U.S. patent application number 14/167693 was filed with the patent office on 2014-12-25 for microorganisms and methods for the co-production of isopropanol with primary alcohols, diols and acids.
This patent application is currently assigned to Genomatica, Inc.. The applicant listed for this patent is Genomatica, Inc.. Invention is credited to Anthony P. BURGARD, Mark J. BURK, Robin E. OSTERHOUT, Priti PHARKYA, Jun SUN.
Application Number | 20140377820 14/167693 |
Document ID | / |
Family ID | 43732800 |
Filed Date | 2014-12-25 |
United States Patent
Application |
20140377820 |
Kind Code |
A1 |
PHARKYA; Priti ; et
al. |
December 25, 2014 |
MICROORGANISMS AND METHODS FOR THE CO-PRODUCTION OF ISOPROPANOL
WITH PRIMARY ALCOHOLS, DIOLS AND ACIDS
Abstract
The invention provides a non-naturally occurring microbial
organism having n-propanol and isopropanol pathways, 1,4-butanediol
(14-BDO) and isopropanol pathways, 1,3-butanediol (13-BDO) and
isopropanol pathways or methylacrylic acid (MAA) and isopropanol
pathways. The microbial organism contains at least one exogenous
nucleic acid encoding an enzyme in each of the respective
n-propanol, 14-BDO, 13-BDO or MAA and isopropanol pathways. The
invention additionally provides a method for co-producing
n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and
isopropanol or MAA and isopropanol. The method can include
culturing an n-propanol and an isopropanol co-producing microbial
organism, where the microbial organism expresses at least one
exogenous nucleic acid encoding an n-propanol, an isopropanol, a
14-BDO, a 13-BDO and/or a MAA pathway enzyme in a sufficient amount
to produce each of the respective products, under conditions and
for a sufficient period of time to produce each of the respective
products.
Inventors: |
PHARKYA; Priti; (San Diego,
CA) ; BURGARD; Anthony P.; (Bellefonte, PA) ;
OSTERHOUT; Robin E.; (San Diego, CA) ; BURK; Mark
J.; (San Diego, CA) ; SUN; Jun; (San Diego,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genomatica, Inc. |
San Diego |
CA |
US |
|
|
Assignee: |
Genomatica, Inc.
San Diego
CA
|
Family ID: |
43732800 |
Appl. No.: |
14/167693 |
Filed: |
January 29, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12878980 |
Sep 9, 2010 |
8715971 |
|
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14167693 |
|
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61254650 |
Oct 23, 2009 |
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61240959 |
Sep 9, 2009 |
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Current U.S.
Class: |
435/136 ;
435/157; 435/158; 435/252.3; 435/252.31; 435/252.33;
435/254.21 |
Current CPC
Class: |
C12P 7/40 20130101; C12N
15/52 20130101; C12P 7/04 20130101; C12P 7/18 20130101 |
Class at
Publication: |
435/136 ;
435/252.33; 435/157; 435/158; 435/254.21; 435/252.3;
435/252.31 |
International
Class: |
C12P 7/40 20060101
C12P007/40; C12P 7/18 20060101 C12P007/18; C12P 7/04 20060101
C12P007/04 |
Claims
1. A non-naturally occurring microbial organism, comprising a
microbial organism having an n-propanol pathway and an isopropanol
pathway, said n-propanol pathway comprising at least one exogenous
nucleic acid encoding an n-propanol pathway enzyme expressed in a
sufficient amount to produce n-propanol, said n-propanol pathway
comprising: a propanol dehydrogenase or a propionaldehyde
dehydrogenase, said isopropanol pathway comprising at least one
exogenous nucleic acid encoding an isopropanol pathway enzyme
expressed in a sufficient amount to produce isopropanol, said
isopropanol pathway comprising: an isopropanol dehydrogenase, an
acetyl-CoA acetyl thiolase, an acetoacetyl-CoA transferase, an
acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase or an
acetoacetate decarboxylase.
2. The non-naturally occurring microbial organism of claim 1,
further comprising an acetyl-CoA pathway comprising at least one
exogenous nucleic acid encoding an acetyl-CoA pathway enzyme
expressed in a sufficient amount to produce acetyl-CoA, said
acetyl-CoA pathway comprising: a pyruvate kinase, a pyruvate
dehydrogenase, or a pyruvate ferredoxin oxidoreductase.
3. The non-naturally occurring microbial organism of claim 1,
further comprising a propionyl-CoA pathway comprising at least one
exogenous nucleic acid encoding a propionyl-CoA pathway enzyme
expressed in a sufficient amount to produce propionyl-CoA, said
propionyl-CoA pathway comprising: a PEP carboxykinase, a PEP
carboxylase, a malate dehydrogenase, a fumarase, a fumarate
reductase, a succinyl-CoA transferase, a succinyl-CoA synthetase, a
methylmalonyl-CoA mutase, a methylmalonyl-CoA epimerase or a
methylmalonyl-CoA decarboxylase.
4. The non-naturally occurring microbial organism of claim 1,
wherein said n-propanol pathway comprises a first set of exogenous
nucleic acids encoding n-propanol pathway enzymes expressed in a
sufficient amount to produce n-propanol, said first set of
exogenous nucleic acids encoding: a propionaldehyde dehydrogenase
and a propanol dehydrogenase, and wherein said isopropanol pathway
comprises a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, said second set of exogenous nucleic acids
encoding: an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase, an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase; an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
5. The non-naturally occurring microbial organism of claim 4,
further comprising an acetyl-CoA pathway comprising a third set of
exogenous nucleic acids encoding acetyl-CoA pathway enzymes
expressed in a sufficient amount to produce acetyl-CoA, said third
set of exogenous nucleic acids encoding: a pyruvate kinase; and a
pyruvate dehydrogenase or a pyruvate ferredoxin oxidoreductase.
6. The non-naturally occurring microbial organism of claim 4,
further comprising a propionyl-CoA pathway comprising a third set
of exogenous nucleic acids encoding propionyl-CoA pathway enzymes
expressed in a sufficient amount to produce propionyl-CoA, said
third set of exogenous nucleic acids encoding: a PEP carboxykinase
or a PEP carboxylase; a malate dehydrogenase; a fumarase; a
fumarate reductase; a succinyl-CoA transferase or a succinyl-CoA
synthetase; a methylmalonyl-CoA mutase; and a methylmalonyl-CoA
decarboxylase.
7. The non-naturally occurring microbial organism of claim 6,
wherein said third set of exogenous nucleic acids further encodes a
methylmalonyl-CoA epimerase.
8. The non-naturally occurring microbial organism of claim 1,
wherein said n-propanol pathway comprises a first set of exogenous
nucleic acids encoding n-propanol pathway enzymes expressed in a
sufficient amount to produce n-propanol, said first set of
exogenous nucleic acids encoding: a PEP carboxykinase or a PEP
carboxylase; a malate dehydrogenase; a fumarase; a fumarate
reductase; a succinyl-CoA transferase or a succinyl-CoA synthetase;
a methylmalonyl-CoA mutase; a methylmalonyl-CoA decarboxylase; a
propionaldehyde dehydrogenase and a propanol dehydrogenase, and
wherein said isopropanol pathway comprises a second set of
exogenous nucleic acids encoding isopropanol pathway enzymes
expressed in a sufficient amount to produce isopropanol, said
second set of exogenous nucleic acids encoding: a pyruvate kinase;
a pyruvate dehydrogenase or a pyruvate ferredoxin oxidoreductase an
acetyl-CoA acetyl thiolase; an acetoacetyl-CoA transferase, an
acetoacetyl-CoA hydrolase or an acetoacetyl-CoA synthethase; an
acetoacetate decarboxylase; and an isopropanol dehydrogenase.
9. The non-naturally occurring microbial organism of claim 8,
wherein said first set of exogenous nucleic acids further encodes a
methylmalonyl-CoA epimerase.
10. The non-naturally occurring microbial organism of claim 1,
wherein said exogenous nucleic acid is a heterologous nucleic
acid.
11. The non-naturally occurring microbial organism of claim 1,
wherein said non-naturally occurring microbial organism is in a
substantially anaerobic culture medium.
12. A method for producing n-propanol and isopropanol, comprising
culturing a non-naturally occurring microbial organism of claim 1
under conditions and for a sufficient period of time to produce
n-propanol and isopropanol.
13. The method of claim 12, wherein said conditions comprise
substantially anaerobic culture conditions.
14. The method of claim 12, wherein said exogenous nucleic acid is
a heterologous nucleic acid.
15. A non-naturally occurring microbial organism, comprising a
microbial organism having an n-propanol pathway, said n-propanol
pathway comprising at least one exogenous nucleic acid encoding an
n-propanol pathway enzyme expressed in a sufficient amount to
produce n-propanol, said n-propanol pathway comprising: a propanol
dehydrogenase or a propionaldehyde dehydrogenase.
16. The non-naturally occurring microbial organism of claim 15,
wherein said n-propanol pathway comprises a set of exogenous
nucleic acids encoding n-propanol pathway enzymes expressed in a
sufficient amount to produce n-propanol, said set of exogenous
nucleic acids encoding: a propionaldehyde dehydrogenase and a
propanol dehydrogenase.
17. The non-naturally occurring microbial organism of claim 15,
further comprising a propionyl-CoA pathway comprising exogenous
nucleic acids encoding propionyl-CoA pathway enzymes expressed in a
sufficient amount to produce propionyl-CoA, wherein the
propionyl-CoA pathway comprises: a PEP carboxykinase, a PEP
carboxylase, a malate dehydrogenase, a fumarase, a fumarate
reductase, a succinyl-CoA transferase, a succinyl-CoA synthetase, a
methylmalonyl-CoA mutase, a methylmalonyl-CoA epimerase or a
methylmalonyl-CoA decarboxylase.
18. The non-naturally occurring microbial organism of claim 15,
wherein said n-propanol pathway comprises a first set of exogenous
nucleic acids encoding n-propanol pathway enzymes expressed in a
sufficient amount to produce n-propanol, said first set of
exogenous nucleic acids encoding: a PEP carboxykinase or a PEP
carboxylase; a malate dehydrogenase; a fumarase; a fumarate
reductase; a succinyl-CoA transferase or a succinyl-CoA synthetase;
a methylmalonyl-CoA mutase; a methylmalonyl-CoA epimerase; a
methylmalonyl-CoA decarboxylase; a propionaldehyde dehydrogenase
and a propanol dehydrogenase.
19. The non-naturally occurring microbial organism of claim 15,
wherein said exogenous nucleic acid is a heterologous nucleic
acid.
20. The non-naturally occurring microbial organism of claim 15,
wherein said non-naturally occurring microbial organism is in a
substantially anaerobic culture medium.
21. A method for producing n-propanol, comprising culturing a
non-naturally occurring microbial organism of claim 15 under
conditions and for a sufficient period of time to produce
n-propanol.
22. The method of claim 21, wherein said conditions comprise
substantially anaerobic culture conditions.
23. The method of claim 21, wherein said exogenous nucleic acid is
a heterologous nucleic acid.
24. A non-naturally occurring microbial organism, comprising a
microbial organism having a 1,4-butanediol pathway and an
isopropanol pathway, said 1,4-butanediol pathway comprising at
least one exogenous nucleic acid encoding a 1,4-butanediol pathway
enzyme expressed in a sufficient amount to produce 1,4-butanediol,
said 1,4-butanediol pathway comprising: a 4-hydroxybutyraldehyde
reductase, a succinyl-CoA reductase, a 4-hydroxybutyrate
dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a
4-hydroxybutyryl-CoA synthetase, or a 4-hydroxybutyryl-CoA
reductase (aldehyde-forming), said isopropanol pathway comprising
at least one exogenous nucleic acid encoding an isopropanol pathway
enzyme expressed in a sufficient amount to produce isopropanol,
said isopropanol pathway comprising: an isopropanol dehydrogenase,
an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA transferase, an
acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, or an
acetoacetate decarboxylase.
25. A non-naturally occurring microbial organism, comprising a
microbial organism having a 1,3-butanediol pathway and an
isopropanol pathway, said 1,3-butanediol pathway comprising at
least one exogenous nucleic acid encoding a 1,3-butanediol pathway
enzyme expressed in a sufficient amount to produce 1,3-butanediol,
said 1,3-butanediol pathway comprising: a 3-hydroxybutyraldehyde
reductase, a succinyl-CoA reductase, a 4-hydroxybutyrate
dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a
4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyryl-CoA
dehydratase, a crotonase, or a 3-hydroxybutyryl-CoA reductase
(aldehyde forming), said isopropanol pathway comprising at least
one exogenous nucleic acid encoding an isopropanol pathway enzyme
expressed in a sufficient amount to produce isopropanol, said
isopropanol pathway comprising: an isopropanol dehydrogenase, an
acetyl-CoA acetyl thiolase, an acetoacetyl-CoA transferase, an
acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, or an
acetoacetate decarboxylase.
26. A non-naturally occurring microbial organism, comprising a
microbial organism having a methylacrylic acid pathway and an
isopropanol pathway, said methylacrylic acid pathway comprising at
least one exogenous nucleic acid encoding a methylacrylic acid
pathway enzyme expressed in a sufficient amount to produce
methylacrylic acid, said methylacrylic acid pathway comprising: a
4-hydroxybutyryl-CoA mutase, a succinyl-CoA reductase, a
4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA
transferase, a 4-hydroxybutyryl-CoA synthetase, a
3-hydroxyisobutyryl-CoA dehydratase, a methacrylyl-CoA transferase,
a methacrylyl-CoA synthetase, a methacrylyl-CoA hydrolase, said
isopropanol pathway comprising at least one exogenous nucleic acid
encoding an isopropanol pathway enzyme expressed in a sufficient
amount to produce isopropanol, said isopropanol pathway comprising:
an isopropanol dehydrogenase, an acetyl-CoA acetyl thiolase, an
acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an
acetoacetyl-CoA synthetase, or an acetoacetate decarboxylase.
27. The non-naturally occurring microbial organism of any one of
claims 24-26, further comprising an acetyl-CoA pathway comprising
at least one exogenous nucleic acid encoding an acetyl-CoA pathway
enzyme expressed in a sufficient amount to produce acetyl-CoA, said
acetyl-CoA pathway comprising: a pyruvate kinase, a pyruvate
dehydrogenase, or a pyruvate ferredoxin oxidoreductase.
28. The non-naturally occurring microbial organism of any one of
claims 24-26, further comprising a succinyl-CoA pathway comprising
at least one exogenous nucleic acid encoding a succinyl-CoA pathway
enzyme expressed in a sufficient amount to produce succinyl-CoA,
said succinyl-CoA pathway comprising: a PEP carboxykinase, a PEP
carboxylase, a malate dehydrogenase, a fumarase, a fumarate
reductase, a succinyl-CoA transferase or a succinyl-CoA
synthetase.
29. The non-naturally occurring microbial organism of claim 24,
wherein said 1,4-butanediol pathway comprises a first set of
exogenous nucleic acids encoding 1,4-butanediol pathway enzymes
expressed in a sufficient amount to produce 1,4-butanediol, said
first set of exogenous nucleic acids encoding: a succinyl-CoA
reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA reductase (aldehyde-forming);
and a 4-hydroxybutyraldehyde reductase, and wherein said
isopropanol pathway comprises a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, said second set of
exogenous nucleic acids encoding: an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
30. The non-naturally occurring microbial organism of claim 25,
wherein said 1,3-butanediol pathway comprises a first set of
exogenous nucleic acids encoding 1,3-butanediol pathway enzymes
expressed in a sufficient amount to produce 1,3-butanediol, said
first set of exogenous nucleic acids encoding: a succinyl-CoA
reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; and a
3-hydroxybutyraldehyde reductase, and wherein said isopropanol
pathway comprises a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, said second set of exogenous nucleic acids
encoding: an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase; an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
31. The non-naturally occurring microbial organism of claim 26,
wherein said methylacrylic acid pathway comprises a first set of
exogenous nucleic acids encoding methylacrylic acid pathway enzymes
expressed in a sufficient amount to produce methylacrylic acid,
said first set of exogenous nucleic acids encoding: a succinyl-CoA
reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA mutase; a
3-hydroxyisobutyryl-CoA dehydratase; and a methacrylyl-CoA
transferase, a methacrylyl-CoA synthetase or a methacrylyl-CoA
hydrolase, and wherein said isopropanol pathway comprises a second
set of exogenous nucleic acids encoding isopropanol pathway enzymes
expressed in a sufficient amount to produce isopropanol, said
second set of exogenous nucleic acids encoding: an acetyl-CoA
acetyl thiolase; an acetoacetyl-CoA transferase or an
acetoacetyl-CoA hydrolase or an acetoacetyl-CoA synthetase; an
acetoacetate decarboxylase; and an isopropanol dehydrogenase.
32. The non-naturally occurring microbial organism of any one of
claims 29-31, further comprising an acetyl-CoA pathway comprising a
third set of exogenous nucleic acids encoding acetyl-CoA pathway
enzymes expressed in a sufficient amount to produce acetyl-CoA,
said third set of exogenous nucleic acids encoding: a pyruvate
kinase; and a pyruvate dehydrogenase or a pyruvate ferredoxin
oxidoreductase.
33. The non-naturally occurring microbial organism of any one of
claims 29-31, further comprising a succinyl-CoA pathway comprising
a third set of exogenous nucleic acids encoding succinyl-CoA
pathway enzymes expressed in a sufficient amount to produce
succinyl-CoA, said third set of exogenous nucleic acids encoding: a
PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a
fumarase, a fumarate reductase, a succinyl-CoA transferase and a
succinyl-CoA synthetase.
34. The non-naturally occurring microbial organism of any one of
claims 24-26, wherein said exogenous nucleic acid is a heterologous
nucleic acid.
35. The non-naturally occurring microbial organism of any one of
claims 24-26, wherein said non-naturally occurring microbial
organism is in a substantially anaerobic culture medium.
36. A method for producing 1,4-butanediol and isopropanol,
comprising culturing a non-naturally occurring microbial organism
of claim 24 under conditions and for a sufficient period of time to
produce 1,4-butanediol and isopropanol.
37. The method of claim 36, wherein said conditions comprise
substantially anaerobic culture conditions.
38. The method of claim 36, wherein said exogenous nucleic acid is
a heterologous nucleic acid.
39. A method for producing 1,3-butanediol and isopropanol,
comprising culturing a non-naturally occurring microbial organism
of claim 25 under conditions and for a sufficient period of time to
produce 1,3-butanediol and isopropanol.
40. The method of claim 39, wherein said conditions comprise
substantially anaerobic culture conditions.
41. The method of claim 39, wherein said exogenous nucleic acid is
a heterologous nucleic acid.
42. A method for producing methylacrylic acid and isopropanol,
comprising culturing a non-naturally occurring microbial organism
of claim 26 under conditions and for a sufficient period of time to
produce methylacrylic acid and isopropanol.
43. The method of claim 42, wherein said conditions comprise
substantially anaerobic culture conditions.
44. The method of claim 42, wherein said exogenous nucleic acid is
a heterologous nucleic acid.
Description
[0001] This application is a continuation of U.S. non-provisional
patent application Ser. No. 12/878,980, filed Sep. 9, 2010, which
claims the benefit of priority to U.S. provisional patent
application No. 61/240,959, filed Sep. 9, 2009 and U.S. provisional
application No. 61/254,650, filed Oct. 23, 2009, which are herein
incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to biosynthetic
processes, and more specifically to organisms having n-propanol and
isopropanol, 1,4-butanediol and isopropanol, 1,3-butanediol and
isopropanol or methylacrylic and isopropanol biosynthetic
capability.
[0003] Isopropanol (IPA) is a colorless, flammable liquid that
mixes completely with most solvents, including water. The largest
use for IPA is as a solvent, including its well known yet small use
as "rubbing alcohol," which is a mixture of IPA and water. As a
solvent, IPA is found in many everyday products such as paints,
lacquers, thinners, inks, adhesives, general-purpose cleaners,
disinfectants, cosmetics, toiletries, de-icers, and
pharmaceuticals. Low-grade IPA is also used in motor oils. The
second largest use is as a chemical intermediate for the production
of isopropylamines, isopropylethers, and isopropyl esters.
Isopropanol can potentially be dehydrated to form propylene, a
polymer precursor with an annual market of more than 2 million
metric tons.
[0004] Current global production capacity of isopropanol (IPA) is
approximately 6 B lb/yr, with approximately 74% of global IPA
capacity concentrated in the US, Europe, and Japan. Isopropanol is
manufactured by two petrochemical routes. The predominant process
entails the hydration of propylene either with or without sulfuric
acid catalysis. Secondarily, IPA is produced via hydrogenation of
acetone, which is a by-product formed in the production of phenol
and propylene oxide. High-priced propylene is currently driving
costs up and margins down throughout the chemical industry
motivating the need for an expanded range of low cost
feedstocks.
[0005] n-Propanol can be potentially used as a gasoline substitute.
It is currently used as a multi-purpose solvent in the
pharmaceutical industry, for surface coatings and in ink
formulations. It is used as a building block for resins and esters,
propyl amines and halides. It is also used for packaging and food
contact applications. Global production of n-propanol in 2005 was
more than 140,000 metric tonnes.
[0006] n-Propanol is manufactured by the catalytic hydrogenation of
propionaldehyde. Propionaldehyde is itself produced via the oxo
process, by hydroformylation of ethylene using carbon monoxide and
hydrogen in the presence of a catalyst such as cobalt octacarbonyl
or a rhodium complex. It is formed naturally in small amounts in
many fermentation processes. For example, microbial production of
very small quantities of n-propanol has been detected from certain
species of Clostridium via threonine catabolism and from yeast in
beer fermentation. No existing microorganism has been reported to
produce 1-propanol from sugars in significant amounts.
[0007] 1,4-Butanediol (14-BDO) is a polymer intermediate and
industrial solvent with a global market of about 3 billion lb/year.
BDO is currently produced from petrochemical precursors, primarily
acetylene, maleic anhydride, and propylene oxide. For example,
acetylene is reacted with 2 molecules of formaldehyde in the Reppe
synthesis reaction (Kroschwitz and Grant, Encyclopedia of Chem.
Tech., John Wiley and Sons, Inc., New York (1999)), followed by
catalytic hydrogenation to form 1,4-butanediol. Downstream, 14-BDO
can be further transformed; for example, by oxidation to
gamma-butyrolactone, which can be further converted to pyrrolidone
and N-methyl-pyrrolidone, or hydrogenolysis to tetrahydrofuran.
These compounds have varied uses as polymer intermediates,
solvents, and additives, and have a combined market of nearly 2
billion lb/year. 1,3-Butanediol (13-BDO) is a four carbon diol
commonly used as an organic solvent for food flavoring agents. It
is also used as a co-monomer for polyurethane and polyester resins
and is widely employed as a hypoglycaemic agent. Optically active
13-BDO is a useful starting material for the synthesis of
biologically active compounds and liquid crystals. A substantial
commercial use of 1,3-butanediol is subsequent dehydration to
afford 1,3-butadiene (Ichikawa, J. Mol. Catalysis. 256:106-112
(2006)), a 25 billion lb/yr petrochemical used to manufacture
synthetic rubbers (e.g., tires), latex, and resins. 13-BDO is
traditionally produced from acetylene via its hydration. The
resulting acetaldehyde is then converted to 3-hydroxybutyraldehdye
which is subsequently reduced to form 1,3-BDO. In more recent
years, acetylene has been replaced by ethylene as a source of
acetaldehyde.
[0008] Methylacrylic acid (MAA) is a key precursor of methyl
methacrylate (MMA), a chemical intermediate with a global demand in
excess of 4.5 billion pounds per year, much of which is converted
to polyacrylates. The conventional process for synthesizing methyl
methacrylate (i.e., the acetone cyanohydrin route) involves the
conversion of hydrogen cyanide (HCN) and acetone to acetone
cyanohydrin which then undergoes acid assisted hydrolysis and
esterification with methanol to give MAA. Difficulties in handling
potentially deadly HCN along with the high costs of byproduct
disposal (1.2 tons of ammonium bisulfate are formed per ton of MAA)
have sparked a great deal of research aimed at cleaner and more
economical processes. As a starting material, MAA can easily be
converted into MAA via esterification with methanol. No existing
microorganism has been reported to produce MAA from sugars in
significant amounts.
[0009] Microbial organisms and methods for effectively co-producing
commercial quantities of n-propanol and isopropanol, 14-BDO and
isopropanol, 13-BDO and isopropanol or MAA and isopropanol are
described herein and include related advantages.
SUMMARY OF THE INVENTION
[0010] The invention provides non-naturally occurring microbial
organisms having an n-propanol pathway and an isopropanol pathway.
In one aspect, the embodiments disclosed herein relate to a
non-naturally occurring microbial organism that includes a
microbial organism having an n-propanol and an isopropanol pathway,
where the n-propanol pathway includes at least one exogenous
nucleic acid encoding an n-propanol pathway enzyme expressed in a
sufficient amount to produce n-propanol and where the isopropanol
pathway includes at least one exogenous nucleic acid encoding an
isopropanol pathway enzyme expressed in a sufficient amount to
produce isopropanol. In one aspect, the n-propanol pathway includes
a propionaldehyde dehydrogenase, a propanol dehydrogenase, a
propionyl-CoA:phosphate propanoyltransferase, a propionyl-CoA
hydrolase, a propionyl-CoA transferase, a propionyl-CoA synthetase,
a propionate kinase, a propionate reductase or a propionyl
phosphate reductase and the isopropanol pathway includes an
acetyl-CoA acetyl thiolase, an acetoacetyl-CoA transferase, an
acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase or an
isopropanol dehydrogenase.
[0011] In another embodiment, the invention provides a
non-naturally occurring microbial organism that includes a
microbial organism having an n-propanol and an isopropanol pathway,
where the n-propanol pathway includes a first set of exogenous
nucleic acids encoding n-propanol pathway enzymes expressed in a
sufficient amount to produce n-propanol and where the isopropanol
pathway includes a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol. In one aspect, the first set encodes
n-propanol pathway enzymes including a propionaldehyde
dehydrogenase and a propanol dehydrogenase; or a
propionyl-CoA:phosphate propanoyltransferase, a propionyl phosphate
reductase and a propanol dehydrogenase; or a propionyl-CoA
hydrolase or a propionyl-CoA transferase or a propionyl-CoA
synthetase, a propionate kinase, a propionyl phosphate reductase
and a propanol dehydrogenase; or a propionyl-CoA hydrolase or a
propionyl-CoA transferase or a propionyl-CoA synthetase, a
propionate reductase and a propanol dehydrogenase. In another
aspect, the second set encodes isopropanol pathway enzymes
including an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA
synthetase or an acetoacetyl-CoA synthetase; an acetoacetate
decarboxylase; and an isopropanol dehydrogenase.
[0012] In another aspect, the invention provides a non-naturally
occurring microbial organism having a first set of exogenous
nucleic acids encoding n-propanol pathway enzymes and a second set
of exogenous nucleic acids encoding isopropanol pathway enzymes,
where the first set encodes a PEP carboxykinase or a PEP
carboxylase; a malate dehydrogenase; a fumarase; a fumarate
reductase; a succinyl-CoA transferase or a succinyl-CoA synthetase;
a methylmalonyl-CoA mutase; a methylmalonyl-CoA decarboxylase; and
a propionaldehyde dehydrogenase and a propanol dehydrogenase; or a
propionyl-CoA:phosphate propanoyltransferase and a propionyl
phosphate reductase; or a propionyl-CoA hydrolase or a
propionyl-CoA transferase or a propionyl-CoA synthetase, a
propionate kinase, a propionyl phosphate reductase and a propanol
dehydrogenase; or a propionyl-CoA hydrolase or a propionyl-CoA
transferase or a propionyl-CoA synthetase, a propionate reductase
and a propanol dehydrogenase, and the second set encodes a pyruvate
kinase; a pyruvate dehydrogenase or a pyruvate ferredoxin
oxidoreductase; or a pyruvate formate lyase, a pyruvate formate
lyase activating enzyme and a formate dehydrogenase; an acetyl-CoA
acetyl thiolase; an acetoacetyl-CoA transferase or an
acetoacetyl-CoA hydrolase or an acetoacetyl-CoA synthetase, an
acetoacetate decarboxylase; and an isopropanol dehydrogenase.
[0013] In another aspect, the invention provides a non-naturally
occurring microbial organism having a first set of exogenous
nucleic acids encoding n-propanol pathway enzymes and a second set
of exogenous nucleic acids encoding isopropanol pathway enzymes,
where the first set encodes a PEP carboxykinase or a PEP
carboxylase; a threonine deaminase; and a 2-oxobutanoate
decarboxylase and a propanol dehydrogenase; or a 2-oxobutanoate
dehydrogenase, a propionaldehyde dehydrogenase and a propanol
dehydrogenase; or a 2-oxobutanoate dehydrogenase, a
propionyl-CoA:phosphate propanoyltransferase, a propionyl phosphate
reductase and a propanol dehydrogenase; or a 2-oxobutanoate
dehydrogenase, a propionyl-CoA hydrolase or a propionyl-CoA
transferase or a propionyl-CoA synthetase, a propionate kinase, a
propionyl phosphate reductase and a propanol dehydrogenase; or a
2-oxobutanoate dehydrogenase, a propionyl-CoA hydrolase or a
propionyl-CoA transferase or a propionyl-CoA synthetase, a
propionate reductase and a propanol dehydrogenase, and the second
set encodes a pyruvate kinase; a pyruvate dehydrogenase or a
pyruvate ferredoxin oxidoreductase; or a pyruvate formate lyase, a
pyruvate formate lyase activating enzyme and a formate
dehydrogenase; an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase, an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0014] In another aspect, the invention provides a non-naturally
occurring microbial organism having a first set of exogenous
nucleic acids encoding n-propanol pathway enzymes and a second set
of exogenous nucleic acids encoding isopropanol pathway enzymes,
where the first set encodes a pyruvate kinase; a pyruvate
dehydrogenase or a pyruvate ferredoxin oxidoreductase; or a
pyruvate formate lyase, a pyruvate formate lyase activating enzyme
and a formate dehydrogenase; an acetyl-CoA carboxylase; a
malonyl-CoA reductase; a malonate semialdehyde reductase;
propionyl-CoA synthase; and a propionaldehyde dehydrogenase and a
propanol dehydrogenase; or a propionyl-CoA:phosphate
propanoyltransferase, a propionyl phosphate reductase and propanol
dehydrogenase; or a propionyl-CoA hydrolase or a propionyl-CoA
transferase or a propionyl-CoA synthetase, a propionate kinase, a
propionyl phosphate reductase and a propanol dehydrogenase; or a
propionyl-CoA hydrolase or a propionyl-CoA transferase or a
propionyl-CoA synthetase, a propionate reductase and a propanol
dehydrogenase and the second set encodes an acetyl-CoA acetyl
thiolase; an acetoacetyl-CoA transferase or an acetoacetyl-CoA
hydrolase or an acetoacetyl-CoA synthetase, an acetoacetate
decarboxylase; and an isopropanol dehydrogenase.
[0015] In another aspect, the invention provides a non-naturally
occurring microbial organism having a first set of exogenous
nucleic acids encoding n-propanol pathway enzymes and a second set
of exogenous nucleic acids encoding isopropanol pathway enzymes,
where the first set encodes a lactate dehydrogenase; a lactate-CoA
transferase; a lactyl-CoA dehydratase; acryloyl CoA reductase; and
a propionaldehyde dehydrogenase and a propanol dehydrogenase; or a
propionyl-CoA:phosphate propanoyltransferase, a propionyl phosphate
reductase and a propanol dehydrogenase; or a propionyl-CoA
hydrolase or a propionyl-CoA transferase or a propionyl-CoA
synthetase, a propionate kinase, a propionyl phosphate reductase
and a propanol dehydrogenase; or a propionyl-CoA hydrolase or a
propionyl-CoA transferase or a propionyl-CoA synthetase, a
propionate reductase and a propanol dehydrogenase and the second
set encodes a pyruvate dehydrogenase or a pyruvate ferredoxin
oxidoreductase; or a pyruvate formate lyase, a pyruvate formate
lyase activating enzyme and a formate dehydrogenase; an acetyl-CoA
acetyl thiolase; an acetoacetyl-CoA transferase or an
acetoacetyl-CoA hydrolase or an acetoacetyl-CoA synthetase, an
acetoacetate decarboxylase; and an isopropanol dehydrogenase.
[0016] In another embodiment, the invention provides a
non-naturally occurring microbial organism having an n-propanol
pathway, the n-propanol pathway including at least one exogenous
nucleic acid encoding an n-propanol pathway enzyme expressed in a
sufficient amount to produce n-propanol. In one aspect the
n-propanol pathway includes a propionaldehyde dehydrogenase, a
propanol dehydrogenase, a propionyl-CoA:phosphate
propanoyltransferase, a propionyl-CoA hydrolase, a propionyl-CoA
transferase, a propionyl-CoA synthetase, a propionate kinase, a
propionate reductase or a propionyl phosphate reductase.
[0017] In another embodiment, the invention provides a
non-naturally occurring microbial organism having an n-propanol
pathway, the n-propanol pathway including a set of exogenous
nucleic acids encoding n-propanol pathway enzymes expressed in a
sufficient amount to produce n-propanol, the set of exogenous
nucleic acids encoding a propionaldehyde dehydrogenase and a
propanol dehydrogenase; or a propionyl-CoA:phosphate
propanoyltransferase, a propionyl phosphate reductase and a
propanol dehydrogenase; or a propionyl-CoA hydrolase or a
propionyl-CoA transferase or a propionyl-CoA synthetase, a
propionate kinase, a propionyl phosphate reductase and a propanol
dehydrogenase; or a propionyl-CoA hydrolase or a propionyl-CoA
transferase or a propionyl-CoA synthetase, a propionate reductase
and a propanol dehydrogenase.
[0018] In still other aspects, embodiments disclosed herein relate
to a method for producing n-propanol and isopropanol that includes
culturing the aforementioned non-naturally occurring microbial
organisms. In still other aspect, embodiments disclosed herein
relate to a method for producing n-propanol that includes culturing
the aforementioned non-naturally occurring microbial organisms.
[0019] In one embodiment, the invention provides non-naturally
occurring microbial organisms having an isopropanol pathway and a
1,4-butanediol (14-BDO) pathway, a 1,3-butanediol (13-BDO) pathway
or a methylacrylic acid (MAA) pathway. In one aspect, the
embodiments disclosed herein relate to a non-naturally occurring
microbial organism that includes a microbial organism having a
1,4-butanediol and an isopropanol pathway, where the 1,4-butanediol
pathway includes at least one exogenous nucleic acid encoding a
1,4-butanediol pathway enzyme expressed in a sufficient amount to
produce 1,4-butanediol and where the isopropanol pathway includes
at least one exogenous nucleic acid encoding an isopropanol pathway
enzyme expressed in a sufficient amount to produce isopropanol. In
one aspect, the embodiments disclosed herein relate to a
non-naturally occurring microbial organism that includes a
microbial organism having a 1,3-butanediol and an isopropanol
pathway, where the 1,3-butanediol pathway includes at least one
exogenous nucleic acid encoding a 1,3-butanediol pathway enzyme
expressed in a sufficient amount to produce 1,3-butanediol and
where the isopropanol pathway includes at least one exogenous
nucleic acid encoding an isopropanol pathway enzyme expressed in a
sufficient amount to produce isopropanol. In one aspect, the
embodiments disclosed herein relate to a non-naturally occurring
microbial organism that includes a microbial organism having a
methylacrylic acid and an isopropanol pathway, where the
methylacrylic acid pathway includes at least one exogenous nucleic
acid encoding a methylacrylic acid pathway enzyme expressed in a
sufficient amount to produce methylacrylic acid and where the
isopropanol pathway includes at least one exogenous nucleic acid
encoding an isopropanol pathway enzyme expressed in a sufficient
amount to produce isopropanol.
[0020] In one embodiment, the isopropanol pathway comprises an
acetyl-CoA acetyl thiolase, an acetoacetyl-CoA transferase, an
acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, an
acetoacetate decarboxylase or an isopropanol dehydrogenase.
[0021] In one embodiment, the 14-BDO pathway comprises a
succinyl-CoA reductase, a succinate reductase, a 4-hydroxybutyrate
dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a
4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyryl-CoA reductase
(aldehyde-forming), a 4-hydroxybutyraldehyde reductase, a
4-hydroxybutyrate reductase; a 4-hydroxybutyrate kinase, a
phosphotrans-4-hydroxybutyrylase, 4-hydroxybutyryl-phosphate
reductase, or a 4-hydroxybutyryl-CoA reductase
(alcohol-forming).
[0022] In one embodiment, the 13-BDO pathway comprises a
succinyl-CoA reductase, a succinate reductase, a 4-hydroxybutyrate
dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a
4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyrate kinase, a
phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA
dehydratase, a crotonase, a 3-hydroxybutyryl-CoA reductase
(aldehyde forming), a 3-hydroxybutyraldehyde reductase, a
3-hydroxybutyryl-CoA reductase (alcohol-forming), a
3-hydroxybutyryl-CoA transferase, a 3-hydroxybutyryl-CoA
synthetase, a 3-hydroxybutyryl-CoA hydrolase, or a
3-hydroxybutyrate reductase.
[0023] In one embodiment, the MAA pathway comprises a succinyl-CoA
reductase, a succinate reductase, a 4-hydroxybutyrate
dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a
4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyrate kinase, a
phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA mutase, a
3-hydroxyisobutyryl-CoA dehydratase, a methacrylyl-CoA transferase,
a methacrylyl-CoA synthetase, a methacrylyl-CoA hydrolase, a
3-hydroxyisobutyryl-CoA transferase, a 3-hydroxyisobutyryl-CoA
synthetase, a 3-hydroxyisobutyryl-CoA hydrolase, a
3-hydroxyisobutyrate dehydratase, a methylmalonyl-CoA mutase, a
methylmalonyl-CoA transferase, a methylmalonyl-CoA synthetase, a
methylmalonyl-CoA hydrolase, a methylmalonate reductase, a
methylmalonyl-CoA reductase (aldehyde forming), a
3-hydroxyisobutyrate dehydrogenase, a methylmalonyl-CoA reductase
(alcohol forming) or a 3-hydroxyisobutyrate dehydratase.
[0024] In one embodiment, the invention provides a non-naturally
occurring microbial organism that includes a microbial organism
having an 14-BDO and an isopropanol pathway, where the 14-BDO
pathway includes a first set of exogenous nucleic acids encoding
14-BDO pathway enzymes expressed in a sufficient amount to produce
14-BDO and where the isopropanol pathway includes a second set of
exogenous nucleic acids encoding isopropanol pathway enzymes
expressed in a sufficient amount to produce isopropanol.
[0025] In one embodiment, the invention provides a non-naturally
occurring microbial organism that includes a microbial organism
having an 13-BDO and an isopropanol pathway, where the 13-BDO
pathway includes a first set of exogenous nucleic acids encoding
13-BDO pathway enzymes expressed in a sufficient amount to produce
13-BDO and where the isopropanol pathway includes a second set of
exogenous nucleic acids encoding isopropanol pathway enzymes
expressed in a sufficient amount to produce isopropanol.
[0026] In one embodiment, the invention provides a non-naturally
occurring microbial organism that includes a microbial organism
having an methylacrylic acid and an isopropanol pathway, where the
methylacrylic acid pathway includes a first set of exogenous
nucleic acids encoding methylacrylic acid pathway enzymes expressed
in a sufficient amount to produce methylacrylic acid and where the
isopropanol pathway includes a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol.
[0027] It is understood that methylacrylic acid pathways passing
through a 3-hydroxyisobutyrate intermediate can be applied for
3-hydroxyisobutyrate production as opposed to methylacrylic acid
production if the downstream enzyme, that is, a dehydratase, is
omitted (see FIGS. 7 and 8). In this case, the non-naturally
occurring organism would produce 3-hydroxyisobutyrate instead of
methylacrylic acid. The non-naturally occurring organism could
alternatively produce a mixture of 3-hydroxyisobutyate and
methylacrylic acid. The maximum molar yields of ATP and product
will be unchanged regardless of whether methylacrylic acid or
3-hydroxyisobutyrate is produced.
[0028] It is further understood that, if desired,
3-hydroxyisobutyric acid expressed by a microbial organism of the
invention can be chemically converted to methylacrylic acid. For
example, 3-hydroxyisobutyric acid, or .beta.-hydroxyisobutyric
acid, can be dehydrated to form methylacrylic acid as described,
for example, in U.S. Pat. No. 7,186,856.
[0029] In still other aspects, embodiments disclosed herein relate
to a method for producing 14-BDO and isopropanol, 13-BDO and
isopropanol or MAA and isopropanol that includes culturing the
aforementioned non-naturally occurring microbial organisms.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 shows an exemplary pathway for co-production of
n-propanol and isopropanol from glucose. Abbreviations:
Glc--glucose, PEP--phosphoenolpyruvate, PYR--pyruvate,
FOR--formate, ACCOA--acetyl-CoA, AACOA--acetoacetyl-CoA,
ACAC--acetoacetate, AC--acetone, PPOH-2--isopropanol,
OAA--oxaloacetate, MAL--malate, FUM--fumarate, SUCC--succinate,
SUCCOA--succinyl--CoA, MMCOA--methylmalonyl-CoA,
PPCOA--propionyl-CoA, PPA--propionate, PPAL--propionaldehyde,
PPPi--propionyl phosphate, PPOH-1--n-propanol.
[0031] FIG. 2 shows an exemplary pathway for co-production of
n-propanol and isopropanol from glucose. Abbreviations:
Glc--glucose, PEP--phosphoenolpyruvate, PYR--pyruvate,
FOR--formate, ACCOA--acetyl-CoA, AACOA--acetoacetyl-CoA,
ACAC--acetoacetate, AC--acetone, PPOH-2--isopropanol,
OAA--oxaloacetate, THR--threonine, 2-OBUT--2-oxobutanoate,
PPCOA--propionyl-CoA, PPA--propionate, PPAL--propionaldehyde,
PPPi--propionyl phosphate, PPOH-1--n-propanol.
[0032] FIG. 3 shows an exemplary pathway for co-production of
n-propanol and isopropanol from glucose. Abbreviations:
Glc--glucose, PEP--phosphoenolpyruvate, PYR--pyruvate,
FOR--formate, ACCOA--acetyl-CoA, AACOA--acetoacetyl-CoA,
ACAC--acetoacetate, AC--acetone, PPOH-2--isopropanol,
MALCOA--malonyl-CoA, MALAL--malonate semialdehyde,
3HP--3-hydroxypropionate, PPCOA--propionyl-CoA, PPA--propionate,
PPAL--propionaldehyde, PPPi--propionyl phosphate,
PPOH-1--n-propanol.
[0033] FIG. 4 shows an exemplary pathway for co-production of
n-propanol and isopropanol from glucose. Abbreviations:
Glc--glucose, PEP--phosphoenolpyruvate, PYR--pyruvate,
FOR--formate, ACCOA--acetyl-CoA, AACOA--acetoacetyl-CoA,
ACAC--acetoacetate, AC--acetone, PPOH-2--isopropanol,
LAC--D-lactate, LACCOA--lactoyl-CoA, ACRYLCOA--acryloyl-CoA,
PPCOA--propionyl-CoA, PPA--propionate, PPAL--propionaldehyde,
PPPi--propionyl phosphate, PPOH-1--n-propanol.
[0034] FIG. 5 shows an exemplary pathway for coproduction of
1,4-BDO and isopropanol from glucose. Abbreviations: Glc--glucose,
PEP--phosphoenolpyruvate, PYR--pyruvate, FOR--formate,
ACCOA--acetyl-CoA, AACOA--acetoacetyl-CoA, ACAC--acetoacetate,
AC--acetone, PPOH-2--isopropanol, OAA--oxaloacetate, MAL--malate,
FUM--fumarate, SUCC--succinate, SUCCOA--succinyl--CoA,
SUCSAL--succinic semialdehyde, 4-HB--4-hydroxybutyrate,
4-HBCOA--4-hydroxybutyryl-CoA, 4-HBALD--4-hydroxybutyraldehyde,
14-BDO--1,4-butanediol, 4-HBP--4-hydroxybutyryl-phosphate.
[0035] FIG. 6 shows an exemplary pathway for coproduction of
1,3-BDO and isopropanol from glucose. Abbreviations: Glc--glucose,
PEP--phosphoenolpyruvate, PYR--pyruvate, FOR--formate,
ACCOA--acetyl-CoA, AACOA--acetoacetyl-CoA, ACAC--acetoacetate,
AC--acetone, PPOH-2--isopropanol, OAA--oxaloacetate, MAL--malate,
FUM--fumarate, SUCC--succinate, SUCCOA--succinyl--CoA,
SUCSAL--succinic semialdehyde, 3-HB--3-hydroxybutyrate,
4-HB--4-hydroxybutyrate, 4-HBCOA--4-hydroxybutyryl-CoA,
CRTCOA--crotonyl-CoA, 3-HBCOA--3-hydroxybutyryl-CoA,
3-HBALD--3-hydroxybutyraldehyde, 13-BDO--1,3-butanediol.
[0036] FIG. 7 shows an exemplary pathway for coproduction of
methyacrylic acid and isopropanol from glucose. Abbreviations:
Glc--glucose, PEP--phosphoenolpyruvate, PYR--pyruvate,
FOR--formate, ACCOA--acetyl-CoA, AACOA--acetoacetyl-CoA,
ACAC--acetoacetate, AC--acetone, PPOH-2--isopropanol,
OAA--oxaloacetate, MACOA--methyacrylyl-CoA, MAL--malate,
FUM--fumarate, SUCC--succinate, SUCCOA--succinyl-CoA,
SUCSAL--succinic semialdehyde, 4-HB--4-hydroxybutyrate,
4-HBCOA--4-hydroxybutyryl-CoA, 3-HIBCOA--3-hydroxyisobutyryl-CoA,
3-HIB--3-hydroxyisobutyrate, MAA--methylacrylic acid.
[0037] FIG. 8 shows an exemplary pathway for coproduction of
methyacrylic acid and isopropanol from glucose. Abbreviations:
Glc--glucose, PEP--phosphoenolpyruvate, PYR--pyruvate,
FOR--formate, ACCOA--acetyl-CoA, AACOA--acetoacetyl-CoA,
ACAC--acetoacetate, AC--acetone, PPOH-2--isopropanol,
OAA--oxaloacetate, MAL--malate, FUM--fumarate, SUCC--succinate,
SUCCOA--succinyl--CoA, MM--methylmalonate,
MMCOA--methylmalonyl-CoA, MMSA--methylmalonate semialdehyde,
3-HIB--3-hydroxyisobutyrate, MAA--methylacrylic acid.
DETAILED DESCRIPTION THE INVENTION
[0038] Embodiments of the present invention provide non-naturally
occurring microbial organisms having redox-balanced anaerobic
pathways for co-production of n-propanol and isopropanol from 3
phosphoenolpyruvate (PEP) molecules as exemplified in FIGS. 1-4.
Some advantages of this co-production strategy include: (1) the
co-production affords the maximum theoretical yield of n-propanol
and isopropanol at 1.33 moles total/mole of glucose; and (2) the
pathway for co-production is completely redox balanced and has a
net positive yield of ATP. This facilitates a completely anaerobic
production of the C3 alcohols as opposed to culturing microbial
organisms having the isopropanol pathway alone, which requires
aeration for regeneration of NAD.
[0039] Embodiments of the present invention also provide
non-naturally occurring microbial organisms that can co-produce
n-propanol and isopropanol from renewable resources as shown in
FIGS. 1-4. Specifically, the organisms include all enzymes utilized
in the co-production of n-propanol and isopropanol from acetyl-CoA
and propionyl-CoA. Formate can be converted to carbon dioxide by a
formate dehydrogenase that provides an additional reducing
equivalent that can be used for n-propanol and isopropanol
syntheses. Additionally, reducing equivalents can be obtained from
other steps in the pathway, such as, the glycolysis pathway during
conversion of glucose to phospheonolpyruvate, pyruvate
dehydrogenase or pyruvate ferredoxin oxidoreductase during
conversion of pyruvate to acetyl-CoA, or 2-oxobutanoate
dehydrogenase during conversion of 2-oxobutanoate to
propionyl-CoA.
[0040] Embodiments of the present invention also provide
non-naturally occurring microbial organisms that can produce
n-propanol via propionyl-CoA. This conversion is carried out by two
different enzymes: an aldehyde and alcohol dehydrogenase or in one
step by a bifunctional aldehyde/alcohol dehydrogenase.
Alternatively, propionyl-CoA can be converted into propionyl
phosphate and then transformed into propionaldehyde by an acyl
phosphate reductase. Alternatively, propionyl-CoA can be converted
to propionate then to propionyl phosphate by a propionyl-CoA
hydrolase, transferase, or synthetase and a propionate dinase,
respectively. Alternatively, propionate can be converted to
propionaldehyde by a propionate reductase. Pathways for production
of propionyl-CoA are exemplified in FIGS. 1-4. In one embodiment,
the pathway for production of propionyl-CoA proceeds via
oxaloacetate as exemplified in FIG. 1. Oxaloacetate is converted to
propionyl-CoA by means of the reductive TCA cycle, a methylmutase,
a decarboxylase, and a decarboxylase. An epimerase may be required
to convert the (R) stereoisomer of methylmalonyl-CoA to the (S)
configuration. In another embodiment, the pathway for production of
propionyl-CoA proceeds via threonine as exemplified in FIG. 2.
Oxaloacetate is converted into threonine by the native threonine
pathway engineered for high yields. It is then deaminated to form
2-oxobutanoate and subsequently converted into propionyl-CoA. In
one alternative, 2-oxobutanoate is converted to propionaldehyde by
a decarboxylase, which is then reduced to n-propanol by a propanol
dehydrogenase. In yet another embodiment, the pathway for
production of propionyl-CoA proceeds via malonyl-CoA as exemplified
in FIG. 3. Acetyl-CoA is carboxylated to form malonyl-CoA. This is
then reduced to malonate semialdehyde, and subsequently transformed
into 3-hydroxypropionate (3HP). 3HP is converted into propionyl-CoA
via propionyl-CoA synthase. In yet another embodiment, the pathway
for production of propionyl-CoA proceeds via lactate as exemplified
in FIG. 4. Pyruvate is reduced to form lactate which is then
activated to form lactoyl-CoA. The lactoyl-CoA is dehydrated to
form acryloyl-CoA and then reduced to generate propionyl-CoA.
[0041] Embodiments of the present invention also provide
non-naturally occurring microbial organisms that can produce
isopropanol via acetyl-CoA. Isopropanol production is achieved via
conversion of acetyl-CoA by an acetoacetyl-CoA thiolase, an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase, and an
isopropanol dehydrogenase as exemplified in FIGS. 1-4. In one
embodiment the pathway for production of acetyl-CoA from glucose
proceeds via phosphoenolpyruvate (PEP). Glucose is converted into
PEP by the native glycolysis pathway of the microbial organism. PEP
is converted to pyruvate by pyruvate kinase and then to acetyl-CoA
by pyruvate dehydrogenase or pyruvate ferredoxin oxidoreductase.
Alternatively, pyruvate is converted to acetyl-CoA and formate by
pyruvate formate lyase. The formate is then converted to carbon
dioxide and hydrogen by a formate dehydrogenase.
[0042] Embodiments of the present invention provide alternate
methods for coproduction of isopropanol with the compounds 14-BDO,
13-BDO and MAA. The production of isopropanol proceeds via
acetyl-CoA as described above. Alone this route is not
redox-balanced and thus requires aeration to achieve high
isopropanol yields. Embodiments described herein use this route and
combine it with pathways for synthesizing the coproducts
1,4-butanediol (14-BDO), 1,3-butanediol (13-BDO) and methylacrylic
acid (MAA). Coproduction routes are redox-balanced under anaerobic
conditions as opposed to the requirement of oxygen if isopropanol
is produced solely through acetone. Coproduction also provides
related advantages, such as, the ease of separating isopropanol
from other fermentation products due it its low boiling point
(82.degree. C.) relative to 14-BDO (230.degree. C.), 13-BDO
(203.degree. C.) and MAA (163.degree. C.) and the coproduction
using any of the microbial organisms described herein provides that
maximum theoretical yield of the carbon from glucose is
afforded.
[0043] Embodiments of the present invention provide non-naturally
occurring microbial organisms that can produce 14-BDO via
succinyl-CoA or in some aspects via succinate. For production of
14-BDO, succinyl-CoA is converted to succinic semialdehyde by a
succinyl-CoA reductase. Alternatively, succinate can be converted
to succinic semialdehyde by a succinate reductase. Next, succinic
semialdehyde is reduced to 4-hydroxybutyrate by 4-hydroxybutyrate
dehydrogenase. Activation of 4-HB to its acyl-CoA is catalyzed by a
CoA transferase or synthetase. Alternatively, 4-HB can be converted
into 4-hydroxybutyryl-phosphate and subsequently transformed into
4-HB-CoA by a phosphotrans-4-hydroxybutyrylase. 4-HB-CoA is then
converted to 14-BDO by either a bifunctional CoA-dependent
aldehyde/alcohol dehydrogenase, or by two separate enzymes with
aldehyde and alcohol dehydrogenase activity. Yet another
alternative that bypasses the 4-HB-CoA intermediate is direct
reduction of 4-HB to 4-hydroxybutyrylaldehyde by a carboxylic acid
reductase. 4-Hydroxybutyrylaldehyde is subsequently reduced to
14-BDO by an alcohol dehydrogenase. Yet another route that bypasses
4-HB-CoA entails reducing 4-hydroxybutyryl-phosphate to
4-hydroxybutyraldehyde by a phosphate reductase.
[0044] Embodiments of the present invention provide non-naturally
occurring microbial organisms that can produce 13-BDO via
succinyl-CoA or in some aspects via succinate. Production of 13-BDO
also proceeds through 4-hydroxybutyryl-CoA, formed as described
above. In this route, 4-hydroxybutyryl-CoA is dehydrated and
isomerized to form crotonyl-CoA. The dehydration and
vinylisomerisation reactions are catalyzed by a bifunctional
enzyme, 4-hydroxybutyryl-CoA dehydratase. Crotonyl-CoA is then
hydrated to 3-hydroxybutyryl-CoA. Removal of the CoA moiety and
concurrent reduction yields 3-hydroxybutyraldehyde. Alternatively,
3-hydroxybutyryl-CoA is converted to 3-hydroxybutyrate by a
3-hydroxybutyryl-CoA transferase, hydrolase, or synthetase and then
reduced by a 3-hydroxybutyrate reductase to yield
3-hydroxybutyraldehyde. Finally reduction of the aldehyde by
3-hydroxybutyraldehyde reductase yields 13-BDO.
[0045] Embodiments of the present invention provide non-naturally
occurring microbial organisms that can produce MAA via two
alternative routes. The first route proceeds through
4-hydroxybutyryl-CoA, formed as described above.
4-Hydroxybutyryl-CoA is converted to 3-hydroxyisobutyryl-CoA by a
methyl mutase. The CoA moiety of 3-Hydroxyisobutyryl-CoA is then
removed by a CoA transferase, hydrolase or synthetase. Finally,
dehydration of the 3-hydroxy group yields MAA. Alternatively,
3-hydroxyisobutyryl-CoA is converted to methyacrylyl-CoA by a
3-hydroxyisobutyryl-CoA dehydratase and then the CoA moiety is
removed by a CoA transferase, hydrolase or synthetase to yield MAA.
In the alternate MAA production route, succinyl-CoA is converted to
methylmalonyl-CoA by methylmalonyl-CoA mutase. An epimerase may be
required to convert the (R) stereoisomer of methylmalonyl-CoA to
the (S) configuration. A CoA-dependent aldehyde dehydrogenase then
converts methylmalonyl-CoA to methylmalonate semialdehyde.
Alternatively, the CoA moiety of (R)-methylmalonyl-CoA or
(S)-methylmalonyl-CoA is removed by a CoA transferase, hydrolase or
synthetase to form methylmalonate, which is then converted to the
semialdehyde by a reductase. Reduction of the aldehyde to
3-hydroxyisobutyrate, followed by dehydration, yields MAA.
Alternately, methylmalonyl-CoA is converted to 3-hydroxyisobutyrate
by an alcohol-forming CoA reductase.
[0046] Embodiments of the present invention provide non-naturally
occurring microbial organisms having pathways for production of
succinyl-CoA as exemplified in FIGS. 5-8. In one embodiment, the
pathway for production of succinyl-CoA proceeds via oxaloacetate.
Oxaloacetate is converted to succinyl-CoA by means of the reductive
TCA cycle, including a malate dehydrogenase, a fumerase, a fumarate
reducatase and a succinyl-CoA transferase or alternatively a
succinyl-CoA synthetase.
[0047] Engineering these pathways into a microorganism involves
cloning an appropriate set of genes encoding a set of enzymes into
a production host described herein, optimizing fermentation
conditions, and assaying product formation following fermentation.
To engineer a production host for the production of n-propanol and
isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or MAA
and isopropanol, one or more exogenous DNA sequence(s) can be
expressed in a microorganism. In addition, the microorganism can
have endogenous gene(s) functionally disrupted, deleted or
overexpressed. The metabolic modifications disclosed herein enable
the production of n-propanol and isopropanol, 14-BDO and
isopropanol, 13-BDO and isopropanol or MAA and isopropanol using
renewable feedstock.
[0048] In some embodiments, the invention provides non-naturally
occurring microbial organisms that include at least one exogenous
nucleic acid that encode an n-propanol pathway enzyme expressed in
a sufficient amount to produce n-propanol.
[0049] In another embodiment, the invention provides non-naturally
occurring microbial organisms that include at least one exogenous
nucleic acid that encode an isopropanol pathway enzyme expressed in
a sufficient amount to produce isopropanol.
[0050] In still other embodiments, the invention provides methods
for co-producing n-propanol and isopropanol, 14-BDO and
isopropanol, 13-BDO and isopropanol or MAA and isopropanol. Such
methods involve culturing the microbial organisms described
herein.
[0051] 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 an
n-propanol, an isopropanol, a 14-BDO, a 13-BDO and/or MAA
biosynthetic pathways.
[0052] 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.
[0053] As used herein, the term "isolated" when used in reference
to a microbial organism are 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.
[0054] As used herein, the terms "microbial," "microbial organism"
or "microorganism" is 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.
[0055] As used herein, "n-propanol" is intended to mean a primary
alcohol with the molecular formula of C.sub.3H.sub.8O and a
molecular mass of 60.1 g/mol. N-propanol is also known in the art
as 1-propanol, 1-propyl alcohol, n-propyl alcohol, propan-1-ol, or
simply propanol. N-propanol is an isomer of isopropanol.
[0056] As used herein, "isopropanol" is intended to mean a
secondary alcohol, with the molecular formula of C.sub.3H.sub.8O
and a molecular mass of 60.1 g/mol, wherein the alcohol carbon is
attached to two other carbons. This attachment is sometimes shown
as (CH.sub.3).sub.2CHOH. Isopropanol is also known in the art as
propan-2-ol, 2-propanol or the abbreviation IPA. Isopropanol is an
isomer of n-propanol.
[0057] As used herein, the term "1,4-butanediol" is intended to
mean an alcohol derivative of the alkane butane, carrying two
hydroxyl groups which has the chemical formula
C.sub.4H.sub.10O.sub.2 and a molecular mass of 90.12 g/mol. The
chemical compound 1,4-butanediol also is known in the art as
1,4-BDO and is a chemical intermediate or precursor for a family of
compounds commonly referred to as the BDO family of compounds.
[0058] As used herein, the term "1,3-butanediol" is intended to
mean one of four stable isomers of butanediol having the chemical
formula C.sub.4H.sub.10O.sub.2 and a molecular mass of 90.12 g/mol.
The chemical compound 1,3-butanediol is known in the art as 13-BDO
or .beta.-butane glycol and is also a chemical intermediate or
precursor for a family of compounds commonly referred to as the BDO
family of compounds.
[0059] As used herein, "methylacrylic acid," having the chemical
formula CH.sub.2.dbd.C(CH.sub.3)CO.sub.2 (also known as methacrylic
acid and IUPAC name 2-methyl-2-propenoic acid), is the acid form of
methylacrylate, and it is understood that methylacrylic acid and
methylacrylate 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. Similarly,
3-hydroxyisobutyrate and 3-hydroxyisobutyric acid can be used
interchangeably throughout to refer to the compound in any of its
neutral or ionized forms, including any salt forms thereof.
[0060] As used herein, the term "CoA" or "coenzyme A" is intended
to mean an organic cofactor or prosthetic group (nonprotein portion
of an enzyme) whose presence is required for the activity of many
enzymes (the apoenzyme) to form an active enzyme system. Coenzyme A
functions in certain condensing enzymes, acts in acetyl or other
acyl group transfer and in fatty acid synthesis and oxidation,
pyruvate oxidation and in other acetylation.
[0061] 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.
[0062] "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.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] Therefore, in identifying and constructing the non-naturally
occurring microbial organisms of the invention having n-propanol
and isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or
MAA and isopropanol 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.
[0070] 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.
[0071] 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.
[0072] In one embodiment, the invention provides a non-naturally
occurring microbial organism, including a microbial organism having
an n-propanol pathway and an isopropanol pathway, the n-propanol
pathway having at least one exogenous nucleic acid encoding an
n-propanol pathway enzyme expressed in a sufficient amount to
produce n-propanol, the n-propanol pathway including a
propionaldehyde dehydrogenase, a propanol dehydrogenase, a
propionyl-CoA:phosphate propanoyltransferase, a propionyl-CoA
hydrolase, a propionyl-CoA transferase, a propionyl-CoA synthetase,
a propionate kinase, a propionate reductase or a propionyl
phosphate reductase, the isopropanol pathway comprising at least
one exogenous nucleic acid encoding an isopropanol pathway enzyme
expressed in a sufficient amount to produce isopropanol, the
isopropanol pathway including an acetyl-CoA acetyl thiolase, an
acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase or an
isopropanol dehydrogenase.
[0073] In a further aspect of the above embodiment, the microbial
organism has an acetyl-CoA pathway having at least one exogenous
nucleic acid encoding an acetyl-CoA pathway enzyme expressed in a
sufficient amount to produce acetyl-CoA, the acetyl-CoA pathway
including a pyruvate kinase, a pyruvate dehydrogenase, a pyruvate
ferredoxin oxidoreductase, a pyruvate formate lyase, a pyruvate
formate lyase activating enzyme, or a formate dehydrogenase.
[0074] In further embodiment, the microbial organism has a
propionyl-CoA pathway having at least one exogenous nucleic acid
encoding a propionyl-CoA pathway enzyme expressed in a sufficient
amount to produce propionyl-CoA, the propionyl-CoA pathway
including a PEP carboxykinase, a PEP carboxylase, a malate
dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA
transferase, a succinyl-CoA synthetase, a methylmalonyl-CoA mutase,
a methylmalonyl-CoA epimerase or a methylmalonyl-CoA decarboxylase.
In a further aspect, the propionyl-CoA pathway includes a pyruvate
carboxylase or a methylmalonyl-CoA carboxytransferase.
[0075] In another further embodiment, the microbial organism has a
propionyl-CoA pathway having at least one exogenous nucleic acid
encoding a propionyl-CoA pathway enzyme expressed in a sufficient
amount to produce propionyl-CoA, the propionyl-CoA pathway
including a PEP carboxykinase, a PEP carboxylase, a threonine
deaminase, or a 2-oxobutanoate dehydrogenase. In a further aspect,
the n-propanol pathway includes 2-oxobutanoate decarboxylase.
[0076] In another further embodiment, the microbial organism has a
propionyl-CoA pathway having at least one exogenous nucleic acid
encoding a propionyl-CoA pathway enzyme expressed in a sufficient
amount to produce propionyl-CoA, the propionyl-CoA pathway
including an acetyl-CoA carboxylase, a malonyl-CoA reductase, a
malonate semialdehyde reductase or propionyl-CoA synthase.
[0077] In another further embodiment, the microbial organism has a
propionyl-CoA pathway having at least one exogenous nucleic acid
encoding a propionyl-CoA pathway enzyme expressed in a sufficient
amount to produce propionyl-CoA, the propionyl-CoA pathway
including a lactate dehydrogenase, a lactate-CoA transferase, a
lactyl-CoA dehydratase or acryloyl CoA reductase.
[0078] In yet another embodiment, the invention provides a
non-naturally occurring microbial organism, including a microbial
organism having an n-propanol pathway and an isopropanol pathway,
the n-propanol pathway having a first set of exogenous nucleic
acids encoding n-propanol pathway enzymes expressed in a sufficient
amount to produce n-propanol, the first set of exogenous nucleic
acids encoding a propionaldehyde dehydrogenase and a propanol
dehydrogenase; or a propionyl-CoA:phosphate propanoyltransferase, a
propionyl phosphate reductase and a propanol dehydrogenase; or a
propionyl-CoA hydrolase or a propionyl-CoA transferase or a
propionyl-CoA synthetase, a propionate kinase, a propionyl
phosphate reductase and a propanol dehydrogenase; or a
propionyl-CoA hydrolase or a propionyl-CoA transferase or a
propionyl-CoA synthetase, a propionate reductase and a propanol
dehydrogenase, and the isopropanol pathway having a second set of
exogenous nucleic acids encoding isopropanol pathway enzymes
expressed in a sufficient amount to produce isopropanol, the second
set of exogenous nucleic acids encoding an acetyl-CoA acetyl
thiolase; an acetoacetyl-CoA transferase or an acetoacetyl-CoA
hydrolase or an acetoacetyl-CoA synthetase, an acetoacetate
decarboxylase; and an isopropanol dehydrogenase.
[0079] In a further aspect of the above embodiment, the microbial
organism has an acetyl-CoA pathway having a third set of exogenous
nucleic acids encoding acetyl-CoA pathway enzymes expressed in a
sufficient amount to produce acetyl-CoA, the third set of exogenous
nucleic acids encoding a pyruvate kinase; and a pyruvate
dehydrogenase or a pyruvate ferredoxin oxidoreductase; or a
pyruvate formate lyase, a pyruvate formate lyase activating enzyme
and a formate dehydrogenase.
[0080] In another further embodiment, the microbial organism has a
propionyl-CoA pathway having a third set of exogenous nucleic acids
encoding propionyl-CoA pathway enzymes expressed in a sufficient
amount to produce propionyl-CoA, the third set of exogenous nucleic
acids encoding a PEP carboxykinase or a PEP carboxylase; a malate
dehydrogenase; a fumarase; a fumarate reductase; a succinyl-CoA
transferase or a succinyl-CoA synthetase; a methylmalonyl-CoA
mutase; and a methylmalonyl-CoA decarboxylase. In a further aspect,
the third set of exogenous nucleic acids further encodes a
methylmalonyl-CoA epimerase, a pyruvate carboxylase or a
methylmalonyl-CoA carboxytransferase.
[0081] In another further embodiment, the microbial organism has a
propionyl-CoA pathway having a third set of exogenous nucleic acids
encoding propionyl-CoA pathway enzymes expressed in a sufficient
amount to produce propionyl-CoA, said third set of exogenous
nucleic acids encoding a PEP carboxykinase or a PEP carboxylase; a
threonine deaminase; and a 2-oxobutanoate dehydrogenase. In a
further aspect, the third set of exogenous nucleic acids further
encodes a methylmalonyl-CoA decarboxylase or a pyruvate
carboxylase. In yet another aspect, the second set of exogenous
nucleic acids further encodes a 2-oxobutanoate decarboxylase.
[0082] In another further embodiment, the microbial organism has a
propionyl-CoA pathway having a third set of exogenous nucleic acids
encoding propionyl-CoA pathway enzymes expressed in a sufficient
amount to produce propionyl-CoA, the third set of exogenous nucleic
acids encoding an acetyl-CoA carboxylase; a malonyl-CoA reductase;
a malonate semialdehyde reductase; and propionyl-CoA synthase.
[0083] In another further embodiment, the microbial organism has a
propionyl-CoA pathway having a third set of exogenous nucleic acids
encoding a lactate dehydrogenase; a lactate-CoA transferase; a
lactyl-CoA dehydratase; and acryloyl CoA reductase.
[0084] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an n-propanol pathway and an isopropanol pathway, the n-propanol
pathway comprising a first set of exogenous nucleic acids encoding
n-propanol pathway enzymes expressed in a sufficient amount to
produce n-propanol, the first set of exogenous nucleic acids
encoding a PEP carboxykinase or a PEP carboxylase; a malate
dehydrogenase; a fumarase; a fumarate reductase; a succinyl-CoA
transferase or a succinyl-CoA synthetase; a methylmalonyl-CoA
mutase; a methylmalonyl-CoA decarboxylase; and a propionaldehyde
dehydrogenase and a propanol dehydrogenase; or a
propionyl-CoA:phosphate propanoyltransferase and a propionyl
phosphate reductase; or a propionyl-CoA hydrolase or a
propionyl-CoA transferase or a propionyl-CoA synthetase, a
propionate kinase, a propionyl phosphate reductase and a propanol
dehydrogenase; or a propionyl-CoA hydrolase or a propionyl-CoA
transferase or a propionyl-CoA synthetase, a propionate reductase
and a propanol dehydrogenase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding a pyruvate kinase; a pyruvate dehydrogenase or a pyruvate
ferredoxin oxidoreductase; or a pyruvate formate lyase, a pyruvate
formate lyase activating enzyme and a formate dehydrogenase; an
acetyl-CoA acetyl thiolase; an acetoacetyl-CoA transferase or an
acetoacetyl-CoA hydrolase or an acetoacetyl-CoA synthetase, an
acetoacetate decarboxylase; and an isopropanol dehydrogenase.
[0085] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an n-propanol pathway and an isopropanol pathway, the n-propanol
pathway comprising a first set of exogenous nucleic acids encoding
n-popanol pathway enzymes expressed in a sufficient amount to
produce n-propanol, the first set of exogenous nucleic acids
encoding a PEP carboxykinase or a PEP carboxylase; a threonine
deaminase; and a 2-oxobutanoate decarboxylase and a propanol
dehydrogenase; or a 2-oxobutanoate dehydrogenase, a propionaldehyde
dehydrogenase and a propanol dehydrogenase; or a 2-oxobutanoate
dehydrogenase, a propionyl-CoA:phosphate propanoyltransferase, a
propionyl phosphate reductase and a propanol dehydrogenase; a
2-oxobutanoate dehydrogenase, a propionyl-CoA hydrolase or a
propionyl-CoA transferase or a propionyl-CoA synthetase, a
propionate kinase, a propionyl phosphate reductase and a propanol
dehydrogenase; or a 2-oxobutanoate dehydrogenase, a propionyl-CoA
hydrolase or a propionyl-CoA transferase or a propionyl-CoA
synthetase, a propionate reductase and a propanol dehydrogenase,
and the isopropanol pathway comprising a second set of exogenous
nucleic acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding a pyruvate kinase; a pyruvate
dehydrogenase or a pyruvate ferredoxin oxidoreductase; or a
pyruvate formate lyase, a pyruvate formate lyase activating enzyme
and a formate dehydrogenase; an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase; and an
isopropanol dehydrogenase. In a further aspect, the second set of
exogenous nucleic acids further encodes a pyruvate carboxylase or a
methylmalonyl-CoA carboxytransferase.
[0086] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an n-propanol pathway and an isopropanol pathway, the n-propanol
pathway comprising a first set of exogenous nucleic acids encoding
n-propanol pathway enzymes expressed in a sufficient amount to
produce n-propanol, the first set of exogenous nucleic acids
encoding a pyruvate kinase; a pyruvate dehydrogenase or a pyruvate
ferredoxin oxidoreductase; or a pyruvate formate lyase, a pyruvate
formate lyase activating enzyme and a formate dehydrogenase; an
acetyl-CoA carboxylase; a malonyl-CoA reductase; a malonate
semialdehyde reductase; propionyl-CoA synthase; and a
propionaldehyde dehydrogenase and a propanol dehydrogenase; or a
propionyl-CoA:phosphate propanoyltransferase, a propionyl phosphate
reductase and propanol dehydrogenase; or a propionyl-CoA hydrolase
or a propionyl-CoA transferase or a propionyl-CoA synthetase, a
propionate kinase, a propionyl phosphate reductase and a propanol
dehydrogenase; or a propionyl-CoA hydrolase or a propionyl-CoA
transferase or a propionyl-CoA synthetase, a propionate reductase
and a propanol dehydrogenase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase, an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0087] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an n-propanol pathway and an isopropanol pathway, the n-propanol
pathway including a first set of exogenous nucleic acids encoding
n-propanol pathway enzymes expressed in a sufficient amount to
produce n-propanol, the first set of exogenous nucleic acids
encoding a lactate dehydrogenase; a lactate-CoA transferase; a
lactyl-CoA dehydratase; acryloyl CoA reductase; and a
propionaldehyde dehydrogenase and a propanol dehydrogenase; or a
propionyl-CoA:phosphate propanoyltransferase, a propionyl phosphate
reductase and a propanol dehydrogenase; or a propionyl-CoA
hydrolase or a propionyl-CoA transferase or a propionyl-CoA
synthetase, a propionate kinase, a propionyl phosphate reductase
and a propanol dehydrogenase; or a propionyl-CoA hydrolase or a
propionyl-CoA transferase or a propionyl-CoA synthetase, a
propionate reductase and a propanol dehydrogenase, and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding a pyruvate dehydrogenase or a
pyruvate ferredoxin oxidoreductase; or a pyruvate formate lyase, a
pyruvate formate lyase activating enzyme and a formate
dehydrogenase; an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase, an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0088] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an n-propanol pathway, the n-propanol pathway comprising at least
one exogenous nucleic acid encoding an n-propanol pathway enzyme
expressed in a sufficient amount to produce n-propanol, the
n-propanol pathway including a propionaldehyde dehydrogenase, a
propanol dehydrogenase, a propionyl-CoA:phosphate
propanoyltransferase, a propionyl-CoA hydrolase, a propionyl-CoA
transferase, a propionyl-CoA synthetase, a propionate kinase, a
propionate reductase, or a propionyl phosphate reductase.
[0089] In another embodiment, the invention provides a
non-naturally occurring microbial organism including a microbial
organism having an n-propanol pathway, the n-propanol pathway
comprising a set of exogenous nucleic acids encoding n-propanol
pathway enzymes expressed in a sufficient amount to produce
n-propanol, the set of exogenous nucleic acids encoding a
propionaldehyde dehydrogenase and a propanol dehydrogenase; or a
propionyl-CoA:phosphate propanoyltransferase, a propionyl phosphate
reductase and a propanol dehydrogenase; or a propionyl-CoA
hydrolase or a propionyl-CoA transferase or a propionyl-CoA
synthetase, a propionate kinase, a propionyl phosphate reductase
and a propanol dehydrogenase; or a propionyl-CoA hydrolase or a
propionyl-CoA transferase or a propionyl-CoA synthetase, a
propionate reductase and a propanol dehydrogenase.
[0090] In a further aspect of the above embodiment, the
non-naturally occurring microbial organism having an n-propanol
pathway also has a propionyl-CoA pathway including exogenous
nucleic acids encoding propionyl-CoA pathway enzymes expressed in a
sufficient amount to produce propionyl-CoA as exemplified herein.
For example, in some aspects the exogenous nucleic acids encode a
PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a
fumarase, a fumarate reductase, a succinyl-CoA transferase, a
succinyl-CoA synthetase, a methylmalonyl-CoA mutase or a
methylmalonyl-CoA decarboxylase. In another aspect, the exogenous
nucleic acids further encode a methylmalonyl-CoA epimerase.
Additionally, in yet another aspect of the above embodiment, the
non-naturally occurring microbial organism having an n-propanol
pathway can have a first set of exogenous nucleic acids encoding
n-propanol pathway enzymes expressed in a sufficient amount to
produce n-propanol, wherein the first set of exogenous nucleic
acids encode a PEP carboxykinase or a PEP carboxylase; a malate
dehydrogenase; a fumarase; a fumarate reductase; a succinyl-CoA
transferase or a succinyl-CoA synthetase; a methylmalonyl-CoA
mutase; a methylmalonyl-CoA epimerase, a methylmalonyl-CoA
decarboxylase; a propionaldehyde dehydrogenase and a propanol
dehydrogenase.
[0091] In one embodiment, the invention provides a non-naturally
occurring microbial organism, including a microbial organism having
an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway
having at least one exogenous nucleic acid encoding an 14-BDO
pathway enzyme expressed in a sufficient amount to produce 14-BDO,
the 14-BDO pathway including a succinyl-CoA reductase, a succinate
reductase, a 4-hydroxybutyrate dehydrogenase, a
4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA
synthetase, a 4-hydroxybutyryl-CoA reductase (aldehyde-forming), a
4-hydroxybutyraldehyde reductase, a 4-hydroxybutyrate reductase; a
4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, a
4-hydroxybutyryl-phosphate reductase or a 4-hydroxybutyryl-CoA
reductase (alcohol-forming), the isopropanol pathway including at
least one exogenous nucleic acid encoding an isopropanol pathway
enzyme expressed in a sufficient amount to produce isopropanol, the
isopropanol pathway including an acetyl-CoA acetyl thiolase, an
acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase or an
isopropanol dehydrogenase.
[0092] In one embodiment, the invention provides a non-naturally
occurring microbial organism, including a microbial organism having
an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway
having at least one exogenous nucleic acid encoding an 13-BDO
pathway enzyme expressed in a sufficient amount to produce 13-BDO,
the 13-BDO pathway including a succinyl-CoA reductase, a succinate
reductase, a 4-hydroxybutyrate dehydrogenase, a
4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA
synthetase, a 4-hydroxybutyrate kinase, a
phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA
dehydratase, a crotonase, a 3-hydroxybutyryl-CoA reductase
(aldehyde forming), a 3-hydroxybutyraldehyde reductase, a
3-hydroxybutyryl-CoA transferase, a 3-hydroxybutyryl-CoA
synthetase, a 3-hydroxybutyryl-CoA hydrolase, or a
3-hydroxybutyrate reductase, or a 3-hydroxybutyryl-CoA reductase
(alcohol-forming), the isopropanol pathway including at least one
exogenous nucleic acid encoding an isopropanol pathway enzyme
expressed in a sufficient amount to produce isopropanol, the
isopropanol pathway including an acetyl-CoA acetyl thiolase, an
acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase or an
isopropanol dehydrogenase.
[0093] In one embodiment, the invention provides a non-naturally
occurring microbial organism, including a microbial organism having
an MAA pathway and an isopropanol pathway, the MAA pathway having
at least one exogenous nucleic acid encoding an MAA pathway enzyme
expressed in a sufficient amount to produce MAA, the MAA pathway
including a succinyl-CoA reductase, a succinate reductase, a
4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA
transferase, a 4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyrate
kinase, a phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA
mutase, a 3-hydroxyisobutyryl-CoA dehydratase, a methacrylyl-CoA
transferase, a methacrylyl-CoA synthetase, a methacrylyl-CoA
hydrolase, a 3-hydroxyisobutyryl-CoA transferase, a
3-hydroxyisobutyryl-CoA synthetase, a 3-hydroxyisobutyryl-CoA
hydrolase, a 3-hydroxyisobutyrate dehydratase, a methylmalonyl-CoA
mutase, a methylmalonyl-CoA epimerase, a methylmalonyl-CoA
transferase, a methylmalonyl-CoA synthetase, a methylmalonyl-CoA
hydrolase, a methylmalonate reductase, a methylmalonyl-CoA
reductase (aldehyde forming), a 3-hydroxyisobutyrate dehydrogenase,
a methylmalonyl-CoA reductase (alcohol forming) or a
3-hydroxyisobutyrate dehydratase, the isopropanol pathway including
at least one exogenous nucleic acid encoding an isopropanol pathway
enzyme expressed in a sufficient amount to produce isopropanol, the
isopropanol pathway including an acetyl-CoA acetyl thiolase, an
acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase or an
isopropanol dehydrogenase.
[0094] In a further aspect of the above embodiments, the microbial
organism has an acetyl-CoA pathway having at least one exogenous
nucleic acid encoding an acetyl-CoA pathway enzyme expressed in a
sufficient amount to produce acetyl-CoA, the acetyl-CoA pathway
including a pyruvate kinase, a pyruvate dehydrogenase, a pyruvate
ferredoxin oxidoreductase, a pyruvate formate lyase, a pyruvate
formate lyase activating enzyme, or a formate dehydrogenase.
[0095] In further aspect of the above embodiments, the microbial
organism has a succinyl-CoA pathway having at least one exogenous
nucleic acid encoding a succinyl-CoA pathway enzyme expressed in a
sufficient amount to produce succinyl-CoA, the succinyl-CoA pathway
including a PEP carboxykinase, a PEP carboxylase, a malate
dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA
transferase or a succinyl-CoA synthetase. In a further aspect, the
succinyl-CoA pathway includes a pyruvate carboxylase or a
methylmalonyl-CoA carboxytransferase.
[0096] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway
including a first set of exogenous nucleic acids encoding 14-BDO
pathway enzymes expressed in a sufficient amount to produce 14-BDO,
the first set of exogenous nucleic acids encoding a succinyl-CoA
reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA reductase (aldehyde-forming);
and a 4-hydroxybutyraldehyde reductase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase; an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0097] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway
including a first set of exogenous nucleic acids encoding 14-BDO
pathway enzymes expressed in a sufficient amount to produce 14-BDO,
the first set of exogenous nucleic acids encoding a succinyl-CoA
reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate
reductase; and a 4-hydroxybutyraldehyde reductase, and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0098] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway
including a first set of exogenous nucleic acids encoding 14-BDO
pathway enzymes expressed in a sufficient amount to produce 14-BDO,
the first set of exogenous nucleic acids encoding a succinyl-CoA
reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate
kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA
reductase (aldehyde-forming); and a 4-hydroxybutyraldehyde
reductase, and the isopropanol pathway comprising a second set of
exogenous nucleic acids encoding isopropanol pathway enzymes
expressed in a sufficient amount to produce isopropanol, the second
set of exogenous nucleic acids encoding an acetyl-CoA acetyl
thiolase; an acetoacetyl-CoA transferase or an acetoacetyl-CoA
hydrolase or an acetoacetyl-CoA synthetase; an acetoacetate
decarboxylase; and an isopropanol dehydrogenase.
[0099] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway
including a first set of exogenous nucleic acids encoding 14-BDO
pathway enzymes expressed in a sufficient amount to produce 14-BDO,
the first set of exogenous nucleic acids encoding a succinyl-CoA
reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate
kinase; a 4-hydroxybutyryl-phosphate reductase; and a
4-hydroxybutyraldehyde reductase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase; an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0100] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway
including a first set of exogenous nucleic acids encoding 14-BDO
pathway enzymes expressed in a sufficient amount to produce 14-BDO,
the first set of exogenous nucleic acids encoding a succinyl-CoA
reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate
kinase; a phosphotrans-4-hydroxybutyrylase; and a
4-hydroxybutyryl-CoA reductase (alcohol-forming), and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0101] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway
including a first set of exogenous nucleic acids encoding 14-BDO
pathway enzymes expressed in a sufficient amount to produce 14-BDO,
the first set of exogenous nucleic acids encoding a succinyl-CoA
reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; and a 4-hydroxybutyryl-CoA reductase (alcohol-forming),
and the isopropanol pathway comprising a second set of exogenous
nucleic acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0102] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway
including a first set of exogenous nucleic acids encoding 14-BDO
pathway enzymes expressed in a sufficient amount to produce 14-BDO,
the first set of exogenous nucleic acids encoding a succinate
reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA reductase (aldehyde-forming);
and a 4-hydroxybutyraldehyde reductase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase; an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0103] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway
including a first set of exogenous nucleic acids encoding 14-BDO
pathway enzymes expressed in a sufficient amount to produce 14-BDO,
the first set of exogenous nucleic acids encoding a succinate
reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate
reductase; and a 4-hydroxybutyraldehyde reductase, and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0104] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway
including a first set of exogenous nucleic acids encoding 14-BDO
pathway enzymes expressed in a sufficient amount to produce 14-BDO,
the first set of exogenous nucleic acids encoding a succinate
reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate
kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA
reductase (aldehyde-forming); and a 4-hydroxybutyraldehyde
reductase, and the isopropanol pathway comprising a second set of
exogenous nucleic acids encoding isopropanol pathway enzymes
expressed in a sufficient amount to produce isopropanol, the second
set of exogenous nucleic acids encoding an acetyl-CoA acetyl
thiolase; an acetoacetyl-CoA transferase or an acetoacetyl-CoA
hydrolase or an acetoacetyl-CoA synthetase; an acetoacetate
decarboxylase; and an isopropanol dehydrogenase.
[0105] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway
including a first set of exogenous nucleic acids encoding 14-BDO
pathway enzymes expressed in a sufficient amount to produce 14-BDO,
the first set of exogenous nucleic acids encoding a succinate
reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate
kinase; a 4-hydroxybutyryl-phosphate reductase; and a
4-hydroxybutyraldehyde reductase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase; an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0106] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway
including a first set of exogenous nucleic acids encoding 14-BDO
pathway enzymes expressed in a sufficient amount to produce 14-BDO,
the first set of exogenous nucleic acids encoding a succinate
reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate
kinase; a phosphotrans-4-hydroxybutyrylase; and a
4-hydroxybutyryl-CoA reductase (alcohol-forming), and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0107] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway
including a first set of exogenous nucleic acids encoding 14-BDO
pathway enzymes expressed in a sufficient amount to produce 14-BDO,
the first set of exogenous nucleic acids encoding a succinate
reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; and a 4-hydroxybutyryl-CoA reductase (alcohol-forming),
and the isopropanol pathway comprising a second set of exogenous
nucleic acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0108] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway
including a first set of exogenous nucleic acids encoding 13-BDO
pathway enzymes expressed in a sufficient amount to produce 13-BDO,
the first set of exogenous nucleic acids encoding a succinyl-CoA
reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; a
3-hydroxybutyryl-CoA reductase (aldehyde forming); and a
3-hydroxybutyraldehyde reductase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase, an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0109] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway
including a first set of exogenous nucleic acids encoding 13-BDO
pathway enzymes expressed in a sufficient amount to produce 13-BDO,
the first set of exogenous nucleic acids encoding a succinyl-CoA
reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; a
3-hydroxybutyryl-CoA transferase or a 3-hydroxybutyryl-CoA
synthetase or a 3-hydroxybutyryl-CoA hydrolase; a 3-hydroxybutyrate
reductase; and a 3 hydroxybutyraldehyde reductase, and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0110] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway
including a first set of exogenous nucleic acids encoding 13-BDO
pathway enzymes expressed in a sufficient amount to produce 13-BDO,
the first set of exogenous nucleic acids encoding a succinyl-CoA
reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate
kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA
dehydratase; a crotonase; a 3-hydroxybutyryl-CoA reductase
(aldehyde forming); and a 3-hydroxybutyraldehyde reductase, and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0111] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway
including a first set of exogenous nucleic acids encoding 13-BDO
pathway enzymes expressed in a sufficient amount to produce 13-BDO,
the first set of exogenous nucleic acids encoding a succinyl-CoA
reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate
kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA
dehydratase; a crotonase; a 3-hydroxybutyryl-CoA transferase or a
3-hydroxybutyryl-CoA synthetase or a 3-hydroxybutyryl-CoA
hydrolase; a 3-hydroxybutyrate reductase; and a
3-hydroxybutyraldehyde reductase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase; an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0112] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway
including a first set of exogenous nucleic acids encoding 13-BDO
pathway enzymes expressed in a sufficient amount to produce 13-BDO,
the first set of exogenous nucleic acids encoding a succinyl-CoA
reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate
kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA
dehydratase; a crotonase; and a 3-hydroxybutyryl-CoA reductase
(alcohol-forming), and the isopropanol pathway comprising a second
set of exogenous nucleic acids encoding isopropanol pathway enzymes
expressed in a sufficient amount to produce isopropanol, the second
set of exogenous nucleic acids encoding an acetyl-CoA acetyl
thiolase; an acetoacetyl-CoA transferase or an acetoacetyl-CoA
hydrolase or an acetoacetyl-CoA synthetase, an acetoacetate
decarboxylase; and an isopropanol dehydrogenase.
[0113] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway
including a first set of exogenous nucleic acids encoding 13-BDO
pathway enzymes expressed in a sufficient amount to produce 13-BDO,
the first set of exogenous nucleic acids encoding a succinyl-CoA
reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; and a
3-hydroxybutyryl-CoA reductase (alcohol-forming), and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0114] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway
including a first set of exogenous nucleic acids encoding 13-BDO
pathway enzymes expressed in a sufficient amount to produce 13-BDO,
the first set of exogenous nucleic acids encoding a succinate
reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; a
3-hydroxybutyryl-CoA reductase (aldehyde forming); and a
3-hydroxybutyraldehyde reductase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase; an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0115] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway
including a first set of exogenous nucleic acids encoding 13-BDO
pathway enzymes expressed in a sufficient amount to produce 13-BDO,
the first set of exogenous nucleic acids encoding a succinate
reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; a
3-hydroxybutyryl-CoA transferase or a 3-hydroxybutyryl-CoA
synthetase or a 3-hydroxybutyryl-CoA hydrolase; a 3-hydroxybutyrate
reductase; and a 3 hydroxybutyraldehyde reductase, and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0116] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway
including a first set of exogenous nucleic acids encoding 13-BDO
pathway enzymes expressed in a sufficient amount to produce 13-BDO,
the first set of exogenous nucleic acids encoding a succinate
reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate
kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA
dehydratase; a crotonase; a 3-hydroxybutyryl-CoA reductase
(aldehyde forming); and a 3-hydroxybutyraldehyde reductase, and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0117] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway
including a first set of exogenous nucleic acids encoding 13-BDO
pathway enzymes expressed in a sufficient amount to produce 13-BDO,
the first set of exogenous nucleic acids encoding a succinate
reductase; a 4-hydroxybutyrate dehydrogenase; a 4 hydroxybutyrate
kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA
dehydratase; a crotonase; a 3-hydroxybutyryl-CoA transferase or a
3-hydroxybutyryl-CoA synthetase or a 3-hydroxybutyryl-CoA
hydrolase; a 3-hydroxybutyrate reductase; and a 3
hydroxybutyraldehyde reductase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase; an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0118] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway
including a first set of exogenous nucleic acids encoding 13-BDO
pathway enzymes expressed in a sufficient amount to produce 13-BDO,
the first set of exogenous nucleic acids encoding a succinate
reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate
kinase; a phosphotrans-4-hydroxybutyrylase; a crotonase; and a
3-hydroxybutyryl-CoA reductase (alcohol-forming), and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0119] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway
including a first set of exogenous nucleic acids encoding 13-BDO
pathway enzymes expressed in a sufficient amount to produce 13-BDO,
the first set of exogenous nucleic acids encoding a succinate
reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; and a
3-hydroxybutyryl-CoA reductase (alcohol-forming), and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0120] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an MAA pathway and an isopropanol pathway, the MAA pathway
including a first set of exogenous nucleic acids encoding MAA
pathway enzymes expressed in a sufficient amount to produce MAA,
the first set of exogenous nucleic acids encoding a succinyl-CoA
reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA mutase; a
3-hydroxyisobutyryl-CoA transferase, a 3-hydroxyisobutyryl-CoA
synthetase or a 3-hydroxyisobutyryl-CoA hydrolase; and a
3-hydroxyisobutyrate dehydratase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase; an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0121] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an MAA pathway and an isopropanol pathway, the MAA pathway
including a first set of exogenous nucleic acids encoding MAA
pathway enzymes expressed in a sufficient amount to produce MAA,
the first set of exogenous nucleic acids encoding a succinyl-CoA
reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA mutase; a
3-hydroxyisobutyryl-CoA dehydratase; and a methacrylyl-CoA
transferase, a methacrylyl-CoA synthetase or a methacrylyl-CoA
hydrolase, and the isopropanol pathway comprising a second set of
exogenous nucleic acids encoding isopropanol pathway enzymes
expressed in a sufficient amount to produce isopropanol, the second
set of exogenous nucleic acids encoding an acetyl-CoA acetyl
thiolase; an acetoacetyl-CoA transferase or an acetoacetyl-CoA
hydrolase or an acetoacetyl-CoA synthetase; an acetoacetate
decarboxylase; and an isopropanol dehydrogenase.
[0122] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an MAA pathway and an isopropanol pathway, the MAA pathway
including a first set of exogenous nucleic acids encoding MAA
pathway enzymes expressed in a sufficient amount to produce MAA,
the first set of exogenous nucleic acids encoding a succinyl-CoA
reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate
kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA
mutase; a 3-hydroxyisobutyryl-CoA transferase, a
3-hydroxyisobutyryl-CoA synthetase or a 3-hydroxyisobutyryl-CoA
hydrolase; and a 3-hydroxyisobutyrate dehydratase, and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0123] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an MAA pathway and an isopropanol pathway, the MAA pathway
including a first set of exogenous nucleic acids encoding MAA
pathway enzymes expressed in a sufficient amount to produce MAA,
the first set of exogenous nucleic acids encoding a succinyl-CoA
reductase; a 4-hydroxybutyrate dehydrogenase; a 4 hydroxybutyrate
kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA
mutase; a 3-hydroxyisobutyryl-CoA dehydratase; and a
methacrylyl-CoA transferase, a methacrylyl-CoA synthetase or a
methacrylyl-CoA hydrolase, and the isopropanol pathway comprising a
second set of exogenous nucleic acids encoding isopropanol pathway
enzymes expressed in a sufficient amount to produce isopropanol,
the second set of exogenous nucleic acids encoding an acetyl-CoA
acetyl thiolase; an acetoacetyl-CoA transferase or an
acetoacetyl-CoA hydrolase or an acetoacetyl-CoA synthetase; an
acetoacetate decarboxylase; and an isopropanol dehydrogenase.
[0124] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an MAA pathway and an isopropanol pathway, the MAA pathway
including a first set of exogenous nucleic acids encoding MAA
pathway enzymes expressed in a sufficient amount to produce MAA,
the first set of exogenous nucleic acids encoding a succinate
reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA mutase; a
3-hydroxyisobutyryl-CoA transferase, a 3-hydroxyisobutyryl-CoA
synthetase or a 3-hydroxyisobutyryl-CoA hydrolase; and a
3-hydroxyisobutyrate dehydratase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase; an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0125] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an MAA pathway and an isopropanol pathway, the MAA pathway
including a first set of exogenous nucleic acids encoding MAA
pathway enzymes expressed in a sufficient amount to produce MAA,
the first set of exogenous nucleic acids encoding a succinate
reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA mutase; a
3-hydroxyisobutyryl-CoA dehydratase; and a methacrylyl-CoA
transferase, a methacrylyl-CoA synthetase or a methacrylyl-CoA
hydrolase, and the isopropanol pathway comprising a second set of
exogenous nucleic acids encoding isopropanol pathway enzymes
expressed in a sufficient amount to produce isopropanol, the second
set of exogenous nucleic acids encoding an acetyl-CoA acetyl
thiolase; an acetoacetyl-CoA transferase or an acetoacetyl-CoA
hydrolase or an acetoacetyl-CoA synthetase; an acetoacetate
decarboxylase; and an isopropanol dehydrogenase.
[0126] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an MAA pathway and an isopropanol pathway, the MAA pathway
including a first set of exogenous nucleic acids encoding MAA
pathway enzymes expressed in a sufficient amount to produce MAA,
the first set of exogenous nucleic acids encoding a succinate
reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate
kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA
mutase; a 3-hydroxyisobutyryl-CoA transferase, a
3-hydroxyisobutyryl-CoA synthetase or a 3-hydroxyisobutyryl-CoA
hydrolase; and a 3-hydroxyisobutyrate dehydratase, and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0127] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an MAA pathway and an isopropanol pathway, the MAA pathway
including a first set of exogenous nucleic acids encoding MAA
pathway enzymes expressed in a sufficient amount to produce MAA,
the first set of exogenous nucleic acids encoding a succinate
reductase; a 4-hydroxybutyrate dehydrogenase; a 4 hydroxybutyrate
kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA
mutase; a 3-hydroxyisobutyryl-CoA dehydratase; and a
methacrylyl-CoA transferase, a methacrylyl-CoA synthetase or a
methacrylyl-CoA hydrolase, and the isopropanol pathway comprising a
second set of exogenous nucleic acids encoding isopropanol pathway
enzymes expressed in a sufficient amount to produce isopropanol,
the second set of exogenous nucleic acids encoding an acetyl-CoA
acetyl thiolase; an acetoacetyl-CoA transferase or an
acetoacetyl-CoA hydrolase or an acetoacetyl-CoA synthetase; an
acetoacetate decarboxylase; and an isopropanol dehydrogenase.
[0128] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an MAA pathway and an isopropanol pathway, the MAA pathway
including a first set of exogenous nucleic acids encoding MAA
pathway enzymes expressed in a sufficient amount to produce MAA,
the first set of exogenous nucleic acids encoding a
methylmalonyl-CoA mutase; a methylmalonyl-CoA reductase (aldehyde
forming); a 3-hydroxyisobutyrate dehydrogenase; and a
3-hydroxyisobutyrate dehydratase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase; an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0129] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an MAA pathway and an isopropanol pathway, the MAA pathway
including a first set of exogenous nucleic acids encoding MAA
pathway enzymes expressed in a sufficient amount to produce MAA,
the first set of exogenous nucleic acids encoding a
methylmalonyl-CoA mutase; a methylmalonyl-CoA epimerase; a
methylmalonyl-CoA transferase, a methylmalonyl-CoA synthetase, or a
methylmalonyl-CoA hydrolase; a methylmalonate reductase; a
3-hydroxyisobutyrate dehydrogenase; and a 3-hydroxyisobutyrate
dehydratase, and the isopropanol pathway comprising a second set of
exogenous nucleic acids encoding isopropanol pathway enzymes
expressed in a sufficient amount to produce isopropanol, the second
set of exogenous nucleic acids encoding an acetyl-CoA acetyl
thiolase; an acetoacetyl-CoA transferase or an acetoacetyl-CoA
hydrolase or an acetoacetyl-CoA synthetase; an acetoacetate
decarboxylase; and an isopropanol dehydrogenase.
[0130] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an MAA pathway and an isopropanol pathway, the MAA pathway
including a first set of exogenous nucleic acids encoding MAA
pathway enzymes expressed in a sufficient amount to produce MAA,
the first set of exogenous nucleic acids encoding a
methylmalonyl-CoA mutase; a methylmalonyl-CoA transferase, a
methylmalonyl-CoA synthetase or a methylmalonyl-CoA hydrolase; a
methylmalonate reductase; a 3-hydroxyisobutyrate dehydrogenase; and
a 3-hydroxyisobutyrate dehydratase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase; an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0131] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an MAA pathway and an isopropanol pathway, the MAA pathway
including a first set of exogenous nucleic acids encoding MAA
pathway enzymes expressed in a sufficient amount to produce MAA,
the first set of exogenous nucleic acids encoding a
methylmalonyl-CoA mutase; a methylmalonyl-CoA epimerase; a
methylmalonyl-CoA reductase (alcohol forming); and a
3-hydroxyisobutyrate dehydratase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase, an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0132] In a further aspect of the above embodiments, the microbial
organism has an acetyl-CoA pathway having a third set of exogenous
nucleic acids encoding acetyl-CoA pathway enzymes expressed in a
sufficient amount to produce acetyl-CoA, the third set of exogenous
nucleic acids encoding a pyruvate kinase; and a pyruvate
dehydrogenase or a pyruvate ferredoxin oxidoreductase; or a
pyruvate formate lyase, a pyruvate formate lyase activating enzyme
and a formate dehydrogenase.
[0133] In another further embodiment, the microbial organism has a
succinyl-CoA pathway having a third set of exogenous nucleic acids
encoding succinyl-CoA pathway enzymes expressed in a sufficient
amount to produce succinyl-CoA, the third set of exogenous nucleic
acids encoding a PEP carboxykinase, a PEP carboxylase, a malate
dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA
transferase and a succinyl-CoA synthetase. In a further aspect, the
third set of exogenous nucleic acids further encodes a pyruvate
carboxylase or a methylmalonyl-CoA carboxytransferase.
[0134] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 14-BDO pathway and an isopropanol pathway, the 14-BDO pathway
including a first set of exogenous nucleic acids encoding 14-BDO
pathway enzymes expressed in a sufficient amount to produce 14-BDO,
the first set of exogenous nucleic acids encoding a PEP
carboxykinase, a PEP carboxylase, a malate dehydrogenase, a
fumarase, a fumarate reductase, a succinyl-CoA transferase, a
succinyl-CoA synthetase, a pyruvate carboxylase, a
methylmalonyl-CoA carboxytransferase, a succinyl-CoA reductase, a
succinate reductase, a 4-hydroxybutyrate dehydrogenase, a
4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA
synthetase, a 4-hydroxybutyryl-CoA reductase (aldehyde-forming), a
4-hydroxybutyraldehyde reductase, a 4-hydroxybutyrate reductase; a
4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, a
4-hydroxybutyryl-phosphate reductase, a 4-hydroxybutyryl-CoA
reductase (alcohol-forming), and a 4-hydroxybutyraldehyde
reductase, and the isopropanol pathway comprising a second set of
exogenous nucleic acids encoding isopropanol pathway enzymes
expressed in a sufficient amount to produce isopropanol, the second
set of exogenous nucleic acids encoding a pyruvate kinase, a
pyruvate dehydrogenase, a pyruvate ferredoxin oxidoreductase, a
pyruvate formate lyase, a pyruvate formate lyase activating enzyme,
a formate dehydrogenase, an acetyl-CoA acetyl thiolase, an
acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase, and an
isopropanol dehydrogenase.
[0135] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an 13-BDO pathway and an isopropanol pathway, the 13-BDO pathway
including a first set of exogenous nucleic acids encoding 13-BDO
pathway enzymes expressed in a sufficient amount to produce 13-BDO,
the first set of exogenous nucleic acids encoding a PEP
carboxykinase, a PEP carboxylase, a malate dehydrogenase, a
fumarase, a fumarate reductase, a succinyl-CoA transferase, a
succinyl-CoA synthetase, a pyruvate carboxylase, a
methylmalonyl-CoA carboxytransferase, a succinyl-CoA reductase, a
succinate reductase, a 4-hydroxybutyrate dehydrogenase, a
4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA
synthetase, a 4-hydroxybutyrate kinase, a
phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA
dehydratase, a crotonase, a 3-hydroxybutyryl-CoA reductase
(aldehyde forming), a 3-hydroxybutyraldehyde reductase, a
3-hydroxybutyryl-CoA transferase, a 3-hydroxybutyryl-CoA
synthetase, a 3-hydroxybutyryl-CoA hydrolase, a 3-hydroxybutyrate
reductase, and a 3-hydroxybutyryl-CoA reductase (alcohol-forming),
and the isopropanol pathway comprising a second set of exogenous
nucleic acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding a pyruvate kinase, a pyruvate
dehydrogenase, a pyruvate ferredoxin oxidoreductase, a pyruvate
formate lyase, a pyruvate formate lyase activating enzyme, a
formate dehydrogenase, an acetyl-CoA acetyl thiolase, an
acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase, and an
isopropanol dehydrogenase.
[0136] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an MAA pathway and an isopropanol pathway, the MAA pathway
including a first set of exogenous nucleic acids encoding MAA
pathway enzymes expressed in a sufficient amount to produce MAA,
the first set of exogenous nucleic acids encoding a PEP
carboxykinase, a PEP carboxylase, a malate dehydrogenase, a
fumarase, a fumarate reductase, a succinyl-CoA transferase, a
succinyl-CoA synthetase, a pyruvate carboxylase, a
methylmalonyl-CoA carboxytransferase, a succinyl-CoA reductase, a
succinate reductase, a 4-hydroxybutyrate dehydrogenase, a
4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA
synthetase, a 4-hydroxybutyrate kinase, a
phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA mutase, a
3-hydroxyisobutyryl-CoA transferase, a 3-hydroxyisobutyryl-CoA
synthetase, a 3-hydroxyisobutyryl-CoA hydrolase,
3-hydroxyisobutyryl-CoA dehydratase, methacrylyl-CoA transferase,
methacrylyl-CoA synthetase, methacrylyl-CoA hydrolase and a
3-hydroxyisobutyrate dehydratase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding a pyruvate kinase, a pyruvate dehydrogenase, a pyruvate
ferredoxin oxidoreductase, a pyruvate formate lyase, a pyruvate
formate lyase activating enzyme, a formate dehydrogenase, an
acetyl-CoA acetyl thiolase, an acetoacetyl-CoA transferase, an
acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, an
acetoacetate decarboxylase, and an isopropanol dehydrogenase.
[0137] In one embodiment, the invention provides a non-naturally
occurring microbial organism including a microbial organism having
an MAA pathway and an isopropanol pathway, the MAA pathway
including a first set of exogenous nucleic acids encoding MAA
pathway enzymes expressed in a sufficient amount to produce MAA,
the first set of exogenous nucleic acids encoding a PEP
carboxykinase, a PEP carboxylase, a malate dehydrogenase, a
fumarase, a fumarate reductase, a succinyl-CoA transferase, a
succinyl-CoA synthetase, a pyruvate carboxylase, a
methylmalonyl-CoA carboxytransferase, a methylmalonyl-CoA mutase, a
methylmalonyl-CoA epimerase, a methylmalonyl-CoA transferase, a
methylmalonyl-CoA synthetase, a methylmalonyl-CoA hydrolase, a
methylmalonate reductase, a methylmalonyl-CoA reductase (aldehyde
forming), a 3-hydroxyisobutyrate dehydrogenase, a methylmalonyl-CoA
reductase (alcohol forming) and a 3-hydroxyisobutyrate dehydratase,
and the isopropanol pathway comprising a second set of exogenous
nucleic acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding a pyruvate kinase, a pyruvate
dehydrogenase, a pyruvate ferredoxin oxidoreductase, a pyruvate
formate lyase, a pyruvate formate lyase activating enzyme, a
formate dehydrogenase, an acetyl-CoA acetyl thiolase, an
acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase, and an
isopropanol dehydrogenase.
[0138] In a further aspect of each of the above embodiments, the
exogenous nucleic acid is a heterologous nucleic acid.
[0139] In a further aspect of each of the above embodiments, the
non-naturally occurring microbial organism is in a substantially
anaerobic culture medium.
[0140] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having an n-propanol and
isopropanol 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 selected
from the group consisting of phosphoenolpyruvate to oxaloacetate,
oxaloacetate to malate, malate to fumarate, fumarate to succinate,
succinate to succinyl-CoA, succinyl-CoA to (R)-methylmalonyl-CoA,
(R)-methylmalonyl-CoA to (S)-methylmalonyl-CoA,
(S)-methylmalonyl-CoA to propionyl-CoA, propionyl-CoA to
propionaldehyde, propionaldehyde to n-propanol, propionyl-CoA to
propionyl phosphate, propionyl-CoA to propionate, propionate to
propionyl phosphate, propionate to propionaldehyde, propionyl
phosphate to propionaldehyde, phosphoenolpyruvate to pyruvate,
pyruvate to oxaloacetate, pyruvate to acetyl-CoA, pyruvate to
acetyl-CoA and formate, formate to CO.sub.2, 2 acetyl-CoA
substrates to 1 acetoacetyl-CoA product, acetoacetyl-CoA to
acetoacetate, acetoacetate to acetone, acetone to isopropanol. 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 an
n-propanol and isopropanol pathway, such as that shown in FIG.
1.
[0141] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having an n-propanol and
isopropanol 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 selected
from the group consisting of phosphoenolpyruvate to oxaloacetate,
oxaloacetate to threonine, threonine to 2-oxobutanoate,
2-oxobutanoate to propionyl-CoA, propionyl-CoA to propionaldehyde,
propionaldehyde to n-propanol, 2-oxobutanoate to propionaldehyde,
propionyl-CoA to propionyl phosphate, propionyl-CoA to propionate,
propionate to propionyl phosphate, propionate to propionaldehyde,
propionyl phosphate to propionaldehyde, phosphoenolpyruvate to
pyruvate, pyruvate to oxaloacetate, pyruvate to acetyl-CoA,
pyruvate to acetyl-CoA and formate, formate to CO.sub.2, 2
acetyl-CoA substrates to 1 acetoacetyl-CoA product, acetoacetyl-CoA
to acetoacetate, acetoacetate to acetone, acetone to isopropanol.
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
an n-propanol and isopropanol pathway, such as that shown in FIG.
2.
[0142] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having an n-propanol and
isopropanol 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 selected
from the group consisting of phosphoenolpyruvate to pyruvate,
pyruvate to acetyl-CoA, pyruvate to acetyl-CoA and formate, formate
to CO.sub.2, acetyl-CoA to malonyl-CoA, malonyl-CoA to malonate
semialdehyde, malonate semialdehyde to 3-hydroxypropionate,
3-hydroxypropionate to propionyl-CoA, propionyl-CoA to
propionaldehyde, propionaldehyde to n-propanol, propionyl-CoA to
propionyl phosphate, propionyl-CoA to propionate, propionate to
propionyl phosphate, propionate to propionaldehyde, propionyl
phosphate to propionaldehyde, 2 acetyl-CoA substrates to 1
acetoacetyl-CoA product, acetoacetyl-CoA to acetoacetate,
acetoacetate to acetone, acetone to isopropanol. 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 an n-propanol and isopropanol pathway,
such as that shown in FIG. 3.
[0143] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having an n-propanol and
isopropanol 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 selected
from the group consisting of pyruvate to D-lactate, D-lactate to
lactoyl-CoA, lactoyl-CoA to acryloyl-CoA, acryloyl-CoA to
propionyl-CoA, propionyl-CoA to propionaldehyde, propionaldehyde to
n-propanol, propionyl-CoA to propionyl phosphate, propionyl-CoA to
propionate, propionate to propionyl phosphate, propionate to
propionaldehyde, propionyl phosphate to propionaldehyde, pyruvate
to acetyl-CoA, pyruvate to acetyl-CoA and formate, formate to
CO.sub.2, 2 acetyl-CoA substrates to 1 acetoacetyl-CoA product,
acetoacetyl-CoA to acetoacetate, acetoacetate to acetone, acetone
to isopropanol. 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 an n-propanol and isopropanol pathway, such as that
shown in FIG. 4.
[0144] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having an n-propanol
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 selected from the
group consisting of propionyl-CoA to propionaldehyde,
propionaldehyde to n-propanol, propionyl-CoA to propionyl
phosphate, propionyl-CoA to propionate, propionate to propionyl
phosphate, propionate to propionaldehyde, and propionyl phosphate
to propionaldehyde. 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 an n-propanol pathway, such as that shown in FIGS.
1-4.
[0145] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having an 14-BDO and an
isopropanol 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 selected
from the group consisting of phosphoenolpyruvate to oxaloacetate,
oxaloacetate to malate, malate to fumarate, fumarate to succinate,
succinate to succinyl-CoA, succinyl-CoA to succinic semialdehyde,
succinic semialdehyde to 4-hydroxybutyrate, 4-hydroxybutyrate to
4-hydroxybutyryl-CoA, 4-hydroxybutyryl-CoA to
4-hydroxybutyraldehyde, 4-hydroxybutyraldehyde to 14-BDO, succinate
to succinic semialdehyde, 4-hydroxybutyrate to
4-hydroxybutyraldehyde, 4-hydroxybutyrate to
4-hydroxybutyryl-phosphate, 4-hydroxybutyryl-phosphate to
4-hydroxybutyryl-CoA, 4-hydroxybutyryl-phosphate to
4-hydroxybutyraldehyde, 4-hydroxybutyryl-CoA to 14-BDO,
propionyl-CoA to propionyl phosphate, propionyl phosphate to
propionaldehyde, phosphoenolpyruvate to pyruvate, pyruvate to
oxaloacetate, pyruvate to acetyl-CoA, pyruvate to acetyl-CoA and
formate, formate to CO.sub.2, 2 acetyl-CoA substrates to 1
acetoacetyl-CoA product, acetoacetyl-CoA to acetoacetate,
acetoacetate to acetone, acetone to isopropanol. 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 an n-propanol and isopropanol pathway,
such as that shown in FIG. 5.
[0146] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having an 13-BDO and an
isopropanol 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 selected
from the group consisting of phosphoenolpyruvate to oxaloacetate,
oxaloacetate to malate, malate to fumarate, fumarate to succinate,
succinate to succinyl-CoA, succinyl-CoA to succinic semialdehyde,
succinic semialdehyde to 4-hydroxybutyrate, 4-hydroxybutyrate to
4-hydroxybutyryl-CoA, succinate to succinic semialdehyde,
4-hydroxybutyrate to 4-hydroxybutyryl-phosphate,
4-hydroxybutyryl-phosphate to 4-hydroxybutyryl-CoA,
4-hydroxybutyryl-CoA to crotonyl-CoA, crotonyl-CoA to
3-hydroxybutyryl-CoA, 3-hydroxybutyryl-CoA to
3-hydroxybutyraldehyde, 3-hydroxybutyryl-CoA to 3-hydroxybutyrate,
3-hydroxybutyrate to.sub.--3-hydroxybutyraldehyde,
3-hydroxybutyraldehyde to 13-BDO, 3-hydroxybutyryl-CoA to 13-BDO,
propionyl-CoA to propionyl phosphate, propionyl phosphate to
propionaldehyde, phosphoenolpyruvate to pyruvate, pyruvate to
oxaloacetate, pyruvate to acetyl-CoA, pyruvate to acetyl-CoA and
formate, formate to CO.sub.2, 2 acetyl-CoA substrates to 1
acetoacetyl-CoA product, acetoacetyl-CoA to acetoacetate,
acetoacetate to acetone, acetone to isopropanol. 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 an n-propanol and isopropanol pathway,
such as that shown in FIG. 6.
[0147] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having an MAA and an
isopropanol 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 selected
from the group consisting of phosphoenolpyruvate to oxaloacetate,
oxaloacetate to malate, malate to fumarate, fumarate to succinate,
succinate to succinyl-CoA, succinyl-CoA to succinic semialdehyde,
succinic semialdehyde to 4-hydroxybutyrate, 4-hydroxybutyrate to
4-hydroxybutyryl-CoA, succinate to succinic semialdehyde,
4-hydroxybutyrate to 4-hydroxybutyryl-phosphate,
4-hydroxybutyryl-phosphate to 4-hydroxybutyryl-CoA,
4-hydroxybutyryl-CoA to 3-hydroxyisobutyryl-CoA,
3-hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate,
3-hydroxyisobutyryl-CoA to methyacrylyl-CoA, methyacrylyl-CoA to
MAA, 3-hydroxyisobutyrate to MAA, succinyl-CoA to
(R)-methylmalonyl-CoA, (R)-methylmalonyl-CoA to
(S)-methylmalonyl-CoA, (S)-methylmalonyl-CoA to methylmalonate
semialdehyde, (S)-methylmalonyl-CoA to 3-hydroxyisobutyrate,
methylmalonate semialdehyde to 3-hydroxyisobutyrate, propionyl-CoA
to propionyl phosphate, propionyl phosphate to propionaldehyde,
phosphoenolpyruvate to pyruvate, pyruvate to oxaloacetate, pyruvate
to acetyl-CoA, pyruvate to acetyl-CoA and formate, formate to
CO.sub.2, 2 acetyl-CoA substrates to 1 acetoacetyl-CoA product,
acetoacetyl-CoA to acetoacetate, acetoacetate to acetone, acetone
to isopropanol. 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 an n-propanol and isopropanol pathway, such as that
shown in FIGS. 7 and 8.
[0148] While generally described herein as a microbial organism
that contains an n-propanol and an isopropanol, a 14-BDO and an
isopropanol, a 13-BDO and an isopropanol or a MAA and an
isopropanol pathway, it is understood that the invention
additionally provides a non-naturally occurring microbial organism
comprising at least one exogenous nucleic acid encoding an
n-propanol, an isopropanol, a 14-BDO, a 13-BDO and/or MAA pathway
enzyme expressed in a sufficient amount to produce an intermediate
of an n-propanol, an isopropanol, a 14-BDO, a 13-BDO and/or MAA
pathway. For example, as disclosed herein, an n-propanol, an
isopropanol, a 14-BDO, a 13-BDO and/or MAA pathway is exemplified
in FIGS. 1-8. Therefore, in addition to a microbial organism
containing an n-propanol and an isopropanol, a 14-BDO and an
isopropanol, a 13-BDO and an isopropanol or a MAA and an
isopropanol pathway that produces n-propanol and isopropanol,
14-BDO and isopropanol, 13-BDO and isopropanol or MAA and
isopropanol, the invention additionally provides a non-naturally
occurring microbial organism comprising at least one exogenous
nucleic acid encoding an n-propanol, an isopropanol, a 14-BDO, a
13-BDO and/or MAA pathway enzyme, where the microbial organism
produces an n-propanol, an isopropanol, a 14-BDO, a 13-BDO and/or
MAA pathway intermediate, for example, acetone, methylmalonyl-CoA,
propionyl phosphate, 2-oxobutanoate, 3-hydroxypropionate,
lactoyl-CoA, 4-hydroxybutyrate, 4-hydroxybutyryl-phosphate,
crotonyl-CoA, succinyl-CoA, succinic semialdehyde or
3-hydroxyisobutyryl-CoA.
[0149] 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. 1-8, 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 an n-propanol, an isopropanol, a 14-BDO, a 13-BDO
and/or MAA intermediate can be utilized to produce the intermediate
as a desired product.
[0150] 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.
[0151] 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 n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA
biosynthetic pathways. Depending on the host microbial organism
chosen for biosynthesis, nucleic acids for some or all of a
particular n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA
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 n-propanol,
isopropanol, 14-BDO, 13-BDO and/or MAA 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 n-propanol,
isopropanol, 14-BDO, 13-BDO and/or MAA.
[0152] Depending on the n-propanol, isopropanol, 14-BDO, 13-BDO
and/or MAA 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
n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAApathway-encoding
nucleic acid and up to all encoding nucleic acids for one or more
n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA biosynthetic
pathways. For example, n-propanol, isopropanol, 14-BDO, 13-BDO
and/or MAA 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 an n-propanol, an isopropanol, a 14-BDO, a
13-BDO and/or a MAApathway, 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 n-propanol and isopropanol can be
included, such as a PEP carboxykinase or a PEP carboxylase; a
malate dehydrogenase; a fumarase; a fumarate reductase; a
succinyl-CoA transferase or a succinyl-CoA synthetase; a
methylmalonyl-CoA mutase; a methylmalonyl-CoA epimerase; a
methylmalonyl-CoA decarboxylase; and a propionaldehyde
dehydrogenase and a propanol dehydrogenase; or a
propionyl-CoA:phosphate propanoyltransferase and a propionyl
phosphate reductase, a pyruvate kinase; a pyruvate dehydrogenase or
a pyruvate ferredoxin oxidoreductase; or a pyruvate formate lyase,
a pyruvate formate lyase activating enzyme and a formate
dehydrogenase; an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase, an acetoacetate decarboxylase; and an isopropanol
dehydrogenase, as exemplified in FIG. 1.
[0153] 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 n-propanol, isopropanol, 14-BDO, 13-BDO and/or
MAApathway 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,
seventeen, eighteen, nineteen, twenty or twenty one, up to all
nucleic acids encoding the enzymes or proteins constituting an
n-propanol, an isopropanol, a 14-BDO, a 13-BDO and/or a MAA
biosynthetic pathway disclosed herein. In some embodiments, the
non-naturally occurring microbial organisms also can include other
genetic modifications that facilitate or optimize n-propanol,
isopropanol, 14-BDO, 13-BDO and/or MAA 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 n-propanol, isopropanol, 14-BDO,
13-BDO and/or MAA pathway precursors such as phosphoenolpyruvate or
pyruvate.
[0154] Generally, a host microbial organism is selected such that
it produces the precursor of an n-propanol, an isopropanol, a
14-BDO, a 13-BDO and/or a MAA 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, phosphoenolpyruvate 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 an n-propanol,
an isopropanol, a 14-BDO, a 13-BDO and/or a MAA pathway.
[0155] In some embodiments, a non-naturally occurring microbial
organism of the invention is generated from a host that contains
the enzymatic capability to synthesize n-propanol, isopropanol,
14-BDO, 13-BDO and/or MAA. In this specific embodiment it can be
useful to increase the synthesis or accumulation of an n-propanol,
an isopropanol, a 14-BDO, a 13-BDO and/or a MAA pathway product to,
for example, drive n-propanol, isopropanol, 14-BDO, 13-BDO and/or
MAA pathway reactions toward n-propanol, isopropanol, 14-BDO,
13-BDO and/or MAA production. Increased synthesis or accumulation
can be accomplished by, for example, overexpression of nucleic
acids encoding one or more of the above-described n-propanol and/or
isopropanol pathway enzymes or proteins. Over expression of the
enzyme or enzymes and/or protein or proteins of the n-propanol,
isopropanol, 14-BDO, 13-BDO and/or MAA 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 n-propanol, isopropanol, 14-BDO,
13-BDO and/or MAA, through overexpression of one, two, three, four,
five, six, seven, eight, nine, ten, eleven, twelve, thirteen,
fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty
or twenty one, that is, up to all nucleic acids encoding
n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA 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 n-propanol,
isopropanol, 14-BDO, 13-BDO and/or MAA biosynthetic pathway.
[0156] 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.
[0157] 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, an n-propanol and isopropanol, 14-BDO
and isopropanol, 13-BDO and isopropanol or MAA and isopropanol
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 n-propanol, isopropanol,
14-BDO, 13-BDO and/or MAA biosynthetic capability. For example, a
non-naturally occurring microbial organism having an n-propanol and
an isopropanol, a 14-BDO and an isopropanol, a 13-BDO and an
isopropanol or a MAA and an isopropanol biosynthetic pathway can
comprise at least two exogenous nucleic acids encoding desired
enzymes or proteins, such as the combination of propionaldehyde
dehydrogenase and isopropanol dehydrogenase, or alternatively
propionyl-CoA synthase and acetyl-CoA acetyl thiolase, or
alternatively lactate dehydrogenase and acetyl-CoA thiolase, or
alternatively a succinyl-CoA reductase and 4-hydroxybutyryl-CoA
reductase (alcohol-forming), or alternatively crotonase and
acetoacetate decarboxylase, or alternatively 4-hydroxybutyrate
kinase and phosphotrans-4-hydroxybutyrylase or alternatively
methylmalonyl-CoA reductase (alcohol forming) and pyruvate kinase
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, PEP carboxykinase, acetyl-CoA acetyl thiolase and propanol
dehydrogenase, or alternatively pyruvate kinase, acetoacetate
decarboxylase and 2-oxobutanoate dehydrogenase, or alternatively
propionyl-CoA:phosphate propanoyltransferase, propionyl phosphate
reductase and isopropanol dehydrogenase, or alternatively
lactate-CoA transferase and lactyl-CoA dehydratase and pyruvate
formate lyase, or alternatively succinyl-CoA dehydrogenase,
4-hydroxybutyrate reductase and 4-hydroxybutyraldehyde reductase,
or alternatively crotonase, PEP carboxylase and acetoacetate
decarboxylase, or alternatively 3-hydroxyisobutyryl-CoA synthetase,
fumarase and isopropanol dehydrogenase, or alternatively acetyl-CoA
acetyl thiolase, acetoacetate decarboxylase and methylmalonyl-CoA
reductase (alcohol forming) 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 or more enzymes
or proteins of a biosynthetic pathway as disclosed herein, for
example, pyruvate carboxylase, malate dehydrogenase,
methylmalonyl-CoA epimerase and acetoacetyl-CoA hydrolase, or
alternatively acetyl-CoA acetyl thiolase, isopropanol
dehydrogenase, propionaldehyde dehydrogenase and propanol
dehydrogenase, or alternatively acetyl-CoA carboxylase, malonyl-CoA
reductase, malonate semialdehyde and acetoacetate decarboxylase, or
alternatively, acryloyl CoA reductase, acetoacetyl-CoA transferase,
acetoacetate decarboxylase, and isopropanol dehydrogenase, or
alternatively succinyl-CoA dehydrogenase, 4-hydroxybutyrate
dehydrogenase, 4-hydroxybutyryl-CoA transferase, and isopropanol
dehydrogenase, or alternatively succinate reductase,
3-hydroxyisobutyryl-CoA synthetase, 3-hydroxyisobutyrate
dehydratase and pyruvate ferredoxin oxidoreductase, or
alternatively acetyl-CoA acetyl thiolase, acetoacetyl-CoA
transferase, methylmalonyl-CoA mutase and hydroxyisobutyrate
dehydratase, 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.
[0158] 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 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.
[0159] In addition to the biosynthesis of n-propanol, isopropanol,
14-BDO, 13-BDO and/or MAA 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 n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA other
than use of the n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA
producers is through addition of another microbial organism capable
of converting an n-propanol, an isopropanol, a 14-BDO, a 13-BDO
and/or a MAA pathway intermediate to n-propanol, isopropanol,
14-BDO, 13-BDO and/or MAA. One such procedure includes, for
example, the fermentation of a microbial organism that produces an
n-propanol, an isopropanol, a 14-BDO, a 13-BDO and/or a MAA pathway
intermediate. The n-propanol and isopropanol, 14-BDO and
isopropanol, 13-BDO and isopropanol or MAA and isopropanol pathway
intermediate can then be used as a substrate for a second microbial
organism that converts the n-propanol, isopropanol, 14-BDO, 13-BDO
and/or MAA pathway intermediate to n-propanol, isopropanol, 14-BDO,
13-BDO and/or MAA. The n-propanol, isopropanol, 14-BDO, 13-BDO
and/or MAA pathway intermediate can be added directly to another
culture of the second organism or the original culture of the
n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA 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.
[0160] 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,
n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and
isopropanol or MAA and isopropanol. 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 n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA
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, n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA
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
propionyl-CoA, succinyl-CoA and/or an acetyl-CoA intermediate and
the second microbial organism converts the intermediate(s) to
n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA.
[0161] 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 n-propanol, isopropanol, 14-BDO,
13-BDO and/or MAA.
[0162] Sources of encoding nucleic acids for an n-propanol, an
isopropanol, a 14-BDO, a 13-BDO and/or a MAA 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, Acetobacter pasteurians,
Acidanus brierleyi, Acinetobacter baylyi Acinetobacter
calcoaceticus, Acinetobacter sp. Strain M-1, Actinobacillus
succinogenes, Anaerobiospirillum succiniciproducens, Anaerostipes
caccae DSM 14662, Arabidopsis thaliana, Bacillus cereus ATCC 14579,
Bacillus subtilis, Bacillus subtilis subsp. subtilis str. 168, Bos
taurus, Bradyrhizobium japonicum USDA110, Caenorhabditis elegans,
Campylobacter jejuni, Chlamydomonas reinhardtii, Chloroflexus
aurantiacus, Clostridium acetobutylicum, Clostridium acetobutylicum
ATCC 824, Clostridium beijerinckii, Clostridium botulinum C str.
Eklund, Clostridium kluyveri, Clostridium kluyveri DSM 555,
Clostridium novyi-NT, Clostridium propionicum, Clostridium
saccharobutylicum, Clostridium saccharoperbutylacetonicum,
Corynebacterium glutamicum, Desulfovibrio africanus, Erythrobacter
sp. NAP1, Escherichia coli K12, Escherichia coli K12 str. MG1655,
Escherichia coli O157:H7, Geobacillus thermoglucosidasius M10EXG,
Haemophilus influenza, Helicobacter pylori, Homo sapiens,
Klebsiella pneumonia MGH78578, Kluyveromyces lactis, Lactobacillus
casei, Lactobacillus plantarum WCFS1, Lactococcus lactis,
Leuconostoc mesenteroides, Mannheimia succiniciproducens, marine
gamma proteobacterium HTCC2080, Mesorhizobium loti, Metallosphaera
sedula, Methylobacterium extorquens, Moorella thermoacetica,
Mycobacterium smegmatis, Mycobacterium tuberculosis, Oryctolagus
cuniculus, Plasmodium ovale, Porphyromonas gingivalis,
Propionibacterium acnes, Propionibacterium fredenreichii sp.
shermanii, Propionibacterium freudenreichii, Propionigenium
modestum, Pseudomonas aeruginosa, Pseudomonas aeruginosa PAO1,
Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas putida
E23, Pseudomonas putida KT2440, Pseudomonas sp, Pseudomonas
stutzeri, Ralstonia eutropha, Ralstonia eutropha H16, Rattus
norvegicus, Rhodobacter spaeroides, Rhodoferax ferrireducens DSM
15236, Rhodospirillum rubrum, Roseiflexus castenholzii,
Saccharomyces cerevisiae, Salmonella enterica, Salmonella
typhimurium, Shigella flexneri, Simmondsia chinensis, Streptococcus
mutans, Sulfolobus acidocaldarius, Sulfolobus solfataricus,
Sulfolobus tokodaii, Syntrophobacter fumaroxidans, Thermococcus
litoralis, Thermotoga maritime, Thermus thermophilus, Trichomonas
vaginalis G3, Trypanosoma brucei, Veillonella parvula, Yersinia
frederiksenii, Zymomonas mobilis, Bacillus megaterium,
butyrate-producing bacterium L2-50, Clostridium aminobutyricum,
Geobacillus thermoglucosidasius, Mycobacterium bovis BCG, Nocardia
farcinica IFM 10152, Nocardia iowensis (sp. NRRL 5646), Penicillium
chrysogenum, Porphyromonas gingivalis ATCC 33277, Pseudomonas
mendocina, Streptomyces griseus subsp. griseus NBRC 13350 as well
as other exemplary species disclosed herein are available as source
organisms for corresponding genes. However, with the complete
genome sequence available for now more than 550 species (with more
than half of these available on public databases such as the NCBI),
including 395 microorganism genomes and a variety of yeast, fungi,
plant, and mammalian genomes, the identification of genes encoding
the requisite n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA
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 n-propanol, isopropanol, 14-BDO, 13-BDO
and/or MAA 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.
[0163] In some instances, such as when an alternative n-propanol,
isopropanol, 14-BDO, 13-BDO and/or MAA biosynthetic pathway exists
in an unrelated species, n-propanol, isopropanol, 14-BDO, 13-BDO
and/or MAA 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 n-propanol, isopropanol, 14-BDO, 13-BDO and/or
MAA.
[0164] Host microbial organisms can be selected from, and the
non-naturally occurring microbial organisms generated in, for
example, bacteria, yeast, fungus or any of a variety of other
microorganisms applicable to fermentation processes. Exemplary
bacteria include species selected from Escherichia coli, Klebsiella
oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus
succinogenes, Mannheimia succiniciproducens, Rhizobium etli,
Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter
oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus
plantarum, Streptomyces coelicolor, Clostridium acetobutylicum,
Pseudomonas fluorescens, and Pseudomonas putida. Exemplary yeasts
or fungi include species selected from Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces
marxianus, Aspergillus terreus, Aspergillus niger and Pichia
pastoris. E. coli is a particularly useful 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. Other particularly useful host
organisms include microbial organisms which naturally produce
sufficient quantities of propionyl-CoA and/or acetyl-CoA for
co-production of n-propanol and isopropanol. Examples of such
organisms include, but are not limited to, Clostrium propionicum,
Escherichia coli and Propionibacterium freudenreichii subsp.
shermanii.
[0165] Methods for constructing and testing the expression levels
of a non-naturally occurring n-propanol-, isopropanol-, 14-BDO-,
13-BDO- and/or MAA-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).
[0166] Exogenous nucleic acid sequences involved in a pathway for
production of n-propanol and isopropanol, 14-BDO and isopropanol,
13-BDO and isopropanol or MAA and isopropanol 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.
[0167] An expression vector or vectors can be constructed to
include one or more n-propanol, isopropanol, 14-BDO, 13-BDO and/or
MAA biosynthetic pathway encoding nucleic acids as exemplified
herein operably linked to expression control sequences functional
in the host organism. Expression vectors applicable for use in the
microbial host organisms of the invention include, for example,
plasmids, phage vectors, viral vectors, episomes and artificial
chromosomes, including vectors and selection sequences or markers
operable for stable integration into a host chromosome.
Additionally, the expression vectors can include one or more
selectable marker genes and appropriate expression control
sequences. Selectable marker genes also can be included that, for
example, provide resistance to antibiotics or toxins, complement
auxotrophic deficiencies, or supply critical nutrients not in the
culture media. Expression control sequences can include
constitutive and inducible promoters, transcription enhancers,
transcription terminators, and the like which are well known in the
art. When two or more exogenous encoding nucleic acids are to be
co-expressed, both nucleic acids can be inserted, for example, into
a single expression vector or in separate expression vectors. For
single vector expression, the encoding nucleic acids can be
operationally linked to one common expression control sequence or
linked to different expression control sequences, such as one
inducible promoter and one constitutive promoter. The
transformation of exogenous nucleic acid sequences involved in a
metabolic or synthetic pathway can be confirmed using methods well
known in the art. Such methods include, for example, nucleic acid
analysis such as Northern blots or polymerase chain reaction (PCR)
amplification of mRNA, or immunoblotting for expression of gene
products, or other suitable analytical methods to test the
expression of an introduced nucleic acid sequence or its
corresponding gene product. It is understood by those skilled in
the art that the exogenous nucleic acid is expressed in a
sufficient amount to produce the desired product, and it is further
understood that expression levels can be optimized to obtain
sufficient expression using methods well known in the art and as
disclosed herein.
[0168] 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 (e.g.,
>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.
[0169] 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.
[0170] 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)--broadens substrate binding to include
non-natural substrates; inhibition (K.sub.i)--to remove inhibition
by products, substrates, or key intermediates; activity
(kcat)--increases enzymatic reaction rates to achieve desired flux;
expression levels--increases 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.
[0171] The following exemplary methods have been developed for the
mutagenesis and diversification of genes to target desired
properties of specific enzymes. Any of these can be used to
alter/optimize activity of a decarboxylase enzyme.
[0172] EpPCR (Pritchard et al., J Theor. Biol 234:497-509 (2005))
introduces random point mutations by reducing the fidelity of DNA
polymerase in PCR reactions by the addition of Mn.sup.2+ ions, by
biasing dNTP concentrations, or by other conditional variations.
The five step cloning process to confine the mutagenesis to the
target gene of interest involves: 1) error-prone PCR amplification
of the gene of interest; 2) restriction enzyme digestion; 3) gel
purification of the desired DNA fragment; 4) ligation into a
vector; 5) transformation of the gene variants into a suitable host
and screening of the library for improved performance. This method
can generate multiple mutations in a single gene simultaneously,
which can be useful. A high number of mutants can be generated by
EpPCR, so a high-throughput screening assay or a selection method
(especially using robotics) is useful to identify those with
desirable characteristics.
[0173] Error-prone Rolling Circle Amplification (epRCA) (Fujii et
al., Nucleic Acids Res 32:e145 (2004); and Fujii et al., Nat.
Protoc. 1:2493-2497 (2006)) has many of the same elements as 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. Adjusting the Mn.sup.2+ concentration can vary
the mutation rate somewhat. This technique uses a simple
error-prone, single-step method to create a full copy of the
plasmid with 3-4 mutations/kbp. No restriction enzyme digestion or
specific primers are required. Additionally, this method is
typically available as a kit.
[0174] DNA or Family Shuffling (Stemmer, Proc Natl Acad Sci U.S.A.
91:10747-10751 (1994); and Stemmer, Nature 370:389-391 (1994))
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. Fragments prime each other and recombination occurs when one
copy primes another copy (template switch). This method can be used
with >1 kbp DNA sequences. In addition to mutational
recombinants created by fragment reassembly, this method introduces
point mutations in the extension steps at a rate similar to
error-prone PCR. The method can be used to remove deleterious,
random and neutral mutations that might confer antigenicity.
[0175] Staggered Extension (StEP) (Zhao et al., Nat. Biotechnol
16:258-261 (1998)) 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). Growing fragments anneal
to different templates and extend further, which is repeated until
full-length sequences are made. Template switching means most
resulting fragments have multiple parents. Combinations of
low-fidelity polymerases (Taq and Mutazyme) reduce error-prone
biases because of opposite mutational spectra.
[0176] In Random Priming Recombination (RPR) 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)) Base misincorporation and mispriming via
epPCR give point mutations. Short DNA fragments prime one another
based on homology and are recombined and reassembled into
full-length by repeated thermocycling. Removal of templates prior
to this step assures low parental recombinants. This method, like
most others, can be performed over multiple iterations to evolve
distinct properties. This technology avoids sequence bias, is
independent of gene length, and requires very little parent DNA for
the application.
[0177] In Heteroduplex Recombination 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)) The mismatch repair step is at
least somewhat mutagenic. Heteroduplexes transform more efficiently
than linear homoduplexes. This method is suitable for large genes
and whole operons.
[0178] Random Chimeragenesis on Transient Templates (RACHITT) (Coco
et al., Nat. Biotechnol 19:354-359 (2001)) employs Dnase I
fragmentation and size fractionation of ssDNA. Homologous fragments
are hybridized in the absence of polymerase to a complementary
ssDNA scaffold. Any overlapping unhybridized fragment ends are
trimmed down by an exonuclease. Gaps between fragments are filled
in, and then ligated to give a pool of full-length diverse strands
hybridized to the scaffold (that contains U to preclude
amplification). The scaffold then is destroyed and is replaced by a
new strand complementary to the diverse strand by PCR
amplification. The method involves one strand (scaffold) that is
from only one parent while the priming fragments derive from other
genes; the parent scaffold is selected against. Thus, no
reannealing with parental fragments occurs. Overlapping fragments
are trimmed with an exonuclease. Otherwise, this is conceptually
similar to DNA shuffling and StEP. Therefore, there should be no
siblings, few inactives, and no unshuffled parentals. This
technique has advantages in that few or no parental genes are
created and many more crossovers can result relative to standard
DNA shuffling.
[0179] Recombined Extension on Truncated templates (RETT) 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)) No
DNA endonucleases are used. Unidirectional ssDNA is made by DNA
polymerase with random primers or serial deletion with exonuclease.
Unidirectional ssDNA are only templates and not primers. Random
priming and exonucleases don't introduce sequence bias as true of
enzymatic cleavage of DNA shuffling/RACHITT. RETT can be easier to
optimize than StEP because it uses normal PCR conditions instead of
very short extensions. Recombination occurs as a component of the
PCR steps--no direct shuffling. This method can also be more random
than StEP due to the absence of pauses.
[0180] In Degenerate Oligonucleotide Gene Shuffling (DOGS)
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)) this can be used to control the
tendency of other methods such as DNA shuffling to regenerate
parental genes. This method can be combined with random mutagenesis
(epPCR) of selected gene segments. This can be a good method to
block the reformation of parental sequences. No endonucleases are
needed. By adjusting input concentrations of segments made, one can
bias towards a desired backbone. This method allows DNA shuffling
from unrelated parents without restriction enzyme digests and
allows a choice of random mutagenesis methods.
[0181] Incremental Truncation for the Creation of Hybrid Enzymes
(ITCHY) creates a combinatorial library with 1 base pair deletions
of a gene or gene fragment of interest. (Ostermeier et al., Proc
Natl Acad Sci U.S.A. 96:3562-3567 (1999); and Ostermeier et al.,
Nat. Biotechnol 17:1205-1209 (1999)) Truncations are introduced in
opposite direction on pieces of 2 different genes. These are
ligated together and the fusions are cloned. This technique does
not require homology between the 2 parental genes. When ITCHY is
combined with DNA shuffling, the system is called SCRATCHY (see
below). A major advantage of both is no need for homology between
parental genes; for example, functional fusions between an E. coli
and a human gene were created via ITCHY. When ITCHY libraries are
made, all possible crossovers are captured.
[0182] Thio-Incremental Truncation for the Creation of Hybrid
Enzymes (THIO-ITCHY) is similar to ITCHY except that phosphothioate
dNTPs are used to generate truncations. (Lutz et al., Nucleic Acids
Res 29:E16 (2001)) Relative to ITCHY, THIO-ITCHY can be easier to
optimize, provide more reproducibility, and adjustability.
[0183] SCRATCHY combines two methods for recombining genes, ITCHY
and DNA shuffling. (Lutz et al., Proc Natl Acad Sci U.S.A.
98:11248-11253 (2001)) SCRATCHY combines the best features of ITCHY
and DNA shuffling. First, ITCHY is used to create a comprehensive
set of fusions between fragments of genes in a DNA
homology-independent fashion. This artificial family is then
subjected to a DNA-shuffling step to augment the number of
crossovers. Computational predictions can be used in optimization.
SCRATCHY is more effective than DNA shuffling when sequence
identity is below 80%.
[0184] In Random Drift Mutagenesis (RNDM) mutations made via epPCR
followed by screening/selection for those retaining usable
activity. (Bergquist et al., Biomol. Eng 22:63-72 (2005)) Then,
these are used in DOGS to generate recombinants with fusions
between multiple active mutants or between active mutants and some
other desirable parent. Designed to promote isolation of neutral
mutations; its purpose is to screen for retained catalytic activity
whether or not this activity is higher or lower than in the
original gene. RNDM is usable in high throughput assays when
screening is capable of detecting activity above background. RNDM
has been used as a front end to DOGS in generating diversity. The
technique imposes a requirement for activity prior to shuffling or
other subsequent steps; neutral drift libraries are indicated to
result in higher/quicker improvements in activity from smaller
libraries. Though published using epPCR, this could be applied to
other large-scale mutagenesis methods.
[0185] Sequence Saturation Mutagenesis (SeSaM) is a random
mutagenesis method that: 1) generates pool of random length
fragments using random incorporation of a phosphothioate nucleotide
and cleavage; this pool is used as a template to 2) extend in the
presence of "universal" bases such as inosine; 3) replication of a
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)) Using this technique it can
be possible to generate a large library of mutants within 2-3 days
using simple methods. This technique is non-directed in comparison
to the mutational bias of DNA polymerases. Differences in this
approach make this technique complementary (or an alternative) to
epPCR.
[0186] In Synthetic Shuffling, overlapping oligonucleotides are
designed to encode "all genetic diversity in targets" and allow a
very high diversity for the shuffled progeny. (Ness et al., Nat.
Biotechnol 20:1251-1255 (2002)) In this technique, one can design
the fragments to be shuffled. This aids in increasing the resulting
diversity of the progeny. One can design sequence/codon biases to
make more distantly related sequences recombine at rates
approaching those observed with more closely related sequences.
Additionally, the technique does not require physically possessing
the template genes.
[0187] Nucleotide Exchange and Excision Technology NexT 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))
The gene is reassembled using internal PCR primer extension with
proofreading polymerase. The sizes for shuffling are directly
controllable using varying dUPT::dTTP ratios. This is an end point
reaction using simple methods for uracil incorporation and
cleavage. Other nucleotide analogs, such as 8-oxo-guanine, can be
used with this method. Additionally, the technique works well with
very short fragments (86 bp) and has a low error rate. The chemical
cleavage of DNA used in this technique results in very few
unshuffled clones.
[0188] In Sequence Homology-Independent Protein Recombination
(SHIPREC) a linker is used to facilitate fusion between two
distantly/unrelated genes. Nuclease treatment is used to generate a
range of chimeras between the two genes. These fusions result in
libraries of single-crossover hybrids. (Sieber et al., Nat.
Biotechnol 19:456-460 (2001)) This produces a limited type of
shuffling and a separate process is required for mutagenesis. In
addition, since no homology is needed this technique can create a
library of chimeras with varying fractions of each of the two
unrelated parent genes. SHIPREC was tested with a heme-binding
domain of a bacterial CP450 fused to N-terminal regions of a
mammalian CP450; this produced mammalian activity in a more soluble
enzyme.
[0189] In Gene Site Saturation Mutagenesis.TM. (GSSM.TM.) the
starting materials are a supercoiled 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)) Primers
carrying the mutation of interest, anneal to the same sequence on
opposite strands of DNA. The mutation is typically in the middle of
the primer and flanked on each side by .about.20 nucleotides of
correct sequence. The sequence in the primer is NNN or NNK (coding)
and MNN (noncoding) (N=all 4, K=G, T, M=A, C). After extension,
DpnI is used to digest dam-methylated DNA to eliminate the
wild-type template. This technique explores all possible amino acid
substitutions at a given locus (i.e., one codon). The technique
facilitates the generation of all possible replacements at a
single-site with no nonsense codons and results in equal to
near-equal representation of most possible alleles. This technique
does not require prior knowledge of the structure, mechanism, or
domains of the target enzyme. If followed by shuffling or Gene
Reassembly, this technology creates a diverse library of
recombinants containing all possible combinations of single-site
up-mutations. The utility of this technology combination has been
demonstrated for the successful evolution of over 50 different
enzymes, and also for more than one property in a given enzyme.
[0190] Combinatorial Cassette Mutagenesis (CCM) 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)) Simultaneous
substitutions at two or three sites are possible using this
technique. Additionally, the method tests a large multiplicity of
possible sequence changes at a limited range of sites. This
technique has been used to explore the information content of the
lambda repressor DNA-binding domain.
[0191] Combinatorial Multiple Cassette Mutagenesis (CMCM) is
essentially similar to CCM except it is employed as part of a
larger program: 1) Use of epPCR at high mutation rate to 2) ID hot
spots and hot regions and then 3) extension by CMCM to cover a
defined region of protein sequence space. (Reetz, M. T., S.
Wilensek, D. Zha, and K. E. Jaeger, 2001, Directed Evolution of an
Enantioselective Enzyme through Combinatorial Multiple-Cassette
Mutagenesis. Angew. Chem. Int. Ed Engl. 40:3589-3591.) As with CCM,
this method can test virtually all possible alterations over a
target region. If used along with methods to create random
mutations and shuffled genes, it provides an excellent means of
generating diverse, shuffled proteins. This approach was successful
in increasing, by 51-fold, the enantioselectivity of an enzyme.
[0192] In the Mutator Strains technique conditional is mutator
plasmids 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)) This technology
is based on a plasmid-derived mutD5 gene, which encodes a mutant
subunit of DNA polymerase III. This subunit binds to endogenous DNA
polymerase III and compromises the proofreading ability of
polymerase III in any strain that harbors the plasmid. A
broad-spectrum of base substitutions and frameshift mutations
occur. In order for effective use, the mutator plasmid should be
removed once the desired phenotype is achieved; this is
accomplished through a temperature sensitive origin of replication,
which allows for plasmid curing at 41.degree. C. It should be noted
that mutator strains have been explored for quite some time (e.g.,
see Low et al., J. Mol. Biol. 260:359-3680 (1996)). In this
technique very high spontaneous mutation rates are observed. The
conditional property minimizes non-desired background mutations.
This technology could be combined with adaptive evolution to
enhance mutagenesis rates and more rapidly achieve desired
phenotypes.
[0193] "Look-Through Mutagenesis (LTM) is a multidimensional
mutagenesis method that assesses and optimizes combinatorial
mutations of selected amino acids." (Rajpal et al., Proc Natl Acad
Sci U.S.A. 102:8466-8471 (2005)) Rather than saturating each site
with all possible amino acid changes, a set of nine is chosen to
cover the range of amino acid R-group chemistry. Fewer changes per
site allows multiple sites to be subjected to this type of
mutagenesis. A >800-fold increase in binding affinity for an
antibody from low nanomolar to picomolar has been achieved through
this method. This is a rational approach to minimize the number of
random combinations and can increase the ability to find improved
traits by greatly decreasing the numbers of clones to be screened.
This has been applied to antibody engineering, specifically to
increase the binding affinity and/or reduce dissociation. The
technique can be combined with either screens or selections.
[0194] Gene Reassembly is a DNA shuffling method that can be
applied to multiple genes at one time or to creating a large
library of chimeras (multiple mutations) of a single gene. (Tunable
GeneReassembly.TM. (TGR.TM.) Technology supplied by Verenium
Corporation) Typically this technology is used in combination with
ultra-high-throughput screening to query the represented sequence
space for desired improvements. This technique allows multiple gene
recombination independent of homology. The exact number and
position of cross-over events can be pre-determined using fragments
designed via bioinformatic analysis. This technology leads to a
very high level of diversity with virtually no parental gene
reformation and a low level of inactive genes. Combined with
GSSM.TM., a large range of mutations can be tested for improved
activity. The method allows "blending" and "fine tuning" of DNA
shuffling, e.g. codon usage can be optimized.
[0195] In Silico Protein Design Automation (PDA) 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. (Hayes et al., Proc Natl Acad Sci U.S.A. 99:15926-15931
(2002)) This technology uses in silico structure-based entropy
predictions in order to search for structural tolerance toward
protein amino acid variations. Statistical mechanics is applied to
calculate coupling interactions at each position. Structural
tolerance toward amino acid substitution is a measure of coupling.
Ultimately, this technology is designed to yield desired
modifications of protein properties while maintaining the integrity
of structural characteristics. The method computationally assesses
and allows filtering of a very large number of possible sequence
variants (10.sup.50). The choice of sequence variants to test is
related to predictions based on the most favorable thermodynamics.
Ostensibly only stability or properties that are linked to
stability can be effectively addressed with this technology. The
method has been successfully used in some therapeutic proteins,
especially in engineering immunoglobulins. In silico predictions
avoid testing extraordinarily large numbers of potential variants.
Predictions based on existing three-dimensional structures are more
likely to succeed than predictions based on hypothetical
structures. This technology can readily predict and allow targeted
screening of multiple simultaneous mutations, something not
possible with purely experimental technologies due to exponential
increases in numbers.
[0196] Iterative Saturation Mutagenesis (ISM) involves: 1) use
knowledge of structure/function to choose a likely site for enzyme
improvement; 2) saturation mutagenesis at chosen site using
Stratagene QuikChange (or other suitable means); 3) screen/select
for desired properties; and 4) with improved clone(s), start over
at another site and continue repeating. (Reetz et al., Nat. Protoc.
2:891-903 (2007); and Reetz et al., Angew. Chem. Int. Ed Engl.
45:7745-7751 (2006)) This is a proven methodology, which assures
all possible replacements at a given position are made for
screening/selection.
[0197] 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.
[0198] In one embodiment, the invention provides a method for
producing n-propanol and isopropanol that includes culturing a
non-naturally occurring microbial organism, including a microbial
organism having an n-propanol pathway and an isopropanol pathway,
the n-propanol pathway having at least one exogenous nucleic acid
encoding an n-propanol pathway enzyme expressed in a sufficient
amount to produce n-propanol, the n-propanol pathway including a
propionaldehyde dehydrogenase, a propanol dehydrogenase, a
propionyl-CoA:phosphate propanoyltransferase, a propionyl-CoA
hydrolase, a propionyl-CoA transferase, a propionyl-CoA synthetase,
a propionate kinase, a propionate reductase or a propionyl
phosphate reductase, the isopropanol pathway comprising at least
one exogenous nucleic acid encoding an isopropanol pathway enzyme
expressed in a sufficient amount to produce isopropanol, the
isopropanol pathway including an acetyl-CoA acetyl thiolase, an
acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase or an
isopropanol dehydrogenase.
[0199] In a further aspect of the above embodiment, the method
includes a microbial organism having an acetyl-CoA pathway having
at least one exogenous nucleic acid encoding an acetyl-CoA pathway
enzyme expressed in a sufficient amount to produce acetyl-CoA, the
acetyl-CoA pathway including a pyruvate kinase, a pyruvate
dehydrogenase, a pyruvate ferredoxin oxidoreductase, a pyruvate
formate lyase, a pyruvate formate lyase activating enzyme, or a
formate dehydrogenase.
[0200] In further embodiment, the method includes a microbial
organism having a propionyl-CoA pathway having at least one
exogenous nucleic acid encoding a propionyl-CoA pathway enzyme
expressed in a sufficient amount to produce propionyl-CoA, the
propionyl-CoA pathway including a PEP carboxykinase, a PEP
carboxylase, a malate dehydrogenase, a fumarase, a fumarate
reductase, a succinyl-CoA transferase, a succinyl-CoA synthetase, a
methylmalonyl-CoA mutase, a methylmalonyl-CoA epimerase or a
methylmalonyl-CoA decarboxylase. In a further aspect, the
propionyl-CoA pathway includes a pyruvate carboxylase or a
methylmalonyl-CoA carboxytransferase.
[0201] In another further embodiment, the method includes a
microbial organism having a propionyl-CoA pathway having at least
one exogenous nucleic acid encoding a propionyl-CoA pathway enzyme
expressed in a sufficient amount to produce propionyl-CoA, the
propionyl-CoA pathway including a PEP carboxykinase, a PEP
carboxylase, a threonine deaminase, or a 2-oxobutanoate
dehydrogenase. In a further aspect, the n-propanol pathway includes
2-oxobutanoate decarboxylase.
[0202] In another further embodiment, the method includes a
microbial organism having a propionyl-CoA pathway having at least
one exogenous nucleic acid encoding a propionyl-CoA pathway enzyme
expressed in a sufficient amount to produce propionyl-CoA, the
propionyl-CoA pathway including an acetyl-CoA carboxylase, a
malonyl-CoA reductase, a malonate semialdehyde reductase or
propionyl-CoA synthase.
[0203] In another further embodiment, the method includes a
microbial organism having a propionyl-CoA pathway having at least
one exogenous nucleic acid encoding a propionyl-CoA pathway enzyme
expressed in a sufficient amount to produce propionyl-CoA, the
propionyl-CoA pathway including a lactate dehydrogenase, a
lactate-CoA transferase, a lactyl-CoA dehydratase or acryloyl CoA
reductase.
[0204] In yet another embodiment, the invention provides a method
for producing n-propanol and isopropanol that includes culturing a
non-naturally occurring microbial organism, including a microbial
organism having an n-propanol pathway and an isopropanol pathway,
the n-propanol pathway having a first set of exogenous nucleic
acids encoding n-propanol pathway enzymes expressed in a sufficient
amount to produce n-propanol, the first set of exogenous nucleic
acids encoding a propionaldehyde dehydrogenase and a propanol
dehydrogenase; or a propionyl-CoA:phosphate propanoyltransferase, a
propionyl phosphate reductase and a propanol dehydrogenase; or a
propionyl-CoA hydrolase or a propionyl-CoA transferase or a
propionyl-CoA synthetase, a propionate kinase, a propionyl
phosphate reductase and a propanol dehydrogenase; or a
propionyl-CoA hydrolase or a propionyl-CoA transferase or a
propionyl-CoA synthetase, a propionate reductase and a propanol
dehydrogenase, and the isopropanol pathway having a second set of
exogenous nucleic acids encoding isopropanol pathway enzymes
expressed in a sufficient amount to produce isopropanol, the second
set of exogenous nucleic acids encoding an acetyl-CoA acetyl
thiolase; an acetoacetyl-CoA transferase or an acetoacetyl-CoA
hydrolase or an acetoacetyl-CoA synthetase, an acetoacetate
decarboxylase; and an isopropanol dehydrogenase.
[0205] In a further aspect of the above embodiment, the method
includes a microbial organism having an acetyl-CoA pathway having a
third set of exogenous nucleic acids encoding acetyl-CoA pathway
enzymes expressed in a sufficient amount to produce acetyl-CoA, the
third set of exogenous nucleic acids encoding a pyruvate kinase;
and a pyruvate dehydrogenase or a pyruvate ferredoxin
oxidoreductase; or a pyruvate formate lyase, a pyruvate formate
lyase activating enzyme and a formate dehydrogenase.
[0206] In another further embodiment, the method includes a
microbial organism having a propionyl-CoA pathway having a third
set of exogenous nucleic acids encoding propionyl-CoA pathway
enzymes expressed in a sufficient amount to produce propionyl-CoA,
the third set of exogenous nucleic acids encoding a PEP
carboxykinase or a PEP carboxylase; a malate dehydrogenase; a
fumarase; a fumarate reductase; a succinyl-CoA transferase or a
succinyl-CoA synthetase; a methylmalonyl-CoA mutase; and a
methylmalonyl-CoA decarboxylase. In a further aspect, the third set
of exogenous nucleic acids further encodes a methylmalonyl-CoA
epimerase or a pyruvate carboxylas.
[0207] In another further embodiment, the method includes a
microbial organism having a propionyl-CoA pathway having a third
set of exogenous nucleic acids encoding propionyl-CoA pathway
enzymes expressed in a sufficient amount to produce propionyl-CoA,
said third set of exogenous nucleic acids encoding a PEP
carboxykinase or a PEP carboxylase; a threonine deaminase; and a
2-oxobutanoate dehydrogenase. In a further aspect, the third set of
exogenous nucleic acids further encodes a methylmalonyl-CoA
decarboxylase or a pyruvate carboxylase. In yet another aspect, the
second set of exogenous nucleic acids further encodes a
2-oxobutanoate decarboxylase.
[0208] In another further embodiment, the method includes a
microbial organism having a propionyl-CoA pathway having a third
set of exogenous nucleic acids encoding propionyl-CoA pathway
enzymes expressed in a sufficient amount to produce propionyl-CoA,
the third set of exogenous nucleic acids encoding an acetyl-CoA
carboxylase; a malonyl-CoA reductase; a malonate semialdehyde
reductase; and propionyl-CoA synthase.
[0209] In another further embodiment, the method includes a
microbial organism having a propionyl-CoA pathway having a third
set of exogenous nucleic acids encoding a lactate dehydrogenase; a
lactate-CoA transferase; a lactyl-CoA dehydratase; and acryloyl CoA
reductase.
[0210] In one embodiment, the invention provides a method for
producing n-propanol and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an n-propanol pathway and an isopropanol pathway,
the n-propanol pathway comprising a first set of exogenous nucleic
acids encoding n-propanol pathway enzymes expressed in a sufficient
amount to produce n-propanol, the first set of exogenous nucleic
acids encoding a PEP carboxykinase or a PEP carboxylase; a malate
dehydrogenase; a fumarase; a fumarate reductase; a succinyl-CoA
transferase or a succinyl-CoA synthetase; a methylmalonyl-CoA
mutase; a methylmalonyl-CoA decarboxylase; and a propionaldehyde
dehydrogenase and a propanol dehydrogenase; or a
propionyl-CoA:phosphate propanoyltransferase and a propionyl
phosphate reductase; or a propionyl-CoA hydrolase or a
propionyl-CoA transferase or a propionyl-CoA synthetase, a
propionate kinase, a propionyl phosphate reductase and a propanol
dehydrogenase; or a propionyl-CoA hydrolase or a propionyl-CoA
transferase or a propionyl-CoA synthetase, a propionate reductase
and a propanol dehydrogenase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding a pyruvate kinase; a pyruvate dehydrogenase or a pyruvate
ferredoxin oxidoreductase; or a pyruvate formate lyase, a pyruvate
formate lyase activating enzyme and a formate dehydrogenase; an
acetyl-CoA acetyl thiolase; an acetoacetyl-CoA transferase or an
acetoacetyl-CoA hydrolase or an acetoacetyl-CoA synthetase, an
acetoacetate decarboxylase; and an isopropanol dehydrogenase.
[0211] In one embodiment, the invention provides a method for
producing n-propanol and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an n-propanol pathway and an isopropanol pathway,
the n-propanol pathway comprising a first set of exogenous nucleic
acids encoding n-propanol pathway enzymes expressed in a sufficient
amount to produce n-propanol, the first set of exogenous nucleic
acids encoding a PEP carboxykinase or a PEP carboxylase; a
threonine deaminase; and a 2-oxobutanoate decarboxylase and a
propanol dehydrogenase; or a 2-oxobutanoate dehydrogenase, a
propionaldehyde dehydrogenase and a propanol dehydrogenase; or a
2-oxobutanoate dehydrogenase, a propionyl-CoA:phosphate
propanoyltransferase, a propionyl phosphate reductase and a
propanol dehydrogenase; or a 2-oxobutanoate dehydrogenase, a
propionyl-CoA hydrolase or a propionyl-CoA transferase or a
propionyl-CoA synthetase, a propionate kinase, a propionyl
phosphate reductase and a propanol dehydrogenase; or a
2-oxobutanoate dehydrogenase, a propionyl-CoA hydrolase or a
propionyl-CoA transferase or a propionyl-CoA synthetase, a
propionate reductase and a propanol dehydrogenase, and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding a pyruvate kinase; a pyruvate
dehydrogenase or a pyruvate ferredoxin oxidoreductase; or a
pyruvate formate lyase, a pyruvate formate lyase activating enzyme
and a formate dehydrogenase; an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase; and an
isopropanol dehydrogenase. In a further aspect, the second set of
exogenous nucleic acids further encodes a pyruvate carboxylase or a
methylmalonyl-CoA carboxytransferase.
[0212] In one embodiment, the invention provides a method for
producing n-propanol and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an n-propanol pathway and an isopropanol pathway,
the n-propanol pathway comprising a first set of exogenous nucleic
acids encoding n-propanol pathway enzymes expressed in a sufficient
amount to produce n-propanol, the first set of exogenous nucleic
acids encoding a pyruvate kinase; a pyruvate dehydrogenase or a
pyruvate ferredoxin oxidoreductase; or a pyruvate formate lyase, a
pyruvate formate lyase activating enzyme and a formate
dehydrogenase; an acetyl-CoA carboxylase; a malonyl-CoA reductase;
a malonate semialdehyde reductase; propionyl-CoA synthase; and a
propionaldehyde dehydrogenase and a propanol dehydrogenase; or a
propionyl-CoA:phosphate propanoyltransferase, a propionyl phosphate
reductase and propanol dehydrogenase; or a propionyl-CoA hydrolase
or a propionyl-CoA transferase or a propionyl-CoA synthetase, a
propionate kinase, a propionyl phosphate reductase and a propanol
dehydrogenase; or a propionyl-CoA hydrolase or a propionyl-CoA
transferase or a propionyl-CoA synthetase, a propionate reductase
and a propanol dehydrogenase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase, an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0213] In one embodiment, the invention provides a method for
producing n-propanol and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an n-propanol pathway and an isopropanol pathway,
the n-propanol pathway including a first set of exogenous nucleic
acids encoding n-propanol pathway enzymes expressed in a sufficient
amount to produce n-propanol, the first set of exogenous nucleic
acids encoding a lactate dehydrogenase; a lactate-CoA transferase;
a lactyl-CoA dehydratase; acryloyl CoA reductase; and a
propionaldehyde dehydrogenase and a propanol dehydrogenase; or a
propionyl-CoA:phosphate propanoyltransferase, a propionyl phosphate
reductase and a propanol dehydrogenase; or a propionyl-CoA
hydrolase or a propionyl-CoA transferase or a propionyl-CoA
synthetase, a propionate kinase, a propionyl phosphate reductase
and a propanol dehydrogenase; or a propionyl-CoA hydrolase or a
propionyl-CoA transferase or a propionyl-CoA synthetase, a
propionate reductase and a propanol dehydrogenase, and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding a pyruvate dehydrogenase or a
pyruvate ferredoxin oxidoreductase; or a pyruvate formate lyase, a
pyruvate formate lyase activating enzyme and a formate
dehydrogenase; an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase, an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0214] In one embodiment, the invention provides a method for
producing n-propanol that includes culturing a non-naturally
occurring microbial organism including a microbial organism having
an n-propanol pathway, the n-propanol pathway comprising at least
one exogenous nucleic acid encoding an n-propanol pathway enzyme
expressed in a sufficient amount to produce n-propanol, the
n-propanol pathway including a propionaldehyde dehydrogenase, a
propanol dehydrogenase, a propionyl-CoA:phosphate
propanoyltransferase, a propionyl-CoA hydrolase, a propionyl-CoA
transferase, a propionyl-CoA synthetase, a propionate kinase, a
propionate reductase, or a propionyl phosphate reductase.
[0215] In a further aspect of the above embodiment, the method for
producing an propanol includes culturing the non-naturally
occurring microbial organism having an n-propanol pathway that also
has a propionyl-CoA pathway including exogenous nucleic acids
encoding propionyl-CoA pathway enzymes expressed in a sufficient
amount to produce propionyl-CoA as exemplified herein. For example,
in some aspects the exogenous nucleic acids encode a PEP
carboxykinase, a PEP carboxylase, a malate dehydrogenase, a
fumarase, a fumarate reductase, a succinyl-CoA transferase, a
succinyl-CoA synthetase, a methylmalonyl-CoA mutase, or a
methylmalonyl-CoA decarboxylase. In another aspect, the exogenous
nucleic acids further encode a methylmalonyl-CoA epimerase.
Additionally, in yet another aspect of the above embodiment, the
method for producing an propanol includes culturing the
non-naturally occurring microbial organism having an n-propanol
pathway that has a first set of exogenous nucleic acids encoding
n-propanol pathway enzymes expressed in a sufficient amount to
produce n-propanol, wherein the first set of exogenous nucleic
acids encode a PEP carboxykinase or a PEP carboxylase; a malate
dehydrogenase; a fumarase; a fumarate reductase; a succinyl-CoA
transferase or a succinyl-CoA synthetase; a methylmalonyl-CoA
mutase; a methylmalonyl-CoA epimerase; a methylmalonyl-CoA
decarboxylase; a propionaldehyde dehydrogenase and a propanol
dehydrogenase.
[0216] In another embodiment, the invention provides a method for
producing n-propanol that includes culturing a non-naturally
occurring microbial organism including a microbial organism having
an n-propanol pathway, the n-propanol pathway comprising a set of
exogenous nucleic acids encoding n-propanol pathway enzymes
expressed in a sufficient amount to produce n-propanol, the set of
exogenous nucleic acids encoding a propionaldehyde dehydrogenase
and a propanol dehydrogenase; or a propionyl-CoA:phosphate
propanoyltransferase, a propionyl phosphate reductase and a
propanol dehydrogenase; or a propionyl-CoA hydrolase or a
propionyl-CoA transferase or a propionyl-CoA synthetase, a
propionate kinase, a propionyl phosphate reductase and a propanol
dehydrogenase; or a propionyl-CoA hydrolase or a propionyl-CoA
transferase or a propionyl-CoA synthetase, a propionate reductase
and a propanol dehydrogenase.
[0217] In one embodiment, the invention provides a method for
producing 14-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism, including a microbial
organism having an 14-BDO pathway and an isopropanol pathway, the
14-BDO pathway having at least one exogenous nucleic acid encoding
an 14-BDO pathway enzyme expressed in a sufficient amount to
produce 14-BDO, the 14-BDO pathway including a succinyl-CoA
reductase, a succinate reductase, a 4-hydroxybutyrate
dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a
4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyryl-CoA reductase
(aldehyde-forming), a 4-hydroxybutyraldehyde reductase, a
4-hydroxybutyrate reductase; a 4-hydroxybutyrate kinase, a
phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-phosphate
reductase or a 4-hydroxybutyryl-CoA reductase (alcohol-forming),
the isopropanol pathway including at least one exogenous nucleic
acid encoding an isopropanol pathway enzyme expressed in a
sufficient amount to produce isopropanol, the isopropanol pathway
including an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA
transferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA
synthetase, an acetoacetate decarboxylase or an isopropanol
dehydrogenase.
[0218] In one embodiment, the invention provides a method for
producing 13-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism, including a microbial
organism having an 13-BDO pathway and an isopropanol pathway, the
13-BDO pathway having at least one exogenous nucleic acid encoding
an 13-BDO pathway enzyme expressed in a sufficient amount to
produce 13-BDO, the 13-BDO pathway including a succinyl-CoA
reductase, a succinate reductase, a 4-hydroxybutyrate
dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a
4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyrate kinase, a
phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA
dehydratase, a crotonase, a 3-hydroxybutyryl-CoA reductase
(aldehyde forming), a 3-hydroxybutyraldehyde reductase, a
3-hydroxybutyryl-CoA transferase, a 3-hydroxybutyryl-CoA
synthetase, a 3-hydroxybutyryl-CoA hydrolase, or a
3-hydroxybutyrate reductase, or a 3-hydroxybutyryl-CoA reductase
(alcohol-forming), the isopropanol pathway including at least one
exogenous nucleic acid encoding an isopropanol pathway enzyme
expressed in a sufficient amount to produce isopropanol, the
isopropanol pathway including an acetyl-CoA acetyl thiolase, an
acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase or an
isopropanol dehydrogenase.
[0219] In one embodiment, the invention provides a method for
producing MAA and isopropanol that includes culturing a
non-naturally occurring microbial organism, including a microbial
organism having an MAA pathway and an isopropanol pathway, the MAA
pathway having at least one exogenous nucleic acid encoding an MAA
pathway enzyme expressed in a sufficient amount to produce MAA, the
MAA pathway including a succinyl-CoA reductase, a succinate
reductase, a 4-hydroxybutyrate dehydrogenase, a
4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA
synthetase, a 4-hydroxybutyrate kinase, a
phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA mutase, a
3-hydroxyisobutyryl-CoA dehydratase, a methacrylyl-CoA transferase,
a methacrylyl-CoA synthetase, a methacrylyl-CoA hydrolase, a
3-hydroxyisobutyryl-CoA transferase, a 3-hydroxyisobutyryl-CoA
synthetase, a 3-hydroxyisobutyryl-CoA hydrolase, a
3-hydroxyisobutyrate dehydratase, a methylmalonyl-CoA mutase, a
methylmalonyl-CoA epimerase, a methylmalonyl-CoA transferase, a
methylmalonyl-CoA synthetase, a methylmalonyl-CoA hydrolase, a
methylmalonate reductase, a methylmalonyl-CoA reductase (aldehyde
forming), a 3-hydroxyisobutyrate dehydrogenase, a methylmalonyl-CoA
reductase (alcohol forming) or a 3-hydroxyisobutyrate dehydratase,
the isopropanol pathway including at least one exogenous nucleic
acid encoding an isopropanol pathway enzyme expressed in a
sufficient amount to produce isopropanol, the isopropanol pathway
including an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA
transferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA
synthetase, an acetoacetate decarboxylase or an isopropanol
dehydrogenase.
[0220] In a further aspect of the above embodiments, the microbial
organism has an acetyl-CoA pathway having at least one exogenous
nucleic acid encoding an acetyl-CoA pathway enzyme expressed in a
sufficient amount to produce acetyl-CoA, the acetyl-CoA pathway
including a pyruvate kinase, a pyruvate dehydrogenase, a pyruvate
ferredoxin oxidoreductase, a pyruvate formate lyase, a pyruvate
formate lyase activating enzyme, or a formate dehydrogenase.
[0221] In further aspect of the above embodiments, the microbial
organism has a succinyl-CoA pathway having at least one exogenous
nucleic acid encoding a succinyl-CoA pathway enzyme expressed in a
sufficient amount to produce succinyl-CoA, the succinyl-CoA pathway
including a PEP carboxykinase, a PEP carboxylase, a malate
dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA
transferase or a succinyl-CoA synthetase. In a further aspect, the
succinyl-CoA pathway includes a pyruvate carboxylase or a
methylmalonyl-CoA carboxytransferase.
[0222] In one embodiment, the invention provides a method for
producing 14-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 14-BDO pathway and an isopropanol pathway, the
14-BDO pathway including a first set of exogenous nucleic acids
encoding 14-BDO pathway enzymes expressed in a sufficient amount to
produce 14-BDO, the first set of exogenous nucleic acids encoding a
succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA reductase (aldehyde-forming);
and a 4-hydroxybutyraldehyde reductase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase, an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0223] In one embodiment, the invention provides a method for
producing 14-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 14-BDO pathway and an isopropanol pathway, the
14-BDO pathway including a first set of exogenous nucleic acids
encoding 14-BDO pathway enzymes expressed in a sufficient amount to
produce 14-BDO, the first set of exogenous nucleic acids encoding a
succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyrate reductase; and a 4-hydroxybutyraldehyde
reductase, and the isopropanol pathway comprising a second set of
exogenous nucleic acids encoding isopropanol pathway enzymes
expressed in a sufficient amount to produce isopropanol, the second
set of exogenous nucleic acids encoding an acetyl-CoA acetyl
thiolase; an acetoacetyl-CoA transferase or an acetoacetyl-CoA
hydrolase or an acetoacetyl-CoA synthetase, an acetoacetate
decarboxylase; and an isopropanol dehydrogenase.
[0224] In one embodiment, the invention provides a method for
producing 14-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 14-BDO pathway and an isopropanol pathway, the
14-BDO pathway including a first set of exogenous nucleic acids
encoding 14-BDO pathway enzymes expressed in a sufficient amount to
produce 14-BDO, the first set of exogenous nucleic acids encoding a
succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a
4-hydroxybutyryl-CoA reductase (aldehyde-forming); and a
4-hydroxybutyraldehyde reductase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase, an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0225] In one embodiment, the invention provides a method for
producing 14-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 14-BDO pathway and an isopropanol pathway, the
14-BDO pathway including a first set of exogenous nucleic acids
encoding 14-BDO pathway enzymes expressed in a sufficient amount to
produce 14-BDO, the first set of exogenous nucleic acids encoding a
succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyrate kinase; a 4-hydroxybutyryl-phosphate reductase;
and a 4-hydroxybutyraldehyde reductase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase; an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0226] In one embodiment, the invention provides a method for
producing 14-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 14-BDO pathway and an isopropanol pathway, the
14-BDO pathway including a first set of exogenous nucleic acids
encoding 14-BDO pathway enzymes expressed in a sufficient amount to
produce 14-BDO, the first set of exogenous nucleic acids encoding a
succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; and a
4-hydroxybutyryl-CoA reductase (alcohol-forming), and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0227] In one embodiment, the invention provides a method for
producing 14-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 14-BDO pathway and an isopropanol pathway, the
14-BDO pathway including a first set of exogenous nucleic acids
encoding 14-BDO pathway enzymes expressed in a sufficient amount to
produce 14-BDO, the first set of exogenous nucleic acids encoding a
succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; and a 4-hydroxybutyryl-CoA reductase (alcohol-forming);
and a 4-hydroxybutyryl-CoA reductase (alcohol-forming), and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0228] In one embodiment, the invention provides a method for
producing 14-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 14-BDO pathway and an isopropanol pathway, the
14-BDO pathway including a first set of exogenous nucleic acids
encoding 14-BDO pathway enzymes expressed in a sufficient amount to
produce 14-BDO, the first set of exogenous nucleic acids encoding a
succinate reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA reductase (aldehyde-forming);
and a 4-hydroxybutyraldehyde reductase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase, an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0229] In one embodiment, the invention provides a method for
producing 14-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 14-BDO pathway and an isopropanol pathway, the
14-BDO pathway including a first set of exogenous nucleic acids
encoding 14-BDO pathway enzymes expressed in a sufficient amount to
produce 14-BDO, the first set of exogenous nucleic acids encoding a
succinate reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyrate reductase; and a 4-hydroxybutyraldehyde
reductase, and the isopropanol pathway comprising a second set of
exogenous nucleic acids encoding isopropanol pathway enzymes
expressed in a sufficient amount to produce isopropanol, the second
set of exogenous nucleic acids encoding an acetyl-CoA acetyl
thiolase; an acetoacetyl-CoA transferase or an acetoacetyl-CoA
hydrolase or an acetoacetyl-CoA synthetase, an acetoacetate
decarboxylase; and an isopropanol dehydrogenase.
[0230] In one embodiment, the invention provides a method for
producing 14-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 14-BDO pathway and an isopropanol pathway, the
14-BDO pathway including a first set of exogenous nucleic acids
encoding 14-BDO pathway enzymes expressed in a sufficient amount to
produce 14-BDO, the first set of exogenous nucleic acids encoding a
succinate reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a
4-hydroxybutyryl-CoA reductase (aldehyde-forming); and a
4-hydroxybutyraldehyde reductase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase, an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0231] In one embodiment, the invention provides a method for
producing 14-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 14-BDO pathway and an isopropanol pathway, the
14-BDO pathway including a first set of exogenous nucleic acids
encoding 14-BDO pathway enzymes expressed in a sufficient amount to
produce 14-BDO, the first set of exogenous nucleic acids encoding a
succinate reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyrate kinase; a 4-hydroxybutyryl-phosphate reductase;
and a 4-hydroxybutyraldehyde reductase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase; an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0232] In one embodiment, the invention provides a method for
producing 14-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 14-BDO pathway and an isopropanol pathway, the
14-BDO pathway including a first set of exogenous nucleic acids
encoding 14-BDO pathway enzymes expressed in a sufficient amount to
produce 14-BDO, the first set of exogenous nucleic acids encoding a
succinate reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; and a
4-hydroxybutyryl-CoA reductase (alcohol-forming), and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0233] In one embodiment, the invention provides a method for
producing 14-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 14-BDO pathway and an isopropanol pathway, the
14-BDO pathway including a first set of exogenous nucleic acids
encoding 14-BDO pathway enzymes expressed in a sufficient amount to
produce 14-BDO, the first set of exogenous nucleic acids encoding a
succinate reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; and a 4-hydroxybutyryl-CoA reductase (alcohol-forming),
and the isopropanol pathway comprising a second set of exogenous
nucleic acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0234] In one embodiment, the invention provides a method for
producing 13-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 13-BDO pathway and an isopropanol pathway, the
13-BDO pathway including a first set of exogenous nucleic acids
encoding 13-BDO pathway enzymes expressed in a sufficient amount to
produce 13-BDO, the first set of exogenous nucleic acids encoding a
succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; a
3-hydroxybutyryl-CoA reductase (aldehyde forming); and a
3-hydroxybutyraldehyde reductase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase, an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0235] In one embodiment, the invention provides a method for
producing 13-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 13-BDO pathway and an isopropanol pathway, the
13-BDO pathway including a first set of exogenous nucleic acids
encoding 13-BDO pathway enzymes expressed in a sufficient amount to
produce 13-BDO, the first set of exogenous nucleic acids encoding a
succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; a
3-hydroxybutyryl-CoA transferase or a 3-hydroxybutyryl-CoA
synthetase or a 3-hydroxybutyryl-CoA hydrolase; a 3-hydroxybutyrate
reductase; and a 3 hydroxybutyraldehyde reductase, and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0236] In one embodiment, the invention provides a method for
producing 13-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 13-BDO pathway and an isopropanol pathway, the
13-BDO pathway including a first set of exogenous nucleic acids
encoding 13-BDO pathway enzymes expressed in a sufficient amount to
produce 13-BDO, the first set of exogenous nucleic acids encoding a
succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a
4-hydroxybutyryl-CoA dehydratase; a crotonase; a
3-hydroxybutyryl-CoA reductase (aldehyde forming); and a
3-hydroxybutyraldehyde reductase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase, an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0237] In one embodiment, the invention provides a method for
producing 13-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 13-BDO pathway and an isopropanol pathway, the
13-BDO pathway including a first set of exogenous nucleic acids
encoding 13-BDO pathway enzymes expressed in a sufficient amount to
produce 13-BDO, the first set of exogenous nucleic acids encoding a
succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a
4-hydroxybutyryl-CoA dehydratase; a crotonase; a
3-hydroxybutyryl-CoA transferase or a 3-hydroxybutyryl-CoA
synthetase or a 3-hydroxybutyryl-CoA hydrolase; a 3-hydroxybutyrate
reductase; and a 3-hydroxybutyraldehyde reductase, and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0238] In one embodiment, the invention provides a method for
producing 13-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 13-BDO pathway and an isopropanol pathway, the
13-BDO pathway including a first set of exogenous nucleic acids
encoding 13-BDO pathway enzymes expressed in a sufficient amount to
produce 13-BDO, the first set of exogenous nucleic acids encoding a
succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a
4-hydroxybutyryl-CoA dehydratase; a crotonase; and a
3-hydroxybutyryl-CoA reductase (alcohol-forming), and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0239] In one embodiment, the invention provides a method for
producing 13-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 13-BDO pathway and an isopropanol pathway, the
13-BDO pathway including a first set of exogenous nucleic acids
encoding 13-BDO pathway enzymes expressed in a sufficient amount to
produce 13-BDO, the first set of exogenous nucleic acids encoding a
succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; and a
3-hydroxybutyryl-CoA reductase (alcohol-forming), and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0240] In one embodiment, the invention provides a method for
producing 13-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 13-BDO pathway and an isopropanol pathway, the
13-BDO pathway including a first set of exogenous nucleic acids
encoding 13-BDO pathway enzymes expressed in a sufficient amount to
produce 13-BDO, the first set of exogenous nucleic acids encoding a
succinate reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; a
3-hydroxybutyryl-CoA reductase (aldehyde forming); and a
3-hydroxybutyraldehyde reductase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase, an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0241] In one embodiment, the invention provides a method for
producing 13-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 13-BDO pathway and an isopropanol pathway, the
13-BDO pathway including a first set of exogenous nucleic acids
encoding 13-BDO pathway enzymes expressed in a sufficient amount to
produce 13-BDO, the first set of exogenous nucleic acids encoding a
succinate reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; a
3-hydroxybutyryl-CoA transferase or a 3-hydroxybutyryl-CoA
synthetase or a 3-hydroxybutyryl-CoA hydrolase; a 3-hydroxybutyrate
reductase; and a 3 hydroxybutyraldehyde reductase, and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0242] In one embodiment, the invention provides a method for
producing 13-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 13-BDO pathway and an isopropanol pathway, the
13-BDO pathway including a first set of exogenous nucleic acids
encoding 13-BDO pathway enzymes expressed in a sufficient amount to
produce 13-BDO, the first set of exogenous nucleic acids encoding a
succinate reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a
4-hydroxybutyryl-CoA dehydratase; a crotonase; a
3-hydroxybutyryl-CoA reductase (aldehyde forming); and a
3-hydroxybutyraldehyde reductase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase, an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0243] In one embodiment, the invention provides a method for
producing 13-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 13-BDO pathway and an isopropanol pathway, the
13-BDO pathway including a first set of exogenous nucleic acids
encoding 13-BDO pathway enzymes expressed in a sufficient amount to
produce 13-BDO, the first set of exogenous nucleic acids encoding a
succinate reductase; a 4-hydroxybutyrate dehydrogenase; a 4
hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a
4-hydroxybutyryl-CoA dehydratase; a crotonase; a
3-hydroxybutyryl-CoA transferase or a 3-hydroxybutyryl-CoA
synthetase or a 3-hydroxybutyryl-CoA hydrolase; a 3-hydroxybutyrate
reductase; and a 3 hydroxybutyraldehyde reductase, and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0244] In one embodiment, the invention provides a method for
producing 13-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 13-BDO pathway and an isopropanol pathway, the
13-BDO pathway including a first set of exogenous nucleic acids
encoding 13-BDO pathway enzymes expressed in a sufficient amount to
produce 13-BDO, the first set of exogenous nucleic acids encoding a
succinate reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a
crotonase; and a 3-hydroxybutyryl-CoA reductase (alcohol-forming),
and the isopropanol pathway comprising a second set of exogenous
nucleic acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0245] In one embodiment, the invention provides a method for
producing 13-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 13-BDO pathway and an isopropanol pathway, the
13-BDO pathway including a first set of exogenous nucleic acids
encoding 13-BDO pathway enzymes expressed in a sufficient amount to
produce 13-BDO, the first set of exogenous nucleic acids encoding a
succinate reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; and a
3-hydroxybutyryl-CoA reductase (alcohol-forming), and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0246] In one embodiment, the invention provides a method for
producing MAA and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an MAA pathway and an isopropanol pathway, the MAA
pathway including a first set of exogenous nucleic acids encoding
MAA pathway enzymes expressed in a sufficient amount to produce
MAA, the first set of exogenous nucleic acids encoding a
succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA mutase; a
3-hydroxyisobutyryl-CoA transferase, a 3-hydroxyisobutyryl-CoA
synthetase or a 3-hydroxyisobutyryl-CoA hydrolase; and a
3-hydroxyisobutyrate dehydratase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase, an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0247] In one embodiment, the invention provides a method for
producing MAA and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an MAA pathway and an isopropanol pathway, the MAA
pathway including a first set of exogenous nucleic acids encoding
MAA pathway enzymes expressed in a sufficient amount to produce
MAA, the first set of exogenous nucleic acids encoding a
succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA mutase; a
3-hydroxyisobutyryl-CoA dehydratase; and a methacrylyl-CoA
transferase, a methacrylyl-CoA synthetase or a methacrylyl-CoA
hydrolase, and the isopropanol pathway comprising a second set of
exogenous nucleic acids encoding isopropanol pathway enzymes
expressed in a sufficient amount to produce isopropanol, the second
set of exogenous nucleic acids encoding an acetyl-CoA acetyl
thiolase; an acetoacetyl-CoA transferase or an acetoacetyl-CoA
hydrolase or an acetoacetyl-CoA synthetase; an acetoacetate
decarboxylase; and an isopropanol dehydrogenase.
[0248] In one embodiment, the invention provides a method for
producing MAA and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an MAA pathway and an isopropanol pathway, the MAA
pathway including a first set of exogenous nucleic acids encoding
MAA pathway enzymes expressed in a sufficient amount to produce
MAA, the first set of exogenous nucleic acids encoding a
succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a
4-hydroxybutyryl-CoA mutase; a 3-hydroxyisobutyryl-CoA transferase,
a 3-hydroxyisobutyryl-CoA synthetase or a 3-hydroxyisobutyryl-CoA
hydrolase; and a 3-hydroxyisobutyrate dehydratase, and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0249] In one embodiment, the invention provides a method for
producing MAA and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an MAA pathway and an isopropanol pathway, the MAA
pathway including a first set of exogenous nucleic acids encoding
MAA pathway enzymes expressed in a sufficient amount to produce
MAA, the first set of exogenous nucleic acids encoding a
succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4
hydroxybutyrate kinase; a phosphotrans-4-hydroxybutyrylase; a
4-hydroxybutyryl-CoA mutase; a 3-hydroxyisobutyryl-CoA dehydratase;
and a methacrylyl-CoA transferase, a methacrylyl-CoA synthetase or
a methacrylyl-CoA hydrolase, and the isopropanol pathway comprising
a second set of exogenous nucleic acids encoding isopropanol
pathway enzymes expressed in a sufficient amount to produce
isopropanol, the second set of exogenous nucleic acids encoding an
acetyl-CoA acetyl thiolase; an acetoacetyl-CoA transferase or an
acetoacetyl-CoA hydrolase or an acetoacetyl-CoA synthetase; an
acetoacetate decarboxylase; and an isopropanol dehydrogenase.
[0250] In one embodiment, the invention provides a method for
producing MAA and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an MAA pathway and an isopropanol pathway, the MAA
pathway including a first set of exogenous nucleic acids encoding
MAA pathway enzymes expressed in a sufficient amount to produce
MAA, the first set of exogenous nucleic acids encoding a succinate
reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA mutase; a
3-hydroxyisobutyryl-CoA transferase, a 3-hydroxyisobutyryl-CoA
synthetase or a 3-hydroxyisobutyryl-CoA hydrolase; and a
3-hydroxyisobutyrate dehydratase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase, an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0251] In one embodiment, the invention provides a method for
producing MAA and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an MAA pathway and an isopropanol pathway, the MAA
pathway including a first set of exogenous nucleic acids encoding
MAA pathway enzymes expressed in a sufficient amount to produce
MAA, the first set of exogenous nucleic acids encoding a succinate
reductase; a 4-hydroxybutyrate dehydrogenase; a
4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA
synthetase; a 4-hydroxybutyryl-CoA mutase; a
3-hydroxyisobutyryl-CoA dehydratase; and a methacrylyl-CoA
transferase, a methacrylyl-CoA synthetase or a methacrylyl-CoA
hydrolase, and the isopropanol pathway comprising a second set of
exogenous nucleic acids encoding isopropanol pathway enzymes
expressed in a sufficient amount to produce isopropanol, the second
set of exogenous nucleic acids encoding an acetyl-CoA acetyl
thiolase; an acetoacetyl-CoA transferase or an acetoacetyl-CoA
hydrolase or an acetoacetyl-CoA synthetase; an acetoacetate
decarboxylase; and an isopropanol dehydrogenase.
[0252] In one embodiment, the invention provides a method for
producing MAA and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an MAA pathway and an isopropanol pathway, the MAA
pathway including a first set of exogenous nucleic acids encoding
MAA pathway enzymes expressed in a sufficient amount to produce
MAA, the first set of exogenous nucleic acids encoding a succinate
reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyrate
kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA
mutase; a 3-hydroxyisobutyryl-CoA transferase, a
3-hydroxyisobutyryl-CoA synthetase or a 3-hydroxyisobutyryl-CoA
hydrolase; and a 3-hydroxyisobutyrate dehydratase, and the
isopropanol pathway comprising a second set of exogenous nucleic
acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding an acetyl-CoA acetyl thiolase; an
acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase; and an
isopropanol dehydrogenase.
[0253] In one embodiment, the invention provides a method for
producing MAA and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an MAA pathway and an isopropanol pathway, the MAA
pathway including a first set of exogenous nucleic acids encoding
MAA pathway enzymes expressed in a sufficient amount to produce
MAA, the first set of exogenous nucleic acids encoding a succinate
reductase; a 4-hydroxybutyrate dehydrogenase; a 4 hydroxybutyrate
kinase; a phosphotrans-4-hydroxybutyrylase; a 4-hydroxybutyryl-CoA
mutase; a 3-hydroxyisobutyryl-CoA dehydratase; and a
methacrylyl-CoA transferase, a methacrylyl-CoA synthetase or a
methacrylyl-CoA hydrolase, and the isopropanol pathway comprising a
second set of exogenous nucleic acids encoding isopropanol pathway
enzymes expressed in a sufficient amount to produce isopropanol,
the second set of exogenous nucleic acids encoding an acetyl-CoA
acetyl thiolase; an acetoacetyl-CoA transferase or an
acetoacetyl-CoA hydrolase or an acetoacetyl-CoA synthetase; an
acetoacetate decarboxylase; and an isopropanol dehydrogenase.
[0254] In one embodiment, the invention provides a method for
producing MAA and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an MAA pathway and an isopropanol pathway, the MAA
pathway including a first set of exogenous nucleic acids encoding
MAA pathway enzymes expressed in a sufficient amount to produce
MAA, the first set of exogenous nucleic acids encoding a
methylmalonyl-CoA mutase; a methylmalonyl-CoA reductase (aldehyde
forming); a 3-hydroxyisobutyrate dehydrogenase; and a
3-hydroxyisobutyrate dehydratase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase; an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0255] In one embodiment, the invention provides a method for
producing MAA and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an MAA pathway and an isopropanol pathway, the MAA
pathway including a first set of exogenous nucleic acids encoding
MAA pathway enzymes expressed in a sufficient amount to produce
MAA, the first set of exogenous nucleic acids encoding a
methylmalonyl-CoA mutase; a methylmalonyl-CoA epimerase; a
methylmalonyl-CoA transferase, a methylmalonyl-CoA synthetase, or a
methylmalonyl-CoA hydrolase; a methylmalonate reductase; a
3-hydroxyisobutyrate dehydrogenase; and a 3-hydroxyisobutyrate
dehydratase, and the isopropanol pathway comprising a second set of
exogenous nucleic acids encoding isopropanol pathway enzymes
expressed in a sufficient amount to produce isopropanol, the second
set of exogenous nucleic acids encoding an acetyl-CoA acetyl
thiolase; an acetoacetyl-CoA transferase or an acetoacetyl-CoA
hydrolase or an acetoacetyl-CoA synthetase; an acetoacetate
decarboxylase; and an isopropanol dehydrogenase.
[0256] In one embodiment, the invention provides a method for
producing MAA and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an MAA pathway and an isopropanol pathway, the MAA
pathway including a first set of exogenous nucleic acids encoding
MAA pathway enzymes expressed in a sufficient amount to produce
MAA, the first set of exogenous nucleic acids encoding a
methylmalonyl-CoA mutase; a methylmalonyl-CoA transferase, a
methylmalonyl-CoA synthetase or a methylmalonyl-CoA hydrolase; a
methylmalonate reductase; a 3-hydroxyisobutyrate dehydrogenase; and
a 3-hydroxyisobutyrate dehydratase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase; an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0257] In one embodiment, the invention provides a method for
producing MAA and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an MAA pathway and an isopropanol pathway, the MAA
pathway including a first set of exogenous nucleic acids encoding
MAA pathway enzymes expressed in a sufficient amount to produce
MAA, the first set of exogenous nucleic acids a methylmalonyl-CoA
mutase; a methylmalonyl-CoA reductase (alcohol forming); and a
3-hydroxyisobutyrate dehydratase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase; an acetoacetate decarboxylase; and an isopropanol
dehydrogenase.
[0258] In a further aspect of the above embodiments, the microbial
organism has an acetyl-CoA pathway having a third set of exogenous
nucleic acids encoding acetyl-CoA pathway enzymes expressed in a
sufficient amount to produce acetyl-CoA, the third set of exogenous
nucleic acids encoding a pyruvate kinase; and a pyruvate
dehydrogenase or a pyruvate ferredoxin oxidoreductase; or a
pyruvate formate lyase, a pyruvate formate lyase activating enzyme
and a formate dehydrogenase.
[0259] In another further embodiment, the microbial organism has a
succinyl-CoA pathway having a third set of exogenous nucleic acids
encoding succinyl-CoA pathway enzymes expressed in a sufficient
amount to produce succinyl-CoA, the third set of exogenous nucleic
acids encoding a PEP carboxykinase, a PEP carboxylase, a malate
dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA
transferase and a succinyl-CoA synthetase. In a further aspect, the
third set of exogenous nucleic acids further encodes a
methylmalonyl-CoA epimerase, a pyruvate carboxylase or a
methylmalonyl-CoA carboxytransferase.
[0260] In one embodiment, the invention provides a method for
producing 14-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 14-BDO pathway and an isopropanol pathway, the
14-BDO pathway including a first set of exogenous nucleic acids
encoding 14-BDO pathway enzymes expressed in a sufficient amount to
produce 14-BDO, the first set of exogenous nucleic acids encoding a
PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a
fumarase, a fumarate reductase, a succinyl-CoA transferase, a
succinyl-CoA synthetase, a pyruvate carboxylase, a
methylmalonyl-CoA carboxytransferase, a succinyl-CoA reductase, a
succinate reductase, a 4-hydroxybutyrate dehydrogenase, a
4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA
synthetase, a 4-hydroxybutyryl-CoA reductase (aldehyde-forming), a
4-hydroxybutyraldehyde reductase, a 4-hydroxybutyrate reductase; a
4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, a
4-hydroxybutyryl-phosphate reductase, a 4-hydroxybutyryl-CoA
reductase (alcohol-forming), and a 4-hydroxybutyraldehyde
reductase, and the isopropanol pathway comprising a second set of
exogenous nucleic acids encoding isopropanol pathway enzymes
expressed in a sufficient amount to produce isopropanol, the second
set of exogenous nucleic acids encoding a pyruvate kinase, a
pyruvate dehydrogenase, a pyruvate ferredoxin oxidoreductase, a
pyruvate formate lyase, a pyruvate formate lyase activating enzyme,
a formate dehydrogenase, an acetyl-CoA acetyl thiolase, an
acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase, and an
isopropanol dehydrogenase.
[0261] In one embodiment, the invention provides a method for
producing 13-BDO and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an 13-BDO pathway and an isopropanol pathway, the
13-BDO pathway including a first set of exogenous nucleic acids
encoding 13-BDO pathway enzymes expressed in a sufficient amount to
produce 13-BDO, the first set of exogenous nucleic acids encoding
PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a
fumarase, a fumarate reductase, a succinyl-CoA transferase, a
succinyl-CoA synthetase, a pyruvate carboxylase, a
methylmalonyl-CoA carboxytransferase, a succinyl-CoA reductase, a
succinate reductase, a 4-hydroxybutyrate dehydrogenase, a
4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA
synthetase, a 4-hydroxybutyrate kinase, a
phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA
dehydratase, a crotonase, a 3-hydroxybutyryl-CoA reductase
(aldehyde forming), a 3-hydroxybutyraldehyde reductase, a
3-hydroxybutyryl-CoA transferase, a 3-hydroxybutyryl-CoA
synthetase, a 3-hydroxybutyryl-CoA hydrolase, a 3-hydroxybutyrate
reductase, and a 3-hydroxybutyryl-CoA reductase (alcohol-forming),
and the isopropanol pathway comprising a second set of exogenous
nucleic acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding a pyruvate kinase, a pyruvate
dehydrogenase, a pyruvate ferredoxin oxidoreductase, a pyruvate
formate lyase, a pyruvate formate lyase activating enzyme, a
formate dehydrogenase, an acetyl-CoA acetyl thiolase, an
acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase, and an
isopropanol dehydrogenase.
[0262] In one embodiment, the invention provides a method for
producing MAA and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an MAA pathway and an isopropanol pathway, the MAA
pathway including a first set of exogenous nucleic acids encoding
MAA pathway enzymes expressed in a sufficient amount to produce
MAA, the first set of exogenous nucleic acids encoding a PEP
carboxykinase, a PEP carboxylase, a malate dehydrogenase, a
fumarase, a fumarate reductase, a succinyl-CoA transferase, a
succinyl-CoA synthetase, a pyruvate carboxylase, a
methylmalonyl-CoA carboxytransferase, a succinyl-CoA reductase, a
succinate reductase, a 4-hydroxybutyrate dehydrogenase, a
4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA
synthetase, a 4-hydroxybutyrate kinase, a
phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA mutase, a
3-hydroxyisobutyryl-CoA transferase, a 3-hydroxyisobutyryl-CoA
synthetase, a 3-hydroxyisobutyryl-CoA hydrolase,
3-hydroxyisobutyryl-CoA dehydratase, methacrylyl-CoA transferase,
methacrylyl-CoA synthetase, methacrylyl-CoA hydrolase and a
3-hydroxyisobutyrate dehydratase, and the isopropanol pathway
comprising a second set of exogenous nucleic acids encoding
isopropanol pathway enzymes expressed in a sufficient amount to
produce isopropanol, the second set of exogenous nucleic acids
encoding a pyruvate kinase, a pyruvate dehydrogenase, a pyruvate
ferredoxin oxidoreductase, a pyruvate formate lyase, a pyruvate
formate lyase activating enzyme, a formate dehydrogenase, an
acetyl-CoA acetyl thiolase, an acetoacetyl-CoA transferase, an
acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, an
acetoacetate decarboxylase, and an isopropanol dehydrogenase.
[0263] In one embodiment, the invention provides a method for
producing MAA and isopropanol that includes culturing a
non-naturally occurring microbial organism including a microbial
organism having an MAA pathway and an isopropanol pathway, the MAA
pathway including a first set of exogenous nucleic acids encoding
MAA pathway enzymes expressed in a sufficient amount to produce
MAA, the first set of exogenous nucleic acids encoding a PEP
carboxykinase, a PEP carboxylase, a malate dehydrogenase, a
fumarase, a fumarate reductase, a succinyl-CoA transferase, a
succinyl-CoA synthetase, a pyruvate carboxylase, a
methylmalonyl-CoA carboxytransferase, a methylmalonyl-CoA mutase, a
methylmalonyl-CoA epimerase, a methylmalonyl-CoA transferase, a
methylmalonyl-CoA synthetase, a methylmalonyl-CoA hydrolase, a
methylmalonate reductase, a methylmalonyl-CoA reductase (aldehyde
forming), a 3-hydroxyisobutyrate dehydrogenase, a methylmalonyl-CoA
reductase (alcohol forming) and a 3-hydroxyisobutyrate dehydratase,
and the isopropanol pathway comprising a second set of exogenous
nucleic acids encoding isopropanol pathway enzymes expressed in a
sufficient amount to produce isopropanol, the second set of
exogenous nucleic acids encoding a pyruvate kinase, a pyruvate
dehydrogenase, a pyruvate ferredoxin oxidoreductase, a pyruvate
formate lyase, a pyruvate formate lyase activating enzyme, a
formate dehydrogenase, an acetyl-CoA acetyl thiolase, an
acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an
acetoacetyl-CoA synthetase, an acetoacetate decarboxylase, and an
isopropanol dehydrogenase.
[0264] In a further aspect of each of the above embodiments, the
exogenous nucleic acid is a heterologous nucleic acid.
[0265] In a further aspect of each of the above embodiments, the
conditions include substantially anaerobic culture conditions.
[0266] Suitable purification and/or assays to test for the
production of n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA
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. Various
alcohols can be quantified by gas chromatography by using a flame
ionization detector as described in Atsumi et al. Metab Eng (2007)
and Hanai et al. Appl Environ Microbiol 73:7814-7818 (2007).
[0267] The n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA 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.
[0268] 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
n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA producers can be
cultured for the biosynthetic production of n-propanol,
isopropanol, 14-BDO, 13-BDO and/or MAA.
[0269] For the production of n-propanol, isopropanol, 14-BDO,
13-BDO and/or MAA, the recombinant strains are cultured in a medium
with a carbon source and other essential nutrients. It is 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
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
U.S. publication 2009/0047719, filed Aug. 10, 2007. Fermentations
can be performed in a batch, fed-batch or continuous manner, as
disclosed herein.
[0270] If desired, the pH of the medium can be maintained at a
desired pH, in particular neutral pH, such as a pH of around 7 by
addition of a base, such as NaOH or other bases, or acid, as needed
to maintain the culture medium at a desirable pH. The growth rate
can be determined by measuring optical density using a
spectrophotometer (600 nm), and the glucose uptake rate by
monitoring carbon source depletion over time.
[0271] The growth medium can include, for example, any carbohydrate
source which can supply a source of carbon to the non-naturally
occurring microorganism. Such sources include, for example, sugars
such as glucose, xylose, arabinose, galactose, mannose, fructose,
sucrose and starch. Other sources of carbohydrate include, for
example, renewable feedstocks and biomass. Exemplary types of
biomasses that can be used as feedstocks in the methods of the
invention include cellulosic biomass, hemicellulosic biomass and
lignin feedstocks or portions of feedstocks. Such biomass
feedstocks contain, for example, carbohydrate substrates useful as
carbon sources such as glucose, xylose, arabinose, galactose,
mannose, fructose and starch. Given the teachings and guidance
provided herein, those skilled in the art will understand that
renewable feedstocks and biomass other than those exemplified above
also can be used for culturing the microbial organisms of the
invention for the production of n-propanol, isopropanol, 14-BDO,
13-BDO and/or MAA.
[0272] In addition to renewable feedstocks such as those
exemplified above, the n-propanol and isopropanol, 14-BDO and
isopropanol, 13-BDO and isopropanol or MAA and isopropanol
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
n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA producing
organisms to provide a metabolic pathway for utilization of syngas
or other gaseous carbon source.
[0273] 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.
[0274] The Wood-Ljungdahl pathway catalyzes the conversion of CO
and H.sub.2 to acetyl-CoA and other products such as acetate.
Organisms capable of utilizing CO and syngas also generally have
the capability of utilizing CO.sub.2 and CO.sub.2/H.sub.2 mixtures
through the same basic set of enzymes and transformations
encompassed by the Wood-Ljungdahl pathway. H.sub.2-dependent
conversion of CO.sub.2 to acetate by microorganisms was recognized
long before it was revealed that CO also could be used by the same
organisms and that the same pathways were involved. Many acetogens
have been shown to grow in the presence of CO.sub.2 and produce
compounds such as acetate as long as hydrogen is present to supply
the necessary reducing equivalents (see for example, Drake,
Acetogenesis, pp. 3-60 Chapman and Hall, New York, (1994)). This
can be summarized by the following equation:
2CO.sub.2+4H.sub.2+nADP+nPi.fwdarw.CH.sub.3COOH+2H.sub.2O+nATP
[0275] 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.
[0276] 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 an n-propanol, an isopropanol, a 14-BDO,
a 13-BDO and/or a MAA 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.
[0277] Additionally, the reductive (reverse) tricarboxylic acid
cycle is 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 an n-propanol, an isopropanol, a 14-BDO, a
13-BDO and/or a MAA 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 an n-propanol, an
isopropanol, a 14-BDO, a 13-BDO and/or a MAA 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 the complete
reductive TCA pathway will confer syngas utilization ability.
[0278] 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, n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA and
any of the intermediate metabolites in the n-propanol, isopropanol,
14-BDO, 13-BDO and/or MAA 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 n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA biosynthetic
pathways. Accordingly, the invention provides a non-naturally
occurring microbial organism that produces and/or secretes
n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA when grown on a
carbohydrate or other carbon source and produces and/or secretes
any of the intermediate metabolites shown in the n-propanol,
isopropanol, 14-BDO, 13-BDO and/or MAA pathway when grown on a
carbohydrate or other carbon source. The n-propanol and
isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or MAA
and isopropanol producing microbial organisms of the invention can
initiate synthesis from an intermediate, for example, succinyl-CoA,
propionyl-CoA and/or acetyl-CoA.
[0279] 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 an n-propanol, an isopropanol, a 14-BDO, a 13-BDO and/or a
MAA pathway enzyme or protein in sufficient amounts to produce
n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA. It is
understood that the microbial organisms of the invention are
cultured under conditions sufficient to produce n-propanol,
isopropanol, 14-BDO, 13-BDO and/or MAA. Following the teachings and
guidance provided herein, the non-naturally occurring microbial
organisms of the invention can achieve biosynthesis of n-propanol,
isopropanol, 14-BDO, 13-BDO and/or MAA resulting in intracellular
concentrations between about 0.1-200 mM or more. Generally, the
intracellular concentration of n-propanol, isopropanol, 14-BDO,
13-BDO and/or MAA 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.
[0280] 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 conditions, the n-propanol,
isopropanol, 14-BDO, 13-BDO and/or MAA producers can synthesize
n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA 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, n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA
producing microbial organisms can produce n-propanol, isopropanol,
14-BDO, 13-BDO and/or MAA intracellularly and/or secrete the
product into the culture medium.
[0281] 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.
[0282] As described herein, one exemplary growth condition for
achieving biosynthesis of n-propanol and isopropanol, 14-BDO and
isopropanol, 13-BDO and isopropanol or MAA and isopropanol includes
anaerobic culture or fermentation conditions. In certain
embodiments, the non-naturally occurring microbial organisms of the
invention can be sustained, cultured or fermented under anaerobic
or substantially anaerobic conditions. Briefly, anaerobic
conditions refers to an environment devoid of oxygen. Substantially
anaerobic conditions include, for example, a culture, batch
fermentation or continuous fermentation such that the dissolved
oxygen concentration in the medium remains between 0 and 10% of
saturation. Substantially anaerobic conditions also includes
growing or resting cells in liquid medium or on solid agar inside a
sealed chamber maintained with an atmosphere of less than 1%
oxygen. The percent of oxygen can be maintained by, for example,
sparging the culture with an N.sub.2/CO.sub.2 mixture or other
suitable non-oxygen gas or gases.
[0283] The culture conditions described herein can be scaled up and
grown continuously for manufacturing of n-propanol and isopropanol,
14-BDO and isopropanol, 13-BDO and isopropanol or MAA and
isopropanol. 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 n-propanol,
isopropanol, 14-BDO, 13-BDO and/or MAA. Generally, and as with
non-continuous culture procedures, the continuous and/or
near-continuous production of n-propanol, isopropanol, 14-BDO,
13-BDO and/or MAA will include culturing a non-naturally occurring
n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and
isopropanol or MAA and isopropanol producing organism of the
invention in sufficient nutrients and medium to sustain and/or
nearly sustain growth in an exponential phase. Continuous culture
under such conditions can be include, for example, growth for 1
day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous
culture can include longer time periods of 1 week, 2, 3, 4 or 5 or
more weeks and up to several months. Alternatively, organisms of
the invention can be cultured for hours, if suitable for a
particular application. It is to be understood that the continuous
and/or near-continuous culture conditions also can include all time
intervals in between these exemplary periods. It is further
understood that the time of culturing the microbial organism of the
invention is for a sufficient period of time to produce a
sufficient amount of product for a desired purpose.
[0284] Fermentation procedures are well known in the art. Briefly,
fermentation for the biosynthetic production of n-propanol and
isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or MAA
and isopropanol 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.
[0285] In addition to the above fermentation procedures using the
n-propanol, isopropanol, 14-BDO, 13-BDO and/or MAA producers of the
invention for continuous production of substantial quantities of
n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and
isopropanol or MAA and isopropanol, the n-propanol, isopropanol,
14-BDO, 13-BDO and/or MAA producers also can be, for example,
simultaneously subjected to chemical synthesis procedures to
convert the product to other compounds or the product can be
separated from the fermentation culture and sequentially subjected
to chemical conversion to convert the product to other compounds,
if desired.
[0286] In addition to the culturing and fermentation conditions
described herein, growth condition for achieving biosynthesis of
n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and
isopropanol or MAA and isopropanol 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 means a
compound that acts as an osmolyte and helps a microbial organism as
described herein survive osmotic stress. Osmoprotectants include,
but are not limited to, betaines, amino acids, and the sugar
trehalose. Non-limiting examples of such are glycine betaine,
praline betaine, dimethylthetin, dimethylslfonioproprionate,
3-dimethylsulfonio-2-methylproprionate, pipecolic acid,
dimethylsulfonioacetate, choline, L-carnitine and ectoine. In one
aspect, the osmoprotectant is glycine betaine. It is understood to
one of ordinary skill in the art that the amount and type of
osmoprotectant suitable for protecting a microbial organism
described herein from osmotic stress will depend on the microbial
organism used. The amount of osmoprotectant in the culturing
conditions can be, for example, no more than about 0.1 mM, no more
than about 0.5 mM, no more than about 1.0 mM, no more than about
1.5 mM, no more than about 2.0 mM, no more than about 2.5 mM, no
more than about 3.0 mM, no more than about 5.0 mM, no more than
about 7.0 mM, no more than about 10 mM, no more than about 50 mM,
no more than about 100 mM or no more than about 500 mM.
[0287] 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 n-propanol, isopropanol, 14-BDO, 13-BDO
and/or MAA.
[0288] 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.
[0289] 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.
[0290] 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.
[0291] 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.
[0292] 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.
[0293] 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.
[0294] 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.
[0295] 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..
[0296] 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.
[0297] 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)).
[0298] 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.
[0299] It is understood that modifications which do not
substantially affect the activity of the various embodiments of
this invention are also provided within the definition of the
invention provided herein. Accordingly, the following examples are
intended to illustrate but not limit the present invention.
Example I
Pathways for Co-Production of n-Propanol and Isopropanol from
Glucose
[0300] This example describes exemplary pathways for co-production
of n-propanol and isopropanol.
[0301] Novel pathways for co-producing n-propanol and isopropanol
and related products are described herein. This invention provides
four alternate methods for co-production of n-propanol and
isopropanol. The production of isopropanol in E. coli has been
described previously (Hanai et al., Appl Environ Microbiol
73:7814-7818 (2007)). Briefly, acetyl CoA is converted into
acetoacetyl CoA, transformed into acetoacetate, decarboxylated to
form acetone and then reduced to form isopropanol (FIGS. 1-4). The
microbial organisms and methods described herein combine this known
route with four novel pathways for synthesizing n-propanol. This
co-production will provide completely redox balanced routes for
production of the C3 alcohols, i.e. n-propanol and isopropanol,
allowing for anaerobic production as opposed to the requirement of
oxygen if isopropanol is produced solely via acetone as described
by Hanai et al., supra. One advantage to the co-production of
n-propanol and isopropanol using any of the pathways described
herein is that the maximum theoretical yield of the C3 alcohols is
afforded:
1 glucose.fwdarw.1.33C.sub.3H.sub.8O+2CO.sub.2+0.67H.sub.2O
[0302] Furthermore, all of these pathways have a net positive yield
of ATP.
Production of Isopropanol Utilizing Acetyl-CoA
[0303] Isopropanol production is achieved via conversion of
acetyl-CoA by an acetoacetyl-CoA thiolase, an acetoacetyl-CoA
transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA
synthetase, an acetoacetate decarboxylase, and an isopropanol
dehydrogenase as exemplified in FIGS. 1-4. Isopropanol production
has been described for recombinant E. coli following expression of
two heterologous genes from C. acetobutylicum (thl and adc encoding
acetoacetyl-CoA thiolase and acetoacetate decarboxylase,
respectively) and one from C. beijerinckii (adh encoding a
secondary alcohol dehydrogenase), along with the increased
expression of the native atoA and atoD genes which encode
acetoacetyl-CoA:acetate:CoA transferase activity (Hanai et al.,
Appl Environ Microbiol 73:7814-7818 (2007)). The conversion of
acetoacetyl-CoA to acetoacetate can alternately be catalyzed by an
enzyme with acetoacetyl-CoA hydrolase or acetoacetyl-CoA synthetase
activities.
Acetoacetyl-CoA Thiolase
[0304] Acetoacetyl-CoA thiolase (also known as acetyl-CoA
acetyltransferase) converts two molecules of acetyl-CoA into one
molecule each of acetoacetyl-CoA and CoA. Exemplary acetoacetyl-CoA
thiolase enzymes include the gene products of atoB from E. coli
(Martin et al., Nat. Biotechnol 21:796-802 (2003)), thlA and thlB
from C. acetobutylicum (Hanai et al., Appl Environ Microbiol
73:7814-7818 (2007); Winzer et al., J. Mol. Microbiol Biotechnol
2:531-541 (2000), and ERG10 from S. cerevisiae Hiser et al., J.
Biol. Chem. 269:31383-31389 (1994)). These genes/proteins are
identified below in Table 1.
TABLE-US-00001 TABLE 1 Gene GenBank ID GI Number Organism AtoB
NP_416728 16130161 Escherichia coli ThlA NP_349476.1 15896127
Clostridium acetobutylicum ThlB NP_149242.1 15004782 Clostridium
acetobutylicum ERG10 NP_015297 6325229 Saccharomyces cerevisiae
Acetoacetyl-CoA Transferase
[0305] Acetoacetyl-CoA transferase catalyzes the conversion of
acetoacetyl-CoA to acetoacetate while transferring the CoA moiety
to a CoA acceptor molecule. Many transferases have broad
specificity and thus may utilize CoA acceptors as diverse as
acetate, succinate, propionate, butyrate, 2-methylacetoacetate,
3-ketohexanoate, 3-ketopentanoate, valerate, crotonate,
3-mercaptopropionate, propionate, vinylacetate, butyrate, among
others.
[0306] Acetoacetyl-CoA:acetate:CoA transferase converts
acetoacetyl-CoA and acetate to acetoacetate and acetyl-CoA.
Exemplary enzymes include the gene products of atoAD from E. coli
(Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007), ctfAB
from C. acetobutylicum (Jojima et al., Appl Microbiol Biotechnol
77:1219-1224 (2008), and ctfAB from Clostridium
saccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol
Biochem. 71:58-68 (2007)) are shown below in Table 2. A
succinyl-CoA:3-ketoacid CoA transferase (SCOT) can also catalyze
the conversion of the 3-ketoacyl-CoA, acetoacetyl-CoA, to the
3-ketoacid, acetoacetate. As opposed to acetoacetyl-CoA:acetate:CoA
transferase, SCOT employs succinate as the CoA acceptor instead of
acetate. Exemplary succinyl-CoA:3:ketoacid-CoA transferases are
present in Helicobacter pylori (Corthesy-Theulaz et al., J Biol
Chem 272:25659-25667 (1997)), Bacillus subtilis (Stols et al.,
Protein Expr Purif 53:396-403 (2007)), and Homo sapiens (Fukao et
al., Genomics 68:144-151 (2000); Tanaka et al., Mol Hum Reprod
8:16-23 (2002)). Yet another transferase capable of this conversion
is butyryl-CoA: acetoacetate CoA-transferase. Exemplary enzymes can
be found in Fusobacterium nucleatum (Barker et al., J Bacteriol
152(1):201-7 (1982)), Clostridium SB4 (Barker et al., J Biol Chem
253(4):1219-25 (1978)), and Clostridium acetobutylicum (Wiesenborn
et al., Appl Environ Microbiol 55(2):323-9 (1989)). Although
specific gene sequences were not provided for
butyryl-CoA:acetoacetate CoA-transferase in these references, the
genes FN0272 and FN0273 have been annotated as a
butyrate-acetoacetate CoA-transferase (Kapatral et al., J Bact
184(7) 2005-2018 (2002)). Homologs in Fusobacterium nucleatum such
as FN1857 and FN1856 also likely have the desired acetoacetyl-CoA
transferase activity. FN1857 and FN1856 are located adjacent to
many other genes involved in lysine fermentation and are thus very
likely to encode an acetoacetate:butyrate CoA transferase
(Kreimeyer, et al., J Biol Chem 282 (10) 7191-7197 (2007)).
Additional candidates from Porphyrmonas gingivalis and
Thermoanaerobacter tengcongensis can be identified in a similar
fashion (Kreimeyer, et al., J Biol Chem 282 (10) 7191-7197 (2007)).
These genes/proteins are identified below in Table 2.
TABLE-US-00002 TABLE 2 Gene GenBank ID GI Number Organism AtoA
NP_416726.1 2492994 Escherichia coli AtoD NP_416725.1 2492990
Escherichia coli CtfA NP_149326.1 15004866 Clostridium
acetobutylicum CtfB NP_149327.1 15004867 Clostridium acetobutylicum
CtfA AAP42564.1 31075384 Clostridium saccharoperbutylacetonicum
CtfB AAP42565.1 31075385 Clostridium saccharoperbutylacetonicum
HPAG1_0676 YP_627417 108563101 Helicobacter pylori HPAG1_0677
YP_627418 108563102 Helicobacter pylori ScoA NP_391778 16080950
Bacillus subtilis ScoB NP_391777 16080949 Bacillus subtilis OXCT1
NP_000427 4557817 Homo sapiens OXCT2 NP_071403 11545841 Homo
sapiens FN0272 NP_603179.1 19703617 Fusobacterium nucleatum FN0273
NP_603180.1 19703618 Fusobacterium nucleatum FN1857 NP_602657.1
19705162 Fusobacterium nucleatum FN1856 NP_602656.1 19705161
Fusobacterium nucleatum PG1066 NP_905281.1 34540802 Porphyromonas
gingivalis W83 PG1075 NP_905290.1 34540811 Porphyromonas gingivalis
W83 TTE0720 NP_622378.1 20807207 Thermoanaerobacter tengcongensis
MB4 TTE0721 NP_622379.1 20807208 Thermoanaerobacter tengcongensis
MB4
Acetoacetyl-CoA Synthetase
[0307] A CoA synthetase can also catalyze the removal of the CoA
moiety from acetoacetyl-CoA. One candidate enzyme, ADP-forming
acetyl-CoA synthetase (ACD, EC 6.2.1.13), couples the conversion of
acyl-CoA esters to their corresponding acids with the concurrent
synthesis of ATP. Several enzymes with broad substrate
specificities have been described in the literature. ACD I from
Archaeoglobus fulgidus, encoded by AF1211, was shown to operate on
a variety of linear and branched-chain substrates including
acetyl-CoA, propionyl-CoA, butyryl-CoA, acetate, propionate,
butyrate, isobutyryate, isovalerate, succinate, fumarate,
phenylacetate, indoleacetate (Musfeldt et al., J Bacteriol
184:636-644 (2002)). The enzyme from Haloarcula marismortui
(annotated as a succinyl-CoA synthetase) accepts propionate,
butyrate, and branched-chain acids (isovalerate and isobutyrate) as
substrates, and was shown to operate in the forward and reverse
directions (Brasen et al., Arch Microbiol 182:277-287 (2004)). The
ACD encoded by PAE3250 from hyperthermophilic crenarchaeon
Pyrobaculum aerophilum showed the broadest substrate range of all
characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA
(preferred substrate) and phenylacetyl-CoA (Brasen et al., supra).
The enzymes from A. fulgidus, H. marismortui and P. aerophilum have
all been cloned, functionally expressed, and characterized in E.
coli (Musfeldt et al., supra; Brasen et al., supra). These
genes/proteins are identified below in Table 3.
TABLE-US-00003 TABLE 3 Gene GenBank ID GI Number Organism AF1211
NP_070039.1 11498810 Archaeoglobus fulgidus DSM 4304 scs
YP_135572.1 55377722 Haloarcula marismortui ATCC 43049 PAE3250
NP_560604.1 18313937 Pyrobaculum aerophilum str. IM2
[0308] Another candidate CoA synthetase is succinyl-CoA synthetase.
The sucCD genes of E. coli form a succinyl-CoA synthetase complex
which naturally catalyzes the formation of succinyl-CoA from
succinate with the concaminant consumption of one ATP, a reaction
which is reversible in vivo (Buck et al., Biochem. 24:6245-6252
(1985)). These genes/proteins are identified below in Table 4.
TABLE-US-00004 TABLE 4 Protein GenBank ID GI Number Organism sucC
NP_415256.1 16128703 Escherichia coli sucD AAC73823.1 1786949
Escherichia coli
[0309] Additional exemplary CoA-ligases include the rat
dicarboxylate-CoA ligase for which the sequence is yet
uncharacterized (Vamecq et al., Biochemical Journal 230:683-693
(1985)), either of the two characterized phenylacetate-CoA ligases
from P. chrysogenum (Lamas-Maceiras et al., Biochem. J. 395:147-155
(2005); Wang et al., Biochem Biophy Res Commun 360(2):453-458
(2007)), the phenylacetate-CoA ligase from Pseudomonas putida
(Martinez-Blanco et al., J. Biol. Chem. 265:7084-7090 (1990)), and
the 6-carboxyhexanoate-CoA ligase from Bacillus subtilis (Bower et
al., J. Bacteriol. 178(14):4122-4130 (1996)). Additional candidate
enzymes are acetoacetyl-CoA synthetases from Mus musculus (Hasegawa
et al., Biochim Biophys Acta 1779:414-419 (2008)) and Homo sapiens
(Ohgami et al., Biochem Pharmacol 65:989-994 (2003)) which
naturally catalyze the ATP-dependant conversion of acetoacetate
into acetoacetyl-CoA. These genes/proteins are identified below in
Table 5.
TABLE-US-00005 TABLE 5 Gene GenBank ID GI Number Organism phl
CAJ15517.1 77019264 Penicillium chrysogenum phlB ABS19624.1
152002983 Penicillium chrysogenum paaF AAC24333.2 22711873
Pseudomonas putida bioW NP_390902.2 50812281 Bacillus subtilis AACS
NP_084486.1 21313520 Mus musculus AACS NP_076417.2 31982927 Homo
sapiens
Acetoacetyl-CoA Hydrolase
[0310] Acetoacetyl-CoA can also be converted to acetoacetate by a
CoA hydrolase. Acetoacetyl-CoA hydrolase enzyme candidates include
acyl-CoA hydrolase, 3-hydroxyisobutyryl-CoA hydrolase, acetyl-CoA
hydrolase, and dicarboxylic acid thioesterase. A short-chain
acyl-CoA hydrolase in rat liver mitochondria was found to accept
acetoacetyl-CoA as a substrate; however, the gene associated with
this enzyme has not been identified to date (Svensson et al. Eur.
J. Biochem., 239:526-531 (1996)).
[0311] 3-Hydroxyisobutyryl-CoA hydrolase efficiently catalyzes the
conversion of 3-hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate
during valine degradation (Shimomura et al., J Biol Chem.
269:14248-14253 (1994)). Genes encoding this enzyme include hibch
of Rattus norvegicus (Shimomura et al., supra; Shimomura et al.,
Methods Enzymol. 324:229-240 (2000)) and Homo sapiens (Shimomura et
al., supra). The H. sapiens enzyme also accepts
3-hydroxybutyryl-CoA and 3-hydroxypropionyl-CoA as substrates
(Shimomura et al., supra). Candidate genes by sequence homology
include hibch of Saccharomyces cerevisiae and BC.sub.--2292 of
Bacillus cereus. These genes/proteins are identified below in Table
6.
TABLE-US-00006 TABLE 6 Gene GenBank ID GI Number Organism hibch
Q5XIE6.2 146324906 Rattus norvegicus hibch Q6NVY1.2 146324905 Homo
sapiens hibch P28817.2 2506374 Saccharomyces cerevisiae BC_2292
AP09256 29895975 Bacillus cereus
[0312] Several eukaryotic acetyl-CoA hydrolases (EC 3.1.2.1) have
broad substrate specificity and thus represent suitable candidate
enzymes. For example, the enzyme from Rattus norvegicus brain
(Robinson et al., Res. Commun. 71:959-965 (1976)) can react with
butyryl-CoA, hexanoyl-CoA and malonyl-CoA. Though its sequence has
not been reported, the enzyme from the mitochondrion of the pea
leaf also has a broad substrate specificity, with demonstrated
activity on acetyl-CoA, propionyl-CoA, butyryl-CoA, palmitoyl-CoA,
oleoyl-CoA, succinyl-CoA, and crotonyl-CoA (Zeiher et al., Plant.
Physiol. 94:20-27 (1990)). The acetyl-CoA hydrolase, ACH1, from S.
cerevisiae represents another candidate hydrolase (Buu et al., J.
Biol. Chem. 278:17203-17209 (2003)). These genes/proteins are
identified below in Table 7.
TABLE-US-00007 TABLE 7 Gene GenBank ID GI Number Organism acot12
NP_570103.1 18543355 Rattus norvegicus ACH1 NP_009538 6319456
Saccharomyces cerevisiae
[0313] Another candidate hydrolase is the human dicarboxylic acid
thioesterase, acot8, which exhibits activity on glutaryl-CoA,
adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA (Westin
et al., J Biol. Chem. 280:38125-38132 (2005)) and the closest E.
coli homolog, tesB, which can also hydrolyze a broad range of CoA
thioesters (Naggert et al., J Biol. Chem. 266:11044-11050 (1991)).
A similar enzyme has also been characterized in the rat liver
(Deana et al., Biochem. Int. 26:767-773 (1992)). Other potential E.
coli thioester hydrolases include the gene products of tesA (Bonner
et al., Chem. 247:3123-3133 (1972)), ybgC (Kuznetsova et al., FEMS
Microbiol Rev 29:263-279 (2005); and (Zhuang et al., FEBS Lett.
516:161-163 (2002)), paaI (Song et al., J Biol. Chem.
281:11028-11038 (2006)), and ybdB (Leduc et al., J Bacteriol.
189:7112-7126 (2007)). These genes/proteins are identified below in
Table 8.
TABLE-US-00008 TABLE 8 Gene GenBank ID GI Number Organism tesB
NP_414986 16128437 Escherichia coli acot8 CAA15502 3191970 Homo
sapiens acot8 NP_570112 51036669 Rattus norvegicus tesA NP_415027
16128478 Escherichia coli ybgC NP_415264 16128711 Escherichia coli
paaI NP_415914 16129357 Escherichia coli ybdB NP_415129 16128580
Escherichia coli
[0314] Yet another candidate hydrolase is the glutaconate
CoA-transferase from Acidaminococcus fermentans. This enzyme was
transformed by site-directed mutagenesis into an acyl-CoA hydrolase
with activity on glutaryl-CoA, acetyl-CoA and 3-butenoyl-CoA (Mack
et al., FEBS. Lett. 405:209-212 (1997)). This suggests that the
enzymes encoding succinyl-CoA:3-ketoacid-CoA transferases and
acetoacetyl-CoA:acetyl-CoA transferases may also serve as
candidates for this reaction step but would require certain
mutations to change their function. These genes/proteins are
identified below in Table 9.
TABLE-US-00009 TABLE 9 Gene GenBank ID GI Number Organism gctA
CAA57199 559392 Acidaminococcus fermentans gctB CAA57200 559393
Acidaminococcus fermentans
Acetoacetate Decarboxylase
[0315] Acetoacetate decarboxylase converts acetoacetate into carbon
dioxide and acetone. Exemplary acetoacetate decarboxylase enzymes
are encoded by the gene products of adc from C. acetobutylicum
(Petersen and Bennett, Appl Environ. Microbiol 56:3491-3498 (1990))
and adc from Clostridium saccharoperbutylacetonicum (Kosaka et al.,
Biosci. Biotechnol Biochem. 71:58-68 (2007)). The enzyme from C.
beijerinkii can be inferred from sequence similarity. These
genes/proteins are identified below in Table 10.
TABLE-US-00010 TABLE 10 Gene GenBank ID GI Number Organism Adc
NP_149328.1 15004868 Clostridium acetobutylicum Adc AAP42566.1
31075386 Clostridium saccharoperbutylacetonicum Adc YP_001310906.1
150018652 Clostridium beijerinckii
Isopropanol Dehydrogenase
[0316] The final step in the isopropanol synthesis pathway involves
the reduction of acetone to isopropanol. Exemplary alcohol
dehydrogenase enzymes capable of this transformation include adh
from C. beijerinckii (Hanai et al., Appl Environ Microbiol
73:7814-7818 (2007); Jojima et al., Appl Microbiol Biotechnol
77:1219-1224 (2008)) and adh from Thermoanaerobacter brockii (Hanai
et al., Appl Environ Microbiol 73:7814-7818 (2007); Peretz et al.,
Anaerobe 3:259-270 (1997)). Additional characterized enzymes
include alcohol dehydrogenases from Ralstonia eutropha (formerly
Alcaligenes eutrophus) (Steinbuchel and Schlegel et al., Eur. J.
Biochem, 141:555-564 (1984)) and Phytomonas species (Uttaro and
Opperdoes et al., Mol. Biochem. Parasitol. 85:213-219 (1997)).
These genes/proteins are identified below in Table 11.
TABLE-US-00011 TABLE 11 Gene GenBank ID GI Number Organism sadh
CAD36475 21615553 Rhodococcus rubber adhA AAC25556 3288810
Pyrococcus furiosus Adh P14941.1 113443 Thermoanaerobobacter
brockii Adh AAA23199.2 60592974 Clostridium beijerinckii
Production of n-Propanol Utilizing Propionyl-CoA
[0317] The pathways described herein for production of n-propanol
utilize reduction of propionyl-CoA into propionaldehyde by a
CoA-dependent aldehyde dehydrogenase that is then reduced further
to form n-propanol (FIGS. 1-4). This conversion is carried out by
two different enzymes: an aldehyde and alcohol dehydrogenase or in
one step by a bifunctional aldehyde/alcohol dehydrogenase.
Alternatively, propionyl CoA can be converted into propionyl
phosphate and then transformed into propionaldehyde by an acyl
phosphate reductase.
Propionaldehyde Dehydrogenase and Propanol Dehydrogenase
[0318] The conversion of propionyl-CoA to propanol is catalyzed by
either a bifunctional enzyme that has both the CoA-dependent
aldehyde dehydrogenase and the alcohol dehydrogenase activities or
by two different enzymes with the aldehyde and alcohol
dehydrogenase activities.
[0319] Exemplary two-step oxidoreductases that convert an acyl-CoA
to alcohol include those that transform substrates such as
acetyl-CoA to ethanol (e.g., adhE from E. coli) (Kessler, FEBS.
Lett. 281:59-63 (1991)) and butyryl-CoA to butanol (e.g. adhE2 from
C. acetobutylicum). (Fontaine et al., J. Bacteriol. 184:821-830
(2002)). In addition to reducing acetyl-CoA to ethanol, the enzyme
encoded by adhE in Leuconostoc mesenteroides has been shown to
oxidize the branched chain compound isobutyraldehyde to
isobutyryl-CoA (Kazahaya, Microbiol. 18:43-55 (1972); and Koo et
al., Biotechnol Lett. 27:505-510 (2005)). These genes/proteins are
identified below in Table 12.
TABLE-US-00012 TABLE 12 Gene GenBank ID GI Number Organism adhE
NP_415757.1 16129202 Escherichia coli adhE2 AAK09379.1 12958626
Clostridium acetobutylicum adhE AAV66076.1 55818563 Leuconostoc
mesenteroides
[0320] Another exemplary enzyme can convert malonyl-CoA to 3-HP. An
NADPH-dependent enzyme with this activity has been characterized in
Chloroflexus aurantiacus where it participates in the
3-hydroxypropionate cycle (Hugler, J. Bacteriol. 184:2404-2410
(2002); and Strauss, Eur. J. Biochem. 215:633-643 (1993)). This
enzyme, with a mass of 300 kDa, is highly substrate-specific and
shows little sequence similarity to other known oxidoreductases
(Hugler, J. Bacteriol. 184:2404-2410 (2002)). No enzymes in other
organisms have been shown to catalyze this specific reaction;
however there is bioinformatic evidence that other organisms may
have similar pathways (Klatt, Environ. Microbiol. 9:2067-2078
(2007)). Enzyme candidates in other organisms including Roseiflexus
castenholzii, Erythrobacter sp. NAP1 and marine gamma
proteobacterium HTCC2080 can be inferred by sequence similarity.
These genes/proteins are identified below in Table 13.
TABLE-US-00013 TABLE 13 Gene GenBank ID GI Number Organism mcr
AAS20429.1 42561982 Chloroflexus aurantiacus Rcas_2929
YP_001433009.1 156742880 Roseiflexus castenholzii NAP1_02720
ZP_01039179.1 85708113 Erythrobacter sp. NAP1 MGP2080_00535
ZP_01626393.1 119504313 marine gamma proteobacterium HTCC2080
[0321] Longer chain acyl-CoA molecules can be reduced by enzymes
such as the jojoba (Simmondsia chinensis) FAR which encodes an
alcohol-forming fatty acyl-CoA reductase. Its overexpression in E.
coli resulted in FAR activity and the accumulation of fatty alcohol
(Metz, Plant Physiology 122:635-644 (2000). These genes/proteins
are identified below in Table 14.
TABLE-US-00014 TABLE 14 Gene GenBank ID GI Number Organism FAR
AAD38039.1 5020215 Simmondsia chinensis
[0322] Several acyl-CoA dehydrogenases are capable of reducing an
acyl-CoA to its corresponding aldehyde. Exemplary genes that encode
such enzymes include the Acinetobacter calcoaceticus acr1 encoding
a fatty acyl-CoA reductase, (Reiser, Journal of Bacteriology
179:2969-2975 (1997)) the Acinetobacter sp. M-1 fatty acyl-CoA
reductase, (Ishige et al., Appl. Environ. Microbiol. 68:1192-1195
(2002)) and a CoA- and NADP-dependent succinate semialdehyde
dehydrogenase encoded by the sucD gene in Clostridium kluyveri
(Sohling, J. Bacteriol. 178:871-880 (1996)). SucD of P. gingivalis
is another succinate semialdehyde dehydrogenase (Takahashi, J.
Bacteriol 182:4704-4710 (2000)). The enzyme acylating acetaldehyde
dehydrogenase in Pseudomonas sp, encoded by bphG, is yet another
candidate as it has been demonstrated to oxidize and acylate
acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde and
formaldehyde (Powlowski, J. Bacteriol. 175:377-385 (1993)). In
addition to reducing acetyl-CoA to ethanol, the enzyme encoded by
adhE in Leuconostoc mesenteroides has been shown to oxidize the
branched chain compound isobutyraldehyde to isobutyryl-CoA
(Kazahaya, J. Gen. Appl. Microbiol. 18:43-55 (1972); and Koo et
al., Biotechnol Lett. 27:505-510 (2005)). These genes/proteins are
identified below in Table 15.
TABLE-US-00015 TABLE 15 Gene GenBank ID GI Number Organism acr1
YP_047869.1 50086359 Acinetobacter calcoaceticus acr1 AAC45217
1684886 Acinetobacter baylyi acr1 BAB85476.1 18857901 Acinetobacter
sp. Strain M-1 sucD P38947.1 172046062 Clostridium kluyveri sucD
NP_904963.1 34540484 Porphyromonas gingivalis bphG BAA03892.1
425213 Pseudomonas sp adhE AAV66076.1 55818563 Leuconostoc
mesenteroides
[0323] An additional enzyme type that converts an acyl-CoA to its
corresponding aldehyde is malonyl-CoA reductase which transforms
malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is a key
enzyme in autotrophic carbon fixation via the 3-hydroxypropionate
cycle in thermoacidophilic archaeal bacteria (Berg, Science
318:1782-1786 (2007); and Thauer, Science 318:1732-1733 (2007)).
The enzyme utilizes NADPH as a cofactor and has been characterized
in Metallosphaera and Sulfolobus spp. (Alber et al., J. Bacteriol.
188:8551-8559 (2006); and Hugler, J. Bacteriol. 184:2404-2410
(2002)). The enzyme is encoded by Msed.sub.--0709 in Metallosphaera
sedula (Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Berg,
Science 318:1782-1786 (2007)). A gene encoding a malonyl-CoA
reductase from Sulfolobus tokodaii was cloned and heterologously
expressed in E. coli (Alber et al., J. Bacteriol 188:8551-8559
(2006). This enzyme has also been shown to catalyze the conversion
of methylmalonyl-CoA to its corresponding aldehyde (WO2007141208
(2007)). Although the aldehyde dehydrogenase functionality of these
enzymes is similar to the bifunctional dehydrogenase from
Chloroflexus aurantiacus, there is little sequence similarity. Both
malonyl-CoA reductase enzyme candidates have high sequence
similarity to aspartate-semialdehyde dehydrogenase, an enzyme
catalyzing the reduction and concurrent dephosphorylation of
aspartyl-4-phosphate to aspartate semialdehyde. Additional gene
candidates can be found by sequence homology to proteins in other
organisms including Sulfolobus solfataricus and Sulfolobus
acidocaldarius and have been listed below. Yet another candidate
for CoA-acylating aldehyde dehydrogenase is the ald gene from
Clostridium beijerinckii (Toth, Appl. Environ. Microbiol.
65:4973-4980 (1999). This enzyme has been reported to reduce
acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This
gene is very similar to cutE that encodes acetaldehyde
dehydrogenase of Salmonella typhimurium and E. coli (Toth, Appl.
Environ. Microbiol. 65:4973-4980 (1999). These genes/proteins are
identified below in Table 16.
TABLE-US-00016 TABLE 16 Gene GenBank ID GI Number Organism
Msed_0709 YP_001190808.1 146303492 Metallosphaera sedula mcr
NP_378167.1 15922498 Sulfolobus tokodaii asd-2 NP_343563.1 15898958
Sulfolobus solfataricus Saci 2370 YP_256941.1 70608071 Sulfolobus
acidocaldarius Ald AAT66436 49473535 Clostridium beijerinckii eutE
AAA80209 687645 Salmonella typhimurium eutE P77445 2498347
Escherichia coli
[0324] Exemplary genes encoding enzymes that catalyze the
conversion of an aldehyde to alcohol (i.e., alcohol dehydrogenase
or equivalently aldehyde reductase) include alrA encoding a
medium-chain alcohol dehydrogenase for C2-C14, (Tani, Appl.
Environ. Microbiol. 66:5231-5235 (2000)) ADH2 from Saccharomyces
cerevisiae, (Atsumi, Nature 451:86-89 (2008)) yqhD from E. coli
which has preference for molecules longer than C3, (Sulzenbacher et
al., Journal of Molecular Biology 342:489-502 (2004)) and bdh I and
bdh II from C. acetobutylicum which converts butyraldehyde into
butanol (Walter, Journal of Bacteriology 174: 7149-7158 (1992)).
The gene product of yqhD catalyzes the reduction of acetaldehyde,
malondialdehyde, propionaldehyde, butyraldehyde, and acrolein using
NADPH as the cofactor (Perez, J. Biol. Chem. 283:7346-7353 (2008)).
ADH1 from Zymomonas mobilis has been demonstrated to have activity
on a number of aldehydes including formaldehyde, acetaldehyde,
propionaldehyde, butyraldehyde, and acrolein (Kinoshita, Appl.
Microbiol. Biotechnol. 22:249-254 (1985)). These genes/proteins are
identified below in Table 17.
TABLE-US-00017 TABLE 17 Gene GenBank ID GI Number Organism alrA
BAB12273.1 9967138 Acinetobacter sp. Strain M-1 ADH2 NP_014032.1
6323961 Saccharomyces cerevisiae yqhD NP_417484.1 16130909
Escherichia coli bdh I NP_349892.1 15896543 Clostridium
acetobutylicum bdh II NP_349891.1 15896542 Clostridium
acetobutylicum adhA YP_162971.1 56552132 Zymomonas mobilis
[0325] Enzymes exhibiting 3-hydroxybutyraldehyde reductase activity
(EC 1.1.1.61) also fall into this category. Such enzymes have been
characterized in Ralstonia eutropha, (Bravo J. Forensic Sci.
49:379-387 (2004)) Clostridium kluyveri (Wolff, Protein Expr. Pur
6:206-212 (1995)) and Arabidopsis thaliana (Breitkreuz et al., J.
Biol. Chem. 278:41552-41556 (2003)). Yet another gene candidate is
the alcohol dehydrogenase adhI from Geobacillus thermoglucosidasius
(Jeon et al., J. Biotechnol 135:127-133 (2008)). These
genes/proteins are identified below in Table 18.
TABLE-US-00018 TABLE 18 Gene GenBank ID GI Number Organism 4hbd
YP_726053.1 113867564 Ralstonia eutropha H16 4hbd L21902.1
146348486 Clostridium kluyveri DSM 555 4hbd Q94B07 75249805
Arabidopsis thaliana adhI AAR91477.1 40795502 Geobacillus
thermoglucosidasius M10EXG
[0326] Another exemplary enzyme is 3-hydroxyisobutyrate
dehydrogenase which catalyzes the reversible oxidation of
3-hydroxyisobutyrate to methylmalonate semialdehyde. This enzyme
participates in valine, leucine and isoleucine degradation and has
been identified in bacteria, eukaryotes, and mammals. The enzyme
encoded by P84067 from Thermus thermophilus HB8 has been
structurally characterized (Lokanath et al., J Mol Biol 352:905-917
(2005)). The reversibility of the human 3-hydroxyisobutyrate
dehydrogenase was demonstrated using isotopically-labeled substrate
(Manning, Biochem J 231:481-484 (1985)). Additional genes encoding
this enzyme include 3hidh in Homo sapiens (Hawes et al., Methods
Enzymol. 324:218-228 (2000)) and Oryctolagus cuniculus, (Hawes et
al., Methods Enzymol. 324:218-228 (2000); and Chowdhury, Biosci.
Biotechnol Biochem. 60:2043-2047 (1996)) (mmsb in Pseudomonas
aeruginosa, and dhat in Pseudomonas putida (Aberhart, J Chem. Soc.
6:1404-1406 (1979); Chowdhury, Biosci. Biotechnol Biochem.
60:2043-2047 (1996) and Chowdhury, Biosci. Biotechnol Biochem.
67:438-441 (2003)). These genes/proteins are identified below in
Table 19.
TABLE-US-00019 TABLE 19 Gene GenBank ID GI Number Organism P84067
P84067 75345323 Thermus thermophilus mmsb P28811.1 127211
Pseudomonas aeruginosa dhat Q59477.1 2842618 Pseudomonas putida
3hidh P31937.2 12643395 Homo sapiens 3hidh P32185.1 416872
Oryctolagus cuniculus
Propionyl-CoA:Phosphate Propanoyltransferase
[0327] The conversion of propanoyl-CoA to propanoyl phosphate can
be catalyzed by a phosphate transferase. Among the phosphate
acetyltransferases (EC 2.3.1.8), several enzymes including those
from Bacillus subtilis, (Rado, Biochem. Biophys. Acta 321:114-125
(1973)) Clostridium kluyveri, (Stadtman, Methods Enzymol 1:596-599
(1955)) and Thermotoga maritima (Bock, J Bacteriol. 181:1861-1867
(1999)) have been shown to have activity on propionyl-CoA.
Therefore, the genes coding for these phosphate acetyltransferases
as well as Escherichia coli pta gene will be utilized to catalyze
this step. These genes/proteins are identified below in Table
20.
TABLE-US-00020 TABLE 20 Gene GenBank ID GI Number Organism pta
P39646 730415 Bacillus subtilis pta A5N801 146346896 Clostridium
kluyveri pta Q9X0L4 6685776 Thermotoga maritima pta POA9M8 71152910
Escherichia coli K12
Propionyl Phosphate Reductase
[0328] The conversion of propanoyl phosphate to propionaldehyde is
catalyzed by the propionyl phosphate reductase. Even though such
direct conversion has not been demonstrated yet, similar
transformations were well documented including
glyceraldehyde-3-phosphate dehydrogenase and aspartate-semialdehyde
dehydrogenase. The following genes encoding
glyceraldehyde-3-phosphate dehydrogenase and aspartate-semialdehyde
dehydrogenase will be considered for catalyzing this step. These
genes/proteins are identified below in Table 21.
TABLE-US-00021 TABLE 21 Gene GenBank ID GI Number Organism asd NP
417891 16131307 Escherichia coli K12 gapA NP 785996 28379104
Lactobacillus plantarum WCFS1 gapA NP 416293 71159358 Escherichia
coli K12 gapA NP 347346 15893997 Clostridium acetobutylicum ATCC
824 gapN NP 350239 15896890 Clostridium acetobutylicum ATCC 824
Propionyl-CoA Hydrolase
[0329] Propionyl-CoA can be converted to propionate by a CoA
hydrolase, synthetase or transferase. The hydrolysis of
propionyl-CoA to propionate occurs in organic acid degradation
pathways that proceed through the intermediate 2-oxobutanoate. This
reaction is catalyzed by acyl-CoA hydrolase enzymes (EC 3.1.2.18).
Propionyl-CoA is the preferred substrate of the short chin acyl-CoA
hydrolase found in rat liver mitochondria (Alexson et al., Biochim
Biophys. Acta., 1105(1):13-9 (1989)). This enzyme has been
characterized but the sequence encoding the gene is not yet
identified (Garras et al., Biochim. Biophys. Acta., 1255:154-160
(1995)). Another enzyme exhibiting CoA hydrolase activity on
propionyl-CoA is found in the mitochondrion of the pea leaf. Though
its sequence has not been reported, this enzyme has a broad
substrate specificity, with demonstrated activity on acetyl-CoA,
propionyl-CoA, butyryl-CoA, palmitoyl-CoA, oleoyl-CoA,
succinyl-CoA, and crotonyl-CoA (Zeiher et al., Plant. Physiol.
94:20-27 (1990)). Additional propionyl-CoA hydrolase candidates
include 3-hydroxyisobutyryl-CoA hydrolase, acetyl-CoA hydrolase,
and dicarboxylic acid thioesterase.
[0330] 3-Hydroxyisobutyryl-CoA hydrolase efficiently catalyzes the
conversion of 3-hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate
during valine degradation (Shimomura et al., J Biol Chem.
269:14248-14253 (1994)). Genes encoding this enzyme include hibch
of Rattus norvegicus (Shimomura et al., supra; Shimomura et al.,
Methods Enzymol. 324:229-240 (2000)) and Homo sapiens (Shimomura et
al., supra). The H. sapiens enzyme also accepts
3-hydroxybutyryl-CoA and 3-hydroxypropionyl-CoA as substrates
(Shimomura et al., supra). Candidate genes by sequence homology
include hibch of Saccharomyces cerevisiae and BC 2292 of Bacillus
cereus. These genes/proteins are identified below in Table 22.
TABLE-US-00022 TABLE 22 Gene GenBank ID GI Number Organism hibch
Q5XIE6.2 146324906 Rattus norvegicus hibch Q6NVY1.2 146324905 Homo
sapiens hibch P28817.2 2506374 Saccharomyces cerevisiae BC_2292
AP09256 29895975 Bacillus cereus
[0331] Several eukaryotic acetyl-CoA hydrolases (EC 3.1.2.1) have
broad substrate specificity and thus represent suitable candidate
enzymes. For example, the enzyme from Rattus norvegicus brain
(Robinson et al., Res. Commun. 71:959-965 (1976)) can react with
butyryl-CoA, hexanoyl-CoA and malonyl-CoA. The acetyl-CoA
hydrolase, ACH1, from S. cerevisiae represents another candidate
hydrolase (Buu et al., J. Biol. Chem. 278:17203-17209 (2003)).
These genes/proteins are identified below in Table 23.
TABLE-US-00023 TABLE 23 Gene GenBank ID GI Number Organism acot12
NP_570103.1 18543355 Rattus norvegicus ACH1 NP_009538 6319456
Saccharomyces cerevisiae
[0332] Another candidate hydrolase is the human dicarboxylic acid
thioesterase, acot8, which exhibits activity on glutaryl-CoA,
adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA (Westin
et al., J Biol. Chem. 280:38125-38132 (2005)) and the closest E.
coli homolog, tesB, which can also hydrolyze a broad range of CoA
thioesters (Naggert et al., J Biol. Chem. 266:11044-11050 (1991)).
A similar enzyme has also been characterized in the rat liver
(Deana et al., Biochem. Int. 26:767-773 (1992)). Other potential E.
coli thioester hydrolases include the gene products of tesA (Bonner
et al., Chem. 247:3123-3133 (1972)), ybgC (Kuznetsova et al., FEMS
Microbiol Rev 29:263-279 (2005); and (Zhuang et al., FEBS Lett.
516:161-163 (2002)), paaI (Song et al., J Biol. Chem.
281:11028-11038 (2006)), and ybdB (Leduc et al., J Bacteriol.
189:7112-7126 (2007)). These genes/proteins are identified below in
Table 24.
TABLE-US-00024 TABLE 24 Gene GenBank ID GI Number Organism tesB
NP_414986 16128437 Escherichia coli acot8 CAA15502 3191970 Homo
sapiens acot8 NP_570112 51036669 Rattus norvegicus tesA NP_415027
16128478 Escherichia coli ybgC NP_415264 16128711 Escherichia coli
paaI NP_415914 16129357 Escherichia coli ybdB NP_415129 16128580
Escherichia coli
[0333] Yet another candidate hydrolase is the glutaconate
CoA-transferase from Acidaminococcus fermentans. This enzyme was
transformed by site-directed mutagenesis into an acyl-CoA hydrolase
with activity on glutaryl-CoA, acetyl-CoA and 3-butenoyl-CoA (Mack
et al., FEBS. Lett. 405:209-212 (1997)). This suggests that the
enzymes encoding succinyl-CoA:3-ketoacid-CoA transferases and
acetoacetyl-CoA:acetyl-CoA transferases may also serve as
candidates for this reaction step but would require certain
mutations to change their function. These genes/proteins are
identified below in Table 25.
TABLE-US-00025 TABLE 25 Gene GenBank ID GI Number Organism gctA
CAA57199 559392 Acidaminococcus fermentans gctB CAA57200 559393
Acidaminococcus fermentans
Propionyl-CoA Synthetase
[0334] A CoA synthetase can also catalyze the removal of the CoA
moiety from propionyl-CoA. One candidate enzyme, ADP-forming
acetyl-CoA synthetase (ACD, EC 6.2.1.13), couples the conversion of
acyl-CoA esters to their corresponding acids with the concurrent
synthesis of ATP. Several enzymes with broad substrate
specificities have been described in the literature. ACD I from
Archaeoglobus fulgidus, encoded by AF1211, was shown to operate on
a variety of linear and branched-chain substrates including
acetyl-CoA, propionyl-CoA, butyryl-CoA, acetate, propionate,
butyrate, isobutyryate, isovalerate, succinate, fumarate,
phenylacetate, indoleacetate (Musfeldt et al., J Bacteriol
184:636-644 (2002)). The enzyme from Haloarcula marismortui
(annotated as a succinyl-CoA synthetase) accepts propionate,
butyrate, and branched-chain acids (isovalerate and isobutyrate) as
substrates, and was shown to operate in the forward and reverse
directions (Brasen et al., Arch Microbiol 182:277-287 (2004)). The
ACD encoded by PAE3250 from hyperthermophilic crenarchaeon
Pyrobaculum aerophilum showed the broadest substrate range of all
characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA
(preferred substrate) and phenylacetyl-CoA (Brasen et al., supra).
The enzymes from A. fulgidus, H. marismortui and P. aerophilum have
all been cloned, functionally expressed, and characterized in E.
coli (Musfeldt et al., supra; Brasen et al., supra). These
genes/proteins are identified below in Table 26.
TABLE-US-00026 TABLE 26 Gene GenBank ID GI Number Organism AF1211
NP_070039.1 11498810 Archaeoglobus fulgidus DSM 4304 scs
YP_135572.1 55377722 Haloarcula marismortui ATCC 43049 PAE3250
NP_560604.1 18313937 Pyrobaculum aerophilum str. IM2
[0335] Another candidate CoA synthetase is succinyl-CoA synthetase.
The sucCD genes of E. coli form a succinyl-CoA synthetase complex
which naturally catalyzes the formation of succinyl-CoA from
succinate with the concaminant consumption of one ATP, a reaction
which is reversible in vivo (Buck et al., Biochem. 24:6245-6252
(1985)). These genes/proteins are identified below in Table 27.
TABLE-US-00027 TABLE 27 Gene GenBank ID GI Number Organism sucC
NP_415256.1 16128703 Escherichia coli sucD AAC73823.1 1786949
Escherichia coli
[0336] Additional exemplary CoA-ligases include the rat
dicarboxylate-CoA ligase for which the sequence is yet
uncharacterized (Vamecq et al., Biochemical Journal 230:683-693
(1985)), either of the two characterized phenylacetate-CoA ligases
from P. chrysogenum (Lamas-Maceiras et al., Biochem. J. 395:147-155
(2005); Wang et al., Biochem Biophy Res Commun 360(2):453-458
(2007)), the phenylacetate-CoA ligase from Pseudomonas putida
(Martinez-Blanco et al., J. Biol. Chem. 265:7084-7090 (1990)), and
the 6-carboxyhexanoate-CoA ligase from Bacillus subtilis (Bower et
al., J. Bacteriol. 178(14):4122-4130 (1996)). Additional candidate
enzymes are acetoacetyl-CoA synthetases from Mus musculus (Hasegawa
et al., Biochim Biophys Acta 1779:414-419 (2008)) and Homo sapiens
(Ohgami et al., Biochem Pharmacol 65:989-994 (2003)) which
naturally catalyze the ATP-dependant conversion of acetoacetate
into acetoacetyl-CoA. These genes/proteins are identified below in
Table 28.
TABLE-US-00028 TABLE 28 Gene GenBank ID GI Number Organism phl
CAJ15517.1 77019264 Penicillium chrysogenum phlB ABS19624.1
152002983 Penicillium chrysogenum paaF AAC24333.2 22711873
Pseudomonas putida bioW NP_390902.2 50812281 Bacillus subtilis AACS
NP_084486.1 21313520 Mus musculus AACS NP_076417.2 31982927 Homo
sapiens
Propionyl-CoA Transferase
[0337] Propionyl-CoA transferase catalyzes the conversion of
propionyl-CoA to propionate while transferring the CoA moiety to a
CoA acceptor molecule. Many transferases have broad specificity and
thus may utilize CoA acceptors as diverse as acetate, succinate,
propionate, butyrate, 2-methylacetoacetate, 3-ketohexanoate,
3-ketopentanoate, valerate, crotonate, 3-mercaptopropionate,
propionate, vinylacetate, butyrate, among others.
[0338] Several genes have been identified that have propionyl-CoA
transferase activity. The enzyme from Roseburia sp. A2-183 was
shown to have butyryl-CoA:acetate:CoA transferase and
propionyl-CoA:acetate:CoA transferase activity (Charrier et al.,
Microbiology 152, 179-185 (2006)). Close homologs can be found in,
for example, Roseburia intestinalis L1-82, Roseburia inulinivorans
DSM 16841, Eubacterium rectale ATCC 33656. Another enzyme with
propionyl-CoA transferase activity can be found in Clostridium
propionicum (Selmer et al., Eur J Biochem 269, 372-380 (2002)).
This enzyme can use acetate, (R)-lactate, (S)-lactate, acrylate,
and butyrate as the CoA acceptor (Selmer et al., Eur J Biochem 269,
372-380 (2002); Schweiger and Buckel, FEBS Letters, 171(1) 79-84
(1984)). Close homologs can be found in, for example, Clostridium
novyi NT, Clostridium beijerinckii NCIMB 8052, and Clostridium
botulinum C str. Eklund. YgfH encodes a propionyl CoA:succinate CoA
transferase in E. coli (Haller et al., Biochemistry, 39(16)
4622-4629). Close homologs can be found in, for example,
Citrobacter youngae ATCC 29220, Salmonella enterica subsp. arizonae
serovar, and Yersinia intermedia ATCC 29909. These genes/proteins
are identified below in Table 29.
TABLE-US-00029 TABLE 29 Gene GenBank ID GI Number Organism Ach1
AAX19660.1 60396828 Roseburia sp. A2-183 ROSINTL182_07121
ZP_04743841.2 257413684 Roseburia intestinalis L1-82
ROSEINA2194_03642 ZP_03755203.1 225377982 Roseburia inulinivorans
DSM 16841 EUBREC_3075 YP_002938937.1 238925420 Eubacterium rectale
ATCC 33656 pct CAB77207.1 7242549 Clostridium propionicum
NT01CX_2372 YP_878445.1 118444712 Clostridium novyi NT Cbei_4543
YP_001311608.1 150019354 Clostridium beijerinckii NCIMB 8052
CBC_A0889 ZP_02621218.1 168186583 Clostridium botulinum C str.
Eklund YgfH NP_417395.1 16130821 Escherichia coli str. K-12 substr.
MG1655 CIT292_04485 ZP_03838384.1 227334728 Citrobacter youngae
ATCC 29220 SARI_04582 YP_001573497.1 161506385 Salmonella enterica
subsp. arizonae serovar yinte0001_14430 ZP_04635364.1 238791727
Yersinia intermedia ATCC 29909
[0339] An additional candidate enzyme is the two-unit enzyme
encoded by pcaI and pcaJ in Pseudomonas, which has been shown to
have 3-oxoadipyl-CoA/succinate transferase activity (Kaschabek et
al., supra). Similar enzymes based on homology exist in
Acinetobacter sp. ADP1 (Kowalchuk et al., Gene 146:23-30 (1994))
and Streptomyces coelicolor. Additional exemplary
succinyl-CoA:3:oxoacid-CoA transferases are present in Helicobacter
pylori (Corthesy-Theulaz et al., J. Biol. Chem. 272:25659-25667
(1997)) and Bacillus subtilis (Stols et al., Protein. Expr. Purif.
53:396-403 (2007)). These genes/proteins are identified below in
Table 30.
TABLE-US-00030 TABLE 30 Gene GenBank ID GI Number Organism pcaI
AAN69545.1 24985644 Pseudomonas putida pcaJ NP_746082.1 26990657
Pseudomonas putida pcaI YP_046368.1 50084858 Acinetobacter sp. ADP1
pcaJ AAC37147.1 141776 Acinetobacter sp. ADP1 pcaI NP_630776.1
21224997 Streptomyces coelicolor pcaJ NP_630775.1 21224996
Streptomyces coelicolor HPAG1_0676 YP_627417 108563101 Helicobacter
pylori HPAG1_0677 YP_627418 108563102 Helicobacter pylori ScoA
NP_391778 16080950 Bacillus subtilis ScoB NP_391777 16080949
Bacillus subtilis
[0340] A CoA transferase that can utilize acetate as the CoA
acceptor is acetoacetyl-CoA transferase, encoded by the E. coli
atoA (alpha subunit) and atoD (beta subunit) genes (Vanderwinkel et
al., Biochem. Biophys. Res Commun. 33:902-908 (1968); Korolev et
al., Acta Crystallogr. D Biol Crystallogr. 58:2116-2121 (2002)).
This enzyme has also been shown to transfer the CoA moiety to
acetate from a variety of branched and linear acyl-CoA substrates,
including isobutyrate (Matthies et al., Appl Environ Microbiol
58:1435-1439 (1992)), valerate (Vanderwinkel et al., supra) and
butanoate (Vanderwinkel et al., supra). Similar enzymes exist in
Corynebacterium glutamicum ATCC 13032 (Duncan et al., Appl Environ
Microbiol 68:5186-5190 (2002)), Clostridium acetobutylicum (Cary et
al., Appl Environ Microbiol 56:1576-1583 (1990)), and Clostridium
saccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol
Biochem. 71:58-68 (2007)). These genes/proteins are identified
below in Table 31.
TABLE-US-00031 TABLE 31 Gene GenBank ID GI Number Organism atoA
P76459.1 2492994 Escherichia coli K12 atoD P76458.1 2492990
Escherichia coli K12 actA YP_226809.1 62391407 Corynebacterium
glutamicum ATCC 13032 cg0592 YP_224801.1 62389399 Corynebacterium
glutamicum ATCC 13032 ctfA NP_149326.1 15004866 Clostridium
acetobutylicum ctfB NP_149327.1 15004867 Clostridium acetobutylicum
ctfA AAP42564.1 31075384 Clostridium saccharoperbutylacetonicum
ctfB AAP42565.1 31075385 Clostridium saccharoperbutylacetonicum
[0341] The above enzymes may also exhibit the desired activities on
propionyl-CoA. Additional exemplary transferase candidates are
catalyzed by the gene products of cat1, cat2, and cat3 of
Clostridium kluyveri which have been shown to exhibit succinyl-CoA,
4-hydroxybutyryl-CoA, and butyryl-CoA transferase activity,
respectively (Seedorf et al., supra; Sohling et al., Eur. J
Biochem. 212:121-127 (1993); Sohling et al., J Bacteriol.
178:871-880 (1996)). Similar CoA transferase activities are also
present in Trichomonas vaginalis (van Grinsven et al., J. Biol.
Chem. 283:1411-1418 (2008)) and Trypanosoma brucei (Riviere et al.,
J. Biol. Chem. 279:45337-45346 (2004)). These genes/proteins are
identified below in Table 32.
TABLE-US-00032 TABLE 32 Gene GenBank ID GI Number Organism cat1
P38946.1 729048 Clostridium kluyveri cat2 P38942.2 172046066
Clostridium kluyveri cat3 EDK35586.1 146349050 Clostridium kluyveri
TVAG_395550 XP_001330176 123975034 Trichomonas vaginalis G3
Tb11.02.0290 XP_828352 71754875 Trypanosoma brucei
[0342] The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from
anaerobic bacterium Acidaminococcus fermentans reacts with diacid
glutaconyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS Lett.
405:209-212 (1997)). The genes encoding this enzyme are gctA and
gctB. This enzyme has reduced but detectable activity with other
CoA derivatives including glutaryl-CoA, 2-hydroxyglutaryl-CoA,
adipyl-CoA and acrylyl-CoA (Buckel et al., Eur. J. Biochem.
118:315-321 (1981)). The enzyme has been cloned and expressed in E.
coli (Mack et al., Eur. J. Biochem. 226:41-51 (1994)). These
genes/proteins are identified below in Table 33.
TABLE-US-00033 TABLE 33 Gene GenBank ID GI Number Organism gctA
CAA57199.1 559392 Acidaminococcus fermentans gctB CAA57200.1 559393
Acidaminococcus fermentans
Propionate Kinase
[0343] Propionate is activated to propionyl-phosphate by an enzyme
with propionate kinase activity. Butyrate kinase (EC 2.7.2.7)
carries out the reversible conversion of butyryl-phosphate to
butyrate during acidogenesis in C. acetobutylicum (Cary et al.,
Appl. Environ. Microbiol 56:1576-1583 (1990)). This enzyme is
encoded by either of the two buk gene products (Huang et al., J
Mol. Microbiol Biotechnol 2:33-38 (2000)). This enzyme was shown to
accept propionate, isobutanoate and valerate as alternate
substrates (Hartmanis, J. Biol. Chem., 262(2):617-21 (1987)). Other
butyrate kinase enzymes are found in C. butyricum and C.
tetanomorphum (Twarog et al., J Bacteriol. 86:112-117 (1963)).
These enzymes also accept propionate, isobutanoate and valerate as
secondary substrates. Related enzyme isobutyrate kinase from
Thermotoga maritima has also been expressed in E. coli and
crystallized (Diao et al., E. Biol. Crystallogr. 59:1100-1102
(2003); and Diao et al., J Bacteriol. 191:2521-2529 (2009)).
Aspartokinase catalyzes the ATP-dependent phosphorylation of
aspartate and participates in the synthesis of several amino acids.
The aspartokinase III enzyme in E. coli, encoded by lysC, has a
broad substrate range and the catalytic residues involved in
substrate specificity have been elucidated (Keng et al., Arch.
Biochem. Biophys. 335:73-81 (1996)). Two additional kinases in E.
coli are also good candidates: acetate kinase and gamma-glutamyl
kinase. The E. coli acetate kinase, encoded by ackA (Skarstedt et
al., J. Biol. Chem. 251:6775-6783 (1976)), phosphorylates
propionate in addition to acetate (Hesslinger et al., Mol.
Microbiol 27:477-492 (1998)). The E. coli gamma-glutamyl kinase,
encoded by proB (Smith et al., J. Bacteriol. 157:545-551 (1984)),
phosphorylates the gamma carbonic acid group of glutamate. These
genes/proteins are identified below in Table 34.
TABLE-US-00034 TABLE 34 Gene GenBank ID GI Number Organism buk1
NP_349675 15896326 Clostridium acetobutylicum buk2 Q97II1 20137415
Clostridium acetobutylicum buk2 Q9X278.1 6685256 Thermotoga
maritima lysC NP_418448.1 16131850 Escherichia coli ackA
NP_416799.1 16130231 Escherichia coli proB NP_414777.1 16128228
Escherichia coli
Propionate Reductase
[0344] The reduction of propionate to propionic semialdehyde is
catalyzed by a carboxylic acid reductase. Exemplary enzyme
candidates for succinate reductase and 4-hydroxybutyrate reductase
enzyme, described below, are also applicable here.
Example II
Pathways for Production of Acetyl-CoA from Glucose
[0345] Further to Example I, the pathway for production of
acetyl-CoA from glucose proceeds via phosphoenolpyruvate (PEP)
(FIGS. 1-4). Glucose is converted into PEP by the native glycolysis
pathway of the microbial organism. PEP is converted to pyruvate by
pyruvate kinase and then to acetyl-CoA by pyruvate dehydrogenase or
pyruvate ferredoxin oxidoreductase. Alternatively, pyruvate is
converted to acetyl-CoA and formate by pyruvate formate lyase.
Formate is then converted to carbon dioxide by a formate
dehydrogenase that also produces NADH. The acetyl-CoA produced by
these pathways are then utilized for production of isopropanol as
described in Example I or utilized for production of both
n-propanol and isopropanol as described in Example V below (FIG.
3).
Pyruvate Dehydrogenase
[0346] The pyruvate dehydrogenase complex, catalyzing the
conversion of pyruvate to acetyl-CoA, has been extensively studied.
The S. cerevisiae complex consists of an E2 (LAT1) core that binds
E1 (PDA1, PDB1), E3 (LPD1), and Protein X (PDX1) components (Pronk,
Yeast 12:1607-1633 (1996)). In the E. coli enzyme, specific
residues in the E1 component are responsible for substrate
specificity (Bisswanger, J. Biol Chem. 256:815-822 (1981); Bremer,
Eur. J Biochem. 8:535-540 (1969) and Gong et al., J. Biol Chem.
275:13645-13653 (2000)). Engineering efforts have improved the E.
coli PDH enzyme activity under anaerobic conditions (Kim, J.
Bacteriol 190:3851-3858 (2008); Kim, Appl. Environ. Microbiol.
73:1766-1771 (2007) and Zhou, Biotechnol. Lett. 30:335-342 (2008)).
In contrast to the E. coli PDH, the B. subtilis complex is active
and required for growth under anaerobic conditions (Nakano, J.
Bacteriol 179:6749-6755 (1997)). The Klebsiella pneumoniae PDH,
characterized during growth on glycerol, is also active under
anaerobic conditions (Menzel, J. Biotechnol. 56:135-142 (1997)).
Crystal structures of the enzyme complex from bovine kidney (Zhou,
Proc. Natl. Acad. Sci. U.S.A. 98:14802-14807 (2001)) and the E2
catalytic domain from Azotobacter vinelandii are available (Mattevi
et al., Science. 255:1544-1550 (1992)). Some mammalian PDH enzymes
complexes can react on alternate substrates such as 2-oxobutanoate
(Paxton, J Bacteriol. 179:5684-5692 (1997)). These genes/proteins
are identified below in Table 35.
TABLE-US-00035 TABLE 35 Gene GenBank ID GI Number Organism LAT1
NP_014328 6324258 Saccharomyces cerevisiae PDA1 NP_011105 37362644
Saccharomyces cerevisiae PDB1 NP_009780 6319698 Saccharomyces
cerevisiae LPD1 NP_116635 14318501 Saccharomyces cerevisiae PDX1
NP_011709 6321632 Saccharomyces cerevisiae aceE NP_414656.1
16128107 Escherichia coli str. K12 substr. MG1655 aceF NP_414657.1
16128108 Escherichia coli str. K12 substr. MG1655 lpd NP_414658.1
16128109 Escherichia coli str. K12 substr. MG1655 pdhA P21881.1
3123238 Bacillus subtilis pdhB P21882.1 129068 Bacillus subtilis
pdhC P21883.2 129054 Bacillus subtilis pdhD P21880.1 118672
Bacillus subtilis aceE YP_001333808.1 152968699 Klebsiella
pneumonia MGH78578 aceF YP_001333809.1 152968700 Klebsiella
pneumonia MGH78578 lpdA YP_001333810.1 152968701 Klebsiella
pneumonia MGH78578 Pdha1 NP_001004072.2 124430510 Rattus norvegicus
Pdha2 NP_446446.1 16758900 Rattus norvegicus Dlat NP_112287.1
78365255 Rattus norvegicus Dld NP_955417.1 40786469 Rattus
norvegicus
Pyruvate Ferredoxin Oxidoreductase
[0347] Pyruvate ferredoxin oxidoreductase (PFOR) catalyzes the
oxidation of pyruvate to form acetyl-CoA. The PFOR from
Desulfovibrio africanus has been cloned and expressed in E. coli
resulting in an active recombinant enzyme that was stable for
several days in the presence of oxygen (Pieulle, J Bacteriol
179:5684-5692 (1997)). Oxygen stability is relatively uncommon in
PFORs and is believed to be conferred by a 60 residue extension in
the polypeptide chain of the D. africanus enzyme. The M.
thermoacetica PFOR is also well characterized (Menon, Biochemistry
36:8484-8494 (1997)) and was even shown to have high activity in
the direction of pyruvate synthesis during autotrophic growth
(Furdui, J Biol Chem. 275:28494-28499 (2000)). Further, E. coli
possesses an uncharacterized open reading frame, ydbK, that encodes
a protein that is 51% identical to the M. thermoacetica PFOR.
Evidence for pyruvate oxidoreductase activity in E. coli has been
described (Blaschkowski, Eur. J Biochem. 123:563-569 (1982)).
Several additional PFOR enzymes are described in the following
review (Ragsdale, Chem. Rev. 103:2333-2346 (2003)). Finally,
flavodoxin reductases (e.g., fqrB from Helicobacter pylori or
Campylobacter jejuni) (St Maurice et al., J. Bacteriol.
189:4764-4773 (2007)) or Rnf-type proteins (Seedorf et al., Proc.
Natl. Acad. Sci. U.S.A. 105:2128-2133 (2008); and Herrmann, J.
Bacteriol 190:784-791 (2008)) provide a means to generate NADH or
NADPH from the reduced ferredoxin generated by PFOR. These
genes/proteins are identified below in Table 36.
TABLE-US-00036 TABLE 36 Gene GenBank ID GI Number Organism por
CAA70873.1 1770208 Desulfovibrio africanus por YP_428946.1 83588937
Moorella thermoacetica ydbK NP_415896.1 16129339 Escherichia coli
fqrB NP_207955.1 15645778 Helicobacter pylori fqrB YP_001482096.1
157414840 Campylobacter jejuni RnfC EDK33306.1 146346770
Clostridium kluyveri RnfD EDK33307.1 146346771 Clostridium kluyveri
RnfG EDK33308.1 146346772 Clostridium kluyveri RnfE EDK33309.1
146346773 Clostridium kluyveri RnfA EDK33310.1 146346774
Clostridium kluyveri RnfB EDK33311.1 146346775 Clostridium
kluyveri
Pyruvate Formate Lyase
[0348] Pyruvate formate lyase is an enzyme that catalyzes the
conversion of pyruvate and CoA into acetyl-CoA and formate.
Pyruvate formate lyase is a common enzyme in prokaryotic organisms
that is used to help modulate anaerobic redox balance. Exemplary
enzymes can be found in Escherichia coli (Knappe, FEMS. Microbiol
Rev. 6:383-398 (1990)), Lactococcus lactis (Melchiorsen, Appl
Microbiol Biotechnol 58:338-344(2002)), and Streptococcus mutans.
(Takahashi-Abbe, Oral. Microbiol Immunol. 18:293-297 (2003)). A
mitochondrial pyruvate formate lyase has also been identified in
the eukaryote, Chlamydomonas reinhardtii. (Hemschemeier, Eukaryot.
Cell 7:518-526 (2008); and Atteia, J. Biol. Chem. 281:9909-9918
(2008)). These genes/proteins are identified below in Table 37.
TABLE-US-00037 TABLE 37 Gene GenBank ID GI Number Organism pflB
NP_415423 16128870 Escherichia coli pfl CAA03993 2407931
Lactococcus lactis pfl BAA09085 1129082 Streptococcus mutans PFL1
EDP09457 158283707 Chlamydomonas reinhardtii
Formate Hydrogen Lyase
[0349] A formate hydrogen lyase enzyme can be employed to convert
formate to carbon dioxide and hydrogen. An exemplary formate
hydrogen lyase enzyme can be found in Escherichia coli. The E. coli
formate hydrogen lyase consists of hydrogenase 3 and formate
dehydrogenase-H (Maeda, Appl Microbiol Biotechnol 77:879-890
(2007)). It is activated by the gene product of fhlA (Maeda, Appl
Microbiol Biotechnol 77:879-890 (2007)). The addition of the trace
elements, selenium, nickel and molybdenum, to a fermentation broth
has been shown to enhance formate hydrogen lyase activity (Soini,
Microb. Cell Fact. 7:26 (2008)). These genes/proteins are
identified below in Table 38.
TABLE-US-00038 TABLE 38 Gene GenBank ID GI Number Organism
Hydrogenase 3: hycD NP_417202 16130629 Escherichia coli hycC
NP_417203 16130630 Escherichia coli hycF NP_417200 16130627
Escherichia coli hycG NP_417199 16130626 Escherichia coli hycB
NP_417204 16130631 Escherichia coli hycE NP_417201 16130628
Escherichia coli Formate dehydrogenase-H: fdhF NP_418503 16131905
Escherichia coli Activator: fhlA NP_417211 16130638 Escherichia
coli
[0350] A formate hydrogen lyase enzyme also exists in the
hyperthermophilic archaeon, Thermococcus litoralis (Takacs et al.,
BMC. Microbiol 8:88 (2008)). These genes/proteins are identified
below in Table 39.
TABLE-US-00039 TABLE 39 Gene GenBank ID GI Number Organism mhyC
ABW05543 157954626 Thermococcus litoralis mhyD ABW05544 157954627
Thermococcus litoralis mhyE ABW05545 157954628 Thermococcus
litoralis myhF ABW05546 157954629 Thermococcus litoralis myhG
ABW05547 157954630 Thermococcus litoralis myhH ABW05548 157954631
Thermococcus litoralis fdhA AAB94932 2746736 Thermococcus litoralis
fdhB AAB94931 157954625 Thermococcus litoralis
[0351] Additional formate hydrogen lyase systems have been found in
Salmonella typhimurium, Klebsiella pneumoniae, Rhodospirillum
rubrum, Methanobacterium formicicum (Vardar-Schara, Microbial
Biotechnology 1:107-125 (2008)).
Formate Dehydrogenase
[0352] Formate dehydrogenase activity is present in both E. coli
and Saccharomyces cerevisiae among other organisms. S. cerevisiae
contains two formate dehydrogenases, FDH1 and FDH2, that catalyze
the oxidation of formate to CO.sub.2. (Overkamp et al., Yeast
19:509-520 (2002)) In Moorella thermoacetica, the loci,
Moth.sub.--2312 and Moth.sub.--2313, are actually one gene that is
responsible for encoding the alpha subunit of formate dehydrogenase
while the beta subunit is encoded by Moth.sub.--2314 (Pierce et
al., Environ. Microbiol (2008); Andreesen, J. Bacteriol.
116:867-873 (1973); Li, J. Bacteriol 92:405-412 (1966) and
Yamamoto, J. Biol. Chem. 258:1826-1832 (1983)) Another set of genes
encoding formate dehydrogenase activity is encoded by
Sfum.sub.--2703 through Sfum.sub.--2706 in Syntrophobacter
fumaroxidans (Reda, Proc. Natl. Acad. Sci. U.S.A. 105:10654-10658
(2008); and de Bok et al., Eur. J. Biochem. 270:2476-2485 (2003)).
Similar to their M. thermoacetica counterparts, Sfum.sub.--2705 and
Sfum.sub.--2706 are actually one gene. E. coli contains multiple
formate dehydrogenases. These genes/proteins are identified below
in Table 40.
TABLE-US-00040 TABLE 40 Gene GenBank ID GI Number Organism FDH1
NP_015033 6324964 Saccharomyces cerevisiae FDH2 Q08987 88909613
Saccharomyces cerevisiae Moth_2312 YP_431142 148283121 Moorella
thermoacetica Moth_2313 YP_431143 83591134 Moorella thermoacetica
Moth_2314 YP_431144 83591135 Moorella thermoacetica Sfum_2703
YP_846816.1 116750129 Syntrophobacter fumaroxidans Sfum_2704
YP_846817.1 116750130 Syntrophobacter fumaroxidans Sfum_2705
YP_846818.1 116750131 Syntrophobacter fumaroxidans Sfum_2706
YP_846819.1 116750132 Syntrophobacter fumaroxidans fdnG, H, I
NP_415991- 16129433 Escherichia coli 993.1 16129434 16129435 fdoG,
H, I NP_418330,29, 16131734 Escherichia coli 28.1 16131733
16131732
Example III
Pathways for Production of Propionyl-CoA from Glucose Utilizing the
Reductive TCA Cycle
[0353] Further to Examples I and II, the pathway for production of
propionyl-CoA proceeds via oxaloacetate (FIG. 1). PEP is converted
into oxaloacetate either via PEP carboxykinase or PEP carboxylase.
Alternatively, PEP is converted first to pyruvate by pyruvate
kinase and then to oxaloacetate by methylmalonyl-CoA
carboxytransferase or pyruvate carboxylase. Oxaloacetate is
converted to propionyl-CoA by means of the reductive TCA cycle, a
methylmutase, a decarboxylase, an epimerase and a
decarboxylase.
PEP Carboxykinase
[0354] Although the net conversion of phosphoenolpyruvate to
oxaloacetate is redox-neutral, the mechanism of this conversion is
important to the overall energetics of the co-production pathway.
The most desirable enzyme for the conversion of PEP to oxaloacetate
is PEP carboxykinase which simultaneously forms an ATP while
carboxylating PEP. In most organisms, however, PEP carboxykinase
serves a gluconeogenic function and converts oxaloacetate to PEP at
the expense of one ATP. S. cerevisiae is one such organism whose
native PEP carboxykinase, PCK1, serves a gluconeogenic role
(Valdes-Hevia, FEBS. Lett. 258:313-316 (1989)). E. coli is another
such organism, as the role of PEP carboxykinase in producing
oxaloacetate is believed to be minor when compared to PEP
carboxylase, which does not form ATP, possibly due to the higher
K.sub.m for bicarbonate of PEP carboxykinase (Kim, Appl Environ
Microbiol 70:1238-1241 (2004)). Nevertheless, activity of the
native E. coli PEP carboxykinase from PEP towards oxaloacetate has
been recently demonstrated in ppc mutants of E. coli K-12 (Kwon,
Journal of Microbiology and Biotechnology 16:1448-1452 (2006)).
These strains exhibited no growth defects and had increased
succinate production at high NaHCO.sub.3 concentrations. In some
organisms, particularly rumen bacteria, PEP carboxykinase is quite
efficient in producing oxaloacetate from PEP and generating ATP.
Examples of PEP carboxykinase genes that have been cloned into E.
coli include those from Mannheimia succiniciproducens (Lee,
Biotechnol. Bioprocess Eng. 7:95-99 (2002)), Anaerobiospirillum
succiniciproducens (Laivenieks, Appl Environ Microbiol 63:2273-2280
(1997)), and Actinobacillus succinogenes (Kim, Appl Environ
Microbiol 70:1238-1241 (2004)). Internal experiments have also
found that the PEP carboxykinase enzyme encoded by Haemophilus
influenza is highly efficient at forming oxaloacetate from PEP.
These genes/proteins are identified below in Table 41.
TABLE-US-00041 TABLE 41 Gene GenBank ID GI Number Organism PCK1
NP_013023 6322950 Saccharomyces cerevisiae pck NP_417862.1 16131280
Escherichia coli pckA YP_089485.1 52426348 Mannheimia
succiniciproducens pckA O09460.1 3122621 Anaerobiospirillum
succiniciproducens pckA Q6W6X5 75440571 Actinobacillus succinogenes
pckA P43923.1 1172573 Haemophilus influenza
[0355] These sequences and sequences for subsequent enzymes listed
in this report can be used to identify homologue proteins in
GenBank or other databases through sequence similarity searches
(e.g. BLASTp). The resulting homologue proteins and their
corresponding gene sequences provide additional DNA sequences for
transformation into the host organism of choice.
PEP Carboxylase
[0356] PEP carboxylase represents an alternative enzyme for the
formation of oxaloacetate from PEP. Since the enzyme does not
generate ATP upon decarboxylating oxaloacetate, its utilization
decreases the maximum ATP yield of the production pathway and
represents a less favorable alternative for converting oxaloacetate
to PEP. Nevertheless, the maximum theoretical C3 alcohols yield of
1.33 mol/mol will remain unchanged if PEP carboxylase is utilized
to convert PEP to oxaloacetate. S. cerevisiae does not naturally
encode a PEP carboxylase, but exemplary organisms that possess
genes that encode PEP carboxylase include E. coli (Kai, Arch.
Biochem. Biophys. 414:170-179 (2003)), Methylobacterium extorquens
AM1 (Arps, J. Bacteriol. 175:3776-3783 (1993)), and Corynebacterium
glutamicum (Eikmanns, Mol. Gen. Genet. 218:330-339 (1989)). These
genes/proteins are identified below in Table 42.
TABLE-US-00042 TABLE 42 Gene GenBank ID GI Number Organism ppc
NP_418391 16131794 Escherichia coli ppcA AAB58883 28572162
Methylobacterium extorquens ppc ABB53270 80973080 Corynebacterium
glutamicum
Pyruvate Kinase and Methylmalonyl-CoA Carboxyltransferase
[0357] An additional energetically efficient route to oxaloacetate
from PEP requires two enzymatic activities: pyruvate kinase and
methylmalonyl-CoA carboxytransferase. Pyruvate kinase catalyzes the
ATP-generating conversion of PEP to pyruvate and is encoded by the
PYK1 (Burke, J. Biol. Chem. 258:2193-2201 (1983)) and PYK2 (Boles
et al., J. Bacteriol. 179:2987-2993 (1997)) genes in S. cerevisiae.
In E. coli, this activity is catalyzed by the gene product of pykF
and pykA. Methylmalonyl-CoA carboxytransferase catalyzes the
conversion of pyruvate to oxaloacetate. Importantly, this reaction
also simultaneously catalyzes the conversion of
(S)-methylmalonyl-CoA to propionyl-CoA (see FIGS. 1 and 2). An
exemplary methylmalonyl-CoA carboxytransferase which is comprised
of 1.3S, 5S, and 12S subunits can be found in Propionibacterium
freudenreichii (Thornton et al., J. Bacteriol 175:5301-5308
(1993)). These genes/proteins are identified below in Table 43.
TABLE-US-00043 TABLE 43 Gene GenBank ID GI Number Organism PYK1
NP_009362 6319279 Saccharomyces cerevisiae PYK2 NP_014992 6324923
Saccharomyces cerevisiae pykF NP_416191.1 16129632 Escherichia coli
pykA NP_416368.1 16129807 Escherichia coli 1.3S subunit P02904
114847 Propionibacterium freudenreichii 5S subunit Q70AC7 62901478
Propionibacterium freudenreichii 12S subunit Q8GBW6 62901481
Propionibacterium freudenreichii
Pyruvate Kinase and Pyruvate Carboxylase
[0358] A combination of enzymes can convert PEP to oxaloacetate
with a stoichiometry identical to that of PEP carboxylase. These
enzymes are encoded by pyruvate kinase, PYK1 (Burke, J. Biol. Chem.
258:2193-2201 (1983)) or PYK2 (Boles et al., J. Bacteriol,
179:2987-2993 (1997)) and pyruvate carboxylase, PYC1 (Walker,
Biochem. Biophys. Res. Commun. 176:1210-1217 (1991)) or PYC2
(Walker, Biochem. Biophys. Res. Commun. 176:1210-1217 (1991)). The
latter genes/proteins are identified below in Table 44.
TABLE-US-00044 TABLE 44 Gene GenBank ID GI Number Organism PYC1
NP_011453 6321376 Saccharomyces cerevisiae PYC2 NP_009777 6319695
Saccharomyces cerevisiae Pyc YP_890857.1 118470447 Mycobacterium
smegmatis
Malate Dehydrogenase, Fumarase, Fumarate Reductase
[0359] Oxaloacetate can be converted to succinate by malate
dehydrogenase, fumarase and fumarate reductase when the TCA cycle
is operating in the reductive cycle. S. cerevisiae possesses three
copies of malate dehydrogenase, MDH1 (McAlister-Henn, J. Bacteriol
169:5157-5166 (1987)) MDH2 (Minard, Mol. Cell. Biol. 11:370-380
(1991); and Gibson, J. Biol. Chem. 278:25628-25636 (2003)), and
MDH3 (Steffan, J. Biol. Chem. 267:24708-24715 (1992)), which
localize to the mitochondrion, cytosol, and peroxisome,
respectively. S. cerevisiae contains one copy of a
fumarase-encoding gene, FUM1, whose product localizes to both the
cytosol and mitochondrion (Sass, J. Biol. Chem. 278:45109-45116
(2003)). Fumarate reductase is encoded by two soluble enzymes,
FRDS1 (Enomoto, DNA. Res. 3:263-267 (1996)) and FRDS2 (Muratsubaki,
Arch. Biochem. Biophys. 352:175-181 (1998)), which localize to the
cytosol and promitochondrion, respectively, and are required for
anaerobic growth on glucose (Arikawa, Microbiol Lett. 165:111-116
(1998)). E. coli is known to have an active malate dehydrogenase.
It has three fumarases encoded byfumA, B and C, each one of which
is active under different conditions of oxygen availability. The
fumarate reductase in E. coli is composed of four subunits. These
genes/proteins are identified below in Table 45.
TABLE-US-00045 TABLE 45 Gene GenBank ID GI Number Organism MDH1
NP_012838 6322765 Saccharomyces cerevisiae MDH2 NP_014515 116006499
Saccharomyces cerevisiae MDH3 NP_010205 6320125 Saccharomyces
cerevisiae FUM1 NP_015061 6324993 Saccharomyces cerevisiae FRDS1
P32614 418423 Saccharomyces cerevisiae FRDS2 NP_012585 6322511
Saccharomyces cerevisiae frdA NP_418578.1 16131979 Escherichia coli
frdB NP_418577.1 16131978 Escherichia coli frdC NP_418576.1
16131977 Escherichia coli frdD NP_418475.1 16131877 Escherichia
coli Mdh NP_417703.1 16131126 Escherichia coli FumA NP_416129.1
16129570 Escherichia coli FumB NP_418546.1 16131948 Escherichia
coli FumC NP_416128.1 16129569 Escherichia coli
Succinyl-CoA Transferase
[0360] Succinyl-CoA transferase catalyzes the conversion of
succinyl-CoA to succinate while transferring the CoA moiety to a
CoA acceptor molecule. Many transferases have broad specificity and
thus may utilize CoA acceptors as diverse as acetate, succinate,
propionate, butyrate, 2-methylacetoacetate, 3-ketohexanoate,
3-ketopentanoate, valerate, crotonate, 3-mercaptopropionate,
propionate, vinylacetate, butyrate, among others.
[0361] The conversion of succinate to succinyl-CoA is ideally
carried by a transferase which does not require the direct
consumption of an ATP or GTP. This type of reaction is common in a
number of organisms. Perhaps the top candidate enzyme for this
reaction step is succinyl-CoA:3-ketoacid-CoA transferase. This
enzyme converts succinate to succinyl-CoA while converting a
3-ketoacyl-CoA to a 3-ketoacid. Exemplary
succinyl-CoA:3:ketoacid-CoA transferases are present in
Helicobacter pylori (Corthesy-Theulaz et al., J. Biol. Chem.
272:25659-25667 (1997)), Bacillus subtilis (Stols et al., Protein.
Expr. Pur 53:396-403 (2007)), and Homo sapiens (Fukao et al.,
Genomics, 68:144-151 (2000); and Tanaka, Mol. Hum. Reprod. 8:16-23
(2002)). These genes/proteins are identified below in Table 46.
TABLE-US-00046 TABLE 46 Gene GenBank ID GI Number Organism
HPAG1_0676 YP_627417 108563101 Helicobacter pylori HPAG1_0677
YP_627418 108563102 Helicobacter pylori ScoA NP_391778 16080950
Bacillus subtilis ScoB NP_391777 16080949 Bacillus subtilis OXCT1
NP_000427 4557817 Homo sapiens OXCT2 NP_071403 11545841 Homo
sapiens
[0362] The conversion of succinate to succinyl-CoA can also be
catalyzed by succinyl-CoA: Acetyl-CoA transferase. The gene product
of cat1 of Clostridium kluyveri has been shown to exhibit
succinyl-CoA: acetyl-CoA transferase activity (Sohling, J
Bacteriol. 178:871-880 (1996)). In addition, the activity is
present in Trichomonas vaginalis (van Grinsven et al., J. Biol.
Chem. 283:1411-1418 (2008)) and Trypanosoma brucei (Riviere et al.,
J. Biol. Chem. 279:45337-45346 (2004)). These genes/proteins are
identified below in Table 47.
TABLE-US-00047 TABLE 47 Gene GenBank ID GI Number Organism cat1
P38946.1 729048 Clostridium kluyveri TVAG_395550 XP_001330176
123975034 Trichomonas vaginalis G3 Tb11.02.0290 XP_828352 71754875
Trypanosoma brucei
[0363] Yet another possible CoA acceptor is benzylsuccinate.
Succinyl-CoA:(R)-Benzylsuccinate CoA-Transferase functions as part
of an anaerobic degradation pathway for toluene in organisms such
as Thauera aromatica (Leutwein and Heider, J. Bact. 183(14)
4288-4295 (2001)). Homologs can be found in Azoarcus sp. T,
Aromatoleum aromaticum EbN1, and Geobacter metallireducens GS-15.
These genes/proteins are identified below in Table 48.
TABLE-US-00048 TABLE 48 Gene GenBank ID GI Number Organism bbsE
AAF89840 9622535 Thauera aromatica bbsf AAF89841 9622536 Thauera
aromatica bbsE AAU45405.1 52421824 Azoarcus sp. T bbsF AAU45406.1
52421825 Azoarcus sp. T bbsE YP_158075.1 56476486 Aromatoleum
aromaticum EbN1 bbsF YP_158074.1 56476485 Aromatoleum aromaticum
EbN1 Gmet_1521 YP_384480.1 78222733 Geobacter metallireducens GS-15
Gmet_1522 YP_384481.1 78222734 Geobacter metallireducens GS-15
[0364] Finally, ygfH encodes a propionyl CoA:succinate CoA
transferase in E. coli (Haller et al., Biochemistry, 39(16)
4622-4629). Close homologs can be found in, for example,
Citrobacter youngae ATCC 29220, Salmonella enterica subsp. arizonae
serovar, and Yersinia intermedia ATCC 29909. These genes/proteins
are identified below in Table 49.
TABLE-US-00049 TABLE 49 Gene GenBank ID GI Number Organism ygfH
NP_417395.1 16130821 Escherichia coli str. K-12 substr. MG1655
CIT292_04485 ZP_03838384.1 227334728 Citrobacter youngae ATCC 29220
SARI_04582 YP_001573497.1 161506385 Salmonella enterica subsp.
arizonae serovar yinte0001_14430 ZP_04635364.1 238791727 Yersinia
intermedia ATCC 29909
Succinyl-CoA Synthetase
[0365] The product of the LSC1 and LSC2 genes of S. cerevisiae and
the sucC and sucD genes of E. coli naturally form a succinyl-CoA
synthetase complex that catalyzes the formation of succinyl-CoA
from succinate with the concomitant consumption of one ATP, a
reaction which is reversible in vivo (Przybyla-Zawilask et al.,
Eur. J. Biochem. 258(2):736-743 (1998) and Buck et al., J. Gen.
Microbiol. 132(6):1753-1762 (1986)). These genes/proteins are
identified below in Table 50.
TABLE-US-00050 TABLE 50 Gene GenBank ID GI Number Organism LSC1
NP_014785 6324716 Saccharomyces cerevisiae LSC2 NP_011760 6321683
Saccharomyces cerevisiae sucC NP_415256.1 16128703 Escherichia coli
sucD AAC73823.1 1786949 Escherichia coli
Methylmalonyl-CoA Mutase
[0366] Succinyl-CoA can be converted into (R)-methylmalonyl-CoA by
methylmalonyl-CoA mutase (MCM). In E. coli, the reversible
adenosylcobalamin-dependant mutase participates in a three-step
pathway leading to the conversion of succinate to propionate
(Haller, Biochemistry 39:4622-9 (2000)). MCM is encoded by genes
scpA in Escherichia coli (Haller, Biochemistry 39: 4622-4629
(2000); and Bobik, Anal. Bioanal. Chem. 375:344-349 (2003)) and
mutA in Homo sapiens (Padovani, Biochemistry 45:9300-9306 (2006)).
In several other organisms MCM contains alpha and beta subunits and
is encoded by two genes. Exemplary gene candidates encoding the
two-subunit protein are Propionibacterium fredenreichii sp.
shermani mutA and mutB (Korotkova, J Biol Chem. 279:13652-13658
(2004)) and Methylobacterium extorquens mcmA and mcmB (Korotkova, J
Biol Chem. 279:13652-13658 (2004)). These genes/proteins are
identified below in Table 51.
TABLE-US-00051 TABLE 51 Gene GenBank ID GI Number Organism scpA
NP_417392.1 16130818 Escherichia coli K12 mutA P22033.3 67469281
Homo sapiens mutA P11652.3 127549 Propionibacterium fredenreichii
sp. shermanii mutB P11653.3 127550 Propionibacterium fredenreichii
sp. shermanii mcmA Q84FZ1 75486201 Methylobacterium extorquens mcmB
Q6TMA2 75493131 Methylobacterium extorquens
[0367] Additional enzyme candidates identified based on high
homology to the E. coli spcA gene product are identified below in
Table 52.
TABLE-US-00052 TABLE 52 Gene GenBank ID GI Number Organism sbm
NP_838397.1 30064226 Shigella flexneri SARI_04585 ABX24358.1
160867735 Salmonella enterica YfreA_01000861 ZP_00830776.1 77975240
Yersinia frederiksenii
[0368] There further exists evidence that genes adjacent to the
methylmalonyl-CoA mutase catalytic genes are also required for
maximum activity. For example, it has been demonstrated that the
meaB gene from M. extorquens forms a complex with methylmalonyl-CoA
mutase, stimulates in vitro mutase activity, and possibly protects
it from irreversible inactivation (Korotkova, J Biol Chem.
279:13652-13658 (2004)). The M. extorquens meaB gene product is
highly similar to the product of the E. coli argK gene (BLASTp: 45%
identity, e-value: 4e-67) which is adjacent to scpA on the
chromosome. No sequence for a meaB homolog in P. freudenreichii is
catalogued in GenBank. However, the Propionibacterium acnes
KPA171202 gene product, YP 055310.1, is 51% identical to the M.
extorquens meaB protein and its gene is also adjacent to the
methylmalonyl-CoA mutase gene on the chromosome. These
genes/proteins are identified below in Table 53.
TABLE-US-00053 TABLE 53 Gene GenBank ID GI Number Organism argK
AAC75955.1 1789285 Escherichia coli K12 KPA171202 YP_055310.1
50842083 Propionibacterium acnes meaB 2QM8_B 158430328
Methylobacterium extorquens
Methylmalonyl-CoA Epimerase
[0369] Methylmalonyl-CoA epimerase (MMCE) is the enzyme that
interconverts (R)-methylmalonyl-CoA and (S)-methylmalonyl-CoA. MMCE
is an essential enzyme in the breakdown of odd-numbered fatty acids
and of the amino acids valine, isoleucine, and methionine.
Methylmalonyl-CoA epimerase is present in organisms such as
Bacillus subtilis (YqjC) (Haller, Biochemistry. 39:4622-4629
(2000)), Homo sapiens (YqjC) (Fuller, Biochem. J 213:643-650
(1983)), Rattus norvegicus (Mcee) (Bobik, J Biol Chem.
276:37194-37198 (2001)), Propionibacterium shermanii (AF454511)
(Haller, Biochemistry 39:4622-9 (2000); McCarthy, Structure
9:637-46 (2001) and (Fuller, Biochem. J 213:643-650 (1983)) and
Caenorhabditis elegans (mmce) (Kuhnl et al., FEBS J 272:1465-1477
(2005)). The additional gene candidate, AE016877 in Bacillus
cereus, has high sequence homology to the other characterized
enzymes. MMCE activity is required if the employed
methylmalonyl-CoA decarboxylase or methylmalonyl-CoA
carboxytransferase requires the (S) stereoisomer of
methylmalonyl-CoA. These genes/proteins are identified below in
Table 54.
TABLE-US-00054 TABLE 54 Gene GenBank ID GI Number Organism YqjC
NP_390273 255767522 Bacillus subtilis MCEE Q96PE7.1 50401130 Homo
sapiens Mcee_predicted NP_001099811.1 157821869 Rattus norvegicus
AF454511 AAL57846.1 18042135 Propionibacterium fredenreichii sp.
shermanii mmce AAT92095.1 51011368 Caenorhabditis elegans AE016877
AAP08811.1 29895524 Bacillus cereus ATCC 14579
Methylmalonyl-CoA Decarboxylase
[0370] Methylmalonyl-CoA decarboxylase, is a biotin-independent
enzyme that catalyzes the conversion of methylmalonyl-CoA to
propionyl-CoA in E. coli (Benning, Biochemistry. 39:4630-4639
(2000); and Haller, Biochemistry. 39:4622-4629 (2000)). The stereo
specificity of the E. coli enzyme was not reported, but the enzyme
in Propionigenium modestum (Bott et al., Eur. J. Biochem.
250:590-599 (1997)) and Veillonella parvula (Huder, J. Biol. Chem.
268:24564-24571 (1993)) catalyzes the decarboxylation of the
(S)-stereoisomer of methylmalonyl-CoA (Hoffmann, FEBS. Lett.
220:121-125 (1987). The enzymes from P. modestum and V. parvula are
comprised of multiple subunits that not only decarboxylate
(S)-methylmalonyl-CoA, but also create a pump that transports
sodium ions across the cell membrane as a means to generate energy.
These genes/proteins are identified below in Table 55.
TABLE-US-00055 TABLE 55 Gene GenBank ID GI Number Organism YgfG
NP_417394 90111512 Escherichia coli mmdA CAA05137 2706398
Propionigenium modestum mmdD CAA05138 2706399 Propionigenium
modestum mmdC CAA05139 2706400 Propionigenium modestum mmdB
CAA05140 2706401 Propionigenium modestum mmdA CAA80872 415915
Veillonella parvula mmdC CAA80873 415916 Veillonella parvula mmdE
CAA80874 415917 Veillonella parvula mmdD CAA80875 415918
Veillonella parvula mmdB CAA80876 415919 Veillonella parvula
Example IV
Pathways for Production of Propionyl-CoA from Glucose Via
Threonine
[0371] Further to Examples I and II, the pathway for production of
propionyl-CoA via threonine is exemplified in FIG. 2. PEP is
converted into oxaloacetate either via PEP carboxykinase or PEP
carboxylase as described in Example III. Alternatively, PEP is
converted first to pyruvate by pyruvate kinase and then to
oxaloacetate by methylmalonyl-CoA carboxytransferase or pyruvate
carboxylase as described in Example III. Oxaloacetate is converted
into threonine by the native threonine pathway engineered for high
yields. It is then deaminated to form 2-oxobutanoate and
subsequently converted into propionyl-CoA. In one alternative,
2-oxobutanoate is converted to propionaldehyde by a decarboxylase,
which is then reduced to n-propanol by a propanol
dehydrogenase.
Threonine Deaminase
[0372] The conversion of threonine to 2-oxobutanoate (or
2-ketobutyrate) can be accomplished by a threonine deaminase. It is
encoded by one or more genes selected from ilvA (Calhoun et al., J.
Biol. Chem. 248(10):3511-6, (1973)) and tdcB (Umbarger et al., J.
Bacteriol. 73(1):105-12, (1957); Datta et al., Proc. Natl. Acad.
Sci. USA 84(2): 393-7(1987)). Rhodospirillum rubrum represents an
additional exemplary organism containing threonine deaminase
(Feldberg et al., Eur. J. Biochem. 21(3): 438-46 (1971); U.S. Pat.
No. 5,958,745). Details for exemplary enzymes for carrying out this
transformation are shown below. These genes/proteins are identified
below in Table 56.
TABLE-US-00056 TABLE 56 Gene GenBank ID GI Number Organism ilvA
AAC77492 1790207 Escherichia coli tdcB AAC76152 1789505 Escherichia
coli Rru_A2877 YP_427961.1 83594209 Rhodospirillum rubrum Rru_A0647
YP_425738.1 83591986 Rhodospirillum rubrum
2-Oxobutanoate Dehydrogenase
[0373] 2-oxobutanoate(2-ketobutyrate) can be converted to
propionyl-CoA via a pyruvate formate lyase and a pyruvate formate
lyase activating enzyme. The pyruvate formate lyase is encoded by
gene selected from pflB and tdcE, while the pyruvate formate lyase
activating enzyme is encoded by a pflA gene. Details for these
exemplary genes for carrying out this transformation are already
listed.
[0374] Alternatively, 2-oxobutanoate can be converted to
propionyl-CoA by means of pyruvate dehydrogenase, pyruvate
ferredoxin oxidoreductase (PFOR), or any other enzyme with
2-ketoacid dehydrogenase functionality. Such enzymes are also
capable of converting pyruvate to acetyl-CoA. Exemplary pyruvate
dehydrogenase enzymes are present in E. coli (Bisswanger, H., J.
Biol. Chem. 256:815-822 (1981); Bremer, J., Eur. J. Biochem.
8:535-540 (1969); Gong et al., J. Biol. Chem. 275:13645-13653
(2000)), B. subtilis (Nakano et al., J. Bacteriol. 179:6749-6755
(1997)), K. pneumonia (Menzel et al., J. Biotechnol. 56:135-142
(1997)), R. norvegicus (Paxton et al., Biochem. J. 234:295-303
(1986)), for example. Exemplary gene information is provided below.
These genes/proteins are identified below in Table 57.
TABLE-US-00057 TABLE 57 Gene GenBank ID GI Number Organism aceE
NP_414656.1 16128107 Escherichia coli str. K12 substr. MG1655 aceF
NP_414657.1 16128108 Escherichia coli str. K12 substr. MG1655 lpd
NP_414658.1 16128109 Escherichia coli str. K12 substr. MG1655 pdhA
P21881.1 3123238 Bacillus subtilis pdhB P21882.1 129068 Bacillus
subtilis pdhC P21883.2 129054 Bacillus subtilis pdhD P21880.1
118672 Bacillus subtilis aceE YP_001333808.1 152968699 Klebsiella
pneumonia MGH78578 aceF YP_001333809.1 152968700 Klebsiella
pneumonia MGH78578 lpdA YP_001333810.1 152968701 Klebsiella
pneumonia MGH78578 Pdha1 NP_001004072.2 124430510 Rattus norvegicus
Pdha2 NP_446446.1 16758900 Rattus norvegicus Dlat NP_112287.1
78365255 Rattus norvegicus Dld NP_955417.1 40786469 Rattus
norvegicus
[0375] Exemplary PFOR enzymes include, for example, the enzyme from
Desulfovibrio africanus which has been cloned and expressed in E.
coli, resulting in an active recombinant enzyme that was stable for
several days in the presence of oxygen (Pieulle et al., J.
Bacteriol. 179:5684-5692 (1997)). Oxygen stability is relatively
uncommon in PFORs and is reported to be conferred by a 60 residue
extension in the polypeptide chain of the D. africanus enzyme. The
M. thermoacetica PFOR is also well characterized (Menon et al.
Biochemistry 36:8484-8494 (1997)) and was shown to have high
activity in the direction of pyruvate synthesis during autotrophic
growth (Furdui et al. J. Biol. Chem. 275:28494-28499 (2000)).
Further, E. coli possesses an uncharacterized open reading frame,
ydbK, that encodes a protein that is 51% identical to the M.
thermoacetica PFOR. Evidence for pyruvate oxidoreductase activity
in E. coli has been described (Blaschkowski et al., Eur. J.
Biochem. 123:563-569 (1982)). The protein sequences of these
exemplary PFOR enzymes can be identified by the following GenBank
accession and/or GI numbers as shown below. Several additional PFOR
enzymes have been described (Ragsdale, Chem. Rev. 103:2333-2346
(2003)). These genes/proteins are identified below in Table 58.
TABLE-US-00058 TABLE 58 Gene GenBank ID GI Number Organism Por
CAA70873.1 1770208 Desulfovibrio africanus Por YP_428946.1 83588937
Moorella thermoacetica YdbK NP_415896.1 16129339 Escherichia
coli
[0376] Additional routes for producing propionyl-CoA are disclosed
in U.S. Pat. No. 5,958,745 which is incorporated by reference
herein in its entirety. One such route involves converting
2-ketobutyrate to propionate by pyruvate oxidase, and converting
propionate to propionyl-CoA via an acyl-CoA synthetase.
2-Oxobutanoate Decarboxylase
[0377] A keto acid decarboxylase can catalyze the conversion of
2-oxobutanoate to propionaldehyde. Several 2-keto acid
decarboxylases have been identified. Enzyme candidates for this
step are pyruvate decarboxylase (EC 4.1.1.1), benzoylformate
decarboxylase (4.1.1.7), alpha-ketoglutarate decarboxylase (EC
4.1.1.71), branched-chain alpha-keto-acid decarboxylase (4.1.1.72),
and indolepyruvate decarboxylase (EC 4.1.1.74). These classes of
decarboxylases are NADH-independent, they utilize thiamine
diphosphate as a cofactor, and the interaction of the substrate
with the enzyme-bound cofactor is thought to be the rate-limiting
step for enzyme activation (Hubner, Eur. J Biochem. 92:175-181
(1978)). Pyruvate decarboxylase and benzoylformate decarboxylase
have broad substrate ranges for diverse keto-acids and have been
characterized in structural detail. Fewer alpha-ketoglutarate and
branched-chain alpha-ketoacid decarboxylases have been
characterized experimentally; however these enzymes also appear to
decarboxylate a variety of keto-acid substrates.
[0378] Pyruvate decarboxylase (PDC), also termed keto-acid
decarboxylase, is a key enzyme in alcoholic fermentation,
catalyzing the decarboxylation of pyruvate to acetaldehyde. The
enzyme from Saccharomyces cerevisiae has a broad substrate range
for aliphatic 2-keto acids including 2-ketobutyrate,
2-ketovalerate, 3-hydroxypyruvate and 2-phenylpyruvate (22). The
PDC from Zymomonas mobilis, encoded by pdc, has been a subject of
directed engineering studies that altered the affinity for
different substrates (Siegert et al., Protein Eng Des Sel
18:345-357 (2005)). The PDC from Saccharomyces cerevisiae has also
been extensively studied, engineered for altered activity, and
functionally expressed in E. coli (Li, Biochemistry. 38:10004-10012
(1999); ter Schure, Appl. Environ. Microbiol. 64:1303-1307 (1998)
and Killenberg-Jabs, Eur. J. Biochem. 268:1698-1704 (2001)). The
crystal structure of this enzyme is available (Killenberg-Jabs,
Eur. J. Biochem. 268:1698-1704 (2001)). Other well-characterized
PDC candidates include the enzymes from Acetobacter pasteurians
(Chandra, Arch. Microbiol. 176:443-451 (2001)) and Kluyveromyces
lactis (Krieger, Eur. J. Biochem. 269:3256-3263 (2002)). These
genes/proteins are identified below in Table 59.
TABLE-US-00059 TABLE 59 Gene GenBank ID GI Number Organism pdc
P06672.1 118391 Zymomonas mobilis pdc1 P06169 30923172
Saccharomyces cerevisiae pdc Q8L388 20385191 Acetobacter
pasteurians pdc1 Q12629 52788279 Kluyveromyces lactis
[0379] Like PDC, benzoylformate decarboxylase has a broad substrate
range and has been the target of enzyme engineering studies. The
enzyme from Pseudomonas putida has been extensively studied and
crystal structures of this enzyme are available (Polovnikova et al,
Biochemistry 42:1820-1830 (2003); and Hasson et al., Biochemistry
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); and
Lingen, Protein Eng 15:585-593 (2002)). The enzyme from Pseudomonas
aeruginosa, encoded by mdlC, has also been characterized
experimentally (Barrowman, FEMS Microbiology Letters 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)). These genes/proteins are
identified below in Table 60.
TABLE-US-00060 TABLE 60 Gene GenBank ID GI Number Organism 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
[0380] A third enzyme capable of decarboxylating 2-oxoacids is
alpha-ketoglutarate decarboxylase (KGD). The substrate range of
this class of enzymes has not been studied to date. The KDC from
Mycobacterium tuberculosis (Tian, Proc Natl Acad Sci U.S.A
102:10670-10675 (2005)) has been cloned and functionally expressed
in other internal projects at Genomatica. However, it is not an
ideal candidate for strain engineering because it is large
(.about.130 kD) and GC-rich. KDC enzyme activity has been detected
in several species of Rhizobia including Bradyrhizobium japonicum
and Mesorhizobium loti (Green, J Bacteriol. 182:2838-2844 (2000)).
Although the KDC-encoding gene(s) have not been isolated in these
organisms, the genome sequences are available and several genes in
each genome are annotated as putative KDCs. A KDC from Euglena
gracilis has also been characterized but the gene associated with
this activity has not been identified to date (Shigeoka, Arch.
Biochem. Biophys. 288:22-28 (1991)). The first twenty amino acids
starting from the N-terminus were sequenced MTYKAPVKDVKFLLDKVFKV
(Shigeoka, Arch. Biochem. Biophys. 288:22-28 (1991)). The gene
could be identified by testing candidate genes containing this
N-terminal sequence for KDC activity. These genes/proteins are
identified below in Table 61.
TABLE-US-00061 TABLE 61 Gene GenBank ID GI Number Organism kgd
O50463.4 160395583 Mycobacterium tuberculosis kgd NP_767092.1
27375563 Bradyrhizobium japonicum USDA110 kgd NP_105204.1 13473636
Mesorhizobium loti
[0381] A fourth candidate enzyme for catalyzing this step is
branched chain alpha-ketoacid decarboxylase (BCKA). This class of
enzyme has been shown to act on a variety of compounds varying in
chain length from 3 to 6 carbons (Oku, J Biol Chem. 263:18386-18396
(1988); and Smit et al., Appl Environ Microbiol 71:303-311 (2005)).
The 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 (Berthold et al., Acta Crystallogr. D Biol
Crystallogr. 63:1217-1224 (2007)). Sequence alignments between the
Lactococcus lactis enzyme and the pyruvate decarboxylase of
Zymomonas mobilis 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. 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, 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. This gene/protein is identified below in Table
62.
TABLE-US-00062 TABLE 62 Gene GenBank ID GI Number Organism kdcA
AAS49166.1 44921617 Lactococcus lactis
[0382] 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 (Wynn, J. Biol. Chem. 267:12400-12403 (1992); Davie, J. Biol.
Chem. 267:16601-16606 (1992) and 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, J. Biol. Chem.
267:12400-12403 (1992)). These enzymes are composed of two alpha
and two beta subunits. These genes/proteins are identified below in
Table 63.
TABLE-US-00063 TABLE 63 Gene GenBank ID GI Number Organism BCKDHB
NP_898871.1 34101272 Homo sapiens BCKDHA NP_000700.1 11386135 Homo
sapiens BCKDHB P21839 115502434 Bos taurus BCKDHA P11178 129030 Bos
taurus
Example V
Pathways for Production of Propionyl-CoA from Glucose Via
Malonyl-CoA
[0383] Further to Examples I and II, the pathway for production of
propionyl-CoA via malonyl-CoA is exemplified in FIG. 3. Acetyl CoA
is carboxylated to form malonyl-CoA. This is then reduced to
malonate semialdehyde, and subsequently transformed into
3-hydroxypropionate (3HP). 3HP is converted into propionyl-CoA via
propionyl-CoA synthase.
Acetyl-CoA Carboxylase
[0384] The multisubunit acetyl-CoA carboxylase complex (ACC),
broadly conserved among bacteria, catalyzes the ATP-dependent
formation of malonyl-CoA by acetyl-CoA and bicarbonate. This
reaction serves as the first committed step in fatty acid
biosynthesis, and the enzyme has been targeted in efforts to
develop antibacterial drugs and inhibitors in E. coli (Freiberg et
al., J. Biol. Chem. 279: 26066-26073 (2004)), yeast (Zhang, Proc.
Natl. Acad. Sci. U S. A. 101:5910-5915 (2004)), Bacillus subtilis
(Freiberg et al., J. Biol. Chem. 279:26066-26073 (2004)) and other
organisms (Barber, Biochim. Biophys. Acta 1733:1-28 (2005)). In E.
coli and many other bacteria, ACC is composed of four subunits
encoded by accA, accB, accC and accD (Choi-Rhee, J. Biol. Chem.
278:30806-30812 (2003)). Expression of two subunits, accB and accC,
is autoregulated by the gene product of accB (James, J. Biol. Chem.
279:2520-2527 (2004)). In yeast, the enzyme is encoded by two
genes, hfa1 and accI. The gene bpl1, encoding a biotin:apoprotein
ligase, is required for enzyme function.
[0385] Autotrophic members of the archael taxonomic group
Sulfolobales exhibit high levels of acetyl-CoA carboxylase activity
in the context of the 3-hydroxypropionate cycle (Chuakrut, J.
Bacteriol. 185:938-947 (2003); and Hugler, Eur. J. Biochem.
270:736-744 (2003)). In Metallosphaera sedula, the acyl-CoA
carboxylase holoenzyme is a multimer composed of subunits encoded
by three genes: Msed.sub.--0148 (biotin/lipoyl attachment),
Msed.sub.--0147 (biotin carboxylase), and Msed.sub.--1375 (carboxyl
transferase). The enzyme has been purified and characterized and
was found to be bifunctional, reacting with acetyl-CoA and
propionyl-CoA (Hugler, Eur. J. Biochem. 270:736-744 (2003)). A
bifunctional archael acetyl-CoA carboxylase enzyme from Acidanus
brierleyi, encoded by three genes, has been cloned into E. coli and
characterized (Chuakrut, J. Bacteriol. 185:938-947 (2003). The
sequences of A. brierleyi acyl-CoA carboxylase genes and flanking
regions were submitted to the DNA Data Bank of Japan (DDBJ) under
accession no. AB088419. Although these archael enzymes exhibit high
activity it should be noted that the optimum temperature is
65.degree. C. (Chuakrut, J. Bacteriol. 185:938-947 (2003)). These
genes/proteins are identified below in Table 64.
TABLE-US-00064 TABLE 64 Gene GenBank ID GI Number Organism accA
NP_414727 16128178 Escherichia coli K12 str. MG1655 accB NP_417721
16131143 Escherichia coli K12 str. MG1655 accC NP_417722 16131144
Escherichia coli K12 str. MG1655 accD NP_416819 16130251
Escherichia coli K12 str. MG1655 accA NP_390798.1 16079972 Bacillus
subtilis subsp. subtilis str. 168 accB NP_390315.1 16079491
Bacillus subtilis subsp. subtilis str. 168 accC NP_390314.1
16079490 Bacillus subtilis subsp. subtilis str. 168 accD
NP_390799.1 16079973 Bacillus subtilis subsp. subtilis str. 168
bpl1 NP_010140.1 6320060 Saccharomyces cerevisiae hfa1 NP_013934.1
6323863 Saccharomyces cerevisiae acc1 NP_014413.1 6324343
Saccharomyces cerevisiae accB Msed_0148 Q8J2Z3 74499802
Metallosphaera sedula accC Msed_0147 Q8J2Z4 74499032 Metallosphaera
sedula pccB Msed_1375 Q8J2Z5 74499033 Metallosphaera sedula accB
BAC55868.1 27877098 Acidanus brierleyi accC BAC55867.1 27877097
Acidanus brierleyi pccB BAC55869.1 27877099 Acidanus brierleyi
Malonyl-CoA Reductase and Malonate Semialdehyde Reductase
[0386] The reduction of malonyl-CoA to 3-HP can be accomplished by
a bifunctional malonyl-CoA reductase with aldehyde dehydrogenase
and alcohol dehydrogenase functionality. An NADPH-dependent enzyme
with this activity has been characterized in Chloroflexus
aurantiacus where it participates in the 3-hydroxypropionate cycle
(Hugler, J. Bacteriol. 184:2404-2410 (2002); and Strauss, Eur. J.
Biochem. 215:633-643 (1993)). This enzyme, with a mass of 300 kDa,
is highly substrate-specific and shows little sequence similarity
to other known oxidoreductases (Hugler, J. Bacteriol. 184:2404-2410
(2002)). No enzymes in other organisms have been shown to catalyze
this specific reaction; however there is bioinformatic evidence
that other organisms may have similar pathways (Klatt, Environ.
Microbiol. 9:2067-2078 (2007). Enzyme candidates in other organisms
including Roseiflexus castenholzii, Erythrobacter sp. NAP1 and
marine gamma proteobacterium HTCC2080 can be inferred by sequence
similarity. These genes/proteins are identified below in Table
65.
TABLE-US-00065 TABLE 65 Gene GenBank ID GI Number Organism mcr
AAS20429.1 42561982 Chloroflexus aurantiacus Rcas_2929
YP_001433009.1 156742880 Roseiflexus castenholzii NAP1_02720
ZP_01039179.1 85708113 Erythrobacter sp. NAP1 MGP2080_00535
ZP_01626393.1 119504313 marine gamma proteobacterium HTCC2080
[0387] Alternatively, the reduction of malonyl-CoA to 3-HP can be
catalyzed by two separate enzymes: a CoA-acylating aldehyde
dehydrogenase and a primary alcohol dehydrogenase. By this route,
malonyl-CoA is first reduced to malonate semialdehyde (MSA) by
malonate-semialdehyde dehydrogenase or malonyl-CoA reductase. MSA
is subsequently converted to 3-HP by 3-HP-dehydrogenase.
[0388] Malonyl-CoA reductase is a key enzyme in autotrophic carbon
fixation via the 3-hydroxypropionate cycle in thermoacidophilic
archael bacteria (Berg, Science. 318:1782-1786 (2007); and Thauer,
Science. 318:1732-1733 (2007)). The enzyme utilizes NADPH as a
cofactor and has been characterized in Metallosphaera and
Sulfolobus spp (Alber et at, J. Bacteriol. 188:8551-8559 (2006);
and Hugler, J. Bacteriol. 184:2404-2410 (2002)). The enzyme encoded
by Msed.sub.--0709 in Metallosphaera sedula is known to convert
malonyl-CoA to malonic semialdehyde and operate in the direction of
interest (Alber et al., J. Bacteriol. 188:8551-8559 (2006); and
(Berg, Science. 318:1782-1786 (2007)). A gene encoding a
malonyl-CoA reductase from Sulfolobus tokodaii was cloned and
heterologously expressed in E. coli (Alber et al., J. Bacteriol.
188:8551-8559 (2006). Although the aldehyde dehydrogenase
functionality of these enzymes is similar to the bifunctional
dehydrogenase from Chloroflexus aurantiacus, there is little
sequence similarity. Both malonyl-CoA reductase enzyme candidates
have high sequence similarity to aspartate-semialdehyde
dehydrogenase, an enzyme catalyzing the reduction and concurrent
dephosphorylation of aspartyl-4-phosphate to aspartate
semialdehyde. Additional gene candidates can be found by sequence
homology to proteins in other organisms including Sulfolobus
solfataricus and Sulfolobus acidocaldarius. These genes/proteins
are identified below in Table 66.
TABLE-US-00066 TABLE 66 Gene GenBank ID GI Number Organism
Msed_0709 YP_001190808.1 146303492 Metallosphaera sedula mcr
NP_378167.1 15922498 Sulfolobus tokodaii asd-2 NP_343563.1 15898958
Sulfolobus solfataricus Saci_2370 YP_256941.1 70608071 Sulfolobus
acidocaldarius
[0389] The subsequent conversion of malonic semialdehyde to 3-HP
can be accomplished by an enzyme with 3-HP dehydrogenase activity.
Three enzymes are known to catalyze this conversion: NADH-dependent
3-hydroxypropionate dehydrogenase, NADPH-dependent malonate
semialdehyde reductase, and NADH-dependent 3-hydroxyisobutyrate
dehydrogenases. An NADH-dependent 3-hydroxypropionate dehydrogenase
is thought to participate in beta-alanine biosynthesis pathways
from propionate in bacteria and plants (Rathinasabapathi, Journal
of Plant Pathology 159:671-674 (2002); and Stadtman, A. J. Am.
Chem. Soc. 77:5765-5766 (1955)). This enzyme has not been
associated with a gene in any organism to date. NADPH-dependent
malonate semialdehyde reductase catalyzes the reverse reaction in
autotrophic CO2-fixing bacteria. Although the enzyme activity has
been detected in Metallosphaera sedula, the identity of the gene is
not known (Alber et al., J. Bacteriol. 188:8551-8559 (2006)).
[0390] Several 3-hydroxyisobutyrate dehydrogenase enzymes have also
been shown to convert malonic semialdehyde to 3-HP. Three gene
candidates exhibiting this activity are mmsB from Pseudomonas
aeruginosa PAO1 (Gokam et al., U.S. Pat. No. 7,393,676 (2008)).
mmsB from Pseudomonas putida KT2440 (Liao, U.S. Patent Publication
2005-0221466 (2005) and mmsB from Pseudomonas putida E23
(Chowdhury, Biosci. Biotechnol. Biochem. 60:2043-2047 (1996)). The
protein from Pseudomonas putida E23 has been characterized and
functionally expressed in E. coli; however, its activity on 3-HP
was relatively low (Chowdhury, Biosci. Biotechnol. Biochem.
60:2043-2047 (1996)). An enzyme with 3-hydroxybutyrate
dehydrogenase activity in Alcaligenes faecalis M3A has also been
identified (Liao, U.S. Patent Publication 2005-0221466 (2005); and
Liao, U.S. Patent Publication 2005-0221466 (2005)). Additional gene
candidates from other organisms including Rhodobacter spaeroides
can be inferred by sequence similarity. These genes/proteins are
identified below in Table 67.
TABLE-US-00067 TABLE 67 Gene GenBank ID GI Number Organism mmsB
AAA25892.1 151363 Pseudomonas aeruginosa mmsB NP_252259.1 15598765
Pseudomonas aeruginosa PAO1 mmsB NP_746775.1 26991350 Pseudomonas
putida KT2440 mmsB JC7926 60729613 Pseudomonas putida E23 orfB1
AAL26884 16588720 Rhodobacter spaeroides
[0391] Enzymes exhibiting a 4-hydroxybutyrate activity (EC
1.1.1.61) may also be able to convert malonic semialdehyde to 3-HP,
as the chemical transformation is very similar. Such enzymes have
been characterized in Ralstonia eutropha (Bravo, J. Forensic Sci.
49:379-387 (2004)), Clostridium kluyveri (Wolff, Protein Expr. Pur
6:206-212 (1995)) and Arabidopsis thaliana (Breitkreuz et al., J.
Biol. Chem. 278:41552-41556 (2003)). Activity of these enzymes on
malonic semialdehyde has not been demonstrated experimentally to
date. However, since these enzymes have been studied in other
internal projects at Genomatica they could easily be tested for
3-HP dehydrogenase activity. These genes/proteins are identified
below in Table 68.
TABLE-US-00068 TABLE 68 Gene GenBank ID GI Number Organism 4hbd
YP_726053.1 113867564 Ralstonia eutropha H16 4hbd L21902.1
146348486 Clostridium kluyveri DSM 555 4hbd Q94B07 75249805
Arabidopsis thaliana
Propionyl-CoA Synthase
[0392] The conversion of 3-hydroxypropionate (3HP) to propionyl-CoA
is accomplished by a propionyl-CoA synthase. This step is known to
be catalyzed by a single fusion protein of 201 KDa in Chloroflexus
aurantiacus (Alber, J Biol. Chem. 277:12137-12143 (2002)). The
protein is comprised of a CoA ligase, an enoyl-CoA hydratase and an
enoyl-CoA reductase. The enzyme has been purified 30-fold to near
homogeneity and has a very large native molecular mass between 500
and 800 kDa. In thermoacidophilic Metallosphaera sedula (and
members of the Sulfolobaceae family), this function is catalyzed by
three different enzymes, a 3-hydroxypropionyl-CoA synthetase that
activates 3HP to its CoA ester, a 3-hydroxypropionyl-CoA
dehydratase that converts 3-HP-CoA to acryloyl-CoA followed by the
reduction of the latter to form propionyl-CoA. A 3-HP-CoA
synthetase had been reported (Alber, J Bacteriol. 190:1383-1389
(2008)). The gene encoding the protein has been sequenced and gene
encoding a homologous protein identified in the genome of
Sulfolobus tokodaii; similar genes were found in S. solfataricus
and S. acidocaldarius. The gene was heterologously expressed in
Escherichia coli. These genes/proteins are identified below in
Table 69.
TABLE-US-00069 TABLE 69 Gene GenBank ID GI Number Organism
Msed_1456 YP_001191537 146304221 M. sedula ST0783 NP_376686
15921017 S. tokodaii acsA-10 NP_344510 15899905 S. solfataricus
Saci_1184 YP_255824 70606954 S. acidocaldarius pcs AAL47820
29126583 C. aurantiacus
[0393] Recently, 3-hydroxypropionyl-CoA dehydratase and
acryloyl-CoA reductase were purified from M. sedula (Teufel, J
Bacteriol. 191:4572-4581 (2009)), the coding genes were identified
from the genome of M. sedula and other members of the Sulfolobales,
and recombinant enzymes were produced as a proof of function. It
was concluded that the genes coding for 3-hydroxypropionyl-CoA
dehydratase and acryloyl-CoA reductase are not clustered on the
Metallosphaera or the Sulfolobus genome. Comparison of the
respective domains of propionyl-CoA synthase in these two organisms
has revealed that the enzyme(s) catalyzing the conversion of 3HP to
propionyl-CoA has evolved independently in these two phyla. The
GenBank accession and/or GI numbers for the 3-HP-CoA dehydratase
from M. sedula are identified below in Table 70.
TABLE-US-00070 TABLE 70 Gene GenBank ID GI Number Organism
Msed_2001 YP_001192065.1 146304749 M. sedula
[0394] The GenBank IDs for acryloyl-CoA reductases are identified
below in Table 71.
TABLE-US-00071 TABLE 71 Gene GenBank ID GI Number Organism
Msed_1426 YP_001191508.1 146304192 M. sedula ST0480 NP_376364
15920695 S. tokodaii
[0395] Other gene candidates encoding these two enzymes can be
obtained by sequence homology searches.
Example VI
Pathways for Production of Propionyl-CoA from Glucose Via
Lactate
[0396] Further to Examples I and II, the pathway for production of
propionyl-CoA via lactate is exemplified in FIG. 4. This pathway
presents yet another redox balanced route for the formation of
propionyl-CoA. Pyruvate is reduced to form lactate which is then
activated to form lactoyl-CoA. The lactoyl-CoA is dehydrated to
form acryloyl-CoA and then reduced to generate propionyl-CoA.
Lactate Dehydrogenase
[0397] The conversion of pyruvate to lactate is catalyzed by
lactate dehydrogenase (EC 1.1.1.27). Many lactate dehydrogenases
have been described in detail (Garvie, Microbiol Rev 44:106-139
(1980)). The fermentative lactate dehydrogenase of Escherichia coli
will be the first candidate to be overexpressed for converting
pyruvate to lactate (Bunch, Microbiology 143 (Pt 1), 187-195
(1997)). Other lactate dehydrogenase candidates will be utilized
for this step including those with low Km for pyruvate that favors
the formation of lactate, such as lactate dehydrogease from:
Lactobacillus casei (Gordon, Eur. J Biochem. 67:543-555 (1976)),
Plasmodium falciparum (Brown et al., Biochemistry 43:6219-6229
(2004)), and Thermotoga maritime (Auerbach et al., Structure.
6:769-781 (1998)). These genes/proteins are identified below in
Table 72.
TABLE-US-00072 TABLE 72 Gene GenBank ID GI Number Organism ldh
P52643 1730102 Escherichia coli ldh P00343 126063 Lactobacillus
casei ldh Q6JH32 74911026 Plasmodium ovale ldh P16115 547837
Thermotoga maritima
Lactate-CoA Transferase
[0398] The activation of lactate to lactoyl-CoA can be catalyzed by
lactate-CoA transferase activity associated with propionate
CoA-transferase (EC 2.8.3.1). Clostridium propionicum ferments
alanine via the nonrandomising pathway with acryloyl-CoA as
characteristic intermediate. In this pathway, lactate is activated
to lactoyl-CoA by the enzyme propionate: acetyl-CoA CoA-transferase
(EC 2.8.3.1, or propionate CoA-transferase) using propionyl-CoA or
acetyl-CoA as a coenzyme A donor (Schweiger, FEBS Lett. 171:79-84
(1984)). The enzyme exhibited rather broad substrate specificities
for monocarboxylic acids including acrylate, propionate and
butyrate whereas dicarboxylic acids were not used. Gene coding for
this enzyme was cloned (Selmer, Eur. J Biochem. 269:372-380
(2002)). Other propionate CoA-transferase can be candidates for
this step include homologues of Clostridium propionicum propionate
CoA-transferase. These genes/proteins are identified below in Table
73.
TABLE-US-00073 TABLE 73 Gene GenBank ID GI Number Organism Pct
Q9L3F7 75416255 Clostridium propionicum Pct YP_002270763.1
209397911 Escherichia coli O157:H7 Pct Q220N6 122479931 Rhodoferax
ferrireducens DSM 15236 Pct Q46MA6 123621528 Ralstonia eutropha
Lactoyl-CoA Dehydratase
[0399] The dehydration of lactoyl-CoA to acryloyl-CoA is catalyzed
by lactoyl-CoA dehydratase (EC 4.2.1.54). Clostridium propionicum
ferments alanine via the nonrandomising pathway with acryloyl-CoA
as characteristic intermediate (Schweiger, FEBS Lett. 171:79-84
(1984)). In this pathway, lactoyl-CoA is dehydrated to acryloyl-CoA
by the lactoyl-CoA dehydratase (Hofmeister, Eur. J Biochem.
206:547-552 (1992)). Cloning of the propionate CoA-transferase also
identified a second ORF (lcdB) likely encoding one subunit of the
lactoyl-CoA dehydratase required in the pathway. The lcdB is
similar to the 2-hydroxyglutaryl-CoA dehydratase .beta. subunit.
Homologues of lcdB will be tested for their activity in this step.
These genes/proteins are identified below in Table 74.
TABLE-US-00074 TABLE 74 Gene GenBank ID GI Number Organism
CBC_A0885 ZP_02621214 168186579 Clostridium botulinum C str. Eklund
CBC_A0886 ZP_02621215 168186580 Clostridium botulinum C str. Eklund
hgdB YP_878441 118444181 Clostridium novyi-NT hgdA YP_878442
118444701 Clostridium novyi-NT
Acryloyl-CoA Reductase
[0400] The conversion of acryloyl-CoA to propionyl-CoA is catalyzed
by the acryloyl-CoA reductase. In alanine-fermenting Clostridium
propionicum, acryloyl-CoA reductase catalyses the irreversible
NADH-dependent formation of propionyl-CoA from acryloyl-CoA. The
enzyme has been purified and the N-termini of the subunits of the
enzyme have been determined (Hetzel et al., Eur. J Biochem.
270:902-910 (2003)). The N-terminus of the dimeric propionyl-CoA
dehydsrogenase subunit is similar to those of butyryl-CoA
dehydrogenases from several Clostridia and related anaerobes (up to
55% sequence identity). The N-termini of the .beta. and .gamma.
subunits share 40% and 35% sequence identities with those of the A
and B subunits of the electron-transferring flavoprotein (ETF) from
Megasphaera elsdenii, respectively, and up to 60% with those of
putative ETFs from other anaerobes. Since the complete genome
sequence of Clostridium propionicum is not available, the
N-terminus of the propionyl-CoA dehydrogenase subunit
"MDFKLTKTQVLQQWLFAEFAGIGIKPIAE" (SEQ ID NO.) was used in similarity
search and resulted in the following homologues of the
propionyl-CoA dehydrogenase for their activities in this step.
These genes/proteins are identified below in Table 75.
TABLE-US-00075 TABLE 75 Gene GenBank ID GI Number Organism bcdA
CAQ53135 188027001 Clostridium saccharobutylicum Cbei_2035 ABR34203
149903370 Clostridium beijerinckii ANACAC_00471 EDR98937 167654808
Anaerostipes caccae DSM 14662
[0401] Additionally, a tri-functional propionyl-CoA synthase (pcs)
gene was identified from the phototrophic green non-sulfur
eubacterium Chloroflexus aurantiacus (Alber, J Biol. Chem.
277:12137-12143 (2002)). The propionyl-CoA synthase is a natural
fusion protein of 201 kDa consisting of a CoA ligase, an enoyl-CoA
hydratase, and an enoyl-CoA reductase. The enzyme catalyzes the
conversion from 3-hydroxypropionate to 3-hydroxypropionyl-CoA to
acryloyl-CoA then to propionyl-CoA. This enzyme can be utilized in
whole or in part for its enoyl-CoA reductase activity. The
gene/protein is identified below in Table 76.
TABLE-US-00076 TABLE 76 Gene GenBank ID GI Number Organism pcs
AAL47820 29126583 Chloroflexus aurantiacus
Example VII
Pathways for Co-Production of 1,4-Butanediol (1,4-BDO) and
Isopropanol from Glucose
[0402] This example describes exemplary pathways for co-production
of 1,4-butanediol (1,4-BDO) and isopropanol.
[0403] Novel pathways for co-producing 1,4-butanediol (1,4-BDO) and
isopropanol and related products are described herein. In the
1,4-butanediol (1,4-BDO) and isopropanol co-production pathway of
FIG. 5, central metabolism intermediates are first channeled into
succinyl-CoA. For formation of succinyl-CoA, phosphoenolpyruvate
(PEP) is converted into oxaloacetate either via PEP carboxykinase
or PEP carboxylase. Alternatively, PEP is converted first to
pyruvate by pyruvate kinase and then to oxaloacetate by
methylmalonyl-CoA carboxytransferase or pyruvate carboxylase.
Oxaloacetate is then converted to succinyl-CoA by means of the
reductive TCA cycle. Succinyl-CoA is then converted to succinic
semialdehyde by a CoA-dependent aldehyde dehydrogenase.
Alternatively, succinate can be converted to succinic semialdehyde
by a succinate reductase. Next, succinic semialdehyde is reduced to
4-hydroxybutyrate by 4-hydroxybutyrate dehydrogenase. Activation of
4-HB to its acyl-CoA is catalyzed by a CoA transferase or
synthetase. Alternatively, 4-HB can be converted into
4-hydroxybutyryl-phosphate and subsequently transformed into
4-HB-CoA by a phosphotrans-4-hydroxybutyrylase. 4-HB-CoA is then
converted to 14-BDO by either a bifunctional CoA-dependent
aldehyde/alcohol dehydrogenase, or by two separate enzymes with
aldehyde and alcohol dehydrogenase activity. Yet another
alternative that bypasses the 4-HB-CoA intermediate is direct
reduction of 4-HB to 4-hydroxybutyrylaldehyde by a carboxylic acid
reductase. 4-Hydroxybutyrylaldehyde is subsequently reduced to
14-BDO by an alcohol dehydrogenase. Yet another route that bypasses
the CoA intermediate is reduction of 4-hydroxybutyryl-phosphate to
4-hydroxybutyryaldehyde by a phosphate reductase. Pathways for
production of isopropanol proceed as described above in Examples I
and II.
[0404] The maximum theoretical yield of a 14-BDO and isopropanol
producing organism is 0.77 moles isopropanol and 0.46 moles 14-BDO
per mole glucose consumed (0.26 g/g IPA and 0.23 g/g 14-BDO), per
the following equation:
13 Glucose.fwdarw.10IPA+6 14-BDO+24CO2+8H.sub.2O
Example VIII
Pathways for Co-Production of 1,3-Butanediol (1,3-BDO) and
Isopropanol from Glucose
[0405] This example describes exemplary pathways for co-production
of 1,3-butanediol (13-BDO) and isopropanol.
[0406] Novel pathways for co-producing 1,3-butanediol (13-BDO) and
isopropanol and related products are described herein. The
coproduction route to 1,3-butanediol (13-BDO) and isopropanol,
shown in FIG. 6, also proceeds through 4-hydroxybutyryl-CoA, formed
as described in Example VI. In this route, 4-hydroxybutyryl-CoA is
dehydrated and isomerized to form crotonyl-CoA. The dehydration and
vinylisomerisation reactions are catalyzed by a bifunctional
enzyme, 4-hydroxybutyryl-CoA dehydratase. Crotonyl-CoA is then
hydrated to 3-hydroxybutyryl-CoA. Removal of the CoA moiety and
concurrent reduction yields 3-hydroxybutyraldehyde. Finally
reduction of the aldehyde by 3-hydroxybutyraldehyde reductase
yields 13-BDO. Alternately, 3-hydroxybutyryl-CoA can be converted
to 13-BDO directly by a 3-hydroxybutyryl-CoA reductase (alcohol
forming). Several other alternate routes are possible in this
pathway. Succinate can be converted to succinic semialdehyde by a
carboxylic acid reductase, bypassing the formation of succinyl-CoA.
4-HB can be phosphorylated to 4-HB-phosphate by a kinase, then
subsequently converted to 4-HB-CoA. Finally 3-hydroxybutyryl-CoA
can be de-acylated by a CoA hydrolase, transferase or synthetase,
then subsequently reduced to 3-hydroxybutyraldehyde by a carboxylic
acid reductase.
[0407] Pathways for production of isopropanol proceed as described
above in Examples I and II.
[0408] The maximum theoretical yield of 13-BDO and isopropanol via
this pathway is 0.77 moles isopropanol and 0.46 moles 13-BDO per
mole glucose consumed (0.26 g/g IPA and 0.23 g/g 13-BDO), per the
following equation:
13 Glucose 10IPA+6 13-BDO+24CO2+8H.sub.2O
Example IX
Pathways for Co-Production of Methylacrylic Acid (MAA) and
Isopropanol from Glucose
[0409] This example describes exemplary pathways for co-production
of methylacrylic acid (MAA) and isopropanol.
[0410] Novel pathways for co-producing methylacrylic acid (MAA) and
isopropanol and related products are described herein. Two
coproduction routes to methylacrylic acid (MAA) are shown in FIGS.
7 and 8. The route shown in FIG. 7 proceeds through
4-hydroxybutyryl-CoA, formed as described previously.
4-Hydroxybutyryl-CoA is converted to 3-hydroxyisobutyryl-CoA by a
methyl mutase. The CoA moiety of 3-Hydroxyisobutyryl-CoA is then
removed by a CoA transferase, hydrolase or synthetase. Finally,
dehydration of the 3-hydroxy group yields MAA. Several of the key
steps in this route can be bypassed by alternate routes. Succinate,
for example, can be directly converted to succinic semialdehyde by
a succinate reductase, bypassing the formation of succinyl-CoA. The
conversion of 4-HB to 4-HB-CoA can proceed through the intermediate
4-hydroxybutyrylphosphate, via the enzymes 4-hydroxybutyrate kinase
and phosphotrans-4-hydroxybutyrylase. 3-HIBCOA can be converted to
MAA via the intermediate methacrylyl-CoA. Pathways for production
of isopropanol proceed as described above in Examples I and II.
[0411] In the alternate MAA coproduction route shown in FIG. 8,
succinyl-CoA is formed through the reductive TCA cycle, then
converted to methylmalonyl-CoA by methylmalonyl-CoA mutase. An
epimerase may be required to convert the (R) stereoisomer of
methylmalonyl-CoA to the (S) configuration. A CoA-dependent
aldehyde dehydrogenase then converts methylmalonyl-CoA to
methylmalonate semialdehyde. Reduction of the aldehyde to
3-hydroxyisobutyrate, followed by dehydration, yields MAA.
Alternately, methylmalonyl-CoA is converted to 3-hydroxyisobutyrate
by an alcohol-forming CoA reductase. In yet another alternate
route, methylmalonyl-CoA is converted to methylmalonate by a CoA
hydrolase, transferase or synthetase. Methylmalonate is
subsequently converted to methylmalonate semialdehyde by a
carboxylic acid reductase. Methylmalonate semialdehyde is converted
to MAA as described previously. Pathways for production of
isopropanol proceed as described above in Examples I and II.
[0412] Both MAA coproduction pathways achieve yields 0.67 moles
each of isopropanol and MAA per mole glucose utilized (0.22 g/g
isopropanol and 0.32 g/g MAA) per the equation:
3 Glucose 2IPA+2MAA+4CO2+4H.sub.2O
Example X
Enzyme Classification System for Production of Isopronaol and
1,4-Butanediol (1,4-BDO), 1,3-Butanediol (1,3-BDO) or Methylacrylic
Acid (MAA)
[0413] This example describes the enzyme classification system for
the exemplary pathways described in Examples VII and IX for
production of 1,4-butanediol (1,4-BDO), 1,3-butanediol (1,3-BDO) or
methylacrylic acid (MAA). Exemplary enzymes for production of
isopropanol from acetyl-CoA are described in Example I and
exemplary enzymes for production acetyl-CoA from glucose are
described in Example II.
PEP Carboxykinase
[0414] Although the net conversion of phosphoenolpyruvate to
oxaloacetate is redox-neutral, the mechanism of this conversion is
important to the overall energetics of the co-production pathway.
The most desirable enzyme for the conversion of PEP to oxaloacetate
is PEP carboxykinase which simultaneously forms an ATP while
carboxylating PEP. In most organisms, however, PEP carboxykinase
serves a gluconeogenic function and converts oxaloacetate to PEP at
the expense of one ATP. S. cerevisiae is one such organism whose
native PEP carboxykinase, PCK1, serves a gluconeogenic role
(Valdes-Hevia et al., FEBS. Lett. 258:313-316 (1989)). E. coli is
another such organism, as the role of PEP carboxykinase in
producing oxaloacetate is believed to be minor when compared to PEP
carboxylase, which does not form ATP, possibly due to the higher
K.sub.m for bicarbonate of PEP carboxykinase (Kim, et al., Appl
Environ Microbiol 70:1238-1241 (2004)). Nevertheless, activity of
the native E. coli PEP carboxykinase from PEP towards oxaloacetate
has been recently demonstrated in ppc mutants of E. coli K-12 (Kwon
et al., Journal of Microbiology and Biotechnology 16:1448-1452
(2006)). These strains exhibited no growth defects and had
increased succinate production at high NaHCO.sub.3 concentrations.
In some organisms, particularly rumen bacteria, PEP carboxykinase
is quite efficient in producing oxaloacetate from PEP and
generating ATP. Examples of PEP carboxykinase genes that have been
cloned into E. coli include those from Mannheimia
succiniciproducens (Lee et al., Gene. Biotechnol. Bioprocess Eng.
7:95-99 (2002)), Anaerobiospirillum succiniciproducens (Laivenieks
et al., Appl Environ Microbiol 63:2273-2280 (1997)), and
Actinobacillus succinogenes (Kim et al., Appl Environ Microbiol
70:1238-1241 (2004)). Internal experiments have also found that the
PEP carboxykinase enzyme encoded by Haemophilus influenza is highly
efficient at forming oxaloacetate from PEP. These genes/proteins
are identified below in Table 77.
TABLE-US-00077 TABLE 77 Gene GenBank ID GI Number Organism PCK1
NP_013023 6322950 Saccharomyces cerevisiae pck NP_417862.1 16131280
Escherichia coli pckA YP_089485.1 52426348 Mannheimia
succiniciproducens pckA O09460.1 3122621 Anaerobiospirillum
succiniciproducens pckA Q6W6X5 75440571 Actinobacillus succinogenes
pckA P43923.1 1172573 Haemophilus influenza
[0415] These sequences and sequences for subsequent enzymes listed
in this report can be used to identify homologue proteins in
GenBank or other databases through sequence similarity searches
(e.g. BLASTp). The resulting homologue proteins and their
corresponding gene sequences provide additional DNA sequences for
transformation into the host organism of our choice.
PEP Carboxylase
[0416] PEP carboxylase represents an alternative enzyme for the
formation of oxaloacetate from PEP. S. cerevisiae does not
naturally encode a PEP carboxylase, but exemplary organisms that
possess genes that encode PEP carboxylase include E. coli (Kai et
al., Arch. BioChem. Biophys. 414:170-179 (2003)), Methylobacterium
extorquens AM1 (Arps et al., J. Bacteriol. 175:3776-3783 (1993)),
and Corynebacterium glutamicum (Eikmanns et al., Mol. Gen. Genet.
218:330-339 (1989)). These genes/proteins are identified below in
Table 78.
TABLE-US-00078 TABLE 78 Gene GenBank ID GI Number Organism ppc
NP_418391 16131794 Escherichia coli ppcA AAB58883 28572162
Methylobacterium extorquens ppc ABB53270 80973080 Corynebacterium
glutamicum
Pyruvate Kinase and Methylmalonyl-CoA Carboxytransferase
[0417] An additional energetically efficient route to oxaloacetate
from PEP requires two enzymatic activities: pyruvate kinase and
methylmalonyl-CoA carboxytransferase. Pyruvate kinase catalyzes the
ATP-generating conversion of PEP to pyruvate and is encoded by the
PYK1 (Burke et al., J. Biol. Chem. 258:2193-2201 (1983)) and PYK2
(Boles et al., J. Bacteriol. 179:2987-2993 (1997)) genes in S.
cerevisiae. In E. coli, this activity is catalyzed by the gene
product of pykF and pykA. Methylmalonyl-CoA carboxytransferase
catalyzes the conversion of pyruvate to oxaloacetate. Importantly,
this reaction also simultaneously catalyzes the conversion of
(S)-methylmalonyl-CoA to propionyl-CoA (see FIGS. 1 and 2). An
exemplary methylmalonyl-CoA carboxytransferase which is comprised
of 1.3S, 5S, and 12S subunits can be found in Propionibacterium
freudenreichii (Thornton et al., J. Bacteriol. 175:5301-5308
(1993)). These genes/proteins are identified below in Table 79.
TABLE-US-00079 TABLE 79 Gene GenBank ID GI Number Organism PYK1
NP_009362 6319279 Saccharomyces cerevisiae PYK2 NP_014992 6324923
Saccharomyces cerevisiae pykF NP_416191.1 16129632 Escherichia coli
pykA NP_416368.1 16129807 Escherichia coli 1.3S subunit P02904
114847 Propionibacterium freudenreichii 5S subunit Q70AC7 62901478
Propionibacterium freudenreichii 12S subunit Q8GBW6 62901481
Propionibacterium freudenreichii
Pyruvate Kinase and Pyruvate Carboxylase
[0418] A combination of enzymes can convert PEP to oxaloacetate
with a stoichiometry identical to that of PEP carboxylase. These
enzymes are encoded by pyruvate kinase, PYK1 (Burke et al., J.
Biol. Chem. 258:2193-2201 (1983)) or PYK2 (Boles et al., J.
Bacteriol. 179:2987-2993 (1997)), and pyruvate carboxylase, PYC1
(Walker et al., BioChem. Biophys. Res. Commun. 176:1210-1217
(1991)) or PYC2 (224). Some candidates for pyruvate carboxylase
function are identified below in Table 80.
TABLE-US-00080 TABLE 80 Gene GenBank ID GI Number Organism PYC1
NP_011453 6321376 Saccharomyces cerevisiae PYC2 NP_009777 6319695
Saccharomyces cerevisiae Pyc YP_890857.1 118470447 Mycobacterium
smegmatis
Malate Dehydrogenase, Fumarase, Fumarate Reductase
[0419] Oxaloacetate can be converted to succinate by malate
dehydrogenase, fumarase and fumarate reductase when the TCA cycle
is operating in the reductive cycle. S. cerevisiae possesses three
copies of malate dehydrogenase, MDH1 (McAlister-Henn et al., J.
Bacteriol. 169:5157-5166 (1987)), MDH2 (Gibson J. Biol. Chem.
278:25628-25636 (2003); and Minard et al., Mol. Cell Biol.
11:370-380 (1991)), and MDH3 (Steffan et al., J. Biol. Chem.
267:24708-24715 (1992)), which localize to the mitochondrion,
cytosol, and peroxisome, respectively. S. cerevisiae contains one
copy of a fumarase-encoding gene, FUM1, whose product localizes to
both the cytosol and mitochondrion (Sass et al., J. Biol. Chem.
278:45109-45116 (2003)). Fumarate reductase is encoded by two
soluble enzymes, FRDS1 (Enomoto et al., DNA. Res. 3:263-267 (1996))
and FRDS2 (Muratsubaki et al., Arch. BioChem. Biophys. 352:175-181
(1998)), which localize to the cytosol and promitochondrion,
respectively, and are required for anaerobic growth on glucose
(Arikawa et al., Microbiol Lett. 165:111-116 (1998)). E. coli is
known to have an active malate dehydrogenase. It has three
fumarases encoded byfumA, B and C, each one of which is active
under different conditions of oxygen availability. The fumarate
reductase in E. coli is composed of four subunits. These
genes/proteins are identified below in Table 81.
TABLE-US-00081 TABLE 81 Gene GenBank ID GI Number Organism MDH1
NP_012838 6322765 Saccharomyces cerevisiae MDH2 NP_014515 116006499
Saccharomyces cerevisiae MDH3 NP_010205 6320125 Saccharomyces
cerevisiae FUM1 NP_015061 6324993 Saccharomyces cerevisiae FRDS1
P32614 418423 Saccharomyces cerevisiae FRDS2 NP_012585 6322511
Saccharomyces cerevisiae frdA NP_418578.1 16131979 Escherichia coli
frdB NP_418577.1 16131978 Escherichia coli frdC NP_418576.1
16131977 Escherichia coli frdD NP_418475.1 16131877 Escherichia
coli Mdh NP_417703.1 16131126 Escherichia coli FumA NP_416129.1
16129570 Escherichia coli FumB NP_418546.1 16131948 Escherichia
coli FumC NP_416128.1 16129569 Escherichia coli
Succinyl-CoA Transferase
[0420] Succinyl-CoA transferase catalyzes the conversion of
succinyl-CoA to succinate while transferring the CoA moiety to a
CoA acceptor molecule. Many transferases have broad specificity and
thus may utilize CoA acceptors as diverse as acetate, succinate,
propionate, butyrate, 2-methylacetoacetate, 3-ketohexanoate,
3-ketopentanoate, valerate, crotonate, 3-mercaptopropionate,
propionate, vinylacetate, butyrate, among others.
[0421] The conversion of succinate to succinyl-CoA is ideally
carried by a transferase which does not require the direct
consumption of an ATP or GTP. This type of reaction is common in a
number of organisms. Perhaps the top candidate enzyme for this
reaction step is succinyl-CoA:3-ketoacid-CoA transferase. This
enzyme converts succinate to succinyl-CoA while converting a
3-ketoacyl-CoA to a 3-ketoacid. Exemplary
succinyl-CoA:3:ketoacid-CoA transferases are present in
Helicobacter pylori (Corthesy-Theulaz et al., J. Biol. Chem.
272:25659-25667 (1997)), Bacillus subtilis (Stols et al., Protein.
Expr. Pur 53:396-403 (2007)), and Homo sapiens (Fukao et al.,
Genomics. 68:144-151 (2000); and Tanaka et al., Mol. Hum. Reprod.
8:16-23 (2002)). These genes/proteins are identified below in Table
82.
TABLE-US-00082 TABLE 82 Gene GenBank ID GI Number Organism
HPAG1_0676 YP_627417 108563101 Helicobacter pylori HPAG1_0677
YP_627418 108563102 Helicobacter pylori ScoA NP_391778 16080950
Bacillus subtilis ScoB NP_391777 16080949 Bacillus subtilis OXCT1
NP_000427 4557817 Homo sapiens OXCT2 NP_071403 11545841 Homo
sapiens
[0422] The conversion of succinate to succinyl-CoA can also be
catalyzed by succinyl-CoA: Acetyl-CoA transferase. The gene product
of cat1 of Clostridium kluyveri has been shown to exhibit
succinyl-CoA: acetyl-CoA transferase activity (Sohling et al., J
Bacteriol. 178:871-880 (1996)). In addition, the activity is
present in Trichomonas vaginalis (van Grinsven et al., J. Biol.
Chem. 283:1411-1418 (2008)) and Trypanosoma brucei (Riviere et al.,
J. Biol. Chem. 279:45337-45346 (2004)). These genes/proteins are
identified below in Table 83.
TABLE-US-00083 TABLE 83 Gene GenBank ID GI Number Organism cat1
P38946.1 729048 Clostridium kluyveri TVAG_395550 XP_001330176
123975034 Trichomonas vaginalis G3 Tb11.02.0290 XP_828352 71754875
Trypanosoma brucei
[0423] Yet another possible CoA acceptor is benzylsuccinate.
Succinyl-CoA:(R)-Benzylsuccinate CoA-Transferase functions as part
of an anaerobic degradation pathway for toluene in organisms such
as Thauera aromatica (Leutwein and Heider, J. Bact. 183(14)
4288-4295 (2001)). Homologs can be found in Azoarcus sp. T,
Aromatoleum aromaticum EbN1, and Geobacter metallireducens GS-15.
These genes/proteins are identified below in Table 84.
TABLE-US-00084 TABLE 84 Gene GenBank ID GI Number Organism bbsE
AAF89840 9622535 Thauera aromatica bbsf AAF89841 9622536 Thauera
aromatica bbsE AAU45405.1 52421824 Azoarcus sp. T bbsF AAU45406.1
52421825 Azoarcus sp. T bbsE YP_158075.1 56476486 Aromatoleum
aromaticum EbN1 bbsF YP_158074.1 56476485 Aromatoleum aromaticum
EbN1 Gmet_1521 YP_384480.1 78222733 Geobacter metallireducens GS-15
Gmet_1522 YP_384481.1 78222734 Geobacter metallireducens GS-15
[0424] Finally, yell encodes a propionyl CoA:succinate CoA
transferase in E. coli (Haller et al., Biochemistry, 39(16)
4622-4629). Close homologs can be found in, for example,
Citrobacter youngae ATCC 29220, Salmonella enterica subsp. arizonae
serovar, and Yersinia intermedia ATCC 29909. These genes/proteins
are identified below in Table 85.
TABLE-US-00085 TABLE 85 Gene GenBank ID GI Number Organism ygfH
NP_417395.1 16130821 Escherichia coli str. K-12 substr. MG1655
CIT292_04485 ZP_03838384.1 227334728 Citrobacter youngae ATCC 29220
SARI_04582 YP_001573497.1 161506385 Salmonella enterica subsp.
arizonae serovar yinte0001_14430 ZP_04635364.1 238791727 Yersinia
intermedia ATCC 29909
Succinyl-CoA Synthetase
[0425] The product of the LSO and LSC2 genes of S. cerevisiae and
the sucC and sucD genes of E. coli naturally form a succinyl-CoA
synthetase complex that catalyzes the formation of succinyl-CoA
from succinate with the concomitant consumption of one ATP, a
reaction which is reversible in vivo (Bravo et al., J. Forensic
Sci. 49:379-387 (2004)). These genes/proteins are identified below
in Table 86.
TABLE-US-00086 TABLE 86 Gene GenBank ID GI Number Organism LSC1
NP_014785 6324716 Saccharomyces cerevisiae LSC2 NP_011760 6321683
Saccharomyces cerevisiae sucC NP_415256.1 16128703 Escherichia coli
sucD AAC73823.1 1786949 Escherichia coli
Pyruvate Formate Lyase
[0426] Pyruvate formate lyase is an enzyme that catalyzes the
conversion of pyruvate and CoA into acetyl-CoA and formate.
Pyruvate formate lyase is a common enzyme in prokaryotic organisms
that is used to help modulate anaerobic redox balance. Exemplary
enzymes can be found in Escherichia coli (Knappe et al., FEMS.
Microbiol Rev. 6:383-398 (1990)), Lactococcus lactis (Melchiorsen
et al., Appl Microbiol Biotechnol 58:338-344 (2002)), and
Streptococcus mutans (Takahashi-Abbe et al., Oral. Microbiol
Immunol. 18:293-297 (2003)). A mitochondrial pyruvate formate lyase
has also been identified in the eukaryote, Chlamydomonas
reinhardtii (Atteia et al., J. Biol. Chem. 281:9909-9918 (2006);
and Hemschemeier et al., Eukaryot. Cell 7:518-526 (2008)). These
genes/proteins are identified below in Table 87.
TABLE-US-00087 TABLE 87 Gene GenBank ID GI Number Organism pflB
NP_415423 16128870 Escherichia coli pfl CAA03993 2407931
Lactococcus lactis pfl BAA09085 1129082 Streptococcus mutans PFL1
EDP09457 158283707 Chlamydomonas reinhardtii
Formate Hydrogen Lyase
[0427] A formate hydrogen lyase enzyme can be employed to convert
formate to carbon dioxide and hydrogen. An exemplary formate
hydrogen lyase enzyme can be found in Escherichia coli. The E. coli
formate hydrogen lyase consists of hydrogenase 3 and formate
dehydrogenase-H (Maeda et al., Appl Microbiol Biotechnol 77:879-890
(2007)). It is activated by the gene product of fhlA (Maeda et al.,
Appl Microbiol Biotechnol 77:879-890 (2007)). The addition of the
trace elements, selenium, nickel and molybdenum, to a fermentation
broth has been shown to enhance formate hydrogen lyase activity
(Soini, et al., Microb. Cell Fact. 7:26 (2008)). These
genes/proteins are identified below in Table 88.
TABLE-US-00088 TABLE 88 Gene GenBank ID GI Number Organism
Hydrogenase 3: hycD NP_417202 16130629 Escherichia coli hycC
NP_417203 16130630 Escherichia coli hycF NP_417200 16130627
Escherichia coli hycG NP_417199 16130626 Escherichia coli hycB
NP_417204 16130631 Escherichia coli hycE NP_417201 16130628
Escherichia coli Formate dehydrogenase-H: fdhF NP_418503 16131905
Escherichia coli Activator: fhlA NP_417211 16130638 Escherichia
coli
[0428] A formate hydrogen lyase enzyme also exists in the
hyperthermophilic archaeon, Thermococcus litoralis (Takacs et al.,
Microbiol 8:88 2008)). These genes/proteins are identified below in
Table 89.
TABLE-US-00089 TABLE 89 Gene GenBank ID GI Number Organism mhyC
ABW05543 157954626 Thermococcus litoralis mhyD ABW05544 157954627
Thermococcus litoralis mhyE ABW05545 157954628 Thermococcus
litoralis myhF ABW05546 157954629 Thermococcus litoralis myhG
ABW05547 157954630 Thermococcus litoralis myhH ABW05548 157954631
VThermococcus litoralis fdhA AAB94932 2746736 Thermococcus
litoralis fdhB AAB94931 157954625 Thermococcus litoralis
[0429] Additional formate hydrogen lyase systems have been found in
Salmonella typhimurium, Klebsiella pneumoniae, Rhodospirillum
rubrum, Methanobacterium formicicum (Vardar-Schara et al.,
Microbial Biotechnology 1:107-125)).
Formate Dehydrogenase
[0430] Formate dehydrogenase activity is present in both E. coli
and Saccharomyces cerevisiae among other organisms. S. cerevisiae
contains two formate dehydrogenases, FDH1 and FDH2, that catalyze
the oxidation of formate to CO.sub.2 (Overkamp et al., Yeast
19:509-520 (2002)). In Moorella thermoacetica, the loci,
Moth.sub.--2312 and Moth.sub.--2313, are actually one gene that is
responsible for encoding the alpha subunit of formate dehydrogenase
while the beta subunit is encoded by Moth.sub.--2314 (Andreesen et
al., J. Bacteriol. 116:867-873 (1973); Li et al., J. Bacteriol.
92:405-412 (1966); Pierce et al., Environ. Microbiol (2008) and
Yamamoto et al., J. Biol. Chem. 258:1826-1832 (1983)). Another set
of genes encoding formate dehydrogenase activity is encoded by
Sfum.sub.--2703 through Sfum.sub.--2706 in Syntrophobacter
fumaroxidans (de Bok, et al., Eur. J. BioChem. 270:2476-2485
(2003); and Reda et al., Proc. Natl. Acad. Sci. US. A.
105:10654-10658 (2008)). Similar to their M. thermoacetica
counterparts, Sfum.sub.--2705 and Sfum.sub.--2706 are actually one
gene. E. coli contains multiple formate dehydrogenases. These
genes/proteins are identified below in Table 90.
TABLE-US-00090 TABLE 90 Gene GenBank ID GI Number Organism FDH1
NP_015033 6324964 Saccharomyces cerevisiae FDH2 Q08987 88909613
Saccharomyces cerevisiae Moth_2312 YP_431142 148283121 Moorella
thermoacetica Moth_2313 YP_431143 83591134 Moorella thermoacetica
Moth_2314 YP_431144 83591135 Moorella thermoacetica Sfum_2703
YP_846816.1 116750129 Syntrophobacter fumaroxidans Sfum_2704
YP_846817.1 116750130 Syntrophobacter fumaroxidans Sfum_2705
YP_846818.1 116750131 Syntrophobacter fumaroxidans Sfum_2706
YP_846819.1 116750132 Syntrophobacter fumaroxidans fdnG, H, I
NP_415991- 16129433 Escherichia coli 993.1 16129434 16129435 fdoG,
H, I NP_418330,29, 16131734 Escherichia coli 28.1 16131733
16131732
Pyruvate Dehydrogenase
[0431] The pyruvate dehydrogenase complex, catalyzing the
conversion of pyruvate to acetyl-CoA, has been extensively studied.
The S. cerevisiae complex consists of an E2 (LAD) core that binds
E1 (PDA1, PDB1), E3 (LPD1), and Protein X (PDX1) components (Pronk
et al., Yeast 12:1607-1633 (1996)). In the E. coli enzyme, specific
residues in the E1 component are responsible for substrate
specificity (Binswanger J Biol Chem. 256:815-822. (1981); Bremer J
BioChem. 8:535-540 (1969) and Gong et al., J Biol Chem.
275:13645-13653 (2000)). Engineering efforts have improved the E.
coli PDH enzyme activity under anaerobic conditions (Kim et al.,
Appl. Environ. Microbiol. 73:1766-1771 (2007); Kim et al., J.
Bacteriol. 190:3851-3858 (2008) and Zhou et al., Biotechnol. Lett.
30:335-342 (2008)). In contrast to the E. coli PDH, the B. subtilis
complex is active and required for growth under anaerobic
conditions (Nakano et al., J. Bacteriol. 179:6749-6755 (1997)). The
Klebsiella pneumoniae PDH, characterized during growth on glycerol,
is also active under anaerobic conditions (Menzel et al., J.
Biotechnol. 56:135-142 (1997)). Crystal structures of the enzyme
complex from bovine kidney (Zhou et al., Proc. Natl. Acad. Sci. U.
S. A 98:14802-14807 (2001)) and the E2 catalytic domain from
Azotobacter vinelandii are available (Mattevi et al., Science.
255:1544-1550 (1992)). Some maMAAlian PDH enzymes complexes can
react on alternate substrates such as 2-oxobutanoate (Paxton et
al., BioChem. J. 234:295-303 (1986)). These genes/proteins are
identified below in Table 91.
TABLE-US-00091 TABLE 91 Gene GenBank ID GI Number Organism LAT1
NP_014328 6324258 Saccharomyces cerevisiae PDA1 NP_011105 37362644
Saccharomyces cerevisiae PDB1 NP_009780 6319698 Saccharomyces
cerevisiae LPD1 NP_116635 14318501 Saccharomyces cerevisiae PDX1
NP_011709 6321632 Saccharomyces cerevisiae aceE NP_414656.1
16128107 Escherichia coli str. K12 substr. MG1655 aceF NP_414657.1
16128108 Escherichia coli str. K12 substr. MG1655 Lpd NP_414658.1
16128109 Escherichia coli str. K12 substr. MG1655 pdhA P21881.1
3123238 Bacillus subtilis pdhB P21882.1 129068 Bacillus subtilis
pdhC P21883.2 129054 Bacillus subtilis pdhD P21880.1 118672
Bacillus subtilis aceE YP_001333808.1 152968699 Klebsiella
pneumonia MGH78578 aceF YP_001333809.1 152968700 Klebsiella
pneumonia MGH78578 lpdA YP_001333810.1 152968701 Klebsiella
pneumonia MGH78578 Pdha1 NP_001004072.2 124430510 Rattus norvegicus
Pdha2 NP_446446.1 16758900 Rattus norvegicus Dlat NP_112287.1
78365255 Rattus norvegicus Dld NP_955417.1 40786469 Rattus
norvegicus
Pyruvate Ferredoxin Oxidoreductase
[0432] Pyruvate ferredoxin oxidoreductase (PFOR) catalyzes the
oxidation of pyruvate to form acetyl-CoA. The PFOR from
Desulfovibrio africanus has been cloned and expressed in E. coli
resulting in an active recombinant enzyme that was stable for
several days in the presence of oxygen (Pieulle et al., J
Bacteriol. 179:5684-5692 (1997)). Oxygen stability is relatively
uncommon in PFORs and is believed to be conferred by a 60 residue
extension in the polypeptide chain of the D. africanus enzyme. The
M. thermoacetica PFOR is also well characterized (Menon et al.,
BioChemistry 36:8484-8494 (1997)) and was even shown to have high
activity in the direction of pyruvate synthesis during autotrophic
growth (Furdui et al., J Biol Chem. 275:28494-28499 (2000)).
Further, E. coli possesses an uncharacterized open reading frame,
ydbK, encoding a protein that is 51% identical to the M.
thermoacetica PFOR. Evidence for pyruvate oxidoreductase activity
in E. coli has been described (Blaschkowski et al., J BioChem.
123:563-569 (1982)). Several additional PFOR enzymes are described
in the following review (Ragsdale, Chem. Rev. 103:2333-2346
(2003)). Finally, flavodoxin reductases (e.g., fqrB from
Helicobacter pylori or Campylobacter jejuni (St Maurice et al., J.
Bacteriol. 189:4764-4773 (2007)) or Rnf-type proteins (Herrmann et
al., J. Bacteriol. 190:784-791 (2008); and Seedorf et al., Proc.
Natl. Acad. Sci. US. A. 105:2128-2133 (2008)) provide a means to
generate NADH or NADPH from the reduced ferredoxin generated by
PFOR. These genes/proteins are identified below in Table 92.
TABLE-US-00092 TABLE 92 Gene GenBank ID GI Number Organism Por
CAA70873.1 1770208 Desulfovibrio africanus Por YP_428946.1 83588937
Moorella thermoacetica ydbK NP_415896.1 16129339 Escherichia coli
fqrB NP_207955.1 15645778 Helicobacter pylori fqrB YP_001482096.1
157414840 Campylobacter jejuni RnfC EDK33306.1 146346770
Clostridium kluyveri RnfD EDK33307.1 146346771 Clostridium kluyveri
RnfG EDK33308.1 146346772 Clostridium kluyveri RnfE EDK33309.1
146346773 Clostridium kluyveri RnfA EDK33310.1 146346774
Clostridium kluyveri RnfB EDK33311.1 146346775 Clostridium
kluyveri
Succinic Semialdehyde Dehydrogenase (CoA-Dependent)
[0433] Succinic semialdehyde dehydrogenase (CoA-dependent), also
referred to as succinyl-CoA reductase, is a CoA- and
NAD(P)H-dependent oxidoreductase that reduces succinyl-CoA to its
corresponding aldehyde. Exemplary enzymes are encoded by the sucD
gene in Clostridium kluyveri (Sohling et al., J Bacteriol
178:871-80 (1996); and Sohling et al., J Bacteriol. 178:871-880
(1996)) and the sucD gene of P. gingivalis (Takahashi et al., J.
Bacteriol. 182:4704-4710 (2000)). Other enzymes that catalyze
similar reactions are the fatty acyl-CoA reductases of
Acinetobacter calcoaceticus (Reiser et al., Journal of Bacteriology
179:2969-2975 (2007)) and Acinetobacter sp. M-1 (Ishige et al.,
Appl. Environ. Microbiol. 68:1192-1195 (2002)), and the acylating
acetaldehyde dehydrogenase in Pseudomonas sp, which has been
demonstrated to oxidize and acylate acetaldehyde, propionaldehyde,
butyraldehyde, isobutyraldehyde and formaldehyde (Powlowski et al.,
J Bacteriol. 175:377-385 (1993)). In addition to reducing
acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc
mesenteroides has been shown to oxidize the branched chain compound
isobutyraldehyde to isobutyryl-CoA (Koo et al., Biotechnol Lett.
27:505-510 (2005)). These genes/proteins are identified below in
Table 93.
TABLE-US-00093 TABLE 93 Gene GenBank ID GI Number Organism sucD
P38947.1 172046062 Clostridium kluyveri sucD NP_904963.1 34540484
Porphyromonas gingivalis acr1 YP_047869.1 50086359 Acinetobacter
calcoaceticus acr1 AAC45217 1684886 Acinetobacter baylyi acr1
BAB85476.1 18857901 Acinetobacter sp. Strain M-1 bphG BAA03892.1
425213 Pseudomonas sp adhE AAV66076.1 55818563 Leuconostoc
mesenteroides
4-Hydroxybutyrate Dehydrogenase
[0434] 4-Hydroxybutyrate dehydrogenase catalyzes the NAD(P)H
dependent reduction of succinic semialdehyde to 4-HB. Enzymes
exhibiting this activity are found in Ralstonia eutropha (Bravo et
al., J. Forensic Sci. 49:379-387 (2004)), Clostridium kluyveri
(Wolff et al., Protein Expr. Pur 6:206-212 (1995)) and Arabidopsis
thaliana (Breitkreuz et al.,. J. Biol. Chem. 278:41552-41556
(2003)). Yet another gene is the alcohol dehydrogenase adhI from
Geobacillus thermoglucosidasius (Jeon et al., J Biotechnol
135:127-133 (2008)). These genes/proteins are identified below in
Table 94.
TABLE-US-00094 TABLE 94 Gene GenBank ID GI Number Organism 4hbd
YP_726053.1 113867564 Ralstonia eutropha H16 4hbd EDK35022.1
146348486 Clostridium kluyveri 4hbd Q94B07 75249805 Arabidopsis
thaliana adhI AAR91477.1 40795502 Geobacillus
thermoglucosidasius
4-Hydroxybutyryl-CoA Transferase
[0435] The conversion of 4-HB to 4-hydroxybutyryl-CoA is catalyzed
by an enzyme with 4-hydroxybutyryl-CoA transferase activity.
Candidate enzymes include the gene products of cat1, cat2, and cat3
of Clostridium kluyveri which have been shown to exhibit
succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA transferase
activity, respectively (Gerhardt et al., Arch. Microbiol
174:189-199 (2000); Arikawa et al., Microbiol Lett. 165:111-116
(1998) and Sohling et al., J Bacteriol. 178:871-880 (1996)).
Similar CoA transferase activities are also present in Trichomonas
vaginalis (van Grinsven et al., J. Biol. Chem. 283:1411-1418
(2008)) and Trypanosoma brucei (Riviere et al., J. Biol. Chem.
279:45337-45346 (2004)). The atoA and atoD genes of E. coli encode
an acetoacetyl-CoA transferase with a broad substrate range (Sramek
et al., Arch. BioChem. Biophys. 171:14-26 (1975)). This enzyme has
been shown to transfer a CoA moiety from acetyl-CoA to a variety of
branched and linear substrates including isobutyrate (Matthies et
al., Appl Environ. Microbiol 58:1435-1439 (1992)), valerate
(Vanderwinkel et al., BioChem. Biophys. Res. Commun. 33:902-908
(1968)) and butanoate (Vanderwinkel et al., BioChem. Biophys. Res.
Commun. 33:902-908 (1968)). These genes/proteins are identified
below in Table 95.
TABLE-US-00095 TABLE 95 Gene GenBank ID GI Number Organism cat1
P38946.1 729048 Clostridium kluyveri cat2 P38942.2 172046066
Clostridium kluyveri cat3 EDK35586.1 146349050 Clostridium kluyveri
TVAG_395550 XP_001330176 123975034 Trichomonas vaginalis G3
Tb11.02.0290 XP_828352 71754875 Trypanosoma brucei atoA P76459.1
2492994 Escherichia coli atoD P76458.1 2492990 Escherichia coli
4-Hydroxybutyryl-CoA synthetase
[0436] The conversion of 4-HB to 4-hydroxybutyryl-CoA can also be
catalyzed by a CoA acid-thiol ligase, also known as a CoA
synthetase. Enzymes catalyzing this exact transformation have not
been characterized to date; however, several enzymes with broad
substrate specificities have been described in the literature. An
exemplary candidate is the enzyme encoded by sucCD in E. coli,
which naturally catalyzes the formation of succinyl-CoA from
succinate with the concomitant consumption of one ATP, a reaction
which is reversible in vivo (Buck et al., BioChemistry 24:6245-6252
(1985)). Additional CoA-ligase candidates include the ADP-forming
phenylacetate-CoA ligases from P. chrysogenum (Lamas-Maceiras et
al., BioChem. J 395:147-155 (2006); and Wang et al., BioChem.
Biophys. Res. Commun. 360:453-458 (2007)) and the pimeloyl-CoA
ligase from Pseudomonas mendocina. The AMP-forming enzyme from
Pseudomonas mendocina, cloned into E. coli, was shown to accept the
alternate substrates hexanedioate and nonanedioate (Binieda et al.,
BioChem. J 340 (Pt 3):793-801 (1999)). These genes/proteins are
identified below in Table 96. CoA synthetase enzyme candidates
identified for acetoacetyl-CoA synthetase, succinyl-CoA synthetase,
propionyl-CoA synthetase, 3-hydroxybutyryl-CoA synthetase,
3-hydroxyisobutyryl-CoA synthetase, methylmalonyl-CoA synthetase
and methacrylyl-CoA synthase are also applicable here.
TABLE-US-00096 TABLE 96 Gene GenBank ID GI Number Organism sucC
NP_415256.1 16128703 Escherichia coli sucD AAC73823.1 1786949
Escherichia coli phl CAJ15517.1 77019264 Penicillium chrysogenum
pauA NP_249708.1 15596214 Pseudomonas mendocina
4-Hydroxybutyryl-CoA Reductase (Aldehyde Forming)
[0437] 4-Hydroxybutyryl-CoA reductase catalyzes the NAD(P)H
dependent reduction of 4-hydroxybutyryl-CoA to
4-hydroxybutyraldehyde. Enzymes that exhibit this activity include
succinate semialdehyde dehydrogenase enzymes encoded by the sucD
gene in Clostridium kluyveri (Sohling et al., J Bacteriol
178:871-80 (1996); and Sohling et al., J Bacteriol. 178:871-880
(1996)) and sucD of P. gingivalis (Takahashi et al., J. Bacteriol.
182:4704-4710 (2000)). Butyraldehyde dehydrogenase enzymes, found
in solventogenic organisms such as Clostridium
saccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol
BioChem. 71:58-68 (2007)), catalyzes a similar reaction: conversion
of butyryl-CoA to butyraldehyde. The enzyme acylating acetaldehyde
dehydrogenase in Pseudomonas sp, encoded by bphG, is yet another
candidate as it has been demonstrated to oxidize and acylate
acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde and
formaldehyde (Powlowski et al., J Bacteriol. 175:377-385 (1993)).
Fatty acyl-CoA reductase enzymes from Acinetobacter calcoaceticus
(Reiser et al., Journal of Bacteriology 179:2969-2975 (1997)) and
the Acinetobacter sp. M-1 (Ishige, et al., Appl. Environ.
Microbiol. 68:1192-1195 (2002)) catalyze similar reactions. In
addition to reducing acetyl-CoA to ethanol, the enzyme encoded by
adhE in Leuconostoc mesenteroides has been shown to oxidize the
branched chain compound isobutyraldehyde to isobutyryl-CoA (Koo et
al., Biotechnol Lett. 27:505-510 (2005)). These genes/proteins are
identified below in Table 97.
TABLE-US-00097 TABLE 97 Gene GenBank ID GI Number Organism sucD
P38947.1 172046062 Clostridium kluyveri sucD NP_904963.1 34540484
Porphyromonas gingivalis bld AAP42563.1 31075383 Clostridium
saccharoperbutylacetonicum bphG BAA03892.1 425213 Pseudomonas sp
acr1 YP_047869.1 50086359 Acinetobacter calcoaceticus acr1 AAC45217
1684886 Acinetobacter baylyi acr1 BAB85476.1 18857901 Acinetobacter
sp. Strain M-1 adhE AAV66076.1 55818563 Leuconostoc
mesenteroides
4-Hydroxybutyraldehyde Reductase
[0438] The conversion of 4-hydroxybutyrylaldehyde to 14-BDO is
catalyzed by an alcohol dehydrogenase. Several native
dehydrogenases in E. coli such as yqhD (Sulzenbacher et al.,
Journal of Molecular Biology 342:489-502 (2004)) exhibit broad
substrate specificity and are able to catalyze this reaction. The
gene product of yqhD catalyzes the reduction of acetaldehyde,
malondialdehyde, propionaldehyde, butyraldehyde, and acrolein using
NADPH as the cofactor (Perez et al., J Biol. Chem. 283:7346-7353
(2008); and Perez et al., J Biol. Chem. 283:7346-7353 (2008)).
Additional enzyme candidates that catalyze the conversion of an
aldehyde to alcohol include alrA encoding a medium-chain alcohol
dehydrogenase for C2-C14 (Tani et al., Appl. Environ. Microbiol.
66:5231-5235 (2000)), ADH2 from Saccharomyces cerevisiae (Atsumi et
al., Nature 451:86-89 (2008)) and bdh I and bdh II from C.
acetobutylicum which converts butyraldehyde into butanol (Walter et
al., Journal of Bacteriology 174:7149-7158 (1992)). The adhA gene
product from Zymomonas mobilis has been demonstrated to have
activity on a number of aldehydes including formaldehyde,
acetaldehyde, propionaldehyde, butyraldehyde, and acrolein
(Kinoshita et al., Appl Microbiol Biotechnol 22:249-254 (1985)).
These genes/proteins are identified below in Table 98.
TABLE-US-00098 TABLE 98 Gene GenBank ID GI Number Organism yqhD
NP_417484.1 16130909 Escherichia coli alrA BAB12273.1 9967138
Acinetobacter sp. Strain M-1 ADH2 NP_014032.1 6323961 Saccharomyces
cerevisiae bdh I NP_349892.1 15896543 Clostridium acetobutylicum
bdh II NP_349891.1 15896542 Clostridium acetobutylicum adhA
YP_162971.1 56552132 Zymomonas mobilis
4-Hydroxybutyryl-CoA Reductase (Alcohol Forming)
[0439] The conversion of 4-hydroxybutyryl-CoA to 14-BDO can also be
catalyzed by a bifunctional oxidoreductase with aldehyde
dehydrogenase and alcohol dehydrogenase capabilities. For example,
the adheE2 gene product from Clostridium acetobutylicum converts
butyryl-CoA to butanol (Fontaine et al., J. Bacteriol. 184:821-830
(2002)). This enzyme also accepts 4-hydroxybutyryl-CoA as a
substrate. Additional bifunctional alcohol-forming reductase
enzymes include the gene products of adhE in Leuconostoc
mesenteroides (Kazahaya et al., J. Gen. Appl. Microbiol. 18:43-55
(1972); and Koo et al., Biotechnol Lett. 27:505-510 (2005)) and FAR
from Simmondsia chinensis (Metz et al., Plant Physiology
122:635-644 (2000)). Another exemplary enzyme is the
NADPH-dependent malonyl-CoA reductase in Chloroflexus aurantiacus
encoded by mcr (Hugler et al., J. Bacteriol. 184:2404-2410 (2002);
and Strauss et al., Eur. J. BioChem. 215:633-643 (1993)). These
genes/proteins are identified below in Table 99.
TABLE-US-00099 TABLE 99 Gene GenBank ID GI Number Organism adhE2
AAK09379.1 12958626 Clostridium acetobutylicum adhE AAV66076.1
55818563 Leuconostoc mesenteroides FAR AAD38039.1 5020215
Simmondsia chinensis mcr AAS20429.1 42561982 Chloroflexus
aurantiacus
4-Hydroxybutyrate Phosphotransferase (aka Kinase)
[0440] 4-Hydroxybutyrate phosphotransferase, also known as
4-hydroxybutyrate kinase, transforms 4-HB to 4-hydroxybutyryl
phosphate with concurrent hydrolysis of one ATP. Candidate enzymes
for catalyzing these transformations include butyrate kinase,
aspartokinase, acetate kinase and gaMAA-glutamyl kinase. Butyrate
kinase (EC 2.7.2.7) enzymes carry out the reversible conversion of
butyryl-phosphate to butyrate during acidogenesis in C.
acetobutylicum (Cary et al., Appl. Environ. Microbiol 56:1576-1583
(1990)). This enzyme is encoded by either of the two buk gene
products (Huang et al., J Mol. Microbiol Biotechnol 2:33-38
(2000)). Other butyrate kinase enzymes are found in C. butyricum
and C. tetanomorphum (TWAROG et al., J Bacteriol. 86:112-117
(1963)). Related enzyme isobutyrate kinase from Thermotoga maritima
has also been expressed in E. coli and crystallized (Diao et al.,
D. Biol. Crystallogr. 59:1100-1102 (2003); and Diao et al., J
Bacteriol. 191:2521-2529 (2009)). Aspartokinase catalyzes the
ATP-dependent phosphorylation of aspartate and participates in the
synthesis of several amino acids. The aspartokinase III enzyme in
E. coli, encoded by lysC, has a broad substrate range and the
catalytic residues involved in substrate specificity have been
elucidated (Keng et al., Arch. BioChem. Biophys. 335:73-81 (1996)).
Two additional kinases in E. coli are also good candidates: acetate
kinase and gaMAA-glutamyl kinase. The E. coli acetate kinase,
encoded by ackA (Skarstedt et al., J. Biol. Chem. 251:6775-6783
(1976)), phosphorylates propionate in addition to acetate
(Hesslinger et al., Mol. Microbiol 27:477-492 (1998)). The E. coli
gaMAA-glutamyl kinase, encoded by proB (Smith et al., J. Bacteriol.
157:545-551 (1984)), phosphorylates the gaMAA carbonic acid group
of glutamate. These genes/proteins are identified below in Table
100.
TABLE-US-00100 TABLE 100 Gene GenBank ID GI Number Organism buk1
NP_349675 15896326 Clostridium acetobutylicum buk2 Q97II1 20137415
Clostridium acetobutylicum buk2 Q9X278.1 6685256 Thermotoga
maritima lysC NP_418448.1 16131850 Escherichia coli ackA
NP_416799.1 16130231 Escherichia coli proB NP_414777.1 16128228
Escherichia coli
Phosphotrans-4-Hydroxybutyrylase
[0441] Phosphotrans-4-hydroxybutyrylase exchanges the phosphate
moiety of 4-hydroxybutyryl-phosphate for a CoA moiety, forming
4-hydroxybutyryl-CoA. A candidate enzyme for this transformation is
phosphotransbutyrylase (EC 2.3.1.19) an enzyme that reversibly
converts butyryl-CoA into butyryl-phosphate. This enzyme is encoded
by ptb genes found in C. acetobutylicum (Walter et al., Gene
134:107-111 (1993); and Wiesenborn et al., Appl Environ. Microbiol
55:317-322 (1989)), butyrate-producing bacterium L2-50 (Louis et
al., J. Bacteriol. 186:2099-2106 (2004)) and Bacillus megaterium
(Vazquez et al., Curr. Microbiol 42:345-349 (2001)). These
genes/proteins are identified below in Table 101.
TABLE-US-00101 TABLE 101 Gene GenBank ID GI Number Organism ptb
NP_349676 34540484 Clostridium acetobutylicum ptb AAR19757.1
38425288 butyrate-producing bacterium L2-50 ptb CAC07932.1 10046659
Bacillus megaterium
4-Hydroxybutyryl-Phosphate Reductase
[0442] The reduction of 4-hydroxybutyryl-phosphate to its
corresponding aldehyde is catalyzed by phosphate reductase. This
reaction is not catalyzed by known enzymes, but a similar reaction
is catalyzed by aspartate semialdehyde dehydrogenase (ASD, EC
1.2.1.11): the NADPH-dependent reduction of 4-aspartyl phosphate to
aspartate-4-semialdehyde. ASD participates in amino acid
biosynthesis and recently has been studied as an antimicrobial
target (Hadfield et al., Biochemistry 40:14475-14483 (2001)). The
E. coli ASD structure has been solved (Hadfield et al., J Mol.
Biol. 289:991-1002 (1999)) and the enzyme has been shown to accept
the alternate substrate beta-3-methylaspartyl phosphate (Shames et
al., J Biol. Chem. 259:15331-15339 (1984)). The Haemophilus
influenzae enzyme has been the subject of enzyme engineering
studies to alter substrate binding affinities at the active site
(Blanco et al., Acta Crystallogr. D. Biol. Crystallogr.
60:1388-1395 (2004); and Blanco et al., Acta Crystallogr. D. Biol.
Crystallogr. 60:1808-1815 (2004)). Other ASD candidates are found
in Mycobacterium tuberculosis (Shafiani et al., J Appl Microbiol
98:832-838 (2005)), Methanococcus jannaschii (Faehnle et al., J
Mol. Biol. 353:1055-1068 (2005)), and the infectious microorganisms
Vibrio cholera and Heliobacter pylori (Moore et al., Protein Expr.
Purif. 25:189-194 (2002)). A related enzyme candidate is
acetylglutamylphosphate reductase (EC 1.2.1.38), an enzyme that
naturally reduces acetylglutamylphosphate to
acetylglutamate-5-semialdehyde, found in S. cerevisiae (Pauwels et
al., Eur. J Biochem. 270:1014-1024 (2003)), B. subtilis (O'Reilly
and Devine, Microbiology 140 (Pt 5):1023-1025 (1994)) and other
organisms. These genes/proteins are identified below in Table
102.
TABLE-US-00102 TABLE 102 Gene GenBank ID GI Number Organism asd
NP_417891.1 16131307 Escherichia coli asd YP_248335.1 68249223
Haemophilus influenzae asd AAB49996 1899206 Mycobacterium
tuberculosis VC2036 NP_231670 15642038 Vibrio cholera asd
YP_002301787.1 210135348 Heliobacter pylori ARG5,6 NP_010992.1
6320913 Saccharomyces cerevisiae argC NP_389001.1 16078184 Bacillus
subtilis
[0443] Other exemplary phosphate reductase enzymes include
glyceraldehyde 3-phosphate dehydrogenase which converts
glyceraldehyde-3-phosphate into D-glycerate 1,3-bisphosphate (e.g.,
E. coli gapA (Branlant et al., Eur. J. Biochem. 150:61-66 (1985))),
N-acetyl-gamma-glutamyl-phosphate reductase which converts
N-acetyl-L-glutamate-5-semialdehyde into
N-acetyl-L-glutamyl-5-phosphate (e.g., E. coli argC (Parsot et al.
Gene, 68: 275-283 (1988))), and glutamate-5-semialdehyde
dehydrogenase which converts L-glutamate-5-semialdehyde into
L-glutamyl-5-phospate (e.g., E. coli proA (Smith et al., J.
Bacteriol., 157:545-551 (1984))). Genes encoding
glutamate-5-semialdehyde dehydrogenase enzymes from Salmonella
typhimurium (Mahan et al., J. Bacteriol., 156: 1249-1262 (1983))
and Campylobacter jejuni (Louie et al., Mol. Gen. Genet., 240:29-35
(1993)) were cloned and expressed in E. coli. These genes/proteins
are identified below in Table 103.
TABLE-US-00103 TABLE 103 Gene GenBank ID GI Number Organism gapA
P0A9B2.2 71159358 Escherichia coli argC NP_418393.1 16131796
Escherichia coli proA NP_414778.1 16128229 Escherichia coli proA
NP_459319.1 16763704 Salmonella typhimurium proA P53000.2 9087222
Campylobacter jejuni
Succinate Reductase and 4-Hydroxybutyrate Reductase
[0444] The direct reduction of succinate to succinic semialdehyde
or 4-HB to 4-hydroxybutyraldehyde can be catalyzed by a carboxylic
acid reductase. The carboxylic acid reductase of Nocardia iowensis,
known equivalently as aryl-aldehyde dehydrogenase, catalyzes the
magnesium, ATP and NADPH-dependent reduction of carboxylic acids to
their corresponding aldehydes (Venkitasubramanian et al., J Biol.
Chem. 282:478-485 (2007)) and is capable of catalyzing the
conversion of 4-hydroxybutyrate to 4-hydroxybutanal. This enzyme,
encoded by car, was cloned and functionally expressed in E. coli
(Venkitasubramanian et al., J Biol. Chem. 282:478-485 (2007)).
Expression of the npt gene product improved activity of the enzyme
via post-transcriptional modification. The npt gene encodes a
specific phosphopantetheine transferase (PPTase) that converts the
inactive apo-enzyme to the active holo-enzyme. The natural
substrate of this enzyme is vanillic acid and the enzyme exhibits
broad acceptance of aromatic and aliphatic substrates
(Venkitasubramanian et al. "Biocatalytic Reduction of Carboxylic
Acids: Mechanism and Applications" Chapter 15 in Biocatalysis in
the Pharmaceutical and Biotechnology Industries, ed. R. N. Patel,
CRC Press LLC, Boca Raton, Fla. (2006)). These genes/proteins are
identified below in Table 104.
TABLE-US-00104 TABLE 104 Gene GenBank ID GI Number Organism car
AAR91681.1 40796035 Nocardia iowensis (sp. NRRL 5646) npt
ABI83656.1 114848891 Nocardia iowensis (sp. NRRL 5646)
[0445] Additional car and npt genes can be identified based on
sequence homology. Non-limiting examples of proteins encoded by
these genes are shown in Table 105.
TABLE-US-00105 TABLE 105 Gene GenBank ID GI Number Organism fadD9
YP_978699.1 121638475 Mycobacterium bovis BCG BCG_2812c YP_978898.1
121638674 Mycobacterium bovis BCG nfa20150 YP_118225.1 54023983
Nocardia farcinica IFM 10152 nfa40540 YP_120266.1 54026024 Nocardia
farcinica IFM 10152 SGR_6790 YP_001828302.1 182440583 Streptomyces
griseus subsp. griseus NBRC 13350 SGR_665 YP_001822177.1 182434458
Streptomyces griseus subsp. griseus NBRC 13350 MSMEG_2956
YP_887275.1 YP_887275.1 Mycobacterium smegmatis MC2155 MSMEG_5739
YP_889972.1 118469671 Mycobacterium smegmatis MC2155 MSMEG_2648
YP_886985.1 118471293 Mycobacterium smegmatis MC2155 MAP1040c
NP_959974.1 41407138 Mycobacterium avium subsp. paratuberculosis
K-10 MAP2899c NP_961833.1 41408997 Mycobacterium avium subsp.
paratuberculosis K-10 MMAR_2117 YP_001850422.1 183982131
Mycobacterium marinum M MMAR_2936 YP_001851230.1 183982939
Mycobacterium marinum M MMAR_1916 YP_001850220.1 183981929
Mycobacterium marinum M TpauDRAFT_33060 ZP_04027864.1 227980601
Tsukamurella paurometabola DSM 20162 TpauDRAFT_20920 ZP_04026660.1
227979396 Tsukamurella paurometabola DSM 20162 CPCC7001_1320
ZP_05045132.1 254431429 Cyanobium PCC7001 DDBDRAFT_0187729
XP_636931.1 66806417 Dictyostelium discoideum AX4
[0446] An additional enzyme candidate found in Streptomyces griseus
is encoded by the griC and griD genes. This enzyme is believed to
convert 3-amino-4-hydroxybenzoic acid to
3-amino-4-hydroxybenzaldehyde as deletion of either griC or griD
led to accumulation of extracellular 3-acetylamino-4-hydroxybenzoic
acid, a shunt product of 3-amino-4-hydroxybenzoic acid metabolism
(Suzuki, et al., J. Antibiot. 60(6):380-387 (2007)). Co-expression
of griC and griD with SGR 665, an enzyme similar in sequence to the
Nocardia iowensis npt, may be beneficial. These genes/proteins are
identified below in Table 106.
TABLE-US-00106 TABLE 106 Gene GenBank ID GI Number Organism griC
YP_001825755.1 182438036 Streptomyces griseus subsp. griseus NBRC
13350 griD YP_001825756.1 182438037 Streptomyces griseus subsp.
griseus NBRC 13350
[0447] An enzyme with similar characteristics, alpha-aminoadipate
reductase (AAR, EC 1.2.1.31), participates in lysine biosynthesis
pathways in some fungal species. This enzyme naturally reduces
alpha-aminoadipate to alpha-aminoadipate semialdehyde. The carboxyl
group is first activated through the ATP-dependent formation of an
adenylate that is then reduced by NAD(P)H to yield the aldehyde and
AMP. Like CAR, this enzyme utilizes magnesium and requires
activation by a PPTase. Enzyme candidates for AAR and its
corresponding PPTase are found in Saccharomyces cerevisiae (Morris
et al., Gene 98:141-145 (1991)), Candida albicans (Guo et al., Mol.
Genet. Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe
(Ford et al., Curr. Genet. 28:131-137 (1995)). The AAR from S.
pombe exhibited significant activity when expressed in E. coli (Guo
et al., Yeast 21:1279-1288 (2004)). The AAR from Penicillium
chrysogenum accepts S-carboxymethyl-L-cysteine as an alternate
substrate, but did not react with adipate, L-glutamate or
diaminopimelate (Hijarrubia et al., J Biol. Chem 278:8250-8256
(2003)). The gene encoding the P. chrysogenum PPTase has not been
identified to date and no high-confidence hits were identified by
sequence comparison homology searching. These genes/proteins are
identified below in Table 107.
TABLE-US-00107 TABLE 107 Gene GenBank ID GI Number Organism LYS2
AAA34747.1 171867 Saccharomyces cerevisiae LYS5 P50113.1 1708896
Saccharomyces cerevisiae LYS2 AAC02241.1 2853226 Candida albicans
LYS5 AAO26020.1 28136195 Candida albicans Lys1p P40976.3 13124791
Schizosaccharomyces pombe Lys7p Q10474.1 1723561
Schizosaccharomyces pombe Lys2 CAA74300.1 3282044 Penicillium
chrysogenum
4-Hydroxybutyryl-CoA Dehydratase
[0448] 4-Hydroxybutyryl-CoA dehydratase catalyzes the reversible
conversion of 4-hydroxybutyryl-CoA to crotonyl-CoA. This enzyme
possesses an intrinsic vinylacetyl-CoA .DELTA.-isomerase activity,
shifting the double bond from the 3,4 position to the 2,3 position
(Scherf et al., Eur. J BioChem. 215:421-429 (1993); and Scherf et
al., Arch. Microbiol 161:239-245 (1994)). 4-Hydroxybutyrul-CoA
dehydratase enzymes from C. aminobutyricum and C. kluyveri were
purified, characterized, and sequenced at the N-terminus (Scherf et
al., Eur. J BioChem. 215:421-429 (1993); and Scherf et al., Arch.
Microbiol 161:239-245 (1994)). The C. kluyveri enzyme, encoded by
abfD, was cloned, sequenced and expressed in E. coli (Gerhardt et
al., Arch. Microbiol 174:189-199 (2000)). The abfD gene product
from Porphyromonas gingivalis ATCC 33277 is closely related by
sequence homology to the Clostridial gene products. These
genes/proteins are identified below in Table 108.
TABLE-US-00108 TABLE 108 Gene GenBank ID GI Number Organism abfD
YP_001396399.1 153955634 Clostridium kluyveri DSM 555 abfD P55792
84028213 Clostridium aminobutyricum abfD YP_001928843 188994591
Porphyromonas gingivalis ATCC 33277
Crotonase
[0449] 3-Hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), also called
crotonase, is an enoyl-CoA hydratase that reversibly dehydrates
3-hydroxyisobutyryl-CoA to form crotonyl-CoA. Crotonase enzymes are
required for n-butanol formation in some organisms, particularly
Clostridial species, and also comprise one step of the
3-hydroxypropionate/4-hydroxybutyrate cycle in thermoacidophilic
Archaea of the genera Sulfolobus, Acidianus, and Metallosphaera.
Exemplary genes encoding crotonase enzymes can be found in C.
acetobutylicum (Atsumi et al., Metab Eng 10:305-311 (2008); and
Boynton et al., J Bacteriol. 178:3015-3024 (1996)), C. kluyveri
(Hillmer et al., FEBS Lett. 21:351-354 (1972)), and Metallosphaera
sedula (Berg et al., Science. 318:1782-1786 (2007)) though the
sequence of the latter gene is not known. The enoyl-CoA hydratase
of Pseudomonas putida, encoded by ech, catalyzes the conversion of
crotonyl-CoA to 3-hydroxybutyryl-CoA (Roberts et al., Arch.
Microbiol 117:99-108 (1978)). Additional enoyl-CoA hydratase
candidates are phaA and phaB, of P. putida, and paaA and paaB from
P. fluorescens (Olivera et al., Proc. Natl. Acad. Sci U. S. A
95:6419-6424 (1998)). Lastly, a number of Escherichia coli genes
have been shown to demonstrate enoyl-CoA hydratase functionality
including maoC (Park et al., J Bacteriol. 185:5391-5397 (2003)),
paaF (Ismail et al., Eur. J BioChem. 270:3047-3054 (2003); Park et
al., Appl. BioChem. Biotechnol 113-116:335-346 (2004) and Park et
al., Biotechnol Bioeng 86:681-686 (2004)) and paaG (Ismail et al.,
Eur. J BioChem. 270:3047-3054 (2003); Park et al., Appl. BioChem.
Biotechnol 113-116:335-346 (2004) and Park et al., Biotechnol
Bioeng 86:681-686 (2004)). These genes/proteins are identified
below in Table 109.
TABLE-US-00109 TABLE 109 Gene GenBank ID GI Number Organism crt
NP_349318.1 15895969 Clostridium acetobutylicum crt1 YP_001393856.1
153953091 Clostridium kluyveri ech NP_745498.1 26990073 Pseudomonas
putida paaA NP_745427.1 26990002 Pseudomonas putida paaB
NP_745426.1 26990001 Pseudomonas putida phaA ABF82233.1 106636093
Pseudomonas fluorescens phaB ABF82234.1 106636094 Pseudomonas
fluorescens maoC NP_415905.1 16129348 Escherichia coli paaF
NP_415911.1 16129354 Escherichia coli paaG NP_415912.1 16129355
Escherichia coli
3-Hydroxybutyryl-CoA Reductase (Aldehyde Forming)
[0450] 3-Hydroxybutyryl-CoA dehydrogenase catalyzes the NAD(P)H
dependent reduction of 3-hydroxybutyryl-CoA to
3-hydroxybutyraldehyde. An enzyme catalyzing this transformation
has not been identified to date. An exemplary CoA-acylating
aldehyde dehydrogenase is the ald gene from Clostridium
beijerinckii (Toth et al., Appl Environ. Microbiol 65:4973-4980
(1999)). This enzyme has been reported to reduce acetyl-CoA and
butyryl-CoA to their corresponding aldehydes. Another enzyme that
converts an acyl-CoA to its corresponding aldehyde is malonyl-CoA
reductase which transforms malonyl-CoA to malonic semialdehyde.
Malonyl-CoA reductase is a key enzyme in autotrophic carbon
fixation via the 3-hydroxypropionate cycle in thermoacidophilic
archael bacteria (Berg et al., Science. 318:1782-1786 (2007); and
Thauer, Science. 318:1732-1733 (2007)). The enzyme utilizes NADPH
as a cofactor and has been characterized in Metallosphaera and
Sulfolobus spp (Alber et al., J. Bacteriol. 188:8551-8559 (2006);
and Hugler et al., J. Bacteriol. 184:2404-2410 (2002)). The enzyme
is encoded by Msed.sub.--0709 in Metallosphaera sedula (Alber et
al., J. Bacteriol. 188:8551-8559 (2006); Berg et al., Science.
318:1782-1786 (2007)). A gene encoding a malonyl-CoA reductase from
Sulfolobus tokodaii was cloned and heterologously expressed in E.
coli (Alber et al., J. Bacteriol. 188:8551-8559 (2006)). This
enzyme has also been shown to catalyze the conversion of
methylmalonyl-CoA to its corresponding aldehyde (WO/2007/141208).
Aldehyde dehydrogenase enzyme candidates for converting
4-hydroxybutyryl-CoA to 4-hydroxybutyraldehyde, described above,
are also applicable here. These genes/proteins are identified below
in Table 110.
TABLE-US-00110 TABLE 110 Gene GenBank ID GI Number Organism Ald
AAT66436 49473535 Clostridium beijerinckii Msed_0709 YP_001190808.1
146303492 Metallosphaera sedula mcr NP_378167.1 15922498 Sulfolobus
tokodaii
3-Hydroxybutyraldehyde Reductase
[0451] An enzyme with 3-hydroxybutyraldehyde reductase activity is
required to convert 3-hydroxybutyraldehyde to 1,3-butanediol.
Exemplary genes encoding enzymes that catalyze the conversion of an
aldehyde to alcohol (i.e., alcohol dehydrogenase or equivalently
aldehyde reductase) include alrA encoding a medium-chain alcohol
dehydrogenase for C2-C14 (Tani et al., Appl. Environ. Microbiol.
66:5231-5235 (2000)), ADH2 from Saccharomyces cerevisiae (Atsumi et
al., Nature 451:86-89 (2008)), yqhD from E. coli which has
preference for molecules longer than C(3) (Sulzenbacher et al.,
Journal of Molecular Biology 342:489-502 (2004)), and bdh I and bdh
II from C. acetobutylicum which converts butyraldehyde into butanol
(Walter et al., Journal of Bacteriology 174:7149-7158 (1992)). The
gene product of yqhD catalyzes the reduction of acetaldehyde,
malondialdehyde, propionaldehyde, butyraldehyde, and acrolein using
NADPH as the cofactor (Perez et al., J Biol. Chem. 283:7346-7353
(2008); and Perez et al., J Biol. Chem. 283:7346-7353 (2008)). The
adhA gene product from Zymomonas mobilis has been demonstrated to
have activity on a number of aldehydes including formaldehyde,
acetaldehyde, propionaldehyde, butyraldehyde, and acrolein
(Kinoshita et al., Appl Microbiol Biotechnol 22:249-254 (1985)).
These genes/proteins are identified below in Table 111.
TABLE-US-00111 TABLE 111 Gene GenBank ID GI Number Organism alrA
BAB12273.1 9967138 Acinetobacter sp. Strain M-1 ADH2 NP_014032.1
6323961 Saccharomyces cerevisiae yqhD NP_417484.1 16130909
Escherichia coli bdh I NP_349892.1 15896543 Clostridium
acetobutylicum bdh II NP_349891.1 15896542 Clostridium
acetobutylicum adhA YP_162971.1 56552132 Zymomonas mobilis
[0452] Additional candidates include 4-hydroxybutyrate
dehydrogenase and 3-hydroxyisobutyrate dehydrogenase enzymes.
4-Hydroxybutyrate dehydrogenase enzymes naturally convert
4-hydroxybutyraldehyde to 4-HB and have been characterized in
Ralstonia eutropha (Bravo et al., J. Forensic Sci. 49:379-387
(2004)), Clostridium kluyveri (Wolff et al., Protein Expr. Pur
6:206-212 (1995)) and Arabidopsis thaliana (Breitkreuz, et al., J.
Biol. Chem. 278:41552-41556 (2003)). 3-Hydroxyisobutyrate
dehydrogenase enzyme candidates include mmsB from Pseudomonas
aeruginosa PAO1 (Gokam et al., U.S. Pat. No. 7,393,676 (2008)),
mmsB from Pseudomonas putida KT2440 (118) and mmsB from Pseudomonas
putida E23 (Chowdhury, et al., Biosci. Biotechnol. BioChem.
60:2043-2047 (1996)). These genes/proteins are identified below in
Table 112.
TABLE-US-00112 TABLE 112 Gene GenBank ID GI Number Organism 4hbd
YP_726053.1 113867564 Ralstonia eutropha H16 4hbd EDK35022.1
146348486 Clostridium kluyveri 4hbd Q94B07 75249805 Arabidopsis
thaliana mmsB NP_252259.1 15598765 Pseudomonas aeruginosa PAO1 mmsB
NP_746775.1 26991350 Pseudomonas putida KT2440 mmsB JC7926 60729613
Pseudomonas putida E23
3-Hydroxybutyryl-CoA Reductase (Alcohol Forming)
[0453] A bifunctional oxidoreductase is required for the direct
conversion of 3-hydroxybutyryl-CoA to 1,3-butanediol. Exemplary
enzymes that convert an acyl-CoA to alcohol include those that
transform substrates such as acetyl-CoA to ethanol (e.g., adhE from
E. coli (Kessler et al., FEBS. Lett. 281:59-63 (1991))),
butyryl-CoA to butanol (e.g. adhE2 from C. acetobutylicum (Fontaine
et al., J. Bacteriol. 184:821-830 (2002))) and 4-hydroxybutyryl-CoA
to 1,4-butanediol (see candidates in previous section). The jojoba
(Simmondsia chinensis) FAR encodes an alcohol-forming fatty
acyl-CoA reductase. This gene was cloned and overexpressed in E.
coli, resulting in FAR activity and the accumulation of fatty
alcohol (Metz et al., Plant Physiology 122:635-644 (2000)). Another
exemplary enzyme convert malonyl-CoA to 3-hydroxypropionate. An
NADPH-dependent enzyme with this activity has characterized in
Chloroflexus aurantiacus where it participates in the
3-hydroxypropionate cycle (Hugler et al., J. Bacteriol.
184:2404-2410 (2002); and Strauss et al., Eur. J. BioChem.
215:633-643 (1993)). This enzyme, with a mass of 300 kDa, is highly
substrate-specific and shows little sequence similarity to other
known oxidoreductases (Hugler et al., J. Bacteriol. 184:2404-2410
(2002)). These genes/proteins are identified below in Table
113.
TABLE-US-00113 TABLE 113 Gene GenBank ID GI Number Organism adhE
NP_415757.1 16129202 Escherichia coli adhE2 AAK09379.1 12958626
Clostridium acetobutylicum adhE AAV66076.1 55818563 Leuconostoc
mesenteroides FAR AAD38039.1 5020215 Simmondsia chinensis mcr
AAS20429.1 42561982 Chloroflexus aurantiacus
3-Hydroxybutyryl-CoA Transferase
[0454] The conversion of 3-hydroxybutyryl-CoA to 3-hydroxybutyrate
(3-HB) is catalyzed by a CoA transferase, hydrolase or synthetase.
A CoA transferase enzyme catalyzing this specific transformation
has not been identified to date. The E. coli enzyme
acyl-CoA:acetate-CoA transferase, also known as acetate-CoA
transferase (EC 2.8.3.8), has been shown to transfer the CoA moiety
to acetate from a variety of branched and linear acyl-CoA
substrates, including isobutyrate (Matthies et al., Appl Environ
Microbiol 58:1435-1439 (1992)), valerate (Vanderwinkel et al.,
BioChem. Biophys. Res Commun. 33:902-908 (1968)) and butanoate
(Vanderwinkel et al., BioChem. Biophys. Res Commun. 33:902-908
(1968)). This enzyme is encoded by atoA (alpha subunit) and atoD
(beta subunit) in E. coli sp. K12 (Korolev et al., D Biol
Crystallogr. 58:2116-2121 (2002); and Vanderwinkel et al., BioChem.
Biophys. Res Commun. 33:902-908 (1968)) and actA and cg0592 in
Corynebacterium glutamicum ATCC 13032 (Duncan et al., Appl Environ
Microbiol 68:5186-5190 (2002)). Similar enzymes exist in
Corynebacterium glutamicum ATCC 13032 (Eikmanns et al., Mol. Gen.
Genet. 218:330-339 (1989)), Clostridium acetobutylicum (Cary et
al., Appl. Environ. Microbiol 56:1576-1583 (1990); and Wiesenborn
et al., Appl. Environ. Microbiol 55:323-329 (1989)), and
Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci.
Biotechnol BioChem. 71:58-68 (2007)). These genes/proteins are
identified below in Table 114.
TABLE-US-00114 TABLE 114 Gene GenBank ID GI Number Organism atoA
P76459.1 2492994 Escherichia coli atoD P76458.1 2492990 Escherichia
coli actA YP_226809.1 62391407 Corynebacterium glutamicum cg0592
YP_224801.1 62389399 Corynebacterium glutamicum ctfA NP_149326.1
15004866 Clostridium acetobutylicum ctfB NP_149327 15004867
Clostridium acetobutylicum ctfA AAP42564.1 31075384 Clostridium
saccharoperbutylacetonicum ctfB AAP42565.1 31075385 Clostridium
saccharoperbutylacetonicum
[0455] CoA transferase gene candidates described for propionyl-CoA
transferase, methylmalonyl-CoA transferase, acetoacetyl-CoA
transferase, methacrylyl-CoA transferase, 3-hydroxyisobutyryl-CoA
transferase, 4-hydroxybutyryl-CoA transferase and succinyl-CoA
transferase are also applicable here.
3-Hydroxybutyryl-CoA Synthetase
[0456] 3-Hydroxybutyryl-CoA can also be converted to 3-HB by a CoA
synthetase (also known as ligase or synthase). A candidate ATP
synthase is ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13),
an enzyme that couples the conversion of acyl-CoA esters to their
corresponding acids with the concurrent synthesis of ATP. Although
this enzyme has not been shown to react with 3-hydroxybutyryl-CoA
as a substrate, several enzymes with broad substrate specificities
have been described in the literature. ACD I from Archaeoglobus
fulgidus, encoded by AF1211, was shown to operate on a variety of
linear and branched-chain substrates including isobutyrate,
isopentanoate, and fumarate (Musfeldt et al., J Bacteriol.
184:636-644 (2002)). A second reversible ACD in Archaeoglobus
fulgidus, encoded by AF1983, was also shown to have a broad
substrate range with high activity on cyclic compounds
phenylacetate and indoleacetate (Musfeldt, et al., J Bacteriol.
184:636-644 (2002)). The enzyme from Haloarcula marismortui
(annotated as a succinyl-CoA synthetase) accepts propionate,
butyrate, and branched-chain acids (isovalerate and isobutyrate) as
substrates, and was shown to operate in the forward and reverse
directions (Brasen et al., Arch. Microbiol 182:277-287 (2004)). The
ACD encoded by PAE3250 from hyperthermophilic crenarchaeon
Pyrobaculum aerophilum showed the broadest substrate range of all
characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA
(preferred substrate) and phenylacetyl-CoA (Brasen et al., Arch.
Microbiol 182:277-287 (2004)). However, directed evolution or
engineering may be necessary for this enzyme to operate at the
physiological temperature of the host organism. The enzymes from A.
fulgidus, H. marismortui and P. aerophilum have all been cloned,
functionally expressed, and characterized in E. coli (Brasen et
al., Arch. Microbiol 182:277-287 (2004); and Musfeldt et al., J
Bacteriol. 184:636-644 (2002)). An additional candidate is the
enzyme encoded by sucCD in E. coli, which naturally catalyzes the
formation of succinyl-CoA from succinate with the concomitant
consumption of one ATP, a reaction which is reversible in vivo
(Buck et al., BioChemistry 24:6245-6252 (1984)). These
genes/proteins are identified below in Table 115.
TABLE-US-00115 TABLE 115 Gene GenBank ID GI Number Organism AF1211
NP_070039.1 11498810 Archaeoglobus fulgidus DSM 4304 AF1983
NP_070807.1 11499565 Archaeoglobus fulgidus DSM 4304 scs
YP_135572.1 55377722 Haloarcula marismortui PAE3250 NP_560604.1
18313937 Pyrobaculum aerophilum str. IM2 sucC NP_415256.1 16128703
Escherichia coli sucD AAC73823.1 1786949 Escherichia coli
[0457] CoA synthetase gene candidates described for propionyl-CoA
synthetase, methylmalonyl-CoA synthetase, methacrylyl-CoA
synthetase, acetoacetyl-CoA synthetase, 3-hydroxyisobutyryl-CoA
synthetase, 4-hydroxybutyryl-CoA synthetase and succinyl-CoA
synthetase are also applicable here.
3-Hydroxybutyryl-CoA Hydrolase
[0458] A 3-hydroxybutyryl-CoA hydrolase is required to convert
3-hydroxybutyryl-CoA to 3-HB. The enzyme 3-hydroxyisobutyryl-CoA
hydrolase (EC 3.1.2.4) catalyzes a related transformation: the
hydrolysis of 3-hydroxyisobutyryl-CoA. The 3-hydroxyisobutyryl-CoA
hydrolase from Homo sapiens also accepts 3-hydroxybutyryl-CoA as a
substrate (Shimomura et al., Methods Enzymol. 324:229-240 (2000)).
This enzyme has also been characterized in Rattus norvegicus
(Shimomura et al., J Biol Chem. 269:14248-14253 (1994); and
Shimomura et al., Methods Enzymol. 324:229-240 (2000)). Candidate
genes by sequence homology include hibch of Saccharomyces
cerevisiae and BC 2292 of Bacillus cereus. These proteins are
identified below in Table 116. Additional CoA hydrolase enzyme
candidates identified for propionyl-CoA hydrolase,
methylmalonyl-CoA hydrolase, methacrylyl-CoA hydrolase,
acetoacetyl-CoA hydrolase and 3-hydroxyisobutyryl-CoA are also
applicable here. These genes/proteins are identified below in Table
116.
TABLE-US-00116 TABLE 116 Gene GenBank ID GI Number Organism hibch
Q5XIE6.2 146324906 Rattus norvegicus hibch Q6NVY1.2 146324905 Homo
sapiens hibch P28817.2 2506374 Saccharomyces cerevisiae BC_2292
AP09256 29895975 Bacillus cereus
3-Hydroxybutyrate Reductase
[0459] The reduction of 3-hydroxybutyrate to 3-hydroxybutyraldehyde
is catalyzed by a carboxylic acid reductase. Exemplary enzyme
candidates for succinate reductase and 4-hydroxybutyrate reductase
enzymes are also applicable here.
4-Hydroxybutyryl-CoA Mutase
[0460] The conversion of 4HB-CoA to 3-hydroxyisobutyryl-CoA is
catalyzed by a methylmutase. Such a conversion has yet to be
demonstrated experimentally. However, two methylmutases (i.e.,
isobutyryl-CoA mutase and methylmalonyl-CoA mutase) that catalyze
similar reactions are promising candidates given the structural
similarity of their corresponding substrates.
[0461] Methylmalonyl-CoA mutase (MCM) is a cobalamin-dependent
enzyme that naturally converts succinyl-CoA to methylmalonyl-CoA.
In E. coli, the reversible adenosylcobalamin-dependant mutase
participates in a three-step pathway leading to the conversion of
succinate to propionate (Haller et al., BioChemistry 39:4622-9
(2000)). MCM is encoded by genes scpA in Escherichia coli (Bobik et
al., Anal. Bioanal. Chem. 375:344-349 (2003); and Haller et al.,
BioChemistry 39:4622-4629 (2000)) and mutA in Homo sapiens
(Padovani et al., BioChemistry 45:9300-9306 (2006)). In several
other organisms MCM contains alpha and beta subunits and is encoded
by two genes. Exemplary gene candidates encoding the two-subunit
protein are Propionibacterium freudenreichii sp. shermanii mutA and
mutB (Korotkova et al., J Biol Chem. 279:13652-13658 (2004)) and
Methylobacterium extorquens mcmA and mcmB (Korotkova et al., J Biol
Chem. 279:13652-13658 (2004)). These genes/proteins are identified
below in Table 117.
TABLE-US-00117 TABLE 117 Gene GenBank ID GI Number Organism scpA
NP_417392.1 16130818 Escherichia coli K12 mutA P22033.3 67469281
Homo sapiens mutA P11652.3 127549 Propionibacterium freudenreichii
sp. shermanii mutB P11653.3 127550 Propionibacterium freudenreichii
sp. shermanii mcmA Q84FZ1 75486201 Methylobacterium extorquens mcmB
Q6TMA2 75493131 Methylobacterium extorquens
Additional enzyme candidates identified based on high homology to
the E. coli spcA gene product include those identified below in
Table 118.
TABLE-US-00118 TABLE 118 Gene GenBank ID GI Number Organism sbm
NP_838397.1 30064226 Shigella flexneri SARI_04585 ABX24358.1
160867735 Salmonella enterica YfreA_01000861 ZP_00830776.1 77975240
Yersinia frederiksenii
[0462] There further exists evidence that genes adjacent to the
methylmalonyl-CoA mutase catalytic genes are also required for
maximum activity. For example, it has been demonstrated that the
meaB gene from M. extorquens forms a complex with methylmalonyl-CoA
mutase, stimulates in vitro mutase activity, and possibly protects
it from irreversible inactivation (Korotkova et al., J Biol Chem.
279:13652-13658 (2004)). The M. extorquens meaB gene product is
highly similar to the product of the E. coli argK gene (BLASTp: 45%
identity, e-value: 4e-67) which is adjacent to scpA on the
chromosome. No sequence for a meaB homolog in P. freudenreichii is
catalogued in GenBank. However, the Propionibacterium acnes
KPA171202 gene product at the locus PPA0597 is 51% identical to the
M. extorquens meaB protein and its gene is also adjacent to the
methylmalonyl-CoA mutase gene on the chromosome. These
genes/proteins are identified below in Table 119.
TABLE-US-00119 TABLE 119 Gene GenBank ID GI Number Organism argK
AAC75955.1 1789285 Escherichia coli K12 PPA0597 YP_055310.1
50842083 Propionibacterium acnes KPA171202 2QM8_B 158430328
Methylobacterium extorquens
[0463] Alternatively, isobutyryl-CoA mutase (ICM) could catalyze
the proposed transformation. ICM is a cobalamin-dependent
methylmutase in the MCM family that reversibly rearranges the
carbon backbone of butyryl-CoA into isobutyryl-CoA (FIG. 7B of
Ratnatilleke, J Biol Chem. 274:31679-31685 (1999)). A recent study
of a novel ICM in Methylibium petroleiphilum, along with previous
work, provides evidence that changing a single amino acid near the
active site alters the substrate specificity of the enzyme
(Ratnatilleke et al., J Biol Chem. 274:31679-31685 (1999); and
Rohwerder et al., Appl Environ Microbiol 72:4128-4135 (2006)). This
implies that if a native enzyme is unable to catalyze the
conversion of 4HB-CoA to 3HIB-CoA, the enzyme could undergo
rational engineering. Exemplary ICM genes encoding homodimeric
enzymes include icmA in Streptomyces coelicolor A3 (2) and Mpe
B0541 in Methylibium petroleiphilum PM1 (Ratnatilleke et al., J
Biol Chem. 274:31679-31685 (1999); and Rohwerder et al., Appl
Environ Microbiol 72:4128-4135 (2006)). Genes encoding
heterodimeric enzymes include icm and icmB in Streptomyces
cinnamonensis (Ratnatilleke, et al., J Biol Chem. 274:31679-31685
(1999); Vrijbloed et al., J Bacteriol. 181:5600-5605 (1999) and
Zerbe-Burkhardt et al., J Biol Chem. 273:6508-6517 (1998)). Enzymes
encoded by icmA and icmB genes in Streptomyces avermitilis MA-4680
show high sequence similarity to known ICMs. These genes/proteins
are identified below in Table 120.
TABLE-US-00120 TABLE 120 Gene GenBank ID GI Number Organism icmA
CAB40912.1 4585853 Streptomyces coelicolor A3(2) Mpe_B0541
YP_001023546.1 124263076 Methylibium petroleiphilum PM1 icm
AAC08713.1 3002492 Streptomyces cinnamonensis icmB CAB59633.1
6137077 Streptomyces cinnamonensis icmA NP_824008.1 29829374
Streptomyces avermitilis icmB NP_824637.1 29830003 Streptomyces
avermitilis
3-Hydroxyisobutyryl-CoA Transferase
[0464] The next step in this pathway entails the conversion of
3-hydroxyisobutyryl-CoA into 3-hydroxyisobutyrate (3-HIB) by a CoA
transferase. An enzyme catalyzing this specific transformation has
not been identified to date. The E. coli enzyme
acyl-CoA:acetate-CoA transferase, also known as acetate-CoA
transferase (EC 2.8.3.8), has been shown to transfer the CoA moiety
to acetate from a variety of branched and linear acyl-CoA
substrates, including isobutyrate (Matthies et al., Appl Environ
Microbiol 58:1435-1439 (1992)), valerate (Vanderwinkel et al.,
BioChem. Biophys. Res Commun. 33:902-908 (1968)) and butanoate
(Vanderwinkel et al., BioChem. Biophys. Res Commun. 33:902-908
(1968)). This enzyme is encoded by atoA (alpha subunit) and atoD
(beta subunit) in E. coli sp. K12 (Korolev et al., D Biol
Crystallogr. 58:2116-2121 (2002); and Vanderwinkel et al., BioChem.
Biophys. Res Commun. 33:902-908 (1968)) and actA and cg0592 in
Corynebacterium glutamicum ATCC 13032 (Duncan et al., Appl Environ
Microbiol 68:5186-5190 (2002)). Similar enzymes exist in
Corynebacterium glutamicum ATCC 13032 (Eikmanns et al., Mol. Gen.
Genet. 218:330-339 (1989)), Clostridium acetobutylicum (Cary et
al., Appl. Environ. Microbiol 56:1576-1583 (1990); and Wiesenborn
et al., Appl. Environ. Microbiol 55:323-329 (1989)), and
Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci.
Biotechnol BioChem. 71:58-68 (2007)). 4-Hydroxybutyryl-CoA
transferase enzyme candidates, described previously, are also
applicable here. These genes/proteins are identified below in Table
121.
TABLE-US-00121 TABLE 121 Gene GenBank ID GI Number Organism atoA
P76459.1 2492994 Escherichia coli atoD P76458.1 2492990 Escherichia
coli actA YP_226809.1 62391407 Corynebacterium glutamicum cg0592
YP_224801.1 62389399 Corynebacterium glutamicum ctfA NP_149326.1
15004866 Clostridium acetobutylicum ctfB NP_149327 15004867
Clostridium acetobutylicum ctfA AAP42564.1 31075384 Clostridium
saccharoperbutylacetonicum ctfB AAP42565.1 31075385 Clostridium
saccharoperbutylacetonicum
3-Hydroxyisobutyryl-CoA Synthetase
[0465] 3-Hydroxyisobutyryl-CoA can also be converted to 3-HIB by a
CoA synthetase (also known as ligase or synthase). A candidate ATP
synthase is ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13),
an enzyme that couples the conversion of acyl-CoA esters to their
corresponding acids with the concurrent synthesis of ATP. Although
this enzyme has not been shown to react with
3-hydroxyisobutyryl-CoA as a substrate, several enzymes with broad
substrate specificities have been described in the literature. ACD
I from Archaeoglobus fulgidus, encoded by AF1211, was shown to
operate on a variety of linear and branched-chain substrates
including isobutyrate, isopentanoate, and fumarate (Musfeldt et
al., J Bacteriol. 184:636-644 (2002)). A second reversible ACD in
Archaeoglobus fulgidus, encoded by AF1983, was also shown to have a
broad substrate range with high activity on cyclic compounds
phenylacetate and indoleacetate (Musfeldt, et al., J Bacteriol.
184:636-644 (2002)). The enzyme from Haloarcula marismortui
(annotated as a succinyl-CoA synthetase) accepts propionate,
butyrate, and branched-chain acids (isovalerate and isobutyrate) as
substrates, and was shown to operate in the forward and reverse
directions (Brasen et al., Arch. Microbiol 182:277-287 (2004)). The
ACD encoded by PAE3250 from hyperthermophilic crenarchaeon
Pyrobaculum aerophilum showed the broadest substrate range of all
characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA
(preferred substrate) and phenylacetyl-CoA (Brasen et al., Arch.
Microbiol 182:277-287 (2004)). However, directed evolution or
engineering may be necessary for this enzyme to operate at the
physiological temperature of the host organism. The enzymes from A.
fulgidus, H. marismortui and P. aerophilum have all been cloned,
functionally expressed, and characterized in E. coli (Brasen et
al., Arch. Microbiol 182:277-287 (2004); and Musfeldt et al., J
Bacteriol. 184:636-644 (2002)). An additional candidate is the
enzyme encoded by sucCD in E. coli, which naturally catalyzes the
formation of succinyl-CoA from succinate with the concomitant
consumption of one ATP, a reaction which is reversible in vivo
(Buck et al., BioChemistry 24:6245-6252 (1984)). These
genes/proteins are identified below in Table 122.
TABLE-US-00122 TABLE 122 Gene GenBank ID GI Number Organism AF1211
NP_070039.1 11498810 Archaeoglobus fulgidus DSM 4304 AF1983
NP_070807.1 11499565 Archaeoglobus fulgidus DSM 4304 scs
YP_135572.1 55377722 Haloarcula marismortui PAE3250 NP_560604.1
18313937 Pyrobaculum aerophilum str. IM2 sucC NP_415256.1 16128703
Escherichia coli sucD AAC73823.1 1786949 Escherichia coli
3-Hydroxyisobutyryl-CoA Hydrolase
[0466] The enzyme 3-hydroxyisobutyryl-CoA hydrolase selectively
converts 3-hydroxyisobutyryl-CoA to 3-HIB during valine degradation
(Shimomura et al., J Biol Chem 269:14248-53 (1994)). Genes encoding
this enzyme were described previously. 3-Hydroxybutyryl-CoA
hydrolase and propionyl-CoA gene candidates, described previously,
are also applicable here.
3-Hydroxyisobutyrate Dehydratase
[0467] The dehydration of 3-hydroxyisobutyrate to methylacrylic
acid is catalyzed by an enzyme with 3-hydroxyisobutyrate
dehydratase activity. No direct evidence for this specific
enzymatic transformation has been identified. However, most
dehydratases catalyze the alpha, beta-elimination of water which
involves activation of the alpha-hydrogen by an
electron-withdrawing carbonyl, carboxylate, or CoA-thiol ester
group and removal of the hydroxyl group from the beta-position
(Buckel et al., J Bacteriol. 117:1248-1260 (1974); and Martins, et
al., Proc Natl Acad Sci USA 101:15645-9 (2004)). This is the exact
type of transformation proposed for the final step in the
methylacrylic acid pathway. The proposed transformation is highly
similar to the 2-(hydroxymethyl)glutarate dehydratase of
Eubacterium barkeri (FIG. 3A). This enzyme has been studied in the
context of nicotinate catabolism and is encoded by hmd (Alhapel et
al., Proc Natl Acad Sci USA 103:12341-6 (2006)). An enzyme with
similar functionality in E. barkeri is dimethylmaleate hydratase, a
reversible Fe.sup.2+-dependent and oxygen-sensitive enzyme in the
aconitase family that hydrates dimethylmaleate to form
(2R,3S)-2,3-dimethylmalate. This enzyme is encoded by dmdAB
(Alhapel et al., Proc Natl Acad Sci USA 103:12341-6 (2006); and
Kollmann-Koch et al., Hoppe Seylers. Z. Physiol Chem. 365:847-857
(1984)). These genes/proteins are identified below in Table
123.
TABLE-US-00123 TABLE 123 Gene GenBank ID GI Number Organism hmd
ABC88407.1 86278275 Eubacterium barkeri dmdA ABC88408 86278276
Eubacterium barkeri dmdB ABC88409.1 86278277 Eubacterium
barkeri
[0468] An additional enzyme candidate is 2-methylmalate
dehydratase, also called citramalate hydrolyase, a reversible
hydrolyase that catalyzes the alpha, beta elimination of water from
citramalate to form mesaconate. This enzyme has been studied in
Methanocaldococcus jannaschii in the context of the pyruvate
pathway to 2-oxobutanoate, where it has been shown to have a broad
substrate specificity (Drevland et al., J Bacteriol. 189:4391-4400
(2007)). This enzyme activity was also detected in Clostridium
tetanomorphum, Morganella morganii, Citrobacter amalonaticus where
it is thought to participate in glutamate degradation (Kato et al.,
Arch. Microbiol 168:457-463 (1997)). The M. jannaschii protein
sequence does not bear significant homology to genes in these
organisms. This genes/proteins is identified below in Table
124.
TABLE-US-00124 TABLE 124 Gene GenBank ID GI Number Organism leuD
Q58673.1 3122345 Methanocaldococcus jannaschii
[0469] Fumarate hydratase enzymes, which naturally catalyze the
dehydration of malate to fumarate, represent an additional set of
candidates. Although the ability of fumarate hydratase to react on
3-hydroxyisobutyrate as a substrate has not been described, a
wealth of structural information is available for this enzyme and
other researchers have successfully engineered the enzyme to alter
activity, inhibition and localization (Weaver, D Biol Crystallogr.
61:1395-1401 (2005)). E. coli has three fumarases: FumA, FumB, and
FumC that are regulated by growth conditions. FumB is oxygen
sensitive and only active under anaerobic conditions. FumA is
active under microanaerobic conditions, and FumC is the only active
enzyme in aerobic growth (Guest et al., J Gen Microbiol
131:2971-2984 (1985); Tseng et al., J Bacteriol 183:461-467 (2001)
and Woods et al., Biochim Biophys Acta 954:14-26 (1988)).
Additional enzyme candidates are found in Campylobacter jejuni
(Smith et al., Int. J BioChem. Cell Biol 31:961-975 (1999)),
Thermus thermophilus (Mizobata et al., Arch. BioChem. Biophys.
355:49-55 (1998)) and Rattus norvegicus (Kobayashi et al., J
BioChem. 89:1923-1931 (1981)). The MmcBC fumarase from
Pelotomaculum thermopropionicum is another class of fumarase with
two subunits (Shimoyama et al., FEMS Microbiol Lett 270:207-213
(2007)). These genes/proteins are identified below in Table
125.
TABLE-US-00125 TABLE 125 Gene GenBank ID GI Number Organism fumA
P0AC33 81175318 Escherichia coli K12 fumB P14407 33112655
Escherichia coli K12 fumC P05042.1 120601 Escherichia coli K12 fumC
O69294.1 9789756 Campylobacter jejuni fumC P84127 75427690 Thermus
thermophilus fumH P14408.1 120605 Rattus norvegicus MmcB
YP_001211906 147677691 Pelotomaculum thermopropionicum MmcC
YP_001211907 147677692 Pelotomaculum thermopropionicum
3-Hydroxyisobutyryl-CoA Dehydratase
[0470] Dehydration of 3-hydroxyisobutyryl-CoA by a CoA dehydratase
yields methacrylyl-CoA. Enoyl-CoA hydratases (EC 4.2.1.17) catalyze
the dehydration of 3-hydroxyacyl-CoA substrates (Agnihotri and
Liu., J. Bacteriol. 188:8551-8559(2003); Conrad et al., J.
Bacteriol. 118:103-111 (1974); and Roberts et al., Arch. Microbiol
117:99-108 (1978)). The enoyl-CoA hydratase (ECH) found in bovine
liver accepts a variety of substrates including methacrylyl-CoA, 2-
and 3-methyl-crotonoyl-CoA, acryloyl-CoA and
1-carboxycyclohexenoyl-CoA (Agnihotri et al., Bioorg Med Chem.,
11(1):9-20 (2003)). A recombinant bovine liver ECH enzyme has been
overexpressed in E. coli and found to have similar catalytic
properties (Dakoji et al., J Am Chem Soc., 123:9749 (2001)). The
enoyl-CoA hydratase of Pseudomonas putida, encoded by ech,
catalyzes the conversion of 3-hydroxybutyryl-CoA to crotonoyl-CoA
(Roberts et al., Arch. Microbiol 117:99-108 (1978)). Additional
enoyl-CoA hydratase candidates are phaA and phaB, of P. putida, and
paaA and paaB from P. fluorescens (Olivera et al., Proc. Natl.
Acad. Sci U.S.A 95:6419-6424 (1998)). The gene product of pimF in
Rhodopseudomonas palustris is predicted to encode an enoyl-CoA
hydratase that participates in pimeloyl-CoA degradation (Harrison
and Harwood, Microbiology 151:727-736 (2005)). Lastly, a number of
Escherichia coli genes have been shown to demonstrate enoyl-CoA
hydratase functionality including maoC (Park and Lee, J. Bacteriol.
185:5391-5397 (2003)), paaF (Ismail et al., J Biochem.
270:3047-3054 (2003); Park and Lee, Appl. Biochem. Biotechnol
113-116:335-346 (2004); and Park and Yup, Biotechnol Bioeng
86:681-686 (2004)) and paaG (Ismail et al., J Biochem.
270:3047-3054(2003); Park and Lee, Appl. Biochem. Biotechnol
113-116:335-346 (2004); and Park and Yup, Biotechnol Bioeng
86:681-686 (2004)). These genes/proteins are identified below in
Table 126.
TABLE-US-00126 TABLE 126 Gene GenBank ID GI Number Organism ECHS1
NP_001020377.2 70778822 Bos taurus ech NP_745498.1 26990073
Pseudomonas putida paaA NP_745427.1 26990002 Pseudomonas putida
paaB NP_745426.1 26990001 Pseudomonas putida phaA ABF82233.1
106636093 Pseudomonas fluorescens phaB ABF82234.1 106636094
Pseudomonas fluorescens pimF CAE29158 39650635 Rhodopseudomonas
palustris maoC NP_415905.1 16129348 Escherichia coli paaF
NP_415911.1 16129354 Escherichia coli paaG NP_415912.1 16129355
Escherichia coli
[0471] Another exemplary enzyme candidate for catalyzing this
reaction is crotonase. Gene candidates for this enzyme are
described above. Alternatively, the E. coli gene products of fadA
and fadB encode a multienzyme complex involved in fatty acid
oxidation that exhibits enoyl-CoA hydratase activity (Nakahigashi
and Inokuchi, Nucleic Acids Res. 18:4937 (1990); Yang, J.
Bacteriol. 173:7405-7406 (1991); and Yang et al., Biochemistry
30:6788-6795 (1991)). Knocking out a negative regulator encoded by
fadR can be utilized to activate the fadB gene product (Sato et
al., J Biosci. Bioeng 103:38-44 (2007)). The fadI and fadJ genes
encode similar functions and are naturally expressed under
anaerobic conditions (Campbell et al., Mol. Microbiol 47:793-805
(2003)). These genes/proteins are identified below in Table
127.
TABLE-US-00127 TABLE 127 Gene GenBank ID GI Number Organism fadA
YP_026272.1 49176430 Escherichia coli fadB NP_418288.1 16131692
Escherichia coli fadI NP_416844.1 16130275 Escherichia coli fadJ
NP_416843.1 16130274 Escherichia coli fadR NP_415705.1 16129150
Escherichia coli
Methacrylyl-CoA Hydrolase
[0472] Conversion of methacrylyl-CoA to MAA is catalyzed by a CoA
transferase, synthetase or hydrolase. CoA hydrolase gene candidates
described for propionyl-CoA hydrolase, methylmalonyl-CoA hydrolase,
acetoacetyl-CoA hydrolase, 3-hydroxybutyryl-CoA hydrolase and
3-hydroxyisobutyryl-CoA hydrolase are also applicable here.
Methacrylyl-CoA Transferase
[0473] Conversion of methacrylyl-CoA to MAA is catalyzed by a CoA
transferase, synthetase or hydrolase. CoA transferase gene
candidates described for propionyl-CoA transferase,
methylmalonyl-CoA transferase, acetoacetyl-CoA transferase,
3-hydroxybutyryl-CoA transferase, 3-hydroxyisobutyryl-CoA
transferase, 4-hydroxybutyryl-CoA transferase and succinyl-CoA
transferase are applicable here.
Methacrylyl-CoA Synthetase
[0474] Conversion of methacrylyl-CoA to MAA is catalyzed by a CoA
transferase, synthetase or hydrolase. CoA synthetase gene
candidates described for propionyl-CoA synthetase,
methylmalonyl-CoA synthetase, acetoacetyl-CoA synthetase,
3-hydroxybutyryl-CoA synthetase, 3-hydroxyisobutyryl-CoA
synthetase, 4-hydroxybutyryl-CoA synthetase and succinyl-CoA
synthetase are applicable here.
Methylmalonyl-CoA Hydrolase
[0475] Methylmalonyl-CoA is converted to methylmalonate by
methylmalonyl-CoA hydrolase (EC 3.1.2.17). This enzyme, isolated
from Rattus norvegicus liver, is also active on malonyl-CoA and
propionyl-CoA as alternative substrates (Kovachy et al., J. Biol.
Chem., 258: 11415-11421 (1983)). The gene associated with this
enzyme is not known. Other CoA hydrolase enzyme candidates for
propionyl-CoA hydrolase, methacrylyl-CoA hydrolase, acetoacetyl-CoA
hydrolase, 3-hydroxybutyryl-CoA hydrolase and
3-hydroxyisobutyryl-CoA hydrolase, described in previous sections,
are applicable here.
Methylmalonyl-CoA Transferase
[0476] Alternately, methylmalonyl-CoA is converted to
methylmalonate by a CoA transferase. CoA transferase gene
candidates described for propionyl-CoA transferase, methacrylyl-CoA
transferase, acetoacetyl-CoA transferase, 3-hydroxybutyryl-CoA
transferase, 3-hydroxyisobutyryl-CoA transferase,
4-hydroxybutyryl-CoA transferase and succinyl-CoA transferase are
also applicable here
Methylmalonyl-CoA Synthetase
[0477] Yet another enzyme that forms methylmalonate from
methylmalonyl-CoA is methylmalonyl-CoA synthetase. CoA synthetase
gene candidates described for propionyl-CoA synthetase,
methacrylyl-CoA synthetase, acetoacetyl-CoA synthetase,
3-hydroxybutyryl-CoA synthetase, 3-hydroxyisobutyryl-CoA
synthetase, 4-hydroxybutyryl-CoA synthetase and succinyl-CoA
synthetase are applicable here.
Methylmalonate Reductase
[0478] The reduction of methylmalonate to methylmalonate
semialdehyde is catalyzed by a carboxylic acid reductase. Exemplary
enzyme candidates for succinate reductase and 4-hydroxybutyrate
reductase enzymes are also applicable here.
[0479] 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.
Sequence CWU 1
1
2129PRTClostridium saccharobutylicum 1Met Asp Phe Lys Leu Thr Lys
Thr Gln Val Leu Gln Gln Trp Leu Phe1 5 10 15 Ala Glu Phe Ala Gly
Ile Gly Ile Lys Pro Ile Ala Glu 20 25 220PRTEuglena gracilisfirst
20 amino acid from N-terminus from KDC gene 2Met Thr Tyr Lys Ala
Pro Val Lys Asp Val Lys Phe Leu Leu Asp Lys1 5 10 15 Val Phe Lys
Val 20
* * * * *