U.S. patent application number 13/774212 was filed with the patent office on 2013-11-28 for engineering microbes and metabolic pathways for the production of ethylene glycol.
This patent application is currently assigned to Massachusetts Institute of Technology. The applicant listed for this patent is Massachusetts Institute of Technology. Invention is credited to Jose Luis Avalos, Marjan De Mey, Deepak Dugar, Brian Pereira, Gregory Stephanopoulos.
Application Number | 20130316416 13/774212 |
Document ID | / |
Family ID | 49006244 |
Filed Date | 2013-11-28 |
United States Patent
Application |
20130316416 |
Kind Code |
A1 |
Stephanopoulos; Gregory ; et
al. |
November 28, 2013 |
ENGINEERING MICROBES AND METABOLIC PATHWAYS FOR THE PRODUCTION OF
ETHYLENE GLYCOL
Abstract
The invention relates to recombinant cells and their use in the
production of ethylene glycol.
Inventors: |
Stephanopoulos; Gregory;
(Winchester, MA) ; Pereira; Brian; (Cambridge,
MA) ; De Mey; Marjan; (Gent, BE) ; Dugar;
Deepak; (Cambridge, MA) ; Avalos; Jose Luis;
(Arlington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Massachusetts Institute of Technology; |
|
|
US |
|
|
Assignee: |
Massachusetts Institute of
Technology
Cambridge
MA
|
Family ID: |
49006244 |
Appl. No.: |
13/774212 |
Filed: |
February 22, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61602322 |
Feb 23, 2012 |
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Current U.S.
Class: |
435/158 ;
435/252.3; 435/254.11; 435/254.2; 435/325; 435/348; 435/419 |
Current CPC
Class: |
C12N 9/16 20130101; C12P
7/24 20130101; C12P 2203/00 20130101; C12P 7/18 20130101; C12N
9/0022 20130101; C12Y 401/02017 20130101; C12Y 207/01017 20130101;
C12Y 102/01052 20130101; C12N 9/1205 20130101; C12N 9/1229
20130101; C12Y 501/03 20130101; C12N 15/70 20130101; C12Y 101/01071
20130101; C12N 9/90 20130101; C12N 9/1096 20130101; C12Y 207/01016
20130101; C12N 9/0006 20130101; C12N 9/88 20130101; C12N 9/0008
20130101; C12Y 102/01021 20130101; C12Y 207/01047 20130101; C12N
15/52 20130101 |
Class at
Publication: |
435/158 ;
435/252.3; 435/254.11; 435/254.2; 435/419; 435/348; 435/325 |
International
Class: |
C12P 7/18 20060101
C12P007/18; C12N 15/70 20060101 C12N015/70 |
Goverment Interests
GOVERNMENT INTEREST
[0002] This invention was made with government support under Grant
No. DE-AR0000059 awarded by the Department of Energy, Office of
ARPA-E. The government has certain rights in this invention.
Claims
1. A cell engineered to produce ethylene glycol, optionally wherein
the cell has reduced or eliminated activity or reduced or
eliminated expression of aldehyde dehydrogenase relative to a wild
type cell, optionally wherein the aldehyde dehydrogenase is
aldehyde dehydrogenase A and optionally wherein the cell is
engineered to produce ethylene glycol from sugar, a pentose,
xylose, L-arabinose, glucose, glycerol, serine, or a metabolite
comprising glycolaldehyde.
2. (canceled)
3. The cell of claim 1, wherein the aldehyde dehydrogenase A is
encoded by an aldA gene, and wherein the cell comprises a deletion
of the aldA gene (.DELTA.aldA).
4. The cell of claim 1, wherein the cell has reduced or eliminated
activity or reduced or eliminated expression of xylulokinase
relative to a wild type cell, and optionally wherein the
xylulokinase is encoded by a xylB gene and the cell comprises a
deletion of the xylB gene (.DELTA.xylB).
5. (canceled)
6. The cell of claim 1, wherein the cell has reduced or eliminated
activity or reduced or eliminated expression of L-ribulokinase
relative to a wild type cell, and optionally wherein the
L-ribulokinase is encoded by a araB gene and the cell comprises a
deletion of the araB gene (.DELTA.araB).
7. (canceled)
8. The cell of claim 1, wherein the cell recombinantly expresses an
enzyme that interconverts D-xylulose and D-ribulose and/or
interconverts L-ribulose and L-xylulose.
9. The cell of claim 8, wherein the enzyme that interconverts
D-xylulose and D-ribulose and/or interconverts L-ribulose and
L-xylulose is D-tagatose 3-epimerase (DTE), and optionally wherein
DTE is encoded by a dte gene, and optionally wherein the dte gene
is from Pseudomonas cichorii.
10-11. (canceled)
12. The cell of claim 9, wherein the cell overexpresses a dte
gene.
13. The cell of claim 1, wherein the cell recombinantly expresses
D-ribulokinase, D-ribulose-phosphate aldolase and glycolaldehyde
reductase, and optionally wherein the D-ribulokinase is encoded by
a fucK gene and/or wherein the D-ribulose-phosphate aldolase is
encoded by a fucA gene and/or wherein the glycolaldehyde reductase
is encoded by a fucO gene.
14. (canceled)
15. The cell of claim 13, wherein the cell overexpresses the fucK
gene and/or the fucA gene and/or the fucO gene.
16. The cell of claim 13, wherein the fucK gene, the fucA gene and
the fucO gene are expressed as part of an operon in conjunction
with a dte gene, and optionally wherein the order of the genes in
the operon is dte-fucA-fucO-fucK.
17. (canceled)
18. The cell of claim 1, wherein the cell recombinantly expresses
ATP:L-xylulose 1-phosphotransferase, L-xylulose-1-phosphate
aldolase, and glycolaldehyde reductase, optionally wherein the
ATP:L-xylulose 1-phosphotransferase is encoded by a rhaB gene
and/or the L-xylulose-1-phosphate aldolase is encoded by a rhaD
gene and/or the glycolaldehyde reductase is encoded by a fucO gene,
and optionally wherein the cell overexpresses the rhaB gene and/or
the rhaD gene and/or the fucO gene.
19-20. (canceled)
21. The cell of claim 18, wherein the rhaB gene, the rhaD gene and
the fucO gene are expressed as part of an operon in conjunction
with a dte gene, and optionally wherein the order of the genes in
the operon is dte-rhaB-rhaD fucO.
22. (canceled)
23. The cell of claim 1, wherein the cell expresses a subunit of
the E1 component of 2-oxoglutarate dehydrogenase within .DELTA.aldA
.DELTA.xylB, optionally wherein the subunit of the E1 component of
2-oxoglutarate dehydrogenase is encoded by a sucA gene, and
optionally wherein the cell overexpresses a sucA gene.
24-25. (canceled)
26. The cell of claim 1, wherein the cell is a bacterial cell, a
fungal cell (including a yeast cell), a plant cell, an insect cell
or an animal cell.
27-28. (canceled)
29. The cell of claim 1, wherein the cell endogenously expresses
the gene encoding D-ribulokinase, D-ribulose-phosphate aldolase,
glycolaldehyde reductase, ATP:L-xylulose 1-phosphotransferase,
L-xylulose-1-phosphate aldolase, and/or subunit of the E1 component
of 2-oxoglutarate dehydrogenase, and wherein endogenous expression
of the gene encoding D-ribulokinase, D-ribulose-phosphate aldolase,
glycolaldehyde reductase, ATP:L-xylulose 1-phosphotransferase,
L-xylulose-1-phosphate aldolase, and/or subunit of the E1 component
of 2-oxoglutarate dehydrogenase is increased through modification
of the gene(s) and/or their promoter(s) and/or their ribosome
binding sites (RBSs).
30-31. (canceled)
32. The cell of claim 1, wherein the cell recombinantly expresses a
3-phosphoglycerate dehydrogenase, a glycerate kinase, a
3-phosphohydroxypyruvate phosphatase, a serine:pyruvate
aminotransferase (SPT), an alanine:glyoxylate aminotransferase
(AGT), a serine decarboxylase, and/or an ethanolamine oxidase.
33. The cell of claim 32, wherein the 3-phosphoglycerate
dehydrogenase is a mutant resistant to inhibition by serine and/or
is encoded by a serA gene of E. coli.
34-35. (canceled)
36. The cell of claim 32, wherein the glycerate kinase is a
glycerate kinase II encoded by a glxK gene of E. coli, or a
glycerate kinase I encoded by a garK gene of E. coli.
37. The cell of claim 1, wherein the expression or activity in the
cell of one or more phosphoglycerate mutases and/or the expression
or activity in the cell of enolase is attenuated, thereby
increasing the amount of 3-phosphoglycerate in the cell by reducing
flux to 2-phosphoglycerate and/or increasing the amount of
2-phosphoglycerate in the cell by reducing flux to
phosphoenolpyruvate, and optionally wherein the one or more
phosphoglycerate mutases is encoded by gpmA, gpmB, and gpmM genes
of E. coli and/or the enolase is encoded by an eno gene from E.
coli.
38. (canceled)
39. The cell of claim 1, wherein the cell recombinantly expresses a
3-phosphoserine aminotransferase to convert
3-phosphohydroxypyruvate to 3-phospho-L-serine and optionally
further recombinantly expresses a phosphoserine phosphatase to
convert 3-phospho-L-serine to L-serine, and optionally wherein the
3-phosphoserine aminotransferase and phosphoserine phosphatase are
encoded by serC and serB genes of E. coli, respectively.
40. (canceled)
41. The cell of claim 1, wherein the expression or activity in the
cell of one or more serine deaminases is attenuated, and optionally
wherein the one or more serine deaminases is encoded by sdaA, sdaB,
tdcB, and tdcG genes of E. coli.
42-43. (canceled)
44. The cell of claim 32, wherein the serine decarboxylase is
encoded by a gene from Arabidopsis thaliana, and/or the
ethanolamine oxidase is encoded by a gene from Arthrobacter sp.,
and optionally wherein the gene from Arabidopsis thaliana is sdc,
and/or the gene from Arthrobacter sp. is aao.
45. (canceled)
46. The cell of claim 44, wherein the serine decarboxylase gene
from A. thaliana is truncated, and optionally wherein the truncated
gene from A. thaliana is t-sdc.
47-48. (canceled)
49. The cell of claim 32, wherein the 3-phosphohydroxypyruvate
phosphatase is encoded by a yeaB gene of E. coli or by GPP2 of S.
cerevisiae.
50. (canceled)
51. The cell of claim 32, wherein the SPT and/or the AGT is encoded
by a gene of Arabidopsis thaliana, Drosophila melanogaster, Canis
lupus familiaris, Homo sapiens or Rattus norvegicus.
52. A method for producing ethylene glycol, comprising culturing
the cell of claim 1 to produce ethylene glycol.
53-62. (canceled)
63. A cell culture produced by culturing the cell of claim 1.
64-65. (canceled)
66. A supernatant of a cell culture produced by culturing the cell
of claim 1.
67-68. (canceled)
69. A method for producing ethylene glycol in a cell, comprising:
reducing or eliminating the activity or expression of aldehyde
dehydrogenase A and/or xylulokinase in the cell, relative to a wild
type cell; increasing the expression of an enzyme that
interconverts D-xylulose and D-ribulose, a D-ribulokinase,
D-ribulose-phosphate aldolase, glycolaldehyde reductase and/or
subunit of the E1 component of 2-oxoglutarate dehydrogenase in the
cell, relative to a wild type cell; and culturing the cell.
70-110. (canceled)
111. A method for producing ethylene glycol in a cell, comprising:
reducing or eliminating the activity or expression of aldehyde
dehydrogenase A and/or L-ribulokinase in the cell, relative to a
wild type cell; increasing the expression of an enzyme that
interconverts L-ribulose and L-xylulose, an ATP:L-xylulose
1-phosphotransferase, L-xylulose-1-phosphate aldolase, and
glycolaldehyde reductase in the cell, relative to a wild type cell;
and culturing the cell.
112-158. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. provisional application No. 61/602,322, filed
Feb. 23, 2012, which is incorporated by reference herein in its
entirety.
FIELD OF THE INVENTION
[0003] The invention relates to the production of ethylene glycol
through recombinant gene expression and metabolic engineering.
BACKGROUND OF THE INVENTION
[0004] Ethylene glycol is an important organic compound commonly
used as a precursor to polymers, primarily polyethylene
terephthalate (PET) which comprises a significant share of the
worlds polymer production. The major end uses of PET are synthetic
fibers, commonly referred to as "polyester," and plastic bottles.
For both of these products, the demand is increasing. Another major
use of ethylene glycol is as a coolant such as automotive
antifreeze. Though less significant, there are also several other
uses for ethylene glycol.
[0005] Currently, ethylene glycol is primarily generated from
ethylene oxide which is derived from fossil fuels. With the growing
issues surrounding fossil fuels, generating ethylene glycol from
renewable sources provides a potential alternative. Thus, ethylene
glycol also is chemically produced from plant-derived ethanol.
However, the direct biological production of ethylene glycol from
renewable sources had not been exhibited.
SUMMARY OF THE INVENTION
[0006] The present invention is based, at least in part, on the
discovery that ethylene glycol can be produced directly from
renewable resources. Of biological sources for the generation of
ethylene glycol, lignocellulosic biomass is the most abundant and
very promising. The cellulose and hemicellulose fractions of
lignocellulose, or other sources of sugars including algae, can be
broken down into their monosaccharide units, primarily glucose,
xylose and other pentoses. Additionally, the production of
biodiesel results in significant amounts of waste glycerol, a sugar
alcohol which can be used as a substrate. Our research shows that
by using engineered microbes we can convert these simple sugars and
glycerol into ethylene glycol.
[0007] According to one aspect, cells engineered to produce
ethylene glycol are provided. The cells in some embodiments have
reduced or eliminated activity or expression of aldehyde
dehydrogenase A relative to a wild type cell. In some embodiments,
the aldehyde dehydrogenase A is encoded by an aldA gene, and the
cell includes a deletion of the aldA gene. This may be referred to
herein as .DELTA.aldA.
[0008] In some embodiments, the cells have reduced or eliminated
activity or expression of xylulokinase relative to wild type cells.
In some embodiments, the xylulokinase is encoded by a xylB gene,
and the cells include a deletion of the xylB gene. This may be
referred to herein as .DELTA.xylB.
[0009] In some embodiments, the cells have reduced or eliminated
activity or expression of L-ribulokinase relative to wild type
cells. In some embodiments, the L-ribulokinase is encoded by an
araB gene, and the cells include a deletion of the araB gene. This
may be referred to herein as .DELTA.araB.
[0010] As will be understood by persons skilled in the art, the
activity or expression of aldehyde dehydrogenase A and/or
xylulokinase and/or L-ribulokinase in the cells can be reduced,
attenuated or eliminated in several ways, including by reducing
expression of the relevant gene(s), disrupting the relevant
gene(s), introducing mutation(s) in the relevant gene(s) that
results in production of a protein with reduced, attenuated or
eliminated enzymatic activity, use of specific enzyme inhibitors to
reduce, attenuate or eliminate the enzymatic activity, etc.
[0011] In some embodiments, the cells recombinantly express an
enzyme that interconverts D-xylulose and D-ribulose. In some
embodiments, the cells recombinantly express an enzyme that
interconverts L-ribulose and L-xylulose. In some embodiments, the
enzyme that interconverts D-xylulose and D-ribulose and/or
L-ribulose and L-xylulose, is D-tagatose 3-epimerase (also referred
to herein as DTE). In some embodiments, the DTE is encoded by a
gene referred to herein as dte. In some embodiments, the dte gene
is from Pseudomonas cichorii. In some embodiments, the cells
overexpress a dte gene.
[0012] In some embodiments, the cells recombinantly express
D-ribulokinase, D-ribulose-phosphate aldolase and glycolaldehyde
reductase. In some embodiments, the D-ribulokinase is encoded by a
fucK gene and/or the D-ribulose-phosphate aldolase is encoded by a
fucA gene and/or the glycolaldehyde reductase is encoded by a fucO
gene. In some embodiments, the cells overexpress the fucK gene
and/or the fucA gene and/or the fucO gene. In some embodiments, the
fucK gene, the fucA gene and the fucO gene are expressed as part of
an operon in conjunction with a dte gene. In some embodiments, the
order of the genes in the operon is dte-fucA-fucO-fucK.
[0013] In some embodiments, the cells recombinantly express
ATP:L-xylulose 1-phosphotransferase, L-xylulose-1-phosphate
aldolase, and glycolaldehyde reductase. In some embodiments, the
ATP:L-xylulose 1-phosphotransferase is encoded by a rhaB gene
and/or the L-xylulose-1-phosphate aldolase is encoded by a rhaD
gene and/or the glycolaldehyde reductase is encoded by a fucO gene.
In some embodiments, the cells overexpress the rhaB gene and/or the
rhaD gene and/or the fucO gene. In some embodiments, the rhaB gene,
the rhaD gene and the fucO gene are expressed as part of an operon
in conjunction with a dte gene. In some embodiments, the order of
the genes in the operon is dte-rhaB-rhaD-fucO.
[0014] In some embodiments, the cells express a subunit of the E1
component of 2-oxoglutarate dehydrogenase within .DELTA.aldA
.DELTA.xylB. In some embodiments, the subunit of the E1 component
of 2-oxoglutarate dehydrogenase is encoded by a sucA gene. In some
embodiments, the cells overexpress a sucA gene.
[0015] In some embodiments, the cells are bacterial cells, fungal
cells (including yeast cells), plant cells, insect cells or animal
cells. In some embodiments, the cells are bacterial cells such as,
for example, Escherichia coli (E. coli) cells.
[0016] In some embodiments, the cells endogenously express the gene
encoding D-ribulokinase, D-ribulose-phosphate aldolase,
glycolaldehyde reductase, ATP:L-xylulose 1-phosphotransferase,
L-xylulose-1-phosphate aldolase, and/or a subunit of the E1
component of 2-oxoglutarate dehydrogenase; in such embodiments,
endogenous expression of the gene encoding D-ribulokinase,
D-ribulose-phosphate aldolase, glycolaldehyde reductase,
ATP:L-xylulose 1-phosphotransferase, L-xylulose-1-phosphate
aldolase, and/or subunit of the E1 component of 2-oxoglutarate
dehydrogenase is increased through modification of the gene(s)
and/or their promoter(s) and/or their ribosome binding sites
(RBSs). In some embodiments, the gene encoding D-ribulokinase,
D-ribulose-phosphate aldolase, glycolaldehyde reductase,
ATP:L-xylulose 1-phosphotransferase, L-xylulose-1-phosphate
aldolase, and/or a subunit of the E1 component of 2-oxoglutarate
dehydrogenase is expressed from a plasmid. In some embodiments, one
or more copies of the gene encoding D-ribulokinase,
D-ribulose-phosphate aldolase, glycolaldehyde reductase,
ATP:L-xylulose 1-phosphotransferase, L-xylulose-1-phosphate
aldolase, and/or a subunit of the E1 component of 2-oxoglutarate
dehydrogenase is integrated into the genome of the cells.
[0017] In some embodiments, the cells recombinantly express a
3-phosphoglycerate dehydrogenase. In some embodiments, the
3-phosphoglycerate dehydrogenase is a mutant resistant to
inhibition by serine. In some embodiments, the 3-phosphoglycerate
dehydrogenase is encoded by a serA gene of E. coli.
[0018] In some embodiments, the cells recombinantly express a
glycerate kinase. In some embodiments, the glycerate kinase is a
glycerate kinase II encoded by a glxK gene of E. coli, or a
glycerate kinase I encoded by a garK gene of E. coli.
[0019] In some embodiments, the expression or activity in the cells
of one or more phosphoglycerate mutases and/or of enolase is
attenuated, thereby increasing the amount of 3-phosphoglycerate in
the cell by reducing flux to 2-phosphoglycerate and/or increasing
the amount of 2-phosphoglycerate in the cell by reducing flux to
phosphoenolpyruvate. In some embodiments, the one or more
phosphoglycerate mutases is encoded by gpmA, gpmB, and gpmM genes
of E. coli and/or the enolase is encoded by an eno gene from E.
coli.
[0020] In some embodiments, the cells recombinantly express a
3-phosphoserine aminotransferase to convert
3-phosphohydroxypyruvate to 3-phospho-L-serine and optionally
further recombinantly express a phosphoserine phosphatase to
convert 3-phospho-L-serine to L-serine. In some embodiments, the
3-phosphoserine aminotransferase and phosphoserine phosphatase are
encoded by serC and serB genes of E. coli, respectively.
[0021] In some embodiments, the expression or activity in the cells
of one or more serine deaminases is attenuated. In some
embodiments, the one or more serine deaminases is encoded by sdaA,
sdaB, tdcB, and tdcG genes of E. coli.
[0022] In some embodiments, the cells recombinantly express a
serine decarboxylase and ethanolamine oxidae. In some embodiments,
the serine decarboxylase is encoded by a gene from Arabidopsis
thaliana (referred to as sdc), and the ethanolamine oxidase is
encoded by a gene from Arthrobacter sp (referred to as aao). In
some embodiments, the serine decarboxylase gene from A. thaliana is
truncated (referred to as t-sdc).
[0023] In some embodiments, the cells recombinantly express a
3-phosphohydroxypyruvate phosphatase. In some embodiments, the
3-phosphohydroxypyruvate phosphatase is encoded by a yeaB gene of
E. coli or by GPP2 of S. cerevisiae.
[0024] In some embodiments, the cells recombinantly express a
serine:pyruvate aminotransferase (SPT) and/or an alanine:glyoxylate
aminotransferase (AGT). In some embodiments, the SPT and/or the AGT
is encoded by a gene of Arabidopsis thaliana, Drosophila
melanogaster, Canis lupus familiaris, Homo sapiens and/or Rattus
norvegicus.
[0025] According to another aspect, methods for producing ethylene
glycol are provided. The methods include culturing any of the cells
described herein to produce ethylene glycol.
[0026] In some embodiments, the cells are cultured in minimal
medium supplemented with a carbon source. In some embodiments, the
carbon source includes D-arabinose and/or L-arabinose and/or
D-glucose and/or glycerol and/or D-xylose and/or a biomass
hydrolysate and/or L-arabinose and/or glycerol and/or serine.
[0027] In some embodiments, the cells are cultured aerobically. In
other embodiments, the cell is cultured anaerobically.
[0028] In some embodiments, the methods further include recovering
the ethylene glycol from the cell culture and/or culture
supernatants.
[0029] In some embodiments, at least 1 g/L ethylene glycol is
produced. In some embodiments, at least 10 g/L ethylene glycol is
produced.
[0030] According to another aspect, cell cultures are provided. The
cell cultures are produced by culturing any of the cells described
herein or by culturing any of the cells described herein according
to any of the methods described herein.
[0031] In some embodiments, the cell culture contains at least 1
g/L ethylene glycol. In some embodiments, the cell culture contains
at least 10 g/L ethylene glycol.
[0032] According to another aspect, supernatants of a cell culture
are provided. The supernatants are produced by culturing any of the
cells described herein or by culturing any of the cells described
herein according to any of the methods described herein.
[0033] In some embodiments, the supernatants contain at least 1 g/L
ethylene glycol. In some embodiments, the supernatants contain at
least 10 g/L ethylene glycol.
[0034] According to another aspect, methods for producing ethylene
glycol in cells are provided. The methods include reducing or
eliminating the activity or expression of aldehyde dehydrogenase A
and/or xylulokinase in the cells, relative to wild type cells;
increasing the expression of an enzyme that interconverts
D-xylulose and D-ribulose, a D-ribulokinase, D-ribulose-phosphate
aldolase, glycolaldehyde reductase and/or subunit of the E1
component of 2-oxoglutarate dehydrogenase in the cells, relative to
wild type cells; and culturing the cells.
[0035] In some embodiments, the aldehyde dehydrogenase A is encoded
by an aldA gene, and the cell comprises a deletion of the aldA gene
(.DELTA.aldA). In certain embodiments, the xylulokinase is encoded
by a xylB gene, and the cell comprises a deletion of the xylB gene
(.DELTA.xylB).
[0036] In some embodiments, the enzyme that interconverts
D-xylulose and D-ribulose is D-tagatose 3-epimerase (DTE). In
certain embodiments, the DTE is encoded by a dte gene. In some
embodiments, the dte gene is from Pseudomonas cichorii. In some
embodiments, the cell overexpresses a dte gene.
[0037] In some embodiments, the D-ribulokinase is encoded by a fucK
gene and/or the D-ribulose-phosphate aldolase is encoded by a fucA
gene and/or the glycolaldehyde reductase is encoded by a fucO gene.
In certain embodiments, the cell overexpresses the fucK gene and/or
the fucA gene and/or the fucO gene. In some embodiments, the fucK
gene, the fucA gene and the fucO gene are expressed as part of an
operon in conjunction with a dte gene. In preferred embodiments,
the order of the genes in the operon is dte-fucA-fucO-fucK.
[0038] In some embodiments, the cell expresses a subunit of the E1
component of 2-oxoglutarate dehydrogenase within .DELTA.aldA
.DELTA.xylB. In certain embodiments, the subunit of the E1
component of 2-oxoglutarate dehydrogenase is encoded by a sucA
gene. In some embodiments, the cell overexpresses a sucA gene.
[0039] In some embodiments, the cell recombinantly expresses a
3-phosphoglycerate dehydrogenase. In certain embodiments, the
3-phosphoglycerate dehydrogenase is encoded by a serA gene of E.
coli.
[0040] In some embodiments, the cell recombinantly expresses a
glycerate kinase. In certain embodiments, the glycerate kinase is a
glycerate kinase II encoded by a glxK gene of E. coli, or a
glycerate kinase I encoded by a garK gene of E. coli.
[0041] In some embodiments, the expression or activity in the cell
of one or more phosphoglycerate mutases and/or of enolase is
attenuated, thereby increasing the amount of 3-phosphoglycerate in
the cell by reducing flux to 2-phosphoglycerate and/or increasing
the amount of 2-phosphoglycerate in the cell by reducing flux to
phosphoenolpyruvate. In certain embodiments, the one or more
phosphoglycerate mutases is encoded by gpmA, gpmB, and gpmM genes
of E. coli and/or of enolase is encoded by an eno gene from E.
coli.
[0042] In some embodiments, the cell recombinantly expresses a
3-phosphoserine aminotransferase to convert
3-phosphohydroxypyruvate to 3-phospho-L-serine and optionally
further recombinantly expresses a phosphoserine phosphatase to
convert 3-phospho-L-serine to L-serine. In certain embodiments, the
3-phosphoserine aminotransferase and phosphoserine phosphatase are
encoded by serC and serB genes of E. coli, respectively.
[0043] In some embodiments, the expression or activity in the cell
of one or more serine deaminases is attenuated. In certain
embodiments, the one or more serine deaminases is encoded by sdaA,
sdaB, tdcB, and tdcG genes of E. coli.
[0044] In some embodiments, the cell recombinantly expresses a
serine decarboxylase.
[0045] In some embodiments, the cell recombinantly expresses a
3-phosphohydroxypyruvate phosphatase. In certain embodiments, the
3-phosphohydroxypyruvate phosphatase is encoded by a yeaB gene of
E. coli or by GPP2 of S. cerevisiae.
[0046] In some embodiments, the cell recombinantly expresses a
serine:pyruvate aminotransferase (SPT) and/or an alanine:glyoxylate
aminotransferase (AGT). In certain embodiments, the SPT and/or AGT
is encoded by a gene of Arabidopsis thaliana, Drosophila
melanogaster, Canis lupus familiaris, Homo sapiens, and Rattus
norvegicus.
[0047] In some embodiments, the cell is a bacterial cell, a fungal
cell (including a yeast cell), a plant cell, an insect cell or an
animal cell. In certain embodiments, the cell is a bacterial cell.
In preferred embodiments, the bacterial cell is an Escherichia coli
cell.
[0048] In some embodiments, the cell is cultured in minimal medium
supplemented with a carbon source. In certain embodiments, the
carbon source comprises D-arabinose and/or D-glucose and/or
D-xylose and/or a biomass hydrolysate and/or L-arabinose and/or
glycerol and/or serine.
[0049] In some embodiments, the cells are cultured in minimal
medium supplemented with serine.
[0050] In some embodiments, the cell is cultured aerobically. In
other embodiments, the cell is cultured anaerobically.
[0051] In some embodiments, the methods further include recovering
the ethylene glycol from the cell culture and/or culture
supernatants. In certain embodiments, at least 1 g/L ethylene
glycol is produced. In preferred embodiments, at least 10 g/L
ethylene glycol is produced.
[0052] According to yet another aspect, methods for producing
ethylene glycol in cells include reducing or eliminating the
activity or expression of aldehyde dehydrogenase A and/or
L-ribulokinase in the cells, relative to wild type cells;
increasing the expression of an enzyme that interconverts
L-ribulose and L-xylulose, an ATP:L-xylulose 1-phosphotransferase,
L-xylulose-1-phosphate aldolase, and glycolaldehyde reductase in
the cells, relative to wild type cells; and culturing the
cells.
[0053] In some embodiments, the aldehyde dehydrogenase A is encoded
by an aldA gene, and wherein the cell comprises a deletion of the
aldA gene (.DELTA.aldA). In some embodiments, the L-ribulokinase is
encoded by a araB gene, and wherein the cell comprises a deletion
of the araB gene (.DELTA.araB).
[0054] In some embodiments, the enzyme that interconverts
L-ribulose and L-xylulose is D-tagatose 3-epimerase (DTE). In some
embodiments, the DTE is encoded by a dte gene. In some embodiments,
the dte gene is from Pseudomonas cichorii. In some embodiments, the
cell overexpresses a dte gene.
[0055] In some embodiments, the ATP:L-xylulose 1-phosphotransferase
is encoded by a rhaB gene and/or the L-xylulose-1-phosphate
aldolase is encoded by a rhaD gene and/or the glycolaldehyde
reductase is encoded by a fucO gene. In some embodiments, the cell
overexpresses the rhaB gene and/or the rhaD gene and/or the fucO
gene. In some embodiments, the rhaB gene, the rhaD gene and the
fucO gene are expressed as part of an operon in conjunction with a
dte gene. In some embodiments, the order of the genes in the operon
is dte-rhaB-rhaD-fucO.
[0056] In some embodiments, the cell recombinantly expresses a
3-phosphoglycerate dehydrogenase. In some embodiments, the
3-phosphoglycerate dehydrogenase is a mutant resistant to
inhibition by serine. In some embodiments, the 3-phosphoglycerate
dehydrogenase is encoded by a serA gene of E. coli.
[0057] In some embodiments, the cell recombinantly expresses a
glycerate kinase. In some embodiments, the glycerate kinase is a
glycerate kinase II encoded by a glxK gene of E. coli, or a
glycerate kinase I encoded by a garK gene of E. coli.
[0058] In some embodiments, the expression or activity in the cell
of one or more phosphoglycerate mutases and/or of enolase is
attenuated, thereby increasing the amount of 3-phosphoglycerate in
the cell by reducing flux to 2-phosphoglycerate and/or increasing
the amount of 2-phosphoglycerate in the cell by reducing flux to
phosphoenolpyruvate. In some embodiments, one or more
phosphoglycerate mutases is encoded by gpmA, gpmB, and gpmM genes
of E. coli and/or of enolase is encoded by an eno gene from E.
coli.
[0059] In some embodiments, the cell recombinantly expresses a
3-phosphoserine aminotransferase to convert
3-phosphohydroxypyruvate to 3-phospho-L-serine and optionally
further recombinantly expresses a phosphoserine phosphatase to
convert 3-phospho-L-serine to L-serine. In some embodiments, the
3-phosphoserine aminotransferase and phosphoserine phosphatase are
encoded by serC and serB genes of E. coli, respectively.
[0060] In some embodiments, the expression or activity in the cell
of one or more serine deaminases is attenuated. In some
embodiments, one or more serine deaminases is encoded by sdaA,
sdaB, tdcB, and tdcG genes of E. coli.
[0061] In some embodiments, the cell recombinantly expresses a
serine decarboxylase and/or ethanolamine oxidase. In some
embodiments, the serine decarboxylase is encoded by a gene from
Arabidopsis thaliana, and the ethanolamine oxidase is encoded by a
gene from Arthrobacter sp. In some embodiments, the gene from
Arabidopsis thaliana is sdc, and the gene from Arthrobacter sp. is
aao. In some embodiments, the serine decarboxylase gene from A.
thaliana is truncated. In some embodiments, the gene from A.
thaliana is t-sdc.
[0062] In some embodiments, the cell recombinantly expresses a
3-phosphohydroxypyruvate phosphatase. In some embodiments, the
3-phosphohydroxypyruvate phosphatase is encoded by a yeaB gene of
E. coli or by GPP2 of S. cerevisiae.
[0063] In some embodiments, the cell recombinantly expresses a
serine:pyruvate aminotransferase (SPT) and/or an alanine:glyoxylate
aminotransferase (AGT). In some embodiments, the SPT and/or AGT is
encoded by a gene of Arabidopsis thaliana, Drosophila melanogaster,
Canis lupus familiaris, Homo sapiens, and Rattus norvegicus.
[0064] In some embodiments, the cell is a bacterial cell, a fungal
cell (including a yeast cell), a plant cell, an insect cell or an
animal cell. In some embodiments, the cell is a bacterial cell. In
some embodiments, the bacterial cell is an Escherichia coli
cell.
[0065] In some embodiments, the cell is cultured in minimal medium
supplemented with a carbon source. In some embodiments, the carbon
source comprises D-arabinose. In some embodiments, the carbon
source comprises D-glucose. In some embodiments, the carbon source
comprises D-xylose. In some embodiments, the carbon source
comprises a biomass hydrolysate. In some embodiments, the carbon
source comprises L-arabinose. In some embodiments, the carbon
source comprises glycerol. In some embodiments, the carbon sources
comprises serine.
[0066] In some embodiments, the cells are cultured in minimal
medium supplemented with serine.
[0067] In some embodiments, the cell is cultured aerobically. In
some embodiments, the cell is cultured anaerobically.
[0068] In some embodiments, the methods further comprise recovering
the ethylene glycol from the cell culture and/or culture
supernatants.
[0069] In some embodiments, at least 1 g/L ethylene glycol is
produced. In some embodiments, at least 10 g/L ethylene glycol is
produced. According to another aspect, a cell culture produced by
any of the foregoing methods is provided. In some embodiments, the
cell culture and/or culture supernatant contains at least 1 g/L
ethylene glycol. In certain embodiments, the cell culture and/or
culture supernatant contains at least 10 g/L ethylene glycol.
[0070] According to another aspect, a supernatant of a cell culture
produced by any of the foregoing methods is provided.
[0071] These and other aspects of the invention are described
further below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0072] FIG. 1: Pathway map of ethylene glycol production from
pentoses. Enzymes that catalyze the depicted reactions are
represented by their corresponding genes (in italics); genes are
from E. coli or from heterologous sources (within brackets).
[0073] FIG. 2: Pathway map of ethylene glycol production from
glycolysis intermediates. Enzymes that catalyze the depicted
reactions are represented by their corresponding genes (in
italics); genes are from E. coli or from heterologous sources
(within brackets).
[0074] FIG. 3: Schematic overview of the construction of
p10_T5T10-dte. bla=Beta-lactamase; cat=chloramphenicol
acetyltransferase; dte=codon-optimized D-tagatose-3-epimerase;
MCS=multi cloning site; RD=restriction digest; HiFi-PCR=High
Fidelity PCR; T5T10=promoter T5T10; rrnBTT=terminator rrnBTT;
oMIT080=Fw-primer 5'-GCTGCCATGGACAAAGTTGGTATGTTCTACACC (SEQ ID
NO:1); 0MIT140=Rv-primer
5'AGTCGTCGACATGAGCTCCGTAGGCCGGCCTAAACGAATTCTTAGGC CAGTTTATCACGG
(SEQ ID NO:2).
[0075] FIGS. 4A and 4B: Schematic overview of the construction of
p10_T5T10-dte-fucA-fucO-fucK. cat=chloramphenicol
acetyltransferase; dte=codon-optimized D-tagatose-3-epimerase;
gDNA=genomic DNA; MCS=multi cloning site; RD=restriction digest;
HiFi-PCR=High Fidelity PCR; T5T10=promoter T5T10; rrnBTT=terminator
rrnBTT; oMIT132=Fw-primer
5'-GAATTCGTTTAGAGCTCTAAATAAGGAGGAATAACCATG GTATCCGGCTATATTGCAGGAG
(SEQ ID NO:3); oMIT133=Rv-primer 5'-ACTGG
TCGACGCTATCTTCACACTTCCTCTATAAATTC (SEQ ID NO:4); oMIT134=Fw-primer
5'-CTGCGGCCGGCCCTTTAATAAGGAGATATACCATGGAACGAAATAA ACTTGC (SEQ ID
NO:5); oMIT135=Rv-primer 5'-GCCGGAGCTCTAAACGAATTCTT
ACCAGGCGGTATGGTAAAGC (SEQ ID NO:6).
[0076] FIG. 5: Schematic overview of the construction of
p5_T5T10-dte-fucA-fucO-fucK. aadA1=gentamycin resistance gene;
cat=chloramphenicol acetyltransferase; dte=codon-optimized
D-tagatose-3-epimerase; MCS=multi cloning site; RD=restriction
digest; HiFi-PCR=High Fidelity PCR; T5T10=promoter T5T10;
rrnBTT=terminator rrnBTT; repA=a plasmid-encoded gene product
required for pSC101 replication in Escherichia coli.
[0077] FIG. 6: Schematic overview of the construction of
p10_T5T10-sucA. bla=Beta-lactamase; cat=chloramphenicol
acetyltransferase; MCS=multi cloning site; RD=restriction digest;
HiFi-PCR=High Fidelity PCR; T5T10=promoter T5T10; rrnBTT=terminator
rrnBTT; oMIT090=Fw-primer 5'-GCTGCCATGGAGAACAGCGCTTTGAAAGC (SEQ ID
NO:7); OMIT91=Rv-primer 5'-CTATGAGCTCCGTAGGCCGGCCTAA
ACGAATTCTTATTCGACGTTCAGCGCGTC (SEQ ID NO:8).
[0078] FIG. 7: Production of ethylene glycol from D-xylose. E. coli
cultures were grown on minimal media supplemented with 10 g/L
sugar, and optical densities and ethylene glycol concentrations
were measured over time. Solid lines and closed symbols correspond
to the optical densities (OD.sub.600), and dashed lines and open
symbols correspond to ethylene glycol concentrations in the culture
supernatants. Triangles are for .DELTA.aldA cultured on
D-arabinose, squares are for .DELTA.aldA .DELTA.xylB cultured on
D-xylose, and diamonds are for .DELTA.aldA
.DELTA.xylB/p10_T5T10-dte cultured on D-xylose.
[0079] FIG. 8: Effect of gene order on production of ethylene
glycol from D-xylose. E. coli cultures were grown on minimal media
supplemented with 10 g/L xylose except for .DELTA.aldA .DELTA.xylB
which was grown on D-arabinose. Ethylene glycol concentrations in
the culture supernatants were measured over time. Closed squares
are .DELTA.aldA .DELTA.xylB; closed diamonds are .DELTA.aldA
.DELTA.xylB/p10_T5T10-dte-fucA-fucO-fucK; open triangles are AaldA
.DELTA.xylB/p10_T5T10-dte-fucK-fucA-fucO; open circles are
.DELTA.aldA .DELTA.xylB/p10_T5T10-fucA-fucO-dte-fucK; open diamonds
are .DELTA.aldA .DELTA.xylB/p10_T5T10-fucA-fucO-fucK-dte; closed
triangles are .DELTA.aldA .DELTA.xylB/p10_T5T10-fucK-dte-fucA-fucO;
closed circles are .DELTA.aldA
.DELTA.xylB/p10_T5T10-fucK-fucA-fucO-dte.
[0080] FIG. 9: Production of EG from D-xylose within a bioreactor.
.DELTA.aldA .DELTA.xylB/p10_T5T10-dte-fucA-fucO-fucK was grown in a
bioreactor in minimal medium supplemented with D-xylose. The
bioreactor was run as fed-batch. EG concentrations of duplicate
bioreactors are shown.
[0081] FIG. 10: Production of EG from L-arabinose. E. coli cultures
were grown on minimal medium supplemented with 15 g/L L-arabinose.
OD.sub.600 (solid lines and closed symbols) and ethylene glycol
concentrations in the culture supernatants (dashed lines and open
symbols) were measured over time. Diamonds represent .DELTA.aldA,
while squares and circles represent duplicate cultures of
.DELTA.aldA .DELTA.araB/p10_T7-dte-rhaB-rhaD-fucO.
[0082] FIG. 11: Production of EG from serine. A culture of E. coli
strain .DELTA.aldA/pCDFDuet_T7-t-sdc+T7-aao/p10_T7-fucO were grown
on minimal medium supplemented with 15 g/L D-glucose and 10 g/L
L-serine. OD.sub.600 (solid lines and closed symbols) and ethylene
glycol concentrations in the culture supernatant (dashed lines and
open symbols) were measured over time.
DETAILED DESCRIPTION OF SOME EMBODIMENTS
[0083] The technology described herein is based on a pathway
engineering scheme consisting of three elements: cleavage of
pentoses to yield glycolaldehyde, generation of glycolaldehyde from
glycolysis intermediates, and conversion of glycolaldehyde to
ethylene glycol. While cleavage of the pentose will depend on the
specific pentose, the latter two elements will be consistent across
glycerol and all sugars.
[0084] In some embodiments, D-arabinose yields glycolaldehyde.
D-arabinose can be transported into the cell through an arabinose
or fucose transporter. D-arabinose isomerase converts D-arabinose
to D-ribulose, and D-ribulose is phosphorylated at the 1 position
by D-ribulokinase. D-ribulose-phosphate aldolase can cleave the
D-ribulose-1-phosphate, thereby resulting in glycolaldehyde and
dihydroxyacetone phosphate. Examples of genes encoding an arabinose
transporter, D-arabinose isomerase, D-ribulokinase, and
D-ribulose-phosphate aldolase are fucP, fucI, fucK, and fucA (all
from E. coli), respectively.
[0085] In some embodiments, D-xylose yields glycolaldehyde. By
means of a xylose transporter, D-xylose enters the cell where it is
converted to D-xylulose. D-xylulose is formed by xylose isomerase
or through the intermediate of D-xylitol by D-xylose reductase and
xylitol dehydrogenase. To prevent flux toward the pentose phosphate
pathway, it is important to attenuate expression or activity of
xylulokinase, if present in the microbe. D-xylulose can then be
converted to D-ribulose by an epimerase. As mentioned above,
D-ribulose can be converted to D-ribulose-1-phosphate which is
subsequently cleaved to glycolaldehyde and dihydroxyacetone
phosphate. Examples of genes encoding a xylose transporter, xylose
isomerase, and xylulokinase are E. coli genes xylE, xylA, and xylB,
respectively. Furthermore, E. coli genes xylF, xylG, and xylH
comprise another xylose transporter. Examples of genes encoding
xylose reductase and xylitol dehydrogenase are Pichia stipitis
genes XYL1 and XYL2, respectively.
[0086] In some embodiments, L-arabinose yields glycolaldehyde.
L-arabinose can be taken in by the cell by a transporter and then
converted to L-ribulose by L-arabinose isomerase. L-ribulose can be
converted to L-xylulose by an epimerase, while a competing
degradation pathway can be reduced by attenuating L-ribulokinase
activity. Alternatively, L-arabinose can be converted to L-xylulose
through the intermediate L-arabitol by L-arabinose reductase and
L-arabitol-4-dehydrogenase. L-xylulose can then be phosphorylated
by ATP:L-xylulose 1-phosphotransferase such as to yield
L-xylulose-1-phosphate, which is subsequently cleaved by
L-xylulose-1-phosphate aldolase to produce glycolaldehyde and
dihydroxyacetone phosphate. Examples of genes encoding an
L-arabinose transporter, L-arabinose isomerase, and L-ribulokinase
include E. coli genes araE, araA and araB, respectively. E. coli
genes araF, araG and araH comprise another L-arabinose transporter.
Examples of genes encoding L-arabinose reductase and
L-arabinose-4-dehydrogenase include Aspergillus niger genes LarA
and LadA. Examples of genes encoding an ATP:L-xylulose
1-phosphotransferase and L-xylulose-1-phosphate aldolase include E.
coli genes rhaB and rhaD, respectively.
[0087] In some embodiments that yield glycolaldehyde from
L-arabinose, L-arabinose is converted to L-xylulose by one of the
pathways described herein. The L-xylulose is then converted to
xylitol by L-xylulose reductase, an example of which is the
Aspergillus niger gene LxrA. Xylitol can be converted to
D-xylulose, which can yield glycolaldehyde, as described
herein.
[0088] In some embodiments, the epimerase which catalyzes the
conversion of D-xylulose to D-ribulose and/or L-ribulose to
L-xylulose is D-tagatose 3-epimerase (DTE). Sources of DTE-encoding
genes (here referred to as dte) include, but are not limited to,
Pseudomonas cichorii, Rhodobacter sphaeroides, Pelagibaca
bermudensis, Desmospora sp. 8437, and Rhizobium loti. In some
embodiments the epimerase is D-psicose 3-epimerase (DPE). Sources
of DPE-encoding genes include, but are not limited to,
Agrobacterium tumefaciens and Clostridium cellulolyticum. In some
embodiments, the epimerase is a DTE-related protein. Such enzymes
include, but are not limited to, TM0416p from Thermotoga maritime
and, D-tagatose 3-epimerase-related protein from Fulvimarina
pelagi. In some embodiments, the epimerase is a ribulose phosphate
epimerase capable of acting on D-xylulose and/or L-ribulose.
[0089] In other embodiments, other pentoses yield glycolaldehyde.
As in the cases for D-arabinose and D-xylose, a given pentose can
yield glycolaldehyde if there exists a pathway to
D-ribulose-1-phosphate. As in the case for L-arabinose, a given
pentose can yield glycolaldehyde if there exists a pathway to
L-xylulose-1-phosphate.
[0090] Glucose, glycerol, and the dihydroxyacetone phosphate
resulting from cleavage of pentoses proceed through glycolysis, so
another element of our pathway engineering scheme is generating
glycolaldehyde from glycolysis intermediates. As shown in FIG. 2,
multiple pathways are possible. In some embodiments, the pathway
begins with conversion of 3-phosphoglycerate to
3-phosphohydroxypyruvate by 3-phosphoglycerate dehydrogenase. One
source of 3-phosphoglycerate dehydrogenase is the serA gene from E.
coli. In some embodiments, the pathway begins with conversion of
3-phosphoglycerate to glycerate by a glycerate kinase. An example
of a glycerate kinase that may be used to convert
3-phosphoglycerate to glycerate is glycerate kinase II, which is
encoded by the glxK gene in E. coli. In some embodiments, the
pathway begins with conversion of 2-phosphoglycerate to glycerate
by a glycerate kinase. An example of a glycerate kinase that may be
used to convert 2-phosphoglycerate to glycerate is glycerate kinase
I, which is encoded by the garK gene in E. coli. In some
embodiments, availability of glycolysis intermediate
3-phosphoglycerate is increased by reducing flux to
2-phosphoglycerate, or availability of 2-phosphoglycerate is
increased by reducing flux to phosphoenolpyruvate. These fluxes can
be reduced by attenuating expression or activity of
phosphoglycerate mutases and of enolase, respectively. The gpmA,
gpmB, and gpmM genes from E. coli are examples of phosphoglycerate
mutase-encoding genes. The eno gene from E. coli is an example of
an enolase-encoding gene.
[0091] In some embodiments, glycolaldehyde will be formed from
tartronate semialdehyde. This reaction has been suggested to occur
non-enzymatically [Hedrick 1961; Kim 2010], though there may be
enzymes capable of catalyzing it.
[0092] In some embodiments, glycolaldehyde will be formed from
hydroxypyruvate. This reaction has been suggested to occur
non-enzymatically, though possibly through formation of tartronate
semialdehyde [Hedrick 1961; Kim 2010]. Additionally, various
enzymes may be used to catalyze the decarboxylation of
hydroxypyruvate. The enzyme can be one specific for
hydroxypyruvate, i.e., hydroxypyruvate decarboxylase; there is
evidence of one such hydroxypyruvate decarboxylase in mammals
[Hedrick 1964]. The enzyme can be a pyruvate decarboxylase with
substrate promiscuity, capable of acting on hydroxypyruvate; an
example of one such pyruvate decarboxylase is that of wheat germ
[Davies 1985]. The enzyme can be a thiamine pyrophosphate-dependent
enzyme with promiscuous activity, capable of decarboxylation of
hydroxypyruvate. Examples of such TPP-dependent enzymes include,
but are not limited to, the subunit of the E1 component of the
2-oxoglutarate dehydrogenase complex (encoded by sucA in E. coli)
and 1-deoxyxylulose-5-phosphate synthase (encoded by dxs in E.
coli) [Kim 2010].
[0093] In some embodiments, glycolaldehyde will be formed from
L-serine. This reaction can be catalyzed by the enzyme
myeloperoxidase (MPO) as part of a MPO--H.sub.2O.sub.2-chloride
system [Anderson 1997]. For example, one source of myeloperoxidase
is the MPO gene from Homo sapiens.
[0094] In some embodiments, glycolaldehyde will be formed from
ethanolamine. This reaction may be catalyzed by ethanolamine
oxidase or by a promiscuous amine oxidase. Evidence of an
ethanolamine oxidase has been found in Phormia regina [Kulkarni
1973]. A promiscuous amine oxidase capable of oxidizing
ethanolamine has been discovered in Arthrobacter sp. [Ota 2008]. In
E. coli, tynA encodes an amine oxidase which may be capable of
oxidizing ethanolamine.
[0095] In some embodiments, glycolaldehyde will be formed from
glycolate. This reaction can be catalyzed by an aldehyde
dehydrogenase. One example of such an aldehyde dehydrogenase is
aldehyde dehydrogenase A (encoded by aldA) in E. coli.
[0096] Formation of 3-phosphohydroxypyruvate or D-glycerate can be
combined with formation of glycolaldehyde from tartronate
semialdehyde, hydroxypyruvate, L-serine, ethanolamine, or glycolate
in several ways through various connecting reactions. In some
embodiments 3-phosphohydroxypyruvate will be converted to
3-phospho-L-serine by 3-phosphoserine aminotransferase, and
3-phospho-L-serine will be further converted to L-serine by
phosphoserine phosphatase. Examples of sources of phosphoserine
aminotransferase and phosphoserine phosphatase are E. coli genes
serC and serB, respectively. In some embodiments, conversion of
L-serine to pyruvate will be reduced by attenuating serine
deaminases. Examples of serine deaminases are those encoded by E.
coli genes sdaA, sdaB, tdcB, and tdcG. In some embodiments,
L-serine will be converted to ethanolamine. This reaction can be
catalyzed by serine decarboxylase (SDC); SDC is found within plants
including, but not limited to, Arabidopsis thaliana [Rontein 2001].
Some enzymes with serine decarboxylase activity may be annotated as
histidine decarboxylase. Activity of SDC may possibly be increased
through truncation of the N-terminal extension of the enzyme.
[0097] In some embodiments, 3-phosphohydroxypyruvate will be
converted to hydroxypyruvate. This reaction can be catalyzed by an
enzyme with 3-phosphohydroxypyruvate phosphatase activity. One
example of such an enzyme is the predicted NUDIX hydrolase encoded
by yeaB in E. coli; this enzyme has been shown to have
3-phosphohydroxypyruvate phosphatase activity [Kim 2010]. Another
enzyme that is hypothesized to have 3-phosphohydroxypyruvate
phosphatase activity, based on substrate similarity, is the
glycerol-3-phosphatase encoded by GPP2 in S. cerevisiae. In some
embodiments, L-serine will be converted to hydroxypyruvate, or
hydroxypyruvate will be converted to L-serine. This reaction can be
catalyzed by serine:pyruvate aminotransferase (SPT) or by
alanine:glyoxylate aminotransferase (AGT). Sources of SPT and AGT
include, but are not limited to, Arabidopsis thaliana, Drosophila
melanogaster, Canis lupus familiaris, Homo sapiens, and Rattus
norvegicus.
[0098] In some embodiments, D-glycerate will be converted to
hydroxypyruvate. Examples of enzymes capable of catalyzing this
reaction include, but are not limited to, glyoxylate reductases
encoded by E. coli genes ghrA and ghrB. In some embodiments,
D-glycerate will be converted to tartronate semialdehyde. Examples
of enzymes capable of catalyzing this reaction include, but are not
limited to, tartronate semialdehyde reductases encoded by E. coli
genes glxR and garR. In some embodiments, hydroxypyruvate will be
converted to tartronate semialdehyde, or tartronate semialdehyde to
hydroxypyruvate. This reaction can be catalyzed by hydroxypyruvate
isomerase. One source of hydroxypyruvate isomerase is the E. coli
gene hyi.
[0099] In some embodiments, tartronate semialdehyde will be
converted to glyoxylate. For example, one enzyme capable of
catalyzing this reaction is tartronate semialdehyde synthase
encoded by the E. coli gene gcl. In some embodiments, glyoxylate
will be converted to glycolate. An example of an enzyme capable of
catalyzing this reaction is glycolate oxidase encoded by E. coli
genes glcD, glcE, and glcF.
[0100] The map presented in FIG. 2, shows the reaction space
available for generating glycolaldehyde from glycolysis
intermediates. Within the reaction space, there exist several
pathways based on combinations of the reactions described
herein.
[0101] Another aspect of this technology includes reducing
glycolaldehyde to ethylene glycol. In some embodiments, this
reaction is catalyzed by glycolaldehyde reductase. An example of
glycolaldehyde reductase includes, but is not limited to, the
enzyme encoded by the E. coli gene fucO. In some embodiments,
glycolaldehyde conversion to ethylene glycol is catalyzed by an
alcohol dehydrogenase. Examples include, but are not limited to,
alcohol dehydrogenases encoded by ADH1 in S. cerevisiae and yqhD in
E. coli. Some organisms can metabolize glycolaldehyde to glycolate,
by an aldehyde dehydrogenase for example. In some embodiments, this
reaction will be attenuated.
[0102] The pathways described herein for the production of ethylene
glycol and ethylene glycol precursors in cells involve several
enzymatic components. In some embodiments, the genes are expressed
as part of an operon. These genes may be placed in any order in the
operon. It should be appreciated that some cells compatible with
the invention may express an endogenous copy of one of more of the
aforementioned enzymatic components as well as a recombinant
copy.
[0103] As one of ordinary skill in the art would be aware,
homologous genes for these enzymes can be obtained from other
species and can be identified by homology searches, for example
through a protein BLAST search, available at the National Center
for Biotechnology Information (NCBI) internet site
(www.ncbi.nlm.nih.gov). Genes associated with the invention can be
cloned, for example by PCR amplification and/or restriction
digestion, from DNA from any source of DNA which contains the given
gene. In some embodiments, a gene associated with the invention is
synthetic. Any means of obtaining a gene encoding for an enzyme
associated with the invention is compatible with the instant
invention.
[0104] Aspects of the invention include strategies to optimize
production of ethylene glycol from a cell. Optimized production of
ethylene glycol refers to producing a higher amount of ethylene
glycol following pursuit of an optimization strategy than would be
achieved in the absence of such a strategy. Optimization of
production of ethylene glycol can involve modifying a gene encoding
for an enzyme before it is recombinantly expressed in a cell. In
some embodiments, such a modification involves codon optimization
for expression in a bacterial cell. For example, this includes the
use of heterologous genes from various sources whose sequence has
been properly modified (including codon optimization) for optimal
expression in the host organism. Codon usages for a variety of
organisms can be accessed in the Codon Usage Database
(kazusa.or.jp/codon/). Codon optimization, including identification
of optimal codons for a variety of organisms, and methods for
achieving codon optimization, are familiar to one of ordinary skill
in the art, and can be achieved using standard methods.
[0105] In some embodiments, modifying a gene encoding for an enzyme
before it is recombinantly expressed in a cell involves making one
or more mutations in the gene encoding for the enzyme before it is
recombinantly expressed in a cell. For example, a mutation can
involve a substitution or deletion of a single nucleotide or
multiple nucleotides. In some embodiments, a mutation of one or
more nucleotides in a gene encoding for an enzyme will result in a
mutation in the enzyme, such as a substitution or deletion of one
or more amino acids.
[0106] Additional changes can include increasing copy numbers of
the gene components of pathways active in production of ethylene
glycol, such as by additional episomal expression. In some
embodiments, screening for mutations in components of the
production of ethylene glycol, or components of other pathways,
that lead to enhanced production of ethylene glycol may be
conducted through a random mutagenesis screen, or through screening
of known mutations. In some embodiments, shotgun cloning of genomic
fragments could be used to identify genomic regions that lead to an
increase in production of ethylene glycol, through screening cells
or organisms that have these fragments for increased production of
ethylene glycol. In some cases one or more mutations may be
combined in the same cell or organism.
[0107] In some embodiments, production of ethylene glycol in a cell
can be increased through manipulation of enzymes that act in the
same pathway as the enzymes associated with the invention. For
example, in some embodiments it may be advantageous to increase
expression of an enzyme or other factor that acts upstream or
downstream of a target enzyme such as an enzyme associated with the
invention. This could be achieved by over-expressing the upstream
or downstream factor using any standard method.
[0108] A further strategy for optimization of production of
ethylene glycol is to increase expression levels of one or more
genes associated with the invention, which can be described as
"pathway balancing". This may be accomplished, for example, through
selection of appropriate promoters and ribosome binding sites. In
some embodiments, the production of ethylene glycol is increased by
balancing expression of the genes such as by selecting promoters of
various strengths to drive expression of the genes. In some
embodiments, this may include the selection of high-copy number
plasmids, or low or medium-copy number plasmids. The step of
transcription termination can also be targeted for regulation of
gene expression, through the introduction or elimination of
structures such as stem-loops.
[0109] The invention also encompasses isolated polypeptides
containing mutations or codon optimizations in residues described
herein, and isolated nucleic acid molecules encoding such
polypeptides. As used herein, the terms "protein" and "polypeptide"
are used interchangeably and thus the term polypeptide may be used
to refer to a full-length polypeptide and may also be used to refer
to a fragment of a full-length polypeptide. As used herein with
respect to polypeptides, proteins, or fragments thereof, "isolated"
means separated from its native environment and present in
sufficient quantity to permit its identification or use. Isolated,
when referring to a protein or polypeptide, means, for example: (i)
selectively produced by expression cloning or (ii) purified as by
chromatography or electrophoresis. Isolated proteins or
polypeptides may be, but need not be, substantially pure. The term
"substantially pure" means that the proteins or polypeptides are
essentially free of other substances with which they may be found
in production, nature, or in vivo systems to an extent practical
and appropriate for their intended use. Substantially pure
polypeptides may be obtained naturally or produced using methods
described herein and may be purified with techniques well known in
the art. Because an isolated protein may be admixed with other
components in a preparation, the protein may comprise only a small
percentage by weight of the preparation. The protein is nonetheless
isolated in that it has been separated from the substances with
which it may be associated in living systems, i.e. isolated from
other proteins.
[0110] The invention also encompasses nucleic acids that encode for
any of the polypeptides described herein, libraries that contain
any of the nucleic acids and/or polypeptides described herein, and
compositions that contain any of the nucleic acids and/or
polypeptides described herein. It should be appreciated that
libraries containing nucleic acids or proteins can be generated
using methods known in the art. A library containing nucleic acids
can contain fragments of genes and/or full-length genes and can
contain wild-type sequences and mutated sequences. A library
containing proteins can contain fragments of proteins and/or full
length proteins and can contain wild-type sequences and mutated
sequences. It should be appreciated that the invention encompasses
codon-optimized forms of any of the nucleic acid and protein
sequences described herein.
[0111] The invention encompasses any type of cell that
recombinantly expresses genes associated with the invention,
including prokaryotic and eukaryotic cells. In some embodiments the
cell is a bacterial cell, such as Escherichia spp., Streptomyces
spp., Zymonas spp., Acetobacter spp., Citrobacter spp.,
Synechocystis spp., Rhizobium spp., Clostridium spp.,
Corynebacterium spp., Streptococcus spp., Xanthomonas spp.,
Lactobacillus spp., Lactococcus spp., Bacillus spp., Alcaligenes
spp., Pseudomonas spp., Aeromonas spp., Azotobacter spp., Comamonas
spp., Mycobacterium spp., Rhodococcus spp., Gluconobacter spp.,
Ralstonia spp., Acidithiobacillus spp., Microlunatus spp.,
Geobacter spp., Geobacillus spp., Arthrobacter spp., Flavobacterium
spp., Serratia spp., Saccharopolyspora spp., Thermus spp.,
Stenotrophomonas spp., Chromobacterium spp., Sinorhizobium spp.,
Saccharopolyspora spp., Agrobacterium spp. and Pantoea spp. The
bacterial cell can be a Gram-negative cell such as an Escherichia
coli (E. coli) cell, or a Gram-positive cell such as a species of
Bacillus. In other embodiments, the cell is a fungal cell such as a
yeast cell, e.g., Saccharomyces spp., Schizosaccharomyces spp.,
Pichia spp., Paffia spp., Kluyveromyces spp., Candida spp.,
Talaromyces spp., Brettanomyces spp., Pachysolen spp., Debaryomyces
spp., Yarrowia spp. and industrial polyploid yeast strains.
Preferably the yeast strain is a S. cerevisiae strain. Other
examples of fungi include Aspergillus spp., Pennicilium spp.,
Fusarium spp., Rhizopus spp., Acremonium spp., Neurospora spp.,
Sordaria spp., Magnaporthe spp., Allomyces spp., Ustilago spp.,
Botrytis spp., and Trichoderma spp. In other embodiments, the cell
is an algal cell, or a plant cell.
[0112] It should be appreciated that some cells compatible with the
invention may express an endogenous copy of one or more of the
genes associated with the invention as well as a recombinant copy.
In some embodiments, if a cell has an endogenous copy of one or
more of the genes associated with the invention then the methods
will not necessarily require adding a recombinant copy of the
gene(s) that are endogenously expressed. In some embodiments the
cell may endogenously express one or more enzymes from the pathways
described herein and may recombinantly express one or more other
enzymes from the pathways described herein for efficient production
of ethylene glycol.
[0113] In some embodiments, one or more of the genes associated
with the invention is expressed in a recombinant expression vector.
In other embodiments, one or more of the genes associated with the
invention is expressed as or from one or more chromosomally
integrated genes.
[0114] As used herein, a "vector" may be any of a number of nucleic
acids into which a desired sequence or sequences may be inserted by
restriction and ligation for transport between different genetic
environments or for expression in a host cell. Vectors are
typically composed of DNA, although RNA vectors are also available.
Vectors include, but are not limited to: plasmids, fosmids,
phagemids, virus genomes and artificial chromosomes.
[0115] A cloning vector is one which is able to replicate
autonomously or integrated in the genome in a host cell, and which
is further characterized by one or more endonuclease restriction
sites at which the vector may be cut in a determinable fashion and
into which a desired DNA sequence may be ligated such that the new
recombinant vector retains its ability to replicate in the host
cell. In the case of plasmids, replication of the desired sequence
may occur many times as the plasmid increases in copy number within
the host cell such as a host bacterium or just a single time per
host before the host reproduces by mitosis. In the case of phage,
replication may occur actively during a lytic phase or passively
during a lysogenic phase.
[0116] An expression vector is one into which a desired DNA
sequence may be inserted by restriction and ligation such that it
is operably joined to regulatory sequences and may be expressed as
an RNA transcript. Vectors may further contain one or more marker
sequences suitable for use in the identification of cells which
have or have not been transformed or transfected with the vector.
Markers include, for example, genes encoding proteins which
increase or decrease either resistance or sensitivity to
antibiotics or other compounds, genes which encode enzymes whose
activities are detectable by standard assays known in the art
(e.g., .beta.-galactosidase, luciferase or alkaline phosphatase),
and genes which visibly affect the phenotype of transformed or
transfected cells, hosts, colonies or plaques (e.g., green
fluorescent protein). Preferred vectors are those capable of
autonomous replication and expression of the structural gene
products present in the DNA segments to which they are operably
joined.
[0117] As used herein, a coding sequence and regulatory sequences
are said to be "operably" joined when they are covalently linked in
such a way as to place the expression or transcription of the
coding sequence under the influence or control of the regulatory
sequences. If it is desired that the coding sequences be translated
into a functional protein, two DNA sequences are said to be
operably joined if induction of a promoter in the 5' regulatory
sequences results in the transcription of the coding sequence and
if the nature of the linkage between the two DNA sequences does not
(1) result in the introduction of a frame-shift mutation, (2)
interfere with the ability of the promoter region to direct the
transcription of the coding sequences, or (3) interfere with the
ability of the corresponding RNA transcript to be translated into a
protein. Thus, a promoter region would be operably joined to a
coding sequence if the promoter region were capable of effecting
transcription of that DNA sequence such that the resulting
transcript can be translated into the desired protein or
polypeptide.
[0118] When the nucleic acid molecule that encodes any of the
enzymes of the claimed invention is expressed in a cell, a variety
of transcription control sequences (e.g., promoter/enhancer
sequences) can be used to direct its expression. The promoter can
be a native promoter, i.e., the promoter of the gene in its
endogenous context, which provides normal regulation of expression
of the gene. In some embodiments the promoter can be constitutive,
i.e., the promoter is unregulated allowing for continual
transcription of its associated gene. A variety of conditional
promoters also can be used, such as promoters controlled by the
presence or absence of a molecule.
[0119] The precise nature of the regulatory sequences needed for
gene expression may vary between species or cell types, but shall
in general include, as necessary, 5' non-transcribed and 5'
non-translated sequences involved with the initiation of
transcription and translation respectively, such as a TATA box,
capping sequence, CAAT sequence, and the like. In particular, such
5' non-transcribed regulatory sequences will include a promoter
region which includes a promoter sequence for transcriptional
control of the operably joined gene. Regulatory sequences may also
include enhancer sequences or upstream activator sequences as
desired. The vectors of the invention may optionally include 5'
leader or signal sequences. The choice and design of an appropriate
vector is within the ability and discretion of one of ordinary
skill in the art.
[0120] Expression vectors containing all the necessary elements for
expression are commercially available and known to those skilled in
the art. See, e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, 1989. Cells are genetically engineered by the introduction
into the cells of heterologous DNA (RNA). That heterologous DNA
(RNA) is placed under operable control of transcriptional elements
to permit the expression of the heterologous DNA in the host cell.
Heterologous expression of genes associated with the invention, for
production of ethylene glycol, is demonstrated in the Examples
using E. coli. The novel method for producing ethylene glycol can
also be expressed in other bacterial cells, fungi (including yeast
cells), plant cells, etc.
[0121] A nucleic acid molecule that encodes the enzyme of the
claimed invention can be introduced into a cell or cells using
methods and techniques that are standard in the art. For example,
nucleic acid molecules can be introduced by standard protocols such
as transformation including chemical transformation and
electroporation, transduction, particle bombardment, etc.
Expressing the nucleic acid molecule encoding the enzymes of the
claimed invention also may be accomplished by integrating the
nucleic acid molecule into the genome.
[0122] In some embodiments one or more genes associated with the
invention is expressed recombinantly in a bacterial cell. Bacterial
cells according to the invention can be cultured in media of any
type (rich or minimal) and any composition. As would be understood
by one of ordinary skill in the art, routine optimization would
allow for use of a variety of types of media. The selected medium
can be supplemented with various additional components. Some
non-limiting examples of supplemental components include one or
more carbon sources such as D-arabinose, D-xylose, D-glucose,
biomass hydrolysates (specifically hemicellulose) that contains
D-xylose, L-arabinose, glycerol and serine; antibiotics; IPTG for
gene induction; ATCC Trace Mineral Supplement; malonate; cerulenin;
and glycolate. Similarly, other aspects of the medium, and growth
conditions of the cells of the invention may be optimized through
routine experimentation. For example, pH and temperature are
non-limiting examples of factors which can be optimized. In some
embodiments, factors such as choice of media, media supplements,
and temperature can influence production levels of ethylene glycol.
In some embodiments the concentration and amount of a supplemental
component may be optimized. In some embodiments, how often the
media is supplemented with one or more supplemental components, and
the amount of time that the media is cultured before harvesting
ethylene glycol, is optimized.
[0123] In some embodiments the temperature of the culture may be
between 25 and 43.degree. C., inclusive. For example it may be 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42
or 43.degree. C., or any value in between. In certain embodiments
the temperature is between 30 and 32.degree. C. including 30, 31
and 32.degree. C. and any value in between. In certain embodiments
the temperature is between 36 and 38.degree. C. including 36, 37
and 38.degree. C. and any value in between. As would be understood
by one of ordinary skill in the art, the optimal temperature in
which to culture a cell for production of ethylene glycol may be
influenced by many factors including the type of cell, the growth
media and the growth conditions.
[0124] Other non-limiting factors that can be varied through
routine experimentation in order to optimize production of ethylene
glycol include the concentration and amount of feedstock and any
supplements provided, how often the media is supplemented, and the
amount of time that the media is cultured before harvesting the
ethylene glycol. In some embodiments the cells may be cultured for
6, 12, 18, 24, 30, 36, 42, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210,
220, 230, 240, 250, 260, 270, 280, 290, 300, including all
intermediate values, or greater than 300 hours. In some embodiments
optimal production is achieved after culturing the cells for
several days such as 3-4 days. However it should be appreciated
that it would be routine experimentation to vary and optimize the
above-mentioned parameters and other such similar parameters.
[0125] According to aspects of the invention, high titers of
ethylene glycol are produced through the recombinant expression of
genes associated with the invention, in a cell. As used herein
"high titer" refers to a titer in the grams per liter (g/L) scale.
The titer produced for a given product will be influenced by
multiple factors including choice of media. In some embodiments the
total ethylene glycol titer is at least 0.5 g/L (500 milligrams per
liter). For example the titer may be 0.5, 0.6, 0.7, 0.8, 0.9, 1.0,
1.1, 1.2, 1.3, 1.4, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0,
7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, 100, including all intermediate values, or more than
100 g/L.
[0126] The liquid cultures used to grow cells associated with the
invention can be housed in any of the culture vessels known and
used in the art. In some embodiments large scale production in an
aerated reaction vessel such as a stirred tank reactor can be used
to produce large quantities of ethylene glycol, which can be
recovered from the cell culture.
EXAMPLES
[0127] The above elements have been combined into engineered
strains of microbial cells. As a proof of concept, one of the
engineered strains is capable of producing small amounts (25 mg/L)
of ethylene glycol from D-glucose, one of the engineered strains is
capable of producing small amounts of ethylene glycol from glycerol
(30 mg/L), and one of the engineered strains is capable of
producing 2 g/L ethylene glycol from L-arabinose. Other of the
engineered strains are capable of converting greater than 30%
(based on mass) of D-arabinose or D-xylose to ethylene glycol.
Furthermore, with the strain engineered to use D-xylose, we have
successfully produced titers of ethylene glycol greater than 40 g/L
and have successfully converted hemicellulose hydrolysate (8.7 g/L
xylose content) to ethylene glycol (2.7 g/L).
[0128] Thus, pathways have been constructed in engineered microbes
for the production of ethylene glycol from simple sugars derived
from biomass. Producing this bulk chemical biologically offers
several advantages over chemical methods, and using sugars as the
source material makes the ethylene glycol a "green" product. Such
technology could therefore be used in the production of "green" PET
products, for example "green" bottles.
Materials and Methods
Media
[0129] Luria Broth (LB) was prepared per instructions (BD, NJ,
USA). M9 minimal medium consisted of M9 salts (BD, NJ, USA; 6.8 g/L
Na.sub.2HPO.sub.4, 3.0 g/L KH.sub.2PO.sub.4, 0.5 g/L NaCl, and 1.0
g/L NH.sub.4Cl), 2 mL/L 1M MgSO.sub.4, 0.1 mL/L 1M CaCl.sub.2, and
specified sugar. In some cases, M9 minimal medium was supplemented
with 0.06 g/L Fe(III) citrate, 4.5 mg/L thiamine, and 1 mL/L trace
elements solution. The trace elements solution consisted of 8.4 g/L
EDTA, 2.5 g/L CoCl.sub.2, 15 g/L MnCl.sub.2, 1.5 g/L CuCl.sub.2, 3
g/L H.sub.3BO.sub.3, 2.5 g/L Na.sub.2MoO.sub.4, and 8 g/L
Zn(CH.sub.3COO).sub.2.
[0130] M9(+) minimal medium consisted of 2.0 g/L NH.sub.4Cl, 5.0
g/L (NH.sub.4).sub.2SO.sub.4, 3.0 g/L KH.sub.2PO.sub.4, 7.3 g/L
K.sub.2HPO.sub.4, 8.4 g/L MOPS, 0.5 g/L NaCl, 2 mL/L 1M MgSO.sub.4,
1 ml/l mineral solution, 0.1 mL/L 4 mM Na.sub.2MoO.sub.4, and
specified sugar. The mineral solution consisted of 3.6 g/l
FeCl.sub.2.4H.sub.2O, 5 g/l CaCl.sub.2.2H.sub.2O, 1.3 g/l
MnCl.sub.2.2H.sub.2O, 0.38 g/l CuCl.sub.2.2H.sub.2O, 0.5 g/l
CoCl.sub.2.6H.sub.2O, 0.94 g/l ZnCl.sub.2, 0.0311 g/l
H.sub.3BO.sub.3, 0.4 g/l Na.sub.2EDTA.2H.sub.2O, and 1.01 g/l
thiamine-HCl. The medium was set to pH 7 with KOH.
[0131] For bioreactor fermentations, minimal medium consisted of
2.0 g/L NH.sub.4Cl, 5.0 g/L (NH.sub.4).sub.2SO.sub.4, 2.0 g/L
KH.sub.2PO.sub.4, 0.5 g/L NaCl, 2 mL/L 1M MgSO.sub.4, 1 ml/l
mineral solution, 0.1 mL/L 4 mM Na.sub.2MoO.sub.4, and specified
sugar.
[0132] Energy cane C5 hydrolysate was pretreated by adding lime
(Ca(OH).sub.2) and heating, while stirring, at 60.degree. C. for 30
minutes; precipitation was then removed by filtration. Pretreated
hydrolysate was diluted in M9(+) minimal medium to yield
hydrolysate medium.
[0133] Media were sterilized by autoclaving (121.degree. C., 21')
or by filtration (0.22 .mu.m Corning, N.Y., USA) where appropriate.
When necessary, the medium was made selective by adding an
antibiotic (ampicillin, chloramphenicol, kanamycin, spectinomycin).
For experiments with strains harboring expression plasmids, IPTG
was added to the medium.
Strains and Plasmids
[0134] Escherichia coli (E. coli) DH5.alpha. (F-,
.phi.80dlacZ.DELTA.M15, .DELTA.(lacZYA-argF)U169, deoR, recA1,
endA1, hsdR17(rk-, mk+), phoA, supE44, .lamda.-, thi-1, gyrA96,
relA1) was used to maintain plasmids. E. coli K-12 MG1655
.DELTA.recA .DELTA.endA DE3 (provided by Professor Kristala
Prather, MIT) and mutants thereof were used as
production/experimental strains.
[0135] Gene disruptions (knock-out, KO) were introduced into E.
coli using the concept of Datsenko and Wanner (2000). Transformants
carrying pKD46 (Red helper plasmid, ampicillin resistance) were
grown in 10 ml LB medium with ampicillin (100 mg/l) and L-arabinose
(10 mM) at 30.degree. C. to an OD.sub.600 nm of 0.6. The cells were
made electro competent by washing them with 50 ml of ice-cold
water, a first time, and with 1 ml ice-cold water, a second time.
Then, the cells were resuspended in 50 .mu.l of ice-cold water.
Electroporation was done with 50 .mu.l of cells and 10-100 ng of
linear double-stranded-DNA product by using a Gene Pulser.RTM.
(Bio-Rad Laboratories, CA, USA) (600.OMEGA., 25 .mu.FD, and 250
volts). After electroporation, cells were added to 1 ml SOC medium
(VWR, PA, USA) incubated 1 h at 37.degree. C., and finally spread
onto LB-agar containing 25 mg/l of chloramphenicol or 50 mg/l of
kanamycin to select antibiotic resistant transformants. Mutants
were verified by PCR with primers upstream and downstream of the
modified region and were grown in LB-agar at 42.degree. C. for the
loss of the helper plasmid. Mutants were tested for ampicillin
sensitivity. The selected mutants (chloramphenicol or kanamycin
resistant) were transformed with the pCP20 plasmid, which is an
ampicillin- and chloramphenicol-resistant plasmid that shows
temperature-sensitive replication and thermal induction of FLP
synthesis. The ampicillin-resistant transformants were selected at
30.degree. C., after which a few were colony purified in LB at
42.degree. C. and then tested for loss of all antibiotic
resistances and of the FLP helper plasmid. The gene knock-outs were
checked with control primers and sequenced.
[0136] For overexpression of enzymes, we utilized
chloramphenicol-resistant p10_T5T10 and p10_T7 plasmids which were
derived from the p15A plasmid, and spectinomycin-resistant p5_T5T10
and p5_T7 plasmids which were derived from the pSC101 plasmid
[Ajikumar, 2010]. "T10" is another promoter region that is not
active in E. coli. Spectinomycin-resistant pCDFDuet (Novagen, EMD
Millipore, Mass., USA) was also utilized. Common cloning methods
using restriction enzymes and ligase enzyme were used to construct
the different plasmids. The construction of several key plasmids is
shown schematically in FIGS. 3-6. All plasmids were checked by PCR
and/or restriction digests.
[0137] Different mutant strains were transformed with the
constructed plasmids by electroporation. The cells were made
electro competent and transformed as above. The selected mutants
were verified by PCR using plasmid specific primers.
Cultivation Conditions
D-Arabinose Degradation Pathway
[0138] A culture, from a single colony on a LB-plate, in 3-ml LB
medium was incubated overnight (o/n) at 37.degree. C. on an orbital
shaker at 200 rpm. This culture was used to inoculate to 1% v/v a
3-mL culture of M9 minimal medium with 4 g/L D-glucose; the culture
was incubated o/n at 37.degree. C., 200 rpm. This culture was used
to inoculate to OD.sub.600 0.01 a 10-mL culture of M9 minimal
medium with 4 g/L D-arabinose and 1 mM L-fucose in a 50-mL Falcon
tube at 37.degree. C., 200 rpm. Samples were taken at various time
points.
[0139] Unsealed Hungate tubes with 10 mL of M9 minimal medium with
4 g/L D-arabinose, with and without 1 mM L-fucose, were placed in
an anaerobic chamber (Coy Laboratory Products, MI, USA) o/n. These
cultures were inoculated to OD.sub.600 0.01 from the aerobic
D-arabinose cultures described herein, sealed with butyl rubber
septa, and incubated at 37.degree. C., 200 rpm. Samples were taken
within the anaerobic chamber at various time points.
D-Arabinose and D-Xylose Comparison
[0140] A culture, from a single colony on a LB-plate, in 3-ml LB
medium was incubated overnight (o/n) at 37.degree. C. on an orbital
shaker at 200 rpm. This culture was used to inoculate to 1% v/v a
3-mL culture of M9 minimal medium with 10 g/L D-glucose and
supplements; the culture was incubated o/n at 37.degree. C., 200
rpm. This culture was used to inoculate to OD.sub.600 0.05 a
10.8-mL culture of M9 minimal medium with supplements, 1 mM
L-fucose, and either 10 g/L D-arabinose or 10 g/L D-xylose. For
strains with plasmids, 1 mM IPTG and appropriate antibiotics were
added to the medium. These cultures were incubated at 37.degree.
C., 200 rpm, and samples were taken at various time points.
Evaluation of Gene Order in Production from D-Xylose
[0141] A culture, from a single colony on a LB-plate, in 3-ml LB
medium was incubated overnight (o/n) at 37.degree. C. on an orbital
shaker at 200 rpm. This culture was used to inoculate to 1% v/v a
3-mL culture of M9 minimal medium with 10 g/L D-glucose and
supplements; the culture was incubated o/n at 37.degree. C., 200
rpm. This culture was used to inoculate to OD.sub.600 0.05 a
10.8-mL culture of M9 minimal medium with supplements, and either
10 g/L D-arabinose and 1 mM L-fucose, or 10 g/L D-xylose. For
strains with plasmids, 1 mM IPTG and appropriate antibiotics were
added to the medium. These cultures were incubated at 37.degree.
C., 200 rpm, and samples were taken at various time points.
Production from D-Xylose in a Bioreactor
[0142] A culture, from a single colony on a LB-plate, in 3-ml LB
medium was incubated overnight (o/n) at 37.degree. C. on an orbital
shaker at 200 rpm. This culture was used to inoculate to 1% v/v
four 50-mL cultures of M9(+) minimal medium with 15 g/L D-glucose
and spectinomycin; the cultures were incubated o/n at 37.degree.
C., 200 rpm. The cultures were combined, and 100 mL of culture was
used to inoculate each bioreactor. Each bioreactor was a two-liter
Bioflo.RTM. culture vessel (New Brunswick, Conn., USA) with 2.0 L
minimal medium with 35 g/L D-xylose, 0.1 mM IPTG, and
spectinomycin. Temperature was maintained at 37.degree. C., and the
pH was maintained at 7.0 with 6N NaOH. Aerobic conditions were
maintained by sparging with air at 0.5 to 1 lpm, and dissolved
oxygen content was maintained at 30% by altering agitation from 400
to 650 rpm. A solution of silicone antifoaming B emulsion
(Sigma-Aldrich, MO, USA) was added when foaming occurred during the
fermentation. All data was logged with the New Brunswick system
(New Brunswick, Conn., USA). When nearly all D-xylose was consumed
(.about.20 h), we initiated pumping of a feed solution into the
bioreactor through a reactor port. The feed solution consisted of
600 g/L D-xylose, 10 g/L (NH.sub.4).sub.2SO.sub.4, 5.0 g/L
MgSO.sub.4, 0.1 mM IPTG, and spectinomycin. The flow rate varied
from 0.05 mL/min to 0.15 mL/min Samples were collected every four
hours via a harvest pipe connected to a reactor port.
Production from Hydrolysate
[0143] A culture, from a single colony on a LB-plate, in 3-mL LB
medium was incubated overnight (o/n) at 37.degree. C. on an orbital
shaker at 200 rpm. This culture was used to inoculate to 1% v/v a
10-mL culture of M9(+) minimal medium with 15 g/L D-glucose and
spectinomycin; the culture was incubated o/n at 37.degree. C., 200
rpm. The culture was used to inoculate to OD.sub.600 0.05 50-mL
hydrolysate cultures; these consisted of pretreated hydrolysate
diluted to 1/5, , 3/5, or 4/5 in M9(+) minimal medium. These
cultures were incubated at 37.degree. C., 200 rpm; samples were
taken at various time points, but growth was only observed in the
1/5 dilution.
Production from L-Arabinose
[0144] A culture, from a single colony on a LB-plate or from a
glycerol stock thereof, in 3-ml LB medium was incubated overnight
(o/n) at 37.degree. C. on an orbital shaker at 200 rpm. This
culture was used to inoculate to 1% v/v a 3-mL culture of M9(+)
minimal medium with 15 g/L D-glucose; the culture was incubated o/n
at 37.degree. C., 200 rpm. This culture was used to inoculate to
OD.sub.600 0.05 a 50-mL culture of M9(+) minimal medium with 15 g/L
L-arabinose. For strains with plasmids, 1 mM IPTG and appropriate
antibiotics were added to the medium. These cultures were incubated
at 37.degree. C., 200 rpm, and samples were taken at various time
points.
Production from Serine
[0145] A culture, from a single colony on a LB-plate or from a
glycerol stock thereof, in 3-ml LB medium was incubated overnight
(o/n) at 37.degree. C. on an orbital shaker at 200 rpm. This
culture was used to inoculate to 1% v/v a 3-mL culture of M9(+)
minimal medium with 15 g/L D-glucose; the culture was incubated o/n
at 37.degree. C., 200 rpm. This culture was used to inoculate to
OD.sub.600 0.1 a 50-mL culture of M9(+) minimal medium with 15 g/L
D-glucose and 10 g/L L-serine. For strains with plasmids, 1 mM IPTG
and appropriate antibiotics were added to the medium. These
cultures were incubated at 22.degree. C., 250 rpm, and samples were
taken at various time points.
Production from D-Glucose or Glycerol
[0146] A culture, from a single colony on a LB-plate, in 3-ml LB
medium was incubated overnight (o/n) at 37.degree. C. on an orbital
shaker at 200 rpm. In some cases, this culture was used to
inoculate to OD.sub.600 0.05 a 3-mL culture of M9(+) minimal medium
with 15 g/L D-glucose, chloramphenicol (as necessary), and 1 mM
IPTG; the culture was incubated o/n at 37.degree. C., 200 rpm.
Samples were taken after 4 days.
[0147] In other cases, the LB culture was used to inoculate to
OD.sub.600 0.05 a 50-mL culture of M9(+) minimal medium with 1 mM
IPTG and either 15 g/L D-glucose or 15.3 g/L glycerol; antibiotics
were added as appropriate, and the culture was incubated o/n at
22.degree. C., 250 rpm. Samples were taken at various time
points.
Analytical Methods
[0148] Cell densities of the cultures were determined by measuring
optical density at 600 nm (Ultrospec 2100 pro, Amersham (GE
Healthcare), NJ, USA). The concentrations of sugars and ethylene
glycol were determined in a Waters HPLC system (Waters, Mass.,
USA), using an Aminex HPX-87H column (Bio-Rad, CA, USA) heated at
50.degree. C., equipped with a 1 cm precolumn, using 14 mM
H.sub.2SO.sub.4 (0.7 ml/min) as mobile phase. A differential
refractive index detector (Waters, Mass., USA) was used for analyte
detection.
Example 1
[0149] Researching E. coli for the biological production of
ethylene glycol (EG) from sugars, we noted the pathway for the
degradation of D-arabinose in K-12 strains. In the pathway, the
pentose intermediate D-ribulose-1-phosphate is cleaved to yield
dihydroxyacetone phosphate (DHAP), which enters into glycolysis,
and glycolaldehyde. Glycolaldehyde is typically oxidized to
glycolate by aldehyde dehydrogenase A (encoded by the aldA gene),
but as in the analogous reaction of L-lactaldehyde to
L-1,2-propanediol, glycolaldehyde can also be reduced by the
oxidoreductase encoded by fucO. The fluxes through these reactions
depend on the availability of oxygen, so we investigated the
degradation of D-arabinose under aerobic and anaerobic
conditions.
[0150] Cultures of E. coli K-12 MG1655 DE3 .DELTA.endA .DELTA.recA
(referred to as wild-type or WT) and E. coli K-12 MG1655 DE3
.DELTA.endA .DELTA.recA .DELTA.aldA (referred to as .DELTA.aldA)
were grown aerobically and anaerobically on minimal medium with 4
g/L D-arabinose as the carbon source. After approximately nine
days, we measured the concentration of ethylene glycol in the
culture supernatants (Table 1). WT yielded 1.0 g/L EG when cultured
anaerobically but only 0.1 g/L EG when cultured aerobically. This
result confirms that E. coli K-12 can generate EG via the
D-arabinose degradation pathway and indicates that the titer of EG
is greater under anaerobic conditions. While deletion of
.DELTA.aldA only slightly improves the anaerobic production of EG
relative to WT, aerobic production significantly improves. The
anaerobic and aerobic titers for .DELTA.aldA are similar, so
.DELTA.aldA can be grown aerobically without sacrificing EG
production.
TABLE-US-00001 TABLE 1 Production of ethylene glycol from
D-arabinose. E. coli cultures were grown on minimal medium with 4
g/L D-arabinose, and ethylene glycol concentrations were measured
at 218 h (post-inoculation) for anaerobic cultures (O.sub.2-) and
209 h for aerobic cultures (O.sub.2+). Strain O.sub.2 Ethylene
Glycol (g/L) WT - 1.0 .DELTA.aldA - 1.3 WT + 0.1 .DELTA.aldA +
1.2
Example 2
[0151] Though we have shown that E. coli is capable of generating
ethylene glycol from D-arabinose, biomass-derived sugars, such as
D-xylose and D-glucose, are much more abundant and therefore are
preferred substrates. Because D-xylose is a pentose, we next
pursued the utilization of D-xylose for EG production through the
previously established D-arabinose degradation pathway. D-tagatose
3-epimerase (DTE) from Pseudomonas cichorii is a promiscuous enzyme
that has been shown to interconvert D-xylulose and D-ribulose
[Izumori 1993], intermediates of the D-xylose degradation and
D-arabinose degradation pathways, respectively. Therefore, DTE
(encoded by the gene here referred to as dte) can provide a path by
which D-xylose can yield EG, however, conversion of D-xylulose to
D-xylulose-5-phosphate, catalyzed by xylulokinase (encoded by
xylB), is a competing reaction. We generated E. coli K-12 MG1655
DE3 .DELTA.endA .DELTA.recA .DELTA.aldA .DELTA.xylB (referred to as
.DELTA.aldA .DELTA.xylB) and transformed the strain with a plasmid
overexpressing DTE (resulting in .DELTA.aldA
.DELTA.xylB/p10_T5T10-dte). These strains were cultured with 10 g/L
D-xylose and compared with .DELTA.aldA grown on 10 g/L D-arabinose
(FIG. 7).
[0152] In this experiment, we observed that .DELTA.aldA .DELTA.xylB
is still capable of growing on D-xylose (FIG. 3, squares), even
though .DELTA.aldA .DELTA.xylB lacks xylulokinase. The .DELTA.aldA
.DELTA.xylB strain grows more slowly than WT (data not shown), and
the pathway by which D-xylose is metabolized does not lead to any
significant EG production. Overexpressing DTE in .DELTA.aldA
.DELTA.xylB provides the connection between D-xylulose and
D-ribulose and consequently leads to EG production (FIG. 7,
diamonds). Though this strain successfully shows the ability to
produce EG, compared to .DELTA.aldA grown on D-arabinose (FIG. 7,
triangles), the final titer is reduced and growth takes much
longer. An improved strain should be able to at least match the
D-arabinose results.
Example 3
[0153] To improve metabolism of D-xylose through the D-arabinose
degradation pathway, we attempted to overexpress the relevant
enzymes: D-ribulokinase (encoded by fucK), D-ribulose-phosphate
aldolase (encoded by fucA), and glycolaldehyde reductase (encoded
by fucO). These enzymes were overexpressed as part of an operon in
conjunction with DTE; the order of the genes in the operon was
varied. All of these were grown on D-xylose, analyzed for EG
production, and compared against .DELTA.aldA cultured on
D-arabinose (FIG. 8). The best strain is .DELTA.aldA
.DELTA.xylB/p10_T5T10-dte-fucA-fucO-fucK, which outperforms the
D-arabinose results. When a plasmid of a lower copy number was used
(p5_T5T10-dte-fucA-fucO-fucK), the results were similar (data not
shown).
[0154] To maximize EG production, we next grew our best strain,
.DELTA.aldA .DELTA.xylB/p5_T5T10-dte-fucA-fucO-fucK, in a
bioreactor. The medium of the initial batch was minimal medium with
30 g/L D-xylose; when nearly all D-xylose was consumed, additional
D-xylose was fed into the bioreactor. Ethylene glycol
concentrations were measured over time (FIG. 9). Our engineered E.
coli produced 42 g/L EG from D-xylose over a 72-h time frame.
[0155] Though the strain is capable of utilizing pure D-xylose as
the substrate, the abundance of D-xylose is based on its presence
in biomass, specifically hemicellulose. D-xylose is made available
by hydrolyzing the hemicellulose, so we tested the ability of our
engineered E. coli to produce EG from such hydrolysate. We prepared
a medium in which pretreated hydrolysate was diluted to 1/5 and
supplemented with minimal medium; the substrate was .about.8.7 g/L
D-xylose contributed by the hydrolysate. .DELTA.aldA
.DELTA.xylB/p10_T5T10-dte-fucA-fucO-fucK was grown in this medium,
and we measured the final EG concentration from the supernatant.
The strain yielded 2.7 g/L EG.
Example 4
[0156] In the engineered strains of Examples 1-3, production of EG
from a pentose (D-xylose or D-arabinose) proceeds through the
intermediate D-ribulose-1-phosphate. Any pentose that can be
converted to D-ribulose-1-phosphate can subsequently be used to
produce EG, however, for production of EG from a pentose, it is not
necessary to convert the pentose to D-ribulose-1-phosphate. In this
example, we show that production of EG from a pentose can proceed
through an alternative pathway.
[0157] As described earlier and as shown in FIG. 1, the pentose
L-lyxose can be converted to L-xylulose-1-phosphate which can be
cleaved by L-xylulose-1-phosphate aldolase. The cleavage yields
dihydroxyacetone phosphate and glycolaldehyde which is subsequently
converted to ethylene glycol. Furthermore, E. coli is capable of
degrading the pentose L-arabinose through the intermediate
L-ribulose (FIG. 1). We thus hypothesized that L-arabinose could
yield glycolaldehyde, and therefore, ethylene glycol: L-arabinose
would be converted to L-ribulose which could be converted to
L-xylulose by DTE; L-xylulose would be phosphorylated to
L-xylulose-1-phosphate which would be cleaved by its respective
aldolase.
[0158] To validate the hypothesis, we first attenuated the native
L-arabinose degradation pathway from E. coli by knocking out the
gene coding for L-ribulokinase, araB. This was done in our
.DELTA.aldA strain, yielding E. coli K-12 MG1655 DE3 .DELTA.endA
.DELTA.recA .DELTA.aldA .DELTA.araB (referred to as .DELTA.aldA
.DELTA.araB). We next constructed a plasmid to overexpress DTE, the
enzymes for L-lyxose degradation, and glycolaldehyde reductase:
p10_T7-dte-rhaB-rhaD-fucO. After transforming
p10_T7-dte-rhaB-rhaD-fucO into .DELTA.aldA .DELTA.araB, we cultured
the strain in M9(+) with L-arabinose. As seen in FIG. 10, our
engineered strain .DELTA.aldA .DELTA.araB/p10_T7-dte-rhaB-rhaD-fucO
produced greater than 2.0 g/L ethylene glycol from L-arabinose; the
control .DELTA.aldA grew but did not produce ethylene glycol. This
experiment confirmed that ethylene glycol can be produced from
L-arabinose and that pentoses can yield ethylene glycolaldehyde
through the intermediate L-xylulose-1-phosphate.
Example 5
[0159] In the above work with D-arabinose, D-xylose, and
L-arabinose, the pentose is cleaved into a two-carbon molecule and
a three-carbon molecule; all of the EG reported has been generated
from the two-carbon molecule resulting from this cleavage. The
three-carbon molecule is dihydroxyacetone phosphate, which enters
into glycolysis. D-glucose is also metabolized through glycolysis,
so that full utilization of D-xylose and utilization of D-glucose
for EG production require an additional pathway by which a
glycolysis intermediate can be converted to EG. There are multiple
possible pathways stemming from either 3-phosphoglycerate or
2-phosphoglycerate. To increase availability of 3-phosphoglycerate,
we knocked out phosphoglycerate mutases from the .DELTA.aldA
.DELTA.xylB strain, generating E. coli K-12 MG1655 DE3 .DELTA.endA
.DELTA.recA .DELTA.aldA .DELTA.xylB .DELTA.gpmA (referred to as
.DELTA.aldA .DELTA.xylB .DELTA.gpmA), E. coli K-12 MG1655 DE3
.DELTA.endA .DELTA.recA .DELTA.aldA .DELTA.xylB .DELTA.gpmB
(referred to as .DELTA.aldA .DELTA.xylB .DELTA.gpmB), and E. coli
K-12 MG1655 DE3 .DELTA.endA .DELTA.recA .DELTA.aldA .DELTA.xylB
.DELTA.gpmA .DELTA.gpmB (referred to as .DELTA.aldA .DELTA.xylB
.DELTA.gpmA .DELTA.gpmB). These strains were cultured on minimal
medium with 15 g/L D-glucose, but no ethylene glycol was detected
(Table 2).
[0160] One pathway from 3-phosphoglycerate to ethylene glycol
proceeds through 3-phosphohydroxypyruvate to hydroxypyruvate to
glycolaldehyde and then to ethylene glycol. The conversion of
3-phosphoglycerate to 3-phosphohydroxypyruvate can be catalyzed by
3-phosphoglycerate dehydrogenase, and conversion of
3-phosphohydroxypyruvate to hydroxypyruvate requires
3-phosphohydroxypyruvate phosphatase activity. On this basis we
generated plasmids containing combinations of E. coli genes serA
and yeaB. These plasmids were transformed into .DELTA.aldA
.DELTA.xylB, and the strains were cultured on minimal medium with
15 g/L D-glucose. Ethylene glycol was not detected for these
strains (Table 2).
[0161] Once hydroxypyruvate is formed, it undergoes a
decarboxylation to form glycolaldehyde. The decarboxylation can be
carried out by an enzyme that acts specifically to decarboxylate
hydroxypyruvate or a decarboxylase that primarily acts on another
substrate but may also be capable of decarboxylating
hydroxypyruvate. To test such enzymes, we overexpressed pyruvate
decarboxylase (encoded by pdc, S. cerevisiae's PDC1 gene
codon-optimized for E. coli), the subunit of the E1 component of
2-oxoglutarate dehydrogenase (encoded by sucA from E. coli), and
1-deoxyxylulose-5-phosphate synthase (encoded by dxs from E. coli).
These enzymes were overexpressed within .DELTA.aldA .DELTA.xylB;
sucA was also overexpressed within .DELTA.aldA .DELTA.xylB
.DELTA.gpmA and .DELTA.aldA .DELTA.xylB .DELTA.gpmB. These strains
were cultured on minimal medium with 15 g/L D-glucose. We were able
to detect 2 mg/L ethylene glycol from .DELTA.aldA
.DELTA.xylB/p10_T5T10-pdc and 30 mg/L from .DELTA.aldA
.DELTA.xylB/p10_T5T10-sucA (Table 2); these results confirm that it
is possible to biologically produce ethylene glycol from
D-glucose.
[0162] After hydroxypyruvate is decarboxylated to glycolaldehyde,
glycolaldehyde is converted to ethylene glycol by glycolaldehyde
reductase or alcohol dehydrogenase. We generated plasmids with E.
coli fucO, alone and in combination with sucA. These plasmids were
transformed into .DELTA.aldA .DELTA.xylB, and the strains were
cultured on minimal medium with 15 g/L D-glucose. Ethylene glycol
was not detected for .DELTA.aldA .DELTA.xylB/p10_T5T10-fucO, but
.DELTA.aldA .DELTA.xylB/p10_T5T10-sucA-fucO yielded 29 mg/L
ethylene glycol (Table 2).
TABLE-US-00002 TABLE 2 Production of ethylene glycol from D-glucose
through hydroxypyruvate. E. coli cultures were grown on minimal
medium with 15 g/L D-glucose, and ethylene glycol concentrations
were measured after 4 days (post-inoculation). ND = Not Detected.
Ethylene Glycol Strain (mg/L) .DELTA.aldA .DELTA.xylB ND
.DELTA.aldA .DELTA.xylB .DELTA.gpmA ND .DELTA.aldA .DELTA.xylB
.DELTA.gpmB ND .DELTA.aldA .DELTA.xylB .DELTA.gpmA .DELTA.gpmB ND
.DELTA.aldA .DELTA.xylB/p10_T5T10-serA ND .DELTA.aldA
.DELTA.xylB/p10_T5T10-serA-yeaB ND .DELTA.aldA
.DELTA.xylB/p10_T5T10-yea5 ND .DELTA.aldA
.DELTA.xylB/p10_T5T10-yeaB-serA ND .DELTA.aldA
.DELTA.xylB/p10_T5T10-dxs ND .DELTA.aldA .DELTA.xylB/p10_T5T10-pdc
2 .DELTA.aldA .DELTA.xylB/p10_T5T10-sucA 30 .DELTA.aldA .DELTA.xylB
.DELTA.gpmA/p10_T5T10-sucA 12 .DELTA.aldA .DELTA.xylB
.DELTA.gpmB/p10_T5T10-sucA 29 .DELTA.aldA
.DELTA.xylB/p10_T5T10-fucO ND .DELTA.aldA
.DELTA.xylB/p10_T5T10-sucA-fucO 29
Example 6
[0163] Another possible pathway to generate glycolaldehyde from the
glycolysis intermediate 3-phosphoglycerate is through serine
biosynthesis followed by serine decarboxylation and then
ethanolamine oxidation. We investigated this pathway by testing
whether an engineered microbe can produce ethylene glycol from
serine. The Arabidopsis thaliana serine decarboxylase gene (sdc)
[Rontein 2001] and the amine oxidase with ethanolamine oxidase
activity from Arthrobacter sp. (aao) [Ota 2008] were
codon-optimized for E. coli. A truncated version of sdc, starting
at Met58 (hereby referred to as t-sdc), and aao were cloned into a
plasmid for overexpression: pCDFDuet_T7-t-sdc+T7-aao. E. coli fucO
was cloned into a separate plasmid for overexpression: p10_T7-fucO.
These plasmids were transformed into the .DELTA.aldA strain, and
the resulting strain was cultured in minimal medium with glucose
and serine. As demonstrated by FIG. 11, the strain
.DELTA.aldA/pCDFDuet_T7-t-sdc+T7-aao/p10_T7-fucO was able to
produce approximately 2 g/L ethylene glycol from serine.
Example 7
[0164] After strain
.DELTA.aldA/pCDFDuet_T7-t-sdc+T7-aao/p10_T7-fucO was confirmed to
produce ethylene glycol from serine, it was tested for production
of ethylene glycol from D-glucose or glycerol. The strain was
cultured independently on minimal medium with glucose and minimal
medium with glycerol.
TABLE-US-00003 TABLE 3 Production of ethylene glycol from D-glucose
and glycerol through ethanolamine. Cultures of E coli strain
AaldA/pCDFDuet_T7-t-sdc+T7-aao/p10_T7-fucO were grown on minimal
medium with 15 g/L D-glucose or minimal medium with 15.3 g/L
glycerol, and ethylene glycol concentrations were measured after 10
days (post-inoculation). Ethylene Glycol Substrate (mg/L) D-Glucose
15 Glycerol 35
[0165] The results presented here show that our engineered microbes
can generate ethylene glycol from D-arabinose, D-xylose,
hemicellulose hydrolysate, L-arabinose, serine, D-glucose, and
glycerol.
REFERENCES
[0166] Ajikumar, P. K., Xiao, W. H., Tyo, K. E. J., Wang, Y.,
Simeon, F., Leonard, E., Mucha, O., Phon, T. H., Pfeifer, B., and
Stephanopoulos, G. (2010). Isoprenoid pathway optimization for
taxol precursor overproduction in Eschericihia coli. Science, 330
(6000), 70-74. [0167] Anderson, M. M., Hazen, S. L., Hsu, F. F.
& Heinecke, J. W. (1997). Human neutrophils employ the
myeloperoxidase-hydrogen peroxide-chloride system to convert
hydroxyl-amino acids into glycolaldehyde, 2-hydroxypropanal, and
acrolein: A mechanism for the generation of highly reactive
.alpha.-hydroxy and .alpha.,.beta.-unsaturated aldehydes by
phagocytes at sites of inflammation. Journal of Clinical
Investigation, 99 (3), 424-432. [0168] Chiu, T. H., Evans, K. L.,
& Feingold, D. S. (1975). L-Rhamnulose-1-phosphate aldolase.
Methods in Enzymology, 42, 264-269. [0169] Davies, D. D. &
Asker, H. (1985). The enzymatic decarboxylation of hydroxypyruvate
associated with purified pyruvate decarboxylase from wheat germ.
Phytochemistry, 24 (2), 231-234. [0170] Hedrick, J. L. &
Sallach, H. J. (1961). The metabolism of hydroxypyruvate: I. The
nonenzymatic decarboxylation and autoxidation of hydroxypyruvate.
The Journal of Biological Chemistry, 236 (7), 1867-1871. [0171]
Hedrick, J. L. & Sallach, H. J. (1964). The nonoxidative
decarboxylation of hydroxypyruvate in mammalian systems. Archives
of Biochemistry and Biophysics, 105 (2), 261-269. [0172] Izumori,
K., Khan, A. R., Okaya, H. & Tsumura, T. (1993). A new enzyme,
D-ketohexose 3-epimerase, from Pseudomonas sp. ST-24. Bioscience
Biotechnology and Biochemistry, 57 (6), 1037-1039. [0173] Kulkarni,
A. P. & Hodgson, E. (1973). Ethanolamine oxidase from the
blowfly Phormia regina (diptera: insecta). Comparative Biochemistry
and Physiology, 44 (2B), 407-422. [0174] Ota, H., Tamezane, H.,
Sasano, Y., Hokazono, E., Yasuda, Y., Sakasegawa, S., Imamura, S.,
Tamura, T. & Osawa, S. (2008). Enzymatic characterization of an
amine oxidase from Arthrobacter sp. used to measure
phosphatidylethanolamine. Bioscience Biotechnology and
Biochemistry, 72 (10), 2732-2738. [0175] Rontein, D., Nishida, I.,
Tashiro, G., Yoshioka, K., Wu, W. I., Voelker, D. R., Basset, G.
& Hanson, A. D. (2001). Plants synthesize ethanolamine by
direct decarboxylation of serine using a pyridoxal phosphate
enzyme. Journal of Biological Chemistry, 276 (38), 35523-35529.
EQUIVALENTS
[0176] Having thus described several aspects of at least one
embodiment of this invention, it is to be appreciated that various
alterations, modifications, and improvements will readily occur to
those skilled in the art. Such alterations, modifications, and
improvements are intended to be part of this disclosure, and are
intended to be within the spirit and scope of the invention.
Accordingly, the foregoing description and drawings are by way of
example only.
[0177] While several inventive embodiments have been described and
illustrated herein, those of ordinary skill in the art will readily
envision a variety of other means and/or structures for performing
the function and/or obtaining the results and/or one or more of the
advantages described herein, and each of such variations and/or
modifications is deemed to be within the scope of the inventive
embodiments described herein. More generally, those skilled in the
art will readily appreciate that all parameters, dimensions,
materials, and configurations described herein are meant to be
exemplary and that the actual parameters, dimensions, materials,
and/or configurations will depend upon the specific application or
applications for which the inventive teachings is/are used. Those
skilled in the art will recognize, or be able to ascertain using no
more than routine experimentation, many equivalents to the specific
inventive embodiments described herein. Such equivalents are
intended to be encompassed by the following claims. It is,
therefore, to be understood that the foregoing embodiments are
presented by way of example only and that, within the scope of the
appended claims and equivalents thereto, inventive embodiments may
be practiced otherwise than as specifically described and claimed.
Inventive embodiments of the present disclosure are directed to
each individual feature, system, article, material, kit, and/or
method described herein. In addition, any combination of two or
more such features, systems, articles, materials, kits, and/or
methods, if such features, systems, articles, materials, kits,
and/or methods are not mutually inconsistent, is included within
the inventive scope of the present disclosure.
[0178] All definitions, as defined and used herein, should be
understood to control over dictionary definitions, definitions in
documents incorporated by reference, and/or ordinary meanings of
the defined terms.
[0179] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0180] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
Multiple elements listed with "and/or" should be construed in the
same fashion, i.e., "one or more" of the elements so conjoined.
Other elements may optionally be present other than the elements
specifically identified by the "and/or" clause, whether related or
unrelated to those elements specifically identified. Thus, as a
non-limiting example, a reference to "A and/or B", when used in
conjunction with open-ended language such as "comprising" can
refer, in one embodiment, to A only (optionally including elements
other than B); in another embodiment, to B only (optionally
including elements other than A); in yet another embodiment, to
both A and B (optionally including other elements); etc.
[0181] As used herein in the specification and in the claims, "or"
should be understood to have the same meaning as "and/or" as
defined above. For example, when separating items in a list, "or"
or "and/or" shall be interpreted as being inclusive, i.e., the
inclusion of at least one, but also including more than one, of a
number or list of elements, and, optionally, additional unlisted
items. Only terms clearly indicated to the contrary, such as "only
one of" or "exactly one of," or, when used in the claims,
"consisting of," will refer to the inclusion of exactly one element
of a number or list of elements. In general, the term "or" as used
herein shall only be interpreted as indicating exclusive
alternatives (i.e. "one or the other but not both") when preceded
by terms of exclusivity, such as "either," "one of," "only one of,"
or "exactly one of." "Consisting essentially of," when used in the
claims, shall have its ordinary meaning as used in the field of
patent law.
[0182] As used herein in the specification and in the claims, the
phrase "at least one," in reference to a list of one or more
elements, should be understood to mean at least one element
selected from any one or more of the elements in the list of
elements, but not necessarily including at least one of each and
every element specifically listed within the list of elements and
not excluding any combinations of elements in the list of elements.
This definition also allows that elements may optionally be present
other than the elements specifically identified within the list of
elements to which the phrase "at least one" refers, whether related
or unrelated to those elements specifically identified. Thus, as a
non-limiting example, "at least one of A and B" (or, equivalently,
"at least one of A or B," or, equivalently "at least one of A
and/or B") can refer, in one embodiment, to at least one,
optionally including more than one, A, with no B present (and
optionally including elements other than B); in another embodiment,
to at least one, optionally including more than one, B, with no A
present (and optionally including elements other than A); in yet
another embodiment, to at least one, optionally including more than
one, A, and at least one, optionally including more than one, B
(and optionally including other elements); etc.
[0183] It should also be understood that, unless clearly indicated
to the contrary, in any methods claimed herein that include more
than one step or act, the order of the steps or acts of the method
is not necessarily limited to the order in which the steps or acts
of the method are recited.
[0184] All references, patents and patent applications disclosed
herein are incorporated by reference with respect to the subject
matter for which each is cited, which in some cases may encompass
the entirety of the document.
[0185] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," "composed of," and
the like are to be understood to be open-ended, i.e., to mean
including but not limited to. Only the transitional phrases
"consisting of" and "consisting essentially of" shall be closed or
semi-closed transitional phrases, respectively, as set forth in the
United States Patent Office Manual of Patent Examining Procedures,
Section 2111.03.
Sequence CWU 1
1
8133DNAArtificial SequenceSynthetic Oligonucleotide 1gctgccatgg
acaaagttgg tatgttctac acc 33260DNAArtificial SequenceSynthetic
Oligonucleotide 2agtcgtcgac atgagctccg taggccggcc taaacgaatt
cttaggccag tttatcacgg 60361DNAArtificial SequenceSynthetic
Oligonucleotide 3gaattcgttt agagctctaa ataaggagga ataaccatgg
tatccggcta tattgcagga 60g 61438DNAArtificial SequenceSynthetic
Oligonucleotide 4actggtcgac gctatcttca cacttcctct ataaattc
38552DNAArtificial SequenceSynthetic Oligonucleotide 5ctgcggccgg
ccctttaata aggagatata ccatggaacg aaataaactt gc 52643DNAArtificial
SequenceSynthetic Oligonucleotide 6gccggagctc taaacgaatt cttaccaggc
ggtatggtaa agc 43729DNAArtificial SequenceSynthetic Oligonucleotide
7gctgccatgg agaacagcgc tttgaaagc 29854DNAArtificial
SequenceSynthetic Oligonucleotide 8ctatgagctc cgtaggccgg cctaaacgaa
ttcttattcg acgttcagcg cgtc 54
* * * * *