U.S. patent application number 13/013704 was filed with the patent office on 2011-08-25 for microorganisms and methods for the biosynthesis of p-toluate and terephthalate.
Invention is credited to Robin E. OSTERHOUT.
Application Number | 20110207185 13/013704 |
Document ID | / |
Family ID | 44319700 |
Filed Date | 2011-08-25 |
United States Patent
Application |
20110207185 |
Kind Code |
A1 |
OSTERHOUT; Robin E. |
August 25, 2011 |
MICROORGANISMS AND METHODS FOR THE BIOSYNTHESIS OF P-TOLUATE AND
TEREPHTHALATE
Abstract
The invention provides non-naturally occurring microbial
organisms having a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway, p-toluate pathway, and/or terephthalate pathway. The
invention additionally provides methods of using such organisms to
produce (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway,
p-toluate pathway or terephthalate pathway.
Inventors: |
OSTERHOUT; Robin E.; (San
Diego, CA) |
Family ID: |
44319700 |
Appl. No.: |
13/013704 |
Filed: |
January 25, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61299794 |
Jan 29, 2010 |
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Current U.S.
Class: |
435/131 ;
435/136; 435/142; 435/243 |
Current CPC
Class: |
C12N 9/0006 20130101;
C12N 9/88 20130101; C12Y 207/01071 20130101; C12N 9/1085 20130101;
C12Y 101/01267 20130101; C12N 15/52 20130101; C12Y 402/01 20130101;
C12Y 402/03005 20130101; C12P 7/40 20130101; C12Y 101/01025
20130101; C12Y 402/0101 20130101; C12Y 402/03004 20130101; C12P
7/44 20130101; C12P 9/00 20130101; C12Y 202/01007 20130101; C12N
9/1022 20130101; C12Y 205/01019 20130101; C12Y 401/0304 20130101;
C12Y 401/03 20130101; C12P 7/24 20130101; C12Y 205/01054 20130101;
C12P 7/62 20130101 |
Class at
Publication: |
435/131 ;
435/243; 435/142; 435/136 |
International
Class: |
C12P 9/00 20060101
C12P009/00; C12N 1/00 20060101 C12N001/00; C12P 7/44 20060101
C12P007/44; C12P 7/40 20060101 C12P007/40 |
Claims
1. A non-naturally occurring microbial organism, comprising a
microbial organism having a
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprising at
least one exogenous nucleic acid encoding a
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme
expressed in a sufficient amount to produce
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, said
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprising
2-C-methyl-D-erythritol-4-phosphate dehydratase.
2. The non-naturally occurring microbial organism of claim 1,
wherein said (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway
further comprises 1-deoxyxylulose-5-phosphate synthase or
1-deoxy-D-xylulose-5-phosphate reductoisomerase.
3. The non-naturally occurring microbial organism of claim 1,
wherein said (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway
further comprises 1-deoxyxylulose-5-phosphate synthase and
1-deoxy-D-xylulose-5-phosphate reductoisomerase.
4. The non-naturally occurring microbial organism of claim 1,
wherein said microbial organism comprises three exogenous nucleic
acids each encoding a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway enzyme.
5. The non-naturally occurring microbial organism of claim 4,
wherein said three exogenous nucleic acids encode
2-C-methyl-D-erythritol-4-phosphate dehydratase,
1-deoxyxylulose-5-phosphate synthase and
1-deoxy-D-xylulose-5-phosphate reductoisomerase.
6. The non-naturally occurring microbial organism of claim 1,
wherein said at least one exogenous nucleic acid is a heterologous
nucleic acid.
7. The non-naturally occurring microbial organism of claim 1,
wherein said non-naturally occurring microbial organism is in a
substantially anaerobic culture medium.
8. A method for producing
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, comprising culturing
the non-naturally occurring microbial organism of claim 1 under
conditions and for a sufficient period of time to produce
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate.
9. The method of claim 8, wherein said non-naturally occurring
microbial organism is in a substantially anaerobic culture
medium.
10. The method of claim 8, wherein said microbial organism
comprises three exogenous nucleic acids each encoding a
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme.
11. The method of claim 10, wherein said three exogenous nucleic
acids encode 2-C-methyl-D-erythritol-4-phosphate dehydratase,
1-deoxyxylulose-5-phosphate synthase and
1-deoxy-D-xylulose-5-phosphate reductoisomerase.
12. The method of claim 8, wherein said at least one exogenous
nucleic acid is a heterologous nucleic acid.
13. A non-naturally occurring microbial organism, comprising a
microbial organism having a p-toluate pathway comprising at least
one exogenous nucleic acid encoding a p-toluate pathway enzyme
expressed in a sufficient amount to produce p-toluate, said
p-toluate pathway comprising 2-dehydro-3-deoxyphosphoheptonate
synthase; 3-dehydroquinate synthase; 3-dehydroquinate dehydratase;
shikimate dehydrogenase; shikimate kinase;
3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase;
or chorismate lyase.
14. The non-naturally occurring microbial organism of claim 13,
wherein said microbial organism comprises two exogenous nucleic
acids each encoding a p-toluate pathway enzyme.
15. The non-naturally occurring microbial organism of claim 13,
wherein said microbial organism comprises three exogenous nucleic
acids each encoding a p-toluate pathway enzyme.
16. The non-naturally occurring microbial organism of claim 13,
wherein said microbial organism comprises four exogenous nucleic
acids each encoding a p-toluate pathway enzyme.
17. The non-naturally occurring microbial organism of claim 13,
wherein said microbial organism comprises five exogenous nucleic
acids each encoding a p-toluate pathway enzyme.
18. The non-naturally occurring microbial organism of claim 13,
wherein said microbial organism comprises six exogenous nucleic
acids each encoding a p-toluate pathway enzyme.
19. The non-naturally occurring microbial organism of claim 13,
wherein said microbial organism comprises seven exogenous nucleic
acids each encoding a p-toluate pathway enzyme.
20. The non-naturally occurring microbial organism of claim 13,
wherein said microbial organism comprises seven exogenous nucleic
acids each encoding a p-toluate pathway enzyme.
21. The non-naturally occurring microbial organism of claim 19,
wherein said eight exogenous nucleic acids encode
2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate
synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase;
shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase;
chorismate synthase; and chorismate lyase.
22. The non-naturally occurring microbial organism of claim 13,
wherein said microbial organism further comprises a
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway.
23. The non-naturally occurring microbial organism of claim 22,
wherein the (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway
comprises 2-C-methyl-D-erythritol-4-phosphate dehydratase,
1-deoxyxylulose-5-phosphate synthase or
1-deoxy-D-xylulose-5-phosphate reductoisomerase.
24. The non-naturally occurring microbial organism of claim 23,
wherein the (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway
comprises 2-C-methyl-D-erythritol-4-phosphate dehydratase,
1-deoxyxylulose-5-phosphate synthase and
1-deoxy-D-xylulose-5-phosphate reductoisomerase.
25. The non-naturally occurring microbial organism of claim 13,
wherein at least one exogenous nucleic acid is a heterologous
nucleic acid.
26. The non-naturally occurring microbial organism of claim 13,
wherein said non-naturally occurring microbial organism is in a
substantially anaerobic culture medium.
27. A method for producing p-toluate, comprising culturing the
non-naturally occurring microbial organism of claim 13 under
conditions and for a sufficient period of time to produce
p-toluate.
28. The method of claim 27, wherein said non-naturally occurring
microbial organism is in a substantially anaerobic culture
medium.
29. The method of claim 27, wherein said microbial organism
comprises seven exogenous nucleic acids each encoding a p-toluate
pathway enzyme.
30. The method of claim 29, wherein said seven exogenous nucleic
acids encode 2-dehydro-3-deoxyphosphoheptonate synthase;
3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate
dehydrogenase; shikimate kinase;
3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase;
and chorismate lyase.
31. The method of claim 27, wherein said microbial organism further
comprises a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway.
32. The method of claim 31, wherein the
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprises
2-C-methyl-D-erythritol-4-phosphate dehydratase,
1-deoxyxylulose-5-phosphate synthase or
1-deoxy-D-xylulose-5-phosphate reductoisomerase.
33. The method of claim 32, wherein the
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprises
2-C-methyl-D-erythritol-4-phosphate dehydratase,
1-deoxyxylulose-5-phosphate synthase and
1-deoxy-D-xylulose-5-phosphate reductoisomerase.
34. The method of claim 27, wherein said at least one exogenous
nucleic acid is a heterologous nucleic acid.
35. A non-naturally occurring microbial organism, comprising a
microbial organism having a terephthalate pathway comprising at
least one exogenous nucleic acid encoding a terephthalate pathway
enzyme expressed in a sufficient amount to produce terephthalate,
said terephthalate pathway comprising p-toluate
methyl-monooxygenase reductase; 4-carboxybenzyl alcohol
dehydrogenase; or 4-carboxybenzyl aldehyde dehydrogenase; and
wherein said microbial organism further comprises a p-toluate
pathway, wherein said p-toluate pathway comprises
2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate
synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase;
shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase;
chorismate synthase; or chorismate lyase.
36. The non-naturally occurring microbial organism of claim 35,
wherein said microbial organism comprises two exogenous nucleic
acids each encoding a terephthalate pathway enzyme.
37. The non-naturally occurring microbial organism of claim 35,
wherein said microbial organism comprises three exogenous nucleic
acids each encoding a terephthalate pathway enzyme.
38. The non-naturally occurring microbial organism of claim 37,
wherein said three exogenous nucleic acids encode p-toluate
methyl-monooxygenase reductase; 4-carboxybenzyl alcohol
dehydrogenase; and 4-carboxybenzyl aldehyde dehydrogenase.
39. The non-naturally occurring microbial organism of claim 35,
wherein said p-toluate pathway comprises
2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate
synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase;
shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase;
chorismate synthase; and chorismate lyase.
40. The non-naturally occurring microbial organism of claim 35,
wherein said microbial organism further comprises a
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway.
41. The non-naturally occurring microbial organism of claim 40,
wherein the (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway
comprises 2-C-methyl-D-erythritol-4-phosphate dehydratase,
1-deoxyxylulose-5-phosphate synthase or
1-deoxy-D-xylulose-5-phosphate reductoisomerase.
42. The non-naturally occurring microbial organism of claim 41,
wherein the (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway
comprises 2-C-methyl-D-erythritol-4-phosphate dehydratase,
1-deoxyxylulose-5-phosphate synthase and
1-deoxy-D-xylulose-5-phosphate reductoisomerase.
43. The non-naturally occurring microbial organism of claim 35,
wherein said at least one exogenous nucleic acid is a heterologous
nucleic acid.
44. The non-naturally occurring microbial organism of claim 35,
wherein said non-naturally occurring microbial organism is in a
substantially anaerobic culture medium.
45. A method for producing terephthalate, comprising culturing the
non-naturally occurring microbial organism of claim 35 under
conditions and for a sufficient period of time to produce
terephthalate.
46. The method of claim 45, wherein said non-naturally occurring
microbial organism is in a substantially anaerobic culture
medium.
47. The method of claim 45, wherein said microbial organism
comprises three exogenous nucleic acids each encoding a
terephthalate pathway enzyme.
48. The method of claim 47, wherein said three exogenous nucleic
acids encode p-toluate methyl-monooxygenase reductase;
4-carboxybenzyl alcohol dehydrogenase; or 4-carboxybenzyl aldehyde
dehydrogenase.
49. The method of claim 45, wherein said p-toluate pathway
comprises 2-dehydro-3-deoxyphosphoheptonate synthase;
3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate
dehydrogenase; shikimate kinase;
3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase;
and chorismate lyase.
50. The method of claim 45, wherein said microbial organism further
comprises a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway.
51. The method of claim 50, wherein the
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprises
2-C-methyl-D-erythritol-4-phosphate dehydratase,
1-deoxyxylulose-5-phosphate synthase or
1-deoxy-D-xylulose-5-phosphate reductoisomerase.
52. The method of claim 51, wherein the
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprises
2-C-methyl-D-erythritol-4-phosphate dehydratase,
1-deoxyxylulose-5-phosphate synthase and
1-deoxy-D-xylulose-5-phosphate reductoisomerase.
53. The method of claim 45, wherein said at least one exogenous
nucleic acid is a heterologous nucleic acid.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates generally to biosynthetic
processes, and more specifically to organisms having p-toluate,
terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
biosynthetic capability.
[0002] Terephthalate (also known as terephthalic acid and PTA) is
the immediate precursor of polyethylene terepthalate (PET), used to
make clothing, resins, plastic bottles and even as a poultry feed
additive. Nearly all PTA is produced from para-xylene by oxidation
in air in a process known as the Mid Century Process. This
oxidation is conducted at high temperature in an acetic acid
solvent with a catalyst composed of cobalt and/or manganese salts.
Para-xylene is derived from petrochemical sources and is formed by
high severity catalytic reforming of naphtha. Xylene is also
obtained from the pyrolysis gasoline stream in a naphtha steam
cracker and by toluene disproportion.
[0003] Cost-effective methods for generating renewable PTA have not
yet been developed to date. PTA, toluene and other aromatic
precursors are naturally degraded by some bacteria. However, these
degradation pathways typically involve monooxygenases that operate
irreversibly in the degradative direction. Hence, biosynthetic
pathways for PTA are severely limited by the properties of known
enzymes to date.
[0004] A promising precursor for PTA is p-toluate, also known as
p-methylbenzoate. P-Toluate is an intermediate in some industrial
processes for the oxidation of p-xylene to PTA. It is also an
intermediate for polymer stabilizers, pesticides, light sensitive
compounds, animal feed supplements and other organic chemicals.
Only slightly soluble in aqueous solution, p-toluate is a solid at
physiological temperatures, with a melting point of 275.degree. C.
Microbial catalysts for synthesizing this compound from sugar
feedstocks have not been described to date.
[0005] Thus, there exists a need for alternative methods for
effectively producing commercial quantities of compounds such as
p-toluate or terephthalate. The present invention satisfies this
need and provides related advantages as well.
SUMMARY OF THE INVENTION
[0006] The invention provides non-naturally occurring microbial
organisms having a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway, p-toluate pathway, and/or terephthalate pathway. The
invention additionally provides methods of using such organisms to
produce (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway,
p-toluate pathway or terephthalate pathway.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 shows a schematic depiction of an exemplary pathway
to (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (2H3M4OP) from
glyceraldehyde-3-phosphate and pyruvate. G3P is
glyceraldehyde-3-phosphate, DXP is 1-deoxy-D-xylulose-5-phosphate
and 2ME4P is C-methyl-D-erythritol-4-phosphate. Enzymes are (A) DXP
synthase; (B) DXP reductoisomerase; and (C) 2ME4P dehydratase.
[0008] FIG. 2 shows a schematic depiction of an exemplary alternate
shikimate pathway to p-toluate. Enzymes are: (A)
2-dehydro-3-deoxyphosphoheptonate synthase; (B) 3-dehydroquinate
synthase; (C) 3-dehydroquinate dehydratase; (D) shikimate
dehydrogenase; (E) Shikimate kinase; (F)
3-phosphoshikimate-2-carboxyvinyltransferase; (G) chorismate
synthase; and (H) chorismate lyase. Compounds are: (1)
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate; (2)
2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate;
(3) 1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate; (4)
5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylate; (5)
3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate; (6)
5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate;
(7)
5-[(1-carboxyeth-1-en-1-yl)oxy]-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-
-carboxylate; (8)
3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate-
; and (9) p-toluate.
[0009] FIG. 3 shows an exemplary pathway for conversion of
p-toluate to terephthalic acid (PTA). Reactions A, B and C are
catalyzed by p-toluate methyl-monooxygenase reductase,
4-carboxybenzyl alcohol dehydrogenase and 4-carboxybenzyl aldehyde
dehydrogenase, respectively. The compounds shown are (1) p-toluic
acid; (2) 4-carboxybenzyl alcohol; (3) 4-carboxybenzaldehyde and
(4) terephthalic acid.
DETAILED DESCRIPTION OF THE INVENTION
[0010] The present invention is directed to the design and
production of cells and organisms having biosynthetic production
capabilities for p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. The results described
herein indicate that metabolic pathways can be designed and
recombinantly engineered to achieve the biosynthesis of p-toluate,
terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate in
Escherichia coli and other cells or organisms. Biosynthetic
production of p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate can be confirmed by
construction of strains having the designed metabolic genotype.
These metabolically engineered cells or organisms also can be
subjected to adaptive evolution to further augment p-toluate,
terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
biosynthesis, including under conditions approaching theoretical
maximum growth.
[0011] The shikimate biosynthesis pathway in E. coli converts
erythrose-4-phosphate to chorismate, an important intermediate that
leads to the biosynthesis of many essential metabolites including
4-hydroxybenzoate. 4-Hydroxybenzoate is structurally similar to
p-toluate, an industrial precursor of terephthalic acid. As
disclosed herein, shikimate pathway enzymes are utilized to accept
the alternate substrate,
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (2H3M4OP) and transform
it to p-toluate. In addition, a pathway is used to synthesize the
2H3M4OP precursor using enzymes from the non-mevalonate pathway for
isoprenoid biosynthesis.
[0012] Disclosed herein are strategies for engineering a
microorganism to produce renewable p-toluate or terephthalate (PTA)
from carbohydrate feedstocks. First, glyceraldehyde-3-phosphate
(G3P) and pyruvate are converted to
2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (2H3M4OP) in three
enzymatic steps (see Example I and FIG. 1). The 2H3M4OP
intermediate is subsequently transformed to p-toluate by enzymes in
the shikimate pathway (see Example II and FIG. 2). P-Toluate can be
further converted to PTA by a microorganism (see Example III and
FIG. 3).
[0013] The conversion of G3P to p-toluate requires one ATP, two
reducing equivalents (NAD(P)H), and two molecules of
phosphoenolpyruvate, according to net reaction below.
G3P+2 PEP+ATP+2 NAD(P)H+2 H.sup.+.fwdarw.p-Toluate+4 Pi+ADP+2
NAD(P).sup.++CO.sub.2+H.sub.2O
[0014] An additional ATP is required to synthesize G3P from
glucose. The maximum theoretical p-toluate yield is 0.67 mol/mol
(0.51 g/g) from glucose minus carbon required for energy. Under the
assumption that 2 ATPs are consumed per p-toluate molecule
synthesized, the predicted p-toluate yield from glucose is 0.62
mol/mol (0.46 g/g) p-toluate.
[0015] If p-toluate is further converted to PTA by enzymes as
described in Example III, the predicted PTA yield from glucose is
0.64 mol/mol (0.58 g/g). In this case, the oxidation of p-toluate
to PTA generates an additional net reducing equivalent according to
the net reaction:
p-toluate+O.sub.2+NAD.sup.+.fwdarw.PTA+NADH+2 H.sup.+
[0016] Enzyme candidates for catalyzing each step of the proposed
pathways are described in the following sections.
[0017] As used herein, the term "non-naturally occurring" when used
in reference to a microbial organism or microorganism of the
invention is intended to mean that the microbial organism has at
least one genetic alteration not normally found in a naturally
occurring strain of the referenced species, including wild-type
strains of the referenced species. Genetic alterations include, for
example, modifications introducing expressible nucleic acids
encoding metabolic polypeptides, other nucleic acid additions,
nucleic acid deletions and/or other functional disruption of the
microbial organism's genetic material. Such modifications include,
for example, coding regions and functional fragments thereof, for
heterologous, homologous or both heterologous and homologous
polypeptides for the referenced species. Additional modifications
include, for example, non-coding regulatory regions in which the
modifications alter expression of a gene or operon. Exemplary
metabolic polypeptides include enzymes or proteins within a
p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthetic
pathway.
[0018] A metabolic modification refers to a biochemical reaction
that is altered from its naturally occurring state. Therefore,
non-naturally occurring microorganisms can have genetic
modifications to nucleic acids encoding metabolic polypeptides, or
functional fragments thereof. Exemplary metabolic modifications are
disclosed herein.
[0019] As used herein, the term "isolated" when used in reference
to a microbial organism is intended to mean an organism that is
substantially free of at least one component as the referenced
microbial organism is found in nature. The term includes a
microbial organism that is removed from some or all components as
it is found in its natural environment. The term also includes a
microbial organism that is removed from some or all components as
the microbial organism is found in non-naturally occurring
environments. Therefore, an isolated microbial organism is partly
or completely separated from other substances as it is found in
nature or as it is grown, stored or subsisted in non-naturally
occurring environments. Specific examples of isolated microbial
organisms include partially pure microbes, substantially pure
microbes and microbes cultured in a medium that is non-naturally
occurring.
[0020] As used herein, the terms "microbial," "microbial organism"
or "microorganism" are intended to mean any organism that exists as
a microscopic cell that is included within the domains of archaea,
bacteria or eukarya. Therefore, the term is intended to encompass
prokaryotic or eukaryotic cells or organisms having a microscopic
size and includes bacteria, archaea and eubacteria of all species
as well as eukaryotic microorganisms such as yeast and fungi. The
term also includes cell cultures of any species that can be
cultured for the production of a biochemical.
[0021] As used herein, the term "CoA" or "coenzyme A" is intended
to mean an organic cofactor or prosthetic group (nonprotein portion
of an enzyme) whose presence is required for the activity of many
enzymes (the apoenzyme) to form an active enzyme system. Coenzyme A
functions in certain condensing enzymes, acts in acetyl or other
acyl group transfer and in fatty acid synthesis and oxidation,
pyruvate oxidation and in other acetylation.
[0022] As used herein, the term
"(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate," abbreviated herein
as 2H3M4OP, has the chemical formula as shown in FIG. 1. Such a
compound can also be described as 3-hydroxy-2-methyl
butanal-4-phosphate.
[0023] As used herein, the term "p-toluate," having the molecular
formula C.sub.8H.sub.7O.sub.2.sup.- (see FIG. 2, compound 9)(IUPAC
name 4-methylbenzoate) is the ionized form of p-toluic acid, and it
is understood that p-toluate and p-toluic acid can be used
interchangeably throughout to refer to the compound in any of its
neutral or ionized forms, including any salt forms thereof. It is
understood by those skilled understand that the specific form will
depend on the pH.
[0024] As used herein, the term "terephthalate," having the
molecular formula C.sub.8H.sub.4O.sub.4.sup.-2 (see FIG. 3,
compound 4)(IUPAC name terephthalate) is the ionized form of
terephthalic acid, also referred to as p-phthalic acid or PTA, and
it is understood that terephthalate and terephthalic acid can be
used interchangeably throughout to refer to the compound in any of
its neutral or ionized forms, including any salt forms thereof It
is understood by those skilled understand that the specific form
will depend on the pH.
[0025] As used herein, the term "substantially anaerobic" when used
in reference to a culture or growth condition is intended to mean
that the amount of oxygen is less than about 10% of saturation for
dissolved oxygen in liquid media. The term also is intended to
include sealed chambers of liquid or solid medium maintained with
an atmosphere of less than about 1% oxygen.
[0026] "Exogenous" as it is used herein is intended to mean that
the referenced molecule or the referenced activity is introduced
into the host microbial organism. The molecule can be introduced,
for example, by introduction of an encoding nucleic acid into the
host genetic material such as by integration into a host chromosome
or as non-chromosomal genetic material such as a plasmid.
Therefore, the term as it is used in reference to expression of an
encoding nucleic acid refers to introduction of the encoding
nucleic acid in an expressible form into the microbial organism.
When used in reference to a biosynthetic activity, the term refers
to an activity that is introduced into the host reference organism.
The source can be, for example, a homologous or heterologous
encoding nucleic acid that expresses the referenced activity
following introduction into the host microbial organism. Therefore,
the term "endogenous" refers to a referenced molecule or activity
that is present in the host. Similarly, the term when used in
reference to expression of an encoding nucleic acid refers to
expression of an encoding nucleic acid contained within the
microbial organism. The term "heterologous" refers to a molecule or
activity derived from a source other than the referenced species
whereas "homologous" refers to a molecule or activity derived from
the host microbial organism. Accordingly, exogenous expression of
an encoding nucleic acid of the invention can utilize either or
both a heterologous or homologous encoding nucleic acid.
[0027] It is understood that when more than one exogenous nucleic
acid is included in a microbial organism that the more than one
exogenous nucleic acids refers to the referenced encoding nucleic
acid or biosynthetic activity, as discussed above. It is further
understood, as disclosed herein, that such more than one exogenous
nucleic acids can be introduced into the host microbial organism on
separate nucleic acid molecules, on polycistronic nucleic acid
molecules, or a combination thereof, and still be considered as
more than one exogenous nucleic acid. For example, as disclosed
herein a microbial organism can be engineered to express two or
more exogenous nucleic acids encoding a desired pathway enzyme or
protein. In the case where two exogenous nucleic acids encoding a
desired activity are introduced into a host microbial organism, it
is understood that the two exogenous nucleic acids can be
introduced as a single nucleic acid, for example, on a single
plasmid, on separate plasmids, can be integrated into the host
chromosome at a single site or multiple sites, and still be
considered as two exogenous nucleic acids. Similarly, it is
understood that more than two exogenous nucleic acids can be
introduced into a host organism in any desired combination, for
example, on a single plasmid, on separate plasmids, can be
integrated into the host chromosome at a single site or multiple
sites, and still be considered as two or more exogenous nucleic
acids, for example three exogenous nucleic acids. Thus, the number
of referenced exogenous nucleic acids or biosynthetic activities
refers to the number of encoding nucleic acids or the number of
biosynthetic activities, not the number of separate nucleic acids
introduced into the host organism.
[0028] The non-naturally occurring microbial organisms of the
invention can contain stable genetic alterations, which refers to
microorganisms that can be cultured for greater than five
generations without loss of the alteration. Generally, stable
genetic alterations include modifications that persist greater than
10 generations, particularly stable modifications will persist more
than about 25 generations, and more particularly, stable genetic
modifications will be greater than 50 generations, including
indefinitely.
[0029] Those skilled in the art will understand that the genetic
alterations, including metabolic modifications exemplified herein,
are described with reference to a suitable host organism such as E.
coli and their corresponding metabolic reactions or a suitable
source organism for desired genetic material such as genes for a
desired metabolic pathway. However, given the complete genome
sequencing of a wide variety of organisms and the high level of
skill in the area of genomics, those skilled in the art will
readily be able to apply the teachings and guidance provided herein
to essentially all other organisms. For example, the E. coli
metabolic alterations exemplified herein can readily be applied to
other species by incorporating the same or analogous encoding
nucleic acid from species other than the referenced species. Such
genetic alterations include, for example, genetic alterations of
species homologs, in general, and in particular, orthologs,
paralogs or nonorthologous gene displacements.
[0030] An ortholog is a gene or genes that are related by vertical
descent and are responsible for substantially the same or identical
functions in different organisms. For example, mouse epoxide
hydrolase and human epoxide hydrolase can be considered orthologs
for the biological function of hydrolysis of epoxides. Genes are
related by vertical descent when, for example, they share sequence
similarity of sufficient amount to indicate they are homologous, or
related by evolution from a common ancestor. Genes can also be
considered orthologs if they share three-dimensional structure but
not necessarily sequence similarity, of a sufficient amount to
indicate that they have evolved from a common ancestor to the
extent that the primary sequence similarity is not identifiable.
Genes that are orthologous can encode proteins with sequence
similarity of about 25% to 100% amino acid sequence identity. Genes
encoding proteins sharing an amino acid similarity less that 25%
can also be considered to have arisen by vertical descent if their
three-dimensional structure also shows similarities. Members of the
serine protease family of enzymes, including tissue plasminogen
activator and elastase, are considered to have arisen by vertical
descent from a common ancestor.
[0031] Orthologs include genes or their encoded gene products that
through, for example, evolution, have diverged in structure or
overall activity. For example, where one species encodes a gene
product exhibiting two functions and where such functions have been
separated into distinct genes in a second species, the three genes
and their corresponding products are considered to be orthologs.
For the production of a biochemical product, those skilled in the
art will understand that the orthologous gene harboring the
metabolic activity to be introduced or disrupted is to be chosen
for construction of the non-naturally occurring microorganism. An
example of orthologs exhibiting separable activities is where
distinct activities have been separated into distinct gene products
between two or more species or within a single species. A specific
example is the separation of elastase proteolysis and plasminogen
proteolysis, two types of serine protease activity, into distinct
molecules as plasminogen activator and elastase. A second example
is the separation of mycoplasma 5'-3' exonuclease and Drosophila
DNA polymerase III activity. The DNA polymerase from the first
species can be considered an ortholog to either or both of the
exonuclease or the polymerase from the second species and vice
versa.
[0032] In contrast, paralogs are homologs related by, for example,
duplication followed by evolutionary divergence and have similar or
common, but not identical functions. Paralogs can originate or
derive from, for example, the same species or from a different
species. For example, microsomal epoxide hydrolase (epoxide
hydrolase I) and soluble epoxide hydrolase (epoxide hydrolase II)
can be considered paralogs because they represent two distinct
enzymes, co-evolved from a common ancestor, that catalyze distinct
reactions and have distinct functions in the same species. Paralogs
are proteins from the same species with significant sequence
similarity to each other suggesting that they are homologous, or
related through co-evolution from a common ancestor. Groups of
paralogous protein families include HipA homologs, luciferase
genes, peptidases, and others.
[0033] A nonorthologous gene displacement is a nonorthologous gene
from one species that can substitute for a referenced gene function
in a different species. Substitution includes, for example, being
able to perform substantially the same or a similar function in the
species of origin compared to the referenced function in the
different species. Although generally, a nonorthologous gene
displacement will be identifiable as structurally related to a
known gene encoding the referenced function, less structurally
related but functionally similar genes and their corresponding gene
products nevertheless will still fall within the meaning of the
term as it is used herein. Functional similarity requires, for
example, at least some structural similarity in the active site or
binding region of a nonorthologous gene product compared to a gene
encoding the function sought to be substituted. Therefore, a
nonorthologous gene includes, for example, a paralog or an
unrelated gene.
[0034] Therefore, in identifying and constructing the non-naturally
occurring microbial organisms of the invention having p-toluate,
terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
biosynthetic capability, those skilled in the art will understand
with applying the teaching and guidance provided herein to a
particular species that the identification of metabolic
modifications can include identification and inclusion or
inactivation of orthologs. To the extent that paralogs and/or
nonorthologous gene displacements are present in the referenced
microorganism that encode an enzyme catalyzing a similar or
substantially similar metabolic reaction, those skilled in the art
also can utilize these evolutionally related genes.
[0035] Orthologs, paralogs and nonorthologous gene displacements
can be determined by methods well known to those skilled in the
art. For example, inspection of nucleic acid or amino acid
sequences for two polypeptides will reveal sequence identity and
similarities between the compared sequences. Based on such
similarities, one skilled in the art can determine if the
similarity is sufficiently high to indicate the proteins are
related through evolution from a common ancestor. Algorithms well
known to those skilled in the art, such as Align, BLAST, Clustal W
and others compare and determine a raw sequence similarity or
identity, and also determine the presence or significance of gaps
in the sequence which can be assigned a weight or score. Such
algorithms also are known in the art and are similarly applicable
for determining nucleotide sequence similarity or identity.
Parameters for sufficient similarity to determine relatedness are
computed based on well known methods for calculating statistical
similarity, or the chance of finding a similar match in a random
polypeptide, and the significance of the match determined. A
computer comparison of two or more sequences can, if desired, also
be optimized visually by those skilled in the art. Related gene
products or proteins can be expected to have a high similarity, for
example, 25% to 100% sequence identity. Proteins that are unrelated
can have an identity which is essentially the same as would be
expected to occur by chance, if a database of sufficient size is
scanned (about 5%). Sequences between 5% and 24% may or may not
represent sufficient homology to conclude that the compared
sequences are related. Additional statistical analysis to determine
the significance of such matches given the size of the data set can
be carried out to determine the relevance of these sequences.
[0036] Exemplary parameters for determining relatedness of two or
more sequences using the BLAST algorithm, for example, can be as
set forth below. Briefly, amino acid sequence alignments can be
performed using BLASTP version 2.0.8 (Jan. 5, 1999) and the
following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap
extension: 1; x_dropoff: 50; expect: 10.0; wordsize: 3; filter: on.
Nucleic acid sequence alignments can be performed using BLASTN
version 2.0.6 (Sep. 16, 1998) and the following parameters: Match:
1; mismatch: -2; gap open: 5; gap extension: 2; x_dropoff: 50;
expect: 10.0; wordsize: 11; filter: off. Those skilled in the art
will know what modifications can be made to the above parameters to
either increase or decrease the stringency of the comparison, for
example, and determine the relatedness of two or more
sequences.
[0037] The invention provides a non-naturally occurring microbial
organism, comprising a microbial organism having a
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprising at
least one exogenous nucleic acid encoding a
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme
expressed in a sufficient amount to produce
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, the
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprising
2-C-methyl-D-erythritol-4-phosphate dehydratase (see Example I and
FIG. 1, step C). A non-naturally occurring microbial organism
comprising a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway
can further comprise 1-deoxyxylulose-5-phosphate synthase or
1-deoxy-D-xylulose-5-phosphate reductoisomerase (see Example I and
FIG. 1, steps A and B). Thus, a
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate can comprise 5
2-C-methyl-D-erythritol-4-phosphate dehydratase,
1-deoxyxylulose-5-phosphate synthase and
1-deoxy-D-xylulose-5-phosphate reductoisomerase.
[0038] The invention also provides a non-naturally occurring
microbial organism, comprising a microbial organism having a
p-toluate pathway comprising at least one exogenous nucleic acid
encoding a p-toluate pathway enzyme expressed in a sufficient
amount to produce p-toluate, the p-toluate pathway comprising
2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate
synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase;
shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase;
chorismate synthase; or chorismate lyase (see Example II and FIG.
2, steps A-H). A non-naturally occurring microbial organism having
a p-toluate pathway can further comprise a
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway (FIG. 1). A
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway can comprise,
for example, 2-C-methyl-D-erythritol-4-phosphate dehydratase,
1-deoxyxylulose-5-phosphate synthase or
1-deoxy-D-xylulose-5-phosphate reductoisomerase (FIG. 1).
[0039] The invention additionally provides a non-naturally
occurring microbial organism, comprising a microbial organism
having a terephthalate pathway comprising at least one exogenous
nucleic acid encoding a terephthalate pathway enzyme expressed in a
sufficient amount to produce terephthalate, the terephthalate
pathway comprising p-toluate methyl-monooxygenase reductase;
4-carboxybenzyl alcohol dehydrogenase; or 4-carboxybenzyl aldehyde
dehydrogenase (see Example III and FIG. 3). Such an organism
containing a terephthalate pathway can additionally comprise a
p-toluate pathway, wherein the p-toluate pathway comprises
2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate
synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase;
shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase;
chorismate synthase; or chorismate lyase (see Examples II and III
and FIGS. 2 and 3). Such a non-naturally occurring
microbialorganism having a terephthalate pathway and a p-toluate
pathway can further comprise a
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway (see Example I
and FIG. 1). A (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway
can comprise, for example, 2-C-methyl-D-erythritol-4-phosphate
dehydratase, 1-deoxyxylulose-5-phosphate synthase or
1-deoxy-D-xylulose-5-phosphate reductoisomerase (see Example I and
FIG. 1).
[0040] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having a p-toluate,
terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway, wherein the non-naturally occurring microbial organism
comprises at least one exogenous nucleic acid encoding an enzyme or
protein that converts a substrate to a product. For example, in a
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, the substrates
and products can be selected from the group consisting of
glyceraldehyde-3-phosphate and pyruvate to
1-deoxy-D-xylulose-5-phosphate; 1-deoxy-D-xylulose-5-phosphate to
C-methyl-D-erythritol-4-phosphate; and
C-methyl-D-erythritol-4-phosphate to
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (see Example I and FIG.
1). In another embodiment, a p-toluate pathway can comprise
substrates and products selected from
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate to
2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate;
2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate
to 1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate;
1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate to
5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylic acid;
5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylic acid to
3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate;
3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate to
5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate;
5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate to
5-[(1-carboxyeth-1-en-1-yl)oxy]-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-
-carboxylate;
5-[(1-carboxyeth-1-en-1-yl)oxy]-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-
-carboxylate to
3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate-
; and
3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carbox-
ylate to p-toluate (see Example II and FIG. 2). In still another
embodiment, a terephthalate pathway can comprise substrates and
products selected from p-toluate to 4-carboxybenzyl alcohol;
4-carboxybenzyl alcohol to 4-carboxybenzaldehyde; and
4-carboxybenzaldehyde to and terephthalic acid (see Example III and
FIG. 3). One skilled in the art will understand that these are
merely exemplary and that any of the substrate-product pairs
disclosed herein suitable to produce a desired product and for
which an appropriate activity is available for the conversion of
the substrate to the product can be readily determined by one
skilled in the art based on the teachings herein. Thus, the
invention provides a non-naturally occurring microbial organism
containing at least one exogenous nucleic acid encoding an enzyme
or protein, where the enzyme or protein converts the substrates and
products of a p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, such as that
shown in FIGS. 1-3.
[0041] While generally described herein as a microbial organism
that contains a p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, it is
understood that the invention additionally provides a non-naturally
occurring microbial organism comprising at least one exogenous
nucleic acid encoding a p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme
expressed in a sufficient amount to produce an intermediate of a
p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway. For example,
as disclosed herein, a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway is exemplified in FIG. 1 (see Example I). Therefore, in
addition to a microbial organism containing a
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway that produces
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, the invention
additionally provides a non-naturally occurring microbial organism
comprising at least one exogenous nucleic acid encoding a
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme, where
the microbial organism produces a
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway intermediate,
for example, 1-deoxy-D-xylulose-5-phosphate or
C-methyl-D-erythritol-4-phosphate. Similarly, the invention also
provides a non-naturally occurring microbial organism containing a
p-toluate pathway that produces p-toluate, wherein the
non-naturally occurring microbial organism comprises at least one
exogenous nucleic acid encoding a p-toluate pathway enzyme, where
the microbial organism produces a p-toluate pathway intermediate,
for example,
2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxyl
ate, 1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate,
5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylate,
3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate,
5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate,
5-[(1-carboxyeth-1-en-1-yl)oxy]-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-
-carboxylate, or
3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate-
. Further, the invention additionally provides a non-naturally
occurring microbial organism containing a terephthalate pathway
enzyme, where the microbial organism produces a terephthalate
pathway intermediate, for example, 4-carboxybenzyl alcohol or
4-carboxybenzaldehyde.
[0042] It is understood that any of the pathways disclosed herein,
as described in the Examples and exemplified in the Figures,
including the pathways of FIGS. 1-3, can be utilized to generate a
non-naturally occurring microbial organism that produces any
pathway intermediate or product, as desired. As disclosed herein,
such a microbial organism that produces an intermediate can be used
in combination with another microbial organism expressing
downstream pathway enzymes to produce a desired product. However,
it is understood that a non-naturally occurring microbial organism
that produces a p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway intermediate
can be utilized to produce the intermediate as a desired
product.
[0043] The invention is described herein with general reference to
the metabolic reaction, reactant or product thereof, or with
specific reference to one or more nucleic acids or genes encoding
an enzyme associated with or catalyzing, or a protein associated
with, the referenced metabolic reaction, reactant or product.
Unless otherwise expressly stated herein, those skilled in the art
will understand that reference to a reaction also constitutes
reference to the reactants and products of the reaction. Similarly,
unless otherwise expressly stated herein, reference to a reactant
or product also references the reaction, and reference to any of
these metabolic constituents also references the gene or genes
encoding the enzymes that catalyze or proteins involved in the
referenced reaction, reactant or product. Likewise, given the well
known fields of metabolic biochemistry, enzymology and genomics,
reference herein to a gene or encoding nucleic acid also
constitutes a reference to the corresponding encoded enzyme and the
reaction it catalyzes or a protein associated with the reaction as
well as the reactants and products of the reaction.
[0044] The non-naturally occurring microbial organisms of the
invention can be produced by introducing expressible nucleic acids
encoding one or more of the enzymes or proteins participating in
one or more p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthetic pathways.
Depending on the host microbial organism chosen for biosynthesis,
nucleic acids for some or all of a particular p-toluate,
terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
biosynthetic pathway can be expressed. For example, if a chosen
host is deficient in one or more enzymes or proteins for a desired
biosynthetic pathway, then expressible nucleic acids for the
deficient enzyme(s) or protein(s) are introduced into the host for
subsequent exogenous expression. Alternatively, if the chosen host
exhibits endogenous expression of some pathway genes, but is
deficient in others, then an encoding nucleic acid is needed for
the deficient enzyme(s) or protein(s) to achieve p-toluate,
terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
biosynthesis. Thus, a non-naturally occurring microbial organism of
the invention can be produced by introducing exogenous enzyme or
protein activities to obtain a desired biosynthetic pathway or a
desired biosynthetic pathway can be obtained by introducing one or
more exogenous enzyme or protein activities that, together with one
or more endogenous enzymes or proteins, produces a desired product
such as p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate.
[0045] Host microbial organisms can be selected from, and the
non-naturally occurring microbial organisms generated in, for
example, bacteria, yeast, fungus or any of a variety of other
microorganisms applicable to fermentation processes. Exemplary
bacteria include species selected from Escherichia coli, Klebsiella
oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus
succinogenes, Mannheimia succiniciproducens, Rhizobium etli,
Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter
oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus
plantarum, Streptomyces coelicolor, Clostridium acetobutylicum,
Pseudomonas fluorescens, and Pseudomonas putida. Exemplary yeasts
or fungi include species selected from Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces
marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris,
Rhizopus arrhizus, Rhizobus oryzae, and the like. E. coli is a
particularly useful host organisms since it is a well characterized
microbial organism suitable for genetic engineering. Other
particularly useful host organisms include yeast such as
Saccharomyces cerevisiae. It is understood that any suitable
microbial host organism can be used to introduce metabolic and/or
genetic modifications to produce a desired product.
[0046] Depending on the p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthetic pathway
constituents of a selected host microbial organism, the
non-naturally occurring microbial organisms of the invention will
include at least one exogenously expressed p-toluate, terephthalate
or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway-encoding
nucleic acid and up to all encoding nucleic acids for one or more
p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthetic pathways.
For example, p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthesis can be
established in a host deficient in a pathway enzyme or protein
through exogenous expression of the corresponding encoding nucleic
acid. In a host deficient in all enzymes or proteins of a
p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, exogenous
expression of all enzyme or proteins in the pathway can be
included, although it is understood that all enzymes or proteins of
a pathway can be expressed even if the host contains at least one
of the pathway enzymes or proteins. For example, exogenous
expression of all enzymes or proteins in a pathway for production
of p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate can be included. For
example, all enzymes in a p-toluate pathway can be included, such
as 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate
synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase;
shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase;
chorismate synthase; and chorismate lyase. In addition, all enzymes
in a terephthalate pathway can be included, such as p-toluate
methyl-monooxygenase reductase; 4-carboxybenzyl alcohol
dehydrogenase; and 4-carboxybenzyl aldehyde dehydrogenase.
Furthermore, all enzymes in a
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway can be
included, such as 2-C-methyl-D-erythritol-4-phosphate dehydratase,
1-deoxyxylulose-5-phosphate synthase and
1-deoxy-D-xylulose-5-phosphate reductoisomerase.
[0047] Given the teachings and guidance provided herein, those
skilled in the art will understand that the number of encoding
nucleic acids to introduce in an expressible form will, at least,
parallel the p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway deficiencies of
the selected host microbial organism. Therefore, a non-naturally
occurring microbial organism of the invention can have one, two,
three, four, five, six, seven, or eight, depending on the
particular pathway, that is, up to all nucleic acids encoding the
enzymes or proteins constituting a p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthetic pathway
disclosed herein. In some embodiments, the non-naturally occurring
microbial organisms also can include other genetic modifications
that facilitate or optimize p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthesis or that
confer other useful functions onto the host microbial organism. One
such other functionality can include, for example, augmentation of
the synthesis of one or more of the p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway precursors such
as glyceraldehyde-3-phosphate, pyruvate,
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate or p-toluate.
Furthermore, as disclosed herein, multiple pathways can be included
in a single organism such as the pathway to produce p-toluate (FIG.
2), terephthalate (FIGS. 3) and
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (FIG. 1), as
desired.
[0048] Generally, a host microbial organism is selected such that
it produces the precursor of a p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, either as a
naturally produced molecule or as an engineered product that either
provides de novo production of a desired precursor or increased
production of a precursor naturally produced by the host microbial
organism. For example, glyceraldehyde-3-phosphate and
phosphoenolpyruvate are produced naturally in a host organism such
as E. coli. A host organism can be engineered to increase
production of a precursor, as disclosed herein. In addition, a
microbial organism that has been engineered to produce a desired
precursor can be used as a host organism and further engineered to
express enzymes or proteins of a p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway.
[0049] In some embodiments, a non-naturally occurring microbial
organism of the invention is generated from a host that contains
the enzymatic capability to synthesize p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. In this specific
embodiment it can be useful to increase the synthesis or
accumulation of a p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway product to, for
example, drive p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway reactions
toward p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate production. Increased
synthesis or accumulation can be accomplished by, for example,
overexpression of nucleic acids encoding one or more of the
above-described p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzymes or
proteins. Over expression the enzyme or enzymes and/or protein or
proteins of the p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway can occur, for
example, through exogenous expression of the endogenous gene or
genes, or through exogenous expression of the heterologous gene or
genes. Therefore, naturally occurring organisms can be readily
generated to be non-naturally occurring microbial organisms of the
invention, for example, producing p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, through overexpression
of one, two, three, four, five, and so forth, that is, up to all
nucleic acids encoding p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthetic pathway
enzymes or proteins. In addition, a non-naturally occurring
organism can be generated by mutagenesis of an endogenous gene that
results in an increase in activity of an enzyme in the p-toluate,
terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
biosynthetic pathway.
[0050] In particularly useful embodiments, exogenous expression of
the encoding nucleic acids is employed. Exogenous expression
confers the ability to custom tailor the expression and/or
regulatory elements to the host and application to achieve a
desired expression level that is controlled by the user. However,
endogenous expression also can be utilized in other embodiments
such as by removing a negative regulatory effector or induction of
the gene's promoter when linked to an inducible promoter or other
regulatory element. Thus, an endogenous gene having a naturally
occurring inducible promoter can be up-regulated by providing the
appropriate inducing agent, or the regulatory region of an
endogenous gene can be engineered to incorporate an inducible
regulatory element, thereby allowing the regulation of increased
expression of an endogenous gene at a desired time. Similarly, an
inducible promoter can be included as a regulatory element for an
exogenous gene introduced into a non-naturally occurring microbial
organism.
[0051] It is understood that, in methods of the invention, any of
the one or more exogenous nucleic acids can be introduced into a
microbial organism to produce a non-naturally occurring microbial
organism of the invention. The nucleic acids can be introduced so
as to confer, for example, a p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthetic pathway
onto the microbial organism. Alternatively, encoding nucleic acids
can be introduced to produce an intermediate microbial organism
having the biosynthetic capability to catalyze some of the required
reactions to confer p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthetic
capability. For example, a non-naturally occurring microbial
organism having a p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthetic pathway
can comprise at least two exogenous nucleic acids encoding desired
enzymes or proteins. For example, in a
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, a combination
of the enzymes expressed can be a combination of
2-C-methyl-D-erythritol-4-phosphate dehydratase and
1-deoxyxylulose-5-phosphate synthase, or
2-C-methyl-D-erythritol-4-phosphate dehydratase and
1-deoxy-D-xylulose-5-phosphate reductoisomerase. In a p-toluate
pathway, a combination of the enzymes expressed can be a
combination of 2-dehydro-3-deoxyphosphoheptonate synthase and
3-dehydroquinate dehydratase; shikimate kinase and
3-phosphoshikimate-2-carboxyvinyltransferase; shikimate kinase and
shikimate dehydrogenase and, and the like. Similarly, in a
terephthalate pathway, a combination of the expressed enzymes can
be p-toluate methyl-monooxygenase reductase and 4-carboxybenzyl
alcohol dehydrogenase; or 4-carboxybenzyl alcohol dehydrogenase and
4-carboxybenzyl aldehyde dehydrogenase, and the like. Thus, it is
understood that any combination of two or more enzymes or proteins
of a biosynthetic pathway can be included in a non-naturally
occurring microbial organism of the invention. Similarly, it is
understood that any combination of three or more enzymes or
proteins of a biosynthetic pathway can be included in a
non-naturally occurring microbial organism of the invention, for
example, 3-dehydroquinate synthase, shikimate dehydrogenase and
shikimate kinase; shikimate kinase, chorismate synthase and
chorismate lyase; 3-dehydroquinate dehydratase, chorismate synthase
and chorismate lyase, and so forth, as desired, so long as the
combination of enzymes and/or proteins of the desired biosynthetic
pathway results in production of the corresponding desired product.
Similarly, any combination of four, five, six, seven or more
enzymes or proteins of a biosynthetic pathway, depending on the
pathway as disclosed herein, can be included in a non-naturally
occurring microbial organism of the invention, as desired, so long
as the combination of enzymes and/or proteins of the desired
biosynthetic pathway results in production of the corresponding
desired product.
[0052] In addition to the biosynthesis of p-toluate, terephthalate
or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate as described herein,
the non-naturally occurring microbial organisms and methods of the
invention also can be utilized in various combinations with each
other and with other microbial organisms and methods well known in
the art to achieve product biosynthesis by other routes. For
example, one alternative to produce p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate other than use of the
p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate producers is through
addition of another microbial organism capable of converting a
p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway intermediate to
p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. One such procedure
includes, for example, the fermentation of a microbial organism
that produces a p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway intermediate.
The p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway intermediate
can then be used as a substrate for a second microbial organism
that converts the p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway intermediate to
p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. The p-toluate,
terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway intermediate can be added directly to another culture of
the second organism or the original culture of the p-toluate,
terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway intermediate producers can be depleted of these microbial
organisms by, for example, cell separation, and then subsequent
addition of the second organism to the fermentation broth can be
utilized to produce the final product without intermediate
purification steps.
[0053] In other embodiments, the non-naturally occurring microbial
organisms and methods of the invention can be assembled in a wide
variety of subpathways to achieve biosynthesis of, for example,
p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. In these embodiments,
biosynthetic pathways for a desired product of the invention can be
segregated into different microbial organisms, and the different
microbial organisms can be co-cultured to produce the final
product. In such a biosynthetic scheme, the product of one
microbial organism is the substrate for a second microbial organism
until the final product is synthesized. For example, the
biosynthesis of p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate can be accomplished by
constructing a microbial organism that contains biosynthetic
pathways for conversion of one pathway intermediate to another
pathway intermediate or the product. Alternatively, p-toluate,
terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate also
can be biosynthetically produced from microbial organisms through
co-culture or co-fermentation using two organisms in the same
vessel, where the first microbial organism produces a p-toluate,
terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
intermediate and the second microbial organism converts the
intermediate to p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate.
[0054] Given the teachings and guidance provided herein, those
skilled in the art will understand that a wide variety of
combinations and permutations exist for the non-naturally occurring
microbial organisms and methods of the invention together with
other microbial organisms, with the co-culture of other
non-naturally occurring microbial organisms having subpathways and
with combinations of other chemical and/or biochemical procedures
well known in the art to produce p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate.
[0055] Sources of encoding nucleic acids for a p-toluate,
terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway enzyme or protein can include, for example, any species
where the encoded gene product is capable of catalyzing the
referenced reaction. Such species include both prokaryotic and
eukaryotic organisms including, but not limited to, bacteria,
including archaea and eubacteria, and eukaryotes, including yeast,
plant, insect, animal, and mammal, including human. Exemplary
species for such sources include, for example, Escherichia coli,
Mycobacterium tuberculosis, Agrobacterium tumefaciens, Bacillus
subtilis, Synechocystis species, Arabidopsis thaliana, Zymomonas
mobilis, Klebsiella oxytoca, Salmonella typhimurium, Salmonella
typhi, Lactobacullus collinoides, Klebsiella pneumoniae,
Clostridium pasteuranum, Citrobacter freundii, Clostridium
butyricum, Roseburia inulinivorans, Sulfolobus solfataricus,
Neurospora crassa, Sinorhizobium fredii, Helicobacter pylori,
Pyrococcus furiosus, Haemophilus influenzae, Erwinia chlysanthemi,
Staphylococcus aureus, Dunaliella saliva, Streptococcus pneumoniae,
Saccharomyces cerevisiae, Aspergillus nidulans, Pneumocystis
carinii, Streptomyces coelicolor, species from the genera
Burkholderia, Alcaligenes, Pseudomonas, Shingomonas and Comamonas,
for example, Comamonas testosteroni, as well as other exemplary
species disclosed herein or available as source organisms for
corresponding genes. However, with the complete genome sequence
available for now more than 550 species (with more than half of
these available on public databases such as the NCBI), including
395 microorganism genomes and a variety of yeast, fungi, plant, and
mammalian genomes, the identification of genes encoding the
requisite p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthetic activity
for one or more genes in related or distant species, including for
example, homologues, orthologs, paralogs and nonorthologous gene
displacements of known genes, and the interchange of genetic
alterations between organisms is routine and well known in the art.
Accordingly, the metabolic alterations allowing biosynthesis of
p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate described herein with
reference to a particular organism such as E. coli can be readily
applied to other microorganisms, including prokaryotic and
eukaryotic organisms alike. Given the teachings and guidance
provided herein, those skilled in the art will know that a
metabolic alteration exemplified in one organism can be applied
equally to other organisms.
[0056] In some instances, such as when an alternative p-toluate,
terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
biosynthetic pathway exists in an unrelated species, p-toluate,
terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
biosynthesis can be conferred onto the host species by, for
example, exogenous expression of a paralog or paralogs from the
unrelated species that catalyzes a similar, yet non-identical
metabolic reaction to replace the referenced reaction. Because
certain differences among metabolic networks exist between
different organisms, those skilled in the art will understand that
the actual gene usage between different organisms may differ.
However, given the teachings and guidance provided herein, those
skilled in the art also will understand that the teachings and
methods of the invention can be applied to all microbial organisms
using the cognate metabolic alterations to those exemplified herein
to construct a microbial organism in a species of interest that
will synthesize p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate.
[0057] Methods for constructing and testing the expression levels
of a non-naturally occurring p-toluate-, terephthalate- or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate-producing host can be
performed, for example, by recombinant and detection methods well
known in the art. Such methods can be found described in, for
example, Sambrook et al., Molecular Cloning: A Laboratory Manual,
Third Ed., Cold Spring Harbor Laboratory, New York (2001); and
Ausubel et al., Current Protocols in Molecular Biology, John Wiley
and Sons, Baltimore, Md. (1999).
[0058] Exogenous nucleic acid sequences involved in a pathway for
production of p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate can be introduced
stably or transiently into a host cell using techniques well known
in the art including, but not limited to, conjugation,
electroporation, chemical transformation, transduction,
transfection, and ultrasound transformation. For exogenous
expression in E. coli or other prokaryotic cells, some nucleic acid
sequences in the genes or cDNAs of eukaryotic nucleic acids can
encode targeting signals such as an N-terminal mitochondrial or
other targeting signal, which can be removed before transformation
into prokaryotic host cells, if desired. For example, removal of a
mitochondrial leader sequence led to increased expression in E.
coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). For
exogenous expression in yeast or other eukaryotic cells, genes can
be expressed in the cytosol without the addition of leader
sequence, or can be targeted to mitochondrion or other organelles,
or targeted for secretion, by the addition of a suitable targeting
sequence such as a mitochondrial targeting or secretion signal
suitable for the host cells. Thus, it is understood that
appropriate modifications to a nucleic acid sequence to remove or
include a targeting sequence can be incorporated into an exogenous
nucleic acid sequence to impart desirable properties. Furthermore,
genes can be subjected to codon optimization with techniques well
known in the art to achieve optimized expression of the
proteins.
[0059] An expression vector or vectors can be constructed to
include one or more p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthetic pathway
encoding nucleic acids as exemplified herein operably linked to
expression control sequences functional in the host organism.
Expression vectors applicable for use in the microbial host
organisms of the invention include, for example, plasmids, phage
vectors, viral vectors, episomes and artificial chromosomes,
including vectors and selection sequences or markers operable for
stable integration into a host chromosome. Additionally, the
expression vectors can include one or more selectable marker genes
and appropriate expression control sequences. Selectable marker
genes also can be included that, for example, provide resistance to
antibiotics or toxins, complement auxotrophic deficiencies, or
supply critical nutrients not in the culture media. Expression
control sequences can include constitutive and inducible promoters,
transcription enhancers, transcription terminators, and the like
which are well known in the art. When two or more exogenous
encoding nucleic acids are to be co-expressed, both nucleic acids
can be inserted, for example, into a single expression vector or in
separate expression vectors. For single vector expression, the
encoding nucleic acids can be operationally linked to one common
expression control sequence or linked to different expression
control sequences, such as one inducible promoter and one
constitutive promoter. The transformation of exogenous nucleic acid
sequences involved in a metabolic or synthetic pathway can be
confirmed using methods well known in the art. Such methods
include, for example, nucleic acid analysis such as Northern blots
or polymerase chain reaction (PCR) amplification of mRNA, or
immunoblotting for expression of gene products, or other suitable
analytical methods to test the expression of an introduced nucleic
acid sequence or its corresponding gene product. It is understood
by those skilled in the art that the exogenous nucleic acid is
expressed in a sufficient amount to produce the desired product,
and it is further understood that expression levels can be
optimized to obtain sufficient expression using methods well known
in the art and as disclosed herein.
[0060] The invention additionally provides a method for producing
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, comprising culturing
the non-naturally occurring microbial organism containing a
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway under
conditions and for a sufficient period of time to produce
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. Such a microbial
organism can have a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway comprising at least one exogenous nucleic acid encoding a
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme
expressed in a sufficient amount to produce
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, the
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprising
2-C-methyl-D-erythritol-4-phosphate dehydratase (see Example I and
FIG. 1, step C). A (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway can optionally further comprise 1-deoxyxylulose-5-phosphate
synthase and/or 1-deoxy-D-xylulose-5-phosphate reductoisomerase
(see Example I and FIG. 1, steps A and B).
[0061] In another embodiment, the invention provides a method for
producing p-toluate, comprising culturing the non-naturally
occurring microbial organism comprising a p-toluate pathway under
conditions and for a sufficient period of time to produce
p-toluate. A p-toluate pathway can comprise at least one exogenous
nucleic acid encoding a p-toluate pathway enzyme expressed in a
sufficient amount to produce p-toluate, the p-toluate pathway
comprising 2-dehydro-3-deoxyphosphoheptonate synthase;
3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate
dehydrogenase; shikimate kinase;
3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase;
and/or chorismate lyase (see Example II and FIG. 2, steps A-H). In
another embodiment, a method of the invention can utilize a
non-naturally occurring microbial organism that further comprises a
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway (see Example I
and FIG. 1). Such a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathway can comprise 2-C-methyl-D-erythritol-4-phosphate
dehydratase, 1-deoxyxylulose-5-phosphate synthase and/or
1-deoxy-D-xylulose-5-phosphate reductoisomerase (see Example I and
FIG. 1).
[0062] The invention further provides a method for producing
terephthalate, comprising culturing a non-naturally occurring
microbial organism containing a terephthalate pathway under
conditions and for a sufficient period of time to produce
terephthalate. Such a terephthalate pathway can comprise at least
one exogenous nucleic acid encoding a terephthalate pathway enzyme
expressed in a sufficient amount to produce terephthalate, the
terephthalate pathway comprising p-toluate methyl-monooxygenase
reductase; 4-carboxybenzyl alcohol dehydrogenase; and/or
4-carboxybenzyl aldehyde dehydrogenase. Such a microbial organism
can further comprise a p-toluate pathway, wherein the p-toluate
pathway comprises 2-dehydro-3-deoxyphosphoheptonate synthase;
3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate
dehydrogenase; shikimate kinase;
3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase;
and/or chorismate lyase (see Examples 2 and 3 and FIGS. 2 and 3).
In another embodiment, the non-naturally occurring microbial
organism can further comprise a
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway (see Example I
and FIG. 1). Thus, in a particular embodiment, the invention
provides a non-naturally occurring microbial organism and methods
of use, in which the microbial organism contains p-toluate,
terephthalate and (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
pathways.
[0063] Suitable purification and/or assays to test for the
production of p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate can be performed using
well known methods. Suitable replicates such as triplicate cultures
can be grown for each engineered strain to be tested. For example,
product and byproduct formation in the engineered production host
can be monitored. The final product and intermediates, and other
organic compounds, can be analyzed by methods such as HPLC (High
Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass
Spectroscopy), LC-MS (Liquid Chromatography-Mass Spectroscopy), and
UV-visible spectroscopy or other suitable analytical methods using
routine procedures well known in the art. The release of product in
the fermentation broth can also be tested with the culture
supernatant. Byproducts and residual glucose can be quantified by
HPLC using, for example, a refractive index detector for glucose
and alcohols, and a UV detector for organic acids (Lin et al.,
Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay and
detection methods well known in the art. The individual enzyme or
protein activities from the exogenous DNA sequences can also be
assayed using methods well known in the art. For example, p-toluate
methyl-monooxygenase activity can be assayed by incubating purified
enzyme with NADH, FeSO.sub.4 and the p-toluate substrate in a water
bath, stopping the reaction by precipitation of the proteins, and
analysis of the products in the supernatant by HPLC (Locher et al.,
J. Bacteriol. 173:3741-3748 (1991)).
[0064] The p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate can be separated from
other components in the culture using a variety of methods well
known in the art. Such separation methods include, for example,
extraction procedures as well as methods that include continuous
liquid-liquid extraction, pervaporation, membrane filtration,
membrane separation, reverse osmosis, electrodialysis,
distillation, crystallization, centrifugation, extractive
filtration, ion exchange chromatography, size exclusion
chromatography, adsorption chromatography, and ultrafiltration. All
of the above methods are well known in the art.
[0065] Any of the non-naturally occurring microbial organisms
described herein can be cultured to produce and/or secrete the
biosynthetic products of the invention. For example, the p-toluate,
terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
producers can be cultured for the biosynthetic production of
p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate.
[0066] For the production of p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, the recombinant
strains are cultured in a medium with carbon source and other
essential nutrients. It is sometimes desirable to maintain
anaerobic conditions in the fermenter to reduce the cost of the
overall process. Such conditions can be obtained, for example, by
first sparging the medium with nitrogen and then sealing the flasks
with a septum and crimp-cap. For strains where growth is not
observed anaerobically, microaerobic conditions can be applied by
perforating the septum with a small hole for limited aeration.
Exemplary anaerobic conditions have been described previously and
are well-known in the art. Exemplary aerobic and anaerobic
conditions are described, for example, in United State publication
2009/0047719, filed Aug. 10, 2007. Fermentations can be performed
in a batch, fed-batch or continuous manner, as disclosed
herein.
[0067] If desired, the pH of the medium can be maintained at a
desired pH, in particular neutral pH, such as a pH of around 7 by
addition of a base, such as NaOH or other bases, or acid, as needed
to maintain the culture medium at a desirable pH. The growth rate
can be determined by measuring optical density using a
spectrophotometer (600 nm), and the glucose uptake rate by
monitoring carbon source depletion over time.
[0068] The growth medium can include, for example, any carbohydrate
source which can supply a source of carbon to the non-naturally
occurring microorganism. Such sources include, for example, sugars
such as glucose, xylose, arabinose, galactose, mannose, fructose,
sucrose and starch. Other sources of carbohydrate include, for
example, renewable feedstocks and biomass. Exemplary types of
biomasses that can be used as feedstocks in the methods of the
invention include cellulosic biomass, hemicellulosic biomass and
lignin feedstocks or portions of feedstocks. Such biomass
feedstocks contain, for example, carbohydrate substrates useful as
carbon sources such as glucose, xylose, arabinose, galactose,
mannose, fructose and starch. Given the teachings and guidance
provided herein, those skilled in the art will understand that
renewable feedstocks and biomass other than those exemplified above
also can be used for culturing the microbial organisms of the
invention for the production of p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate.
[0069] In addition to renewable feedstocks such as those
exemplified above, the p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate microbial organisms of
the invention also can be modified for growth on syngas as its
source of carbon. In this specific embodiment, one or more proteins
or enzymes are expressed in the p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate producing organisms to
provide a metabolic pathway for utilization of syngas or other
gaseous carbon source.
[0070] Synthesis gas, also known as syngas or producer gas, is the
major product of gasification of coal and of carbonaceous materials
such as biomass materials, including agricultural crops and
residues. Syngas is a mixture primarily of H.sub.2 and CO and can
be obtained from the gasification of any organic feedstock,
including but not limited to coal, coal oil, natural gas, biomass,
and waste organic matter. Gasification is generally carried out
under a high fuel to oxygen ratio. Although largely H.sub.2 and CO,
syngas can also include CO.sub.2 and other gases in smaller
quantities. Thus, synthesis gas provides a cost effective source of
gaseous carbon such as CO and, additionally, CO.sub.2.
[0071] The Wood-Ljungdahl pathway catalyzes the conversion of CO
and H.sub.2 to acetyl-CoA and other products such as acetate.
Organisms capable of utilizing CO and syngas also generally have
the capability of utilizing CO.sub.2 and CO.sub.2/H.sub.2 mixtures
through the same basic set of enzymes and transformations
encompassed by the Wood-Ljungdahl pathway. H.sub.2-dependent
conversion of CO.sub.2 to acetate by microorganisms was recognized
long before it was revealed that CO also could be used by the same
organisms and that the same pathways were involved. Many acetogens
have been shown to grow in the presence of CO.sub.2 and produce
compounds such as acetate as long as hydrogen is present to supply
the necessary reducing equivalents (see for example, Drake,
Acetogenesis, pp. 3-60 Chapman and Hall, New York, (1994)). This
can be summarized by the following equation:
2CO.sub.2+4H.sub.2+n ADP+n Pi.fwdarw.CH.sub.3COOH+2H.sub.2O+n
ATP
Hence, non-naturally occurring microorganisms possessing the
Wood-Ljungdahl pathway can utilize CO.sub.2 and H.sub.2 mixtures as
well for the production of acetyl-CoA and other desired
products.
[0072] The Wood-Ljungdahl pathway is well known in the art and
consists of 12 reactions which can be separated into two branches:
(1) methyl branch and (2) carbonyl branch. The methyl branch
converts syngas to methyl-tetrahydrofolate (methyl-THF) whereas the
carbonyl branch converts methyl-THF to acetyl-CoA. The reactions in
the methyl branch are catalyzed in order by the following enzymes
or proteins: ferredoxin oxidoreductase, formate dehydrogenase,
formyltetrahydrofolate synthetase, methenyltetrahydrofolate
cyclodehydratase, methylenetetrahydrofolate dehydrogenase and
methylenetetrahydrofolate reductase. The reactions in the carbonyl
branch are catalyzed in order by the following enzymes or proteins:
methyltetrahydrofolate:corrinoid protein methyltransferase (for
example, AcsE), corrinoid iron-sulfur protein, nickel-protein
assembly protein (for example, AcsF), ferredoxin, acetyl-CoA
synthase, carbon monoxide dehydrogenase and nickel-protein assembly
protein (for example, CooC). Following the teachings and guidance
provided herein for introducing a sufficient number of encoding
nucleic acids to generate a p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, those skilled
in the art will understand that the same engineering design also
can be performed with respect to introducing at least the nucleic
acids encoding the Wood-Ljungdahl enzymes or proteins absent in the
host organism. Therefore, introduction of one or more encoding
nucleic acids into the microbial organisms of the invention such
that the modified organism contains the complete Wood-Ljungdahl
pathway will confer syngas utilization ability.
[0073] The reductive tricarboxylic acid cycle coupled with carbon
monoxide dehydrogenase and/or hydrogenase activities can also allow
the conversion of CO, CO.sub.2 and/or H.sub.2 to acetyl-CoA and
other products such as acetate. Organisms capable of fixing carbon
via the reductive TCA pathway can utilize one or more of the
following enzymes: ATP citrate-lyase, citrate lyase, aconitase,
isocitrate dehydrogenase, alpha-ketoglutarate:ferredoxin
oxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase,
fumarate reductase, fumarase, malate dehydrogenase,
NAD(P)H:ferredoxin oxidoreductase, carbon monoxide dehydrogenase,
and hydrogenase. Specifically, the reducing equivalents extracted
from CO and/or H.sub.2 by carbon monoxide dehydrogenase and
hydrogenase are utilized to fix CO.sub.2 via the reductive TCA
cycle into acetyl-CoA or acetate. Acetate can be converted to
acetyl-CoA by enzymes such as acetyl-CoA transferase, acetate
kinase/phosphotransacetylase, and acetyl-CoA synthetase. Acetyl-CoA
can be converted to the p-toluate, terepathalate, or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate precursors,
glyceraldehyde-3-phosphate, phosphoenolpyruvate, and pyruvate, by
pyruvate:ferredoxin oxidoreductase and the enzymes of
gluconeogenesis. Following the teachings and guidance provided
herein for introducing a sufficient number of encoding nucleic
acids to generate a p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, those skilled
in the art will understand that a similar engineering design also
can be performed with respect to introducing at least the nucleic
acids encoding the reductive TCA pathway enzymes or proteins absent
in the host organism. Therefore, introduction of one or more
encoding nucleic acids into the microbial organisms of the
invention such that the modified organism contains the complete
reductive TCA pathway will confer syngas utilization ability.
[0074] Accordingly, given the teachings and guidance provided
herein, those skilled in the art will understand that a
non-naturally occurring microbial organism can be produced that
secretes the biosynthesized compounds of the invention when grown
on a carbon source such as a carbohydrate. Such compounds include,
for example, p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate and any of the
intermediate metabolites in the p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway. All that is
required is to engineer in one or more of the required enzyme or
protein activities to achieve biosynthesis of the desired compound
or intermediate including, for example, inclusion of some or all of
the p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate biosynthetic pathways.
Accordingly, the invention provides a non-naturally occurring
microbial organism that produces and/or secretes p-toluate,
terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate when
grown on a carbohydrate or other carbon source and produces and/or
secretes any of the intermediate metabolites shown in the
p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway when grown on a
carbohydrate or other carbon source. The p-toluate, terephthalate
or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate producing microbial
organisms of the invention can initiate synthesis from an
intermediate. For example, a
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway intermediate
can be 1-deoxy-D-xylulose-5-phosphate or
C-methyl-D-erythritol-4-phosphate (see Example I and FIG. 1). A
p-toluate pathway intermediate can be, for example,
2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate,
1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate,
5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylate,
3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate,
5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate,
5-[(1-carboxyeth-1-en-1-yl)oxy]-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-
-carboxylate, or
3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate
(see Example II and FIG. 2). A terephthalate intermediate can be,
for example, 4-carboxybenzyl alcohol or 4-carboxybenzaldehyde (see
Example III and FIG. 3).
[0075] The non-naturally occurring microbial organisms of the
invention are constructed using methods well known in the art as
exemplified herein to exogenously express at least one nucleic acid
encoding a p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme or
protein in sufficient amounts to produce p-toluate, terephthalate
or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. It is understood
that the microbial organisms of the invention are cultured under
conditions sufficient to produce p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. Following the
teachings and guidance provided herein, the non-naturally occurring
microbial organisms of the invention can achieve biosynthesis of
p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate resulting in
intracellular concentrations between about 0.1-200 mM or more.
Generally, the intracellular concentration of p-toluate,
terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate is
between about 3-150 mM, particularly between about 5-125 mM and
more particularly between about 8-100 mM, including about 10 mM, 20
mM, 50 mM, 80 mM, or more. Intracellular concentrations between and
above each of these exemplary ranges also can be achieved from the
non-naturally occurring microbial organisms of the invention.
[0076] In some embodiments, culture conditions include anaerobic or
substantially anaerobic growth or maintenance conditions. Exemplary
anaerobic conditions have been described previously and are well
known in the art. Exemplary anaerobic conditions for fermentation
processes are described herein and are described, for example, in
U.S. publication 2009/0047719, filed Aug. 10, 2007. Any of these
conditions can be employed with the non-naturally occurring
microbial organisms as well as other anaerobic conditions well
known in the art. The p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate producers can
synthesize p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate at intracellular
concentrations of 5-10 mM or more as well as all other
concentrations exemplified herein under substantially anaerobic
conditions. It is understood that, even though the above
description refers to intracellular concentrations, p-toluate,
terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
producing microbial organisms can produce p-toluate, terephthalate
or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate intracellularly
and/or secrete the product into the culture medium.
[0077] In addition to the culturing and fermentation conditions
disclosed herein, growth conditions for achieving biosynthesis of
p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate can include the
addition of an osmoprotectant to the culturing conditions. In
certain embodiments, the non-naturally occurring microbial
organisms of the invention can be sustained, cultured or fermented
as described herein in the presence of an osmoprotectant. Briefly,
an osmoprotectant refers to a compound that acts as an osmolyte and
helps a microbial organism as described herein survive osmotic
stress. Osmoprotectants include, but are not limited to, betaines,
amino acids, and the sugar trehalose. Non-limiting examples of such
are glycine betaine, praline betaine, dimethylthetin,
dimethylslfonioproprionate, 3-dimethylsulfonio-2-methylproprionate,
pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine and
ectoine. In one aspect, the osmoprotectant is glycine betaine. It
is understood to one of ordinary skill in the art that the amount
and type of osmoprotectant suitable for protecting a microbial
organism described herein from osmotic stress will depend on the
microbial organism used. The amount of osmoprotectant in the
culturing conditions can be, for example, no more than about 0.1
mM, no more than about 0.5 mM, no more than about 1.0 mM, no more
than about 1.5 mM, no more than about 2.0 mM, no more than about
2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no
more than about 7.0 mM, no more than about 10 mM, no more than
about 50 mM, no more than about 100 mM or no more than about 500
mM.
[0078] The culture conditions can include, for example, liquid
culture procedures as well as fermentation and other large scale
culture procedures. As described herein, particularly useful yields
of the biosynthetic products of the invention can be obtained under
anaerobic or substantially anaerobic culture conditions.
[0079] As described herein, one exemplary growth condition for
achieving biosynthesis of p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate includes anaerobic
culture or fermentation conditions. In certain embodiments, the
non-naturally occurring microbial organisms of the invention can be
sustained, cultured or fermented under anaerobic or substantially
anaerobic conditions. Briefly, anaerobic conditions refers to an
environment devoid of oxygen. Substantially anaerobic conditions
include, for example, a culture, batch fermentation or continuous
fermentation such that the dissolved oxygen concentration in the
medium remains between 0 and 10% of saturation. Substantially
anaerobic conditions also includes growing or resting cells in
liquid medium or on solid agar inside a sealed chamber maintained
with an atmosphere of less than 1% oxygen. The percent of oxygen
can be maintained by, for example, sparging the culture with an
N.sub.2/CO.sub.2 mixture or other suitable non-oxygen gas or
gases.
[0080] The culture conditions described herein can be scaled up and
grown continuously for manufacturing of p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. Exemplary growth
procedures include, for example, fed-batch fermentation and batch
separation; fed-batch fermentation and continuous separation, or
continuous fermentation and continuous separation. All of these
processes are well known in the art. Fermentation procedures are
particularly useful for the biosynthetic production of commercial
quantities of p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. Generally, and as with
non-continuous culture procedures, the continuous and/or
near-continuous production of p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate will include culturing
a non-naturally occurring p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate producing organism of
the invention in sufficient nutrients and medium to sustain and/or
nearly sustain growth in an exponential phase. Continuous culture
under such conditions can be include, for example, growth for 1
day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous
culture can include longer time periods of 1 week, 2, 3, 4 or 5 or
more weeks and up to several months. Alternatively, organisms of
the invention can be cultured for hours, if suitable for a
particular application. It is to be understood that the continuous
and/or near-continuous culture conditions also can include all time
intervals in between these exemplary periods. It is further
understood that the time of culturing the microbial organism of the
invention is for a sufficient period of time to produce a
sufficient amount of product for a desired purpose.
[0081] Fermentation procedures are well known in the art. Briefly,
fermentation for the biosynthetic production of p-toluate,
terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate can be
utilized in, for example, fed-batch fermentation and batch
separation; fed-batch fermentation and continuous separation, or
continuous fermentation and continuous separation. Examples of
batch and continuous fermentation procedures are well known in the
art.
[0082] In addition to the above fermentation procedures using the
p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate producers of the
invention for continuous production of substantial quantities of
p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, the p-toluate,
terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
producers also can be, for example, simultaneously subjected to
chemical synthesis procedures to convert the product to other
compounds or the product can be separated from the fermentation
culture and sequentially subjected to chemical conversion to
convert the product to other compounds, if desired.
[0083] To generate better producers, metabolic modeling can be
utilized to optimize growth conditions. Modeling can also be used
to design gene knockouts that additionally optimize utilization of
the pathway (see, for example, U.S. patent publications US
2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US
2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Pat.
No. 7,127,379). Modeling analysis allows reliable predictions of
the effects on cell growth of shifting the metabolism towards more
efficient production of p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate.
[0084] One computational method for identifying and designing
metabolic alterations favoring biosynthesis of a desired product is
the OptKnock computational framework (Burgard et al., Biotechnol.
Bioeng. 84:647-657 (2003)). OptKnock is a metabolic modeling and
simulation program that suggests gene deletion or disruption
strategies that result in genetically stable microorganisms which
overproduce the target product. Specifically, the framework
examines the complete metabolic and/or biochemical network of a
microorganism in order to suggest genetic manipulations that force
the desired biochemical to become an obligatory byproduct of cell
growth. By coupling biochemical production with cell growth through
strategically placed gene deletions or other functional gene
disruption, the growth selection pressures imposed on the
engineered strains after long periods of time in a bioreactor lead
to improvements in performance as a result of the compulsory
growth-coupled biochemical production. Lastly, when gene deletions
are constructed there is a negligible possibility of the designed
strains reverting to their wild-type states because the genes
selected by OptKnock are to be completely removed from the genome.
Therefore, this computational methodology can be used to either
identify alternative pathways that lead to biosynthesis of a
desired product or used in connection with the non-naturally
occurring microbial organisms for further optimization of
biosynthesis of a desired product.
[0085] Briefly, OptKnock is a term used herein to refer to a
computational method and system for modeling cellular metabolism.
The OptKnock program relates to a framework of models and methods
that incorporate particular constraints into flux balance analysis
(FBA) models. These constraints include, for example, qualitative
kinetic information, qualitative regulatory information, and/or DNA
microarray experimental data. OptKnock also computes solutions to
various metabolic problems by, for example, tightening the flux
boundaries derived through flux balance models and subsequently
probing the performance limits of metabolic networks in the
presence of gene additions or deletions. OptKnock computational
framework allows the construction of model formulations that allow
an effective query of the performance limits of metabolic networks
and provides methods for solving the resulting mixed-integer linear
programming problems. The metabolic modeling and simulation methods
referred to herein as OptKnock are described in, for example, U.S.
publication 2002/0168654, filed Jan. 10, 2002, in International
Patent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S.
publication 2009/0047719, filed Aug. 10, 2007.
[0086] Another computational method for identifying and designing
metabolic alterations favoring biosynthetic production of a product
is a metabolic modeling and simulation system termed SimPheny.RTM..
This computational method and system is described in, for example,
U.S. publication 2003/0233218, filed Jun. 14, 2002, and in
International Patent Application No. PCT/US03/18838, filed Jun. 13,
2003. SimPheny.RTM. is a computational system that can be used to
produce a network model in silico and to simulate the flux of mass,
energy or charge through the chemical reactions of a biological
system to define a solution space that contains any and all
possible functionalities of the chemical reactions in the system,
thereby determining a range of allowed activities for the
biological system. This approach is referred to as
constraints-based modeling because the solution space is defined by
constraints such as the known stoichiometry of the included
reactions as well as reaction thermodynamic and capacity
constraints associated with maximum fluxes through reactions. The
space defined by these constraints can be interrogated to determine
the phenotypic capabilities and behavior of the biological system
or of its biochemical components.
[0087] These computational approaches are consistent with
biological realities because biological systems are flexible and
can reach the same result in many different ways. Biological
systems are designed through evolutionary mechanisms that have been
restricted by fundamental constraints that all living systems must
face. Therefore, constraints-based modeling strategy embraces these
general realities. Further, the ability to continuously impose
further restrictions on a network model via the tightening of
constraints results in a reduction in the size of the solution
space, thereby enhancing the precision with which physiological
performance or phenotype can be predicted.
[0088] Given the teachings and guidance provided herein, those
skilled in the art will be able to apply various computational
frameworks for metabolic modeling and simulation to design and
implement biosynthesis of a desired compound in host microbial
organisms. Such metabolic modeling and simulation methods include,
for example, the computational systems exemplified above as
SimPheny.RTM. and OptKnock. For illustration of the invention, some
methods are described herein with reference to the OptKnock
computation framework for modeling and simulation. Those skilled in
the art will know how to apply the identification, design and
implementation of the metabolic alterations using OptKnock to any
of such other metabolic modeling and simulation computational
frameworks and methods well known in the art.
[0089] The methods described above will provide one set of
metabolic reactions to disrupt. Elimination of each reaction within
the set or metabolic modification can result in a desired product
as an obligatory product during the growth phase of the organism.
Because the reactions are known, a solution to the bilevel OptKnock
problem also will provide the associated gene or genes encoding one
or more enzymes that catalyze each reaction within the set of
reactions. Identification of a set of reactions and their
corresponding genes encoding the enzymes participating in each
reaction is generally an automated process, accomplished through
correlation of the reactions with a reaction database having a
relationship between enzymes and encoding genes.
[0090] Once identified, the set of reactions that are to be
disrupted in order to achieve production of a desired product are
implemented in the target cell or organism by functional disruption
of at least one gene encoding each metabolic reaction within the
set. One particularly useful means to achieve functional disruption
of the reaction set is by deletion of each encoding gene. However,
in some instances, it can be beneficial to disrupt the reaction by
other genetic aberrations including, for example, mutation,
deletion of regulatory regions such as promoters or cis binding
sites for regulatory factors, or by truncation of the coding
sequence at any of a number of locations. These latter aberrations,
resulting in less than total deletion of the gene set can be
useful, for example, when rapid assessments of the coupling of a
product are desired or when genetic reversion is less likely to
occur.
[0091] To identify additional productive solutions to the above
described bilevel OptKnock problem which lead to further sets of
reactions to disrupt or metabolic modifications that can result in
the biosynthesis, including growth-coupled biosynthesis of a
desired product, an optimization method, termed integer cuts, can
be implemented. This method proceeds by iteratively solving the
OptKnock problem exemplified above with the incorporation of an
additional constraint referred to as an integer cut at each
iteration. Integer cut constraints effectively prevent the solution
procedure from choosing the exact same set of reactions identified
in any previous iteration that obligatorily couples product
biosynthesis to growth. For example, if a previously identified
growth-coupled metabolic modification specifies reactions 1, 2, and
3 for disruption, then the following constraint prevents the same
reactions from being simultaneously considered in subsequent
solutions. The integer cut method is well known in the art and can
be found described in, for example, Burgard et al., Biotechnol.
Prog. 17:791-797 (2001). As with all methods described herein with
reference to their use in combination with the OptKnock
computational framework for metabolic modeling and simulation, the
integer cut method of reducing redundancy in iterative
computational analysis also can be applied with other computational
frameworks well known in the art including, for example,
SimPheny.RTM..
[0092] The methods exemplified herein allow the construction of
cells and organisms that biosynthetically produce a desired
product, including the obligatory coupling of production of a
target biochemical product to growth of the cell or organism
engineered to harbor the identified genetic alterations. Therefore,
the computational methods described herein allow the identification
and implementation of metabolic modifications that are identified
by an in silico method selected from OptKnock or SimPheny.RTM.. The
set of metabolic modifications can include, for example, addition
of one or more biosynthetic pathway enzymes and/or functional
disruption of one or more metabolic reactions including, for
example, disruption by gene deletion.
[0093] As discussed above, the OptKnock methodology was developed
on the premise that mutant microbial networks can be evolved
towards their computationally predicted maximum-growth phenotypes
when subjected to long periods of growth selection. In other words,
the approach leverages an organism's ability to self-optimize under
selective pressures. The OptKnock framework allows for the
exhaustive enumeration of gene deletion combinations that force a
coupling between biochemical production and cell growth based on
network stoichiometry. The identification of optimal gene/reaction
knockouts requires the solution of a bilevel optimization problem
that chooses the set of active reactions such that an optimal
growth solution for the resulting network overproduces the
biochemical of interest (Burgard et al., Biotechnol. Bioeng.
84:647-657 (2003)).
[0094] An in silico stoichiometric model of E. coli metabolism can
be employed to identify essential genes for metabolic pathways as
exemplified previously and described in, for example, U.S. patent
publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US
2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,
and in U.S. Pat. No. 7,127,379. As disclosed herein, the OptKnock
mathematical framework can be applied to pinpoint gene deletions
leading to the growth-coupled production of a desired product.
Further, the solution of the bilevel OptKnock problem provides only
one set of deletions. To enumerate all meaningful solutions, that
is, all sets of knockouts leading to growth-coupled production
formation, an optimization technique, termed integer cuts, can be
implemented. This entails iteratively solving the OptKnock problem
with the incorporation of an additional constraint referred to as
an integer cut at each iteration, as discussed above.
[0095] As disclosed herein, a nucleic acid encoding a desired
activity of a p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway can be
introduced into a host organism. In some cases, it can be desirable
to modify an activity of a p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme or
protein to increase production of p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. For example, known
mutations that increase the activity of a protein or enzyme can be
introduced into an encoding nucleic acid molecule. Additionally,
optimization methods can be applied to increase the activity of an
enzyme or protein and/or decrease an inhibitory activity, for
example, decrease the activity of a negative regulator.
[0096] One such optimization method is directed evolution. Directed
evolution is a powerful approach that involves the introduction of
mutations targeted to a specific gene in order to improve and/or
alter the properties of an enzyme. Improved and/or altered enzymes
can be identified through the development and implementation of
sensitive high-throughput screening assays that allow the automated
screening of many enzyme variants (for example, >10.sup.4).
Iterative rounds of mutagenesis and screening typically are
performed to afford an enzyme with optimized properties.
Computational algorithms that can help to identify areas of the
gene for mutagenesis also have been developed and can significantly
reduce the number of enzyme variants that need to be generated and
screened. Numerous directed evolution technologies have been
developed (for reviews, see Hibbert et al., Biomol. Eng 22:11-19
(2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical
and biotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC
Press; Otten and Quax. Biomol. Eng 22:1-9 (2005).; and Sen et al.,
Appl Biochem. Biotechnol 143:212-223 (2007)) to be effective at
creating diverse variant libraries, and these methods have been
successfully applied to the improvement of a wide range of
properties across many enzyme classes. Enzyme characteristics that
have been improved and/or altered by directed evolution
technologies include, for example: selectivity/specificity, for
conversion of non-natural substrates; temperature stability, for
robust high temperature processing; pH stability, for bioprocessing
under lower or higher pH conditions; substrate or product
tolerance, so that high product titers can be achieved; binding
(K.sub.m), including broadening substrate binding to include
non-natural substrates; inhibition (K.sub.i), to remove inhibition
by products, substrates, or key intermediates; activity (kcat), to
increases enzymatic reaction rates to achieve desired flux;
expression levels, to increase protein yields and overall pathway
flux; oxygen stability, for operation of air sensitive enzymes
under aerobic conditions; and anaerobic activity, for operation of
an aerobic enzyme in the absence of oxygen.
[0097] A number of exemplary methods have been developed for the
mutagenesis and diversification of genes to target desired
properties of specific enzymes. Such methods are well known to
those skilled in the art. Any of these can be used to alter and/or
optimize the activity of a p-toluate, terephthalate or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme or
protein. Such methods include, but are not limited to EpPCR, which
introduces random point mutations by reducing the fidelity of DNA
polymerase in PCR reactions (Pritchard et al., J Theor. Biol.
234:497-509 (2005)); Error-prone Rolling Circle Amplification
(epRCA), which is similar to epPCR except a whole circular plasmid
is used as the template and random 6-mers with exonuclease
resistant thiophosphate linkages on the last 2 nucleotides are used
to amplify the plasmid followed by transformation into cells in
which the plasmid is re-circularized at tandem repeats (Fujii et
al., Nucleic Acids Res. 32:e145 (2004); and Fujii et al., Nat.
Protoc. 1:2493-2497 (2006)); DNA or Family Shuffling, which
typically involves digestion of two or more variant genes with
nucleases such as Dnase I or EndoV to generate a pool of random
fragments that are reassembled by cycles of annealing and extension
in the presence of DNA polymerase to create a library of chimeric
genes (Stemmer, Proc Natl Acad Sci USA 91:10747-10751 (1994); and
Stemmer, Nature 370:389-391 (1994)); Staggered Extension (StEP),
which entails template priming followed by repeated cycles of 2
step PCR with denaturation and very short duration of
annealing/extension (as short as 5 sec) (Zhao et al., Nat.
Biotechnol. 16:258-261 (1998)); Random Priming Recombination (RPR),
in which random sequence primers are used to generate many short
DNA fragments complementary to different segments of the template
(Shao et al., Nucleic Acids Res 26:681-683 (1998)).
[0098] Additional methods include Heteroduplex Recombination, in
which linearized plasmid DNA is used to form heteroduplexes that
are repaired by mismatch repair (Volkov et al, Nucleic Acids Res.
27:el 8 (1999); and Volkov et al., Methods Enzymol. 328:456-463
(2000)); Random Chimeragenesis on Transient Templates (RACHITT),
which employs Dnase I fragmentation and size fractionation of
single stranded DNA (ssDNA) (Coco et al., Nat. Biotechnol.
19:354-359 (2001)); Recombined Extension on Truncated templates
(RETT), which entails template switching of unidirectionally
growing strands from primers in the presence of unidirectional
ssDNA fragments used as a pool of templates (Lee et al., J. Molec.
Catalysis 26:119-129 (2003)); Degenerate Oligonucleotide Gene
Shuffling (DOGS), in which degenerate primers are used to control
recombination between molecules; (Bergquist and Gibbs, Methods Mol.
Biol 352:191-204 (2007); Bergquist et al., Biomol. Eng 22:63-72
(2005); Gibbs et al., Gene 271:13-20 (2001)); Incremental
Truncation for the Creation of Hybrid Enzymes (ITCHY), which
creates a combinatorial library with 1 base pair deletions of a
gene or gene fragment of interest (Ostermeier et al., Proc. Natl.
Acad. Sci. USA 96:3562-3567 (1999); and Ostermeier et al., Nat.
Biotechnol. 17:1205-1209 (1999)); Thio-Incremental Truncation for
the Creation of Hybrid Enzymes (THIO-ITCHY), which is similar to
ITCHY except that phosphothioate dNTPs are used to generate
truncations (Lutz et al., Nucleic Acids Res 29:E16 (2001));
SCRATCHY, which combines two methods for recombining genes, ITCHY
and DNA shuffling (Lutz et al., Proc. Natl. Acad. Sci. USA
98:11248-11253 (2001)); Random Drift Mutagenesis (RNDM), in which
mutations made via epPCR are followed by screening/selection for
those retaining usable activity (Bergquist et al., Biomol. Eng.
22:63-72 (2005)); Sequence Saturation Mutagenesis (SeSaM), a random
mutagenesis method that generates a pool of random length fragments
using random incorporation of a phosphothioate nucleotide and
cleavage, which is used as a template to extend in the presence of
"universal" bases such as inosine, and replication of an
inosine-containing complement gives random base incorporation and,
consequently, mutagenesis (Wong et al., Biotechnol. J. 3:74-82
(2008); Wong et al., Nucleic Acids Res. 32:e26 (2004); and Wong et
al., Anal. Biochem. 341:187-189 (2005)); Synthetic Shuffling, which
uses overlapping oligonucleotides designed to encode "all genetic
diversity in targets" and allows a very high diversity for the
shuffled progeny (Ness et al., Nat. Biotechnol. 20:1251-1255
(2002)); Nucleotide Exchange and Excision Technology NexT, which
exploits a combination of dUTP incorporation followed by treatment
with uracil DNA glycosylase and then piperidine to perform endpoint
DNA fragmentation (Muller et al., Nucleic Acids Res. 33:e117
(2005)).
[0099] Further methods include Sequence Homology-Independent
Protein Recombination (SHIPREC), in which a linker is used to
facilitate fusion between two distantly related or unrelated genes,
and a range of chimeras is generated between the two genes,
resulting in libraries of single-crossover hybrids (Sieber et al.,
Nat. Biotechnol. 19:456-460 (2001)); Gene Site Saturation
Mutagenesis.TM. (GSSM.TM.), in which the starting materials include
a supercoiled double stranded DNA (dsDNA) plasmid containing an
insert and two primers which are degenerate at the desired site of
mutations (Kretz et al., Methods Enzymol. 388:3-11 (2004));
Combinatorial Cassette Mutagenesis (CCM), which involves the use of
short oligonucleotide cassettes to replace limited regions with a
large number of possible amino acid sequence alterations
(Reidhaar-Olson et al. Methods Enzymol. 208:564-586 (1991); and
Reidhaar-Olson et al. Science 241:53-57 (1988)); Combinatorial
Multiple Cassette Mutagenesis (CMCM), which is essentially similar
to CCM and uses epPCR at high mutation rate to identify hot spots
and hot regions and then extension by CMCM to cover a defined
region of protein sequence space (Reetz et al., Angew. Chem. Int.
Ed Engl. 40:3589-3591 (2001)); the Mutator Strains technique, in
which conditional is mutator plasmids, utilizing the mutD5 gene,
which encodes a mutant subunit of DNA polymerase III, to allow
increases of 20 to 4000-X in random and natural mutation frequency
during selection and block accumulation of deleterious mutations
when selection is not required (Selifonova et al., Appl. Environ.
Microbiol. 67:3645-3649 (2001)); Low et al., J. Mol. Biol.
260:359-3680 (1996)).
[0100] Additional exemplary methods include Look-Through
Mutagenesis (LTM), which is a multidimensional mutagenesis method
that assesses and optimizes combinatorial mutations of selected
amino acids (Rajpal et al., Proc. Natl. Acad. Sci. USA
102:8466-8471 (2005)); Gene Reassembly, which is a DNA shuffling
method that can be applied to multiple genes at one time or to
create a large library of chimeras (multiple mutations) of a single
gene (Tunable GeneReassembly.TM. (TGR.TM.) Technology supplied by
Verenium Corporation), in Silico Protein Design Automation (PDA),
which is an optimization algorithm that anchors the structurally
defined protein backbone possessing a particular fold, and searches
sequence space for amino acid substitutions that can stabilize the
fold and overall protein energetics, and generally works most
effectively on proteins with known three-dimensional structures
(Hayes et al., Proc. Natl. Acad. Sci. USA 99:15926-15931 (2002));
and Iterative Saturation Mutagenesis (ISM), which involves using
knowledge of structure/function to choose a likely site for enzyme
improvement, performing saturation mutagenesis at chosen site using
a mutagenesis method such as Stratagene QuikChange (Stratagene; San
Diego Calif.), screening/selecting for desired properties, and,
using improved clone(s), starting over at another site and continue
repeating until a desired activity is achieved (Reetz et al., Nat.
Protoc. 2:891-903 (2007); and Reetz et al., Angew. Chem. Int. Ed
Engl. 45:7745-7751 (2006)).
[0101] Any of the aforementioned methods for mutagenesis can be
used alone or in any combination. Additionally, any one or
combination of the directed evolution methods can be used in
conjunction with adaptive evolution techniques, as described
herein.
[0102] It is understood that modifications which do not
substantially affect the activity of the various embodiments of
this invention are also provided within the definition of the
invention provided herein. Accordingly, the following examples are
intended to illustrate but not limit the present invention.
EXAMPLE I
Exemplary Pathway for Producing
(2-Hydroxy-3-methyl-4-oxobutoxy)phosphonate
[0103] This example describes an exemplary pathway for producing
the terephthalic acid (PTA) precursor
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (2H3M4OP).
[0104] The precursor to the p-toluate and PTA pathways is 2H3M4OP.
This chemical can be derived from central metabolites
glyceraldehyde-3-phosphate (G3P) and pyruvate in three enzymatic
steps as shown in FIG. 1. The first two steps are native to E. coli
and other organisms that utilize the methylerythritol phosphate
(non-mevalonate) pathway for isoprenoid biosynthesis. Pyruvate and
G3P are first condensed to form 1-deoxy-D-xylulose 5-phosphate
(DXP) by DXP synthase. Subsequent reduction and rearrangement of
the carbon backbone is catalyzed by DXP reductoisomerase. Finally,
a novel diol dehydratase transforms
2-C-methyl-D-erythritol-4-phosphate to the p-toluate precursor
2H3M4OP.
[0105] A. 1-Deoxyxylulose-5-phosphate (DXP) synthase. Pyruvate and
G3P are condensed to form DXP by DXP synthase (EC 2.2.1.7). This
enzyme catalyzes the first step in the non-mevalonate pathway of
isoprenoid biosynthesis. The enzyme requires thiamine diphosphate
as a cofactor, and also requires reduced FAD, although there is no
net redox change. A crystal structure of the E. coli enzyme is
available (Xiang et al., J. Biol. Chem. 282:2676-2682
(2007)(doi:M610235200, pii;10.1074/jbc.M610235200 doi). Other
enzymes have been cloned and characterized in M. tuberculosis
(Bailey et al., Glycobiology 12:813-820 (2002) and Agrobacterium
tumefaciens (Lee et al., J. Biotechnol. 128:555-566
(2007)(doi:S0168-1656(06)00966-7,
pii;10.1016/j.jbiotec.2006.11.009, doi). DXP synthase enzymes from
B. subtilis and Synechocystis sp. PCC 6803 were cloned into E. coli
(Harker and Bramley, FEBS Lett. 448:115-119
(1999)(doi:S0014-5793(99)00360-9, pii).
TABLE-US-00001 GenBank Gene Accession No. GI No. Organism dxs
AAC73523.1 1786622 Escherichia coli dxs P0A554.1 61222979 M.
tuberculosis dxs11 AAP56243.1 37903541 Agrobacterium tumefaciens
dxs P54523.1 1731052 Bacillus subtilis sll1945 BAA17089.1 1652165
Synechocystis sp. PCC 6803
[0106] B. 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (EC
1.1.1.267). The NAD(P)H-dependent reduction and rearrangement of
1-deoxy-D-xylulose-5-phosphate (DXP) to
2-C-methyl-D-erythritol-4-phosphate is catalyzed by DXP
reductoisomerase (DXR, EC 1.1.1.267) in the second step of the
non-mevalonate pathway for isoprenoid biosynthesis. The
NADPH-dependent E. coli enzyme is encoded by dxr (Takahashi et al.,
Proc. Natl. Acad. Sci. USA 95:9879-9884 (1998)). A recombinant
enzyme from Arabidopsis thaliana was functionally expressed in E.
coli (Carretero-Paulet et al., Plant Physiol. 129:1581-1591
(2002)(doi:10.1104/pp.003798 (doi). DXR enzymes from Zymomonas
mobilis and Mycobacterium tuberculosis have been characterized and
crystal structures are available (Grolle et al., FEMS Microbiol.
Lett. 191:131-137 (2000)(doi:S0378-1097(00)00382-7, pii);
Henriksson et al., Acta Crystallogr. D. Biol. Crystallogr.
62:807-813 (2006)(doi:S0907444906019196,
pii;10.1107/S0907444906019196, doi). Most characterized DXR enzymes
are strictly NADPH dependent, but the enzymes from A. thaliana and
M. tuberculosis react with NADH at a reduced rate (Argyrou and
Blanchard, Biochemistry 43:4375-4384 (2004)(doi:10.1021/bi049974k,
doi); Rohdich et al., FEBS J. 273:4446-4458 (2006)(doi:EJB5446,
pii;10.1111/j.1742-4658.2006.05446.x, doi.
TABLE-US-00002 GenBank Gene Accession No. GI No. Organism dxr
AAC73284.1 1786369 Escherichia coli dxr AAF73140.1 8131928
Arabisopsis thaliana dxr CAB60758.1 6434139 Zymomonas mobilis dxr
NP_217386.2 57117032 Mycobacterium tuberculosis
[0107] C. 2-C-Methyl-D-erythritol-4-phosphate dehydratase. A diol
dehydratase is required to convert
2-C-methyl-D-erythritol-4-phosphate into the p-toluate precursor
(Aitmiller and Wagner, Arch. Biochem. Biophys. 138:160-170 (1970)).
Although this transformation has not been demonstrated
experimentally, several enzymes catalyze similar transformations
including dihydroxy-acid dehydratase (EC 4.2.1.9), propanediol
dehydratase (EC 4.2.1.28), glycerol dehydratase (EC 4.2.1.30) and
myo-inositose dehydratase (EC 4.2.1.44).
[0108] Diol dehydratase or propanediol dehydratase enzymes (EC
4.2.1.28) capable of converting the secondary diol 2,3-butanediol
to 2-butanone are excellent candidates for this transformation.
Adenosylcobalamin-dependent diol dehydratases contain alpha, beta
and gamma subunits, which are all required for enzyme function.
Exemplary gene candidates are found in Klebsiella pneumoniae
(Tobimatsu et al., Biosci. Biotechnol. Biochem. 62:1774-1777
(1998); Toraya et al.,. Biochem. Biophys. Res. Commun. 69:475-480
(1976)), Salmonella typhimurium (Bobik et al., J. Bacteriol.
179:6633-6639 (1997)), Klebsiella oxytoca (Tobimatsu et al., J.
Biol. Chem. 270:7142-7148 (1995)) and Lactobacillus collinoides
(Sauvageot et al., FEMS Microbiol. Lett. 209:69-74 (2002)). Methods
for isolating diol dehydratase gene candidates in other organisms
are well known in the art (see, for example, U.S. Pat. No.
5,686,276).
TABLE-US-00003 GenBank Gene Accession No. GI No. Organism pddA
BAA08099.1 868006 Klebsiella oxytoca pddB BAA08100.1 868007
Klebsiella oxytoca pddC BAA08101.1 868008 Klebsiella oxytoca pduC
AAB84102.1 2587029 Salmonella typhimurium pduD AAB84103.1 2587030
Salmonella typhimurium pduE AAB84104.1 2587031 Salmonella
typhimurium pduC CAC82541.1 18857678 Lactobacullus collinoides pduD
CAC82542.1 18857679 Lactobacullus collinoides pduE CAD01091.1
18857680 Lactobacullus collinoides pddA AAC98384.1 4063702
Klebsiella pneumoniae pddB AAC98385.1 4063703 Klebsiella pneumoniae
pddC AAC98386.1 4063704 Klebsiella pneumoniae
[0109] Enzymes in the glycerol dehydratase family (EC 4.2.1.30) can
also be used to dehydrate 2-C-methyl-D-erythritol-4-phosphate.
Exemplary gene candidates encoded by gldABC and dhaB123 in
Klebsiella pneumoniae (WO 2008/137403) and (Toraya et al., Biochem.
Biophys. Res. Commun. 69:475-480 (1976)), dhaBCE in Clostridium
pasteuranum (Macis et al., FEM Microbiol Lett. 164:21-28 (1998))
and dhaBCE in Citrobacter freundii (Seyfried et al., J. Bacteriol.
178:5793-5796 (1996)). Variants of the B12-dependent diol
dehydratase from K. pneumoniae with 80- to 336-fold enhanced
activity were recently engineered by introducing mutations in two
residues of the beta subunit (Qi et al., J. Biotechnol. 144:43-50
(2009)(doi:S0168-1656(09)00258-2, pii;10.1016/j
jbiotec.2009.06.015, doi). Diol dehydratase enzymes with reduced
inactivation kinetics were developed by DuPont using error-prone
PCR (WO 2004/056963).
TABLE-US-00004 GenBank Gene Accession No. GI No. Organism gldA
AAB96343.1 1778022 Klebsiella pneumoniae gldB AAB96344.1 1778023
Klebsiella pneumoniae gldC AAB96345.1 1778024 Klebsiella pneumoniae
dhaB1 ABR78884.1 150956854 Klebsiella pneumoniae dhaB2 ABR78883.1
150956853 Klebsiella pneumoniae dhaB3 ABR78882.1 150956852
Klebsiella pneumoniae dhaB AAC27922.1 3360389 Clostridium
pasteuranum dhaC AAC27923.1 3360390 Clostridium pasteuranum dhaE
AAC27924.1 3360391 Clostridium pasteuranum dhaB P45514.1 1169287
Citrobacter freundii dhaC AAB48851.1 1229154 Citrobacter freundii
dhaE AAB48852.1 1229155 Citrobacter freundii
[0110] If a B 12-dependent diol dehydratase is utilized,
heterologous expression of the corresponding reactivating factor is
recommended. B12-dependent diol dehydratases are subject to
mechanism-based suicide activation by substrates and some
downstream products. Inactivation, caused by a tight association
with inactive cobalamin, can be partially overcome by diol
dehydratase reactivating factors in an ATP-dependent process.
Regeneration of the B12 cofactor requires an additional ATP. Diol
dehydratase regenerating factors are two-subunit proteins.
Exemplary candidates are found in Klebsiella oxytoca (Mori et al.,
J. Biol. Chem. 272:32034-32041 (1997)), Salmonella typhimurium
(Bobik et al., J. Bacteriol. 179:6633-6639 (1997); Chen et al., J.
Bacteriol. 176:5474-5482 (1994)), Lactobacillus collinoides
(Sauvageot et al., FEMS Microbiol. Lett. 209:69-74 (2002)), and
Klebsiella pneumonia (WO 2008/137403).
TABLE-US-00005 GenBank Gene Accession No. GI No. Organism ddrA
AAC15871.1 3115376 Klebsiella oxytoca ddrB AAC15872.1 3115377
Klebsiella oxytoca pduG AAL20947.1 16420573 Salmonella typhimurium
pduH AAL20948.1 16420574 Salmonella typhimurium pduG YP_002236779
206579698 Klebsiella pneumonia pduH YP_002236778 206579863
Klebsiella pneumonia pduG CAD01092 29335724 Lactobacillus
collinoides pduH CAD01093 29335725 Lactobacillus collinoides
[0111] B 12-independent diol dehydratase enzymes utilize
S-adenosylmethionine (SAM) as a cofactor, function under strictly
anaerobic conditions, and require activation by a specific
activating enzyme (Frey et al., Chem. Rev. 103:2129-2148 (2003)).
The glycerol dehydrogenase and corresponding activating factor of
Clostridium butyricum, encoded by dhaB1 and dhaB2, have been
well-characterized (O'Brien et al., Biochemistry 43:4635-4645
(2004); Raynaud et al., Proc. Natl. Acad. Sci USA 100:5010-5015
(2003)). This enzyme was recently employed in a 1,3-propanediol
overproducing strain of E. coli and was able to achieve very high
titers of product (Tang et al., Appl. Environ. Microbiol.
75:1628-1634 (2009)(doi:AEM.02376-08, pii;10.1128/AEM.02376-08,
doi). An additional B 12-independent diol dehydratase enzyme and
activating factor from Roseburia inulinivorans was shown to
catalyze the conversion of 2,3-butanediol to 2-butanone (US
publication 2009/09155870).
TABLE-US-00006 GenBank Gene Accession No. GI No. Organism dhaB1
AAM54728.1 27461255 Clostridium butyricum dhaB2 AAM54729.1 27461256
Clostridium butyricum rdhtA ABC25539.1 83596382 Roseburia
inulinivorans rdhtB ABC25540.1 83596383 Roseburia inulinivorans
[0112] Dihydroxy-acid dehydratase (DHAD, EC 4.2.1.9) is a
B12-independent enzyme participating in branched-chain amino acid
biosynthesis. In its native role, it converts
2,3-dihydroxy-3-methylvalerate to 2-keto-3-methyl-valerate, a
precursor of isoleucine. In valine biosynthesis, the enzyme
catalyzes the dehydration of 2,3-dihydroxy-isovalerate to
2-oxoisovalerate. The DHAD from Sulfolobus solfataricus has a broad
substrate range, and activity of a recombinant enzyme expressed in
E. coli was demonstrated on a variety of aldonic acids (Kim and
Lee, J. Biochem. 139:591-596 (2006)(doi:139/3/591,
pii;10.1093/jb/mvj057, doi). The S. solfataricus enzyme is tolerant
of oxygen unlike many diol dehydratase enzymes. The E. coli enzyme,
encoded by ilvD, is sensitive to oxygen, which inactivates its
iron-sulfur cluster (Flint et al., J. Biol. Chem. 268:14732-14742
(1993)). Similar enzymes have been characterized in Neurospora
crassa (Altmiller and Wagner, Arch. Biochem. Biophys. 138:160-170
(1970)) and Salmonella typhimurium (Armstrong et al., Biochim.
Biophys. Acta 498:282-293 (1977)).
TABLE-US-00007 GenBank Gene Accession No. GI No. Organism ilvD
NP_344419.1 15899814 Sulfolobus solfataricus ilvD AAT48208.1
48994964 Escherichia coli ilvD NP_462795.1 16767180 Salmonella
typhimurium ilvD XP_958280.1 85090149 Neurospora crassa
[0113] The diol dehydratase myo-inosose-2-dehydratase (EC 4.2.1.44)
is another exemplary candidate. Myo-inosose is a six-membered ring
containing adjacent alcohol groups. A purified enzyme encoding
myo-inosose-2-dehydratase functionality has been studied in
Klebsiella aerogenes in the context of myo-inositol degradation
(Berman and Magasanik, J. Biol. Chem. 241:800-806 (1966)), but has
not been associated with a gene to date. The
myo-inosose-2-dehydratase of Sinorhizobium fredii was cloned and
functionally expressed in E. coli (Yoshida et al., Biosci.
Biotechnol. Biochem. 70:2957-2964 (2006)(doi:JST.JSTAGE/bbb/60362,
pii). A similar enzyme from B. subtilis, encoded by iolE, has also
been studied (Yoshida et al., Microbiology 150:571-580 (2004)).
TABLE-US-00008 GenBank Gene Accession No. GI No. Organism ME
P42416.1 1176989 Bacillus subtilis ME AAX24114.1 60549621
Sinorhizobium fredii
EXAMPLE II
Exemplary Pathway for Synthesis of p-Toluate from
(2-Hydroxy-3-methyl-4-oxobutoxy)phosphonate by Shikimate Pathway
Enzymes
[0114] This example describes exemplary pathways for synthesis of
p-toluate using shikimate pathway enzymes.
[0115] The chemical structure of p-toluate closely resembles
p-hydroxybenzoate, a precursor of the electron carrier ubiquinone.
4-Hydroxybenzoate is synthesized from central metabolic precursors
by enzymes in the shikimate pathway, found in bacteria, plants and
fungi. The shikimate pathway is comprised of seven enzymatic steps
that transform D-erythrose-4-phosphate (E4P) and
phosphoenolpyruvate (PEP) to chorismate. Pathway enzymes include
2-dehydro-3-deoxyphosphoheptonate (DAHP) synthase, dehydroquinate
(DHQ) synthase, DHQ dehydratase, shikimate dehydrogenase, shikimate
kinase, 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase and
chorismate synthase. In the first step of the pathway,
D-erythrose-4-phosphate and phosphoenolpyruvate are joined by DAHP
synthase to form 3-deoxy-D-arabino-heptulosonate-7-phosphate. This
compound is then dephosphorylated, dehydrated and reduced to form
shikimate. Shikimate is converted to chorismate by the actions of
three enzymes: shikimate kinase,
3-phosphoshikimate-2-carboxyvinyltransferase and chorismate
synthase. Subsequent conversion of chorismate to 4-hydroxybenzoate
is catalyzed by chorismate lyase.
[0116] The synthesis of p-toluate proceeds in an analogous manner
as shown in FIG. 2. The pathway originates with PEP and 2H3M4OP, a
compound analogous to E4P with a methyl group in place of the
3-hydroxyl group of E4P. The hydroxyl group of E4P does not
directly participate in the chemistry of the shikimate pathway
reactions, so the methyl-substituted 2H3M4OP precursor is expected
to react as an alternate substrate. Directed or adaptive evolution
can be used to improve preference for 2H3M4OP and downstream
derivatives as substrates. Such methods are well-known in the
art.
[0117] Strain engineering strategies for improving the efficiency
of flux through shikimate pathway enzymes are also applicable here.
The availability of the pathway precursor PEP can be increased by
altering glucose transport systems (Yi et al., Biotechnol. Prog.
19:1450-1459 (2003)(doi:10.1021/bp0340584, doi).
4-Hydroxybenzoate-overproducing strains were engineered to improve
flux through the shikimate pathway by means of overexpression of a
feedback-insensitive isozyme of 3-deoxy-D-arabinoheptulosonic
acid-7-phosphate synthase (Barker and Frost, Biotechnol. Bioeng.
76:376-390 (2001)(doi:10.1002/bit.10160, pii). Additionally,
expression levels of shikimate pathway enzymes and chorismate lyase
were enhanced. Similar strategies can be employed in a strain for
overproducing p-toluate.
[0118] A. 2-Dehydro-3-deoxyphosphoheptonate synthase (EC 2.5.1.54).
The condensation of D-erythrose-4-phosphate and phosphoenolpyruvate
is catalyzed by 2-dehydro-3-deoxyphosphoheptonate (DAHP) synthase
(EC 2.5.1.54). Three isozymes of this enzyme are encoded in the E.
coli genome by aroG, aroF and aroH and are subject to feedback
inhibition by phenylalanine, tyrosine and tryptophan, respectively.
In wild-type cells grown on minimal medium, the aroG, aroF and aroH
gene products contributed 80%, 20% and 1% of DAHP synthase
activity, respectively (Hudson and Davidson, J. Mol. Biol.
180:1023-1051 (1984)(doi:0022-2836(84)90269-9, pii). Two residues
of AroG were found to relieve inhibition by phenylalanine (Kikuchi
et al., Appl. Environ. Microbiol. 63:761-762 (1997)). The feedback
inhibition of AroF by tyrosine was removed by a single base-pair
change (Weaver and Herrmann, J. Bacteriol. 172:6581-6584 (1990)).
The tyrosine-insensitive DAHP synthase was overexpressed in a
4-hydroxybenzoate-overproducing strain of E. coli (Barker and
Frost, Biotechnol. Bioeng. 76:376-390 (2001)(doi:10.1002/bit.10160,
pii). The aroG gene product was shown to accept a variety of
alternate 4- and 5-carbon length substrates (Sheflyan et al., J.
Am. Chem. Soc. 120(43):11027-11032 (1998); Williamson et al.,
Bioorg. Med. Chem. Lett. 15:2339-2342
(2005)(doi:S0960-894X(05)00273-8, pii;10.1016/j.bmc1.2005.02.080,
doi). The enzyme reacts efficiently with
(3S)-2-deoxyerythrose-4-phosphate, a substrate analogous to
D-erythrose-4-phosphate but lacking the alcohol at the 2-position
(Williamson et al., supra 2005). Enzymes from Helicobacter pylori
and Pyrococcus furiosus also accept this alternate substrate
(Schofield et al., Biochemistry 44:11950-11962
(2005)(doi:10.1021/bi050577z, doi; Webby et al., Biochem. J.
390:223-230 2005)(doi:BJ20050259, pii;10.1042/BJ20050259, doi) and
have been expressed in E. coli. An evolved variant of DAHP
synthase, differing from the wild type E. coli AroG enzyme by 7
amino acids, was shown to exhibit a 60-fold improvement in
Kcat/K.sub.M (Ran and Frost, J. Am. Chem. Soc. 129:6130-6139
(2007)(doi:10.1021/ja067330p, doi).
TABLE-US-00009 GenBank Gene Accession No. GI No. Organism aroG
AAC73841.1 1786969 Escherichia coli aroF AAC75650.1 1788953
Escherichia coli aroH AAC74774.1 1787996 Escherichia coli aroF
Q9ZMU5 81555637 Helicobacter pylori PF1690 NP_579419.1 18978062
Pyrococcus furiosus
[0119] B. 3-Dehydroquinate synthase (EC 4.2.3.4). The
dephosphorylation of substrate
(2)(2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carb-
oxylate) to substrate
(3)(1,3-dihydroxy-4-methylcylohex-1-ene-1-carboxylate) as shown in
FIG. 2 is analogous to the dephosphorylation of
3-deoxy-arabino-heptulonate-7-phosphate by 3-dehydroquinate
synthase. The enzyme has been characterized in E. coli (Mehdi et
al., Methods Enzymol. 142:306-314 (1987), B. subtilis (Hasan and
Nester, J. Biol. Chem. 253:4999-5004 (1978)) and Mycobacterium
tuberculosis H3 7Rv (de Mendonca et al., J. Bacteriol.
189:6246-6252 (2007)(doi:JB.00425-07, pii;10.1128/JB.00425-07,
doi). The E. coli enzyme is subject to inhibition by L-tyrosine
(Barker and Frost, Biotechnol. Bioeng. 76:376-390
2001)(doi:10.1002/bit.10160, pii).
TABLE-US-00010 GenBank Gene Accession No. GI No. Organism aroB
AAC76414.1 1789791 Escherichia coli aroB NP_390151.1 16079327
Bacillus subtilis aroB CAB06200.1 1781064 Mycobacterium
tuberculosis
[0120] C. 3-Dehydroquinate dehydratase (EC 4.2.1.10).
3-Dehydroquinate dehydratase, also termed 3-dehydroquinase
(DHQase), naturally catalyzes the dehydration of 3-dehydroquinate
to 3-dehydroshikimate, analogous to step C in the p-toluate pathway
of FIG. 2. DHQase enzymes can be divided into two classes based on
mechanism, stereochemistry and sequence homology (Gourley et al.,
Nat. Struct. Biol. 6:521-525. (1999)(doi:10.1038/9287, doi).
Generally the type 1 enzymes are involved in biosynthesis, while
the type 2 enzymes operate in the reverse (degradative) direction.
Type 1 enzymes from E. coli (Kinghorn et al., Gene 14:73-80.
1981)(doi:0378-1119(81)90149-9, pii), Salmonella typhi (Kinghorn et
al., supra 1981; Servos et al., J. Gen. Microbiol. 137:147-152
(1991)) and B. subtilis (Warburg et al., Gene 32:57-66
1984)(doi:0378-1119(84)90032-5, pii) have been cloned and
characterized. Exemplary type II 3-dehydroquinate dehydratase
enzymes are found in Mycobacterium tuberculosis, Streptomyces
coelicolor (Evans et al., FEBS Lett. 530:24-30 (2002)) and
Helicobacter pylori (Lee et al., Proteins 51:616-7 (2003)).
TABLE-US-00011 GenBank Gene Accession No. GI No. Organism aroD
AAC74763.1 1787984 Escherichia coli aroD P24670.2 17433709
Salmonella typhi aroC NP_390189.1 16079365 Bacillus subtilis aroD
P0A4Z6.2 61219243 Mycobacterium tuberculosis aroQ P15474.3 8039781
Streptomyces coelicolor aroQ Q48255.2 2492957 Helicobacter
pylori
[0121] D. Shikimate dehydrogenase (EC 1.1.1.25). Shikimate
dehydrogenase catalyzes the NAD(P)H dependent reduction of
3-dehydroshikimate to shikimate, analogous to Step D of FIG. 2. The
E. coli genome encodes two shikimate dehydrogenase paralogs with
different cofactor specificities. The enzyme encoded by aroE is
NADPH specific, whereas the ydiB gene product is a
quinate/shikimate dehydrogenase which can utilize NADH (preferred)
or NADPH as a cofactor (Michel et al., J. Biol. Chem.
278:19463-19472 (2003)(doi:10.1074/jbc.M300794200, doi;M300794200,
pii). NADPH-dependent enzymes from Mycobacterium tuberculosis
(Zhang et al., J. Biochem. Mol. Biol. 38:624-631 (2005)),
Haemophilus influenzae (Ye et al., J. Bacteriol. 185:4144-4151
(2003)) and Helicobacter pylori (Han et al., FEBS J. 273:4682-4692
(2006)(doi:EJB5469, pii;10.1111/j.1742-4658.2006.05469.x, doi) have
been functionally expressed in E. coli.
TABLE-US-00012 GenBank Gene Accession No. GI No. Organism aroE
AAC76306.1 1789675 Escherichia coli ydiB AAC74762.1 1787983
Escherichia coli aroE NP_217068.1 15609689 Mycobacterium
tuberculosis aroE P43876.1 1168510 Haemophilus influenzae aroE
AAW22052.1 56684731 Helicobacter pylori
[0122] E. Shikimate kinase (EC 2.7.1.71). Shikimate kinase
catalyzes the ATP-dependent phosphorylation of the 3-hydroxyl group
of shikimate analogous to Step E of FIG. 2. Two shikimate kinase
enzymes are encoded by aroK (SKI) and aroL (SK2) in E. coli
(DeFeyter and Pittard, J. Bacteriol. 165:331-333 (1986);
Lobner-Olesen and Marinus, J. Bacteriol. 174:525-529 (1992)). The
Km of SK2, encoded by aroL, is 100-fold lower than that of SK1,
indicating that this enzyme is responsible for aromatic
biosynthesis (DeFeyter et al., supra 1986). Additional shikimate
kinase enzymes from Mycobacterium tuberculosis (Gu et al., J. Mol.
Biol. 319:779-789 (2002)(doi:10.1016/S0022-2836(02)00339-X,
doi;S0022-2836(02)00339-X, pii) Oliveira et al., Protein Expr.
Purif. 22:430-435 (2001)(doi:10.1006/prep.2001.1457,
doi;S1046-5928(01)91457-3, pii), Helicobacter pylori (Cheng et al.,
J. Bacterial. 187:8156-8163 (2005)(doi:187/23/8156,
pii;10.1128/JB.187.23.8156-8163.2005, doi) and Erwinia chrysanthemi
(Krell et al., Protein Sci. 10:1137-1149
(2001)(doi:10.1110/ps.52501, doi) have been cloned in E. coli.
TABLE-US-00013 GenBank Gene Accession No. GI No. Organism aroK
YP_026215.2 90111581 Escherichia coli aroL NP_414922.1 16128373
Escherichia coli aroK CAB06199.1 1781063 Mycobacterium tuberculosis
aroK NP_206956.1 15644786 Helicobacter pylori SK CAA32883.1 42966
Erwinia chrysanthemi
[0123] F. 3-Phosphoshikimate-2-carboxyvinyltransferase (EC
2.5.1.19). 3-Phosphoshikimate-2-carboxyvinyltransferase, also known
as 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), catalyzes
the transfer of the enolpyruvyl moiety of phosphoenolpyruvate to
the 5-hydroxyl of shikimate-3-phosphate. The enzyme is encoded by
aroA in E. coli (Anderson et al., Biochemistry 27:1604-1610
(1988)). EPSPS enzymes from Mycobacterium tuberculosis (Oliveira et
al., Protein Expr. Purif. 22:430-435
(2001)(doi:10.1006/prep.2001.1457, doi;S1046-5928(01)91457-3, pii),
Dunaliella salina (Yi et al., J. Microbiol. 45:153-157
(2007)(doi:2519, pii) and Staphylococcus aureus (Priestman et al.,
FEBS Lett. 579:728-732 (2005)(doi:S0014-5793(05)00012-8,
pii;10.1016/j.febslet.2004.12.057, doi) have been cloned and
functionally expressed in E. coli.
TABLE-US-00014 GenBank Gene Accession No. GI No. Organism aroA
AAC73994.1 1787137 Escherichia coli aroA AAA25356.1 149928
Mycobacterium tuberculosis aroA AAA71897.1 152956 Staphylococcus
aureus aroA ABM68632.1 122937807 Dunaliella salina
[0124] G. Chorismate synthase (EC 4.2.3.5). Chorismate synthase is
the seventh enzyme in the shikimate pathway, catalyzing the
transformation of 5-enolpyruvylshikimate-3-phosphate to chorismate.
The enzyme requires reduced flavin mononucleotide (FMN) as a
cofactor, although the net reaction of the enzyme does not involve
a redox change. In contrast to the enzyme found in plants and
bacteria, the chorismate synthase in fungi is also able to reduce
FMN at the expense of NADPH (Macheroux et al., Planta 207:325-334
(1999)). Representative monofunctional enzymes are encoded by aroC
of E. coli (White et al., Biochem. J. 251:313-322 (1988)) and
Streptococcus pneumoniae (Maclean and Ali, Structure 11:1499-1511
(2003)(doi:S0969212603002648, pii). Bifunctional fungal enzymes are
found in Neurospora crassa (Kitzing et al., J. Biol. Chem.
276:42658-42666 (2001)(doi:10.1074/jbc.M107249200, doi;M107249200,
pii) and Saccharomyces cerevisiae (Jones et al., Mol. Microbiol.
5:2143-2152 (1991)).
TABLE-US-00015 GenBank Gene Accession No. GI No. Organism aroC
NP_416832.1 16130264 Escherichia coli aroC ACH47980.1 197205483
Streptococcus pneumoniae U25818.1: AAC49056.1 976375 Neurospora
crassa 19 . . . 1317 ARO2 CAA42745.1 3387 Saccharomyces
cerevisiae
[0125] H. Chorismate lyase (EC 4.1.3.40). Chorismate lyase
catalyzes the first committed step in ubiquinone biosynthesis: the
removal of pyruvate from chorismate to form 4-hydroxybenzoate. The
enzymatic reaction is rate-limited by the slow release of the
4-hydroxybenzoate product (Gallagher et al., Proteins 44:304-311
(2001)(doi:10.1002/prot.1095, pii), which is thought to play a role
in delivery of 4-hydroxybenzoate to downstream membrane-bound
enzymes. The chorismate lyase of E. coli was cloned and
characterized and the enzyme has been crystallized (Gallagher et
al., supra 2001; Siebert et al., FEBS Lett. 307:347-350
(1992)(doi:0014-5793(92)80710-X, pii). Structural studies implicate
the G90 residue as contributing to product inhibition (Smith et
al., Arch. Biochem. Biophys. 445:72-80
(2006)(doi:S0003-9861(05)00446-7, pii;10.1016/j.abb.2005.10.026,
doi). Modification of two surface-active cysteine residues reduced
protein aggregation (Holden et al., Biochim. Biophys. Acta
1594:160-167 (2002)(doi:S0167483801003028, pii). A recombinant form
of the Mycobacterium tuberculosis chorismate lyase was cloned and
characterized in E. coli (Stadthagen et al., J. Biol. Chem.
280:40699-40706 2005)(doi:M508332200, pii;10.1074/jbc.M508332200,
doi).
TABLE-US-00016 GenBank Gene Accession No. GI No. Organism ubiC
AAC77009.2 87082361 Escherichia coli Rv2949c NP_217465.1 15610086
Mycobacterium tuberculosis
[0126] B-F. Multifunctional AROM protein. In most bacteria, the
enzymes of the shikimate pathway are encoded by separate
polypeptides. In microbial eukaryotes, five enzymatic functions are
catalyzed by a polyfunctional protein encoded by a pentafunctional
supergene (Campbell et al., Int. J. Parasitol. 34:5-13
(2004)(doi:S0020751903003102, pii). The multifunctional AROM
protein complex catalyzes reactions analogous to reactions B-F of
FIG. 2. The AROM protein complex has been characterized in fungi
including Aspergillus nidulans, Neurospora crassa, Saccharomyces
cerevisiae and Pneumocystis carinii (Banerji et al., J. Gen.
Microbiol. 139:2901-2914 (1993); Charles et al., Nucleic Acids Res.
14:2201-2213 (1986); Coggins et al., Methods Enzymol. 142:325-341
(1987); Duncan, K., Biochem. J. 246:375-386 (1987)). Several
components of AROM have been shown to function independently as
individual polypeptides. For example, dehydroquinate synthase
(DHQS) forms the amino-terminal domain of AROM, and can function
independently when cloned into E. coli (Moore et al., Biochem. J.
301 (Pt 1):297-304 (1994)). Several crystal structures of AROM
components from Aspergillus nidulans provide insight into the
catalytic mechanism (Carpenter et al., Nature 394:299-302
(1998)(doi:10.1038/28431, doi).
TABLE-US-00017 GenBank Gene Accession No. GI No. Organism AROM
P07547.3 238054389 Aspergillus nidulans AROM P08566.1 114166
Saccharomyces cerevisiae AROM P07547.3 238054389 Aspergillus
nidulans AROM Q12659.1 2492977 Pneumocystis carinii
EXAMPLE III
Exemplary Pathway for Enzymatic Transformation of p-Toluate to
Terephthalic Acid
[0127] This example describes exemplary pathways for conversion of
p-toluate to terephthalic acid (PTA).
[0128] P-toluate can be further transformed to PTA by oxidation of
the methyl group to an acid in three enzymatic steps as shown in
FIG. 3. The pathway is comprised of a p-toluate
methyl-monooxygenase reductase, a 4-carboxybenzyl alcohol
dehydrogenase and a 4-carboxybenzyl aldehyde dehydrogenase. In the
first step, p-toluate methyl-monooxyngenase oxidizes p-toluate to
4-carboxybenzyl alcohol in the presence of O.sub.2. The Comamonas
testosteroni enzyme (tsaBM), which also reacts with 4-toluene
sulfonate as a substrate, has been purified and characterized
(Locher et al., J. Bacteriol. 173:3741-3748 (1991)).
4-Carboxybenzyl alcohol is subsequently converted to an aldehyde by
4-carboxybenzyl alcohol dehydrogenase (tsaC). The aldehyde to acid
transformation is catalyzed by 4-carboxybenzaldehyde dehydrogenase
(tsaD). Enzymes catalyzing these reactions are found in Comamonas
testosteroni T-2, an organism capable of utilizing p-toluate as the
sole source of carbon and energy (Junker et al., J. Bacteriol.
179:919-927 (1997)). Additional genes to transform p-toluate to PTA
can be found by sequence homology, in particular to proteobacteria
in the genera Burkholderia, Alcaligenes, Pseudomonas, Shingomonas
and Comamonas (U.S. Pat. No. 6,187,569 and US publication
2003/0170836). Genbank identifiers associated with the Comamonas
testosteroni enzymes are listed below.
TABLE-US-00018 GenBank Gene Accession No. GI No. Organism tsaB
AAC44805.1 1790868 Comamonas testosteroni tsaM AAC44804.1 1790867
Comamonas testosteroni tsaC AAC44807.1 1790870 Comamonas
testosteroni tsaD AAC44808.1 1790871 Comamonas testosteroni
[0129] Throughout this application various publications have been
referenced. The disclosures of these publications in their
entireties, including GenBank and GI number publications, are
hereby incorporated by reference in this application in order to
more fully describe the state of the art to which this invention
pertains. Although the invention has been described with reference
to the examples provided above, it should be understood that
various modifications can be made without departing from the spirit
of the invention.
* * * * *