U.S. patent application number 16/041271 was filed with the patent office on 2019-01-31 for microorganisms and processes for producing terephthalic acid and its salts.
The applicant listed for this patent is Genomatica, Inc.. Invention is credited to Anthony P. Burgard, Mark J. Burk, Robin E. Osterhout.
Application Number | 20190032095 16/041271 |
Document ID | / |
Family ID | 47628457 |
Filed Date | 2019-01-31 |
View All Diagrams
United States Patent
Application |
20190032095 |
Kind Code |
A1 |
Osterhout; Robin E. ; et
al. |
January 31, 2019 |
MICROORGANISMS AND PROCESSES FOR PRODUCING TEREPHTHALIC ACID AND
ITS SALTS
Abstract
The invention provides non-naturally occurring microbial
organisms having a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
(2H3M4OP) pathway, p-toluate pathway, and/or terephthalate pathway.
The invention additionally provides methods of using such organisms
to produce 2H3M4OP, p-toluate or terephthalate. Also provided
herein are processes for isolating bio-based aromatic carboxylic
acid, in particular, p-toluic acid or terephthalic acid, from a
culture medium, wherein the processes involve contacting the
culture medium with sufficient carbon dioxide (CO.sub.2) to lower
the pH of the culture medium to produce a precipitate comprised of
the aromatic carboxylic acid.
Inventors: |
Osterhout; Robin E.; (San
Diego, CA) ; Burgard; Anthony P.; (San Diego, CA)
; Burk; Mark J.; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genomatica, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
47628457 |
Appl. No.: |
16/041271 |
Filed: |
July 20, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14373160 |
Jul 18, 2014 |
10059967 |
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PCT/US2013/022113 |
Jan 18, 2013 |
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16041271 |
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61598743 |
Feb 14, 2012 |
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61589081 |
Jan 20, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07F 9/091 20130101;
C12N 9/1205 20130101; C12P 7/62 20130101; C12P 7/40 20130101; C08G
63/183 20130101; C12N 9/88 20130101; C12N 9/1007 20130101; C12P
7/44 20130101; C12N 9/1085 20130101; C12N 9/0004 20130101; C07C
51/43 20130101; C12N 9/0006 20130101; C12N 9/0008 20130101; C12P
7/24 20130101; C12N 9/1022 20130101; C12P 9/00 20130101; C07C 51/43
20130101; C07C 63/26 20130101; C07C 51/43 20130101; C07C 63/04
20130101 |
International
Class: |
C12P 7/44 20060101
C12P007/44; C12P 7/24 20060101 C12P007/24; C12N 9/10 20060101
C12N009/10; C12P 9/00 20060101 C12P009/00; C12N 9/02 20060101
C12N009/02; C07C 51/43 20060101 C07C051/43; C12N 9/12 20060101
C12N009/12; C08G 63/183 20060101 C08G063/183; C12N 9/88 20060101
C12N009/88; C12N 9/04 20060101 C12N009/04; C12P 7/62 20060101
C12P007/62; C07F 9/09 20060101 C07F009/09; C12P 7/40 20060101
C12P007/40; C07C 63/26 20060101 C07C063/26; C07C 63/04 20060101
C07C063/04 |
Claims
1. A non-naturally occurring microbial organism, said microbial
organism having a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate
(2H3M4OP) pathway a p-toluate pathway and a terephthalate pathway
and comprising at least one exogenous nucleic acid encoding a
2H3M4OP pathway enzyme expressed in a sufficient amount to produce
2H3M4OP, wherein said 2H3M4OP pathway comprises a pathway selected
from: (1) 1A, 1B, 1C, 1D, 1E and 1F; (2) 2A, 2B and 2C; and (3) 2D,
2E and 2C, wherein 1A is an erythrose-4-phosphate dehydrogenase,
wherein 1B is a 4-phosphoerythronate dehydrogenase, wherein 1C is a
2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase, wherein 1D is a
2-acetyl-2,3-dihydroxy-4-phosphobutanoate reductoisomerase, wherein
1E is a 2,3,4-trihydroxy-3-methyl-5-phosphopentanoate dehydratase,
wherein 1F is a 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate
decarboxylase, wherein 2A is a 4,5-dihydroxy-2-oxopentanoate
methyltransferase, wherein 2B is a
4,5-dihydroxy-3-methyl-2-oxopentanoate kinase, wherein 2C is a
4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase, wherein
2D is a 4,5-dihydroxy-2-oxopentanoate kinase, wherein 2E is a
4-hydroxy-2-oxo-5-phosphopentanoate methyltransferase, wherein said
p-toluate pathway comprises at least one exogenous nucleic acid
encoding a p-toluate pathway enzyme expressed in a sufficient
amount to produce p-toluate, wherein said p-toluate pathway
comprises 4A, 4B, 4C, 4D, 4E, 4F, 4G and 4H, wherein 4A is a
2-dehydro-3-deoxyphosphoheptonate synthase; wherein 4B is a
3-dehydroquinate synthase; wherein 4C is a 3-dehydroquinate
dehydratase; wherein 4D is a shikimate dehydrogenase; wherein 4E is
a shikimate kinase; wherein 4F is a
3-phosphoshikimate-2-carboxyvinyltransferase; wherein 4G is a
chorismate synthase and wherein 4H is a chorismate lyase; and
wherein said microbial organism has a terephthalate pathway and
comprises at least one exogenous nucleic acid encoding a
terephthalate pathway enzyme expressed in a sufficient amount to
produce terephthalate, wherein said terephthalate pathway comprises
5A, 5B and 5C, wherein 5A is a p-toluate methyl-monooxygenase
reductase, wherein 5B is a 4-carboxybenzyl alcohol dehydrogenase
and wherein 5C is a 4-carboxybenzyl aldehyde dehydrogenase.
2. The non-naturally occurring microbial organism of claim 1,
wherein said microbial organism comprises two, three, four, five or
six exogenous nucleic acids each encoding at least one enzyme from
a 2H3M4OP, a p-toluate or a terephthalate pathway enzyme.
3. The non-naturally occurring microbial organism of claim 2,
wherein said microbial organism comprises exogenous nucleic acids
encoding each of the enzymes of at least one of the pathways
selected from the 2H3M4OP (1)-(3).
4. The non-naturally occurring microbial organism of claim 1,
wherein said exogenous nucleic acid is a heterologous nucleic
acid.
5. The non-naturally occurring microbial organism of claim 1,
wherein said non-naturally occurring microbial organism is in a
substantially anaerobic culture medium.
6.-15. (canceled)
16. The non-naturally occurring microbial organism of claim 1,
wherein said microbial organism is a species of bacteria, yeast or
fungus.
17.-21. (canceled)
22. Culture medium comprising bio-based terephthalate, wherein said
culture medium is separated from said microbial organism of claim
1.
23.-24. (canceled)
25. A composition comprising bio-based terephthalate and a compound
other than said bio-based terephthalate, wherein said compound is a
trace amount of a cellular portion of said microbial organism of
claim 1.
26. A process for isolating a bio-based aromatic carboxylic acid
from a culture medium comprising: a) culturing a non-naturally
occurring microbial organism in a culture medium to produce an
aromatic carboxylate anion at a pH sufficient to maintain the
aromatic carboxylate anion in soluble form; b) separating the
culture medium from non-soluble materials; c) contacting the
culture medium with sufficient carbon dioxide (CO.sub.2) to lower
the pH of the culture medium to produce an aromatic carboxylic acid
precipitate, wherein the culture medium is substantially depleted
of the aromatic carboxylate anion; and d) separating the aromatic
carboxylic acid from the culture medium; wherein the aromatic
carboxylic acid is p-toluic acid or terephthalic acid.
27.-52. (canceled)
53. A process for obtaining a bio-derived polymer using said
bio-based terephthalate produced by the non-naturally occurring
microbial organism of claim 1.
54. The bio-derived polymer of claim 53, where said polymer is
polyethylene terephthalate (PET), polybutyl terephthalate (PBT), or
polytrimethylene terephthalate (PTT).
55.-88. (canceled)
Description
[0001] This application claims the priority of U.S. application
Ser. No. 14/373,160 filed Jul. 18, 2014, which claims priority to
International Application No. PCT/US2013/022113, which was filed on
Jan. 18, 2013, which claims the benefit of U.S. Provisional
application Ser. No. 61/589,081, filed Jan. 20, 2012, and U.S.
Provisional application Ser. No. 61/598,743, filed Feb. 14, 2012,
the entire contents of which are incorporated herein by
reference.
TECHNICAL FIELD
[0002] The present invention relates generally to biosynthetic
processes, and more specifically to organisms having
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (2H3M4OP), p-toluate or
terephthalate biosynthetic capability. Also provided herein are
processes for isolating a bio-based aromatic carboxylic acid, for
example, p-toluic acid or terephthalic acid, from a culture medium,
wherein the processes involve lowering the pH of the culture medium
to produce a precipitate comprised of the aromatic carboxylic
acid.
BACKGROUND OF THE INVENTION
[0003] Terephthalate (also known as terephthalic acid and PTA) is
the immediate precursor of polyethylene terephthalate (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.
[0004] 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.
[0005] 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.
[0006] Petrochemical based chemical syntheses for making
terephthalic acid are known (see, e.g., U.S. Pat. Nos. 2,905,709;
3,023,234; 3,042,717; 3,043,846; 3,064,041; 3,096,366; and
6,441,225). Alternative technologies for the production of
terephthalic acid have been made feasible with the advent of
molecular recombinant technologies used to modify biosynthetic
pathways in microbial organisms. For example, microbial organisms
have been described which produce precursors useful for the
synthesis of bio-based terephthalic acid. Exemplary indirect
semi-synthetic routes of producing bio-based terephthalic acid are
described in U.S. Patent Publication No. 2011/0124911 A1. Direct
biosynthetic routes, wherein terephthalate is itself biosynthesized
in microbial organisms have been described in U.S. Patent
Publication No. 2011/0207185 A1. Even with advantages that
bio-based production of terephthalic acid offers, improvements
and/or additional processes are sought, for example, to improve
recovery of terephthalic acid in terms of yields and purity, and to
increase efficiency and scalability of the processes including, for
instance, reducing the number of manufacturing steps, lowering
energy usage, recapturing and recycling materials and reducing
environmental discharges.
[0007] Thus, there exists a need for alternative methods for
effectively producing and isolating commercial quantities of
compounds such as p-toluate or terephthalate. The present invention
satisfies this need and provides related advantages as well.
SUMMARY OF INVENTION
[0008] The invention provides non-naturally occurring microbial
organisms having a 2H3M4OP pathway, p-toluate pathway, and/or
terephthalate pathway. The invention additionally provides methods
of using such organisms to produce 2H3M4OP, p-toluate or
terephthalate.
[0009] The invention also provides a process for isolating a
bio-based aromatic carboxylic acid from a culture medium. In
certain embodiments, the process comprises the steps of: (a)
culturing a non-naturally occurring microbial organism in a culture
medium to produce an aromatic carboxylate anion at a pH sufficient
to maintain the aromatic carboxylate anion in soluble form; (b)
lowering the pH of the culture medium to produce an aromatic
carboxylic acid precipitate from the aromatic carboxylate anion. In
certain embodiments, lowering the pH of the culture medium
comprises contacting the culture medium with carbon dioxide
(CO.sub.2). In certain embodiments, the culture medium is
substantially depleted of the aromatic carboxylate anion. In
certain embodiments, the process further comprises separating the
culture medium from non-soluble materials, for example, cells, cell
debris, cellulosic material, feed stock, etc., prior to lowering
the pH. In certain embodiments, the process further comprises
separating the aromatic carboxylic acid from the culture medium,
for example, by centrifugation or membrane filtration, etc.
[0010] In certain embodiments of the isolation process, the
aromatic carboxylic acid is p-toluic acid. In certain embodiments,
the aromatic carboxylic acid is terephthalic acid.
[0011] In certain embodiments of the isolation process, a counter
ion to the aromatic carboxylate anion in the culture medium is an
ammonium, sodium or potassium cation. In certain embodiments, the
counter ion is an ammonium cation.
[0012] In certain embodiments of the isolation process, the pH
sufficient to maintain the aromatic carboxylate anion in soluble
form is between about 5.0-9.0 pH units. In some embodiments, the pH
is about 6.0 to about 8.0 pH units or about 6.2 to about 7.8 pH
units. In certain embodiments, the pH sufficient to maintain the
aromatic carboxylate anion in soluble form is about 7.0 pH units.
In certain embodiments, base is added to the culture medium in the
culturing step to maintain the aromatic carboxylate anion in
soluble form. In certain embodiments, the base is ammonia.
[0013] In certain embodiments of the isolation process, when
lowering the pH of the culture medium, the pH is lowered to less
than about 5.0 pH units, less than about 4.5 pH units, less than
about 4.0 pH units, less than about 3.5 pH units, less than about
3.0 pH units, less than about 2.5 pH units, less than about 2.0 pH
units, less than about 1.5 pH units, or less than about 1.0 pH
units. In certain embodiments, the pH of the culture medium
following the first separation step is lowered to less than about
3.0 pH units. In certain embodiments, the pH is lowered in the
culture medium following separation of culture medium from
non-soluble materials such as cells, cell debris, feedstock, and
the like, which, for example, can be present when culturing a
non-naturally occurring microbial organism.
[0014] In certain embodiments of the isolation process, wherein the
culture medium is contacted with CO.sub.2, the CO.sub.2 used in the
contacting step is gaseous. In certain embodiments, the gaseous
CO.sub.2 is pure CO.sub.2 gas. In certain embodiments, the gaseous
CO.sub.2 is in a mixture with one or more additional gases. In
certain embodiments, the additional gas is nitrogen gas. In certain
embodiments, CO.sub.2 generated from the culturing step can be used
to lower the pH of the culture medium.
[0015] In certain embodiments of the isolation process, the culture
medium is contacted with CO.sub.2 in the range of 0.1 to 30 atm. In
certain embodiments, the culture medium is stirred at temperatures
between 0.degree. C. and 80.degree. C. for up to 24 hours during
this contacting step.
[0016] In certain embodiments of the isolation process, when
present, the second separation step comprises filtering and
recovering of the aromatic carboxylic acid from the culture
medium.
[0017] In certain embodiments of the isolation process, the process
further comprises purifying the separated aromatic carboxylic acid.
In certain embodiments, the purification step comprises
crystallizing the aromatic carboxylic acid.
[0018] In certain embodiments of the isolation process, the
non-naturally occurring microbial organism produces p-toluate and
the aromatic carboxylic acid is p-toluic acid.
[0019] In certain embodiments of the isolation process, the
non-naturally occurring microbial organism produces terephthalate
and the aromatic carboxylic acid is terephthalic acid.
[0020] In certain embodiments of the isolation process, the
non-naturally occurring microbial organism has a 2H3M4OP pathway,
p-toluate pathway, and/or terephthalate pathway.
[0021] In certain embodiments of the isolation process, the
non-naturally occurring microbial organism produces muconate, and
the process further comprises contacting muconate with acetylene to
form a cyclohexadiene adduct, and oxidizing the cyclohexadiene
adduct to form the aromatic carboxylate anion. In certain
embodiments, wherein the non-naturally occurring microbial organism
produces muconate, the process for isolating a bio-based aromatic
carboxylic acid from a culture medium comprises the steps of: (a)
culturing a non-naturally occurring microbial organism in a culture
medium to produce muconate at a pH sufficient to maintain muconate
in soluble form; (b) contacting muconate with acetylene to form a
cyclohexadiene adduct; (c) oxidizing the cyclohexadiene adduct to
form the aromatic carboxylate anion; and (d) contacting the culture
medium with sufficient carbon dioxide (CO.sub.2) to lower the pH of
the culture medium to produce an aromatic carboxylic acid
precipitate, wherein the culture medium is substantially depleted
of the aromatic carboxylate anion. In certain embodiments, the
process further comprises separating the culture medium from
non-soluble materials, for example, cells, cell debris, cellulosic
material, feed stock, etc., prior to lowering the pH. In certain
embodiments, the process further comprises separating the aromatic
carboxylic acid from the culture medium, for example, by
centrifugation or membrane filtration, etc. In certain embodiments,
the aromatic carboxylic acid is terephthalic acid.
[0022] In another aspect of the isolation process, an isolated
aromatic carboxylic acid produced by the processes disclosed herein
is provided. In certain embodiments, isolated bio-based p-toluic
acid is produced. In certain embodiments, isolated bio-based
terephthalic acid is produced.
[0023] Terephthalate produced by a microorganism or isolation
process of the invention can be used as a precursor for production
of a polymer, including polyethylene terephthalate (PET), polybutyl
terephthalate (PBT) or polytrimethylene terephthalate (PTT). PET
can be produced by reacting ethylene glycol with dimethyl
terephthalate of the invention by transesterification or by
reacting ethylene glycol with terephthalate of the invention by
esterification. PBT can be produced by reacting 1,4-butanediol with
terephthalate of the invention. PTT can be produced by reacting
1,3-propanediol with terephthalate of the invention. Accordingly,
in certain embodiments, the invention provides PET, PBT or PTT
comprising, obtained by or manufactured using the terephthalate
produced by a microorganism of the invention or isolated by a
process described herein. Furthermore, PET can be used to
manufacture bulk materials such as, for example, chips (e.g. PET
bottle chips), resins and fibers, which in turn can be used to make
cloth, clothing and plastic bottles, or even used as a poultry feed
additive. PBT can be used to manufacture several products, such as,
for example, molded articles, injection-molded products,
injection-molded parts, such as an automotive part, extrusion
resins, electrical parts or casings. PTT can also be used to
manufacture several products, including, for example, fibers,
cloth, carpets or bottles.
[0024] In certain embodiments, the invention provides a process for
obtaining PET by reacting ethylene glycol with dimethyl
terephthalate, wherein the dimethyl terephthalate is produced from
terephthalate produced by a microorganism of the invention or
isolated by a process described herein. In another aspect, the
invention provides a process for obtaining PET by reacting ethylene
glycol with terephthalic acid, wherein the terephthalic acid is
produced by a microorganism of the invention or isolated by a
process described herein.
[0025] In certain embodiments, the invention provides a process for
obtaining PBT by reacting 1,4-butanediol with terephthalate
produced by a microorganism of the invention or isolated by a
process described herein.
[0026] In certain embodiments, the invention provides a process for
obtaining PTT by reacting 1,3-propanediol with terephthalate
produced by a microorganism of the invention or isolated by a
process described herein, or reacting 1,3-propanediol with dimethyl
terephthalate, wherein the dimethyl terephthalate is produced from
the terephthalate produced by a microorganism of the invention or
isolated by a process described herein.
[0027] In certain embodiments, the invention provides a polyester
fiber, a polyester cloth or a polyester carpet comprising, obtained
by or manufactured using PET or PTT, wherein the PET or PTT
comprises, was obtained by or was manufactured using the
terephthalate produced by a microorganism of the invention or
isolated by a process described herein.
[0028] In certain embodiments, the invention provides a chip
comprising, obtained by or manufactured using PET or PTT, wherein
the PET or PTT comprises, was obtained by or was manufactured using
the terephthalate produced by a microorganism of the invention or
isolated by a process described herein. In another aspect, the
invention provides a PET or PTT bottle comprising, obtained by or
manufactured using the chips described herein.
[0029] In certain embodiments, the invention provides a packaging
container comprising, obtained by or manufactured using PET,
wherein the PET comprises, was obtained by or was manufactured
using the terephthalate produced by a microorganism of the
invention or a process described herein.
[0030] In certain embodiments, the invention provides a film
comprising, obtained by or manufactured using PET, wherein the PET
comprises, was obtained by or was manufactured using the
terephthalate produced by a microorganism of the invention or a
process described herein.
[0031] In certain embodiments, the invention provides a molded
article comprising, obtained by or manufactured using PET, PBT or
PTT, wherein the PET, PBT or PTT comprises, was obtained by or was
manufactured using the terephthalate produced by a microorganism of
the invention or a process described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 shows a schematic depiction of an exemplary pathway
from erythrose-4-phosphate to
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate ("2H3M4OP"). In steps
A-F, the enzymes can be: A. erythrose-4-phosphate dehydrogenase; B.
4-phosphoerythronate dehydrogenase; C.
2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase; D.
2-acetyl-2,3-dihydroxy-4-phosphobutanoate reductoisomerase; E.
2,3,4-trihydroxy-3-methyl-5-phosphopentanoate dehydratase; and F.
4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase.
[0033] FIG. 2 shows a schematic depiction of exemplary pathways to
2H3M4OP from 4,5-dihydroxy-2-oxopentanoate. In steps A-E, the
enzymes can be: A. 4,5-dihydroxy-2-oxopentanoate methyltransferase;
B. 4,5-dihydroxy-3-methyl-2-oxopentanoate kinase; C.
4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase; D.
4,5-dihydroxy-2-oxopentanoate kinase; and E.
4-hydroxy-2-oxo-5-phosphopentanoate methyltransferase. 2H3M4OP is
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate.
[0034] FIG. 3 shows a schematic depiction of an exemplary pathway
to 2H3M4OP from glyceraldehyde-3-phosphate and pyruvate. G3P is
glyceraldehyde-3-phosphate, DXP is 1-deoxy-D-xylulose-5-phosphate,
2ME4P is C-methyl-D-erythritol-4-phosphate and 2H3M4OP is
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. In steps A-C, the
enzymes can be: A. DXP synthase; B. DXP reductoisomerase; and C.
2ME4P dehydratase.
[0035] FIG. 4 shows a schematic depiction of an exemplary alternate
shikimate pathway to p-toluate. In steps A-H, the enzymes can be:
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.
[0036] FIG. 5 shows a schematic depiction of 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.
[0037] FIG. 6 depicts an exemplary process for preparing bio-based
terephthalic acid.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The present invention is directed to, at least in part, the
design and production of cells and organisms having biosynthetic
production capabilities for 2H3M4OP, p-toluate or terephthalate.
The results described herein indicate that metabolic pathways can
be designed and recombinantly engineered to achieve the
biosynthesis of 2H3M4OP, p-toluate or terephthalate in Escherichia
coli and other cells or organisms. Biosynthetic production of
2H3M4OP, p-toluate or terephthalate 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 2H3M4OP,
p-toluate or terephthalate biosynthesis, including under conditions
approaching theoretical maximum growth.
[0039] 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 can be utilized to
accept the alternate substrate, 2H3M4OP and transform it to
p-toluate. In addition, various pathway enzymes can be used to
synthesize the 2H3M4OP precursor from erythrose-4-phosphate using
enzymes for P5C biosynthesis and enzymes analogous to the
isoleucine biosynthesis pathway, or alternatively from
4,5-dihydroxy-2-oxopentanoate using a methyl transferase, a kinase
and a decarboxylase. Synthesis of the 2H3M4OP precursor from
glyceraldehydes-3-phosphate and pyruvate can also be done using
enzymes from the non-mevalonate pathway for isoprenoid
biosynthesis.
[0040] Disclosed herein are strategies for engineering a
microorganism to produce renewable p-toluate or terephthalate (PTA)
from carbohydrate feedstocks. The substrate
4,5-dihydroxy-2-oxopentanoate is naturally derived from sugars such
as arabinose and xylose. Additionally, this substrate can be formed
enzymatically by condensation of pyruvate and glycolaldehyde by
aldolase enzymes such as 2-dehydro-3-deoxypentonate aldolase,
2-dehydro-3-deoxyglucarate aldolase, or other enzymes in EC class
4.1.2 or 4.1.3. The substrate erythrose-4-phosphate is an
intermediate in the pentose phosphate pathway and the Calvin cycle.
Additionally, erythrose-4-phosphate can serve as a precursor to the
biosynthesis of the aromatic amino acids tyrosine, phenylalanine
and tryptophan. First, erythrose-4-phosphate can be converted to
2H3M4OP in six enzymatic steps (see Example I and FIG. 1). In one
alternative, 4,5-dihydroxy-2-oxopentanoate is converted to 2H3M4OP
using one or both of the pathways described in Example I and FIG.
2. In another alternative, glyceraldehyde-3-phosphate (G3P) and
pyruvate are converted to 2H3M4OP in three enzymatic steps (see
Example II and FIG. 3). The 2H3M4OP intermediate can be
subsequently transformed to p-toluate by enzymes in the shikimate
pathway (see Example III and FIG. 4). p-Toluate can be further
converted to PTA (terephthalate) by a microorganism (see Example IV
and FIG. 5).
[0041] The purification method of the invention, e.g. exemplified
in FIG. 6, can also be applied to biosynthetic pathways for
aromatic carboxylic acid and terephthalic acid production described
in WIPO patent publications WO/2009/120457A2 entitled "Bio-Based
Polyethylene Terephthalate Polymer And Method Of Making The Same",
WO/2011/094131A1 entitled "Microorganisms And Methods For The
Biosynthesis Of P-Toluate And Terephthalate", and WO/2011/017560A1
entitled "Semi-Synthetic Terephthalic Acid Via Microorganisms That
Produce Muconic Acid" and U.S. Pat. No. 6,461,840 entitled
"Terephthalic acid producing proteobacteria" and U.S. Pat. No.
6,187,569 entitled "Microbial production of terephthalic acid and
isophthalic acid."
[0042] The maximum theoretical PTA yield from glucose via the
proposed erythrose-4-phosphate pathway in FIG. 1, in conjunction
with the pathways from 2H3M4OP to PTA in FIGS. 4 and 5, is 0.6
moles of PTA per mole of glucose utilized (0.55 g/g). Increasing
product yields to 0.61 mol/mol (0.56 g/g) is possible if cells are
capable of fixing CO.sub.2 through pathways such as the reductive
TCA cycle or the Wood-Ljungdahl pathway.
[0043] The maximum theoretical PTA yield from xylose via the
4,5-dihydroxy-2-oxopentanoate pathway of FIG. 2 is 0.46 moles PTA
per mole xylose utilized (0.51 g/g).
[0044] 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+2H.sup.+.fwdarw.p-Toluate+4 Pi+ADP+2
NAD(P).sup.++CO.sub.2+H.sub.2O
One equivalent of CO.sub.2 is generated in this net reaction.
[0045] 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.
[0046] If p-toluate is further converted to PTA by enzymes as
described in Example IV, 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+2H.sup.+
[0047] Enzyme candidates for catalyzing each step of the proposed
pathways are described in the following sections.
[0048] 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
muconate, 2H3M4OP, p-toluate and/or terephthalate biosynthetic
pathway.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] As used herein, the term "p-toluate," having the molecular
formula C.sub.8H.sub.7O.sub.2.sup.- (see FIG. 4, 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.
[0054] As used herein, the term "terephthalate," having the
molecular formula C.sub.8H.sub.4O.sub.4.sup.-2 (see FIG. 5,
compound 4)(IUPAC name terephthalate) is the ionized form or
dianionic form of terephthalic acid, also referred to as p-phthalic
acid or PTA, depicted below.
##STR00001##
Those skilled in the art will understand that occurrences of
terephthalate and terephthalic acid in solution will depend on pH,
moreover the terms "terephthalate" and "terephthalic acid," unless
otherwise indicated in the context that they are used, can be used
interchangeably throughout to refer to the compound in any of its
neutral or ionized forms, including any salt forms thereof and are
not intended to be limiting to one specific form, e.g., its neutral
or acid form, or ionized forms, including any salt forms thereof.
As terephthalic acid is a dicarboxylic acid, it can exist in a
partially protonated monoacid form or a fully protonated diacid
form, dependent on the pH. Unless otherwise specified, as used
herein "terephthalic acid" will refer to the fully protonated
diacid form.
[0055] As used herein, the term "bio-based" means derived from or
synthesized by a biological organism and can be considered a
renewable resource since it can be generated by a biological
organism. Such a biological organism, in particular the microbial
organisms of the invention disclosed herein, can utilize feedstock
or biomass, such as sugars, glycerol or carbohydrates obtained from
an agricultural, plant, bacterial, or animal source. Alternatively,
the biological organism can utilize atmospheric carbon, carbon
dioxide, formate, methane, methanol, carbon in the form of syngas
or a carbon source generated from electrochemical conversion of
carbon dioxide.
[0056] As used herein, the term "bio-derived" means a product as
described herein that is composed, in whole or in part, of a
bio-based compound of the invention. A bio-derived or bio-based
product is in contrast to a petroleum derived product, wherein such
a product is derived from or synthesized from petroleum or a
petrochemical feedstock.
[0057] As used herein, the term "muconate" is an ionized or anionic
form of muconic acid depicted below.
##STR00002##
Those skilled in the art will understand that occurrences of
muconate and muconic acid in solution will depend on pH, moreover
the terms "muconate" and "muconic acid," unless otherwise indicated
in the context that they are used, are not intended to be limiting
to one specific form, e.g., its neutral or acid form, or ionized
forms, including any salt forms thereof.
[0058] As used herein, the term "aromatic carboxylic acid" refers
to a compound that contains one or more carboxylate (COOH) groups,
bonded to an aromatic ring. Examples of aromatic carboxylic acids
include benzoic acid, salicylic acid, gallic acid, o-toluic acid,
m-toluic acid, p-toluic acid, phthalic acid, isopthalic acid, and
terephthalic acid, as depicted below.
##STR00003##
[0059] Those skilled in the art will understand that certain
aromatic carboxylic acids are mono carboxylic acids (monoacids),
such as p-toluic acid, whereas certain aromatic carboxylic acids
are di carboxylic acids (diacids), such as terephthalic acid.
[0060] As used herein, the term "aromatic carboxylate anion" refers
to the conjugate base of the aromatic carboxylic acid. Those
skilled in the art will understand that when a carboxyl group is
deprotonated, the carboxylate anion is formed. Those skilled in the
art will further understand that the specific form of the aromatic
carboxylic acid (i.e., whether protonated as an acid, or
deprotonated as an anion) will depend on the pH.
[0061] As used herein, the term "p-toluate" is the ionized or
anionic form of p-toluic acid, as depicted below.
##STR00004##
Those skilled in the art will understand that occurrences of
p-toluate and p-toluic acid in solution will depend on pH, moreover
the terms "p-toluate" and "p-toluic acid," unless otherwise
indicated in the context that they are used, are not intended to be
limiting to one specific form, e.g., its neutral or acid form, or
ionized forms, including any salt forms thereof.
[0062] As used herein, the terms "about" or "approximately" means
an acceptable error for a particular value as determined by those
of skill in the art, which depends in part on how the value is
measured or determined. In certain embodiments, the terms "about"
or "approximately" means within 1, 2, 3, or 4 standard deviations.
In certain embodiments, the term "about" or "approximately" means
within above or below 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,
2%, 1%, 0.5%, or 0.05% of a given value or range.
[0063] 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.
[0064] "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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] Therefore, in identifying and constructing the non-naturally
occurring microbial organisms of the invention having 2H3M4OP,
p-toluate or terephthalate 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.
[0073] 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.
[0074] 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.
[0075] In one embodiment, the invention provides a non-naturally
occurring microbial organism having a 2H3M4OP pathway and including
at least one exogenous nucleic acid encoding a 2H3M4OP pathway
enzyme expressed in a sufficient amount to produce 2H3M4OP. The
2H3M4OP pathway of the microbial organism can include a pathway
selected from: (1) 1A, 1B, 1C, 1D, 1E and 1F; (2) 2A, 2B and 2C;
and (3) 2D, 2E and 2C, wherein 1A is an erythrose-4-phosphate
dehydrogenase, wherein 1B is a 4-phosphoerythronate dehydrogenase,
wherein 1C is a 2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase,
wherein 1D is a 2-acetyl-2,3-dihydroxy-4-phosphobutanoate
reductoisomerase, wherein 1E is a
2,3,4-trihydroxy-3-methyl-5-phosphopentanoate dehydratase, wherein
1F is a 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase,
wherein 2A is a 4,5-dihydroxy-2-oxopentanoate methyltransferase,
wherein 2B is a 4,5-dihydroxy-3-methyl-2-oxopentanoate kinase,
wherein 2C is a 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate
decarboxylase, wherein 2D is a 4,5-dihydroxy-2-oxopentanoate
kinase, wherein 2E is a 4-hydroxy-2-oxo-5-phosphopentanoate
methyltransferase (see FIGS. 1 and 2 and Example I). Additionally,
in some aspects, the microbial organism of the invention can
include two, three, four, five or six exogenous nucleic acids,
wherein each exogenous nucleic acid encodes a 2H3M4OP pathway
enzyme as described herein. In some aspects, the invention provides
a microbial organism of the invention having exogenous nucleic
acids encoding each of the 2H3M4OP pathway enzymes of at least one
of the 2H3M4OP pathways selected from (1)-(3), as described
above.
[0076] In some aspects, the invention provides the 2H3M4OP pathway
of the microbial organisms includes a
2-C-methyl-D-erythritol-4-phosphate dehydratase (see Example II and
FIG. 3, step C). A non-naturally occurring microbial organism
having a 2H3M4OP pathway can further have a
1-deoxyxylulose-5-phosphate synthase or a
1-deoxy-D-xylulose-5-phosphate reductoisomerase (see Example II and
FIG. 3, steps A and B). Thus, a 2H3M4OP pathway can include a
2-C-methyl-D-erythritol-4-phosphate dehydratase, a
1-deoxyxylulose-5-phosphate synthase and a
1-deoxy-D-xylulose-5-phosphate reductoisomerase.
[0077] The invention also provides a non-naturally occurring
microbial organism having a 2H3M4OP pathway as described herein
and/or a p-toluate pathway. In this aspect, the p-toluate pathway
can include at least one exogenous nucleic acid encoding a
p-toluate pathway enzyme expressed in a sufficient amount to
produce p-toluate. In some aspects, the p-toluate pathway includes
4A, 4B, 4C, 4D, 4E, 4F, 4G and/or 4H, wherein 4A is a
2-dehydro-3-deoxyphosphoheptonate synthase; wherein 4B is a
3-dehydroquinate synthase; wherein 4C is a 3-dehydroquinate
dehydratase; wherein 4D is a shikimate dehydrogenase; wherein 4E is
a shikimate kinase; wherein 4F is a
3-phosphoshikimate-2-carboxyvinyltransferase; wherein 4G is a
chorismate synthase and wherein 4H is a chorismate lyase (see
Example III and FIG. 4, steps A-H). A non-naturally occurring
microbial organism having a p-toluate pathway can further include a
2H3M4OP pathway as described herein (see Examples I and II and
FIGS. 1-3). For example, a 2H3M4OP pathway can include a pathway
selected from: (1) 1A, 1B, 1C, 1D, 1E and 1F; (2) 2A, 2B and 2C;
and (3) 2D, 2E and 2C, wherein 1A is an erythrose-4-phosphate
dehydrogenase, wherein 1B is a 4-phosphoerythronate dehydrogenase,
wherein 1C is a 2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase,
wherein 1D is a 2-acetyl-2,3-dihydroxy-4-phosphobutanoate
reductoisomerase, wherein 1E is a
2,3,4-trihydroxy-3-methyl-5-phosphopentanoate dehydratase, wherein
1F is a 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase,
wherein 2A is a 4,5-dihydroxy-2-oxopentanoate methyltransferase,
wherein 2B is a 4,5-dihydroxy-3-methyl-2-oxopentanoate kinase,
wherein 2C is a 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate
decarboxylase, wherein 2D is a 4,5-dihydroxy-2-oxopentanoate
kinase, wherein 2E is a 4-hydroxy-2-oxo-5-phosphopentanoate
methyltransferase (see FIGS. 1 and 2 and Example I). Alternatively,
a 2H3M4OP pathway can include a 2-C-methyl-D-erythritol-4-phosphate
dehydratase, a 1-deoxyxylulose-5-phosphate synthase and/or a
1-deoxy-D-xylulose-5-phosphate reductoisomerase (see FIG. 3 and
Example II).
[0078] In some aspects, the microbial organism of the invention
includes two, three, four, five, six, seven or eight exogenous
nucleic acids, wherein each nucleic acid encodes a p-toluate
pathway enzyme. Additionally, in some aspects, the invention
provides that the microbial organism of the invention includes
exogenous nucleic acids encoding each of the enzymes of the
p-toluate pathway disclosed herein.
[0079] The invention additionally provides a non-naturally
occurring microbial organism having a 2H3M4OP pathway and/or a
p-toluate pathway as disclosed herein and/or a terephthalate
pathway. In this aspect, the terephthalate pathway can include at
least one exogenous nucleic acid encoding a terephthalate pathway
enzyme expressed in a sufficient amount to produce terephthalate.
In some aspects of the invention, the terephthalate pathway can
include 5A, 5B and 5C, wherein 5A is a p-toluate
methyl-monooxygenase reductase, wherein 5B is a 4-carboxybenzyl
alcohol dehydrogenase and wherein 5C is a 4-carboxybenzyl aldehyde
dehydrogenase (see Example IV and FIG. 5). Such an organism
containing a terephthalate pathway can additionally include a
p-toluate pathway, wherein the p-toluate pathway includes 4A, 4B,
4C, 4D, 4E, 4F, 4G and/or 4H, wherein 4A is a
2-dehydro-3-deoxyphosphoheptonate synthase; wherein 4B is a
3-dehydroquinate synthase; wherein 4C is a 3-dehydroquinate
dehydratase; wherein 4D is a shikimate dehydrogenase; wherein 4E is
a shikimate kinase; wherein 4F is a
3-phosphoshikimate-2-carboxyvinyltransferase; wherein 4G is a
chorismate synthase and wherein 4H is a chorismate lyase (see
Examples III and IV and FIGS. 4 and 5). Such a non-naturally
occurring microbial organism having a terephthalate pathway and a
p-toluate pathway can further include a 2H3M4OP pathway as
described herein (see Examples I and II and FIGS. 1-3). For
example, a 2H3M4OP pathway can include a pathway selected from: (1)
1A, 1B, 1C, 1D, 1E and 1F; (2) 2A, 2B and 2C; and (3) 2D, 2E and
2C, wherein 1A is an erythrose-4-phosphate dehydrogenase, wherein
1B is a 4-phosphoerythronate dehydrogenase, wherein 1C is a
2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase, wherein 1D is a
2-acetyl-2,3-dihydroxy-4-phosphobutanoate reductoisomerase, wherein
1E is a 2,3,4-trihydroxy-3-methyl-5-phosphopentanoate dehydratase,
wherein 1F is a 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate
decarboxylase, wherein 2A is a 4,5-dihydroxy-2-oxopentanoate
methyltransferase, wherein 2B is a
4,5-dihydroxy-3-methyl-2-oxopentanoate kinase, wherein 2C is a
4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase, wherein
2D is a 4,5-dihydroxy-2-oxopentanoate kinase, wherein 2E is a
4-hydroxy-2-oxo-5-phosphopentanoate methyltransferase (see FIGS. 1
and 2 and Example I). Alternatively, a 2H3M4OP pathway can include
a 2-C-methyl-D-erythritol-4-phosphate dehydratase, a
1-deoxyxylulose-5-phosphate synthase and/or a
1-deoxy-D-xylulose-5-phosphate reductoisomerase (see FIG. 3 and
Example II).
[0080] In some aspects, the microbial organism of the invention
includes two or three exogenous nucleic acids, wherein each nucleic
acid encodes a terephthalate pathway enzyme. Additionally, in some
aspects, the invention provides that the microbial organism of the
invention includes exogenous nucleic acids encoding each of the
enzymes of the terephthalate pathway disclosed herein.
[0081] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having a 2H3M4OP,
p-toluate and/or terephthalate 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 2H3M4OP pathway, the
substrates and products can be selected from the group consisting
of erythrose-4-phosphate to 4-phosphoerythronate;
4-phosphoerythronate to 2-oxo-3-hydroxy-4-phosphobutanoate;
2-oxo-3-hydroxy-4-phosphobutanoate to
2-acetyl-2,3-dihydroxy-4-phosphobutanoate;
2-acetyl-2,3-dihydroxy-4-phosphobutanoate to
2,3,4-trihydroxy-3-methyl-5-phosphopentanoate;
2,3,4-trihydroxy-3-methyl-5-phosphopentanoate to
4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate;
4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate to
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate;
4,5-dihydroxy-2-oxopentanoate to
4,5-dihydroxy-3-methyl-2-oxopentanoate;
4,5-dihydroxy-2-oxopentanoate to
4-hydroxy-2-oxo-5-phosphopentanoate;
4-hydroxy-2-oxo-5-phosphopentanoate to
4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate;
4,5-dihydroxy-3-methyl-2-oxopentanoate to
4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate;
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 Examples I and II
and FIGS. 1-3). 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 2H3M4OP, p-toluate or terephthalate pathway, such as
that shown in FIGS. 1-5.
[0082] While generally described herein as a microbial organism
that contains a 2H3M4OP, p-toluate or terephthalate pathway, it is
understood that the invention additionally provides a non-naturally
occurring microbial organism comprising at least one exogenous
nucleic acid encoding a 2H3M4OP, p-toluate or terephthalate pathway
enzyme expressed in a sufficient amount to produce an intermediate
of a 2H3M4OP, p-toluate or terephthalate pathway. For example, as
disclosed herein, a 2H3M4OP, p-toluate or terephthalate pathway is
exemplified in FIGS. 1-5. Therefore, in addition to a microbial
organism containing a 2H3M4OP, p-toluate or terephthalate pathway
that produces 2H3M4OP, p-toluate or terephthalate, the invention
additionally provides a non-naturally occurring microbial organism
comprising at least one exogenous nucleic acid encoding a 2H3M4OP,
p-toluate or terephthalate pathway enzyme, where the microbial
organism produces a 2H3M4OP, p-toluate or terephthalate pathway
intermediate, for example, 4-phosphoerythronate,
2-oxo-3-hydroxy-4-phosphobutanoate, 2-acetyl-2,3-phosphodutanoate,
2-acetyl-2,3-dihydroxy-4-phosphobutanoate,
2,3,4-trihydroxy-3-methyl-5-phosphopentanoate,
4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate,
4,5-dihydroxy-3-methyl-2-oxopentanoate,
4-hydroxy-2-oxo-5-phosphopentanoate,
1-deoxy-D-xylulose-5-phosphate, C-methyl-D-erythritol-4-phosphate,
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,
3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate-
, 4-carboxybenzyl alcohol or 4-carboxybenzaldehyde.
[0083] 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. 1-5, 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 2H3M4OP, p-toluate or terephthalate pathway
intermediate can be utilized to produce the intermediate as a
desired product.
[0084] 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.
[0085] As disclosed herein, the product p-toluate or terephthalate
or the intermediate 4-phosphoerythronate,
2-oxo-3-hydroxy-4-phosphobutanoate, 2-acetyl-2,3-phosphodutanoate,
2-acetyl-2,3-dihydroxy-4-phosphobutanoate,
2,3,4-trihydroxy-3-methyl-5-phosphopentanoate,
4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate,
4,5-dihydroxy-3-methyl-2-oxopentanoate,
4-hydroxy-2-oxo-5-phosphopentanoate,
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,
3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate-
, 4-carboxybenzyl alcohol or 4-carboxybenzaldehyde, as well as
other intermediates, are carboxylic acids, which can occur in
various ionized forms, including fully protonated, partially
protonated, and fully deprotonated forms. Accordingly, the suffix
"-ate," or the acid form, can be used interchangeably to describe
both the free acid form as well as any deprotonated form, in
particular since the ionized form is known to depend on the pH in
which the compound is found. It is understood that carboxylate
products or intermediates includes ester forms of carboxylate
products or pathway intermediates, such as O-carboxylate and
S-carboxylate esters. O- and S-carboxylates can include lower
alkyl, that is C1 to C6, branched or straight chain carboxylates.
Some such O- or S-carboxylates include, without limitation, methyl,
ethyl, n-propyl, n-butyl, i-propyl, sec-butyl, and tert-butyl,
pentyl, hexyl O- or S-carboxylates, any of which can further
possess an unsaturation, providing for example, propenyl, butenyl,
pentyl, and hexenyl O- or S-carboxylates. O-carboxylates can be the
product of a biosynthetic pathway. Exemplary O-carboxylates
accessed via biosynthetic pathways can include, without limitation,
methyl terephthalate, ethyl terephthalate, and n-propyl
terephthalate. Other biosynthetically accessible O-carboxylates can
include medium to long chain groups, that is C7-C22, O-carboxylate
esters derived from fatty alcohols, such heptyl, octyl, nonyl,
decyl, undecyl, lauryl, tridecyl, myristyl, pentadecyl, cetyl,
palmitolyl, heptadecyl, stearyl, nonadecyl, arachidyl, heneicosyl,
and behenyl alcohols, any one of which can be optionally branched
and/or contain unsaturations. O-carboxylate esters can also be
accessed via a biochemical or chemical process, such as
esterification of a free carboxylic acid product or
transesterification of an O- or S-carboxylate. S-carboxylates are
exemplified by CoA S-esters, cysteinyl S-esters, alkylthioesters,
and various aryl and heteroaryl thioesters.
[0086] 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 2H3M4OP, p-toluate or terephthalate biosynthetic
pathways. Depending on the host microbial organism chosen for
biosynthesis, nucleic acids for some or all of a particular
2H3M4OP, p-toluate or terephthalate 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 2H3M4OP, p-toluate or terephthalate 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 2H3M4OP,
p-toluate or terephthalate.
[0087] 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 or suitable to fermentation processes.
Exemplary bacteria include any species selected from the order
Enterobacteriales, family Enterobacteriaceae, including the genera
Escherichia and Klebsiella; the order Aeromonadales, family
Succinivibrionaceae, including the genus Anaerobiospirillum; the
order Pasteurellales, family Pasteurellaceae, including the genera
Actinobacillus and Mannheimia; the order Rhizobiales, family
Bradyrhizobiaceae, including the genus Rhizobium; the order
Bacillales, family Bacillaceae, including the genus Bacillus; the
order Actinomycetales, families Corynebacteriaceae and
Streptomycetaceae, including the genus Corynebacterium and the
genus Streptomyces, respectively; order Rhodospirillales, family
Acetobacteraceae, including the genus Gluconobacter; the order
Sphingomonadales, family Sphingomonadaceae, including the genus
Zymomonas; the order Lactobacillales, families Lactobacillaceae and
Streptococcaceae, including the genus Lactobacillus and the genus
Lactococcus, respectively; the order Clostridiales, family
Clostridiaceae, genus Clostridium; and the order Pseudomonadales,
family Pseudomonadaceae, including the genus Pseudomonas.
Non-limiting species of host bacteria include 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.
[0088] Similarly, exemplary species of yeast or fungi species
include any species selected from the order Saccharomycetales,
family Saccaromycetaceae, including the genera Saccharomyces,
Kluyveromyces and Pichia; the order Saccharomycetales, family
Dipodascaceae, including the genus Yarrowia; the order
Schizosaccharomycetales, family Schizosaccaromycetaceae, including
the genus Schizosaccharomyces; the order Eurotiales, family
Trichocomaceae, including the genus Aspergillus; and the order
Mucorales, family Mucoraceae, including the genus Rhizopus.
Non-limiting species of host yeast or fungi include Saccharomyces
cerevisiae, Schizosaccharomyces pombe, Candida albicans,
Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus,
Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizopus
oryzae, Yarrowia lipolytica, and the like. Preferred 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, as disclosed in U.S. Patent Publication No.
US 2011/0207185 A1. E. coli is a particularly useful and preferred
host organism 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.
[0089] Depending on the 2H3M4OP, p-toluate or terephthalate
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 2H3M4OP,
p-toluate or terephthalate pathway-encoding nucleic acid and up to
all encoding nucleic acids for one or more 2H3M4OP, p-toluate or
terephthalate biosynthetic pathways. For example, 2H3M4OP,
p-toluate or terephthalate 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 2H3M4OP, p-toluate or
terephthalate 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 2H3M4OP, p-toluate or terephthalate can
be included. A non-limiting example of all enzymes in a p-toluate
pathway includes a 2-dehydro-3-deoxyphosphoheptonate synthase; a
3-dehydroquinate synthase; a 3-dehydroquinate dehydratase; a
shikimate dehydrogenase; shikimate kinase; a
3-phosphoshikimate-2-carboxyvinyltransferase; a chorismate
synthase; and a chorismate lyase. In addition, a non-limiting
example of all enzymes in a terephthalate pathway included a
p-toluate methyl-monooxygenase reductase; a 4-carboxybenzyl alcohol
dehydrogenase; and a 4-carboxybenzyl aldehyde dehydrogenase.
Furthermore, a non-limiting example of all enzymes in a 2H3M4OP
pathway include an erythrose-4-phosphate dehydrogenase, a
4-phosphoerythronate dehydrogenase, a
2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase, a
2-acetyl-2,3-dihydroxy-4-phosphobutanoate reductoisomerase, a
2,3,4-trihydroxy-3-methyl-5-phosphopentanoate dehydratase and a
4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase.
[0090] 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 2H3M4OP, p-toluate or terephthalate 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, eight, nine, ten,
eleven, twelve, thirteen, fourteen, fifteen, sixteen or seventeen,
up to all nucleic acids encoding the enzymes or proteins
constituting a 2H3M4OP, p-toluate or terephthalate biosynthetic
pathway disclosed herein. In some embodiments, the non-naturally
occurring microbial organisms also can include other genetic
modifications that facilitate or optimize 2H3M4OP, p-toluate or
terephthalate 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 2H3M4OP, p-toluate or terephthalate pathway precursors such
as erythrose-4-phosphate, 4,5-dihydroxy-2-oxopentanoate,
glyceraldehyde-3-phosphate and pyruvate.
[0091] Generally, a host microbial organism is selected such that
it produces the precursor of a 2H3M4OP, p-toluate or terephthalate
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,
erythrose-4-phosphate, 4,5-dihydroxy-2-oxopentanoate,
glyceraldehyde-3-phosphate and pyruvate 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 2H3M4OP, p-toluate
or terephthalate pathway.
[0092] In some embodiments, a non-naturally occurring microbial
organism of the invention is generated from a host that contains
the enzymatic capability to synthesize 2H3M4OP, p-toluate or
terephthalate. In this specific embodiment it can be useful to
increase the synthesis or accumulation of a 2H3M4OP, p-toluate or
terephthalate pathway product to, for example, drive 2H3M4OP,
p-toluate or terephthalate pathway reactions toward 2H3M4OP,
p-toluate or terephthalate production. Increased synthesis or
accumulation can be accomplished by, for example, overexpression of
nucleic acids encoding one or more of the above-described 2H3M4OP,
p-toluate or terephthalate pathway enzymes or proteins.
Overexpression of the enzyme or enzymes and/or protein or proteins
of the 2H3M4OP, p-toluate or terephthalate 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 2H3M4OP, p-toluate or
terephthalate, through overexpression of one, two, three, four,
five one, two, three, four, five, six, seven, eight, nine, ten,
eleven, twelve, thirteen, fourteen, fifteen, sixteen or seventeen,
that is, up to all nucleic acids encoding 2H3M4OP, p-toluate or
terephthalate 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 2H3M4OP, p-toluate or terephthalate
biosynthetic pathway.
[0093] 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.
[0094] 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 2H3M4OP, p-toluate or terephthalate
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 2H3M4OP, p-toluate or
terephthalate biosynthetic capability. For example, a non-naturally
occurring microbial organism having a 2H3M4OP, p-toluate or
terephthalate biosynthetic pathway can comprise at least two
exogenous nucleic acids encoding desired enzymes or proteins, such
as the combination of a erythrose-4-phosphate dehydrogenase and a
4-phosphoerythronate dehydrogenase, or alternatively a
2-acetyl-2,3-dihydroxy-4-phosphobutanoate reductoisomerase and a
4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase, or
alternatively a 4,5-dihydroxy-3-methyl-2-oxopentanoate kinase and a
4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase, or
alternatively a 2-dehydro-3-deoxyphosphoheptonate synthase and a
shikimate kinase, or alternatively a 4-carboxybenzyl alcohol
dehydrogenase and a 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, a 2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase, a
2-acetyl-2,3-dihydroxy-4-phosphobutanoate reductoisomerase and a
2,3,4-trihydroxy-3-methyl-5-phosphopentanoate dehydratase, or
alternatively a 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate
decarboxylase, a 4,5-dihydroxy-2-oxopentanoate kinase and a
4-hydroxy-2-oxo-5-phosphopentanoate methyltransferase, or
alternatively a shikimate dehydrogenase, a shikimate kinase and a
3-phosphoshikimate-2-carboxyvinyltransferase, 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, eight, nine, ten, eleven, twelve, thirteen,
fourteen, fifteen, sixteen or seventeen or more enzymes or proteins
of a biosynthetic 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.
[0095] In addition to the biosynthesis of 2H3M4OP, p-toluate or
terephthalate 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 2H3M4OP, p-toluate or terephthalate other than use of
the 2H3M4OP, p-toluate or terephthalate producers is through
addition of another microbial organism capable of converting a
2H3M4OP, p-toluate or terephthalate pathway intermediate to
2H3M4OP, p-toluate or terephthalate. One such procedure includes,
for example, the fermentation of a microbial organism that produces
a 2H3M4OP, p-toluate or terephthalate pathway intermediate. The
2H3M4OP, p-toluate or terephthalate pathway intermediate can then
be used as a substrate for a second microbial organism that
converts the 2H3M4OP, p-toluate or terephthalate pathway
intermediate to 2H3M4OP, p-toluate or terephthalate. The 2H3M4OP,
p-toluate or terephthalate pathway intermediate can be added
directly to another culture of the second organism or the original
culture of the 2H3M4OP, p-toluate or terephthalate 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.
[0096] 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,
2H3M4OP, p-toluate or terephthalate. 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 2H3M4OP, p-toluate or terephthalate 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,
2H3M4OP, p-toluate or terephthalate 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 2H3M4OP intermediate, p-toluate
intermediate or terephthalate intermediate and the second microbial
organism converts the intermediate to 2H3M4OP, p-toluate or
terephthalate.
[0097] 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 2H3M4OP, p-toluate or
terephthalate.
[0098] Sources of encoding nucleic acids for a 2H3M4OP, p-toluate
or terephthalate 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, Arabidopsis thaliana, Azospirillum brasilense,
Bacillus subtilis, Bacteroides fragilis, Bos taurus, Bradyrhizobium
japonicum USDA110, Burkholderia ambifaria, Burkholderia
cenocepacia, Corynebacterium glutamicum, Homo sapiens, Lactococcus
lactis, Mesorhizobium loti, Methanococcus aeolicus, Mycobacterium
tuberculosis, Neurospora crassa, Pseudomonas aeruginosa,
Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri,
Pseudomonas syringae, Ralstonia eutropha, Rattus norvegicus,
Saccharomyces cerevisiae, Salmonella typhimurium, Sinorhizobium
melitoti, Streptomyces coelicolor, Streptomyces fradiae,
Streptomyces luridus, Streptomyces roseosporus, Streptomyces
viridochromogenes, Streptomyces wedmorensis, Sulfolobus
solfataricus, Synechococcus sp. PCC 7002, Thermoproteus tenax,
Vibrio cholera, Xanthomonas oryzae and Zymomonas mobilis, as well
as other exemplary species disclosed herein or available as source
organisms for corresponding genes. Exemplary species for such
sources further include, for example, Escherichia coli, as well as
other exemplary species disclosed in U.S. Patent Publication No. US
2011/0207185 A1. 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 2H3M4OP, p-toluate or terephthalate 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
2H3M4OP, p-toluate or terephthalate 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.
[0099] In some instances, such as when an alternative 2H3M4OP,
p-toluate or terephthalate biosynthetic pathway exists in an
unrelated species, 2H3M4OP, p-toluate or terephthalate 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 2H3M4OP,
p-toluate or terephthalate.
[0100] Methods for constructing and testing the expression levels
of a non-naturally occurring 2H3M4OP, p-toluate or terephthalate
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).
[0101] Exogenous nucleic acid sequences involved in a pathway for
production of 2H3M4OP, p-toluate or terephthalate 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.
[0102] An expression vector or vectors can be constructed to
include one or more muconate, 2H3M4OP, p-toluate or terephthalate
biosynthetic pathway encoding nucleic acids as exemplified herein
operably linked to expression control sequences functional in the
host organism. An expression vector or vectors can be constructed
to include one or more muconate biosynthetic pathway encoding
nucleic acids as exemplified herein operably linked to expression
control sequences functional in the host organism, as disclosed in
U.S. Patent Publication No. US 2011/0207185 A1. 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.
[0103] The invention additionally provides a method for producing
2H3M4OP, by culturing the non-naturally occurring microbial
organism containing a 2H3M4OP pathway under conditions and for a
sufficient period of time to produce 2H3M4OP. Such a microbial
organism can have a 2H3M4OP pathway and include at least one
exogenous nucleic acid encoding a 2H3M4OP pathway enzyme expressed
in a sufficient amount to produce 2H3M4OP. The 2H3M4OP pathway of
the microbial organism can include a pathway selected from: (1) 1A,
1B, 1C, 1D, 1E and 1F; (2) 2A, 2B and 2C; and (3) 2D, 2E and 2C,
wherein 1A is an erythrose-4-phosphate dehydrogenase, wherein 1B is
a 4-phosphoerythronate dehydrogenase, wherein 1C is a
2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase, wherein 1D is a
2-acetyl-2,3-dihydroxy-4-phosphobutanoate reductoisomerase, wherein
1E is a 2,3,4-trihydroxy-3-methyl-5-phosphopentanoate dehydratase,
wherein 1F is a 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate
decarboxylase, wherein 2A is a 4,5-dihydroxy-2-oxopentanoate
methyltransferase, wherein 2B is a
4,5-dihydroxy-3-methyl-2-oxopentanoate kinase, wherein 2C is a
4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase, wherein
2D is a 4,5-dihydroxy-2-oxopentanoate kinase, wherein 2E is a
4-hydroxy-2-oxo-5-phosphopentanoate methyltransferase (see FIGS. 1
and 2 and Example I). Additionally, in some aspects, the microbial
organism cultured in the methods of the invention can include two,
three, four, five or six exogenous nucleic acids, wherein each
exogenous nucleic acid encodes a 2H3M4OP pathway enzyme as
described herein. In some aspects, the invention provides a method
for producing 2H3M4OP by culturing a microbial organism of the
invention having exogenous nucleic acids encoding each of the
2H3M4OP pathway enzymes of at least one of the 2H3M4OP pathways
selected from (1)-(3), as described above.
[0104] In some aspects, the invention provides the 2H3M4OP pathway
of the microbial organism cultured in the invention methods
includes a 2-C-methyl-D-erythritol-4-phosphate dehydratase (see
Example II and FIG. 3, step C). The non-naturally occurring
microbial organism having a 2H3M4OP pathway can further have a
1-deoxyxylulose-5-phosphate synthase or a
1-deoxy-D-xylulose-5-phosphate reductoisomerase (see Example II and
FIG. 3, steps A and B). Thus, a 2H3M4OP pathway can include a
2-C-methyl-D-erythritol-4-phosphate dehydratase, a
1-deoxyxylulose-5-phosphate synthase and a
1-deoxy-D-xylulose-5-phosphate reductoisomerase.
[0105] The invention also provides a method for producing p-toluate
by culturing the non-naturally occurring microbial organism
containing a 2H3M4OP pathway and/or a p-toluate pathway under
conditions and for a sufficient period of time to produce
p-toluate. Such a microbial organism can include at least one
exogenous nucleic acid encoding a p-toluate pathway enzyme
expressed in a sufficient amount to produce p-toluate. In some
aspects, the p-toluate pathway includes 4A, 4B, 4C, 4D, 4E, 4F, 4G
and/or 4H, wherein 4A is a 2-dehydro-3-deoxyphosphoheptonate
synthase; wherein 4B is a 3-dehydroquinate synthase; wherein 4C is
a 3-dehydroquinate dehydratase; wherein 4D is a shikimate
dehydrogenase; wherein 4E is a shikimate kinase; wherein 4F is a
3-phosphoshikimate-2-carboxyvinyltransferase; wherein 4G is a
chorismate synthase and wherein 4H is a chorismate lyase (see
Example III and FIG. 4, steps A-H). A non-naturally occurring
microbial organism having a p-toluate pathway can further include a
2H3M4OP pathway as described herein (see Examples I and II and
FIGS. 1-3). For example, a 2H3M4OP pathway can include a pathway
selected from: (1) 1A, 1B, 1C, 1D, 1E and 1F; (2) 2A, 2B and 2C;
and (3) 2D, 2E and 2C, wherein 1A is an erythrose-4-phosphate
dehydrogenase, wherein 1B is a 4-phosphoerythronate dehydrogenase,
wherein 1C is a 2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase,
wherein 1D is a 2-acetyl-2,3-dihydroxy-4-phosphobutanoate
reductoisomerase, wherein 1E is a
2,3,4-trihydroxy-3-methyl-5-phosphopentanoate dehydratase, wherein
1F is a 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase,
wherein 2A is a 4,5-dihydroxy-2-oxopentanoate methyltransferase,
wherein 2B is a 4,5-dihydroxy-3-methyl-2-oxopentanoate kinase,
wherein 2C is a 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate
decarboxylase, wherein 2D is a 4,5-dihydroxy-2-oxopentanoate
kinase, wherein 2E is a 4-hydroxy-2-oxo-5-phosphopentanoate
methyltransferase (see FIGS. 1 and 2 and Example I). Alternatively,
a 2H3M4OP pathway can include a 2-C-methyl-D-erythritol-4-phosphate
dehydratase, a 1-deoxyxylulose-5-phosphate synthase and/or a
1-deoxy-D-xylulose-5-phosphate reductoisomerase (see FIG. 3 and
Example II).
[0106] In some aspects, the microbial organism cultured in the
invention methods include two, three, four, five, six, seven or
eight exogenous nucleic acids, wherein each nucleic acid encodes a
p-toluate pathway enzyme. Additionally, in some aspects, the
invention provides that the microbial organism cultured in the
invention methods includes exogenous nucleic acids encoding each of
the enzymes of the p-toluate pathway disclosed herein.
[0107] The invention also provides a method for producing
terephthalate by culturing the non-naturally occurring microbial
organism containing a 2H3M4OP pathway, a p-toluate pathway and/or a
terephthalate pathway under conditions and for a sufficient period
of time to produce terephthalate. Such a microbial organism can
include at least one exogenous nucleic acid encoding a
terephthalate pathway enzyme expressed in a sufficient amount to
produce terephthalate. In some aspects of the invention, the
terephthalate pathway can include 5A, 5B and 5C, wherein 5A is a
p-toluate methyl-monooxygenase reductase, wherein 5B is a
4-carboxybenzyl alcohol dehydrogenase and wherein 5C is a
4-carboxybenzyl aldehyde dehydrogenase (see Example IV and FIG. 5).
Such an organism containing a terephthalate pathway can
additionally include a p-toluate pathway, wherein the p-toluate
pathway includes 4A, 4B, 4C, 4D, 4E, 4F, 4G and/or 4H, wherein 4A
is a 2-dehydro-3-deoxyphosphoheptonate synthase; wherein 4B is a
3-dehydroquinate synthase; wherein 4C is a 3-dehydroquinate
dehydratase; wherein 4D is a shikimate dehydrogenase; wherein 4E is
a shikimate kinase; wherein 4F is a
3-phosphoshikimate-2-carboxyvinyltransferase; wherein 4G is a
chorismate synthase and wherein 4H is a chorismate lyase (see
Examples III and IV and FIGS. 4 and 5). Such a non-naturally
occurring microbial organism having a terephthalate pathway and a
p-toluate pathway can further include a 2H3M4OP pathway as
described herein (see Examples I and II and FIGS. 1-3). For
example, a 2H3M4OP pathway can include a pathway selected from: (1)
1A, 1B, 1C, 1D, 1E and 1F; (2) 2A, 2B and 2C; and (3) 2D, 2E and
2C, wherein 1A is an erythrose-4-phosphate dehydrogenase, wherein
1B is a 4-phosphoerythronate dehydrogenase, wherein 1C is a
2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase, wherein 1D is a
2-acetyl-2,3-dihydroxy-4-phosphobutanoate reductoisomerase, wherein
1E is a 2,3,4-trihydroxy-3-methyl-5-phosphopentanoate dehydratase,
wherein 1F is a 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate
decarboxylase, wherein 2A is a 4,5-dihydroxy-2-oxopentanoate
methyltransferase, wherein 2B is a
4,5-dihydroxy-3-methyl-2-oxopentanoate kinase, wherein 2C is a
4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase, wherein
2D is a 4,5-dihydroxy-2-oxopentanoate kinase, wherein 2E is a
4-hydroxy-2-oxo-5-phosphopentanoate methyltransferase (see FIGS. 1
and 2 and Example I). Alternatively, a 2H3M4OP pathway can include
a 2-C-methyl-D-erythritol-4-phosphate dehydratase, a
1-deoxyxylulose-5-phosphate synthase and/or a
1-deoxy-D-xylulose-5-phosphate reductoisomerase (see FIG. 3 and
Example II).
[0108] In some aspects, the microbial organism cultured in the
invention methods includes two or three exogenous nucleic acids,
wherein each nucleic acid encodes a terephthalate pathway enzyme.
Additionally, in some aspects, the invention provides that the
microbial organism cultured in the invention methods includes
exogenous nucleic acids encoding each of the enzymes of the
terephthalate pathway disclosed herein.
[0109] Suitable purification and/or assays to test for the
production of 2H3M4OP, p-toluate or terephthalate 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) and LC-MS (Liquid
Chromatography-Mass 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)).
[0110] The 2H3M4OP, p-toluate or terephthalate 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.
[0111] 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 muconate,
2H3M4OP, p-toluate or terephthalate producers can be cultured for
the biosynthetic production of muconate, 2H3M4OP, p-toluate or
terephthalate. Accordingly, in some embodiments, the invention
provides culture medium having muconate, 2H3M4OP, p-toluate or
terephthalate or a muconate, 2H3M4OP, p-toluate or terephthalate
pathway intermediate described herein. In some aspects, the culture
mediums can also be separated from the non-naturally occurring
microbial organisms of the invention that produced the muconate,
2H3M4OP, p-toluate or terephthalate or muconate, 2H3M4OP, p-toluate
or terephthalate pathway intermediate. Methods for separating a
microbial organism from culture medium are well known in the art.
Exemplary methods include filtration, flocculation, precipitation,
centrifugation, sedimentation, and the like.
[0112] For the production of 2H3M4OP, p-toluate or terephthalate,
the recombinant strains are cultured in a medium with carbon source
and other essential nutrients. It is sometimes desirable and can be
highly 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 or
substantially anaerobic 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. Exemplary cell growth procedures
and fermentations used in the production of a compound of interest,
such as, for example, muconate, 2H3M4OP, p-toluate or
terephthalate, include, batch fermentation, fed-batch fermentation
with batch separation; fed-batch fermentation with continuous
separation, and continuous fermentation with continuous separation.
All of these processes are well known in the art. Depending on the
non-naturally occurring microbial organism's design, the
fermentations can be carried out under aerobic or anaerobic
conditions. In certain embodiments, the temperature of the cultures
are kept between about 30 and about 45.degree. C., including 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, and 44.degree.
C.
[0113] In batch fermentation, a tank fermenter (or bioreactor) is
filled with the prepared media to support growth. The temperature
and pH for microbial fermentation is properly adjusted, and any
additional supplements are added. An inoculum of a producing
non-naturally occurring microbial organism is added to the
fermenter. In batch fermentation the fermentation will generally
run for a fixed period and then the products from the fermentation
are isolated. The process can be repeated in batch runs.
[0114] In fed-batch fermentation fresh media is continuously or
periodically added to the fermentation bioreactor. Fixed-volume
fed-batch fermentation is a type of fed-batch fermentation in which
a carbon source is fed without diluting the culture. The culture
volume can also be maintained nearly constant by feeding the growth
carbon source as a concentrated liquid or gas. In another type of
fixed-volume fed-batch culture, sometimes called a cyclic fed-batch
culture, a portion of the culture is periodically withdrawn and
used as the starting point for a further fed-batch process. Once
the fermentation reaches a certain stage, the culture is removed
and the biomass is diluted to the original volume with sterile
water or medium containing the carbon feed substrate. The dilution
decreases the biomass concentration and results in an increase in
the specific growth rate. Subsequently, as feeding continues, the
growth rate will decline gradually as biomass increases and
approaches the maximum sustainable in the vessel once more, at
which point the culture can be diluted again. Alternatively, a
fed-batch fermentation can be variable volume. In variable-volume
mode the volume of the fermentation broth changes with the
fermentation time as nutrient and media are continually added to
the culture without removal of a portion of the fermentation
broth.
[0115] In a continuous fermentation, fresh media is generally
continually added with continuous separation of spent medium, which
can include the product of interest, such as, for example,
muconate, 2H3M4OP, p-toluate or terephthalate, when the product is
secreted. One feature of the continuous culture is that a
time-independent steady-state can be obtained which enables one to
determine the relations between microbial behavior and the
environmental conditions. Achieving this steady-state is
accomplished by means of a chemostat, or similar bioreactor. A
chemostat allows for the continual addition of fresh medium while
culture liquid is continuously removed to keep the culture volume
constant. By altering the rate at which medium is added to the
chemostat, the growth rate of the non-naturally occurring microbial
organism can be controlled.
[0116] The continuous and/or near-continuous production of a
compound of interest, such as, for example, muconate, 2H3M4OP,
p-toluate or terephthalate, can include culturing a
compound-producing non-naturally occurring microbial organism in
sufficient nutrients and medium to sustain and/or nearly sustain
growth in an exponential phase. Continuous culture under such
conditions can include, for example, 1 day, 2, 3, 4, 5, 6 or 7 days
or more. Additionally, continuous culture can include 1 week, 2, 3,
4 or 5 or more weeks and up to several months. Alternatively,
organisms that produce a compound of interest 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 compound-producing non-naturally occurring microbial
organism is for a sufficient period of time to produce a sufficient
amount of product for a desired purpose.
[0117] In certain embodiments, the culture can be conducted under
aerobic conditions. An oxygen feed to the culture can be
controlled. Oxygen can be supplied as air, enriched oxygen, pure
oxygen or any combination thereof. Methods of monitoring oxygen
concentration are known in the art. Oxygen can be delivered at a
certain feed rate or can be delivered on demand by measuring the
dissolved oxygen content of the culture and feeding accordingly
with the intention of maintaining a constant dissolved oxygen
content.
[0118] Fermentations can be performed under anaerobic conditions.
For example, as explained above, the culture can be rendered
substantially free of oxygen by first sparging the medium with
nitrogen and then sealing culture vessel (e.g., flasks can be
sealed with a septum and crimp-cap). Microaerobic conditions also
can be utilized by providing a small hole for limited aeration. On
a commercial scale, microaerobic conditions are achieved by
sparging a fermentor with air or oxygen as in the aerobic case, but
at a much lower rate and with tightly controlled agitation.
[0119] 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.
[0120] 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.
[0121] 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; glycerol; carbon dioxide; formate; methane;
methanol; or a carbon source generated from electrochemical
conversion of carbon dioxide, such as formate or methanol, alone as
the sole source of carbon or in combination with other carbon
sources described herein or known in the art. 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 2H3M4OP,
p-toluate or terephthalate.
[0122] In addition to renewable feedstocks such as those
exemplified above, the 2H3M4OP, p-toluate or terephthalate
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
2H3M4OP, p-toluate or terephthalate producing organisms to provide
a metabolic pathway for utilization of syngas or other gaseous
carbon source.
[0123] 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.
[0124] 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:
2 CO.sub.2+4 H.sub.2+n ADP+n Pi.fwdarw.CH.sub.3COOH+2H.sub.2O+n
ATP
[0125] 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.
[0126] 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 2H3M4OP, p-toluate or terephthalate
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.
[0127] Additionally, the reductive (reverse) tricarboxylic acid
cycle coupled with carbon monoxide dehydrogenase and/or hydrogenase
activities can also be used for 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 2H3M4OP, p-toluate or
terephthalate 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 2H3M4OP, p-toluate
or terephthalate 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 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 a reductive TCA pathway can confer syngas
utilization ability.
[0128] 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, 2H3M4OP, p-toluate or terephthalate and any of the
intermediate metabolites in the 2H3M4OP, p-toluate or terephthalate
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 2H3M4OP, p-toluate or terephthalate
biosynthetic pathways. Accordingly, the invention provides a
non-naturally occurring microbial organism that produces and/or
secretes 2H3M4OP, p-toluate or terephthalate when grown on a
carbohydrate or other carbon source and produces and/or secretes
any of the intermediate metabolites shown in the 2H3M4OP, p-toluate
or terephthalate pathway when grown on a carbohydrate or other
carbon source. The 2H3M4OP, p-toluate or terephthalate producing
microbial organisms of the invention can initiate synthesis from an
intermediate. For example, a 2H3M4OP pathway intermediate can be
4-phosphoerythronate, 2-oxo-3-hydroxy-4-phosphobutanoate,
2-acetyl-2,3-phosphodutanoate,
2-acetyl-2,3-dihydroxy-4-phosphobutanoate,
2,3,4-trihydroxy-3-methyl-5-phosphopentanoate,
4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate,
4,5-dihydroxy-3-methyl-2-oxopentanoate,
4-hydroxy-2-oxo-5-phosphopentanoate, 1-deoxy-D-xylulose-5-phosphate
or C-methyl-D-erythritol-4-phosphate (see Examples I and II and
FIGS. 1-3). 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).
[0129] 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 2H3M4OP, p-toluate or terephthalate pathway enzyme or
protein in sufficient amounts to produce 2H3M4OP, p-toluate or
terephthalate. It is understood that the microbial organisms of the
invention are cultured under conditions sufficient to produce
2H3M4OP, p-toluate or terephthalate. Following the teachings and
guidance provided herein, the non-naturally occurring microbial
organisms of the invention can achieve biosynthesis of 2H3M4OP,
p-toluate or terephthalate resulting in intracellular
concentrations between about 0.1-200 mM or more. Generally, the
intracellular concentration of 2H3M4OP, p-toluate or terephthalate
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.
[0130] 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. Under such anaerobic or substantially anaerobic
conditions, the 2H3M4OP, p-toluate or terephthalate producers can
synthesize 2H3M4OP, p-toluate or terephthalate at intracellular
concentrations of 5-10 mM or more as well as all other
concentrations exemplified herein. It is understood that, even
though the above description refers to intracellular
concentrations, 2H3M4OP, p-toluate or terephthalate producing
microbial organisms can produce 2H3M4OP, p-toluate or terephthalate
intracellularly and/or secrete the product into the culture
medium.
[0131] Exemplary fermentation processes include, but are not
limited to, fed-batch fermentation and batch separation; fed-batch
fermentation and continuous separation; and continuous fermentation
and continuous separation. In an exemplary batch fermentation
protocol, the production organism is grown in a suitably sized
bioreactor sparged with an appropriate gas. Under anaerobic
conditions, the culture is sparged with an inert gas or combination
of gases, for example, nitrogen, N.sub.2/CO.sub.2 mixture, argon,
helium, and the like. As the cells grow and utilize the carbon
source, additional carbon source(s) and/or other nutrients are fed
into the bioreactor at a rate approximately balancing consumption
of the carbon source and/or nutrients. The temperature of the
bioreactor is maintained at a desired temperature, generally in the
range of 22-37 degrees C., but the temperature can be maintained at
a higher or lower temperature depending on the the growth
characteristics of the production organism and/or desired
conditions for the fermentation process. Growth continues for a
desired period of time to achieve desired characteristics of the
culture in the fermenter, for example, cell density, product
concentration, and the like. In a batch fermentation process, the
time period for the fermentation is generally in the range of
several hours to several days, for example, 8 to 24 hours, or 1, 2,
3, 4 or 5 days, or up to a week, depending on the desired culture
conditions. The pH can be controlled or not, as desired, in which
case a culture in which pH is not controlled will typically
decrease to pH 3-6 by the end of the run. Upon completion of the
cultivation period, the fermenter contents can be passed through a
cell separation unit, for example, a centrifuge, filtration unit,
and the like, to remove cells and cell debris. In the case where
the desired product is expressed intracellularly, the cells can be
lysed or disrupted enzymatically or chemically prior to or after
separation of cells from the fermentation broth, as desired, in
order to release additional product. The fermentation broth can be
transferred to a product separations unit. Isolation of product
occurs by standard separations procedures employed in the art to
separate a desired product from dilute aqueous solutions. Such
methods include, but are not limited to, liquid-liquid extraction
using a water immiscible organic solvent (e.g, toluene or other
suitable solvents, including but not limited to diethyl ether,
ethyl acetate, tetrahydrofuran (THF), methylene chloride,
chloroform, benzene, pentane, hexane, heptane, petroleum ether,
methyl tertiary butyl ether (MTBE), dioxane, dimethylformamide
(DMF), dimethyl sulfoxide (DMSO), and the like) to provide an
organic solution of the product, if appropriate, standard
distillation methods, and the like, depending on the chemical
characteristics of the product of the fermenation process.
[0132] In an exemplary fully continuous fermentation protocol, the
production organism is generally first grown up in batch mode in
order to achieve a desired cell density. When the carbon source
and/or other nutrients are exhausted, feed medium of the same
composition is supplied continuously at a desired rate, and
fermentation liquid is withdrawn at the same rate. Under such
conditions, the product concentration in the bioreactor generally
remains constant, as well as the cell density. The temperature of
the fermenter is maintained at a desired temperature, as discussed
above. During the continuous fermentation phase, it is generally
desirable to maintain a suitable pH range for optimized production.
The pH can be monitored and maintained using routine methods,
including the addition of suitable acids or bases to maintain a
desired pH range. The bioreactor is operated continuously for
extended periods of time, generally at least one week to several
weeks and up to one month, or longer, as appropriate and desired.
The fermentation liquid and/or culture is monitored periodically,
including sampling up to every day, as desired, to assure
consistency of product concentration and/or cell density. In
continuous mode, fermenter contents are constantly removed as new
feed medium is supplied. The exit stream, containing cells, medium,
and product, are generally subjected to a continuous product
separations procedure, with or without removing cells and cell
debris, as desired. Continuous separations methods employed in the
art can be used to separate the product from dilute aqueous
solutions, including but not limited to continuous liquid-liquid
extraction using a water immiscible organic solvent (e.g., toluene
or other suitable solvents, including but not limited to diethyl
ether, ethyl acetate, tetrahydrofuran (THF), methylene chloride,
chloroform, benzene, pentane, hexane, heptane, petroleum ether,
methyl tertiary butyl ether (MTBE), dioxane, dimethylformamide
(DMF), dimethyl sulfoxide (DMSO), and the like), standard
continuous distillation methods, and the like, or other methods
well known in the art.
[0133] In addition to the culturing and fermentation conditions
disclosed herein, growth conditions for achieving biosynthesis of
2H3M4OP, p-toluate or terephthalate can include the addition of an
osmoprotectant to the culturing conditions, as described, for
example, in U.S. Patent Publication No. US 2011/0207185 A1.
[0134] In addition to the culturing and fermentation conditions
disclosed herein, growth condition for achieving biosynthesis of
2H3M4OP, p-toluate or terephthalate 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-camitine 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.
[0135] In some embodiments, the carbon feedstock and other cellular
uptake sources such as phosphate, ammonia, sulfate, chloride and
other halogens can be chosen to alter the isotopic distribution of
the atoms present in 2H3M4OP, p-toluate or terephthalate or any
2H3M4OP, p-toluate or terephthalate pathway intermediate. The
various carbon feedstock and other uptake sources enumerated above
will be referred to herein, collectively, as "uptake sources."
Uptake sources can provide isotopic enrichment for any atom present
in the product 2H3M4OP, p-toluate or terephthalate or 2H3M4OP,
p-toluate or terephthalate pathway intermediate, or for side
products generated in reactions diverging away from a 2H3M4OP,
p-toluate or terephthalate pathway. Isotopic enrichment can be
achieved for any target atom including, for example, carbon,
hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or other
halogens.
[0136] In some embodiments, the uptake sources can be selected to
alter the carbon-12, carbon-13, and carbon-14 ratios. In some
embodiments, the uptake sources can be selected to alter the
oxygen-16, oxygen-17, and oxygen-18 ratios. In some embodiments,
the uptake sources can be selected to alter the hydrogen,
deuterium, and tritium ratios. In some embodiments, the uptake
sources can be selected to alter the nitrogen-14 and nitrogen-15
ratios. In some embodiments, the uptake sources can be selected to
alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35 ratios. In
some embodiments, the uptake sources can be selected to alter the
phosphorus-31, phosphorus-32, and phosphorus-33 ratios. In some
embodiments, the uptake sources can be selected to alter the
chlorine-35, chlorine-36, and chlorine-37 ratios.
[0137] In some embodiments, the isotopic ratio of a target atom can
be varied to a desired ratio by selecting one or more uptake
sources. An uptake source can be derived from a natural source, as
found in nature, or from a man-made source, and one skilled in the
art can select a natural source, a man-made source, or a
combination thereof, to achieve a desired isotopic ratio of a
target atom. An example of a man-made uptake source includes, for
example, an uptake source that is at least partially derived from a
chemical synthetic reaction. Such isotopically enriched uptake
sources can be purchased commercially or prepared in the laboratory
and/or optionally mixed with a natural source of the uptake source
to achieve a desired isotopic ratio. In some embodiments, a target
atom isotopic ratio of an uptake source can be achieved by
selecting a desired origin of the uptake source as found in nature.
For example, as discussed herein, a natural source can be derived
from or synthesized by a biological organism or a source such as
petroleum-based products or the atmosphere. In some such
embodiments, a source of carbon, for example, can be selected from
a fossil fuel-derived carbon source, which can be relatively
depleted of carbon-14, or an environmental or atmospheric carbon
source, such as CO.sub.2, which can possess a larger amount of
carbon-14 than its petroleum-derived counterpart.
[0138] The unstable carbon isotope carbon-14 or radiocarbon makes
up for roughly 1 in 10.sup.12 carbon atoms in the earth's
atmosphere and has a half-life of about 5700 years. The stock of
carbon is replenished in the upper atmosphere by a nuclear reaction
involving cosmic rays and ordinary nitrogen (.sup.14N). Fossil
fuels contain no carbon-14, as it decayed long ago. Burning of
fossil fuels lowers the atmospheric carbon-14 fraction, the
so-called "Suess effect".
[0139] Methods of determining the isotopic ratios of atoms in a
compound are well known to those skilled in the art. Isotopic
enrichment is readily assessed by mass spectrometry using
techniques known in the art such as accelerated mass spectrometry
(AMS), Stable Isotope Ratio Mass Spectrometry (SIRMS) and
Site-Specific Natural Isotopic Fractionation by Nuclear Magnetic
Resonance (SNIF-NMR). Such mass spectral techniques can be
integrated with separation techniques such as liquid chromatography
(LC), high performance liquid chromatography (HPLC) and/or gas
chromatography, and the like.
[0140] In the case of carbon, ASTM D6866 was developed in the
United States as a standardized analytical method for determining
the bio-based content of solid, liquid, and gaseous samples using
radiocarbon dating by the American Society for Testing and
Materials (ASTM) International. The standard is based on the use of
radiocarbon dating for the determination of a product's bio-derived
content. ASTM D6866 was first published in 2004, and the current
active version of the standard is ASTM D6866-11 (effective Apr. 1,
2011). Radiocarbon dating techniques are well known to those
skilled in the art, including those described herein.
[0141] The bio-based content of a compound is estimated by the
ratio of carbon-14 (.sup.14C) to carbon-12 (.sup.12C).
Specifically, the Fraction Modem (Fm) is computed from the
expression: Fm=(S-B)/(M-B), where B, S and M represent the
.sup.14C/.sup.12C ratios of the blank, the sample and the modem
reference, respectively. Fraction Modem is a measurement of the
deviation of the .sup.14C/.sup.12C ratio of a sample from "Modem."
Modem is defined as 95% of the radiocarbon concentration (in AD
1950) of National Bureau of Standards (NBS) Oxalic Acid I (i.e.,
standard reference materials (SRM) 4990b) normalized to
.delta..sup.13C.sub.VPDB=-19 per mil (Olsson, The use of Oxalic
acid as a Standard. in, Radiocarbon Variations and Absolute
Chronology, Nobel Symposium, 12th Proc., John Wiley & Sons, New
York (1970)). Mass spectrometry results, for example, measured by
ASM, are calculated using the internationally agreed upon
definition of 0.95 times the specific activity of NBS Oxalic Acid I
(SRM 4990b) normalized to .delta..sup.13C.sub.VPDB=-19 per mil.
This is equivalent to an absolute (AD 1950).sup.14C/.sup.12C ratio
of 1.176.+-.0.010.times.10.sup.-12 (Karlen et al., Arkiv Geofysik,
4:465-471 (1968)). The standard calculations take into account the
differential uptake of one isotope with respect to another, for
example, the preferential uptake in biological systems of C.sup.12
over C.sup.13 over C.sup.14, and these corrections are reflected as
a Fm corrected for .delta..sup.13.
[0142] An oxalic acid standard (SRM 4990b or HOx 1) was made from a
crop of 1955 sugar beet. Although there were 1000 lbs made, this
oxalic acid standard is no longer commercially available. The
Oxalic Acid II standard (HOx 2; N.I.S.T designation SRM 4990 C) was
made from a crop of 1977 French beet molasses. In the early 1980's,
a group of 12 laboratories measured the ratios of the two
standards. The ratio of the activity of Oxalic acid II to 1 is
1.2933.+-.0.001 (the weighted mean). The isotopic ratio of HOx II
is -17.8 per mille. ASTM D6866-11 suggests use of the available
Oxalic Acid II standard SRM 4990 C (Hox2) for the modem standard
(see discussion of original vs. currently available oxalic acid
standards in Mann, Radiocarbon, 25(2):519-527 (1983)). A Fm=0%
represents the entire lack of carbon-14 atoms in a material, thus
indicating a fossil (for example, petroleum based) carbon source. A
Fm=100%, after correction for the post-1950 injection of carbon-14
into the atmosphere from nuclear bomb testing, indicates an
entirely modem carbon source. As described herein, such a "modem"
source includes bio-based sources.
[0143] As described in ASTM D6866, the percent modem carbon (pMC)
can be greater than 100% because of the continuing but diminishing
effects of the 1950s nuclear testing programs, which resulted in a
considerable enrichment of carbon-14 in the atmosphere as described
in ASTM D6866-11. Because all sample carbon-14 activities are
referenced to a "pre-bomb" standard, and because nearly all new
bio-based products are produced in a post-bomb environment, all pMC
values (after correction for isotopic fraction) must be multiplied
by 0.95 (as of 2010) to better reflect the true bio-based content
of the sample. A bio-based content that is greater than 103%
suggests that either an analytical error has occurred, or that the
source of bio-based carbon is more than several years old.
[0144] ASTM D6866 quantifies the bio-based content relative to the
material's total organic content and does not consider the
inorganic carbon and other non-carbon containing substances
present. For example, a product that is 50% starch-based material
and 50% water would be considered to have a Bio-derived
Content=100% (50% organic content that is 100% bio-based) based on
ASTM D6866. In another example, a product that is 50% starch-based
material, 25% petroleum-based, and 25% water would have a
Bio-derived Content=66.7% (75% organic content but only 50% of the
product is bio-based). In another example, a product that is 50%
organic carbon and is a petroleum-based product would be considered
to have a Bio-derived Content=0% (50% organic carbon but from
fossil sources). Thus, based on the well known methods and known
standards for determining the bio-based content of a compound or
material, one skilled in the art can readily determine the
bio-derived content and/or prepared downstream products that
utilize of the invention having a desired bio-based content.
[0145] Applications of carbon-14 dating techniques to quantify
bio-based content of materials are known in the art (Currie et al.,
Nuclear Instruments and Methods in Physics Research B, 172:281-287
(2000)). For example, carbon-14 dating has been used to quantify
bio-based content in terephthalate-containing materials (Colonna et
al., Green Chemistry, 13:2543-2548 (2011)). Notably, polypropylene
terephthalate (PPT) polymers derived from renewable 1,3-propanediol
and petroleum-derived terephthalic acid resulted in Fm values near
30% (i.e., since 3/11 of the polymeric carbon derives from
renewable 1,3-propanediol and 8/11 from the fossil end member
terephthalic acid) (Currie et al., supra, 2000). In contrast,
polybutylene terephthalate polymer derived from both renewable
1,4-butanediol and renewable terephthalic acid resulted in
bio-based content exceeding 90% (Colonna et al., supra, 2011).
[0146] Accordingly, in some embodiments, the present invention
provides 2H3M4OP, p-toluate or terephthalate or a 2H3M4OP,
p-toluate or terephthalate pathway intermediate that has a
carbon-12, carbon-13, and carbon-14 ratio that reflects an
atmospheric carbon, also referred to as environmental carbon,
uptake source. For example, in some aspects the 2H3M4OP, p-toluate
or terephthalate or a 2H3M4OP, p-toluate or terephthalate pathway
intermediate can have an Fm value of at least 10%, at least 15%, at
least 20%, at least 25%, at least 30%, at least 35%, at least 40%,
at least 45%, at least 50%, at least 55%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 98% or as much as 100%. In some
such embodiments, the uptake source is CO.sub.2. In some
embodiments, the present invention provides 2H3M4OP, p-toluate or
terephthalate or a 2H3M4OP, p-toluate or terephthalate pathway
intermediate that has a carbon-12, carbon-13, and carbon-14 ratio
that reflects petroleum-based carbon uptake source. In this aspect,
the 2H3M4OP, p-toluate or terephthalate or a 2H3M4OP, p-toluate or
terephthalate pathway intermediate can have an Fm value of less
than 95%, less than 90%, less than 85%, less than 80%, less than
75%, less than 70%, less than 65%, less than 60%, less than 55%,
less than 50%, less than 45%, less than 40%, less than 35%, less
than 30%, less than 25%, less than 20%, less than 15%, less than
10%, less than 5%, less than 2% or less than 1%. In some
embodiments, the present invention provides 2H3M4OP, p-toluate or
terephthalate or a 2H3M4OP, p-toluate or terephthalate pathway
intermediate that has a carbon-12, carbon-13, and carbon-14 ratio
that is obtained by a combination of an atmospheric carbon uptake
source with a petroleum-based uptake source. Using such a
combination of uptake sources is one way by which the carbon-12,
carbon-13, and carbon-14 ratio can be varied, and the respective
ratios would reflect the proportions of the uptake sources.
[0147] Further, the present invention relates to the biologically
produced 2H3M4OP, p-toluate or terephthalate or a 2H3M4OP,
p-toluate or terephthalate pathway intermediate as disclosed
herein, and to the products derived therefrom, wherein the 2H3M4OP,
p-toluate or terephthalate or a 2H3M4OP, p-toluate or terephthalate
pathway intermediate has a carbon-12, carbon-13, and carbon-14
isotope ratio of about the same value as the CO.sub.2 that occurs
in the environment. For example, in some aspects the invention
provides bio-based 2H3M4OP, p-toluate or terephthalate or a
bio-based 2H3M4OP, p-toluate or terephthalate intermediate having a
carbon-12 versus carbon-13 versus carbon-14 isotope ratio of about
the same value as the CO.sub.2 that occurs in the environment, or
any of the other ratios disclosed herein. It is understood, as
disclosed herein, that a product can have a carbon-12 versus
carbon-13 versus carbon-14 isotope ratio of about the same value as
the CO.sub.2 that occurs in the environment, or any of the ratios
disclosed herein, wherein the product is generated from bio-based
2H3M4OP, p-toluate or terephthalate or a bio-based 2H3M4OP,
p-toluate or terephthalate pathway intermediate as disclosed
herein, wherein the bio-based product is chemically modified to
generate a final product. Methods of chemically modifying a
bio-based product of 2H3M4OP, p-toluate or terephthalate, or an
intermediate thereof, to generate a desired product are well known
to those skilled in the art, as described herein. The invention
further provides a chip, resin, fiber, film or any other product
described herein having a carbon-12 versus carbon-13 versus
carbon-14 isotope ratio of about the same value as the CO.sub.2
that occurs in the environment, wherein the chip, resin, fiber,
film or any other product described herein is generated directly
from or in combination with bio-based 2H3M4OP, p-toluate or
terephthalate or a bio-based 2H3M4OP, p-toluate or terephthalate
pathway intermediate as disclosed herein.
[0148] The terephthalate produced by the microbial organisms,
pathways, fermentation process and/or isolation or purification
processes described herein can be used as a precursor of polymers
including PET, polybutyl terephthalate (PBT), polytrimethylene
terephthalate (PTT), and polyethylene naphthalate (PEN).
Accordingly, the resulting bio-derived polymer can contain
terephthalate, comprise terephthalate, be obtained with
terephthalate, be obtained by using terephthalate, be made by using
terephthalate, be obtained by converting terephthalate, or be
obtained by a reaction using terephthalate, wherein the
terephthalate is produced by a microbial organism, pathway,
fermentation process and/or isolation or purification process
described herein. Moreover, in some aspects, the invention provides
use of a bio-derived polymer described herein to make a product
disclosed herein. Still further, the invention provides, in some
aspects, use of a product disclosed herein in making a composition,
product or article.
[0149] When both a bio-based terephthalate of the invention and a
second non-bio-based terephthalat, such as a petroleum-derived
terephthalate or a therephthalate not made or obtained according to
the present invention, are present in the same composition or
product, such as a polymer, chip, fiber, resin, film or any other
product described herein, the bio-based terephthalate can be
present in an amount from 5% by weight to 100% by weight based on
the total weight of the bio-based terephthalate and the second
non-bio-based terephthalate present in the composition or product.
In some aspects, the bio-based terephthalate can be from 25% by
weight to 100% by weight; or alternatively, from 50% by weight to
100% by weight.
[0150] When both a polymer of the invention, i.e. a bio-derived
polymer, and a second polymer such as a petroleum based polymer or
a polymer not made or obtained by the bio-based terephthalate of
the invention are present in the same composition or product, e.g.
resin, chip, fiber, film, or any other product described herein,
the bio-derived polymer can be present in an amount from 5% by
weight to 100% by weight based on the total weight of the
bio-derived polymer and the second polymer present in the
composition or product. In some aspects, the bio-derived polymer
can be present in an amount from 25% by weight to 100% by weight,
or alternatively, from 50% by weight to 100% by weight.
[0151] In certain embodiments, the invention provides a PET polymer
used in polyester products and methods described herein. PET can
also be referred to as melt-phase PET resin, reactor-grade
polyester or polyester chip, e.g. PET bottle chips. A PET based
polymer can be used in the production of polyester fibers or
filaments, polyester film, solid-state (bottle-grade) resins and
PET engineering resins of the invention. Polyethylene terephthalate
used to make synthetic fibers is often referred to as polyester and
can be used to make bottle and packaging, which are often referred
to by its acronym PET. Products of the invention using polyester
fibers include cloth, clothing, bedding, furniture, and carpet.
Products of the invention using Bottle Grade PET include beverage,
food and pharmaceutical containers and packaging, including
beverage bottles. Polyester films or sheets include those having a
single layer or a multilayer, a heat-shrinkable film, hollow
containers, formed articles, fibers, having a coating material of
the solution type, having a coating of the powder type, having a
toner and having an adhesive. Products of the invention using
polyester film include coated and uncoated film, and includes an
Adhesion Film, a Barrier Film, a Coated Film, a Heat Sealable Film,
a Lidding Film, a Matte Film, a Siliconized Release Film and/or a
UV Stabilized film. Polyester film used for packaging can be coated
or uncoated, and can be a Matte Film, High COF (coefficient of
friction) Film, Direct Extrudable Film, UV Stabilized Film, Cold
Seal Film, White Film, Weldable Film, thermal lamination film, and
Peelable Film. Polyester film can also be used in products and uses
including: a balloon; as electrical shrink tubing, and electrical
wire and cable wraps; as a release liner in multilayer ceramic
capacitor (MLCC) and ceramic casting; as a layer or backing for a
photovoltaic apparatus such as a photovoltaic solar module or used
as a part of the construction for coated flexible photovoltaic
solar module; in presentation and display media, printing and
pre-press film, overhead transparencies, photo-tool, medical and
x-ray film and plates, printing plates, reprographic films, and
digital imaging film, for example as used with a printer; for
industrial applications as a Roll Leaf, Protective Face Shield or
Window Box; for construction industry products including a shingle
release liner, peel and stick release liner, flexible ductwork,
fiberglass roofing, fiberglass tubing, carpet backing, flooring
foam substrate, adhesive pads for carpet tile installation,
acoustical panel, ceiling panel, and solar window and safety film;
and a Thermal Transfer Ribbons (TTR) for use in thermal transfer
printing. An example of such films include those under trade name
Hostaphan.RTM. (Mitsubishi Polyester Film, Japan) and Mylar.RTM.,
Melinex.RTM., and Tetoron.RTM. (DuPont USA and Teijin Japan).
[0152] PET of the invention can be produced by polycondensation of
ethylene glycol with either dimethyl terephthalate or terephthalic
acid of the invention. For example, with dimethyl terephthalate, a
transesterification reaction can be sued, or with terephthalic
acid, an esterification reaction can be sued (Kopnick et al.
"Polyesters" in Ullmann's Encyclopedia of Industrial Chemistry,
A21, Wiley-VCH, Weinheim, 2000). Dimethyl terephthalate can also be
produced by di-esterification of terephthalate of the invention and
methanol (Sheehan, "Terephthalic Acid, Dimethyl Terephthalate, and
Isophthalic Acid" in Ullmann's Encyclopedia of Industrial
Chemistry, Wiley-VCH, Weinheim, 2005). Accordingly, in certain
embodiments, the invention provides a process for obtaining
polyethylene terephthalate by reacting ethylene glycol with
dimethyl terephthalate, wherein the dimethyl terephthalate is
produced from terephthalate produced by or obtained from a
microorganism, pathway, fermentation process and/or isolation or
purification process described herein. In another embodiment is a
process for obtaining polyethylene terephthalate by reacting
ethylene glycol with terephthalate, wherein the terephthalate is
produced by or obtained from a microorganism, pathway, fermentation
process and/or isolation or purification process described
herein.
[0153] Accordingly, in certain embodiments, the invention provides
polyethylene terephthalate obtained by a process using any of the
microorganisms, processes and embodiments described herein. Also
provided is polyethylene terephthalate polymer obtained from
terephthalate obtained by a process with any of the microorganisms,
processes and embodiments described herein claims. Still further
provided is polyester chip, resin or fiber comprising or obtained
by using polyethylene terephthalate obtained by a process with any
of the microorganisms, processes and embodiments described herein
claims. Also provided is a product, as well as a process to obtain
that product, as described herein above, e.g. a polyester cloth,
carpet, film, comprising or obtained by using polyester fiber
obtained by terephthalate as disclosed herein. Also provided is a
product, as well as a process to obtain that product, as described
herein, for example, a PET bottle or PET container or packaging,
comprising or obtained by using PET chip or resin obtained by
terephthalate as disclosed herein. In another embodiment are PET
bottle chips, or method of their making, comprising or obtained by
using PET obtained by a process as described herein. Further
disclosed is a PET bottle, container or package comprising or
obtained by using PET bottle chips obtained as described
herein.
[0154] In certain embodiments, the invention provides a polybutyl
terephthalate (PBT) polymer that can be used in PBT products and
processes of the invention as described herein. PBT polymer can be
used in molded articles, for example, injection-molded products and
parts, including automotive parts, in extrusion resin, in
electrical and automotive parts, and in casings, for example, power
tool casings. Products include those containing PBT sold under the
trade name Advanite (SASA), Anjacom (Almaak International), Amite
(DSM), Celanex, Vandar polyester alloy (Ticona), Duranex
(Polyplastics), Crastin (DuPont), Pocan (Lanxess), Ultradur (BASF),
Valox (SABIC Innovative Plastics), Schuladur (A. Schulman), Later
(LATI), Kebater (BARLOG plastics), VESTODUR (Evonik Degussa), and
ENVIRON.RTM. (Enviroplas). A PBT polymer of the invention can be
produced by reaction of 1,4-butanediol with terephthalic acid of
the invention, for example by esterification. Accordingly, in one
embodiment, the invention provides a process for obtaining PBT by
reacting 1,4-butanediol with terephthalate where the terephthalate
is obtained by or produced from a microrganism, pathway,
fermentation process and/or isolation or purification process
described herein.
[0155] In certain embodiments, the invention provides
polytrimethylene terephthalate (PTT) polymer that can be used in
PTT products and processes of the invention as described herein. A
PTT polymer can be used in fibers to make products, such as cloth,
clothing and carpet, or bulk products such as, for example, chips
for the manufacture of bottles. PTT can be prepared by the
esterification of 1,3-propanediol with terephthalic acid, or by
transesterification of dimethyl terephthalate, where the
terephthalate is obtained by or produced from a microorganism,
pathway, fermentation process and/or isolation or purification
process described herein. Products and processes of the invention
include those that comprise PBT polymer that is sold under the
trade name Sorona (DuPont), and includes co-polymers, for example,
a poly(trimethylene terephthalate) copolymer (e.g. Tritan by
Eastman) and as used to produce Nalgene bottles.
[0156] The polymers of the present invention may further comprise
additional compounds. Additional compounds include a solvent; one
or more other bio-derived polymers described herein; one or more
resins including polyester, aliphatic polyester resins,
thermoplastic polyester elastomers, polyolefins, polystyrenes,
acrylonitrile-butadiene-styrene copolymers, polymethyl
methacrylate, polysulfones, polyethers, phenoxy resins,
polyphenylene oxides, thermoplastic resins such as polyethylene,
polypropylene, acrylic resins, polycarbonates, polyamides,
polyphenylene sulfide, polyethylene terephthalate, liquid
crystalline polyesters, polyacetals and polyphenylene oxide and
thermosetting resins such as phenol resins, melamine resins,
silicone resins and epoxy resins (which may be added when a molded
article is prepared); and/or one or more auxiliary agents.
Auxiliary agents can be added during the polymerization to form the
bio-derived polymer or after the polymerization. Auxiliary agents
include those for molding and additives such as fillers, coloring
agents, reinforcing materials, surface-smoothing agents, leveling
agents, accelerators for a curing reaction, photostabilizers,
ultraviolet absorbers, plasticizers, antioxidants, extenders,
delustering agents, agents for adjusting drying, antistatic agents,
agents for preventing precipitating, surfactants, agents for
improving flow, drying oils, waxes and thermoplastic oligomers,
stabilizers including heat and UV stabilizers, lubricants, catalyst
deactivators, nucleating agents for crystallization and promoters
for crystallization. To provide the bio-polymer with desired
properties, ultraviolet absorbents, stabilizers such as weathering
stabilizers, coloring agents such as dyes, antistatic agents,
foaming agents, plasticizers and impact resistance improvers can be
added. Reinforcing fillers added to the resin of the present
invention are not particularly limited, examples include inorganic
fibers such as glass fiber, carbon fiber, silica-alumina fiber,
zirconia fiber, boron fiber, boron nitride fiber, silicon nitride
potassium titanate fiber and metal fibers; and organic fibers such
as aromatic polyamide fibers and fluoro-resin fibers. Other fillers
include inorganic fillers having a plate shape, ceramic beads,
wollastonite, talc, clay, mica, zeolite, kaolin, potassium
titanate, barium sulfate, titanium oxide, silicon oxide, aluminum
oxide and magnesium hydroxide. By adding an inorganic filler having
a plate shape, anisotropy and warp in the molded article can be
decreased. Preferable examples of the inorganic filler having a
plate shape include glass flakes, mica and metal foils. Among these
inorganic fillers having a plate shape, glass flakes are more
preferably used. Antioxidants include phenolic antioxidants,
sterically hindered phenols and/or phosphites, hydroquinones,
aromatic secondary amines, such as diphenylamines, and various
substituted representatives of these groups. UV stabilizers include
various substituted resorcinols, salicylates, benzotriazoles, and
benzophenones. Coloring agents that can be added comprise inorganic
and organic pigments, and also dyes, such as nigrosin and
anthraquinones. Lubricants and mold-release agents include
long-chain fatty acids (e.g. stearic acid or behenic acid), salts
of these (e.g. Ca stearate or Zn stearate), or montan waxes
(mixtures made of straight-chain, saturated carboxylic acids having
chain lengths of from 28 to 32 carbon atoms), Ca montanate or Na
montanate, low-molecular-weight polyethylene waxes or
low-molecular-weight polypropylene waxes.
[0157] The bio-derived polymers of the present invention can be
molded in accordance with a conventional process suitable for each
application. For example, molded articles can be prepared in
accordance with the conventional injection molding process and
sheets and films can be prepared in accordance with the extrusion
process or the casting process. Formed articles can be prepared in
accordance with the extrusion-expansion process or the
expansion-in-mold process. A molding process conventionally used
for thermoplastic resins such as the injection molding, the blow
molding, the extrusion molding and the compression molding can be
applied.
[0158] In some embodiments, the invention provides chip, resin,
fiber, film or any other product described herein comprising
bio-based 2H3M4OP, p-toluate or terephthalate, or a bio-based
2H3M4OP, p-toluate or terephthalate pathway intermediate, wherein
the bio-based 2H3M4OP, p-toluate or terephthalate, or bio-based
2H3M4OP, p-toluate or terephthalate pathway intermediate includes
all or part of the 2H3M4OP, p-toluate or terephthalate, or 2H3M4OP,
p-toluate or terephthalate pathway intermediate used in the
production of chip, resin, fiber, film or any other product
described herein. For example, the final chip, resin, fiber, film
or any other product described herein can contain the bio-based
2H3M4OP, p-toluate or terephthalate, 2H3M4OP, p-toluate or
terephthalate pathway intermediate, or a portion thereof that is
the result of the manufacturing of chip, resin, fiber, film or any
other product described herein. Such manufacturing can include
chemically reacting the bio-based 2H3M4OP, p-toluate or
terephthalate or bio-based 2H3M4OP, p-toluate or terephthalate
pathway intermediate (e.g. chemical conversion, chemical
functionalization, chemical coupling, oxidation, reduction,
polymerization, copolymerization and the like) into the final chip,
resin, fiber, film or any other product described herein. Thus, in
some aspects, the invention provides a bio-derived chip, resin,
fiber, film or any other product described herein comprising at
least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at
least 20%, at least 25%, at least 30%, at least 35%, at least 40%,
at least 50%, at least 60%, at least 70%, at least 80%, at least
90%, at least 95%, at least 98% or 100% bio-based 2H3M4OP,
p-toluate or terephthalate or bio-based 2H3M4OP, p-toluate or
terephthalate pathway intermediate as disclosed herein.
[0159] Additionally, in some embodiments, the invention provides a
composition having a bio-based 2H3M4OP, p-toluate or terephthalate
or 2H3M4OP, p-toluate or terephthalate pathway intermediate
disclosed herein and a compound other than the bio-based 2H3M4OP,
p-toluate or terephthalate or 2H3M4OP, p-toluate or terephthalate
pathway intermediate. For example, in some aspects, the invention
provides a bio-derived chip, resin, fiber, film or any other
product described herein wherein the 2H3M4OP, p-toluate or
terephthalate, or 2H3M4OP, p-toluate or terephthalate pathway
intermediate used in its production is a combination of bio-based
and petroleum derived 2H3M4OP, p-toluate or terephthalate, or
2H3M4OP, p-toluate or terephthalate pathway intermediate. For
example, a bio-derived chip, resin, fiber, film or any other
product described herein can be produced using 50% bio-based
2H3M4OP, p-toluate or terephthalate and 50% petroleum derived
2H3M4OP, p-toluate or terephthalate or other desired ratios such as
60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%,
30%/70%, 20%/80%, 10%/90% of bio-based/petroleum derived
precursors, so long as at least a portion of the product comprises
a bio-based product produced by the microbial organisms disclosed
herein. It is understood that methods for producing chip, resin,
fiber, film or any other product described herein using the
bio-based 2H3M4OP, p-toluate or terephthalate or bio-based 2H3M4OP,
p-toluate or terephthalate pathway intermediate of the invention
are well known in the art.
[0160] 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.
[0161] As described herein, one exemplary growth condition for
achieving biosynthesis of 2H3M4OP, p-toluate or terephthalate
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, an anaerobic
condition 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.
[0162] The culture conditions described herein can be scaled up and
grown continuously for manufacturing of 2H3M4OP, p-toluate or
terephthalate. 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 2H3M4OP,
p-toluate or terephthalate. Generally, and as with non-continuous
culture procedures, the continuous and/or near-continuous
production of 2H3M4OP, p-toluate or terephthalate will include
culturing a non-naturally occurring 2H3M4OP, p-toluate or
terephthalate 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
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.
[0163] Fermentation procedures are well known in the art. Briefly,
fermentation for the biosynthetic production of 2H3M4OP, p-toluate
or terephthalate 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.
[0164] In addition to the above fermentation procedures using the
2H3M4OP, p-toluate or terephthalate producers of the invention for
continuous production of substantial quantities of 2H3M4OP,
p-toluate or terephthalate, the 2H3M4OP, p-toluate or terephthalate
producers also can be, for example, simultaneously subjected to
chemical synthesis and/or enzymatic procedures to convert the
product to other compounds or the product can be separated from the
fermentation culture and sequentially subjected to chemical and/or
enzymatic conversion to convert the product to other compounds, if
desired.
[0165] 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 2H3M4OP, p-toluate or terephthalate.
[0166] 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.
[0167] 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.
[0168] 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.
[0169] 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.
[0170] 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.
[0171] 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.
[0172] 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.
[0173] 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..
[0174] 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.
[0175] 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)).
[0176] 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.
[0177] As disclosed herein, a nucleic acid encoding a desired
activity of a 2H3M4OP, p-toluate or terephthalate pathway can be
introduced into a host organism. In some cases, it can be desirable
to modify an activity of a 2H3M4OP, p-toluate or terephthalate
pathway enzyme or protein to increase production of 2H3M4OP,
p-toluate or terephthalate. 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.
[0178] 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.
[0179] 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 2H3M4OP, p-toluate or terephthalate
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)).
[0180] 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:e18 (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)).
[0181] 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 ts 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)).
[0182] 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)).
[0183] 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.
Isolation (Purification) Process of the Invention
[0184] Provided herein are processes for isolating a bio-based
aromatic carboxylic acid such as, for example, p-toluic acid or
terephthalic acid, from a culture medium, wherein the processes
comprise lowering the pH of the culture medium to produce an
aromatic carboxylic acid precipitate. In certain embodiments,
lowering the pH of the culture medium comprises contacting the
culture medium with carbon dioxide to lower the pH of the culture
medium. In certain embodiments, a process as provided herein
further comprises culturing a non-naturally occurring microbial
organism in a culture medium at a pH sufficient to produce a
bio-based aromatic carboxylic acid which will be in an anionic,
soluble form in the culture medium.
[0185] As used herein, a "bio-based aromatic carboxylic acid" means
an aromatic carboxylic acid made biosynthetically from a
non-naturally occurring microbial organism.
[0186] An aromatic carboxylic acid such as terephthalic acid can be
precipitated directly out of a culture medium to substantially
deplete the medium of the acid or its conjugate anion. In certain
embodiments, the bio-based aromatic carboxylic acid anion can have
an ammonium counterion in the culture medium. Moreover, such
embodiments where carbon dioxide is used to lower the pH to produce
a bio-based aromatic acid precipitate, it will be understood that
ammonium carbonate will result which can be easily recovered as
ammonia and carbon dioxide, which components may be separated,
recovered and recycled. In certain embodiments, the carbon dioxide
source for acidifying the culture medium will be that collected as
produced from culturing the non-naturally occurring microbial
organism.
Culturing the Microbial Organism to Produce the Aromatic
Carboxylate Anion
[0187] In certain embodiments, the aromatic carboxylate anion is
produced by an indirect semi-synthetic route, whereby muconate is
biosynthesized in a non-naturally occurring microbial organism from
simple carbohydrate feedstocks, which in turn provides a viable
synthetic route to the aromatic carboxylate anion, for example,
terephthalate, as disclosed in U.S. Patent Publication No. US
2011/0124911 A1, hereby incorporated by reference in its entirety
for all purposes. In particular, these pathways provide trans,
trans-muconate or cis, trans-muconate biocatalytically from simple
sugars. The all trans or cis, trans isomer of muconate may then be
converted to terephthalate in a two step process via inverse
electron demand Diels-Alder reaction with acetylene followed by
oxidation in air or oxygen. The Diels-Alder reaction between
muconate and acetylene proceeds to form
cyclohexa-2,5-diene-1,4-dicarboxylate (P1) (see FIG. 1 of U.S.
Patent Publication No. US 2011/0124911 A1, hereby incorporated by
reference). Subsequent exposure to air or oxygen rapidly converts
P1 to terephthalate.
[0188] In certain embodiments, the Diels-Alder reaction between
muconate and acetylene can be performed in the culture medium.
Optionally, the culture medium can be filtered, for example, to
remove cells of the organism, prior to adding acetylene.
[0189] In certain embodiments, the pH sufficient to maintain the
muconate in soluble form is between about 5.0-9.0, between about
5.5-9.0, between about 6.0-9.0, between about 6.5-9.0, between
about 7.0-9.0, between about 5.5-8.0, between about 6.0-8.0, or
between about 6.5-8.0 pH units. In certain embodiments, the pH
sufficient to maintain the muconate in soluble form is about 5.5,
about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, or about 8.5
pH units. In certain embodiments, the pH sufficient to maintain the
muconate in soluble form is about 7.0 pH units. In certain
embodiments, the pH sufficient to maintain the muconate in soluble
form is a neutral pH, for example a pH of about 7.0 typically
bounded on its lower end by pH of about 5.0, 5.5 or 6.0 and on its
upper end by a pH of about 9.0, 8.5 or 8.0.
[0190] The pH of the culture medium can be maintained at a desired
pH, for example, a pH between about 5.0-9.0, by addition of a base,
such as NaOH or other bases, or acid, as needed to maintain the
culture medium at the desired pH. It will be understood that a pH
between about 5.0-9.0 can, for example, be conducive for optimal
culturing conditions to produce a desired product (e.g., a
bio-based aromatic carboxylate anion or a precursor) and/or to
produce an anionic form in a soluble form in culture medium. In
certain embodiments, a base is added to the culture medium in
sufficient quantities to maintain muconate in soluble form. In
certain embodiments, a base is added to the culture medium in
sufficient quantities to maintain the culture medium at a pH
between about 5.0-9.0.
[0191] In certain embodiments, following the Diels-Alder reaction,
the culture medium can be separated from the cells and/or any
non-soluble material by, for example, centrifugation or membrane
filtration, to provide a cell-free medium or broth comprising the
terephthalate, prior to the contacting step (described herein and
in section "Acidification of the Culture Medium to Precipitate the
Aromatic Carboxylic Acid"). For convenience, the culture medium or
broth separated from the non-soluble materials will be termed
"cell-free." Those skilled in the art will understand that medium
and broth can be used interchangeably throughout to refer to a
liquid or gel designed to support the growth of the non-naturally
occurring microbial organism.
[0192] In certain embodiments, the aromatic carboxylate anion is
produced by a direct biosynthetic route, whereby the aromatic
carboxylate anion, for example, p-toluate or terephthalate, is
biosynthesized in a non-naturally occurring microbial organism from
simple carbohydrate feedstocks, as disclosed in U.S. Patent
Publication No. US 2011/0207185 A1, which is hereby incorporated by
reference in its entirety for all purposes.
[0193] Exemplary biosynthetic pathways include, for example, the
conversion of erythrose-4-phosphate to
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (2H3M4OP), also known
as 3-hydroxy-2-methyl butanal-4-phosphate, in six enzymatic steps
(see FIG. 1). In one alternative, 4,5-dihydroxy-2-oxopentanoate is
converted to 2H3M4OP using one or both of the pathways described in
FIG. 2. In another alternative, glyceraldehyde-3-phosphate (G3P)
and pyruvate are converted to 2H3M4OP in three enzymatic steps (see
FIG. 3). The 2H3M4OP intermediate can be subsequently transformed
to p-toluate by enzymes in the shikimate pathway (see FIG. 4).
p-Toluate can be further converted to terephthalate by a
microorganism (see FIG. 5). The purification method of the
invention (exemplified in FIG. 6) can also be applied to
biosynthetic pathways for aromatic carboxylic acid and terephthalic
acid production described in WIPO patent publications
WO/2009/120457A2 entitled "Bio-Based Polyethylene Terephthalate
Polymer And Method Of Making The Same", WO/2011/094131A1 entitled
"Microorganisms And Methods For The Biosynthesis Of P-Toluate And
Terephthalate", and WO/2011/017560A1 entitled "Semi-Synthetic
Terephthalic Acid Via Microorganisms That Produce Muconic Acid" and
U.S. Pat. No. 6,461,840 entitled "Terephthalic acid producing
proteobacteria" and 6187569 entitled "Microbial production of
terephthalic acid and isophthalic acid."
[0194] In certain embodiments, the pH sufficient to maintain the
p-toluate or terephthalate in soluble form is between about
5.0-9.0, between about 5.5-9.0, between about 6.0-9.0, between
about 6.5-9.0, between about 7.0-9.0, between about 5.5-8.0,
between about 6.0-8.0, or between about 6.5-8.0 pH units. In
certain embodiments, the pH sufficient to maintain the p-toluate or
terephthalate in soluble form is about 5.5, about 6.0, about 6.5,
about 7.0, about 7.5, about 8.0, or about 8.5 pH units. In certain
embodiments, the pH sufficient to maintain the p-toluate or
terephthalate in soluble form is about 7.0 pH units. In certain
embodiments, the pH sufficient to maintain p-toluate or
terephthalate in soluble form is a neutral pH, for example a pH of
about 7.0 typically bounded on its lower end by pH of about 5.0,
5.5 or 6.0 and on its upper end by a pH of about 9.0, 8.5 or
8.0.
[0195] The pH of the culture medium can be maintained at a desired
pH, which can, for example, be a pH between about 5.0-9.0, by
addition of a base or acid, as needed to maintain the culture
medium at the desired pH.
[0196] Those of skill in the art will understand that the dianionic
salts of terephthalic acid are soluble at concentrations >100
g/L (up to 13% by weight) at 25.degree. C. In certain embodiments,
a base is added to the culture medium in sufficient quantities to
maintain terephthalic acid in soluble form. In certain embodiments,
a base is added to the culture medium in sufficient quantities to
maintain the culture medium at a pH between about 5.0-9.0.
[0197] It will be understood that counterions, including for
instance, sodium, potassium, ammonium, among others, can be added
to the culture medium to produce a desired counterion to an
aromatic carboxylate anion, to the extent that the form of the
counterion, when added, maintains a desired pH. In certain
embodiments, a base, such as sodium hydroxide (NaOH), potassium
hydroxide (KOH), sodium bicarbonate (NaHCO.sub.3), or other bases,
or acid, are added as needed to maintain the culture medium at a
desirable pH.
[0198] In certain embodiments, anhydrous ammonia (NH.sub.3) is
added as a base to the culture medium to maintain the pH around 7.
In certain embodiments, a solution of ammonia, also known as
ammonium hydroxide (NH.sub.4OH), ammonia water, ammonical liquor,
ammonia liquor, aqua ammonia, aqueous ammonia, or ammonia, is added
as a base to the culture medium to maintain the pH around 7. Use of
ammonia as a base offers certain advantages for recovery and
recycling of salts in the processes disclosed herein (described
herein and in section entitled "Recovery of Salts for
Recycling").
[0199] Those skilled in the art will understand that the aromatic
carboxylate anions produced by the biosynthetic routes described
herein, i.e., p-toluate and terephthalate, will be in the form of
soluble carboxylate salts, i.e., the carboxylate anions will be
solvated in solution with a counter ion (a cation) in the culture
medium. The choice of base used to maintain the culture medium at a
neutral pH will determine the counter ion and the type of salt. For
example, where a sodium base, such as NaOH or NaHCO.sub.3 is used
to maintain neutral pH, the counter ion will be sodium (Na.sup.+),
and a soluble sodium carboxylate salt will be formed. Similarly,
where KOH is used, the counter ion will be potassium (K.sup.+) and
a soluble potassium carboxylate salt will be formed. Where ammonia
is used, the counter ion will be ammonium (NH.sub.4.sup.+), and a
soluble ammonium carboxylate salt will be formed. Where the
aromatic carboxylic anion is a diacid, for example, terephthalate,
a disalt (e.g., disodium, dipotassium, or diammonium terephthalate)
will be formed.
[0200] In certain embodiments, following fermentation, the culture
medium can be separated from the cells and/or any non-soluble
material by, for example, centrifugation, filtration, or technique
used in the art, to provide a cell-free medium or broth comprising
the aromatic carboxylate anion, prior to the contacting step
(described herein and in section entitled "Acidification of the
Culture Medium to Precipitate the Aromatic Carboxylic Acid").
Exemplary filtrations can be micro, nano or ultra filtration.
Centrifugation and filtration methods are well known to those
skilled in the art.
Acidification of the Culture Medium to Precipitate the Aromatic
Carboxylic Acid
[0201] In certain embodiments, the aromatic carboxylate anion
produced by the biosynthetic routes described herein can be
isolated by acidification of the culture medium. For example, in
certain embodiments, a process is provided comprising lowering the
pH of the culture medium to produce an aromatic carboxylic acid
precipitate.
[0202] It will be understood that the expressions "acidifying" or
"acidification" as used herein mean the addition of substance, acid
or otherwise, to an aqueous solution, for example, a culture
medium, to result in that solution having a lower pH. Thus, for
instance, it will be recognized that contacting a culture medium
with CO.sub.2 is an acidification of the culture medium.
[0203] In certain embodiments, the pH of the culture medium
comprising the aromatic carboxylate anion is lowered to less than
about 5.0 pH units, less than about 4.5 pH units, less than about
4.0 pH units, less than about 3.5 pH units, less than about 3.0 pH
units, less than about 2.5 pH units, less than about 2.0 pH units,
less than about 1.5 pH units, or less than about 1.0 pH units. In
certain embodiments, the pH of the culture medium is lowered to
less than about 3.0 pH units. In certain embodiments, the pH of the
culture medium is lowered to less than about 2.0 pH units.
[0204] Those skilled in the art will understand that the pH of the
culture medium is optimally lowered to a pH value less than the pKa
value of the aromatic carboxylic acid to be isolated.
[0205] In certain embodiments, the aqueous solubility of the
aromatic carboxylate anions and/or salts disclosed herein is
greater than about 100 g/L at room temperature. In certain
embodiments, the aqueous solubility of the aromatic carboxylic
acids disclosed herein is less than 1 g/L at room temperature.
[0206] In certain embodiments, acidification of the culture medium
results in precipitation of the aromatic carboxylic acid. In
certain embodiments, a decrease in pH of the culture medium results
in precipitation of the aromatic carboxylic acid.
[0207] In certain embodiments, the precipitate is comprised of a
monoacid, for example, p-toluic acid. In certain embodiments, the
precipitate is comprised of a diacid, for example, terephthalic
acid.
[0208] In certain embodiments, acidification results in the culture
medium being substantially depleted of the aromatic carboxylate
anion. In certain embodiments, a decrease in pH of the culture
medium results in the culture medium being substantially depleted
of the aromatic carboxylate anion. In certain embodiments,
precipitation of the aromatic carboxylic acid results in the
culture medium being substantially depleted of the aromatic
carboxylate anion.
[0209] As used herein, the term "substantially depleted" is
understood to mean that less than about 50% of the total amount of
aromatic carboxylate anion produced biosynthetically remains in the
culture medium following acidification. In certain embodiments,
less than about 50%, less than about 40%, less than about 30%, less
than about 20%, less than about 10%, less than about 5%, or less
than about 1% of the aromatic carboxylate anion remains in the
culture medium following acidification. In certain embodiments,
"substantially depleted" means that the amount of aromatic
carboxylate anion in the culture medium is less than that which can
measurably detected.
[0210] In certain embodiments, the pH of the culture medium is
lowered by contacting the culture medium with an acid. Such acids
can, for example, be those known in the art, including, for
instance, hydrochloric, phosphoric, sulfuric, and so forth. In
certain embodiments, the acid is carbon dioxide. It is believed use
of carbon dioxide is advantageous in that, it can, for example, be
obtained as a product from the cultured microbial organism and/or
easily recovered and recycled from the acidified culture
medium.
[0211] In certain embodiments, the pH of the culture medium is
lowered through contact with sufficient carbon dioxide (CO.sub.2)
to lower the pH of the culture medium to produce the precipitate
comprising the aromatic carboxylic acid.
[0212] Those skilled in the art will understand that when carbon
dioxide dissolves in water it exists in chemical equilibrium
producing carbonic acid (H.sub.2CO.sub.3):
CO.sub.2+H.sub.2O H.sub.2CO.sub.2,
which in sufficient quantities can be used to lower the pH of the
culture medium.
[0213] In certain embodiments, the culture medium is contacted with
a solution of CO.sub.2 in water, for example, a solution of
carbonic acid (H.sub.2CO.sub.3). In certain embodiments, the
culture medium is contacted with gaseous CO.sub.2. In certain
embodiments, the gaseous CO.sub.2 is pure CO.sub.2 gas. In certain
embodiments, the gaseous CO.sub.2 is in a mixture with one or more
additional gases. In certain embodiments, the one or more
additional gases include nitrogen, an inert gas (helium, argon,
etc.), or the like.
[0214] In certain embodiments, the culture medium is stirred while
it is contacted with gaseous CO.sub.2.
[0215] In certain embodiments, the culture medium is contacted with
gaseous CO.sub.2 for up to 20 hours.
[0216] To provide sufficient H.sub.2CO.sub.3 in solution in the
culture medium, the temperature and pressure of the culture medium
can be adjusted. Those skilled in the art will understand that the
optimum temperature and pressure will depend on the pKa of the
aromatic carboxylic acid to be precipitated, the concentration of
the aromatic carboxylate anion, and the fraction of aromatic
carboxylate anion required to be converted to the aromatic
carboxylic acid.
[0217] In certain embodiments, the culture medium is pressurized in
the range of about 1 to 300 atm with CO.sub.2. In certain
embodiments, the culture medium is pressurized in the range of
about 50 to 200 atm with CO.sub.2. In certain embodiments, the
culture medium is pressurized in the range of about 1 to 100 atm
with CO.sub.2. In certain embodiments, the culture medium is
pressurized in the range of about 20 to 50 atm with CO.sub.2. In
certain embodiments, the culture medium is pressurized in the range
of about 10 to 50 atm with CO.sub.2.
[0218] More particularly, lower atmospheres of CO.sub.2, between,
for example, 1 to 30 atm with CO.sub.2 may be useful. In certain
embodiments, the culture medium is pressurized in the range of
about 1 to 30 atm with CO.sub.2. In certain embodiments, the
culture medium is contacted with CO.sub.2 under pressure in the
range of about 1 to about 20 atm, about 2 to about 25 atm, about 5
to about 20 atm, about 10 to about 20 atm, about 13 to about 20
atm, about 14 to about 20 atm, about 15 to about 20 atm, about 14
to about 16 atm, about 16 to about 20 atm, about 17 to about 25
atm, about 2 to about 20 atm, or about 10 to about 16 atm. In
certain embodiments, the culture medium is contacted with CO.sub.2
under about 0.01, 0.1, 0.5, 1.0, 2, 5, 10, 14, 15, 16, or 20 atm
with CO.sub.2.
[0219] Where the culture media is contacted with "sufficient"
CO.sub.2, by "sufficient," it is meant that the amount CO.sub.2
lowers the pH of the culture medium to result in an aromatic
carboxylic acid precipitate to substantially deplete the culture
medium of the aromatic carboxyate anion. In certain embodiments,
"sufficient" CO.sub.2 comprises contacting the culture media with
CO.sub.2 under a pressure and/or temperatures as described in the
paragraphs above and below.
[0220] In certain embodiments, the temperature of the culture
medium is in the range of about 0.degree. C. to 90.degree. C. In
certain embodiments, the temperature of the culture medium is in
the range of about 0.degree. C. to 80.degree. C. In certain
embodiments, the temperature of the culture medium is in the range
of about 0.degree. C. to 60.degree. C. In certain embodiments, the
temperature of the culture medium is in the range of about
5.degree. C. to 50.degree. C. In certain embodiments, the
temperature of the culture medium is in the range of about
5.degree. C. to 10.degree. C., 10.degree. C. to 20.degree. C.,
20.degree. C. to 30.degree. C., 30.degree. C. to 40.degree. C.,
40.degree. C. to 50.degree. C., or 40.degree. C. to 60.degree. C.
In certain embodiments, the temperature of the culture medium is
about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50.degree. C.
[0221] In certain embodiments, the contacting step takes place in a
vessel. In such embodiments, the pressures and temperatures, and
ranges thereof, provided above are those inside the vessel.
[0222] In certain embodiments, the vessel is pressurized in the
range of about 0.1 to 30 atm with CO.sub.2 and the pressurized
culture medium in the vessel is stirred at temperatures between
about 0.degree. C. and about 80.degree. C. for up to 24 hours.
Trapping and Recycling Side Product from Culturing Step
[0223] In certain embodiments, a side product of the culturing step
is trapped and recycled. In certain embodiments, the side product
of the culturing step is CO.sub.2. For example, the biosynthetic
pathways to 2H3M4OP, exemplified in FIGS. 1-3, each generate one
equivalent of CO.sub.2. Carbon dioxide may also be produced in
other metabolic pathways when culturing the non-naturally occurring
microbial organism. The production of this CO.sub.2 if not
collected and reused represents inefficiency and, if released, has
a potential to contribute to Green House Gas emissions. Production
of CO.sub.2 by the culture medium is considered bio-based CO.sub.2
by virtue of the fact that is has the expected .sup.14C content
measured by, for example, ASTM D6866-05 analysis methods for dating
radioactive carbon.
[0224] In certain embodiments, a process as provided herein further
comprises collecting CO.sub.2 produced by culturing the
non-naturally occurring organism. The collected CO.sub.2 can, for
example, be used to lower the pH of the culture medium to produce
the aromatic carboxylic acid precipitate, or, for example, to
supplement the CO.sub.2 used to lower the pH of the culture
medium.
[0225] Accordingly, one objective is to improve overall carbon
capture and/or improve overall costs and efficiencies of the
processes disclosed herein for isolating an aromatic carboxylic
acid.
[0226] In certain embodiments, CO.sub.2 generated in the
biosynthesis of the aromatic carboxylic acid can be trapped and
recycled for later use, using, for example, a fermentation trap or
CO.sub.2 capture device.
[0227] In certain embodiments, CO.sub.2 generated in the
biosynthesis of the aromatic carboxylic acid can be trapped and
recycled for use to lower the pH of the culture medium to
precipitate the aromatic carboxylic acid.
[0228] In certain embodiments, CO.sub.2 generated, for example, in
culturing organisms in the biosynthesis of the aromatic carboxylic
acid can be trapped and recycled for use to maintain anaerobic or
substantially anaerobic conditions in the culture medium.
[0229] Technologies for collecting (also termed trapping,
sequestering, recovering) for CO.sub.2 are known including those
applicable to collecting CO.sub.2 from culturing organisms. It will
understood that such methods can, for example, be employed in
connection with collecting CO.sub.2 from the cultured organisms as
described herein.
[0230] In certain embodiments, CO.sub.2 generated in the
biosynthesis of the aromatic carboxylic acid can be trapped and
recycled for use as a source of carbon to the non-naturally
occurring microorganism in the culture medium. In certain
embodiments, CO.sub.2 can be trapped and mixed with Synthesis gas,
also known as syngas or producer gas, for use as a source of carbon
to the non-naturally occurring microorganism in the culture medium,
as disclosed in U.S. Patent Publication No. US 2011/0207185 A1.
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. 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.
Recovery of Salts for Recycling
[0231] The aqueous solution remaining after the aromatic carboxylic
acid is precipitated from the culture medium contains solvated
salts as counter ions, which can be recovered for recycling in the
processes disclosed herein. For example, where CO.sub.2 is used to
lower the pH of the culture medium, the aqueous solution will
contain solvated carbonate salts. Where ammonia (NH.sub.3) is used
as a base to maintain the aromatic carboxylate anion in soluble
form, the aqueous solution will contain solvated ammonium salts.
Where both NH.sub.3 and CO.sub.2 are used, the aqueous solution
will contain solvated ammonium carbonate
((NH.sub.4).sub.2CO.sub.3).
[0232] In certain advantageous embodiments, it is contemplated that
solvated (NH.sub.4).sub.2CO.sub.3 salt remaining in the aqueous
solution following precipitation of the aromatic carboxylic acid
can be recovered and recycled. For example, heating the aqueous
solution will decompose the (NH.sub.4).sub.2CO.sub.3 into NH.sub.3
and CO.sub.2, as depicted below.
(NH.sub.4).sub.2CO.sub.3.fwdarw.2NH.sub.3+CO.sub.2+H.sub.2O
The resultant gaseous NH.sub.3 and CO.sub.2 can be separated,
recovered and/or recycled using, for example, a fermentation trap
or gas capture device. Recovery/recycling of gaseous NH.sub.3 and
CO.sub.2 will reduce the costs associated with use of these
materials and will also reduce environmental discharges, for
example, Green House Gas emissions in the case of the CO.sub.2 gas.
This will further improve overall carbon capture and/or improve
overall costs and efficiencies of the processes disclosed herein
for isolating an aromatic carboxylic acid.
[0233] In certain embodiments, the NH.sub.3 recovered from
decomposition of (NH.sub.4).sub.2CO.sub.3 can be recycled for use
as a base to maintain the aromatic carboxylate anion in soluble
form in the culture medium.
[0234] In certain embodiments, the CO.sub.2 recovered from
decomposition of (NH.sub.4).sub.2CO.sub.3 can be recycled for use
to lower the pH of the culture medium to precipitate the aromatic
carboxylic acid.
[0235] In certain embodiments, the CO.sub.2 recovered from
decomposition of (NH.sub.4).sub.2CO.sub.3 can be recycled for use
to maintain anaerobic or substantially anaerobic conditions in the
culture medium used to produce the aromatic carboxylate anion.
[0236] In certain embodiments, the CO.sub.2 recovered from
decomposition of (NH.sub.4).sub.2CO.sub.3 can be recycled for use
as a source of carbon to the non-naturally occurring microorganism
in the culture medium.
[0237] The remaining aqueous solution will be at a higher pH, and
may also be recycled for use in, for example, the culture medium.
In certain embodiments, enough CO.sub.2 will be removed so that the
aqueous solution returns to a neutral pH to provide a neutral
solution for the culture medium.
[0238] In certain embodiments, following acidification and
precipitation of the aromatic carboxylic acid, the aqueous solution
can be filtered to provide an aqueous filtrate from which the
solvated salts may be recovered for recycling.
Separating the Aromatic Carboxylic Acid from the Culture Medium
[0239] Once precipitated, the aromatic carboxylic acid may be
separated from the other components in the culture medium by
removing the culture medium. In certain embodiments, the separation
of the aromatic carboxylic acid from the other components in the
culture medium comprises filtering and recovering of the aromatic
carboxylic acid from the culture medium. In certain embodiments the
precipitated aromatic carboxylic acid is resolubilized and
subjected to extraction or separation procedures well know in the
art, which can, for example, 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.
[0240] In certain embodiments, the aromatic carboxylic acid is
purified following separation from the other components in the
culture medium. In certain embodiments, the aromatic carboxylic
acid is purified using crystallization.
[0241] Therefore, provided herein is an isolated bio-based p-toluic
acid produced by the processes described herein. Also provided
herein is an isolated bio-based terephthalic acid produced by the
processes described herein.
Exemplary Calculations
[0242] Dependence of Terephthalic Acid Forms on pH
[0243] The following describes exemplary calculations of dependence
of terephthalic acid forms on pH.
TABLE-US-00001 TABLE 1 Dependence of terephthalic acid forms on pH
Total acid Undissociated acid Monoanionic acid Dianioninc acid pH
(mol/L) (mol/L) Percent (mol/L) Percent (mol/L) Percent 2 0.354314
0.344349 97.2% 0.009931 2.8% 3.44E-05 0.0% 3 0.00421 0.003243 77.0%
0.000935 22.2% 3.24E-05 0.8% 4 1E-04 2.05E-05 20.5% 5.91E-05 59.1%
2.05E-05 20.5% 5 5.67E-06 4.37E-08 0.8% 1.26E-06 22.2% 4.37E-06
77.0% 6 5.02E-07 4.88E-11 0.0% 1.41E-08 2.8% 4.88E-07 97.2% 6.5
1.43E-07 1.42E-12 0.0% 1.29E-09 0.9% 1.42E-07 99.1%
[0244] Table 1 demonstrates that at a pH value of 4, terephthalic
acid forms exists in a ratio of about 20.5/59/20.5 anion to
monoacid to diacid; at a pH value of 3, terephthalic acid forms
exists in a ratio of about 1/22/77 anion to monoacid to diacid; at
a pH value of 2, terephthalic acid forms exists in a ratio of about
0/3/97 anion to monoacid to diacid, as illustrated below.
TABLE-US-00002 ##STR00005## ##STR00006## ##STR00007## pH Ratios 2 0
3 97 3 1 22 77 4 20.5 59 20.5
[0245] Table 1 further demonstrates that at pH 7, close to 100% of
terephthalic acid will be in the anionic form. Thus, in the
processes provided herein, where the non-naturally occurring
microbial organism produces terephthalate, performing the culturing
step at a neutral pH will produce the anion, that is, close to 100%
of terephthalic acid will be in the anionic form
(terephthalate).
[0246] Similarly, a culture medium that is acidified to less than
about 3.0 pH units will afford predominantly the diacid form of
terephthalic acid. It will be understood that terephthalic acid is
virtually insoluble in aqueous solutions (0.017 g/L solubility in
water at 25.degree. C.). In light that terephthalic acid is not
soluble in aqueous solution, it is quite clear from Table 1 that
acidification of the culture medium to values less than about 3.0
pH units will result in a substantial depletion of terephthalate in
the culture medium, because at values less than about 3.0 pH units,
there will no soluble form of terephthalic acid remaining in the
culture medium, no matter what the form (anion, monoacid or
diacid).
[0247] Pressure-pH Relationship for CO.sub.2 in Water
[0248] The following describes exemplary calculations of
pressure-pH relationship for CO.sub.2 in water.
TABLE-US-00003 TABLE 2 Pressure-pH relationship for CO.sub.2 in
water. [H.sub.2CO.sub.3] (atm) pH [CO.sub.2] (mol/L) (mol/L)
[HCO.sub.3.sup.-] (mol/L) [CO.sub.3.sup.2-] (mol/L) 1.0 .times.
10.sup.-8 7.00 3.36 .times. 10.sup.-10 5.71 .times. 10.sup.-13 1.42
.times. 10.sup.-09 7.90 .times. 10.sup.-13 1.0 .times. 10.sup.-7
6.94 3.36 .times. 10.sup.-09 5.71 .times. 10.sup.-12 5.90 .times.
10.sup.-09 1.90 .times. 10.sup.-12 1.0 .times. 10.sup.-6 6.81 3.36
.times. 10.sup.-08 5.71 .times. 10.sup.-11 9.16 .times. 10.sup.-08
3.30 .times. 10.sup.-11 1.0 .times. 10.sup.-5 6.42 3.36 .times.
10.sup.-07 5.71 .times. 10.sup.-09 3.78 .times. 10.sup.-07 4.53
.times. 10.sup.-11 1.0 .times. 10.sup.-4 5.92 3.36 .times.
10.sup.-06 5.71 .times. 10.sup.-09 1.19 .times. 10.sup.-06 5.57
.times. 10.sup.-11 3.5 .times. 10.sup.-4 5.65 1.18 .times.
10.sup.-05 2.00 .times. 10.sup.-08 2.23 .times. 10.sup.-06 5.60
.times. 10.sup.-11 1.0 .times. 10.sup.-3 5.42 3.36 .times.
10.sup.-05 5.71 .times. 10.sup.-08 3.78 .times. 10.sup.-06 5.61
.times. 10.sup.-11 1.0 .times. 10.sup.-2 4.92 3.36 .times.
10.sup.-04 5.71 .times. 10.sup.-07 1.19 .times. 10.sup.-05 5.61
.times. 10.sup.-11 1.0 .times. 10.sup.-1 4.42 3.36 .times.
10.sup.-03 5.71 .times. 10.sup.-06 3.78 .times. 10.sup.-05 5.61
.times. 10.sup.-11 1.0 .times. 10.sup.+0 3.92 3.36 .times.
10.sup.-02 5.71 .times. 10.sup.-05 1.20 .times. 10.sup.-04 5.61
.times. 10.sup.-11 2.5 .times. 10.sup.+0 3.72 8.40 .times.
10.sup.-02 1.43 .times. 10.sup.-04 1.89 .times. 10.sup.-04 5.61
.times. 10.sup.-11 1.0 .times. 10.sup.+1 3.42 3.36 .times.
10.sup.-01 5.71 .times. 10.sup.-04 3.78 .times. 10.sup.-04 5.61
.times. 10.sup.-11
[0249] As demonstrated in Table 2, CO.sub.2 pressures between 1-10
atmospheres correspond to pH values as low as 3.42. Thus,
acidification of aqueous culture media with CO.sub.2 pressures
greater than 2.5 atmospheres can, for example, reduce pH values to
less than 4.0.
[0250] 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.
Examples
Example I. Exemplary Pathways for Producing 2H3M4OP
[0251] This example describes an exemplary pathway for producing
the terephthalic acid (PTA) precursor 2H3M4OP.
[0252] The precursor to the p-toluate and PTA pathways is 2H3M4OP.
FIG. 1 shows a pathway from erythrose-4-phosphate to 2H3M4OP. The
first two steps of the pathway employ enzymes of P5C biosynthesis.
In the first step, oxidation of erythrose-4-phosphate to
4-phosphoerythronate is catalyzed by erythrose-4-phosphate
dehydrogenase (EC 1.2.1.72). In step B, 4-phosphoerythronate is
further oxidized to 2-oxo-3-hydroxy-4-phosphobutanoate by
4-phosphoerythronate dehydrogenase (EC 1.1.1.290). The next three
steps of the pathway are analogous to the isoleucine biosynthesis
pathway comprising a synthase, a ketol-acid reductoisomerase and a
diol dehydratase. In step C, a
2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase converts
pyruvate and 2-oxo-3-hydroxy-4-phosphobutanoate to
2-acetyl-2,3-dihydroxy-4-phosphobutanoate, releasing CO.sub.2. This
intermediate is then converted to
2,3,4-trihydroxy-3-methyl-5-phosphopentanoate by a
reductoisomerase. Dehydration by a diol dehydratase forms the
2H3M4OP precursor, 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate.
2H3M4OP is then formed by decarboxylation of the keto-acid.
[0253] Pathways from 4,5-dihydroxy-2-oxopentanoate to 2H3M4OP are
depicted in FIG. 2. 4,5-dihydroxy-2-oxopentanoate is derived from
sugars such as arabinose and xylose. It can also be formed
enzymatically by condensation of pyruvate and glycolaldehyde by
aldolase enzymes such as 2-dehydro-3-deoxypentonate aldolase,
2-dehydro-3-deoxyglucarate aldolase, or other enzymes in EC class
4.1.2 or 4.1.3. In one 2H3M4OP pathway,
4,5-dihydroxy-2-oxopentanoate is converted to
4,5-dihydroxy-3-methyl-2-oxopentanoate by a methyltransferase (step
A). The methylated product is then phosphorylated and
decarboxylated (steps B/C). In an alternate pathway, the
4,5-dihydroxy-2-oxopentanoate substrate is phosphorylated to
4-hydroxy-2-oxo-5-phosphopentanoate (step D), then methylated by a
methyltransferase (step E) to the
4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate intermediate. Both
pathways share the final decarboxylation of
4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate to 2H3M4OP.
Enzyme Candidates
TABLE-US-00004 [0254] EC Class Description Pathway step 1.1.1.a
Oxidoreductase (alcohol to oxo) 1B 1.1.1.b Ketol-acid
reductoisomerase 1D 1.2.1.a Oxidoreductase (aldehyde to acid) 1A
2.2.1.a Synthase 1C 2.7.1.a Kinase 2B, 2D 4.1.1.a Decarboxylase 1F,
2C 4.2.1.b Diol dehydratase 1E No EC Methyltransferase 2A, 2E
EC 1.1.1.a
[0255] The NAD(P)+ dependent oxidation of erythronate-4-phosphate
to 2-oxo-3-hydroxy-4-phosphobutanoate is catalyzed by
4-phosphoerythronate dehydrogenase (Step 1B, EC 1.1.1.290). This
enzyme, encoded by the pdxB gene of E. coli, participates in
pyridoxine biosynthesis (Zhao et al, J Bacteriol 177:2804-12
(1995)). An analogous enzyme is encoded by pdxB of Pseudomonas
aeruginosa (Ha et. al, J Mol Biol 366:1294-1304 (2007)). The E.
coli and P. aeruginosa enzymes utilize NAD+ as a cofactor. The pdxR
gene from Sinorhizobium melitoti utilizes FAD+ as a cofactor
(Hoshino et al, WO/2004/029250). Alcohol dehydrogenases that
convert 2-hydroxyacids to 2-ketoacids, such as malate dehydrogenase
(mdh) and lactate dehydrogenase (ldhA) of E. coli, are also
suitable candidates. The lactate dehydrogenase from Ralstonia
eutropha has been shown to demonstrate high activities on
2-ketoacids of various chain lengths includings lactate,
2-oxobutyrate, 2-oxopentanoate and 2-oxoglutarate (Steinbuchel et
al., Eur. J. Biochem. 130:329-334 (1983)).
TABLE-US-00005 Gene Accession No. GI No. Organism pdxB NP_416823.1
16130255 Escherichia coli pdxB Q9I3W9.1 46396520 Pseudomonas
aeruginosa pdxR AEH77803.1 336031871 Sinorhizobium melitoti mdh
AAC76268.1 1789632 Escherichia coli ldhA NP_415898.1 16129341
Escherichia coli ldh YP_725182.1 113866693 Ralstonia eutropha
EC 1.1.1.b
[0256] The reduction and isomerization of
2-acetyl-2,3-dihydroxy-4-phosphobutanoate to
2,3,4-trihydroxy-3-methyl-5-phosphopentanoate is catalyzed by a
bifunctional enzyme with 2-acetyl-2,3-dihydroxy-4-phosphobutanoate
reductoisomerase activity (Step 1D). An analogous transformation is
catalyzed by ketol-acid reductoisomerase (EC 1.1.1.86), an enzyme
involved in branched chain amino acid biosynthesis. This enzyme,
encoded by ilvC of Escherichia coli, is active on multiple
substrates including 2-hydroxy-2-methyl-3-oxobutanoate,
2,3-dihydroxy-3-methylpentanoate and 2,3-dihydroxy-3-isopentanoate.
Crystal structure of enzymes from E. coli and Pseudomonas
aeruginosa are available (Tyagi et al., Prot Sci 14:3089-3100
(2005); Ahn et al., J Mol Biol 328:505-15 (2003)). Additional
candidates are ilvC of C. glutamicum (Lewal et al, J Biotechnol
104:241-52 (2003)) and ilv5p of Saccharomyces cerevisiae (Omura,
Appl Microbiol Biotechnol 78:503-13 (2008))
TABLE-US-00006 Gene Accession No. GI No. Organism ilvC NP_418222.1
16131632 Escherichia coli ilvC YP_793157.1 116052840 Pseudomonas
aeruginosa ilvC EHE83601.1 354510679 Corynebacterium glutamicum
Ilv5p NP_013459.1 6323387 Saccharomyces cerevisiae
[0257] The oxidation of erythrose-4-phosphate to
4-phosphoerythronate is catalyzed by an oxidoreductase that
converts an aldehyde to an acid. An enzyme with this activity is
erythrose-4-phosphate dehydrogenase (EC 1.2.1.72). An NAD+
dependent erythrose-4-dehydrogenase is encoded by the epd gene of
E. coli (Yang et al, J Bacteriol 180:4294-99 (1998)). A similar
enzyme has been characterized in Vibrio cholera (Carroll et al, J
Bacteriol 179:293-6 (1997)). Additional candidates are
NAD+-dependent aldehyde dehydrogenases (EC 1.2.1.-) and NAD(P)+
dependent glyceraldehyde-3-phosphate dehydrogenases (EC 1.2.1.3 and
1.2.1.9). Two aldehyde dehydrogenases found in human liver, ALDH-1
and ALDH-2, have broad substrate ranges for a variety of aliphatic,
aromatic and polycyclic aldehydes (Klyosov, Biochemistry
35:4457-4467 (1996a)). Active ALDH-2 has been efficiently expressed
in E. coli using the GroEL proteins as chaperonins (Lee et al.,
Biochem. Biophys. Res. Commun. 298:216-224 (2002)). The rat
mitochondrial aldehyde dehydrogenase also has a broad substrate
range (Siew et al., Arch. Biochem. Biophys. 176:638-649 (1976)).
The NAD+ dependent gapN gene of Thermoproteus tenax oxidizes
glyceraldehyde-3-phosphate to its corresponding acid (Brunner et
al, J Biol Chem 273:6149-56 (1998)). An exemplary NADP+ dependent
glyceraldehyde-3-phosphate dehydrogenase is the GAPN gene product
of Arabidopsis thaliana (Rius et al Plant Mol Biol 61:945-57
(2006))
TABLE-US-00007 Gene Accession No. GI No. Organism epd NP_417402.1
16130828 Escherichia coli epd YP_001216003.2 229259769 Vibrio
cholera ALDH-2 P05091.2 118504 Homo sapiens ALDH-2 NP_115792.1
14192933 Raffia norvegicus gapN CAA71651.1 3059159 Thermoproteus
tenax GAPN ABB83822.1 82570696 Arabidopsis thaliana
EC 2.2.1.a
[0258] Formation of 2-acetyl-2,3-dihydroxy-4-phosphobutanoate in
Step 1C is catalyzed by a synthase enzyme in EC class 2.2.1. A
suitable enzyme for catalyzing this transformation is
acetohydroxyacid synthase (EC 2.2.1.6), which condenses pyruvate
and 2-oxobutanoate to 2-aceto-2-hydroxybutanoate and carbon
dioxide. Alternately, two pyruvates are condensed forming
acetolactate. The enzyme operates in branched chain amino acid
biosynthesis pathways. Two isozymes are active in E. coli: ilvBN
and ilvHI (Vyazmensky et al Biochem, 35:10339-46 (1996); Engel et
al, Biotechnol bioeng 88:825-31 (2004)). The IlvBN enzyme has
activity on a broad range of aldehyde substrates in vitro. The
Methanococcus aeolicus ilvBN gene product is similar to the E. coli
IlvBN enzyme (Xing et al, J Bacteriol 176:1207-13 (1994)). The
acetolactate synthase from Bacillus subtilis (AlsS), which
naturally catalyzes the condensation of two molecules of pyruvate
to form 2-acetolactate, is also able to catalyze the
decarboxylation of 2-ketoisovalerate like KDC both in vivo and in
vitro (Atsumi and Liao, AEM 75:6306-11 (2009)).
TABLE-US-00008 Gene Accession No. GI No. Organism ilvB NP_418127.1
16131541 Escherichia coli ilvN NP_418126.1 16131540 Escherichia
coli ilvH NP_414620.1 16128071 Escherichia coli ilvI YP_025294.2
90111084 Escherichia coli ilvB AAB53488.1 2065479 Methanococcus
aeolicus ilvN AAB53489.1 2065480 Methanococcus aeolicus alsS
Q04789.3 239938889 Bacillus subtilis
EC 2.7.1.a
[0259] Kinases catalyze the ATP-dependent transfer of a phosphate
group to an alcohol. Kinase enzymes are required to catalyze the
phosphorylation of 4,5-dihydroxy-3-methyl-2-oxopentanoate (Step 2B)
and 4,5-dihydroxy-2-oxopentanoate (Step 2D). The enzymes described
below naturally possess such activity or can be engineered to
exhibit this activity. Kinases that catalyze the transfer of a
phosphate group to an alcohol group are members of the EC 2.7.1
enzyme class. The table below lists several useful kinase enzymes
in the EC 2.7.1 enzyme class.
TABLE-US-00009 Enzyme Commission Number Enzyme Name 2.7.1.1
hexokinase 2.7.1.2 glucokinase 2.7.1.3 ketohexokinase 2.7.1.4
fructokinase 2.7.1.5 rhamnulokinase 2.7.1.6 galactokinase 2.7.1.7
mannokinase 2.7.1.8 glucosamine kinase 2.7.1.10 phosphoglucokinase
2.7.1.11 6-phosphofructokinase 2.7.1.12 gluconokinase 2.7.1.13
dehydrogluconokinase 2.7.1.14 sedoheptulokinase 2.7.1.15 ribokinase
2.7.1.16 ribulokinase 2.7.1.17 xylulokinase 2.7.1.18
phosphoribokinase 2.7.1.19 phosphoribulokinase 2.7.1.20 adenosine
kinase 2.7.1.21 thymidine kinase 2.7.1.22 ribosylnicotinamide
kinase 2.7.1.23 NAD+ kinase 2.7.1.24 dephospho-CoA kinase 2.7.1.25
adenylyl-sulfate kinase 2.7.1.26 riboflavin kinase 2.7.1.27
erythritol kinase 2.7.1.28 triokinase 2.7.1.29 glycerone kinase
2.7.1.30 glycerol kinase 2.7.1.31 glycerate kinase 2.7.1.32 choline
kinase 2.7.1.33 pantothenate kinase 2.7.1.34 pantetheine kinase
2.7.1.35 pyridoxal kinase 2.7.1.36 mevalonate kinase 2.7.1.39
homoserine kinase 2.7.1.40 pyruvate kinase 2.7.1.41
glucose-1-phosphate phosphodismutase 2.7.1.42 riboflavin
phosphotransferase 2.7.1.43 glucuronokinase 2.7.1.44
galacturonokinase 2.7.1.45 2-dehydro-3- deoxygluconokinase 2.7.1.46
L-arabinokinase 2.7.1.47 D-ribulokinase 2.7.1.48 uridine kinase
2.7.1.49 hydroxymethylpyrimidine kinase 2.7.1.50
hydroxyethylthiazole kinase 2.7.1.51 L-fuculokinase 2.7.1.52
fucokinase 2.7.1.53 L-xylulokinase 2.7.1.54 D-arabinokinase
2.7.1.55 allose kinase 2.7.1.56 1-phosphofructokinase 2.7.1.58
2-dehydro-3- deoxygalactonokinase 2.7.1.59 N-acetylglucosamine
kinase 2.7.1.60 N-acylmannosamine kinase 2.7.1.61
acyl-phosphate-hexose phosphotransferase 2.7.1.62
phosphoramidate-hexose phosphotransferase 2.7.1.63
polyphosphate-glucose phosphotransferase 2.7.1.64 inositol 3-kinase
2.7.1.65 scyllo-inosamine 4-kinase 2.7.1.66 undecaprenol kinase
2.7.1.67 1-phosphatidylinositol 4- kinase 2.7.1.68
1-phosphatidylinositol-4- phosphate 5-kinase 2.7.1.69 protein-Np-
phosphohistidine-sugar phosphotransferase 2.7.1.70 identical to EC
2.7.1.37. 2.7.1.71 shikimate kinase 2.7.1.72 streptomycin 6-kinase
2.7.1.73 inosine kinase 2.7.1.74 deoxycytidine kinase 2.7.1.76
deoxyadenosine kinase 2.7.1.77 nucleoside phosphotransferase
2.7.1.78 polynucleotide 5'- hydroxyl-kinase 2.7.1.79
diphosphate-glycerol phosphotransferase 2.7.1.80 diphosphate-serine
phosphotransferase 2.7.1.81 hydroxylysine kinase 2.7.1.82
ethanolamine kinase 2.7.1.83 pseudouridine kinase 2.7.1.84
alkylglycerone kinase 2.7.1.85 .beta.-glucoside kinase 2.7.1.86
NADH kinase 2.7.1.87 streptomycin 3''-kinase 2.7.1.88
dihydrostreptomycin-6- phosphate 3'a-kinase 2.7.1.89 thiamine
kinase 2.7.1.90 diphosphate-fructose-6- phosphate 1-
phosphotransferase 2.7.1.91 sphinganine kinase 2.7.1.92
5-dehydro-2- deoxygluconokinase 2.7.1.93 alkylglycerol kinase
2.7.1.94 acylglycerol kinase 2.7.1.95 kanamycin kinase 2.7.1.100
S-methyl-5-thioribose kinase 2.7.1.101 tagatose kinase 2.7.1.102
hamamelose kinase 2.7.1.103 viomycin kinase 2.7.1.105
6-phosphofructo-2-kinase 2.7.1.106 glucose-1,6-bisphosphate
synthase 2.7.1.107 diacylglycerol kinase 2.7.1.108 dolichol kinase
2.7.1.113 deoxyguanosine kinase 2.7.1.114 AMP-thymidine kinase
2.7.1.118 ADP-thymidine kinase 2.7.1.119 hygromycin-B 7''-O- kinase
2.7.1.121 phosphoenolpyruvate- glycerone phosphotransferase
2.7.1.122 xylitol kinase 2.7.1.127 inositol-trisphosphate 3- kinase
2.7.1.130 tetraacyldisaccharide 4'- kinase 2.7.1.134
inositol-tetrakisphosphate 1-kinase 2.7.1.136 macrolide 2'-kinase
2.7.1.137 phosphatidylinositol 3- kinase 2.7.1.138 ceramide kinase
2.7.1.140 inositol-tetrakisphosphate 5-kinase 2.7.1.142
glycerol-3-phosphate- glucose phosphotransferase 2.7.1.143
diphosphate-purine nucleoside kinase 2.7.1.144 tagatose-6-phosphate
kinase 2.7.1.145 deoxynucleoside kinase 2.7.1.146 ADP-dependent
phosphofructokinase 2.7.1.147 ADP-dependent glucokinase 2.7.1.148
4-(cytidine 5'-diphospho)- 2-C-methyl-D-erythritol kinase 2.7.1.149
1-phosphatidylinositol-5- phosphate 4-kinase 2.7.1.150
1-phosphatidylinositol-3- phosphate 5-kinase 2.7.1.151
inositol-polyphosphate multikinase 2.7.1.153
phosphatidylinositol-4,5- bisphosphate 3-kinase 2.7.1.154
phosphatidylinositol-4- phosphate 3-kinase 2.7.1.156
adenosylcobinamide kinase 2.7.1.157 N-acetylgalactosamine kinase
2.7.1.158 inositol- pentakisphosphate 2- kinase 2.7.1.159
inositol-1,3,4- trisphosphate 5/6-kinase 2.7.1.160
2'-phosphotransferase 2.7.1.161 CTP-dependent riboflavin kinase
2.7.1.162 N-acetylhexosamine 1- kinase 2.7.1.163 hygromycin B
4-O-kinase 2.7.1.164 O-phosphoseryl-tRNASec kinase
[0260] Particularly useful kinase enzymes for catalyzing steps 2B
and 2D are mevalonate kinase, glycerol kinase, homoserine kinase,
glycerate kinase and erythritol kinase. A good candidate for this
step is mevalonate kinase (EC 2.7.1.36) that phosphorylates the
terminal hydroxyl group of mevalonate. Some gene candidates for
this step are erg12 from S. cerevisiae, mvk from Methanocaldococcus
jannaschi, MVK from Homo sapiens, and mvk from Arabidopsis thaliana
col.
TABLE-US-00010 Protein GenBank ID GI Number Organism erg12
CAA39359.1 3684 Saccharomyces cerevisiae mvk Q58487.1 2497517
Methanocaldococcus jannaschii mvk AAH16140.1 16359371 Homo sapiens
mvk NP_851084.1 30690651 Arabidopsis thaliana
[0261] Glycerol kinase also phosphorylates the terminal hydroxyl
group in glycerol to form glycerol-3-phosphate. This reaction
occurs in several species, including Escherichia coli,
Saccharomyces cerevisiae, and Thermotoga maritima. The E. coli
glycerol kinase has been shown to accept alternate substrates such
as dihydroxyacetone and glyceraldehyde (Hayashi et al., J Biol.
Chem. 242:1030-1035 (1967)). T, maritime has two glycerol kinases
(Nelson et al., Nature 399:323-329 (1999)). Glycerol kinases have
been shown to have a wide range of substrate specificity. Crans and
Whiteside studied glycerol kinases from four different organisms
(Escherichia coli, S. cerevisiae, Bacillus stearothermophilus, and
Candida mycoderma) (Crans et al., J. Am. Chem. Soc. 107:7008-7018
(2010); Nelson et al., Nature 399:323-329 (1999)). They studied 66
different analogs of glycerol and concluded that the enzyme could
accept a range of substituents in place of one terminal hydroxyl
group and that the hydrogen atom at C2 could be replaced by a
methyl group. Interestingly, the kinetic constants of the enzyme
from all four organisms were very similar.
TABLE-US-00011 Protein GenBank ID GI Number Organism glpK
AP_003883.1 89110103 Escherichia coli glpK1 NP_228760.1 15642775
Thermotoga maritime glpK2 NP_229230.1 15642775 Thermotoga maritime
Gut1 NP_011831.1 82795252 Saccharomyces cerevisiae
[0262] Homoserine kinase is another candidate kinase. This enzyme
is present in a number of organisms including E. coli, Streptomyces
sp, and S. cerevisiae. Homoserine kinase from E. coli has been
shown to have activity on numerous substrates, including,
L-2-amino,1,4-butanediol, aspartate semialdehyde, and
2-amino-5-hydroxyvalerate (Huo et al., Biochemistry 35:16180-16185
(1996); Huo et al., Arch. Biochem. Biophys. 330:373-379 (1996)).
This enzyme can act on substrates where the carboxyl group at the
alpha position has been replaced by an ester or by a hydroxymethyl
group.
TABLE-US-00012 Protein GenBank ID GI Number Organism thrB
BAB96580.2 85674277 Escherichia coli SACT1DRAFT_4809 ZP_06280784.1
282871792 Streptomyces sp. ACT-1 Thr1 AAA35154.1 172978
Saccharomyces serevisiae
[0263] The interconversion of 3-phosphoglycerate and glycerate is
catalyzed by glycerate kinase (EC 2.7.1.31). Three classes of
glycerate kinase have been identified. Enzymes in class I and II
produce glycerate-2-phosphate, whereas the class III enzymes found
in plants and yeast produce glycerate-3-phosphate (Bartsch et al.,
FEBS Lett. 582:3025-3028 (2008)). In a recent study, class III
glycerate kinase enzymes from Saccharomyces cerevisiae, Oryza
sativa and Arabidopsis thaliana were heterologously expressed in E.
coli and characterized (Bartsch et al., FEBS Lett. 582:3025-3028
(2008)).
TABLE-US-00013 Protein GenBank ID GI Number Organism glxK
AAC73616.1 1786724 Escherichia coli YGR205W AAS56599.1 45270436
Saccharomyces cerevisiae Os01g0682500 BAF05800.1 113533417 Oryza
sativa At1g80380 BAH57057.1 227204411 Arabidopsis thaliana
[0264] Erythritol is converted to erythritol-4-phosphate by the
erythritol kinase. Erythritol kinase (EC 2.7.1.27) catalyzes the
phosphorylation of erythritol. Erythritol kinase was characterized
in erythritol utilizing bacteria such as Brucella abortus (Sperry
et al., J Bacteriol. 121:619-630 (1975)). The eryA gene of Brucella
abortus has been functionally expressed in Escherichia coli and the
resultant EryA was shown to catalyze the ATP-dependent conversion
of erythritol to erythritol-4-phosphate (Lillo et al., Bioorg. Med.
Chem. Lett. 13:737-739 (2003)).
TABLE-US-00014 Protein GenBank ID GI Number Organism eryA Q8YCU8
81850596 Brucella melitensis eriA Q92NH0 81774560 Sinorhizobium
meliloti eryA YP_001108625.1 134102964 Saccharopolyspora erythraea
NRRL 2338
EC 4.1.1.a
[0265] Decarboxylation of
4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate to 2H3M4OP (Steps 1F
and 2C) is catalyzed by a keto-acid decarboxylase. Although an
enzyme with 2H3M4OP-forming activity has not been described in the
literature, the decarboxylation of keto-acids is catalyzed by a
variety of enzymes with varied substrate specificities, including
pyruvate decarboxylase (EC 4.1.1.1), benzoylformate decarboxylase
(EC 4.1.1.7), alpha-ketoglutarate decarboxylase and branched-chain
alpha-ketoacid decarboxylase, phosphonopyruvate decarboxylase,
sulfopyruvate decarboxylase, acetohydroxy acid
synthase/acetolactate synthase, glyoxylate carboligase and indole
pyruvate decarboxylase.
[0266] Branched chain alpha-ketoacid decarboxylase (BCKAD, EC
4.1.1.72) is a particularly useful enzyme for the invention, as the
pathway substrate is also a branched alpha-ketoacid. This class of
enzyme has been shown to decarboxylate a variety of compounds
varying in chain length from 3 to 6 carbons (Oku et al., J Biol
Chem. 263:18386-18396 (1988); Smit et al., Appl Environ Microbiol
71:303-311 (2005)). The BCKAD enzyme in Lactococcus lactis has been
characterized on a variety of branched and linear substrates
including 2-oxobutanoate, 2-oxohexanoate, 2-oxopentanoate,
3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate and isocaproate
(Smit et al., Appl Environ Microbiol 71:303-311 (2005)). The enzyme
has been structurally characterized (Berg et al., Science.
318:1782-1786 (2007)). Sequence alignments between the Lactococcus
lactis enzyme and the pyruvate decarboxylase of Zymomonas mobilus
indicate that the catalytic and substrate recognition residues are
nearly identical (Siegert et al., Protein Eng Des Sel 18:345-357
(2005)), so this enzyme would be a promising candidate for directed
engineering of substrate specificity. Several ketoacid
decarboxylases of Saccharomyces cerevisiae catalyze the
decarboxylation of branched substrates, including ARO10, PDC6,
PDC5, PDC1 and THI3 (Dickenson et al, J Biol Chem 275:10937-42
(2000)). Yet another BCKAD enzyme is encoded by rv0853c of
Mycobacterium tuberculosis (Werther et al, J Biol Chem 283:5344-54
(2008)). This enzyme is subject to allosteric activation by
alpha-ketoacid substrates. Decarboxylation of alpha-ketoglutarate
by a BCKA was detected in Bacillus subtilis; however, this activity
was low (5%) relative to activity on other branched-chain
substrates (Oku and Kaneda, J Biol Chem. 263:18386-18396 (1988))
and the gene encoding this enzyme has not been identified to date.
Additional BCKA gene candidates can be identified by homology to
the Lactococcus lactis protein sequence. Many of the high-scoring
BLASTp hits to this enzyme are annotated as indolepyruvate
decarboxylases (EC 4.1.1.74). Indolepyruvate decarboxylase (IPDA)
is an enzyme that catalyzes the decarboxylation of indolepyruvate
to indoleacetaldehyde in plants and plant bacteria. Recombinant
branched chain alpha-keto acid decarboxylase enzymes derived from
the E1 subunits of the mitochondrial branched-chain keto acid
dehydrogenase complex from Homo sapiens and Bos taurus have been
cloned and functionally expressed in E. coli (Davie et al., J.
Biol. Chem. 267:16601-16606 (1992); Wynn et al., J. Biol. Chem.
267:12400-12403 (1992); Wynn et al., J. Biol. Chem. 267:1881-1887
(1992)). In these studies, the authors found that co-expression of
chaperonins GroEL and GroES enhanced the specific activity of the
decarboxylase by 500-fold (Wynn et al., J. Biol. Chem.
267:12400-12403 (1992)).
TABLE-US-00015 Protein GenBank ID GI Number Organism kdcA
AAS49166.1 44921617 Lactococcus lactis PDC6 NP_010366.1 6320286
Saccharomyces cerevisiae PDC5 NP_013235.1 6323163 Saccharomyces
cerevisiae PDC1 P06169 30923172 Saccharomyces cerevisiae ARO10
NP_010668.1 6320588 Saccharomyces cerevisiae THI3 NP_010203.1
6320123 Saccharomyces cerevisiae rv0853c O53865.1 81343167
Mycobacterium tuberculosis BCKDHB NP_898871.1 34101272 Homo sapiens
BCKDHA NP_000700.1 11386135 Homo sapiens BCKDHB P21839 115502434
Bos taurus BCKDHA P11178 129030 Bos taurus
[0267] Another class of enzymes suitable for decarboxylating a
phosphorylated alpha-ketoacid such as
4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate is phosphonopyruvate
decarboxylase (EC 4.1.1.82). This enzyme catalyzes the
decarboxylation of 3-phosphonopyruvate to 2-phosphonoacetaldehyde.
Exemplary phosphonopyruvate decarboxylase enzymes are encoded by
dhpF of Streptomyces luridus, ppd of Streptomyces
viridochromogenes, fom2 of Streptomyces wedmorensis (Circello et
al, Chem Biol 17:402-11 (2010); Blodgett et al, FEMS Microbiol Lett
163:149-57 (2005); Hidaka et al, Mol Gen Genet 249:274-80 (1995)).
The Bacteroidesfragilis enzyme, encoded by aepY, also
decarboxylates pyruvate and sulfopyruvate (Zhang et al, J Biol Chem
278:41302-8 (2003)).
TABLE-US-00016 Protein GenBank ID GI Number Organism dhpF
ACZ13457.1 268628095 Streptomyces luridus Ppd CAJ14045.1 68697716
Streptomyces viridochromogenes Fom2 BAA32496.1 1061008 Streptomyces
wedmorensis aepY AAG26466.1 11023509 Bacteroides fragilis
[0268] Other useful ketoacid decarboxylases include pyruvate
decarboxylase enzymes such as the pdc gene product of Zymomonas
mobilis. This enzyme has a broad substrate range and has been a
subject of directed engineering studies to alter the affinity for
different substrates (Siegert et al., Protein Eng Des Sel
18:345-357 (2005)). The crystal structure of this enzyme is
available (Killenberg-Jabs et al., Eur. J. Biochem. 268:1698-1704
(2001)). The benzoylformate decarboxylase from Pseudomonas putida
has a broad substrate range and has been the target of enzyme
engineering studies (Polovnikova et al., 42:1820-1830 (2003);
Hasson et al., 37:9918-9930 (1998)). Site-directed mutagenesis of
two residues in the active site of the Pseudomonas putida enzyme
altered the affinity (Km) of naturally and non-naturally occurring
substrates (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)).
The properties of this enzyme have been further modified by
directed engineering (Lingen et al., Chembiochem. 4:721-726 (2003);
Lingen et al., Protein Eng 15:585-593 (2002)). The benzoylformate
decarboxylase from Pseudomonas aeruginosa, encoded by mdlC, has
also been characterized experimentally (Barrowman et al., 34:57-60
(1986)). Additional gene candidates from Pseudomonas stutzeri,
Pseudomonas fluorescens and other organisms can be inferred by
sequence homology or identified using a growth selection system
developed in Pseudomonas putida (Henning et al., Appl. Environ.
Microbiol. 72:7510-7517 (2006)). Alpha-ketoglutarate decarboxylases
are also relevant to the invention. An exemplary KDC is encoded by
kad in Mycobacterium tuberculosis (Tian et al., PNAS
102:10670-10675 (2005)). KDC enzyme activity has also been detected
in several species of rhizobia including Bradyrhizobium japonicum
and Mesorhizobium loti (Green et al., J Bacteriol 182:2838-2844
(2000)). A novel class of AKG decarboxylase enzymes has recently
been identified in cyanobacteria such as Synechococcus sp. PCC 7002
and homologs (Zhang and Bryant, Science 334:1551-3 (2011)).
TABLE-US-00017 Protein GenBank ID GI Number Organism pdc P06672.1
118391 Zymomonas mobilis mdlC P20906.2 3915757 Pseudomonas putida
mdlC Q9HUR2.1 81539678 Pseudomonas aeruginosa dpgB ABN80423.1
126202187 Pseudomonas stutzeri ilvB-1 YP_260581.1 70730840
Pseudomonas fluorescens kgd O50463.4 160395583 Mycobacterium
tuberculosis kgd NP_767092.1 27375563 Bradyrhizobium japonicum
USDA110 kgd NP_105204.1 13473636 Mesorhizobium loti ilvB ACB00744.1
169887030 Synechococcus sp. PCC 7002
EC 4.2.1.a
[0269] Formation of 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate
from 2,3,4-trihydroxy-3-methyl-5-phosphopentanoate (Step 1E) is
catalyzed by a diol dehydratase enzyme in EC class 4.2.1. Exemplary
diol dehydratase enzymes are listed in the table below.
TABLE-US-00018 Enzyme Commission Number Enzyme Name 4.2.1.5
arabinonate dehydratase 4.2.1.6 galactonate dehydratase 4.2.1.7
altronate dehydratase 4.2.1.8 mannonate dehydratase 4.2.1.9
dihydroxy-acid dehydratase 4.2.1.12 phosphogluconate dehydratase
4.2.1.25 L-arabinonate dehydratase 4.2.1.28 propanediol dehydratase
4.2.1.30 glycerol dehydratase 4.2.1.32 L(+)-tartrate dehydratase
4.2.1.39 gluconate dehydratase 4.2.1.40 glucarate dehydratase
4.2.1.41 5-dehydro-4-deoxyglucarate dehydratase 4.2.1.42
galactarate dehydratase 4.2.1.43 2-dehydro-3-deoxy-L- arabinonate
dehydratase 4.2.1.44 myo-inosose-2 dehydratase 4.2.1.45 CDP-glucose
4,6-dehydratase 4.2.1.46 dTDP-glucose 4,6-dehydratase 4.2.1.47
GDP-mannose 4,6-dehydratase 4.2.1.76 UDP-glucose 4,6-dehydratase
4.2.1.81 D(-)-tartrate dehydratase 4.2.1.82 xylonate dehydratase
4.2.1.90 L-rhamnonate dehydratase 4.2.1.109 methylthioribulose 1-
phosphate dehydratase
[0270] Particularly useful diol dehydratase enzymes include
dihydroxy-acid dehydratase (EC 4.2.1.9), phosphogluconate
dehydratase (EC 4.2.1.12) and arabonate dehydratase (EC 4.2.1.25).
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 et al., J. Biochem. 139:591-596
(2006)). 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 et
al., Arch. Biochem. Biophys. 138:160-170 (1970)), Salmonella
typhimurium (Armstrong et al., Biochim. Biophys. Acta 498:282-293
(1977)) and Corynebacterium glutamicum (Holatko et al, J Biotechnol
139:203-10 (2009)). Other groups have shown that the overexpression
of one or more Aft proteins or homologs thereof improves DHAD
activity (US Patent Application 2011/0183393. In Saccharomyces
cerevisiae, the Aft1 and Aft2 proteins are transcriptional
activators that regulate numerous proteins related to the
acquisition, compartmentalization, and utilization of iron.
Phosphogluconate dehydratase is another keto-acid forming diol
dehydratase that is active on a phosphorylated substrate. This
enzyme participates in the pentose phosphate pathway catalyzing the
conversion of 6-phosphogluconate to
2-dehydro-3-deoxygluconate-6-phosphate. These enzymes are
B12-independent and contain an Fe--S cluster in the active site.
Exemplary enzymes are the edd gene products of E. coli (Egan et al,
J Bacteriol 174:4638-46 (1992)), Pseudomonas aeruginosa (Cuskey et
al, J Bacteriol 162:865-71 (1985)) and Xanthomonas oryzae (Kim et
al, Biotechnol Lett 32:527-31 (2010)). Another suitable enzyme is
arabonate dehydratase. A gene encoding this activity is araC of
Azospirillum brasilense (Watanabe et al, J Biol Chem 287:33521-36
(2006)). Additional diol dehydratase candidates are described in
Example II.
TABLE-US-00019 Protein GenBank ID GI Number 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 ilvD
CAB57218.1 6010023 Corynebacterium glutamicum Aft1 P22149.2 1168370
Saccharomyces cerevisiae Aft2 Q08957.1 74583775 Saccharomyces
cerevisiae edd NP_416365.1 16129804 Escherichia coli edd
NP_251884.1 15598390 Pseudomonas aeruginosa edd YP_001913412.1
188576483 Xanthomonas oryzae araC BAE94269.1 95102048 Azospirillum
brasilense
No EC
[0271] Step 2A depicts 4,5-dihydroxy-2-oxopentanoate
methyltransferase which catalyzes the methylation of
4.5-dihydroxy-2-oxopentanoate to form
4,5-dihydroxy-3-methyl-2-oxopentanoate. A similar transformation is
catalyzed by 4-hydroxy-2-oxo-5-phosphopentanoate methyltransferase
in Step 2E. Although enzymes with these activities have not been
identified to date, methyltransferase enzymes that react with
similar substrates include alpha-ketoglutarate methyltransferase
and keto-arginine methyltransferase. Alpha-ketoglutarate
methyltransferase is catalyzed by the products of genes glmT from
Streptomyces coelicolor, dptI from Streptomyces roseosporus, and
lptI from Streptomyces fradiae (Mahlert et al., J. Am. Chem. Soc.,
2007, 129 (39), 12011-12018). Keto-arginine methyltransferase is
encoded by mrsA of Pseudomonas syringae (Braun et al, AEM 76:2500-8
(2010)).
TABLE-US-00020 Protein GenBank ID GI Number Organism glmT
NP_627429.1 21221650 Streptomyces coelicolor dptI ZP_04706744.1
239986080 Streptomyces roseosporus lptI AAZ23087.1 71068232
Streptomyces fradiae mrsA ACY54549.1 262342060 Pseudomonas
syringae
Example II. Exemplary Pathway for Producing 2H3M4OP from
Glyceraldehyde-3-Phosphate (G3P) and Pyruvate
[0272] This example describes an exemplary pathway for producing
the terephthalic acid (PTA) precursor 2H3M4OP.
[0273] 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. 3. 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.
[0274] A. 1-Deoxyxylulose-5-phosphate (DXP) synthase.
[0275] 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)). 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). 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).
TABLE-US-00021 GenBank Accession Gene 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
[0276] 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).
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);
Henriksson et al., Acta Crystallogr. D. Biol. Crystallogr.
62:807-813 (2006). 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); Rohdich et al., FEBS
J. 273:4446-4458 (2006)).
TABLE-US-00022 GenBank Accession Gene 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
[0277] C. 2-C-Methyl-D-erythritol-4-phosphate dehydratase.
[0278] A diol dehydratase is required to convert
2-C-methyl-D-erythritol-4-phosphate into the p-toluate precursor
(Altmiller 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),
myo-inositose dehydratase (EC 4.2.1.44), 2-keto-3-deoxyarabonate
dehydratase (EC 4.2.1.43), phosphogluconate dehydratase (EC
4.2.1.12) and arabonate dehydratase. These enzymes are described in
further detail below. Additional diol dehydratase enzyme candidates
are described above in Example 1.
[0279] 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-00023 GenBank Accession Gene 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
[0280] 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., FEMS 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)). Diol dehydratase enzymes with reduced inactivation
kinetics were developed by DuPont using error-prone PCR (WO
2004/056963)
TABLE-US-00024 GenBank Accession Gene 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
[0281] If a B12-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-00025 GenBank Accession Gene 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
[0282] B12-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)). An additional B12-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-00026 GenBank Accession Gene 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
[0283] 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)). 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-00027 GenBank Accession Gene 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
[0284] 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)). A similar enzyme from B.
subtilis, encoded by iolE, has also been studied (Yoshida et al.,
Microbiology 150:571-580 (2004)).
TABLE-US-00028 GenBank Accession Gene No. GI No. Organism iolE
P42416.1 1176989 Bacillus subtilis iolE AAX24114.1 60549621
Sinorhizobium fredii
[0285] 2-Keto-3-deoxyarabonate dehydratase participates in
arabinose degradation in Azospirillum brasilense, converting
2-keto-3-deoxyarabonate to alpha-ketoglutarate semialdehyde
(Watanabe et al, J Biol Chem 287:33521-36 (2006)). Similar enzymes
can be identified by sequence homology. Phosphogluconate
dehydratase is a diol dehydratase that participates in the pentose
phosphate pathway catalyzing the conversion of 6-phosphogluconate
to 2-dehydro-3-deoxygluconate-6-phosphate. Phosphogluconate
dehydratase enzymes are B12-independent and contain an Fe--S
cluster in the active site. Exemplary enzymes are the edd gene
products of E. coli (Egan et al, J Bacteriol 174:4638-46 (1992)),
Pseudomonas aeruginosa (Cuskey et al, J Bacteriol 162:865-71
(1985)) and Xanthomonas oryzae (Kim et al, Biotechnol Lett
32:527-31 (2010)). Another suitable enzyme is arabonate
dehydratase. A gene encoding this activity is araC of Azospirillum
brasilense (Watanabe et al, J Biol Chem 287:33521-36 (2006)).
TABLE-US-00029 Protein GenBank ID GI Number Organism araD
BAE94270.1 95102049 Azospirillum brasilense dapA ZP_02907581.1
171318425 Burkholderia ambifaria BCAM2800 P_002235400.1 206564637
Burkholderia cenocepacia edd NP_416365.1 16129804 Escherichia coli
edd NP_251884.1 15598390 Pseudomonas aeruginosa edd YP_001913412.1
188576483 Xanthomonas oryzae araC BAE94269.1 95102048 Azospirillum
brasilense
Example III. Exemplary Pathway for Synthesis of p-Toluate from
2H3M4OP by Shikimate Pathway Enzymes
[0286] This example describes exemplary pathways for synthesis of
p-toluate using shikimate pathway enzymes.
[0287] 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.
[0288] The synthesis of p-toluate proceeds in an analogous manner
as shown in FIG. 4. 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.
[0289] 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)). 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)). Additionally,
expression levels of shikimate pathway enzymes and chorismate lyase
were enhanced. Similar strategies can be employed in a strain for
overproducing p-toluate.
[0290] A. 2-Dehydro-3-deoxyphosphoheptonate synthase (EC
2.5.1.54).
[0291] 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)). 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)). 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)). 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); Webby et
al., Biochem. J. 390:223-230 2005)) 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)).
TABLE-US-00030 GenBank Accession Gene 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
[0292] B. 3-Dehydroquinate synthase (EC 4.2.3.4).
[0293] The dephosphorylation of substrate
(2)(2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate)
to substrate (3)(1,3-dihydroxy-4-methylcylohex-1-ene-1-carboxylate)
as shown in FIG. 4 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 H37Rv (de Mendonca et al., J. Bacteriol. 189:6246-6252
(2007)). The E. coli enzyme is subject to inhibition by L-tyrosine
(Barker and Frost, Biotechnol. Bioeng. 76:376-390 2001)).
TABLE-US-00031 GenBank Accession Gene No. GI No. Organism aroB
AAC76414.1 1789791 Escherichia coli aroB NP_390151.1 16079327
Bacillus subtilis aroB CAB06200.1 1781064 Mycobacterium
tuberculosis
[0294] C. 3-Dehydroquinate dehydratase (EC 4.2.1.10).
[0295] 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. 4. 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)). 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
(Kinghom et al., Gene 14:73-80. 1981)), Salmonella typhi (Kinghom
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)) 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-00032 GenBank Accession Gene 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
[0296] D. Shikimate dehydrogenase (EC 1.1.1.25).
[0297] Shikimate dehydrogenase catalyzes the NAD(P)H dependent
reduction of 3-dehydroshikimate to shikimate, analogous to Step D
of FIG. 4. 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)( ). 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)) have been functionally expressed
in E. coli.
TABLE-US-00033 GenBank Accession Gene 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
[0298] E. Shikimate kinase (EC 2.7.1.71).
[0299] Shikimate kinase catalyzes the ATP-dependent phosphorylation
of the 3-hydroxyl group of shikimate analogous to Step E of FIG. 4.
Two shikimate kinase enzymes are encoded by aroK (SK1) 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)); Oliveira et al., Protein
Expr. Purif 22:430-435 (2001)), Helicobacter pylori (Cheng et al.,
J. Bacteriol. 187:8156-8163 (2005)) and Erwinia chrysanthemi (Krell
et al., Protein Sci. 10:1137-1149 (2001)) have been cloned in E.
coli.
TABLE-US-00034 GenBank Accession Gene 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
[0300] F. 3-Phosphoshikimate-2-carboxyvinyltransferase (EC
2.5.1.19).
[0301] 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)), Dunaliella salina (Yi et
al., J. Microbiol. 45:153-157 (2007)) and Staphylococcus aureus
(Priestman et al., FEBS Lett. 579:728-732 (2005)) have been cloned
and functionally expressed in E. coli.
TABLE-US-00035 GenBank Accession Gene 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
[0302] G. Chorismate Synthase (EC 4.2.3.5).
[0303] 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)). Bifunctional fungal enzymes are found in Neurospora crassa
(Kitzing et al., J. Biol. Chem. 276:42658-42666 (2001)) and
Saccharomyces cerevisiae (Jones et al., Mol. Microbiol. 5:2143-2152
(1991)).
TABLE-US-00036 GenBank Gene Accession No. GI No. Organism aroC
NP_416832.1 16130264 Escherichia coli aroC ACH47980.1 197205483
Streptococcus pneumoniae U25818.1:19..1317 AAC49056.1 976375
Neurospora crassa ARO2 CAA42745.1 3387 Saccharomyces cerevisiae
[0304] H. Chorismate Lyase (EC 4.1.3.40).
[0305] 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)), 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)).
Structural studies implicate the G90 residue as contributing to
product inhibition (Smith et al., Arch. Biochem. Biophys. 445:72-80
(2006)). Modification of two surface-active cysteine residues
reduced protein aggregation (Holden et al., Biochim. Biophys. Acta
1594:160-167 (2002)). 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)).
TABLE-US-00037 GenBank Gene Accession No. GI No. Organism ubiC
AAC77009.2 87082361 Escherichia coli Rv2949c NP_217465.1 15610086
Mycobacterium tuberculosis
[0306] B-F. Multifunctional AROM Protein.
[0307] 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)). The multifunctional AROM protein
complex catalyzes reactions analogous to reactions B-F of FIG. 4.
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)).
TABLE-US-00038 GenBank Accession Gene 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 IV. Exemplary Pathway for Enzymatic Transformation of
p-Toluate to Terephthalic Acid
[0308] This example describes exemplary pathways for conversion of
p-toluate to terephthalic acid (PTA).
[0309] 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. 5. 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-00039 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
Example V. Exemplary Process for Production and Recovery of
Terephthalic Acid
[0310] The following example describes an exemplary process for
production and recovery of terephthalic acid from a culture medium,
as exemplified and illustrated in FIG. 6.
Culturing the Non-Naturally Occurring Microbial Organism to Produce
Terephthalate:
[0311] Sugars can be fermented in a culture medium at neutral pH
using a non-naturally occurring microbial organism containing a
functional terephthalate pathway. Under these conditions,
terephthalate thus produced will be in the culture medium of the
cells.
[0312] If conventional sugars are employed, cells can be removed by
centrifugation or membrane filtration. If biomass is used as a
primary feedstock, a pretreated stream of biomass can be introduced
into the fermenter along with cellulolytic enzymes in order to
engage in a simultaneous saccharification and fermentation
process.
[0313] Ammonia can be used as a base to maintain pH at 7, in which
case the fermentation process will produce diammonium
terephthalate, which is soluble in the aqueous culture medium.
[0314] Following fermentation, once sufficient quantities of
terephthalate are produced, the culture medium can be separated
from any non-soluble materials by centrifugation or membrane
filtration to provide a cell-free broth containing the
terephthalate salt. Where biomass is used as a primary feedstock,
both cells and any non-soluble lignocellulosic material remaining
can be removed together through centrifugation or membrane
filtration to provide a cell-free broth containing the
terephthalate salt, which is soluble in the cell-free broth.
Lowering the pH with CO.sub.2 to Precipitate the Terephthalic
Acid:
[0315] The cell-free broth can be acidified by adding gaseous
carbon dioxide (CO.sub.2). The vessel can be pressurized in the
range of 0.1 to 30 atm with CO.sub.2 and allowed to stir at
temperatures between 0.degree. C. and 50.degree. C. for up to 20
hours. Lowering the pH of the cell-free broth leads to the
insoluble terephthalic acid precipitating from solution, where the
resulting ammonium carbonate ((NH.sub.4).sub.2CO.sub.3) remains in
solution.
Filtering and Recovering the Terephthalic Acid:
[0316] The crude terephthalic acid can be filtered from the
solution and recovered. The remaining aqueous filtrate contains
(NH.sub.4).sub.2CO.sub.3, and this solution can be heated to
decompose (NH.sub.4).sub.2CO.sub.3 into NH.sub.3 and CO.sub.2 for
separation, recovery, and recycling of both or either of these
materials. Crude terephthalic acid solid can be recrystallized
using standard techniques and solvents to afford purified
terephthalic acid.
[0317] Using this exemplary process, a high predicted fermentation
yield of up to 0.58 lb terephthalic acid/lb sugar may be
obtained.
[0318] 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.
* * * * *