Of particular use were
the plasmids pBR322 (Sutcliffe, J. G. (1979) Proc. Natl. Acad. Sci.
USA, 75:3737-3741); pACYC177 (Change & Cohen (1978) J.
Bacteriol 134:1141-1156), plasmids of the pBS series (pBSSK+,
pBSSK- and others; Stratagene, LaJolla, USA), or cosmids as
SuperCosl (Stratagene, LaJolla, USA) or Lorist6 (Gibson, T. J.,
Rosenthal A. and Waterson, R. H. (1987) Gene 53:283-286. Gene
libraries specifically for use in C. glutamicum may be constructed
using plasmid pSL109 (Lee, H.-S. and A. J. Sinskey (1994) J.
Microbiol. Biotechnol. 4: 256-263).
EXAMPLE 3
DNA Sequencing and Computational Functional Analysis
[0172] Genomic libraries as described in Example 2 were used for
DNA sequencing according to standard methods, in particular by the
chain termination method using ABI377 sequencing machines (see
e.g., Fleischman, R. D. et al (1995) "Whole-genome Random
Sequencing and Assembly of Haemophilus Influenzae Rd., Science,
269:496-512). Sequencing primers with the following nucleotide
sequences were used: 5'-GGAAACAGTATGACCATG-3' (SEQ ID NO:441) or
5'-GTAAAACGACGGCCAGT-3' (SEQ ID NO:442).
EXAMPLE 4
In Vivo Mutagenesis
[0173] 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 genes for the DNA
repair system (e.g., mutHLS, mutd, mutT, etc.; for reference, see
Rupp, W. D. (1996) DNA repair mechanisms, in: Escherichia coli and
Salmonella, p. 2277-2294, ASM: Washington.) Such strains are well
known to those of ordinary skill in the art. The use of such
strains is illustrated, for example, in Greener, A. and Callahan,
M. (1994) Strategies 7: 32-34.
EXAMPLE 5
DNA Transfer Between Escherichia coli and Corynebacterium
glutamicum
[0174] 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).
[0175] 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
Schafer, 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).
[0176] 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).
[0177] 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
[0178] 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.
[0179] 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 calorimetric label which may he 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
[0180] 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.
[0181] 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.
[0182] 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.
[0183] 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.
[0184] 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.
[0185] 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 0.5-1.5 using cells grown on agar plates, such
as CM plates (10 .mu.l glucose, 2.5 g/l NaCl, 2 .mu.l urea, 10 g/l
polypeptone, 5 g/l yeast extract, 5 g/l meat extract, 22 g/l NaCl,
2 .mu.l urea, 10 g/l polypeptone, 5 .mu.l yeast extract, 5 .mu.l
meat extract, 22 .mu.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
[0186] 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, .sub.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.l, 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.
[0187] 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.
[0188] 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
[0189] The effect of the genetic modification in C. glutamicum an
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.)
[0190] In addition to the measurement of the final product of
fermentation, it is also possible to analyze other components of
the metabolic pathways utilized for the production of the desired
compound, such as intermediates and side-products, to determine the
overall efficiency of production of the compound. Analysis methods
include measurements of nutrient levels in the medium (e.g.,
sugars, hydrocarbons, nitrogen sources, phosphate, and other ions),
measurements of biomass composition and growth, analysis of the
production of common metabolites of biosynthetic pathways, and
measurement of gasses produced during fermentation. Standard
methods for these measurements are outlined in Applied Microbial
Physiology, A Practical Approach, P. M. Rhodes and P. F. Stanbury,
eds., IRL Press, p. 103-129; 131-163; and 165-192 (ISBN:
0199635773) and references cited therein.
EXAMPLE 10
Purification of the Desired Product from C. glutamicum Culture
[0191] 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.
[0192] 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.
[0193] 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).
[0194] 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 5 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
[0195] 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 HA 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 HA 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.
[0196] 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.
[0197] 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.
[0198] A comparative analysis of the gene sequences of the
invention with those present in Genbank has been performed using
techniques known in the art (see, e.g., Bexevanis and Ouellette,
eds. (1998) Bioinformatics: A Practical Guide to the Analysis of
Genes and Proteins. John Wiley and Sons: New York). The gene
sequences of the invention were compared to genes present in
Genbank in a three-step process. In a first step, a BLASTN analysis
(e.g., a local alignment analysis) was performed for each of the
sequences of the invention against the nucleotide sequences present
in Genbank, and the 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
[0199] 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).
[0200] 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).
[0201] 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:
467470).
[0202] 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.
[0203] 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.
[0204] 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).
[0205] 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)
[0206] 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.
[0207] 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 a. (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.
[0208] 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.
[0209] 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.
[0210] 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.
[0211] 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.
[0212] Equivalents
[0213] 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.
1TABLE 1 Genes in the Application Nucleic Acid Amino Acid
Identification SEQ ID NO SEQ ID NO Code Contig. NT Start NT Stop
Function 1 2 RXA02548 GR00727 3 293 SULFATE ADENYLATE TRANSFERASE
SUBUNIT 2 (EC 2.7.7.4) 3 4 RXN00249 VV0057 36825 35869
ADENYLYLSULFATE KINASE (EC 2.7.1.25) 5 6 F RXA00249 GR00037 8837
7884 ADENYLYLSULFATE KINASE (EC 2.7.1.25) 7 8 RXA01073 GR00300 1274
2104 NH(3)-DEPENDENT NAD(+) SYNTHETASE (EC 6.3.5.1) Urease 9 10
RXN02913 VV0020 8998 8513 UREASE BETA SUBUNIT (EC 3.5.1.5) 11 12 F
RXA02264 GR00655 123 4 UREASE ALPHA SUBUNIT (EC 3.5.1.5) 13 14
RXN02274 VV0020 8509 6800 UREASE ALPHA SUBUNIT (EC 3.5.1.5) 15 16 F
RXA02274 GR00656 3 1604 UREASE ALPHA SUBUNIT (EC 3.5.1.5) 17 18
RXA02265 GR00655 452 153 UREASE GAMMA SUBUNIT (EC 3.5.1.5) 19 20
RXA02278 GR00656 3420 4268 UREASE OPERON URED PROTEIN 21 22
RXA02275 GR00656 1632 2102 UREASE ACCESSORY PROTEIN UREE 23 24
RXA02276 GR00656 2105 2782 UREASE ACCESSORY PROTEIN UREF 25 26
RXA02277 GR00656 2802 3416 UREASE ACCESSORY PROTEIN UREG 27 28
RXA02603 GR00742 7742 8737 4-HYDROXYBENZOATE OCTAPRENYLTRANSFERASE
(EC 2.5.1.--) 29 30 RXA01385 GR00406 5320 3440 PHENOL 2
MONOOXYGENASE (EC 1.14.13.7) Proteolysis 31 32 RXN00675 VV0005
33258 34049 METHIONINE AMINOPEPTIDASE (EC 3.4.11.18) 33 34 F
RXA00675 GR00178 2 484 METHIONINE AMINOPEPTIDASE (EC 3.4.11.18) 35
36 RXA01609 GR00449 2740 3612 METHIONINE AMINOPEPTIDASE (EC
3.4.11.18) 37 38 RXA01358 GR00393 5337 6857 ATP-DEPENDENT PROTEASE
LA (EC 3.4.21.53) 39 40 RXA01458 GR00420 3225 2176 ATP-DEPENDENT
PROTEASE LA (EC 3.4.21.53) 41 42 RXA01654 GR00459 986 1981
(AL022121) putative alkaline serine protease [Mycobacterium
tuberculosis] 43 44 RXN01868 VV0127 9980 11905 ZINC METALLOPROTEASE
(EC 3.4.24.--) 45 46 F RXA01868 GR00534 1640 30 ZINC
METALLOPROTEASE (EC 3.4.24.--) 47 48 F RXA01869 GR00534 1954 1652
ZINC METALLOPROTEASE (EC 3.4.24.--) 49 50 RXN03028 VV0008 41156
43930 ATP-DEPENDENT CLP PROTEASE ATP-BINDING SUBUNIT CLPA 51 52 F
RXA02470 GR00715 2216 3196 ATP-DEPENDENT CLP PROTEASE ATP-BINDING
SUBUNIT CLPA 53 54 F RXA02471 GR00715 3159 4991 ATP-DEPENDENT CLP
PROTEASE ATP-BINDING SUBUNIT CLPA 55 56 RXA02630 GR00748 2654 1332
(AL021999) putative serine protease [Mycobacterium tuberculosis] 57
58 RXA02834 GR00823 3 497 ATPases with chaperone activity,
ATP-dependent protease subunit 59 60 RXA00112 GR00016 3687 2497
PROBABLE PERIPLASMIC SERINE PROTEASE DO-LIKE PRECURSOR 61 62
RXA00566 GR00152 742 137 ATP-DEPENDENT CLP PROTEASE PROTEOLYTIC
SUBUNIT (EC 3.4.21.92) 63 64 RXA00567 GR00152 1388 798
ATP-DEPENDENT CLP PROTEASE PROTEOLYTIC SUBUNIT (EC 3.4.21.92) 65 66
RXN03094 VV0057 1794 43 CLPB PROTEIN 67 68 F RXA01668 GR00464 2205
3920 CLPB PROTEIN 69 70 RXN01120 VV0182 5678 4401 ATP-DEPENDENT CLP
PROTEASE ATP-BINDING SUBUNIT CLPX 71 72 F RXA01120 GR00310 2349
1072 ATP-DEPENDENT CLP PROTEASE ATP-BINDING SUBUNIT CLPX 73 74
RXA00744 GR00202 10722 9781 Periplasmic serine proteases 75 76
RXA00844 GR00228 3620 4453 Hypothetical Secretory Serine Protease
(EC 3.4.21.--) 77 78 RXA01151 GR00324 862 5 ATP-dependent Zn
proteases 79 80 RXA02317 GR00665 9664 9053 PEPTIDASE E (EC
3.4.--.--) 81 82 RXA02644 GR00751 767 117 XAA-PRO DIPEPTIDASE (EC
3.4.13.9) 83 84 RXN02820 VV0131 4799 6109
GAMMA-GLUTAMYLTRANSPEPTIDASE (EC 2.3.2.2) 85 86 F RXA02820 GR00801
1 507 GAMMA-GLUTAMYLTRANSPEPTIDASE (EC 2.3.2.2) 87 88 F RXA02000
GR00589 3430 3933 GAMMA-GLUTAMYLTRANSPEPTIDASE (EC 2.3.2.2) 89 90
RXN03178 VV0334 921 121 PENICILLIN-BINDING PROTEIN 5* PRECURSOR
(D-ALANYL- D-ALANINE CARBOXYPEPTIDASE) (EC 3.4.16.4) 91 92 F
RXA02859 GR10005 846 121 PENICILLIN-BINDING PROTEIN 5* PRECURSOR
(D-ALANYL- D-ALANINE CARBOXYPEPTIDASE) (EC 3.4.16.4) 93 94 RXA00137
GR00022 738 1826 XAA-PRO AMINOPEPTIDASE (EC 3.4.11.9) 95 96
RXN00499 VV0086 8158 9438 PROLINE IMINOPEPTIDASE (EC 3.4.11.5) 97
98 F RXA00499 GR00125 3 959 PROLINE IMINOPEPTIDASE 99 100 RXN00877
VV0099 2221 3885 PEPTIDYL-DIPEPTIDASE DCP (EC 3.4.15.5) 101 102 F
RXA00877 GR00242 3 1067 PEPTIDYL-DIPEPTIDASE DCP (EC 3.4.15.5) 103
104 RXN01014 VV0209 13328 10728 AMINOPEPTIDASE N (EC 3.4.11.2) 105
106 F RXA01014 GR00289 3 1580 AMINOPEPTIDASE N (EC 3.4.11.2) 107
108 F RXA01018 GR00290 2289 3152 AMINOPEPTIDASE N (EC 3.4.11.2) 109
110 RXA01147 GR00323 1353 94 VACUOLAR AMINOPEPTIDASE I PRECURSOR
(EC 3.4.11.1) 111 112 RXA01161 GR00329 1253 117 XAA-PRO
AMINOPEPTIDASE (EC 3.4.11.9) 113 114 RXN01181 VV0065 1 957
AMINOPEPTIDASE A/I (EC 3.4.11.1) 115 116 F RXA01181 GR00337 1 957
AMINOPEPTIDASE 117 118 RXN01277 VV0009 32155 34158 PROLYL
ENDOPEPTIDASE (EC 3.4.21.26) 119 120 F RXA01277 GR00368 1738 50
PROLYL ENDOPEPTIDASE (EC 3.4.21.26) 121 122 RXA01914 GR00548 125
550 AMINOPEPTIDASE 123 124 RXA02048 GR00624 207 1580 AMINOPEPTIDASE
N (EC 3.4.11.2) 125 126 RXN00621 VV0135 5853 5071 PROTEASE II (EC
3.4.21.83) 127 128 F RXA00621 GR00163 4075 4857 PTRB periplasmic
protease 129 130 RXN00622 VV0135 5150 3735 PROTEASE II (EC
3.4.21.83) 131 132 F RXA00622 GR00163 4778 6193 PTRB periplasmic
protease 133 134 RXN00982 VV0149 7596 6091 (L42758) proteinase
[Streptomyces lividans] 135 136 F RXA00977 GR00275 1647 2660
(L42758) proteinase [Streptomyces lividans] 137 138 F RXA00982
GR00276 5194 4949 (L42758) proteinase [Streptomyces lividans] 139
140 RXA00152 GR00023 7175 5880 HFLC PROTEIN (EC 3.4.--.--) 141 142
RXA02558 GR00731 4939 3965 HFLC PROTEIN (EC 3.4.--.--) 143 144
RXA00500 GR00125 969 1643 O-SIALOGLYCOPROTEIN ENDOPEPTIDASE (EC
3.4.24.57) 145 146 RXA00501 GR00125 1643 2149 O-SIALOGLYCOPROTEIN
ENDOPEPTIDASE (EC 3.4.24.57) 147 148 RXA00502 GR00125 2156 3187
O-SIALOGLYCOPROTEIN ENDOPEPTIDASE (EC 3.4.24.57) Enzymes in general
149 150 RXN02589 VV0098 16346 17110 Hypothetical Methyltransferase
(EC 2.1.1.--) 151 152 F RXA02589 GR00741 13804 13040 Predicted
S-adenosylmethionine-dependent methyltransferase 153 154 RXA00226
GR00032 26836 26012 SAM-dependent methyltransferases 155 156
RXN01885 VV0184 2004 2804 Hypothetical Methyltransferase (EC
2.1.1.--) 157 158 F RXA01885 GR00539 1589 2389 SAM-dependent
methyltransferases 159 160 RXA02592 GR00741 18477 17707
SAM-dependent methyltransferases 161 162 RXN01795 VV0093 722 1318
MODIFIKATION METHYLASE (EC 2.1.1.73) 163 164 F RXA01795 GR00507 706
1140 MODIFICATION METHYLASE (EC 2.1.1.73) 165 166 RXA01214 GR00351
1640 3130 LACCASE 1 PRECURSOR (EC 1.10.3.2) 167 168 RXA01250
GR00364 592 5 LACCASE 1 PRECURSOR (EC 1.10.3.2) 169 170 RXA02477
GR00715 10581 11201 CARBONIC ANHYDRASE (EC 4.2.1.1) 171 172
RXN00833 GR00225 374 6 THIOL PEROXIDASE (EC 1.11.1.--) 173 174 F
RXA00833 GR00225 374 6 THIOL PEROXIDASE (EC 1.11.1.--) 175 176
RXA01224 GR00354 4186 5208 2-NITROPROPANE DIOXYGENASE (EC
1.13.11.32) 177 178 RXA01182 GR00337 1363 971 Hypothetical
Oxidoreductase 179 180 RXA02531 GR00726 1226 1936 Hypothetical
Oxidoreductase 181 182 RXN00689 VV0005 22416 20926 BETAINE-ALDEHYDE
DEHYDROGENASE PRECURSOR (EC 1.2.1.8) 183 184 F RXA00689 GR00180
1401 775 BETAINE-ALDEHYDE DEHYDROGENASE PRECURSOR (EC 1.2.1.8) 185
186 RXN03128 VV0120 3 857 MORPHINE 6-DEHYDROGENASE (EC 1.1.1.218)
187 188 F RXA02192 GR00643 2 523 MORPHINE 6-DEHYDROGENASE (EC
1.1.1.218) 189 190 RXA02351 GR00679 132 1070 NITRILOTRIACETATE
MONOOXYGENASE COMPONENT A (EC 1.14.13.--) 191 192 RXN00905 VV0238
8075 8875 N-ACYL-L-AMINO ACID AMIDOHYDROLASE (EC 3.5.1.14) 193 194
F RXA00905 GR00247 2 694 N-ACYL-L-AMINO ACID AMIDOHYDROLASE (EC
3.5.1.14) 195 196 RXA00906 GR00247 630 1133 N-ACYL-L-AMINO ACID
AMIDOHYDROLASE (EC 3.5.1.14) 197 198 RXA00907 GR00247 1143 1265
N-ACYL-L-AMINO ACID AMIDOHYDROLASE (EC 3.5.1.14) 199 200 RXA02101
GR00631 3104 1842 N-ACYL-L-AMINO ACID AMIDOHYDROLASE (EC 3.5.1.14)
201 202 RXN02565 VV0154 14299 13034 N-ACYL-L-AMINO ACID
AMIDOHYDROLASE (EC 3.5.1.14) 203 204 F RXA02565 GR00733 1 342
N-ACYL-L-AMINO ACID AMIDOHYDROLASE (EC 3.5.1.14) 205 206 F RXA02567
GR00734 3 740 N-ACYL-L-AMINO ACID AMIDOHYDROLASE (EC 3.5.1.14) 207
208 RXN03077 VV0043 1729 2913 N-ACYL-L-AMINO ACID AMIDOHYDROLASE
(EC 3.5.1.14) 209 210 F RXA02855 GR10002 1693 2877 N-ACYL-L-AMINO
ACID AMIDOHYDROLASE (EC 3.5.1.14), hippurate hydrolase 211 212
RXA00026 GR00003 3657 5042 Hypothetical Amidohydrolase (EC
3.5.1.--) 213 214 RXA01971 GR00569 963 133 Hypothetical
Metal-Dependent Hydrolase 215 216 RXA01802 GR00509 3461 4291
Predicted hydrolases (HAD superfamily) 217 218 RXN00866 VV0258 3557
4522 Predicted Zn-dependent hydrolases 219 220 F RXA00866 GR00236
3555 4499 Predicted Zn-dependent hydrolases 221 222 RXA02410
GR00703 792 127 Predicted Zn-dependent hydrolases 223 224 RXA00961
GR00267 2 433 SALICYLATE HYDROXYLASE (EC 1.14.13.1) 225 226
RXA00111 GR00016 930 1922 SOLUBLE EPOXIDE HYDROLASE (SEH) (EC
3.3.2.3) 227 228 RXA01932 GR00555 6479 5583 ACETYL-HYDROLASE (EC
3.1.1.--) 229 230 RXA02574 GR00739 833 1840 PUTATIVE SECRETED
HYDROLASE 231 232 RXN00983 VV0231 1796 321 SIALIDASE PRECURSOR (EC
3.2.1.18) 233 234 F RXA00983 GR00278 1200 4 SIALIDASE PRECURSOR (EC
3.2.1.18) 235 236 RXA00984 GR00278 1716 1300 SIALIDASE PRECURSOR
(EC 3.2.1.18) 237 238 RXN02513 VV0193 737 6 SIALIDASE PRECURSOR (EC
3.2.1.18) 239 240 F RXA02513 GR00722 93 824 SIALIDASE PRECURSOR (EC
3.2.1.18) 241 242 RXA00903 GR00246 637 5 Putative epimerase 243 244
RXA01224 GR00354 4186 5208 2-NITROPROPANE DIOXYGENASE (EC
1.13.11.32) 245 246 RXA01571 GR00438 1360 1959 ALCOHOL
DEHYDROGENASE (EC 1.1.1.1) 247 248 RXN02478 VV0119 7564 6350
SIALIDASE PRECURSOR (EC 3.2.1.18) 249 250 RXN00343 VV0125 1118 6
3-OXOSTEROID 1-DEHYDROGENASE (EC 1.3.99.4) 251 252 RXN01555 VV0135
29820 28861 3-OXOSTEROID 1-DEHYDROGENASE (EC 1.3.99.4) 253 254
RXN01166 VV0117 18142 16838 EXTRACELLULAR LIPASE PRECURSOR (EC
3.1.1.3) 255 256 RXN02001 VV0326 630 1787 N-ACYL-L-AMINO ACID
AMIDOHYDROLASE (EC 3.5.1.14) 257 258 RXN03145 VV0142 7561 7115
4-OXALOCROTONATE TAUTOMERASE (EC 5.3.2.--) 259 260 RXN01466 VV0019
7050 6091 ARYLESTERASE (EC 3.1.1.2) 261 262 RXN01145 VV0077 7538
6525 KETOL-ACID REDUCTOISOMERASE (EC 1.1.1.86) 263 264 RXN03088
VV0052 3431 3817 Hypothetical Methyltransferase (EC 2.1.1.--) 265
266 RXN02952 VV0320 1032 1547 PUTATIVE REDUCTASE 267 268 RXN00513
VV0092 1573 653 CARBOXYVINYL-CARBOXYPHOSPHONATE PHOSPHORYLMUTASE
(EC 2.7.8.23) 269 270 RXN01152 VV0136 1740 907
PROTEIN-L-ISOASPARTATE O-METHYLTRANSFERASE (EC 2.1.1.77) 271 272
RXN00787 VV0321 3736 5637 D-AMINO ACID DEHYDROGENASE LARGE SUBUNIT
(EC 1.4.99.1) N-metabolism 273 274 RXN01302 VV0148 2837 2385
NITRATE REDUCTASE ALPHA CHAIN (EC 1.7.99.4) 275 276 F RXA01302
GR00376 370 5 NITRATE REDUCTASE ALPHA CHAIN (EC 1.7.99.4) 277 278
RXN01308 VV0148 2406 4 NITRATE REDUCTASE ALPHA CHAIN (EC 1.7.99.4)
279 280 F RXA01307 GR00377 686 6 NITRATE REDUCTASE ALPHA CHAIN (EC
1.7.99.4) 281 282 F RXA01308 GR00378 1211 6 NITRATE REDUCTASE ALPHA
CHAIN (EC 1.7.99.4) 283 284 RXN01309 VV0158 1 801 NITRATE REDUCTASE
ALPHA CHAIN (EC 1.7.99.4) 285 286 F RXA01309 GR00379 719 51 NITRATE
REDUCTASE ALPHA CHAIN (EC 1.7.99.4) 287 288 RXA02017 GR00610 1731
1048 NITRATE REDUCTASE ALPHA CHAIN (EC 1.7.99.4) 289 290 RXA02018
GR00610 2788 1739 NITRATE REDUCTASE BETA CHAIN (EC 1.7.99.4) 291
292 RXA02016 GR00610 1036 260 NITRATE REDUCTASE GAMMA CHAIN (EC
1.7.99.4) 293 294 RXA00471 GR00119 2997 3686 NITRATE/NITRITE
RESPONSE REGULATOR PROTEIN NARL 295 296 RXA00133 GR00021 201 1013
NITRATE/NITRITE RESPONSE REGULATOR PROTEIN NARP 297 298 RXA00650
GR00169 4017 3382 NITRATE/NITRITE RESPONSE REGULATOR PROTEIN NARP
299 300 RXA01189 GR00339 2545 1937 NITRATE/NITRITE RESPONSE
REGULATOR PROTEIN NARP 301 302 RXA01607 GR00449 123 752
NITRATE/NITRITE RESPONSE REGULATOR PROTEIN NARP 303 304 RXN00470
VV0086 27401 28669 NITRATE/NITRITE SENSOR PROTEIN NARX (EC
2.7.3.--) 305 306 F RXA00470 GR00119 1752 2951 NITRATE/NITRITE
SENSOR PROTEIN NARX (EC 2.7.3.--) 307 308 RXA00756 GR00203 2932
1937 N UTILIZATION SUBSTANCE PROTEIN A 309 310 RXA00139 GR00022
2514 3224 N UTILIZATION SUBSTANCE PROTEIN B 311 312 RXA01303
GR00376 1724 390 NITRITE EXTRUSION PROTEIN 313 314 RXA01412 GR00412
620 417 NITROGEN FIXATION PROTEIN FIXI (PROBABLE E1-E2 TYPE CATION
ATPASE) (EC 3.6.1.--) 315 316 RXA00773 GR00205 3208 4350 NITROGEN
REGULATION PROTEIN NIFR3 317 318 RXA02746 GR00764 1 267 NITROGEN
REGULATORY PROTEIN P-II 319 320 RXA02745 GR00763 15350 14472
NODULATION ATP-BINDING PROTEIN I 321 322 RXN00820 VV0054 19455
19817 NODULATION PROTEIN N 323 324 F RXA00820 GR00221 1007 1369
NODULATION PROTEIN N 325 326 RXA01059 GR00296 8782 9390
OXYGEN-INSENSITIVE NAD(P)H NITROREDUCTASE (EC 1.--.--.--) 327 328
RXN01386 VV0008 39246 38317 NITRILASE REGULATOR 329 330 RXN00073
VV0154 2369 687 FERREDOXIN--NITRITE REDUCTASE (EC 1.7.7.1) 331 332
RXN03131 VV0127 276 4 RHIZOPINE CATABOLISM PROTEIN MOCC 333 334
RXS00153 VV0167 4195 4620 NODULATION PROTEIN Urease Phosphate and
Phosphonate metabolism 335 336 RXN01716 VV0319 3259 2774
EXOPOLYPHOSPHATASE (EC 3.6.1.11) 337 338 RXN02972 VV0319 2763 2353
EXOPOLYPHOSPHATASE (EC 3.6.1.11) 339 340 RXN00663 VV0142 10120
11493 PHOH PROTEIN HOMOLOG 341 342 RXN00778 VV0103 18126 19250
PHOSPHATE-BINDING PERIPLASMIC PROTEIN PRECURSOR 343 344 RXN00250
VV0189 286 1032 DEDA PROTEIN-ALKALINE PHOSPHATASE LIKE PROTEIN
Sulfate metabolism 345 346 RXA00072 GR00012 446 6 PHOSPHOADENOSINE
PHOSPHOSULFATE REDUCTASE (EC 1.8.99.4) 347 348 RXA00793 GR00211
1469 2644 SULFATE STARVATION-INDUCED PROTEIN 6 349 350 RXA01192
GR00342 161 733 SULFATE STARVATION-INDUCED PROTEIN 6 351 352
RXA00715 GR00188 2120 2914 THIOSULFATE SULFURTRANSFERASE (EC
2.8.1.1) 353 354 RXA01664 GR00463 1306 485 THIOSULFATE
SULFURTRANSFERASE (EC 2.8.1.1) 355 356 RXN02334 VV0141 7939 7217
THIOSULFATE SULFURTRANSFERASE (EC 2.8.1.1) 357 358 F RXA02334
GR00672 2 355 THIOSULFATE SULFURTRANSFERASE (EC 2.8.1.1)
Fe-Metabolism 359 360 RXN01499 VV0008 7034 3213 ENTEROBACTIN
SYNTHETASE COMPONENT F 361 362 RXN01997 VV0084 33308 33793 FERRITIN
Mg Metabolism 363 364 RXA01848 GR00524 1532 789 MAGNESIUM-CHELATASE
SUBUNIT CHLI 365 366 RXN01849 VV0139 16415 17515
MAGNESIUM-CHELATASE SUBUNIT CHLI 367 368 F RXA01849 GR00524 2004
1555 MAGNESIUM-CHELATASE SUBUNIT CHLI 369 370 F RXA01691 GR00474
570 4 MAGNESIUM-CHELATASE SUBUNIT CHLI 371 372 RXN00665 VV0252 135
635 MG2+/CITRATE COMPLEX SECONDARY TRANSPORTER Modification and
degradation of aromatic compounds 373 374 RXN03026 VV0007 28635
28901 3-DEHYDROQUINATE DEHYDRATASE (EC 4.2.1.10) 375 376 RXN02908
VV0025 8507 8247 O-SUCCINYLBENZOIC ACID-COA LIGASE (EC 6.2.1.26)
377 378 RXN03000 VV0235 570 4 SALICYLATE HYDROXYLASE (EC 1.14.13.1)
379 380 RXN03036 VV0014 671 6 PROTOCATECHUATE 3,4-DIOXYGENASE BETA
CHAIN (EC 1.13.11.3) 381 382 RXN02974 VV0229 12631 12437
4-NITROPHENYLPHOSPHATASE (EC 3.1.3.41) 383 384 RXN00393 VV0025 7241
6348 1,4-DIHYDROXY-2-NAPHTHOATE OCTAPRENYLTRANSFERASE (EC
2.5.--.--) 385 386 RXN00948 VV0107 4266 5384 12-oxophytodienoate
reductase (EC 1.3.1.42) 387 388 RXN01923 VV0020 3384 4133
2-HYDROXY-6-OXO-6-PHENYLHEXA-2,4-DIENOATE HYDROLASE (EC 3.7.1.--)
389 390 RXN00398 VV0025 14633 13884 2-PYRONE-4,6-DICARBOXYLATE
LACTONASE (EC 3.1.1.57) 391 392 RXN02813 VV0128 13120 14118
3-CARBOXY-CIS,CIS-MUCONATE CYCLOISOMERASE HOMOLOG (EC 5.5.1.2) 393
394 RXN00136 VV0134 13373 14467 3-DEHYDROQUINATE SYNTHASE (EC
4.6.1.3) 395 396 RXN02508 VV0007 26733 28586 3-DEHYDROSHIKIMATE
DEHYDRATASE (EC 4.2.1.--) 397 398 RXN02839 VV0362 3 449
4-HYDROXYBENZOATE OCTAPRENYLTRANSFERASE (EC 2.5.1.--) 399 400
RXN00639 VV0128 7858 8712 CATECHOL 1,2-DIOXYGENASE (EC 1.13.11.1)
401 402 RXN02530 VV0057 5469 6125 DIMETHYLANILINE MONOOXYGENASE
(N-OXIDE FORMING) 1 (EC 1.14.13.8) 403 404 RXN00434 VV0112 12078
11212 QUINONE OXIDOREDUCTASE (EC 1.6.5.5) 405 406 RXN01619 VV0050
24649 23675 QUINONE OXIDOREDUCTASE (EC 1.6.5.5) 407 408 RXN01842
VV0234 1615 2532 QUINONE OXIDOREDUCTASE (EC 1.6.5.5) 409 410
RXN00641 VV0128 7440 5950 TOLUATE 1,2-DIOXYGENASE ALPHA SUBUNIT (EC
1.14.12.--) 411 412 RXN01993 VV0182 16 1143 VANILLATE DEMETHYLASE
(EC 1.14.--.--) 413 414 RXN00658 VV0083 15705 16397 PHENOL
2-MONOOXYGENASE (EC 1.14.13.7) 415 416 RXN00178 VV0174 14670 15554
hydroxyquinol 1,2-dioxygenase (EC 1.13.11.37) 417 418 RXN01461
VV0128 12414 13025 PROTOCATECHUATE 3,4-DIOXYGENASE ALPHA CHAIN (EC
1.13.11.3) 419 420 RXN01653 VV0321 12867 11407 DIBENZOTHIOPHENE
DESULFURIZATION ENZYME A 421 422 RXN02053 VV0009 39448 40026 DRGA
PROTEIN 423 424 RXN00177 VV0174 13589 14656 MALEYLACETATE REDUCTASE
(EC 1.3.1.32) 425 426 RXC00963 VV0249 1816 2652 PROTEIN involved in
degradation of aromatic compounds Modification and degradation of
alphatic compounds 427 428 RXN00299 VV0176 43379 42402 ALKANAL
MONOOXYGENASE ALPHA CHAIN (EC 1.14.14.3) 429 430 F RXA00299 GR00048
7376 6633 ALKANAL MONOOXYGENASE ALPHA CHAIN (EC 1.14.14.3) 431 432
RXA00332 GR00057 16086 15385 ALKANAL MONOOXYGENASE ALPHA CHAIN (EC
1.14.14.3) 433 434 RXA01838 GR00519 2 820 ALKANAL MONOOXYGENASE
ALPHA CHAIN (EC 1.14.14.3) 435 436 RXA02643 GR00750 1603 560
ALKANAL MONOOXYGENASE ALPHA CHAIN (EC 1.14.14.3) 437 438 RXA01933
GR00555 6590 7192 2-HALOALKANOIC ACID DEHALOGENASE I (EC 3.8.1.2)
439 440 RXA02351 GR00679 132 1070 NITRILOTRIACETATE MONOOXYGENASE
COMPONENT A (EC 1.14.13.--)
[0214]
2TABLE 2 GENES IDENTIFIED FROM GENBANK GenBank .TM. Accession No.
Gene Name Gene Function Reference A09073 ppg Phosphoenol Bachmann,
B. et al. "DNA fragment coding for phosphoenolpyruvat pyruvate
carboxylase 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;
ftsQ; ftsZ Kobayashi, M. et al. "Cloning, sequencing, and
characterization of the ftsZ gene from coryneform bacteria,"
Biochem. Biophys. Res. Commun., 236(2): 383-388 (1997) AB015023
murC; ftsQ Wachi, M. et al. "A murC gene from Coryneform bacteria,"
Appl. Microbiol. 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; apt; rel
Dipeptide-binding Wehmeier, L. et al. "The role of the
Corynebacterium glutamicum rel gene in protein; adenine (p)ppGpp
metabolism," Microbiology, 144: 1853-1862 1998)
phosphoribosyltransferase; GTP pyrophosphokinase AF041436 argR
Arginine repressor AF045998 impA Inositol monophosphate phosphatase
AF048764 argH Argininosuccinate lyase AF049897 argC; argJ; argB; N-
argD; argF; argR; acetylglutamylphosphate argG; argH reductase;
ornithine acetyltransferase; N- acetylglutamate kinase;
acetylornithine transminase; ornithine carbamoyltransferase;
arginine repressor; argininosuccinate synthase; argininosuccinate
lyase AF050109 inhA Enoyl-acyl carrier protein reductase AF050166
hisG ATP phosphoribosyltransferase AF051846 hisA
Phosphoribosylformimino- 5- amino-1- phosphoribosyl-
4-imidazolecarboxamide isomerase AF052652 metA Homoserine Park, S.
et al. "Isolation and analysis of metA, a methionine biosynthetic
gene O-acetyltransferase 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- Dusch, N. et al.
"Expression of the Corynebacterium glutamicum panD gene
alpha-decarboxylase encoding L-aspartate-alpha-decarboxylase leads
to pantothenate precursor overproduction in Escherichia coli,"
Appl. Environ. Microbiol., 65(4)1530-1539 (1999) AF124518 aroD;
aroE 3-dehydroquinase; shikimate dehydrogenase AF124600 aroC; aroK;
aroB; Chorismate synthase; pepQ shikimate kinase; 3- dehydroquinate
synthase; putative cytoplasmic peptidase AF145897 inhA AF145898
inhA AJ001436 ectP Transport Peter, H. et al. "Corynebacterium
glutamicum is equipped with four secondary of ectoine, carriers for
compatible solutes: Identification, sequencing, and
characterization glycine betaine, of the proline/ectoine uptake
system, ProP, and the ectoine/proline/glycine proline betaine
carrier, EctP," J. Bacteriol., 180(22): 6005-6012 (1998) AJ004934
dapD Tetrahydrodipicolinate Wehrmann, A. et al. "Different modes of
diaminopimelate synthesis and their succinylase (incomplete.sup.i)
role in cell wall integrity: A study with Corynebacterium
glutamicum," J. Bacteriol., 180(12): 3159-3165 (1998) AJ007732 ppc;
secG; Phosphoenolpyruvate- amt; ocd; soxA carboxylase; ?; high
affinity ammonium uptake protein; putative ornithine-
cyclodecarboxylase; sarcosine oxidase AJ010319 ftsY, glnB, Involved
in cell Jakoby, M. et al. "Nitrogen regulation in Corynebacterium
glutamicum; glnD; srp; amtP division; PII protein; Isolation of
genes involved in biochemical characterization of corresponding
uridylyltransferase proteins," FEMS Microbiol., 173(2): 303-310
(1999) (uridylyl- removing enzmye); signal recognition particle;
low affinity ammonium uptake protein AJ132968 cat Chloramphenicol
aceteyl transferase AJ224946 mqo L-malate: quinone Molenaar, D. et
al. "Biochemical and genetic characterization of the oxidoreductase
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 Vertes, A.
A. et al. "Isolation and characterization of IS31831, a
transposable IS31831 element from Corynebacterium glutamicum," Mol.
Microbiol., 11(4): 739-746 (1994) D84102 odhA 2-oxoglutarate Usuda,
Y. et al. "Molecular cloning of the Corynebacterium glutamicum
dehydrogenase (Brevibacterium lactofermentum AJ12036) odhA gene
encoding a novel type of 2-oxoglutarate dehydrogenase,"
Microbiology, 142: 3347-3354 (1996) E01358 hdh; hk Homoserine
Katsumata, R. et al. "Production of L-thereonine and L-isoleucine,"
Patent: JP dehydrogenase; homoserine 1987232392-A 1 Oct. 12, 1987
kinase E01359 Upstream of the start Katsumata, R. et al.
"Production of L-thereonine and L-isoleucine," Patent: JP codon of
homoserine 1987232392-A 2 Oct. 12, 1987 kinase gene E01375
Tryptophan operon E01376 trpL; trpE Leader peptide; Matsui, K. et
al. "Tryptophan operon, peptide and protein coded thereby,
anthranilate synthase utilization of tryptophan operon gene
expression and production of tryptophan," Patent: JP 1987244382-A 1
Oct. 24, 1987 E01377 Promoter and Matsui, K. et al. "Tryptophan
operon, peptide and protein coded thereby, operator regions of
utilization of tryptophan operon gene expression and production of
tryptophan operon 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 Kohama, K. et al. "Gene coding diaminopelargonic
acid aminotransferase and acid aminotransferase 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 Katsumata, R. et al. "Gene manifestation
controlling DNA," Patent: JP lyase N-terminal fragment 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
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synthetase synthetase and its use," Patent: JP 1993184371-A 1 Jul.
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coding Diaminopimelic acid dehydrogenase 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 Kikuchi, T. et al. "Production of
L-phenylalanine by fermentation method," dehydratase Patent: JP
1993344881-A 1 Dec. 27, 1993 E06146 Acetohydroxy acid Inui, M. et
al. "Gene capable of coding Acetohydroxy acid synthetase and its
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Aspartokinase Sugimoto, M. et al. "Mutant aspartokinase gene,"
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aspartokinase Sugimoto, M. et al. "Mutant aspartokinase gene,"
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Mutated aspartokinase Sugimoto, M. et al. "Mutant aspartokinase
gene," patent: JP 1994062866-A 1 alpha subunit 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,
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Aspartokinase released from E08179, released Aspartokinase feedback
inhibition and its utilization," Patent: JP 1994261766-A 1 Sep. 20,
1994 E08180, E08181, E08182 E08232 Acetohydroxy-acid Inui, M. et
al. "Gene DNA coding acetohydroxy acid isomeroreductase,"
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 Hatakeyama, K. et al. "DNA fragment having
promoter function in and desthiobiotin coryneform bacterium,"
Patent: JP 1995031476-A 1 Feb. 03, 1995 synthetase promoter region
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 Madori, M.
et al. "DNA fragment containing gene coding Dihydrodipicolinate
reductase acid reductase and utilization thereof," Patent: JP
1995075578-A 1 Mar. 20, 1995 E08901 Diaminopimelic acid Madori, M.
et al. "DNA fragment containing gene coding Diaminopimelic acid
decarboxylase decarboxylase and utilization thereof," Patent: JP
1995075579-A 1 Mar. 20, 1995 E12594 Serine Hatakeyama, K. et al.
"Production of L-trypophan," Patent: JP 1997028391-A
hydroxymethyltransferase 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; Moriya, M. et al. "Amplification of
gene using artificial transposon," Patent: diaminopimelic JP
1997070291-A Mar. 18, 1997 acid decarboxylase E12767
Dihydrodipicolinic Moriya, M. et al. "Amplification of gene using
artificial transposon," Patent: acid synthetase JP 1997070291-A
Mar. 18, 1997 E12770 aspartokinase Moriya, M. et al. "Amplification
of gene using artificial transposon," Patent: JP 1997070291-A
03/18/97 E12773 Dihydrodipicolinic Moriya, M. et al. "Amplification
of gene using artificial transposon," Patent: acid reductase JP
1997070291-A Mar. 18, 1997 E13655 Glucose-6-phosphate Hatakeyama,
K. et al. "Glucose-6-phosphate dehydrogenase and DNA capable
dehydrogenase of coding the same," Patent: JP 1997224661-A 1 Sep.
02, 1997 L01508 IlvA Threonine dehydratase Moeckel, B. et al.
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Corynebacterium glutamicum," J. Bacteriol., 174: 8065-8072 (1992)
L07603 EC 4.2.1.15 3-deoxy-D- Chen, C. et al. "The cloning and
nucleotide sequence of Corynebacterium arabinoheptulosonate-7-
glutamicum 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase
gene," phosphate synthase FEMS Microbiol. Lett., 107: 223-230
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small subunit; Acetohydroxy acid isomeroreductase L18874 PtsM
Phosphoenolpyruvate sugar Fouet, A et al. "Bacillus subtilis
sucrose-specific enzyme II of the phosphotransferase
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gene encoding the Corynebacterium glutamicum mannose enzyme II and
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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
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Follettie, M. T. et al. "Molecular cloning and nucleotide sequence
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et al. "Structure and function of the trp operon control regions of
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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 O'Regan, M. et al.
"Cloning and nucleotide sequence of the carboxylase
Phosphoenolpyruvate carboxylase-coding gene of Corynebacterium
glutamicum ATCC13032," Gene, 77(2): 237-251 (1989) M85106 23S rRNA
gene Roller, C. et al. "Gram-positive bacteria with a high DNA G +
C content are insertion sequence characterized by a common
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"Gram-positive bacteria with a high DNA G + C content are M85108
insertion sequence characterized by a common insertion within their
23S
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brnQ; Beta C-S lyase; Rossol, I. et al. "The Corynebacterium
glutamicum aecD gene encodes a C-S yhbw branched-chain amino acid
lyase with alpha, beta-elimination activity that degrades
aminoethylcysteine," uptake carrier; J. Bacteriol., 174(9):
2968-2977 (1992); Tauch, A. et al. "Isoleucine uptake in
hypothetical protein yhbw Corynebacterium glutamicum ATCC 13032 is
directed by the brnQ gene product," Arch. Microbiol., 169(4):
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hyperproducing strain of Corynebacterium glutamicum: identification
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J. P. and Dunican, L. K. (1994) Complete nucleotide sequence of the
phosphoribosyltransferase Corynebacterium glutamicum ATCC 21850 tpD
gene." Thesis, Microbiology Department, University College Galway,
Ireland. U13922 cglIM; cglIR; Putative Schafer, A. et al. "Cloning
and characterization of a DNA region encoding a clgIIR type II
5-cytosoine stress-sensitive restriction system from
Corynebacterium glutamicum ATCC methyltransferase; 13032 and
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Schafer, A. et al. "The restriction endonuclease; Corynebacterium
glutamicum cglIM gene encoding a 5-cytosine in an McrBC- putative
type I or type III deficient Escherichia coli strain," Gene,
203(2): 95-101 (1997) restriction endonuclease 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: Ankri, S. et al. "Mutations in the Corynebacterium
glutamicumproline NADP+ 5- biosynthetic pathway: A natural bypass
of the proA step," J. Bacteriol., oxidoreductase 178(15): 4412-4419
(1996) U31230 obg; proB; unkdh ?; gamma glutamyl Ankri, S. et al.
"Mutations in the Corynebacterium glutamicumproline kinase; similar
to D- biosynthetic pathway: A natural bypass of the proA step," J.
Bacteriol., isomer specific 2-hydroxyacid 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;
accBC Thiosulfate Jager, W. et al. "A Corynebacterium glutamicum
gene encoding a two-domain sulfurtransferase; protein similar to
biotin carboxylases and biotin-carboxyl-carrier proteins," acyl CoA
Arch. Microbiol., 166(2); 76-82 (1996) carboxylase U43535 cmr
Multidrug resistance protein Jager, W. et al. "A Corynebacterium
glutamicum gene conferring multidrug resistance in the heterologous
host Escherichia coli," J. Bacteriol., 179(7): 2449-2451 (1997)
U43536 clpB Heat shock ATP-binding protein U53587 aphA-3
3'5"-aminoglycoside phosphotransferase U89648 Corynebacterium
glutamicum unidentified sequence involved in histidine
biosynthesis, partial sequence X04960 trpA; trpB; Tryptophan operon
Matsui, K. et al. "Complete nucleotide and deduced amino acid
sequences of trpC; trpD; the Brevibacterium lactofermentum
tryptophan operon," Nucleic Acids Res., trpE; trpG; trpL 14(24):
10113-10114 (1986) X07563 lys A DAP decarboxylase Yeh, P. et al.
"Nucleic sequence of the lysA gene of Corynebacterium
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modulation of its expression," Mol. decarboxylase, Gen. Genet.,
212(1): 112-119 (1988) EC 4.1.1.20) X14234 EC 4.1.1.31
Phosphoenolpyruvate Eikmanns, B. J. et al. "The Phosphoenolpyruvate
carboxylase gene of carboxylase Corynebacterium glutamicum:
Molecular cloning, nucleotide sequence, and expression," Mol. Gen.
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Phosphoenolpyruvate carboxylase gene family: structure, function
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X17313 fda Fructose-bisphosphate Von der Osten, C. H. et al.
"Molecular cloning, nucleotide sequence and fine- aldolase
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 Bonnassie, S. et al. "Nucleic
sequence of the dapA gene from synthetase (EC Corynebacterium
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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 Marcel, T. et al.
"Nucleotide sequence and organization of the upstream region
synthetase; of the Corynebacterium glutamicum lysA gene," Mol.
Microbiol., 4(11): 1819-1830 Diaminopimelate (1990) decarboxylase
X55994 trpL; trpE Putative leader Heery, D. M. et al. "Nucleotide
sequence of the Corynebacterium glutamicum peptide; anthranilate
trpE gene," Nucleic Acids Res., 18(23): 7138 (1990) synthase
component 1 X56037 thrC Threonine synthase Han, K. S. et al. "The
molecular structure of the Corynebacterium glutamicum threonine
synthase gene," Mol. Microbiol, 4(10): 1693-1702 (1990) X56075
attB-related site Attachment site Cianciotto, N. et al. "DNA
sequence homology between att B-related sites of Corynebacterium
diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum,
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66: 299-302 (1990) X57226 lysC-alpha; Aspartokinase- Kalinowski, J.
et al. "Genetic and biochemical analysis of the Aspartokinase
lysC-beta; alpha subunit; from Corynebacterium glutamicum," Mol.
Microbiol., 5(5): 1197-1204 (1991); asd Aspartokinase-beta subunit;
Kalinowski, J. et al. "Aspartokinase genes lysC alpha and lysC beta
overlap aspartate beta and are adjacent to the aspertate
beta-semialdehyde dehydrogenase gene asd in semialdehyde
Corynebacterium glutamicum," Mol. Gen. Genet., 224(3): 317-324
(1990) dehydrogenase X59403 gap; pgk; tpi Glyceraldehyde- Eikmanns,
B. J. "Identification, sequence analysis, and expression of a
3-phosphate; Corynebacterium glutamicum gene cluster encoding the
three glycolytic phosphoglycerate kinase; enzymes
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Bacteriol., 174(19): 6076-6086 isomerase (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 PsI
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 Bonamy, C. et al. "Identification of IS1206, a
Corynebacterium glutamicum insertion element 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 Eikmanns, B. J. et al. "Cloning sequence
analysis, expression, and inactivation dehydrogenase (NADP+) 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 Heery, D. M.
et al. "A sequence from a tryptophan-hyperproducing strain of
X70584 resistance 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; gluB; gluC; Glutamate uptake system
Kronemeyer, W. et al. "Structure of the gluABCD cluster encoding
the gluD glutamate uptake system of Corynebacterium glutamicum," J.
Bacteriol., 177(5): 1152-1158 (1995) X81379 dapE
Succinyldiaminopimelate Wehrmann, A. et al. "Analysis of different
DNA fragments of desuccinylase 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
Serebrijski, I. et al. "Multicopy suppression by asd gene and
osmotic stress- dehydrogenase; ? dependent complementation by
heterologous proA in proA mutants," J. Bacteriol., 177(24):
7255-7260 (1995) X82929 proA Gamma-glutamyl Serebrijski, I. et al.
"Multicopy suppression by asd gene and osmotic stress- phosphate
reductase 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; dapE Aromatic
amino Wehrmann, A. et al. "Functional analysis of sequences
adjacent to dapE of acid permease; ? Corynebacterium
glutamicumproline reveals the presence of aroP, which encodes the
aromatic amino acid transporter," J. Bacteriol., 177(20): 5991-5993
(1995) X86157 argB; argC; argD; Acetylglutamate Sakanyan, V. et at.
"Genes and enzymes of the acetyl cycle of arginine argF; argJ
kinase; N-acetyl-gamma- biosynthesis in Corynebacterium glutamicum:
enzyme evolution in the early glutamyl-phosphate steps of the
arginine pathway," Microbiology, 142: 99-108 (1996) reductase;
acetylornithine aminotransferase; ornithine carbamoyltransferase;
glutamate N- acetyltransferase X89084 pta, ackA Phosphate
acetyltransferase; Reinscheid, D. J. et al. "Cloning, sequence
analysis, expression and inactivation acetate kinase 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 at.
"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 Siewe, R. M. et al.
"Functional and genetic characterization of the (methyl) transport
system ammonium uptake carrier of Corynebacterium glutamicum," J.
Biol. Chem., 271(10): 5398-5403 (1996) X93514 betP Glycine
betaine
Peter, H. et al. "Isolation, characterization, and expression of
the transport system 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 Vrljic, M. et al. "A new
type of transporter with a new type of cellular exporter protein;
function: L-lysine export from Corynebacterium glutamicum," Mol.
Lysine export Microbiol., 22(5): 815-826 (1996) regulator protein
X96580 panB; panC; xylB 3-methyl-2-oxobutanoate Sahm, H. et al.
"D-pantothenate synthesis in Corynebacterium glutamicum and
hydroxymethyltransferase; use of panBC and genes encoding L-valine
synthesis for D-pantothenate pantoate-beta- overproduction," Appl.
Environ. Microbiol., 65(5): 1973-1979 (1999) alanine ligase;
xylulokinase 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 Ishino, S. et al. "Nucleotide sequence of the
meso-diaminopimelate D- D-dehydrogenase dehydrogenase gene from
Corynebacterium glutamicum," Nucleic Acids Res., (EC 1.4.1.16)
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 Peoples, O.
P. et al. "Nucleotide sequence and fine structural analysis of the
dehydrogenase; Corynebacterium glutamicum hom-thrB operon," Mol.
Microbiol., 2(1): 63-72 homoserine kinase (1988) Y08964 murC;
UPD-N-acetylmuramate- Honrubia, M. P. et al. "Identification,
characterization, and chromosomal ftsQ/divD; ftsZ alanine ligase;
organization of the ftsZ gene from Brevibacterium lactofermentum,"
Mol. Gen. division initiation protein Genet., 259(1): 97-104 (1998)
or cell division protein; cell division protein Y09163 putP High
affinity proline Peter, H. et al. "Isolation of the putP gene of
Corynebacterium transport system 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 Patek, M. et al.
"Analysis of the leuB gene from Corynebacterium dehydrogenase
glutamicum," Appl. Microbiol. Biotechnol., 50(1): 42-47 (1998)
Y12472 Attachment site Moreau, S. et al. "Site-specific integration
of corynephage Phi-16: The bacteriophage Phi-16 construction of an
integration vector," Microbiol., 145: 539-548 (1999) Y12537 proP
Proline/ectoine uptake Peter, H. et al. "Corynebacterium glutamicum
is equipped with four secondary system protein 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 Moreau, S. et al. "Analysis of the
integration functions of φ 304L: An Corynephage 304L integrase
module among corynephages," Virology, 255(1): 150-159 (1999) Z21501
argS; lysA Arginyl-tRNA Oguiza, J. A. et al. "A gene encoding
arginyl-tRNA synthetase is located in the synthetase; upstream
region of the lysA gene in Brevibacterium lactofermentum:
diaminopimelate Regulation of argS-lysA cluster expression by
arginine," J. decarboxylase (partial) Bacteriol., 175(22):
7356-7362 (1993) Z21502 dapA; dapB Dihydrodipicolinate Pisabarro,
A. et al. "A cluster of three genes (dapA, orf2, and dapB) of
synthase; Brevibacterium lactofermentum encodes dihydrodipicolinate
reductase, and a dihydrodipicolinate third polypeptide of unknown
function," J. Bacteriol., 175(9): 2743-2749 reductase (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 Oguiza,
J. A. et al "The galE gene encoding the UDP-galactose 4-epimerase
of UDP-galactose 4- Brevibacterium lactofermentum is coupled
transcriptionally to the dmdR epimerase; diphtheria gene," Gene,
177: 103-107 (1996) toxin regulatory protein Z49824 orfl; 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.
[0215]
3TABLE 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.
[0216]
4TABLE 4 ALIGNMENT RESULTS length % homology Date of ID # (NT)
Genbank Hit Length Accession Name of Genbank Hit Source of Genbank
Hit (GAP) Deposit rxa00026 1509 GB_RO: MMHC310M6 158405 AF109906
Mus musculus MHC class III region RD gene, partial cds; Bf, C2,
G9A, Mus musculus 38,003 10-DEC-1998 NG22, G9, HSP70, HSP70,
HSC70t, and smRNP genes, complete cds; G7A gene, partial cds; and
unknown genes. GB_HTG2: AC007029 119007 AC007029 Homo sapiens clone
DJ0855F16, *** SEQUENCING IN PROGRESS Homo sapiens 37,943 7-Apr-99
***, 1 unordered pieces. GB_HTG2: AC007029 119007 AC007029 Homo
sapiens clone DJ0855F16, *** SEQUENCING IN PROGRESS Homo sapiens
37,943 7-Apr-99 ***, 1 unordered pieces. rxa00072 rxa00111 1116
GB_BA1: SAUSIGA 2748 M94370 Stigmatella aurantiaca sigma factor
(sigA) gene, complete cds. Stigmatella aurantiaca 40,435 16-Aug-94
GB_BA1: SC5B8 28500 AL022374 Streptomyces coelicolor cosmid 5B8.
Streptomyces coelicolor 40,090 22-Apr-98 GB_BA2: AE001767 9086
AE001767 Thermotoga maritima section 79 of 136 of the complete
genome. Thermotoga maritima 35,091 2-Jun-99 rxa00112 1314 GB_EST35:
AU075536 418 AU075536 AU075536 Rice shoot Oryza sativa cDNA clone
S0028_2Z, mRNA Oryza sativa 39,423 7-Jul-99 sequence. GB_GSS9:
AQ157585 647 AQ157585 nbxb0009B16r CUGI Rice BAC Library Oryza
sativa genomic clone Oryza sativa 40,867 12-Sep-98 nbxb0009B16r,
genomic survey sequence. GB_GSS14: AQ510314 542 AQ510314
nbxb0095O05f CUGI Rice BAC Library Oryza sativa genomic clone Oryza
sativa 39,372 04-MAY-1999 nbxb0095O05f, genomic survey sequence.
rxa00133 936 GB_BA1: SC2G5 38404 AL035478 Streptomyces coelicolor
cosmid 2G5. Streptomyces coelicolor 41,170 11-Jun-99 GB_EST7:
W64291 515 W64291 md98h12.r1 Soares mouse embryo NbME13.5 14.5 Mus
musculus Mus musculus 35,306 10-Jun-96 cDNA clone IMAGE: 386087 5'
similar to gb: L26528 Mus musculus Rab11b mRNA, complete cds
(MOUSE);, mRNA sequence. GB_PR3: AC005624 39594 AC005624 Homo
sapiens chromosome 19, cosmid R30017, complete sequence. Homo
sapiens 39,054 6-Sep-98 rxa00137 1212 GB_BA2: AF124600 4115
AF124600 Corynebacterium glutamicum chorismate synthase (aroC),
shikimate Corynebacterium 99,867 04-MAY-1999 kinase (aroK), and
3-dehydroquinate synthase (aroB) genes, complete glutamicum cds;
and putative cytoplasmic peptidase (pepQ) gene, partial cds.
GB_BA1: MTCY159 33818 Z83863 Mycobacterium tuberculosis H37Rv
complete genome; segment Mycobacterium 40,959 17-Jun-98 111/162.
tuberculosis GB_BA1: MT3DEHQ 3437 X59509 M. tuberculosis, genes for
3-dehydroquinate synthase and 3- Mycobacterium 52,583 30-Jun-93
dehydroquinase. tuberculosis rxa00139 834 GB_BA1: BLELONP 738
X99289 B. lactofermentum gene encoding elongation factor P.
Corynebacterium 100,000 1-Nov-97 glutamicum GB_PL1: SPAC24C9 38666
Z98601 S. pombe chromosome I cosmid c24C9. Schizosaccharomyces
35,230 24-Feb-99 pombe GB_HTG1: CEY102A5_1 110000 Z99711
Caenorhabditis elegans chromosome V clone Y102A5, ***
Caenorhabditis elegans 37,775 Z99711 SEQUENCING IN PROGRESS ***, in
unordered pieces. rxa00152 1419 GB_BA1: MTCY277 38300 Z79701
Mycobacterium tuberculosis H37Rv complete genome; segment
Mycobacterium 58,500 17-Jun-98 65/162. tuberculosis GB_BA1: MSGY456
37316 AD000001 Mycobacterium tuberculosis sequence from clone y456.
Mycobacterium 38,913 03-DEC-1996 tuberculosis GB_BA2: AF002133
15437 AF002133 Mycobacterium avium strain GIR10 transcriptional
regulator (mav81) Mycobacterium avium 64,009 26-MAR-1998 gene,
partial cds, aconitase (acn), invasin 1 (inv1), invasin 2 (inv2),
transcriptional regulator (moxR), ketoacyl-reductase (fabG), enoyl-
reductase (inhA) and ferrochelatase (mav272) genes, complete cds.
rxa00226 948 GB_PR3: AC005756 43299 AC005756 Homo sapiens
chromosome 19, fosmid 39347, complete sequence, Homo sapiens 36,209
02-OCT-1998 GB_GSS5: AQ818463 413 AQ818463 HS_5250_A2_B08_SP6E
RPCI-11 Human Male BAC Library Homo Homo sapiens 37,288 26-Aug-99
sapiens genomic clone Plate = 826 Col = 16 Row = C, genomic survey
sequence. GB_GSS5: AQ782337 832 AQ782337 HS_3184_B1_H12_T7C CIT
Approved Human Genomic Sperm Homo sapiens 35,917 2-Aug-99 Library D
Homo sapiens genomic clone Plate = 3184 Col = 23 Row = P, genomic
survey sequence. rxa00249 980 GB_BA2: AF035608 3614 AF035608
Pseudomonas aeruginosa ATP sulfurylase small subunit (cysD) and
Pseudomonas aeruginosa 50,205 1-Jun-98 ATP sulfurylase GTP-binding
subunit/APS kinase (cysN) genes, complete cds. GB_BA1: AB017641
17101 AB017641 Micromonospora griseorubida gene for polyketide
synthase, complete Micromonospora 40,266 2-Apr-99 cds. griseorubida
GB_BA2: AF002133 15437 AF002133 Mycobacterium avium strain GIR10
transcriptional regulator (mav81) Mycobacterium avium 38,429
26-MAR-1998 gene, partial cds, aconitase (acn), invasin 1 (inv1),
invasin 2 (inv2), transcriptional regulator (moxR),
ketoacyl-reductase (fabG), enoyl- reductase (inhA) and
ferrochelatase (mav272) genes, complete cds. rxa00299 1101 GB_BA2:
CORCSLYS 2821 M89931 Corynebacterium glutamicum beta C-S lyase
(aecD) and branched- Corynebacterium 100,000 4-Jun-98 chain amino
acid uptake carrier (brnQ) genes, complete cds, and glutamicum
hypothetical protein Yhbw (yhbw) gene, partial cds. GB_BA1: CGECTP
2719 AJ001436 Corynebacterium glutamicum ectP gene. Corynebacterium
41,143 20-Nov-98 glutamicum GB_BA2: AF181035 5922 AF181035
Rhodobacter sphaeroides glycogen utilization operon, complete
Rhodobacter sphaeroides 36,701 7-Sep-99 sequence. rxa00332 825
GB_BA1: CGTHRC 3120 X56037 Corynebacterium glutamicum thrC gene for
threonine synthase (EC Corynebacterium 37,730 17-Jun-97 4.2.99.2).
glutamicum GB_PAT: I09078 3146 I09078 Sequence 4 from Patent WO
8809819. Unknown. 38,700 02-DEC-1994 GB_PR3: HSJ333B15 73666
AL109954 Human DNA sequence from clone 333B15 on chromosome 20,
Homo sapiens 37,203 23-Nov-99 complete sequence. rxa00470 1392
GB_PL2: DCPCNAM 865 X62977 D. carota mRNA for proliferating cell
nuclear antigen (PCNA). Daucus carota 37,914 30-Sep-99 GB_PL2:
AC006267 101644 AC006267 Arabidopsis thaliana BAC F9M13 from
chromosome IV near 21.5 cM, Arabidopsis thaliana 36,158 27-Apr-99
complete sequence. GB_BA1: TT10SARNA 721 Y15063 Thermus
thermophilus 10Sa RNA gene. Thermus thermophilus 39,494 18-Aug-98
rxa00471 813 GB_BA1: SERERYAA 11219 M63676 S. erythraea first ORF
of eryA gene, complete cds. Saccharopolyspora 38,781 26-Apr-93
erythraea GB_PAT: AR049367 11219 AR049367 Sequence 1 from patent
U.S. Pat. No. 5824513. Unknown. 38,781 29-Sep-99 GB_BA1: SERERYAA
11219 M63676 S. erythraea first ORF of eryA gene, complete cds.
Saccharopolyspora 38,205 26-Apr-93 erythraea rxa00499 1404 GB_PR4:
AC007206 42732 AC007206 Homo sapiens chromosome 19, cosmid R27370,
complete sequence. Homo sapiens 34,982 4-Apr-99 GB_EST26: AI344735
462 AI344735 qp05a10.x1 NCI_CGAP_Kid5 Homo sapiens cDNA clone Homo
sapiens 42,675 2-Feb-99 IMAGE: 1917114 3' similar to gb: M15800
T-LYMPHOCYTE MATURATION-ASSOCIATED PROTEIN (HUMAN);, mRNA sequence.
GB_PR4: AC006479 161837 AC006479 Homo sapiens clone DJ1051J04,
complete sequence. Homo sapiens 38,462 11-Nov-99 rxa00500 798
GB_PR4: AC006111 190825 AC006111 Homo sapiens chromosome 16 clone
RPCI-11_461A8, complete Homo sapiens 40,736 3-Jul-99 sequence.
GB_HTG2: AF128834 196589 AF128834 Homo sapiens chromosome 8 clone
BAC 57G24 map 8p12, *** Homo sapiens 34,062 28-Feb-99 SEQUENCING IN
PROGRESS ***, in unordered pieces. GB_HTG2: AF128834 196589
AF128834 Homo sapiens chromosome 8 clone BAC 57G24 map 8p12, ***
Homo sapiens 34,062 28-Feb-99 SEQUENCING IN PROGRESS ***, in
unordered pieces. rxa00501 630 GB_BA1: D86429 5925 D86429
Saccharopolyspora rectivirgula gene for beta-galactosidase,
complete Saccharopolyspora 53,871 09-DEC-1998 cds. rectivirgula
GB_HTG1: HS1099D15 1301 AL035456 Homo sapiens chromosome 20 clone
RP5-1099D15, *** Homo sapiens 33,546 23-Nov-99 SEQUENCING IN
PROGRESS ***, in unordered pieces. GB_HTG1: HS1099D15 1301 AL035456
Homo sapiens chromosome 20 clone RP5-1099D15, *** Homo sapiens
33,546 23-Nov-99 SEQUENCING IN PROGRESS ***, in unordered pieces.
rxa00502 1155 GB_BA2: U00015 42325 U00015 Mycobacterium leprae
cosmid B1620. Mycobacterium leprae 34,783 01-MAR-1994 GB_BA1:
U00020 36947 U00020 Mycobacterium leprae cosmid B229. Mycobacterium
leprae 34,900 01-MAR-1994 GB_HTG1: HS179I15 210672 Z84464 Homo
sapiens chromosome 13 clone 179I15, *** SEQUENCING IN Homo sapiens
32,898 22-Jan-97 PROGRESS ***, in unordered pieces. rxa00566 729
GB_BA1: MTV008 63033 AL021246 Mycobacterium tuberculosis H37Rv
complete genome; segment Mycobacterium 37,011 17-Jun-98 108/162.
tuberculosis GB_BA2: AF071885 2188 AF071885 Streptomyces coelicolor
ATP-dependent Clp protease proteolytic Streptomyces coelicolor
62,963 29-Jun-99 subunit 1 (clpP1) and ATP-dependent Clp protease
proteolytic subunit 2 (clpP2) genes, complete cds; and
ATP-dependent Clp protease ATP-binding subunit Clpx (clpX) gene,
partial cds. GB_BA2: AF013216 15742 AF013216 Myxococcus xanthus Dog
(dog), isocitrate lyase (icl), Mls (mls), Ufo Myxococcus xanthus
54,683 28-Jan-98 (ufo), fumarate hydratase (fhy), and proteosome
major subunit (clpP) genes, complete cds, and acyl-CoA oxidase
(aco) gene, partial cds. rxa00567 714 GB_BA1: MTV008 63033 AL021246
Mycobacterium tuberculosis H37Rv complete genome; segment
Mycobacterium 42,090 17-Jun-98 108/162. tuberculosis GB_BA1:
CGBPHI16 962 Y12472 C. glutamicum DNA, attachment site
bacteriophage Phi-16. Corynebacterium 40,000 05-MAR-1999 glutamicum
GB_BA1: ECOCLPPA 1236 J05534 Escherichia coli ATP-dependent clp
protease proteolytic component Escherichia coli 52,119 26-Apr-93
(clpP) gene, complete cds. rxa00621 906 GB_EST1: D36491 360 D36491
CELK033GYF Yuji Kohara unpublished cDNA Caenorhabditis elegans
Caenorhabditis elegans 40,390 8-Aug-94 cDNA clone yk33g11 5', mRNA
sequence. GB_IN2: CELC16A3 34968 U41534 Caenorhabditis elegans
cosmid C16A3. Caenorhabditis elegans 35,477 18-MAY-1999 GB_HTG3:
AC009311 160198 AC009311 Homo sapiens clone NH0311L03, ***
SEQUENCING IN PROGRESS Homo sapiens 38,636 13-Aug-99 ***, 3
unordered pieces. rxa00622 1539 GB_BA1: AB004795 3039 AB004795
Pseudomonas sp. gene for dipeptidyl aminopeptidase, complete cds.
Pseudomonas sp. 54,721 5-Feb-99 GB_BA1: MBOPII 2392 D38405
Moraxella lacunata gene for protease II, complete cds. Moraxella
lacunata 50,167 8-Feb-99 GB_IN2: AF078916 2960 AF078916 Trypanosoma
brucei brucei oligopeptidase B (opb) gene, complete Trypanosoma
brucei 48,076 08-OCT-1999 cds. brucei rxa00650 759 GB_BA2: AF161327
2021 AF161327 Corynebacterium diphtheriae histidine kinase ChrS
(chrS) and Corynebacterium 51,319 9-Sep-99 response regulator ChrA
(chrA) genes, complete cds. diphtheriae GB_PL2: ATAC006533 99188
AC006533 Arabidopsis thaliana chromosome II BAC F20M17 genomic
sequence, Arabidopsis thaliana 38,051 26-MAY-1999 complete
sequence. GB_PL2: ATAC006533 99188 AC006533 Arabidopsis thaliana
chromosome II BAC F20M17 genomic sequence, Arabidopsis thaliana
35,403 26-MAY-1999 complete sequence. rxa00675 915 GB_BA1: SC3C8
33095 AL023861 Streptomyces coelicolor cosmid 3C8. Streptomyces
coelicolor 36,836 15-Jan-99 GB_PR3: AC005736 215441 AC005736 Homo
sapiens chromosome 16, BAC clone 462G18 (LANL), complete Homo
sapiens 42,027 01-OCT-1998 sequence. GB_IN2: AC005719 188357
AC005719 Drosophila melanogaster, chromosome 2L, region 38A5-38B4,
BAC Drosophila melanogaster 35,531 27-OCT-1999 clone BACR48M05,
complete sequence. rxa00689 1614 GB_PAT: E07294 2975 E07294 genomic
DNA encoding dehydrogenase of Bacillus Bacillus 45,677 29-Sep-97
stearothermophilus. stearothermophilus GB_BA1: BACALDHT 1975 D13846
B. stearothermophilus aldhT gene for aldehyde dehydrogenase,
Bacillus 45,677 20-Feb-99 complete cds. stearothermophilus GB_BA2:
PPU96338 5276 U96338 Pseudomonas putida NCIMB 9866 plasmid pRA4000
p-cresol Pseudomonas putida 44,317 13-MAY-1999 degradative pathway
genes, p-hydroxybenzaldehyde dehydrogenase (pchA), p-cresol
methylhydroxylase, cytochrome subunit precursor (pchC), unknown
(pchX) and p-cresol methylhydroxylase, flavoprotein subunit (pchF)
genes, complete cds. rxa00715 918 GB_EST30: AI647104 218 AI647104
vn15c01.y1 Stratagene mouse heart (#937316) Mus musculus cDNA Mus
musculus 58,511 29-Apr-99 clone IMAGE: 1021248 5', mRNA sequence.
GB_EST17: AA636159 447 AA636159 vn15c01.r1 Stratagene mouse heart
(#937316) Mus musculus cDNA Mus musculus 41,195 22-OCT-1997 clone
IMAGE: 1021248 5', mRNA sequence. GB_EST10: AA184468 583 AA184468
mt52h05.r1 Stratagene mouse embryonic carcinoma (#937317) Mus Mus
musculus 40,426 12-Feb-97 musculus cDNA clone IMAGE: 633561 5'
similar to gb: D10918 Mouse mRNA for ubiquitin like protein,
partial sequence (MOUSE);, mRNA sequence. rxa00744 1065 GB_HTG3:
AC009855 167592 AC009855 Homo sapiens clone 1_C_5, *** SEQUENCING
IN PROGRESS ***, Homo sapiens 36,673 3-Sep-99 13 unordered pieces.
GB_HTG3: AC009855 167592 AC009855 Homo sapiens clone 1_C_5, ***
SEQUENCING IN PROGRESS ***, Homo sapiens 36,673 3-Sep-99 13
unordered pieces. GB_PR4: AC005082 169739 AC005082 Homo sapiens
clone RG271G13, complete sequence. Homo sapiens 39,557 8-Sep-99
rxa00756 1119 GB_BA1: MLCB596 38426 AL035472 Mycobacterium leprae
cosmid B596. Mycobacterium leprae 54,562 27-Aug-99 GB_GSS12:
AQ368028 652 AQ368028 toxb0001N11rCUGI Tomato BAC Library
Lycopersicon esculentum Lycopersicon esculentum 42,657 5-Feb-99
genomic clone toxb0001N11r, genomic survey sequence. GB_HTG3:
AC008067 151242 AC008067 Homo sapiens clone NH0303104, ***
SEQUENCING IN PROGRESS Homo sapiens 37,239 8-Sep-99 ***, 2
unordered pieces. rxa00773 1266 GB_BA1: MLU15182 40123 U15182
Mycobacterium leprae cosmid B2266. Mycobacterium leprae 36,616
09-MAR-1995 GB_BA1: MSGL611CS 37769 L78822 Mycobacterium leprae
cosmid L611 DNA sequence. Mycobacterium leprae 35,714 15-Jun-96
GB_GSS14: AQ578181 728 AQ578181 nbxb0083P08r CUGI Rice BAC Library
Oryza sativa genomic clone Oryza sativa 39,246 2-Jun-99
nbxb0083P08r, genomic survey sequence. rxa00793 1299 GB_GSS5:
AQ769737 519 AQ769737 HS_3160_A2_G04_T7C CIT Approved Human Genomic
Sperm Homo sapiens 37,765 28-Jul-99 Library D Homo sapiens genomic
clone Plate = 3160 Col = 8 Row = M, genomic survey sequence.
GB_BA1: RTU08434 2400 U08434 Rhizobium trifolii orotate
phosphoribosyltransferase (pyrE) and Rhizobium trifolii 40,700
16-Apr-97 fructokinase (frk) genes, complete cds. GB_EST31: F33810
243 F33810 HSPD27491 HM3 Homo sapiens cDNA clone s3000041E12, mRNA
Homo sapiens 41,564 13-MAY-1999 sequence. rxa00820 486 GB_PR4:
AC005868 96180 AC005868 Homo sapiens 12q24.2 PAC RPCI5-944M2
(Roswell Park Cancer Homo sapiens 32,298 27-Feb-99 Institute Human
PAC Library) complete sequence. GB_EST8: AA000903 396 AA000903
mg38b04.r1 Soares mouse embryo NbME13.5 14.5 Mus musculus Mus
musculus 42,045 18-Jul-96 cDNA clone IMAGE: 426031 5', mRNA
sequence. GB_EST25: AI317789 696 AI317789 uj20g09.y1 Sugano mouse
embryo mewa Mus musculus cDNA clone Mus musculus 38,557 17-DEC-1998
IMAGE: 1920544 5' similar to WP: C13C4.5 CE08130 SUGAR
TRANSPORTER;, mRNA sequence. rxa00833 618 GB_PH: BPH6589 41489
AJ006589 Bacteriophage phi-C31 complete genome. Bacteriophage
phi-C31 41,806 29-Apr-99 GB_HTG2: AC006887 215801 AC006887
Caenorhabditis elegans clone Y59H11, *** SEQUENCING IN
Caenorhabditis elegans 35,798 24-Feb-99 PROGRESS ***, 3 unordered
pieces. GB_HTG2: AC006887 215801 AC006887 Caenorhabditis elegans
clone Y59H11, *** SEQUENCING IN Caenorhabditis elegans 35,798
24-Feb-99 PROGRESS ***, 3 unordered pieces. rxa00844 957 GB_GSS15:
AQ605195 459 AQ605195 HS_2136_B1_C12_T7C CIT Approved Human Genomic
Sperm Homo sapiens 38,074 10-Jun-99 Library D Homo sapiens genomic
clone Plate = 2136 Col = 23 Row = F, genomic survey sequence.
GB_HTG1: CNS00M8S 214599 AL079302
Homo sapiens chromosome 14 clone R-1089B7, *** SEQUENCING Homo
sapiens 38,120 15-OCT-1999 IN PROGRESS ***, in ordered pieces.
GB_HTG1: CNS00M8S 214599 AL079302 Homo sapiens chromosome 14 clone
R-1089B7, *** SEQUENCING Homo sapiens 38,120 15-OCT-1999 IN
PROGRESS ***, in ordered pieces. rxa00866 1066 GB_BA1: CGORF4GEN
2398 X95649 C. glutamicum ORF4 gene. Corynebacterium 99,273
10-MAR-1998 glutamicum GB_BA1: BLDAPAB 3572 Z21502 B.
lactofermentum dapA and dapB genes for dihydrodipicolinate
Corynebacterium 99,301 16-Aug-93 synthase and dihydrodipicolinate
reductase. glutamicum GB_PAT: E14517 1411 E14517 DNA encoding
Brevibacterium dihydrodipicolinic acid reductase. Corynebacterium
99,659 28-Jul-99 glutamicum rxa00877 1788 GB_PAT: I92050 567 I92050
Sequence 17 from patent US 5726299. Unknown. 62,787 01-DEC-1998
GB_PAT: I78760 567 I78760 Sequence 16 from patent US 5693781.
Unknown. 62,787 3-Apr-98 GB_BA2: AE000426 10240 AE000426
Escherichia coli K-12 MG1655 section 316 of 400 of the complete
Escherichia coli 36,456 12-Nov-98 genome. rxa00903 733 GB_BA2:
AE001598 11136 AE001598 Chlamydia pneumoniae section 14 of 103 of
the complete genome. Chlamydophila 32,782 08-MAR-1999 pneumoniae
GB_PL2: AF079370 2897 AF079370 Kluyveromyces lactis invertase
(INV1) gene, complete cds. Kluyveromyces lactis 35,849 4-Aug-99
GB_BA2: AE001598 11136 AE001598 Chlamydia pneumoniae section 14 of
103 of the complete genome. Chlamydophila 40,138 08-MAR-1999
pneumoniae rxa00905 924 GB_PR2: HSQ15C24 73192 AJ239325 Homo
sapiens chromosome 21 from cosmids LLNLc116 1C16 and Homo sapiens
35,076 28-Sep-99 LLNLc116 15C24 map 21q22.3 region D21S171-LA161,
complete sequence. GB_GSS4: AQ691923 446 AQ691923
HS_5400_B2_G04_SP6E RPCI-11 Human Male BAC Library Homo Homo
sapiens 33,500 6-Jul-99 sapiens genomic clone Plate = 976 Col = 8
Row = N, genomic survey sequence. GB_EST37: AI967802 479 AI967802
Ljirnpest12-930-d6 Ljirnp Lambda HybriZap two-hybrid library Lotus
Lotus japonicus 41,127 24-Aug-99 japonicus cDNA clone LP930-12-d6
5' similar to 60S ribosomal protein L7A, mRNA sequence. rxa00906
627 GB_PAT: I78750 588 I78750 Sequence 6 from patent US 5693781.
Unknown. 97,071 3-Apr-98 GB_PAT: I92039 588 I92039 Sequence 6 from
patent US 5726299. Unknown. 97,071 01-DEC-1998 GB_PR3: HS929C8
139190 AL020994 Human DNA sequence from clone 929C8 on chromosome
22q12.1-12.3 Homo sapiens 39,016 23-Nov-99 Contains CA repeat, GSS,
STS, complete sequence. rxa00907 246 GB_PAT: I78750 588 I78750
Sequence 6 from patent US 5693781. Unknown. 97,561 3-Apr-98 GB_PAT:
I92039 588 I92039 Sequence 6 from patent US 5726299. Unknown.
97,561 01-DEC-1998 GB_PAT: I78750 588 I78750 Sequence 6 from patent
US 5693781. Unknown. 37,222 3-Apr-98 rxa00961 455 GB_BA1: AB032799
9077 AB032799 Chromobacterium violaceum violacein biosynthetic gene
cluster (vio Chromobacterium 39,868 02-OCT-1999 A, vio B, vio C,
vio D), complete cds. violaceum GB_BA2: AF172851 10094 AF172851
Chromobacterium violaceum violacein biosynthetic gene cluster,
Chromobacterium 42,760 30-Aug-99 complete sequence. violaceum
GB_BA1: AB032799 9077 AB032799 Chromobacterium violaceum violacein
biosynthetic gene cluster (vio Chromobacterium 39,551 02-OCT-1999
A, vio B, vio C, vio D), complete cds. violaceum rxa00982 1629
GB_BA1: BLARGS 2501 Z21501 B. lactofermentum argS and lysA genes
for arginyl-tRNA synthetase Corynebacterium 39,003 28-DEC-1993 and
diaminopimelate decarboxylase (partial). glutamicum GB_BA1: CGXLYSA
2344 X54740 Corynebacterium glutamicum argS-lysA operon gene for
the upstream Corynebacterium 41,435 30-Jun-93 region of the
arginyl-tRNA synthetase and diaminopimelate glutamicum
decarboxylase (EC 4.1.1.20). GB_PAT: E14508 3579 E14508 DNA
encoding Brevibacterium diaminopimelic acid decarboxylase and
Corynebacterium 40,566 28-Jul-99 arginyl-tRNA synthase. glutamicum
rxa00983 1599 GB_HTG2: AC008152 24000 AC008152 Leishmania major
chromosome 35 clone L7936 strain Friedlin, *** Leishmania major
38,658 28-Jul-99 SEQUENCING IN PROGRESS ***, 4 unordered pieces.
GB_HTG2: AC008152 24000 AC008152 Leishmania major chromosome 35
clone L7936 strain Friedlin, *** Leishmania major 38,658 28-Jul-99
SEQUENCING IN PROGRESS ***, 4 unordered pieces. GB_HTG3: AC008648
87249 AC008648 Homo sapiens chromosome 5 clone CIT978SKB_186E14,
*** Homo sapiens 36,102 3-Aug-99 SEQUENCING IN PROGRESS ***, 22
unordered pieces. rxa00984 440 GB_BA1: MVINED 3098 D01045
Micromonospora viridifaciens DNA for nedR protein and
Micromonospora 59,226 2-Feb-99 neuraminidase, complete cds.
viridifaciens GB_PAT: E02375 1881 E02375 Neuraminidase gene.
Micromonospora 59,226 29-Sep-97 viridifaciens GB_PR4: HUAC004513
101311 AC004513 Homo sapiens Chromosome 16 BAC clone
CIT987SK-A-926E7, Homo sapiens 41,204 23-Nov-99 complete sequence.
rxa01014 2724 GB_BA1: MTV008 63033 AL021246 Mycobacterium
tuberculosis H37Rv complete genome; segment Mycobacterium 56,167
17-Jun-98 108/162. tuberculosis GB_BA1: STMAMPEPN 2849 L23172
Streptomyces lividans aminopeptidase N gene, complete cds.
Streptomyces lividans 57,067 18-MAY-1994 GB_BA1: SC7H2 42655
AL109732 Streptomyces coelicolor cosmid 7H2. Streptomyces
coelicolor 37,551 2-Aug-99 A3(2) rxa01059 732 GB_HTG3: AC008154
172241 AC008154 Homo sapiens chromosome 7, *** SEQUENCING IN
PROGRESS ***, Homo sapiens 39,499 8-Sep-99 26 unordered pieces.
GB_HTG3: AC008154 172241 AC008154 Homo sapiens chromosome 7, ***
SEQUENCING IN PROGRESS ***, Homo sapiens 39,499 8-Sep-99 26
unordered pieces. GB_EST32: AI756574 299 AI756574 ea02f10.y1
Eimeria M5-6 Merozoite stage Eimeria tenella cDNA 5', Eimeria
tenella 37,793 23-Jun-99 mRNA sequence. rxa01073 954 GB_BA1:
BACOUTB 1004 M15811 Bacillus subtilis outB gene encoding a
sporulation protein, complete Bacillus subtilis 53,723 26-Apr-93
cds. GB_PR4: AC007938 167237 AC007938 Homo sapiens clone UWGC:
djs201 from 7q31, complete sequence. Homo sapiens 34,322 1-Jul-99
GB_PL2: ATAC006282 92577 AC006282 Arabidopsis thaliana chromosome
II BAC F13K3 genomic sequence, Arabidopsis thaliana 36,181
13-MAR-1999 complete sequence. rxa01120 1401 GB_BA1: MTV008 63033
AL021246 Mycobacterium tuberculosis H37Rv complete genome; segment
Mycobacterium 36,715 17-Jun-98 108/162. tuberculosis GB_BA1:
CAJ10321 6710 AJ010321 Caulobacter crescentus partial tig gene and
clpP, cicA, clpX, Ion Caulobacter crescentus 63,311 01-OCT-1998
genes. GB_BA2: AF150957 4440 AF150957 Azospirillum brasilense
trigger factor (tig), heat-shock protein ClpP Azospirillum
brasilense 60,613 7-Jun-99 (clpP), and heat-shock protein ClpX
(clpX) genes, complete cds; and Lon protease (lon) gene, partial
cds. rxa01147 1383 GB_PR3: HS408N23 97916 Z98048 Human DNA sequence
from PAC 408N23 on chromosome 22q13. Homo sapiens 34,567 23-Nov-99
Contains HIP, HSC70-INTERACTING PROTEIN (PROGESTERONE
RECEPTOR-ASSOCIATED P48 PROTEIN), ESTs and STS. GB_BA2: AE001227
26849 AE001227 Treponema pallidum section 43 of 87 of the complete
genome. Treponema pallidum 37,564 16-Jul-98 GB_PR3: HS408N23 97916
Z98048 Human DNA sequence from PAC 408N23 on chromosome 22q13. Homo
sapiens 34,911 23-Nov-99 Contains HIP, HSC70-INTERACTING PROTEIN
(PROGESTERONE RECEPTOR-ASSOCIATED P48 PROTEIN), ESTs and STS.
rxa01151 958 GB_BA1: MTCY261 27322 Z97559 Mycobacterium
tuberculosis H37Rv complete genome; segment Mycobacterium 38,789
17-Jun-98 95/162. tuberculosis GB_HTG4: AC009849 114993 AC009849
Drosophila melanogaster chromosome 2 clone BACR07H08 (D864)
Drosophila melanogaster 39,213 25-OCT-1999 RPCI-98 07.H.8 map
31B-31C strain y; cn bw sp, *** SEQUENCING IN PROGRESS ***, 55
unordered pieces. GB_HTG4: AC009849 114993 AC009849 Drosophila
melanogaster chromosome 2 clone BACR07H08 (D864) Drosophila
melanogaster 39,213 25-OCT-1999 RPCI-98 07.H.8 map 31B-31C strain
y; cn bw sp, *** SEQUENCING IN PROGRESS ***, 55 unordered pieces.
rxa01161 1260 GB_BA2: AF176799 2943 AF176799 Lactobacillus pentosus
PepQ (pepQ) and catabolite control protein A Lactobacillus pentosus
37,043 5-Sep-99 (ccpA) genes, complete cds. GB_BA2: AF012084 3082
AF012084 Lactobacillus helveticus prolidase (pepQ) gene, complete
cds. Lactobacillus helveticus 46,796 1-Jul-98 GB_EST32: AI728955
611 AI728955 BNLGHi12114 Six-day Cotton fiber Gossypium hirsutum
cDNA 5' Gossypium hirsutum 37,647 11-Jun-99 similar to (AC004481)
putative permease [Arabidopsis thaliana], mRNA sequence. rxa01181
980 GB_BA1: MLCB22 40281 Z98741 Mycobacterium leprae cosmid B22.
Mycobacterium leprae 61,570 22-Aug-97 GB_BA1: MTCY190 34150 Z70283
Mycobacterium tuberculosis H37Rv complete genome; segment
Mycobacterium 60,434 17-Jun-98 98/162. tuberculosis GB_BA1: SC5F7
40024 AL096872 Streptomyces coelicolor cosmid 5F7. Streptomyces
coelicolor 57,011 22-Jul-99 A3(2) rxa01182 516 GB_HTG1: CEY116A8_2
110000 Z98858 Caenorhabditis elegans chromosome IV clone Y116A8,
*** Caenorhabditis elegans 34,843 26-Oct-99 SEQUENCING IN PROGRESS
***, in unordered pieces. GB_HTG1: CEY116A8_2 110000 Z98858
Caenorhabditis elegans chromosome IV clone Y116A8, ***
Caenorhabditis elegans 34,843 26-Oct-99 SEQUENCING IN PROGRESS ***,
in unordered pieces. GB_IN1: CEY116A8C 260341 AL117204
Caenorhabditis elegans cosmid Y116A8C, complete sequence.
Caenorhabditis elegans 34,843 19-Nov-99 rxa01189 732 GB_BA1: D90915
130001 D90915 Synechocystis sp. PCC6803 complete genome, 17/27,
2137259-2267259. Synechocystis sp. 36,538 7-Feb-99 GB_BA1: D90915
130001 D90915 Synechocystis sp. PCC6803 complete genome, 17/27,
2137259-2267259. Synechocystis sp. 34,512 7-Feb-99 GB_HTG3:
AC010515 41038 AC010515 Homo sapiens chromosome 19 clone
LLNL-R_249H9, *** Homo sapiens 33,564 15-Sep-99 SEQUENCING IN
PROGRESS ***, 31 unordered pieces. rxa01192 681 GB_OM: CFP180RRC
5425 X87224 Canis familiaris mRNA for ribosome receptor, p180.
Canis familiaris 41,229 22-Jan-99 GB_OM: CFP180RRC 5425 X87224
Canis familiaris mRNA for ribosome receptor, p180. Canis familiaris
38,187 22-Jan-99 rxa01214 1614 GB_IN1: CEY47D3A 199814 AL117202
Caenorhabditis elegans cosmid Y47D3A, complete sequence.
Caenorhabditis elegans 36,604 19-Nov-99 GB_PR4: AC006039 176257
AC006039 Homo sapiens clone NH0319F03, complete sequence. Homo
sapiens 34,984 05-MAY-1999 GB_PR4: AC006039 176257 AC006039 Homo
sapiens clone NH0319F03, complete sequence. Homo sapiens 35,951
05-MAY-1999 rxa01224 1146 GB_E5T22: AI070047 479 AI070047
UI-R-C1-In-f-08-0-UI.s1 UI-R-C1 Rattus norvegicus cDNA clone UI-R-
Rattus norvegicus 36,975 5-Jul-99 C1-ln-f-08-0-UI 3', mRNA
sequence. GB_RO: S75965 625 S75965 THP = Tamm-Horsfall protein
{promoter} [rats, Genomic, 625 nt]. Rattus sp. 34,400 27-Jul-95
GB_EST5: H96951 459 H96951 yu01g03.r1 Soares_pineal_gland_N3HPG
Homo sapiens cDNA clone Homo sapiens 32,969 11-DEC-1995 IMAGE:
232564 5', mRNA sequence. rxa01250 588 GB_PL1: NEULCCB 2656 M18334
N. crassa (strain TS) laccase gene, complete cds. Neurospora crassa
44,330 03-MAY-1994 GB_OV: MTRACOMPL 16714 Y16884 Rhea americana
complete mitochondrial genome. Mitochondrion Rhea 35,094 19-Jul-99
americana GB_OV: AF090339 16704 AF090339 Rhea americana
mitochondrion, complete genome. Mitochondrion Rhea 35,094
27-MAY-1999 americana rxa01277 2127 GB_PL2: AF111709 52684 AF111709
Oryza sativa subsp. indica Retrosat 1 retrotransposon and Ty3-Gypsy
Oryza sativa subsp. indica 37,410 26-Apr-99 type Retrosat 2
retrotransposon, complete sequences; and unknown genes. GB_IN1:
CELZC250 34372 AF003383 Caenorhabditis elegans cosmid ZC250.
Caenorhabditis elegans 35,506 14-MAY-1997 GB_EST1: Z14808 331
Z14808 CEL5E4 Chris Martin sorted cDNA library Caenorhabditis
elegans Caenorhabditis elegans 36,890 19-Jun-97 cDNA clone cm5e4
5', mRNA sequence. rxa01302 576 GB_BA1: MTCI65 34331 Z95584
Mycobacterium tuberculosis H37Rv complete genome; segment
Mycobacterium 59,298 17-Jun-98 50/162. tuberculosis GB_BA1: MSGY348
40056 AD000020 Mycobacterium tuberculosis sequence from clone y348.
Mycobacterium 59,227 10-DEC-1996 tuberculosis GB_BA1: SC5C7 41906
AL031515 Streptomyces coelicolor cosmid 5C7. Streptomyces
coelicolor 39,261 7-Sep-98 rxa01303 1458 GB_BA1: TTAJ5043 837
AJ225043 Thermus thermophilus partial narK gene. Thermus
thermophilus 55,245 18-Jun-98 GB_PL2: AC010675 84723 AC010675
Arabidopsis thaliana chromosome I BAC T17F3 genomic sequence,
Arabidopsis thaliana 37,058 11-Nov-99 complete sequence. GB_GSS9:
AQ170862 518 AQ170862 HS_3165_B2_F03_T7 CIT Approved Human Genomic
Sperm Library Homo sapiens 38,610 17-OCT-1998 D Homo sapiens
genomic clone Plate = 3165 Col = 6 Row = L, genomic survey
sequence. rxa01308 2503 GB_BA1: D90757 17621 D90757 Escherichia
coli genomic DNA. (27.3-27.7 min). Escherichia coli 55,445 7-Feb-99
GB_BA1: D90787 15942 D90787 E. coli genomic DNA, Kohara clone
#276(33.0-33.3 min.). Escherichia coli 36,815 29-MAY-1997 GB_BA1:
90758 13860 D90758 Escherichia coli genomic DNA. (27.6-27.9 min),
Escherichia coli 54,942 7-Feb-99 rxa01309 824 GB_BA1: SCJ12 35302
AL109989 Streptomyces coelicolor cosmid J12. Streptomyces
coelicolor 62,423 24-Aug-99 A3(2) GB_BA1: BSNARYWI 12450 Z49884 B.
subtilis nar[G, H, I, J, K], ywi[C, D, E] and argS genes. Bacillus
subtilis 57,447 24-Jun-98 GB_BA1: BSUB0020 212150 Z99123 Bacillus
subtilis complete genome (section 20 of 21): from 3798401 to
Bacillus subtilis 37,129 26-Nov-97 4010550. rxa01358 1644 GB_GSS11:
AQ260413 453 AQ260413 CITBI-E1-2510B12.TF CITBI-E1 Homo sapiens
genomic clone Homo sapiens 41,531 24-OCT-1998 2510B12, genomic
survey sequence. GB_EST20: AA840582 326 AA840582 vw77h07.r1
Stratagene mouse heart (#937316) Mus musculus cDNA Mus musculus
42,901 27-Feb-98 clone IMAGE: 1261021 5' similar to gb: J04181
Mouse A-X actin mRNA, complete cds (MOUSE);, mRNA sequence. GB_PAT:
A39944 3836 A39944 Sequence 1 from Patent WO9421807. unidentified
38,764 05-MAR-1997 rxa01385 2004 GB_BA1: FVBPENTA 2519 M98557
Flavobacterium sp. pentachlorophenol 4-monooxygenase gene,
Flavobacterium sp. 40,855 26-Apr-93 complete mRNA. GB_PAT: I19994
2516 I19994 Sequence 2 from patent US 5512478. Unknown. 40,855
07-OCT-1996 GB_BA2: AF059680 2410 AF059680 Sphingomonas sp. UG30
pentachlorophenol 4-monooxygenase Sphingomonas sp. UG30 42,993
27-Apr-99 (pcpB) gene, complete cds; and pentachlorophenol
4-monooxygenase reductase (pcpD) gene, partial cds. rxa01412 327
GB_GSS12: AQ332469 459 AQ332469 HS_5003_A1_H08_SP6E RPCI11 Human
Male BAC Library Homo Homo sapiens 38,208 06-MAR-1999 sapiens
genomic clone Plate = 579 Col = 15 Row = O, genomic survey
sequence. GB_EST27: AA998532 453 AA998532 UI-R-C0-ic-d-11-0-UI.s1
UI-R-C0 Rattus norvegicus cDNA clone UI-R- Rattus norvegicus 39,336
09-MAR-1999 C0-ic-d-11-0-UI 3', mRNA sequence. GB_HTG1: HSA342D11
178183 AL121748 Homo sapiens chromosome 10 clone RP11-342D11, ***
Homo sapiens 40,550 23-Nov-99 SEQUENCING IN PROGRESS ***, in
unordered pieces. rxa01458 1173 GB_BA2: AE000745 15085 AE000745
Aquifex aeolicus section 77 of 109 of the complete genome. Aquifex
aeolicus 37,694 25-MAR-1998 GB_BA2: AE000745 15085 AE000745 Aquifex
aeolicus section 77 of 109 of the complete genome. Aquifex aeolicus
35,567 25-MAR-1998 rxa01571 723 GB_BA1: AB011413 12070 AB011413
Streptomyces griseus genes for Orf2, Orf3, Orf4, Orf5, AfsA, Orf8,
Streptomyces griseus 57,500 7-Aug-98 partial and complete cds.
GB_BA1: AB011413 12070 AB011413 Streptomyces griseus genes for
Orf2, Orf3, Orf4, Orf5, AfsA, Orf8, Streptomyces griseus 35,655
7-Aug-98 partial and complete cds. rxa01607 753 GB_PR4: AC005005
133893 AC005005 Homo sapiens PAC clone DJ412A9 from 22, complete
sequence. Homo sapiens 38,399 02-MAR-1999 GB_HTG3: AC008257 109187
AC008257 Drosophila melanogaster chromosome 2 clone BACR08A11
(D916) Drosophila melanogaster 33,741 08-OCT-1999 RPCI-98 08.A.11
map 42A-42A strain y; cn bw sp, *** SEQUENCING IN PROGRESS ***, 93
unordered pieces. GB_HTG3: AC008257 109187
AC008257 Drosophila melanogaster chromosome 2 clone BACR08A11
(D916) Drosophila melanogaster 33,741 08-OCT-1999 RPCI-98 08.A.11
map 42A-42A strain y; cn bw sp, *** SEQUENCING IN PROGRESS ***, 93
unordered pieces: rxa01609 996 GB_BA1: MTV003 13246 AL008883
Mycobacterium tuberculosis H37Rv complete genome; segment
Mycobacterium 39,369 17-Jun-98 125/162. tuberculosis GB_BA1:
MSGB1529CS 36985 L78824 Mycobacterium leprae cosmid B1529 DNA
sequence. Mycobacterium leprae 60,624 15-Jun-96 GB_BA1: AB024601
14807 AB024601 Pseudomonas aeruginosa dapD gene for
tetrahydrodipicolinate N- Pseudomonas aeruginosa 41,603 12-MAR-1999
succinyletransferase, complete cds, strain PAO1. rxa01654 1119
GB_GSS4: AQ704352 532 AQ704352 HS_2147_A2_H04_MR CIT Approved Human
Genomic Sperm Homo sapiens 37,838 7-Jul-99 Library D Homo sapiens
genomic clone Plate = 2147 Col = 8 Row = O, genomic survey
sequence. GB_RO: MMAE000663 250611 AE000663 Mus musculus TCR beta
locus from bases 1 to 250611 (section 1 of Mus musculus 35,799
4-Sep-97 3) of the complete sequence. GB_EST23: AI158428 511
AI158428 ud24f12.r1 Soares 2NbMT Mus musculus cDNA clone Mus
musculus 41,337 30-Sep-98 IMAGE: 1446863 5', mRNA sequence.
rxa01664 945 GB_OV: AF026198 63155 AF026198 Fugu rubripes neural
cell adhesion molecule L1 homolog (L1-CAM) Fugu rubripes 35,187
02-MAY-1998 gene, complete cds; putative protein 1 (PUT1) gene,
partial cds; mitosis-specific chromosome segregation protein SMC1
homolog (SMC1) gene, complete cds; and calcium channel alpha-1
subunit homolog (CCA1) and putative protein 2 (PUT2) genes, partial
cds, complete sequence. GB_PR3: AC004466 122186 AC004466 Homo
sapiens 12q13.1 PAC RPCI5-1057I20 (Roswell Park Cancer Homo sapiens
37,382 17-Sep-98 Institute Human PAC library) complete sequence.
GB_PR3: AC004466 122186 AC004466 Homo sapiens 12q13.1 PAC
RPCI5-1057I20 (Roswell Park Cancer Homo sapiens 37,325 17-Sep-98
Institute Human PAC library) complete sequence. rxa01795 720
GB_BA2: CGU13922 4412 U13922 Corynebacterium glutamicum putative
type II 5-cytosoine Corynebacterium 99,444 3-Feb-98
methyltransferase (cgIIM) and putative type II restriction
endonuclease glutamicum (cgIIR) and putative type I or type III
restriction endonuclease (cIgIIR) genes, complete cds. GB_BA1:
S86113 1044 S86113 ORF 1 [Neisseria gonorrhoeae, Genomic, 1044 nt].
Neisseria gonorrhoeae 58,320 07-MAY-1993 GB_PAT: I22080 850 I22080
Sequence 1 from patent US 5525717. Unknown. 57,722 07-OCT-1996
rxa01802 954 GB_BA2: AE001519 14062 AE001519 Helicobacter pylori,
strain J99 section 80 of 132 of the complete Helicobacter pylori
J99 33,510 20-Jan-99 genome. GB_GSS5: AQ774071 552 AQ774071
HS_2269_B1_C10_T7C CIT Approved Human Genomic Sperm Homo sapiens
37,967 29-Jul-99 Library D Homo sapiens genomic clone Plate = 2269
Col = 19 Row = F, genomic survey sequence. GB_PR4: AC007459 40907
AC007459 Homo sapiens chromosome 16 clone 306C6, complete sequence.
Homo sapiens 39,140 04-MAY-1999 rxa01838 842 GB_BA1: SCE15 26440
AL049707 Streptomyces coelicolor cosmid E15. Streptomyces
coelicolor 36,297 22-Apr-99 GB_HTG3: AC009545 165042 AC009545 Homo
sapiens chromosome 11 clone 131_J_04 map 11, *** Homo sapiens
37,651 01-OCT-1999 SEQUENCING IN PROGRESS ***, 8 unordered pieces.
GB_HTG3: AC009545 165042 AC009545 Homo sapiens chromosome 11 clone
131_J_04 map 11, *** Homo sapiens 37,651 01-OCT-1999 SEQUENCING IN
PROGRESS ***, 8 unordered pieces. rxa01848 867 GB_BA1: MTCY24A1
20270 Z95207 Mycobacterium tuberculosis H37Rv complete genome;
segment Mycobacterium 38,270 17-Jun-98 124/162. tuberculosis
GB_EST21: C89252 587 C89252 C89252 Mouse early blastocyst cDNA Mus
musculus cDNA clone Mus musculus 37,219 28-MAY-1998 01B00061JC08,
mRNA sequence. GB_EST14: AA423340 457 AA423340 ve39d04.r1 Soares
mouse mammary gland NbMMG Mus musculus Mus musculus 38,377
16-OCT-1997 cDNA clone IMAGE: 820519 5', mRNA sequence. rxa01849
1224 GB_BA1: MTCY24A1 20270 Z95207 Mycobacterium tuberculosis H37Rv
complete genome; segment Mycobacterium 39,950 17-Jun-98 124/162.
tuberculosis GB_BA2: RCPHSYNG 45959 Z11165 R. capsulatus complete
photosynthesis gene cluster. Rhodobacter capsulatus 37,344 2-Sep-99
GB_BA1: RSP010302 40707 AJ010302 Rhodobacter sphaeroides
photosynthetic gene cluster. Rhodobacter sphaeroides 40,898
27-Aug-99 rxa01868 2049 GB_BA1: MTV033 21620 AL021928 Mycobacterium
tuberculosis H37Rv complete genome; segment Mycobacterium 38,679
17-Jun-98 11/162. tuberculosis GB_BA1: MLCL622 42498 Z95398
Mycobacterium leprae cosmid L622. Mycobacterium leprae 38,911
24-Jun-97 GB_BA1: MSGB983CS 36788 L78828 Mycobacterium leprae
cosmid B983 DNA sequence. Mycobacterium leprae 38,933 15-Jun-96
rxa01885 924 GB_BA1: MTCY1A10 25949 Z95387 Mycobacterium
tuberculosis H37Rv complete genome; segment Mycobacterium 51,094
17-Jun-98 117/162. tuberculosis GB_PR3: HSU220B11 41247 Z69908
Human DNA sequence from cosmid cU220B11, between markers Homo
sapiens 39,038 23-Nov-99 DXS6791 and DX58038 on chromosome X.
GB_BA1: PDU17435 993 U17435 Paracoccus denitrificans Fnr-like
transcriptional activator (nnr) gene, Paracoccus denitrificans
39,390 19-Jul-95 complete cds. rxa01914 526 GB_PR3: AC005796 43843
AC005796 Homo sapiens chromosome 19, cosmid R31408, complete
sequence. Homo sapiens 34,961 06-OCT-1998 GB_PR3: HS390C10 114231
AL008721 Homo sapiens DNA sequence from BAC 390C10 on chromosome
Homo sapiens 39,600 23-Nov-99 22q11.21-12.1. Contains an
Immunoglobulin LIKE gene and a pseudogene similar to Beta
Crystallin. Contains ESTs, STSs, GSSs and taga and tat repeat
polymorphisms, complete sequence. GB_PR3: AC005796 43843 AC005796
Homo sapiens chromosome 19, cosmid R31408, complete sequence. Homo
sapiens 37,725 06-OCT-1998 rxa01932 1020 GB_PR3: AC003025 112309
AC003025 Human Chromosome 11p12.2 PAC clone pDJ466a11, complete
Homo sapiens 35,585 23-Jul-98 sequence. GB_GSS3: B78728 312 B78728
CIT-HSP-431E3.TV CIT-HSP Homo sapiens genomic clone 431E3, Homo
sapiens 38,907 25-Jun-98 genomic survey sequence. GB_PR3: AC003025
112309 AC003025 Human Chromosome 11p12.2 PAC clone pDJ466a11,
complete Homo sapiens 35,859 23-Jul-98 sequence. rxa01933 726
GB_HTG1: HS74O16 169401 AL110119 Homo sapiens chromosome 21 clone
RPCIP704O1674 map 21q21, Homo sapiens 35,302 27-Aug-99 ***
SEQUENCING IN PROGRESS ***, in unordered pieces. GB_HTG1: HS74O16
169401 AL110119 Homo sapiens chromosome 21 clone RPCIP704O1674 map
21q21, Homo sapiens 35,302 27-Aug-99 *** SEQUENCING IN PROGRESS
***, in unordered pieces. GB_PR4: AC006032 170282 AC006032 Homo
sapiens BAC clone NH0115E20 from Y, complete sequence. Homo sapiens
37,640 27-Feb-99 rxa01971 954 GB_HTG3: AC008230 108469 AC008230
Drosophila melanogaster chromosome 2 clone BACR17I17 (D934)
Drosophila melanogaster 35,466 10-Aug-99 RPCI-98 17.I.17 map
53A-53C strain y; cn bw sp, *** SEQUENCING IN PROGRESS ***, 108
unordered pieces. GB_HTG3: AC008230 108469 AC008230 Drosophila
melanogaster chromosome 2 clone BACR17I17 (D934) Drosophila
melanogaster 35,466 10-Aug-99 RPCI-98 17.I.17 map 53A-53C strain y;
cn bw sp, *** SEQUENCING IN PROGRESS***, 108 unordered pieces.
GB_PR3: AF064860 165382 AF064860 Homo sapiens chromosome 21q22.3
PAC 70I24, complete sequence. Homo sapiens 39,716 2-Jun-98 rxa02016
900 GB_EST2: D48846 459 D48846 RICS15292A Rice green shoot Oryza
sativa cDNA, mRNA sequence. Oryza sativa 37,118 2-Aug-95 GB_GSS10:
AQ195886 595 AQ195886 RPCI11-66O13.TJ RPCI-11 Homo sapiens genomic
clone RPCI-11- Homo sapiens 41,000 20-Apr-99 66O13, genomic survey
sequence. GB_GSS10: AQ195886 595 AQ195886 RPCI11-66O13.TJ RPCI-11
Homo sapiens genomic clone RPCI-11- Homo sapiens 34,790 20-Apr-99
66O13, genomic survey sequence. rxa02017 807 GB_EST20: AA855266 406
AA855266 vw70b08.r1 Stratagene mouse heart (#937316) Mus musculus
cDNA Mus musculus 42,638 06-MAR-1998 clone IMAGE: 1260279 5', mRNA
sequence. GB_EST20: AA855266 406 AA855266 vw70b08.r1 Stratagene
mouse heart (#937316) Mus musculus cDNA Mus musculus 37,183
06-MAR-1998 clone IMAGE: 1260279 5', mRNA sequence. rxa02018 1073
GB_BA1: SC5C7 41906 AL031515 Streptomyces coelicolor cosmid 5C7.
Streptomyces coelicolor 41,732 7-Sep-98 GB_BA1: MTCI65 34331 Z95584
Mycobacterium tuberculosis H37Rv complete genome; segment
Mycobacterium 62,395 17-Jun-98 50/162. tuberculosis GB_BA1: SCJ12
35302 AL109989 Streptomyces coelicolor cosmid J12. Streptomyces
coelicolor 61,603 24-Aug-99 A3(2) rxa02048 1497 GB_PAT: E15823 2323
E15823 DNA encoding cell surface protein from Corynebacterium
Corynebacterium 53,942 28-Jul-99 ammoniagenes. ammoniagenes GB_OM:
SSAMPTDN 3387 Z29522 S. scrofa mRNA for aminopeptidase N. Sus
scrofa 42,672 26-Sep-94 GB_OV: D87992 3181 D87992 Gallus gallus
mRNA for aminopeptidase Ey, complete cds. Gallus gallus 41,554
5-Jun-99 rxa02101 1386 GB_BA1: AP000064 247695 AP000064 Aeropyrum
pernix genomic DNA, section 7/7. Aeropyrum pernix 39,882 22-Jun-99
GB_PL2: ATAC006587 79262 AC006587 Arabidopsis thaliana chromosome
II BAC T17D12 genomic sequence, Arabidopsis thaliana 38,490
23-MAR-1999 complete sequence. GB_PL2: ATAC006587 79262 AC006587
Arabidopsis thaliana chromosome II BAC T17D12 genomic sequence,
Arabidopsis thaliana 34,863 23-MAR-1999 complete sequence. rxa02265
423 GB_BA2: AF120718 4137 AF120718 Lactobacillus fermentum urease
operon, partial sequence. Lactobacillus fermentum 56,265
31-MAR-1999 GB_PAT: E03531 2896 E03531 DNA sequence coding for acid
urease. Lactobacillus fermentum 56,265 29-Sep-97 GB_BA1: LBAAURE
2896 D10605 L. fermentum gene for acid urease. Lactobacillus
fermentum 56,265 2-Feb-99 rxa02276 801 GB_GSS10: AQ242920 451
AQ242920 HS_2061_A1_E08_MR CIT Approved Human Genomic Sperm Homo
sapiens 37,916 03-OCT-1998 Library D Homo sapiens genomic clone
Plate = 2061 Col = 15 Row = I, genomic survey sequence. GB_IN1:
SLMMTPMF 14503 D29637 Physarum polycephalum mitochondrial DNA.
Mitochondrion Physarum 40,335 12-MAY-1999 polycephalum GB_IN2:
AF012249 5542 AF012249 Physarum polycephalum strain aux2-S region
of mitochondria derived Mitochondrion Physarum 40,335 08-MAY-1998
from mF plasmid, including URFA', URFC, URFD, URFE, URFF, and
polycephalum URFG genes, complete cds, and URFH gene, partial cds.
rxa02277 738 GB_BA2: AF048784 681 AF048784 Actinomyces naeslundii
urease accessory protein (ureG) gene, Actinomyces naeslundii 66,814
9-Feb-99 complete cds. GB_BA2: AF056321 5482 AF056321 Actinomyces
naeslundii urease gamma subunit UreA (ureA), urease Actinomyces
naeslundii 63,686 9-Feb-99 beta subunit UreB (ureB), urease alpha
subunit UreC (ureC), urease accessory protein UreE (ureE), urease
accessory protein UreF (ureF), urease accessory protein UreG
(ureG), and urease accessory protein UreD (ureD) genes, complete
cds. GB_BA2: SSU35248 5773 U35248 Streptococcus salivarius ure
cluster nickel transporter homolog (urel) Streptococcus salivarius
61,931 26-Jan-96 gene, partial cds, and urease beta subunit (ureA),
gamma subunit (ureB), alpha subunit (ureC), and accessory proteins
(ureE), (ureF), (ureG), and (ureD) genes, complete cds. rxa02278
972 GB_GSS3: B49054 543 B49054 RPCI11-4I13.TV RPCI-11 Homo sapiens
genomic clone RPCI-11- Homo sapiens 39,161 8-Apr-99 4I13, genomic
survey sequence. GB_PL1: PMCMSGI 3363 L27092 Pneumocystis carinii
B-cell receptor (msgl) gene, 3' end. Pneumocystis carinii 39,819
26-Sep-94 GB_PL2: AF038556 12792 AF038556 Pneumocystis carinii f.
sp. hominis variant regions of major surface Pneumocystis carinii
f. sp. 33,832 10-Sep-98 glycoproteins (msg1, msg3, msg4) genes,
partial cds. hominis rxa02317 735 GB_GSS8: AQ051031 914 AQ051031
nbxb0004dG10r CUGI Rice BAC Library Oryza sativa genomic clone
Oryza sativa 32,299 24-MAR-1999 nbxb0004N20r, genomic survey
sequence. GB_GSS8: AQ051031 914 AQ051031 nbxb0004dG10r CUGI Rice
BAC Library Oryza sativa genomic clone Oryza sativa 34,573
24-MAR-1999 nbxb0004N20r, genomic survey sequence. rxa02334 746
GB_BA1: CGU35023 3195 U35023 Corynebacterium glutamicum thiosulfate
sulfurtransferase (thtR) gene, Corynebacterium 100,000 16-Jan-97
partial cds, acyl CoA carboxylase (accBC) gene, complete cds.
glutamicum GB_BA1: MTCY71 42729 Z92771 Mycobacterium tuberculosis
H37Rv complete genome; segment Mycobacterium 60,380 10-Feb-99
141/162. tuberculosis GB_BA1: U00012 33312 U00012 Mycobacterium
leprae cosmid B1308. Mycobacterium leprae 37,660 30-Jan-96 rxa02351
1039 GB_HTG2: HS225E12 126464 AL031772 Homo sapiens chromosome 6
clone RP1-225E12 map q24, *** Homo sapiens 35,973 03-DEC-1999
SEQUENCING IN PROGRESS ***, in unordered pieces. GB_HTG2: HS225E12
126464 AL031772 Homo sapiens chromosome 6 clone RP1-225E12 map q24,
*** Homo sapiens 35,973 03-DEC-1999 SEQUENCING IN PROGRESS ***, in
unordered pieces. GB_HTG2: HS225E12 126464 AL031772 Homo sapiens
chromosome 6 clone RP1-225E12 map q24, *** Homo sapiens 36,992
03-DEC-1999 SEQUENCING IN PROGRESS ***, in unordered pieces.
rxa02410 789 GB_BA1: AB020624 1605 AB020624 Corynebacterium
glutamicum murl gene for D-glutamate racemase, Corynebacterium
99,227 24-Jul-99 complete cds. glutamicum GB_EST4: H51527 294
H51527 yo33b09.s1 Soares adult brain N2b4HB55Y Homo sapiens cDNA
Homo sapiens 40,411 18-Sep-95 clone IMAGE: 179705 3', mRNA
sequence. GB_GSS1: CNS003CM 1101 AL064136 Drosophila melanogaster
genome survey sequence T7 end of BAC # Drosophila melanogaster
37,674 3-Jun-99 BACR08C19 of RPCI-98 library from Drosophila
melanogaster (fruit fly), genomic survey sequence. rxa02477 744
GB_HTG4: AC010054 130191 AC010054 Drosophila melanogaster
chromosome 3L/74E2 clone RPCI98-15E10, Drosophila melanogaster
37,466 16-OCT-1999 *** SEQUENCING IN PROGRESS ***, 70 unordered
pieces. GB_HTG4: AC010054 130191 AC010054 Drosophila melanogaster
chromosome 3L/74E2 clone RPCI98-15E10, Drosophila melanogaster
37,466 16-OCT-1999 *** SEQUENCING IN PROGRESS ***, 70 unordered
pieces. GB_HTG4: AC009375 137069 AC009375 Drosophila melanogaster
chromosome 3L/75A1 clone RPCI98-44L18, Drosophila melanogaster
39,118 16-OCT-1999 *** SEQUENCING IN PROGRESS ***, 59 unordered
pieces. rxa02513 832 GB_BA1: MTER260 373 X92572 M. terrae gene for
32 kDa protein (partial). Mycobacterium terrae 42,895 15-Jan-98
GB_PL1: AB019229 84294 AB019229 Arabidopsis thaliana genomic DNA,
chromosome 3, P1 clone: Arabidopsis thaliana 36,084 20-Nov-99
MDC16, complete sequence. GB_PL1: AB019229 84294 AB019229
Arabidopsis thaliana genomic DNA, chromosome 3, P1 clone:
Arabidopsis thaliana 35,244 20-Nov-99 MDC16, complete sequence.
rxa02531 834 GB_BA1: CGLATTB 271 X89850 C. glutamicum DNA for attB
region. Corynebacterium 40,590 8-Aug-96 glutamicum GB_EST11:
AA239557 423 AA239557 mv25f04.r1 GuayWoodford Beier mouse kidney
day 0 Mus musculus Mus musculus 38,760 12-MAR-1997 cDNA clone
IMAGE: 656095 5' similar to gb: X52634 Murine tlm oncogene for tlm
protein (MOUSE);, mRNA sequence. GB_BA1: RSPYPPCL 6500 AJ002398
Rhodobacter sphaeroides pyp and pcl genes, and orfA, orfB, orfC,
Rhodobacter sphaeroides 37,091 17-DEC-1998 orfD, orfE, orfF.
rxa02548 314 GB_BA2: AF127374 63734 AF127374 Streptomyces
lavendulae LinA homolog, cytochrome P450 Streptomyces lavendulae
66,242 27-MAY-1999 hydroxylase ORF4, cytochrome P450 hydroxylase
ORF3, MitT (mitT), MitS (mitS), MitR (mitR), MitQ (mitQ), MitP
(mitP), MitO (mitO), MitN (mitN), MitM (mitM), MitL (mitL), MitK
(mitK), MitJ (mitJ), MitI (mitI), MitH (mitH), MitG (mitG), MitF
(mitF), MitE (mitE), MitD (mitD), MitC (mitC), MitB (mitB), MitA
(mitA), MmcA (mmcA), MmcB (mmcB), MmcC (mmcC), MmcD (mmcD), MmcE
(mmcE), MmcF (mmcF), MmcG (mmcG), MmcH (mmcH), MmcI (mmcI), MmcJ
(mmcJ), MmcK (mmcK), MmcL (mmcL), MmcM (mmcM), MmcN (mmcN), MmcO
(mmcO), Mrd (mrd), MmcP (mmcP), MmcQ (mmcQ), MmcR (mmcR), MmcS
(mmcS), MmcT (mmcT), MmcU (mmcU), MmcV (mmcV), Mct (mct), MmcW
(mmcW), MmcX (mmcX), and MmcY (mmcY) genes, complete cds; and
unknown genes. GB_BA2: AF127374
63734 AF127374 Streptomyces lavendulae LinA homolog, cytochrome
P450 Streptomyces lavendulae 38,411 27-MAY-1999 hydroxylase ORF4,
cytochrome P450 hydroxylase ORF3, MitT (mitT), MitS (mitS), MitR
(mitR), MitQ (mitQ), MitP (mitP), MitO (mitO), MitN (mitN), MitM
(mitM), MitL (mitL), MitK (mitK), MitJ (mitJ), MitI (mitI), MitH
(mitH), MitG (mitG), MitF (mitF), MitE (mitE), MitD (mitD), MitC
(mitC), MitB (mitB), MitA (mitA), MmcA (mmcA), MmcB (mmcB), MmcC
(mmcC), MmcD (mmcD), MmcE (mmcE), MmcF (mmcF), MmcG (mmcG), MmcH
(mmcH), MmcI (mmcI), MmcJ (mmcJ), MmcK (mmcK), MmcL (mmcL), MmcM
(mmcM), MmcN (mmcN), MmcO (mmcO), Mrd (mrd), MmcP (mmcP), MmcQ
(mmcQ), MmcR (mmcR), MmcS (mmcS), MmcT (mmcT), MmcU (mmcU), MmcV
(mmcV), Mct (mct), MmcW (mmcW), MmcX (mmcX), and MmcY (mmcY) genes,
complete cds; and unknown genes. GB_GSS4: AQ741886 742 AQ741886
HS_5569_B2_B02_SP6 RPCI-11 Human Male BAC Library Homo Homo sapiens
38,907 16-Jul-99 sapiens genomic clone Plate = 1145 Col = 4 Row =
D, genomic survey sequence. rxa02558 1098 GB_EST18: AA567307 741
AA567307 HL01004.5prime HL Drosophila melanogaster head BlueScript
Drosophila melanogaster 38,736 28-Nov-98 Drosophila melanogaster
cDNA clone HL01004 5prime, mRNA sequence. GB_EST27: AI402394 630
AI402394 GH21610.5prime GH Drosophila melanogaster head pOT2
Drosophila Drosophila melanogaster 41,308 8-Feb-99 melanogaster
cDNA clone GH21610 5prime, mRNA sequence. GB_GSS10: AQ237646 715
AQ237646 RPCI11-61I9.TJB RPCI-11 Homo sapiens genomic clone
RPCI-11- Homo sapiens 44,340 21-Apr-99 61I9, genomic survey
sequence. rxa02565 1389 GB_EST32: AI726448 562 AI726448 BNLGHi5854
Six-day Cotton fiber Gossypium hirsutum cDNA 5' Gossypium hirsutum
37,003 11-Jun-99 similar to (U53418) UDP-glucose dehydrogenase
[Glycine max], mRNA sequence. GB_EST32: AI726198 608 AI726198
BNLGHi5243 Six-day Cotton fiber Gossypium hirsutum cDNA 5'
Gossypium hirsutum 40,925 11-Jun-99 similar to (U53418) UDP-glucose
dehydrogenase [Glycine max], mRNA sequence. GB_PR4: AC002992 154848
AC002992 Homo sapiens chromosome Y, clone 203M13, complete
sequence. Homo sapiens 38,039 13-OCT-1999 rxa02574 1131 GB_EST4:
H29653 415 H29653 ym58f01.r1 Soares infant brain 1NIB Homo sapiens
cDNA clone Homo sapiens 39,036 17-Jul-95 IMAGE: 52678 5' similar to
SP: OXDD_BOVIN P31228 D-ASPARTATE OXIDASE;, mRNA sequence. GB_PR3:
HSDJ261K5 131974 AL050350 Human DNA sequence from clone 261K5 on
chromosome 6q21-22.1. Homo sapiens 35,957 23-Nov-99 Contains the 3'
part of the gene for a novel organic cation transporter (BAC ORF
RG331P03), the DDO gene for D-aspartate oxidase (EC 1.4.3.1), ESTs,
STSs, GSSs and two putative CpG islands, complete sequence.
GB_EST2: R20147 494 R20147 yg18h02.r1 Soares infant brain 1NIB Homo
sapiens cDNA clone Homo sapiens 36,437 17-Apr-95 IMAGE: 32866 5'
similar to SP: OXDD_BOVIN P31228 D-ASPARTATE OXIDASE;, mRNA
sequence. rxa02589 888 GB_HTG1: CEY6E2 186306 Z96799 Caenorhabditis
elegans chromosome V clone Y6E2, *** Caenorhabditis elegans 37,979
02-OCT-1997 SEQUENCING IN PROGRESS ***, in unordered pieces.
GB_HTG1: CEY6E2 186306 Z96799 Caenorhabditis elegans chromosome V
clone Y6E2, *** Caenorhabditis elegans 37,979 02-OCT-1997
SEQUENCING IN PROGRESS ***, in unordered pieces. GB_HTG3: AC011690
72277 AC011690 Homo sapiens clone 17_E_13, LOW-PASS SEQUENCE
SAMPLING. Homo sapiens 35,814 10-OCT-1999 rxa02592 894 GB_BA1:
MSGB983CS 36788 L78828 Mycobacterium leprae cosmid B983 DNA
sequence. Mycobacterium leprae 53,235 15-Jun-96 GB_GSS9: AQ170723
487 AQ170723 HS_2270_B2_F05_MR CIT Approved Human Genomic Sperm
Library Homo sapiens 39,666 16-OCT-1998 D Homo sapiens genomic
clone Plate = 2270 Col = 10 Row = L, genomic survey sequence.
GB_GSS12: AQ349397 791 AQ349397 RPCI11-118H16.TJ RPCI-11 Homo
sapiens genomic clone RPCI-11- Homo sapiens 34,204 07-MAY-1999
118H16, genomic survey sequence. rxa02603 1119 GB_BA1: MTV026 23740
AL022076 Mycobacterium tuberculosis H37Rv complete genome; segment
Mycobacterium 37,975 24-Jun-99 157/162. tuberculosis GB_IN2:
AC005714 177740 AC005714 Drosophila melanogaster, chromosome 2R,
region 58D4-58E2, BAC Drosophila melanogaster 41,226 01-MAY-1999
clone BACR48M13, complete sequence. GB_EST19: AA775050 218 AA775050
ac76e10.s1 Stratagene lung (#937210) Homo sapiens cDNA clone Homo
sapiens 40,826 5-Feb-98 IMAGE: 868554 3' similar to gb: Y00371_rna1
HEAT SHOCK COGNATE 71 KD PROTEIN (HUMAN);, mRNA sequence. rxa02630
1446 GB_BA1: MLCL373 37304 AL035500 Mycobacterium leprae cosmid
L373. Mycobacterium leprae 49,015 27-Aug-99 GB_BA1: MTV044 16150
AL021999 Mycobacterium tuberculosis H37Rv complete genome; segment
Mycobacterium 49,192 17-Jun-98 45/162. tuberculosis GB_BA1:
MLU15180 38675 U15180 Mycobacterium leprae cosmid B1756.
Mycobacterium leprae 45,621 09-MAR-1995 rxa02643 1167 GB_EST37:
AI950576 308 AI950576 wx52e08.x1 NCI_CGAP_Lu28 Homo sapiens cDNA
clone Homo sapiens 40,909 6-Sep-99 IMAGE: 2547302 3', mRNA
sequence. GB_EST37: AI950576 308 AI950576 wx52e08.x1 NCI_CGAP_Lu28
Homo sapiens cDNA clone Homo sapiens 40,288 6-Sep-99 IMAGE: 2547302
3', mRNA sequence. rxa02644 774 GB_EST34: AV149547 302 AV149547
AV149547 Mus musculus C57BL/6J 10-11 day embryo Mus musculus Mus
musculus 38,627 5-Jul-99 cDNA clone 2810489D03, mRNA sequence.
GB_EST35: AV156221 271 AV156221 AV156221 Mus musculus head C57BL/6J
12-day embryo Mus Mus musculus 33,990 7-Jul-99 musculus cDNA clone
3000001C24, mRNA sequence. GB_EST32: AV054919 274 AV054919 AV054919
Mus musculus pancreas C57BL/6J adult Mus musculus Mus musculus
36,585 23-Jun-99 cDNA clone 1810033C08, mRNA sequence. rxa02745 902
GB_BA1: MTV007 32806 AL021184 Mycobacterium tuberculosis H37Rv
complete genome; segment Mycobacterium 39,298 17-Jun-98 64/162.
tuberculosis GB_BA2: AF027770 30683 AF027770 Mycobacterium
smegmatis FxbA (fxbA) gene, partial cds; FxbB (fxbB), Mycobacterium
smegmatis 55,125 03-DEC-1998 FxbC (fxbC), and FxuD (fxtD) genes,
complete cds; and unknown genes. GB_BA2: SAU43537 3938 U43537
Streptomyces argillaceus mithramycin resistance determinant, ATP-
Streptomyces argillaceus 46,868 5-Sep-96 binding protein (mtrA) and
membrane protein (mtrB) genes, complete cds. rxa02746 290 GB_BA1:
CAJ10319 5368 AJ010319 Corynebacterium glutamicum amtP, glnB, glnD
genes and partial ftsY Corynebacterium 100,000 14-MAY-1999 and srp
genes. glutamicum GB_BA1: MTCY338 29372 Z74697 Mycobacterium
tuberculosis H37Rv complete genome; segment Mycobacterium 39,785
17-Jun-98 127/162. tuberculosis GB_HTG3: AC008733 216140 AC008733
Homo sapiens chromosome 19 clone CITB-E1_2525J15, *** Homo sapiens
35,688 3-Aug-99 SEQUENCING IN PROGRESS ***, 72 unordered pieces.
rxa02820 1411 GB_BA1: BFU64514 3837 U64514 Bacillus firmus dppABC
operon, dipeptide transporter protein dppA Bacillus firmus 36,859
1-Feb-97 gene, partial cds, and dipeptide transporter proteins dppB
and dppC genes, complete cds. GB_IN1: CET04C10 20958 Z69885
Caenorhabditis elegans cosmid T04C10, complete sequence.
Caenorhabditis elegans 35,934 2-Sep-99 GB_EST35: AI823090 720
AI823090 L30-944T3 Ice plant Lambda Uni-Zap XR expression library,
30 hours Mesembryanthemum 35,770 21-Jul-99 NaCl treatment
Mesembryanthemum crystallinum cDNA clone L30- crystallinum 944 5'
similar to 60S ribosomal protein L36 (AC004684)[Arabidopsis
thaliana], mRNA sequence. rxa02834 518 GB_BA1: CJY13333 3315 Y13333
Campylobacter jejuni clpB gene. Campylobacter jejuni 53,400
12-Apr-99 GB_BA2: AF065404 181654 AF065404 Bacillus anthracis
virulence plasmid PX01, complete sequence. Bacillus anthracis
45,168 20-OCT-1999 GB_PL2: AC006601 110684 AC006601 Arabidopsis
thaliana chromosome V map near 60.5 cM, complete Arabidopsis
thaliana 36,680 22-Feb-99 sequence.
[0217]
Sequence CWU 0
0
* * * * *
References