U.S. patent application number 11/767394 was filed with the patent office on 2009-01-15 for process and materials for production of glucosamine.
This patent application is currently assigned to Arkion Life Sciences LLC d/b/a Bio-Technical Resources, Arkion Life Sciences LLC d/b/a Bio-Technical Resources. Invention is credited to Alan Berry, Richard P. Burlingame, James R. Millis.
Application Number | 20090017520 11/767394 |
Document ID | / |
Family ID | 22361646 |
Filed Date | 2009-01-15 |
United States Patent
Application |
20090017520 |
Kind Code |
A1 |
Berry; Alan ; et
al. |
January 15, 2009 |
Process and Materials for Production of Glucosamine
Abstract
The present invention relates to a method and materials for
producing glucosamine by fermentation of a genetically modified
microorganism. Included in the present invention are genetically
modified microorganisms useful in the present method for producing
glucosamine, as well as recombinant nucleic acid molecules and the
proteins produces by such recombinant nucleic acid molecules.
Inventors: |
Berry; Alan; (Manitowoc,
WI) ; Burlingame; Richard P.; (Manitowoc, WI)
; Millis; James R.; (Kohler, WI) |
Correspondence
Address: |
SHERIDAN ROSS PC
1560 BROADWAY, SUITE 1200
DENVER
CO
80202
US
|
Assignee: |
Arkion Life Sciences LLC d/b/a
Bio-Technical Resources
Manitowoc
WI
|
Family ID: |
22361646 |
Appl. No.: |
11/767394 |
Filed: |
June 22, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11245473 |
Oct 5, 2005 |
|
|
|
11767394 |
|
|
|
|
10024460 |
Dec 17, 2001 |
|
|
|
11245473 |
|
|
|
|
09115475 |
Jul 15, 1998 |
6372457 |
|
|
10024460 |
|
|
|
|
PCT/US98/00800 |
Jan 14, 1998 |
|
|
|
09115475 |
|
|
|
|
60035494 |
Jan 14, 1997 |
|
|
|
Current U.S.
Class: |
435/183 ;
536/23.2 |
Current CPC
Class: |
C12P 19/26 20130101;
C12N 15/70 20130101; C12N 9/1096 20130101; C07H 11/04 20130101;
C12Y 206/01016 20130101; C07H 5/06 20130101 |
Class at
Publication: |
435/183 ;
536/23.2 |
International
Class: |
C12N 9/00 20060101
C12N009/00; C07H 21/04 20060101 C07H021/04 |
Claims
1-55. (canceled)
56. A glucosamine-6-phosphate synthase which has
glucosamine-6-phosphate synthase action, said synthase being
encoded by a nucleic acid sequence having a genetic modification
that results in increased glucosamine-6-phosphate synthase
action.
57. The glucosamine-6-phosphate synthase of claim 56, wherein said
synthase comprises at least one amino acid modification selected
from the group consisting of deletion, insertion, inversion,
substitution and derivatization of at least one amino acid
residue.
58. The glucosamine-6-phosphate synthase of claim 56, wherein said
synthase is encoded by a nucleic acid sequence selected from the
group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ
ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27,
SEQ ID NO:29 and SEQ ID NO:30.
59. The glucosamine-6-phosphate synthase of claim 56, wherein said
synthase comprises an amino acid sequence selected from the group
consisting of SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID
NO:28 and SEQ ID NO:31.
60. (canceled)
61. The glucosamine-6-phosphate synthase of claim 56, wherein the
genetic modification reduces glucosamine-6-phosphate product
inhibition of the glucosamine-6-phosphate synthase.
62. The glucosamine-6-phosphate synthase of claim 56, wherein the
genetic modification also results in increased in vivo
glucosamine-6-phosphate synthase action.
63. The nucleic acid sequence of claim 56, wherein the nucleic acid
sequence comprises a recombinant nucleic acid molecule.
64. The recombinant nucleic acid molecule of claim 63, wherein the
recombinant nucleic acid molecule is selected from the group
consisting of pKLN23-49, pKLN23-54, pKLN23-124, pKLN23-149,
pKLN23-151, nglmS-49.sub.2184, nglmS-49.sub.1830,
nglmS-54.sub.2184, nglmS-54.sub.1830, nglmS-124.sub.2184,
nglmS-124.sub.1830, nglmS-149.sub.2184, nglmS-149.sub.1830,
nglmS-151.sub.2184 and nglmS-151.sub.1830.
65. The recombinant nucleic acid molecule of claim 63, wherein the
recombinant nucleic acid molecule is operatively linked to a
transcription control sequence.
66. The glucosamine-6-phosphate synthase of claim 56, wherein the
genetic modification increases glucosamine-6-phosphate synthase
action as compared to a recombinant nucleic acid molecule encoding
a naturally occurring glucosamine-6-phosphate synthase that does
not have the genetic modification.
67. The glucosamine-6-phosphate synthase of claim 57, wherein the
at least one amino acid modification is at an amino acid sequence
position, corresponding to amino acid sequence SEQ ID NO:16,
selected from the group consisting of Ile(4), Ile(272), Ser(450),
Ala(39), Arg(250), Gly(472), Leu(469), and combinations
thereof.
68. The glucosamine-6-phosphate synthase of claim 57, wherein the
at least one amino acid modification is at an amino acid sequence
position, corresponding to amino acid sequence SEQ ID NO:16,
selected from the group consisting of: (a) an amino acid residue
having an aliphatic hydroxyl side group for Ile(4); (b) an amino
acid residue having an aliphatic hydroxyl side group for Ile(272);
(c) an amino acid residue having an aliphatic side group for
Ser(450); (d) an amino acid residue having an aliphatic hydroxyl
side group for Ala(39); (e) an amino acid residue having a
sulfur-containing side group for Arg(250); (f) an amino acid
residue having an aliphatic hydroxyl side group for Gly(472); (g)
an amino acid residue having an aliphatic side group for Leu(469);
(h) and combinations of (a)-(g).
69. The glucosamine-6-phosphate synthase of claim 57, wherein the
at least one amino acid modification is at an amino acid sequence
position, corresponding to amino acid sequence SEQ ID NO:16,
selected from the group consisting of: Ile(4) to Thr, Ile(272) to
Thr, Ser(450) to Pro, Ala(39) to Thr, Arg(250) to Cys, Gly(472) to
Ser, Leu(469) to Pro, and combinations thereof.
70. The glucosamine-6-phosphate synthase of claim 57, wherein the
at least one amino acid modification is a substitution at an amino
acid sequence position, corresponding to amino acid sequence SEQ ID
NO:16, of a proline residue for a leucine residue at amino acid
sequence position Leu(469).
71. The glucosamine-6-phosphate synthase of claim 57, wherein the
at least one amino acid modification is at an amino acid sequence
position, corresponding to amino acid sequence SEQ ID NO:16,
selected from the group consisting of: (a) a threonine residue for
an alanine residue at position Ala(39); (b) a cysteine residue for
an arginine residue at position Arg(250); (c) a serine residue for
a glycine residue at position Gly(472); and (d) any combination of
(a), (b), or (c).
72. The glucosamine-6-phosphate synthase of claim 57, wherein the
at least one amino acid modification is a substitution of an amino
acid residue selected from the group consisting of: (a) a threonine
residue for an isoleucine residue at position Ile(4); (b) a
threonine residue for an isoleucine residue at position Ile(272);
(c) a proline residue for a serine residue at position Ser(450);
and (d) any combination of (a), (b), or (c).
73. The glucosamine-6-phosphate synthase of claim 56, wherein the
increased glucosamine-6-phosphate synthase action results from the
glucosamine-6-phosphate synthase having improved affinity for its
substrates.
74. The recombinant nucleic acid molecule of claim 63, wherein the
recombinant nucleic acid molecule is selected from the group
consisting of plasmids pKLN23-49, pKLN23-54, pKLN23-124, pKLN23-149
and pKLN23-151.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part application of
PCT Application No. PCT/US98/00800, filed Jan. 14, 1998, which
designates the United States. PCT/US98/00800 claims priority under
35 U.S.C. .sctn. 119(e) from U.S. Provisional Application Ser. No.
60/035,494, filed Jan. 14, 1997. Both PCT Application No.
PCT/US98/00800 and U.S. Provisional Application Ser. No. 60/035,494
are incorporated herein by reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to a method for producing
glucosamine by fermentation. The present invention also relates to
genetically modified strains of microorganisms useful for producing
glucosamine.
BACKGROUND OF THE INVENTION
[0003] Amino sugars are usually found as monomer residues in
complex oligosaccharides and polysaccharides. Glucosamine is an
amino derivative of the simple sugar, glucose. Glucosamine and
other amino sugars are important constituents of many natural
polysaccharides. For example, polysaccharides containing amino
sugars can form structural materials for cells, analogous to
structural proteins.
[0004] Glucosamine is manufactured as a nutraceutical product with
applications in the treatment of osteoarthritic conditions in
animals and humans. The market for glucosamine is experiencing
tremendous growth. Furthermore, significant erosion of the world
market price for glucosamine is not expected.
[0005] Glucosamine is currently obtained by acid hydrolysis of
chitin, a complex carbohydrate derived from N-acetyl-D-glucosamine.
Alternatively, glucosamine can also be produced by acid hydrolysis
of variously acetylated chitosans. These processes suffer from poor
product yields (in the range of 50% conversion of substrate to
glucosamine). Moreover, the availability of raw material (i.e., a
source of chitin, such as crab shells) is becoming increasingly
limited. Therefore, there is a need in the industry for a
cost-effective method for producing high yields of glucosamine for
commercial sale and use.
SUMMARY OF THE INVENTION
[0006] One embodiment of the present invention relates to a method
to produce glucosamine by fermentation of a microorganism. This
method includes the steps of: (a) culturing in a fermentation
medium a microorganism having a genetic modification in an amino
sugar metabolic pathway; and (b) recovering a product produced from
the step of culturing which is selected from the group of
glucosamine-6-phosphate and glucosamine. Such an amino sugar
metabolic pathway is selected from the group of a pathway for
converting glucosamine-6-phosphate into another compound, a pathway
for synthesizing glucosamine-6-phosphate, a pathway for transport
of glucosamine or glucosamine-6-phosphate out of the microorganism,
a pathway for transport of glucosamine into the microorganism, and
a pathway which competes for substrates involved in the production
of glucosamine-6-phosphate. The fermentation medium includes
assimilable sources of carbon, nitrogen and phosphate. In a
preferred embodiment, the microorganism is a bacterium or a yeast,
and more preferably, a bacterium of the genus Escherichia, and even
more preferably, Escherichia coli.
[0007] In one embodiment, the product can be recovered by
recovering intracellular glucosamine-6-phosphate from the
microorganism and/or recovering extracellular glucosamine from the
fermentation medium. In further embodiments, the step of recovering
can include purifying glucosamine from the fermentation medium,
isolating glucosamine-6-phosphate from the microorganism, and/or
dephosphorylating the glucosamine-6-phosphate to produce
glucosamine. In one embodiment, at least about 1 g/L of product is
recovered.
[0008] In yet another embodiment, the step of culturing includes
the step of maintaining the carbon source at a concentration of
from about 0.5% to about 5% in the fermentation medium. In another
embodiment, the step of culturing is performed at a temperature of
from about 28.degree. C. to about 37.degree. C. In yet another
embodiment, the step of culturing is performed in a fermentor.
[0009] In one embodiment of the present invention, the
microorganism has a modification in a gene which encodes a protein
including, but not limited to, N-acetylglucosamine-6-phosphate
deacetylase, glucosamine-6-phosphate deaminase,
N-acetyl-glucosamine-specific enzyme II.sup.Nag,
glucosamine-6-phosphate synthase, phosphoglucosamine mutase,
glucosamine-1-phosphate
acetyltransferase-N-acetylglucosamine-1-phosphate
uridyltransferase, phosphofructokinase, enzyme II.sup.Glc of the
PEP:glucose PTS, EIIM, P/III.sup.Man of the PEP:mannose PTS, and/or
a phosphatase.
[0010] In another embodiment, the genetic modification includes a
genetic modification which increases the action of
glucosamine-6-phosphate synthase in the microorganism. Such a
genetic modification includes the transformation of the
microorganism with a recombinant nucleic acid molecule encoding
glucosamine-6-phosphate synthase to increase the action of
glucosamine-6-phosphate synthase and/or to overexpress the
glucosamine-6-phosphate synthase by the microorganism. In one
embodiment, the recombinant nucleic acid molecule is operatively
linked to a transcription control sequence. In a further
embodiment, the recombinant nucleic acid molecule is integrated
into the genome of the microorganism. In yet another embodiment,
the recombinant nucleic acid molecule encoding
glucosamine-6-phosphate synthase has a genetic modification which
increases the action of the synthase. Such genetic modifications
can result in reduced glucosamine-6-phosphate product inhibition of
the glucosamine-6-phosphate synthase, for example.
[0011] In one embodiment, a recombinant nucleic acid molecule of
the present invention which comprises a nucleic acid sequence
encoding a glucosamine-6-phosphate synthase encodes an amino acid
sequence SEQ ID NO:16. In another embodiment, such a recombinant
nucleic acid molecule comprises a nucleic acid sequence selected
from the group of SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15.
Preferred recombinant nucleic acid molecules of the present
invention include pKLN23-28, nglmS-28.sub.2184 and
nglmS-28.sub.1830.
[0012] Also included in the present invention are recombinant
nucleic acid molecules encoding a glucosamine-6-phosphate synthase
which comprises a genetic modification which increases the action
of the glucosamine-6-phosphate synthase (i.e., a
glucosamine-6-phosphate synthase homologue). Such a genetic
modification can reduce glucosamine-6-phosphate product inhibition
of the synthase, for example. In one embodiment, such a genetic
modification in a recombinant nucleic acid molecule of the present
invention which encodes a glucosamine-6-phosphate synthase results
in at least one amino acid modification selected from the group of
an addition, substitution, deletion, and/or derivatization of an
amino acid residue of the glucosamine-6-phosphate synthase. In one
embodiment, such an amino acid modification is in an amino acid
sequence position in the modified protein (i.e., homologue) which
corresponds to one or more of the following amino acid positions in
amino acid sequence SEQ ID NO:16 Ile(4), Ile(272), Ser(450),
Ala(39), Arg(250), Gly(472), Leu(469). In another embodiment, such
an amino acid modification is selected from the group of a
substitution of: (a) an amino acid residue having an aliphatic
hydroxyl side group for Ile(4); (b) an amino acid residue having an
aliphatic hydroxyl side group for Ile(272); (c) an amino acid
residue having an aliphatic side group for Ser(450); (d) an amino
acid residue having an aliphatic hydroxyl side group for Ala(39);
(e) an amino acid residue having a sulfur-containing side group for
Arg(250); (f) an amino acid residue having an aliphatic hydroxyl
side group for Gly(472); (g) an amino acid residue having an
aliphatic side group for Leu(469); and, (h) combinations of
(a)-(g).
[0013] In yet another embodiment of the present invention, an amino
acid modification as described above is preferably a substitution
selected from the group of: Ile(4) to Thr, Ile(272) to Thr,
Ser(450) to Pro, Ala(39) to Thr, Arg(250) to Cys, Gly(472) to Ser,
Leu(469) to Pro, and combinations thereof.
[0014] In another embodiment, a genetically modified recombinant
nucleic acid molecule of the present invention comprises a nucleic
acid sequence encoding glucosamine-6-phosphate synthase comprising
an amino acid sequence selected from the group of SEQ ID NO:19, SEQ
ID NO:22, SEQ ID NO:25, SEQ ID NO:28 or SEQ ID NO:31. In another
embodiment, a genetically modified recombinant nucleic acid
molecule of the present invention comprises a nucleic acid sequence
selected from the group of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:
20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID
NO:27, SEQ ID NO:29 and SEQ ID NO:30. Preferred genetically
modified recombinant nucleic acid molecule of the present invention
include pKLN23-49, pKLN23-54, pKLN23-124, pKLN23-149, pKLN23-151,
nglmS-49.sub.2184, nglmS-49.sub.1830, nglmS-54.sub.2184,
nglmS-54.sub.1830, nglmS-124.sub.2184, nglmS-124.sub.1830,
nglmS-149.sub.2184, nglmS-149.sub.1830, nglmS-151.sub.2184 and
nglmS-151.sub.1830.
[0015] Another embodiment of the present invention relates to a
glucosamine-6-phosphate synthase which has glucosamine-6-phosphate
synthase action, such synthase being encoded by a nucleic acid
sequence having a genetic modification that results in increased
glucosamine-6-phosphate synthase action. Such a nucleic acid
sequence has been describe above with regard to recombinant nucleic
acid molecules of the present invention.
[0016] Yet another embodiment of the present invention relates to a
method to produce glucosamine by fermentation, such method
comprising: (a) culturing in a fermentation medium comprising
assimilable sources of carbon, nitrogen and phosphate, a
genetically modified microorganism having increased
glucosamine-6-phosphate synthase action, wherein the genetically
modified microorganism is produced by a process comprising the
steps of: 1) generating modifications in an isolated nucleic acid
molecule comprising a nucleic acid sequence encoding
glucosamine-6-phosphate synthase to create a plurality of modified
nucleic acid sequences; (2) transforming microorganisms with the
modified nucleic acid sequences to produce genetically modified
microorganisms; (3) screening the genetically modified
microorganisms for glucosamine-6-phosphate synthase action; and,
(4) selecting the genetically modified microorganisms which have
increased glucosamine-6-phosphate synthase action; and, (b)
recovering the product. The step of culturing produces a product
selected from the group of glucosamine-6-phosphate and glucosamine
from the microorganism.
[0017] In another embodiment, a microorganism of the present
invention has an additional genetic modification in genes encoding
N-acetylglucosamine-6-phosphate deacetylase,
glucosamine-6-phosphate deaminase, N-acetyl-glucosamine-specific
enzyme II.sup.Nag, phosphoglucosamine mutase,
glucosamine-1-phosphate
acetyltransferase-N-acetylglucosamine-1-phosphate
uridyltransferase, phosphofructokinase, Enzyme II.sup.Glc of the
PEP:glucose PTS, EIIM, P/III.sup.Man of the PEP:mannose PTS,
wherein the modification decreases the action of such proteins. In
another embodiment, a microorganism of the present invention has an
additional genetic modification in a gene encoding a phosphatase,
wherein the modification increases the action of the phosphatase.
In a preferred embodiment, a microorganism of the present invention
has an additional genetic modification in the genes encoding the
following proteins: N-acetylglucosamine-6-phosphate deacetylase,
glucosamine-6-phosphate deaminase and N-acetyl-glucosamine-specific
enzyme II.sup.Nag, such modifications including, but not limited
to, a deletion of at least a portion of such genes.
[0018] Another embodiment of the present invention relates to a
method to produce glucosamine by fermentation which includes the
steps of (a) culturing an Escherichia coli transformed with a
recombinant nucleic acid molecule encoding glucosamine-6-phosphate
synthase in a fermentation medium comprising assimilable sources of
carbon, nitrogen and phosphate to produce a product, and (b)
recovering the product. The product includes intracellular
glucosamine-6-phosphate which is recovered from the Escherichia
coli and/or extracellular glucosamine which is recovered from the
fermentation medium. In this embodiment, the recombinant nucleic
acid molecule increases expression of the glucosamine-6-phosphate
synthase by the Escherichia coli, and is operatively linked to a
transcription control sequence. In one embodiment, the recombinant
nucleic acid molecule comprises a genetic modification which
reduces glucosamine-6-phosphate product inhibition of the
glucosamine-6-phosphate synthase. In another embodiment, the
Escherichia coli has an additional genetic modification in at least
one gene selected from the group of nagA, nagB, nagC, nagD, nagE,
manXYZ, glmM, pfkB, pfkA, glmU, glmS, ptsG and/or a phosphatase
gene. In yet another embodiment, the additional modification
comprises a deletion of nagA, nagB, nagC, nagD, nagE, and a
mutation in manXYZ, wherein the modification results in decreased
enzymatic activity of N-acetylglucosamine-6-phosphate deacetylase,
glucosamine-6-phosphate deaminase and N-acetyl-glucosamine-specific
enzyme II.sup.Nag.
[0019] Yet another embodiment of the present invention relates to a
microorganism for producing glucosamine by a biosynthetic process.
The microorganism is transformed with a recombinant nucleic acid
molecule encoding glucosamine-6-phosphate synthase, wherein the
recombinant nucleic acid molecule is operatively linked to a
transcription control sequence. The recombinant nucleic acid
molecule further comprises a genetic modification which increases
the action of the glucosamine-6-phosphate synthase. The expression
of the recombinant nucleic acid molecule increases production of
the glucosamine by the microorganism. In a preferred embodiment,
the recombinant nucleic acid molecule is integrated into the genome
of the microorganism. In yet another embodiment, the microorganism
has at least one additional genetic modification in a gene encoding
a protein selected from the group consisting of
N-acetylglucosamine-6-phosphate deacetylase,
glucosamine-6-phosphate deaminase, N-acetyl-glucosamine-specific
enzyme II.sup.Nag, phosphoglucosamine mutase,
glucosamine-1-phosphate
acetyltransferase-N-acetylglucosamine-1-phosphate
uridyltransferase, phosphofructokinase, Enzyme II.sup.Glc of the
PEP:glucose PTS, and/or EIIM, P/III.sup.Man of the PEP:mannose PTS,
wherein the genetic modification decreases the action of the
protein. In another embodiment, the microorganism has a
modification in a gene encoding a phosphatase, wherein the genetic
modification increases the action of the phosphatase. In yet
another embodiment, the microorganism has a modification in genes
encoding N-acetylglucosamine-6-phosphate deacetylase,
glucosamine-6-phosphate deaminase and N-acetyl-glucosamine-specific
enzyme II Nag wherein the genetic modification decreases enzymatic
activity of the protein. In a preferred embodiment, the genetic
modification is a deletion of at least a portion of the genes.
[0020] In a further embodiment, the microorganism is Escherichia
coli, having a modification in a gene selected from the group of
nagA, nagB, nagC, nagD, nagE, manXYZ, glmM, pfkB, pfkA, glmu, ptsG
and/or a phosphatase gene. In one embodiment, such an Escherichia
coli has a deletion of nag regulon genes, and in another
embodiment, such an Escherichia coli has a deletion of nag regulon
genes and a genetic modification in manXYZ genes such that the
proteins encoded by the manXYZ genes have decreased action.
[0021] Yet another embodiment of the present invention is a
microorganism as described above which produces at least about 1
g/L of glucosamine when cultured for about 10-60 hours at from
about 28.degree. C. to about 37.degree. C. to a cell density of at
least about 8 g/L by dry cell weight, in a pH 7.0 fermentation
medium comprising: 14 g/L K.sub.2HPO.sub.4, 16 g/L
KH.sub.2PO.sub.4, 1 g/L Na.sub.3Citrate 2H.sub.2O, 5 g/L
(NH.sub.4).sub.2SO.sub.4, 20 g/L glucose, 10 mM MgSO.sub.4, 1 mM
CaCl.sub.2, and from about 0.2 mM to about 1 mM IPTG.
[0022] Another embodiment of the present invention is a
microorganism for producing glucosamine by a biosynthetic process,
which includes: (a) a recombinant nucleic acid molecule encoding
glucosamine-6-phosphate synthase operatively linked to a
transcription control sequence; and, (b) at least one genetic
modification in a gene encoding a protein selected from the group
of N-acetylglucosamine-6-phosphate deacetylase,
glucosamine-6-phosphate deaminase, N-acetyl-glucosamine-specific
enzyme II.sup.Nag, phosphoglucosamine mutase,
glucosamine-1-phosphate
acetyltransferase-N-acetylglucosamine-1-phosphate
uridyltransferase, phosphofructokinase, Enzyme II.sup.Glc of the
PEP:glucose PTS, and/or EIIM, P/III.sup.man of the PEP:mannose PTS,
wherein the genetic modification decreases the action of the
protein. In another embodiment, the microorganism includes at least
one genetic modification in a gene encoding a phosphatase, wherein
the genetic modification increases the action of the phosphatase.
Expression of the recombinant nucleic acid molecule increases the
action of the glucosamine-6-phosphate synthase in the
microorganism. In a further embodiment, the recombinant nucleic
acid molecule is integrated into the genome of the
microorganism.
BRIEF DESCRIPTION OF THE FIGURES OF THE INVENTION
[0023] FIG. 1 is a schematic representation of the pathways for the
biosynthesis and catabolism of glucosamine and N-acetyl-glucosamine
and their phosphorylated derivatives in Escherichia coli.
[0024] FIG. 2 is a schematic representation of the modifications to
the pathways related to amino sugar metabolism for the
overproduction of glucosamine in Escherichia coli.
[0025] FIG. 3 is a schematic representation of the production of
Escherichia coli strains containing combinations of the manXYZ,
ptsG, and .DELTA.nag mutations.
[0026] FIG. 4 is a line graph illustrating the effects on
glucosamine accumulation of feeding additional glucose and ammonium
sulfate to cultures.
[0027] FIG. 5 is a line graph which shows that
glucosamine-6-phosphate synthase is inhibited by
glucosamine-6-phosphate and glucosamine.
[0028] FIG. 6 is a line graph illustrating product inhibition of
glucosamine-6-phosphate synthase activity in mutant glmS
clones.
[0029] FIG. 7 is a schematic representation of the strategy for
constructions of Escherichia coli strains containing mutant glmS
genes.
[0030] FIG. 8 is a line graph illustrating product inhibition of
glucosamine-6-phosphate synthase in Escherichia coli strains with
integrated mutant glmS genes.
[0031] FIG. 9 is a line graph showing glucosamine production in
mutant Escherichia coli strains with integrated mutant glmS
genes.
[0032] FIG. 10 is a line graph showing inhibition of
glucosamine-6-phosphate synthase in glucosamine-producing
strains.
[0033] FIG. 11A is a line graph showing the thermal stability at
45.degree. C. of glucosamine-6-phosphate synthase in
glucosamine-producing strains.
[0034] FIG. 11B is a line graph illustrating the thermal stability
at 50.degree. C. of glucosamine-6-phosphate synthase in
glucosamine-producing strains.
[0035] FIG. 12 is a line graph showing the effect of IPTG
concentration on glucosamine production.
[0036] FIG. 13 is a line graph demonstrating the effects of IPTG
concentration and temperature on glucosamine production.
[0037] FIG. 14A is a line graph illustrating growth and glucosamine
production by glucosamine-producing strain 2123-54 at 30.degree.
C.
[0038] FIG. 14B is a line graph illustrating growth and glucosamine
production by glucosamine-producing strain 2123-54 at 37.degree.
C.
[0039] FIG. 15A is a line graph showing glucosamine production by
strain 2123-49 at 30.degree. C.
[0040] FIG. 15B is a line graph showing glucosamine production by
strain 2123-124 at 30.degree. C.
[0041] FIG. 16A is a line graph illustrating glucosamine production
by a glucosamine-producing strain in a glucose limited fermentor at
37.degree. C.
[0042] FIG. 16B is a line graph illustrating glucosamine production
by a glucosamine-producing strain in a glucose limited fermentor at
30.degree. C.
[0043] FIG. 16A is a line graph illustrating glucosamine production
by a glucosamine-producing strain in a glucose excess fermentor at
30.degree. C.
DETAILED DESCRIPTION OF THE INVENTION
[0044] The present invention relates to a biosynthetic method for
producing glucosamine. Such a method includes fermentation of a
genetically modified microorganism to produce glucosamine. The
present invention also relates to genetically modified
microorganisms, such as strains of Escherichia coli, useful for
producing glucosamine. As used herein, the terms glucosamine and
N-glucosamine can be used interchangeably. Similarly, the terms
glucosamine-6-phosphate and N-glucosamine-6-phosphate can be used
interchangeably. Glucosamine can also be abbreviated as GlcN and
glucosamine-6-phosphate can also be abbreviated as GlcN-6-P.
[0045] The novel method of the present invention for production of
glucosamine by fermentation is inexpensive and can produce a yield
of glucosamine that exceeds the yield per cost of glucosamine
produced by current hydrolysis methods. In addition, by using a
genetically modified microorganism as described herein, the method
of the present invention can be easily modified to adapt to
particular problems or changing needs relative to the production of
glucosamine.
[0046] The amino sugars, N-acetylglucosamine (GlcNAc) and
glucosamine (GlcN), are fundamentally important molecules in
microorganisms, because they are the precursors for the
biosynthesis of major macromolecules, and in particular,
glycoconjugates (i.e., macromolecules containing covalently bound
oligosaccharide chains). For example, in Escherichia coli,
N-acetylglucosamine and glucosamine are precursors for two
macromolecules of the cell envelope, peptidoglycan and
lipopolysaccharide. Mutations that block the biosynthesis of
peptidoglycan or lipopolysaccharide are lethal, resulting in loss
of integrity of the cell envelope and ultimately in cell lysis.
[0047] One embodiment of the present invention relates to a method
to produce glucosamine by fermentation of a microorganism. This
method includes the steps of (a) culturing in a fermentation medium
a microorganism having a genetic modification in an amino sugar
metabolic pathway which includes: a pathway for converting
glucosamine-6-phosphate into another compound, a pathway for
synthesizing glucosamine-6-phosphate, a pathway for transport of
glucosamine or glucosamine-6-phosphate out of said microorganism, a
pathway for transport of glucosamine into said microorganism, and a
pathway which competes for substrates involved in the production of
glucosamine-6-phosphate, to produce a product which can include
intracellular glucosamine-6-phosphate and/or extracellular
glucosamine from the microorganism; and (b) recovering the product
by recovering intracellular glucosamine-6-phosphate from the
microorganism and/or recovering extracellular glucosamine from the
fermentation medium. The fermentation medium includes assimilable
sources of carbon, nitrogen and phosphate.
[0048] Another embodiment of the present invention relates to a
method to produce glucosamine by fermentation. Such method includes
the steps of: (a) culturing in a fermentation medium comprising
assimilable sources of carbon, nitrogen and phosphate, an
Escherichia coli transformed with a recombinant nucleic acid
molecule encoding glucosamine-6-phosphate synthase operatively
linked to a transcription control sequence; and (b) recovering a
product selected from the group of glucosamine-6-phosphate and
glucosamine. The recombinant nucleic acid molecule increases
expression of the glucosamine-6-phosphate synthase by the
Escherichia coli. In a further embodiment, the recombinant nucleic
acid molecule comprises a genetic modification which reduces
glucosamine-6-phosphate product inhibition of the
glucosamine-6-phosphate synthase. In yet another embodiment, the
Escherichia coli has an additional genetic modification in at least
one gene selected from the group of nagA, nagB, nagC, nagD, nagE,
manXYZ, glmM, pfkB, pfkA, glmu, glmS, ptsG and/or a phosphatase
gene.
[0049] To produce significantly high yields of glucosamine by the
fermentation method of the present invention, a microorganism is
genetically modified to enhance production of glucosamine. As used
herein, a genetically modified microorganism, such as Escherichia
coli, has a genome which is modified (i.e., mutated or changed)
from its normal (i.e., wild-type or naturally occurring) form.
Genetic modification of a microorganism can be accomplished using
classical strain development and/or molecular genetic techniques.
Such techniques are generally disclosed, for example, in Sambrook
et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Labs Press. The reference Sambrook et al., ibid., is
incorporated by reference herein in its entirety. Additionally,
techniques for genetic modification of a microorganism are
described in detail in the Examples section. A genetically modified
microorganism can include a natural genetic variant as well as a
microorganism in which nucleic acid molecules have been inserted,
deleted or modified (i.e., mutated; e.g., by insertion, deletion,
substitution, and/or inversion of nucleotides), in such a manner
that such modifications provide the desired effect within the
microorganism. According to the present invention, a genetically
modified microorganism includes a microorganism that has been
modified using recombinant technology. As used herein, genetic
modifications which result in a decrease in gene expression, in the
function of the gene, or in the function of the gene product (i.e.,
the protein encoded by the gene) can be referred to as inactivation
(complete or partial), deletion, interruption, blockage or
down-regulation of a gene. For example, a genetic modification in a
gene which results in a decrease in the function of the protein
encoded by such gene, can be the result of a complete deletion of
the gene (i.e., the gene does not exist, and therefore the protein
does not exist), a mutation in the gene which results in incomplete
or no translation of the protein (e.g., the protein is not
expressed), or a mutation in the gene which decreases or abolishes
the natural function of the protein (e.g., a protein is expressed
which has decreased or no enzymatic activity or action). Genetic
modifications which result in an increase in gene expression or
function can be referred to as amplification, overproduction,
overexpression, activation, enhancement, addition, or up-regulation
of a gene.
[0050] In one embodiment of the present invention, a genetic
modification of a microorganism increases or decreases the action
of a protein involved in an amino sugar metabolic pathway according
to the present invention. Such a genetic modification includes any
type of modification and specifically includes modifications made
by recombinant technology and by classical mutagenesis. For
example, in one embodiment, a microorganism of the present
invention has a genetic modification that increases the action of
glucosamine-6-phosphate synthase. It should be noted that reference
to increasing the action (or activity) of glucosamine-6-phosphate
synthase and other enzymes discussed herein refers to any genetic
modification in the microorganism in question which results in
increased functionality of the enzymes and includes higher activity
of the enzymes (e.g., specific activity or in vivo enzymatic
activity), reduced inhibition or degradation of the enzymes and
overexpression of the enzymes. For example, gene copy number can be
increased, expression levels can be increased by use of a promoter
that gives higher levels of expression than that of the native
promoter, or a gene can be altered by genetic engineering or
classical mutagenesis to increase the action of an enzyme. Examples
of nucleic acid molecules encoding glucosamine-6-phosphate synthase
which have been genetically modified to increase the action of the
glucosamine-6-phosphate synthase are described in the Examples
section. Similarly, reference to decreasing the action of enzymes
discussed herein refers to any genetic modification in the
microorganism in question which results in decreased functionality
of the enzymes and includes decreased activity of the enzymes
(e.g., specific activity), increased inhibition or degradation of
the enzymes and a reduction or elimination of expression of the
enzymes. For example, the action of an enzyme of the present
invention can be decreased by blocking or reducing the production
of the enzyme, reducing enzyme activity, or inhibiting the activity
of the enzyme. Blocking or reducing the production of an enzyme can
include placing the gene encoding the enzyme under the control of a
promoter that requires the presence of an inducing compound in the
growth medium. By establishing conditions such that the inducer
becomes depleted from the medium, the expression of the gene
encoding the enzyme (and therefore, of enzyme synthesis) could be
turned off. Blocking or reducing the activity of an enzyme could
also include using an excision technology approach similar to that
described in U.S. Pat. No. 4,743,546, incorporated herein by
reference. To use this approach, the gene encoding the enzyme of
interest is cloned between specific genetic sequences that allow
specific, controlled excision of the gene from the genome. Excision
could be prompted by, for example, a shift in the cultivation
temperature of the culture, as in U.S. Pat. No. 4,743,546, or by
some other physical or nutritional signal.
[0051] An amino sugar is an amino derivative of a saccharide (e.g.,
a saccharide having an amino group in place of a hydroxyl group).
According to the present invention, an amino sugar metabolic
pathway is any biochemical pathway involved in, or affecting, the
biosynthesis, anabolism or catabolism of an amino sugar. As used
herein, amino sugar metabolic pathways include pathways involved in
the transport of amino sugars and their precursors into and out of
a cell, and can also include biochemical pathways which compete for
substrates involved in the biosynthesis or catabolism of an amino
sugar. For example, the immediate precursor to one of the earliest
formed amino sugars is fructose-6-phosphate (F-6-P), which, in a
biochemical reaction with glutamine (Gln, the amino group donor),
forms glucosamine-6-phosphate. Fructose-6-phosphate is also an
intermediate in the glycolysis pathway. Therefore, the glycolytic
pathway competes with the glucosamine-6-phosphate biosynthetic
pathway by competing for a substrate, fructose-6-phosphate. In
addition, glucosamine-6-phosphate can be converted to other amino
sugars and form constituents in various macromolecules by a series
of biochemical reactions. As such, the
fructose-6-phosphate/glucosamine-6-phosphate pathway, the
fructose-6-phosphate glycolytic pathway, to the extent that it
affects the biosynthesis of glucosamine-6-phosphate, and the
glucosamine-6-phosphate/macromolecule biosynthesis pathway are all
considered to be amino sugar metabolic pathways in the present
invention.
[0052] In general, a microorganism having a genetically modified
amino sugar metabolic pathway has at least one genetic
modification, as discussed above, which results in a change in one
or more amino sugar metabolic pathways as described above as
compared to a wild-type microorganism cultured under the same
conditions. Such a modification in an amino sugar metabolic pathway
changes the ability of the microorganism to produce an amino sugar.
According to the present invention, a genetically modified
microorganism preferably has an enhanced ability to produce
glucosamine compared to a wild-type microorganism cultured under
the same conditions. An amino sugar metabolic pathway which affects
the production of glucosamine can generally be categorized into at
least one of the following kinds of pathways: (a) pathways for
converting glucosamine-6-phosphate into other compounds, (b)
pathways for synthesizing glucosamine-6-phosphate, (c) pathways for
transporting glucosamine into a cell, (d) pathways for transporting
glucosamine or glucosamine-6-phosphate out of a cell, and (e)
pathways which compete for substrates involved in the production of
glucosamine-6-phosphate.
[0053] A genetically modified microorganism useful in a method of
the present invention typically has at least one modified gene
involved in at least one amino sugar metabolic pathway which
results in (a) reduced ability to convert glucosamine-6-phosphate
into other compounds (i.e., inhibition of glucosamine-6-phosphate
catabolic or anabolic pathways), (b) an enhanced ability to produce
(i.e., synthesize) glucosamine-6-phosphate, (c) a reduced ability
to transport glucosamine into the cell, (d) an enhanced ability to
transport glucosamine-6-phosphate or glucosamine out of the cell,
and/or (e) a reduced ability to use substrates involved in the
production of glucosamine-6-P for competing biochemical
reactions.
[0054] It is to be understood that the present invention discloses
a method comprising the use of a microorganism with an ability to
produce commercially useful amounts of glucosamine in a
fermentation process (i.e., preferably an enhanced ability to
produce glucosamine compared to a wild-type microorganism cultured
under the same conditions). This method is achieved by the genetic
modification of one or more genes encoding a protein involved in an
amino sugar metabolic pathway which results in the production
(expression) of a protein having an altered (e.g., increased or
decreased) function as compared to the corresponding wild-type
protein. Such an altered function enhances the ability of the
genetically engineered microorganism to produce glucosamine. It
will be appreciated by those of skill in the art that production of
genetically modified microorganisms having a particular altered
function as described elsewhere herein (e.g., an enhanced ability
to produce glucosamine-6-phosphate) such as by the specific
selection techniques described in the Examples, can produce many
organisms meeting the given functional requirement, albeit by
virtue of a variety of different genetic modifications. For
example, different random nucleotide deletions and/or substitutions
in a given nucleic acid sequence may all give rise to the same
phenotypic result (e.g., decreased action of the protein encoded by
the sequence). The present invention contemplates any such genetic
modification which results in the production of a microorganism
having the characteristics set forth herein.
[0055] For a variety of microorganisms, many of the amino sugar
metabolic pathways have been elucidated. In particular, pathways
for the biosynthesis and catabolism of glucosamine and
N-acetylglucosamine and their phosphorylated derivatives have been
elucidated in Escherichia coli. These pathways include the multiple
transport systems for the utilization of these amino sugars as
carbon sources. Genes encoding the enzymes and proteins directly
related to the transport, catabolism and biosynthesis of amino
sugars in Escherichia coli have been cloned and sequenced. In
addition, mutant strains of Escherichia coli blocked in
substantially every step of amino sugar metabolism have been
isolated. The known pathways for amino sugar metabolism for
Escherichia coli are illustrated in FIG. 1.
[0056] As will be discussed in detail below, even though many of
the pathways and genes involved in the amino sugar metabolic
pathways have been elucidated, until the present invention, it was
not known which of the many possible genetic modifications would be
necessary to generate a microorganism that can produce commercially
significant amounts of glucosamine. Indeed, the present inventors
are the first to design and engineer a glucosamine-producing
microorganism that has glucosamine production capabilities that far
exceed the glucosamine production capability of any known wild-type
or mutant microorganism. The present inventors are also the first
to appreciate that such a genetically modified microorganism is
useful in a method to produce glucosamine for commercial use.
[0057] A microorganism to be used in the fermentation method of the
present invention is preferably a bacterium or a yeast. More
preferably, such a microorganism is a bacterium of the genus
Escherichia. Escherichia coli is the most preferred microorganism
to use in the fermentation method of the present invention.
Particularly preferred strains of Escherichia coli include K-12, B
and W, and most preferably, K-12. Although Escherichia coli is most
preferred, it is to be understood that any microorganism that
produces glucosamine and can be genetically modified to enhance
production of glucosamine can be used in the method of the present
invention. A microorganism for use in the fermentation method of
the present invention can also be referred to as a production
organism.
[0058] The amino sugar metabolic pathways of the microorganism,
Escherichia coli, will be addressed as specific embodiments of the
present invention are described below. It will be appreciated that
other microorganisms and in particular, other bacteria, have
similar amino sugar metabolic pathways and genes and proteins
having similar structure and function within such pathways. As
such, the principles discussed below with regard to Escherichia
coli are applicable to other microorganisms.
[0059] In one embodiment of the present invention, a genetically
modified microorganism includes a microorganism which has an
enhanced ability to synthesize glucosamine-6-phosphate. According
to the present invention, "an enhanced ability to synthesize" a
product refers to any enhancement, or up-regulation, in an amino
sugar metabolic pathway related to the synthesis of the product
such that the microorganism produces an increased amount of the
product compared to the wild-type microorganism cultured under the
same conditions. In one embodiment of the present invention,
enhancement of the ability of a microorganism to synthesize
glucosamine-6-phosphate is accomplished by amplification of the
expression of the glucose-6-phosphate synthase gene, which in
Escherichia coli is the glmS gene, the product of which is
glucosamine-6-phosphate synthase. Glucosamine-6-phosphate synthase
catalyzes the reaction in which fructose-6-phosphate and glutamine
form glucosamine-6-phosphate and glutamic acid. Amplification of
the expression of glucosamine-6-phosphate synthase can be
accomplished in Escherichia coli, for example, by introduction of a
recombinant nucleic acid molecule encoding the glmS gene.
[0060] Overexpression of glmS is crucial for the intracellular
accumulation of glucosamine-6-phosphate and ultimately for
production of glucosamine, since the level of
glucosamine-6-phosphate synthase in the cell will control the
redirection of carbon flow away from glycolysis and into
glucosamine-6-phosphate synthesis. The glmS gene is located at 84
min on the Escherichia coli chromosome, and sequence analysis of
this region of the chromosome reveals that glmS resides in an
operon with the glmU gene, which encodes the bifunctional enzyme,
glucosamine-1-phosphate
acetyltransferase-N-acetylglucosamine-1-phosphate
uridyltransferase. Glucosamine-1-phosphate
acetyltransferase-N-acetylglucosamine-1-phosphate uridyltransferase
functions within the amino sugar metabolic pathway in which
glucosamine-6-phosphate is incorporated, through a series of
biochemical reactions, into macromolecules. No obvious promoter
sequence is detected upstream of glmS; transcription of the glmUS
operon is initiated from two promoter sequences upstream of glmU.
Thus, it is preferred that the glmS gene be cloned under control of
an artificial promoter. The promoter can be any suitable promoter
that will provide a level of glmS expression required to maintain a
sufficient level of glucosamine-6-phosphate synthase in the
production organism. Preferred promoters are constitutive (rather
than inducible) promoters, since the need for addition of expensive
inducers is therefore obviated. Such promoters include normally
inducible promoter systems that have been made functionally
constitutive or "leaky" by genetic modification, such as by using a
weaker, mutant repressor gene. Particularly preferred promoters to
be used with glmS are lac, .lamda.P.sub.L and T7. The gene dosage
(copy number) of glmS can be varied according to the requirements
for maximum product formation. In one embodiment, the recombinant
glmS gene is integrated into the E. coli chromosome.
[0061] Therefore, it is an embodiment of the present invention to
provide a microorganism, such as E. coli, which is transformed with
a recombinant nucleic acid molecule comprising a nucleic acid
sequence encoding a glucosamine-6-phosphate synthase, which in E.
coli, for example, is encoded by the glmS gene. Preferred
recombinant nucleic acid molecules comprising such a nucleic acid
sequence include recombinant nucleic acid molecules comprising a
nucleic acid sequence which encodes a glucosamine-6-phosphate
synthase comprising an amino acid sequence SEQ ID NO:16. Other
preferred recombinant nucleic acid molecules of the present
invention include nucleic acid molecules which comprise a nucleic
acid sequence selected from the group of SEQ ID NO:13, SEQ ID NO:14
and/or SEQ ID NO:15. Particularly preferred recombinant nucleic
acid molecules of the present invention include nucleic acid
molecules comprising nucleic acid molecules nglmS-28.sub.2184
and/or nglmS-28.sub.1830. One recombinant molecule of the present
invention, referred to herein as plasmid pKLN23-28, includes SEQ ID
NOs:13, 14 and 15 and is particularly useful for expressing
glucosamine-6-phosphate synthase in a microorganism. The above
identified nucleic acid molecules represent nucleic acid molecules
comprising wild-type (i.e., naturally occurring or endogenous)
nucleic acid sequences encoding glucosamine-6-phosphate synthase
proteins. Genetically modified nucleic acid molecules which include
nucleic acid sequences encoding homologues (i.e., modified and/or
mutated) glucosamine-6-phosphate synthase proteins are also
encompassed by the present invention and are described in detail
below.
[0062] The reported K.sub.m's of glucosamine-6-phosphate synthase
from Escherichia coli are 2 mM and 0.4 mM for fructose-6-phosphate
and glutamine, respectively. These are relatively high values
(i.e., the affinity of the enzyme for its substrates is rather
weak). It is therefore another embodiment of the present invention
to provide a microorganism having a glucosamine-6-phosphate
synthase with improved affinity for its substrates. A
glucosamine-6-phosphate synthase with improved affinity for its
substrates can be produced by any suitable method of genetic
modification or protein engineering. For example, computer-based
protein engineering can be used to design a glucosamine-6-phosphate
synthase protein with greater stability and better affinity for its
substrate. See for example, Maulik et al., 1997, Molecular
Biotechnology Therapeutic Applications and Strategies, Wiley-Liss,
Inc., which is incorporated herein by reference in its
entirety.
[0063] White (1968, Biochem. J., 106:847-858) first demonstrated
that glucosamine-6-phosphate synthase was inhibited by
glucosamine-6-phosphate. The present inventors determined that this
inhibition was a key factor which limits glucosamine accumulation
in glucosamine production strains of the present invention, which
have been designed for commercial use. Therefore, it is yet another
embodiment of the present invention to provide a microorganism
having a glucosamine-6-phosphate synthase with reduced
glucosamine-6-phosphate product feedback inhibition. A
glucosamine-6-phosphate synthase with reduced product inhibition
can be a mutated (i.e., genetically modified)
glucosamine-6-phosphate synthase gene, for example, and can be
produced by any suitable method of genetic modification. For
example, a recombinant nucleic acid molecule encoding
glucosamine-6-phosphate synthase can be modified by any method for
inserting, deleting, and/or substituting nucleotides, such as by
error-prone PCR. In this method, the gene is amplified under
conditions that lead to a high frequency of misincorporation errors
by the DNA polymerase used for the amplification. As a result, a
high frequency of mutations are obtained in the PCR products. This
method is described in detail in Example 5. The resulting
glucosamine-6-phosphate synthase gene mutants can then be screened
for reduced product inhibition by testing the mutant genes for the
ability to confer increased glucosamine production onto a test
microorganism, as compared to a microorganism carrying the
non-mutated recombinant glucosamine-6-phosphate synthase nucleic
acid molecule. It should be noted that decreased product inhibition
of glucosamine-6-phosphate synthase typically results in a
glucosamine-6-phosphate synthase with increased action, even when
the specific activity of the enzyme is remains the same, or
actually is decreased, relative to a naturally occurring
glucosamine-6-phosphate enzyme. Therefore, it is an embodiment of
the present invention to produce a genetically modified
glucosamine-6-phosphate synthase with increased action and
increased in vivo enzymatic activity, which has unmodified or even
decreased specific activity as compared to a naturally occurring
glucosamine-6-phosphate synthase. Also encompassed by the present
invention are genetically modified glucosamine-6-phosphate
synthases with increased specific activity.
[0064] Therefore, it is an embodiment of the present invention to
provide a microorganism, such as E. coli, which is transformed with
a genetically modified recombinant nucleic acid molecule comprising
a nucleic acid sequence encoding a mutant, or homologue,
glucosamine-6-phosphate synthase protein. Such
glucosamine-6-phosphate synthase proteins can be referred to herein
as glucosamine-6-phosphate synthase homologues. Protein homologues
are described in detail below. Preferred recombinant nucleic acid
molecules comprising such a nucleic acid sequence include
recombinant nucleic acid molecules comprising a nucleic acid
sequence which encodes a glucosamine-6-phosphate synthase
comprising an amino acid sequence selected from the group of SEQ ID
NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28 and/or SEQ ID
NO:31. Other preferred recombinant nucleic acid molecules comprise
a nucleic acid sequence selected from the group of SEQ ID NO:17,
SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID
NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29 and/or SEQ ID
NO:30. Particularly preferred genetically modified recombinant
nucleic acid molecules useful in the present invention include
nucleic acid molecules comprising nucleic acid molecules selected
from the group of nglmS-49.sub.2184, nglmS-49.sub.1830,
nglmS-54.sub.2184, nglmS-54.sub.1830, nglmS-124.sub.2184,
nglmS-124.sub.1830, nglmS-149.sub.2184, nglmS-149.sub.1830,
nglmS-151.sub.2184 and nglmS-151.sub.1830. Plasmids pKLN23-49,
pKLN23-54, pKLN23-124, pKLN23-149 and pKLN23-151 are recombinant
nucleic acid molecules of the present invention which are
particularly useful for expressing glucosamine-6-phosphate synthase
homologues in a microorganism.
[0065] An adequate intracellular supply of glutamine (Gln) is
critical for the glucosamine-6-phosphate synthase reaction.
Inspection of the synthetic and degradative pathways for
glucosamine-6-phosphate reveals the presence of a potential futile
cycle whereby continuous interconversion of fructose-6-phosphate
and glucosamine-6-phosphate results in wasteful depletion of
glutamine. In one embodiment of the present invention, the supply
of glutamine can be increased either by genetic modification of the
production organism to increase glutamine production in the cell,
or by modifying the fermentation medium (i.e., adding glutamine to
the fermentation medium), to ensure that the supply of glutamine
will not limit the production of glucosamine-6-phosphate.
[0066] In another embodiment of the present invention, the
potential futile cycling of fructose-6-phosphate and
glucosamine-6-phosphate is addressed by inhibiting, or blocking,
the reverse reaction in which glucosamine-6-phosphate is converted
into fructose-6-phosphate. In this embodiment, a microorganism is
genetically modified to have an inactivation or deletion of the
gene which catalyzes this conversion, glucosamine-6-phosphate
deaminase, which in Escherichia coli is the nagB gene. nagB is one
of several nag genes which are part of the nag regulon. The nag
genes involved in the degradation of glucosamine and
N-acetyl-glucosamine exist as a regulon located at 15 min on the
Escherichia coli chromosome. In another embodiment, the entire nag
regulon is inactivated or deleted. The advantages of deleting the
entire nag regulon are discussed in detail below.
[0067] As discussed above, overproduction of
glucosamine-6-phosphate synthase results in diversion of
fructose-6-phosphate synthesis to glucosamine-6-phosphate
synthesis. However, many other enzymes can compete for the
substrate, fructose-6-phosphate. Therefore, one embodiment of the
present invention includes a microorganism in which these
competitive side reactions are blocked. In a preferred embodiment,
a microorganism having complete or partial inactivation of the gene
encoding phosphofructokinase is provided. The second step in the
glycolytic pathway is the conversion of fructose-6-phosphate to
fructose-1,6-diphosphate by phosphofructokinase, which in
Escherichia coli exists as two isozymes encoded by the pfkA and
pfkB genes. Complete or partial inactivation of either the pfkA or
pfkB genes slows the entry of fructose-6-phosphate into the
glycolytic pathway and enhances the conversion of
fructose-6-phosphate to glucosamine-6-phosphate. As used herein,
inactivation of a gene can refer to any modification of a gene
which results in a decrease in the activity (i.e., expression or
function) of such a gene, including attenuation of activity or
complete deletion of activity.
[0068] In a further embodiment of the present invention, a
genetically modified microorganism has a decreased ability to
convert glucosamine-6-phosphate into other compounds. Inactivation
of glucosamine-6-phosphate deaminase, as described above,
represents one such modification, however, glucosamine-6-phosphate
serves as a substrate for other biochemical reactions. The first
committed step in the pathway leading to production of
macromolecules such as lipopolysaccharide and peptidoglycan in
Escherichia coli is the conversion of glucosamine-6-phosphate to
glucosamine-1-phosphate by phosphoglucosamine mutase, which in
Escherichia coli is the product of the glmM gene. The involvement
of this enzyme activity in the pathway of lipopolysaccharide and
peptidoglycan biosynthesis was recently confirmed with the cloning
of the glmM gene. Consequently, the regulation of glmM gene, and
its cognate product, phosphoglucosamine mutase, has not been
studied in detail. It has been shown, however, that the
phosphoglucosamine mutase, like all other hexosephosphate mutase
enzymes studied, is regulated by phosphorylation. This type of
regulation at the enzyme level is typically exquisitely sensitive
to levels of the pathway end products. Thus, carbon flow through
phosphoglucosamine mutase can be self-regulating and may not be a
problem as glucosamine-6-phosphate accumulates. Since the sequence
of the glmM gene is known, however, it is a preferred embodiment of
the present invention to provide a microorganism in which the gene
encoding phosphoglucosamine mutase is interrupted or deleted. More
preferably, the gene encoding phosphoglucosamine mutase is
down-regulated, but not completely inactivated, by a mutation, so
as not to completely block the biosynthesis of the critical cell
envelope components.
[0069] Another pathway which results in the conversion of
glucosamine-6-phosphate to another compound is catalyzed by the
enzyme, N-acetylglucosamine-6-phosphate deacetylase.
N-acetylglucosamine-6-phosphate deacetylase is capable of
catalyzing the reverse reaction of converting
glucosamine-6-phosphate (plus acetyl CoA) to
N-acetyl-glucosamine-6-phosphate. This could result in futile
cycling of glucosamine-6-phosphate and
N-acetyl-glucosamine-6-phosphate and result in a product composed
of a mixture of glucosamine and N-acetyl-glucosamine. Therefore, it
is a further embodiment of the present invention to provide a
genetically modified microorganism in which the gene encoding
N-acetylglucosamine-6-phosphate deacetylase, which is the nagA gene
in Escherichia coli, is inactivated.
[0070] It is a further embodiment of the present invention to
inactivate the transport systems for glucosamine in a microorganism
such that once the glucosamine is excreted by the cell it is not
taken back up. This modification is helpful for avoiding a high
intracellular level of glucosamine which could be toxic to the
cells, and facilitates recovery of the product, since the product
remains extracellular. In a preferred embodiment of the present
invention, the transportation systems for glucosamine are
inactivated to keep glucosamine outside of the microorganism once
it is excreted by the microorganism. During growth of Escherichia
coli on glucosamine as sole carbon source, glucosamine is
transported into the cell by the PEP:mannose phosphotransferase
(PTS) system, which is not only capable of transporting glucosamine
into the cell, but is also induced by glucosamine. It is therefore
an embodiment of the present invention to provide a microorganism
lacking the ability to transport glucosamine into the cell. For
example, a manXYZ mutant (i.e., an Escherichia coli lacking or
having a mutation in the genes encoding EIIM, P/III.sup.man of the
PEP:mannose PTS) can not transport glucosamine into the cell by
this mechanism. The PEP:glucose PTS of Escherichia coli, on the
other hand, is capable of transporting both glucose and glucosamine
into the cell, but glucosamine cannot induce this system. Thus, in
order to grow a manXYZ mutant on glucosamine, the cells must first
be grown on glucose to induce expression of the (alternate) glucose
transport system and allow glucose (the preferred carbon source) to
be transported into the cell. These induced cells are then capable
of transporting glucosamine into the cell via the glucose
transporter. A similar situation exists for transport of
glucosamine by the PEP:fructose PTS, although in this case
glucosamine transport by the enzyme II Fr is poor. Methods to
inhibit these secondary glucosamine transport pathways are
discussed below. It is yet another embodiment of the present
invention to provide a microorganism having a decreased function in
the PEP:glucose PTS (described above). Such a modification may be
necessary to avoid "reabsorption of glucosamine from the culture
medium. For example, a ptsG mutant (i.e., an Escherichia coli
lacking or having a mutation in the genes encoding enzyme
II.sup.Glc of the PEP:glucose PTS). Since such microorganisms will
have reduced ability to grow using glucose as a carbon source, such
organisms can be further genetically modified to take up glucose by
a PEP:glucose PTS-independent mechanism. It is has been shown, for
example, that mutant microorganisms can be selected which are
defective in the PEP:glucose PTS and still have an ability to grow
on glucose (Flores et al., 1996, Nature Biotechnology
14:620-623).
[0071] DNA sequencing of the nag regulon in Escherichia coli
reveals that the nagE gene, encoding the
N-acetyl-glucosamine-specific enzyme II.sup.Nag protein of the
PEP:sugar phosphotransferase (PTS) system, which is involved in
glucosamine transport into the cell, resides on one arm of the
regulon and is transcribed divergently from the other nag genes
(nagBACD) located on the other arm of the regulon. Therefore,
another genetic modification that would result in decreased ability
of an Escherichia coli to transport glucosamine into the cell is an
inactivation or deletion of the nagE gene, or a gene encoding a
similar enzyme in any microorganism used in a method of the present
invention.
[0072] As discussed above, in one embodiment of the present
invention, a genetically modified Escherichia coli microorganism
useful in a method of the present invention has a deletion of the
entire nag regulon. Deletion of the entire chromosomal nag regulon
is preferred, because many genes which are deleterious to the
production of glucosamine-6-phosphate are inactivated together. The
genes, nagA, nagB and nagE, have been discussed in detail above.
The nagC gene encodes a regulatory protein that acts as a repressor
of the nag regulon as well as both an activator and repressor of
the glmUS operon. The glm genes are discussed in detail above. The
function of the nagD gene is not known, but is believed to be
related to amino sugar metabolism as it resides within the nag
regulon. Thus, in Escherichia coli, a complete deletion of the nag
regulon avoids catabolism of the initial intracellular product
(glucosamine-6-phosphate) in a strain of Escherichia coli designed
to overproduce glucosamine. A preferred Escherichia coli mutant
strain having a deletion of the nag regulon is an Escherichia coli
having a .DELTA.nagEBACD::tc deletion/insertion.
[0073] With regard to activation of the glmUS operon (a function of
nagC), although activation of the glmS gene, encoding
glucosamine-6-phosphate synthase, is desirable, an increase in the
level of the glmu gene product, glucosamine-1-phosphate
acetyltransferase-N-acetylglucosamine-1-phosphate uridyltransferase
could be deleterious to accumulation of glucosamine-6-phosphate as
it could lead to siphoning off of carbon flow toward cell envelope
components. It is therefore an embodiment of the present invention
to inactivate glucosamine-1-phosphate
acetyltransferase-N-acetylglucosamine-1-phosphate uridyltransferase
in a microorganism useful in a method of the present invention. In
a microorganism in which the glmUS operon, or its equivalent, has
been inactivated or deleted, it is a further embodiment of the
present invention to genetically modify the microorganism by
recombinantly producing the gene encoding glucosamine-6-phosphate
synthase under control of an artificial promoter in the
microorganism.
[0074] The initial intracellular product in the genetically
modified microorganism described herein is glucosamine-6-phosphate.
In many microorganisms, including Escherichia coli,
glucosamine-6-phosphate is typically dephosphorylated to
glucosamine prior to transport out of the cell. Nonetheless, it is
yet another embodiment of the present invention to provide a
microorganism which is genetically modified to have a suitable
phosphatase activity for the conversion of glucosamine-6-phosphate
to glucosamine. Such a phosphatase can include, but is not limited
to, for example, alkaline phosphatase. In a preferred embodiment,
such an Escherichia coli has an enhanced (i.e., increased) level of
phosphatase activity (i.e., phosphatase action).
[0075] As noted above, in the method for production of glucosamine
of the present invention, a microorganism having a genetically
modified amino sugar metabolic pathway is cultured in a
fermentation medium for production of glucosamine. An appropriate,
or effective, fermentation medium refers to any medium in which a
genetically modified microorganism of the present invention, when
cultured, is capable of producing glucosamine. Such a medium is
typically an aqueous medium comprising assimilable carbon, nitrogen
and phosphate sources. Such a medium can also include appropriate
salts, minerals, metals and other nutrients. One advantage of the
genetic modifications to a microorganism described herein is that
although such genetic modifications significantly alter the
metabolism of amino sugars, they do not create any nutritional
requirements for the production organism. Thus, a minimal-salts
medium containing glucose as the sole carbon source is preferably
used as the fermentation medium. The use of a minimal-salts-glucose
medium for the glucosamine fermentation will also facilitate
recovery and purification of the glucosamine product.
[0076] Microorganisms of the present invention can be cultured in
conventional fermentation bioreactors. The microorganisms can be
cultured by any fermentation process which includes, but is not
limited to, batch, fed-batch, cell recycle, and continuous
fermentation. Preferably, microorganisms of the present invention
are grown by batch or fed-batch fermentation processes.
[0077] In one embodiment of the present invention, before
inoculation, the fermentation medium is brought up to the desired
temperature, typically from about 20.degree. C. to about 40.degree.
C., preferably from about 25.degree. C. to about 40.degree. C.,
with temperatures of from about 28.degree. C. to about 37.degree.
C., and in some embodiments, about 30.degree. C. or about
37.degree. C. being more preferred. The present inventors have
discovered that glucosamine production in microorganisms of the
present invention transfected with a nucleic acid molecule under
control of the T7-lac promoter (see Examples section) continues
after growth has ceased when the microorganisms are cultured at
30.degree. C., while at 37.degree. C., growth and glucosamine
production occur in concert. Growth at 37.degree. C. is slightly
better than at 30.degree. C., but glucosamine production at
30.degree. C. is significantly better than at 37.degree. C. It is
noted that the optimum temperature for growth and glucosamine
production by a microorganism of the present invention can vary
according to a variety of factors. For example, the selection of a
particular promoter for expression of a recombinant nucleic acid
molecule in the microorganism can affect the optimum culture
temperature. One of ordinary skill in the art can readily determine
the optimum growth and glucosamine production temperature for any
microorganism of the present invention using standard techniques,
such as those described in the Examples section for one
microorganism of the present invention.
[0078] The medium is inoculated with an actively growing culture of
the genetically modified microorganism in an amount sufficient to
produce, after a reasonable growth period, a high cell density. The
cells are grown to a cell density of at least about 10 g/l,
preferably between about 10 g/l and about 40 g/l, and more
preferably at least about 40 g/l. This process typically requires
about 10-60 hours.
[0079] Sufficient oxygen must be added to the medium during the
course of the fermentation to maintain cell growth during the
initial cell growth and to maintain metabolism and glucosamine
production. Oxygen is conveniently provided by agitation and
aeration of the medium. Conventional methods, such as stirring or
shaking, may be used to agitate and aerate the medium. Preferably
the oxygen concentration in the medium is greater than about 15% of
the saturation value (i.e., the solubility of oxygen in the medium
at atmospheric pressure and about 30-40.degree. C.) and more
preferably greater than about 20% of the saturation value, although
excursions to lower concentrations may occur if fermentation is not
adversely affected. The oxygen concentration of the medium can be
monitored by conventional methods, such as with an oxygen
electrode. Other sources of oxygen, such as undiluted oxygen gas
and oxygen gas diluted with inert gas other than nitrogen, can be
used.
[0080] Since the production of glucosamine by fermentation is
preferably based on using glucose as the sole carbon source, in a
preferred embodiment, in Escherichia coli, the PEP:glucose PTS will
be induced. Accordingly, even in the absence of a functional EIIM,
P/III.sup.Man of the PEP:mannose PTS (e.g., in an Escherichia coli
having a manXYZ mutation), the product, glucosamine, will still be
taken up by the cells via the induced glucose transport system. In
the presence of excess glucose, however, uptake of glucosamine is
severely repressed. Thus, it is one embodiment of the present
invention to prevent uptake of the glucosamine product by
maintaining an excess of glucose in the fermentation bioreactor. As
used herein, "an excess" of glucose refers to an amount of glucose
above that which is required to maintain the growth of the
microorganism under normal conditions, such as the culturing
conditions described above. Preferably, the glucose concentration
is maintained at a concentration of from about 0.5% to about 5%
weight/volume of the fermentation medium. In another embodiment,
the glucose concentration is maintained at a concentration of from
about 5 g/L to about 50 g/L of the fermentation medium, and even
more preferably, from about 5 g/L to about 20 g/L of the
fermentation medium. In one embodiment, the glucose concentration
of the fermentation medium is monitored by any suitable method
(e.g., by using glucose test strips), and when the glucose
concentration is at or near depletion, additional glucose can be
added to the medium. In another embodiment, the glucose
concentration is maintained by semi-continuous or continuous
feeding of the fermentation medium. The parameters disclosed herein
for glucose can be applied to any carbon source used in the
fermentation medium of the present invention. It is further
understood that the carbon source can be allowed to reach
undetectable levels for any appropriate amount of time during the
fermentation if it enhances the glucosamine production process.
[0081] It is a further embodiment of the present invention to
supplement and/or control other components and parameters of the
fermentation medium, as necessary to maintain and/or enhance the
production of glucosamine by a production organism. For example, in
one embodiment, the fermentation medium includes ammonium sulfate,
and the ammonium sulfate concentration in the culture medium is
supplemented by the addition of excess ammonium sulfate.
Preferably, the amount of ammonium sulfate is maintained at a level
of from about 0.1% to about 1% (weight/volume) in the fermentation
medium, and preferably, at about 0.5%. In yet another embodiment,
the pH of the fermentation medium is monitored for fluctuations in
pH. In the fermentation method of the present invention, the pH is
preferably maintained at a pH of from about pH 6.0 to about pH 8.0,
and more preferably, at about pH 7.0. In the method of the present
invention, if the starting pH of the fermentation medium is pH 7.0,
the pH of the fermentation medium is monitored for significant
variations from pH 7.0, and is adjusted accordingly, for example,
by the addition of sodium hydroxide.
[0082] A further embodiment of the present invention is to redirect
carbon flux from acetate production to the production of less toxic
byproducts. By such methods, problems of toxicity associated with
an excess of glucose in the fermentation medium can be avoided.
Methods to redirect carbon flux from acetate production are known
in the art.
[0083] In a batch fermentation process of the present invention,
fermentation is continued until the formation of glucosamine, as
evidenced by the accumulation of extracellular glucosamine,
essentially ceases. The total fermentation time is typically from
about 40 to about 60 hours, and more preferably, about 48 hours. In
a continuous fermentation process, glucosamine can be removed from
the bioreactor as it accumulates in the medium. The method of the
present invention results in production of a product which can
include intracellular or extracellular glucosamine-6-phosphate and
intracellular or extracellular glucosamine.
[0084] The method of the present invention further includes
recovering the product, which can be intracellular
glucosamine-6-phosphate or extracellular glucosamine. The phrase
"recovering glucosamine" refers simply to collecting the product
from the fermentation bioreactor and need not imply additional
steps of separation or purification. For example, the step of
recovering can refer to removing the entire culture (i.e., the
microorganism and the fermentation medium) from the bioreactor,
removing the fermentation medium containing extracellular
glucosamine from the bioreactor, and/or removing the microorganism
containing intracellular glucosamine-6-phosphate from the
bioreactor. These steps can be followed by further purification
steps. Glucosamine is preferably recovered in substantially pure
form. As used herein, "substantially pure" refers to a purity that
allows for the effective use of the glucosamine as a nutriceutical
compound for commercial sale. In one embodiment, the glucosamine
product is preferably separated from the production organism and
other fermentation medium constituents. Methods to accomplish such
separation are described below.
[0085] Preferably, by the method of the present invention, at least
about 1 g/L of product (i.e., glucosamine and/or
glucosamine-6-phosphate) are recovered from the microorganism
and/or fermentation medium. More preferably, by the method of the
present invention, at least about 5 g/L, and even more preferably,
at least about 10 g/L, and even more preferably, at least about 20
g/L and even more preferably, at least about 50 g/L of product are
recovered. In one embodiment, glucosamine product is recovered in
an amount from about 1 g/L to about 50 g/L.
[0086] Typically, most of the glucosamine produced in the present
process is extracellular. The microorganism can be removed from the
fermentation medium by conventional methods, such as by filtration
or centrifugation. In one embodiment, the step of recovering the
product includes the purification of glucosamine from the
fermentation medium. Glucosamine can be recovered from the
cell-free fermentation medium by conventional methods, such as
chromatography, extraction, crystallization (e.g., evaporative
crystallization), membrane separation, reverse osmosis and
distillation. In a preferred embodiment, glucosamine is recovered
from the cell-free fermentation medium by crystallization. In
another embodiment, the step of recovering the product includes the
step of concentrating the extracellular glucosamine.
[0087] In one embodiment, glucosamine-6-phosphate accumulates
intracellularly, the step of recovering the product includes
isolating glucosamine-6-phosphate from the microorganism. For
example, the product can be recovered by lysing the microorganism
cells by a method which does not degrade the glucosamine product,
centrifuging the lysate to remove insoluble cellular debris, and
then recovering the glucosamine and/or glucosamine-6-phosphate
product by a conventional method as described above.
[0088] The initial intracellular product in the genetically
modified microorganism described herein is glucosamine-6-phosphate.
It is generally accepted that phosphorylated intermediates are
dephosphorylated during export from the microorganism, most likely
due to the presence of alkaline phosphatase in the periplasmic
space of the microorganism. In one embodiment of the present
invention, glucosamine-6-phosphate is dephosphorylated before or
during export from the cell by naturally occurring phosphatases in
order to facilitate the production of the desired product,
glucosamine. In this embodiment, the need for amplification of a
recombinantly provided phosphatase activity in the cell or
treatment of the fermentation medium with a phosphatase is
obviated. In another embodiment, the level of phosphatase in the
production organism is increased by a method including, but not
limited to, genetic modification of an endogenous phosphatase gene
or by recombinant modification of the microorganism to express a
phosphatase gene. In yet another embodiment, the recovered
fermentation medium is treated with a phosphatase after
glucosamine-6-phosphate is released into the medium, such as when
cells are lysed as described above.
[0089] As noted above, the process of the present invention
produces significant amounts of extracellular glucosamine. In
particular, the process produces extracellular glucosamine such
that greater than about 50% of total glucosamine is extracellular,
more preferably greater than about 75% of total glucosamine is
extracellular, and most preferably greater than about 90% of total
glucosamine is extracellular. By the method of the present
invention, production of an extracellular glucosamine concentration
can be achieved which is greater than about 1 g/l, more preferably
greater than about 5 g/l, even more preferably greater than about
10 g/l, and even more preferably greater than about 20 g/L and even
more preferably greater than about 50 g/l.
[0090] One embodiment of the present invention relates to a method
to produce glucosamine by fermentation which includes the steps of
(a) culturing an Escherichia coli having a genetically modified
amino sugar metabolic pathway in a fermentation medium comprising
assimilable sources of carbon, nitrogen and phosphate to produce a
product, and (b) recovering the product. The product includes
intracellular glucosamine-6-phosphate which is recovered from the
Escherichia coli and/or extracellular glucosamine which is
recovered from the fermentation medium.
[0091] One embodiment of the present invention relates to a
microorganism for producing glucosamine by a biosynthetic process.
The microorganism is transformed with a recombinant nucleic acid
molecule encoding glucosamine-6-phosphate synthase operatively
linked to a transcription control sequence. The recombinant nucleic
acid molecule has a genetic modification which reduces
glucosamine-6-phosphate product inhibition of the
glucosamine-6-phosphate synthase. Expression of the recombinant
nucleic acid molecule increases expression of the
glucosamine-6-phosphate synthase by the microorganism. In a
preferred embodiment, the recombinant nucleic acid molecule is
integrated into the genome of the microorganism. In a further
embodiment, the microorganism has at least one additional genetic
modification in a gene encoding a protein selected from the group
of N-acetylglucosamine-6-phosphate deacetylase,
glucosamine-6-phosphate deaminase, N-acetyl-glucosamine-specific
enzyme II.sup.Nag, phosphoglucosamine mutase,
glucosamine-1-phosphate
acetyltransferase-N-acetylglucosamine-1-phosphate
uridyltransferase, phosphofructokinase, Enzyme II.sup.Glc of the
PEP:glucose PTS, EIIM, P/III.sup.man of the PEP:mannose PTS, and/or
a phosphatase. The genetic modification decreases the action of the
protein, except in the case of the phosphatase, in which the action
of the phosphatase is preferably increased. In another preferred
embodiment, the microorganism has a modification in genes encoding
N-acetylglucosamine-6-phosphate deacetylase,
glucosamine-6-phosphate deaminase and N-acetyl-glucosamine-specific
enzyme II.sup.Nag wherein the genetic modification decreases action
of the protein. In one embodiment, the genetic modification is a
deletion of at least a portion of the genes.
[0092] In a preferred embodiment, the genetically modified
microorganism is a bacterium or a yeast, and more preferably, a
bacterium of the genus Escherichia, and even more preferably,
Escherichia coli. A genetically modified Escherichia coli
preferably has a modification in a gene which includes, but is not
limited to, nagA, nagB, nagC, nagD, nagE, manXYZ, glmM, pfkB, pfkA,
glmu, glmS, ptsG or a phosphatase gene. In another embodiment, such
a genetically modified Escherichia coli has a deletion of nag
regulon genes, and in yet another embodiment, a deletion of nag
regulon genes and a genetic modification in manXYZ genes such that
the proteins encoded by the manXYZ genes have decreased action.
[0093] Yet another embodiment of the present invention relates to a
microorganism for producing glucosamine by a biosynthetic process
which has a recombinant nucleic acid molecule encoding
glucosamine-6-phosphate synthase operatively linked to a
transcription control sequence; and at least one genetic
modification in a gene encoding a protein selected from the group
of N-acetylglucosamine-6-phosphate deacetylase,
glucosamine-6-phosphate deaminase, N-acetyl-glucosamine-specific
enzyme II.sup.Nag, phosphoglucosamine mutase,
glucosamine-1-phosphate
acetyltransferase-N-acetylglucosamine-1-phosphate
uridyltransferase, phosphofructokinase, Enzyme II.sup.Glc of the
PEP:glucose PTS, and/or EIIM, P/III.sup.man of the PEP:mannose PTS.
The genetic modification decreases action of said protein and
expression of the recombinant nucleic acid molecule increases
expression of the glucosamine-6-phosphate synthase by the
microorganism. In another embodiment, the microorganism has at
least one genetic modification in a phosphatase gene, such that the
phosphatase encoded by such gene has increased action. In a
preferred embodiment, the recombinant nucleic acid molecule is
integrated into the genome of the microorganism.
[0094] Another embodiment of the present invention relates to any
of the above-described microorganisms which produces at least about
1 g/L of glucosamine when cultured for about 24 hours at 37.degree.
C. to a cell density of at least about 8 g/L by dry cell weight, in
a pH 7.0 fermentation medium comprising: 14 g/L K.sub.2HPO.sub.4,
16 g/L KH.sub.2PO.sub.4, 1 g/L Na.sub.3Citrate-2H.sub.2O, 5 g/L
(NH.sub.4).sub.2SO.sub.4, 20 g/L glucose, 10 mM MgSO.sub.4, 1 mM
CaCl.sub.2, and 1 mM IPTG.
[0095] A more preferred embodiment of the present invention relates
to any of the above-described microorganisms which produces at
least about 1 g/L of glucosamine when cultured for about 10 to
about 60 hours at from about 28.degree. C. to about 37.degree. C.
to a cell density of at least about 8 g/L by dry cell weight, in a
pH 7.0 fermentation medium comprising: 14 g/L K.sub.2HPO.sub.4, 16
g/L KH.sub.2PO.sub.4, 1 g/L Na.sub.3Citrate 2H.sub.2O, 5 g/L
(NH.sub.4).sub.2SO.sub.4, 20 g/L glucose, 10 mM MgSO.sub.4, 1 mM
CaCl.sub.2, and from about 0.2 mM to about 1 mM IPTG. In a
preferred embodiment, the amount of IPTG is about 0.2 mM.
[0096] Yet another embodiment of the present invention relates to
any of the above-described genetically modified microorganisms
which produce at least about 1 g/L, and preferably at least about 5
g/L, and more preferably, at least about 10 g/L, and even more
preferably, at least about 20 g/L, and even more preferably, at
least about 50 g/L of glucosamine and/or glucosamine-6-phosphate
when cultured under the culture conditions as described herein.
Another embodiment of the present invention relates to any of the
above-described genetically modified microorganisms which produce
at least about 2-fold more glucosamine and/or
glucosamine-6-phosphate, and preferably at least about 5-fold, and
more preferably at least about 10-fold, and more preferably at
least about 25-fold, and more preferably at least about 50-fold,
and even more preferably at least about 100-fold, and even more
preferably, at least about 200-fold more glucosamine and/or
glucosamine-6-phosphate synthase than a wild-type (i.e.,
non-modified, naturally occurring) microorganism cultured under the
same conditions as the genetically modified microorganism.
[0097] A number of specific microorganisms are identified in the
Examples section. Additional embodiments of the present invention
include these microorganisms and microorganisms having the
identifying characteristics of the microorganisms specifically
identified in the Examples. Such microorganisms are preferably
yeast or bacteria, more preferably, are bacteria, and most
preferably are E. coli. Such identifying characteristics can
include any or all genotypic and/or phenotypic characteristics of
the microorganisms in the Examples, including their abilities to
produce glucosamine.
[0098] Preferred microorganisms of the present invention include
strains of Escherichia coli which have been transformed with a
recombinant nucleic acid molecule encoding glucosamine-6-phosphate
synthase. Preferably, such a nucleic acid molecule is integrated
into the genome of the microorganism. A particularly preferred
microorganism is Escherichia coli strain 2123-12. Strain 2123-12
has integrated into its genome a recombinant nucleic acid molecule
comprising a nucleic acid sequence SEQ ID NO:15, which represents
the coding region of a wild-type (i.e., normal, unmodified, or
naturally occurring) glucosamine-6-phosphate synthase enzyme having
amino acid sequence SEQ ID NO:16. Particularly preferred
microorganisms of the present invention have been transformed with
a nucleic acid molecule comprising a nucleic acid sequence encoding
a glucosamine-6-phosphate synthase that has been genetically
modified such that the synthase has increased action (described
above). Most preferably, such genetic modification enhances the
ability of the microorganism to produce glucosamine as compared to
a microorganism which has not been transformed with such a nucleic
acid molecule. Particularly preferred genetically modified
microorganisms of the present invention are described in the
Examples section, and include E. coli strains 2123-49, 2123-54,
2123-124, 2123-149 and 2123-151.
[0099] Development of a microorganism with enhanced ability to
produce glucosamine by genetic modification can be accomplished
using both classical strain development and molecular genetic
techniques. In general, the strategy for creating a microorganism
with enhanced glucosamine production is to (1) inactivate or delete
at least one, and preferably more than one of the amino sugar
metabolic pathways in which production of glucosamine-6-phosphate
is negatively affected (e.g., inhibited), and (2) amplify at least
one, and preferably more than one of the amino sugar metabolic
pathways in which glucosamine-6-phosphate production is enhanced.
As such, genetically modified microorganisms of the present
invention have a (a) reduced ability to convert
glucosamine-6-phosphate into other compounds (i.e., inhibition of
glucosamine-6-phosphate catabolic or anabolic pathways), (b) an
enhanced ability to produce (i.e., synthesize)
glucosamine-6-phosphate, (c) a reduced ability to transport
glucosamine into the cell, (d) an enhanced ability to transport
glucosamine-6-phosphate or glucosamine out of the cell, and/or (e)
a reduced ability to use substrates involved in the production of
glucosamine-6-P for competing biochemical reactions.
[0100] As previously discussed herein, in one embodiment, a
genetically modified microorganism can be a microorganism in which
nucleic acid molecules have been deleted, inserted or modified,
such as by insertion, deletion, substitution, and/or inversion of
nucleotides, in such a manner that such modifications provide the
desired effect within the microorganism. Such genetic modifications
can, in some embodiments, be within the coding region for a protein
encoded by the nucleic acid molecule which results in amino acid
modifications such as insertions, deletions, substitutions in the
amino acid sequence of the protein which provide the desired effect
within the microorganisms. A genetically modified microorganism can
be modified by recombinant technology, such as by introduction of
an isolated nucleic acid molecule into a microorganism. For
example, a genetically modified microorganism can be transfected
with a recombinant nucleic acid molecule encoding a protein of
interest, such as a protein for which increased expression is
desired. The transfected nucleic acid molecule can remain
extrachromosomal or can integrate into one or more sites within a
chromosome of the transfected (i.e., recombinant) host cell in such
a manner that its ability to be expressed is retained. Preferably,
once a host cell of the present invention is transfected with a
nucleic acid molecule, the nucleic acid molecule is integrated into
the host cell genome. A significant advantage of integration is
that the nucleic acid molecule is stably maintained in the cell. In
a preferred embodiment, the integrated nucleic acid molecule is
operatively linked to a transcription control sequence (described
below) which can be induced to control expression of the nucleic
acid molecule.
[0101] A nucleic acid molecule can be integrated into the genome of
the host cell either by random or targeted integration. Such
methods of integration are known in the art. For example, as
described in detail in Example 2, E. coli strain ATCC 47002 (Table
1) contains mutations that confer upon it an inability to maintain
plasmids which contain a ColE1 origin of replication. When such
plasmids are transferred to this strain, selection for genetic
markers contained on the plasmid results in integration of the
plasmid into the chromosome. This strain can be transformed, for
example, with plasmids containing the gene of interest and a
selectable marker flanked by the 5'- and 3'-termini of the E. coli
lacZ gene. The lacZ sequences target the incoming DNA to the lacZ
gene contained in the chromosome. Integration at the lacZ locus
replaces the intact lacZ gene, which encodes the enzyme
.beta.-galactosidase, with a partial lacZ gene interrupted by the
gene of interest. Successful integrants can be selected for
.beta.-galactosidase negativity. A genetically modified
microorganism can also be produced by introducing nucleic acid
molecules into a recipient cell genome by a method such as by using
a transducing bacteriophage. The use of recombinant technology and
transducing bacteriophage technology to produce several different
genetically modified microorganism of the present invention is
known in the art and is described in detail in the Examples
section. According to the present invention, a gene, for example
the pstG gene, includes all nucleic acid sequences related to a
natural pstG gene such as regulatory regions that control
production of the pstG protein (Enzyme II.sup.Glc of the
PEP:glucose PTS) encoded by that gene (such as, but not limited to,
transcription, translation or post-translation control regions) as
well as the coding region itself. In another embodiment, a gene,
for example the pstG gene, can be an allelic variant (i.e., a
naturally occurring allelic variant) that includes a similar but
not identical sequence to the nucleic acid sequence encoding a
given pstG gene. An allelic variant of a pstG gene which has a
given nucleic acid sequence is a gene that occurs at essentially
the same locus (or loci) in the genome as the gene having the given
nucleic acid sequence, but which, due to natural variations caused
by, for example, mutation or recombination, has a similar but not
identical sequence. Allelic variants typically encode proteins
having similar activity to that of the protein encoded by the gene
to which they are being compared. Allelic variants can also
comprise alterations in the 5' or 3' untranslated regions of the
gene (e.g., in regulatory control regions). Allelic variants are
well known to those skilled in the art and would be expected to be
found within a given microorganism, such as an E. coli, and/or
among a group of two or more microorganisms.
[0102] Although the phrase "nucleic acid molecule" primarily refers
to the physical nucleic acid molecule and the phrase "nucleic acid
sequence" primarily refers to the sequence of nucleotides on the
nucleic acid molecule, the two phrases can be used interchangeably,
especially with respect to a nucleic acid molecule, or a nucleic
acid sequence, being capable of encoding a gene involved in an
amino sugar metabolic pathway. In addition, the phrase "recombinant
molecule" primarily refers to a nucleic acid molecule operatively
linked to a transcription control sequence, but can be used
interchangeably with the phrase "nucleic acid molecule" which is
isolated and expressed in a host cell.
[0103] Knowing the nucleic acid sequences of certain nucleic acid
molecules of the present invention, and particularly Escherichia
coli nucleic acid molecules, allows one skilled in the art to, for
example, (a) make copies of those nucleic acid molecules and/or (b)
obtain nucleic acid molecules including at least a portion of such
nucleic acid molecules (e.g., nucleic acid molecules including
full-length genes, full-length coding regions, regulatory control
sequences, truncated coding regions). Such nucleic acid molecules
can be obtained in a variety of ways including traditional cloning
techniques using oligonucleotide probes of to screen appropriate
libraries or DNA and PCR amplification of appropriate libraries or
DNA using oligonucleotide primers. Preferred libraries to screen or
from which to amplify nucleic acid molecule include bacterial and
yeast genomic DNA libraries, and in particular, Escherichia coli
genomic DNA libraries. Techniques to clone and amplify genes are
disclosed, for example, in Sambrook et al., ibid.
[0104] In accordance with the present invention, an isolated
nucleic acid molecule is a nucleic acid molecule that has been
removed from its natural milieu (i.e., that has been subject to
human manipulation). As such, "isolated" does not reflect the
extent to which the nucleic acid molecule has been purified. An
isolated nucleic acid molecule can include DNA, RNA, or derivatives
of either DNA or RNA. There is no limit, other than a practical
limit, on the maximal size of a nucleic acid molecule in that the
nucleic acid molecule can include a portion of a gene, an entire
gene, or multiple genes, or portions thereof.
[0105] An isolated nucleic acid molecule of the present invention
can be obtained from its natural source either as an entire (i.e.,
complete) gene or a portion thereof capable of forming a stable
hybrid with that gene. An isolated nucleic acid molecule can also
be produced using recombinant DNA technology (e.g., polymerase
chain reaction (PCR) amplification, cloning) or chemical synthesis.
Isolated nucleic acid molecules include natural nucleic acid
molecules and homologues thereof, including, but not limited to,
natural allelic variants and modified nucleic acid molecules in
which nucleotides have been inserted, deleted, substituted, and/or
inverted in such a manner that such modifications provide the
desired effect within the microorganism.
[0106] A nucleic acid molecule homologue can be produced using a
number of methods known to those skilled in the art (see, for
example, Sambrook et al., ibid.). For example, nucleic acid
molecules can be modified using a variety of techniques including,
but not limited to, classic mutagenesis techniques and recombinant
DNA techniques, such as site-directed mutagenesis, chemical
treatment of a nucleic acid molecule to induce mutations,
restriction enzyme cleavage of a nucleic acid fragment, ligation of
nucleic acid fragments, PCR amplification and/or mutagenesis of
selected regions of a nucleic acid sequence, synthesis of
oligonucleotide mixtures and ligation of mixture groups to "build"
a mixture of nucleic acid molecules and combinations thereof.
Nucleic acid molecule homologues can be selected from a mixture of
modified nucleic acids by screening for the function of the protein
encoded by the nucleic acid and/or by hybridization with a
wild-type gene. Examples of such techniques are described in detail
in the Examples section.
[0107] In one embodiment of the present invention, a nucleic acid
homologue of a nucleic acid molecule of the present invention
preferably comprises a genetic modification which results in an
modification of the action of the protein encoded by the nucleic
acid homologue. For example, in one embodiment of the present
invention, a genetically modified recombinant nucleic acid molecule
is provided which comprises a nucleic acid sequence encoding a
glucosamine-6-phosphate synthase protein homologue, wherein the
genetic modification increases the action of the
glucosamine-6-phosphate synthase homologue, preferably as compared
to a recombinant nucleic acid molecule encoding a naturally
occurring glucosamine-6-phosphate synthase in the absence of such
genetic modification. Such a genetic modification can increase the
action of the glucosamine-6-phosphate synthase, for example, by
encoding a glucosamine-6-phosphate synthase having reduced
glucosamine-6-phosphate product inhibition and/or increased
specific activity. Such recombinant nucleic acid molecules having
genetic modifications are referred to herein as nucleic acid
homologues of wild-type nucleic acid molecules encoding
glucosamine-6-phosphate synthase. According to the present
invention, proteins having modifications as a result of genetic
modifications in the nucleic acid molecules encoding the proteins
are referred to herein as protein homologues, or homologues of the
given protein.
[0108] Accordingly, a glucosamine-6-phosphate synthase protein, for
example, which has glucosamine-6-phosphate synthase activity and is
useful in the present invention, can be a full-length
glucosamine-6-phosphate synthase protein, an enzymatically active
portion of a full-length glucosamine-6-phosphate synthase protein,
or any homologue of such proteins, such as a
glucosamine-6-phosphate synthase protein having at least one or a
few amino acid modifications in which amino acid residues have been
deleted (e.g., a truncated version of the protein, such as a
peptide), inserted, inverted, substituted and/or derivatized (e.g.,
by glycosylation, phosphorylation, acetylation, myristoylation,
prenylation, palmitation, amidation and/or addition of
glycosylphosphatidyl inositol).
[0109] A protein homologue of any of the proteins within the amino
sugar metabolic pathways as described in the present invention is a
protein having an amino acid sequence that is sufficiently similar
to a natural protein amino acid sequence (i.e., naturally
occurring, unmodified, or wild-type) that a nucleic acid sequence
encoding the homologue is capable of hybridizing under stringent
conditions to (i.e., with) a nucleic acid molecule encoding the
natural protein (i.e., to the complement of the nucleic acid strand
encoding the natural protein amino acid sequence). A nucleic acid
sequence complement of any nucleic acid sequence of the present
invention refers to the nucleic acid sequence of the nucleic acid
strand that is complementary to (i.e., can form a double helix with
the entire molecule) the strand for which the sequence is cited. It
is to be noted that a double-stranded nucleic acid molecule of the
present invention for which a nucleic acid sequence has been
determined for one strand that is represented by a SEQ ID NO also
comprises a complementary strand having a sequence that is a
complement of that SEQ ID NO. As such, nucleic acid molecules of
the present invention, which can be either double-stranded or
single-stranded, include those nucleic acid molecules that form
stable hybrids under stringent hybridization conditions with either
a given SEQ ID NO denoted herein and/or with the complement of that
SEQ ID NO, which may or may not be denoted herein. Methods to
deduce a complementary sequence are known to those skilled in the
art. The minimal size of a protein homologue of the present
invention is a size sufficient to be encoded by a nucleic acid
molecule capable of forming a stable hybrid with the complementary
sequence of a nucleic acid molecule encoding the corresponding
natural protein. Additionally, the minimal size of a protein
homologue of the present invention is a size sufficient to have
glucosamine-6-phosphate synthase action (e.g., a catalytically or
enzymatically active portion), and preferably, increased
glucosamine-6-phosphate synthase action. As such, the size of the
nucleic acid molecule encoding such a protein homologue is
dependent on nucleic acid composition and percent homology between
the nucleic acid molecule and complementary sequence as well as
upon hybridization conditions per se (e.g., temperature, salt
concentration, and formamide concentration). There is no limit,
other than a practical limit, on the maximal size of such a nucleic
acid molecule in that the nucleic acid molecule can include a
portion of a gene, an entire gene, or multiple genes, or portions
thereof. Similarly, the minimal size of a protein homologue of the
present invention is from about 4 to about 6 amino acids in length,
with preferred sizes depending on whether a full-length,
multivalent (i.e., fusion protein having more than one domain each
of which has a function), or functional portions of such proteins
are desired.
[0110] As used herein, stringent hybridization conditions refer to
standard hybridization conditions under which nucleic acid
molecules are used to identify similar nucleic acid molecules. Such
standard conditions are disclosed, for example, in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs
Press, 1989. Sambrook et al., ibid., is incorporated by reference
herein in its entirety (see specifically, pages 9.31-9.62, 11.7 and
11.45-11.61). In addition, formulae to calculate the appropriate
hybridization and wash conditions to achieve hybridization
permitting varying degrees of mismatch of nucleotides are
disclosed, for example, in Meinkoth et al., 1984, Anal. Biochem.
138, 267-284; Meinkoth et al., ibid., is incorporated by reference
herein in its entirety.
[0111] More particularly, stringent hybridization conditions, as
referred to herein, refer to conditions which permit isolation of
nucleic acid molecules having at least about 70% nucleic acid
sequence identity with the nucleic acid molecule being used to
probe in the hybridization reaction, more particularly at least
about 75%, and most particularly at least about 80%. Such
conditions will vary, depending on whether DNA:RNA or DNA:DNA
hybrids are being formed. Calculated melting temperatures for
DNA:DNA hybrids are 10.degree. C. less than for DNA:RNA hybrids. In
particular embodiments, stringent hybridization conditions for
DNA:DNA hybrids include hybridization at an ionic strength of
0.1.times.SSC (0.157 M Na.sup.+) at a temperature of between about
20.degree. C. and about 35.degree. C., more preferably, between
about 28.degree. C. and about 40.degree. C., and even more
preferably, between about 35.degree. C. and about 45.degree. C. In
particular embodiments, stringent hybridization conditions for
DNA:RNA hybrids include hybridization at an ionic strength of
0.1.times.SSC (0.157 M Na.sup.+) at a temperature of between about
30.degree. C. and about 45.degree. C., more preferably, between
about 38.degree. C. and about 50.degree. C., and even more
preferably, between about 45.degree. C. and about 55.degree. C.
These values are based on calculations of a melting temperature for
molecules larger than about 100 nucleotides, 0% formamide and a G+C
content of about 50%. Alternatively, T.sub.m can be calculated
empirically as set forth in Sambrook et al., supra, pages 11.55 to
11.57.
[0112] Protein homologues of proteins involved in an amino sugar
metabolic pathway according to the present invention can be the
result of natural allelic variation or natural mutation. Protein
homologues of the present invention can also be produced using
techniques known in the art including, but not limited to, direct
modifications to the protein or modifications to the gene encoding
the protein using, for example, classic or recombinant DNA
techniques to effect random or targeted mutagenesis, as discussed
above.
[0113] In one embodiment of the present invention, a genetic
modification in a recombinant nucleic acid molecule of the present
invention which encodes a glucosamine-6-phosphate synthase results
in at least one amino acid modification (i.e., modification in the
amino acid sequence of the encoded protein) selected from the group
of an addition, substitution, deletion, and/or derivatization of an
amino acid residue of the glucosamine-6-phosphate synthase. Such a
modification in the amino acid sequence of the encoded protein can
be determined as compared to a wild-type, or naturally occurring
glucosamine-6-phosphate synthase, such as a glucosamine-6-phosphate
synthase having an amino acid sequence SEQ ID NO:16. One or more of
such amino acid modifications results in increased action of
glucosamine-6-phosphate synthase as compared to the naturally
occurring glucosamine-6-phosphate synthase having amino acid
sequence SEQ ID NO:16. In one embodiment, such an amino acid
modification is in an amino acid sequence position in the modified
protein (i.e., homologue) which corresponds to one or more of the
following amino acid positions in amino acid sequence SEQ ID NO:16:
Ile(4), Ile(272), Ser(450), Ala(39), Arg(250), Gly(472),
Leu(469).
[0114] In another embodiment, such an amino acid modification is
selected from the group of a substitution of: (a) an amino acid
residue having an aliphatic hydroxyl side group for Ile(4); (b) an
amino acid residue having an aliphatic hydroxyl side group for
Ile(272); (c) an amino acid residue having an aliphatic side group
for Ser(450); (d) an amino acid residue having an aliphatic
hydroxyl side group for Ala(39); (e) an amino acid residue having a
sulfur-containing side group for Arg(250); (f) an amino acid
residue having an aliphatic hydroxyl side group for Gly(472); (g)
an amino acid residue having an aliphatic side group for Leu(469);
and, (h) combinations of (a)-(g). According to the present
invention, amino acid residues having an aliphatic hydroxyl group
include serine and threonine, and amino acid residues having
aliphatic side groups include glycine, alanine, valine, leucine,
isoleucine and proline.
[0115] In yet another embodiment of the present invention, an amino
acid modification as described above is preferably a substitution
selected from the group of: Ile(4) to Thr, Ile(272) to Thr,
Ser(450) to Pro, Ala(39) to Thr, Arg(250) to Cys, Gly(472) to Ser,
Leu(469) to Pro, and combinations thereof. Specific examples of
recombinant nucleic acid molecules having genetic modifications
resulting in such amino acid modifications are described in detail
in the Examples section.
[0116] Preferred genetically modified recombinant nucleic acid
molecules comprising a nucleic acid sequence encoding a
glucosamine-6-phosphate synthase having increased action include
recombinant nucleic acid molecules comprising a nucleic acid
sequence which encodes a glucosamine-6-phosphate synthase
comprising an amino acid sequence selected from the group of SEQ ID
NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28 and/or SEQ ID
NO:31. Other preferred genetically modified recombinant nucleic
acid molecules comprise a nucleic acid sequence selected from the
group of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21,
SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID
NO:29 and/or SEQ ID NO:30. Particularly preferred genetically
modified recombinant nucleic acid molecules useful in the present
invention include nucleic acid molecules comprising nucleic acid
molecules selected from the group of pKLN23-49, pKLN23-54,
pKLN23-124, pKLN23-149, pKLN23-151, nglmS-49.sub.2184,
nglmS-49.sub.1830, nglmS-54.sub.2184, nglmS-54.sub.1830,
nglmS-124.sub.2184, nglmS-124.sub.1830, nglmS-149.sub.2184,
nglmS-149.sub.1830, nglmS-151.sub.2184 and nglmS-151.sub.1830.
[0117] The present invention includes a recombinant vector, which
includes at least one isolated nucleic acid molecule of the present
invention, inserted into any vector capable of delivering the
nucleic acid molecule into a bacterial cell. Such a vector can
contain bacterial nucleic acid sequences that are not naturally
found adjacent to the isolated nucleic acid molecules to be
inserted into the vector. The vector can be either RNA or DNA and
typically is a plasmid. Recombinant vectors can be used in the
cloning, sequencing, and/or otherwise manipulating of nucleic acid
molecules. One type of recombinant vector, referred to herein as a
recombinant nucleic acid molecule and described in more detail
below, can be used in the expression of nucleic acid molecules.
Preferred recombinant vectors are capable of replicating in a
transformed bacterial or yeast cell, and in particular, in an
Escherichia coli cell.
[0118] Transformation of a nucleic acid molecule into a cell can be
accomplished by any method by which a nucleic acid molecule can be
inserted into the cell. Transformation techniques include, but are
not limited to, transfection, electroporation and
microinjection.
[0119] Recombinant molecules of the present invention, which can be
either DNA or RNA, can also contain additional regulatory
sequences, such as translation regulatory sequences, origins of
replication, and other regulatory sequences that are compatible
with the recombinant cell. One or more recombinant molecules of the
present invention can be used to produce an encoded product (e.g.,
a glucosamine-6-phosphate synthase). In one embodiment, an encoded
product is produced by expressing a nucleic acid molecule of the
present invention under conditions effective to produce the
protein. Such conditions (i.e., culture conditions) have been
described above and are further discussed in the Examples section.
A preferred method to produce an encoded protein is by transfecting
a host cell with one or more recombinant molecules of the present
invention to form a recombinant cell.
[0120] As discussed above, preferred recombinant molecules of the
present invention include, nglmS-28.sub.2184, nglmS-28.sub.1830,
nglmS-49.sub.2184, nglmS-49.sub.1830, nglmS-54.sub.2184,
nglmS-54.sub.1830, nglmS-124.sub.2184, nglmS-124.sub.1830,
nglmS-149.sub.2184, nglmS-149.sub.1830, nglmS-151.sub.2184,
nglmS-151.sub.1830, pKLN23-28, pKLN23-49, pKLN23-54, pKLN23-124,
pKLN23-149 and/or pKLN23-151.
[0121] A recombinant cell is preferably produced by transforming a
bacterial cell (i.e., a host cell) with one or more recombinant
molecules, each comprising one or more nucleic acid molecules
operatively linked to an expression vector containing one or more
transcription control sequences. The phrase, operatively linked,
refers to insertion of a nucleic acid molecule into an expression
vector in a manner such that the molecule is able to be expressed
when transformed into a host cell. As used herein, an expression
vector is a DNA or RNA vector that is capable of transforming a
host cell and of effecting expression of a specified nucleic acid
molecule. Preferably, the expression vector is also capable of
replicating within the host cell. In the present invention,
expression vectors are typically plasmids. Expression vectors of
the present invention include any vectors that function (i.e.,
direct gene expression) in a yeast host cell or a bacterial host
cell, preferably an Escherichia coli host cell. Preferred
recombinant cells of the present invention are set forth in the
Examples section.
[0122] Nucleic acid molecules of the present invention can be
operatively linked to expression vectors containing regulatory
sequences such as transcription control sequences, translation
control sequences, origins of replication, and other regulatory
sequences that are compatible with the recombinant cell and that
control the expression of nucleic acid molecules of the present
invention. In particular, recombinant molecules of the present
invention include transcription control sequences. Transcription
control sequences are sequences which control the initiation,
elongation, and termination of transcription. Particularly
important transcription control sequences are those which control
transcription initiation, such as promoter, enhancer, operator and
repressor sequences. Suitable transcription control sequences
include any transcription control sequence that can function in
yeast or bacterial cells and preferably, Escherichia coli. A
variety of such transcription control sequences are known to those
skilled in the art.
[0123] It may be appreciated by one skilled in the art that use of
recombinant DNA technologies can improve expression of transformed
nucleic acid molecules by manipulating, for example, the number of
copies of the nucleic acid molecules within a host cell, the
efficiency with which those nucleic acid molecules are transcribed,
the efficiency with which the resultant transcripts are translated,
and the efficiency of post-translational modifications. Recombinant
techniques useful for increasing the expression of nucleic acid
molecules of the present invention include, but are not limited to,
operatively linking nucleic acid molecules to high-copy number
plasmids, integration of the nucleic acid molecules into the host
cell chromosome, addition of vector stability sequences to
plasmids, substitutions or modifications of transcription control
signals (e.g., promoters, operators, enhancers), substitutions or
modifications of translational control signals, modification of
nucleic acid molecules of the present invention to correspond to
the codon usage of the host cell, deletion of sequences that
destabilize transcripts, and use of control signals that temporally
separate recombinant cell growth from recombinant enzyme production
during fermentation. The activity of an expressed recombinant
protein of the present invention may be improved by fragmenting,
modifying, or derivatizing nucleic acid molecules encoding such a
protein. Such modifications are described in detail in the Examples
section.
[0124] The following experimental results are provided for the
purposes of illustration and are not intended to limit the scope of
the invention.
EXAMPLES
Example 1
[0125] The following example describes the production of mutant
Escherichia coli strains which are blocked in amino acid sugar
metabolic pathways involving degradation of glucosamine.
[0126] The starting strain for the construction of all glucosamine
overproducing strains described herein was E. coli W3110 (publicly
available from the American Type Culture Collection as ATCC No.
25947), a prototrophic, F.sup.- .lamda..sup.- derivative of E. coli
K-12 (Bachmann, 1987, "Escherichia coli and Salmonella
typhimurium", Cellular and Molecular Biology, pp. 1190-1219;
incorporated herein by reference in its entirety). A list of all
strains used and produced in the following examples is provided in
Table 1.
TABLE-US-00001 TABLE 1 Bacterial strains. Strain Alias Genotype
Source/Reference W3110 F.sup.- mcrA mcrB IN(rrnD-rrnE) 1
.lamda..sup.- ATCC IBPC 522 thi-1 argG6 argE3 his-4 mtl-1 xyl-5
rpsL J. Plumbridge tsx-29? .DELTA.lacX74 manXYZ8 nagE47 ptsG22
zcf-229::Tn10 IBPC 566 thi-1 argG6 argE3 his-4 mtl-1 xyl-5 rpsL J.
Plumbridge tsx-29? .DELTA.lacX74 manXYZ8 zdj-225::Tn10 IBPC 590
thi-1 argG6 argE3 his-4 mtl-1 xyl-5 rpsL J. Plumbridge tsx-29?
.DELTA.lacX74 .DELTA.nag::TcR 7101-6 W3110 ptsM F.sup.- mcrA mcrB
IN(rrnD-rrnE) 1 .lamda..sup.- W3110 x P1.sub.vir(IBPC566) manXYZ8
zdj-225::Tn10 7101-7 W3110 ptsM F.sup.- mcrA mcrB IN(rrnD-rrnE) 1
.lamda..sup.- W3110 x P1.sub.vir(IBPC566) manXYZ8 zdj-225::Tn10
7101-9 W3110 .DELTA.nag F.sup.- mcrA mcrB IN(rrnD-rrnE) 1
.lamda..sup.- W3110 x P1.sub.vir(IBPC590) .DELTA.nag::TcR 7101-13
W3110 ptsM F.sup.- mcrA mcrB IN(rrnD-rrnE) 1 .lamda..sup.- 7101-6
selected on TCS medium TcS manXYZ8 zdj-225::Tn10? TcS 7101-14 W3110
ptsM F.sup.- mcrA mcrB IN(rrnD-rrnE) 1 .lamda..sup.- 7101-7
selected on TCS medium TcS manXYZ8 zdj-225::Tn10? TcS 7101-15 W3110
ptsM F.sup.- mcrA mcrB IN(rrnD-rrnE) 1 .lamda..sup.- 7101-14 x
P1.sub.vir(IBPC522) ptsG manXYZ8 zdj-225::Tn10? ptsG22 zcf-
229::Tn10 7101-17 W3110 ptsM F.sup.- mcrA mcrB IN(rrnD-rrnE) 1
.lamda..sup.- 7101-13 x P1.sub.vir(IBPC590) .DELTA.nag manXYZ8
zdj-225::Tn10? TcS .DELTA.nag::TcR 7101-22 W3110 ptsM F.sup.- mcrA
mcrB IN(rrnD-rrnE) 1 .lamda..sup.- 7101-15 selected on TCS medium
ptsG TcS manXYZ8 zdj-225::Tn10? ptsG22 zcf- 229::Tn10? TcS 2123-4
W3110 ptsM F.sup.- mcrA mcrB IN(rrnD-rrnE) 1 .lamda..sup.- 7101-22
x P1.sub.vir(IBPC590) ptsG .DELTA.nag manXYZ8 zdj-225::Tn10? ptsG22
zcf- 229::Tn10? TcS .DELTA.nag::TcR W3110(DE3) F.sup.- mcrA mcrB
IN(rrnD-rrnE) 1 .lamda.DE3 W3110 lysogenized with .lamda.DE3
7101-9(DE3) F.sup.- mcrA mcrB IN(rrnD-rrnE) 1 .lamda.DE3 7101-9
lysogenized with .lamda.DE3 .DELTA.nag::TcR 7101-17(DE3) F.sup.-
mcrA mcrB IN(rrnD-rrnE) 1 .lamda.DE3 7101-17 lysogenized with
.lamda.DE3 manXYZ8 zdj-225::Tn10? TcS .DELTA.nag::TcR 2123-4(DE3)
F.sup.- mcrA mcrB IN(rrnD-rrnE) 1 .lamda.DE3 2123-4 lysogenized
with .lamda.DE3 manXYZ8 zdj-225::Tn10? ptsG22 zcf- 229::Tn10 TcS
.DELTA.nag::TcR BL21(DE3) F.sup.- ompT hsdS.sub.B gal dcm
.lamda.DE3 Novagen, Inc. ATCC 47002 JC7623 F.sup.- recB21 recC22
sbcB15 leu-6 ara-14 ATCC his-4 .lamda..sup.- T-71 F.sup.- recB21
recC22 sbcB15 leu-6 ara-14 Integration of pT7-glmS-Cm into lacZ of
his-4 .lamda..sup.- lacZ::pT7-glmS-Cm8H7 ATCC47002 by
transformation with pKLN23-28 T-81 F.sup.- recB21 recC22 sbcB15
leu-6 ara-14 Integration of pT7-glmS-Cm into lacZ of his-4
.lamda..sup.- lacZ::pT7-glmS-Cm8H8 ATCC47002 by transformation with
pKLN23-28 2123-5 W3110(DE3) lacZ::pT7-glmS-Cm8H7 W3110(DE3) x P1
.sub.vir(T-71) 2123-6 W3110(DE3) lacZ::pT7-glmS-Cm8H8 W3110(DE3) x
P1 .sub.vir(T-81) 2123-7 W3110(DE3) lacZ::pT7-glmS-Cm8H7 W3110(DE3)
x P1 .sub.vir(T-71) 2123-8 W3110(DE3) lacZ::pT7-glmS-Cm8H8
W3110(DE3) x P1 .sub.vir(T-81) 2123-9 7101-9(DE3)
lacZ::pT7-glmS-Cm8H7 7101-9(DE3) x P1 .sub.vir(T-71) 2123-10
7101-9(DE3) lacZ::pT7-glmS-Cm8H8 7101-9(DE3) x P1 .sub.vir(T-81)
2123-11 7101-17(DE3) lacZ::pT7-glmS-Cm8H7 7101-17(DE3) x P1
.sub.vir(T-71) 2123-12 7101-17(DE3) lacZ::pT7-glmS-Cm8H8
7101-17(DE3) x P1 .sub.vir(T-81) 2123-13 2123-4(DE3)
lacZ::pT7-glmS-Cm8H7 2123-4(DE3) x P1 .sub.vir(T-71) 2123-14
2123-4(DE3) lacZ::pT7-glmS-Cm8H8 2123-4(DE3) x P1 .sub.vir(T-81)
NovaBlue endA1 hsdR17 supE44 thi-1 recA1 Novagen gyrA96 relA1 lac
[F' proA.sup.+B.sup.+ lacl.sup.qZ.DELTA.M15::Tn10] LE392 F.sup.-
e14.sup.- (McrA.sup.-) hsdR514(r.sup.-m.sup.+) supE44 Lab
collection supF58 lacY1 or .DELTA.lac(IZY)6 galK2 galT22 metB1
trpR55 2123-16 LE392 glmS13 NG mutagenesis of LE392 2123-49
7101-17(DE3) lacZ::pT7-glmS11C- Error-prone PCR with pKLN23-28;
Cm8H8 integration of mutant glmS into ATCC47002; transfer to
7101-17(DE3) by P1 transduction 2123-51 7101-17(DE3)
lacZ::pT7-glmS52B- Error-prone PCR with pKLN23-28; Cm8H8
integration of mutant glmS into ATCC47002; transfer to 7101-17(DE3)
by P1 transduction 2123-54 7101-17(DE3) lacZ::pT7-glmS8A-
Error-prone PCR with pKLN23-28; Cm8H8 integration of mutant glmS
into ATCC47002; transfer to 7101-17(DE3) by P1 transduction
2123-124 7101-17(DE3) Error-prone PCR with pKLN23-28;
lacZ::pT7-glmS94A integration of mutant glmS into S-Cm8H8
ATCC47002; transfer to 7107-17(DE3) by P1 transduction 2123-149
7101-17(DE3) pKLN23-54 EcoRI-HindIII (1.0 kb) .times.
lacZ::pT7-glmS149-Cm8H8 pKLN23-28 EcoRI-HindIII (6.4 kb);
integration of mutant glmS into ATCC47002; transfer to 7101-17(DE3)
by P1 transduction 2123-151 7101-17(DE3) pKLN23-54 EcoRI-HindIII
(1.0 kb) .times. lacZ::pT7-glmS151-Cm8H8 pKLN23-28 EcoRI-HindIII
(6.4 kb); integration of mutant glmS into ATCC47002; transfer to
7101-17(DE3) by P1 transduction
[0127] Host strains blocked for glucosamine uptake and degradation
were constructed by introducing mutations in the nagE, manXYZ and
ptsG genes, which block transport of glucosamine, and the nagA, -B,
-C, and -D genes, which are involved in metabolism of
glucosamine-6-phosphate. Each of these genes has been described in
detail previously herein. Mutations in these genes were introduced
into strains using the transducing bacteriophage Pl.sub.vir (as
described in Miller, 1972, "Experiments in Molecular Genetics",
Cold Spring Harbor Laboratory, which is incorporated herein by
reference in its entirety).
[0128] In this technique, genes or mutations from one strain (the
donor strain) are transferred to a recipient strain using the
bacteriophage Pl.sub.vir. When bacteriophage Pl.sub.vir is grown on
the donor strain, a small portion of the phage particles that are
produced contain chromosomal DNA from the donor rather than the
normal complement of phage DNA. Upon infection of the recipient
strain with bacteriophage grown on the donor strain, those
bacteriophage particles containing chromosomal DNA from the donor
strain can transfer that DNA to the recipient strain. If there is a
strong selection for the DNA from the donor strain, recipient
strains containing the appropriate gene or mutation from the donor
strain can be selected.
[0129] To grow Pl.sub.vir on a donor strain, an existing
bacteriophage stock was used to infect a culture of that strain.
The recipient strain was grown at 37.degree. C. in LBMC medium (10
g/L Bacto tryptone, 5 g/L yeast extract, 10 g/L NaCl, 1 mM
MgCl.sub.2, 5 mM CaCl.sub.2) until the absorbance at 600 nm was
approximately 1.0, corresponding to approximately 6.times.10.sup.8
cells per mL of culture. One mL of the culture was then infected
with a dilution of the phage stock at a ratio of approximately one
phage per 10 cells. The mixture was incubated without shaking for
20 minutes at 37.degree. C., then transferred to 10 mL of prewarmed
LBMC broth in a 125 mL baffled Erlenmeyer flask. The resulting
culture was shaken vigorously for 2-3 hours at 37.degree. C. During
this period, it was generally observed that the culture would
become more turbid, indicating bacterial growth. Toward the end of
this incubation period, the culture would become clear, indicating
cell lysis due to bacteriophage growth. After lysis had occurred,
the culture was cooled on ice, a few drops of chloroform were
added, and the flask was shaken briefly. The contents of the flask
were then centrifuged to remove the cell debris and chloroform, and
the resulting supernatant generally contained between 10.sup.8 and
10.sup.9 bacteriophage per mL.
[0130] Mutations were transferred to recipient strains by
transduction with Pl.sub.vir grown on the appropriate donor strain
as described above. For transduction with Pl.sub.vir, a culture of
the recipient strain was grown overnight at 37.degree. C. in LBMC
broth. 0.1 mL of culture was mixed with 0.1 mL of bacteriophage
lysate or a serial dilution of the lysate in a sterile test tube
and incubated at 37.degree. C. for 20 minutes. 0.2 mL of 1 M sodium
citrate was added to the tube, and the mixture was plated to
selective medium. For each transduction, controls containing
uninfected cells and bacteriophage lysates without cells were
performed as described above. For the production of strains blocked
in glucosamine degradation, selections were for tetracycline
resistance as described below. Tetracycline resistant mutants were
selected by plating to LB medium (10 g/L Bacto tryptone, 5 g/L
yeast extract, 10 g/L NaCl) containing 12.5 .mu.g/mL tetracycline
and 10 mM sodium citrate.
[0131] The mutations in the nag genes were introduced
simultaneously as a deletion mutation (.DELTA.nag::Tc.sup.R). In
strain IBPC590 (Plumbridge, Table 1), which contains this mutation,
the nag genes have been replaced by a tetracycline-resistance
(Tc.sup.R) determinant. As a result, the mutation which removes the
nag functions was transferred to appropriate recipient hosts by
selection for tetracycline resistance. In this case, since the
Tc.sup.R determinant was contained within the mutation of interest,
the .DELTA.nag and Tc.sup.R mutations were 100% linked. That is,
all of the recipient strains receiving the Tc.sup.R determinant
from IBPC590 also received the .DELTA.nag mutation. This was
confirmed by examining the growth phenotype of the tetracycline
resistant strains resulting from infection with Pl.sub.vir grown on
IBPC590. All such strains were unable to grow on media containing
glucosamine or N-acetylglucosamine as carbon sources, indicating
the presence of the .DELTA.nag mutation.
[0132] Mutations in the manXYZ and ptsG genes were also introduced
by Pl.sub.vir transduction using phage grown on strains IBPC566 and
IBPC522 (Plumbridge, Table 1), respectively. These strains also
contained tetracycline-resistance determinants linked to the
mutations of interest (designated zdj-225::Tn10 and zcf-229::Tn10,
respectively). In these strains, the Tc.sup.R determinants were not
within the gene but were linked to the gene. Accordingly, not all
recipient strains receiving the Tc.sup.R determinant contained the
mutations of interest. The degree of linkage is indicative of the
distance on the chromosome between the Tc.sup.R determinant and the
mutation of interest. As a result, it was necessary to screen
tetracycline resistant strains for manXYZ and ptsG. The manXYZ
strains grew slowly on mannose and failed to grow on glucosamine as
sole carbon sources for growth. The ptsG strains grew slowly on
glucose as sole carbon source.
[0133] Because all of the selections for the mutations described
above were for tetracycline resistance, it was necessary to render
strains tetracycline sensitive between steps if multiple mutations
were to be introduced. To accomplish this, tetracycline-resistant
strains were plated to TCS medium (15 g/L agar, 5 g/L Bacto
tryptone, 5 g/L yeast extract, 50 mg/L chlortetracycline
hydrochloride, 10 g/L NaCl, 10 g/L NaH.sub.2PO.sub.4H.sub.2O, 12
mg/L fusaric acid, and 0.1 mM ZnCl.sub.2) which selects for
tetracycline sensitive mutants (described in Maloy and Nunn, 1981,
J. Bacteriol., 145:1110-1112, which is incorporated herein by
reference in its entirety). Colonies arising on this medium were
purified by restreaking to the same medium, then checking
individual colonies for tetracycline sensitivity by plating to LB
media with and without 12.5 .mu.g/mL tetracycline.
[0134] The scheme described above for the production of strains
containing combinations of the manXYZ, ptsG, and .DELTA.nag
mutations is presented schematically in FIG. 3.
Example 2
[0135] The following Example describes the cloning and
overexpression of the glmS gene and the integration of the T7-glmS
gene cassette into the E. coli chromosome.
[0136] Cloning and Overexpression of the glmS Gene.
[0137] Using information obtained from the published sequence of
the glmS gene (Walker et al., 1984, Biochem. J., 224:799-815, which
is incorporated herein by reference in its entirety), primers were
synthesized to amplify the gene from genomic DNA isolated from
strain W3110 (Table 1) using the polymerase chain reaction (PCR).
The primers used for PCR amplification were designated Up1 and Lo8
and had the following sequences:
TABLE-US-00002 (SEQ ID NO: 1) Up1:
5'-CGGTCTCCCATGTGTGGAATTGTTGGCGC-3' (SEQ ID NO: 2) Lo8:
5'-CTCTAGAGCGTTGATATTCAGTCAATTACAAACA-3'
[0138] The Up1 primer contained sequences corresponding to the
first twenty nucleotides of the glmS gene (represented in
nucleotides 10-29 of SEQ ID NO:1) preceded by a BsaI restriction
endonuclease recognition site (GGTCTC, represented in nucleotides
2-7 of SEQ ID NO:1). The Lo8 primer contained sequences
corresponding to positions between 145 and 171 bases downstream of
the glmS gene (represented in nucleotides 8-34 of SEQ ID NO:2)
preceded by a XbaI restriction endonuclease site (TCTAGA,
represented in nucleotides 2-7 of SEQ ID NO:2). PCR amplification
was conducted using a standard protocol to generate a fragment of
DNA containing the glmS gene with 171 base pairs of DNA downstream
of the gene flanked by BsaI and XbaI sites. This DNA fragment was
cloned into the vector pCR-Script.TM.SK(+) (Stratagene Cloning
Systems, La Jolla, Calif.) using materials and instructions
supplied by the manufacturer. The resulting plasmid was designated
pKLN23-20.
[0139] One consequence of this cloning was that it placed a unique
SacI restriction endonuclease site downstream of the gene. This
allowed excision of a fragment of DNA containing the glmS gene from
pKLN23-20 using the restriction endonucleases BsaI and SacI. This
fragment was then cloned between the NcoI and SacI sites of the
expression vector pET-24d(+) (Novagen, Inc., Madison, Wis.) to
generate plasmid pKLN23-23. The pET-24d(+) expression vector is
based on the T7 promoter system (Studier and Moffatt, 1986, J. Mol.
Biol., 189:113-130). Cloning in this manner resulted in placement
of the glmS gene behind the T7-lac promoter contained on
pET-24d(+). The T7-lac promoter is specifically recognized by the
T7 RNA polymerase and is only expressed in strains containing a
cloned T7 gene 1, which encodes the T7 RNA polymerase. The cloned
T7 polymerase gene is contained on a defective bacteriophage
.lamda. phage designated .lamda.DE3. Strains in which the
.lamda.DE3 element is integrated into the chromosome contain the T7
RNA polymerase gene driven by the lacUV5 promoter. In those
strains, expression of the T7 RNA polymerase gene can be induced
using the lactose analog isopropylthio-.beta.-D-galactopyranoside
(IPTG). Accordingly, addition of IPTG to such cultures results in
induction of the T7 RNA polymerase gene and expression of any genes
controlled by the T7 or T7-lac promoter.
[0140] To verify that pKLN23-23 contained the glmS gene driven by
the T7-lac promoter, the plasmid was transferred to strain
BL21(DE3) (Novagen, Inc.) (Table 1). Strain BL21(DE3)/pKLN23-23 was
grown in duplicate in LB medium containing 50 mg/L kanamycin
(kanamycin resistance is encoded by the plasmid). One of the
duplicates was induced with 1 mM IPTG; the other was not. When the
total proteins were examined from these two cultures by sodium
dodecyl sulfate polyacrylamide gel electrophoresis, a prominent
protein of approximately 70,000 molecular weight, corresponding to
the predicted size for the glmS gene product, was observed in cells
from the induced culture but not in cells from the uninduced
culture. A preliminary enzyme assay from an induced culture
indicated several hundred fold higher glucosamine-6-phosphate
synthase activity in the induced culture than in what had typically
been observed in a wild type strain.
[0141] Integration of the T7-glmS Gene Cassette into the E. coli
Chromosome.
[0142] The glmS gene driven by the T7-lac (the T7-glmS gene
cassette) promoter was transferred to the chromosome of E. coli
strains by a multistep process. First, the cassette was cloned into
plasmid pBRINT-Cm (Balbas et al., 1996, Gene 96:65-69), which is
incorporated herein by reference in its entirety). The gene
cassette was then integrated into the chromosome of strain
ATCC47002 (Table 1) by the techniques described by Balbas et al.,
1996, supra, to generate strains T-71 and T-81 (Table 1). Finally,
the gene cassette was transferred to strains of interest by
transduction with Pl.sub.vir, as described below.
[0143] The T7-glmS cassette was excised from pKLN23-23 by
performing a partial digest of the plasmid with restriction
endonuclease BglII and a complete digest with restriction
endonuclease HinDIII. Plasmid pKLN23-23 contains a BglII site
approximately 20 base pairs upstream of the T7 promoter. The glmS
gene also contains two BglII sites. A partial digest with this
enzyme was necessary to cut the plasmid upstream of the T7 promoter
while avoiding the two internal sites. Plasmid pKLN23-23 also
contains a unique HinDIII site downstream of the glmS gene. The
excised T7-glmS cassette was then cloned between the unique BamHI
and HinDIII sites of pBRINT-Cm. This resulted in the production of
plasmids designated pKLN23-27 and pKLN23-28. Plasmids pKLN23-27 and
pKLN23-28 contain the T7-glmS cassette in addition to a
chloramphenicol resistance determinant flanked by the 5'- and
3'-termini of the E. coli lacZ gene.
[0144] Strain ATCC 47002 (Table 1) contains mutations that confer
upon it an inability to maintain plasmids such as pBRINT-Cm which
contain a ColE1 origin of replication. When such plasmids are
transferred to this strain, selection for genetic markers contained
on the plasmid results in integration of the plasmid into the
chromosome (Balbas et al., 1996, supra). As mentioned above,
plasmids pKLN23-27 and -28 contain the T7-glmS cassette and a
chloramphenicol resistance determinant flanked by the 5'- and
3'-termini of the E. coli lacZ gene. The lacZ sequences target the
incoming DNA to the lacZ gene contained in the chromosome.
Integration at the lacZ locus replaces the intact lacZ gene, which
encodes the enzyme .beta.-galactosidase, with a partial lacZ gene
interrupted by the T7-glmS-Cm cassette. As a result, integration at
lacZ results in the strain becoming .beta.-galactosidase negative.
The plasmid also contains an ampicillin resistance determinant
remote from the 5'-lacZ-T7-glmS-Cm-lacZ-3' cassette. Integration at
lacZ and plasmid loss also results in ampicillin sensitivity.
[0145] Plasmids pKLN23-27 and -28 were transferred to strain ATCC
47002, and cells were plated to LB medium containing 10 .mu.g/mL
chloramphenicol, 1 mM IPTG, and 40 .mu.g/mL
5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside (X-gal). The
X-gal contained in the medium is a chromogenic .beta.-galactosidase
substrate. Hydrolysis of X-gal by .beta.-galactosidase results in a
blue derivative. Inclusion of X-gal and IPTG, which induces the
native lacZ gene, results in blue lacZ-positive colonies and white
lacZ-negative colonies. White (lacZ-negative) chloramphenicol
resistant colonies were then selected and purified. The strains
were then verified for sensitivity to ampicillin by plating to LB
media with and without 100 .mu.g/mL ampicillin. DNA integration was
further confirmed using a PCR scheme as described by Balbas et al.,
1996, supra. Integrants T-71 and T-81 (Table 1) resulted from the
integration of the desired segments of plasmids pKLN23-27 and
pKLN23-28, respectively, into the chromosome of ATCC 47002.
[0146] The T7-glmS-Cm cassette was then transferred to strains
W3110(DE3), 7101-9(DE3), 7101-17(DE3), and 2123-4(DE3) by
Pl.sub.vir transduction, as described in Example 1, using lysates
prepared on strains T-71 and T-81. These strains contain various
combinations of the .DELTA.nag, manXYZ, and ptsG mutations in
addition to the .lamda.DE3 element necessary for expression from
the T7-lac promoter. The .lamda.DE3 element was introduced to these
strains using the .lamda.DE3 lysogenization kit produced by
Novagen, Inc. (Madison, Wis.). Transductants were selected on LB
agar plates containing 30 .mu.g/mL chloramphenicol and 10 mM sodium
citrate. Loss of .beta.-galactosidase activity was verified on
plates containing X-gal and IPTG and DNA integration was further
confirmed using a PCR scheme as described by Balbas et al., 1996,
supra.
[0147] Glucosamine-6-phosphate synthase activity was measured in
production strains containing integrated T7-glmS cassettes after
growth in LB medium with and without IPTG (Table 2).
Glucosamine-6-phosphate synthase was assayed in crude cell extracts
using either colorimetric or spectrophotometric assays (Badet et
al., 1987, Biochemistry 26:1940-1948) as described below. The
extracts used for those assays were prepared by suspending washed
cell pellets in 5 mL of 0.1 M KH.sub.2PO.sub.4/K.sub.2HPO.sub.4, pH
7.5 per gram of wet cell paste, passing the suspension through a
French press at 16,000 psi, and centrifuging the disrupted cell
suspension at 35,000-40,000.times.g for 15 to 20 minutes. The
supernatant was used as the source of enzyme for the assay.
[0148] For a colorimetric assay, 1 mL reactions were prepared
containing 45 mM KH.sub.2PO.sub.4/K.sub.2HPO.sub.4, 20 mM
fructose-6-phosphate, 15 mM L-glutamine, 2.5 mM EDTA, pH 7.5, and
cell extract. The reactions were incubated at 37.degree. C. for 20
minutes and stopped by boiling for 4 minutes. The resulting
precipitate was removed by centrifugation and the supernatant was
assayed for glucosamine-6-phosphate by a modification of the method
of Elson and Morgan (1933, Biochem. J. 27:1824-1828) essentially as
described by Zalkin (1985, Meth. Enzymol. 113:278-281), both
publications of which are incorporated herein by reference in their
entireties. To 100 .mu.L of the above supernatant was added 12.5
.mu.L of saturated NaHCO.sub.3 and 12.5 .mu.L of cold, freshly
prepared 5% aqueous acetic anhydride. After incubating for 3
minutes at room temperature, the mixture was boiled for 3 minutes
to drive off excess acetic anhydride. After cooling to room
temperature, 150 .mu.L of 0.8 M potassium borate, pH 9.2 (0.8 M
H.sub.3BO.sub.3 adjusted to pH 9.2 with KOH) was added and the
mixture was boiled for 3 minutes. After cooling to room
temperature, 1.25 mL Ehrlich's reagent (1%
p-dimethylaminobenzaldehyde in glacial acetic acid containing 0.125
N HCl) was added to each tube. After incubating at 37.degree. C.
for 30 minutes, the absorbance at 585 nm was measured and the
amount of glucosamine-6-phosphate formed was determined using a
standard curve. In the absence of the substrate,
fructose-6-phosphate, or when boiled extract was used in the assay,
no significant absorbance at 585 nm was observed.
[0149] In the spectrophotometric assay, 1 mL reactions containing
50 mM KH.sub.2PO.sub.4/K.sub.2HPO.sub.4, 10 mM
fructose-6-phosphate, 6 mM L-glutamine, 10 mM KCl, 0.6 mM
acetylpyridine adenine dinucleotide (APAD), and 50-60 Units of
L-glutamic dehydrogenase (Sigma Type II from bovine liver) were run
at room temperature. The activity was followed by monitoring the
absorbance at 365 nm after the addition of extract and corrected
for the small absorbance increase observed in the absence of
extract. The activity was calculated using a molar extinction
coefficient for APAD of 9100.
TABLE-US-00003 TABLE 2 Glucosamine 6-Phosphate Synthase Activity in
Production Strains Containing Integrated T7-glmS Cassettes
Activity, (.mu.mole per minute per mL of extract) Strain Host
Genotype -IPTG +IPTG 2123-5 DE3 23 64 2123-6 DE3 4 4 2123-7 DE3 23
96 2123-8 DE3 25 89 2123-9 DE3 .DELTA.nag 26 58 2123-10 DE3
.DELTA.nag 33 67 2123-11 DE3 .DELTA.nag manXYZ 32 59 2123-12 DE3
.DELTA.nag manXYZ 17 67 2123-13 DE3 .DELTA.nag manXYZ ptsG 21 68
2123-14 DE3 .DELTA.nag manXYZ ptsG 20 88
[0150] Table 2 shows that, on average, the activity of
glucosamine-6-phosphate synthase in production strains containing
integrated T7-glmS cassettes was about three- to four-fold higher
with IPTG induction than without. The activities were substantially
higher than those obtained with a wild type glmS strain driven by
its native promoter, which typically were in the range of 0.05-0.1
.mu.mole per minute per mL of extract. One of the strains, 2123-6,
apparently suffered an aberrant integration event since the
activity was lower than that observed in the other strains and was
not influenced by the presence of IPTG in the medium.
Example 3
[0151] The following example shows the effect of strain genotype on
glucosamine accumulation.
[0152] Strains with T7-glmS integrants, produced as described in
Example 2, as well as the corresponding parent strains without
integrated DNA, were grown in shake flasks containing M9A medium
(14 g/L K.sub.2HPO.sub.4, 16 g/L KH.sub.2PO.sub.4, 1 g/L
Na.sub.3Citrate-2H.sub.2O, 5 g/L (NH.sub.4).sub.2SO.sub.4, pH 7.0)
supplemented with 20 g/L glucose, 10 mM MgSO.sub.4, 1 mM
CaCl.sub.2, and 1 mM IPTG. Samples were taken periodically over the
course of two days, and the glucosamine concentration in the
culture supernatant was measured using the modified Elson-Morgan
assay as described in Example 2. Samples were assayed with and
without acetic anhydride treatment, and the amount of glucosamine
present was determined from the net absorbance using a standard
curve.
[0153] Glucosamine concentrations after 24 hours of cultivation, at
which time the concentration peaked, are indicated in Table 3. The
results shown in Table 3 indicate that for significant glucosamine
production to occur, the T7-glmS gene cassette must be present. The
data also indicate that the presence of the .DELTA.nag mutation in
the host results in a significant increase in glucosamine
accumulation compared with its absence. Little effect of the manXYZ
mutation was observed in this experiment. In further shake flask
experiments, however, strain 2123-12 accumulated slightly higher
glucosamine concentrations on a consistent basis.
TABLE-US-00004 TABLE 3 Glucosamine in Culture Supernatants of
Production Strains Glucosamine Concentration, Strain Genotype mg/L
(24 hours) 2123-5 DE3, T-71 integrant 21 2123-7 DE3, T-71 integrant
23 2123-9 DE3 .DELTA.nag, T-71 integrant 67 2123-10 DE3 .DELTA.nag,
T-81 integrant 80 2123-11 DE3 .DELTA.nag manXYZ, T-71 integrant 65
2123-12 DE3 .DELTA.nag manXYZ, T-81 integrant 63 W3110(DE3) DE3, no
integrant 4 7101-9(DE3) DE3 .DELTA.nag, no integrant 0 7101-17(DE3)
DE3 .DELTA.nag manXYZ, no integrant 0
Example 4
[0154] The following example demonstrates the effect feeding
nutrients to the cultures has on glucosamine accumulation.
[0155] In early experiments, it was observed that glucosamine
accumulation ceased when glucose was depleted from cultures. In the
experiment summarized by Table 4 and FIG. 4, it was found that
increased glucosamine accumulation could be accomplished by feeding
additional glucose and ammonium sulfate as they became depleted.
For this experiment, strain 2123-12 was grown in M9A medium
supplemented with 10 mM MgSO.sub.4, 1 mM CaCl.sub.2, and 1 mM IPTG.
Initial glucose concentrations and feeding regimens were varied as
indicated in Table 4. In one of the flasks, the initial ammonium
sulfate concentration was 10 g/L rather than the 5 g/L normally
used in M9A medium. Glucose concentration was monitored in shake
flasks during cultivation using Diastix.RTM. glucose test strips
(Bayer Corporation Diagnostics Division, Elkhart, Ind.). When the
glucose concentration was at or near depletion (<5 g/L
remaining), glucose and/or ammonium sulfate were supplemented as
indicated in Table 4. pH was also monitored during cultivation.
When the pH varied significantly from the initial pH of 7.0, it was
adjusted to 7.0 with sodium hydroxide.
TABLE-US-00005 TABLE 4 Shake Flask Experiment to Examine the Effect
of Glucose Feeding Initial Initial Ammonium Flask No. Glucose, g/L
Sulfate, g/L Feed 1 20 5 None 2 50 5 None 3 50 10 None 4 20 5 20
g/L Glucose 5 20 5 20 g/L Glucose + 5 g/L AmSO.sub.4
[0156] As FIG. 4 indicates, increasing the supply of glucose had a
positive effect on glucosamine accumulation. By periodically
feeding with glucose and ammonium sulfate (20 g/L and 5 g/L
additions, respectively), a maximum accumulation of 0.4 g/L of
glucosamine was observed, approximately four-fold higher than what
was observed in the absence of feeding.
Example 5
[0157] The following example describes the isolation of mutant glmS
genes encoding glucosamine-6-phosphate synthase enzymes with
decreased sensitivity to glucosamine-6-phosphate product
inhibition.
[0158] White (1968, Biochem. J., 106:847-858) first demonstrated
that glucosamine-6-phosphate synthase was inhibited by
glucosamine-6-phosphate. Using the spectrophotometric assay for
glucosamine-6-phosphate synthase as described in Example 2, the
effects of glucosamine-6-phosphate and glucosamine on
glucosamine-6-phosphate synthase were measured. For determination
of product inhibition, assays were run in the presence of various
concentrations of added glucosamine-6-phosphate.
[0159] As indicated in FIG. 5, the enzyme is significantly
inhibited by glucosamine-6-phosphate and slightly inhibited by
glucosamine. These results are similar to those obtained by White,
1968, supra. This inhibition may be a key factor in limiting
glucosamine accumulation in the glucosamine production strains.
[0160] To further increase glucosamine synthesis in production
strains, efforts were made to isolate mutants of the glmS gene
encoding glucosamine-6-phosphate synthase variants with reduced
product inhibition. To accomplish this, the cloned gene was
amplified using the technique of error-prone PCR. In this method,
the gene is amplified under conditions that lead to a high
frequency of misincorporation errors by the DNA polymerase used for
the amplification. As a result, a high frequency of mutations are
obtained in the PCR products.
[0161] Plasmid pKLN23-28 contains a unique SpeI restriction
endonuclease site 25 base pairs upstream of the T7 promoter and 111
base pairs upstream of the start of the glmS gene. The plasmid also
contains a unique HinDIII site 177 base pairs downstream of the
glmS gene. PCR primers of the following sequences were synthesized
to correspond to regions just upstream of the SpeI and downstream
of the HinDIII sites, respectively:
TABLE-US-00006 5'-ATGGATGAGCAGACGATGGT-3' (SEQ ID NO: 3)
5'-CCTCGAGGTCGACGGTATC-3' (SEQ ID NO: 4)
[0162] Amplification with these primers (SEQ ID NO:3 and SEQ ID
NO:4) allowed mutagenesis of a 2247 base pair region that included
the entire glmS gene. PCR conditions were as described by Moore and
Arnold, 1996, Nature Biotechnology 14:458-467, which is
incorporated herein by reference in its entirety. Briefly, a 100
.mu.L solution was prepared containing 1 mM each of the four
deoxynucleotide triphosphates, 16.6 mM ammonium sulfate, 67 mM
Tris-HCl, pH 8.8, 6.1 mM MgCl.sub.2, 6.7 .mu.M EDTA, 10 mM
.beta.-mercaptoethanol, 10 .mu.L DMSO, 30 ng each of the primers
(SEQ ID NO:3 and SEQ ID NO:4), either 7 or 35 ng of plasmid
pKLN23-28 linearized with Kpn I, and 2.5 Units of Taq DNA
polymerase (Perkin Elmer-Cetus, Emeryville, Calif.). The reaction
mixture was covered with 70 .mu.L of mineral oil and placed in a
thermocycler, where the following steps were repeated for 25
cycles:
1 minute at 94.degree. C. 1 minute at 42.degree. C. 2 minutes at
72.degree. C.
[0163] Under these conditions, an error frequency of approximately
one mutation per 1000 base pairs has been reported (Moore and
Arnold, 1996, supra). The product of the reaction was recovered,
purified, and digested with SpeI and HinDIII, and cloned into the
SpeI-HinDIII backbone fragment of pKLN23-28, which effectively
substitutes for the wild type glmS gene on the SpeI-HinDIII
fragment of pKLN23-28. The cloned DNA was used to transform strain
NovaBlue (Novagen, Inc., Madison, Wis.), and the transformed cells
were plated to LB agar containing ampicillin. A total of 9000
plasmid-containing colonies were pooled from the ampicillin plates
and plasmid DNA was prepared from the pooled cells to generate a
library of pKLN23-28 derivative plasmids containing mutations in
the cloned glmS gene.
[0164] The mutant plasmids generated by error-prone PCR were
screened for their ability to confer increased glucosamine
production in a .DELTA.nag manXYZ DE3 host background. This screen
was in the form of a bioassay in which the ability of
plasmid-containing strains to crossfeed glucosamine-requiring
strains of E. coli was assessed.
[0165] Strains of E. coli (Sarvas, 1971, J. Bacteriol. 105:467-471;
Wu and Wu, 1971, J. Bacteriol. 105:455-466) and Bacillus subtilis
(Freese et al., 1970, J. Bacteriol. 101:1046-1062) which are
defective for glucosamine-6-phosphate synthase require glucosamine
or N-acetylglucosamine for growth. A glucosamine-requiring strain
of E. coli was isolated after mutagenesis with
N-methyl-N'-nitro-N-nitrosoguanidine (NG). Strain LE392 (Table 1)
was grown in LB medium to a cell density of 6.times.10.sup.8 cells
per mL. 50 .mu.L of 2.5 mg/mL NG dissolved in methanol was added to
2 mL of this culture and the mixture was incubated at 37.degree. C.
for 20 minutes. This treatment resulted in about 10% survival of
the strain. The mutagenized cells were harvested by centrifugation,
and the cells were washed twice by suspension in 0.9% NaCl and
recentrifugation. The washed cells were diluted and plated to
nutrient agar medium (NA; 5 g/L Bacto peptone, 3 g/L beef extract,
15 g/L agar) containing 0.2 g/L N-acetylglucosamine at a density of
between 50 and 200 colony forming units per plate. Approximately
13,000 colonies were plated. These colonies were replica-plated to
NA agar with and without 0.2 g/L N-acetylglucosamine. Twenty-two
colonies grew on NA with 0.2 g/L N-acetylglucosamine but not on NA
without 0.2 g/L N-acetylglucosamine. These colonies were purified
by streaking to NA with 0.2 g/L N-acetylglucosamine, and their
growth phenotype was rechecked. Of the original 22 colonies
selected, five had the phenotype expected of a glmS mutant of
LE392. They failed to grow on NA but grew on NA supplemented with
0.2 g/L of glucosamine or 0.2 g/L N-acetylglucosamine. They also
failed to grow on glucose minimal agar, but grew on glucose minimal
agar supplemented with 0.2 g/L N-acetylglucosamine. One of these
mutants was designated 2123-16 (Table 1).
[0166] For the cross-feeding assay, agar plates containing either
glycerol or fructose as the principle carbon source for growth were
overlaid with cells from a culture of strain 2123-16, the
glucosamine-requiring strain isolated as described above.
Glucosamine-producing strains were stabbed into the agar and the
ability to produce glucosamine was assessed based on the size of
the "halo" of growth of the indicator strain surrounding the stab.
Those stabs surrounded by larger halos were considered to produce
greater amounts of glucosamine.
[0167] The media used for the cross-feeding assays consisted of M9
minimal medium (6 g/L Na.sub.2HPO.sub.4, 3 g/L KH.sub.2PO.sub.4,
0.5 g/L NaCl, 1 g/L NH.sub.4Cl, 1 mM MgSO.sub.4, 0.1 mM CaCl.sub.2)
supplemented with 40 mg/L of L-methionine (required for growth of
strains LE392 or 2123-16) and 2 g/L of either glycerol or fructose.
These plates were overlaid with strain 2123-16 as follows. A
culture of strain 2123-16 was grown overnight at 37.degree. C. in
LB medium containing 1 g/L N-acetylglucosamine. The culture was
harvested by centrifugation, and the cells were washed twice by
suspension in 0.9% NaCl and recentrifugation. The washed cells were
suspended in the original volume of 0.9% NaCl. For each plate to be
overlaid, 0.1 mL of washed cell suspension was mixed with 3 mL of
molten (50.degree. C.) F-top agar (8 g/L NaCl, 8 g/L agar) and
poured onto the plate.
[0168] The library of pKLN23-28 mutant plasmids was transferred to
strain 7101-17(DE3) and transformed cells were plated to LB agar
containing 100 .mu.g/mL ampicillin. Each colony arising on these
plates contained an individual member of the mutant plasmid
library. The colonies were screened by picking them from the
LB+ampicillin plates and stabbing them sequentially into:
(1) LB agar+ampicillin; (2) glycerol minimal agar overlaid with
strain 2123-16; and, (3) fructose minimal agar overlaid with strain
2123-16
[0169] All plates were incubated for about 24 hours at 37.degree.
C. After this incubation period, halos of growth of the 2123-16
indicator strain could be observed surrounding the stabs in the
overlaid plates. Those colonies giving rise to the larger halos
were picked from the corresponding LB+ampicillin plate and streaked
for purification. In an initial screen, 4368 mutant candidates were
screened, and 96 initial candidates were identified. Upon
rescreening those, 30 appeared to be superior to the rest, i.e.
resulted in larger halos of the indicator strain.
[0170] Enzyme assays performed with six of the plasmid-containing
strains isolated as described above indicated that three of the
strains were less sensitive to inhibition by
glucosamine-6-phosphate than the enzyme from the control strain
7101-17(DE3)/pKLN23-28. The strains were grown overnight in LB
broth containing 100 .mu.g/mL ampicillin and 1 mM IPTG. Extracts
prepared from cells harvested from those cultures were assayed for
glucosamine-6-phosphate synthase using the spectrophotometric assay
(described in Example 2) in the presence and absence of added
glucosamine-6-phosphate. The mutant clones designated 11C, 65A, and
8A were significantly less sensitive to glucosamine-6-phosphate
than the control strain (FIG. 6). Other mutants were not
distinguishable from the control by this assay.
Example 6
[0171] The following example describes the construction and
characterization of glucosamine production strains with mutations
in glmS which result in reduced product inhibition.
[0172] Plasmid DNA isolated from clones 11C, 52B, and 8A described
above were transferred to strain ATCC 47002, which had been used
previously to integrate the cloned T7-glmS construct into the E.
coli chromosome. Integration was readily accomplished using the
methods described in Example 2, and the integrated DNA was
transferred to strain 7101-17(DE3) by P1 transduction as described
in Example 1. These procedures produced strains that have the same
genotype as strain 2123-12 except for the presence of mutations in
the glmS gene generated by PCR. These new mutant production strains
were designated 2123-49, 2323-51, and 2123-54, respectively. A
summary of the strain construction strategy is presented in FIG.
7.
[0173] Strains 2123-12, 2123-49, 2123-51, and 2123-54 were grown
overnight in LB broth containing 1 mM IPTG. Extracts prepared from
cells harvested from those cultures were assayed for
glucosamine-6-phosphate synthase using the spectrophotometric assay
described in Example 2 in the presence and absence of added
glucosamine-6-phosphate. The results of these assays are shown in
FIG. 8.
[0174] Glucosamine production in these mutants was significantly
elevated compared to that in 2123-12. When glucosamine production
was assayed in shake flask cultures grown using the glucose and
ammonium sulfate feeding protocol previously described in Example
4, when the cultures were grown to a cell density (measured as
O.D..sub.600) of about 14 (about 8.4 g/L by dry cell weight),
strains 2123-49, 2123-51, and 2123-54 produced 1.5, 2.4, and 5.8
g/L glucosamine, respectively (FIG. 9) compared with 0.3 g/L for
2123-12.
Example 7
[0175] The following example describes the production of yet
another strain with a mutation in glmS which results in reduced
product inhibition.
[0176] An additional 6,344 colonies containing mutant plasmids
generated by error-prone PCR as described in Example 5 were
screened using the cross-feeding assay, also described in Example
5. Fifty four colonies resulted in larger halos than the rest of
the colonies. DNA was isolated from all 54 colonies and strains
isogenic to 2123-12 except for the mutations in glmS were
constructed as described in Example 6.
[0177] Glucosamine production in most of these mutants was
significantly elevated compared to strain 2123-12. Among the newly
isolated mutants, the strain that produced the most glucosamine was
a strain designated 2123-124. This strain produced 3.6 g/L of
glucosamine when production was assayed in shake flasks using the
glucose and ammonium sulfate feeding protocol described in Example
4 compared with 4.3 g/L for strain 2123-54 in a side-by-side
experiment.
Example 8
[0178] The following example describes the sequencing of the cloned
wild type glmS gene present in plasmid pKLN23-28. In addition, the
sequences present in plasmids pKLN23-49, pKLN23-54, and pKLN23-124,
containing the mutant glmS genes used to construct strains 2123-49,
2123-54, and 2123-124, respectively were sequenced and are
described.
[0179] DNA sequencing reactions were performed using the Applied
Biosystems (ABI) Prism Dye Terminator Cycle sequencing method with
AmpliTaq DNA polymerase. The extended products were separated by
gel electrophoresis on an ABI DNA sequencer 373A or 377. Sequences
were analyzed using ABI Sequencing Analysis 3.0 software from ABI
and Sequencher 3.1 from Gene Codes.
[0180] The primers used for sequencing were as follows:
TABLE-US-00007 PK-1: 5'-TGGATGAGCAGACGATGG-3' (SEQ ID NO: 5) PK-2:
5'-TCCGTCACAGGTATTTATTC-3' (SEQ ID NO: 6) PK-3:
5'-AGCTGCGTGGTGCGTAC-3' (SEQ ID NO: 7) PK-4:
5'-GGACCGTGTTTCAGTTCG-3' (SEQ ID NO: 8) PK-5A:
5'-GCCGTGGCGATCAGTAC-3' (SEQ ID NO: 9) PK-6A:
5'-GCCAATCACCAGCGGAC-3' (SEQ ID NO: 10) PK-7:
5'-ATGGTTTCCCGCTACTGG-3' (SEQ ID NO: 11) PK-8:
5'-GAACCAAGGTAACCCAGC-3' (SEQ ID NO: 12)
[0181] The nucleotide sequence of plasmid pKLN23-28, containing the
wild-type glmS gene, was determined to be a 7408 bp nucleic acid
sequence represented herein as SEQ ID NO:13. The 2184 base pairs
between positions 1130 and 3313 of SEQ ID NO:13 were determined
using the primers described above. The nucleic acid molecule
representing positions 1130-3313 of SEQ ID NO:13 is referred to
herein as nglmS-28.sub.2184 and is further identified as SEQ ID
NO:14. nglmS-28.sub.2184 was shown to include the SpeI and HinDIII
sites used to construct the mutant plasmids as described in Example
5. The remaining DNA sequence of SEQ ID NO:13 is based on the known
sequences of the vectors used for the construction of pKLN23-28.
The same 2184 base pair region was sequenced in plasmids pKLN23-49,
pKLN23-54, and pKLN23-124. It is noted that for the discussion of
the mutant glmS genes of these plasmids (Table 5), the specific
nucleotide position of mutations in the nucleotide sequence of the
mutant glmS-containing plasmids will be described using SEQ ID
NO:13 as a reference.
[0182] SEQ ID NOs:13 and 14 contain an open reading frame that
encodes the glmS gene product (i.e., GlcN6P synthase enzyme) which
is a nucleic acid molecule referred to herein as nglmS-28.sub.1830,
the nucleic acid sequence of which is represented by SEQ ID NO:15.
SEQ ID NO:15 spans nucleotides 1253 to 3082 of SEQ ID NO:13, with
an initiation codon spanning from nucleotides 1253-1255 and a
termination codon spanning from nucleotides 3080-3082. SEQ ID NO:15
encodes a protein of 609 amino acids referred to herein as
GlcN6P-S-28, the deduced amino acid sequence of which is
represented herein as SEQ ID NO:16. It is noted that for the
discussion of the mutant glmS gene products produced by the mutant
strains described herein, specific mutations in the amino acid
sequence of the mutant glmS gene products will be described using
SEQ ID NO:16 as a reference.
[0183] The primers described above correspond to the following
nucleotide positions of SEQ ID NO:13:
PK-1 (SEQ ID NO:5): positions 1087-1104 of SEQ ID NO:13; PK-2 (SEQ
ID NO:6): positions 3378-3359 of a nucleic acid sequence
complementary to SEQ ID NO:13; PK-3 (SEQ ID NO:7): positions
1707-1723 of SEQ ID NO:13; PK-4 (SEQ ID NO:8): positions 2772-2755
of a nucleic acid sequence complementary to SEQ ID NO:13; PK-5A
(SEQ ID NO:9): positions 2667-2683 of SEQ ID NO:13; PK-6A (SEQ ID
NO:10): positions 1798-1782 of a nucleic acid sequence
complementary to SEQ ID NO:13; PK-7 (SEQ ID NO:11): positions
2177-2194 of SEQ ID NO:13; PK-8 (SEQ ID NO:12): positions 2364-2347
of a nucleic acid sequence complementary to SEQ ID NO:13.
[0184] The nucleic acid sequence of nucleic acid molecule
nglmS-28.sub.1830 (SEQ ID NO:15, or positions 1253-3082 of SEQ ID
NO:13) from pKLN23-28, differs from the published sequence (Walker,
J. E, et al., 1984, "DNA sequence around the Escherichia coli unc
operon", Biochem. J. 224:799-815) at positions 2509 and 2510 (with
reference to SEQ ID NO:13). The nucleotides for pKLN23-28 at these
positions as determined in this example were G and C, respectively,
while those reported in the published sequence were C and G.
Otherwise, the published and determined sequences of the glmS gene
were identical. The sequences determined upstream and downstream
from the glmS gene were those expected based on the known sequences
of the vectors used for the construction of pKLN23-28 and the
methods used to construct the plasmid.
[0185] The nucleotide sequences for the mutant glmS genes for
plasmids pKLN23-49, pKLN23-54, and pKLN23-124 were determined as
described above for pKLN23-28. Mutations were found in each of
those plasmids. The mutations and the predicted amino acid changes
in the corresponding mutant glmS gene products, as compared to the
wild-type sequence determined for pKLN23-28 (SEQ ID NO:13) are
summarized in Table 5.
TABLE-US-00008 TABLE 5 Mutations in glmS Genes of
Glucosamine-Overproducing strains. Amino Acid Change Plasmid
Position* Base Change (Position**) pKLN23-49 1263 T to C Ile to Thr
(4) 2067 T to C Ile to Thr (272) 2600 T to C Ser to Pro (450)
pKLN23-54 1367 G to A Ala to Thr (39) 2000 C to T Arg to Cys (250)
2239 T to C Silent (329) 2666 G to A Gly to Ser (472) 3264 A to G
Outside gene pKLN23-124 1525 T to C Silent (91) 2658 T to C Leu to
Pro (469) 3280 G to A Outside gene *Refers to nucleic acid position
as indicated in the sequence of pKLN23-28 (SEQ ID NO: 13) **The
glmS gene (nglmS-28.sub.1830; SEQ ID NO: 15) encodes a protein of
609 amino acids in length (SEQ ID NO: 16); the methionine residue
at position 1 is removed by a hydrolase.
[0186] Plasmid pKLN23-49 contains a 2184 bp nucleic acid molecule
referred to herein as nglmS-49.sub.2184, which comprises a mutant
glmS gene. The nucleic acid sequence of nglmS-49.sub.2184 is
represented herein as SEQ ID NO:17. A nucleic acid molecule
spanning from nucleotide 124 through 1953 of SEQ ID NO:17, referred
to herein as nglmS-49.sub.1830, represents an open reading frame
encoding a mutant glucosamine-6-phosphate synthase of the present
invention, with an initiation codon spanning from nucleotides
124-126 and a termination codon spanning from nucleotides 1951-1953
of SEQ ID NO:17. The nucleic acid sequence of nglmS-49.sub.1830 is
represented herein as SEQ ID NO:18. SEQ ID NO:18 encodes a mutant
glucosamine-6-phosphate synthase protein of 609 amino acids
referred to herein as GlcN6P-S-49, the deduced amino acid sequence
of which is represented herein as SEQ ID NO:19. SEQ ID NO:17 has a
nucleic acid sequence that is identical to positions 1130 through
3313 of SEQ ID NO:13 (i.e., SEQ ID NO:14), except for the mutations
as indicated for plasmid pKLN23-49 in Table 5. SEQ ID NO:18 has a
nucleic acid sequence that is identical to positions 1253 through
3082 of SEQ ID NO:13 (i.e., SEQ ID NO:15), except for the mutations
as indicated for plasmid pKLN23-49 in Table 5.
[0187] Plasmid pKLN23-54 contains a 2184 bp nucleic acid molecule
referred to herein as nglmS-54.sub.2184, which comprises a mutant
glmS gene. The nucleic acid sequence of nglmS-54.sub.2184 is
represented herein as SEQ ID NO:20. A nucleic acid molecule
spanning from nucleotide 124 through 1953 of SEQ ID NO:20, referred
to herein as nglmS-54.sub.1830, represents an open reading frame
encoding a mutant glucosamine-6-phosphate synthase of the present
invention, with an initiation codon spanning from nucleotides
124-126 and a termination codon spanning from nucleotides 1951-1953
of SEQ ID NO:20. The nucleic acid sequence of nglmS-54.sub.1830 is
represented herein as SEQ ID NO:21. SEQ ID NO:21 encodes a mutant
glucosamine-6-phosphate synthase protein of 609 amino acids
referred to herein as GlcN6P-S-54, the deduced amino acid sequence
of which is represented herein as SEQ ID NO:22. SEQ ID NO:20 has a
nucleic acid sequence that is identical to positions 1130 through
3313 of SEQ ID NO:13 (i.e., SEQ ID NO:14), except for the mutations
as indicated for plasmid pKLN23-54 in Table 5. SEQ ID NO:21 has a
nucleic acid sequence that is identical to positions 1253 through
3082 of SEQ ID NO:13 (i.e., SEQ ID NO:15), except for the mutations
as indicated for plasmid pKLN23-54 in Table 5.
[0188] Plasmid pKLN23-124 contains a 2184 bp nucleic acid molecule
referred to herein as nglmS-124.sub.2184, which comprises a mutant
glmS gene. The nucleic acid sequence of nglmS-124.sub.2184 is
represented herein as SEQ ID NO:23. A nucleic acid molecule
spanning from nucleotide 124 through 1953 of SEQ ID NO:23, referred
to herein as nglmS-124.sub.1830, represents an open reading frame
encoding a mutant glucosamine-6-phosphate synthase of the present
invention, with an initiation codon spanning from nucleotides
124-126 and a termination codon spanning from nucleotides 1951-1953
of SEQ ID NO:23. The nucleic acid sequence of nglmS-124.sub.1830 is
represented herein as SEQ ID NO:24. SEQ ID NO:24 encodes a mutant
glucosamine-6-phosphate synthase protein of 609 amino acids
referred to herein as GlcN6P-S-124, the deduced amino acid sequence
of which is represented herein as SEQ ID NO:25. SEQ ID NO:23 has a
nucleic acid sequence that is identical to positions 1130 through
3313 of SEQ ID NO:13 (i.e., SEQ ID NO:14), except for the mutations
as indicated for plasmid pKLN23-124 in Table 5. SEQ ID NO:24 has a
nucleic acid sequence that is identical to positions 1253 through
3082 of SEQ ID NO:13 (i.e., SEQ ID NO:15), except for the mutations
as indicated for plasmid pKLN23-124 in Table 5.
[0189] To verify that the same mutations were present in the
strains into which the mutant glmS genes were integrated into the
chromosome, PCR products were generated from genomic DNA isolated
from strains 2123-49, 2123-54, and 2123-124. For PCR amplification,
the primers listed in Example 3 for the mutagenesis of the gene
(SEQ ID NO:3 and SEQ ID NO:4) were used. PCR reactions were carried
out in 50 .mu.L reactions consisting of 20 mM Tris.HCl (pH 8.8), 10
mM KCl, 10 mM (NH.sub.4).sub.2SO.sub.4, 20 mM MgSO.sub.4, 0.1%
Triton X-100, 0.1 mg/mL nuclease-free bovine serum albumin, 0.05 mM
each deoxynucleotide triphosphate, 2 .mu.M each primer, 1.25 U
cloned Pfu DNA polymerase (Stratagene), and 160 ng of genomic DNA.
The complete reactions were placed in a RoboCycler Gradient 96
Temperature Cycler (Stratagene). After 3 minutes at 94.degree. C.,
the following three steps were repeated for 30 cycles: (1) 30
seconds at 94.degree. C.; (2) 30 seconds at 47.degree. C.; and (3)
2 minutes at 72.degree.. This was followed with a 7 minute
incubation at 72.degree. C.
[0190] The resulting DNA contained the expected amplification
product in addition to extraneous products. The product containing
the glmS gene was purified using a QIAquick PCR purification kit,
followed by electrophoresis of the purified product on an agarose
gel, isolation of the correct band using a QIAquick gel extraction
kit, and reamplification using this isolated DNA as a template. The
reactions with the isolated DNA were amplified in a similar fashion
as the original amplification described above, except that 40 ng of
DNA was used as template, and only 20 cycles of amplification were
performed. The product from this second amplification reaction was
recovered as described above.
[0191] The presence of mutations in the genomic DNA was verified
using primers specific for the DNA regions containing the mutations
identified in the plasmids. For 2123-49, these included primers
PK-1 (SEQ ID NO:5), PK-3 (SEQ ID NO:7), PK-4 (SEQ ID NO:8), and
PK-5A (SEQ ID NO:9). For 2123-124, primers PK-1 (SEQ ID NO:5), PK-4
(SEQ ID NO:8) and PK-5 (SEQ ID NO:9) were used. For 2123-54, the
entire PCR product was sequenced using all eight primers described
earlier (SEQ ID NOs:5-12). Sequencing of the PCR products confirmed
the presence of the mutations identified from the plasmids and
listed in Table 5.
Example 9
[0192] This example describes the construction of strains
containing a mutant glmS gene encoding a product containing only
the glycine to serine alteration at position 472 (SEQ ID NO:22)
from strain 2123-54.
[0193] As indicated in Table 5, the only amino acid change in the
GlcN6P synthase enzyme for strain 2123-124 (GlcN6P-S-124) is a
leucine to proline alteration at position 469 (SEQ ID NO:25),
unambiguously defining this mutation as being responsible for the
overproduction of glucosamine by strain 2123-124. This would
suggest the possibility that the glycine to serine alteration at
position 472 (gly.fwdarw.ser472; SEQ ID NO:22) of GlcN6P-S-54 in
strain 2123-54 was likewise responsible for the glucosamine
overproduction phenotype for this strain. In an effort to
demonstrate this, the alteration was isolated away from the other
two amino acid alterations in the GlcN6P-S-54 amino acid sequence
(SEQ ID NO:22) of strain 2123-54 (i.e., Ala.fwdarw.Thr39 and
Arg.fwdarw.Cys250) by digesting plasmid pKLN23-54 with EcoRI and
HinDIII. These enzymes each have unique cleavage sites on the
plasmid and cut at positions 2241 and 3305, respectively (positions
indicated with respect to the equivalent positions in SEQ ID NO:13
for pKLN23-28), resulting in fragments of 1064 and 6344 base pairs.
The smaller fragment contains mutations in which the
gly.fwdarw.ser472 alteration is the only amino acid change in this
portion of GlcN6P-S-54. This smaller fragment was ligated to the
corresponding larger fragment from pKLN23-28 containing the wild
type glmS gene.
[0194] Two plasmids resulting from this ligation were designated
pKLN23-149 and pKLN23-151. Sequencing the DNA from these plasmids
using primers PK-1 (SEQ ID NO:5), PK-3 (SEQ ID NO:7), and PK-4 (SEQ
ID NO:8) verified that these plasmids contained the mutation at
position 2666 present in plasmid pKLN23-54 but not the mutations at
positions 1367 and 2000 (Table 5 with reference to SEQ ID
NO:13).
[0195] The nucleic acid sequence of the 2184 base pairs between
positions 1130 and 3313 of plasmid pKLN23-149 (these positions
being determined relative to the equivalent positions in SEQ ID
NO:13) are referred to herein as nucleic acid molecule
nglmS-149.sub.2184, the nucleic acid sequence of which is
represented by SEQ ID NO:26. SEQ ID NO:26 contains a nucleic acid
sequence spanning nucleotides 124 through 1953, referred to herein
as nglmS-149.sub.1830, which represents an open reading frame
encoding a mutant glucosamine-6-phosphate synthase of the present
invention, with an initiation codon spanning from nucleotides
124-126 and a termination codon spanning from nucleotides 1951-1953
of SEQ ID NO:26. The nucleic acid sequence of nglmS-149.sub.1830 is
represented herein as SEQ ID NO:27. SEQ ID NO:27 encodes a mutant
glucosamine-6-phosphate synthase protein of 609 amino acids
referred to herein as GlcN6P-S-149, the deduced amino acid sequence
of which is represented herein as SEQ ID NO:28.
[0196] The nucleic acid sequence of the 2184 base pairs between
positions 1130 and 3313 of plasmid pKLN23-151 (these positions
being determined relative to the equivalent positions in SEQ ID
NO:13) are referred to herein as nucleic acid molecule
nglmS-151.sub.2184, the nucleic acid sequence of which is
represented by SEQ ID NO:29. SEQ ID NO:29 contains a nucleic acid
sequence spanning nucleotides 124 to 1953, referred to herein as
nglmS-151.sub.1830, which represents an open reading frame encoding
a mutant glucosamine-6-phosphate synthase of the present invention,
with an initiation codon spanning from nucleotides 124-126 and a
termination codon spanning from nucleotides 1951-1953 of SEQ ID
NO:29. The nucleic acid sequence of nglmS-151.sub.1830 is
represented herein as SEQ ID NO:30. SEQ ID NO:30 encodes a mutant
glucosamine-6-phosphate synthase protein of 609 amino acids
referred to herein as GlcN6P-S-151, the deduced amino acid sequence
of which is represented herein as SEQ ID NO:31.
[0197] Strains isogenic to strain 2123-12 except for mutations
conferring the gly.fwdarw.ser472 alteration were constructed using
the scheme indicated in FIG. 7. Strains 2123-149 and 2123-151 were
generated from plasmids pKLN23-149 and pKLN23-151, respectively.
The presence of the mutation at position 2666 (SEQ ID NO:13) and
the absence of mutations at positions 1367 and 2000 were verified
by sequencing of PCR products from genomic DNA of these strains
using the methods described in Example 8.
Example 10
[0198] This example compares properties of GlcN6P synthase enzymes
from strains 2123-12, 2123-49, 2123-54, 2123-124, 2123-149, and
2123-151.
[0199] Strains 2123-12, 2123-49, 2123-54, 2123-124, 2123-149, and
2123-151, described in the examples above, were grown overnight in
LB broth at 37.degree. C. then transferred to fresh LB broth.
Cultures were grown to an absorbance at 600 nm of 0.8 to 0.9, then
induced for GlcN6P synthase production by the addition of 1 mM
IPTG. The cultures were grown for an additional three hours at
37.degree. and harvested. Extracts were prepared from cells
harvested from those cultures as described in Example 2 and were
assayed for glucosamine-6-phosphate synthase using the
spectrophotometric assay as described in Example 2 except that a
fructose-6-phosphate concentration of 20 mM was used. The enzyme
was assayed in the presence and absence of added
glucosamine-6-phosphate. In the absence of glucosamine-6-phosphate,
the specific activities measured for these enzymes were similar
except for that from strain 2123-124. The data from Table 6
suggests that the latter strain encodes a less active variant of
the enzyme.
TABLE-US-00009 TABLE 6 Specific Activities of GlcN6P Synthase from
Glucosamine-Producing Strains Strain Specific Activity, .mu.mol
min.sup.-1 mg.sup.-1 2123-12 0.385 2123-49 0.375 2123-54 0.416
2123-124 0.0076 2123-149 0.494 2123-151 0.515
[0200] FIG. 10 shows that the GlcN6P synthase enzymes from strains
2123-49, 2123-54, and 2123-124 are significantly less inhibited by
GlcN6P than the enzyme from strain 2123-12. Enzymes from strains
2123-149 and 2123-151 are slightly less inhibited by GlcN6P than
the enzyme from 2123-12. Thermal stability of the enzymes was also
examined using these extracts. The extracts were incubated at
45.degree. C. (FIG. 11A) or 50.degree. C. (FIG. 11B) for various
periods then assayed using the spectrophotometric assay. FIGS. 11A
and 11B show that the enzymes from 2123-49 and 2123-54 are much
less stable than the wild type enzyme from strain 2123-12. The
enzyme from strain 2123-124 is comparable in stability to the wild
type enzyme, and the enzymes from 2123-149 and 2123-151 are
slightly less stable under the incubation conditions described
here.
Example 11
[0201] The following example illustrates the effects of
isopropylthio-.beta.-D-galactoside (IPTG) concentration and
temperature on glucosamine production.
[0202] Cultures of strains 2123-54 and 2123-124 were grown for 20
hours at 37.degree. C. on M9A medium (14 g/L K.sub.2HPO.sub.4, 16
g/L KH.sub.2PO.sub.4, 1 g/L Na.sub.3Citrate-2H.sub.2O, 5 g/L
(NH.sub.4).sub.2SO.sub.4, pH 7.0) supplemented with 20 g/L glucose,
1 mM MgSO.sub.4, 0.1 mM CaCl.sub.2, and varying amounts of IPTG. At
the end of the growth period, a sample was taken and the
glucosamine concentration in the culture supernatant was assayed
using the Elson-Morgan assay described in Example 2. The results
shown in FIG. 12 indicate that the optimum IPTG concentration for
production is about 0.2 mM.
[0203] Subsequently, strain 2123-54 was grown in the same medium as
described above in shake flasks with either 0.2 or 1 mM IPTG and at
30.degree. C. or 37.degree. C. These flask cultures were also fed
glucose and ammonium sulfate as described in Example 4. At various
intervals, samples were taken and the glucosamine concentrations in
culture supernatants were assayed using the Elson-Morgan assay
described in Example 2. FIG. 13 shows that under the conditions of
this experiment, there was little difference in glucosamine
production associated with the differences in IPTG concentration.
However, growth at 30.degree. C. resulted in higher glucosamine
production than did growth at 37.degree. C. Results shown in FIGS.
14A and 14B further indicated that at 30.degree. C. (FIG. 14A),
glucosamine production continued after growth had ceased, while at
37.degree. C. (FIG. 14B), growth and glucosamine production
occurred in concert.
[0204] When strains 2123-49 and 2123-124 were grown with 0.2 mM
IPTG at 30.degree. C., glucosamine production also occurred after
growth had ceased, as shown in FIGS. 15A (2123-49) and 15B
(2123-124). As observed at 37.degree. C., the highest
concentrations of glucosamine were obtained with strain 2123-54,
followed by 2123-124 and 2123-49. Also tested were strains 2123-149
and 2123-151 which produced negligibly higher concentrations of
glucosamine than did 2123-12 (Table 7).
TABLE-US-00010 TABLE 7 Production of Glucosamine at 30.degree.
Strain Maximum Glucosamine Production, g/L 2123-12 0.3 2123-49 4.6
2123-54 7.2 2123-124 5.3 2123-149 0.6 2123-151 0.6
Example 12
[0205] The following example illustrates that glucosamine can be
produced at higher concentrations in fermentor cultures of strain
2123-54 as compared to shake flasks. This example also illustrates
that in fermentors, strain 2123-54 produces more glucosamine at
30.degree. C. than at 37.degree. C.
[0206] Fermentation cultures of strain 2123-54 were cultivated in
the medium shown in Table 8. Fermentations were run using NaOH for
pH control to pH 6.7 and were fed a mixture of 33% glucose, 8%
ammonium sulfate. Aeration and agitation were adjusted to maintain
a dissolved oxygen concentration of greater than 20% of air
saturation.
TABLE-US-00011 TABLE 8 Fermentation Medium Component Amount, g/L
K.sub.2HPO.sub.4 14 KH.sub.2PO.sub.4 16 Na.sub.3Citrate 2H.sub.2O 1
(NH.sub.4).sub.2SO.sub.4 5 MgSO.sub.4 0.12 CaCl.sub.2 0.011 Mazu
204 Antifoam 0.5 mL/L IPTG 0.048 Glucose 20 Trace Metals * *Trace
metal composition is 0.7 mg/L CoCl.sub.2, 1.7 mg/L H.sub.3BO.sub.3,
0.6 mg/L CuCl.sub.2.cndot.2H.sub.2O, 10.5 mg/L
FeCl.sub.3.cndot.6H.sub.2O, 12 mg/L MnCl.sub.2.cndot.4H.sub.2O, 1.5
mg/L Na.sub.2Mo.sub.4.cndot.2H.sub.2O, 1.5 mg/L ZnCl.sub.2.
[0207] In the following experiment, three fermentations were run in
one-liter vessels containing an initial volume of 600 mL. Variables
tested were as follows.
[0208] Fermentor #1: The mixture of 33% glucose and 8% ammonium
sulfate was fed at such a rate that no glucose accumulated in the
fermentor. Growth was at 37.degree..
[0209] Fermentor #2: As with fermentation #1 except that growth was
at 30.degree..
[0210] Fermentor #3: As with fermentation #2 except that the feed
rate was increased to maintain a constant glucose concentration in
the fermentor of 5 to 10 g/L.
[0211] Results from these fermentations are shown in FIGS. 16A, 16B
and 16C. Comparison of the results from fermentors 1 (FIG. 16A) and
2 (FIG. 16B) shows that glucosamine titers are markedly higher at
30.degree. C. than they are at 37.degree. C., as observed in shake
flasks. The maximum glucosamine concentration observed was in the
glucose-excess fermentor 3 grown at 30.degree. C. (FIG. 16C), at
10.9 g/L. At 30.degree. C., growth and glucosamine concentration
appeared to coincide, and there appeared to be a slight advantage
to growth under glucose-excess. In subsequent fermentation
experiments, run under conditions similar to fermentor #3,
glucosamine concentrations in excess of 12 g/L have been obtained
(data not shown).
[0212] In summary, the present inventors have described herein the
use of metabolic engineering to create the first glucosamine
overproducing strains of E. coli. The concept, proven here, will be
generally applicable to any microorganism having a pathway for the
production of amino sugars, or to any recombinant microorganism
into which a pathway for the production of amino sugars has been
introduced. In addition to the present strategy for creating a
glucosamine-producing strain (i.e., eliminating glucosamine
degradation and uptake and increasing expression of the glmS gene),
the present inventors have also established that reducing product
inhibition of glucosamine-6-phosphate synthase by
glucosamine-6-phosphate improves glucosamine production.
[0213] While various embodiments of the present invention have been
described in detail, it is apparent that modifications and
adaptations of those embodiments will occur to those skilled in the
art. However, it is to be expressly understood that such
modifications and adaptations are within the spirit and scope of
the present invention, as set forth in the following claims.
Sequence CWU 1
1
18122DNAArtificial SequenceDescription of Artificial Sequence
Primer 1aggtsmarct gcagsagtcw gg 22257DNAArtificial
SequenceDescription of Artificial Sequence Primer 2catgccatga
ctcgcggccc agccggccat ggccsaggts marctgcags agtcwgg
57353DNAArtificial SequenceDescription of Artificial Sequence
Primer 3aacagttaag cttccgcttg cggccgcgga gctggggtct tcgctgtggt gcg
53453DNAArtificial SequenceDescription of Artificial Sequence
Primer 4aacagttaag cttccgcttg cggccgctgg ttgtggtttt ggtgtcttgg gtt
535117PRTLama glama 5Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Ala Gly Asp 1 5 10 15Phe Leu Arg Phe Ser Cys Ala Ala Leu
Gly Ala Arg Phe Ser Ser Asp 20 25 30Val Met Gly Trp Phe Arg Gln Ala
Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ser Ser Trp Asn Gly
Asp Thr Thr His Tyr Ser Asp Ser Val 50 55 60Glu Gly Gln Phe Thr Ile
Ser Arg Asp Ile Ala Lys Asn Thr Ser Tyr 65 70 75 80Leu Gln Met Asn
Arg Leu Gln Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Arg Trp Cys
Arg Pro Pro Arg Pro Lys Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val
Thr Val Ser Ser 1156115PRTLama glama 6Gln Val Gln Leu Gln Gln Ser
Gly Gly Gly Leu Val Gln Ala Gly Ser 1 5 10 15Phe Leu Ser Phe Ser
Cys Thr Ala Ser Gly Arg Thr Phe Ser Asn Tyr 20 25 30Ala Met Gly Trp
Phe Arg Gln Ala Ser Gly Asn Gln Arg Ala Phe Val 35 40 45Ala Ala Ile
Gly Arg Asn Gly Asp Thr His Tyr Ile Asp Ser Val Lys 50 55 60Gly Arg
Phe Thr Ile Ser Arg Asp Asn Gly Lys Asp Thr Val Tyr Leu 65 70 75
80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Arg
85 90 95Ile Trp Val Gly Ala Arg Asp Tyr Trp Gly Gln Gly Thr Gln Val
Thr 100 105 110Val Ser Ser 1157116PRTLama glama 7Gln Val Gln Leu
Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15Phe Leu
Arg Phe Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Arg Tyr 20 25 30Thr
Met Gly Trp Phe Arg Gln Ala Pro Gly Asn Glu Arg Lys Phe Val 35 40
45Ala Ala Val Ser Thr Ser Gly Asn Thr His Tyr Thr Gly Ser Val Lys
50 55 60Gly Arg Phe Thr Ile Phe Arg Gln Asn Ala Lys Asn Thr Val Tyr
Leu 65 70 75 80Gln Met Ser Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Ala Arg Phe Gly Gly Met Asn Trp Lys Tyr Trp Gly
Gln Gly Ile Gln 100 105 110Val Thr Val Ser 1158121PRTLama glama
8Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Pro 1
5 10 15Phe Leu Asn Val Ser Cys Val Val Ser Gly Gly Ile Phe Ser Asp
Tyr 20 25 30Thr Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Lys
Phe Val 35 40 45Ala Ala Val Ser Ser Gly Gly Ser Thr His Tyr Thr Gly
Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Ala Asn
Thr Met Tyr Leu 65 70 75 80Gln Met Ser Ser Leu Lys Pro Asp Asp Thr
Ala Val Tyr Tyr Cys Asn 85 90 95Ala Ile Val Pro Pro Thr Arg Thr Phe
Cys Gly Arg Thr Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val
Ser Ser 115 1209112PRTLama glama 9Gln Val Gln Leu Gln Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Asp 1 5 10 15Phe Val Arg Leu Ser Cys
Ala Ala Ser Arg Arg Ala Ser Ser Thr Tyr 20 25 30Ala Val Gly Trp Phe
Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Gly Arg Ile His
Arg Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Thr Gln Asn Thr Val Tyr 65 70 75 80Leu
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Asn Val Arg Ser Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
100 105 11010117PRTLama glama 10Gln Val Gln Leu Gln Glu Ser Gly Gly
Gly Leu Val Gln Ala Gly Gly 1 5 10 15Phe Leu Arg Phe Ser Cys Ala
Ala Ser Asn Ala Leu Phe Ser Gly Tyr 20 25 30Ala Met Gly Cys Phe Arg
Gln Ala Val Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Thr Trp
Asn Asn Arg Asn Thr His Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80Leu Gln
Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr
Ser Gly Met Arg Arg Leu Gly Asp Tyr Trp Gly Gln Gly Thr Gln 100 105
110Val Thr Val Ser Ser 11511124PRTLama glama 11Gln Val Lys Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Lys Tyr 20 25 30Ala Ile
Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45Ala
Gly Ile Ser Thr Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp Thr Val Tyr Leu
65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95Ala Gly Arg Arg Ile Ser Ser Ser Tyr Tyr Ser Arg Gly
Leu Tyr Ala 100 105 110Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser
Ser 115 12012124PRTLama glama 12Gln Val Gln Leu Gln Glu Ser Gly Gly
Gly Leu Val Gln Ala Gly Asp 1 5 10 15Ser Leu Arg Leu Ser Cys Glu
Ala Ser Gly Arg Ser Phe Ser Asn Phe 20 25 30Ala Met Ala Trp Phe Arg
Gln Thr Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Gly Ile Ser Trp
Arg Gly Gly Arg Thr Tyr Tyr Ala Ala Ser Val 50 55 60Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Gly Lys Asn Thr Val Tyr 65 70 75 80Leu Gln
Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Thr Ala Tyr Gly Gln Gly Pro Ile Thr Val Pro Lys Phe Tyr Thr 100 105
110Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
12013121PRTLama glama 13Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Ala Gly Gly 1 5 10 15Cys Val Arg Leu Ser Cys Ala Ala Ser
Gly Arg Thr Phe Ser Arg Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln Ala
Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Ser Trp Arg Ser
Gly Gly Ile Lys Ile Tyr Gly Asp Ser 50 55 60Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asp Thr Val 65 70 75 80Tyr Val Gln Met
Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 85 90 95Cys Asn Ser
Arg Pro Arg Ile Tyr Arg Gly Asn Val Val Tyr Trp Gly 100 105 110Gln
Gly Thr Gln Val Thr Val Ser Ser 115 1201434DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 14ggcccagccg gccatggccc aggtgcagct gcag
341511PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 15Ala Gln Pro Ala Met Ala Gln Val Gln Leu Gln 1 5
101639DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 16gcggccgccc atcaccatca ccatcacggg
gccgcagaa 391713PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 17Ala Ala Ala His His His His His His
Gly Ala Ala Glu 1 5 101811PRTUnknown OrganismDescription of Unknown
Organism Myc peptide sequence 18Glu Gln Lys Leu Ile Ser Glu Glu Asp
Leu Asn 1 5 10
* * * * *