U.S. patent application number 14/180075 was filed with the patent office on 2014-06-12 for biological production of pentose sugars using recombinant cells.
This patent application is currently assigned to DANISCO US INC.. The applicant listed for this patent is Danisco US Inc.. Invention is credited to Joseph C. MCAULIFFE, Rachel E. MUIR.
Application Number | 20140162322 14/180075 |
Document ID | / |
Family ID | 45476685 |
Filed Date | 2014-06-12 |
United States Patent
Application |
20140162322 |
Kind Code |
A1 |
MCAULIFFE; Joseph C. ; et
al. |
June 12, 2014 |
BIOLOGICAL PRODUCTION OF PENTOSE SUGARS USING RECOMBINANT CELLS
Abstract
The invention provides, inter alia, compositions and methods for
the biological production of pentose sugars, such as
2-methylerythritol (2-ME), 1-deoxyxylulose (1-DX), and
monoacetylated-2-C-methylerythritols, using recombinant cells.
Inventors: |
MCAULIFFE; Joseph C.;
(Sunnyvale, CA) ; MUIR; Rachel E.; (Redwood City,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Danisco US Inc. |
Palo Alto |
CA |
US |
|
|
Assignee: |
DANISCO US INC.
Palo Alto
CA
|
Family ID: |
45476685 |
Appl. No.: |
14/180075 |
Filed: |
February 13, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13335792 |
Dec 22, 2011 |
8691541 |
|
|
14180075 |
|
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|
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61426483 |
Dec 22, 2010 |
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Current U.S.
Class: |
435/94 ;
435/252.3; 435/252.31; 435/252.33; 435/252.34; 435/252.35;
435/254.11; 435/254.2; 435/257.2 |
Current CPC
Class: |
C12P 19/24 20130101;
C12N 1/12 20130101; C12N 1/16 20130101; C12N 1/20 20130101; C12P
19/02 20130101; C12N 1/18 20130101; C12N 1/14 20130101 |
Class at
Publication: |
435/94 ;
435/252.3; 435/254.11; 435/254.2; 435/257.2; 435/252.33;
435/252.31; 435/252.34; 435/252.35 |
International
Class: |
C12P 19/02 20060101
C12P019/02; C12P 19/24 20060101 C12P019/24 |
Claims
1. A method of producing a pentose sugar, the method comprising:
(a) culturing recombinant cells that overexpress
1-deoxyxylulose-5-phosphate synthase and/or
1-deoxy-D-xylulose-5-phosphate reductoisomerase, wherein said
recombinant cells comprise (i) a heterologous nucleic acid encoding
a 1-deoxyxylulose-5-phosphate synthase and/or
1-deoxy-D-xylulose-5-phosphate reductoisomerase polypeptide
introduced into the cells and/or (ii) one or more copies of an
endogenous nucleic acid encoding a 1-deoxyxylulose-5-phosphate
synthase and/or 1-deoxy-D-xylulose-5-phosphate reductoisomerase
polypeptide introduced into the cells; and (b) producing said
pentose sugar.
2. The method of claim 1 wherein the recombinant cells further
comprise (iii) a heterologous nucleic acid encoding a phosphatase
and/or (iv) one or more copies of an endogenous nucleic acid
encoding a phosphatase.
3. The method of claim 2, wherein the pentose sugar is selected
from the group consisting of 2-methylerythritol (2-ME) and
1-deoxyxylulose (1-DX).
4. The method of claim 3, wherein the pentose sugar is
2-methylerythritol (2-ME).
5. The method of claim 4, wherein the recombinant cells are capable
of producing a cumulative titer of 2-ME of at least about 20
g/L.
6. The method of claim 4, wherein the recombinant cells are capable
of producing a cumulative titer of 2-ME of at least about 30
g/L.
7. The method of claim 4, wherein the recombinant cells are capable
of producing a cumulative titer of 2-ME of at least about 45
g/L.
8. The method of claim 1, wherein the recombinant cells further
comprise (iii) one or more heterologous nucleic acids encoding a
deoxyxylulose phosphate pathway polypeptide other than a
1-deoxyxylulose-5-phosphate synthase and/or
1-deoxy-D-xylulose-5-phosphate reductoisomerase polypeptide and/or
(iv) one or more copies of an endogenous nucleic acid encoding a
deoxyxylulose phosphate pathway polypeptide other than a
1-deoxyxylulose-5-phosphate synthase and/or
1-deoxy-D-xylulose-5-phosphate reductoisomerase polypeptide.
9. The method of claim 8, wherein the deoxyxylulose phosphate
pathway polypeptide is
1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase.
10. The method of claim 9, wherein the
1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase polypeptide
is a T. elongatus 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate
synthase polypeptide.
11. The method of claim 9, wherein the
1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase polypeptide
is an E. coli 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate
synthase polypeptide.
12. The method of claim 8, wherein the recombinant cells further
comprise a nucleic acid encoding an iron-sulfur cluster-interacting
redox polypeptide.
13. The method of claim 12, wherein the iron-sulfur
cluster-interacting redox polypeptide is selected from ferrodoxin
and flavodoxin.
14. The method of claim 1, wherein the recombinant cells further
comprise at least one heterologous nucleic acid encoding an
isopentenyl-diphosphate delta-isomerase polypeptide or at least one
copy of an endogenous nucleic acid encoding an
isopentenyl-diphosphate delta-isomerase polypeptide.
15. The method of claim 1, wherein the recombinant cells are
bacterial, algal, fungal or yeast cells.
16. The method of claim 15, wherein the cells are bacterial
cells.
17. The method of claim 16, wherein the bacterial cells are
gram-positive bacterial cells or gram-negative bacterial cells.
18. The method claim 17, wherein the bacterial cells are selected
from the group consisting of E. coli, P. citrea, B. subtilis, B.
licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.
alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B.
megaterium, B. coagulans, B. circulans, B. lautus, B.
thuringiensis, S. albus, S. lividans, S. coelicolor, S. griseus,
Pseudomonas sp., and P. alcaligenes cells.
19. The method of claim 18, wherein the bacterial cells is an E.
coli cells.
20. The method of claim 8, wherein the deoxyxylulose phosphate
pathway polypeptide is selected from the group of
4-diphosphocytidyl-2C-methyl-D-erythritol synthase,
4-diphosphocytidyl-2C-methyl-D-erythritol kinase,
2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase,
1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase, and
isopentenyl-diphosphate delta-isomerase.
21. The method of claim 20, wherein the additional deoxyxylulose
phosphate pathway polypeptide is selected from the group of
1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase, and
isopentenyl-diphosphate delta-isomerase.
22. A cell culture comprising the recombinant cells of claim 1,
wherein the cell culture produces at least about 45 g/L of 2-ME.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/335,792, filed Dec. 22, 2011, which claims
priority benefit of U.S. Provisional Application No. 61/426,483,
filed Dec. 22, 2010, the disclosures of which are hereby
incorporated by reference herein in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the biological
production of pentose sugars, such as 2-methylerythritol (2-ME),
1-deoxyxylulose (1-DX), and monoacetylated-2-C-methylerythritols,
using recombinant cells.
BACKGROUND OF THE INVENTION
[0003] Production of biochemicals from renewable resources is of
strategic interest as society seeks to move to sustainable
industrial processes. Fermentation using engineered microorganisms
allows the direct conversion of a range of carbon-sources (sugars,
lipids etc.) to compounds of greater value under mild conditions,
such as pentose sugars.
[0004] Pentose sugars are found in a number of industries, for
example as food additives/preservatives, sweeteners, in cosmetic
formulations, as chiral precursors for pharmaceuticals and as
building blocks for detergents and other chemicals. There exists a
need for more commercially efficient ways of producing pentose
sugars in a sustainable manner. The invention described herein
addresses this need and provides additional benefits as well.
[0005] All publications, patent applications, and patents cited in
this specification are herein incorporated by reference as if each
individual publication, patent application, or patent were
specifically and individually indicated to be incorporated by
reference. In particular, all publications cited herein are
expressly incorporated herein by reference for the purpose of
describing and disclosing compositions and methodologies which
might be used in connection with the invention.
BRIEF SUMMARY OF THE INVENTION
[0006] The invention provides, inter alia, compositions and methods
for the biological production of pentose sugars, such as
2-methylerythritol (2-ME), 1-deoxyxylulose (1-DX), and
monoacetylated-2-C-methylerythritols, using recombinant cells.
[0007] Accordingly, in one aspect, the invention provides for
methods of producing a pentose sugar by (a) culturing recombinant
cells comprising (i) a heterologous nucleic acid encoding a DXS
and/or DXR polypeptide and/or (ii) one or more copies of an
endogenous nucleic acid encoding a DXS and/or DXR polypeptide,
wherein the cells are cultured under conditions suitable for
producing a pentose sugar and (b) producing said pentose sugar. In
one aspect, the recombinant cells further comprise (iii) a
heterologous nucleic acid encoding a phosphatase and/or (ii) one or
more copies of an endogenous nucleic acid encoding a phosphatase.
In another aspect, the pentose sugar is selected from the group
consisting of 2-methylerythritol (2-ME) and 1-deoxyxylulose (1-DX).
In another aspect, the pentose sugar is 2-methylerythritol (2-ME).
In another aspect, the recombinant cells are capable of producing a
cumulative titer of 2-ME of at least about 20 g/L. In another
aspect, the recombinant cells are capable of producing a cumulative
titer of 2-ME of at least about 30 g/L. In another aspect, the
recombinant cells are capable of producing a cumulative titer of
2-ME of at least about 45 g/L.
[0008] In another aspect, the invention provides for methods of
producing 2-methylerythritol (2-ME), the method comprising: (a)
culturing recombinant cells comprising (i) a heterologous nucleic
acid encoding a DXS and/or DXR polypeptide and/or (ii) one or more
copies of an endogenous nucleic acid encoding a DXS and/or DXR
polypeptide, wherein the cells are cultured under conditions
suitable for producing 2-ME and (b) producing 2-ME. In one aspect,
the cells are capable of producing at least about 45 g/L of
2-ME.
[0009] In another aspect, the invention provides for methods of
producing 2-methylerythritol (2-ME), the method comprising: (a)
culturing recombinant cells comprising (i) a heterologous nucleic
acid encoding a DXS and/or DXR polypeptide and/or (ii) one or more
copies of an endogenous nucleic acid encoding a DXS and/or DXR
polypeptide and/or (iii) a heterologous nucleic acid encoding a
phosphatase and/or (ii) one or more copies of an endogenous nucleic
acid encoding a phosphatase, wherein the cells are cultured under
conditions suitable for producing 2-ME and (b) producing 2-ME. In
one aspect, the cells are capable of producing at least about 45
g/L of 2-ME.
[0010] In another aspect, the invention provides for methods for
producing at least one pentose sugar, the method comprising: (a)
culturing recombinant cells described herein under conditions
suitable for producing a pentose sugar and (b) producing the
pentose sugar, wherein the pentose sugar is selected from the group
consisting of 2-methylerythritol (2-ME), 1-deoxyxylulose (1-DX),
and monoacetylated-2-C-methylerythritol. In one aspect, the cells
are capable of producing at least about 45 g/L of 2-ME. In other
aspects, any of the methods for production also include recovering
the pentose sugar.
[0011] In other aspects, the invention provides for recombinant
cells capable of producing a pentose sugar, the cell comprising (a)
culturing recombinant cells comprising (i) a heterologous nucleic
acid encoding a DXS and/or DXR polypeptide and/or (ii) one or more
copies of an endogenous nucleic acid encoding a DXS and/or DXR
polypeptide and optionally (iii) a heterologous nucleic acid
encoding a phosphatase and/or (ii) one or more copies of an
endogenous nucleic acid encoding a phosphatase. In one aspect, the
cells are capable of producing at least about 45 g/L of 2-ME.
[0012] In any of the aspects above, the recombinant cells can also
include one or more heterologous nucleic acids encoding a DXP
pathway polypeptide (other than a DXS and/or DXR polypeptide)
and/or (ii) one or more copies of an endogenous nucleic acid
encoding a DXP pathway polypeptide (other than a DXS and/or DXR
polypeptide).
[0013] In any of the aspects herein, the recombinant cells can be
bacterial, algal, fungal or yeast cells. In one aspect, the cells
are bacterial cells. In another aspect, the bacterial cells are
gram-positive bacterial cells or gram-negative bacterial cells. In
another aspect, the bacterial cells are selected from the group
consisting of E. coli, P. citrea, B. subtilis, B. licheniformis, B.
lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B.
amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B.
coagulans, B. circulans, B. lautus, B. thuringiensis, S. albus, S.
lividans, S. coelicolor, S. griseus, Pseudomonas sp., and P.
alcaligenes cells. In another aspect, the recombinant cells can be
a bacterial cell. In any of the aspects above, the recombinant
cells can be E. coli.
[0014] In any of the aspects above, the DXP pathway polypeptide can
be IspG. In some aspects, only one IspG is used. In other aspects,
two types of IspG are used. In another aspect, the IspG
polypeptides are from T. elongatus or E. coli. In another aspect,
the DXP pathway enzyme is selected from the group of DXS, DXR, MCT,
CMK, MCS, HDR (IspH), and IDI. In another aspect, the additional
DXP pathway enzyme is selected from the group of DXS, DXR, HDR
(IspH), and IDI.
[0015] In any of the aspects above, the recombinant cells can also
include an iron-sulfur cluster-interacting redox polypeptide. Such
iron-sulfur cluster-interacting redox polypeptides can be
ferredoxins and flavodoxins.
[0016] In any of the aspects above, the recombinant cells can also
include a heterologous nucleic acid encoding for PGL polypeptide or
one or more copies of endogenous nucleic acid encoding for PGL
polypeptide. In any of the aspects above, the PGL nucleic acid is
integrated into the host cell's chromosome.
[0017] In any of the aspects above, the recombinant cells can also
include one or more heterologous nucleic acid encoding an
isopentenyl-diphosphate delta-isomerase (IDI) polypeptide or one or
more copies of an endogenous nucleic acid encoding an IDI
polypeptide.
[0018] In another aspect, the invention provides for a cell culture
comprising the recombinant cell as described herein. In one aspect,
the cell culture produces at least about 45 g/L of 2-ME.
[0019] In another aspect, the invention also provides for systems
for making pentose sugars, such as 2-methylerythritol (2-ME),
1-deoxyxylulose (1-DX), and monoacetylated-2-C-methylerythritols,
using recombinant cells as described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 shows MVA and DXP metabolic pathways for isoprene
(based on F. Bouvier et al., Progress in Lipid Res. 44:357-429,
2005). The following description includes alternative names for
each polypeptide in the pathways and a reference that discloses an
assay for measuring the activity of the indicated polypeptide (each
of these references are each hereby incorporated herein by
reference in their entireties). Mevalonate Pathway: AACT;
Acetyl-CoA acetyltransferase, MvaE, EC 2.3.1.9. Assay: J.
Bacteriol. 184:2116-2122, 2002; HMGS; Hydroxymethylglutaryl-CoA
synthase, MvaS, EC 2.3.3.10. Assay: J. Bacteriol. 184:4065-4070,
2002; HMGR; 3-Hydroxy-3-methylglutaryl-CoA reductase, MvaE, EC
1.1.1.34. Assay: J. Bacteriol. 184:2116-2122, 2002; MVK; Mevalonate
kinase, ERG12, EC 2.7.1.36. Assay: Curr Genet 19:9-14, 1991. PMK;
Phosphomevalonate kinase, ERGS, EC 2.7.4.2, Assay: Mol. Cell Biol.
11:620-631, 1991; DPMDC; Diphosphomevalonate decarboxylase, MVD1,
EC 4.1.1.33. Assay:Biochemistry 33:13355-13362, 1994; IDI;
Isopentenyl-diphosphate delta-isomerase, IDI1, EC 5.3.3.2. Assay:
J. Biol. Chem. 264:19169-19175, 1989. DXP Pathway: DXS;
1-Deoxyxylulose-5-phosphate synthase, dxs, EC 2.2.1.7. Assay:PNAS
94:12857-62, 1997; DXR; 1-Deoxy-D-xylulose 5-phosphate
reductoisomerase, dxr, EC 2.2.1.7. Assay: Eur. J. Biochem.
269:4446-4457, 2002; MCT; 4-Diphosphocytidyl-2C-methyl-D-erythritol
synthase, IspD, EC 2.7.7.60. Assay: PNAS 97: 6451-6456, 2000; CMK;
4-Diphosphocytidyl-2-C-methyl-D-erythritol kinase, IspE, EC
2.7.1.148. Assay: PNAS 97:1062-1067, 2000; MCS;
2C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase, IspF, EC
4.6.1.12. Assay:PNAS 96:11758-11763, 1999; HDS;
1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, ispG, EC
1.17.4.3. Assay: J. Org. Chem. 70:9168-9174, 2005; HDR;
1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase, IspH, EC
1.17.1.2. Assay:JACS 126:12847-12855, 2004. FIG. 1 also depicts how
1-deoxy-D-xylulose (or 1-DX) and 2-C-Methyl-D-erythritol (or 2-ME)
can be obtained the removal of the phospatate group by
phosphatase.
[0021] FIG. 2 shows 2-Methyl-D-erythritol (ME) titers plotted
against time. Shown are the data trends for run 20100703 (open
triangles), run 20100917 (black diamonds), and 20101011 (open
circles). The X and Y axes are labeled in the figure.
[0022] FIG. 3 shows 1-Deoxy-D-xylulose (1-DX) titers plotted
against time. Shown are the data trends for run 20101011 (open
circles) and run 20100785 (black diamonds). The X and Y axes are
labeled in the figure.
[0023] FIG. 4 shows a comparison of the values for
2-C-Methyl-D-erythritol as analyzed by the two different HPLC
methods. The data shown are the values for 2-C-Methyl-D-erythritol
in 20101011 as analyzed by Organic Acids Column HPLC analysis
(black triangles) in comparison to the Amino Propyl Column HPLC
analysis (open circles).
DETAILED DESCRIPTION OF THE INVENTION
[0024] The invention provides, inter alia, systems, compositions
and methods for the biological production of pentose sugars, such
as 2-methylerythritol (2-ME), 1-deoxyxylulose (1-DX), and
monoacetylated-2-C-methylerythritols, using recombinant cells. As
further detailed herein, the inventors have observed that
engineered microorganisms (e.g., E. coli BL21 strain) are capable
of producing significant amounts of the pentose (five-carbon)
sugars, such as 2-C-methyl-D-erythritol or 2-methylerythritol
(2-ME) and 1-deoxy-D-xylulose (1-DX). The production of these
pentose sugars can be achieved as part of a process for the
conversion of glucose to isoprene (2-methyl-1,3-butadiene) via the
deoxyxylulose phosphate (DXP) pathway. These pentose sugars have
utility in a number of industries, for example as food
additives/preservatives, sweeteners, in cosmetic formulations, as
chiral precursors for pharmaceuticals and as building blocks for
detergents and other chemicals.
DEFINITIONS
[0025] Unless defined otherwise, the meanings of all technical and
scientific terms used herein are those commonly understood by one
of skill in the art to which this invention belongs. Singleton, et
al., Dictionary of Microbiology and Molecular Biology, 2nd ed.,
John Wiley and Sons, New York (1994), and Hale & Marham, The
Harper Collins Dictionary of Biology, Harper Perennial, N.Y. (1991)
provide one of skill with a general dictionary of many of the terms
used in this invention. It is to be understood that this invention
is not limited to the particular methodology, protocols, and
reagents described, as these may vary. One of skill in the art will
also appreciate that any methods and materials similar or
equivalent to those described herein can also be used to practice
or test the invention. The headings provided herein are not
limitations of the various aspects or aspects of the invention
which can be had by reference to the specification as a whole.
[0026] As used herein, the term "polypeptides" includes
polypeptides, proteins, peptides, fragments of polypeptides, and
fusion polypeptides.
[0027] As used herein, an "isolated polypeptide" is not part of a
library of polypeptides, such as a library of 2, 5, 10, 20, 50 or
more different polypeptides and is separated from at least one
component with which it occurs in nature. An isolated polypeptide
can be obtained, for example, by expression of a recombinant
nucleic acid encoding the polypeptide. An isolated polypeptide can
be a non-naturally occurring polypeptide.
[0028] By "heterologous polypeptide" is meant a polypeptide encoded
by a nucleic acid sequence derived from a different organism,
species, or strain than the host cell. In some aspects, a
heterologous polypeptide is not identical to a wild-type
polypeptide that is found in the same host cell in nature.
[0029] As used herein, a "nucleic acid" refers to two or more
deoxyribonucleotides and/or ribonucleotides covalently joined
together in either single or double-stranded form.
[0030] By "recombinant nucleic acid" is meant a nucleic acid of
interest that is free of one or more nucleic acids (e.g., genes)
which, in the genome occurring in nature of the organism from which
the nucleic acid of interest is derived, flank the nucleic acid of
interest. The term therefore includes, for example, a recombinant
DNA which is incorporated into a vector, into an autonomously
replicating plasmid or virus, or into the genomic DNA of a
prokaryote or eukaryote, or which exists as a separate molecule
(e.g., a cDNA, a genomic DNA fragment, or a cDNA fragment produced
by PCR or restriction endonuclease digestion) independent of other
sequences. In some cases, a recombinant nucleic acid is a nucleic
acid that encodes a non-naturally occurring polypeptide.
[0031] By "heterologous nucleic acid" is meant a nucleic acid
sequence derived from a different organism, species or strain than
the host cell. In some aspects, the heterologous nucleic acid is
not identical to a wild-type nucleic acid that is found in the same
host cell in nature.
[0032] As used herein, the phrase, "various genes and polypeptides
associated with the DXP pathway," or "DXP pathway associated
nucleic acid(s) or polypeptide(s)" refers to any nucleic acid or
polypeptide that interacts with DXP pathway polypeptides or nucleic
acids, including, but not limited to, a terpene synthase (e.g.,
ocimene synthase, farnesene synthase, and artemesinin synthase),
either directly or indirectly.
[0033] For use herein, unless clearly indicated otherwise, use of
the terms "a", "an," and the like refers to one or more.
[0034] Reference to "about" a value or parameter herein includes
(and describes) aspects that are directed to that value or
parameter per se. For example, description referring to "about X"
includes description of "X." Numeric ranges are inclusive of the
numbers defining the range.
[0035] It is understood that aspects and aspects of the invention
described herein include "comprising," "consisting," and
"consisting essentially of" aspects and aspects.
Compositions and Methods Involving Recombinant Cells Capable of
Producing Pentose Sugars
[0036] As described in greater detail and further exemplified
herein, the inventions provides for compositions and methods for
the biological production of pentose sugars using recombinant
cells. Pentose sugars that can be made include, but are not limited
to, 2-methylerythritol (2-ME), 1-deoxyxylulose (1-DX), and
monoacetylated-2-C-methylerythritols. In one aspect, the invention
provides for compositions of and methods for producing pentose
sugars using recombinant cells that contain (i) a heterologous
nucleic acid encoding a DXS and/or DXR polypeptide and/or (ii) one
or more copies of an endogenous nucleic acid encoding a DXS and/or
DXR polypeptide and optionally (iii) a heterologous nucleic acid
encoding a phosphatase and/or (ii) one or more copies of an
endogenous nucleic acid encoding a phosphatase.
[0037] Some recombinant cells (e.g., BL21 E. coli strain) have been
engineered to utilize the DXP pathway for making isoprene, however,
the inventors have observed that the production of pentose sugars
(such as 2-ME) is decreased when isoprene production is increased.
Thus, for commercial production of pentose sugars, it is
recommended that the system be engineered as to decrease the amount
of isoprene production so that more pentose sugars, such as 2-ME,
can be made. A non-limiting way of accomplishing this is to not
introduce heterologous nucleic acids encoding for isoprene synthase
or additional copies of endogenous isoprene synthase into the host
cell. In one aspect, a recombinant cell is made such that it
contains one or more of the DXP pathway polypeptides, such as IspG,
and does not include a heterologous nucleic acid encoding for
isoprene synthase or additional copies of endogenous isoprene
synthase.
[0038] As shown in FIG. 1, 1-deoxy-D-xylulose (1-DX) can be made by
the removal of a phosphate group from the DXP. Accordingly, the
production of 1-DX can be achieved by overexpressing the DXS
enzyme. This overexpression can be achieved by the introduction of
a heterologous nucleic acid encoding a DXS polypeptide and/or (ii)
one or more copies of an endogenous nucleic acid encoding a DXS
polypeptide. For increasing production of 1-DX as a end product,
one of skill in the art can increase 1-DX by increasing the amount
of DXP along with increasing the amount of phosphatase in the
system. This can be achieved by the introduction of a heterologous
nucleic acid encoding a phosphatase and/or (ii) one or more copies
of an endogenous nucleic acid encoding a phosphatase.
[0039] As shown in FIG. 1, 2-C-methyl-D-erythritol (2-ME) can be
made by the removal of a phosphate group from the MEP compound. The
production of 2-ME can be achieved by overexpressing the DXR
enzyme. This overexpression can be achieved by the introduction of
a heterologous nucleic acid encoding a DXR polypeptide and/or (ii)
one or more copies of an endogenous nucleic acid encoding a DXR
polypeptide. For increasing production of 2-ME as a end product,
one of skill in the art can increase 2-ME by increasing the amount
of DXP along with increasing the amount of phosphatase in the
system. This can be achieved by the introduction of a heterologous
nucleic acid encoding a phosphatase and/or (ii) one or more copies
of an endogenous nucleic acid encoding a phosphatase.
[0040] In some cases, production of both 1-DX and 2-ME is desired.
In that case, the overexpression of both DXS and DXR enzymes should
be used. Production of both 1-DX and 2-ME can be achieved by using
recombinant cells that contain (i) a heterologous nucleic acid
encoding a DXS and/or DXR polypeptide and/or (ii) one or more
copies of an endogenous nucleic acid encoding a DXS and/or DXR
polypeptide and optionally (iii) a heterologous nucleic acid
encoding a phosphatase and/or (ii) one or more copies of an
endogenous nucleic acid encoding a phosphatase. The same would be
equally applicable for the production of
monoacetylated-2-C-methylerythritols and other pentose sugars.
[0041] In some instances, the other DXP pathway enzymes may be
overexpressed, in conjunction with the expression or overexpression
with DXS and/or DXP, to achieve production of pentose sugars. The
DXP pathway enzymes are described in greater detail below. One such
DXP enzyme that can be used is IspG or HDS.
IspG Enzymes and Systems
[0042] IspG enzymes are part of the lower DXP pathway. IspG genes
code for HDS polypeptides, which convert 2-C-methyl-D-erythritol
2,4-cyclodiphoshphate (ME-CPP or cMEPP) into
(E)-4-hydroxy-3-methylbut-2-en-1-yl-diphosphate (HMBPP or
HDMAPP).
[0043] For increasing IspG activity, one option is to express more
of the endogenous E. coli IspG system. The systems, compositions of
recombinant cells, and methods described herein utilize a different
approach where IspG activity and subsequent pentose sugar
production is enhanced by over-expression of two types of IspG
genes. In one aspect, the two types of IspG are E. coli and T.
elongatus IspG system.
[0044] The E. coli IspG system includes, but is not limited to, the
enzyme IspG (encoded by the gene ispG) and the required flavodoxin
redox partner FldA (encoded by the gene fldA). The T. elongatus
IspG system includes, but is not limited to, the enzyme IspG
(encoded by the gene gcpE) and the required ferredoxin redox
partner Fd (encoded by the petF gene), as well as the nonessential
ferredoxin-NADP(+) oxidoreductase redox partner Fpr (encoded by the
petH gene). In some instances, Fpr activity is not required for the
T. elongatus IspG to function within E. coli where the activity of
the T. elongatus IspG was found to be dependent on the Fd cofactor.
The fpr gene of E. coli is nonessential and the activity of the T.
elongatus IspG within E. coli depends on co-expression of the T.
elongatus Fd.
[0045] Without being bound by theory, the E. coli IspG system and
the T. elongatus IspG system are believed to ultimately obtain the
electrons necessary to perform their catalytic function from NADPH
via some flavodoxin/ferredoxin-NADP(+) oxidoreductase activity.
Enzymes with flavodoxin/ferredoxin-NADP(+) oxidoreductase activity
have been demonstrated in vitro to fulfill the role of electron
transport to the required flavodoxin and ferredoxin cofactors
essential for IspG activity, however the in vivo physiological
relevance of these reductases has not been shown and, as such,
cannot be predictable.
Exemplary Polypeptides and Nucleic Acids
[0046] As noted above, recombinant cells of the invention and their
progeny are engineered to have one or more heterologous nucleic
acids encoding a DXS and/or DXR polypeptide and/or one or more
copies of an endogenous nucleic acid encoding a DXS and/or DXR
polypeptide. In one aspect, the recombinant cells can have one IspG
enzyme or two types of IspG enzymes and one or more DXP pathway
polypeptide(s). In some aspect, the cell can further contain
various iron-sulfur cluster-interacting redox polypeptides and
nucleic acids, DXP pathway associated polypeptide, MVA pathway
polypeptides and nucleic acids, PGL polypeptides and nucleic acids
and IDI polypeptides and nucleic acids.
[0047] Polypeptides includes polypeptides, proteins, peptides,
fragments of polypeptides, and fusion polypeptides. In some
aspects, the fusion polypeptide includes part or all of a first
polypeptide (e.g., an iron-sulfur cluster-interacting redox
polypeptide, DXP pathway polypeptide, DXP pathway associated
polypeptide, and IDI polypeptide, or catalytically active fragment
thereof) and may optionally include part or all of a second
polypeptide (e.g., a peptide that facilitates purification or
detection of the fusion polypeptide, such as a His-tag). In some
aspects, the fusion polypeptide has an activity of two or more DXP
pathway polypeptides.
[0048] In particular aspects, the nucleic acid includes a segment
of or the entire nucleic acid sequence of any iron-sulfur
cluster-interacting redox nucleic acid, IspG, DXP pathway nucleic
acid, DXP pathway associated nucleic acid, or IDI nucleic acid. In
some aspects, the nucleic acid includes at least or about 50, 100,
150, 200, 300, 400, 500, 600, 700, 800, or more contiguous
nucleotides from a iron-sulfur cluster-interacting redox nucleic
acid, IspG, DXP pathway nucleic acid, DXP pathway associated
nucleic acid, or IDI nucleic acid. In some aspects, the nucleic
acid has one or more mutations compared to the sequence of a
wild-type (i.e., a sequence occurring in nature) IspG, iron-sulfur
cluster-interacting redox nucleic acid, DXP pathway nucleic acid,
DXP pathway associated nucleic acid, or IDI nucleic acid. In some
aspects, the nucleic acid has one or more mutations (e.g., a silent
mutation) that increase the transcription or translation of IspG,
iron-sulfur cluster-interacting redox nucleic acid, DXP pathway
nucleic acid, DXP pathway associated nucleic acid, or IDI nucleic
acid. In some aspects, the nucleic acid is a degenerate variant of
any nucleic acid encoding an iron-sulfur cluster-interacting redox
polypeptide, DXP pathway polypeptide, DXP pathway associated
polypeptide, or IDI polypeptide.
[0049] The accession numbers of exemplary DXP pathway polypeptides
and nucleic acids are listed in Appendix 1 of WO 2009/076676.
Exemplary Iron-sulfur Cluster-Interacting Redox Polypeptides and
Nucleic Acids
[0050] Iron-sulfur cluster-interacting redox polypeptide plays an
essential role in the DXP pathway for isoprenoid biosynthesis.
Exemplary iron-sulfur cluster-interacting redox polypeptides
include polypeptides, fragments of polypeptides, peptides, and
fusions polypeptides that have at least one activity of a
iron-sulfur cluster-interacting redox polypeptide. Standard methods
can be used to determine whether a polypeptide has iron-sulfur
cluster-interacting redox polypeptide activity by using a
hydrogenase-linked assay measuring the rate of
metronidazole[1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole]
reduction (Chen and Blanchard, Analytical Biochem, 93:216-222
(1979)).
[0051] Exemplary iron-sulfur cluster-interacting redox polypeptide
nucleic acids include nucleic acids that encode a polypeptide,
fragment of a polypeptide, peptide, or fusion polypeptide that has
at least one activity of an iron-sulfur cluster-interacting redox
polypeptide. Exemplary iron-sulfur cluster-interacting redox
polypeptides and nucleic acids include naturally-occurring
polypeptides and nucleic acids from any of the source organisms
described herein as well as mutant polypeptides and nucleic acids
derived from any of the source organisms described herein.
[0052] Iron-sulfur cluster-interacting redox polypeptide is a
polypeptide that is capable of transferring electrons to a
polypeptide containing an iron-sulfur cluster. An iron-sulfur
cluster-interacting redox polypeptide includes, but is not limited
to, flavodoxin (e.g., flavodoxin I), flavodoxin reductase,
ferredoxin (e.g., ferredoxin I), ferredoxin-NADP+ oxidoreductase,
and genes or polypeptides encoding thereof (e.g., fpr or fldA). For
example, DXP pathway polypeptide HDS (GcpE) is a metallo-enzyme
possessing a [4Fe-4S].sup.2+ center and catalyzes the reduction of
cMEPP into HMBPP via two successive one-electron transfers mediated
by the reduction of [4Fe-4S].sup.2+ center in the presence of
flavodoxin/flavodoxin reductase (see, Wolff et al., FEBS Letters,
541:115-120 (2003)). Similarly, DXP pathway polypeptide HDR (LytB)
is also a Fe/S protein catalyzing the reduction of HMBPP into IPP
or DMAPP via two successive one-electron transfers in the presence
of flavodoxin/flavodoxin reductase/NADPH system. See, for example,
Seemann, M. et al. Agnew. Chem. Int. Ed., 41: 4337-4339 (2002);
Wolff, M. et al., FEBS Letters, 541: 115-120 (2003)).
[0053] Flavodoxin is a protein that is capable of transferring
electrons and contains the prosthetic group flavin mononucleotide.
In Escherichia coli (E. coli), flavodoxin is encoded by the fldA
gene and reduced by the FAD-containing protein NADPH:ferredoxin
oxidoreductase, and plays an essential role in the DXP pathway for
isoprenoid biosynthesis (see, example, Kia-Joo, P. et al. FEBS
Letters, 579: 3802-3806, 2005).
[0054] Ferredoxin is a protein that is capable of transferring
electron and contains iron and labile sulfur in equal amounts and
plays an essential role in the DXP pathway for isoprenoid
biosynthesis. For example, HDS from plants and cyanobacteria have
been shown to be ferredoxin, rather than flavodoxin-dependent,
enzymes (Seemann et al., FEBS Lett., 580(6):1547-52 (2006)).
[0055] Fpr encodes flavodoxin/ferredoxin NADPH-oxidoreductase and
provides the necessary electron derived from NADPH via FldA for HDS
and HDR to perform their catalytic functions (reviewed in report by
L. A. Furgerson, The Mevalonate-Independent Pathway to Isoprenoid
Compounds: Discovery, Elucidation, and Reaction Mechanisms,
published Feb. 13, 2006).
Exemplary DXP Pathway Polypeptides and Nucleic Acids
[0056] Exemplary DXP pathways polypeptides include, but are not
limited to any of the following polypeptides: DXS polypeptides, DXR
polypeptides, MCT polypeptides, CMK polypeptides, MCS polypeptides,
HDS polypeptides, HDR polypeptides, IDI polypeptides, and
polypeptides (e.g., fusion polypeptides) having an activity of one,
two, or more of the DXP pathway polypeptides. In particular, DXP
pathway polypeptides include polypeptides, fragments of
polypeptides, peptides, and fusions polypeptides that have at least
one activity of a DXP pathway polypeptide. Exemplary DXP pathway
nucleic acids include nucleic acids that encode a polypeptide,
fragment of a polypeptide, peptide, or fusion polypeptide that has
at least one activity of a DXP pathway polypeptide. Exemplary DXP
pathway polypeptides and nucleic acids include naturally-occurring
polypeptides and nucleic acids from any of the source organisms
described herein as well as mutant polypeptides and nucleic acids
derived from any of the source organisms described herein. In some
aspects, the heterologous nucleic acid encoding a DXP pathway
polypeptide is operably linked to a constitutive promoter. In some
aspects, the heterologous nucleic acid encoding an DXP pathway
polypeptide is operably linked to a strong promoter.
[0057] In particular, DXS polypeptides convert pyruvate and
D-glyceraldehyde 3-phosphate into 1-deoxy-d-xylulose 5-phosphate
(DXP). Standard methods can be used to determine whether a
polypeptide has DXS polypeptide activity by measuring the ability
of the polypeptide to convert pyruvate and D-glyceraldehyde
3-phosphate in vitro, in a cell extract, or in vivo.
[0058] DXR polypeptides convert 1-deoxy-D-xylulose 5-phosphate
(DXP) into 2-C-methyl-D-erythritol 4-phosphate (MEP). Standard
methods can be used to determine whether a polypeptide has DXR
polypeptides activity by measuring the ability of the polypeptide
to convert DXP in vitro, in a cell extract, or in vivo.
[0059] MCT polypeptides convert 2-C-methyl-D-erythritol 4-phosphate
(MEP) into 4-(cytidine 5'-diphospho)-2-methyl-D-erythritol
(CDP-ME). Standard methods can be used to determine whether a
polypeptide has MCT polypeptides activity by measuring the ability
of the polypeptide to convert MEP in vitro, in a cell extract, or
in vivo.
[0060] CMK polypeptides convert 4-(cytidine
5'-diphospho)-2-C-methyl-D-erythritol (CDP-ME) into
2-phospho-4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
(CDP-MEP). Standard methods can be used to determine whether a
polypeptide has CMK polypeptides activity by measuring the ability
of the polypeptide to convert CDP-ME in vitro, in a cell extract,
or in vivo.
[0061] MCS polypeptides convert 2-phospho-4-(cytidine
5'-diphospho)-2-C-methyl-D-erythritol (CDP-MEP) into
2-C-methyl-D-erythritol 2,4-cyclodiphosphate (ME-CPP or cMEPP).
Standard methods can be used to determine whether a polypeptide has
MCS polypeptides activity by measuring the ability of the
polypeptide to convert CDP-MEP in vitro, in a cell extract, or in
vivo.
[0062] HDS polypeptides convert 2-C-methyl-D-erythritol
2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-en-1-yl
diphosphate (HMBPP or HDMAPP). Standard methods can be used to
determine whether a polypeptide has HDS polypeptides activity by
measuring the ability of the polypeptide to convert ME-CPP in
vitro, in a cell extract, or in vivo.
[0063] HDR polypeptides convert (E)-4-hydroxy-3-methylbut-2-en-1-yl
diphosphate into isopentenyl diphosphate (IPP) and dimethylallyl
diphosphate (DMAPP). In one embodiment, the ispH gene can be used
to encode for HDR polypeptides. IspH is also known as
1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase, 4Fe-4S
protein, ECK0030, JW0027, lytB, yaaE, and b0029. Standard methods
can be used to determine whether a polypeptide has HDR polypeptides
activity by measuring the ability of the polypeptide to convert
HMBPP in vitro, in a cell extract, or in vivo.
[0064] IDI polypeptides convert isopentenyl diphosphate into
dimethylallyl diphosphate. Standard methods can be used to
determine whether a polypeptide has IDI polypeptides activity by
measuring the ability of the polypeptide to convert isopentenyl
diphosphate in vitro, in a cell extract, or in vivo.
Exemplary MVA Pathway Polypeptides and Nucleic Acids
[0065] In some aspects of the invention, the cells described in any
of the compositions or methods described herein can also include a
nucleic acid encoding an MVA pathway polypeptide. In some aspects,
the MVA pathway polypeptide is an endogenous polypeptide. In some
aspects, the MVA pathway polypeptide is an heterologous
polypeptide. In some aspects, the cells comprise one or more
additional copies of a heterologous nucleic acid encoding an MVA
pathway polypeptide. In some aspects, the cells comprise one or
more additional copies of an endogenous nucleic acid encoding an
MVA pathway polypeptide. In some aspects, the endogenous nucleic
acid encoding an MVA pathway polypeptide operably linked to a
constitutive promoter. In some aspects, the endogenous nucleic acid
encoding an MVA pathway polypeptide operably linked to a
constitutive promoter. In some aspects, the endogenous nucleic acid
encoding an MVA pathway polypeptide is operably linked to a strong
promoter. In a particular aspect, the cells are engineered to
over-express the endogenous MVA pathway polypeptide relative to
wild-type cells.
[0066] In some aspects, the MVA pathway polypeptide is a
heterologous polypeptide. In some aspects, the cells comprise more
than one copy of a heterologous nucleic acid encoding an MVA
pathway polypeptide. In some aspects, the heterologous nucleic acid
encoding an MVA pathway polypeptide is operably linked to a
constitutive promoter. In some aspects, the heterologous nucleic
acid encoding an MVA pathway polypeptide is operably linked to a
strong promoter.
[0067] Exemplary MVA pathway polypeptides include acetyl-CoA
acetyltransferase (AA-CoA thiolase) polypeptides,
3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase)
polypeptides, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA
reductase) polypeptides, mevalonate kinase (MVK) polypeptides,
phosphomevalonate kinase (PMK) polypeptides, diphosphomevalonate
decarboxylase (MVD) polypeptides, phosphomevalonate decarboxylase
(PMDC) polypeptides, isopentenyl phosphate kinase (IPK)
polypeptides, IDI polypeptides, and polypeptides (e.g., fusion
polypeptides) having an activity of two or more MVA pathway
polypeptides. In particular, MVA pathway polypeptides include
polypeptides, fragments of polypeptides, peptides, and fusions
polypeptides that have at least one activity of an MVA pathway
polypeptide. Exemplary MVA pathway nucleic acids include nucleic
acids that encode a polypeptide, fragment of a polypeptide,
peptide, or fusion polypeptide that has at least one activity of an
MVA pathway polypeptide. Exemplary MVA pathway polypeptides and
nucleic acids include naturally-occurring polypeptides and nucleic
acids from any of the source organisms described herein. In
addition, variants of MVA pathway polypeptide that confer the
result of better pentose sugar production can also be used as
well.
[0068] Types of MVA pathway polypeptides and/or DXP pathway
polypeptides which can be used and methods of making microorganisms
(e.g., E. coli) encoding MVA pathway polypeptides and/or DXP
pathway polypeptides are also described in International Patent
Application Publication No. WO 2009/076676 and WO 2010/003007.
[0069] One of skill in the art can readily select and/or use
suitable promoters to optimize the expression of any of the DXP
pathway polypeptides (such as DXS, DXR, HDS or IspG), PGL
polypeptides and/or MVA pathway polypeptides. Similarly, one of
skill in the art can readily select and/or use suitable vectors (or
transfer vehicle) to optimize the expression of these polypeptides.
In some aspects, the vector contains a selective marker. Examples
of selectable markers include, but are not limited to, antibiotic
resistance nucleic acids (e.g., kanamycin, ampicillin,
carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin,
neomycin, or chloramphenicol) and/or nucleic acids that confer a
metabolic advantage, such as a nutritional advantage on the host
cell.
[0070] In some aspects, the nucleic acid encoding for any of the
DXP pathway polypeptides (such as DXS, DXR, HDS or IspG), PGL
polypeptides, or MVA pathway polypeptides integrates into a
chromosome of the cells without a selective marker. One of skill in
the art would appreciate that integration should occur at a
location that is not essential to the host organism. For example,
in a bacterial cell (e.g., E. coli cell), integration into the
origin of replication (or any other essential region of the
chromosome) would render the bacteria unable to replicate. Thus,
care should be taken to avoid integrating into essential locations
of the chromosome in the host organism.
Exemplary Source Organisms
[0071] DXP pathway nucleic acid, PGL nucleic acid, iron-sulfur
cluster-interacting redox nucleic acid, DXP pathway associated
nucleic acid, or IDI nucleic acid (and their encoded polypeptides)
can be obtained from any organism that naturally contains these
nucleic acids. Thus, DXS, DXR, MCT, CMK, MCS, HDS (IspG), or HDR
nucleic acids can be obtained, e.g., from any organism that
contains the DXP pathway or contains both the MVA and DXP pathways.
IDI, and PGL nucleic acid nucleic acids can be obtained, e.g., from
any organism that contains the MVA pathway, DXP pathway, or both
the MVA and DXP pathways.
[0072] In some aspects, the nucleic acid sequence of the
iron-sulfur cluster-interacting redox nucleic acid, DXP pathway
nucleic acid, DXP pathway associated nucleic acid, or IDI nucleic
acid is identical to the sequence of a nucleic acid that is
produced by any of the following organisms in nature. In some
aspects, the amino acid sequence of iron-sulfur cluster-interacting
redox polypeptide, DXP pathway polypeptide, DXP pathway associated
polypeptide, or IDI polypeptide is identical to the sequence of a
polypeptide that is produced by any of the following organisms in
nature. In some aspects, the iron-sulfur cluster-interacting redox
nucleic acid, DXP pathway nucleic acid, DXP pathway associated
nucleic acid, or IDI nucleic acid or its encoded polypeptide is a
mutant nucleic acid or polypeptide derived from any of the
organisms described herein. As used herein, "derived from" refers
to the source of the nucleic acid or polypeptide into which one or
more mutations is introduced. For example, a polypeptide that is
"derived from a plant polypeptide" refers to polypeptide of
interest that results from introducing one or more mutations into
the sequence of a wild-type (i.e., a sequence occurring in nature)
plant polypeptide.
[0073] In some aspects, the source organism is a fungus, examples
of which are species of Aspergillus such as A. oryzae and A. niger,
species of Saccharomyces such as S. cerevisiae, species of
Schizosaccharomyces such as S. pombe, and species of Trichoderma
such as T. reesei. In some aspects, the source organism is a
filamentous fungal cell. The term "filamentous fungi" refers to all
filamentous forms of the subdivision Eumycotina (see, Alexopoulos,
C. J. (1962), Introductory Mycology, Wiley, New York). These fungi
are characterized by a vegetative mycelium with a cell wall
composed of chitin, cellulose, and other complex polysaccharides.
The filamentous fungi are morphologically, physiologically, and
genetically distinct from yeasts. Vegetative growth by filamentous
fungi is by hyphal elongation and carbon catabolism is obligatory
aerobic. The filamentous fungal parent cell may be a cell of a
species of, but not limited to, Trichoderma, (e.g., Trichoderma
reesei, the asexual morph of Hypocrea jecorina, previously
classified as T. longibrachiatum, Trichoderma viride, Trichoderma
koningii, Trichoderma harzianum) (Sheir-Neirs et al., Appl.
Microbiol. Biotechnol 20: 46-53, 1984; ATCC No. 56765 and ATCC No.
26921); Penicillium sp., Humicola sp. (e.g., H. insolens, H.
lanuginose, or H. grisea); Chrysosporium sp. (e.g., C.
lucknowense), Gliocladium sp., Aspergillus sp. (e.g., A. oryzae, A.
niger, A sojae, A. japonicus, A. nidulans, or A. awamori) (Ward et
al., Appl. Microbiol. Biotechnol. 39: 7380743, 1993 and Goedegebuur
et al., Genet 41: 89-98, 2002), Fusarium sp., (e.g., F. roseum, F.
graminum F. cerealis, F. oxysporuim, or F. venenatum), Neurospora
sp., (e.g., N. crassa), Hypocrea sp., Mucor sp., (e.g., M. miehei),
Rhizopus sp. and Emericella sp. (see also, Innis et al., Sci. 228:
21-26, 1985). The term "Trichoderma" or "Trichoderma sp." or
"Trichoderma spp." refer to any fungal genus previously or
currently classified as Trichoderma.
[0074] In some aspects, the fungus is A. nidulans, A. awamori, A.
oryzae, A. aculeatus, A. niger, A. japonicus, T. reesei, T. viride,
F. oxysporum, or F. solani. Aspergillus strains are disclosed in
Ward et al., Appl. Microbiol. Biotechnol. 39:738-743, 1993 and
Goedegebuur et al., Curr Gene 41:89-98, 2002. In particular
aspects, the fungus is a strain of Trichoderma, such as a strain of
T. reesei. Strains of T. reesei are known and non-limiting examples
include ATCC No. 13631, ATCC No. 26921, ATCC No. 56764, ATCC No.
56765, ATCC No. 56767, and NRRL 15709. In some aspects, the host
strain is a derivative of RL-P37. RL-P37 is disclosed in
Sheir-Neiss et al., Appl. Microbiol. Biotechnology 20:46-53,
1984.
[0075] In some aspects, the source organism is a yeast, such as
Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., or Candida
sp.
[0076] In some aspects, the source organism is a bacterium, such as
strains of Bacillus such as B. licheniformis or B. subtilis,
strains of Pantoea such as P. citrea, strains of Pseudomonas such
as P. alcaligenes, strains of Streptomyces such as S. lividans or
S. rubiginosus, strains of Thermosynechococcus such as T.
elongatus, strains of Sinorhizobium such as S. meliloti, strains of
Helicobacter such as H. pylori, strains of Agrobacterium such as A.
tumefaciens, strains of Deinococcus such as D. radiodurans, strains
of Listeria such as L. monocytogenes, strains of Lactobacillus such
as L. spp, or strains of Escherichia such as E. coli.
[0077] As used herein, "the genus Bacillus" includes all species
within the genus "Bacillus," as known to those of skill in the art,
including but not limited to B. subtilis, B. licheniformis, B.
lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B.
amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B.
coagulans, B. circulans, B. lautus, and B. thuringiensis. It is
recognized that the genus Bacillus continues to undergo taxonomical
reorganization. Thus, it is intended that the genus include species
that have been reclassified, including but not limited to such
organisms as B. stearothermophilus, which is now named "Geobacillus
stearothermophilus." The production of resistant endospores in the
presence of oxygen is considered the defining feature of the genus
Bacillus, although this characteristic also applies to the recently
named Alicyclobacillus, Amphibacillus, Aneurinibacillus,
Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus,
Halobacillus, Paenibacillus, Salibacillus, Thermobacillus,
Ureibacillus, and Virgibacillus.
[0078] In some aspects, the source organism is a gram-positive
bacterium. Non-limiting examples include strains of Streptomyces
(e.g., S. lividans, S. coelicolor, or S. griseus), Bacillus,
Listeria (e.g., L. monocytogenes) or Lactobacillus (e.g., L. spp).
In some aspects, the source organism is a gram-negative bacterium,
such as E. coli, Pseudomonas sp, or H. pylori.
[0079] In some aspects, the source organism is a plant, such as a
plant from the family Fabaceae, such as the Faboideae subfamily. In
some aspects, the source organism is kudzu, poplar (such as Populus
alba.times.tremula CAC35696), aspen (such as Populus tremuloides),
Quercus robur, Arabidopsis (such as A. thaliana), or Zea (such as
Z. mays).
[0080] In some aspects, the source organism is an algae, such as a
green algae, red algae, glaucophytes, chlorarachniophytes,
euglenids, chromista, or dinoflagellates.
[0081] In some aspects, the source organism is a cyanobacterium,
such such as a cyanobacterium, classified into any of the following
groups based on morphology: Chroococcales, Pleurocapsales,
Oscillatoriales, Nostocales, or Stigonematales. In some aspects,
the cyanobacterium is Thermosynechococcus elongates.
Exemplary Host Cells
[0082] A variety of host cells can be used to express iron-sulfur
cluster-interacting redox polypeptide, DXP pathway polypeptide
(e.g., DXS, DXR, and/or IspG), DXP pathway associated polypeptide,
MVA pathway polypeptide, MVA pathway associated polypeptide, PGL
polypeptide or IDI polypeptide and to produce pentose sugars in the
methods of the claimed invention. Exemplary host cells include
cells from any of the organisms listed in the prior section under
the heading "Exemplary Source Organisms." The host cell may be a
cell that naturally produces isoprene or a cell that does not
naturally produce isoprene. In some aspects, the host cell
naturally produces pentose sugars using the DXP pathway, and one or
more DXP pathway polypeptide and iron-sulfur cluster-interacting
redox polypeptides are added to enhance production of pentose sugar
using this pathway. In some aspects, the host cell naturally
produces pentose sugars using the DXP pathway, and one or more DXP
pathway nucleic acids, one or more iron-sulfur cluster-interacting
redox nucleic acids, and IDI are added to enhance production of
pentose sugars using this pathway.
Exemplary Transformation Methods
[0083] IspG nucleic acids, iron-sulfur cluster-interacting redox
nucleic acid, DXP pathway nucleic acid, DXP pathway associated
nucleic acid, or IDI nucleic acid or its vectors containing them
can be inserted into a host cell (e.g., E. coli cell, a plant cell,
a fungal cell, a yeast cell, or a bacterial cell described herein)
using standard techniques known to one of skill in the art. The
introduced nucleic acids may be integrated into chromosomal DNA (as
described above) or maintained as extrachromosomal replicating
sequences.
Exemplary Cell Culture Media and Conditions
[0084] The invention also includes a cell or a population of cells
in culture that produce pentose sugar(s). By "cells in culture" is
meant two or more cells in a solution (e.g., a cell medium) that
allows the cells to undergo one or more cell divisions. "Cells in
culture" do not include plant cells that are part of a living,
multicellular plant containing cells that have differentiated into
plant tissues. In various aspects, the cell culture includes at
least or about 10, 20, 50, 100, 200, 500, 1,000, 5,000, 10,000 or
more cells.
[0085] Carbon source that can be used to cultivate the host cells
are described in WO 2009/076676, WO 2010/003007, and WO
2009/132220. In one aspect, the recombinant cells of the invention
can be grown in a fed-batch culture at the 15-L scale using the
following reagents:
Medium Recipe (Per Liter Fermentation Medium):
[0086] K2HPO4 7.5 g, MgSO4*7H2O 2 g, citric acid monohydrate 2 g,
ferric ammonium citrate 0.3 g, yeast extract 0.5 g, 50% H2S04 2.25
mL. All of the components were added together and dissolved in Di
H2O. This solution was heat sterilized (123.degree. C. for 20
minutes). The pH was adjusted to 7.0 with ammonium hydroxide (28%)
and q.s. to volume. Glucose 10 g, Vitamin Solution 12 mL, and
antibiotics were added after sterilization and pH adjustment.
1000.times. Trace Metal Solution (per Liter):
[0087] Citric Acids*H2O 40 g, MnSO4*H2O 30 g, NaCl 10 g, FeSO4*7H2O
1 g, CoCl2*6H2O 1 g, ZnSO*7H2O 1 g, CuSO4*5H2O 100 mg, H.sub.3BO3
100 mg, NaMoO4*2H2O 100 mg. Each component was dissolved one at a
time in Di H2O, pH was adjusted to 3.0 with HCl/NaOH, and then the
solution was q.s. to volume and filter sterilized with a 0.22
micron filter.
Vitamin Solution (Per Liter):
[0088] Thiamine hydrochloride 1.0 g, D-(+)-biotin 1.0 g, nicotinic
acid 1.0 g, D-pantothenic acid 4.8 g, pyridoxine hydrochloride 4.0
g. Each component was dissolved one at a time in Di H2O, pH was
adjusted to 3.0 with HCl/NaOH, and then the solution was q.s. to
volume and filter sterilized with 0.22 micron filter.
Macro Salt Solution (Per Liter):
[0089] MgSO4*7H2O 296 g, citric acid monohydrate 296 g, ferric
ammonium citrate 49.6 g. All components were dissolved in water,
q.s. to volume and filter sterilized with 0.22 micron filter.
Feed Solution (Per Kilogram):
[0090] Glucose 0.57 kg, Di H2O 0.38 kg, K2HPO4 7.5 g, 100%
Foamblast 10 g. All components were mixed together and autoclaved.
0.82 mL 1000.times. Trace Metal Solution, 6.5 mLVitamin Solution
and 5.5 mL Macro Salt Solution were added once the feed was
cooled.
[0091] Other methods can be used to culture the recombinant cells
of this invention are also described in the Examples section.
Materials and methods suitable for the maintenance and growth of
bacterial cultures are well known in the art. Exemplary techniques
may be found in WO 2009/076676, WO 2010/003007, and WO 2009/132220,
and Manual of Methods for General Bacteriology Gerhardt et al.,
eds), American Society for Microbiology, Washington, D.C. (1994) or
Brock in Biotechnology: A Textbook of Industrial Microbiology,
Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass.
In some aspects, the cells are cultured in a culture medium under
conditions permitting the expression of DXP pathway polypeptide
(e.g., DXS, DXR, and/or IspG), iron-sulfur cluster-interacting
redox polypeptide, DXP pathway associated polypeptide, or IDI
polypeptide encoded by a nucleic acid inserted into the host
cells.
Exemplary Methods for Decoupling Pentose Sugar Production from Cell
Growth.
[0092] The recombinant cells of the invention can be grown in a way
as to decouple the pentose sugar production from cell growth. When
feedstock is used, it is desirable for the carbon from the
feedstock to be converted to pentose sugar(s) rather than to the
growth and maintenance of the cells. In some aspects, the cells are
grown to a low to medium OD.sub.600, then production of pentose
sugar(s) is started or increased. This strategy permits a large
portion of the carbon to be converted to pentose sugar(s). One of
skill in the art can grow the recombinant cells of the invention by
following the teaching in WO 2010/003007.
[0093] In some aspects, pentose sugar(s) are only produced in
stationary phase. In some aspects, pentose sugar(s) is produced in
both the growth phase and stationary phase. In some aspects,
pentose sugar(s) is only produced in the growth phase. In some
aspects, the nucleic acids encoding the various enzymes and
polypeptides described herein are placed under the control of a
promoter or factor that is more active in stationary phase than in
the growth phase. For example, one or more iron-sulfur
cluster-interacting redox nucleic acid, DXP pathway nucleic acid,
DXP pathway associated nucleic acid, and/or IDI nucleic acid may be
placed under control of a stationary phase sigma factor, such as
RpoS. In some aspects, one or more iron-sulfur cluster-interacting
redox nucleic acid, DXP pathway nucleic acid, DXP pathway
associated nucleic acid, and/or IDI nucleic acid are placed under
control of a promoter inducible in stationary phase, such as a
promoter inducible by a response regulator active in stationary
phase.
Exemplary Production of Pentose Sugar(s)
[0094] The invention provides, inter alia, compositions and methods
for increasing the production of pentose sugar(s) from recombinant
cells comprising (i) a heterologous nucleic acid encoding a DXS
and/or DXR polypeptide and/or (ii) one or more copies of an
endogenous nucleic acid encoding a DXS and/or DXR polypeptide,
optionally with (iii) a heterologous nucleic acid encoding a
phosphatase and/or (ii) one or more copies of an endogenous nucleic
acid encoding a phosphatase. In one aspect, cultured cells using
one IspG enzyme or two types of IspG enzymes, one or more DXP
pathway enzymes (e.g., DXS and/or DXR), optionally in combination
with iron-sulfur cluster-interacting redox genes or polypeptides,
PGL genes and polypeptides, and IDI genes and polypeptides can be
used. In some aspects, the recombinant cells produce a cumulative
titer (total amount) of pentose sugar at greater than or about 10,
20, 25, 30, 35, 40, 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 or 300 g/L.sub.broth. In one
aspect, 2-methylerythritol (2-ME) is produced at greater than or
about 10, 20, 25, 30, 35, 40, 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 or 300 g/L.sub.broth. In
another aspect, 1-deoxyxylulose (1-DX) is produced at greater than
or about 10, 20, 25, 30, 35, 40, 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 or 300 g/L.sub.broth.
In another aspect, monoacetylated-2-C-methylerythritol is produced
at greater than or about 10, 20, 25, 30, 35, 40, 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 or 300
g/L.sub.broth. In another aspect, 1-DX and 2-ME are produced at
greater than or about 10, 20, 25, 30, 35, 40, 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 or 300
g/L.sub.broth. In another aspect, 1-DX and
2-monoacetylated-2-C-methylerythritol are produced at greater than
or about 10, 20, 25, 30, 35, 40, 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 or 300 g/L.sub.broth.
In another aspect, 2-ME and 2-monoacetylated-2-C-methylerythritol
are produced at greater than or about 10, 20, 25, 30, 35, 40, 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 or 300 g/L.sub.broth. In another aspect, 1-DX, 2-ME and
monoacetylated-2-C-methylerythritol are produced at greater than or
about 10, 20, 25, 30, 35, 40, 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 or 300 g/L.sub.broth.
[0095] In other aspects, pentose sugars such as 1-DX, 2-ME and/or
monoacetylated-2-C-methylerythritol are produced with an upper
limit of 10, 20, 25, 30, 35, 40, 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 or 300
g/L.sub.broth.
[0096] In other aspects, pentose sugars such as 1-DX, 2-ME and/or
monoacetylated-2-C-methylerythritol are produced with a lower limit
of 10, 20, 25, 30, 35, 40, 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 or 300 g/L.sub.broth.
[0097] Various measurement for pentose sugar production can be
measured by methods known to one of skill in the art, for example,
using GC/MS, GC/FID, NMR, and/or HPLC as exemplified herein.
[0098] The invention also contemplates cell cultures of recombinant
cells that are capable of producing pentose sugars (e.g., 1-DX,
2-ME, and/or monoacetylated-2-C-methylerythritol) in any of the
amount described above. Systems for producing pentose sugars using
the recombinant cells described herein are also contemplated within
the scope of the invention. Such system can include, but are not
limited to, recombinant cells, fermentation unit(s), recovery tools
and/or purification tools.
Exemplary Purification Methods
[0099] In some aspects, any of the methods described herein further
include recovering the pentose sugars. In contrast to isoprene,
which is mostly present in the off-gas, pentose sugars such as
2-ME, 1-DX, and monoacetylated-2-C-methylerythritol, are found in
the broth. Standard techniques of recovering a biochemical from
fermentation broth are known to those of skill in the art.
Non-limiting examples of how to recover pentose sugars such as
2-ME, 1-DX, and monoacetylated-2-C-methylerythritol, from the
fermentation broth are also described below.
EXAMPLES
[0100] The examples, which are intended to be purely exemplary of
the invention and should therefore not be considered to limit the
invention in any way, also describe and detail aspects and aspects
of the invention discussed above. Unless indicated otherwise,
temperature is in degrees Centigrade and pressure is at or near
atmospheric. The foregoing examples and detailed description are
offered by way of illustration and not by way of limitation.
Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of
understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
Example 1
Construction of strains with an engineered DXP pathway that produce
1-deoxy-D-xylulose (1-DX) and 2-C-methyl-D-erythritol (2-ME)
[0101] Construction of Strain REM F9.sub.--17 with a Modified DXP
Pathway
[0102] The isoprene producing parental strain REM D6.sub.--12 was
described previously (see U.S. patent application Ser. No.
12/817,134, Example 26). The REM D6.sub.--12 strain harbors
increased expression from the chromosomally encoded DXP pathway
gene dxs. Using a standard electroporation method (BIO RAD), the
Ptac Anabaena ispH aspA term/pEWL454 plasmid was moved into strain
REM D6.sub.--12. The BIO RAD Gene Pulser system (0.1 cm cuvette
cat. #165-2089) was used for the electroporation described.
Transformants were recovered in LB broth for 1 hour at 37.degree.
C. before plating onto LB agar containing carbenicillin (50
.mu.g/ml), spectinomycin (50 .mu.g/ml) and kanamycin (50 .mu.g/ml).
The resulting strain was named REM F9.sub.--17.
Construction of Strain REM H8.sub.--12 with a Modified DXP
Pathway
[0103] The REM H8.sub.--12 strain (see U.S. patent application Ser.
No. 12/817,134, Example 29) was constructed from an E. coli BL21
strain that overexpressed the first two enzymes in the DXP pathway
(PL.6-dxs and GI1.6-dxr, both chromosomally encoded), the last
enzyme in the DXP pathway (GI1.6-yIDI, chromosomally encoded),
other plasmid encoded genes involved in the DXP pathway
(GI1.6-fldA-ispG/pCL, PTac-Anabaena ispH aspA term/pEWL454), the
lower MVA pathway (PL.2-mKKDyI, integrated within the genome) and
truncated isoprene synthase from P. alba (pDW33, plasmid encoded).
The strain also contained a restored chromosomal pgl gene (t
ybgS::frt). The REM H8.sub.--12 strain has increased expression of
both dxs and dxr relative to the REM F9.sub.--17 strain, which is a
result of varied promoter strengths governing expression of the DXP
genes. Varied accumulation of dxs and dxr driven by the
aforementioned promoters has been confirmed by immunoblot.
Construction of Strain REM F2.sub.--18. With a Modified DXP
Pathway
[0104] The isoprene producing parental strain REM I7.sub.--11 was
described previously (see U.S. patent application Ser. No.
12/817,134, example 29) was used to produce REM F2.sub.--18. The
REM I7.sub.--11 strain harbors plasmid encoded copies of both fldA
and ispG as well as the fldA and ispG loci present within the BL21
genome in addition to increased expression from chromosomally
encoded DXP pathway genes dxs and dxr. The Ptac Anabaena ispH-T
elong ispG system aspA term/pEWL454 plasmid was introduced by
electroporation into strain REM I7.sub.--11. Electroporation was
performed using a Bio-Rad Gene Pulser system with a 0.1 cm cuvette,
cat. #165-2089. Transformation was achieved by following the
manufacturer's suggested protocol. Transformants were recovered in
LB broth for 1 hour at 37.degree. C. before plating onto LB agar
containing spectinomycin (50 .mu.g/ml), carbenicillin (50
.mu.g/ml), and kanamycin (50 .mu.g/ml). The resulting strain was
named REM F2.sub.--18.
Example 2
Fermentation of strains with an engineered DXP pathway that produce
1-Deoxy-D-xylulose and 2-C-Methyl-D-erythritol
Large Scale Fermentation of the REM F9.sub.--17 Strain
[0105] 2-C-Methyl-D-erythritol was produced from an E. coli strain
expressing genes from the DXP pathway, grown in fed-batch culture
at the 15-L scale. The following media compositions were used:
Medium Recipe (Per Liter Fermentation Medium):
[0106] K2HPO4 7.5 g, MgSO4*7H2O 2 g, citric acid monohydrate 2 g,
ferric ammonium citrate 0.3 g, yeast extract 0.5 g, 50% H2S04 2.25
mL. All of the components were added together and dissolved in Di
H2O. This solution was heat sterilized (123.degree. C. for 20
minutes). The pH was adjusted to 7.0 with ammonium hydroxide (28%)
and q.s. to volume. Glucose 10 g, Vitamin Solution 12 mL, and
antibiotics were added after sterilization and pH adjustment.
1000.times. Trace Metal Solution (per Liter):
[0107] Citric Acids*H2O 40 g, MnSO4*H2O 30 g, NaCl 10 g, FeSO4*7H2O
1 g, CoCl2*6H2O 1 g, ZnSO*7H2O 1 g, CuSO4*5H2O 100 mg, H3BO3 100
mg, NaMoO4*2H2O 100 mg. Each component was dissolved one at a time
in Di H2O, pH was adjusted to 3.0 with HCl/NaOH, and then the
solution was q.s. to volume and filter sterilized with a 0.22
micron filter.
Vitamin Solution (Per Liter):
[0108] Thiamine hydrochloride 1.0 g, D-(+)-biotin 1.0 g, nicotinic
acid 1.0 g, D-pantothenic acid 4.8 g, pyridoxine hydrochloride 4.0
g. Each component was dissolved one at a time in Di H2O, pH was
adjusted to 3.0 with HCl/NaOH, and then the solution was q.s. to
volume and filter sterilized with 0.22 micron filter.
Macro Salt Solution (per Liter):
[0109] MgSO4*7H2O 296 g, citric acid monohydrate 296 g, ferric
ammonium citrate 49.6 g. All components were dissolved in water,
q.s. to volume and filter sterilized with 0.22 micron filter.
Feed Solution (Per Kilogram):
[0110] Glucose 0.57 kg, Di H2O 0.38 kg, K2HPO4 7.5 g, 100%
Foamblast 10 g. All components were mixed together and autoclaved.
0.82 mL 1000.times. Trace Metal Solution, 6.5 mLVitamin Solution
and 5.5 mL Macro Salt Solution were added once the feed was
cooled.
[0111] Fermentation was performed in a 15-L bioreactor with strain
REMF9.sub.--17. This particular fermentation is referred to as run
20100703 (see FIG. 2). This experiment was carried out at a
fermentation pH of 7.0 and temperature of 34.degree. C. A frozen
vial of the E. coli strain was thawed and inoculated into
tryptone-yeast extract medium for the bioreactor. After the culture
grew to optical density 1.0, measured at 550 nm (OD.sub.550), 500
mL was used to inoculate a 15-L bioreactor and bring the initial
tank volume to 5 L. The antibiotics carbenicillin, spectinomycin
and kanamycin were each present at a concentration of 50 ug/mL,
respectively, in the seed flask and fermentation tank.
[0112] Once the batch glucose was depleted, a glucose feed was
initiated. There was an initial bolus of 3 g/min for 20 min.
Afterwards the tank was pulse fed with pulses lasting 30 min.
Pulses were induced by a pH rise above 7.05. Pulse rates were
calculated by determining the total carbon dioxide evolution rate
(mmol/h) divided by a factor of 300. The highest feed rate of
glucose achieved for a given pulse was 8.2 g/min over the 50 hour
fermentation.
[0113] Induction was achieved by adding
isopropyl-beta-D-1-thiogalactopyranoside (IPTG) from a 10 mg/mL
stock. At time zero, 3 mL was added (25 uM). Subsequent additions
were at a carbon dioxide evolution rate (CER) of 25 mmol/L/h (3
mL), CER of 50 mmol/L/h (6 mL) and CER of 100 mmol/Lh (6 mL). ME
titer was determined by the Organic acids column HPLC quantitation
method (see method description below) and is depicted in FIG.
2.
Large Scale Fermentation of the REM H8.sub.--12 Strain
[0114] 2-C-Methyl-D-erythritol was produced by an E. coli strain
expressing genes from the DXP pathway, grown in fed-batch culture
at the 15-L scale. The following media compositions were used:
Medium Recipe (Per Liter Fermentation Medium):
[0115] K2HPO4 7.5 g, MgSO4*7H2O 2 g, citric acid monohydrate 2 g,
ferric ammonium citrate 0.3 g, yeast extract 0.5 g, 1000.times.
Trace Metal Solution 1.5 ml, Macro Salt solution 3.4 mL, 50% H2S04
2.25 mL. All of the components were added together and dissolved in
Di H2O. This solution was heat sterilized (123.degree. C. for 20
minutes). The pH was adjusted to 7.0 with ammonium hydroxide (28%)
and q.s. to volume. Glucose 10 g, Mercury Vitamin Solution 12 mL,
and antibiotics were added after sterilization and pH
adjustment.
1000.times. Trace Metal Solution (per Liter):
[0116] Citric Acids*H2O 40 g, MnSO4*H2O 30 g, NaCl 10 g, FeSO4*7H2O
1 g, CoCl2*6H2O 1 g, ZnSO*7H2O 1 g, CuSO4*5H2O 100 mg, H.sub.3BO3
100 mg, NaMoO4*2H2O 100 mg. Each component was dissolved one at a
time in Di H2O, pH was adjusted to 3.0 with HCl/NaOH, and then the
solution was q.s. to volume and filter sterilized with a 0.22
micron filter.
Vitamin Solution (per Liter):
[0117] Thiamine hydrochloride 1.0 g, D-(+)-biotin 1.0 g, nicotinic
acid 1.0 g, D-pantothenic acid 4.8 g, pyridoxine hydrochloride 4.0
g. Each component was dissolved one at a time in Di H2O, pH was
adjusted to 3.0 with HCl/NaOH, and then the solution was q.s. to
volume and filter sterilized with 0.22 micron filter.
Macro Salt Solution (per Liter):
[0118] MgSO4*7H2O 296 g, citric acid monohydrate 296 g, ferric
ammonium citrate 49.6 g. All components were dissolved in water,
q.s. to volume and filter sterilized with 0.22 micron filter.
Feed Solution (Per Kilogram):
[0119] Glucose 0.57 kg, Di H2O 0.38 kg, K2HPO4 7.5 g, and 100%
Foamblast 10 g. All components were mixed together and
autoclaved.
[0120] Fermentation was performed in a 15-L bioreactor with strain
REM H8.sub.--12. This particular fermentation is referred to as run
20100917 (see FIGS. 2-4). This experiment was carried out at a
fermentation pH of 7.0 and temperature of 34.degree. C. A frozen
vial of the E. coli strain was thawed and inoculated into
tryptone-yeast extract medium for the bioreactor. After the culture
grew to optical density 1.0, measured at 550 nm (OD.sub.550), 500
mL was used to inoculate a 15-L bioreactor and bring the initial
tank volume to 5 L. Carbenicillin, spectinomycin and kanamycin were
each present at a concentration of 50 ug/mL, respectively, in the
seed flask and fermentation tank.
[0121] Once the batch glucose was depleted, the glucose feed
solution was fed at an exponential rate from 0.35 g/min until the
feed rate reached 2.72 g/min. This was immediately followed by a
linear ramp that lasted the duration of the fermentation and
brought the feed rate up to 3.75 g/min at 53 h. The total amount of
glucose delivered to the bioreactor during the 53 h fermentation
was 2.6 kg.
[0122] Induction was achieved by adding
isopropyl-beta-D-1-thiogalactopyranoside (IPTG) from a 10 mg/mL
stock. At time zero, 3 mL was added (25 uM). Subsequent additions
were at a carbon dioxide evolution rate (CER) of 25 mmol/L/h (3
mL), CER of 50 mmol/L/h (6 mL) and CER of 100 mmol/Lh (6 mL).
[0123] 1 L of broth was centrifuged and the supernatant was
provided for methylerythritol (ME) recovery; described below. The
titer of 2-ME was determined by the Organic acids column HPLC
quantitation method (see FIG. 2).
Large Scale Fermentation of REM F2.sub.--18 Strain
[0124] 2-C-Methyl-D-erythritol production from E. coli expressing
genes from the DXP pathway and isoprene synthase, grown in
fed-batch culture at the 15-L scale. The following media
compositions were used:
Medium Recipe (Per Liter Fermentation Medium):
[0125] K2HPO4 7.5 g, MgSO4*7H2O 2 g, citric acid monohydrate 2 g,
ferric ammonium citrate 0.3 g, yeast extract 0.5 g, 1000.times.
Trace Metal Solution 1.0 ml, 50% H2S04 2.25 mL. All of the
components were added together and dissolved in Di H2O. This
solution was heat sterilized (123.degree. C. for 20 minutes). The
pH was adjusted to 7.0 with ammonium hydroxide (28%) and q.s. to
volume. Glucose 10 g, Mercury Vitamin Solution 8 mL, and
antibiotics were added after sterilization and pH adjustment.
1000.times. Trace Metal Solution (per Liter):
[0126] Citric Acids*H2O 40 g, MnSO4*H2O 30 g, NaCl 10 g, FeSO4*7H2O
1 g, CoCl2*6H2O 1 g, ZnSO4*7H2O 1 g, CuSO4*5H2O 100 mg, H.sub.3BO3
100 mg, NaMoO4*2H2O 100 mg. Each component was dissolved one at a
time in Di H2O, pH was adjusted to 3.0 with HCl/NaOH, and then the
solution was q.s. to volume and filter sterilized with a 0.22
micron filter.
Vitamin Solution (per Liter):
[0127] Thiamine hydrochloride 1.0 g, D-(+)-biotin 1.0 g, nicotinic
acid 1.0 g, D-pantothenic acid 4.8 g, pyridoxine hydrochloride 4.0
g. Each component was dissolved one at a time in Di H2O, pH was
adjusted to 3.0 with HCl/NaOH, and then the solution was q.s. to
volume and filter sterilized with 0.22 micron filter.
Macro Salt Solution (per Liter):
[0128] MgSO4*7H2O 296 g, citric acid monohydrate 296 g, ferric
ammonium citrate 49.6 g. All components were dissolved in water,
q.s. to volume and filter sterilized with 0.22 micron filter.
Feed Solution (Per Kilogram):
[0129] Glucose 0.57 kg, Di H2O 0.38 kg, K2HPO4 7.5 g, and 100%
Foamblast 10 g. All components were mixed together and autoclaved.
Macro Salt Solution 11.1 mL, 1000.times. Trace Metal Solution 1.6
ml and Vitamin Solution 13.1 mL were added after the solution had
cooled to 25.degree. C.
Phosphate Solution (per Liter):
[0130] KH2PO4 68 g, K2HPO4 68 g. All components were dissolved in
water, q.s. to volume and autoclaved for 30 min.
[0131] Fermentation was performed in a 15-L bioreactor with strain
REM F2.sub.--18. This particular fermentation is referred to as run
20101011 (see FIGS. 2-4). This experiment was carried out at the
desired fermentation pH 7.0 and temperature 34.degree. C. A frozen
vial of the E. coli strain was thawed and inoculated into
tryptone-yeast extract medium for the bioreactor. After the culture
grew to optical density 1.0, measured at 550 nm (OD.sub.550), 500
mL was used to inoculate a 15-L bioreactor and bring the initial
tank volume to 5 L. Carbenicillin, spectinomycin and kanamycin were
each present at a concentration of 50 ug/mL, respectively, in the
seed flask and fermentation tank.
[0132] Once the batch glucose was depleted, the glucose feed
solution was fed at an exponential rate from 0.35 g/min until the
feed rate reached 2.75 g/min. This was immediately followed by a
linear ramp. The top rate was fixed at 4 g/min at 62.4 h EFT.
[0133] The phosphate solution described above was fed at 0.21 g/min
starting at a carbon dioxide evolution rate (CER) of 50 mmol/L/h,
and at 16 h feed time, was stepped down to 0.11 g/min and fed for
the duration of the experiment.
[0134] Induction was achieved by adding
isopropyl-.beta.-D-1-thiogalactopyranoside (IPTG) from a 10 mg/mL
stock. At time zero, 3 mL was added (25 uM). Subsequent additions
were at a carbon dioxide evolution rate (CER) of 25 mmol/L/h (3
mL), CER of 50 mmol/L/h (6 mL) and CER of 100 mmol/L/h (6 mL). ME
titer was determined by both the Organic acids column HPLC
quantitation method and the amino propyl column HPLC quantitation
method described below. FIGS. 2 and 4 demonstrate the agreement in
ME determination resolved by the two methods.
Large Scale Fermentation of REM I4.sub.--18
[0135] The genotype of REM I4.sub.--18 is BL21 pgl+PL.6-dxs
GI1.6-dxr GI1.6-yidi PL.2-lower MVA+pDW33 (carb 50)+Ptac-Anabaena
ispH-T.elong. ispG-fd-fnr/pEWL454 (kan50). Isoprene production from
E. coli expressing genes from the DXP pathway and isoprene
synthase, grown in fed-batch culture at the 15-L scale.
Medium Recipe (Per Liter Fermentation Medium):
[0136] K2HPO4 7.5 g, MgSO4*7H2O 2 g, citric acid monohydrate 2 g,
ferric ammonium citrate 0.3 g, yeast extract 0.5 g, 50% H2S04 2.25
mL. All of the components were added together and dissolved in Di
H2O. This solution was heat sterilized (123.degree. C. for 20
minutes). The pH was adjusted to 7.0 with ammonium hydroxide (28%)
and q.s. to volume. Glucose 10 g, Vitamin Solution 12 mL, and
antibiotics were added after sterilization and pH adjustment.
1000.times. Trace Metal Solution (per Liter):
[0137] Citric Acids*H2O 40 g, MnSO4*H2O 30 g, NaCl 10 g, FeSO4*7H2O
1 g, CoCl2*6H2O 1 g, ZnSO*7H2O 1 g, CuSO4*5H2O 100 mg, H.sub.3BO3
100 mg, NaMoO4*2H2O 100 mg. Each component was dissolved one at a
time in Di H2O, pH was adjusted to 3.0 with HCl/NaOH, and then the
solution was q.s. to volume and filter sterilized with a 0.22
micron filter.
Vitamin Solution (per Liter):
[0138] Thiamine hydrochloride 1.0 g, D-(+)-biotin 1.0 g, nicotinic
acid 1.0 g, D-pantothenic acid 4.8 g, pyridoxine hydrochloride 4.0
g. Each component was dissolved one at a time in Di H2O, pH was
adjusted to 3.0 with HCl/NaOH, and then the solution was q.s. to
volume and filter sterilized with 0.22 micron filter.
Macro Salt Solution (per Liter):
[0139] MgSO4*7H2O 296 g, citric acid monohydrate 296 g, ferric
ammonium citrate 49.6 g. All components were dissolved in water,
q.s. to volume and filter sterilized with 0.22 micron filter.
Feed Solution (Per Kilogram):
[0140] Glucose 0.57 kg, Di H2O 0.38 kg, K2HPO4 7.5 g, 100%
Foamblast 10 g. All components were mixed together and autoclaved.
0.82 mL 1000.times. Trace Metal Solution, 6.5 mLVitamin Solution
and 5.5 mL Macro Salt Solution were added once the feed was
cooled.
[0141] Fermentation was performed in a 15-L bioreactor with E. coli
BL21 cells (strain name REM I4.sub.--18). This particular
fermentation is referred to as run 20100785 (see FIG. 3).
[0142] This experiment was carried out at a fermentation pH of 7.0
and temperature of 34.degree. C. A frozen vial of the E. coli
strain was thawed and inoculated into tryptone-yeast extract medium
for the bioreactor. After the culture grew to optical density 1.0,
measured at 550 nm (OD.sub.550), 500 mL was used to inoculate a
15-L bioreactor and bring the initial tank volume to 5 L.
Carbenicillin and kanamycin were each present at a concentration of
50 ug/mL in the seed flask and fermentation tank, respectively.
[0143] Once the batch glucose was depleted, the glucose feed
solution was fed. There was an initial bolus of 3 g/min for 20 min.
Afterwards the tank was pulse fed with pulses lasting 30 min for 20
h. Pulses were induced by a pH rise above 7.05. Pulse rates were
calculated by determining the total carbon dioxide evolution rate
(mmol/h) divided by a factor between 400 and 600. After 27 h, the
feed was constant at 3.5 g/min until the end of the fermentation at
51 h.
[0144] Induction was achieved by adding
isopropyl-beta-D-1-thiogalactopyranoside (IPTG) from a 10 mg/mL
stock. 24 mL was added (200 uM) at a carbon dioxide evolution rate
of 25.
Example 3
Methods for Carbohydrate and Organic Acid Analysis--Analysis Using
an Aminopropyl HPLC Column
Sample Preparation and Metabolite Extraction
[0145] For the carbohydrates analysis by HPLC, fermentation broth
samples were heat-treated at 65.degree. C. for 5 minutes to lyse
the cells. The samples were kept on wet ice for the remainder of
the experiment. The samples were then centrifuged at 16000 RPM and
-9.degree. C. for 5 minutes. The supernatant was collected and the
cell pellets were resuspended in deionized water in a volume equal
to the removed supernatant. The suspension was centrifuged at 16000
RPM and -9.degree. C. for 5 minutes. The supernatant was combined
with the first collected supernatant portion, and the pellet was
discarded. The samples were subject to lyophilization overnight.
The lyophilized extracts were resuspended in an equal or lesser
volume of 85% acetonitrile. The samples were centrifuged to remove
any insoluble material and the supernatant was kept at a maximum
temperature of 4.degree. C. until the HPLC analysis.
HPLC Analytical Method
[0146] The HPLC analysis was performed using an Amino Propyl column
(Phenomenex, Luna NH2-250 mm.times.2.0 mm.times.5 .mu.m). The
mobile phase was comprised of 85% Acetonitrile. 20 uL of sample was
injected onto the column and run for 15 minutes isocratically with
the column temperature set at 40.degree. C. The RID (Refractive
Index Detector) was used at 40.degree. C. in positive mode to
detect 2-C-methylerythritol. The peak for 2-C-methylerythritol was
observed at approximately 2.3 minutes. 2-C-Methylerythritol was
quantified by a standard curve generated with 2-C-methylerythritol
purchased from Echelon Biosciences, Incorporated (Catalog
#I-M051A). The linear standard curve generated covered a range of
0.1 g/L to 5 g/L. The limit of quantitation was observed as 0.1 g/L
while the limit of detection was approximately 0.05 g/L.
Example 4
Methods for Carbohydrate and Organic Acid Analysis--Analysis Using
an Organic Acids Column
Sample Preparation and Metabolite Extraction
[0147] An aliquot of 500 uL of 2% H2S04 was added to 2 mL tubes.
167 uL of whole broth is transferred to each tube and mixed with
the 2% H.sub.2SO.sub.4. Tubes were centrifuged at 14000 RPM for 5
minutes to remove cell debris. The supernatant was decanted into a
300 uL conical bottom HPLC vial and the vials were checked for air
bubbles. The samples were kept at a maximum temperature of
4.degree. C. until the HPLC analysis.
HPLC Analytical Method
[0148] The HPLC analysis was performed using an Ion Exclusion
column (BioRad, Aminex HPX-87H-300 mm.times.7.8 mm) with a
Microguard Cation guard column (BioRad, Microguard Cation-30
mm.times.4.6 mm). 0.01N H.sub.2SO.sub.4 buffer (equivalent to 5 mM)
was prepared as the mobile phase using Sulfuric Acid from
Mallinckrodt Chemicals (Catalog #H378-07) and run at 0.6 mL/min.
The column temperature was set to 50.degree. C. and 20 .mu.L of
sample was injected onto the column.
Example 5
Isolation of 2-C-methyl-D-erythritol and acetylated
2-C-methyl-D-erythritols from fermentation broth
[0149] A clarified fermentation broth sample (.about.1.5 L) was
concentrated under reduced pressure to produce a brown oily
suspension. Dilution with dichloromethane:methanol
(CH.sub.2Cl.sub.2:MeOH) (80:20, 0.5 L) and filtration through a
silica gel plug (17 cm.times.17 cm) was followed by further washing
of the silica plug with dichloromethane:methanol (80:20, 5.times.1
L). The yellow filtrate was concentrated under reduced pressure to
afford a brown viscous oil. Purification over a silica gel column
(silica gel 200-400 mesh, 60 .ANG., 60 cm.times.6 cm) using a
gradient elution (90:10 dichloromethane:methanol to 85:15
dichloromethane:methanol) afforded two products. Product #1 was a
brown viscous liquid (.about.9 g) and was identified as consisting
of a mixture of 1-O-acetyl-2-C-methyl-D-erythritol (1a) and
4-O-acetyl-2-C-methyl-D-erythritol (1b) in a 1:2 ratio by .sup.1H
NMR. In order to further confirm this assignment, a small sample
(100 mg) of product #1 was subjected to deacetylating conditions
with sodium hydride (2.6 mg, 0.11 mmol, 0.2 eq) in methanol (3 mL)
at room temperature for 1 hour. This treatment produced a more
polar product which co-eluted with an authentic sample of
2-C-methyl-D-erythritol (2) as determined by TLC (eluted with
CH.sub.2Cl.sub.2/MeOH, 9:1). Product #2 was a yellow waxy solid
(approximately 24 g) and had a .sup.1H NMR spectrum identical to
that reported in the literature..sup.1
##STR00001##
[0150] 1-O-Acetyl-2-C-methyl-D-erythritol (1a): .sup.1H NMR (500
MHz, D.sub.2O) .delta.4.13, 4.02 (2H, ABq, J.sub.1,1', =11.5 Hz,
H1,1'); 3.85 (1H, dd, J.sub.3,4=2.4 Hz, J.sub.3,4'=11.4 Hz, H-3);
3.71 (1H, dd, J.sub.3,4=2.4 Hz, J.sub.4,4'=11.4 Hz, H-4); 3.58 (1H,
d, J.sub.4,4'=11.4 Hz, H-4'); 2.13 (3H, s, COCH.sub.3); 1.16 (3H,
s, CH.sub.3).
[0151] 4-O-Acetyl-2-C-methyl-D-erythritol (1b): .sup.1H NMR (500
MHz, D.sub.2O) .delta.4.38 (1H, dd, J.sub.3,4=2.4 Hz,
J.sub.4,4'=11.4 Hz, H-4); 4.12 (1H, d, J.sub.4,4'=11.4 Hz, H-4');
3.86 (1H, dd, J.sub.3,4=2.4 Hz, J.sub.3,4'=11.4 Hz, H-3); 3.60,
3.49 (2H, ABq, J.sub.1,1'=11.5 Hz, H1,1'); 2.12 (3H, s,
COCH.sub.3); 1.15 (3H, s, CH.sub.3).
[0152] 2-C-Methyl-D-erythritol (2): .sup.1H NMR (500 MHz, D.sub.2O)
.delta.3.83 (1H, dd, J.sub.3,4=2.4 Hz, J.sub.3,4'=11.4 Hz, H-3);
3.66 (1H, dd, J.sub.3,4=2.4 Hz, J.sub.4,4'=11.4 Hz, H-4); 3.60 (1H,
d, J.sub.4,4'=11.4 Hz, H-4'); 3.58, 3.47 (2H, ABq, J.sub.1,1'=11.5
Hz, H1,1'), 1.13 (3H, s, CH.sub.3).
[0153] Sakamoto, I., Ichimura, K., and Ohrui, H., Biosci.
Biotechnol. Biochem. (2000), 64(9), 1915-1922.
Example 6
Improving Production of 2-Methyl-D-erythritol
[0154] To improve production of 2-Methyl-D-erythritol using current
strains with engineered DXP pathways, various protocols are carried
out.
[0155] First, further upregulation of dxs and dxr genes, with
downregulation of IspDF and other genes that utilize
2-methyl-D-erythritol-5-phosphate (MEP) is done. A complete ispDF
knockout is done in strains that also expressed the MVA pathway at
a level sufficient to support the IPP/DMAPP levels needed to
support cell growth. For example, reduced levels of ispDF is
accomplished by inserting the GI1.0 promoter in place of the
endogenous promoter.
[0156] Secondly, strains are run under low phosphate conditions so
as to induce phosphatase expression. Phosphatases are required to
convert MEP, and perhaps other DXP metabolites (cMEPP) to
2-C-methyl-D-erythritol.
[0157] Third, heterologous phosphatase (e.g. bovine phosphatase)
with a sufficiently high Km so as to not disrupt normal cell
metabolism is overexpressed. In some cases, this system is able to
dephosphorylate pooled MEP intermediate.
[0158] Fourth, the endogenous E. coli phosphatases that are
responsible for 2-ME production in the current strains is
identified and overexpressed. The MEP phosphatase is identified via
a genomic linrary or ASKA collection approach.
[0159] Fifth, the acetyl transferases responsible for converting
2-ME to the 1- and 4-monoacetyl derivatives are knocked out or
downregulated to increase the yield of 2-ME. Conversely, if these
monoacetates are desired, these acetyltransferases are
overexpressed. In one instance, the LacA gene, a high Km acetly
transferase is further over expressed.
Example 7
Improving Production of 1-deoxy-D-xylulose
[0160] For improving production of 1-deoxy-D-xylulose, strategies
similar to those described in Example 6 are used to improve
production of 1-deoxy-D-xylulose using current strains with
engineered DXP pathways except in this case, the dxr gene is
knocked out or downregulated so as to accumulate
1-deoxy-D-xylulose-5-phosphate (DXP), which is subsequently
dephosphorylated to 1-deoxy-D-xylulose.
* * * * *