U.S. patent application number 10/579690 was filed with the patent office on 2008-02-07 for method for the preparation of lysine by fermentation of corynebacterium glutamicum.
This patent application is currently assigned to Basf Aktiengesellschaft. Invention is credited to Stefan Hafner, Elmar Heinzle, Patrick Kiefer, Corinna Klopprogge, Burkhard Kroger, Hartwig Schroder, Christoph Wittmann, Oskar Zelder.
Application Number | 20080032374 10/579690 |
Document ID | / |
Family ID | 34685565 |
Filed Date | 2008-02-07 |
United States Patent
Application |
20080032374 |
Kind Code |
A1 |
Zelder; Oskar ; et
al. |
February 7, 2008 |
Method for the Preparation of Lysine by Fermentation of
Corynebacterium Glutamicum
Abstract
The present invention features methods of increasing the
production of a fine chemical, e.g., lysine from a microorganism,
e.g., Corynebacterium by way of deregulating an enzyme encoding
gene, i.e., fructose-1,6-bisphosphatase. In a preferred embodiment,
the invention provides methods of increasing the production of
lysine in Corynebacterium glutamicum by way of increasing the
expression of fructose-1,6-bisphosphatase activity. The invention
also provides a novel process for the production of lysine by way
of regulating carbon flux towards oxaloacetate (OAA). In a
preferred embodiment, the invention provides methods for the
production of lysine by way of utilizing fructose or sucrose as a
carbon source.
Inventors: |
Zelder; Oskar; (Speyer,
DE) ; Klopprogge; Corinna; (Ludwigshafen, DE)
; Schroder; Hartwig; (Nussloch, DE) ; Hafner;
Stefan; (Ludwigshafen, DE) ; Kroger; Burkhard;
(Limburgerhof, DE) ; Kiefer; Patrick;
(Saarbrucken, DE) ; Heinzle; Elmar; (Saarbrucken,
DE) ; Wittmann; Christoph; (Saarbrucken, DE) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP
ONE POST OFFICE SQUARE
BOSTON
MA
02109-2127
US
|
Assignee: |
Basf Aktiengesellschaft
Ludwigshafen
DE
|
Family ID: |
34685565 |
Appl. No.: |
10/579690 |
Filed: |
December 17, 2004 |
PCT Filed: |
December 17, 2004 |
PCT NO: |
PCT/IB04/04429 |
371 Date: |
March 6, 2007 |
Current U.S.
Class: |
435/170 ;
435/196; 435/243; 435/252.3 |
Current CPC
Class: |
C12P 13/08 20130101;
C12N 9/16 20130101 |
Class at
Publication: |
435/170 ;
435/196; 435/243; 435/252.3 |
International
Class: |
C12P 1/04 20060101
C12P001/04; C12N 1/00 20060101 C12N001/00; C12N 1/21 20060101
C12N001/21; C12N 9/16 20060101 C12N009/16 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 18, 2003 |
IB |
IB2003/006456 |
Claims
1. A method for increasing metabolic flux through the pentose
phosphate pathway in a microorganism comprising culturing a
microorganism comprising a gene which is deregulated under
conditions such that metabolic flux through the pentose phosphate
pathway is increased.
2. The method of claim 1, wherein fructose or sucrose is used as a
carbon source.
3. The method of claim 1, wherein fructose is used as a carbon
source.
4. The method of claim 1, wherein the gene is
fructose-1,6-bisphosphatase.
5. The method of claim 4, wherein the fructose-1,6-bisphosphatase
gene is derived from Corynebacterium.
6. The method of claim 4, wherein the fructose-1,6-bisphosphatase
gene is overexpressed.
7. The method of claim 1, wherein the gene encodes
fructose-1,6-bisphosphatase.
8. The method of claim 7, wherein fructose-1,6-bisphosphatase has
increased activity.
9. The method of claim 1, wherein the microorganism is a Gram
positive microorganism.
10. The method of claim 1, wherein the microorganism belongs to the
genus Corynebacterium.
11. The method of claim 10, wherein the microorganism is
Corynebacterium glutamicum.
12. The method of claiim 1, wherein the microorganism is fermented
to produce a fine chemical.
13. The method of claim 1, wherein the microorganism further
comprises one or more additional deregulated gene.
14. The method of claim 13, wherein the one or more additional
deregulated gene is selected from the group consisting of an ask
gene, a dapA gene, an asd gene, a dapB gene, a ddh gene, a lysA
gene, a lysE gene, a pycA gene, a zwf gene, a pepCL gene, a gap
gene, a zwal gene, a tkt gene, a tad gene, a mqo gene, a tpi gene,
a pgk gene, and a sigC gene.
15. The method of claim 14, wherein the one or more additional
deregulated gene is overexpressed.
16. The method of claim 13, wherein the one or more additional
deregulated gene encodes a protein selected from the group
consisting of a feed-back resistant aspartokinase, a
dihydrodipicolinate synthase, an aspartate semialdehyde
dehydrogenase, a dihydrodipicolinate reductase, a diaminopimelate
dehydrogenase, a diaminopimelate epimerase, a lysine exporter, a
pyruvate carboxylase, a glucose-6-phosphate dehydrogenase, a
phosphoenolpyruvate carboxylase, a glyceraldedyde-3-phosphate
dehydrogenase, an RPF protein precursor, a transketolase, a
transaldolase, a menaquinine oxidoreductase, a triosephosphate
isomerase, a 3-phosphoglycerate kinase, and an RNA-polymerase sigma
factor sigC.
17. The method of claim 16, wherein the protein has increased
activity.
18. The method of claim 13, wherein the one or more additional
deregulated gene is selected from the group consisting of a pepCK
gene, a mal E gene, a glgA gene, a pgi gene, a dead gene, a menE
gene, a citE gene, a mikE17 gene, a poxB gene, a zwa2 gene, and a
sucC gene.
19. The method of claim 18, wherein the one or more additional
deregulated gene is attenuated, decreased or repressed.
20. The method of claim 13, wherein the one or more additional
deregulated gene encodes a protein selected from the group
consisting of a phosphoenolpyruvate carboxykinase, a malic enzyme,
a glycogen synthase, a glucose-6-phosphate isomerase, an ATP
dependent RNA helicase, an o-succinylbenzoic acid-CoA ligase, a
citrate lyase beta chain, a transcriptional regulator, a pyruvate
dehydrogenase, an RPF protein precursor, and a
Succinyl-CoA-Synthetase.
21. The method of claim 20, wherein the protein has. decreased
activity.
22. A method for producing a fine chemical comprising: a) culturing
a microorganism in which fructose-1,6-bisphosphatase is
deregulated; and b) accumulating the fine chemical in the medium or
in the cells of the microorganisms, thereby producing a fine
chemical.
23. A method for producing a fine chemical comprising culturing a
microorganism in which at least one pentose phosphosphate
biosynthetic pathway gene or enzyme is deregulated under conditions
such that the fine chemical is produced.
24. The method of claim 23, wherein said biosynthetic pathway gene
is fructose-1,6-bisphosphatase.
25. The method of claim 23, wherein said biosynthetic pathway
enzyme is fructose-1,6-bisphosphatase.
26. The method of claim 22 or 24, wherein
fructose-1,6-bisphosphatase expression is increased.
27. The method of claim 22 or 25, wherein
fructose-1,6-bisphosphatase activity is increased.
28. The method of claim 22 or 23, fur comprising recovering the
fine chemical.
29. The method of.claim 22 or 23, wherein one or more additional
gene is deregulated.
30. The method of claim 29, wherein the one or more additional
deregulated gene is selected from the group consisting of an ask
gene, a dapA gene, an asd gene, a dapB gene, a ddh gene, a lysA
gene, a lysE gene, a pycA gene, a zwf gene, a pepCL gene, a gap
gene, a zwal gene, a tkt gene, a tad gene, a mqo gene, a tpi gene,
a pgk gene, and a sigC gene.
31. The method of claim 30, wherein the one or more additional
deregulated gene is overexpressed.
32. The method of claim 29, wherein the one or more additional
deregulated gene encodes a protein selected from the group
consisting of a feed-back resistant aspartokinase, a
dihydrodipicolinate synthase, an aspartate semialdehyde
dehydrogenase, a dihydrodipicolinate reductase, a diaminopimelate
dehydrogenase, a diaminopimelate epinerase, a lysine exporter, a
pyruvate carboxylase, a glucose-6-phosphate dehydrogenase, a
phosphoenolpyruvate carboxylase, a glyceraldedyde-3-phosphate
dehydrogenase, an RPF protein precursor, a transketolase, a
transaldolase, a menaquinine oxidoreductase, a triosephosphate
isomerase, a 3-phosphoglycerate kinase, and an RNA-polymerase sigma
factor sigC.
33. The method of claim 32, wherein the protein has increased
activity.
34. The method of claim 29, wherein the one or more additional
deregulated gene is selected from the group consisting of a pepCK
gene, a mal E gene, a glgA gene, a pgi gene, a dead gene, a menE
gene, a cit gene, a mikE17 gene, apoxB gene, a zwa2 gene, and a
sucC gene.
35. The method of claim 34, wherein the one or more additional
deregulated gene is attenuated, decreased or repressed.
36. The method of claim 29, wherein the one or more additional
deregulated gene encodes a protein selected from the group
consisting of a phdsphoenolpyruvate carboxykinase, a malic enzyme,
a glycogen synthase, a glucose-6-phosphate isomerase, an ATP
dependent RNA helicase, an o-succinylbenzoic acid-CoA ligase, a
citrate lyase beta-chain, a transcriptional regulator, a
pyruvate-dehydrogenase, an RPF protein precursor, and a
Succinyl-CoA-Synthetase.
37. The method of claim 36, wherein the protein has decreased
activity.
38. The method of claim 22 or 23, wherein the microorganism is a
Gram positive microorganism.
39. The method of claim 22 or 23, wherein the microorganism belongs
to the genus Corynebacterium.
40. The method of claim 39, wherein the microorganism is
Corynebacterium glutamicum.
41. The method of claim 22 or 23, wherein the fine chemical is
lysine.
42. The method of claim 41, wherein lysine is produced at a yield
of at least 100 g/L.
43. The method of claim 41, wherein lysine is produced at a yield
of at least 150 g/L.
44. The method of claim 22 or 23, wherein fructose or sucrose is
used as a carbon source.
45. The method of claim 22 or 23, wherein fructose is used as a
carbon source.
46. The method of claim 22 or 24, wherein
fructose-1,6-bisphosphatase comprises the nucleotide sequence of
SEQ ID NO:1.
47. The method of claim 22 or 24, wherein
fructose-1,6-bisphosphatase encodes a polypeptide comprising
theaamino acid sequence of SEQ ID NO:2.
48. A recombinant microorganism which has a deregulated pentose
phosphate biosynthesis pathway.
49. A recombinant microorganism comprising a deregulated pentose
phosphate biosynthesis gene.
50. The recombinant microorganism of claim 49, wherein said
deregulated gene is fructose-1,6-bisphosphatase.
51. The recombinant microorganism of claim 50, wherein
fructose-1,6-bisphosphatase expression is increased.
52. The recombinant microorganism of claim 50, wherein said
fructose-1,6-bisphosphatase gene encodes a
fructose-1,6-bisphosphatase protein having increased activity.
53. The recombinant microorganism of claim 49, wherein the
microorganism belongs to the genus Corynebacterium.
54. The recombinant microorganism of claim 53, wherein the
microorganism is Corynebacterium glutamicum.
55. A polypeptide encoded by the nucleotide sequence of SEQ ID
NO:1, wherein said polypeptide has fructose-1,6-bisphosphatase
activity.
Description
BACKGROUND OF THE INVENTION
[0001] The industrial production of the amino acid lysine has
became an economically important industrial process. Lysirme is
used commercially as an animal feed supplement, because of its
ability to improve the quality of feed by increasing the absorption
of other amino acids, in human medicine, particularly as
ingredients of infusion solutions, and in the pharmaceutical
industry.
[0002] Commercial production of this lysine is principally done
utilizing the grain positive Corynebacterium glutamicum,
Brevibacterium flavunt and Brevibacterium lactofermentuwn
(KIeemann, A., et. al., "Amino Acids," in ULLMANN'S ENCYCLOPEDIA OF
INDUSTRIAL CHEMISTRY, vol. A2, pp. 57-97, Weinham:
VCH-Verlagsgesellschaft (1985)). These organisms presently account
for the approximately 250,000 tons of lysine produced annually. A
significant amount of research has gone into isolating mutant
bacterial strains which produce larger amounts of lysine.
Microorganisms employed in microbial process for amino acid
production are divided into 4 classes: wild-type strain,
auxotrophic mutant, regulatory mutant and auxotrophic regulatory
mutant (K. Nakayama et al., in Nutritional Improvement of Food and
Feed Proteins, M. Friedman, ed., (1978), pp. 649-661). Mutants of
Corynebacterium and related organisms enable inexpensive production
of amino acids from cheap carbon sources, e.g., molasses, acetic
acid and ethanol, by direct fermentation. In addition, the
stereospecificity of the amino acids produced by fermentation (the
L isomer) makes the process advantageous compared with synthetic
processes.
[0003] Another method in improving the efficiency of the commercial
production of lysine is by investigating the correlation between
lysine production and metabolic flux through the pentose phosphate
pathway. Given the economic importance of lysine production by the
fermentive process, the biochemical pathway for lysine synthesis
has been intensively investigated, ostensibly for the purpose of
increasing the total amount of lysine produced and decreasing
production costs (reviewed by Sahm et al., (1996) Ann. N.Y. Acad.
Sci. 782:25-39). There has been some success in using metabolic
engineering to direct the flux of glucose derived carbons toward
aromatic amino acid formation (Flores, N. et al., (1996) Nature
Biotechnol. 14:620-623). Upon cellular absorption, glucose is
phosphorylated with consumption of phosphoenolpyiuvate
(phosphotransferase system) (Malin & Bourd, (1991) Journal of
Applied Bacteriology 71, 517-523) and is then available to the cell
as glucose-6-phosphate. Sucrose is converted into fructose and
glucose-6-phosphate by a phosphotransferase system (Shio et al.,
(1990) Agricultural and Biological Chemistry 54, 1513-1519) and
invertase reaction (Yamamoto et al., (1986) Journal of Fermentation
Technology 64, 285-291).
[0004] During glucose catabolsm, the enzymes glucose-6-phosphate
dehydrogenase (EC 1.1.14.9) and glucose-6-phosphate isomerase (EC
5.3.1.9) compete for the substrate glucose-6-phosphate. The enzyme
glucose-6-phosphate isomerase catalyses the first reaction step off
the Embden-Meyerhof-Parnas pathway, or glycolysis, namely
conversion into fructose-6-phosphate. The enzyme
glucose-6-phosphate dehydrogenase catalyses the first reaction step
of the oxidative portion of the pentose phosphate cycle, namely
conversion into 6-phosphogluconolactone.
[0005] In the oxidative portion of the pentose phosphate cycle,
glucose-6-phosphate is converted into ribulose-5-phosphate, so
producing reduction equivalents in the ftrm of NADPH. As the
pentose phosphate cycle proceeds further, pentose phosphates,
hexose phosphates and triose phosphates are interconverted. Pentose
phosphates, such as for exarmple 5-phosphoribosyl-1-pyrophosphate
are required, for example, in nucleotide biosynthesis.
5-Phosphoribosyl-1-pyrophosphate is moreover a precursor for
aromatic amino acids and the amino acid L-histidine. NADPH acts as
a reduction equivalent in numerous anabolic biosyntheses. Four
molecules of NADPH are thus consumedafor the biosynthesis of one
molecule of lysine from oxalacetic acid. Thus, carbon flux towards
oxaloacetate (OAA) remain s constant regardless of system
perturbations (J. Vallino et al., (1993) Biotechnol. Bioeng., 41,
633-646).
SUMMARY OF THE INVENTION
[0006] The present invention is based, at least in part, on the
discovery of key enzyme-encodin genes, e.g.,
fructose-1,6-bisphosphatase, of the pentose phosphate pathway in
Corynebacterium glutamicum, and the discovery that deregulation,
e.g., increasing expression or activity of
fructose-1,6-bisphosphatase results in increased lysine production.
Furthermore, it has been found that increasing the carbon yield
during production of lysine by deregulating, e.g., increasing,
fructose-1,6 bisphosphatase expression or activity leads to
increased lysine production. In one embodiment, the carbon source
is fructose or sucrose. Accordingly, the present invention provides
methods for increasing production of lysine by microorganisms,
e.g., C. glutamicum, where fructose or sucrose is the
substrate.
[0007] Accordingly, in one aspect, the invention provides methods
for increasing metabolic flux through the pentose phosphate pathway
in a microorganism comprising culturing amicroorganism comprising a
gene which is deregulated under conditions such that metabolic flux
through the pentose phosphate pathway is increased. In one
embodiment, the microorganism is fermented to produce a fine
chemical, e.g., lysine. In another embodiment, fructose or sucrose
is used as a carbon source. In still another embodimeit, the gene
is fructose-1,6-bisphosphatase. In a related embodiment, the
fructose-1,6-bisphosphatase gene is derived from Corynebacterium,
e.g., Corynebacterium glutamicum. In another embodiment,
fructose-1,6 bisphosphatase gene is overexpressed. In a further
embodiment, the protein encoded by the fructose-1,6-bisphosphatase
gene has increased activity.
[0008] In another embodiment, the microorganism further comprises
one or more additional deregulated genes. The one or more
additional deregulated gene can include, but is not limited to, an
ask gene, a dapA gene, an asd gene, a dapb gene, a ddh gene, a lysA
gene, a lysE gene, a pycA gene, a zwf gene, a pepCL gene, a gap
gene, a zwal gene, a tkt gene, a tad gene, a mqo gene, a tpi gene,
a pgk gene, and a sigC gene. In a particular embodiment, the gene
may be overexpressed or underexpressed. Moreover, the deregulated
gene can encode a protein selected from the group consisting of a
feed-back resistant as partokinase, a dihydrodipicolinate synthase,
an aspartate semialdehyde dehydrogenase, a dihydrodipicolinate
reductase, a diaminopimelate dehydrogenase, a diaminopimelate
epimerase, a lysine exporter, a pyruvate carboxylase, a
glucose-6-phosphate dehydrogenase, a phosphoenolpyruvate
carboxylase, a glyceraldedyde-3-phosphate dehydrogenase, an RPF
protein precursor, a transketolase, a transaldolase, a menaqurine
oxidoreductase, a triosephosphate isomerase, a 3-phosphoglycerate
kinase, and an RNA-polymerase sigma factor sigC. In a particular
embodiment, the protein may have an increased or a decreased
activity.
[0009] In accordance with the methods of the present invention, the
one or more additional deregulated genes can also include, but is
not limited to, a pepCK gene, a mal E gene, a glgA gene, a pgi
gene, a dead gene, a nienE gene, a citE gene, a mikE17 gene, a poxB
gene, a zwa2 gene, and a sucC gene. In a particular embodiment the
expression of the at least one gene is upregulated, attenuated,
decreased, downregulated or repressed. Moreover, the deregulated
gene can encode a protein selected from the group consisting of a
phosphoenolpyruvate carboxykinase, a malic enzyme, a glycogen
synthase, a glucose-6-phosphate isomerase, an ATP. dependent RNA
helicase, an o-succinylbenzoic acid-CoA ligase, a citrate lyase
beta chain, a transcriptional regulator, a pyruvate dehydrogenase,
an RPF protein precursor, and a Succinyl-CoA-Synthetase. In a
particular embodiment, the protein has a decreased or an increased
activity.
[0010] In one embodiment, the microorganisms used in the methods of
the invention belong to the genus Corynebacterium, e.g.,
Corynebacterium glutamicum.
[0011] In another aspect, the invention provides methods for
producing a fine chemical comprising fermenting a microorganism in
which fructose-1,6-bisphosphatase is deregulated and accumulating
the fine chemical, e.g., lysine, in the medium or in the cells of
the microorganisms, thereby producing a fine chemical. In one
embodiment, the methods include recovenrng the fine chemical. In
another embodiment, the fructose-1,6-bisphosphatase gene is
overexpressed. In yet another embodiment, fructose or sucrose is
used as a carbon source.
[0012] In one aspect, fuctose-1,6-bisphosphatase is derived from
Corynebacterium glutamicum and comprises the nucleotide sequence of
SEQ ID NO:1 and the amino acid sequence of SEQ ID NO:2.
[0013] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1: is a schematic representation of the pentose
biosynthetic pathway.
[0015] FIG. 2: Comparison of relative mass isotopomer fractions of
secreted lysine and trehalose measured by GC/MS in tracer
experiments of Corynebacterium glutamicum ATCC 21526 during lysine
production on glucose and fructose.
[0016] FIG. 3: In vivo carbon flux distribution in the central
metabolism of Corynebacterium glutamicum ATCC -21526 during, lysine
production on glucose estimated from the best fit to the
experimental results using a comprehensive approach of combined
metabolite balancing and isotopboer modeling for .sup.13C tracer
experiments with labeling measurement of secreted lysine and
trehalose by GC/MS, respectively. Net fluxes are given in square
symbols, whereby for reversible reactions the direction of the net
flux is indicated by an arrow aside the corresponding black box.
Numbers in brackets below the fluxes of transaldolase,
transketolase and glucose 6-phosphate isomerase indicate flux
reversibilities. All fluxes are expressed as a molar percentage of
the mean specific glucose uptake rate (1.77 mmol
g.sup.-1h.sup.-1).
[0017] FIG. 4: In vivo carbon flux distribution in the central
metabolism of Corynebacterium glutainicum ATCC 21526 during lysine
production on fructose estimated from the best fit to the
experimental results using a comprehensive approach of combined
metabolite balancing and isotopomer modeling for .sup.13C tracer
experiments with labeling measurement of secreted lysine and
trehalose by GC/MS, respectively. Net fluxes are given in square
symbols, whereby for reversible reactions the direction of the net
flux is indicated by an arrow aside the corresponding black box.
Numbers in brackets below the fluxes of transaldolase,
transketolase and glucose 6-phosphate isomerase indicate flux
reversibilities. All fluxes are expressed as a molar percentage of
the mean specific fructose uptake rate (1.93 mmol
g.sup.-1h.sup.-1).
[0018] FIG. 5: Metabolic network of the central metabolism for
glucose-grown (A) and fructose-grown (B) lysine producing
Corynebacterium glutamicum including transport fluxes, anabolic
fluxes and fluxes between intermediary metabolite pools.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention is based at least in part, on the
identification of genes, e.g., Corynebacterium glutamicum genes,
which encode essential enzymes of the pentose phosphate pathway.
The present invention features methods comprising manipulating the
pentose phosphate biosynthetic paihway in a microorganism, e.g.,
Corynebacterium glutamicum such that the carbon yield is increased
and certain desirable fine chemicals, e.g., lysine, are produced,
e.g., produced-at increased yields. In particular, the invention
includes methods for producing fine chemicals, e.g., lysine, by
fermentation of a microorganism, e.g., Corynebacterium glutamicum,
having deregulated, e.g., increased, fructose-1,6-bisphosphatase,
expression or activity. In one embodiment, fructose or sacdharose
is used as a carbon source in the fermentation of the
microorganism. Fructose has been established to be a less efficient
substrate for the production of fine chemicals, e. g., lysine, from
microorganisms. However, the present invention prbvides methods for
optimizing production of lysine by microorganisms, e.g., C.
glutamicum where fructose or sucrose is the substrate.
Deregulation, e.g., amplification, of fructose-1,6-bisphosphatase
expression or activity leads to a higher flux through the pentose
phosphate pathway, resulting in increased NADPH generation and
increased lysine yield.
[0020] The term "pentose phosphate pathway" includes the pathway
involving pentose phosphate enzymes (e.g., polypeptides encoded by
biosynthetic enzyme-encoding genes), compounds (e.g., precursors,
substrates, intermediates or products), cofactors and the like
utilized in the formation or synthesis of fine chemicals, e.g.,
lysine. The pentose phosphate pathway converts glucose molecules
into biochemically useful smaller molecule.
[0021] In order that the present invention may be more readily
understood, certain terms are first defined herein.
[0022] The term "pentose phosphosphate biosynthetic pathway"
includes the biosynthetic pathway involving pentose phosphate
biosynthetic genes, enzymes (e.g., polypeptides encoded by
biosynthetic enzyme-encoding genes), compounds (e.g., precursors,
substrates, intermediates or products), cofactors and the like
utilized in the formation or synthesis of fine chemicals, e.g,
lysine. The term "pentose phosphosphate biosynthetic pathway"
includes the biosynthetic pathway leading to the synthesis of fine
chemicals, e.g., lysine, in a microorganisms (e.g., in vivo) as
well as the biosynthetic pathway leading to the synthesis df fine
chemicals, e.g., lysine, in vitro. The term "pentbse phosphosphate
biosynthetic pathway protein" or "pentose phosphosphate
biosynthetic pathway enzyme" includes a those peptides,
polypeptides, proteins, enzymes, and fragments thereof which are
directly or indirectly involved in the pentose phposphsphate
biosynthetic pathway, e.g., the fructose-1,6-bisphosphatase
enzyme.
[0023] The term "pentpse phosphosphate biosynthetic pathway gene"
includes a those genes and gene fragments encoding peptides,
polypeptides, proteins, and enzymes which are directly or
indirectly involved in the pentose phosphosphate biosynthetic
pathway, e.g., the fructose-1,6-bisphosphatase gene.
[0024] The term "amino acid-biosynthetic pathway gene" is meant to
include those genes and gene fragments encoding peptides,
polypeptides, proteins, and enzymes, which are directly involved in
the synthesis of amino acids, e.g., fructose-1,6-bisphosphatase.
These genes may be identical to those which naturally occur within
a host cell and are involved in the synthesis of any amino acid,
and particularly lysine, within that host cell.
[0025] The term "lysine biosynthetic pathway, gene" includes those
genes and genes fragments encoding peptides, polypeptides,
proteins, and enzymes, which are directly involved in the synthesis
of lysine, e.g., fructos-1,6-bisphosphatase. These genes can be
identical to those which naturally occur within a host cell and are
involved in the, synthesis of lysine within that host cell.
Alternatively, there can be modifications or mutations of such
genes, for example, the genes can contain modifications or
mutations which do not significantly affect the biological activity
of the encoded protein.
[0026] For example, the natural gene can be modified by mutagenesis
or by introducing or substituting one or more nucleotides or by
removing nonessential regions of the gene. Such modifications are
readily performed by standard, techniques.
[0027] The term "lysine biosynthetic pathway protein" is meant to
include those peptides, polypeptides, proteins, enzymes, and
fragments thereof which are directly involved in the synthesis of
lysine. These proteins can be identical to those which naturally
occur within a host cell and are involved in the synthesis of
lysine within that host cell. Alternatively, there can be
modifications or mutations of such proteins, for example, the
proteins can contain modifications or mutations which do not
significantly affect the biological activity of the protein. For
example, the natural protein can be modified by mutagenesis or by
introducing or substituting one or more amino acid, preferably by
conservative amino acid substitution, or by removing nonessential
regions of the protein. Such modifications are readily performed by
standard techniques. Alternatively, lysine biosynthetic proteins
can be heterologous to the particular host cell. Such proteins can
be from any organism having genes encoding proteins having the
same, or similar, biosynthetic roles.
[0028] The term, "carbon flux" refers to the number of glucose
molecules which proceed down a particular metabolic path relative
to competing paths. In particular, increased NADPH within a
microorganism is achieved by altering the carbon flux distribution
between the glycolytic and pentose phosphate pathways of that
organism.
[0029] "Fructose-1,6-bisphosphatase activity" includes any activity
exerted by a fructose-1,6-bisphosphatase protein, polypeptide or
nucleic acid molecule as determined in vivo, or in vitro, according
to standard techniques. Fructose-1,6-bisphosphatase is involved in
many different metabolic pathways and found in most organisms.
Preferably, a fructose-1,6-bisphosphatase acitivity includes the
catalysis of the hydrolysis of fructose 1,6-bisphosphate to
fructose 6-phosphate.
[0030] 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-protein6genic amino acids, pine and pyrimidine bases,
nucleosides, and nucleotides (as described e.g. in Kuiinaka, A.
(1996) Nucleotides and related compounds, p. 561-612, in
Biotechnology vol. 6, Rehm et al., eds. VCH: Weinheim, and
referdnces 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), land 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.
Amino Acid Metabolism and Uses
[0031] 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:
Weiheim (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
(1998)). 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.
[0032] 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.
[0033] 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 pyruvte, 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.
[0034] 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. 24: "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. Biochemicsty, 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.
Vitamin, Cofactor, and Nutraceutical Metabolism and Uses
[0035] Vitamins, cofactors, and nutraceuticals comprise another
group of molecules which the higher animals have lost the ability
to synthesize and so must ingest, although they are readily
synthesized by other organisms such as bacteria. These molecules
are either bioactive substances themselves, or are precursors of
biologically active substances which may serve as electron carriers
or intermediates in a variety of metabolic pathways. Aside from
their nutritive value, these compounds also have significant
industrial value as coloring agents, antioxidants, and catalysts or
other processing aids. (For an overview of the structure, activity,
and industrial applications of these compounds, see, for example,
Ullman's Encyclopedia of Industrial Chemistry, "Vitamins" vol. A27,
p. 443-613, VCH: Weinheim, 1996). The term "vitamin" is
art-recognized, and includes nutrients which are required by an
organism for normal functioing, but which that organism cannot
synthesize by itself. The group of vitamins may encompass cofactors
and nutraceutical compounds. The language "cofactor" includes
nonproteinaceous compounds required for a normal enzymatic activity
to occur. Such compounds may be organic or inorganic; the cofactor
molecules of the invention are preferably organic. The term
"nutraceutical" includes dietary supplements having health benefits
in plants and animals, particularly humans. Examples of such
molecules are vitamins, antioxidants, and also certain lipids
(e.g., polyunsaturated fatty acids).
[0036] The biosynthesis of these molecules in organisms capable of
producing them, such as bacteria, has been largely characterized
(Ullman's Encyclopedia of Industrial Chemistry, "Vitamins" vol.
A27, p. 443-613, VCH: Weinheim, 1996; Michal, G. (1999) Biochemical
Pathways: An Atlas of Biochemistry and Molecular Biology, John
Wiley & Sons; Ong, A. S., Nild, 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).
[0037] Thiamin (vitamin-B.sub.1) is produced by the chemical
coupling of pyrimidine and thiazole moieties. Riboflavin (vitamin
B.sub.2) is synthesized from guanosine-5'-triphosphate (GTP) and
ribose-5'-phosphate. Riboflavin, in turn, is utilized for the
synthesis of flavin mononucleotide (FMN) and flavin adenine
dinucleotide (FAD). The family of compounds collectively termed
`vitamin B.sub.6`(e.g., pyridoxine, pyridoxamine,
pyridoxa-5'-phosphate, and the commercially used pyridoxin
hydrochloride) are all derivatives of the common structural unit,
5-hydroxy-6-methylpyridine. Pantothenate (pantothenic acid,
(R)-(+)-N-(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)-.beta.-alanine)
can be produced either by chemical synthesis or by fermentation.
The final steps in pantothenate biosynthesis consist of the
ATP-driven condensation of .beta.-alanine and pantoic acid. The
enzymes responsible for the biosynthesis steps for the conversion
to pantoic acid, to .beta.-alanine and for the condensation to
panthotenic acid are known. The metabolically active form of
pantothenate is Coenzyme A, for which the biosynthesis proceeds in
5 enzymatic steps. Pantothenate, pyridoxal-5'-phosphate, cysteine
and ATP are the precursors of Coenzyme A. These enzymes not only
catalyze the formation of panthothante, but also the production of
(R)-pantoic acid, (R)-pantolacton, (R)-panthenol (provitamin
B.sub.5), pantetheine (and its derivatives) and coenzyme A.
[0038] Biotin biosynthesis from the precursor molecule pimeloyl-CoA
in microorganisms has been studied in detail and several of the
genes involved have been identified. Many of the corresponding
proteins have been found to also be involved in Fe-cluster
synthesis and are members of the nifS class of proteins. Lipoic
acid is derived from octanoic acid, and serves as a coenzyme in
energy metabolism, where it becomes part of the pyruvate
dehydrogenase complex and the .alpha.-ketoglutarate dehydrogenase
complex. The folates are a group of substances which are all
derivatives of folic acid, which is turn is derived from L-glutamic
acid, p-amino-benzoic acid and 6-methylpterin. The biosynthesis of
folic acid and its derivatives, starting from the metabolism
intermediates guanosine-5'-triphosphate (GTP), L-glutamic acid and
p-amino-benzoic acid has been studied in detail in certain
microorganisms.
[0039] Corrinoids (such as the cobalamines and particularly vitamin
B.sub.12) and porphyrines belong to a group of chemicals
characterized by a tetrapyrole ring system. The biosynthesis of
vitamin B.sub.12 is sufficiently complex that it has not yet been
completely characterized, but many of the enzymes and substrates
involved are now known.
[0040] Nicotinic acid (nicotinate), and nicotinamide are pyridine
derivatives which are also termed `niacin`. Niacin is the precursor
of the important coenzymes NAD (nicotinamide and adenine
dinucleotide) and NADP (nicotinamide adenine dinucleotide
phosphate) and their reduced forms.
[0041] 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.
Purine, Pyrimidine, Nucleoside and Nucleotide Metabolism and
Uses
[0042] Purine and pyrimidine metabolism genes and their
corresponding proteins are important targets for the therapy of
tumor diseases and viral infections. The language "purine" or
"pyrimidine" includes the nitrogenous bases which are constituents
of nucleic acids, co-enzymes, and nucleotides. The term
"nucleotide" includes the basic structural units of nucleic acid
molecules, which are comprised of a nitrogenous base, a pentose
sugar (in the case of RNA, the sugar is ribose; in the case of DNA,
the sugar is D-deoxyribose), and phosphoric acid. The language
"nucleoside" includes molecules which serve as precursors to
nucleotides, but which are lacking the phosphoric acid moiety that
nucleotides possess. By inhibiting the biosynthesis of these
molecules, or their mobilization to form nucleic acid molecules, it
is possible to inhibit RNA and DNA synthesis; by inhibiting this
activity in a fashion targeted to cancerous cells, the ability of
tumor cells to divide and replicate may be inhibited. Additionally,
there are nucleotides which do not form nucleic acid molecules, but
rather serve as energy stores (i.e., AMP) or as coenzymes (i.e.,
FAD and NAD).
[0043] Several publications have described the use of these
chemicals for these medical indications, by influencing purine
and/or pyrimidine metabolism (e.g. Christopherson, R. I. and Lyons,
S. D. (1990) "Potent inhibitors of de novo pyrimidine and purine
biosynthesis as chemotherapeutic agents." Med. Res. Reviews 10:
505-548). Studies of enzymes involved in purine and pyrimidine
metabolism have been focused on the development of new drags which
can be used, for example, as immunosuppressants or
anti-proliferants (Smith, J. L., (1995) "Enzymes in nucleotide
synthesis." Curr. Opin. Struct. Biol. 5: 752-757; (1995) Biochem
Soc. Transact. 23: 877-902). However, purine and pyrimidine bases,
nucleosides and nucleotides have other utilities: as intermediates
in the biosynthesis of several fine chemicals (e.g., thiamine,
S-adenosyl-methionine, folates, or riboflavin), as energy carriers
for the cell (e.g., ATP or GTP), and for chemicals themselves,
commonly used as flavor enhancers (e.g., IMP or GMP) or for several
medicinal applications (see, for example, Kuninaka, A. (1996)
Nucleotides and Related Compounds in Biotechnology vol. 6, Rehm et
al., eds. VCH: Weinheim, p. 561-612). Also, enzymes involved in
punine, pyrimidine, nucleoside, or nucledtide metabolism are
increasingly serving as targets against which chemicals for crop
protection, including fungicides, herbicides and insecticides, are
developed.
[0044] The metabolism of these compounds in bacteria has been
characterized (for reviews see, for example, Zalkin, H. and Dixon,
J. E. (1992) "de novo purine nucleotide biosynthesis", in: Progress
in Nucleic Acid Research and Molecular Biology, vol. 42, Academic
Press, p. 259-287; and Michal, G. (1999) "Nucleotides and
Nucleosides", Chapter 8 in: Biochemical Pathways: An Atlas of
Biochemistry and Molecular Biology, Wiley, N.Y.). Purine metabolism
has been the subject of intensive research, and is essential to the
normal functioning of the cell. Impaired purine metabbolism 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 nucledtides 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.
Trehalose Metabolism and Uses
[0045] Trehalose consists of two glucose molecules, bound in
.alpha.,.alpha.-1,1 linkage. It is commonly used in the food
industry as a sweetener, an additive for dried or frozen foods, and
in beverages: However, it also has applications in the
pharmaceutical, cosmetics and biotechnology industries (see, for
example, Nishimoto et al., (1998) U.S. Pat. No. 5,759,610; Singer,
M. A. and Lindquist, S. (1998) Trends Biotech. 16: 460-467; Paiva,
C. L. A. and Panek, A. D. (1996) Biotech. Ann. Rev. 2: 293-314; and
Shiosaka, M. (1997) J. Japan 172: 97-102). Trehalose is produced by
enzymes from many microorganisms and is naturally released into the
surrounding medium, from which it can be collected using methods
known in the art.
I. Recombinait Microorganisms and Methods for Culturing
Microorganisms Such That A Fine Chemical Is Produced
[0046] The methodologies of the present invention feature
microorganisms, e.g., recombinant microorganisms, preferably
including vectors or genes (e.g., wild-type and/or mutated, genes)
as described herein and/or cultured in a manner which results in
the production of a desired fine chemical, e.g. lysine. The term
"recombinant" microorganism includes a microorganism (e.g.,
bacteria, yeast cell, fungal cell, etc.) which has been genetically
altered, modified or engineered (e.g., genetically engineered) such
that it exhibits an altered, modified or different genotype and/or
phenotype (e.g., when the genetic modification affects coding
nucleic acid sequences of the microorganism) as compared to the
naturally-occurring microorganism from which it was derived.
Preferably, a "recombinant" microorganizm of the present invention
has been genetically engineered such that it overexpresses at least
one bacterial gene or gene product as described herein, preferably
a biosynthetic enzyme encoding-gene, e.g.,
fructose-1,6-bisphosphatase, included within a recombinant vector
as described herein and/or biosynthetic enzyme, e.g.,
fructose-1,6-bisphosphatase expressed from a recombinant vector.
The ordinary skilled will appreciate that a microorganism
expressing or overexpressing a gene product produces or
overproduces the gene product as a result of expression or
overexpression of nucleic acid sequences and/or genes encoding the
gene product. In one embodiment, the recombinant microorganism has
increased biosynthetic enzyme, e.g., fructose-1,6-bisphosphatase,
activity.
[0047] In certain embodiments of the present invention, at least
one gene or protein may be deregulated, in addition to the
fructose-1,6-bisphosphatase gene or enzyme, so as to enhance the
production of L-amino acids. For example, a gene or an enzyme of
the biosynthesis pathways, for example, of glycolysis, of
anaplerosis, of the citric acid cycle, of the pentose phosphate
cycle, or of amino acids export may be deregulated. Additionally, a
regulatory gene or protein may be deregulated.
[0048] In various embodiments, expression of a gene may be
increased so as to increase the intracellular activity or
concentration of the protein encoded by the gene, thereby
ultimately improving the production of the desired amino acid. One
skilled in the art may use various techniques to achieve the
desired result. For example, a skilled practitioner may increase
the number of copies of the gene or genes, use a potent promoter,
and/or use a gene or allele which codes for the corresponding
enzyme having high activity. Using the methods of the present
invention, for example, overexpressing a particular gene, the
activity or concentration of the corresponding protein can be
increased by at least about 10%, 25%, 50%, 75%, 100%, 150%, 200%,
300%, 400%, 500%, 1000% or 2000%, based on the starting activity or
concentration.
[0049] In various embodiments, the deregulated gene may include,
but is not limited to, at least one of the following genes or
proteins: [0050] the ask gene which encodes a feed-back resistant
aspartokinase (as disclosed in Iternational Publication No.
WO2004069996); [0051] the dapA gene which encodes
dihydrodipicolinate synthase (as disclosed in SEQ ID NOs:55 and 56,
respectively, in International Publication No. WO200100843); [0052]
the asd gene which encodes an aspartate semialdehyde dehydrogenase
(as disclosed in SEQ ID NOs:3435 and 6935, respectively, in
European Publication No. 1108790); [0053] the dapB gene which
encodes a dihydrodipicolinate reductase (as disclosed in SEQ ID
NOs:35 and 36, respectively, in International Publication No.
WO200100843); [0054] the ddh gene which encodes a diaminopimelate
dehydrogenase (as disclosed in SEQ ID NOs:3444 and 6944,
respectively, in European Publication No. 1108790); [0055] the lysA
gene which encodes a diaminopimelate epimerase (as disclosed in SEQ
ID NOs:3451 and 6951, respectively, in European Publication No.
1108790); [0056] the lysE gene which encodes a lysine exporter (as
disclosed in SEQ ID NOs:3455 and 6955, respectively, in European
Publication No. 1108790); [0057] the pycA gene which encodes a
pyruvate carboxylase (as disclosed in SEQ ID NOs:765 and 4265,
respectively, in European Publication No. 1108790); [0058] the zwf
gene which encodes a glucose-6-phosphate dehydrogenase (as
disclosed in SEQ ID NOs:243 and 244, respectively, in International
Publication No. WO200100844); [0059] the pepCL gene which encodes a
phosphoenolpyruvate carboxylase (as disclosed in SEQ ID NOs:3470
and 6970, respectively, in European Publication No. 1108790);
[0060] the gap gene which encodes a glyceraldedyde-3-phosphate
dehydrogenase (as disclosed in SEQ ID NOs:67 and 68, respectively,
in International Publication No. WO200100844); [0061] the zwa1 gene
which encodes an RPF protein precursor (as disclosed in SEQ ID
NOs:917 and 4417, respectively in European Publication No.
1108790); [0062] the tkt gene which encodes a transketolase (as
disclosed in SEQ ID NOs:247 and 248, respectively in International
Publication No. WO200100844); [0063] the tad gene which encodes a
transaldolase (as disclosed in SEQ ID NOs:245 and 246,
respectively, in International Publiction No. WO20010084); [0064]
mqo gene which codes for a menaquinine oxidoreductase (as disclosed
in SEQ ID NOs:569 and 770, respectively in International
Publication No. WO200100844); [0065] the tpi gene which codes for a
triosephosphate isomerase (as disclosed in SEQ ID NOs:61 and 62,
respectively, in International Publication No. WO200100844); [0066]
the pgk gene which codes for a 3-phosphoglycerate kinase (as
disclosed in SEQ ID NOs:69 and 70, respectively, in International
Publication No. WO200100844); and [0067] the sigC gene which codes
for an RNA-polymerase sigma factor sigC (as disclosed in SEQ ID
NOs:284 and 3784, respectively in European Publication No.
1108790). particular embodiments, the gene may be overexpressed
and/or the activity of the protein may be increased.
[0068] Alternatively, in other embodiments, expression of a gene
may be attenuated, decreased or repressed so as to decrease, for
example, eliminate, the intracellular activity or concentration of
the protein encoded by the gene, thereby ultimately improving the
production of the desired amino acid. For example, one skilled in
the art may use a weak promoter. Alternatively or in combination, a
skilled practitioner may use a gene or allele that either codes for
the corresponding enzyme having low activity or inactivates the
corresponding gene or enzyme. Using the methods of the present
invention, the activity or concentration of the corresponding
protein can be reduced to about 0 to 50%, 0 to 25%, 0 to 10%, 0 to
9%, 0 to 8%, 0 to 7%, 0 to 6%, 0 to 5%, 0 to 4%, 0 to 3%, 0 to 2%
or 0 to 1% of the activity or concentration of the wild-type
protein.
[0069] In certain embodiments, the deregulated gene may include,
but is not limited to, at least one of the following genes or
proteins: [0070] the pepCK gene which codes for the
phosphoenolpyruvate carboxykinase (as disclosed in SEQ ID NOs:179
and 180, respectively, in International Publication No.
WO200100844); [0071] the mal E gene which codes for the malic
enzyme (as disclosed in SEQ ID NOs:3328 and 6828, respectively, in
European Publication No. 1108790); [0072] the glgA gene which codes
for the glycogen synthase (as disclosed in SEQ ID NOs:1239 and
4739, respectively, in European Publication No. 1108790); [0073]
the pgi gene which codes for the glucose-6-phosphate isomerase (as
disclosed in SEQ ID NOs:41 and 42, respectively, in International
Publication No. WO200100844); [0074] the dead gene which codes for
the ATP dependent RNA helicase (as disclosed in SEQ ID NOs:1278 and
4778, respectively, in European Publication No. 1108790); [0075]
the menE gene which codes for the 0-succinylbenzoic acid-CoA ligase
(as disclosed in SEQ ID NOs:505 and 4005, respectively, in European
Publication No. 1108790); [0076] the citE gene which codes for the
citrate lyase beta chain (as disclosed in SEQ ID NOs:547 and 548,
respectively, in International Publication No. WO200100844); [0077]
the mikE17 gene which codes for a transcriptional regulator (as
disclosed in SEQ ID NOs:411 and 3911, respectively, in European
Publication No. 1108790); [0078] the poxB gene which codes for the
pyruvate dehydrogenase (as disclosed in SEQ ID NOs:85 and 86,
respectively, in International Publication No. WO200100844); [0079]
the zwa2 gene which codes for an RPF protein precursor (as
disclosed in European Publication No. 1106693); and [0080] the sucC
gene which codes for the Succinyl-CoA-Synthetase (as disclosed in
European Publication No. 1103611).
In particular embodiments, the expression of the gene may be
attenuated, decreased or repressed and/or the activity of the
protein may be decreased.
[0081] The term"manipulated microorganism" includes a microorganism
that has been engineered (e.g., genetically engineered) or modified
such that results in the disruption or alteration of a metabolic
pathway so as to cause a change in the metabolism of carbon. An
enzyme is "overexpressed" in a metabolically engineered cell when
the enzyme is expressed in the metabolically engineered cell at a
level higher than the level at which it is expressed in a
comparable wild-type cell. In cells that do not endogenously
express a particular enzyme, any level of expression of that enzyme
in the cell is deemed an "overexpression" of that enzyme for
purposes of the present invention. Over expression may lead to
increased activity of the protein encoded by the gene, e.g.,
fructose-1,6-bisphosphatase.
[0082] Modification or engineering of such microorganisms can be
according to any methodology described herein including, but not
limited to, deregulation of a biosynthetic pathway and/or
overexpression of at least one biosynthetic enzyme. A "manipulated"
enzyme (e.g., a "manipulated" biosynthetic enzyme) includes an
enzyme, the expression or production of which has been altered or
modified such that at least one upstream or downstream precursor,
substrate or product of the enzyme is altered or modified, e.g.,
has increased activity, for example, as compared to a corresponding
wild-type or naturally-occurring enzyme.
[0083] The term "overexpressed" or "overexpression" includes
expression of a gene product (e.g., apentose phosphate biosynthetic
enzyme) at a level greater than that expressed prior to
manipulation of the microorganism or in a comparable microorganism
which has not been manipulated. In one embodiment, the
microorganism can be genetically manipulated (e.g., genetically
engineered) to overexpress a level of gene product greater than
that expressed prior to manipulation of the microorganism or in a
comparable microorganism which has not been manipulated. Genetic
manipulation,can include, but is not limited to, altering or
modifying regulatory sequences or sites associated with expression
of a particular gene (e.g. by adding strohg promoters, inducible
promoters or multiple promoters or by removing regulatory sequences
such that expression is constitutive), modifying the chromosomal
location of aparticular gene, altering nucleic acid sequences
adjacent to a particular gene such as a ribosome binding site or
transcription terminator, increasing the copy number of a
particular gene, modifying proteins (e.g., regulatory proteins,
suppressors, enhancers, transcriptional activators and the like)
involved in transcription of a particular gene and/or translation
of a particular gene product, or any other conventional means of
deregulating expression of a particular gene routine in the art
(including but not limited to use of antisense nucleic acid
molecules, for example, to block expression of repressor
proteins).
[0084] In another embodiment, the microorganism can be physically
or environmentally manipulated to overexpress a level of gene
product greater than that expressed prior to manipulation of the
microorganism or in a comparable microorganism which has not been
manipulated. For example, a microorganism can be treated with or
cultured in the presence of an agent known or suspected to increase
transcription of a particular gene and/or translation of a
particular gene product such that transcription and/or translation
are enhanced or increased. Alternatively, a microorganism can be
cultured at a temperature selected to increase transcription of a
particular gene and/or translation of a particular gene product
such that transcription and/or translation are enhanced or
increased.
[0085] The term "deregulated" or "deregulation" includes the
alteration or modification of at least one gene in a microorganism
that encodes an enzyme in a biosynthetic pathway, such that the
level or activity of the biosynthetic enzyme in the microorganism
is altered or modified. Preferably, at least one gene that encodes
an enzyme in a biosynthetic pathway is altered or modified such
that the gene product is enhanced or increased, thereby enhancing
or increasing the activity of the gene product. The phrase
"deregulated pathway" can also include a biosynthetic pathway in
which more than one gene that encodes an enzyme in a biosynthetic
pathway is altered or modified such that the level or activity of
more than one biosynthetic enzyme is altered or modified. The
ability to "deregulate" apathway (e.g., to simultaneously
deregulate more than one gene in a given biosynthetic pathway) in a
microorganism arises from the particular phenomenon of
microorganisms in which more than one enzyme (e.g., two or three
biosynthetic enzymes) are encoded by genes occurring adjacent to
one another on a contiguous piece of genetic material termed an
"operon".
[0086] The term "operon" includes a coordinated unit of gene
expression that contains a promoter and possibly a regulatory
element associated with one or more, preferably at least two,
structural genes (e.g., genes encoding enzymes, for example,
biosynthetic enzymes). Expression of the structural genes can be
coordinately regulated, for example by regulatory proteins binding
to the regulatory element or by anti-termination of transcription.
The structural genes can be transcribed to give a single mRNA that
encodes all of the structural proteins. Due to the coordinated
regulation of genes included in an operon, alteration or
modification of the single promoter and/or regulatory element can
result in alteration or modification of each gene product encoded
by the operon. Alteration or modification of the regulatory element
can include, but is not limited to removing the endogenous promoter
and/or regulatory element(s), adding strong promoters, inducible
promoters or multiple promoters or removing regulatory. sequences
such that expression of the gene products is modified, modifying
the chromosomal location of the operon, altering nucleic acid
sequences adjacent to the operon or within the operon such as a
ribosome binding site, increasing the copy number of the operon,
modifying proteins (e.g., regulatory proteins, suppressors,
enhancers, transcriptional activators and the like) involved in
transcription of the operon and/or translation of the gene products
of the operon, or any other conventional means of deregulating
expression of genes routine in the art (including but not limited
to use of antisense nucleic acid molecules, for example, to block
expression of repressor proteins). Deregulation can also involve
altering the coding region of one or more genes to yield, for
example, an enzyme that is feedback resistant or has a higher or
lower specific activity.
[0087] A partcularly preferred "recombinant" microorganism of the
present invention has been genetically engineered to overexpress a
bacterially-derived gene or gene product. The term
"bacterially-derived" or "derived-from", for example bacteria,
includes a gene which is naturally found in bacteria or a gene
product which is encoded by a bacterial gene (e.g., encoded by
fructose-1,6-bisphosphatase).
[0088] The methodologies of the present invention feature
recombinant microorganisms which overexpress one or more genes,
e.g., the fructose-1,6-bisphosphatase, gene or have increased or
enhanced the fructose-1,6-bisphosphatase activity. A particularly
preferred recombinant microorganism of the present invention (e.g.,
Corynebacterium glutamicium, Corynebacterium acetoglutamicum,
Coryebacterium acetoacidophilum, and Corynebacterium
thermoaminogenes, etc.) has been genetically engineered to
overexpress a biosynthetic enzyme (e.g.
fructose-1,6-bisphosphatase, the amino acid sequence of SEQ ID NO:2
or encoded by the nucleic acid sequence of SEQ ID NO:1).
[0089] Other preferred "recombinant" microorganisms of the present
invention have an enzyme deregulated in the pentose phosphate
pathway. The phrase "microorganism having a deregulated pentose
phosphate pathway" includes a microorganism having an alteration or
modification in at least one gene encoding an enzyme of the pentose
phosphate pathway or having an alteration or modification in an
operon including more than one gene encoding an enzyme of the
pentose phosphate pathway. A preferred "microorganism having a
deregulated pentose phosphate pathway" has been genetically
engineered to overexpress a Cornynebacterium (e.g., C. glutamicium)
biosynthetic enzyme (e.g., has been engineered to overexpress
fructose-1,6-bisphosphatase).
[0090] In another preferred embodiment, a recombinant microorganism
is designed or engineered such that one or more pentose phosphate
biosynthetic enzyme is overexpressed or deregulated.
[0091] In another preferred embodiment, a microorganism of the
present invention overexpresses or is mutated for a gene or
biosynthetic enzyme (e.g., a pentose phosphate biosynthetic enzyme)
which is bacterially-derived. The term "bacterially-derived" or
"derived-from", for example bacteria, includes a gene product
(e.g., fructose-1,6-bisphosphatase) which is encoded by a bacterial
gene.
[0092] In one embodiment, a recombinant microorganism of the
present invention is a Gram positive organism (e.g., a
microorganism which retains basic dye, for example, crystal violet,
due to the presence of a Gram-positive wall surrounding the
microorganism). In a preferred embodiment, the recombinant
microorganism is a microorganism belonging to a genus selected from
the group consisting of Bacillus, Brevibacterium, Cornyebacterium,
Lactobacillus, Lactococci and Streptomyces. In a more preferred
embodiment, the recombinant microorganism is of the genus
Cornyebacterium. In another preferred embodiment, the recombinant
microorganism is selected from the group consisting of
Cornynebacterium glutamicium, Corynebacterium acetoglutamicum,
Corynebacterium acetoacidophilum or Corynebacterium
thermoaminogenes. In a particularly preferred embodiment, the
recombinant microorganism is Cornynebacterium glutamicium.
[0093] An important aspect of the present invention involves
culturing the recombinant microorganisms described herein, such
that a desired compound (e.g., a desired fine chemical) is
produced. The term "culturing" includes maintaining and/or growing
a living microorganism of the present invention (e.g. maintaining
and/or growing a culture or strain). In one embodiment, a
microorganism of the invention is cultured in liquid media. In
another embodiment, a microorganism of the invention is cultured in
solid media or semi-solid media. In a preferred embodiment, a
microorganism of the invention is cultured in media (e.g., a
sterile, liquid media) comprising nutrients essential or beneficial
to the maintenance and/or growth of the microorganism. Carbon
sources which may be used include sugars and carbohydrates, such as
for example glucose, sucrose, lactose, fructose, maltose, molasses,
starch and cellulose, oils and fats, such as for example soy oil,
sunflower oil, peanut oil and, coconut oil, fatty acids, such as
for example palmitic acid, stearic acid and linoleic acid,
alcohols, such as for example glycerol and ethanol, and organic
acids, such as for example acetic acid. In a preferred embodiment,
fructose or saccharose. These substances may be used individually
or as a mixture.
[0094] Nitrogen sources which may be used comprise organic
compounds containing nitrogen, such as peptones, yeast extract,
meat extract, malt extract, corn steep liquor, soya flour and urea
or inorganic compounds, such as ammonium sulfate, ammonium
chloride, ammonium phosphate, ammonium carbonate and ammonium
nitrate. The nitrogen sources may be used individually or as a
mixture. Phosphorus sources which may be used are phosphoric acid,
potassium dihydrogen phosphate or dipotassium hydrogen phosphate or
the corresponding salts containing sodium. The culture medium must
furthermore contain metal salts, such as for example magnesium
sulfate or iron sulfate, which are necessary for growth. Finally,
essential growth-promoting substances such as amino acids and
vitamins may also be used in addition to the above-stated
substances. Suitable precursors may furthermore be added to the
culture medium. The stated feed substances may be added to the
culture as a single batch or be fed appropriately during
cultivation.
[0095] Preferably, microorganisms of the present invention are
cultured under controlled pH. The term "controlled pH" includes any
pH which results in production of the desired fine chemical, e.g.,
lysine. In one embodiment, microorganisms are cultured at a pH of
about 7. In another embodiment, microorganisms are cultured at a pH
of between 6.0 and 8.5. The desired pH may be maintained by any
number of methods known to those skilled in the art. For example,
basic compounds such as sodium hydroxide, potassium hydroxide,
ammonia, or ammonia water, or acidic compounds, such as phosphoric
acid or suluic acid, are used to appropriately control the pH of
the culture.
[0096] Also preferably, microorganisms of the present invention are
cultured under controlled aeration. The term "controlled aeration"
includes sufficient aeration (e.g., oxygen) to result in production
of the desired fine chemical, e.g., lysine. In one embodiment,
aeration is controlled by regulating oxygen levels in the culture,
for example, by regulating the amount of oxygen dissolved in
culture media. Preferably, aeration of the culture is controlled by
agitating the culture. Agitation may be provided by a propeller or
similar mechanical agitation equipment, by revolving or shaking the
growth vessel (e.g., fermentor) or by various pumping equipment.
Aeration may be further controlled by the passage of sterile air or
oxygen through the medium (e.g., through the fermentation mixture).
Also preferably, microorganisms of the present invention are
cultured without excess foaming (e.g., via addition of antifoaming
agents such as fatty acid polyglycol esters).
[0097] Moreover, microorganisms of the present invention can be
cultured under controlled temperatures. The term "controlled
temperature" includes any temperature which results in production
of the desired fine chemical, e.g., lysine. In one embodiment,
controlled temperatures include temperatures between 15.degree. C.
and 95.degree. C. In another embodiment, controlled temperatures
include temperatures between 15.degree. C. and 70.degree. C.
Preferred temperatures are between 20.degree. C. and 55.degree. C.,
more preferably between 30.degree. C. and 45.degree. C. or between
30.degree. C. and 50.degree. C.
[0098] Microorganisms can be cultured (e.g., maintained and/or
grown) in liquid media and preferably are cultured, either
continuously or intermittently, by conventional culturing methods
such as standing culture, test tube culture, shaking culture (e.g.,
rotary shaking culture, shake flask culture, etc.), aeration
spinner culture, or fermentation. In a preferred embodiment, the
microorganisms are cultured in shake flasks. In a more preferred
embodiment, the microorganisms are cultured in a fermentor (e.g., a
fermentation process). Fermentation processes of the present
invention include, but are not limited to, batch, fed-batch and
continuous methods of fermentation. The phrase "batch process" or
"batch fermentation" refers to a closed system in which the
composition of media, nutrients, supplemental additives and the
like is set at the beginning of the fermentation and not subject to
alteration during the fermentation, however, attempts may be made
to control such factors as pH and oxygen concentration to prevent
excess media acidification and/or microorganism death. The phrase
"fed-batch process" or "fed-batch" fermentation refers to a batch
fermentation with the exception that one or more substrates or
supplements are added (e.g., added in increments or continuously)
as the fermentation progresses. The phrase "continuous process" or
"continuous fermentation" refers to a system in which a defined
fermentation media is added continuously to a fermentor and an
equal amount of used or "conditioned" media is simultaneously
removed, preferably for recovery of the desired fine chemical,
e.g., lysine. A variety of such processes have been developed and
are well-known in the art.
[0099] The phrase "culturing under conditions such that a desired
fine chemical, e.g., lysine is produced" includes maintaining
and/or growing microorganisms under conditions (e.g., temperature,
pressure, pH, duration, etc.) appropriate or sufficient to obtain
production of the desired fine chemical or to obtain desired yields
of the particularifine chemical, e.g., lysine, being produced. For
example, culturing is continued for a time sufficient to produce
the desired amount of a fine chemical (e.g., lysine). Preferably,
culturing is continued for a time sufficient to substantially reach
maximal production of the fine chemical. In one embodiment,
culturing is continued for about 12 to 24 hours. In another
embodiment, culturing is continued for about 24 to 36 hours, 36 to
48 hours, 48 to 72 hours, 72 to 96 hours, 96 to 120 hours, 120 to
144 hours, or greater than 144 hours. In another embodiment,
culturing is continued for a time sufficient to reach production
yields of a fine chemical, for example, cells are cultured such
that at least about 15 to 20 g/L of a fine chemical are produced,
at least about 20 to 25 g/L of a fine chemical are produced, at
least about 25 to 30 g/L of a fine chemical are produced, at least
about 30 to 35 g/L of a fine chemical are produced, at least about
35 to 40 g/L of a fine chemical are produced, at least about 40 to
50 g/L of a fine chermical are produced, at least about 50 to 60
g/L of a fine chemical are produced, at least about 60 to 70 g/L of
a fine chemical are produced, at least about 70 to 80 g/L of a fine
chemical are produced, at least about 80 to 90 g/L of a fine
chemical are produced, at least about 90 to 100 g/L of a fine
chemical are produced, at least about 100 to 110 g/L of a fine
chemical are produced, at least about 110 to 120 g/L of a fine
chemical are produced, at least about 120 to 130 g/L of a fine
chemical are produced, at least about 130 to 140 g/L of a fine
chemical are produced, or at least about 140 to 160 g/L of a fine
chemical are produced. In yet another embodiment, microorganisms
are cultured under conditions such that a preferred yield of a fine
chemical, for example, a yield within a range set forth above, is
produced in about 24 hours, in about 36 hours, in about 40 hours,
in about 48 hours, in about 72 hours, in about 96 hours, in about
108 hours, in about 122 hours, or in about 144 hours.
[0100] The methodology of the present invention can further include
a step of recovering a desired fine chemical, e.g., lysine. The
term "recovering" a desired fine chemical, e.g., lysine includes,
extracting, harvesting, isolating or purifying the compound from
culture media. Recovering the compound can be performed according
to any conventional isolation or purification methodology known in
the art including, but not limited to, treatment with a
conventional resin (e.g., anion or cation exchange resin, non-ionic
adsorption resin, etc.), treatment with a conventional adsorbent
(e.g., activated charcoal, silicic acid, silica gel, cellulose,
alumina, etc.), alteration of pH, solvent extraction (e.g., with a
conventional solvent such as an alcohol, ethyl acetate, hexane and
the like), dialysis, filtration, concentration, crystallization,
recrystallization, pH adjustment, lyophilization and the like. For
example, a fine chemical, e.g., lysine, can be recovered from
culture media by first removing the microorganisms from the
culture. Media is then passed through or over a cation exchange
resin to remove unwanted cations and then through or over an anion
exchange resin to remove unwanted inorganic anions and organic
acids having stronger acidities than the fine chemical of interest
(e.g., lysine).
[0101] Preferably, a desired fine chemical of the present invention
is "extracted", "isolated" or "purified" such that the resulting
preparation is substantially free of other components (e.g., free
of media components and/or fermentation byproducts). The language
"substantially free of other components" includes preparations of
desired compound in which the compound is separated (e.g., purified
or partially purified) from media components or fermentation
byproducts of the culture from which it is produced.
[0102] In one embodiment, the preparation has greater than about
80% (by dry weight) of the desired compound (e.g., less than about
20% of other media components or fermentation byproducts), more
preferably greater than about 90% of the desired compound (e.g.,
less than about 10% of other media components or fermentation
byproducts), still more preferably greater than about 95% of the
desired compound (e.g., less than about 5% of other media
components or fermentation byproducts), and most preferably greater
than about 98-99% desired compound (e.g., less than about 1-2%
other media components or fermentation byproducts).
[0103] In an alternative embodiment, the desired fine chemical,
e.g., lysine, is not purified from the microorganism, for example,
when the microorganism is biologically non-hazardous (e.g., safe).
For example, the entire culture (or culture supernatant) can be
used as a source of product (e.g., crude product). In one
embodiment, the culture (or culture supernatant) supernatant is
used without modification. In another embodiment, the culture (or
culture supernatant) is concentrated. In yet another embodiment,
the culture (or culture supernatant) is dried or lyophilized.
II. Methods of Producing A Fine Chemical Independent of Precursor
Feed Requirements
[0104] Depending on the biosynthetic enzyme or combination of
biosynthetic enzymes manipulated, it maybe desirable or necessary
to provide (e.g., feed) microorganisms of the present invention at
least one pentose phosphase pathway biosynthetic precursor sudh
that fine chemicals, e.g., lysine, are produced. The term "pentose
phosphase pathway biosynthetic precursor" or "precursor" includes
an agent or compound which, when provided to, brought into contact
with, or included in the culture medium of a microorganism, serves
to enhance or increase pentose phosphate biosynthesis. In one
embodiment, the pentose phosphate biosynthetic precursor or
precursor is glucose. In another embodiment, the pentose phosphate
biosynthetic precursor is fructose. The amount of glucose or
fructose added is preferably an amount that results in a
concentration in the culture medium sufficient to enhance
productivity of the microorganism (e.g., a concentration sufficient
to enhance production of a fine chemical e.g., lysine). Penitose
phosphate biosynthetic precursors of the present invention can be
added in:theform of a concentrated solution or suspension (e.g., in
a suitable solvent such as water or buffer) or in the form of a
solid (e.g., in the form of a powder). Moreover, pentose phosphate
biosynthetic precursors of the present invention can be added as
asingle aliquot, continuously or intermittently over a given period
of time.
[0105] Providing pent,ose phosphate biosynthetic precursors in the
pentose phosphate biosynthetic methodologies of the present
invention, can be associated with high costs, for example, when the
methodologies are used to produce high yields of a fine chemical.
Accordingly, preferred methodologies of the present invention
feature microorganisms having at least one biosynthetic enzyme or
combination of biosynthetic enzymes (e.g., at least one pentose
phosphate biosynthetic enzyme) manipulated such that lysine or
other desired fine chemicals are produced in a manner independent
of precursor feed. The phrase "a manner independent of precursor
feed", for example, when referring to a method for producing a
desired compound includes an approach to or a mode of producing the
desired compound that does not depend or rely on precursors being
provided (e.g., fed) to the microorganism being utilized to produce
the desired compound. For example, microorganisms featured in the
methodologies of the present invention can be used to produce fine
chemicals in a manner requning no feeding of the precursors glucose
or fructose.
[0106] Alternative preferred methodologies of the present invention
feature microorganisms having at least one biosynthetic enzyme or
combination of biosynthetic enzymes manipulated such that L-Lysine
or other fine chemicals are produced in a manner substantially
independent of precursor feed. The phrase "a manner substantially
independent of precursor feed", includes an approach to or a method
of producing the desired compound that depends or relies to a
lesser extent on precursors being provided (e.g., fed) to the
microorganism being utilized. For example, microorganisms featured
in the methodologies of the present invention can be used to
produce fine chemicals in a manner requiring feeding of
substantially reduced amounts of the precursors glucose or
fructose.
[0107] Preferred methods of producing desired fine chemicals in a
manner independent of precursor feed or alternatively, in a manner
substantially independent of precursor feed, involve culturing
microorganisms which have been manipulated (e.g., designed or
engineered for example, genetically engineered) such that
expression of at least one pentose phosphate biosynthetic enzyme is
modified. For example, in one embodiment, a microorganism is
manipulated (e.g., designed or engineered) such that the production
of at least one pentose phosphate biosynthetic enzyme is
deregulated. In a preferred embodiment, a microorganism is
manipulated (e.g. designed or engineered) such that it has a
deregulated biosynthetic pathway, for example, a deregulated
pentose phosphate biosynthesis pathway, as defined herein. In
another preferred embodiment, a microorganism is manipulated (e.g.,
designed or engineered) such that at least one pentose phosphate
biosynthetic enzyme, e.g., fructose-1,6-bisphosphatase is
overexpressed.
III. High Yield Production Methodologies
[0108] A particularly preferred embodiment of the present invention
is a high yield production method for producing a fine chemical,
e.g., lysine, comprising culturing a manipulated microorganism
under conditions such that lysine is produced at a significantly
high yield. The phrase "high yield production method", for example,
a high yield production method for producing a desired fine
chemical, e.g. lysine, includes a method that results in production
of the desired fine chemical at a level which is elevated or above
what is usual for comparable production methods. Preferably, a high
yield production method results in production of the desired
compound at a significaiitly high yield. The phrase "significantly
high yield" includes a level of production or yield which is
sufficiently elevated or above what is usual for comparable
production methods, for example, which is elevated to a level
sufficient for commercial production of the desired product (e.g.,
production of the product at a commercially feasible cost). In one
embodiment, the invention features a high yield production method
of producing lysine that includes culturing a manipulated
microorganism under conditions such that lysine is produced at a
level greater than 2 g/L, 10 g/L, 15 g/L, 20 g/L, 25 g/L, 30 g/L,
35 g/L, 40 g/L, 45 g/L, 50 g/L, 55 g/L, 60 g/L, 65 g/L, 70 g/L, 75
g/L, 80 g/L, 85 g/L, 90 g/L, 95 g/L, 100 g/L, 110 g/L, 120 g/L, 130
g/L 140 g/L, 150 g/L, 160 g/L, 170 g/L, 180 g/L, 190 g/L, or 200
g/L.
[0109] The invention firther features a high yield production
method for producing a desied fine chemical, e.g., lysine, that
involves culturing a manipulated microorganism under conditions
such that a sufficiently elevated level of compound is produced
within a commercially desirable period of time. In an exemplary
embodiment, the invention features a high yield production method
of producing lysine that includes culturing a manipulated
microorganism under conditions such that lysine is produced at a
level greater than 15-20 g/L in 5 hours. In another embodiment, the
invention features a high yield production method of producing
lysine that includes culturing a manipulated microorganism .under
conditions such that lysine is produced at a level. greater than
25-40 g/L, in 10 hours. In another embodiment, the invention
features a high yield production method of producing lysine that
includes culturing a manipulated microorganism under conditions
suchi that lysine is produced at a level greater than 50-100 g/L in
20 hours. In another embodiment, the invention features a high
yield production method of producing lysine that includes culturing
a manipulated microorgarusm under conditions such that lysine is
produced at alevel greater than 140-160 g/L in 40 hours, for
example greater than 150 g/L in 40 hours. (In another embodiment,
the invention features a high yield production method of producing
lysine that includes culturing a manipulated microorganism under
conditions such that lysine is produced at a level greater than
130-160 g/L in 40 hours, for example, greater than 135, 145 or 150
g/L in 40 hours. Values and ranges included and/or intermediate
within the ranges set forth herein are also intended to be within
the scope of the present invention. For example, lysine production
at levels of at least 140, 141, 142, 143, 144, 145, 146, 147, 148,
149, and 150 g/L in 40 hours are intended to be included within the
range of 140-150 g/L in 40 hours. In another example, ranges of
140-145 g/L or 145-150 g/L are intended to be included within the
range of 140-150 g/L in 40 hours. Moreover, the skilled artisan
will appreciate that culturing a manipulated microorganism to
achieve a production level of, for example, "140-150 g/L in 40
hours" includes culturing the microorganism for additional time
periods (e.g., time periods longer than 40 hours), optionally
resulting in even higher yields of lysine being produced.
IV. Isolated Nucleic Acid Molecules and Genes
[0110] Another aspect of the present invention features isolated
nucleic acid molecules that encode proteins (e.g., C. glutamicium
proteins), for example, Corynebactrium pentose phosphate
biosynthetic enzymes (e.g., C. glutamicium pentose phosphate
enzymes) for use in the methods of the invention. In one
embodiment, the isolated nucleic acid molecules used in the methods
of the invention are fructose-1,6-bisphosphatase nucleic acid
molecules.
[0111] The term "nucleic acid molecule" includes DNA molecules
(e.g., linear, circular, cDNA or chromosomal DNA) and RNA molecules
(e.g., tRNA, rRNA, mRNA) and analogs of the DNA or RNA generated
using nucleotide analogs. The nucleic acid molecule can be
single-stranded or double-stranded, but preferably is
double-stranded DNA. The term "isolated" nucleic acid molecule
includes a nucleic acid molecule which is free of sequences which
naturally flank the nucleic acid molecule (i.e., sequences located
at the 5' and 3' ends of the nucleic acid molecule) in the
chromnosomal DNA of the organism from which the nucleic acid is
derived. In various embodiments, an isolated nucleic acid molecule
can contain less than about 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,
0.5 kb, 0.1 kb, 50 bp, 25 bp or 10 bp of nucleotide sequences which
naturally flank the nucleic acid molecule in chromosomal DNA of the
microorganism from which the nucleic acid molecule is derived.
Moreover, an "isolated" nucleic acid molecule, such as a cDNA
molecule, can be substantially free of other cellular materials
when produced by recombinant techiiques, or substantially free of
chemical precursors or other chemicals when chemically
synthesized.
[0112] The term "gene," as used herein, includes a nucleic acid
molecule (e:g., a DNA molecule or segment thereof), for example, a
protein or RNA-encoding nucleic acid mnolecule, that in an
organism, is separated from another gene or other genes, by
intergenic DNA (i.e., intervening or spacer DNA which naturally
flanks the gene and/or separates genes inthe chromosomal DNA of the
organism). A gene may direct synthesis of an enzyme or. other
protein molecule (e.g., may comprise coding sequences, for example,
a contiguous, open reading frame (ORF) which encodes a protein) or
may itself be functional in the organism. A gene in an organism,
may be clustered in an operon, as defined herein, said operon being
separated from other genes and/or operons by the intergenic DNA.
Individual genes contained within an operon may overlap without
intergenic DNA between said individual genes. An "isolated gene",
as used herein, includes a gene which is essentially free of
sequences which naturally flank the gene in the chromosomal DNA of
the organism from which the gene is derived (i.e., is free of
adjacent coding sequences which encode a second or distinct protein
or RNA molecule, adjacent structural sequences or the like) and
optionally includes 5' and 3' regulatory sequences, for example
promoter sequences and/or terminator sequences. In one embodiment,
an isolated gene includes predominantly coding sequences for a
protein (e.g., sequences which encode Corynebactrium proteins). In
another embodiment, an isolated gene includes coding sequences for
a protein (e.g., for a Corynebactrium protein) and adjacent 5'
and/or 3' regulatory sequences from the chromosomal DNA of the
organism from which the gene is derived (e.g., adjacent 5' and/or
3' Corynebactrium regulatory sequences). Preferably, an isolated
gene contains less than about 10 kb, 5 kb, 2 kb, 1 kb, 0.5 kb, 0.2
kb, 0.1 kb, 50 bp, 25 bp or 10 bp of nucleotide sequences which
naturally flank the gene in the chromosomal DNA of the organism
from which the gene is derived.
[0113] In one aspect, the methods of the present invention features
use of isolated fructose-1,6-bisphosphatate nucleic acid sequences
or genes.
[0114] In a preferred embodiment, the nucleic acid or gene is
derived from Bacillus (e.g., is Corynebacetrium-derived). The term
"derived from Corynebactrium" or "Corynebactrium-derived" includes
a nucleic acid or gene which is naturally found in microorganisms
of the genus Corynebactrium. Preferably, the nucleic acid or gene
is derived from a microorganism selected fromithe group consisting
of Cornyebacterium glutamicium, Corynebacterium acetoglutamicum,
Corynebacterium acetoacidophilum or Corynebacterium
thermoaminogenes. In a particularly preferred embodiment, the
nucleic acid or gene is derived from Corynebacterium glutamicium
(e.g., is Cornynebacterium glutamicium-derived). In yet another
preferred embodiment, the nucleic acid or gene is a
Cornynebacterium gene homologue (e.g., is derived from a species
distinct from Cornynebacterium but having significant homology to a
Cornynebacterium gene of the present invention, for example, a
Cornynebacterium fructose-1,6-bisphosphatase gene).
[0115] Included within the scope of the present invention are
bacterial-derived nucleic acid molecules or genes and/or
Cornynebacterium-derived nucleic acid Molecules or genes (e.g.,
Cornynebacterium-derived nucleic acid molecules or genes), for
example, the genes identified by the present inventors, for
example, Cornynebacterium or C. glutamicium
fructose-1,6-bisphosphatase genes. Further included within the
scope of the present invention are bacterial-derived nucleic acid
molecules or genes and/or Cornynebacterium-derived nucleic acid
molecules or genes (e.g., C. glutamicium-derived nucleic acid
molecules or genes) (e.g., C. glutamicium nucleic acid molecules or
genes) which differ from naturally-occurning bacterial and/or
Cornynebacterium nucleic acid molecules or genes (e.g., C.
glutamicium nucleic acid molecules or genes), for example, nucleic
acid molecules or genes which have nucleic acids that are
substituted, inserted or deleted, but which encode proteins
substantially similar to the naturally-occurring gene products of
the present invention. In one embodiment, an isolated nucleic acid
molecule comprises the nucleotide sequences set forth as SEQ ID
NO:1, or encodes the amino acid sequence set forth in SEQ ID
NO:2.
[0116] In another embodiment, an isolated nucleic acid molecule of
the present invention comprises a nucleotide sequence which is at
least about 60-65%, preferably at least about 70-75%, more
preferable at least about 80-85%, and even more preferably at least
about 90-95% or more identical to a nucleotide sequence set forth
as SEQ ID NO:1. another embodiment, an isolated nucleic acid
molecule hybridizes under stringent conditions to a nucleic acid
molecule having a nucleotide sequence set forth as SEQ ID NO:1.
Such stringent conditions are known to those skilled 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 (e.g. high stringency) hybridization
conditions are hybridization in 6.times. sodium chloride/sodium
citrate (SSC) at about 45.degree. C., folldowed 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 the sequence of SEQ ID NO:1
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.
[0117] A nucleic acid molecule of the present invention (e.g., a
nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1
can be isolated using standard molecular biology techniques and the
sequence information provided herein. For example, nucleic acid
molecules can be isolated using standard hybridization and cloning
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;
Could Spring, Harbor, N.Y., 1989) or can be isolated by the
polymerase chain reaction using synthetic oligonucleotide primers
designed based upon the sequence of SEQ ID NO:1. A nucleic acid of
the invention can be amplified using cDNA, mRNA or alternatively,
genomic DNA, as a template and appropriate oligonuceotide primers
according to standard PCR amplification techniques. In another
preferred embodiment, an isolated nucleic acid molecule of the
invention comprises a nucleic acid molecule which is a complement
of the nucleotide sequence shown in SEQ ID NO:1.
[0118] In another embodiment, an isolated nucleic acid molecule is
or includes a fructose-1,6-bisphosphatase gene, or portion
orfragment thereof. In one embodiment, an isolated
fructose-1,6-bisphosphatase nucleic acid molecule or gene comprises
the nucleotide sequence as set forth in SEQ ID NO:1 (e.g.,
comprises the C. glutamicium frucfose-1,6-bisphosphatase nucleotide
sequence). In another embodiment, an isolated
fructose-1,6-bisphosphatase nucleic acid molecule or gene comprises
a nucleotide sequence that encodes the amino acid sequence as set
forth in SEQ ID NO:2 (e.g., encodes the C. glutamicium
fructose-1,6-bisphosphatase amino acid sequence). In yet another
embodiment, an isolated fructose-1,6-bisphosphatase nucleic acid
molecule or gene encodes a homologue of the
fructose-1,6-bisphosphatase protein having the amino acid sequence
of SEQ ID NO:2. As used herein, the term "homologue" includes a
protein or polypeptide sharing at least about 30-35%, preferably at
least about 35-40%, more preferably; at least about 40-50 %, and
even more preferably at least about 60%, 70%, 80%, 90% or more
identity with the amino acid sequence of a wild-type protein or
pdlypeptide described herein and having a substantially equivalent
functional or biological activity as said wild-type protein or
polypeptide. For example, a fructose-1,6-bisphosphatase homologue
shares at least about 30-35%, preferably at least about 35-40%,
more preferably at least about 40-50 %, and even more preferably at
least about 60%, 70%, 80%, 90% or more identity with the protein
having the amino acid sequence set forth as SEQ ID NO:2 and has a
substantially equivalent functional or biological activity (i.e.,
is a functional equivalent) of the protein having the amino acid
sequence set forth as SEQ ID NO:2 (e.g., has a substantially
equivalent pantothenate kinase activity). In a preferred
embodiment, an isolated fructose-1,6-bisphosphatase nucleic acid
molecule or gene comprises a nucleotide sequence that encodes a
polypeptide as set forth in SEQ ID NO:2. In another embodiment, an
isolated fructose-1,6-bisphosphatase nucleic acid molecule
hybridizes to all or a portion of a nucleic acid molecule having
the nucleotide sequence set forth in SEQ ID NO:1 or hybridizes to
all or a portion of a nucleic acid molecule having a nucleotide
sequence that encodes a polypeptide having the amino acid sequence
of SEQ ID NOs:2. Such hybridization conditions are known to those
skilled in the art and can be found in Current Protocols in
Molecular Biology, Ausubel et al., eds., John Wiley & Sons,
Inc. (1995), sections 2, 4 and 6. Additional stringent conditions
can be found in Molecular Cloning: A Laboratory Manual, Sambrook et
al., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989),
chapters 7; 9 and 11. A preferred, non-limiting example of
stringent hybridization conditions includes hybridization in
4.times. sodium chloride/sodium citrate (SSC), at about
65-70.degree. C. (or hybridization in 4.times.SSC plus 50%
formamide at about 42-50.degree. C.) followed by one or more washes
in 1.times.SSC, at about 65-70.degree. C. A preferred, non-limiting
example of highly stringent hybridization conditions includes
hybridization in 1.times.SSC, at about 65-70.degree. C. (or
hybridization in 1.times.SSC plus 50% formamide at about
42-50.degree. C.) followed by one or more washes in 0.3.times.SSC,
at about 65-70.degree. C. A preferred, non-limiting example of
reduced stringency hybridization conditions includes hybridization
in 4.times.SSC, at about 50-60.degree. C. (or alternatively
hybridization in 6.times.SSC plus 50% formamide at about
40-45.degree. C.) followed by one or more washes in 2.times.SSC, at
about 50-60.degree. C. Ranges intermediate to the above-recited
values, e.g., at 65-70.degree. C. or at 42-50.degree. C. are also
intended to be encompassed by the present invention. SSPE
(1.times.SSPE is 0.15 M NaCl, 10 mM NaH.sub.2PO.sub.4, and 1.25 mM
EDTA, pH 7.4) can be substituted for SSC (1.times.SSC is 0.15 M
NaCl and 15 mM sodium citrate) in the hybridization and wash
buffers; washes are performed for 15 minutes each after
hybridization is complete. The hybridization temperature for
hybrids anticipated to be less than 50 base pairs in length should
be 5-10.degree. C. less than the melting temperature (T.sub.m) of
the hybrid, where T.sub.m is determined according to the followimg
equations. For hybrids less thah 18 base pairs in length,
T.sub.m(.degree. C.)=2(#of A+T bases)+4(# of G+C bases). For
hybrids between 18 and 49 base pairs in length, T.sub.m(.degree.
C.)=81.5+16.6(log.sub.10[Na.sup.+])+0.41(%G+C)-(600/N), where N is
the number of bases in the hybrid, and [Na.sup.+] is the
concentration of sodium ions in the hybridization buffer
([Na.sup.+] for 1.times.SSC=0.165 M). It will also be recognized by
the skilled practitionerthat additional reagents may be added to
hybridization and/or wash buffers to decrease non-specific
hybridization of nucleic acid molecules to membranes, for example,
nitrocellulose or nylon membranes, including but not limited to
blocking agents (e.g., BSA or salmon or herring sperm carrier DNA),
detergents (e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP
and the like. When using nylon membranes, in particular, an
additional preferred, non-limiting example of stringent
hybridization conditions is hybridization in 0.25-0.5M
NaH.sub.2PO.sub.4, 7% SDS at about 65.degree. C., followed by one
or more washes at 0.02M NaH.sub.2PO.sub.4, 1% SDS at 65.degree. C.,
see e.g., Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA
81:1991-1995, (or, alternatively, 0.2.times.SSC, 1% SDS). In
another preferred embodiment, an isolated nucleic acid molecule
comprises a nucleotide, sequence that is complementary to a
fructose-1,6-bisphosphatase nucleotide sequence as set forth herein
(e.g., is the full complement of the nucleotide sequence set forth
as SEQ ID NO:1).
[0119] A nucleic acid molecle of the present invention (e.g., a
fructose-1,6-bisphosphatase nucleic acid molecule or gene), can be
isolated using standard molecular biology techniques and the
sequence information provided herein. For example, nucleic acid
molecules can be isolated using standard hybridization and cloning
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) or can be isolated by the polymerase
chain reaction using synthetic oligonucleotide primers designed
based upon the fructose-1,6-bisphosphatase nucleotide sequences set
forth herein, or flanking sequences thereof. A nucleic acid of the
invention (e.g., a fructose-1,6-bisphosphatase nucleic acid
molecule or gene), can be amplified using cDNA, mRNA or
alternatively, chromosomal DNA, as a template and appropriate
oligonucleotide primers according to standard PCR amplification
techniques.
[0120] Yet another embodiment of the present invention features
mutant fructose-1,6-bisphosphatase nucleic acid molecules or genes.
The phrase "mutant nucleic acid molecule" or "mutant gene" as used
herein, includes a nucleic acid molecule or gene having a
nucleotide sequence which includes at least one alteration (e.g.,
substitution, insertion, deletion) such that the polypeptide or
protein that may be encoded by said mutant exhibits an activity
that differs from the polypeptide or protein encoded by the
wild-type nucleic acid molecule or gene. Preferably, a mutant
nucleic acid molecule or mutant gene (e.g., a mutant
fructoseb-1,6-bisphosphatase gene) encodes a polypeptide or protein
having an increased activity (e.g., having an increased
fructose-1,6-bisphosphatase activity) as compared to the
polypeptide or protein encoded by the wild-type nucleic acid
molecule or gene, for example, when assayed under similar
conditions (e.g., assayed in microorganisms cultured at the same
temperature). A mutant gene also can have an increased level of
production of the wild-type polypeptide.
[0121] As used herein, an "increased or enhanced activity" or
"increased or enhanced enzymatic activity" is one that is at least
5% greater than that of the polypeptide or protein encoded by the
wild-type nucleic acid molecule or gene, preferably at least 5-10%
more, more preferably at least 10-25% more and even more preferably
at least 25-50%, 50-75% or 75-100% more than that of the
polypeptide or protein encoded by the wild-type nucleic acid
molecule or gene. Ranges intermediate to the above-recited values
e.g., 75-85%, 85-90%, 90-95%, are also intended to be encompassed
by the present invention. Activity can be determined according to
any well accepted assay for measuring activity of a particular
protein of interest. Activity can be measured or assayed directly,
for example, measuring an activity of a protein isolated or
purified from a cell. Alternatively, an activity can be measured or
assayed within a cell or in an extracellular medium.
[0122] It will be appreciated by the skilled artisan that even a
single substitution in a nucleic acid or gene sequence (e.g., a
base substitution that encodes an amino acid change in the
corresponding amino acid sequence) can dramatically affect the
activity of an encoded polypeptide or protein as compared to the
corresponding wild-type polypeptide or protein. A mutant nucleic
acid or mutant gene (e.g., encoding a mutant polypeptide or
protein), as defined herein, is readily distinguishable from a
nucleic acid or gene encoding a protein homologue, as described
above, in that a mutant nucleic acid or mutant gene encodes a
protein or polypeptide having an altered activity, optionally
observable as a different or distinct phenotype in a microorganism
expressing said mutant gene or nucleic acid or producing said
mutant protein or polypeptide (i.e., a mutant microorganism) as
compared to a corresponding microorganism expressing the wild-type
gene or nucleic acid or producing said mutant protein or
polypeptide. By contrast, a protein honidlogue has an identical or
substantially similar activity, optionally phenotypically
indiscernable when produced in a microorganism, as compared to a
corresponding microorganism expressing the wild-type gene or
nucleic acid. Accordingly it is not, for example, the degree of
sequence identity between nucleic acid molecules, genes, protein or
polypeptides that serves to distinguish between homologues and
mutants, rather it is the activity of the encoded protein or
polypeptide that distinguishes between homologues and mutants:
homologues having, for example, low (e.g., 30-50% sequence
identity) sequence identity yet having substantially equivalent
functional activities, and mutants, for example sharing 99%
sequence identity yet having dramatically different or altered
functional activities.
V. Recombinant Nucleic Acid Molecules and Vectors
[0123] The present invention further features recombinant nucleic
acid molecules (e.g., recombinant DNA molecules) that include
nucleic acid molecules and/or genes described herein (e.g.,
isolated nucleic acid molecules and/or genes), preferably
Cornynebacterium genes, more preferably Cornynebacterium
glutamicium genes, even more preferably Cornynebacterium
glutamicium fructose-1,6-bisphosphatase genes.
[0124] The present invention further features vectors (e.g.,
recombinant vectors) that include nucleic acid molecules (e.g.,
isolated or recombinant nucleic acid molecules and/or genes)
described herein. In particular, recombinant vectors are featured
that include nucleic acid sequences that encode bacterial gene
products as described herein, preferably Cornynebacterium gene
products, more preferably Cornynebacterium glutamicium gene
products (e.g., pentose phosphate enzymes, for example,
fructose-1,6-bisphosphatase).
[0125] The term "recombinant nucleic acid molecule" includes, a
nucleic acid molecule, (e.g., a DNA molecule) that has been altered
modified or engineered such that it differs in nucleotide sequence
from the native or natural nucleic acid molecule from which the
recombinant nucleic acid molecule was derived (e.g., by addition,
deletion or substitution of one or more nucleotides). Preferably, a
recombinant nucleic acid molecule (e.g., a recombinant DNA
molecule) includes an isolated nucleic acid molecule or gene ofthe
present invention (e.g., an isolated fructose-1,6-bisphosphatase
gene) operably linked to regulatory sequences.
[0126] The term "recombinant vector" includes a vector (e.g.,
plasmid, phage, phasmid, virus, cosmid or other purified nucleic
acid vector) that has been altered, modified or engineered such
that it contains grelater, fewer or different nucleic acid
sequences than those included in the native or natural nucleic acid
molecule from which the recombinant vector was derived. Preferably,
the recombinant vector includes a fructose-1,6-bisphosphatase gene
or recombinant nucleic acid molecule including such
fructose-1,6-bisphosphatase gene, operably linked to regulatory
sequences, for example, promoter sequences, terminator sequences
andior artificial ribosome binding sites (RBSs).
[0127] The phrase "operably linked to regulatory sequence(s)" means
that the nucleotide sequence of the nucleic acid molecule or gene
of interest is linked to the regulatory sequence(s) in a manner
which allows for expression (e.g., enhanced, increased,
constitutive, basal, attenuated, decreased or repressed expression)
of the nucleotide sequence, preferably expression of a gene product
encoded by the nucleotide sequence (e.g., when the recombinant
nucleic acid molecule is included in a recombinant vector, as
defined herein, and is introduced into a microorganism).
[0128] The term "regulatory sequence" includes nucleic acid
sequences which affect (e.g., modulate or regulate) expression of
other nucleic acid sequences. In one embodiment, a regulatory
sequence is included in a recombinant nucleic acid molecule or
recombinant vector in a similar or identical position and/or
orientation relative to a particular gene of interest as is
observed for the regulatory sequence and gene of interest as it
appears in nature, e.g., in a native position and/or orientation.
For example, a gene of internest can be included in a recombinant
nucleic acid molecule or recombinant vector operably linked to a
regulatory sequence which accompanies or is adjacent to the gene of
interest in the natural organism (e.g., operably linked to "native"
regulatory sequences, for example, to the "native" promoter).
Alternatively, a gene of interest can be included in a recombinant
nucleic acid molecule or recombinant vector operably linked to a
regulatory sequence which accompanies or is adjacent to another
(e.g., a different) gene in the natural organism. Alternatively, a
gene of interest can be included in a redombinant nucleic acid
molecule or recombinant vector operably linked to a regulatory
sequence from another organism. For example, regulatory sequences
from other microbes (e.g., other bacterial regulatory sequences,
bacteriophage regulatory sequences and the like) can be operably
linked to a particular gene of interest.
[0129] In one embodiment, a regulatory sequence is a non-native or
non-naturally-occurring sequence (e.g., a sequence which has been
modified, mutated, substituted, derivatized, deleted including
sequences which are chemically synthesized).
[0130] Preferred regulatory sequences include promoters, enhancers,
termination signals, anti-termination signals and other expression
control elements (e.g., sequences to which repressors or inducers
bind and/or binding sites for transcriptional and/or translational
regulatory proteins, for example, in the transcribed mRNA). Such
regulatory sequences are described, for example, 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. Regulatory
sequences include those which direct constitutive expression of a
nucleotide sequence in a microorganism (e.g., constitutive
promoters and strong constitutive promoters), those which direct
inducible expression of a nucleotide sequence in a microorganism
(e.g., inducible promoters, for example, xylose inducible
promoters) and those which attenuate or repress expression of a
nucleotide sequence in a microorganism (e.g., attenuation signals
or repressor sequences). It is also within the scope of the present
invention to regulate expression of a gene of interest by removing
or deleting regulatory sequences. For example, sequences involved
in the negative regulation of transcription can be removed such
that expression of a gene, of interest is enhanced.
[0131] In one embodiment, a recombinant nucleic acid molecule or
recombinant vector of the present invention includes a nucleic acid
sequence or gene that encodes at least one bacterial gene product
(e.g., a pentose phosphate biosynthetic enzyme, for example
fructose-1,6-bisphosphatase) operably linked to a promoter or
promoter sequence. Preferred promoters of the present invention
iniclude Corynebacterium promoters and/or bacteriophage promoters
(e.g., bacteriophage which infect Corynebacterium). In one
embodiment, a promoter is a Corynebacterium promoter, preferably a
strong Corynebacterium promoter (e.g., a promoter associated with a
biochemical housekeeping gene in Corynebacterium or a promoter
associated with a glycolytic pathway gene in Corynebacterium). In
another embodiment, a promoter is a bacteriophage promoter.
[0132] In another embodiment, a recombinant nucleic acid molecule
or recombinant vector of the present invention includes a
terminator sequence or terminator sequences (e.g., transcription
terminator sequences). The term "terminator sequences" includes
regulatory sequences which serve to terminate transcription of a
gene. Terminator sequences (or tandem transcription terminators)
can further serve to stabilize mRNA (e.g., by adding structure to
mRNA) for example, against nucleases.
[0133] In yet another embodiment, a recombinant nucleic acid
molecule or recombinant vector of the present invention includes
sequences which allow for detection of the vector containing said
sequences (i.e., detectable and/or selectable markers), for
example, sequences that overcome auxotrophic mutations, for
example, ura3 or ilvE, fluorescent markers, and/or colorimetric
markers (e.g., lacZ/.beta.-galactosidase), and/or antibiotic
resistance genes (e.g., amp or tet).
[0134] In yet another embodiment, a recombinant vector of the
present invention includes antibiotic resistance genes. The term
"antibiotic resistance genes" includes sequences which promote or
confer resistance to antibiotics on the host organism (e.g.,
Bacillus). In one embodiment, the antibiotic resistance genes are
selected from the group consisting of cat (chloramphenicol
resistance) genes, tet (tetracycline resistance) genes, erm
(erythromycin resistance) genes, neo (neomycin resistance) genes
and spec (spectinomycin resistance) genes. Recombinant vectors of
the present invention can fruther include homologous recombination
sequences (e.g., sequences designed to allow recombination of the
gene of interest into the chromosome of the host organism). For
example, amyE sequences can be used as homology targets for
recombination into the host, chromosome.
[0135] It will further be appreciated by one of skill in the art
that the design of a vector can be tailored depending on such
factors as the choice of microorganism to be genetically
engineered, the level of expression of gene prodauct desired and
the like.
VI. Isolated Proteins
[0136] Another aspect of the present invention features isolated
proteins (e.g. isolated pertose phosphate biosynthetic enzymes, for
example isolated fructose-1,6-bisphosphatase). In one embodiment,
proteins (e.g., isolated pentose phosphate enzymes, for example
isolated fructose-1,6-bisphosphatase) are produced by recombinant
DNA techniques and can be isolated from microorganisms of the
present invention by an appropriate purification scheme using
standard protein purification techniques. In another embodiment,
proteins are synthesized chemically using standard peptide
synthesis techniques.
[0137] An "isolated" or "purified" protein (e.g., an isolated or
purified biosynthetic enzyme) is substantially free of cellular
material or other contaminating proteins from the microorganism
from which the protein is derived, or substantially free from
chemical precursors or other chemicals when chemically synthesized.
In one embodiment, an isolated or purified proteinihas less than
about 30% (by dry weight) of contaminatihg protein or chemicals,
more preferably less than about 20% of contaminating protein or
chemicals, still more preferably less than about 10% of
contaminating protein or chemicals, and most preferably less than
about 5% contaminating protein or chemicals.
[0138] In a preferred embodiment, the protein or gene product is
derived from Cornynebacterium (e.g., is Cornynebacterium-derived).
The term "derived from Cornynebacterium" or
"Cornynebacterium-derived" includes a protein or gene product which
is encoded by a Cornynebacterium gene. Preferably, the gene product
is derived from a microorganism selected from the group consisting
of Cornynebacterium glutamicium, Corynebacterium acetoglutamicum,
Corynebacterium acetoacidophilum or Corynebacterium
thermoaminogenes. In a particularly preferred embodiment, the
protein or gene product is derived from Cornynebacterium
glutamicium (e.g., is Cornynebacterium glutamicium-derived). The
term "derived from Cornynebacterium glutamicium" or
"Cornynebacterium glutamicium-derived" includes a protein or gene
product which is encoded by a Cornynebacterium glutamicium gene. In
yet another preferred embodiment, the protein or gene product is
encoded by a Cornynebacterium gene homologue (e.g., a gene derived
from a species distinct from Cornynebacterium but having
significant homology to a Cornynebacterium gene of the present
invention, for example, a Cornynebacterium
fructose-1,6-bisphosphatase gene).
[0139] Included within the scope of the present invention are
bacterial-derived proteins or gene products and/or
Cornynebacterium-derived proteins or gene products (e.g., C.
glutamicium-derived gene products) that are encoded by
naturally-occurring bacterial and/or Cornynebacterium genes (e.g.,
C. glutamicium genes), for example, the genes identified by the
present inventors, for example, Cornynebacterium or C. glutamicium
fructose-1,6-bisphosphatase genes. Further included within the
scope of the present invention are bacterial-derived proteins or
gene products and/or Cornynebacterium-derived proteins or gene
products (e.g., C. glutamicium-derived gene products) that are
encoded bacterial and/or Cornynebacterium genes (e.g., C.
glutamicium genes) which differ from naturally-occurring bacterial
and/or Cornynebacterium genes (e.g., C. glutamicium genes), for
example, genes which have nucleic acids that are mutated, inserted
or deleted, but which encode proteins substantially similar to the
naturally-occurring gene products of the present invention. For
example, it is well understood that one of skill in the art can
mutate (e.g., substitute) nucleic acids which, due to the
degeneracy of the genetic code, encode for an identical amino acid
as that encoded by the naturally-occurring gene. Moreover, it is
weil understood that one of skill in the art can mutate (e.g.,
substitute) nucleic acids which encode for conservative amino acid
substitutions. It is further well understood that one of skill in
the art can substitute, add or delete amino acids to acertain
degree without substantially affecting the function of a gene
product as compared with a naturally-occrring gene product, each
instance of which is intended to be included within the scope of
the present invention.
[0140] In a preferred embodiment, an isolated protein of the
present invention (e.g., an isolated pentose phosphate biosynthetic
enzyme, for example isolated fructose-1,6-bisphosphatase) has an
amino acid sequence shown in SEQ ID NO:2. In other embodiments, an
isolated protein of the present invention is a homologue of the
protein set forth as SEQ ID NO:2, (e.g., comprises an amino acid
sequence at least about 30-40% identical, preferably about 40-50%
identical, more preferably about 50-60% identical, and even more
preferably about 60-70%, 70-80%, 80-90%, 90-95% or more identical
to the amino acid sequence of SEQ ID NO:2, and has an activity that
is substantially similar to that of the protein encoded by the
amino acid sequence of SEQ ID NO:2.
[0141] To determine the percent homology of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in the
sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a second amino or nucleic acid sequence). When a
position in the first sequence is occupied by the same amino acid
residue or nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position. The
percent identity between the two sequences is a function of the
number of identical positions shared by the sequences (i.e., %
identity=# of identical positions/total # of positions .times.100),
preferably taking into account the number of gaps and size of said
gaps necessary to produce an optimal alignment.
[0142] The comparison of sequences and determination of percent
homology between two sequences can be accomplished using a
mathematical algorithm. A preferred, non-limiting example of a
mathematical algorithm utilized for the comparison of sequences is
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 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 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
Research 25(17):3389-3402. When utilizing BLAST and Gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
Another preferred, non-limiting example of a mathematical algorithm
utilized for the comparison of sequences is the algoritbhm of Myers
and Miller (1988) Comput Appl Biosci. 4:11-17. Such an algorithm is
incorporated into the ALIGN program available, for example, at the
GENESTREAM network server, IGH Montpellier, FRANCE
(http://vega.igh.cnrs.fr) or at the ISREC server
(http://www.ch.embnet.org). 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.
[0143] In another preferred embodiment, the percent homology
between two amino acid sequences can be determined using the GAP
program in the GCG software package:(available at
http://www.gcg.com), using either a Blossom 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. In yet another preferred embodiment, the percent
homology between two nucleic acid sequences can be accomplished
using the GAP program in the GCG software package (available at
http://www.gcg.com), using a gap weight of 50 and a length weight
of 3.
[0144] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents, Sequence Listing, Figures, and published
patent applications cited throughout this application are
incorporated herein by reference.
EXAMPLES
General Methodology
[0145] Strains. Corynebacterium glutamicum ATCC 21526 was obtained
from the American Type and Culture Collection Manassas, USA). This
homoserine auxptrophic strain excretes lysine during L-threonine
limitation due to the bypass of concerted aspartate kinase
inhibition. Precultures were grown in complex medium containing 5 g
L.sup.-1 of either fructose or glucose. For agar plates the complex
medium was additionally amended with 12 g L.sup.-1 agar. For the
production of cells as inoculum for the tracer experiments and the
tracer studies itself a minimal medium amended with 1 mg ml.sup.-1
calcium panthotenate HCl was used (Wittmann, C. and E. Heinzle 2002
Appl. Environ. Microbiol. 68:5843-5859). In this medium
concentrations of carbon source glucose or fructose, of the
essential amino acids threonine, methionine and leucine and of
citrate were varied as specified below.
[0146] Cultivation. Precultivation consisted of three steps
involving (i) a starter cultivation in complex medium with cells
from agar plate as inoculum, (ii) a short cultivation for adaption
to minimal medium, and (iii) a prolonged cultivaion on minimal
medium with elevated concentrations of essential amino acids.
Pre-cultures inoculated from agar plates were grown overnight in
100 ml baffled shake flasks on 10 ml complex medium. Afterwards
cells were harvested by centrifugation (8800 g, 2 min, 30.degree.
C.), inoculated into minimal medium, and grown-up to an optical
density of 2 to obtain exponentially growing cells adapted to
minimal medium. Afterwards cells were harvested by centrifugation
(8800 g, 30.degree. C., and 2 min) including a washing step with
sterile 0.9% NaCl. They were then inoculated into 6 ml minimal
medium in 50 ml baffled shake flasks with initial concentrations of
0.30 g L.sup.-1 threonine, 0.08 g L.sup.-1 methionine, 0.20 g
L.sup.-1 leucine, and 0.57 g L.sup.-1 citrate. As carbon source 70
mM glucose or 80 mM fructose were added, respectively. Cells were
grown until depletion of the essential amino acids, which was
checked by HPLC analysis. At the end of the growth phase cells were
harvested, and washed with sterile NaCl (0.9%). Subsequently they
were transferred into 4 ml minimal tracer medium in 25 ml baffled
shake flasks for metabolic flux analysis under lysine producing
conditions. The tracer medium did not contain any threonine,
methionine, leucine and citrate. For each carbon source two
parallel flasks were incubated containing (i) 40 mM [1.sup.13C]
labeled substrate, and (ii) 20 mM [.sup.13C.sub.6] labeled
substrate plus 20 mM of naturally labeled substrate, respectively.
All cultivations were carried out on a rotary shaker (Inova 4230,
New Brunswick, Edison, N.J., USA) at 30.degree. C. and 150 rpm.
[0147] Chenmicals. 99% [1-.sup.13C] glucose,99% [1-.sup.13C]
fructose, 99% [.sup.13C.sub.6] glucose and 99% [C.sup.13C.sub.6]
fructose were purchased from Campro Scientific (Veenendaal,
Netherlands). Yeast extract and tryptone were obtained from Difco
Laboratories (Detroit, Mich. USA). All other applied chemicals were
from Sigia (St. Louis, Mich. USA), Merck (Darmstadt, Germany) or
Fluka (Buchs, Switzerland), respectively, and of analytical
grade
[0148] Substrate and prodiuct analysis. Cell concentration was
determined by measurement of cell density at 660 nm (OD.sub.660 nm)
using a photometer (Marsha Pharmacia biotech, Freiburg, Germany) or
by gravimetry. The latter was determined by harvesting 100 ml of
cells from cultivation broth at room temperature for 10 min at 3700
g, including a washing step with water. Washed cells were dried at
80.degree. C. until weight constancy. The correlation factor (g
biomass/OD.sub.660nm) between dry cell dry mass and OD.sub.660nm
was determined as 0.353.
[0149] Concentrations of extracellular substrates and products were
determined in cultivation supernatants, obtained via 3 miin
centrifugation at 16000 g. Fructose, glucose, sucrose, and
trehalose were quantified by GC after derivatization into oxime
trimethylsilyl derivatives. For this purpose a HP 6890 gas
chromatograph (Hewlett Packard, Palo Alto, USA) with an HP 5MS
column (5% phenyl-methyl-siloxane-diphenyldimnethylpolysiloxane, 30
m.times.250 .mu.m, Hewlett Packard, Paolo Alto, Calif., USA), and a
quadrupole mass selective detector with electron impact ionizatiori
at 70 eV (Agilent Technologies, Waldbronn, Germany) was applied.
Sample preparation included lyophilization of the culture
supernatant, dissolution in pyridine, and subsequent two-step
derivatization of the sugars with hydroxylamine and
(trimethylsilyl)trifluoroacetamide (BSTFA) Macherey & Nagel,
Duren, Germany) (13, 14). .beta.-D-ribose was used as internal
standard for quantification. The injected sample volume was 0.2
.mu.l. The time program for GC analysis was as follows: 150.degree.
C. (0-5 min), 8.degree. C. min.sup.-1 (5-25 min), 310.degree. C.
(25-35 min). Helium was used as carrier gas with a flow of 1.5 1
min.sup.-1. The inlet temperature was 310.degree. C. and the
detector temperature was 320.degree. C. Acetate, lactate, pyruvate,
2-oxoglutarate, and dihydroxyacetone were determined by BPLC
utilizing an Aminex-HPX-87H Biorad Column (300.times.7.8 mm,
Hercules, Calif., USA) with 4 mM sulfuric acid as mobile phase at a
flow rate of 0.8 ml min.sup.-1, and UV-detection at 210 nm.
Glycerol was quantified by enzymatic measurement (Boehringer,
Mannheim, Germany). Amino acids were analyzed by HPLC (Agilent
Technologies, Waldbronn, Germany) utilizing a Zorbax Eclypse-AAA
column (150.times.4.6 mm, 5 .mu.m, Agilent Technologies, Waldbronn
Germany), with automated online derivatization (o-phtaldialdehyde
+3-mercaptopropionic acid) at a flow rate of 2 ml min.sup.-1, and
fluorescence detection. Details are given in the instruction manual
.alpha.-amino butyrate was used as internal standard for
quantification.
[0150] .sup.13C labeling analysis. The labeling patterns of lysine
and trehalose in cultivation supernatants were quantified by GC-MS.
Hereby single mass isotopomer fractions were determined. In the
current work they are defined as M.sub.0 (relative amount of
non-labelled mass isotopomer fraction), M.sub.1 (relative amount of
single labelled mass isotopomer fraction) and corresponding terms
for higher labelling. GC-MS analysis of lysine was performed after
conversion into the t-butyl-dimethylsilyl (TBDMS) derivate as
described previously (Rubino, F. M. 1989. J. Chromatogr.
473:125-133). Quantification of mass isotopomer distributions was
performed in selective ion monitoring (SIM) mode for the ion
cluster m/z 431-437. This ion cluster corresponds to a fragment
ion, which is formed by loss of a t-butyl group from the
derivatization residue, and thus includes the complete carbon
skeleton of lysine (Wittmann, C., M. Hans and E. Heinzle. 2002.
Analytical Biochem. 307:379-382). The labeling pattern of trehalose
was determined from its trnethylsilyl (TMS) derivate as described
previously (Wittmann, C., H. M. Kim and E. Heinzle 2003. Metabolic
flux analysis at miniaturized scale submitted). The labeling
pattern of trehalose was estimated via the ion cluster at m/z
361-367 corresponding to a fragment ion that contained an entire
monomer unit of trehalose and thus a carbon skeleton equal to that
of glucose 6-phosphate. All samples were measured first in scan
mode therewith excluding isobaric interference between analyzed
products and other sample components. All measurements by SIM were
performed in duplicate. The experimental errors of single mass
isotopomer fractions in the tracer experiments on fructose were
0.85% (M.sub.0), 0.16% (M.sub.1), 0.27% (M.sub.2), 0.35% (M.sub.3),
0.45% (M.sub.4) for lysine on [1-.sup.13C] fructose, 0.87%
M.sub.0), 0.19% (M.sub.1), 0.44% (M.sub.2), 0.45% (M.sub.3), 0.88%
M.sub.4) for trehalose on [1-.sup.13C] fructose, and 0.44%
(M.sub.0), 0.54% (M.sub.1), 0.34% (M.sub.2), 0.34% (M.sub.3) 0.19%
(M.sub.4) 0.14% (M.sub.5) and 0.52% (M.sub.6) for trehalose on 50%
[.sup.13C.sub.6] fructose, respectively. The experimental errors of
MS measurements in glucose tracer experiments were 0.47% (M.sub.0),
0.44% (M.sub.1), 0.21% (M.sub.2), 0.26% (M.sub.3), 0.77% (M.sub.4)
for lysine on [1-.sup.13C] glucose, 0.71% (M.sub.0), 0.85%
(M.sub.1), 0.17% (M.sub.2), 0.32 % (M.sub.3), 0.46% (M.sub.4) for
trehalose on [1-.sup.13C] glucose, and 1.29% (M.sub.0), 0.50%
(M.sub.1), 0.83% (M.sub.2), 0.84% (M.sub.3), 1.71% (M.sub.4), 1.84%
(M.sub.5) and 0.58% (M.sub.6) for trehalose on 50% [.sup.13C.sub.6]
glucose, respectively.
[0151] Metabolic modelling and parameter estimation. All metabolic
simulations were carried out on a personal computer. Metabolic
network of lysine-producing C. glutamicum was implemented in Matlab
6.1 and Simulink 3.0 (Mathworks, Inc., Natick, Mass. USA). The
software implementation included an isotopomer model in Simulink to
calculate the .sup.13 C labeling distribution in the network. For
parameter estimation the isotopomer model was coupled with an
iterative optiniation algorithm in Matlab. Details on the applied
computational:tools are given by Wittmann and Heinzle (Wittmann, C.
and E. Heinzle 2002 Appl. Environ. Microbiol. 68:5843-5859).
[0152] The metabolic network was based on previous work and
comprised glycolysis, pentose phosphate pathway (PPP),
tricarboxylic acid (TCA) cycle, anaplerotic carboxylation of
pyruvate, biosynthesis of lysine and other secreted products (Tab.
1), and anabolic fluxes from intermediary precursors into biomass.
In addition uptake systems for glucose and fructose were
alternatively implemented. Uptake of glucose involved
phosphorylation to glucose 6-phosphate via a PTS (Ohnishi, J., S.
Mitsuhashi, M. Hayashi, S. Ando, H. Yokoi, K. Ochiai and M. A.
Ikeda 2002 Appl. Microbiol. Biotechno1 58:217-223). For fructose
two uptake systems were considered (i) uptake by PTSFructose and
conversion of fructose into fructose 1,6-bisphosphatase via
fructose 1-phosphate and (ii) uptake by PTS.sub.Mannose leading to
fructose 6-phosphate, respectively (Dominguez, H., C. Rollin, A.
Guyonvarch, J. L. Guerquin-Kern, M. Cocaign-Bousquet and N. D.
Lindley. 1998. Eur. J. Biochem. 254:96-102). In addition
fructose-1,6-bisphosphatase was implemented into the model to allow
carbon flux in both directions in the upper glycolysis. Reactions
regarded reversible were transaldolase and transketolases in the
PPP. Additionally glucose 6-phosphate isomerase was considered
reversible for the experiments on glucose, whereby the trehalose
labeling sensitively reflected the reversibility of this enzyme. In
contrast the reversibility of glucose 6-phosphate isomerase could
not be determined on fructose. In fructose-grown cells, glucose
6-phosphate is exclusively formed from fructose 6-phosphate leading
to identical labeling patterns for the two pools. Therefore
interconversion between glucose 6-phosphate and fructose
6-phosphate by a reversible glucose 6-phosphate isomerase does not
result in labeling differences that could be used for the
estimation of glucose 6-phosphate isomerase reversibility. The
measured labeling of lysine and trehalose was not sensitive towards
(i) the reversibility of the flux between the lumped pools of
phosphoenolpyruvate/pyruvate and malate/oxaloacetate and (ii) the
reversibility of malate dehydrogenase and fumarate hydratase in the
TCA cycle. Accordingly these reactions were regarded irreversible.
The labeling of alanine from a mixture of naturally labeled and
[.sup.13C.sub.6] labeled substrate, which is sensitive for these
flux parameters, was not available in this study. Based on previous
results the glyoxylate pathway was assumed to be inactive
(Wittmann, C. and E. Heinzle 2002 Appl. Environ. Microbiol.
68:5843-5859).
[0153] Stoichiometric data on growth, product formation, and
biomass composition of C. glutamicum together with mass
spectrometric labeling data of secreted lysine and trehalose were
used to calculate metabolic flux distributions. The set of fluxes
that gave minimum deviation between experimental (M.sub.i, exp) and
simulated (M.sub.i, calc) mass isotopomer fractions of lysine and
trehalose of the two parallel experiments was taken as best
estimate for the intracellular flux distribution. As described in
the appendix the two networks of glucose-grown and fructose-grown
cells were over determined. A least square approach was therefore
possible. As error criterion a weighted sum of least squares (SLS)
was used, where S.sub.i, exp is the standard deviation of the
measurements (Eq. 1).
S L S = i ( M i , exp - M i , calc ) 2 S i , exp 2 ( Equation 1 )
##EQU00001##
Multiple parameter initializations were applied to investigate
whether an obtained flux distribution represented a global optimum.
For all strains the glucose uptake flux during lysine production
was set to 100% and the other fluxes in the network are given as
relative molar fluxes normalized to the glucose uptake flux.
[0154] Statisdcal evaluation. Statistical analysis of the obtained
metabolic fluxes was carried out by a Monte-Carlo approach as
described previously (Wittmann, C. and E. Heinzle 2002 Appl.
Environ. Microbiol. 68:5843-5859). For each strain, the statistical
analysis was carried out by 100 parameter estimation runs, whereby
the experimental data, comprising measured mass isotopomer ratios
and measured flukes, were varied statisically. From the obtained
data 90% confidence limits for the single parameters were
calculated.
Example I
Lysine Production By C. glutamicum on Fructose and Glucose
[0155] Metabolic fluxes of lysine producing C. glutamicum were
analyzed in comparative batch cultures on glucose and fructose. For
this purpose pre-grown cells were transferred into tracer medium
and incubated for about 5 hours. The analysis of substrates and
products at the beginning and the end of the tracer experiment
revealed drastic differences between the two carbon sources.
Overall 11.1 mM lysine was produced on glucose, whereas a lower
concentration of only 8.6 mM was reached on fructose. During the
incubation over 5 hours, the cell concentration increased from 3.9
g L-1 to 6.0 g L.sup.-1 (glucose) and from 3.5 g L-1 to 4.4 g, L-1
(fructose). Due to the fact that threonine and methionine were not
present in the medium, internal sources were probably utilized by
the cells for biomass synthesis. The mean specific sugar uptake
rate was higher on fructose (1.93 mmol g-1 h-1) compared, to
glucose (1.71 mmol g-1 h-1). As depicted in Table 1, the obtained
yields of C. glutamicum ATCC 2152.6 differed drastically between
fructose and glucose. This involved the main product lysine and
various byproducts. Concerning lysine, the yield on fructose was
244 mmol mol-1 and thus was lower compared to the yield on glucose
(281 mmol mol-1). Additionally the carbon source had a drastic
influence on.the biomass yield, which was reduced by almost50% on
fructose in comparison to glucose. The most significant influence
of the carbon source on byproduct formation was observed for
dihydroxyacetone, glycerol, and lactate. On fructose, accumulation
of these byproducts was strongly enhanced. The yield for glycerol
was 10 fold higher, whereas dihydroxyacetone and lactate secretion
were increased by a factor of six. Dihydroxyacetone was the
dominating byproduct on fructose. Due to the lower biomass yield a
significantly reduced demand for anabolic precursors resulted for
fructose-grown cells (Table 2).
TABLE-US-00001 TABLE 1 Biomass and metabolites in the stage of
lysine production by Corynebacterium glutamicum ATCC 21526 from
glucose (left) and fructose (right). Experimental yields are mean
values of two parallel incubations on (i) 40 mM [1-.sup.13C]
labeled substrate and (ii) 20 mM [.sup.13C.sub.6] labeled substrate
plus 20 mM naturally labeled substrate with corresponding
deviations between the two incubations. All yields are given in
(mmol product) (mol).sup.-1 except the yield for biomass, which is
given in (mg of dry biomass) (mmol).sup.-1. Lysine Lysine Yield
production on glucose production on fructose Biomass 54.1 .+-. 0.8
28.5 .+-. 0.0 Lysine 281.0 .+-. 2.0 244.4 .+-. 23.3 Valine 0.1 .+-.
0.0 0.0 .+-. 0.0 Alanine 0.1 .+-. 0.0 0.4 .+-. 0.1 Glycine 6.6 .+-.
0.0 7.1 .+-. 0.4 Dihydroxyacetone 26.3 .+-. 15.3 156.6 .+-. 25.8
Glycerol 3.8 .+-. 2.4 38.4 .+-. 3.9 Trehalose 3.3 .+-. 0.5 0.9 .+-.
0.1 .alpha.-Ketoglutarate 1.6 .+-. 0.4 6.5 .+-. 0.3 Acetate 45.1
.+-. 0.3 36.2 .+-. 5.7 Pyruvate 1.2 .+-. 0.4 2.1 .+-. 0.5 Lactate
7.1 .+-. 1.7 38.3 .+-. 3.5
TABLE-US-00002 TABLE 2 Anabolic demand of Corynebacterium
glutamicum ATCC 21526 for intracellular metabolites in the stage of
lysine production from glucose (left) and fructose (right).
Experimental data are mean values of two parallel incubations on
(i) [1-.sup.13C] labeled substrate and (ii) a 1:1 mixture of
naturally labeled and [.sup.13C.sub.6] substrate with deviation
between the two incubations. Precursor Demand* Lysine production
Lysine production mmol (mol glucose).sup.-1 on glucose on fructose
Glucose 6-phosphate 11.09 .+-. 0.16 5.84 .+-. 0.05 Fructose
6-phosphate 3.84 .+-. 0.06 2.02 .+-. 0.02 Pentose 5-phosphate 47.50
.+-. 0.70 25.05 .+-. 0.21 Erythrose 4-phosphate 14.50 .+-. 0.22
7.64 .+-. 0.06 Glyceraldehyde 3-phosphate 6.98 .+-. 0.10 3.68 .+-.
0.03 3-Phosphoglycerate 59.95 .+-. 0.89 36.85 .+-. 0.31
Pyruvate/Phosphoenolpyruvate 107.80 .+-. 1.60 56.80 .+-. 0.48
.alpha.-Ketoglutarate 92.51 .+-. 1.37 48.73 .+-. 0.41 Oxaloacetate
48.91 .+-. 0.72 45.76 .+-. 0.38 Acetyl CoA 135.30 .+-. 2.00 71.25
.+-. 0.60 Diaminopimelate + Lysine** 18.83 .+-. 0.28 9.92 .+-. 0.08
*The estimation of precursor demands was based on the experimental
biomass yield obtained for each strain (Tab. 1) and the biomass
composition previously measured for C. glutamicum (Marx, A., A. A.
de Graaf, W. Wiechert, L. Eggeling and H. Sahm. 1996. Biotechnol.
Bioeng. 49:111-129). **Draminopimelate and lysine are regarded as
separate anabolic precursors. This is due to the fact that anabolic
fluxes from pyruvate and oxaloacetate into diaminopimelate (cell
wall) and lysine (protein) contribute in addition to the flux of
lysine secretion to the overall flux through the lysine
biosynthetic pathway.
Example II
Manual Inspection of .sup.13C-Labeling Patterns in Tracer
Experiments
[0156] Relative mass isotopomer fractions of secreted lysine and
trehalose were quantified with GC-MS. These-mass isotoppmer
fractions are sensitive towards intracellular fluxes and therefore
display fingerprints for the fluxome of the investigated biological
system. As shown in FIG. 2, labeling patterns of secreted lysine
and trehalose differed, significantly between glucose and
fructose-grown cells of C. gluiamicum. The differences were found
for both applied tracer labelings and for both measured products.
This indicates substantial differences in the carbon flux pattern
depending on the applied carbon source. As previously shown, mass
isotopomer fractions from two parallel cultivations of C.
glutainicum on a mixture of [1-.sup.13C] and [.sup.13C.sub.6]
glucose were almost identical (Wittmann, C., H. M. Kim and E.
Heinzle 2003. Metabolic flux analysis at miniaturized scale
submitted). Therefore, the differences observed can be clearly
related to substrate specific differences in metabolic fluxes.
Example III
Estimation of Intracellular Fluxes
[0157] A central issue of the performed studies was the comparative
investigation of intracellular fluxes of C glutamicum during lysine
production on glucose and fructose as carbon source, respectively.
For this purpose, the experimental data obtained from the tracer
experiments were used to calculate metabolic flux distributions for
each substrate applying the flux estimation software as described
above The parameter estimation was carried out by minimizing the
deviation between experimental and calculated mass isotopomer
fractions. The performed approach utilize metabolite balancing
during each step of the optimization. This included (i)
stoichiometric data on product secretion (Table 2) and (ii)
stoichiometric data on anabolic demand for biomass precursors
(Table 3). The set of intracellular fluxes that gave the minimum
deviation between experimental and simulated labeling patterns was
taken as best estimate for the intracellular flux distribution. For
both scenarios, identical flux distributions were obtained with
multiple initialization values, suggesting that global minima were
identified. Obviously, good agreement between experimentally
determined and calculated mass isotopomer ratios was achieved
(Table 4).
TABLE-US-00003 TABLE 3 Relative mass isotopomer fractions of
secreted lysine and trehalose of lysine producing Corynebacterium
glutamicum ATCC 21526 cultivated on glucose and fructose,
respectively. For both carbon sources two parallel tracer
experiments on (i) [1-.sup.13C] labeled and (ii) a 1:1 mixture of
naturally .sup.13C labeled, and [.sup.13C.sub.6] labeled tracer
substrate were carried out. Experimental GC/MS data (exp) and
values predicted by the solution of the mathematical model
corresponding to the optimized set of fluxes (calc). M.sub.0
denotes the relative amount of non-labelled mass isotopomer
fraction, M.sub.1 the relative amount of the single labelled mass
isotopomer fraction, and corresponding terms stand for higher
labelling Lysine (on [1-.sup.13C] Trehalose (on [1-.sup.13C]
Trehalose (on 50% [.sup.13C.sub.6] labeled substrate) labeled
substrate) labeled substrate) M.sub.0 M.sub.1 M.sub.2 M.sub.3
M.sub.4 M.sub.0 M.sub.1 M.sub.2 M.sub.3 M.sub.4 M.sub.0 M.sub.1
M.sub.2 M.sub.3 M.sub.4 M.sub.5 M.sub.6 glucose exp 0.234 0.360
0.247 0.110 0.037 0.110 0.551 0.216 0.094 0.023 0.271 0.114 0.087
0.115 0.069 0.066 0.279 calc 0.242 0.355 0.245 0.110 0.037 0.114
0.549 0.212 0.094 0.023 0.268 0.113 0.085 0.113 0.068 0.064 0.289
fructose exp 0.133 0.316 0.304 0.162 0.062 0.212 0.412 0.244 0.092
0.030 0.141 0.103 0.104 0.250 0.133 0.110 0.159 calc 0.139 0.321
0.298 0.159 0.061 0.195 0.419 0.254 0.094 0.030 0.144 0.103 0.102
0.245 0.131 0.111 0.164
Example IV
Metabolic Fluxes on Fructose and Glucose During Lysine
Production
[0158] The obtained infracellular flux distributions for
lysine-producing C. glutamicum on glucose and fructose are shown in
FIGS. (4, 5). Obviously, the intracellular fluxes differed
tremendously depending on the carbon source applied. On glucose,
62% of the carbon flux was directed towards the PPP, whereas only
36% were channeled through the glycolytic chain (FIG. 4) Due to
this a relatively high amount, 124% NADPH was generated by the PPP
enzymes glucose 6-phosphate dehydrogenase and 6-phosphogluconate
dehydrogenase. The situation on fructose was completely different
(FIG. 5). The performed flux analysis revealed the in vivo activity
of two PTS for uptake of fructose, whereby 92.3% of fructose were
taken up by fructose specific PTS.sub.Fructose. A comparably small
fraction of 7.7% of fructose was taken up by PTS.sub.Mannose. Thus,
the majority of fructose entered the glycolysis at the level of
fructose 1,6-bisphosphatase, whereas only a small fraction was
channeled upstream at fructose 6-phosphate into the glycolytic
chain. In comparison to glucose-grown cells, the PPP exhibited a
dramatically reduced activity of only 14.4%. Glucose 6-phosphate
isomerase operated in opposite directions on the two carbon
sources. In glucose-grown cells 36.2% net flux were directed from
glucose 6-phosphate to fructose 6-phosphate, whereas a backward net
flux of 1:5.2% was observed on fructose.
[0159] On fructose, the flux through glucose 6-phosphate isomerase
and PPP was about twice as high as the flux through the
PTS.sub.Mannose. However this was not due to a gluconeogenetic flux
of carbon from fructose-1,6-bisphosphatase to fructose 6-phosphate,
which could have supplied extra carbon flux towards the PPP. In
fact flux through fructose 1,6-bisphosphatase catalyzing this
reaction was zero. The metabolic reactions responsible for the
additional flux towards the PPP are the reversible enzymes
transaldolase and transketolasein the PPP. About 3.5% of this
additional flux was supplied by transketolase 2, which recycled
carbon stemming from the PPP back into this pathway. Moreover 4.2%
of flux was directed towards fructose 6-phosphate and the PPP by
the action of transaldolase.
[0160] Depending on the carbon source, completely different flux
patterns in lysine producing C. glutamicum were also observed
around the pyruvate node (FIGS. 4, 5). On glucose the flux into the
lysine pathway was 30.0%, whereas a reduced flux of 25.4% was found
on fructose. The elevated lysine yield on glucose compared to
fructose is the major reason for this flux difference, but also the
higher biomass yield resulting in a higher demand for
diaminopimelate for cell wall synthesis and lysine for protein
synthesis contributes to it. The anaplerotic flux on glucose was
44.5% and thus markedly higher compared to the flux on fructose
(33.5%). This is mainly due to the higher demand for oxaloacetate
for lysine production, but also to the higher anabolic demandas for
oxaloacetate and 2-oxoglutarate on glucose. On the other hand, flux
through pyruvate dehydrogenase was substantially lower on glucose
(70.9%) compared to fructose (95.2%). This reduced.carbon flux into
the TCA cycle resulted in more than 30% reduced fluxes through TCA
cycle enzymes on glucose (FIGS. 3, 4).
[0161] Statistical evaluation of the obtained fluxes by a
Monte-Carlo approach was used to calculate 90% confidence intervals
for the determined flux parameters. As shown for various key fluxes
in Table 5, the confidence intervals were generally narrow. As
example the confidence interval for the flux through glucose
6-phosphate dehydrogenase was only 1.2% for glucose-grown and 3.5%
for fructose-grown cells. The chosen approach therefore allowed
precise flux estimation. It can be concluded that the flux
differences observed on glucose and fructose, respectively, are
clearly caused by the applied carbon source.
[0162] It has to be noticed that the mean specific substrate uptake
of 1.93 mmol g.sup.-1 h.sup.1 onrifructose was slightly higher than
that of 1.77 mmol g.sup.-1 h.sup.-1 found on glucose. Due to this
the absolute intracellular fluxes expressed in mmol g.sup.-1
h.sup.-1 are slightly increased in relation to glucose compared to
the relative fluxes discussed above. The flux distributions of
lysine producing C. glutamicum on fructose and glucose,
respectively, are however so completely different, that all
comparisons drawn above also hold for absolute carbon fluxes.
TABLE-US-00004 TABLE 4 Statistical evaluation of metabolic fluxes
of lysine producing Corynebacterium glutamicum ATCC 21526 grown on
fructose (left) and glucose (right) determined by .sup.13C tracer
studies, with mass spectrometry and metabolite balancing: 90%
confidence intervals of key flux parameters were obtained by a
Monte-Carlo approach including 100 independent parameter estimation
runs for each substrate with statistically varied experimental
data. Flux parameter Glucose Fructose Net Flux fructose uptake by
PTS.sub.Frc -- [90.0 96.1] fructose uptake by PTS.sub.Man -- [3.9
10.0] glucose 6-phosphate isomerase [35.7 36.8] [13.4 16.9]
phosphofructokinase [35.7 36.8] -- fructose 1,6-bisphosphatase* --
[-2.1 3.4] fructose 1,6-bisphosphatase [73.7 73.8] [91.7 92.9]
aldolase glucose 6-phosphate [62.5 63.7] [12.6 16.1] dehydrogenase
transaldolase [19.4 19.8] [3.6 4.1] transketolase 1 [19.4 19.8]
[3.6 4.1] transketolase 2 [17.9 18.3] [2.9 4.0] glyceraldehyde
3-phosphate [158.1 164.5] [163.3 174.6] dehydrogenase pyruvate
kinase [156.2 167.4] [158.9 168.2] pyruvate dehydrogenase [69.5
72.5] [87.1 102.3] pyruvate carboxylase [43.7 44.8] [29.9 37.3]
citrate synthase [51.2 54.8] [76.5 91.5] isocitrate dehydrogenase
[51.2 54.8] [76.5 91.5] oxoglutarate dehydrogenase [41.6 45.6]
[70.9 86.0] aspartokinase [29.6 30.3] [21.8 29.2] Flux
Reversibility** glucose 6-phosphate isomerase [4.5 5.1] --
transaldolase [4.3 4.9] [14.5 18.2] transketolase 1 [0.0 0.0] [0.0
0.1] transketolase 2 [0.4 0.6] [0.0 0.1] *The negative flux for the
lower confidence boundary is equal to a positive flux in the
reverse direction (through phosphofructokinase). **Flux
reversibility is defined as ratio of back flux to net flux.
Discussion of Examples I-IV
[0163] A. Substrate Specific Culture Characteristics
[0164] Cultivation of lysine producing C. glutamicum, on fructose
and on glucose, respectively, revealed that growth and product
formation strongly depend on the carbon source applied.
Significantly reduced yields of lysine and biomass on fructose were
previously also reported for another strain of C. glutamicum, where
lysine and biomass yield were 30% and 20% less, respectively,
compared to glucose (Kiefer, P., E. Heinzle and C. Wittmann 2002.
J. Ind. Microbiol. Biotechnol. 28:338-43). Cultivation of C.
glutamicum and C. melassecola on fructose is linked to higher
carbon dioxide production rates in comparison to glucose
(Dominguez, H., C. Rollin, A. Guyonvarch, J. L. Gudrquin-Kern, M.
Cocaign-Bousquet and N. D. Lindley. 1998. Eur. J. Biocheim
254:96-102; Kiefer, P., E. Heinzle and C. Wittmann. 2002. J. Ind.
Microbiol. Biotechnol. 28:338-43). This coincides with the elevated
flux through the TCA cycle observed in the present work for this
carbon source. Substrate specific differences were also observed
for byproducts. The formation of trehalose was lower on fructose
compared to glucose. This may be related to different entry points
of glucose and fructose into glycolysis (Kiefer, P., E. Heinzle and
C. Wittmann 2002. J. Ind. Microbiol. Biotechnol. 28:338-43).
Considering the uptake systems in C. glutamicum, utilization of
glucose leads to the formation of the trehalose precursor glucose
6-phosphate, whereas fructose is converted into fructose
1,6-bisphosphatase and thus enters the central metabolism
downstream from glucose 6-phosphate (Dominguez, H., C. Rollin, A.
Guyonvarch, J. L. Guerquin-Kern, M. Cocaign-Bousquet and N. D.
Lindley 1998. Eur. J. Biochem. 254:96-102). Other byproducts such
as dihydroxyacetone, glycerol, and lactate were strongly increased,
when fructose was applied as carbon source. From the viewpoint of
lysine production, this is not desired, because a substantial
fraction of carbon is withdrawn from the central metabolism into
the formed byproducts. The specific substrate uptake on fructose
(1.93 inmol g.sup.-1 h.sup.-1) was higher than on glucose (1.77
mmol g.sup.-1 h.sup.-1). This result differs from a previous study
on exponentially growing C. melassecola ATCC 17965 (Dominguez, H.,
C. Rollin, A. Guyohvarch, J. L. Guerquin-Kern, M. Cocaign-Bousquet
and N. D. Lindley 1998. Eur. J. Biochem. 254:96-102), where similar
specific uptake rates on fructose and glucose were observed. The
higher uptake rate for fructose observed in our study might be due
to the fact that the studied strains are different. C. melassecola
and C. glutamicum are related species, but might differ in certain
metabolic properties. The strain studied in the present work was
previously derived by classical strain optimization. This could
have introduced mutations influencing substrate uptake. Another
explanation is the difference in cultivation conditions. Fructose
might be more effectively utilized under conditions of limited
growth and lysine production.
[0165] B. Metabolic Flux Distributions
[0166] The obtained intracellular flux distributions for
lysine-producing C. gtutamicum on glucose and fructose revealed
tremendous differences. Statistical evaluation of the obtained
fluxes revealed narrow 90% confidence intervals, so that the
observed flux differences can be clearly attributed to the applied
carbon sources. One of the most remarkable differences concerns the
flux partitioning between glycolysis and PPP. On glucose 62.3% of
carbon was channeled through the PPP. The predominance of the PPP
of lysine-producing C. glutamicum on this substrate has been
previously observed in different studies (Marx, A., A. A. de Graaf,
W. Wiechert, L. Eggeling and H. Sahm. 1996. Biotechnol. Bioeng.
49:111-129; Wittmann, C. and E. Heinzle. 2001. Eur. J. Biochem.
268:2441-2455; Wittmann, C. and E. Heinzle. 2002. Appl. Environ.
Microbiol. 68:5843-5859). On fructose the flux into the PPP was
reduced to 14.4%. As identified by the performed metabolic flux
analysis, this was mainly due to the unfavourable combination of
the entry of fructose at the level of fructose 1,6-bisphosphatate
and the inactivity of fructose-1,6-bisphosphatase. The observed
inactivity of fructose-1,6-bisphos phatase agrees well with
enzymatic measurements of C. melassecola ATCC 17965 during
exponential growth on fructose and on glucose, respectively
(Dominguez, H., C. Rollin, A. Guyonvarch, J. L. Guerquin-Kern, M.
Cocaign-Bousquet and N. D. Lindley. 1998. Eur. J. Biochem
254:96-102). Surprisingly, the flux through glucose 6-phosphate
isomerase and PPP was about twice as high as the flux through the
PTS.sub.Mannose, when C. glutamicum was cultivated on fructose. Due
to the inactivity of fructose-1,6-bisphosphatase this was not
caused by a gluconeogenetic flux. In fact, C. glutamicum possesses
an operating meitabolic cycle via fructose 6-phosphate, glucose
6-phosphate, and ribose 5-phosphate. Additional flux into the PPP
was supplied by transketolase 2, which recycled carbon stemming
from the PPP back into this pathway, and by the action of
transaldolase, which redirected glyceraldehyde 3-phosphate back
into the PPP, thus bypassing gluconeogenesis. This cycling activity
may help the cell to overcome NADPH limitation on fructose. The
drastically reduced flux arriving at glucose 6-phosphate for
fructose-grown C. glutamicum might also explain the reduced
formation of trehalose on this substrate (Kiefer, P., E. Heinzle
and C. Wittmann. 2002. J. Ind. Microbiol. Biotechnol. 28:338-43).
Glucose. 6-phosphate isomerase operated in opposite directions
depending on the carbon source. In glucose-grown net flux was
directed from glucose 6-phosphate to fructose 6-phosphate, whereas
an inverse net flux, was observed on fructose. This underlines the
importance of the reversibility of this enzyme for metabolic
flexibility in C. glutamicum.
[0167] C. NADPH Metabolism
[0168] The following calculations provide a comparison of the NADPH
metabolism of lysine producing C. glutamicum on fructose and
glucose. The overall supply of NADPH. was calculated from the
estimated flux through glucose 6-phosphate dehydrogenase,
6-phosphogluconate dehydrogenase, and isocitrate dehydrogenase. On
glucose, the PPP enzymes glucose 6-phosphate dehydrogenase (62.0%)
and glucose 6-phosphate dehydrogenase (62.0%) supplied the major
fraction of NADPH. Isocitrate dehydrogenase. (52.9%) contributed
only to a minor extent. A completely different contribution of PPP
and TCA cycle to NADPH supply was observed on fructose, where
isocitrate dehydrogenase (83.3%) was the major source for NADPH.
Glucose 6-phosphate dehydrogenase (14.4%) and glucose 6-phosphate
dehydrogenase (14.4%) produced much less NADPH on fructose. NADPH
is required for growth and formation of lysine. The NADPH
requirement for growth was calculated from a stoichiometric demand
of 11.51 mmol NAPDH (g biomass).sup.-1, which was assumed to be
identical for glucose and fructose (Dominguez, H., C. Rollin, A.
Guyonvarch, J. L. Guerquin-Kern, M. Cocaign-Bousquet and N. D.
Lindley. 1998. Eur. J. Biochem. 254:96-102), and the experimenital
biomass yield of the present work (Tab.1). C. glutamicum consumed
62.3% of NADPH for biomass production on glucose, which was much
higher as compared to fructose as carbon source (32.8%). The amount
of NADPH required for product synthesis was determined from the
estimated flux into lysine (Tab. 1) and the corresponding
stoichiometric NADPH demand of 4 mol (mol lysine)-1. It was 112.4%
for lysine production from glucose and 97.6% for lysine production
from fructose. The overall NADPH supply on glucose was
significantly higher (176.9%) compared to fructose (112.1%), which
can be mainly attributed to the increased PPP flux on glucose.
[0169] The NADPH balance was almost closed on glucose. In contrast
a significant apparent deficiency for NADPH of 18.3% was observed
on fructose. This raises the question for enzymes catalyzing
metabolic reactions that could supply NADPH in addition to the
above mentioned enzymes glucose 6-phosphate dehydrogenase,
6-phosphogluconate dehydrogenase and isocitrate dehydrogenase. A
likely candidate seems NADPH-dependent malic enzyme. Previously an
increased specific activity of this enzyme was detected oh
fructose-grown C. melassecola in comparison to glucose-grown cells
(Dominguez, H., C. Rollin, A. Guydnvarch, J. L. Guerquin-Kern, M.
Cocaign-Bousquet and N. D. Lindley. 1998. Eur. J. Biochem.
254:96-102). However, the flux through this particular enzyme could
not be resolved by the experimental setup in the present work.
Assuming malic enzyme as missing NADPH generating enzyme; a flux of
18.3% would be sufficient to supply the apparently missing NADPH.
Detailed flux studies of C. glutamicum with glucose as carbon
source revealed no significant activity of malic enzyme (Petersen,
S., A. A. de Graaf, L. Eggeling, M. Mollney, W. Wiechert and H.
Sahm. 2000. J. Biol. Chem. 75:35932-35941). The situation on
fructose might however be coupled to elevated in vivo activity of
this enzyme.
[0170] D. NADH Metabolism
[0171] On fructose C. glutamicum revealed increased activity of
NADH forming enzymes. 421.2% NADH were formed on fructose by
glyceraldehyde 3-phosphate dehydrogenase, pyruvate dehydrogenase,
2-oxoglutarate dehydrqgenase, and malate dehydrogenase. On glucose
the NADH production was only 322.4%. Additionally, the anabolic
NADH demjaand was significantly lower on fructose than on glucose.
The significantly enhanced NADH production coupled to a reduced
metabolic demand could lead to an increased NADH/NAD ratio. For C.
melassecola it was previously shown that fructose leads to
increased NADH/NAD ratio compared to glucose (Dominguez, H., C.
Rollin, A. Guyonvarch, J. L. Guerquin-Kern, M. Cocaign-Bousquet and
N. D. Lindley. 1998. Eur. J. Biochem. 254:96-102). This raises the
question for NADH regenerating mechanisms during lysine production
on fructose. Fructose-grown cells exhibited an enhanced secretion
of dihydroxyacetone, glycerol, and lactate. The increased formation
of dihydroxyacetone and glycerol could be due a higher NADH/NAD
ratio. NADH was previously shown to inhibit glyceraldehyde
dehydrogenase, so that overflow of dihydroxyacetone and glycerol
might be related to a reduction of the flux capacity of this
enzyme. The reduction of dihydroxyacetone to glycerol could
additionally be favored:by the high NADH/NAD ratio and thus
contribute to regeneration of excess NADH. The NADH demanding
lactate formation from pyruvate could have a similar background as
the production of glycerol. In comparison to exponential growth,
NADH excess under lysine producing conditions, characterized by
relatively high TCA cycle activity and reduced biomass yield, might
be even higher.
[0172] E. Potential Targets for Optimization of Lysine-producing C.
glutamicum on Fructose
[0173] Based on the obtained flux patterns, several potential
targets for the optimization of lysine production by C. glutamicum
on fructose can be formulated. A central point is the supply of
NADPH. Fructose-1,6-bisphosphatase is one target for increasing the
supply of NADPH. Deregulation, e.g., amplification of its activity
leads to a higher flux through the PPP, resulting in increased
NADPH generation and increased lysine yield. An increase of the
fluxthrough the PPP via amplification of fructose
1,6-bisphosphatase is also be beneficial for aromatic amino acid
production (Ikeda, M. 2003. Adv. Biochem. Eng. Biotechnol.
79:1-36). The inactivity of fructose 1,6-bisphosphatase during
growth on fructose is detrimental from the viewpoint of lysine
production but not surprising, because this gluconeogenetic enzyme
is not required during growth on sugars and probably suppressed. In
prokaryotes, this enzyme is under efficient metabolic control by
e.g. fructose 1,6-bisphosphatase, fructose-2,6-bisphosphatase,
metal ions and AMP (Skrypal, I. G. and O. V. Iastrebova. 2002.
Miobiol Z. 64:82-94). It is known that C. glutamicum can grow on
acetate (Wendisch, V. F., A. A. de Graaf, H. Sahm H. and B.
Eikmans. 2000. J. Bacteriol. 182:3088-3096), where this enzyme is
essential to maintain gluconeogenesis. Another potential target to
increase the flux through the PPP is the PTS for fructose uptake.
Modification of flux partitioning between PTS.sub.Fructose and
PTS.sub.Mannose could yield a higher proportion of fructose, which
enters at the level of fructose 6-phosphate and thus also lead to
an increased PPP flux. Additionally amplification of malic enzyme
that probably contributes significantly to NADPH supply on fructose
could be an interesting target.
[0174] Another bottleneck comprises the strong secretion of
dihydroxyacetone, glycerol, and lactate. The formation of
dihydroxyacetone and glycerol could be blocked by deregulation,
e.g., deletion of the corresponding enzymes. The conversion of
dihydroxyacetone phosphate to dihydroxyacetone could be catalyzed
by a corresponding phosphatase. A dihydroxyacetone phosphatase has
however yet not been annotated in C. glutamicum (see the National
Center for Biotecbnology Information (NCBI) Taxonomy website:
http://www3.ncbi.nlm.nih.gov/Taxonomy/). This reaction may be also
catalyzed by a kinase, e.g., glycerol kinase. Currently two entries
in the genome data base of C. glutamicum relate to dihydroxyacetone
kinase (see the National Center for Biotechnology Information
(NCBI) Taxonomy website:
http://www3.ncbi.nlm.nih.gov/Taxonomy/).
[0175] Lactate secretion can also be avoided by deregulation, e.g.,
knockout, of lactate dehydrogenase. Since glycerol and lactate
formation could be important for NADH regeneration, negative
effects on the overall performance of the organism can however not
be excluded. In case carbon flux through the lower glycolytic chain
is limited by the capacity of glyceraldehyde 3-phosphate
dehydrogenase as previously speculated (Dominguez, H., C. Rollin,
A. Guyonvarch, J. L. Guerquin-Kern, M. Cocaign-Bousquet and N. D.
Lindley. 1998. Eur. J. Biochem. 254:96-102), the suppression of
dihydroxyacetone and glycerol production could eventually lead to
an activation of fructose-1,6-bisphosphatase and a redirection of
carbon flux through the PPP. It should be noticed that
dihydroxyacetone is not reutilized during the cultivation of C.
glutamicum and thus displays wasted carbon with respect to product
synthesis, whereas thisis not the case for lactate
(Cocaign-Bousquet, M. and N. D. Lindley. 1995. Enz. Microbiol.
Technol. 17:260-267).
[0176] In one embodiment, deregulation of one or more of the above
genes in combination is useful in the production of a fine
chemical, e.g., lysine.
[0177] In addition, sucrose is also useful as carbon source for
lysine production by C. glutamicum, e.g., used in conjunction with
the methods of the invention. Sucrose is the major carbon source in
molasses. As shown previously, the fructose unit of sucrose enters
glycolysis at the level of fructose 1,6-bisphosphatase (Dominguez,
H. and N. D. Lindley. 1996. Appl. Environ. Microbiol.
62:3878-3880). Therefore this part of the sucrose
molecule--assuming an inactive fructose
1,6-bisphosphatase--probably does not enter into the PPP, so that
NADPH supply in lysine producing strains could be limited.
Example V
Construction of Plasmid PCIS LysC
[0178] The first step of strain construction calls for an allelic
replacement of the lysC wild-type gene in C. glutamicum ATCC13032.
In it, a nucleotide replacement in the lysC gene is carried out, so
that, the resulting protein, the amino acid Thr in position 311 is
replaced by an Ile. Starting from the chromosomal DNA from
ATCC13032 as template for a PCR reaction and using the
oligonucleotide primers SEQ D NO:3 and SEQ ID NO:4, lysC is
amplified by use of the Pfu Turbo PCR system (Stratagene USA) in
accordance with the instructions of the manufacturer. Chromosomal
DNA from C. glutamicum ATCC13031 is prepared according to Tauch et
al. (1995) Plasmid 33:168-179 or Eikmanns et al. (1994)
Microbiology 140:1817-1828. The amplified fragment is flanked at
its 5' end by a SalI restriction cut and at its 3' end by aMluI
restriction cut. Prior to the cloning, the amplified fragment is
digested by these two restriction enzymes and purified using the
GFX.TM. PCR DNA and Gel Band Purification Kit (Amersham Pharmacia,
Freiburg).
TABLE-US-00005 SEQ ID NO:3
5'-GAGAGAGAGACGCGTCCCAGTGGCTGAGACGCATC-3' SEQ ID NO:4
5'-CTCTCTCTGTCGACGAATTCAATCTTACGGCCTG-3'
[0179] The obtained polynucleotide is cloned through the SalI and
MluI restriction cuts in pCLIK5 MCS with integrated SacB, referred
to in the following as pCIS (SEQ ID NO:5) and transformed in E.
coli XL-1 blue. A selection for plasmid-carrying cells is
accomplished by plating out on kanamycin (20 .mu.g/mL)--containing
LB agar (Lennox, 1955, Virology, 1:190). The plasmid is isolated
and the expected nucleotide sequence is confirmed by sequencing.
The preparation of the plasmid DNA is carried out according to
methods of and using materials of the company Quiagen. Sequencing
reactions are carried out according to Sanger et al. (1977)
Proceedings of the National Academy of Sciences USA 74:5463-5467.
The sequencing reactions are separated by means of ABI-Prism 377
(PE Applied Biosystems, Weiterstadt) and analyzed. The obtained
plasmid pCIS lysC is listed as SEQ ID NO:6.
Example VI
Mutagenesis of the LysC Gene from C. glutamicum
[0180] The targeted mutagenesis of the lysC gene from C. glutamicum
is carried out using the QuickChange Kit (Company: Stratagene/USA)
in accordance with the instructions of the manufacturer. The
mutagenesis is carried out in the plasmid pCIS lysC, SEQ ID NO:6.
The following oligonucleotide, primers are synthesized for the
replacement of thr 311 by 311 ile by use of the QuickChange method
(Stratagene):
TABLE-US-00006 SEQ ID NO:7
5'-CGGCACCACCGACATCATCTTCACCTGCCCTCGTTCCG-3' SEQ ID NO:8
5'-CGGAACGAGGGCAGGTGAAGATGATGTCGGTGGTGCCG-3'
[0181] The use of these bligonucleotide priners in the QuickChange
reaction leads, in the lysC gene (SEQ ID NO:9), to the replacement
of the nucleotide in position 932 (from C to T). The resulting
amino acid replacement Thr311Ile in the lysC gene is confirmed,
after transformation in E. coil XL1-blue and plasmid preparation,
by [a] sequencing reaction. The plasmid is given the designation
pCIS lysC thr311 ile and is listed as SEQ ID NO:10.
[0182] The plasnid pCIS lysC thr311 ile is transformed in C.
glutamicum ATCC13632 by means of electroporation, as described in
Liebl, et al. (1989) FEMS. Microbiology Letters 53:299-303.
Modifications of the protocol are described in DE 10046870. The
chromosomal arrangement of the lysC locus of individual
transformants is checked using standard methods by Southern blot
and hybridization, as described in Sambrook et al. (1989),
Molecular Cloning. A Laboratory Manual, Cold Spring Harbor. It is
thereby established that the transformants involved are those that
have integrated the transformed plasmid by homologous recombination
at the lysC locus. After growth of such colonies overnight in media
containing no antibiotic, the cells are plated out on a saccharose
CM agar medium (10% saccharose) and incubated at 30.degree. C. for
24 hours. Because the sacB gene contained in the vectbr pCIS ilysC
thr311ile converts saccharose into a toxic product, only those
colonies can grow that have deleted the sacB gene by a second
homologous recombination step between the wild-type lysC gene and
the mutated gene lysC thr311ile. During the homologous
recombination, either the wild-type gene or the mutated gene
together with the sacB gene can be deleted. If the sacB gene
together with the wild-type gene is removed, a mutated transformant
results.
[0183] Growing colonies are picked and examined for a
kanamycin-sensitive phenotype. Clones with deleted SacB gene must
simultaneously show kanamycin-sensitive growth behavior. Such
kanamycin-sensitive clones are investigated in a shaking flask for
their lysine productivity (see Example 6). For comparison, the
non-treated C. glutamicum ATCC13032 is taken. Clones with an
elevated lysine production in comparison to the control are
selected, chromosomal DNA are recovered, and the corresponding
region of the lysC gene is amplified by a PCR reaction and
sequenced. One such clone with the property of elevated lysine
synthesis and detected mutation in lysC at position 932 is
designated as ATCC13032 lysCfbr.
Example VII
Preparation of the Plasmid PK19 MOB SACB PEFTU
Fructose-1,6Bisphosphatase
[0184] Chromosomal DNA from C. glutamicum ATCC13032 is prepared
according to Tauch et al. (1995) Plasmid 33:168-179 or Eikmanns et
al. (1994) Microbiology 140:1817-1828.
[0185] PCR 1: With the oligonucleotide primers SEQ ID NO 11 and SEQ
ID NO 12, the chromos6mal DNA as template, and Pfu Turbo polymerase
(Company: Stratagene), a region lying upstream of the start codon
of the elongation factor TU is amplified by use of the polymerase
chain reaction (PCR) according to standard methods, as described in
Innis et al. (1990) PCR Protocols. A Guide to Methods and
Applications, Academic Press.
TABLE-US-00007 5'-TGGCCGTTACCCTGCGAATG-3' SEQ ID NO 11 and
5'-TGTATGTCCTCCTGGACTTC-3' SEQ ID NO 12
[0186] The obtained DNA fragment of approximately 200 bp size is
purified using the GFX.TM. PCR DNA and Gel Band Purification Kit
(Amersham Pharmacia, Freiburg) in accordance with the instructions
of the manufacturer.
[0187] PCR 2: With the oligonucleotide primers SEQ ID NO 13 and SEQ
ID NO 14, the chromosomal DNA as template, and Pfu Turbo
polymerase. (Company: Stratagene); the 5' region of the gene for
fructose-1,6-bisphosphatase is amplified by use of the, polymerase
chain reaction (PCR) according to standard methods, as described in
Innis et al. (1990) PCR Protocols. A Guide to Methods and
Applications, Academic Press.
TABLE-US-00008 SEQ ID NO 13
5'-GAAGTCCAGGAGGACATACAATGAACCTAAAGAACCCCGA-3' and SEQ ID NO 14
5'-ATCTACGTCGACCCAGGATGCCCTGGATTTC-3'
The obtained DNA fragment of approximately 740 bp size is purified
using the GFX.TM. PCR DNA and Gel Band Purification Kit (Amersham
Pharmacia, Freiburg) in accordance with the instructions of the
manufacturer.
[0188] PCR 3: With the oligonucleotide primers SEQ ID NO 15 and SEQ
ID NO 16, the chromosomal DNA as template, and Pfu Turbo polymerase
(Company: Stratagene), a region lying upstream of the start codon
of fructose-1,6-bisphosphatase is amplified by use of the
polymerase chain reaction (PCR) according to standard methods, as
described in Innis et al. (1990) PCR Protocols. A Guide to Methods
and Applications, Academic Press.
TABLE-US-00009 SEQ ID NO 15 5'-TATCAACGCGTTCTTCATCGGTAGCAGCACC-3'
and SEQ ID NO 16 5'-CATTCGCAGGGTAACGGCCACTGAAGGGCCTCCTGGG-3'
[0189] The obtained DNA fragment of approximately 720 bp size is
purified using the GFX.TM. PCR DNA and Gel Band Purification Kit
(Amersham Pharmacia, Freiburg) in accordance with the instructions
of the manufacturer.
[0190] PCR 4: With the oligonucleotide primers SEQ ID NO 17 and SEQ
ID NO 14, the PCR products from PCR 1 and 2 as template, and Pfu
Turbo polymerase (Company: Stratagene), a fusion PCR is carried out
by use of the polymerase chain reaction (PCR) according to standard
methods, as described in Innis et al. (1990) PCR Protocols. A Guide
to Methods and Applications, Academic Press.
[0191] The obtained DNA fragment of approximately 920 bp size is
purified using the GFX.TM. PCR DNA and Gel Band Purification Kit
(Amersham Pharmacia, Freiburg) in accordance with the instructions
of the manufacturer.
[0192] PCR 5: With the oligonucledtide primers SEQ ID NO 15 and SEQ
ID NO 14, the PCR products from PCR 3 and 4 as template, and Pfu
Turbo polymerase (Company: Stratagene), a fuision PCR is carried
out by use of the polymerase chain reaction (PCR) according to
standard methods, as described in Innis et al. (1990) PCR
Protocols. A Guide to Methods and Applications, Academic Press.
[0193] The obtained DNA fragment of approximately 1640 bp size is
purified using the GFX.TM. PCR DNA and Gel Band Purification Kit
(Amersham Pharmacia, Freiburg) in accordance with the instructions
of the manufacturer. Following this, it is cleaved using the
restriction enzymes MluI and SalI (Roche Diagnostics, Mannheim) and
the DNA fragment is purified using the GFX.TM. PCR DNA and Gel Band
Purification Kit.
[0194] The vector pCIS is cut with the restriction enzymes MluI and
SalI and a fragment of 4.3 kb size is isolated, after
electrophoretic separation, by use of the GFX.TM. PCR DNA and Gel
Band Purification Kit.
[0195] The vector fragment is ligated together with the PCR
fragment from PCR 5 by use of the Rapid DNA Ligation Kit (Roche
Diagnostics, Mannheim) in accordance with the instructions of the
manufacturer and the ligation batch is transformed in competent E.
coli XL-1 Blue (Stratagene, La Jolla, USA) according to standard
methods, as described in Sambrook et al. (Molecular Cloning. A
Laboratory Manual, Cold Spring Harbor, (1989)). A selection for
plasmid-carrying cells is accomplished by plating out on kanamycin
(20 .mu.g/mL)--containing LB agar (Lennox, 1955, Virology,
1:190).
[0196] The preparation of the plasmid DNA is carried out according
to methods of and using materials of the company Qiagen. Sequencing
reactions are carried out according to Sanger et al. (1977)
Proceedings of the National Academy of Sciences USA 74:5463-5467.
The sequencing reactions are separated by means of ABI Prism 377
(PE Applied Biosystems, Weiterstadt) and analyzed.
[0197] The resulting plasmid pCIS Peftu fructose-1,6-bisphosphatase
is listed as SEQ ID NO:17.
Example VIII
Production of Lysine
[0198] The plasmid pCIS Peftu fructose-1,6-bisphosphatase is
transformed in C. glutamicum ATCC13032 lysCfbr by means of
electroporation, as described in Liebl, et al. (1989) FEMS
Microbiology Letters 53:299-303. Modifications of the protocol are
described in DE 10046870. The chromosomal arrangement of the
fructose-1,6-bisphosphatase gene locus of individual transformants
is checked using standard methods by Southern blot and
hybridization, as described in Sambrook et al. (1989), Molecular
Cloning. A Laboratory-Manual, Crold Spring Harbor. It is thereby
established that the transformants involve those that have
integrated the transformed plasmid by homologous recombination at
the fructose-1,6-bisphosphatase gene locus. After growth of such
colonies overnight in media containing no antibiotic, the cells are
plated out on a saccharose CM agar medium (10% saccharose) and
incubated at 30.degree. C. for 24 hours.
[0199] Because the sacB gene contained in the vector pCIS Peftu
fructose-1,6-bisphosphatase converts saccharose into a toxic
product, only those colonies can grow that have deleted the sacB
gene by a second homologous recombination step between the
wild-type fructose-1,6-bisphosphatase gene and the Peftu
fructose-1,6-bisphosphatase fusion. During the homologous
recombination, either the wild-type gene or the fusion together
with the sacB gene can be deleted. If the sacB gene together with
the wild-type gene is removed, a mutated transformant results.
[0200] Growing colonies are picked and examined for a
kanamycin-sensitive phenotype. Clones with deleted SacB gene must
simultaneously show kanamycin-sensitive growth behavior. Whether
the desired replacement of the natural promoter by the Peftu
promoter had also taken place is checked by means of the polymerase
chain reaction (PCR). For this analysis, chromosomal DNA from the
starting strain and the resulting clones is isolated. To this end,
the respective clones are removed from the agar plate with a
toothpick and suspended in 100 gL of H2O and boiled up for 10 min
at 95.degree. C. In each case, 10 .mu.L of the resulting solution
is used as template in the PCR. Used as primers are
oligonucleotides that are homologous to the Peftu promoter and to
the fructose-1,6-bisphosphatase gene. The PCR conditions are
selected as follows: initial denaturation: 5 min at 95.degree. C.;
denaturation 30 sec at 95.degree. C.; hybridization 30 sec at
55.degree. C.; amplification 2 min at 72.degree. C.; 30 cycles; end
extension 5 min at 72.degree. C. In the batch with the DNA of the
starting strain, no PCR product could form owing to the selection
of the oligonucleotide. Only for clones that had completed the
replacement of the natural promoter by Peftu through the 2nd
recombination are a band with a size of 340 bp expected. Overall,
of the tested clones, 2 clones are positive. The clones are
designated as ATCC13032 lysCfbr Peftu fructose-1,6-bisphosphatasel
1 and 2.
[0201] In order to investigate the effect of the Peftu
fructose-1,6-bisphosphatase construction the lysine production, the
strains ATCC13032, ATCC13032 lysCfbr, and ATCC13032 lysCfbr Peftu
fructose-1,6-bisphosphatase 1 are cultivated on CM plates (10.0 g/L
D-glucose, 2.5 g/L NaCl, 2.0 g/L urea, 10.0 g/L bacto pepton
(Difco), 5.0 g/L yeast extract (Difco), 5.0 g/L beef extract
(Difco), 22.0 g/L agar (Difco), autoclaved (20 min. 121.degree.
C.)) for 2 days at 30.degree. C. Subsequently, the cells are
scraped off the plate and resuspended in saline. For the main
culture, 10 mL of medium 1 and 0.5 g of autoclaved CaCO3 (Riedel de
Haen) are inoculated in a 100 mL Erlenmeyer flask with the cell
suspension up to an OD600 of 1.5 and incubated for 39 h on a
[shaking incubator] of the type Infors AJ118 (Company: Infors,
Bottmingen, Switzerland) at 220 rpm. Subsequently, the
concentration of the lysine that separated out in the medium is
determined.
Medium I:
TABLE-US-00010 [0202] 40 g/L saccharose 60 g/L Molasses (calculated
with respect to 100% sugar content) 10 g/L (NH.sub.4).sub.2SO.sub.4
0.4 g/L MgSO.sub.4*7H.sub.2O 0.6 g/L KH.sub.2PO.sub.4 0.3 mg/L
thiamine*HCl 1 mg/L biotin (from a 1 mg/mL sterile-filtered stock
solution that is adjusted with NH.sub.4OH to pH 8.0) 2 mg/L
FeSO.sub.4 2 mg/L MnSO.sub.4 adjusted with NH.sub.4OH to pH 7.8,
autoclaved (121.degree. C., 20 min).
In addition, vitamin B12 (hydroxycobalamin Sigma Chemicals) from a
stock solution (200 .mu.g/mL, sterile-filtered) is added up to a
final concentration of 100 .mu.g/L.
[0203] The determination of the amino acid concentration is
conducted by means of high pressure liquid chromatography according
to Agilent on an Agilent 1100 Series LC System HPLC. A precolumn
derivatization with ortho-phthalaldehyde permits the quantification
of the amino acids that are formed; the separation of the amino
acid mixture takes place on a Hypersil AA columnn (Agilent).
Example IX
Preparation of the Plasmid PCIS PSOD Fructose-1,6
Bisphosphatase
[0204] Chromosomal DNA from C. glutamicum ATCC 13032 is prepared
according to Tauch et al. (1995) Plasmid 33:168-179 or Eikanns et
al. (1994) Microbiology 140:1817-1828.
[0205] PCR 1: With the oligonucleotide primers SEQ ID NO 18 and SEQ
ID NO 19, the chromosomal DNA as template, and Pfu Turbo polymerase
(Company: Stratagene), a region lying upstream of the start codon
of the superoxid dismutase is amplified by use of the polymerase
chain reaction (PCR) according to standard methods, as described in
Innis et al. (1990) PCR Protocols. A Guide to Methods and
Applications, Academic Press.
TABLE-US-00011 5'-tagctgccaattattccggg-3' SEQ ID NO 18 and
5'-GGGTAAAAAATCCTTTCGTA-3' SEQ ID NO 19
[0206] The obtained DNA fragment of approximately 200 bp size is
purified using the GFX.TM. PCR DNA and Gel Band Purification Kit
(Amersham Pharmacia, Freiburg) in accordance with the instructions
of the manufacturer.
[0207] PCR 2: With the oligonucleotide primers SEQ ID NO 20 and SEQ
ID NO 21, the chromosomal DNA as template, and Pfu Turbo polymerase
(Company: Stratagene), the 5' region of the gene for
fructose-1,6-bisphosphatase is amplified by use of the polymerase
chain reaction (PCR) according to standard methods, as described in
Innis et al. (1990) PCR Protocols. A Guide to Methods and
Applications, Academic Press.
TABLE-US-00012 SEQ ID NO 20
5'-CCCGGAATAATTGGCAGCTACTGAAGGGCCTCCTGGG-3' and SEQ ID NO 21
5'-TATCAACGCGTTCTTCATCGGTAGCAGCACC-3'
[0208] The obtained DNA fragment of approximately 720 bp size is
purified usig the GFX.TM. PCR DNA and Gel Band Purification Kit
(Amersham Pharmacia, Freiburg) in accordance with the instructions
of the manufacturer.
[0209] PCR 3: With the oligonucleotide primers SEQ ID NO 22 and SEQ
ID NO 23, the chromosomal DNA as template, and Pfu Turbo polymerase
(Company: Stratagene), a region lying upstream of the start codon
of fructose-1,6-bisphosphatase is amplified by use of the
polymerase chain reaction (PCR) according to standard methods, as
described in Innis et al. (1990) PCR Protocols. A Guide to Methods
and Applications, Academic Press.
TABLE-US-00013 SEQ ID NO 22
5'-TACGAAAGGATTTTTTACCCATGAACCTAAAGAACCCCGA-3' and SEQ ID NO 23
5'-ATCTACGTCGACCCAGGATGCCCTGGATTTC-3'
[0210] The obtained DNA fragment of approximately 740 bp size is
purified using the GFX.TM. PCR DNA and Gel Band Purification Kit
(Amersham Pharmacia, Freiburg) in accordance with the instructions
of the manufacturer.
[0211] PCR 4: With the oligonucleotide primers SEQ ID NO 18 and SEQ
ID NO 23, the PCR products from PCR 1 and 3 as template, and Pfu
Turbo polymerase (Company: Stratagene), a fusion PCR is carried out
by use of the polymerase chain reaction (PCR) according to standard
methods, as described in Innis et al. (1990) PCR Protocols. A Guide
to Methods and Applications, Academic Press. The obtained DNA
fragment of approximately 930 bp size is purified using the GFX.TM.
PCR DNA and Gel Band Purification Kit (Amersham Pharmacia,
Freiburg) in accordance with the instructions of the
manufacturer.
[0212] PCR 5: With the oligonucleotide primers SEQ ID NO 21 and SEQ
ID NO 23, the PCR products from PCR 2 and 4 as template, and Pfu
Turbo polymerase (Company: Stratagene), a fusion PCR is carried out
by use of the polymerase chain reaction (PCR) according to standard
methods, as described in Innis et al. (1990) PCR Protocols. A Guide
to Methods and Applications, Academic Press.
[0213] The obtained DNA fragment of approximately 1650 bp size is
purified using the GFX.TM. PCR DNA and Gel Band Purification Kit
(Amersham Pharmacia, Freiburg) in accordance with the instructions
of the manufacturer. Following this, it is cleaved using the
restriction enzymes MluI and SalI (Roche Diagnostics, Mannheim) and
the DNA fragment is purified using the GFX.TM. PCR DNA and Gel Band
Purification Kit.
[0214] The vector pCIS is cut with the restriction enzymes MluI and
SalI and a fragment of 4.3 kb size is isolated, after
elecfrophoretic separation, by use of the GFX.TM. PCR DNA and Gel
Band Purification Kit.
[0215] The vector fragment is ligated together with the PCR
fragment from PCR 5 by use of the Rapid DNA Ligation Kit (Roche
Diagnostics, Mannheim) in accordance with the instructions of the
manufacturer and the ligation batch is transformed in competent E.
coli XL-1 Blue (Stratagene, La Jolla, USA) according to standard
methods, as described in Sambrook et.al. (Molecular Cloning. A
Laboratory Manual, Cold Spring Harbor, (1989)). A selection for
plasmid-carrying cells is accomplished by plating out on kanamycin
(20 .mu.g/mL)--containing LB agar (Lennox, 1955, Virology,
1:190).
[0216] The preparation of the plasmid DNA is carried out according
to methods of and using materials of the company Qiagen. Sequencing
reactions are carried out according to Sanger et al. (1977)
Proceedings of the National Academy of Sciences USA 74:5463-5467.
The sequencing reactions are separated by means of ABI Prism 377
(PE Applied Biosystems, Weiterstadt) and analyzed.
[0217] The resulting plasmid pCIS Psod fructose-1,6-bisphosphatase
is listed as SEQ ID NO: 24.
Example X
Production of Lysine
[0218] The plasmid pCIS Psod fructose-1,6-bisphosphatase is
transformed in C. glutamicum ATCC13032 lysCfbr by means of
electroporation, as described in Liebl, et al. (1989) FEMS
Microbiology Letters 53:299-303. Modifications of the protocol are
described in DE 10046870. The chromosomal arrangement of the
fructose-1,6-bisphosphatase gene locus of individual transformants
is checked using standard methods by Southern blot and
hybridization, as described in Sambrook et al. (1989), Molecular
Cloning. A Laboratory Manual, Cold Spring Harbor. It is thereby
established that the transformants involve those that have
integrated the transformed plasmid by homologous recombination at
the fructose-1,6-bisphosphatase gene locus. After growth of such
colonies overnight in media containing no antibiotic, the cells are
plated out on a saccharose CM agar medium (10% saccharose) and
incubated at 30.degree. C. for 24 hours.
[0219] Because the sacB gene contained in the vector pCIS Psod
fructose-1,6-bisphosphatase converts saccharose into a toxic
product, only those colonies can grow that have deleted the sacB
gene by a second homologous recombination step between the
wild-type fructose-1,6-bisphosphatase gene and the Psod
fructose-1,6-bisphosphatase fusion. During the homologous
recombination, either the wild-type gene or the fusion together
with the sacB gene can be deleted. If the sacB gene together with
the wild-type gene is removed, a mutated transformant results.
[0220] Growing colonies are picked and examined for a
kanamycin-sensitive phenotype. Clones with deleted SacB gene must
simultaneously show kanamycin-sensitive growth behavior. Whether
the desired replacement of the natural promoter by the Psod
promoter had also taken place is checked by means of the polymerase
chain reaction (PCR). For this analysis, chromosomal DNA from the
starting strain and the resulting clones is isolated. To this end,
the respective clones are removed from the agar plate with a
toothpick and suspended in 100 .mu.L of H2O and boiled up for 10
min at 95.degree. C. In each case, 10 .mu.L of the resulting
solution is used as-template in the PCR. Used as primers are
oligonucleotides that are homologous to the Psod promoter and to
the fructose-1,6-bisphosphatase gene. The PCR conditions are
selected as follows: initial denaturation: 5 min at 95.degree. C.;
denaturation 30 sec it 95.degree. C.; hybridization 30 sec at
55.degree. C.; amplification 2 min at 72.degree. C.; 30 cycles; end
extension 5 min at 72.degree. C. In the batch with the DNA of the
starting strain, no PCR product could form owing to the selection
of the oligonucleotide. Only three clones that had completed the
replacement of the natural promoter by Psod through the 2nd
recombination are a band with a size of 350 bp expected. Overall,
of the tested clones, 3 clones are positive. The clones are
designated as ATCC13032 lyscfbr Psod fructose-1,6-bisphosphatase 1,
2 and 3.
[0221] In order to investigate the effect of the Psod
fructose-1,6-bisphosphatase construct on the lysine production, the
strains ATCC13032, ATCC13032 lysCfbr, and ATCC13032 lysCfbr Psod
fructose-1,6-bisphosphatase 1 are cultivated on CM plates (10.0 g/L
D-glucose, 2.5 g/L NaCl, 2.0 g/L urea, 10.0 g/L bacto pepton
(Difco), 5.0 g/L yeast extract (Difco), 5.0 g/L beef extract
(Difco), 22.0 g/L agar (Difco), autoclaved (20 min. 121.degree.
C.)) for 2 days at 30.degree. C. Subsequently, the cells are
scraped off the plate and resuspended in saline. For the main
culture, 10 mL of medium I and 0.5 g of autoclaved CaCO3 (Riedel de
Haen) are inoculated in a 100 mL Erlenmeyer flask with the cell
suspension up to an OD600 of 1.5 and incubated for 39 h on a
shaking incubator of the type Infors AJ118 (Company: Infors,
Bottmingen, Switzerland) at 220 rpm. Subsequently, the
concentration of the lysine that separated out in the medium is
determined.
Medium I:
TABLE-US-00014 [0222] 40 g/L saccharose 60 g/L Molasses (calculated
with respect to 100% sugar content) 10 g/L (NH.sub.4).sub.2SO.sub.4
0.4 g/L MgSO.sub.4*7H.sub.2O 0.6 g/L KH.sub.2PO.sub.4 0.3 mg/L
thiamine*HCl 1 mg/L biotin (from a 1 mg/mL sterile-filtered stock
solution that is adjusted with NH.sub.4OH to pH 8.0) 2 mg/L
FeSO.sub.4 2 mg/L MnSO.sub.4 adjusted with NH.sub.4OH to pH 7.8,
autoclaved (121.degree. C., 20 min).
In addition, vitamin B12 (hydroxycobalani Sigma Chemicals) from a
stock solution (200 .mu.g/mL, sterile-filtered) is added up to a
final concentration of 100 .mu.g/L.
[0223] The determination of the amino acid concentration is
conducted by means of high pressure liquid chromatography according
to Agilent on an Agilent 1100 Series LC System HPLC. A precolumn
derivatization with ortho-phthalaldehyde permits the quantification
of the amino acids that are formed; the separation of the amino
acid mixture takes place on a Hypersil AA column (Agilent).
Equivalents
[0224] Those skilled in the art will recoginze, or 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.
Sequence CWU 1
1
2411070DNACorynebacterium glutamicumCDS(22)...(1029) 1gtgccccagg
aggcccttca g atg aac cta aag aac ccc gaa acg cca gac 51 Met Asn Leu
Lys Asn Pro Glu Thr Pro Asp 1 5 10cgt aac ctt gct atg gag ctg gtg
cga gtt acg gaa gca gct gca ctg 99Arg Asn Leu Ala Met Glu Leu Val
Arg Val Thr Glu Ala Ala Ala Leu 15 20 25gct tct gga cgt tgg gtt gga
cgt ggc atg aag aat gaa ggc gac ggt 147Ala Ser Gly Arg Trp Val Gly
Arg Gly Met Lys Asn Glu Gly Asp Gly 30 35 40gcc gct gtt gac gcc atg
cgc cag ctc atc aac tca gtg acc atg aag 195Ala Ala Val Asp Ala Met
Arg Gln Leu Ile Asn Ser Val Thr Met Lys 45 50 55ggc gtc gtt gtt atc
ggc gag ggc gaa aaa gac gaa gct cca atg ctg 243Gly Val Val Val Ile
Gly Glu Gly Glu Lys Asp Glu Ala Pro Met Leu 60 65 70tac aac ggc gaa
gag gtc gga acc ggc ttt gga cct gag gtt gat atc 291Tyr Asn Gly Glu
Glu Val Gly Thr Gly Phe Gly Pro Glu Val Asp Ile 75 80 85 90gca gtt
gac cca gtt gac ggc acc acc ctg atg gct gag ggt cgc ccc 339Ala Val
Asp Pro Val Asp Gly Thr Thr Leu Met Ala Glu Gly Arg Pro 95 100
105aac gca att tcc att ctc gca gct gca gag cgt ggc acc atg tac gat
387Asn Ala Ile Ser Ile Leu Ala Ala Ala Glu Arg Gly Thr Met Tyr Asp
110 115 120cca tcc tcc gtc ttc tac atg aag aag atc gcc gtg gga cct
gag gcc 435Pro Ser Ser Val Phe Tyr Met Lys Lys Ile Ala Val Gly Pro
Glu Ala 125 130 135gca ggc aag atc gac atc gaa gct cca gtt gcc cac
aac atc aac gcg 483Ala Gly Lys Ile Asp Ile Glu Ala Pro Val Ala His
Asn Ile Asn Ala 140 145 150gtg gca aag tcc aag gga atc aac cct tcc
gac gtc acc gtt gtc gtg 531Val Ala Lys Ser Lys Gly Ile Asn Pro Ser
Asp Val Thr Val Val Val155 160 165 170ctt gac cgt cct cgc cac atc
gaa ctg atc gca gac att cgt cgt gca 579Leu Asp Arg Pro Arg His Ile
Glu Leu Ile Ala Asp Ile Arg Arg Ala 175 180 185ggc gca aag gtt cgt
ctc atc tcc gac ggc gac gtt gca ggt gca gtt 627Gly Ala Lys Val Arg
Leu Ile Ser Asp Gly Asp Val Ala Gly Ala Val 190 195 200gca gca gct
cag gat tcc aac tcc gtg gac atc atg atg ggc acc ggc 675Ala Ala Ala
Gln Asp Ser Asn Ser Val Asp Ile Met Met Gly Thr Gly 205 210 215gga
acc cca gaa ggc atc atc act gcg tgc gcc atg aag tgc atg ggt 723Gly
Thr Pro Glu Gly Ile Ile Thr Ala Cys Ala Met Lys Cys Met Gly 220 225
230ggc gaa atc cag ggc atc ctg gcc cca atg aac gat ttc gag cgc cag
771Gly Glu Ile Gln Gly Ile Leu Ala Pro Met Asn Asp Phe Glu Arg
Gln235 240 245 250aag gca cac gac gct ggt ctg gtt ctt gat cag gtt
ctg cac acc aac 819Lys Ala His Asp Ala Gly Leu Val Leu Asp Gln Val
Leu His Thr Asn 255 260 265gat ctg gtg agc tcc gac aac tgc tac ttc
gtg gca acc ggt gtg acc 867Asp Leu Val Ser Ser Asp Asn Cys Tyr Phe
Val Ala Thr Gly Val Thr 270 275 280aac ggt gac atg ctc cgt ggc gtt
tcc tac cgc gca aac ggc gca acc 915Asn Gly Asp Met Leu Arg Gly Val
Ser Tyr Arg Ala Asn Gly Ala Thr 285 290 295acc cgt tcc ctg gtt atg
cgc gca aag tca ggc acc atc cgc cac atc 963Thr Arg Ser Leu Val Met
Arg Ala Lys Ser Gly Thr Ile Arg His Ile 300 305 310gag tct gtc cac
cag ctg tcc aag ctg cag gaa tac tcc gtg gtt gac 1011Glu Ser Val His
Gln Leu Ser Lys Leu Gln Glu Tyr Ser Val Val Asp315 320 325 330tac
acc acc gcg acc taa gagctcttag ttcgaaaaac cgccggccat 1059Tyr Thr
Thr Ala Thr * 335tgtggtcggc g 10702335PRTCorynebacterium glutamicum
2Met Asn Leu Lys Asn Pro Glu Thr Pro Asp Arg Asn Leu Ala Met Glu 1
5 10 15Leu Val Arg Val Thr Glu Ala Ala Ala Leu Ala Ser Gly Arg Trp
Val 20 25 30Gly Arg Gly Met Lys Asn Glu Gly Asp Gly Ala Ala Val Asp
Ala Met 35 40 45Arg Gln Leu Ile Asn Ser Val Thr Met Lys Gly Val Val
Val Ile Gly 50 55 60Glu Gly Glu Lys Asp Glu Ala Pro Met Leu Tyr Asn
Gly Glu Glu Val65 70 75 80Gly Thr Gly Phe Gly Pro Glu Val Asp Ile
Ala Val Asp Pro Val Asp 85 90 95Gly Thr Thr Leu Met Ala Glu Gly Arg
Pro Asn Ala Ile Ser Ile Leu 100 105 110Ala Ala Ala Glu Arg Gly Thr
Met Tyr Asp Pro Ser Ser Val Phe Tyr 115 120 125Met Lys Lys Ile Ala
Val Gly Pro Glu Ala Ala Gly Lys Ile Asp Ile 130 135 140Glu Ala Pro
Val Ala His Asn Ile Asn Ala Val Ala Lys Ser Lys Gly145 150 155
160Ile Asn Pro Ser Asp Val Thr Val Val Val Leu Asp Arg Pro Arg His
165 170 175Ile Glu Leu Ile Ala Asp Ile Arg Arg Ala Gly Ala Lys Val
Arg Leu 180 185 190Ile Ser Asp Gly Asp Val Ala Gly Ala Val Ala Ala
Ala Gln Asp Ser 195 200 205Asn Ser Val Asp Ile Met Met Gly Thr Gly
Gly Thr Pro Glu Gly Ile 210 215 220Ile Thr Ala Cys Ala Met Lys Cys
Met Gly Gly Glu Ile Gln Gly Ile225 230 235 240Leu Ala Pro Met Asn
Asp Phe Glu Arg Gln Lys Ala His Asp Ala Gly 245 250 255Leu Val Leu
Asp Gln Val Leu His Thr Asn Asp Leu Val Ser Ser Asp 260 265 270Asn
Cys Tyr Phe Val Ala Thr Gly Val Thr Asn Gly Asp Met Leu Arg 275 280
285Gly Val Ser Tyr Arg Ala Asn Gly Ala Thr Thr Arg Ser Leu Val Met
290 295 300Arg Ala Lys Ser Gly Thr Ile Arg His Ile Glu Ser Val His
Gln Leu305 310 315 320Ser Lys Leu Gln Glu Tyr Ser Val Val Asp Tyr
Thr Thr Ala Thr 325 330 335335DNAArtificial SequenceSynthetic
construct 3gagagagaga cgcgtcccag tggctgagac gcatc
35434DNAArtificial SequenceSynthetic construct 4ctctctctgt
cgacgaattc aatcttacgg cctg 3454323DNACorynebacterium glutamicum
5tcgagaggcc tgacgtcggg cccggtacca cgcgtcatat gactagttcg gacctaggga
60tatcgtcgac atcgatgctc ttctgcgtta attaacaatt gggatcctct agacccggga
120tttaaatcgc tagcgggctg ctaaaggaag cggaacacgt agaaagccag
tccgcagaaa 180cggtgctgac cccggatgaa tgtcagctac tgggctatct
ggacaaggga aaacgcaagc 240gcaaagagaa agcaggtagc ttgcagtggg
cttacatggc gatagctaga ctgggcggtt 300ttatggacag caagcgaacc
ggaattgcca gctggggcgc cctctggtaa ggttgggaag 360ccctgcaaag
taaactggat ggctttcttg ccgccaagga tctgatggcg caggggatca
420agatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
tggattgcac 480gcaggttctc cggccgcttg ggtggagagg ctattcggct
atgactgggc acaacagaca 540atcggctgct ctgatgccgc cgtgttccgg
ctgtcagcgc aggggcgccc ggttcttttt 600gtcaagaccg acctgtccgg
tgccctgaat gaactgcagg acgaggcagc gcggctatcg 660tggctggcca
cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
720agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
tcaccttgct 780cctgccgaga aagtatccat catggctgat gcaatgcggc
ggctgcatac gcttgatccg 840gctacctgcc cattcgacca ccaagcgaaa
catcgcatcg agcgagcacg tactcggatg 900gaagccggtc ttgtcgatca
ggatgatctg gacgaagagc atcaggggct cgcgccagcc 960gaactgttcg
ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
1020ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
attcatcgac 1080tgtggccggc tgggtgtggc ggaccgctat caggacatag
cgttggctac ccgtgatatt 1140gctgaagagc ttggcggcga atgggctgac
cgcttcctcg tgctttacgg tatcgccgct 1200cccgattcgc agcgcatcgc
cttctatcgc cttcttgacg agttcttctg agcgggactc 1260tggggttcga
aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
1320ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
ggctggatga 1380tcctccagcg cggggatctc atgctggagt tcttcgccca
cgctagcggc gcgccggccg 1440gcccggtgtg aaataccgca cagatgcgta
aggagaaaat accgcatcag gcgctcttcc 1500gcttcctcgc tcactgactc
gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 1560cactcaaagg
cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
1620tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
ggcgtttttc 1680cataggctcc gcccccctga cgagcatcac aaaaatcgac
gctcaagtca gaggtggcga 1740aacccgacag gactataaag ataccaggcg
tttccccctg gaagctccct cgtgcgctct 1800cctgttccga ccctgccgct
taccggatac ctgtccgcct ttctcccttc gggaagcgtg 1860gcgctttctc
atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
1920ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
cggtaactat 1980cgtcttgagt ccaacccggt aagacacgac ttatcgccac
tggcagcagc cactggtaac 2040aggattagca gagcgaggta tgtaggcggt
gctacagagt tcttgaagtg gtggcctaac 2100tacggctaca ctagaaggac
agtatttggt atctgcgctc tgctgaagcc agttaccttc 2160ggaaaaagag
ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
2220tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
tcctttgatc 2280ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
gttaagggat tttggtcatg 2340agattatcaa aaaggatctt cacctagatc
cttttaaagg ccggccgcgg ccgccatcgg 2400cattttcttt tgcgttttta
tttgttaact gttaattgtc cttgttcaag gatgctgtct 2460ttgacaacag
atgttttctt gcctttgatg ttcagcagga agctcggcgc aaacgttgat
2520tgtttgtctg cgtagaatcc tctgtttgtc atatagcttg taatcacgac
attgtttcct 2580ttcgcttgag gtacagcgaa gtgtgagtaa gtaaaggtta
catcgttagg atcaagatcc 2640atttttaaca caaggccagt tttgttcagc
ggcttgtatg ggccagttaa agaattagaa 2700acataaccaa gcatgtaaat
atcgttagac gtaatgccgt caatcgtcat ttttgatccg 2760cgggagtcag
tgaacaggta ccatttgccg ttcattttaa agacgttcgc gcgttcaatt
2820tcatctgtta ctgtgttaga tgcaatcagc ggtttcatca cttttttcag
tgtgtaatca 2880tcgtttagct caatcatacc gagagcgccg tttgctaact
cagccgtgcg ttttttatcg 2940ctttgcagaa gtttttgact ttcttgacgg
aagaatgatg tgcttttgcc atagtatgct 3000ttgttaaata aagattcttc
gccttggtag ccatcttcag ttccagtgtt tgcttcaaat 3060actaagtatt
tgtggccttt atcttctacg tagtgaggat ctctcagcgt atggttgtcg
3120cctgagctgt agttgccttc atcgatgaac tgctgtacat tttgatacgt
ttttccgtca 3180ccgtcaaaga ttgatttata atcctctaca ccgttgatgt
tcaaagagct gtctgatgct 3240gatacgttaa cttgtgcagt tgtcagtgtt
tgtttgccgt aatgtttacc ggagaaatca 3300gtgtagaata aacggatttt
tccgtcagat gtaaatgtgg ctgaacctga ccattcttgt 3360gtttggtctt
ttaggataga atcatttgca tcgaatttgt cgctgtcttt aaagacgcgg
3420ccagcgtttt tccagctgtc aatagaagtt tcgccgactt tttgatagaa
catgtaaatc 3480gatgtgtcat ccgcattttt aggatctccg gctaatgcaa
agacgatgtg gtagccgtga 3540tagtttgcga cagtgccgtc agcgttttgt
aatggccagc tgtcccaaac gtccaggcct 3600tttgcagaag agatattttt
aattgtggac gaatcaaatt cagaaacttg atatttttca 3660tttttttgct
gttcagggat ttgcagcata tcatggcgtg taatatggga aatgccgtat
3720gtttccttat atggcttttg gttcgtttct ttcgcaaacg cttgagttgc
gcctcctgcc 3780agcagtgcgg tagtaaaggt taatactgtt gcttgttttg
caaacttttt gatgttcatc 3840gttcatgtct ccttttttat gtactgtgtt
agcggtctgc ttcttccagc cctcctgttt 3900gaagatggca agttagttac
gcacaataaa aaaagaccta aaatatgtaa ggggtgacgc 3960caaagtatac
actttgccct ttacacattt taggtcttgc ctgctttatc agtaacaaac
4020ccgcgcgatt tacttttcga cctcattcta ttagactctc gtttggattg
caactggtct 4080attttcctct tttgtttgat agaaaatcat aaaaggattt
gcagactacg ggcctaaaga 4140actaaaaaat ctatctgttt cttttcattc
tctgtatttt ttatagtttc tgttgcatgg 4200gcataaagtt gcctttttaa
tcacaattca gaaaatatca taatatctca tttcactaaa 4260taatagtgaa
cggcaggtat atgtgatggg ttaaaaagga tcggcggccg ctcgatttaa 4320atc
432365860DNACorynebacterium glutamicum 6cccggtacca cgcgtcccag
tggctgagac gcatccgcta aagccccagg aaccctgtgc 60agaaagaaaa cactcctctg
gctaggtaga cacagtttat aaaggtagag ttgagcgggt 120aactgtcagc
acgtagatcg aaaggtgcac aaaggtggcc ctggtcgtac agaaatatgg
180cggttcctcg cttgagagtg cggaacgcat tagaaacgtc gctgaacgga
tcgttgccac 240caagaaggct ggaaatgatg tcgtggttgt ctgctccgca
atgggagaca ccacggatga 300acttctagaa cttgcagcgg cagtgaatcc
cgttccgcca gctcgtgaaa tggatatgct 360cctgactgct ggtgagcgta
tttctaacgc tctcgtcgcc atggctattg agtcccttgg 420cgcagaagcc
caatctttca cgggctctca ggctggtgtg ctcaccaccg agcgccacgg
480aaacgcacgc attgttgatg tcactccagg tcgtgtgcgt gaagcactcg
atgagggcaa 540gatctgcatt gttgctggtt tccagggtgt taataaagaa
acccgcgatg tcaccacgtt 600gggtcgtggt ggttctgaca ccactgcagt
tgcgttggca gctgctttga acgctgatgt 660gtgtgagatt tactcggacg
ttgacggtgt gtataccgct gacccgcgca tcgttcctaa 720tgcacagaag
ctggaaaagc tcagcttcga agaaatgctg gaacttgctg ctgttggctc
780caagattttg gtgctgcgca gtgttgaata cgctcgtgca ttcaatgtgc
cacttcgcgt 840acgctcgtct tatagtaatg atcccggcac tttgattgcc
ggctctatgg aggatattcc 900tgtggaagaa gcagtcctta ccggtgtcgc
aaccgacaag tccgaagcca aagtaaccgt 960tctgggtatt tccgataagc
caggcgaggc tgcgaaggtt ttccgtgcgt tggctgatgc 1020agaaatcaac
attgacatgg ttctgcagaa cgtctcttct gtagaagacg gcaccaccga
1080catcaccttc acctgccctc gttccgacgg ccgccgcgcg atggagatct
tgaagaagct 1140tcaggttcag ggcaactgga ccaatgtgct ttacgacgac
caggtcggca aagtctccct 1200cgtgggtgct ggcatgaagt ctcacccagg
tgttaccgca gagttcatgg aagctctgcg 1260cgatgtcaac gtgaacatcg
aattgatttc cacctctgag attcgtattt ccgtgctgat 1320ccgtgaagat
gatctggatg ctgctgcacg tgcattgcat gagcagttcc agctgggcgg
1380cgaagacgaa gccgtcgttt atgcaggcac cggacgctaa agttttaaag
gagtagtttt 1440acaatgacca ccatcgcagt tgttggtgca accggccagg
tcggccaggt tatgcgcacc 1500cttttggaag agcgcaattt cccagctgac
actgttcgtt tctttgcttc cccacgttcc 1560gcaggccgta agattgaatt
cgtcgacatc gatgctcttc tgcgttaatt aacaattggg 1620atcctctaga
cccgggattt aaatcgctag cgggctgcta aaggaagcgg aacacgtaga
1680aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg
gctatctgga 1740caagggaaaa cgcaagcgca aagagaaagc aggtagcttg
cagtgggctt acatggcgat 1800agctagactg ggcggtttta tggacagcaa
gcgaaccgga attgccagct ggggcgccct 1860ctggtaaggt tgggaagccc
tgcaaagtaa actggatggc tttcttgccg ccaaggatct 1920gatggcgcag
gggatcaaga tctgatcaag agacaggatg aggatcgttt cgcatgattg
1980aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
ttcggctatg 2040actgggcaca acagacaatc ggctgctctg atgccgccgt
gttccggctg tcagcgcagg 2100ggcgcccggt tctttttgtc aagaccgacc
tgtccggtgc cctgaatgaa ctgcaggacg 2160aggcagcgcg gctatcgtgg
ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg 2220ttgtcactga
agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
2280tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
atgcggcggc 2340tgcatacgct tgatccggct acctgcccat tcgaccacca
agcgaaacat cgcatcgagc 2400gagcacgtac tcggatggaa gccggtcttg
tcgatcagga tgatctggac gaagagcatc 2460aggggctcgc gccagccgaa
ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg 2520atctcgtcgt
gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
2580tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
gacatagcgt 2640tggctacccg tgatattgct gaagagcttg gcggcgaatg
ggctgaccgc ttcctcgtgc 2700tttacggtat cgccgctccc gattcgcagc
gcatcgcctt ctatcgcctt cttgacgagt 2760tcttctgagc gggactctgg
ggttcgaaat gaccgaccaa gcgacgccca acctgccatc 2820acgagatttc
gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
2880ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
tcgcccacgc 2940tagcggcgcg ccggccggcc cggtgtgaaa taccgcacag
atgcgtaagg agaaaatacc 3000gcatcaggcg ctcttccgct tcctcgctca
ctgactcgct gcgctcggtc gttcggctgc 3060ggcgagcggt atcagctcac
tcaaaggcgg taatacggtt atccacagaa tcaggggata 3120acgcaggaaa
gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
3180cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
aatcgacgct 3240caagtcagag gtggcgaaac ccgacaggac tataaagata
ccaggcgttt ccccctggaa 3300gctccctcgt gcgctctcct gttccgaccc
tgccgcttac cggatacctg tccgcctttc 3360tcccttcggg aagcgtggcg
ctttctcata gctcacgctg taggtatctc agttcggtgt 3420aggtcgttcg
ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
3480ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
tcgccactgg 3540cagcagccac tggtaacagg attagcagag cgaggtatgt
aggcggtgct acagagttct 3600tgaagtggtg gcctaactac ggctacacta
gaaggacagt atttggtatc tgcgctctgc 3660tgaagccagt taccttcgga
aaaagagttg gtagctcttg atccggcaaa caaaccaccg 3720ctggtagcgg
tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
3780aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
aactcacgtt 3840aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
ctagatcctt ttaaaggccg 3900gccgcggccg ccatcggcat tttcttttgc
gtttttattt gttaactgtt aattgtcctt 3960gttcaaggat gctgtctttg
acaacagatg ttttcttgcc tttgatgttc agcaggaagc 4020tcggcgcaaa
cgttgattgt ttgtctgcgt agaatcctct gtttgtcata tagcttgtaa
4080tcacgacatt gtttcctttc gcttgaggta cagcgaagtg tgagtaagta
aaggttacat 4140cgttaggatc aagatccatt tttaacacaa ggccagtttt
gttcagcggc ttgtatgggc 4200cagttaaaga attagaaaca taaccaagca
tgtaaatatc gttagacgta atgccgtcaa 4260tcgtcatttt tgatccgcgg
gagtcagtga acaggtacca tttgccgttc attttaaaga 4320cgttcgcgcg
ttcaatttca tctgttactg tgttagatgc aatcagcggt ttcatcactt
4380ttttcagtgt gtaatcatcg tttagctcaa tcataccgag agcgccgttt
gctaactcag 4440ccgtgcgttt tttatcgctt tgcagaagtt tttgactttc
ttgacggaag aatgatgtgc 4500ttttgccata gtatgctttg ttaaataaag
attcttcgcc ttggtagcca tcttcagttc 4560cagtgtttgc ttcaaatact
aagtatttgt ggcctttatc ttctacgtag tgaggatctc 4620tcagcgtatg
gttgtcgcct gagctgtagt tgccttcatc gatgaactgc tgtacatttt
4680gatacgtttt tccgtcaccg tcaaagattg atttataatc ctctacaccg
ttgatgttca 4740aagagctgtc tgatgctgat acgttaactt gtgcagttgt
cagtgtttgt ttgccgtaat 4800gtttaccgga gaaatcagtg tagaataaac
ggatttttcc gtcagatgta aatgtggctg
4860aacctgacca ttcttgtgtt tggtctttta ggatagaatc atttgcatcg
aatttgtcgc 4920tgtctttaaa gacgcggcca gcgtttttcc agctgtcaat
agaagtttcg ccgacttttt 4980gatagaacat gtaaatcgat gtgtcatccg
catttttagg atctccggct aatgcaaaga 5040cgatgtggta gccgtgatag
tttgcgacag tgccgtcagc gttttgtaat ggccagctgt 5100cccaaacgtc
caggcctttt gcagaagaga tatttttaat tgtggacgaa tcaaattcag
5160aaacttgata tttttcattt ttttgctgtt cagggatttg cagcatatca
tggcgtgtaa 5220tatgggaaat gccgtatgtt tccttatatg gcttttggtt
cgtttctttc gcaaacgctt 5280gagttgcgcc tcctgccagc agtgcggtag
taaaggttaa tactgttgct tgttttgcaa 5340actttttgat gttcatcgtt
catgtctcct tttttatgta ctgtgttagc ggtctgcttc 5400ttccagccct
cctgtttgaa gatggcaagt tagttacgca caataaaaaa agacctaaaa
5460tatgtaaggg gtgacgccaa agtatacact ttgcccttta cacattttag
gtcttgcctg 5520ctttatcagt aacaaacccg cgcgatttac ttttcgacct
cattctatta gactctcgtt 5580tggattgcaa ctggtctatt ttcctctttt
gtttgataga aaatcataaa aggatttgca 5640gactacgggc ctaaagaact
aaaaaatcta tctgtttctt ttcattctct gtatttttta 5700tagtttctgt
tgcatgggca taaagttgcc tttttaatca caattcagaa aatatcataa
5760tatctcattt cactaaataa tagtgaacgg caggtatatg tgatgggtta
aaaaggatcg 5820gcggccgctc gatttaaatc tcgagaggcc tgacgtcggg
5860738DNAArtificial SequenceSynthetic construct 7cggcaccacc
gacatcatct tcacctgccc tcgttccg 38838DNAArtificial SequenceSynthetic
construct 8cggaacgagg gcaggtgaag atgatgtcgg tggtgccg
3891263DNACorynebacterium glutamicum 9gtggccctgg tcgtacagaa
atatggcggt tcctcgcttg agagtgcgga acgcattaga 60aacgtcgctg aacggatcgt
tgccaccaag aaggctggaa atgatgtcgt ggttgtctgc 120tccgcaatgg
gagacaccac ggatgaactt ctagaacttg cagcggcagt gaatcccgtt
180ccgccagctc gtgaaatgga tatgctcctg actgctggtg agcgtatttc
taacgctctc 240gtcgccatgg ctattgagtc ccttggcgca gaagcccaat
ctttcacggg ctctcaggct 300ggtgtgctca ccaccgagcg ccacggaaac
gcacgcattg ttgatgtcac tccaggtcgt 360gtgcgtgaag cactcgatga
gggcaagatc tgcattgttg ctggtttcca gggtgttaat 420aaagaaaccc
gcgatgtcac cacgttgggt cgtggtggtt ctgacaccac tgcagttgcg
480ttggcagctg ctttgaacgc tgatgtgtgt gagatttact cggacgttga
cggtgtgtat 540accgctgacc cgcgcatcgt tcctaatgca cagaagctgg
aaaagctcag cttcgaagaa 600atgctggaac ttgctgctgt tggctccaag
attttggtgc tgcgcagtgt tgaatacgct 660cgtgcattca atgtgccact
tcgcgtacgc tcgtcttata gtaatgatcc cggcactttg 720attgccggct
ctatggagga tattcctgtg gaagaagcag tccttaccgg tgtcgcaacc
780gacaagtccg aagccaaagt aaccgttctg ggtatttccg ataagccagg
cgaggctgcg 840aaggttttcc gtgcgttggc tgatgcagaa atcaacattg
acatggttct gcagaacgtc 900tcttctgtag aagacggcac caccgacatc
accttcacct gccctcgttc cgacggccgc 960cgcgcgatgg agatcttgaa
gaagcttcag gttcagggca actggaccaa tgtgctttac 1020gacgaccagg
tcggcaaagt ctccctcgtg ggtgctggca tgaagtctca cccaggtgtt
1080accgcagagt tcatggaagc tctgcgcgat gtcaacgtga acatcgaatt
gatttccacc 1140tctgagattc gtatttccgt gctgatccgt gaagatgatc
tggatgctgc tgcacgtgca 1200ttgcatgagc agttccagct gggcggcgaa
gacgaagccg tcgtttatgc aggcaccgga 1260cgc
1263105860DNACorynebacterium glutamicum 10cccggtacca cgcgtcccag
tggctgagac gcatccgcta aagccccagg aaccctgtgc 60agaaagaaaa cactcctctg
gctaggtaga cacagtttat aaaggtagag ttgagcgggt 120aactgtcagc
acgtagatcg aaaggtgcac aaaggtggcc ctggtcgtac agaaatatgg
180cggttcctcg cttgagagtg cggaacgcat tagaaacgtc gctgaacgga
tcgttgccac 240caagaaggct ggaaatgatg tcgtggttgt ctgctccgca
atgggagaca ccacggatga 300acttctagaa cttgcagcgg cagtgaatcc
cgttccgcca gctcgtgaaa tggatatgct 360cctgactgct ggtgagcgta
tttctaacgc tctcgtcgcc atggctattg agtcccttgg 420cgcagaagcc
caatctttca cgggctctca ggctggtgtg ctcaccaccg agcgccacgg
480aaacgcacgc attgttgatg tcactccagg tcgtgtgcgt gaagcactcg
atgagggcaa 540gatctgcatt gttgctggtt tccagggtgt taataaagaa
acccgcgatg tcaccacgtt 600gggtcgtggt ggttctgaca ccactgcagt
tgcgttggca gctgctttga acgctgatgt 660gtgtgagatt tactcggacg
ttgacggtgt gtataccgct gacccgcgca tcgttcctaa 720tgcacagaag
ctggaaaagc tcagcttcga agaaatgctg gaacttgctg ctgttggctc
780caagattttg gtgctgcgca gtgttgaata cgctcgtgca ttcaatgtgc
cacttcgcgt 840acgctcgtct tatagtaatg atcccggcac tttgattgcc
ggctctatgg aggatattcc 900tgtggaagaa gcagtcctta ccggtgtcgc
aaccgacaag tccgaagcca aagtaaccgt 960tctgggtatt tccgataagc
caggcgaggc tgcgaaggtt ttccgtgcgt tggctgatgc 1020agaaatcaac
attgacatgg ttctgcagaa cgtctcttct gtagaagacg gcaccaccga
1080catcatcttc acctgccctc gttccgacgg ccgccgcgcg atggagatct
tgaagaagct 1140tcaggttcag ggcaactgga ccaatgtgct ttacgacgac
caggtcggca aagtctccct 1200cgtgggtgct ggcatgaagt ctcacccagg
tgttaccgca gagttcatgg aagctctgcg 1260cgatgtcaac gtgaacatcg
aattgatttc cacctctgag attcgtattt ccgtgctgat 1320ccgtgaagat
gatctggatg ctgctgcacg tgcattgcat gagcagttcc agctgggcgg
1380cgaagacgaa gccgtcgttt atgcaggcac cggacgctaa agttttaaag
gagtagtttt 1440acaatgacca ccatcgcagt tgttggtgca accggccagg
tcggccaggt tatgcgcacc 1500cttttggaag agcgcaattt cccagctgac
actgttcgtt tctttgcttc cccacgttcc 1560gcaggccgta agattgaatt
cgtcgacatc gatgctcttc tgcgttaatt aacaattggg 1620atcctctaga
cccgggattt aaatcgctag cgggctgcta aaggaagcgg aacacgtaga
1680aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg
gctatctgga 1740caagggaaaa cgcaagcgca aagagaaagc aggtagcttg
cagtgggctt acatggcgat 1800agctagactg ggcggtttta tggacagcaa
gcgaaccgga attgccagct ggggcgccct 1860ctggtaaggt tgggaagccc
tgcaaagtaa actggatggc tttcttgccg ccaaggatct 1920gatggcgcag
gggatcaaga tctgatcaag agacaggatg aggatcgttt cgcatgattg
1980aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
ttcggctatg 2040actgggcaca acagacaatc ggctgctctg atgccgccgt
gttccggctg tcagcgcagg 2100ggcgcccggt tctttttgtc aagaccgacc
tgtccggtgc cctgaatgaa ctgcaggacg 2160aggcagcgcg gctatcgtgg
ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg 2220ttgtcactga
agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
2280tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
atgcggcggc 2340tgcatacgct tgatccggct acctgcccat tcgaccacca
agcgaaacat cgcatcgagc 2400gagcacgtac tcggatggaa gccggtcttg
tcgatcagga tgatctggac gaagagcatc 2460aggggctcgc gccagccgaa
ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg 2520atctcgtcgt
gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
2580tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
gacatagcgt 2640tggctacccg tgatattgct gaagagcttg gcggcgaatg
ggctgaccgc ttcctcgtgc 2700tttacggtat cgccgctccc gattcgcagc
gcatcgcctt ctatcgcctt cttgacgagt 2760tcttctgagc gggactctgg
ggttcgaaat gaccgaccaa gcgacgccca acctgccatc 2820acgagatttc
gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
2880ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
tcgcccacgc 2940tagcggcgcg ccggccggcc cggtgtgaaa taccgcacag
atgcgtaagg agaaaatacc 3000gcatcaggcg ctcttccgct tcctcgctca
ctgactcgct gcgctcggtc gttcggctgc 3060ggcgagcggt atcagctcac
tcaaaggcgg taatacggtt atccacagaa tcaggggata 3120acgcaggaaa
gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
3180cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
aatcgacgct 3240caagtcagag gtggcgaaac ccgacaggac tataaagata
ccaggcgttt ccccctggaa 3300gctccctcgt gcgctctcct gttccgaccc
tgccgcttac cggatacctg tccgcctttc 3360tcccttcggg aagcgtggcg
ctttctcata gctcacgctg taggtatctc agttcggtgt 3420aggtcgttcg
ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
3480ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
tcgccactgg 3540cagcagccac tggtaacagg attagcagag cgaggtatgt
aggcggtgct acagagttct 3600tgaagtggtg gcctaactac ggctacacta
gaaggacagt atttggtatc tgcgctctgc 3660tgaagccagt taccttcgga
aaaagagttg gtagctcttg atccggcaaa caaaccaccg 3720ctggtagcgg
tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
3780aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
aactcacgtt 3840aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
ctagatcctt ttaaaggccg 3900gccgcggccg ccatcggcat tttcttttgc
gtttttattt gttaactgtt aattgtcctt 3960gttcaaggat gctgtctttg
acaacagatg ttttcttgcc tttgatgttc agcaggaagc 4020tcggcgcaaa
cgttgattgt ttgtctgcgt agaatcctct gtttgtcata tagcttgtaa
4080tcacgacatt gtttcctttc gcttgaggta cagcgaagtg tgagtaagta
aaggttacat 4140cgttaggatc aagatccatt tttaacacaa ggccagtttt
gttcagcggc ttgtatgggc 4200cagttaaaga attagaaaca taaccaagca
tgtaaatatc gttagacgta atgccgtcaa 4260tcgtcatttt tgatccgcgg
gagtcagtga acaggtacca tttgccgttc attttaaaga 4320cgttcgcgcg
ttcaatttca tctgttactg tgttagatgc aatcagcggt ttcatcactt
4380ttttcagtgt gtaatcatcg tttagctcaa tcataccgag agcgccgttt
gctaactcag 4440ccgtgcgttt tttatcgctt tgcagaagtt tttgactttc
ttgacggaag aatgatgtgc 4500ttttgccata gtatgctttg ttaaataaag
attcttcgcc ttggtagcca tcttcagttc 4560cagtgtttgc ttcaaatact
aagtatttgt ggcctttatc ttctacgtag tgaggatctc 4620tcagcgtatg
gttgtcgcct gagctgtagt tgccttcatc gatgaactgc tgtacatttt
4680gatacgtttt tccgtcaccg tcaaagattg atttataatc ctctacaccg
ttgatgttca 4740aagagctgtc tgatgctgat acgttaactt gtgcagttgt
cagtgtttgt ttgccgtaat 4800gtttaccgga gaaatcagtg tagaataaac
ggatttttcc gtcagatgta aatgtggctg 4860aacctgacca ttcttgtgtt
tggtctttta ggatagaatc atttgcatcg aatttgtcgc 4920tgtctttaaa
gacgcggcca gcgtttttcc agctgtcaat agaagtttcg ccgacttttt
4980gatagaacat gtaaatcgat gtgtcatccg catttttagg atctccggct
aatgcaaaga 5040cgatgtggta gccgtgatag tttgcgacag tgccgtcagc
gttttgtaat ggccagctgt 5100cccaaacgtc caggcctttt gcagaagaga
tatttttaat tgtggacgaa tcaaattcag 5160aaacttgata tttttcattt
ttttgctgtt cagggatttg cagcatatca tggcgtgtaa 5220tatgggaaat
gccgtatgtt tccttatatg gcttttggtt cgtttctttc gcaaacgctt
5280gagttgcgcc tcctgccagc agtgcggtag taaaggttaa tactgttgct
tgttttgcaa 5340actttttgat gttcatcgtt catgtctcct tttttatgta
ctgtgttagc ggtctgcttc 5400ttccagccct cctgtttgaa gatggcaagt
tagttacgca caataaaaaa agacctaaaa 5460tatgtaaggg gtgacgccaa
agtatacact ttgcccttta cacattttag gtcttgcctg 5520ctttatcagt
aacaaacccg cgcgatttac ttttcgacct cattctatta gactctcgtt
5580tggattgcaa ctggtctatt ttcctctttt gtttgataga aaatcataaa
aggatttgca 5640gactacgggc ctaaagaact aaaaaatcta tctgtttctt
ttcattctct gtatttttta 5700tagtttctgt tgcatgggca taaagttgcc
tttttaatca caattcagaa aatatcataa 5760tatctcattt cactaaataa
tagtgaacgg caggtatatg tgatgggtta aaaaggatcg 5820gcggccgctc
gatttaaatc tcgagaggcc tgacgtcggg 58601120DNAArtificial
SequenceSynthetic construct 11tggccgttac cctgcgaatg
201220DNAArtificial SequenceSynthetic construct 12tgtatgtcct
cctggacttc 201340DNAArtificial SequenceSynthetic construct
13gaagtccagg aggacataca atgaacctaa agaaccccga 401431DNAArtificial
SequenceSynthetic construct 14atctacgtcg acccaggatg ccctggattt c
311531DNAArtificial SequenceSynthetic construct 15tatcaacgcg
ttcttcatcg gtagcagcac c 311637DNAArtificial SequenceSynthetic
construct 16cattcgcagg gtaacggcca ctgaagggcc tcctggg
37175928DNACorynebacterium glutamicum 17tcgagaggcc tgacgtcggg
cccggtacca cgcgttcttc atcggtagca gcacccgaga 60ccatgacgcg ggcatcgccc
agatccatca cacgcagatc acgcacatca gattcctgtg 120aggtgtaaat
tcccacgtcg tggccatcaa gatcataaga ctcagaaaga tcacgccagc
180gagtatcata accagccaca gcatcctcaa cggtttcacc agtttgagtg
agctgaatat 240agccctcatc tgcggtgaca tatccaacta cagatgccgg
ggtgtcatcc accatggtgc 300gtcgagctga atttgtggtc cagccttcag
gagtttccgg caacctagtt gcatgatcag 360tcattgcgcg cgcttccatt
gacataaaag tggaagcatc aacttcaggt acctgcccat 420tttcagggga
tcctgtattg aaagaacaca ttcccgtgaa tcccaccgct accaacatga
480tgatcgcgga gactaccaac gagataatca tgtctcgact gccatcaaaa
attttcggtc 540gtttctcagc cacccgccta gtatgtcacg agtttggtac
gaaaccccct tttgggtgtc 600cagaatccaa aattccgggc acaaaagtgc
aacaatagat gacgtgcggg ttgatacagc 660ccaagcgccg atacatttat
aatgcgccta gatacgtgca acccacgtaa ccaggtcaga 720tcaagtgccc
caggaggccc ttcagtggcc gttaccctgc gaatgtccac agggtagctg
780gtagtttgaa aatcaacgcc gttgccctta ggattcagta actggcacat
tttgtaatgc 840gctagatctg tgtgctcagt cttccaggct gcttatcaca
gtgaaagcaa aaccaattcg 900tggctgcgaa agtcgtagcc accacgaagt
ccaggaggac atacaatgaa cctaaagaac 960cccgaaacgc cagaccgtaa
ccttgctatg gagctggtgc gagttacgga agcagctgca 1020ctggcttctg
gacgttgggt tggacgtggc atgaagaatg aaggcgacgg tgccgctgtt
1080gacgccatgc gccagctcat caactcagtg accatgaagg gcgtcgttgt
tatcggcgag 1140ggcgaaaaag acgaagctcc aatgctgtac aacggcgaag
aggtcggaac cggctttgga 1200cctgaggttg atatcgcagt tgacccagtt
gacggcacca ccctgatggc tgagggtcgc 1260cccaacgcaa tttccattct
cgcagctgca gagcgtggca ccatgtacga tccatcctcc 1320gtcttctaca
tgaagaagat cgccgtggga cctgaggccg caggcaagat cgacatcgaa
1380gctccagttg cccacaacat caacgcggtg gcaaagtcca agggaatcaa
cccttccgac 1440gtcaccgttg tcgtgcttga ccgtcctcgc cacatcgaac
tgatcgcaga cattcgtcgt 1500gcaggcgcaa aggttcgtct catctccgac
ggcgacgttg caggtgcagt tgcagcagct 1560caggattcca actccgtgga
catcatgatg ggcaccggcg gaaccccaga aggcatcatc 1620actgcgtgcg
ccatgaagtg catgggtggc gaaatccagg gcatcctggg tcgacatcga
1680tgctcttctg cgttaattaa caattgggat cctctagacc cgggatttaa
atcgctagcg 1740ggctgctaaa ggaagcggaa cacgtagaaa gccagtccgc
agaaacggtg ctgaccccgg 1800atgaatgtca gctactgggc tatctggaca
agggaaaacg caagcgcaaa gagaaagcag 1860gtagcttgca gtgggcttac
atggcgatag ctagactggg cggttttatg gacagcaagc 1920gaaccggaat
tgccagctgg ggcgccctct ggtaaggttg ggaagccctg caaagtaaac
1980tggatggctt tcttgccgcc aaggatctga tggcgcaggg gatcaagatc
tgatcaagag 2040acaggatgag gatcgtttcg catgattgaa caagatggat
tgcacgcagg ttctccggcc 2100gcttgggtgg agaggctatt cggctatgac
tgggcacaac agacaatcgg ctgctctgat 2160gccgccgtgt tccggctgtc
agcgcagggg cgcccggttc tttttgtcaa gaccgacctg 2220tccggtgccc
tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
2280ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
ctggctgcta 2340ttgggcgaag tgccggggca ggatctcctg tcatctcacc
ttgctcctgc cgagaaagta 2400tccatcatgg ctgatgcaat gcggcggctg
catacgcttg atccggctac ctgcccattc 2460gaccaccaag cgaaacatcg
catcgagcga gcacgtactc ggatggaagc cggtcttgtc 2520gatcaggatg
atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
2580ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
tgcctgcttg 2640ccgaatatca tggtggaaaa tggccgcttt tctggattca
tcgactgtgg ccggctgggt 2700gtggcggacc gctatcagga catagcgttg
gctacccgtg atattgctga agagcttggc 2760ggcgaatggg ctgaccgctt
cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc 2820atcgccttct
atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
2880ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
gccttctatg 2940aaaggttggg cttcggaatc gttttccggg acgccggctg
gatgatcctc cagcgcgggg 3000atctcatgct ggagttcttc gcccacgcta
gcggcgcgcc ggccggcccg gtgtgaaata 3060ccgcacagat gcgtaaggag
aaaataccgc atcaggcgct cttccgcttc ctcgctcact 3120gactcgctgc
gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
3180atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
aaaaggccag 3240caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
ttttccatag gctccgcccc 3300cctgacgagc atcacaaaaa tcgacgctca
agtcagaggt ggcgaaaccc gacaggacta 3360taaagatacc aggcgtttcc
ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 3420ccgcttaccg
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
3480tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
ctgtgtgcac 3540gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
actatcgtct tgagtccaac 3600ccggtaagac acgacttatc gccactggca
gcagccactg gtaacaggat tagcagagcg 3660aggtatgtag gcggtgctac
agagttcttg aagtggtggc ctaactacgg ctacactaga 3720aggacagtat
ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
3780agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
ttgcaagcag 3840cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
tgatcttttc tacggggtct 3900gacgctcagt ggaacgaaaa ctcacgttaa
gggattttgg tcatgagatt atcaaaaagg 3960atcttcacct agatcctttt
aaaggccggc cgcggccgcc atcggcattt tcttttgcgt 4020ttttatttgt
taactgttaa ttgtccttgt tcaaggatgc tgtctttgac aacagatgtt
4080ttcttgcctt tgatgttcag caggaagctc ggcgcaaacg ttgattgttt
gtctgcgtag 4140aatcctctgt ttgtcatata gcttgtaatc acgacattgt
ttcctttcgc ttgaggtaca 4200gcgaagtgtg agtaagtaaa ggttacatcg
ttaggatcaa gatccatttt taacacaagg 4260ccagttttgt tcagcggctt
gtatgggcca gttaaagaat tagaaacata accaagcatg 4320taaatatcgt
tagacgtaat gccgtcaatc gtcatttttg atccgcggga gtcagtgaac
4380aggtaccatt tgccgttcat tttaaagacg ttcgcgcgtt caatttcatc
tgttactgtg 4440ttagatgcaa tcagcggttt catcactttt ttcagtgtgt
aatcatcgtt tagctcaatc 4500ataccgagag cgccgtttgc taactcagcc
gtgcgttttt tatcgctttg cagaagtttt 4560tgactttctt gacggaagaa
tgatgtgctt ttgccatagt atgctttgtt aaataaagat 4620tcttcgcctt
ggtagccatc ttcagttcca gtgtttgctt caaatactaa gtatttgtgg
4680cctttatctt ctacgtagtg aggatctctc agcgtatggt tgtcgcctga
gctgtagttg 4740ccttcatcga tgaactgctg tacattttga tacgtttttc
cgtcaccgtc aaagattgat 4800ttataatcct ctacaccgtt gatgttcaaa
gagctgtctg atgctgatac gttaacttgt 4860gcagttgtca gtgtttgttt
gccgtaatgt ttaccggaga aatcagtgta gaataaacgg 4920atttttccgt
cagatgtaaa tgtggctgaa cctgaccatt cttgtgtttg gtcttttagg
4980atagaatcat ttgcatcgaa tttgtcgctg tctttaaaga cgcggccagc
gtttttccag 5040ctgtcaatag aagtttcgcc gactttttga tagaacatgt
aaatcgatgt gtcatccgca 5100tttttaggat ctccggctaa tgcaaagacg
atgtggtagc cgtgatagtt tgcgacagtg 5160ccgtcagcgt tttgtaatgg
ccagctgtcc caaacgtcca ggccttttgc agaagagata 5220tttttaattg
tggacgaatc aaattcagaa acttgatatt tttcattttt ttgctgttca
5280gggatttgca gcatatcatg gcgtgtaata tgggaaatgc cgtatgtttc
cttatatggc 5340ttttggttcg tttctttcgc aaacgcttga gttgcgcctc
ctgccagcag tgcggtagta 5400aaggttaata ctgttgcttg ttttgcaaac
tttttgatgt tcatcgttca tgtctccttt 5460tttatgtact gtgttagcgg
tctgcttctt ccagccctcc tgtttgaaga tggcaagtta 5520gttacgcaca
ataaaaaaag acctaaaata tgtaaggggt gacgccaaag tatacacttt
5580gccctttaca cattttaggt cttgcctgct ttatcagtaa caaacccgcg
cgatttactt 5640ttcgacctca ttctattaga ctctcgtttg gattgcaact
ggtctatttt cctcttttgt 5700ttgatagaaa atcataaaag gatttgcaga
ctacgggcct aaagaactaa aaaatctatc 5760tgtttctttt cattctctgt
attttttata gtttctgttg catgggcata aagttgcctt 5820tttaatcaca
attcagaaaa tatcataata tctcatttca ctaaataata gtgaacggca
5880ggtatatgtg atgggttaaa aaggatcggc
ggccgctcga tttaaatc 59281820DNAArtificial Sequencesynthetic
construct 18tagctgccaa ttattccggg 201920DNAArtificial
Sequencesynthetic construct 19gggtaaaaaa tcctttcgta
202037DNAArtificial Sequencesynthetic construct 20cccggaataa
ttggcagcta ctgaagggcc tcctggg 372131DNAArtificial Sequencesynthetic
construct 21tatcaacgcg ttcttcatcg gtagcagcac c 312240DNAArtificial
Sequencesynthetic construct 22tacgaaagga ttttttaccc atgaacctaa
agaaccccga 402331DNAArtificial Sequencesynthetic construct
23atctacgtcg acccaggatg ccctggattt c 31245920DNAArtificial
Sequencesynthetic construct 24cgcgttcttc atcggtagca gcacccgaga
ccatgacgcg ggcatcgccc agatccatca 60cacgcagatc acgcacatca gattcctgtg
aggtgtaaat tcccacgtcg tggccatcaa 120gatcataaga ctcagaaaga
tcacgccagc gagtatcata accagccaca gcatcctcaa 180cggtttcacc
agtttgagtg agctgaatat agccctcatc tgcggtgaca tatccaacta
240cagatgccgg ggtgtcatcc accatggtgc gtcgagctga atttgtggtc
cagccttcag 300gagtttccgg caacctagtt gcatgatcag tcattgcgcg
cgcttccatt gacataaaag 360tggaagcatc aacttcaggt acctgcccat
tttcagggga tcctgtattg aaagaacaca 420ttcccgtgaa tcccaccgct
accaacatga tgatcgcgga gactaccaac gagataatca 480tgtctcgact
gccatcaaaa attttcggtc gtttctcagc cacccgccta gtatgtcacg
540agtttggtac gaaaccccct tttgggtgtc cagaatccaa aattccgggc
acaaaagtgc 600aacaatagat gacgtgcggg ttgatacagc ccaagcgccg
atacatttat aatgcgccta 660gatacgtgca acccacgtaa ccaggtcaga
tcaagtgccc caggaggccc ttcagtagct 720gccaattatt ccgggcttgt
gacccgctac ccgataaata ggtcggctga aaaatttcgt 780tgcaatatca
acaaaaaggc ctatcattgg gaggtgtcgc accaagtact tttgcgaagc
840gccatctgac ggattttcaa aagatgtata tgctcggtgc ggaaacctac
gaaaggattt 900tttacccatg aacctaaaga accccgaaac gccagaccgt
aaccttgcta tggagctggt 960gcgagttacg gaagcagctg cactggcttc
tggacgttgg gttggacgtg gcatgaagaa 1020tgaaggcgac ggtgccgctg
ttgacgccat gcgccagctc atcaactcag tgaccatgaa 1080gggcgtcgtt
gttatcggcg agggcgaaaa agacgaagct ccaatgctgt acaacggcga
1140agaggtcgga accggctttg gacctgaggt tgatatcgca gttgacccag
ttgacggcac 1200caccctgatg gctgagggtc gccccaacgc aatttccatt
ctcgcagctg cagagcgtgg 1260caccatgtac gatccatcct ccgtcttcta
catgaagaag atcgccgtgg gacctgaggc 1320cgcaggcaag atcgacatcg
aagctccagt tgcccacaac atcaacgcgg tggcaaagtc 1380caagggaatc
aacccttccg acgtcaccgt tgtcgtgctt gaccgtcctc gccacatcga
1440actgatcgca gacattcgtc gtgcaggcgc aaaggttcgt ctcatctccg
acggcgacgt 1500tgcaggtgca gttgcagcag ctcaggattc caactccgtg
gacatcatga tgggcaccgg 1560cggaacccca gaaggcatca tcactgcgtg
cgccatgaag tgcatgggtg gcgaaatcca 1620gggcatcctg ggtcgacatc
gatgctcttc tgcgttaatt aacaattggg atcctctaga 1680cccgggattt
aaatcgctag cgggctgcta aaggaagcgg aacacgtaga aagccagtcc
1740gcagaaacgg tgctgacccc ggatgaatgt cagctactgg gctatctgga
caagggaaaa 1800cgcaagcgca aagagaaagc aggtagcttg cagtgggctt
acatggcgat agctagactg 1860ggcggtttta tggacagcaa gcgaaccgga
attgccagct ggggcgccct ctggtaaggt 1920tgggaagccc tgcaaagtaa
actggatggc tttcttgccg ccaaggatct gatggcgcag 1980gggatcaaga
tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
2040attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
actgggcaca 2100acagacaatc ggctgctctg atgccgccgt gttccggctg
tcagcgcagg ggcgcccggt 2160tctttttgtc aagaccgacc tgtccggtgc
cctgaatgaa ctgcaggacg aggcagcgcg 2220gctatcgtgg ctggccacga
cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga 2280agcgggaagg
gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
2340ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
tgcatacgct 2400tgatccggct acctgcccat tcgaccacca agcgaaacat
cgcatcgagc gagcacgtac 2460tcggatggaa gccggtcttg tcgatcagga
tgatctggac gaagagcatc aggggctcgc 2520gccagccgaa ctgttcgcca
ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt 2580gacccatggc
gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
2640catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
tggctacccg 2700tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
ttcctcgtgc tttacggtat 2760cgccgctccc gattcgcagc gcatcgcctt
ctatcgcctt cttgacgagt tcttctgagc 2820gggactctgg ggttcgaaat
gaccgaccaa gcgacgccca acctgccatc acgagatttc 2880gattccaccg
ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
2940tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccacgc
tagcggcgcg 3000ccggccggcc cggtgtgaaa taccgcacag atgcgtaagg
agaaaatacc gcatcaggcg 3060ctcttccgct tcctcgctca ctgactcgct
gcgctcggtc gttcggctgc ggcgagcggt 3120atcagctcac tcaaaggcgg
taatacggtt atccacagaa tcaggggata acgcaggaaa 3180gaacatgtga
gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
3240gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
caagtcagag 3300gtggcgaaac ccgacaggac tataaagata ccaggcgttt
ccccctggaa gctccctcgt 3360gcgctctcct gttccgaccc tgccgcttac
cggatacctg tccgcctttc tcccttcggg 3420aagcgtggcg ctttctcata
gctcacgctg taggtatctc agttcggtgt aggtcgttcg 3480ctccaagctg
ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
3540taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
cagcagccac 3600tggtaacagg attagcagag cgaggtatgt aggcggtgct
acagagttct tgaagtggtg 3660gcctaactac ggctacacta gaaggacagt
atttggtatc tgcgctctgc tgaagccagt 3720taccttcgga aaaagagttg
gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 3780tggttttttt
gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
3840tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
aagggatttt 3900ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
ttaaaggccg gccgcggccg 3960ccatcggcat tttcttttgc gtttttattt
gttaactgtt aattgtcctt gttcaaggat 4020gctgtctttg acaacagatg
ttttcttgcc tttgatgttc agcaggaagc tcggcgcaaa 4080cgttgattgt
ttgtctgcgt agaatcctct gtttgtcata tagcttgtaa tcacgacatt
4140gtttcctttc gcttgaggta cagcgaagtg tgagtaagta aaggttacat
cgttaggatc 4200aagatccatt tttaacacaa ggccagtttt gttcagcggc
ttgtatgggc cagttaaaga 4260attagaaaca taaccaagca tgtaaatatc
gttagacgta atgccgtcaa tcgtcatttt 4320tgatccgcgg gagtcagtga
acaggtacca tttgccgttc attttaaaga cgttcgcgcg 4380ttcaatttca
tctgttactg tgttagatgc aatcagcggt ttcatcactt ttttcagtgt
4440gtaatcatcg tttagctcaa tcataccgag agcgccgttt gctaactcag
ccgtgcgttt 4500tttatcgctt tgcagaagtt tttgactttc ttgacggaag
aatgatgtgc ttttgccata 4560gtatgctttg ttaaataaag attcttcgcc
ttggtagcca tcttcagttc cagtgtttgc 4620ttcaaatact aagtatttgt
ggcctttatc ttctacgtag tgaggatctc tcagcgtatg 4680gttgtcgcct
gagctgtagt tgccttcatc gatgaactgc tgtacatttt gatacgtttt
4740tccgtcaccg tcaaagattg atttataatc ctctacaccg ttgatgttca
aagagctgtc 4800tgatgctgat acgttaactt gtgcagttgt cagtgtttgt
ttgccgtaat gtttaccgga 4860gaaatcagtg tagaataaac ggatttttcc
gtcagatgta aatgtggctg aacctgacca 4920ttcttgtgtt tggtctttta
ggatagaatc atttgcatcg aatttgtcgc tgtctttaaa 4980gacgcggcca
gcgtttttcc agctgtcaat agaagtttcg ccgacttttt gatagaacat
5040gtaaatcgat gtgtcatccg catttttagg atctccggct aatgcaaaga
cgatgtggta 5100gccgtgatag tttgcgacag tgccgtcagc gttttgtaat
ggccagctgt cccaaacgtc 5160caggcctttt gcagaagaga tatttttaat
tgtggacgaa tcaaattcag aaacttgata 5220tttttcattt ttttgctgtt
cagggatttg cagcatatca tggcgtgtaa tatgggaaat 5280gccgtatgtt
tccttatatg gcttttggtt cgtttctttc gcaaacgctt gagttgcgcc
5340tcctgccagc agtgcggtag taaaggttaa tactgttgct tgttttgcaa
actttttgat 5400gttcatcgtt catgtctcct tttttatgta ctgtgttagc
ggtctgcttc ttccagccct 5460cctgtttgaa gatggcaagt tagttacgca
caataaaaaa agacctaaaa tatgtaaggg 5520gtgacgccaa agtatacact
ttgcccttta cacattttag gtcttgcctg ctttatcagt 5580aacaaacccg
cgcgatttac ttttcgacct cattctatta gactctcgtt tggattgcaa
5640ctggtctatt ttcctctttt gtttgataga aaatcataaa aggatttgca
gactacgggc 5700ctaaagaact aaaaaatcta tctgtttctt ttcattctct
gtatttttta tagtttctgt 5760tgcatgggca taaagttgcc tttttaatca
caattcagaa aatatcataa tatctcattt 5820cactaaataa tagtgaacgg
caggtatatg tgatgggtta aaaaggatcg gcggccgctc 5880gatttaaatc
tcgagaggcc tgacgtcggg cccggtacca 5920
* * * * *
References