U.S. patent application number 16/004285 was filed with the patent office on 2019-05-02 for microorganisms for producing butadiene and methods related thereto.
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 | 20190127764 16/004285 |
Document ID | / |
Family ID | 47422903 |
Filed Date | 2019-05-02 |
View All Diagrams
United States Patent
Application |
20190127764 |
Kind Code |
A1 |
Burk; Mark J. ; et
al. |
May 2, 2019 |
MICROORGANISMS FOR PRODUCING BUTADIENE AND METHODS RELATED
THERETO
Abstract
The invention provides non-naturally occurring microbial
organisms having a butadiene or crotyl alcohol pathway. The
invention additionally provides methods of using such organisms to
produce butadiene or crotyl alcohol.
Inventors: |
Burk; Mark J.; (San Diego,
CA) ; Burgard; Anthony P.; (Elizabeth, PA) ;
Osterhout; Robin E.; (San Diego, CA) ; Sun; Jun;
(San Diego, CA) ; Pharkya; Priti; (San Diego,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genomatica, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
47422903 |
Appl. No.: |
16/004285 |
Filed: |
June 8, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14869872 |
Sep 29, 2015 |
10006055 |
|
|
16004285 |
|
|
|
|
13527440 |
Jun 19, 2012 |
9169486 |
|
|
14869872 |
|
|
|
|
61502264 |
Jun 28, 2011 |
|
|
|
61500130 |
Jun 22, 2011 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 5/026 20130101;
C12N 15/52 20130101; C12N 15/70 20130101; C12P 7/16 20130101; C12P
7/04 20130101 |
International
Class: |
C12P 7/04 20060101
C12P007/04; C12N 15/70 20060101 C12N015/70; C12N 15/52 20060101
C12N015/52; C12P 5/02 20060101 C12P005/02 |
Claims
1. A non-naturally occurring microbial organism, comprising a
microbial organism having a butadiene pathway comprising at least
one exogenous nucleic acid encoding a butadiene pathway enzyme
expressed in a sufficient amount to produce butadiene; said
non-naturally occurring microbial organism further comprising: (a)
a reductive TCA pathway comprising at least one exogenous nucleic
acid encoding a reductive TCA pathway enzyme, wherein said at least
one exogenous nucleic acid is selected from an ATP-citrate lyase, a
citrate lyase, a citryl-CoA synthetase, a citryl-CoAlyase, a
fumarate reductase, and an alpha-ketoglutarate:ferredoxin
oxidoreductase; (b) a reductive TCA pathway comprising at least one
exogenous nucleic acid encoding a reductive TCA pathway enzyme,
wherein said at least one exogenous nucleic acid is selected from a
pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate
carboxylase, a phosphoenolpyruvate carboxykinase, a CO
dehydrogenase, and an H.sub.2 hydrogenase; or (c) at least one
exogenous nucleic acid encodes an enzyme selected from a CO
dehydrogenase, an H.sub.2 hydrogenase, and combinations thereof,
wherein said butadiene pathway comprises a pathway selected from:
(i) an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA
reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA
reductase (aldehyde forming), a crotonaldehyde reductase (alcohol
forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase
and a butadiene synthase; (ii) an acetyl-CoA:acetyl-CoA
acyltransferase, an acetoacetyl-CoA reductase, a
3-hydroxybutyryl-CoA dehydratase, a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA
reductase (alcohol forming); (iii) an acetyl-CoA:acetyl-CoA
acyltransferase, an acetoacetyl-CoA reductase, a
3-hydroxybutyryl-CoA dehydratase, a butadiene synthase, a
crotonyl-CoA reductase (alcohol forming) and a crotyl alcohol
diphosphokinase; (iv) an acetyl-CoA:acetyl-CoA acyltransferase, an
acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a
crotonaldehyde reductase (alcohol forming), a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a
crotonyl-CoA hydrolase, synthetase or transferase and a crotonate
reductase; (v) an acetyl-CoA:acetyl-CoA acyltransferase, an
acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a
crotonaldehyde reductase (alcohol forming), a butadiene synthase, a
crotonyl-CoA hydrolase, synthetase or transferase, a crotonate
reductase and a crotyl alcohol diphosphokinase; (vi) an
acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA
reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA
reductase (aldehyde forming), a crotonaldehyde reductase (alcohol
forming), a butadiene synthase and a crotyl alcohol
diphosphokinase. (vii) a glutaconyl-CoA decarboxylase, a
crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase and a butadiene synthase. (viii) a
glutaconyl-CoA decarboxylase, a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA
reductase (alcohol forming); (ix) a glutaconyl-CoA decarboxylase, a
butadiene synthase, a crotonyl-CoA reductase (alcohol forming) and
a crotyl alcohol diphosphokinase; (x) a glutaconyl-CoA
decarboxylase, a crotonaldehyde reductase (alcohol forming), a
crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene
synthase, a crotonyl-CoA hydrolase, synthetase, or transferase and
a crotonate reductase; (xi) a glutaconyl-CoA decarboxylase, a
crotonaldehyde reductase (alcohol forming), a butadiene synthase, a
crotonyl-CoA hydrolase, synthetase or transferase, a crotonate
reductase and a crotyl alcohol diphosphokinase; (xii) a
3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
butadiene a glutaconyl-CoA decarboxylase and a crotyl alcohol
diphosphokinase; (xiii) a glutaryl-CoA dehydrogenase, a
crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase and a butadiene synthase; (xiv) a
glutaryl-CoA dehydrogenase, a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA
reductase (alcohol forming); (xv) a glutaryl-CoA dehydrogenase, a
butadiene synthase, a crotonyl-CoA reductase (alcohol forming) and
a crotyl alcohol diphosphokinase; (xvi) a glutaryl-CoA
dehydrogenase, a crotonaldehyde reductase (alcohol forming), a
crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene
synthase, a crotonyl-CoA hydrolase, synthetase, or transferase and
a crotonate reductase; (xvii) a glutaryl-CoA dehydrogenase, a
crotonaldehyde reductase (alcohol forming), a butadiene synthase, a
crotonyl-CoA hydrolase, synthetase or transferase, a crotonate
reductase and a crotyl alcohol diphosphokinase; (xviii) a
3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
butadiene synthase, a glutaryl-CoA dehydrogenase and a crotyl
alcohol diphosphokinase; (xix) an 3-aminobutyryl-CoA deaminase, a
crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase and a butadiene synthase; (xx) an
3-aminobutyryl-CoA deaminase, a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA
reductase (alcohol forming); (xxi) an 3-aminobutyryl-CoA deaminase,
a butadiene synthase, a crotonyl-CoA reductase (alcohol forming)
and a crotyl alcohol diphosphokinase; (xxii) an 3-aminobutyryl-CoA
deaminase, a crotonaldehyde reductase (alcohol forming), a crotyl
alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene
synthase, a crotonyl-CoA hydrolase, synthetase or transferase and a
crotonate reductase; (xxiii) an 3-aminobutyryl-CoA deaminase, a
crotonaldehyde reductase (alcohol forming), a butadiene synthase, a
crotonyl-CoA hydrolase, synthetase or transferase, a crotonate
reductase and a crotyl alcohol diphosphokinase; (xxiv) a
3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
butadiene synthase, a 3-aminobutyryl-CoA deaminase and a crotyl
alcohol diphosphokinase; (xxv) a 4-hydroxybutyryl-CoA dehydratase,
a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase and a butadiene synthase; (xxvi) a
4-hydroxybutyryl-CoA dehydratase, a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA
reductase (alcohol forming); (xxvii) a 4-hydroxybutyryl-CoA
dehydratase, a butadiene synthase, a crotonyl-CoA reductase
(alcohol forming) and a crotyl alcohol diphosphokinase; (xxviii) a
4-hydroxybutyryl-CoA dehydratase, a crotonaldehyde reductase
(alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate
kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase
or transferase and a crotonate reductase; (xxix) a
4-hydroxybutyryl-CoA dehydratase, a crotonaldehyde reductase
(alcohol forming), a butadiene synthase, a crotonyl-CoA hydrolase,
synthetase or transferase, a crotonate reductase and a crotyl
alcohol diphosphokinase; (xxx) a 3-hydroxybutyryl-CoA dehydratase,
a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a butadiene synthase, a
4-hydroxybutyryl-CoA dehydratase and a crotyl alcohol
diphosphokinase; (xxxi) an erythrose-4-phosphate reductase, an
erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate reductase and a butadiene
synthase; (xxxii) an erythrose-4-phosphate reductase, an
erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate reductase, a butenyl
4-diphosphate isomerase and a butadiene synthase; (xxxiii) an
erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate reductase, a butadiene synthase,
an erythrose-4-phosphate kinase, an erythrose reductase and a
erythritol kinase; (xxxiv) an erythritol-4-phospate
cytidylyltransferase, a 4-(cytidine 5'-diphospho)-erythritol
kinase, an erythritol 2,4-cyclodiphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate synthase, a 1-hydroxy-2-butenyl
4-diphosphate reductase, a butenyl 4-diphosphate isomerase, a
butadiene synthase, an erythrose-4-phosphate kinase, an erythrose
reductase and an erythritol kinase; (xxxv) a malonyl-CoA:acetyl-CoA
acyltransferase, an 3-oxoglutaryl-CoA reductase (ketone-reducing),
a 3-hydroxyglutaryl-CoA reductase (aldehyde forming), a
3-hydroxy-5-oxopentanoate reductase, a 3,5-dihydroxypentanoate
kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase and a butadiene
synthase; (xxxvi) a malonyl-CoA:acetyl-CoA acyltransferase, a
3,5-dihydroxypentanoate kinase, a
3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, an 3-oxoglutaryl-CoA reductase (aldehyde forming), a
3,5-dioxopentanoate reductase (aldehyde reducing) and a
5-hydroxy-3-oxopentanoate reductase; (xxxvii) a
malonyl-CoA:acetyl-CoA acyltransferase, a 3-hydroxy-5-oxopentanoate
reductase, a 3,5-dihydroxypentanoate kinase, a
3-Hydroxy-5-phosphonatooxypentanoate kinase, a
3-Hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, an 3-oxoglutaryl-CoA reductase (aldehyde forming) and a
3,5-dioxopentanoate reductase (ketone reducing); (xxxviii) a
malonyl-CoA:acetyl-CoA acyltransferase, a 3,5-dihydroxypentanoate
kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, a 5-hydroxy-3-oxopentanoate reductase and a
3-oxo-glutaryl-CoA reductase (CoA reducing and alcohol forming);
and (xxxix) a butadiene pathway comprising a malonyl-CoA:acetyl-CoA
acyltransferase, an 3-oxoglutaryl-CoA reductase (ketone-reducing),
a 3,5-dihydroxypentanoate kinase, a
3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase and a 3-hydroxyglutaryl-CoA reductase (alcohol
forming).
2. The non-naturally occurring microbial organism of claim 1,
wherein said microbial organism comprising (a) further comprises an
exogenous nucleic acid encoding an enzyme selected from a
pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate
dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA
transferase, a fumarase, a malate dehydrogenase, an acetate kinase,
a phosphotransacetylase, an acetyl-CoA synthetase, an
NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations
thereof.
3. The non-naturally occurring microbial organism of claim 1,
wherein said microbial organism comprising (b) further comprises an
exogenous nucleic acid encoding an enzyme selected from an
aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase,
a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and
combinations thereof.
4. The non-naturally occurring microbial organism of claim 1,
wherein said microbial organism comprises two, three, four, five,
six or seven exogenous nucleic acids each encoding a butadiene
pathway enzyme.
5. The non-naturally occurring microbial organism of claim 4,
wherein said microbial organism comprises exogenous nucleic acids
encoding each of the enzymes selected from: (i) an
acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA
reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA
reductase (aldehyde forming), a crotonaldehyde reductase (alcohol
forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase
and a butadiene synthase; (ii) an acetyl-CoA:acetyl-CoA
acyltransferase, an acetoacetyl-CoA reductase, a
3-hydroxybutyryl-CoA dehydratase, a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA
reductase (alcohol forming); (iii) an acetyl-CoA:acetyl-CoA
acyltransferase, an acetoacetyl-CoA reductase, a
3-hydroxybutyryl-CoA dehydratase, a butadiene synthase, a
crotonyl-CoA reductase (alcohol forming) and a crotyl alcohol
diphosphokinase; (iv) an acetyl-CoA:acetyl-CoA acyltransferase, an
acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a
crotonaldehyde reductase (alcohol forming), a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a
crotonyl-CoA hydrolase, synthetase or transferase and a crotonate
reductase; (v) an acetyl-CoA:acetyl-CoA acyltransferase, an
acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a
crotonaldehyde reductase (alcohol forming), a butadiene synthase, a
crotonyl-CoA hydrolase, synthetase or transferase, a crotonate
reductase and a crotyl alcohol diphosphokinase; (vi) an
acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA
reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA
reductase (aldehyde forming), a crotonaldehyde reductase (alcohol
forming), a butadiene synthase and a crotyl alcohol
diphosphokinase. (vii) a glutaconyl-CoA decarboxylase, a
crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase and a butadiene synthase. (viii) a
glutaconyl-CoA decarboxylase, a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA
reductase (alcohol forming); (ix) a glutaconyl-CoA decarboxylase, a
butadiene synthase, a crotonyl-CoA reductase (alcohol forming) and
a crotyl alcohol diphosphokinase; (x) a glutaconyl-CoA
decarboxylase, a crotonaldehyde reductase (alcohol forming), a
crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene
synthase, a crotonyl-CoA hydrolase, synthetase, or transferase and
a crotonate reductase; (xi) a glutaconyl-CoA decarboxylase, a
crotonaldehyde reductase (alcohol forming), a butadiene synthase, a
crotonyl-CoA hydrolase, synthetase or transferase, a crotonate
reductase and a crotyl alcohol diphosphokinase; (xii) a
3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
butadiene a glutaconyl-CoA decarboxylase and a crotyl alcohol
diphosphokinase; (xiii) a glutaryl-CoA dehydrogenase, a
crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase and a butadiene synthase; (xiv) a
glutaryl-CoA dehydrogenase, a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA
reductase (alcohol forming); (xv) a glutaryl-CoA dehydrogenase, a
butadiene synthase, a crotonyl-CoA reductase (alcohol forming) and
a crotyl alcohol diphosphokinase; (xvi) a glutaryl-CoA
dehydrogenase, a crotonaldehyde reductase (alcohol forming), a
crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene
synthase, a crotonyl-CoA hydrolase, synthetase, or transferase and
a crotonate reductase; (xvii) a glutaryl-CoA dehydrogenase, a
crotonaldehyde reductase (alcohol forming), a butadiene synthase, a
crotonyl-CoA hydrolase, synthetase or transferase, a crotonate
reductase and a crotyl alcohol diphosphokinase; (xviii) a
3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
butadiene synthase, a glutaryl-CoA dehydrogenase and a crotyl
alcohol diphosphokinase; (xix) an 3-aminobutyryl-CoA deaminase, a
crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase and a butadiene synthase; (xx) an
3-aminobutyryl-CoA deaminase, a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA
reductase (alcohol forming); (xxi) an 3-aminobutyryl-CoA deaminase,
a butadiene synthase, a crotonyl-CoA reductase (alcohol forming)
and a crotyl alcohol diphosphokinase; (xxii) an 3-aminobutyryl-CoA
deaminase, a crotonaldehyde reductase (alcohol forming), a crotyl
alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene
synthase, a crotonyl-CoA hydrolase, synthetase or transferase and a
crotonate reductase; (xxiii) an 3-aminobutyryl-CoA deaminase, a
crotonaldehyde reductase (alcohol forming), a butadiene synthase, a
crotonyl-CoA hydrolase, synthetase or transferase, a crotonate
reductase and a crotyl alcohol diphosphokinase; (xxiv) a
3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
butadiene synthase, a 3-aminobutyryl-CoA deaminase and a crotyl
alcohol diphosphokinase; (xxv) a 4-hydroxybutyryl-CoA dehydratase,
a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase and a butadiene synthase; (xxvi) a
4-hydroxybutyryl-CoA dehydratase, a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA
reductase (alcohol forming); (xxvii) a 4-hydroxybutyryl-CoA
dehydratase, a butadiene synthase, a crotonyl-CoA reductase
(alcohol forming) and a crotyl alcohol diphosphokinase; (xxviii) a
4-hydroxybutyryl-CoA dehydratase, a crotonaldehyde reductase
(alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate
kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase
or transferase and a crotonate reductase; (xxix) a
4-hydroxybutyryl-CoA dehydratase, a crotonaldehyde reductase
(alcohol forming), a butadiene synthase, a crotonyl-CoA hydrolase,
synthetase or transferase, a crotonate reductase and a crotyl
alcohol diphosphokinase; (xxx) a 3-hydroxybutyryl-CoA dehydratase,
a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a butadiene synthase, a
4-hydroxybutyryl-CoA dehydratase and a crotyl alcohol
diphosphokinase; (xxxi) an erythrose-4-phosphate reductase, an
erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate reductase and a butadiene
synthase; (xxxii) an erythrose-4-phosphate reductase, an
erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate reductase, a butenyl
4-diphosphate isomerase and a butadiene synthase; (xxxiii) an
erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate reductase, a butadiene synthase,
an erythrose-4-phosphate kinase, an erythrose reductase and a
erythritol kinase; (xxxiv) an erythritol-4-phospate
cytidylyltransferase, a 4-(cytidine 5'-diphospho)-erythritol
kinase, an erythritol 2,4-cyclodiphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate synthase, a 1-hydroxy-2-butenyl
4-diphosphate reductase, a butenyl 4-diphosphate isomerase, a
butadiene synthase, an erythrose-4-phosphate kinase, an erythrose
reductase and an erythritol kinase; (xxxv) a malonyl-CoA:acetyl-CoA
acyltransferase, an 3-oxoglutaryl-CoA reductase (ketone-reducing),
a 3-hydroxyglutaryl-CoA reductase (aldehyde forming), a
3-hydroxy-5-oxopentanoate reductase, a 3,5-dihydroxypentanoate
kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase and a butadiene
synthase; (xxxvi) a malonyl-CoA:acetyl-CoA acyltransferase, a
3,5-dihydroxypentanoate kinase, a
3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, an 3-oxoglutaryl-CoA reductase (aldehyde forming), a
3,5-dioxopentanoate reductase (aldehyde reducing) and a
5-hydroxy-3-oxopentanoate reductase; (xxxvii) a
malonyl-CoA:acetyl-CoA acyltransferase, a 3-hydroxy-5-oxopentanoate
reductase, a 3,5-dihydroxypentanoate kinase, a
3-Hydroxy-5-phosphonatooxypentanoate kinase, a
3-Hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, an 3-oxoglutaryl-CoA reductase (aldehyde forming) and a
3,5-dioxopentanoate reductase (ketone reducing); (xxxviii) a
malonyl-CoA:acetyl-CoA acyltransferase, a 3,5-dihydroxypentanoate
kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, a 5-hydroxy-3-oxopentanoate reductase and a
3-oxo-glutaryl-CoA reductase (CoA reducing and alcohol forming);
and (xxxix) a butadiene pathway comprising a malonyl-CoA:acetyl-CoA
acyltransferase, an 3-oxoglutaryl-CoA reductase (ketone-reducing),
a 3,5-dihydroxypentanoate kinase, a
3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase and a 3-hydroxyglutaryl-CoA reductase (alcohol
forming).
6. The non-naturally occurring microbial organism of claim 1,
wherein said microbial organism comprises two, three, four or five
exogenous nucleic acids each encoding enzymes of (a), (b) or
(c).
7. The non-naturally occurring microbial organism of claim 6,
wherein said microbial organism comprising (a) comprises three
exogenous nucleic acids encoding ATP-citrate lyase or citrate
lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin
oxidoreductase; wherein said microbial organism comprising (b)
comprises four exogenous nucleic acids encoding a
pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate
carboxylase or a phosphoenolpyruvate carboxykinase, a CO
dehydrogenase, and an H2 hydrogenase; or wherein said microbial
organism comprising (c) comprises two exogenous nucleic acids
encoding CO dehydrogenase and H2 hydrogenase.
8. The non-naturally occurring microbial organism of claim 1,
wherein said at least one exogenous nucleic acid is a heterologous
nucleic acid.
9. The non-naturally occurring microbial organism of claim 1,
wherein said non-naturally occurring microbial organism is in a
substantially anaerobic culture medium.
10. A method for producing butadiene, comprising culturing the
non-naturally occurring microbial organism of claim 1 under
conditions and for a sufficient period of time to produce
butadiene.
11. A non-naturally occurring microbial organism, comprising a
microbial organism having a crotyl alcohol pathway comprising at
least one exogenous nucleic acid encoding a crotyl alcohol pathway
enzyme expressed in a sufficient amount to produce crotyl alcohol;
said non-naturally occurring microbial organism further comprising:
(a) a reductive TCA pathway comprising at least one exogenous
nucleic acid encoding a reductive TCA pathway enzyme, wherein said
at least one exogenous nucleic acid is selected from an ATP-citrate
lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA
lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin
oxidoreductase; (b) a reductive TCA pathway comprising at least one
exogenous nucleic acid encoding a reductive TCA pathway enzyme,
wherein said at least one exogenous nucleic acid is selected from a
pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate
carboxylase, a phosphoenolpyruvate carboxykinase, a CO
dehydrogenase, and an H.sub.2 hydrogenase; or (c) at least one
exogenous nucleic acid encodes an enzyme selected from a CO
dehydrogenase, an H.sub.2 hydrogenase, and combinations thereof;
wherein said crotyl alcohol pathway comprises a pathway selected
from: (i) an acetyl-CoA:acetyl-CoA acyltransferase; an
acetoacetyl-CoA reductase; a 3-hydroxybutyryl-CoA dehydratase; a
crotonyl-CoA hydrolase, synthase, or transferase; a crotonate
reductase; and a crotonaldehyde reductase (alcohol forming); (ii)
an acetyl-CoA:acetyl-CoA acyltransferase; an acetoacetyl-CoA
reductase; a 3-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA
reductase (aldehyde forming); and a crotonaldehyde reductase
(alcohol forming); (iii) an acetyl-CoA:acetyl-CoA acyltransferase;
an acetoacetyl-CoA reductase; a 3-hydroxybutyryl-CoA dehydratase;
and a crotonyl-CoA reductase (alcohol forming); (iv) a
glutaconyl-CoA decarboxylase; a crotonyl-CoA hydrolase, synthase,
or transferase; a crotonate reductase; and a crotonaldehyde
reductase (alcohol forming); (v) a glutaconyl-CoA decarboxylase; a
crotonyl-CoA reductase (aldehyde forming); and a crotonaldehyde
reductase (alcohol forming); and (vi) a glutaconyl-CoA
decarboxylase; and a crotonyl-CoA reductase (alcohol forming).
(vii) a glutaryl-CoA dehydrogenase; a crotonyl-CoA hydrolase,
synthase, or transferase; a crotonate reductase; and a
crotonaldehyde reductase (alcohol forming); (viii) a glutaryl-CoA
dehydrogenase; a crotonyl-CoA reductase (aldehyde forming); and a
crotonaldehyde reductase (alcohol forming); (ix) a glutaryl-CoA
dehydrogenase; and a crotonyl-CoA reductase (alcohol forming). (x)
a 3-aminobutyryl-CoA deaminase; a crotonyl-CoA hydrolase, synthase,
or transferase; a crotonate reductase; and a crotonaldehyde
reductase (alcohol forming); (xi) a 3-aminobutyryl-CoA deaminase; a
crotonyl-CoA reductase (aldehyde forming); and a crotonaldehyde
reductase (alcohol forming); (xii) a 3-aminobutyryl-CoA deaminase;
and a crotonyl-CoA reductase (alcohol forming). (xiii) a
4-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA hydrolase,
synthase, or transferase; a crotonate reductase; and a
crotonaldehyde reductase (alcohol forming); (xiv) a
4-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA reductase
(aldehyde forming); and a crotonaldehyde reductase (alcohol
forming); and (xv) a 4-hydroxybutyryl-CoA dehydratase; and a
crotonyl-CoA reductase (alcohol forming).
12. The non-naturally occurring microbial organism of claim 11,
wherein said microbial organism comprising (a) further comprises an
exogenous nucleic acid encoding an enzyme selected from a
pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate
dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA
transferase, a fumarase, a malate dehydrogenase, an acetate kinase,
a phosphotransacetylase, an acetyl-CoA synthetase, an
NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations
thereof.
13. The non-naturally occurring microbial organism of claim 11,
wherein said microbial organism comprising (b) further comprises an
exogenous nucleic acid encoding an enzyme selected from an
aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase,
a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and
combinations thereof.
14. The non-naturally occurring microbial organism of claim 11,
wherein said microbial organism comprises two, three, four, five,
six or seven exogenous nucleic acids each encoding a crotyl alcohol
pathway enzyme.
15. The non-naturally occurring microbial organism of claim 14,
wherein said microbial organism comprises exogenous nucleic acids
encoding each of the enzymes selected from: (i) an
acetyl-CoA:acetyl-CoA acyltransferase; an acetoacetyl-CoA
reductase; a 3-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA
hydrolase, synthase, or transferase; a crotonate reductase; and a
crotonaldehyde reductase (alcohol forming); (ii) an
acetyl-CoA:acetyl-CoA acyltransferase; an acetoacetyl-CoA
reductase; a 3-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA
reductase (aldehyde forming); and a crotonaldehyde reductase
(alcohol forming); (iii) an acetyl-CoA:acetyl-CoA acyltransferase;
an acetoacetyl-CoA reductase; a 3-hydroxybutyryl-CoA dehydratase;
and a crotonyl-CoA reductase (alcohol forming); (iv) a
glutaconyl-CoA decarboxylase; a crotonyl-CoA hydrolase, synthase,
or transferase; a crotonate reductase; and a crotonaldehyde
reductase (alcohol forming); (v) a glutaconyl-CoA decarboxylase; a
crotonyl-CoA reductase (aldehyde forming); and a crotonaldehyde
reductase (alcohol forming); and (vi) a glutaconyl-CoA
decarboxylase; and a crotonyl-CoA reductase (alcohol forming).
(vii) a glutaryl-CoA dehydrogenase; a crotonyl-CoA hydrolase,
synthase, or transferase; a crotonate reductase; and a
crotonaldehyde reductase (alcohol forming); (viii) a glutaryl-CoA
dehydrogenase; a crotonyl-CoA reductase (aldehyde forming); and a
crotonaldehyde reductase (alcohol forming); (ix) a glutaryl-CoA
dehydrogenase; and a crotonyl-CoA reductase (alcohol forming). (x)
a 3-aminobutyryl-CoA deaminase; a crotonyl-CoA hydrolase, synthase,
or transferase; a crotonate reductase; and a crotonaldehyde
reductase (alcohol forming); (xi) a 3-aminobutyryl-CoA deaminase; a
crotonyl-CoA reductase (aldehyde forming); and a crotonaldehyde
reductase (alcohol forming); (xii) a 3-aminobutyryl-CoA deaminase;
and a crotonyl-CoA reductase (alcohol forming). (xiii) a
4-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA hydrolase,
synthase, or transferase; a crotonate reductase; and a
crotonaldehyde reductase (alcohol forming); (xiv) a
4-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA reductase
(aldehyde forming); and a crotonaldehyde reductase (alcohol
forming); and (xv) a 4-hydroxybutyryl-CoA dehydratase; and a
crotonyl-CoA reductase (alcohol forming).
16. The non-naturally occurring microbial organism of claim 11,
wherein said microbial organism comprises two, three, four or five
exogenous nucleic acids each encoding enzymes of (a), (b) or
(c).
17. The non-naturally occurring microbial organism of claim 16,
wherein said microbial organism comprising (a) comprises three
exogenous nucleic acids encoding ATP-citrate lyase or citrate
lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin
oxidoreductase; wherein said microbial organism comprising (b)
comprises four exogenous nucleic acids encoding a
pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate
carboxylase or a phosphoenolpyruvate carboxykinase, a CO
dehydrogenase, and an H.sub.2 hydrogenase; or wherein said
microbial organism comprising (c) comprises two exogenous nucleic
acids encoding a CO dehydrogenase and an H2 hydrogenase.
18. The non-naturally occurring microbial organism of claim 11,
wherein said at least one exogenous nucleic acid is a heterologous
nucleic acid.
19. The non-naturally occurring microbial organism of claim 11,
wherein said non-naturally occurring microbial organism is in a
substantially anaerobic culture medium.
20. A method for producing crotyl alcohol, comprising culturing the
non-naturally occurring microbial organism of claim 11 under
conditions and for a sufficient period of time to produce crotyl
alcohol.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/869,872, filed Sep. 29, 2015, which is a divisional of
U.S. patent application Ser. No. 13/527,440, filed Jun. 19, 2012,
now U.S. Pat. No. 9,169,486, issued on Oct. 27, 2015, which claims
the benefit of priority of U.S. Provisional Patent Application No.
61/500,130, filed Jun. 22, 2011, and U.S. Provisional Patent
Application No. 61/502,264, filed Jun. 28, 2011, the entire
contents of each of which are incorporated herein by reference.
[0002] The instant application contains a Sequence Listing which
has been submitted via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Jun. 8, 2018
is named 12956-450-999-SEQ.txt and is 77,874 bytes in size.
BACKGROUND OF THE INVENTION
[0003] The present invention relates generally to biosynthetic
processes, and more specifically to organisms having butadiene or
crotyl alcohol biosynthetic capability.
[0004] Over 25 billion pounds of butadiene (1,3-butadiene, BD) are
produced annually and is applied in the manufacture of polymers
such as synthetic rubbers and ABS resins, and chemicals such as
hexamethylenediamine and 1,4-butanediol. Butadiene is typically
produced as a by-product of the steam cracking process for
conversion of petroleum feedstocks such as naphtha, liquefied
petroleum gas, ethane or natural gas to ethylene and other olefins.
The ability to manufacture butadiene from alternative and/or
renewable feedstocks would represent a major advance in the quest
for more sustainable chemical production processes
[0005] One possible way to produce butadiene renewably involves
fermentation of sugars or other feedstocks to produce diols, such
as 1,4-butanediol or 1,3-butanediol, which are separated, purified,
and then dehydrated to butadiene in a second step involving
metal-based catalysis. Direct fermentative production of butadiene
from renewable feedstocks would obviate the need for dehydration
steps and butadiene gas (bp -4.4.degree. C.) would be continuously
emitted from the fermenter and readily condensed and collected.
Developing a fermentative production process would eliminate the
need for fossil-based butadiene and would allow substantial savings
in cost, energy, and harmful waste and emissions relative to
petrochemically-derived butadiene.
[0006] Microbial organisms and methods for effectively producing
butadiene or crotyl alcohol from cheap renewable feedstocks such as
molasses, sugar cane juice, and sugars derived from biomass
sources, including agricultural and wood waste, as well as C1
feedstocks such as syngas and carbon dioxide, are described herein
and include related advantages.
SUMMARY OF THE INVENTION
[0007] The invention provides non-naturally occurring microbial
organisms containing butadiene or crotyl alcohol pathways
comprising at least one exogenous nucleic acid encoding a butadiene
or crotyl alcohol pathway enzyme expressed in a sufficient amount
to produce butadiene or crotyl alcohol. The invention additionally
provides methods of using such microbial organisms to produce
butadiene or crotyl alcohol, by culturing a non-naturally occurring
microbial organism containing butadiene or crotyl alcohol pathways
as described herein under conditions and for a sufficient period of
time to produce butadiene or crotyl alcohol.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 shows a natural pathway to isoprenoids and terpenes.
Enzymes for transformation of the identified substrates to products
include: A. acetyl-CoA:acetyl-CoA acyltransferase, B.
hydroxymethylglutaryl-CoA synthase, C.
3-hydroxy-3-methylglutaryl-CoA reductase (alcohol forming), D.
mevalonate kinase, E. phosphomevalonate kinase, F.
diphosphomevalonate decarboxylase, G. isopentenyl-diphosphate
isomerase, H. isoprene synthase.
[0009] FIG. 2 shows exemplary pathways for production of butadiene
from acetyl-CoA, glutaconyl-CoA, glutaryl-CoA, 3-aminobutyryl-CoA
or 4-hydroxybutyryl-CoA via crotyl alcohol. Enzymes for
transformation of the identified substrates to products include: A.
acetyl-CoA:acetyl-CoA acyltransferase, B. acetoacetyl-CoA
reductase, C. 3-hydroxybutyryl-CoA dehydratase, D. crotonyl-CoA
reductase (aldehyde forming), E. crotonaldehyde reductase (alcohol
forming), F. crotyl alcohol kinase, G. 2-butenyl-4-phosphate
kinase, H. butadiene synthase, I. crotonyl-CoA hydrolase,
synthetase, transferase, J. crotonate reductase, K. crotonyl-CoA
reductase (alcohol forming), L. glutaconyl-CoA decarboxylase, M.,
glutaryl-CoA dehydrogenase, N. 3-aminobutyryl-CoA deaminase, O.
4-hydroxybutyryl-CoA dehydratase, P. crotyl alcohol
diphosphokinase.
[0010] FIG. 3 shows exemplary pathways for production of butadiene
from erythrose-4-phosphate. Enzymes for transformation of the
identified substrates to products include: A. Erythrose-4-phosphate
reductase, B. Erythritol-4-phospate cytidylyltransferase, C.
4-(cytidine 5'-diphospho)-erythritol kinase, D. Erythritol
2,4-cyclodiphosphate synthase, E. 1-Hydroxy-2-butenyl 4-diphosphate
synthase, F. 1-Hydroxy-2-butenyl 4-diphosphate reductase, G.
Butenyl 4-diphosphate isomerase, H. Butadiene synthase I.
Erythrose-4-phosphate kinase, J. Erythrose reductase, K. Erythritol
kinase.
[0011] FIG. 4 shows an exemplary pathway for production of
butadiene from malonyl-CoA plus acetyl-CoA. Enzymes for
transformation of the identified substrates to products include: A.
malonyl-CoA:acetyl-CoA acyltransferase, B. 3-oxoglutaryl-CoA
reductase (ketone-reducing), C. 3-hydroxyglutaryl-CoA reductase
(aldehyde forming), D. 3-hydroxy-5-oxopentanoate reductase, E.
3,5-dihydroxypentanoate kinase, F. 3H5PP kinase, G. 3H5PDP
decarboxylase, H. butenyl 4-diphosphate isomerase, I. butadiene
synthase, J. 3-hydroxyglutaryl-CoA reductase (alcohol forming), K.
3-oxoglutaryl-CoA reductase (aldehyde forming), L.
3,5-dioxopentanoate reductase (ketone reducing), M.
3,5-dioxopentanoate reductase (aldehyde reducing), N.
5-hydroxy-3-oxopentanoate reductase, O. 3-oxo-glutaryl-CoA
reductase (CoA reducing and alcohol forming). Compound
abbreviations include: 3H5PP=3-Hydroxy-5-phosphonatooxypentanoate
and 3H5PDP=3-Hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy
pentanoate.
[0012] FIG. 5 shows an exemplary pathway for production of crotyl
alcohol from acetyl-CoA. Enzymes for transformation of the
identified substrates to products include: A. acetyl-CoA:acetyl-CoA
acyltransferase, B. acetoacetyl-CoA reductase, C.
3-hydroxybutyryl-CoA dehydratase, D. crotonyl-CoA reductase
(aldehyde forming), E. crotonaldehyde reductase (alcohol forming),
F. crotonyl-CoA reductase (alcohol forming), G. crotonyl-CoA
hydrolase, synthetase, transferase, and H. crotonate reductase.
[0013] FIG. 6 shows the reverse TCA cycle for fixation of CO.sub.2
on carbohydrates as substrates. The enzymatic transformations are
carried out by the enzymes as shown.
[0014] FIG. 7 shows the pathway for the reverse TCA cycle coupled
with carbon monoxide dehydrogenase and hydrogenase for the
conversion of syngas to acetyl-CoA.
[0015] FIG. 8 shows Western blots of 10 micrograms ACS90 (lane 1),
ACS91 (lane2), Mta98/99 (lanes 3 and 4) cell extracts with size
standards (lane 5) and controls of M. thermoacetica CODH
(Moth_1202/1203) or Mtr (Moth_1197) proteins (50, 150, 250, 350,
450, 500, 750, 900, and 1000 ng).
[0016] FIG. 9 shows CO oxidation assay results. Cells (M.
thermoacetica or E. coli with the CODH/ACS operon; ACS90 or ACS91
or empty vector: pZA33S) were grown and extracts prepared. Assays
were performed at 55.degree. C. at various times on the day the
extracts were prepared. Reduction of methylviologen was followed at
578 nm over a 120 sec time course.
[0017] FIGS. 10A and 10B show exemplary pathways to crotyl alcohol.
FIG. 10A shows the pathways for fixation of CO.sub.2 to acetyl-CoA
using the reductive TCA cycle. FIG. 10B shows exemplary pathways
for the biosynthesis of crotyl alcohol from acetyl-CoA; the
enzymatic transformations shown are carried out by the following
enzymes: A. acetyl-CoA:acetyl-CoA acyltransferase, B.
acetoacetyl-CoA reductase, C. 3-hydroxybutyryl-CoA dehydratase, D.
crotonyl-CoA reductase (aldehyde forming), E. crotonaldehyde
reductase (alcohol forming), F. crotonyl-CoA reductase (alcohol
forming), G. crotonyl-CoA hydrolase, synthetase, transferase, and
H. crotonate reductase.
[0018] FIGS. 11A and 11B show exemplary pathways to butadiene. FIG.
11A shows the pathways for fixation of CO.sub.2 to acetyl-CoA using
the reductive TCA cycle. FIG. 11B shows exemplary pathways for the
biosynthesis of butadiene from acetyl-CoA; the enzymatic
transformations shown are carried out by the following enzymes: A.
acetyl-CoA:acetyl-CoA acyltransferase, B. acetoacetyl-CoA
reductase, C. 3-hydroxybutyryl-CoA dehydratase, D. crotonyl-CoA
reductase (aldehyde forming), E. crotonaldehyde reductase (alcohol
forming), F. crotonyl-CoA reductase (alcohol forming), G.
crotonyl-CoA hydrolase, synthetase, transferase, H. crotonate
reductase, I. crotyl alcohol kinase, J. 2-butenyl-4-phosphate
kinase, K. butadiene synthase, L. crotyl alcohol
diphosphokinase.
[0019] FIG. 12A shows the nucleotide sequence (SEQ ID NO: 1) of
carboxylic acid reductase from Nocardia iowensis (GNM_720), and
FIG. 12B shows the encoded amino acid sequence (SEQ ID NO: 2).
[0020] FIG. 13A shows the nucleotide sequence (SEQ ID NO: 3) of
phosphpantetheine transferase, which was codon optimized, and FIG.
13B shows the encoded amino acid sequence (SEQ ID NO: 4).
[0021] FIG. 14A shows the nucleotide sequence (SEQ ID NO: 5) of
carboxylic acid reductase from Mycobacterium smegmatis mc(2)155
(designated 890), and FIG. 14B shows the encoded amino acid
sequence (SEQ ID NO: 6).
[0022] FIG. 15A shows the nucleotide sequence (SEQ ID NO: 7) of
carboxylic acid reductase from Mycobacterium avium subspecies
paratuberculosis K-10 (designated 891), and FIG. 15B shows the
encoded amino acid sequence (SEQ ID NO: 8).
[0023] FIG. 16A shows the nucleotide sequence (SEQ ID NO: 9) of
carboxylic acid reductase from Mycobacterium marinum M (designated
892), and FIG. 16B shows the encoded amino acid sequence (SEQ ID
NO: 10).
[0024] FIG. 17A shows the nucleotide sequence (SEQ ID NO: 11) of
carboxylic acid reductase designated 891GA, and FIG. 17B shows the
encoded amino acid sequence (SEQ ID NO: 12).
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention is directed to the design and
production of cells and organisms having biosynthetic production
capabilities for butadiene or crotyl alcohol. The invention, in
particular, relates to the design of microbial organism capable of
producing butadiene or crotyl alcohol by introducing one or more
nucleic acids encoding a butadiene or a crotyl alcohol pathway
enzyme.
[0026] In one embodiment, the invention utilizes in silico
stoichiometric models of Escherichia coli metabolism that identify
metabolic designs for biosynthetic production of butadiene or
crotyl alcohol. The results described herein indicate that
metabolic pathways can be designed and recombinantly engineered to
achieve the biosynthesis of butadiene or crotyl alcohol in
Escherichia coli and other cells or organisms. Biosynthetic
production of butadiene or crotyl alcohol, for example, for the in
silico designs can be confirmed by construction of strains having
the designed metabolic genotype. These metabolically engineered
cells or organisms also can be subjected to adaptive evolution to
further augment butadiene or crotyl alcohol biosynthesis, including
under conditions approaching theoretical maximum growth.
[0027] In certain embodiments, the butadiene biosynthesis
characteristics of the designed strains make them genetically
stable and particularly useful in continuous bioprocesses. Separate
strain design strategies were identified with incorporation of
different non-native or heterologous reaction capabilities into E.
coli or other host organisms leading to butadiene producing
metabolic pathways from acetyl-CoA, glutaconyl-CoA, glutaryl-CoA,
3-aminobutyryl-CoA, 4-hydroxybutyryl-CoA, erythrose-4-phosphate or
malonyl-CoA plus acetyl-CoA. In silico metabolic designs were
identified that resulted in the biosynthesis of butadiene in
microorganisms from each of these substrates or metabolic
intermediates.
[0028] Strains identified via the computational component of the
platform can be put into actual production by genetically
engineering any of the predicted metabolic alterations, which lead
to the biosynthetic production of butadiene or other intermediate
and/or downstream products. In yet a further embodiment, strains
exhibiting biosynthetic production of these compounds can be
further subjected to adaptive evolution to further augment product
biosynthesis. The levels of product biosynthesis yield following
adaptive evolution also can be predicted by the computational
component of the system.
[0029] The maximum theoretical butadiene yield from glucose is 1.09
mol/mol (0.33 g/g).
11C.sub.6H.sub.12O.sub.6=12C.sub.4H.sub.6+18CO.sub.2+30H.sub.2O
[0030] The pathways presented in FIGS. 2 and 4 achieve a yield of
1.0 moles butadiene per mole of glucose utilized. Increasing
product yields to theoretical maximum value is possible if cells
are capable of fixing CO.sub.2 through pathways such as the
reductive (or reverse) TCA cycle or the Wood-Ljungdahl pathway.
Organisms engineered to possess the pathway depicted in FIG. 3 are
also capable of reaching near theoretical maximum yields of
butadiene.
[0031] As used herein, the term "non-naturally occurring" when used
in reference to a microbial organism or microorganism of the
invention is intended to mean that the microbial organism has at
least one genetic alteration not normally found in a naturally
occurring strain of the referenced species, including wild-type
strains of the referenced species. Genetic alterations include, for
example, modifications introducing expressible nucleic acids
encoding metabolic polypeptides, other nucleic acid additions,
nucleic acid deletions and/or other functional disruption of the
microbial organism's genetic material. Such modifications include,
for example, coding regions and functional fragments thereof, for
heterologous, homologous or both heterologous and homologous
polypeptides for the referenced species. Additional modifications
include, for example, non-coding regulatory regions in which the
modifications alter expression of a gene or operon. Exemplary
metabolic polypeptides include enzymes or proteins within a
butadiene or crotyl alcohol biosynthetic pathway.
[0032] 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.
[0033] As used herein, the term "butadiene," having the molecular
formula C.sub.4H6 and a molecular mass of 54.09 g/mol (see FIGS.
2-4) (IUPAC name Buta-1,3-diene) is used interchangeably throughout
with 1,3-butadiene, biethylene, erythrene, divinyl, vinylethylene.
Butadiene is a colorless, non corrosive liquefied gas with a mild
aromatic or gasoline-like odor. Butadiene is both explosive and
flammable because of its low flash point.
[0034] As used herein, the term "isolated" when used in reference
to a microbial organism is intended to mean an organism that is
substantially free of at least one component as the referenced
microbial organism is found in nature. The term includes a
microbial organism that is removed from some or all components as
it is found in its natural environment. The term also includes a
microbial organism that is removed from some or all components as
the microbial organism is found in non-naturally occurring
environments. Therefore, an isolated microbial organism is partly
or completely separated from other substances as it is found in
nature or as it is grown, stored or subsisted in non-naturally
occurring environments. Specific examples of isolated microbial
organisms include partially pure microbes, substantially pure
microbes and microbes cultured in a medium that is non-naturally
occurring.
[0035] As used herein, the terms "microbial," "microbial organism"
or "microorganism" are intended to mean any organism that exists as
a microscopic cell that is included within the domains of archaea,
bacteria or eukarya. Therefore, the term is intended to encompass
prokaryotic or eukaryotic cells or organisms having a microscopic
size and includes bacteria, archaea and eubacteria of all species
as well as eukaryotic microorganisms such as yeast and fungi. The
term also includes cell cultures of any species that can be
cultured for the production of a biochemical.
[0036] 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.
[0037] 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.
[0038] "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.
[0039] It is understood that when more than one exogenous nucleic
acid is included in a microbial organism that the more than one
exogenous nucleic acids refers to the referenced encoding nucleic
acid or biosynthetic activity, as discussed above. It is further
understood, as disclosed herein, that such more than one exogenous
nucleic acids can be introduced into the host microbial organism on
separate nucleic acid molecules, on polycistronic nucleic acid
molecules, or a combination thereof, and still be considered as
more than one exogenous nucleic acid. For example, as disclosed
herein a microbial organism can be engineered to express two or
more exogenous nucleic acids encoding a desired pathway enzyme or
protein. In the case where two exogenous nucleic acids encoding a
desired activity are introduced into a host microbial organism, it
is understood that the two exogenous nucleic acids can be
introduced as a single nucleic acid, for example, on a single
plasmid, on separate plasmids, can be integrated into the host
chromosome at a single site or multiple sites, and still be
considered as two exogenous nucleic acids. Similarly, it is
understood that more than two exogenous nucleic acids can be
introduced into a host organism in any desired combination, for
example, on a single plasmid, on separate plasmids, can be
integrated into the host chromosome at a single site or multiple
sites, and still be considered as two or more exogenous nucleic
acids, for example three exogenous nucleic acids. Thus, the number
of referenced exogenous nucleic acids or biosynthetic activities
refers to the number of encoding nucleic acids or the number of
biosynthetic activities, not the number of separate nucleic acids
introduced into the host organism.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] Therefore, in identifying and constructing the non-naturally
occurring microbial organisms of the invention having butadiene or
crotyl alcohol 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.
[0047] 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.
[0048] 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.
[0049] In some embodiments, the invention provides a non-naturally
occurring microbial organism, including a microbial organism having
a butadiene pathway having at least one exogenous nucleic acid
encoding a butadiene pathway enzyme expressed in a sufficient
amount to produce butadiene, the butadiene pathway including an
acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA
reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA
reductase (aldehyde forming), a crotonaldehyde reductase (alcohol
forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase,
a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or
transferase, a crotonate reductase, a crotonyl-CoA reductase
(alcohol forming), a glutaconyl-CoA decarboxylase, a glutaryl-CoA
dehydrogenase, an 3-aminobutyryl-CoA deaminase, a
4-hydroxybutyryl-CoA dehydratase or a crotyl alcohol
diphosphokinase (FIG. 2). In one aspect, the non-naturally
occurring microbial organism includes a microbial organism having a
butadiene pathway having at least one exogenous nucleic acid
encoding butadiene pathway enzymes expressed in a sufficient amount
to produce butadiene, the butadiene pathway including an
acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA
reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA
reductase (aldehyde forming), a crotonaldehyde reductase (alcohol
forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase
and a butadiene synthase (FIG. 2, steps A-H). In one aspect, the
non-naturally occurring microbial organism includes a microbial
organism having a butadiene pathway having at least one exogenous
nucleic acid encoding butadiene pathway enzymes expressed in a
sufficient amount to produce butadiene, the butadiene pathway
including an acetyl-CoA:acetyl-CoA acyltransferase, an
acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a
crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene
synthase and crotonyl-CoA reductase (alcohol forming) (FIG. 2,
steps A-C, K, F, G, H). In one aspect, the non-naturally occurring
microbial organism includes a microbial organism having a butadiene
pathway having at least one exogenous nucleic acid encoding
butadiene pathway enzymes expressed in a sufficient amount to
produce butadiene, the butadiene pathway including an
acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA
reductase, a 3-hydroxybutyryl-CoA dehydratase, a butadiene
synthase, a crotonyl-CoA reductase (alcohol forming) and a crotyl
alcohol diphosphokinase (FIG. 2, steps A-C, K, P, H). In one
aspect, the non-naturally occurring microbial organism includes a
microbial organism having a butadiene pathway having at least one
exogenous nucleic acid encoding butadiene pathway enzymes expressed
in a sufficient amount to produce butadiene, the butadiene pathway
including an acetyl-CoA:acetyl-CoA acyltransferase, an
acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a
crotonaldehyde reductase (alcohol forming), a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a
crotonyl-CoA hydrolase, synthetase, or transferase and a crotonate
reductase, (FIG. 2, steps A-C, I, J, E, F, G, H). In one aspect,
the non-naturally occurring microbial organism includes a microbial
organism having a butadiene pathway having at least one exogenous
nucleic acid encoding butadiene pathway enzymes expressed in a
sufficient amount to produce butadiene, the butadiene pathway
including an acetyl-CoA:acetyl-CoA acyltransferase, an
acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a
crotonaldehyde reductase (alcohol forming), a butadiene synthase, a
crotonyl-CoA hydrolase, synthetase or transferase, a crotonate
reductase and a crotyl alcohol diphosphokinase (FIG. 2, steps A-C,
I, J, E, P, H). In one aspect, the non-naturally occurring
microbial organism includes a microbial organism having a butadiene
pathway having at least one exogenous nucleic acid encoding
butadiene pathway enzymes expressed in a sufficient amount to
produce butadiene, the butadiene pathway including an
acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA
reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA
reductase (aldehyde forming), a crotonaldehyde reductase (alcohol
forming), a butadiene synthase and a crotyl alcohol diphosphokinase
(FIG. 2, steps A-E, P, H). In one aspect, the non-naturally
occurring microbial organism includes a microbial organism having a
butadiene pathway having at least one exogenous nucleic acid
encoding butadiene pathway enzymes expressed in a sufficient amount
to produce butadiene, the butadiene pathway including a
glutaconyl-CoA decarboxylase, a crotonyl-CoA reductase (aldehyde
forming), a crotonaldehyde reductase (alcohol forming), a crotyl
alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene
synthase (FIG. 2, steps L, D-H). In one aspect, the non-naturally
occurring microbial organism includes a microbial organism having a
butadiene pathway having at least one exogenous nucleic acid
encoding butadiene pathway enzymes expressed in a sufficient amount
to produce butadiene, the butadiene pathway including a
glutaconyl-CoA decarboxylase, a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA
reductase (alcohol forming) (FIG. 2, steps L, K, F, G, H). In one
aspect, the non-naturally occurring microbial organism includes a
microbial organism having a butadiene pathway having at least one
exogenous nucleic acid encoding butadiene pathway enzymes expressed
in a sufficient amount to produce butadiene, the butadiene pathway
including a glutaconyl-CoA decarboxylase, a butadiene synthase, a
crotonyl-CoA reductase (alcohol forming) and a crotyl alcohol
diphosphokinase (FIG. 2, steps L, K, P, H). In one aspect, the
non-naturally occurring microbial organism includes a microbial
organism having a butadiene pathway having at least one exogenous
nucleic acid encoding butadiene pathway enzymes expressed in a
sufficient amount to produce butadiene, the butadiene pathway
including a glutaconyl-CoA decarboxylase, a crotonaldehyde
reductase (alcohol forming), a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase, a butadiene synthase, a crotonyl-CoA
hydrolase, synthetase, or transferase and a crotonate reductase
(FIG. 2, steps L, I, J, E, F, G, H). In one aspect, the
non-naturally occurring microbial organism includes a microbial
organism having a butadiene pathway having at least one exogenous
nucleic acid encoding butadiene pathway enzymes expressed in a
sufficient amount to produce butadiene, the butadiene pathway
including a glutaconyl-CoA decarboxylase, a crotonaldehyde
reductase (alcohol forming), a butadiene synthase, a crotonyl-CoA
hydrolase, synthetase or transferase, a crotonate reductase and a
crotyl alcohol diphosphokinase (FIG. 2, steps L, I, J, E, P, H). In
one aspect, the non-naturally occurring microbial organism includes
a microbial organism having a butadiene pathway having at least one
exogenous nucleic acid encoding butadiene pathway enzymes expressed
in a sufficient amount to produce butadiene, the butadiene pathway
including a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA
reductase (aldehyde forming), a crotonaldehyde reductase (alcohol
forming), a butadiene a glutaconyl-CoA decarboxylase and a crotyl
alcohol diphosphokinase (FIG. 2, steps L, C, D, E, P, H). In one
aspect, the non-naturally occurring microbial organism includes a
microbial organism having a butadiene pathway having at least one
exogenous nucleic acid encoding butadiene pathway enzymes expressed
in a sufficient amount to produce butadiene, the butadiene pathway
including a glutaryl-CoA dehydrogenase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a
butadiene synthase (FIG. 2, steps M, D-H). In one aspect, the
non-naturally occurring microbial organism includes a microbial
organism having a butadiene pathway having at least one exogenous
nucleic acid encoding butadiene pathway enzymes expressed in a
sufficient amount to produce butadiene, the butadiene pathway
including a glutaryl-CoA dehydrogenase, a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA
reductase (alcohol forming) (FIG. 2, steps M, K, F, G, H). In one
aspect, the non-naturally occurring microbial organism includes a
microbial organism having a butadiene pathway having at least one
exogenous nucleic acid encoding butadiene pathway enzymes expressed
in a sufficient amount to produce butadiene, the butadiene pathway
including a glutaryl-CoA dehydrogenase, a butadiene synthase, a
crotonyl-CoA reductase (alcohol forming) and a crotyl alcohol
diphosphokinase (FIG. 2, steps M, K, P, H). In one aspect, the
non-naturally occurring microbial organism includes a microbial
organism having a butadiene pathway having at least one exogenous
nucleic acid encoding butadiene pathway enzymes expressed in a
sufficient amount to produce butadiene, the butadiene pathway
including a glutaryl-CoA dehydrogenase, a crotonaldehyde reductase
(alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate
kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase,
or transferase and a crotonate reductase (FIG. 2, steps M, I, J, E,
F, G, H). In one aspect, the non-naturally occurring microbial
organism includes a microbial organism having a butadiene pathway
having at least one exogenous nucleic acid encoding butadiene
pathway enzymes expressed in a sufficient amount to produce
butadiene, the butadiene pathway including a glutaryl-CoA
dehydrogenase, a crotonaldehyde reductase (alcohol forming), a
butadiene synthase, a crotonyl-CoA hydrolase, synthetase or
transferase, a crotonate reductase and a crotyl alcohol
diphosphokinase (FIG. 2, steps M, I, J, E, P, H). In one aspect,
the non-naturally occurring microbial organism includes a microbial
organism having a butadiene pathway having at least one exogenous
nucleic acid encoding butadiene pathway enzymes expressed in a
sufficient amount to produce butadiene, the butadiene pathway
including a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA
reductase (aldehyde forming), a crotonaldehyde reductase (alcohol
forming), a butadiene synthase, a glutaryl-CoA dehydrogenase and a
crotyl alcohol diphosphokinase (FIG. 2, steps M, C, D, E, P, H). In
one aspect, the non-naturally occurring microbial organism includes
a microbial organism having a butadiene pathway having at least one
exogenous nucleic acid encoding butadiene pathway enzymes expressed
in a sufficient amount to produce butadiene, the butadiene pathway
including an 3-aminobutyryl-CoA deaminase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a
butadiene synthase (FIG. 2, steps N, D-H). In one aspect, the
non-naturally occurring microbial organism includes a microbial
organism having a butadiene pathway having at least one exogenous
nucleic acid encoding butadiene pathway enzymes expressed in a
sufficient amount to produce butadiene, the butadiene pathway
including an 3-aminobutyryl-CoA deaminase, a crotyl alcohol kinase,
a 2-butenyl-4-phosphate kinase, a butadiene synthase and
crotonyl-CoA reductase (alcohol forming) (FIG. 2, steps N, K, F, G,
H). In one aspect, the non-naturally occurring microbial organism
includes a microbial organism having a butadiene pathway having at
least one exogenous nucleic acid encoding butadiene pathway enzymes
expressed in a sufficient amount to produce butadiene, the
butadiene pathway including an 3-aminobutyryl-CoA deaminase, a
butadiene synthase, a crotonyl-CoA reductase (alcohol forming) and
a crotyl alcohol diphosphokinase (FIG. 2, steps N, K, P, H). In one
aspect, the non-naturally occurring microbial organism includes a
microbial organism having a butadiene pathway having at least one
exogenous nucleic acid encoding butadiene pathway enzymes expressed
in a sufficient amount to produce butadiene, the butadiene pathway
including an 3-aminobutyryl-CoA deaminase, a crotonaldehyde
reductase (alcohol forming), a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase, a butadiene synthase, a crotonyl-CoA
hydrolase, synthetase, or transferase and a crotonate reductase
(FIG. 2, steps N, I, J, E, F, G, H). In one aspect, the
non-naturally occurring microbial organism includes a microbial
organism having a butadiene pathway having at least one exogenous
nucleic acid encoding butadiene pathway enzymes expressed in a
sufficient amount to produce butadiene, the butadiene pathway
including an 3-aminobutyryl-CoA deaminase, a crotonaldehyde
reductase (alcohol forming), a butadiene synthase, a crotonyl-CoA
hydrolase, synthetase or transferase, a crotonate reductase and a
crotyl alcohol diphosphokinase (FIG. 2, steps N, I, J, E, P, H). In
one aspect, the non-naturally occurring microbial organism includes
a microbial organism having a butadiene pathway having at least one
exogenous nucleic acid encoding butadiene pathway enzymes expressed
in a sufficient amount to produce butadiene, the butadiene pathway
including a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA
reductase (aldehyde forming), a crotonaldehyde reductase (alcohol
forming), a butadiene synthase, a 3-aminobutyryl-CoA deaminase and
a crotyl alcohol diphosphokinase (FIG. 2, steps N, C, D, E, P, H).
In one aspect, the non-naturally occurring microbial organism
includes a microbial organism having a butadiene pathway having at
least one exogenous nucleic acid encoding butadiene pathway enzymes
expressed in a sufficient amount to produce butadiene, the
butadiene pathway including a 4-hydroxybutyryl-CoA dehydratase, a
crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase and a butadiene synthase (FIG. 2,
steps O, D-H). In one aspect, the non-naturally occurring microbial
organism includes a microbial organism having a butadiene pathway
having at least one exogenous nucleic acid encoding butadiene
pathway enzymes expressed in a sufficient amount to produce
butadiene, the butadiene pathway including a 4-hydroxybutyryl-CoA
dehydratase, a crotyl alcohol kinase, a 2-butenyl-4-phosphate
kinase, a butadiene synthase and crotonyl-CoA reductase (alcohol
forming) (FIG. 2, steps O, K, F, G, H). In one aspect, the
non-naturally occurring microbial organism includes a microbial
organism having a butadiene pathway having at least one exogenous
nucleic acid encoding butadiene pathway enzymes expressed in a
sufficient amount to produce butadiene, the butadiene pathway
including a 4-hydroxybutyryl-CoA dehydratase, a butadiene synthase,
a crotonyl-CoA reductase (alcohol forming) and a crotyl alcohol
diphosphokinase (FIG. 2, steps O, K, P, H). In one aspect, the
non-naturally occurring microbial organism includes a microbial
organism having a butadiene pathway having at least one exogenous
nucleic acid encoding butadiene pathway enzymes expressed in a
sufficient amount to produce butadiene, the butadiene pathway
including a 4-hydroxybutyryl-CoA dehydratase, a crotonaldehyde
reductase (alcohol forming), a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase, a butadiene synthase, a crotonyl-CoA
hydrolase, synthetase, or transferase and a crotonate reductase
(
FIG. 2, steps O, I, J, E, F, G, H). In one aspect, the
non-naturally occurring microbial organism includes a microbial
organism having a butadiene pathway having at least one exogenous
nucleic acid encoding butadiene pathway enzymes expressed in a
sufficient amount to produce butadiene, the butadiene pathway
including a 4-hydroxybutyryl-CoA dehydratase, a crotonaldehyde
reductase (alcohol forming), a butadiene synthase, a crotonyl-CoA
hydrolase, synthetase or transferase, a crotonate reductase and a
crotyl alcohol diphosphokinase (FIG. 2, steps O, I, J, E, P, H). In
one aspect, the non-naturally occurring microbial organism includes
a microbial organism having a butadiene pathway having at least one
exogenous nucleic acid encoding butadiene pathway enzymes expressed
in a sufficient amount to produce butadiene, the butadiene pathway
including a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA
reductase (aldehyde forming), a crotonaldehyde reductase (alcohol
forming), a butadiene synthase, a 4-hydroxybutyryl-CoA dehydratase
and a crotyl alcohol diphosphokinase (FIG. 2, steps L, C, D, E, P,
H).
[0050] In some embodiments, the invention provides a non-naturally
occurring microbial organism, including a microbial organism having
a butadiene pathway having at least one exogenous nucleic acid
encoding a butadiene pathway enzyme expressed in a sufficient
amount to produce butadiene, the butadiene pathway including an
erythrose-4-phosphate reductase, an erythritol-4-phospate
cytidylyltransferase, a 4-(cytidine 5'-diphospho)-erythritol
kinase, an erythritol 2,4-cyclodiphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate synthase, a 1-hydroxy-2-butenyl
4-diphosphate reductase, a butenyl 4-diphosphate isomerase, a
butadiene synthase, an erythrose-4-phosphate kinase, an erythrose
reductase or an erythritol kinase (FIG. 3). In one aspect, the
non-naturally occurring microbial organism includes a microbial
organism having a butadiene pathway having at least one exogenous
nucleic acid encoding butadiene pathway enzymes expressed in a
sufficient amount to produce butadiene, the butadiene pathway
including an erythrose-4-phosphate reductase, an
erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate reductase and a butadiene
synthase (FIG. 3, steps A-F, and H). In one aspect, the
non-naturally occurring microbial organism includes a microbial
organism having a butadiene pathway having at least one exogenous
nucleic acid encoding butadiene pathway enzymes expressed in a
sufficient amount to produce butadiene, the butadiene pathway
including an erythrose-4-phosphate reductase, an
erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate reductase, a butenyl
4-diphosphate isomerase and butadiene synthase (FIG. 3, steps A-H).
In one aspect, the non-naturally occurring microbial organism
includes a microbial organism having a butadiene pathway having at
least one exogenous nucleic acid encoding butadiene pathway enzymes
expressed in a sufficient amount to produce butadiene, the
butadiene pathway including an erythritol-4-phospate
cytidylyltransferase, a 4-(cytidine 5'-diphospho)-erythritol
kinase, an erythritol 2,4-cyclodiphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate synthase, a 1-hydroxy-2-butenyl
4-diphosphate reductase, a butadiene synthase, an
erythrose-4-phosphate kinase, an erythrose reductase and a
erythritol kinase (FIG. 3, steps I, J, K, B-F, H). In one aspect,
the non-naturally occurring microbial organism includes a microbial
organism having a butadiene pathway having at least one exogenous
nucleic acid encoding butadiene pathway enzymes expressed in a
sufficient amount to produce butadiene, the butadiene pathway
including an erythritol-4-phospate cytidylyltransferase, a
4-(cytidine 5'-diphospho)-erythritol kinase, an erythritol
2,4-cyclodiphosphate synthase, a 1-hydroxy-2-butenyl 4-diphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate reductase, a butenyl
4-diphosphate isomerase, a butadiene synthase, an
erythrose-4-phosphate kinase, an erythrose reductase and an
erythritol kinase (FIG. 3, steps I, J, K, B-H).
[0051] In some embodiments, the invention provides a non-naturally
occurring microbial organism, including a microbial organism having
a butadiene pathway having at least one exogenous nucleic acid
encoding a butadiene pathway enzyme expressed in a sufficient
amount to produce butadiene, the butadiene pathway including a
malonyl-CoA:acetyl-CoA acyltransferase, an 3-oxoglutaryl-CoA
reductase (ketone-reducing), a 3-hydroxyglutaryl-CoA reductase
(aldehyde forming), a 3-hydroxy-5-oxopentanoate reductase, a
3,5-dihydroxypentanoate kinase, a
3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, a 3-hydroxyglutaryl-CoA reductase (alcohol forming), an
3-oxoglutaryl-CoA reductase (aldehyde forming), a
3,5-dioxopentanoate reductase (ketone reducing), a
3,5-dioxopentanoate reductase (aldehyde reducing), a
5-hydroxy-3-oxopentanoate reductase or an 3-oxo-glutaryl-CoA
reductase (CoA reducing and alcohol forming) (FIG. 4). In one
aspect, the non-naturally occurring microbial organism includes a
microbial organism having a butadiene pathway having at least one
exogenous nucleic acid encoding butadiene pathway enzymes expressed
in a sufficient amount to produce butadiene, the butadiene pathway
including a malonyl-CoA:acetyl-CoA acyltransferase, an
3-oxoglutaryl-CoA reductase (ketone-reducing), a
3-hydroxyglutaryl-CoA reductase (aldehyde forming), a
3-hydroxy-5-oxopentanoate reductase, a 3,5-dihydroxypentanoate
kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase and a butadiene
synthase (FIG. 4, steps A-I). In one aspect, the non-naturally
occurring microbial organism includes a microbial organism having a
butadiene pathway having at least one exogenous nucleic acid
encoding butadiene pathway enzymes expressed in a sufficient amount
to produce butadiene, the butadiene pathway including a
malonyl-CoA:acetyl-CoA acyltransferase, a 3,5-dihydroxypentanoate
kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, an 3-oxoglutaryl-CoA reductase (aldehyde forming), a
3,5-dioxopentanoate reductase (aldehyde reducing) and a
5-hydroxy-3-oxopentanoate reductase. (FIG. 4, steps A, K, M, N, E,
F, G, H, I). In one aspect, the non-naturally occurring microbial
organism includes a microbial organism having a butadiene pathway
having at least one exogenous nucleic acid encoding butadiene
pathway enzymes expressed in a sufficient amount to produce
butadiene, the butadiene pathway including a malonyl-CoA:acetyl-CoA
acyltransferase, a 3-hydroxy-5-oxopentanoate reductase, a
3,5-dihydroxypentanoate kinase, a
3-Hydroxy-5-phosphonatooxypentanoate kinase, a
3-Hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, an 3-oxoglutaryl-CoA reductase (aldehyde forming) and a
3,5-dioxopentanoate reductase (ketone reducing). (FIG. 4, steps A,
K, L, D, E, F, G, H, I). In one aspect, the non-naturally occurring
microbial organism includes a microbial organism having a butadiene
pathway having at least one exogenous nucleic acid encoding
butadiene pathway enzymes expressed in a sufficient amount to
produce butadiene, the butadiene pathway including a
malonyl-CoA:acetyl-CoA acyltransferase, a 3,5-dihydroxypentanoate
kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, a 5-hydroxy-3-oxopentanoate reductase and a
3-oxo-glutaryl-CoA reductase (CoA reducing and alcohol forming).
(FIG. 4, steps A, O, N, E, F, G, H, I). In one aspect, the
non-naturally occurring microbial organism includes a microbial
organism having a butadiene pathway having at least one exogenous
nucleic acid encoding butadiene pathway enzymes expressed in a
sufficient amount to produce butadiene, the butadiene pathway
including a malonyl-CoA:acetyl-CoA acyltransferase, an
3-oxoglutaryl-CoA reductase (ketone-reducing), a
3,5-dihydroxypentanoate kinase, a
3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase and a 3-hydroxyglutaryl-CoA reductase (alcohol forming).
(FIG. 4, steps A, B, J, E, F, G, H, I).
[0052] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having a butadiene or a
crotyl alcohol 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 acetyl-CoA to
acetoacetyl-CoA, acetoacetyl-CoA to 3-hydroxybutyryl-CoA,
3-hydroxybutyryl-CoA to crotonyl-CoA, crotonyl-CoA to
crotonaldehyde, crotonaldehyde to crotyl alcohol, crotyl alcohol to
2-betenyl-phosphate, 2-betenyl-phosphate to
2-butenyl-4-diphosphate, 2-butenyl-4-diphosphate to butadiene,
erythrose-4-phosphate to erythritol-4-phosphate,
erythritol-4-phosphate to 4-(cytidine 5'-diphospho)-erythritol,
4-(cytidine 5'-diphospho)-erythritol to 2-phospho-4-(cytidine
5'-diphospho)-erythritol, 2-phospho-4-(cytidine
5'-diphospho)-erythritol to erythritol-2,4-cyclodiphosphate,
erythritol-2,4-cyclodiphosphate to 1-hydroxy-2-butenyl
4-diphosphate, 1-hydroxy-2-butenyl 4-diphosphate to butenyl
4-diphosphate, butenyl 4-diphosphate to 2-butenyl 4-diphosphate,
1-hydroxy-2-butenyl 4-diphosphate to 2-butenyl 4-diphosphate,
2-butenyl 4-diphosphate to butadiene, malonyl-CoA and acetyl-CoA to
3-oxoglutaryl-CoA, 3-oxoglutaryl-CoA to 3-hydroxyglutaryl-CoA to
3-hydroxy-5-oxopentanoate, 3-hydroxy-5-oxopentanoate to
3,5-dihydroxy pentanoate, 3,5-dihydroxy pentanoate to
3-hydroxy-5-phosphonatooxypentanoate,
3-hydroxy-5-phosphonatooxypentanoate to
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate,
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate to
butenyl 4-biphosphate, glutaconyl-CoA to crotonyl-CoA, glutaryl-CoA
to crotonyl-CoA, 3-aminobutyryl-CoA to crotonyl-CoA,
4-hydroxybutyryl-CoA to crotonyl-CoA, crotonyl-CoA to crotonate,
crotonate to crotonaldehyde, crotonyl-CoA to crotyl alcohol, crotyl
alcohol to 2-butenyl-4-diphosphate, erythrose-4-phosphate to
erythrose, erythrose to erythritol, erythritol to
erythritol-4-phosphate, 3-oxoglutaryl-CoA to 3,5-dioxopentanoate,
3,5-dioxopentanoate to 5-hydroxy-3-oxopentanoate,
5-hydroxy-3-oxopentanoate to 3,5-dihydroxypentanoate,
3-oxoglutaryl-CoA to 5-hydroxy-3-oxopentanoate, 3,5-dioxopentanoate
to 3-hydroxy-5-oxopentanoate, 3-hydroxyglutaryl-CoA to
3,5-dihydroxypentanoate and oxaloacetate to malate, malate to
fumarate, fumarate to succiniate, succinate to succinyl-CoA,
succinyl-CoA to .alpha.-ketoglutarate, .alpha.-ketoglutarate to
D-isocitrate, D-isocitrate to succinate, D-isocitrate to
glyoxylate, glyoxylate and acetyl-CoA to malate, D-isocitrate to
citrate, citrate to acetate, citrate to oxaloacetate, citrate to
acetyl-CoA, acetyl-CoA to pyruvate, pyruvate to
phosphoenolpyruvate, pyruvate to oxaloacetate, pyruvate to malate,
phhosphoenolpyruvate to oxaloacetate. One skilled in the art will
understand that these are merely exemplary and that any of the
substrate-product pairs disclosed herein suitable to produce a
desired product and for which an appropriate activity is available
for the conversion of the substrate to the product can be readily
determined by one skilled in the art based on the teachings herein.
Thus, the invention provides a non-naturally occurring microbial
organism containing at least one exogenous nucleic acid encoding an
enzyme or protein, where the enzyme or protein converts the
substrates and products of a butadiene or a crotyl alcohol pathway,
such as that shown in FIGS. 2-7 and 10-11.
[0053] While generally described herein as a microbial organism
that contains a butadiene or a crotyl alcohol pathway, it is
understood that the invention additionally provides a non-naturally
occurring microbial organism comprising at least one exogenous
nucleic acid encoding a butadiene or a crotyl alcohol pathway
enzyme expressed in a sufficient amount to produce an intermediate
of a butadiene or a crotyl alcohol pathway. For example, as
disclosed herein, a butadiene pathway is exemplified in FIGS. 2-4.
Therefore, in addition to a microbial organism containing a
butadiene pathway that produces butadiene, the invention
additionally provides a non-naturally occurring microbial organism
comprising at least one exogenous nucleic acid encoding a butadiene
pathway enzyme, where the microbial organism produces a butadiene
pathway intermediate, for example, acetoacetyl-CoA,
3-hydroxybutyryl-CoA, crotonyl-CoA, crotonaldehyde, crotyl alcohol,
2-betenyl-phosphate, 2-butenyl-4-diphosphate,
erythritol-4-phosphate, 4-(cytidine 5'-diphospho)-erythritol,
2-phospho-4-(cytidine 5'-diphospho)-erythritol,
erythritol-2,4-cyclodiphosphate, 1-hydroxy-2-butenyl 4-diphosphate,
butenyl 4-diphosphate, 2-butenyl 4-diphosphate, 3-oxoglutaryl-CoA,
3-hydroxyglutaryl-CoA, 3-hydroxy-5-oxopentanoate, 3,5-dihydroxy
pentanoate, 3-hydroxy-5-phosphonatooxypentanoate,
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate,
crotonate, erythrose, erythritol, 3,5-dioxopentanoate or
5-hydroxy-3-oxopentanoate.
[0054] 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. 2-7 and 10-11, can be utilized to
generate a non-naturally occurring microbial organism that produces
any pathway intermediate or product, as desired. As disclosed
herein, such a microbial organism that produces an intermediate can
be used in combination with another microbial organism expressing
downstream pathway enzymes to produce a desired product. However,
it is understood that a non-naturally occurring microbial organism
that produces a butadiene or crotyl alcohol pathway intermediate
can be utilized to produce the intermediate as a desired
product.
[0055] This invention is also directed, in part to engineered
biosynthetic pathways to improve carbon flux through a central
metabolism intermediate en route to butadiene or crotyl alcohol.
The present invention provides non-naturally occurring microbial
organisms having one or more exogenous genes encoding enzymes that
can catalyze various enzymatic transformations en route to
butadiene or crotyl alcohol. In some embodiments, these enzymatic
transformations are part of the reductive tricarboxylic acid (RTCA)
cycle and are used to improve product yields, including but not
limited to, from carbohydrate-based carbon feedstock.
[0056] In numerous engineered pathways, realization of maximum
product yields based on carbohydrate feedstock is hampered by
insufficient reducing equivalents or by loss of reducing
equivalents and/or carbon to byproducts. In accordance with some
embodiments, the present invention increases the yields of
butadiene or crotyl alcohol by (a) enhancing carbon fixation via
the reductive TCA cycle, and/or (b) accessing additional reducing
equivalents from gaseous carbon sources and/or syngas components
such as CO, CO.sub.2, and/or H2. In addition to syngas, other
sources of such gases include, but are not limited to, the
atmosphere, either as found in nature or generated.
[0057] The CO.sub.2-fixing reductive tricarboxylic acid (RTCA)
cycle is an endergenic anabolic pathway of CO.sub.2 assimilation
which uses reducing equivalents and ATP (FIG. 6). One turn of the
RTCA cycle assimilates two moles of CO.sub.2 into one mole of
acetyl-CoA, or four moles of CO.sub.2 into one mole of
oxaloacetate. This additional availability of acetyl-CoA improves
the maximum theoretical yield of product molecules derived from
carbohydrate-based carbon feedstock. Exemplary carbohydrates
include but are not limited to glucose, sucrose, xylose, arabinose
and glycerol.
[0058] In some embodiments, the reductive TCA cycle, coupled with
carbon monoxide dehydrogenase and/or hydrogenase enzymes, can be
employed to allow syngas, CO.sub.2, CO, H.sub.2, and/or other
gaseous carbon source utilization by microorganisms. Synthesis gas
(syngas), in particular is a mixture of primarily H.sub.2 and CO,
sometimes including some amounts of CO.sub.2, that can be obtained
via gasification of any organic feedstock, such as coal, coal oil,
natural gas, biomass, or waste organic matter. Numerous
gasification processes have been developed, and most designs are
based on partial oxidation, where limiting oxygen avoids full
combustion, of organic materials at high temperatures
(500-1500.degree. C.) to provide syngas as a 0.5:1-3:1 H.sub.2/CO
mixture. In addition to coal, biomass of many types has been used
for syngas production and represents an inexpensive and flexible
feedstock for the biological production of renewable chemicals and
fuels. Carbon dioxide can be provided from the atmosphere or in
condensed from, for example, from a tank cylinder, or via
sublimation of solid CO.sub.2. Similarly, CO and hydrogen gas can
be provided in reagent form and/or mixed in any desired ratio.
Other gaseous carbon forms can include, for example, methanol or
similar volatile organic solvents.
[0059] The components of synthesis gas and/or other carbon sources
can provide sufficient CO.sub.2, reducing equivalents, and ATP for
the reductive TCA cycle to operate. One turn of the RTCA cycle
assimilates two moles of CO.sub.2 into one mole of acetyl-CoA and
requires 2 ATP and 4 reducing equivalents. CO and/or H.sub.2 can
provide reducing equivalents by means of carbon monoxide
dehydrogenase and hydrogenase enzymes, respectively. Reducing
equivalents can come in the form ofNADH, NADPH, FADH, reduced
quinones, reduced ferredoxins, thioredoxins and reduced
flavodoxins. The reducing equivalents, particularly NADH, NADPH,
and reduced ferredoxin, can serve as cofactors for the RTCA cycle
enzymes, for example, malate dehydrogenase, fumarate reductase,
alpha-ketoglutarate:ferredoxin oxidoreductase (alternatively known
as 2-oxoglutarate:ferredoxin oxidoreductase, alpha-ketoglutarate
synthase, or 2-oxoglutarate synthase), pyruvate:ferredoxin
oxidoreductase and isocitrate dehydrogenase. The electrons from
these reducing equivalents can alternatively pass through an
ion-gradient producing electron transport chain where they are
passed to an acceptor such as oxygen, nitrate, oxidized metal ions,
protons, or an electrode. The ion-gradient can then be used for ATP
generation via an ATP synthase or similar enzyme.
[0060] The reductive TCA cycle was first reported in the green
sulfur photosynthetic bacterium Chlorobium limicola (Evans et al.,
Proc. Natl. Acad. Sci. U.S.A. 55:928-934 (1966)). Similar pathways
have been characterized in some prokaryotes (proteobacteria, green
sulfur bacteria and thermophillic Knallgas bacteria) and
sulfur-dependent archaea (Hugler et al., J. Bacteriol.
187:3020-3027 (2005; Hugler et al., Environ. Microbiol. 9:81-92
(2007). In some cases, reductive and oxidative (Krebs) TCA cycles
are present in the same organism (Hugler et al., supra (2007);
Siebers et al., J. Bacteriol. 186:2179-2194 (2004)). Some
methanogens and obligate anaerobes possess incomplete oxidative or
reductive TCA cycles that may function to synthesize biosynthetic
intermediates (Ekiel et al., J. Bacteriol. 162:905-908 (1985); Wood
et al., FEMS Microbiol. Rev. 28:335-352 (2004)).
[0061] The key carbon-fixing enzymes of the reductive TCA cycle are
alpha-ketoglutarate:ferredoxin oxidoreductase, pyruvate:ferredoxin
oxidoreductase and isocitrate dehydrogenase. Additional carbon may
be fixed during the conversion of phosphoenolpyruvate to
oxaloacetate by phosphoenolpyruvate carboxylase or
phosphoenolpyruvate carboxykinase or by conversion of pyruvate to
malate by malic enzyme.
[0062] Many of the enzymes in the TCA cycle are reversible and can
catalyze reactions in the reductive and oxidative directions.
However, some TCA cycle reactions are irreversible in vivo and thus
different enzymes are used to catalyze these reactions in the
directions required for the reverse TCA cycle. These reactions are:
(1) conversion of citrate to oxaloacetate and acetyl-CoA, (2)
conversion of fumarate to succinate, and (3) conversion of
succinyl-CoA to alpha-ketoglutarate. In the TCA cycle, citrate is
formed from the condensation of oxaloacetate and acetyl-CoA. The
reverse reaction, cleavage of citrate to oxaloacetate and
acetyl-CoA, is ATP-dependent and catalyzed by ATP-citrate lyase, or
citryl-CoA synthetase and citryl-CoA lyase. Alternatively, citrate
lyase can be coupled to acetyl-CoA synthetase, an acetyl-CoA
transferase, or phosphotransacetylase and acetate kinase to form
acetyl-CoA and oxaloacetate from citrate. The conversion of
succinate to fumarate is catalyzed by succinate dehydrogenase while
the reverse reaction is catalyzed by fumarate reductase. In the TCA
cycle succinyl-CoA is formed from the NAD(P).sup.+ dependent
decarboxylation of alpha-ketoglutarate by the alpha-ketoglutarate
dehydrogenase complex. The reverse reaction is catalyzed by
alpha-ketoglutarate:ferredoxin oxidoreductase.
[0063] An organism capable of utilizing the reverse tricarboxylic
acid cycle to enable production of acetyl-CoA-derived products on
1) CO, 2) CO.sub.2 and H.sub.2, 3) CO and CO.sub.2, 4) synthesis
gas comprising CO and H.sub.2, and 5) synthesis gas or other
gaseous carbon sources comprising CO, CO.sub.2, and H.sub.2 can
include any of the following enzyme activities: ATP-citrate lyase,
citrate lyase, aconitase, isocitrate dehydrogenase,
alpha-ketoglutarate:ferredoxin oxidoreductase, succinyl-CoA
synthetase, succinyl-CoA transferase, fumarate reductase, fumarase,
malate dehydrogenase, acetate kinase, phosphotransacetylase,
acetyl-CoA synthetase, acetyl-CoA transferase, pyruvate:ferredoxin
oxidoreductase, NAD(P)H:ferredoxin oxidoreductase, carbon monoxide
dehydrogenase, hydrogenase, and ferredoxin (see FIG. 7). Enzymes
and the corresponding genes required for these activities are
described herein.
[0064] Carbon from syngas or other gaseous carbon sources can be
fixed via the reverse TCA cycle and components thereof.
Specifically, the combination of certain carbon gas-utilization
pathway components with the pathways for formation of butadiene or
crotyl alcohol from acetyl-CoA results in high yields of these
products by providing an efficient mechanism for fixing the carbon
present in carbon dioxide, fed exogenously or produced endogenously
from CO, into acetyl-CoA.
[0065] In some embodiments, a butadiene or crotyl alcohol pathway
in a non-naturally occurring microbial organism of the invention
can utilize any combination of (1) CO, (2) CO.sub.2, (3) H.sub.2,
or mixtures thereof to enhance the yields of biosynthetic steps
involving reduction, including addition to driving the reductive
TCA cycle.
[0066] In some embodiments a non-naturally occurring microbial
organism having a butadiene or crotyl alcohol pathway includes at
least one exogenous nucleic acid encoding a reductive TCA pathway
enzyme. The at least one exogenous nucleic acid is selected from an
ATP-citrate lyase, citrate lyase, a fumarate reductase, isocitrate
dehydrogenase, aconitase, and an alpha-ketoglutarate:ferredoxin
oxidoreductase; and at least one exogenous enzyme selected from a
carbon monoxide dehydrogenase, a hydrogenase, a NAD(P)H:ferredoxin
oxidoreductase, and a ferredoxin, expressed in a sufficient amount
to allow the utilization of (1) CO, (2) CO.sub.2, (3) H.sub.2, (4)
CO.sub.2 and H.sub.2, (5) CO and CO.sub.2, (6) CO and H.sub.2, or
(7) CO, CO.sub.2, and H.sub.2.
[0067] In some embodiments a method includes culturing a
non-naturally occurring microbial organism having a butadiene or
crotyl alcohol pathway also comprising at least one exogenous
nucleic acid encoding a reductive TCA pathway enzyme. The at least
one exogenous nucleic acid is selected from an ATP-citrate lyase,
citrate lyase, a fumarate reductase, isocitrate dehydrogenase,
aconitase, and an alpha-ketoglutarate:ferredoxin oxidoreductase.
Additionally, such an organism can also include at least one
exogenous enzyme selected from a carbon monoxide dehydrogenase, a
hydrogenase, a NAD(P)H:ferredoxin oxidoreductase, and a ferredoxin,
expressed in a sufficient amount to allow the utilization of (1)
CO, (2) CO.sub.2, (3) H.sub.2, (4) CO.sub.2 and H.sub.2, (5) CO and
CO.sub.2, (6) CO and H.sub.2, or (7) CO, CO.sub.2, and H.sub.2 to
produce a product.
[0068] In some embodiments a non-naturally occurring microbial
organism having a butadiene or crotyl alcohol pathway further
includes at least one exogenous nucleic acid encoding a reductive
TCA pathway enzyme expressed in a sufficient amount to enhance
carbon flux through acetyl-CoA. The at least one exogenous nucleic
acid is selected from an ATP-citrate lyase, citrate lyase, a
fumarate reductase, a pyruvate:ferredoxin oxidoreductase,
isocitrate dehydrogenase, aconitase, and an
alpha-ketoglutarate:ferredoxin oxidoreductase.
[0069] In some embodiments a non-naturally occurring microbial
organism having a butadiene or crotyl alcohol pathway includes at
least one exogenous nucleic acid encoding an enzyme expressed in a
sufficient amount to enhance the availability of reducing
equivalents in the presence of carbon monoxide and/or hydrogen,
thereby increasing the yield of redox-limited products via
carbohydrate-based carbon feedstock. The at least one exogenous
nucleic acid is selected from a carbon monoxide dehydrogenase, a
hydrogenase, an NAD(P)H:ferredoxin oxidoreductase, and a
ferredoxin. In some embodiments, the present invention provides a
method for enhancing the availability of reducing equivalents in
the presence of carbon monoxide or hydrogen thereby increasing the
yield of redox-limited products via carbohydrate-based carbon
feedstock, such as sugars or gaseous carbon sources, the method
includes culturing this non-naturally occurring microbial organism
under conditions and for a sufficient period of time to produce
butadiene or crotyl alcohol.
[0070] In some embodiments, the non-naturally occurring microbial
organism having a butadiene or crotyl alcohol pathway includes two
exogenous nucleic acids, each encoding a reductive TCA pathway
enzyme. In some embodiments, the non-naturally occurring microbial
organism having a butadiene or crotyl alcohol pathway includes
three exogenous nucleic acids each encoding a reductive TCA pathway
enzyme. In some embodiments, the non-naturally occurring microbial
organism includes three exogenous nucleic acids encoding an
ATP-citrate lyase, a fumarate reductase, and an
alpha-ketoglutarate:ferredoxin oxidoreductase. In some embodiments,
the non-naturally occurring microbial organism includes three
exogenous nucleic acids encoding a citrate lyase, a fumarate
reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase. In
some embodiments, the non-naturally occurring microbial organism
includes four exogenous nucleic acids encoding a
pyruvate:ferredoxin oxidoreductase; a phosphoenolpyruvate
carboxylase or a phosphoenolpyruvate carboxykinase, a CO
dehydrogenase; and an H.sub.2 hydrogenase. In some embodiments, the
non-naturally occurring microbial organism includes two exogenous
nucleic acids encoding a CO dehydrogenase and an H.sub.2
hydrogenase.
[0071] In some embodiments, the non-naturally occurring microbial
organisms having a butadiene or crotyl alcohol pathway further
include an exogenous nucleic acid encoding an enzyme selected from
a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate
dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA
transferase, a fumarase, a malate dehydrogenase, an acetate kinase,
a phosphotransacetylase, an acetyl-CoA synthetase, an
NAD(P)H:ferredoxin oxidoreductase, and combinations thereof.
[0072] In some embodiments, the non-naturally occurring microbial
organism having a butadiene or crotyl alcohol pathway further
includes an exogenous nucleic acid encoding an enzyme selected from
carbon monoxide dehydrogenase, acetyl-CoA synthase, ferredoxin,
NAD(P)H:ferredoxin oxidoreductase and combinations thereof.
[0073] In some embodiments, the non-naturally occurring microbial
organism having a butadiene or crotyl alcohol pathway utilizes a
carbon feedstock selected from (1) CO, (2) CO.sub.2, (3) CO.sub.2
and H.sub.2, (4) CO and H.sub.2, or (5) CO, CO.sub.2, and H.sub.2.
In some embodiments, the non-naturally occurring microbial organism
having a butadiene or crotyl alcohol pathway utilizes hydrogen for
reducing equivalents. In some embodiments, the non-naturally
occurring microbial organism having a butadiene or crotyl alcohol
pathway utilizes CO for reducing equivalents. In some embodiments,
the non-naturally occurring microbial organism having a butadiene
or crotyl alcohol pathway utilizes combinations of CO and hydrogen
for reducing equivalents.
[0074] In some embodiments, the non-naturally occurring microbial
organism having a butadiene or crotyl alcohol pathway further
includes one or more nucleic acids encoding an enzyme selected from
a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate
carboxykinase, a pyruvate carboxylase, and a malic enzyme.
[0075] In some embodiments, the non-naturally occurring microbial
organism having a butadiene or crotyl alcohol pathway further
includes one or more nucleic acids encoding an enzyme selected from
a malate dehydrogenase, a fumarase, a fumarate reductase, a
succinyl-CoA synthetase, and a succinyl-CoA transferase.
[0076] In some embodiments, the non-naturally occurring microbial
organism having a butadiene or crotyl alcohol pathway further
includes at least one exogenous nucleic acid encoding a citrate
lyase, an ATP-citrate lyase, a citryl-CoA synthetase, a citryl-CoA
lyase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA
synthetase, a succinyl-CoA transferase, a fumarase, a malate
dehydrogenase, an acetate kinase, a phosphotransacetylase, an
acetyl-CoA synthetase, and a ferredoxin.
[0077] It is understood by those skilled in the art that the
above-described pathways for increasing product yield can be
combined with any of the pathways disclosed herein, including those
pathways depicted in the figures. One skilled in the art will
understand that, depending on the pathway to a desired product and
the precursors and intermediates of that pathway, a particular
pathway for improving product yield, as discussed herein above and
in the examples, or combination of such pathways, can be used in
combination with a pathway to a desired product to increase the
yield of that product or a pathway intermediate.
[0078] In one embodiment, the invention provides a non-naturally
occurring microbial organism, comprising a microbial organism
having a butadiene pathway comprising at least one exogenous
nucleic acid encoding a butadiene pathway enzyme expressed in a
sufficient amount to produce butadiene. Such a microbial organism
can further comprise (a) a reductive TCA pathway comprising at
least one exogenous nucleic acid encoding a reductive TCA pathway
enzyme, wherein the at least one exogenous nucleic acid is selected
from an ATP-citrate lyase, a citrate lyase, a citryl-CoA
synthetase, a citryl-CoA lyase, a fumarate reductase, and an
alpha-ketoglutarate:ferredoxin oxidoreductase; (b) a reductive TCA
pathway comprising at least one exogenous nucleic acid encoding a
reductive TCA pathway enzyme, wherein the at least one exogenous
nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase,
a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate
carboxykinase, a CO dehydrogenase, and an H.sub.2 hydrogenase; or
(c) at least one exogenous nucleic acid encodes an enzyme selected
from a CO dehydrogenase, an H.sub.2 hydrogenase, and combinations
thereof. In such a microbial organism, a butadiene pathway can
comprise a butadiene pathway disclosed herein. For example, the
butadien pathway can be selected from: (i) an acetyl-CoA:acetyl-CoA
acyltransferase, an acetoacetyl-CoA reductase, a
3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a
butadiene synthase; (ii) an acetyl-CoA:acetyl-CoA acyltransferase,
an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a
crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene
synthase and crotonyl-CoA reductase (alcohol forming); (iii) an
acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA
reductase, a 3-hydroxybutyryl-CoA dehydratase, a butadiene
synthase, a crotonyl-CoA reductase (alcohol forming) and a crotyl
alcohol diphosphokinase; (iv) an acetyl-CoA:acetyl-CoA
acyltransferase, an acetoacetyl-CoA reductase, a
3-hydroxybutyryl-CoA dehydratase, a crotonaldehyde reductase
(alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate
kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase
or transferase and a crotonate reductase; (v) an
acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA
reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonaldehyde
reductase (alcohol forming), a butadiene synthase, a crotonyl-CoA
hydrolase, synthetase or transferase, a crotonate reductase and a
crotyl alcohol diphosphokinase; (vi) an acetyl-CoA:acetyl-CoA
acyltransferase, an acetoacetyl-CoA reductase, a
3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
butadiene synthase and a crotyl alcohol diphosphokinase. (vii) a
glutaconyl-CoA decarboxylase, a crotonyl-CoA reductase (aldehyde
forming), a crotonaldehyde reductase (alcohol forming), a crotyl
alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene
synthase. (viii) a glutaconyl-CoA decarboxylase, a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase and
crotonyl-CoA reductase (alcohol forming); (ix) a glutaconyl-CoA
decarboxylase, a butadiene synthase, a crotonyl-CoA reductase
(alcohol forming) and a crotyl alcohol diphosphokinase; (x) a
glutaconyl-CoA decarboxylase, a crotonaldehyde reductase (alcohol
forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase,
a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or
transferase and a crotonate reductase; (xi) a glutaconyl-CoA
decarboxylase, a crotonaldehyde reductase (alcohol forming), a
butadiene synthase, a crotonyl-CoA hydrolase, synthetase or
transferase, a crotonate reductase and a crotyl alcohol
diphosphokinase; (xii) a 3-hydroxybutyryl-CoA dehydratase, a
crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a butadiene a glutaconyl-CoA
decarboxylase and a crotyl alcohol diphosphokinase; (xiii) a
glutaryl-CoA dehydrogenase, a crotonyl-CoA reductase (aldehyde
forming), a crotonaldehyde reductase (alcohol forming), a crotyl
alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene
synthase; (xiv) a glutaryl-CoA dehydrogenase, a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase and
crotonyl-CoA reductase (alcohol forming); (xv) a glutaryl-CoA
dehydrogenase, a butadiene synthase, a crotonyl-CoA reductase
(alcohol forming) and a crotyl alcohol diphosphokinase; (xvi) a
glutaryl-CoA dehydrogenase, a crotonaldehyde reductase (alcohol
forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase,
a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or
transferase and a crotonate reductase; (xvii) a glutaryl-CoA
dehydrogenase, a crotonaldehyde reductase (alcohol forming), a
butadiene synthase, a crotonyl-CoA hydrolase, synthetase or
transferase, a crotonate reductase and a crotyl alcohol
diphosphokinase; (xviii) a 3-hydroxybutyryl-CoA dehydratase, a
crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a butadiene synthase, a glutaryl-CoA
dehydrogenase and a crotyl alcohol diphosphokinase; (xix) an
3-aminobutyryl-CoA deaminase, a crotonyl-CoA reductase (aldehyde
forming), a crotonaldehyde reductase (alcohol forming), a crotyl
alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene
synthase; (xx) an 3-aminobutyryl-CoA deaminase, a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase and
crotonyl-CoA reductase (alcohol forming); (xxi) an
3-aminobutyryl-CoA deaminase, a butadiene synthase, a crotonyl-CoA
reductase (alcohol forming) and a crotyl alcohol diphosphokinase;
(xxii) an 3-aminobutyryl-CoA deaminase, a crotonaldehyde reductase
(alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate
kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase
or transferase and a crotonate reductase; (xxiii) an
3-aminobutyryl-CoA deaminase, a crotonaldehyde reductase (alcohol
forming), a butadiene synthase, a crotonyl-CoA hydrolase,
synthetase or transferase, a crotonate reductase and a crotyl
alcohol diphosphokinase; (xxiv) a 3-hydroxybutyryl-CoA dehydratase,
a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a butadiene synthase, a
3-aminobutyryl-CoA deaminase and a crotyl alcohol diphosphokinase;
(xxv) a 4-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a
butadiene synthase; (xxvi) a 4-hydroxybutyryl-CoA dehydratase, a
crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene
synthase and crotonyl-CoA reductase (alcohol forming); (xxvii) a
4-hydroxybutyryl-CoA dehydratase, a butadiene synthase, a
crotonyl-CoA reductase (alcohol forming) and a crotyl alcohol
diphosphokinase; (xxviii) a 4-hydroxybutyryl-CoA dehydratase, a
crotonaldehyde reductase (alcohol forming), a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a
crotonyl-CoA hydrolase, synthetase or transferase and a crotonate
reductase; (xxix) a 4-hydroxybutyryl-CoA dehydratase, a
crotonaldehyde reductase (alcohol forming), a butadiene synthase, a
crotonyl-CoA hydrolase, synthetase or transferase, a crotonate
reductase and a crotyl alcohol diphosphokinase; (xxx) a
3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
butadiene synthase, a 4-hydroxybutyryl-CoA dehydratase and a crotyl
alcohol diphosphokinase; (xxxi) an erythrose-4-phosphate reductase,
an erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate reductase and a butadiene
synthase; (xxxii) an erythrose-4-phosphate reductase, an
erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate reductase, a butenyl
4-diphosphate isomerase and a butadiene synthase; (xxxiii) an
erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate reductase, a butadiene synthase,
an erythrose-4-phosphate kinase, an erythrose reductase and a
erythritol kinase; (xxxiv) an erythritol-4-phospate
cytidylyltransferase, a 4-(cytidine 5'-diphospho)-erythritol
kinase, an erythritol 2,4-cyclodiphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate synthase, a 1-hydroxy-2-butenyl
4-diphosphate reductase, a butenyl 4-diphosphate isomerase, a
butadiene synthase, an erythrose-4-phosphate kinase, an erythrose
reductase and an erythritol kinase; (xxxv) a malonyl-CoA:acetyl-CoA
acyltransferase, an 3-oxoglutaryl-CoA reductase (ketone-reducing),
a 3-hydroxyglutaryl-CoA reductase (aldehyde forming), a
3-hydroxy-5-oxopentanoate reductase, a 3,5-dihydroxypentanoate
kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase and a butadiene
synthase; (xxxvi) a malonyl-CoA:acetyl-CoA acyltransferase, a
3,5-dihydroxypentanoate kinase, a
3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, an 3-oxoglutaryl-CoA reductase (aldehyde forming), a
3,5-dioxopentanoate reductase (aldehyde reducing) and a
5-hydroxy-3-oxopentanoate reductase; (xxxvii) a
malonyl-CoA:acetyl-CoA acyltransferase, a 3-hydroxy-5-oxopentanoate
reductase, a 3,5-dihydroxypentanoate kinase, a
3-Hydroxy-5-phosphonatooxypentanoate kinase, a
3-Hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, an 3-oxoglutaryl-CoA reductase (aldehyde forming) and a
3,5-dioxopentanoate reductase (ketone reducing); (xxxviii) a
malonyl-CoA:acetyl-CoA acyltransferase, a 3,5-dihydroxypentanoate
kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, a 5-hydroxy-3-oxopentanoate reductase and a
3-oxo-glutaryl-CoA reductase (CoA reducing and alcohol forming);
and (xxxix) a butadiene pathway comprising a malonyl-CoA:acetyl-CoA
acyltransferase, an 3-oxoglutaryl-CoA reductase (ketone-reducing),
a 3,5-dihydroxypentanoate kinase, a
3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase and a 3-hydroxyglutaryl-CoA reductase (alcohol
forming).
[0079] In such microbial organisms of the invention, a microbial
organism comprising (a) can further comprise an exogenous nucleic
acid encoding an enzyme selected from a pyruvate:ferredoxin
oxidoreductase, an aconitase, an isocitrate dehydrogenase, a
succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a
malate dehydrogenase, an acetate kinase, a phosphotransacetylase,
an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase,
ferredoxin, and combinations thereof. In addition, a microbial
organism comprising (b) can further comprise an exogenous nucleic
acid encoding an enzyme selected from an aconitase, an isocitrate
dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA
transferase, a fumarase, a malate dehydrogenase, and combinations
thereof.
[0080] In a particular embodiment, such a microbial organism can
comprise two, three, four, five, six or seven exogenous nucleic
acids each encoding a butadiene pathway enzyme. For example, such a
microbial organism can comprise exogenous nucleic acids encoding
each of the enzymes selected from: (i) an acetyl-CoA:acetyl-CoA
acyltransferase, an acetoacetyl-CoA reductase, a
3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a
butadiene synthase; (ii) an acetyl-CoA:acetyl-CoA acyltransferase,
an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a
crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene
synthase and crotonyl-CoA reductase (alcohol forming); (iii) an
acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA
reductase, a 3-hydroxybutyryl-CoA dehydratase, a butadiene
synthase, a crotonyl-CoA reductase (alcohol forming) and a crotyl
alcohol diphosphokinase; (iv) an acetyl-CoA:acetyl-CoA
acyltransferase, an acetoacetyl-CoA reductase, a
3-hydroxybutyryl-CoA dehydratase, a crotonaldehyde reductase
(alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate
kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase
or transferase and a crotonate reductase; (v) an
acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA
reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonaldehyde
reductase (alcohol forming), a butadiene synthase, a crotonyl-CoA
hydrolase, synthetase or transferase, a crotonate reductase and a
crotyl alcohol diphosphokinase; (vi) an acetyl-CoA:acetyl-CoA
acyltransferase, an acetoacetyl-CoA reductase, a
3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
butadiene synthase and a crotyl alcohol diphosphokinase; (vii) a
glutaconyl-CoA decarboxylase, a crotonyl-CoA reductase (aldehyde
forming), a crotonaldehyde reductase (alcohol forming), a crotyl
alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene
synthase; (viii) a glutaconyl-CoA decarboxylase, a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase and
crotonyl-CoA reductase (alcohol forming); (ix) a glutaconyl-CoA
decarboxylase, a butadiene synthase, a crotonyl-CoA reductase
(alcohol forming) and a crotyl alcohol diphosphokinase; (x) a
glutaconyl-CoA decarboxylase, a crotonaldehyde reductase (alcohol
forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase,
a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or
transferase and a crotonate reductase; (xi) a glutaconyl-CoA
decarboxylase, a crotonaldehyde reductase (alcohol forming), a
butadiene synthase, a crotonyl-CoA hydrolase, synthetase or
transferase, a crotonate reductase and a crotyl alcohol
diphosphokinase; (xii) a 3-hydroxybutyryl-CoA dehydratase, a
crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a butadiene a glutaconyl-CoA
decarboxylase and a crotyl alcohol diphosphokinase; (xiii) a
glutaryl-CoA dehydrogenase, a crotonyl-CoA reductase (aldehyde
forming), a crotonaldehyde reductase (alcohol forming), a crotyl
alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene
synthase; (xiv) a glutaryl-CoA dehydrogenase, a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase and
crotonyl-CoA reductase (alcohol forming); (xv) a glutaryl-CoA
dehydrogenase, a butadiene synthase, a crotonyl-CoA reductase
(alcohol forming) and a crotyl alcohol diphosphokinase; (xvi) a
glutaryl-CoA dehydrogenase, a crotonaldehyde reductase (alcohol
forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase,
a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or
transferase and a crotonate reductase; (xvii) a glutaryl-CoA
dehydrogenase, a crotonaldehyde reductase (alcohol forming), a
butadiene synthase, a crotonyl-CoA hydrolase, synthetase or
transferase, a crotonate reductase and a crotyl alcohol
diphosphokinase; (xviii) a 3-hydroxybutyryl-CoA dehydratase, a
crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a butadiene synthase, a glutaryl-CoA
dehydrogenase and a crotyl alcohol diphosphokinase; (xix) an
3-aminobutyryl-CoA deaminase, a crotonyl-CoA reductase (aldehyde
forming), a crotonaldehyde reductase (alcohol forming), a crotyl
alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene
synthase; (xx) an 3-aminobutyryl-CoA deaminase, a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase and
crotonyl-CoA reductase (alcohol forming); (xxi) an
3-aminobutyryl-CoA deaminase, a butadiene synthase, a crotonyl-CoA
reductase (alcohol forming) and a crotyl alcohol diphosphokinase;
(xxii) an 3-aminobutyryl-CoA deaminase, a crotonaldehyde reductase
(alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate
kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase
or transferase and a crotonate reductase; (xxiii) an
3-aminobutyryl-CoA deaminase, a crotonaldehyde reductase (alcohol
forming), a butadiene synthase, a crotonyl-CoA hydrolase,
synthetase or transferase, a crotonate reductase and a crotyl
alcohol diphosphokinase; (xxiv) a 3-hydroxybutyryl-CoA dehydratase,
a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a butadiene synthase, a
3-aminobutyryl-CoA deaminase and a crotyl alcohol diphosphokinase;
(xxv) a 4-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a
butadiene synthase; (xxvi) a 4-hydroxybutyryl-CoA dehydratase, a
crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene
synthase and crotonyl-CoA reductase (alcohol forming); (xxvii) a
4-hydroxybutyryl-CoA dehydratase, a butadiene synthase, a
crotonyl-CoA reductase (alcohol forming) and a crotyl alcohol
diphosphokinase; (xxviii) a 4-hydroxybutyryl-CoA dehydratase, a
crotonaldehyde reductase (alcohol forming), a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a
crotonyl-CoA hydrolase, synthetase or transferase and a crotonate
reductase; (xxix) a 4-hydroxybutyryl-CoA dehydratase, a
crotonaldehyde reductase (alcohol forming), a butadiene synthase, a
crotonyl-CoA hydrolase, synthetase or transferase, a crotonate
reductase and a crotyl alcohol diphosphokinase; (xxx) a
3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
butadiene synthase, a 4-hydroxybutyryl-CoA dehydratase and a crotyl
alcohol diphosphokinase; (xxxi) an erythrose-4-phosphate reductase,
an erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate reductase and a butadiene
synthase; (xxxii) an erythrose-4-phosphate reductase, an
erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate reductase, a butenyl
4-diphosphate isomerase and a butadiene synthase; (xxxiii) an
erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate reductase, a butadiene synthase,
an erythrose-4-phosphate kinase, an erythrose reductase and a
erythritol kinase; (xxxiv) an erythritol-4-phospate
cytidylyltransferase, a 4-(cytidine 5'-diphospho)-erythritol
kinase, an erythritol 2,4-cyclodiphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate synthase, a 1-hydroxy-2-butenyl
4-diphosphate reductase, a butenyl 4-diphosphate isomerase, a
butadiene synthase, an erythrose-4-phosphate kinase, an erythrose
reductase and an erythritol kinase; (xxxv) a malonyl-CoA:acetyl-CoA
acyltransferase, an 3-oxoglutaryl-CoA reductase (ketone-reducing),
a 3-hydroxyglutaryl-CoA reductase (aldehyde forming), a
3-hydroxy-5-oxopentanoate reductase, a 3,5-dihydroxypentanoate
kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase and a butadiene
synthase; (xxxvi) a malonyl-CoA:acetyl-CoA acyltransferase, a
3,5-dihydroxypentanoate kinase, a
3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, an 3-oxoglutaryl-CoA reductase (aldehyde forming), a
3,5-dioxopentanoate reductase (aldehyde reducing) and a
5-hydroxy-3-oxopentanoate reductase; (xxxvii) a
malonyl-CoA:acetyl-CoA acyltransferase, a 3-hydroxy-5-oxopentanoate
reductase, a 3,5-dihydroxypentanoate kinase, a
3-Hydroxy-5-phosphonatooxypentanoate kinase, a
3-Hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, an 3-oxoglutaryl-CoA reductase (aldehyde forming) and a
3,5-dioxopentanoate reductase (ketone reducing); (xxxviii) a
malonyl-CoA:acetyl-CoA acyltransferase, a 3,5-dihydroxypentanoate
kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, a 5-hydroxy-3-oxopentanoate reductase and a
3-oxo-glutaryl-CoA reductase (CoA reducing and alcohol forming);
and (xxxix) a butadiene pathway comprising a malonyl-CoA:acetyl-CoA
acyltransferase, an 3-oxoglutaryl-CoA reductase (ketone-reducing),
a 3,5-dihydroxypentanoate kinase, a
3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase and a 3-hydroxyglutaryl-CoA reductase (alcohol
forming).
[0081] Such microbial organisms of the invention can comprise two,
three, four or five exogenous nucleic acids each encoding enzymes
of (a), (b) or (c). For example, a microbial organism comprising
(a) can comprise three exogenous nucleic acids encoding ATP-citrate
lyase or citrate lyase, a fumarate reductase, and an
alpha-ketoglutarate:ferredoxin oxidoreductase; a microbial organism
comprising (b) can comprise four exogenous nucleic acids encoding
pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate
carboxylase or a phosphoenolpyruvate carboxykinase, a CO
dehydrogenase, and an H.sub.2 hydrogenase; or a microbial organism
comprising (c) can comprise two exogenous nucleic acids encoding CO
dehydrogenase and H.sub.2 hydrogenase. The invention further
provides methods for producing butadiene by culturing such
non-naturally occurring microbial organisms under conditions and
for a sufficient period of time to produce butadiene.
[0082] The invention additionally provides a non-naturally
occurring microbial organism, comprising a microbial organism
having a crotyl alcohol pathway comprising at least one exogenous
nucleic acid encoding a crotyl alcohol pathway enzyme expressed in
a sufficient amount to produce crotyl alcohol. Such a microbial
organism can further comprise (a) a reductive TCA pathway
comprising at least one exogenous nucleic acid encoding a reductive
TCA pathway enzyme, wherein the at least one exogenous nucleic acid
is selected from an ATP-citrate lyase, a citrate lyase, a
citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase,
and an alpha-ketoglutarate:ferredoxin oxidoreductase; (b) a
reductive TCA pathway comprising at least one exogenous nucleic
acid encoding a reductive TCA pathway enzyme, wherein the at least
one exogenous nucleic acid is selected from a pyruvate:ferredoxin
oxidoreductase, a phosphoenolpyruvate carboxylase, a
phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an
H.sub.2 hydrogenase; or (c) at least one exogenous nucleic acid
encodes an enzyme selected from a CO dehydrogenase, an H.sub.2
hydrogenase, and combinations thereof.
[0083] In such a microbial organism, the crotyl alcohol pathway can
be selected from any of those disclosed herein and in the figures.
For example, the crtoyl alcohol pathway can be selected from (i) an
acetyl-CoA:acetyl-CoA acyltransferase; an acetoacetyl-CoA
reductase; a 3-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA
hydrolase, synthase, or transferase; a crotonate reductase; and a
crotonaldehyde reductase (alcohol forming); (ii) an
acetyl-CoA:acetyl-CoA acyltransferase; an acetoacetyl-CoA
reductase; a 3-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA
reductase (aldehyde forming); and a crotonaldehyde reductase
(alcohol forming); (iii) an acetyl-CoA:acetyl-CoA acyltransferase;
an acetoacetyl-CoA reductase; a 3-hydroxybutyryl-CoA dehydratase;
and a crotonyl-CoA reductase (alcohol forming); (iv) a
glutaconyl-CoA decarboxylase; a crotonyl-CoA hydrolase, synthase,
or transferase; a crotonate reductase; and a crotonaldehyde
reductase (alcohol forming); (v) a glutaconyl-CoA decarboxylase; a
crotonyl-CoA reductase (aldehyde forming); and a crotonaldehyde
reductase (alcohol forming); and (vi) a glutaconyl-CoA
decarboxylase; and a crotonyl-CoA reductase (alcohol forming).
(vii) a glutaryl-CoA dehydrogenase; a crotonyl-CoA hydrolase,
synthase, or transferase; a crotonate reductase; and a
crotonaldehyde reductase (alcohol forming); (viii) a glutaryl-CoA
dehydrogenase; a crotonyl-CoA reductase (aldehyde forming); and a
crotonaldehyde reductase (alcohol forming); (ix) a glutaryl-CoA
dehydrogenase; and a crotonyl-CoA reductase (alcohol forming); (x)
a 3-aminobutyryl-CoA deaminase; a crotonyl-CoA hydrolase, synthase,
or transferase; a crotonate reductase; and a crotonaldehyde
reductase (alcohol forming); (xi) a 3-aminobutyryl-CoA deaminase; a
crotonyl-CoA reductase (aldehyde forming); and a crotonaldehyde
reductase (alcohol forming); (xii) a 3-aminobutyryl-CoA deaminase;
and a crotonyl-CoA reductase (alcohol forming); (xiii) a
4-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA hydrolase,
synthase, or transferase; a crotonate reductase; and a
crotonaldehyde reductase (alcohol forming); (xiv) a
4-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA reductase
(aldehyde forming); and a crotonaldehyde reductase (alcohol
forming); and (xv) a 4-hydroxybutyryl-CoA dehydratase; and a
crotonyl-CoA reductase (alcohol forming).
[0084] Such a microbial organism of the invention comprising (a)
can further comprise an exogenous nucleic acid encoding an enzyme
selected from a pyruvate:ferredoxin oxidoreductase, an aconitase,
an isocitrate dehydrogenase, a succinyl-CoA synthetase, a
succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an
acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase,
an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations
thereof. Such a microbial organism comprising (b) can further
comprise an exogenous nucleic acid encoding an enzyme selected from
an aconitase, an isocitrate dehydrogenase, a succinyl-CoA
synthetase, a succinyl-CoA transferase, a fumarase, a malate
dehydrogenase, and combinations thereof. Such a microbial organism
can comprise two, three, four, five, six or seven exogenous nucleic
acids each encoding a crotyl alcohol pathway enzyme.
[0085] For example, the microbial organism can comprise exogenous
nucleic acids encoding each of the enzymes selected from (i) an
acetyl-CoA:acetyl-CoA acyltransferase; an acetoacetyl-CoA
reductase; a 3-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA
hydrolase, synthase, or transferase; a crotonate reductase; and a
crotonaldehyde reductase (alcohol forming); (ii) an
acetyl-CoA:acetyl-CoA acyltransferase; an acetoacetyl-CoA
reductase; a 3-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA
reductase (aldehyde forming); and a crotonaldehyde reductase
(alcohol forming); (iii) an acetyl-CoA:acetyl-CoA acyltransferase;
an acetoacetyl-CoA reductase; a 3-hydroxybutyryl-CoA dehydratase;
and a crotonyl-CoA reductase (alcohol forming); (iv) a
glutaconyl-CoA decarboxylase; a crotonyl-CoA hydrolase, synthase,
or transferase; a crotonate reductase; and a crotonaldehyde
reductase (alcohol forming); (v) a glutaconyl-CoA decarboxylase; a
crotonyl-CoA reductase (aldehyde forming); and a crotonaldehyde
reductase (alcohol forming); (vi) a glutaconyl-CoA decarboxylase;
and a crotonyl-CoA reductase (alcohol forming); (vii) a
glutaryl-CoA dehydrogenase; a crotonyl-CoA hydrolase, synthase, or
transferase; a crotonate reductase; and a crotonaldehyde reductase
(alcohol forming); (viii) a glutaryl-CoA dehydrogenase; a
crotonyl-CoA reductase (aldehyde forming); and a crotonaldehyde
reductase (alcohol forming); (ix) a glutaryl-CoA dehydrogenase; and
a crotonyl-CoA reductase (alcohol forming); (x) a
3-aminobutyryl-CoA deaminase; a crotonyl-CoA hydrolase, synthase,
or transferase; a crotonate reductase; and a crotonaldehyde
reductase (alcohol forming); (xi) a 3-aminobutyryl-CoA deaminase; a
crotonyl-CoA reductase (aldehyde forming); and a crotonaldehyde
reductase (alcohol forming); (xii) a 3-aminobutyryl-CoA deaminase;
and a crotonyl-CoA reductase (alcohol forming). (xiii) a
4-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA hydrolase,
synthase, or transferase; a crotonate reductase; and a
crotonaldehyde reductase (alcohol forming); (xiv) a
4-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA reductase
(aldehyde forming); and a crotonaldehyde reductase (alcohol
forming); and (xv) a 4-hydroxybutyryl-CoA dehydratase; and a
crotonyl-CoA reductase (alcohol forming).
[0086] Such microbial organisms of the invention can comprise two,
three, four or five exogenous nucleic acids each encoding enzymes
of (a), (b) or (c). For example, a microbial organism comprising
(a) can comprise three exogenous nucleic acids encoding ATP-citrate
lyase or citrate lyase, a fumarate reductase, and an
alpha-ketoglutarate:ferredoxin oxidoreductase; a microbial organism
comprising (b) can comprise four exogenous nucleic acids encoding
pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate
carboxylase or a phosphoenolpyruvate carboxykinase, a CO
dehydrogenase, and an H.sub.2 hydrogenase; or a microbial organism
comprising (c) can comprise two exogenous nucleic acids encoding CO
dehydrogenase and H.sub.2 hydrogenase. The invention additionally
provides methods for producing crotyl alcohol, comprising culturing
the non-naturally occurring microbial organism under conditions and
for a sufficient period of time to produce crotyl alcohol.
[0087] In some embodiments, the carbon feedstock and other cellular
uptake sources such as phosphate, ammonia, sulfate, chloride and
other halogens can be chosen to alter the isotopic distribution of
the atoms present in butadiene or crotyl alcohol or any butadiene
or crotyl alcohol pathway intermediate. The various carbon
feedstock and other uptake sources enumerated above will be
referred to herein, collectively, as "uptake sources." Uptake
sources can provide isotopic enrichment for any atom present in the
product butadiene or crotyl alcohol or butadiene or crotyl alcohol
pathway intermediate, or for side products generated in reactions
diverging away from a butadiene or crotyl alcohol pathway. Isotopic
enrichment can be achieved for any target atom including, for
example, carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus,
chloride or other halogens.
[0088] In some embodiments, the uptake sources can be selected to
alter the carbon-12, carbon-13, and carbon-14 ratios. In some
embodiments, the uptake sources can be selected to alter the
oxygen-16, oxygen-17, and oxygen-18 ratios. In some embodiments,
the uptake sources can be selected to alter the hydrogen,
deuterium, and tritium ratios. In some embodiments, the uptake
sources can be selected to alter the nitrogen-14 and nitrogen-15
ratios. In some embodiments, the uptake sources can be selected to
alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35 ratios. In
some embodiments, the uptake sources can be selected to alter the
phosphorus-31, phosphorus-32, and phosphorus-33 ratios. In some
embodiments, the uptake sources can be selected to alter the
chlorine-35, chlorine-36, and chlorine-37 ratios.
[0089] In some embodiments, a target isotopic ratio of an uptake
source can be obtained via synthetic chemical enrichment of the
uptake source. Such isotopically enriched uptake sources can be
purchased commercially or prepared in the laboratory. In some
embodiments, a target isotopic ratio of an uptake source can be
obtained by choice of origin of the uptake source in nature. In
some such embodiments, a source of carbon, for example, can be
selected from a fossil fuel-derived carbon source, which can be
relatively depleted of carbon-14, or an environmental carbon
source, such as CO.sub.2, which can possess a larger amount of
carbon-14 than its petroleum-derived counterpart.
[0090] Isotopic enrichment is readily assessed by mass spectrometry
using techniques known in the art such as Stable Isotope Ratio Mass
Spectrometry (SIRMS) and Site-Specific Natural Isotopic
Fractionation by Nuclear Magnetic Resonance (SNIF-NMR). Such mass
spectral techniques can be integrated with separation techniques
such as liquid chromatography (LC) and/or high performance liquid
chromatography (HPLC).
[0091] In some embodiments, the present invention provides
butadiene or crotyl alcohol or a butadiene or crotyl alcohol
intermediate that has a carbon-12, carbon-13, and carbon-14 ratio
that reflects an atmospheric carbon uptake source. In some such
embodiments, the uptake source is CO.sub.2. In some embodiments,
the present invention provides butadiene or crotyl alcohol or a
butadiene or crotyl alcohol intermediate that has a carbon-12,
carbon-13, and carbon-14 ratio that reflects petroleum-based carbon
uptake source. In some embodiments, the present invention provides
butadiene or crotyl alcohol or a butadiene or crotyl alcohol
intermediate that has a carbon-12, carbon-13, and carbon-14 ratio
that is obtained by a combination of an atmospheric carbon uptake
source with a petroleum-based uptake source. Such combination of
uptake sources is one means by which the carbon-12, carbon-13, and
carbon-14 ratio can be varied.
[0092] 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.
[0093] As disclosed herein, the intermediates crotonate,
3,5-dioxopentanoate, 5-hydroxy-3-oxopentanoate,
3-hydroxy-5-oxopentanoate, 3-oxoglutaryl-CoA and
3-hydroxyglutaryl-CoA, as well as other intermediates, are
carboxylic acids, which can occur in various ionized forms,
including fully protonated, partially protonated, and fully
deprotonated forms. Accordingly, the suffix "-ate," or the acid
form, can be used interchangeably to describe both the free acid
form as well as any deprotonated form, in particular since the
ionized form is known to depend on the pH in which the compound is
found. It is understood that carboxylate products or intermediates
includes ester forms of carboxylate products or pathway
intermediates, such as O-carboxylate and S-carboxylate esters. O-
and S-carboxylates can include lower alkyl, that is C1 to C6,
branched or straight chain carboxylates. Some such O- or
S-carboxylates include, without limitation, methyl, ethyl,
n-propyl, n-butyl, i-propyl, sec-butyl, and tert-butyl, pentyl,
hexyl O- or S-carboxylates, any of which can further possess an
unsaturation, providing for example, propenyl, butenyl, pentyl, and
hexenyl O- or S-carboxylates. O-carboxylates can be the product of
a biosynthetic pathway. Exemplary O-carboxylates accessed via
biosynthetic pathways can include, without limitation: methyl
crotanate; methy-3,5-dioxopentanoate;
methyl-5-hydroxy-3-oxopentanoate; methyl-3-hydroxy-5-oxopentanoate;
3-oxoglutaryl-CoA, methyl ester; 3-hydroxyglutaryl-CoA, methyl
ester; ethyl crotanate; ethyl-3,5-dioxopentanoate;
ethyl-5-hydroxy-3-xopentanoate; ethyl-3-hydroxy-5-oxopentanoate;
3-oxoglutaryl-CoA, ethyl ester; 3-hydroxyglutaryl-CoA, ethyl ester;
n-propyl crotanate; n-propyl-3,5-dioxopentanoate;
n-propyl-5-hydroxy-3-oxopentanoate;
n-propyl-3-hydroxy-5-oxopentanoate; 3-oxoglutaryl-CoA, n-propyl
ester; and 3-hydroxyglutaryl-CoA, n-propyl ester. Other
biosynthetically accessible O-carboxylates can include medium to
long chain groups, that is C7-C22, O-carboxylate esters derived
from fatty alcohols, such heptyl, octyl, nonyl, decyl, undecyl,
lauryl, tridecyl, myristyl, pentadecyl, cetyl, palmitolyl,
heptadecyl, stearyl, nonadecyl, arachidyl, heneicosyl, and behenyl
alcohols, any one of which can be optionally branched and/or
contain unsaturations. O-carboxylate esters can also be accessed
via a biochemical or chemical process, such as esterification of a
free carboxylic acid product or transesterification of an O- or
S-carboxylate. S-carboxylates are exemplified by CoA S-esters,
cysteinyl S-esters, alkylthioesters, and various aryl and
heteroaryl thioesters.
[0094] 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 butadiene or crotyl alcohol biosynthetic pathways.
Depending on the host microbial organism chosen for biosynthesis,
nucleic acids for some or all of a particular butadiene or crotyl
alcohol 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 butadiene
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 butadiene.
[0095] Host microbial organisms can be selected from, and the
non-naturally occurring microbial organisms generated in, for
example, bacteria, yeast, fungus or any of a variety of other
microorganisms applicable to fermentation processes. Exemplary
bacteria include species selected from Escherichia coli, Klebsiella
oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus
succinogenes, Mannheimia succiniciproducens, Rhizobium etli,
Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter
oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus
plantarum, Streptomyces coelicolor, Clostridium acetobutylicum,
Pseudomonas fluorescens, and Pseudomonas putida. Exemplary yeasts
or fungi include species selected from Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces
marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris,
Rhizopus arrhizus, Rhizopus oryzae, Yarrowia lipolytica, and the
like. 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. It is understood that any
suitable microbial host organism can be used to introduce metabolic
and/or genetic modifications to produce a desired product.
[0096] Depending on the butadiene or crotyl alcohol 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 butadiene or crotyl
alcohol pathway-encoding nucleic acid and up to all encoding
nucleic acids for one or more butadiene or crotyl alcohol
biosynthetic pathways. For example, butadiene biosynthesis can be
established in a host deficient in a pathway enzyme or protein
through exogenous expression of the corresponding encoding nucleic
acid. In a host deficient in all enzymes or proteins of a butadiene
pathway, exogenous expression of all enzyme or proteins in the
pathway can be included, although it is understood that all enzymes
or proteins of a pathway can be expressed even if the host contains
at least one of the pathway enzymes or proteins. For example,
exogenous expression of all enzymes or proteins in a pathway for
production of butadiene can be included, such as an
acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA
reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA
reductase (aldehyde forming), a crotonaldehyde reductase (alcohol
forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase
and a butadiene synthase (FIG. 2, steps A-H).
[0097] 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 butadiene or crotyl alcohol pathway deficiencies of
the selected host microbial organism. Therefore, a non-naturally
occurring microbial organism of the invention can have one, two,
three, four, five, six, seven, eight, nine or ten, up to all
nucleic acids encoding the enzymes or proteins constituting a
butadiene or crotyl alcohol biosynthetic pathway disclosed herein.
In some embodiments, the non-naturally occurring microbial
organisms also can include other genetic modifications that
facilitate or optimize butadiene or crotyl alcohol 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 butadiene or
crotyl alcohol pathway precursors such as acetyl-CoA,
glutaconyl-CoA, glutaryl-CoA, 3-aminobutyryl-CoA,
4-hydroxybutyryl-CoA, erythrose-4-phosphate or malonyl-CoA.
[0098] Generally, a host microbial organism is selected such that
it produces the precursor of a butadiene or crotyl alcohol 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, acetyl-CoA, glutaconyl-CoA,
glutaryl-CoA, 3-aminobutyryl-CoA, 4-hydroxybutyryl-CoA,
erythrose-4-phosphate or malonyl-CoA are produced naturally in a
host organism such as E. coli. A host organism can be engineered to
increase production of a precursor, as disclosed herein. In
addition, a microbial organism that has been engineered to produce
a desired precursor can be used as a host organism and further
engineered to express enzymes or proteins of a butadiene or crotyl
alcohol pathway.
[0099] In some embodiments, a non-naturally occurring microbial
organism of the invention is generated from a host that contains
the enzymatic capability to synthesize butadiene or crotyl alcohol.
In this specific embodiment it can be useful to increase the
synthesis or accumulation of a butadiene or a crotyl alcohol
pathway product to, for example, drive butadiene or crotyl alcohol
pathway reactions toward butadiene or crotyl alcohol production.
Increased synthesis or accumulation can be accomplished by, for
example, overexpression of nucleic acids encoding one or more of
the above-described butadiene or crotyl alcohol pathway enzymes or
proteins. Overexpression the enzyme or enzymes and/or protein or
proteins of the butadiene or crotyl alcohol 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 butadiene or crotyl alcohol,
through overexpression of one, two, three, four, five, six, seven,
eight, nine, or ten, that is, up to all nucleic acids encoding
butadiene or crotyl alcohol 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 butadiene or crotyl
alcohol biosynthetic pathway.
[0100] 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.
[0101] It is understood that, in methods of the invention, any of
the one or more exogenous nucleic acids can be introduced into a
microbial organism to produce a non-naturally occurring microbial
organism of the invention. The nucleic acids can be introduced so
as to confer, for example, a butadiene or crotyl alcohol
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 butadiene or crotyl
alcohol biosynthetic capability. For example, a non-naturally
occurring microbial organism having a butadiene biosynthetic
pathway can comprise at least two exogenous nucleic acids encoding
desired enzymes or proteins, such as the combination of a crotyl
alcohol kinase and a butadiene synthase, or alternatively a
4-(cytidine 5'-diphospho)-erythritol kinase and a butadiene
synthase, or alternatively a 1-hydroxy-2-butenyl 4-diphosphate
synthase and a butadiene synthase, or alternatively a
3-hydroxy-5-phosphonatooxypentanoate kinase and a butadiene
synthase, or alternatively a crotonyl-CoA hydrolase and a crotyl
alcohol diphosphokinase, or alternatively an erythrose reductase
and butadiene synthase, or alternatively an 3-oxo-glutaryl-CoA
reductase (CoA reducing and alcohol forming) and
3-Hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, or alternative an ATP-citrate lyase and butadiene
synthase, or alternatively a pyruvate:ferredoxin oxidoreductase and
a crotyl alcohol diphosphokinase, or alternatively a CO
dehydrogenase and a butadiene synthase, and the like. Thus, it is
understood that any combination of two or more enzymes or proteins
of a biosynthetic pathway can be included in a non-naturally
occurring microbial organism of the invention. Similarly, it is
understood that any combination of three or more enzymes or
proteins of a biosynthetic pathway can be included in a
non-naturally occurring microbial organism of the invention, for
example, a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase
and a butadiene synthase, or alternatively a 1-hydroxy-2-butenyl
4-diphosphate synthase, a 1-hydroxy-2-butenyl 4-diphosphate
reductase, and a butadiene synthase, or alternatively an
3-oxoglutaryl-CoA reductase, a 3-hydroxy-5-oxopentanoate reductase,
and a butadiene synthase, or alternatively an acetyl-CoA:acetyl-CoA
acyltransferase, a crotyl alcohol kinase and a butadiene synthase,
or alternatively a glutaconyl-CoA decarboxylase, a crotonyl-CoA
reductase (alcohol forming), and a crotyl alcohol diphosphokinase,
or alternatively a an erythrose-4-phosphate kinase, a 4-(cytidine
5'-diphospho)-erythritol kinase and a 1-hydroxy-2-butenyl
4-diphosphate synthase, or alternatively a 3,5-dioxopentanoate
reductase (aldehyde reducing), a butenyl 4-diphosphate isomerase,
and a butadiene synthase, or alternatively a citrate lyase, a
fumarate reductase, and a butadiene synthase, or alternatively a
phosphoenolpyruvate carboxylase, a CO dehydrogenase, and a
butadiene synthase, or alternatively an
alpha-ketoglutarate:ferredoxin oxidoreductase, an H.sub.2
hydrogenase, and a crotyl alcohol diphosphokinase, 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,
such as a crotonaldehyde reductase (alcohol forming), a crotyl
alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene
synthase, or alternatively a 1-hydroxy-2-butenyl 4-diphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate reductase, a butenyl
4-diphosphate isomerase and butadiene synthase, or alternatively a
3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate kinase,
a butenyl 4-diphosphate isomerase and a butadiene synthase, or
alternatively an erythrose-4-phosphate reductase, an
erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase and a butadiene synthase, or
alternatively an 3-aminobutyryl-CoA deaminase, a crotonyl-CoA
reductase (alcohol forming), a crotyl alcohol diphosphokinase and a
butadiene synthase, or alternatively an erythrose reductase, a
4-(cytidine 5'-diphospho)-erythritol kinase, an erythritol
2,4-cyclodiphosphate synthase and a 1-hydroxy-2-butenyl
4-diphosphate reductase, or alternatively a malonyl-CoA:acetyl-CoA
acyltransferase, a 3-hydroxyglutaryl-CoA reductase (alcohol
forming), a butenyl 4-diphosphate isomerase and a butadiene
synthase, or alternatively citrate lyase, a fumarate reductase, an
alpha-ketoglutarate:ferredoxin oxidoreductase, and a butadiene
synthase, or alternatively a phosphoenolpyruvate carboxykinase, a
CO dehydrogenase, an H.sub.2 hydrogenase and a crotyl alcohol
diphosphokinase, or alternatively a pyruvate:ferredoxin
oxidoreductase, a phosphoenolpyruvate carboxylase, a
phosphoenolpyruvate carboxykinase, and a glutaconyl-CoA
decarboxylase, or more enzymes or proteins of a biosynthetic
pathway as disclosed herein can be included in a non-naturally
occurring microbial organism of the invention, as desired, so long
as the combination of enzymes and/or proteins of the desired
biosynthetic pathway results in production of the corresponding
desired product.
[0102] In addition to the biosynthesis of butadiene or crotyl
alcohol 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 butadiene other than use of the butadiene producers is
through addition of another microbial organism capable of
converting a butadiene pathway intermediate to butadiene. One such
procedure includes, for example, the fermentation of a microbial
organism that produces a butadiene pathway intermediate. The
butadiene pathway intermediate can then be used as a substrate for
a second microbial organism that converts the butadiene pathway
intermediate to butadiene. The butadiene pathway intermediate can
be added directly to another culture of the second organism or the
original culture of the butadiene 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.
[0103] 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,
butadiene or crotyl alcohol. 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 butadiene 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,
butadiene 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
butadiene intermediate and the second microbial organism converts
the intermediate to butadiene.
[0104] 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 butadiene or crotyl alcohol.
[0105] Sources of encoding nucleic acids for a butadiene or crotyl
alcohol 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 aceti, Acidaminococcus fermentans, Acinetobacter
baylyi, Acinetobacter calcoaceticus, Acinetobacter sp. ADP1,
Acinetobacter sp. Strain M-1, Actinobacillus succinogenes,
Aeropyrum pernix, Allochromatium vinosum DSM 180,
Anaerobiospirillum succiniciproducens, Aquifex aeolicus, Aquifex
aeolicus, Arabidopsis thaliana, Arabidopsis thaliana col,
Archaeoglobus fulgidus, Archaeoglobus fulgidus DSM 4304,
Aromatoleum aromaticum EbN1, Ascaris suum, Aspergillus nidulans,
Azoarcus sp. CIB, Azoarcus sp. T, Azotobacter vinelandii DJ,
Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Balnearium
lithotrophicum, Bos Taurus, BRC 13350, Brucella melitensis,
Burkholderia ambifaria AMMD, Burkholderia phymatum,
butyrate-producing bacterium L2-50, Campylobacter curvus 525.92,
Campylobacter jejuni, Candida albicans, Candida magnolia,
Carboxydothermus hydrogenoformans, Chlorobium phaeobacteroides DSM
266, Chlorobium limicola, Chlorobium tepidum, Chloroflexus
aurantiacus, Citrobacter youngae ATCC 29220, Clostridium
acetobutylicum, Clostridium aminobutyricum, Clostridium
beijerinckii, Clostridium beijerinckii NCIMB 8052, Clostridium
beijerinckii NRRL B593, Clostridium botulinum C str. Eklund,
Clostridium carboxidivorans P7, Clostridium cellulolyticum H10,
Clostridium kluyveri, Clostridium kluyveri DSM 555, Clostridium
novyi NT, Clostridium pasteurianum, Clostridium
saccharoperbutylacetonicum, Corynebacterium glutamicum,
Corynebacterium glutamicum ATCC 13032, Cupriavidus taiwanensis,
Cyanobium PCC7001, Desulfovibrio africanus, DesulfoVibrio
desulfuricans G20, Desulfovibrio desulfuricans subsp. desulfuricans
str. ATCC 27774, Desulfovibrio fructosovorans JJ, Desulfovibrio
vulgaris str. Hildenborough, Dictyostelium discoideum AX4 DSM 266,
Enterococcus faecalis, Erythrobacter sp. NAP1, Escherichia coli
K12, Escherichia coli str. K-12 substr. MG1655, Eubacterium rectale
ATCC 33656, Fusobacterium nucleatum, Fusobacterium nucleatum subsp.
nucleatum ATCC 25586, Geobacillus thermoglucosidasius, Geobacter
metallireducens GS-15, Geobacter sulfurreducens, Haematococcus
pluvialis, Haemophilus influenza, Haloarcula marismortui,
Haloarcula marismortui ATCC 43049, Helicobacter pylori,
Helicobacter pylori 26695, Homo sapiens, Hydrogenobacter
thermophilus, Klebsiella pneumonia, Klebsiella pneumonia,
Lactobacillus plantarum, Leuconostoc mesenteroides, Leuconostoc
mesenteroides, Mannheimia succiniciproducens, marine gamma
proteobacterium HTCC2080, Metallosphaera sedula, Methanocaldococcus
jannaschii, Methanosarcina thermophila, Methanothermobacter
thermautotrophicus, Methylobacterium extorquens, Moorella
thermoacetica, Mus musculus, Mycobacterium avium subsp.
paratuberculosis K-10, Mycobacterium bovis BCG, Mycobacterium
marinum M, Mycobacterium smegmatis, Mycobacterium smegmatis MC2
155, Mycobacterium tuberculosis, Mycoplasma pneumoniae M129,
Nocardia farcinica IFM 10152, Nocardia iowensis (sp. NRRL 5646),
Nostoc sp. PCC 7120, Oryctolagus cuniculus, Paracoccus
denitrificans, Pelobacter carbinolicus DSM 2380, Pelotomaculum
thermopropionicum, Penicillium chrysogenum, Populus alba, Populus
tremula.times.Populus alba, Porphyromonas ingivalis, Porphyromonas
gingivalis W83, Pseudomonas aeruginosa, Pseudomonas aeruginosa
PA01, Pseudomonas fluorescens, Pseudomonas fluorescens Pf-5,
Pseudomonas knackmussii (B13), Pseudomonas putida, Pseudomonas
putida E23, Pseudomonas putida KT2440, Pseudomonas sp, Pueraria
Montana, Pyrobaculum aerophilum str. IM2, Pyrococcus furiosus,
Ralstonia eutropha, Ralstonia eutropha H16, Ralstonia
metallidurans, Rattus norvegicus, Rhodobacter capsulatus,
Rhodobacter spaeroides, Rhodococcus rubber, Rhodopseudomonas
palustris, Rhodopseudomonas palustris, Rhodopseudomonas palustris
CGA009, Rhodospirillum rubrum, Roseburia intestinalis L1-82,
Roseburia inulinivorans DSM 16841, Roseburia sp. A2-183,
Roseiflexus castenholzii, Saccharomyces cerevisiae, Saccharomyces
cerevisiae, Saccharopolyspora rythraea NRRL 2338, Salmonella
enteric, Salmonella enterica subsp., rizonae serovar, Salmonella
typhimurium, Schizosaccharomyces pombe, Simmondsia chinensis,
Sinorhizobium meliloti, Sordaria macrospora, Staphylococcus ureus,
Streptococcus pneumonia, Streptomyces coelicolor, Streptomyces
griseus subsp. griseus, Streptomyces griseus subsp. griseus NBRC
13350, Streptomyces sp. ACT-1, Sulfolobus acidocalarius, Sulfolobus
shibatae, Sulfolobus solfataricus, Sulfolobus sp. strain 7,
Sulfolobus tokodaii, Sulfurihydrogenibium subterraneum,
Sulfurimonas denitrificans, Synechocystis sp. strain PCC6803,
Syntrophus, ciditrophicus, Thauera aromatica, Thermoanaerobacter
brockii HTD4, Thermoanaerobacter tengcongensis MB4, Thermocrinis
albus, Thermosynechococcus elongates, Thermotoga maritime,
Thermotoga maritime MSB8, Thermus hermophilus HB8, Thermus
thermophilus, Thermus thermophilus, Thiobacillus denitrificans,
Thiocapsa roseopersicina, Trichomonas vaginalis G3,
Trichosporonoides megachiliensis, Trypanosoma brucei, Tsukamurella
paurometabola DSM 20162, Yarrowia lipolytica, Yersinia intermedia
ATCC 29909, Zea mays, Zoogloea ramigera, Zygosaccharomyces rouxii,
Zymomonas mobilis, 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 butadiene or crotyl
alcohol 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 butadiene or crotyl alcohol 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.
[0106] In some instances, such as when an alternative butadiene or
crotyl alcohol biosynthetic pathway exists in an unrelated species,
butadiene or crotyl alcohol 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 butadiene or crotyl
alcohol.
[0107] Methods for constructing and testing the expression levels
of a non-naturally occurring butadiene or crotyl alcohol-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).
[0108] Exogenous nucleic acid sequences involved in a pathway for
production of butadiene or crotyl alcohol 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.
[0109] An expression vector or vectors can be constructed to
include one or more butadiene or crotyl alcohol 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.
[0110] In some embodiments, the invention provides a method for
producing butadiene that includes culturing a non-naturally
occurring microbial organism, including a microbial organism having
a butadiene pathway, the butadiene pathway including at least one
exogenous nucleic acid encoding a butadiene pathway enzyme
expressed in a sufficient amount to produce butadiene, the
butadiene pathway including an acetyl-CoA:acetyl-CoA
acyltransferase, an acetoacetyl-CoA reductase, a
3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene
synthase, a crotonyl-CoA hydrolase, synthetase, or transferase, a
crotonate reductase, a crotonyl-CoA reductase (alcohol forming), a
glutaconyl-CoA decarboxylase, a glutaryl-CoA dehydrogenase, an
3-aminobutyryl-CoA deaminase, a 4-hydroxybutyryl-CoA dehydratase or
a crotyl alcohol diphosphokinase (FIG. 2). In one aspect, the
method includes a microbial organism having a butadiene pathway
including an acetyl-CoA:acetyl-CoA acyltransferase, an
acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a
crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase and a butadiene synthase (FIG. 2,
steps A-H). In one aspect, the method includes a microbial organism
having a butadiene pathway including an acetyl-CoA:acetyl-CoA
acyltransferase, an acetoacetyl-CoA reductase, a
3-hydroxybutyryl-CoA dehydratase, a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA
reductase (alcohol forming) (FIG. 2, steps A-C, K, F, G, H). In one
aspect, the method includes a microbial organism having a butadiene
pathway including an acetyl-CoA:acetyl-CoA acyltransferase, an
acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a
butadiene synthase, a crotonyl-CoA reductase (alcohol forming) and
a crotyl alcohol diphosphokinase (FIG. 2, steps A-C, K, P, H). In
one aspect, the method includes a microbial organism having a
butadiene pathway including an acetyl-CoA:acetyl-CoA
acyltransferase, an acetoacetyl-CoA reductase, a
3-hydroxybutyryl-CoA dehydratase, a crotonaldehyde reductase
(alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate
kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase,
or transferase and a crotonate reductase, (FIG. 2, steps A-C, I, J,
E, F, G, H). In one aspect, the method includes a microbial
organism having a butadiene pathway including an
acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA
reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonaldehyde
reductase (alcohol forming), a butadiene synthase, a crotonyl-CoA
hydrolase, synthetase or transferase, a crotonate reductase and a
crotyl alcohol diphosphokinase (FIG. 2, steps A-C, I, J, E, P, H).
In one aspect, the method includes a microbial organism having a
butadiene pathway including an acetyl-CoA:acetyl-CoA
acyltransferase, an acetoacetyl-CoA reductase, a
3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
butadiene synthase and a crotyl alcohol diphosphokinase (FIG. 2,
steps A-E, P, H). In one aspect, the method includes a microbial
organism having a butadiene pathway including a glutaconyl-CoA
decarboxylase, a crotonyl-CoA reductase (aldehyde forming), a
crotonaldehyde reductase (alcohol forming), a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase
(FIG. 2, steps L, D-H). In one aspect, the method includes a
microbial organism having a butadiene pathway including a
glutaconyl-CoA decarboxylase, a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA
reductase (alcohol forming) (FIG. 2, steps L, K, F, G, H). In one
aspect, the method includes a microbial organism having a butadiene
pathway including a glutaconyl-CoA decarboxylase, a butadiene
synthase, a crotonyl-CoA reductase (alcohol forming) and a crotyl
alcohol diphosphokinase (FIG. 2, steps L, K, P, H). In one aspect,
the method includes a microbial organism having a butadiene pathway
including a glutaconyl-CoA decarboxylase, a crotonaldehyde
reductase (alcohol forming), a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase, a butadiene synthase, a crotonyl-CoA
hydrolase, synthetase, or transferase and a crotonate reductase
(FIG. 2, steps L, I, J, E, F, G, H). In one aspect, the method
includes a microbial organism having a butadiene pathway including
a glutaconyl-CoA decarboxylase, a crotonaldehyde reductase (alcohol
forming), a butadiene synthase, a crotonyl-CoA hydrolase,
synthetase or transferase, a crotonate reductase and a crotyl
alcohol diphosphokinase (FIG. 2, steps L, I, J, E, P, H). In one
aspect, the method includes a microbial organism having a butadiene
pathway including a 3-hydroxybutyryl-CoA dehydratase, a
crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a butadiene a glutaconyl-CoA
decarboxylase and a crotyl alcohol diphosphokinase (FIG. 2, steps
L, C, D, E, P, H). In one aspect, the method includes a microbial
organism having a butadiene pathway including a glutaryl-CoA
dehydrogenase, a crotonyl-CoA reductase (aldehyde forming), a
crotonaldehyde reductase (alcohol forming), a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase
(FIG. 2, steps M, D-H). In one aspect, the method includes a
microbial organism having a butadiene pathway including a
glutaryl-CoA dehydrogenase, a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA
reductase (alcohol forming) (FIG. 2, steps M, K, F, G, H). In one
aspect, the method includes a microbial organism having a butadiene
pathway including a glutaryl-CoA dehydrogenase, a butadiene
synthase, a crotonyl-CoA reductase (alcohol forming) and a crotyl
alcohol diphosphokinase (FIG. 2, steps M, K, P, H). In one aspect,
the method includes a microbial organism having a butadiene pathway
including a glutaryl-CoA dehydrogenase, a crotonaldehyde reductase
(alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate
kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase,
or transferase and a crotonate reductase (FIG. 2, steps M, I, J, E,
F, G, H). In one aspect, the method includes a microbial organism
having a butadiene pathway including a glutaryl-CoA dehydrogenase,
a crotonaldehyde reductase (alcohol forming), a butadiene synthase,
a crotonyl-CoA hydrolase, synthetase or transferase, a crotonate
reductase and a crotyl alcohol diphosphokinase (FIG. 2, steps M, I,
J, E, P, H). In one aspect, the method includes a microbial
organism having a butadiene pathway including a
3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
butadiene synthase, a glutaryl-CoA dehydrogenase and a crotyl
alcohol diphosphokinase (FIG. 2, steps M, C, D, E, P, H). In one
aspect, the method includes a microbial organism having a butadiene
pathway including an 3-aminobutyryl-CoA deaminase, a crotonyl-CoA
reductase (aldehyde forming), a crotonaldehyde reductase (alcohol
forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase
and a butadiene synthase (FIG. 2, steps N, D-H). In one aspect, the
method includes a microbial organism having a butadiene pathway
including an 3-aminobutyryl-CoA deaminase, a crotyl alcohol kinase,
a 2-butenyl-4-phosphate kinase, a butadiene synthase and
crotonyl-CoA reductase (alcohol forming) (FIG. 2, steps N, K, F, G,
H). In one aspect, the method includes a microbial organism having
a butadiene pathway including an 3-aminobutyryl-CoA deaminase, a
butadiene synthase, a crotonyl-CoA reductase (alcohol forming) and
a crotyl alcohol diphosphokinase (FIG. 2, steps N, K, P, H). In one
aspect, the method includes a microbial organism having a butadiene
pathway including an 3-aminobutyryl-CoA deaminase, a crotonaldehyde
reductase (alcohol forming), a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase, a butadiene synthase, a crotonyl-CoA
hydrolase, synthetase, or transferase and a crotonate reductase
(FIG. 2, steps N, I, J, E, F, G, H). In one aspect, the method
includes a microbial organism having a butadiene pathway including
an 3-aminobutyryl-CoA deaminase, a crotonaldehyde reductase
(alcohol forming), a butadiene synthase, a crotonyl-CoA hydrolase,
synthetase or transferase, a crotonate reductase and a crotyl
alcohol diphosphokinase (FIG. 2, steps N, I, J, E, P, H). In one
aspect, the method includes a microbial organism having a butadiene
pathway including a 3-hydroxybutyryl-CoA dehydratase, a
crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a butadiene synthase, a
3-aminobutyryl-CoA deaminase and a crotyl alcohol diphosphokinase
(FIG. 2, steps N, C, D, E, P, H). In one aspect, the method
includes a microbial organism having a butadiene pathway including
a 4-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a
butadiene synthase (FIG. 2, steps O, D-H). In one aspect, the
method includes a microbial organism having a butadiene pathway
including a 4-hydroxybutyryl-CoA dehydratase, a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase and
crotonyl-CoA reductase (alcohol forming) (FIG. 2, steps O, K, F, G,
H). In one aspect, the method includes a microbial organism having
a butadiene pathway including a 4-hydroxybutyryl-CoA dehydratase, a
butadiene synthase, a crotonyl-CoA reductase (alcohol forming) and
a crotyl alcohol diphosphokinase (FIG. 2, steps O, K, P, H). In one
aspect, the method includes a microbial organism having a butadiene
pathway including a 4-hydroxybutyryl-CoA dehydratase, a
crotonaldehyde reductase (alcohol forming), a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a
crotonyl-CoA hydrolase, synthetase, or transferase and a crotonate
reductase (FIG. 2, steps O, I, J, E, F, G, H). In one aspect, the
method includes a microbial organism having a butadiene pathway
including a 4-hydroxybutyryl-CoA dehydratase, a crotonaldehyde
reductase (alcohol forming), a butadiene synthase, a crotonyl-CoA
hydrolase, synthetase or transferase, a crotonate reductase and a
crotyl alcohol diphosphokinase (FIG. 2, steps O, I, J, E, P, H). In
one aspect, the method includes a microbial organism having a
butadiene pathway including a 3-hydroxybutyryl-CoA dehydratase, a
crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a butadiene synthase, a
4-hydroxybutyryl-CoA dehydratase and a crotyl alcohol
diphosphokinase (FIG. 2, steps O, C, D, E, P, H).
[0111] In some embodiments, the invention provides a method for
producing butadiene that includes culturing a non-naturally
occurring microbial organism, including a microbial organism having
a butadiene pathway, the butadiene pathway including at least one
exogenous nucleic acid encoding a butadiene pathway enzyme
expressed in a sufficient amount to produce butadiene, the
butadiene pathway including an erythrose-4-phosphate reductase, an
erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate reductase, a butenyl
4-diphosphate isomerase, a butadiene synthase, an
erythrose-4-phosphate kinase, an erythrose reductase or an
erythritol kinase (FIG. 3). In one aspect, the method includes a
microbial organism having a butadiene pathway including an
erythrose-4-phosphate reductase, an erythritol-4-phospate
cytidylyltransferase, a 4-(cytidine 5'-diphospho)-erythritol
kinase, an erythritol 2,4-cyclodiphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate synthase, a 1-hydroxy-2-butenyl
4-diphosphate reductase and a butadiene synthase (FIG. 3, steps
A-F, and H). In one aspect, the method includes a microbial
organism having a butadiene pathway including an
erythrose-4-phosphate reductase, an erythritol-4-phospate
cytidylyltransferase, a 4-(cytidine 5'-diphospho)-erythritol
kinase, an erythritol 2,4-cyclodiphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate synthase, a 1-hydroxy-2-butenyl
4-diphosphate reductase, a butenyl 4-diphosphate isomerase and
butadiene synthase (FIG. 3, steps A-H). In one aspect, the method
includes a microbial organism having a butadiene pathway including
an erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate reductase, a butadiene synthase,
an erythrose-4-phosphate kinase, an erythrose reductase and a
erythritol kinase (FIG. 3, steps I, J, K, B-F, H). In one aspect,
the method includes a microbial organism having a butadiene pathway
including an erythritol-4-phospate cytidylyltransferase, a
4-(cytidine 5'-diphospho)-erythritol kinase, an erythritol
2,4-cyclodiphosphate synthase, a 1-hydroxy-2-butenyl 4-diphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate reductase, a butenyl
4-diphosphate isomerase, a butadiene synthase, an
erythrose-4-phosphate kinase, an erythrose reductase and an
erythritol kinase (FIG. 3, steps I, J, K, B-H).
[0112] In some embodiments, the invention provides a method for
producing butadiene that includes culturing a non-naturally
occurring microbial organism, including a microbial organism having
a butadiene pathway, the butadiene pathway including at least one
exogenous nucleic acid encoding a butadiene pathway enzyme
expressed in a sufficient amount to produce butadiene, the
butadiene pathway including a malonyl-CoA:acetyl-CoA
acyltransferase, an 3-oxoglutaryl-CoA reductase (ketone-reducing),
a 3-hydroxyglutaryl-CoA reductase (aldehyde forming), a
3-hydroxy-5-oxopentanoate reductase, a 3,5-dihydroxypentanoate
kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, a 3-hydroxyglutaryl-CoA reductase (alcohol forming), an
3-oxoglutaryl-CoA reductase (aldehyde forming), a
3,5-dioxopentanoate reductase (ketone reducing), a
3,5-dioxopentanoate reductase (aldehyde reducing), a
5-hydroxy-3-oxopentanoate reductase or an 3-oxo-glutaryl-CoA
reductase (CoA reducing and alcohol forming) (FIG. 4). In one
aspect, the method includes a microbial organism having a butadiene
pathway including a malonyl-CoA:acetyl-CoA acyltransferase, an
3-oxoglutaryl-CoA reductase (ketone-reducing), a
3-hydroxyglutaryl-CoA reductase (aldehyde forming), a
3-hydroxy-5-oxopentanoate reductase, a 3,5-dihydroxypentanoate
kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase and a butadiene
synthase (FIG. 4, steps A-I). In one aspect, the method includes a
microbial organism having a butadiene pathway including a
malonyl-CoA:acetyl-CoA acyltransferase, a 3,5-dihydroxypentanoate
kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, an 3-oxoglutaryl-CoA reductase (aldehyde forming), a
3,5-dioxopentanoate reductase (aldehyde reducing) and a
5-hydroxy-3-oxopentanoate reductase. (FIG. 4, steps A, K, M, N, E,
F, G, H, I). In one aspect, the method includes a microbial
organism having a butadiene pathway including a
malonyl-CoA:acetyl-CoA acyltransferase, a 3-hydroxy-5-oxopentanoate
reductase, a 3,5-dihydroxypentanoate kinase, a
3-Hydroxy-5-phosphonatooxypentanoate kinase, a
3-Hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, an 3-oxoglutaryl-CoA reductase (aldehyde forming) and a
3,5-dioxopentanoate reductase (ketone reducing). (FIG. 4, steps A,
K, L, D, E, F, G, H, I). In one aspect, the method includes a
microbial organism having a butadiene pathway including a
malonyl-CoA:acetyl-CoA acyltransferase, a 3,5-dihydroxypentanoate
kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, a 5-hydroxy-3-oxopentanoate reductase and a
3-oxo-glutaryl-CoA reductase (CoA reducing and alcohol forming).
(FIG. 4, steps A, O, N, E, F, G, H, I). In one aspect, the method
includes a microbial organism having a butadiene pathway including
a malonyl-CoA:acetyl-CoA acyltransferase, an 3-oxoglutaryl-CoA
reductase (ketone-reducing), a 3,5-dihydroxypentanoate kinase, a
3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase and a 3-hydroxyglutaryl-CoA reductase (alcohol forming).
(FIG. 4, steps A, B, J, E, F, G, H, I).
[0113] In some embodiments, the invention provides a method for
producing butadiene that includes culturing a non-naturally
occurring microbial organism as described herein, including a
microbial organism having a butadiene pathway comprising at least
one exogenous nucleic acid encoding a butadiene pathway enzyme
expressed in a sufficient amount to produce butadiene. Such a
microbial organism can further comprise (a) a reductive TCA pathway
comprising at least one exogenous nucleic acid encoding a reductive
TCA pathway enzyme, wherein the at least one exogenous nucleic acid
is selected from an ATP-citrate lyase, a citrate lyase, a
citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase,
and an alpha-ketoglutarate:ferredoxin oxidoreductase; (b) a
reductive TCA pathway comprising at least one exogenous nucleic
acid encoding a reductive TCA pathway enzyme, wherein the at least
one exogenous nucleic acid is selected from a pyruvate:ferredoxin
oxidoreductase, a phosphoenolpyruvate carboxylase, a
phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an
H.sub.2 hydrogenase; or (c) at least one exogenous nucleic acid
encodes an enzyme selected from a CO dehydrogenase, an H.sub.2
hydrogenase, and combinations thereof. In such a microbial
organism, a butadiene pathway can comprise a butadiene pathway
disclosed herein. For example, the butadien pathway can be selected
from: (i) an acetyl-CoA:acetyl-CoA acyltransferase, an
acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a
crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase and a butadiene synthase; (ii) an
acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA
reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase and
crotonyl-CoA reductase (alcohol forming); (iii) an
acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA
reductase, a 3-hydroxybutyryl-CoA dehydratase, a butadiene
synthase, a crotonyl-CoA reductase (alcohol forming) and a crotyl
alcohol diphosphokinase; (iv) an acetyl-CoA:acetyl-CoA
acyltransferase, an acetoacetyl-CoA reductase, a
3-hydroxybutyryl-CoA dehydratase, a crotonaldehyde reductase
(alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate
kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase
or transferase and a crotonate reductase; (v) an
acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA
reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonaldehyde
reductase (alcohol forming), a butadiene synthase, a crotonyl-CoA
hydrolase, synthetase or transferase, a crotonate reductase and a
crotyl alcohol diphosphokinase; (vi) an acetyl-CoA:acetyl-CoA
acyltransferase, an acetoacetyl-CoA reductase, a
3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
butadiene synthase and a crotyl alcohol diphosphokinase. (vii) a
glutaconyl-CoA decarboxylase, a crotonyl-CoA reductase (aldehyde
forming), a crotonaldehyde reductase (alcohol forming), a crotyl
alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene
synthase. (viii) a glutaconyl-CoA decarboxylase, a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase and
crotonyl-CoA reductase (alcohol forming); (ix) a glutaconyl-CoA
decarboxylase, a butadiene synthase, a crotonyl-CoA reductase
(alcohol forming) and a crotyl alcohol diphosphokinase; (x) a
glutaconyl-CoA decarboxylase, a crotonaldehyde reductase (alcohol
forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase,
a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or
transferase and a crotonate reductase; (xi) a glutaconyl-CoA
decarboxylase, a crotonaldehyde reductase (alcohol forming), a
butadiene synthase, a crotonyl-CoA hydrolase, synthetase or
transferase, a crotonate reductase and a crotyl alcohol
diphosphokinase; (xii) a 3-hydroxybutyryl-CoA dehydratase, a
crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a butadiene a glutaconyl-CoA
decarboxylase and a crotyl alcohol diphosphokinase; (xiii) a
glutaryl-CoA dehydrogenase, a crotonyl-CoA reductase (aldehyde
forming), a crotonaldehyde reductase (alcohol forming), a crotyl
alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene
synthase; (xiv) a glutaryl-CoA dehydrogenase, a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase and
crotonyl-CoA reductase (alcohol forming); (xv) a glutaryl-CoA
dehydrogenase, a butadiene synthase, a crotonyl-CoA reductase
(alcohol forming) and a crotyl alcohol diphosphokinase; (xvi) a
glutaryl-CoA dehydrogenase, a crotonaldehyde reductase (alcohol
forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase,
a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or
transferase and a crotonate reductase; (xvii) a glutaryl-CoA
dehydrogenase, a crotonaldehyde reductase (alcohol forming), a
butadiene synthase, a crotonyl-CoA hydrolase, synthetase or
transferase, a crotonate reductase and a crotyl alcohol
diphosphokinase; (xviii) a 3-hydroxybutyryl-CoA dehydratase, a
crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a butadiene synthase, a glutaryl-CoA
dehydrogenase and a crotyl alcohol diphosphokinase; (xix) an
3-aminobutyryl-CoA deaminase, a crotonyl-CoA reductase (aldehyde
forming), a crotonaldehyde reductase (alcohol forming), a crotyl
alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene
synthase; (xx) an 3-aminobutyryl-CoA deaminase, a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase and
crotonyl-CoA reductase (alcohol forming); (xxi) an
3-aminobutyryl-CoA deaminase, a butadiene synthase, a crotonyl-CoA
reductase (alcohol forming) and a crotyl alcohol diphosphokinase;
(xxii) an 3-aminobutyryl-CoA deaminase, a crotonaldehyde reductase
(alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate
kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase
or transferase and a crotonate reductase; (xxiii) an
3-aminobutyryl-CoA deaminase, a crotonaldehyde reductase (alcohol
forming), a butadiene synthase, a crotonyl-CoA hydrolase,
synthetase or transferase, a crotonate reductase and a crotyl
alcohol diphosphokinase; (xxiv) a 3-hydroxybutyryl-CoA dehydratase,
a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a butadiene synthase, a
3-aminobutyryl-CoA deaminase and a crotyl alcohol diphosphokinase;
(xxv) a 4-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a
butadiene synthase; (xxvi) a 4-hydroxybutyryl-CoA dehydratase, a
crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene
synthase and crotonyl-CoA reductase (alcohol forming); (xxvii) a
4-hydroxybutyryl-CoA dehydratase, a butadiene synthase, a
crotonyl-CoA reductase (alcohol forming) and a crotyl alcohol
diphosphokinase; (xxviii) a 4-hydroxybutyryl-CoA dehydratase, a
crotonaldehyde reductase (alcohol forming), a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a
crotonyl-CoA hydrolase, synthetase or transferase and a crotonate
reductase; (xxix) a 4-hydroxybutyryl-CoA dehydratase, a
crotonaldehyde reductase (alcohol forming), a butadiene synthase, a
crotonyl-CoA hydrolase, synthetase or transferase, a crotonate
reductase and a crotyl alcohol diphosphokinase; (xxx) a
3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
butadiene synthase, a 4-hydroxybutyryl-CoA dehydratase and a crotyl
alcohol diphosphokinase; (xxxi) an erythrose-4-phosphate reductase,
an erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate reductase and a butadiene
synthase; (xxxii) an erythrose-4-phosphate reductase, an
erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate reductase, a butenyl
4-diphosphate isomerase and a butadiene synthase; (xxxiii) an
erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate reductase, a butadiene synthase,
an erythrose-4-phosphate kinase, an erythrose reductase and a
erythritol kinase; (xxxiv) an erythritol-4-phospate
cytidylyltransferase, a 4-(cytidine 5'-diphospho)-erythritol
kinase, an erythritol 2,4-cyclodiphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate synthase, a 1-hydroxy-2-butenyl
4-diphosphate reductase, a butenyl 4-diphosphate isomerase, a
butadiene synthase, an erythrose-4-phosphate kinase, an erythrose
reductase and an erythritol kinase; (xxxv) a malonyl-CoA:acetyl-CoA
acyltransferase, an 3-oxoglutaryl-CoA reductase (ketone-reducing),
a 3-hydroxyglutaryl-CoA reductase (aldehyde forming), a
3-hydroxy-5-oxopentanoate reductase, a 3,5-dihydroxypentanoate
kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase and a butadiene
synthase; (xxxvi) a malonyl-CoA:acetyl-CoA acyltransferase, a
3,5-dihydroxypentanoate kinase, a
3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, an 3-oxoglutaryl-CoA reductase (aldehyde forming), a
3,5-dioxopentanoate reductase (aldehyde reducing) and a
5-hydroxy-3-oxopentanoate reductase; (xxxvii) a
malonyl-CoA:acetyl-CoA acyltransferase, a 3-hydroxy-5-oxopentanoate
reductase, a 3,5-dihydroxypentanoate kinase, a
3-Hydroxy-5-phosphonatooxypentanoate kinase, a
3-Hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, an 3-oxoglutaryl-CoA reductase (aldehyde forming) and a
3,5-dioxopentanoate reductase (ketone reducing); (xxxviii) a
malonyl-CoA:acetyl-CoA acyltransferase, a 3,5-dihydroxypentanoate
kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, a 5-hydroxy-3-oxopentanoate reductase and a
3-oxo-glutaryl-CoA reductase (CoA reducing and alcohol forming);
and (xxxix) a butadiene pathway comprising a malonyl-CoA:acetyl-CoA
acyltransferase, an 3-oxoglutaryl-CoA reductase (ketone-reducing),
a 3,5-dihydroxypentanoate kinase, a
3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase and a 3-hydroxyglutaryl-CoA reductase (alcohol
forming).
[0114] In some embodiments, the invention provides a method for
producing butadiene that includes culturing a non-naturally
occurring microbial organism as described herein, including a
microbial organism comprising (a) as described above, which can
further comprise an exogenous nucleic acid encoding an enzyme
selected from a pyruvate:ferredoxin oxidoreductase, an aconitase,
an isocitrate dehydrogenase, a succinyl-CoA synthetase, a
succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an
acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase,
an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations
thereof. In addition, a microbial organism comprising (b) as
described above can further comprise an exogenous nucleic acid
encoding an enzyme selected from an aconitase, an isocitrate
dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA
transferase, a fumarase, a malate dehydrogenase, and combinations
thereof.
[0115] In a particular embodiment, such a microbial organism used
in a method for producing butadiene can comprise two, three, four,
five, six or seven exogenous nucleic acids each encoding a
butadiene pathway enzyme. For example, such a microbial organism
can comprise exogenous nucleic acids encoding each of the enzymes
selected from: (i) an acetyl-CoA:acetyl-CoA acyltransferase, an
acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a
crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a crotyl alcohol kinase, a
2-butenyl-4-phosphate kinase and a butadiene synthase; (ii) an
acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA
reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase and
crotonyl-CoA reductase (alcohol forming); (iii) an
acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA
reductase, a 3-hydroxybutyryl-CoA dehydratase, a butadiene
synthase, a crotonyl-CoA reductase (alcohol forming) and a crotyl
alcohol diphosphokinase; (iv) an acetyl-CoA:acetyl-CoA
acyltransferase, an acetoacetyl-CoA reductase, a
3-hydroxybutyryl-CoA dehydratase, a crotonaldehyde reductase
(alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate
kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase
or transferase and a crotonate reductase; (v) an
acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA
reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonaldehyde
reductase (alcohol forming), a butadiene synthase, a crotonyl-CoA
hydrolase, synthetase or transferase, a crotonate reductase and a
crotyl alcohol diphosphokinase; (vi) an acetyl-CoA:acetyl-CoA
acyltransferase, an acetoacetyl-CoA reductase, a
3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
butadiene synthase and a crotyl alcohol diphosphokinase; (vii) a
glutaconyl-CoA decarboxylase, a crotonyl-CoA reductase (aldehyde
forming), a crotonaldehyde reductase (alcohol forming), a crotyl
alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene
synthase; (viii) a glutaconyl-CoA decarboxylase, a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase and
crotonyl-CoA reductase (alcohol forming); (ix) a glutaconyl-CoA
decarboxylase, a butadiene synthase, a crotonyl-CoA reductase
(alcohol forming) and a crotyl alcohol diphosphokinase; (x) a
glutaconyl-CoA decarboxylase, a crotonaldehyde reductase (alcohol
forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase,
a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or
transferase and a crotonate reductase; (xi) a glutaconyl-CoA
decarboxylase, a crotonaldehyde reductase (alcohol forming), a
butadiene synthase, a crotonyl-CoA hydrolase, synthetase or
transferase, a crotonate reductase and a crotyl alcohol
diphosphokinase; (xii) a 3-hydroxybutyryl-CoA dehydratase, a
crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a butadiene a glutaconyl-CoA
decarboxylase and a crotyl alcohol diphosphokinase; (xiii) a
glutaryl-CoA dehydrogenase, a crotonyl-CoA reductase (aldehyde
forming), a crotonaldehyde reductase (alcohol forming), a crotyl
alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene
synthase; (xiv) a glutaryl-CoA dehydrogenase, a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase and
crotonyl-CoA reductase (alcohol forming); (xv) a glutaryl-CoA
dehydrogenase, a butadiene synthase, a crotonyl-CoA reductase
(alcohol forming) and a crotyl alcohol diphosphokinase; (xvi) a
glutaryl-CoA dehydrogenase, a crotonaldehyde reductase (alcohol
forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase,
a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or
transferase and a crotonate reductase; (xvii) a glutaryl-CoA
dehydrogenase, a crotonaldehyde reductase (alcohol forming), a
butadiene synthase, a crotonyl-CoA hydrolase, synthetase or
transferase, a crotonate reductase and a crotyl alcohol
diphosphokinase; (xviii) a 3-hydroxybutyryl-CoA dehydratase, a
crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a butadiene synthase, a glutaryl-CoA
dehydrogenase and a crotyl alcohol diphosphokinase; (xix) an
3-aminobutyryl-CoA deaminase, a crotonyl-CoA reductase (aldehyde
forming), a crotonaldehyde reductase (alcohol forming), a crotyl
alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene
synthase; (xx) an 3-aminobutyryl-CoA deaminase, a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase and
crotonyl-CoA reductase (alcohol forming); (xxi) an
3-aminobutyryl-CoA deaminase, a butadiene synthase, a crotonyl-CoA
reductase (alcohol forming) and a crotyl alcohol diphosphokinase;
(xxii) an 3-aminobutyryl-CoA deaminase, a crotonaldehyde reductase
(alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate
kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase
or transferase and a crotonate reductase; (xxiii) an
3-aminobutyryl-CoA deaminase, a crotonaldehyde reductase (alcohol
forming), a butadiene synthase, a crotonyl-CoA hydrolase,
synthetase or transferase, a crotonate reductase and a crotyl
alcohol diphosphokinase; (xxiv) a 3-hydroxybutyryl-CoA dehydratase,
a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde
reductase (alcohol forming), a butadiene synthase, a
3-aminobutyryl-CoA deaminase and a crotyl alcohol diphosphokinase;
(xxv) a 4-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a
butadiene synthase; (xxvi) a 4-hydroxybutyryl-CoA dehydratase, a
crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene
synthase and crotonyl-CoA reductase (alcohol forming); (xxvii) a
4-hydroxybutyryl-CoA dehydratase, a butadiene synthase, a
crotonyl-CoA reductase (alcohol forming) and a crotyl alcohol
diphosphokinase; (xxviii) a 4-hydroxybutyryl-CoA dehydratase, a
crotonaldehyde reductase (alcohol forming), a crotyl alcohol
kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a
crotonyl-CoA hydrolase, synthetase or transferase and a crotonate
reductase; (xxix) a 4-hydroxybutyryl-CoA dehydratase, a
crotonaldehyde reductase (alcohol forming), a butadiene synthase, a
crotonyl-CoA hydrolase, synthetase or transferase, a crotonate
reductase and a crotyl alcohol diphosphokinase; (xxx) a
3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase
(aldehyde forming), a crotonaldehyde reductase (alcohol forming), a
butadiene synthase, a 4-hydroxybutyryl-CoA dehydratase and a crotyl
alcohol diphosphokinase; (xxxi) an erythrose-4-phosphate reductase,
an erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate reductase and a butadiene
synthase; (xxxii) an erythrose-4-phosphate reductase, an
erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate reductase, a butenyl
4-diphosphate isomerase and a butadiene synthase; (xxxiii) an
erythritol-4-phospate cytidylyltransferase, a 4-(cytidine
5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate
synthase, a 1-hydroxy-2-butenyl 4-diphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate reductase, a butadiene synthase,
an erythrose-4-phosphate kinase, an erythrose reductase and a
erythritol kinase; (xxxiv) an erythritol-4-phospate
cytidylyltransferase, a 4-(cytidine 5'-diphospho)-erythritol
kinase, an erythritol 2,4-cyclodiphosphate synthase, a
1-hydroxy-2-butenyl 4-diphosphate synthase, a 1-hydroxy-2-butenyl
4-diphosphate reductase, a butenyl 4-diphosphate isomerase, a
butadiene synthase, an erythrose-4-phosphate kinase, an erythrose
reductase and an erythritol kinase; (xxxv) a malonyl-CoA:acetyl-CoA
acyltransferase, an 3-oxoglutaryl-CoA reductase (ketone-reducing),
a 3-hydroxyglutaryl-CoA reductase (aldehyde forming), a
3-hydroxy-5-oxopentanoate reductase, a 3,5-dihydroxypentanoate
kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase and a butadiene
synthase; (xxxvi) a malonyl-CoA:acetyl-CoA acyltransferase, a
3,5-dihydroxypentanoate kinase, a
3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, an 3-oxoglutaryl-CoA reductase (aldehyde forming), a
3,5-dioxopentanoate reductase (aldehyde reducing) and a
5-hydroxy-3-oxopentanoate reductase; (xxxvii) a
malonyl-CoA:acetyl-CoA acyltransferase, a 3-hydroxy-5-oxopentanoate
reductase, a 3,5-dihydroxypentanoate kinase, a
3-Hydroxy-5-phosphonatooxypentanoate kinase, a
3-Hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, an 3-oxoglutaryl-CoA reductase (aldehyde forming) and a
3,5-dioxopentanoate reductase (ketone reducing); (xxxviii) a
malonyl-CoA:acetyl-CoA acyltransferase, a 3,5-dihydroxypentanoate
kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase, a 5-hydroxy-3-oxopentanoate reductase and a
3-oxo-glutaryl-CoA reductase (CoA reducing and alcohol forming);
and (xxxix) a butadiene pathway comprising a malonyl-CoA:acetyl-CoA
acyltransferase, an 3-oxoglutaryl-CoA reductase (ketone-reducing),
a 3,5-dihydroxypentanoate kinase, a
3-hydroxy-5-phosphonatooxypentanoate kinase, a
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate
decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene
synthase and a 3-hydroxyglutaryl-CoA reductase (alcohol
forming).
[0116] In some aspects, the invention provides a method for
producing butatiene, wherein the microbial organisms of the
invention comprise two, three, four or five exogenous nucleic acids
each encoding enzymes of (a), (b) or (c) as described above. For
example, a microbial organism comprising (a) can comprise the
exogenous nucleic acids encoding ATP-citrate lyase or citrate
lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin
oxidoreductase; a microbial organism comprising (b) can comprise
four exogenous nucleic acids encoding pyruvate:ferredoxin
oxidoreductase, a phosphoenolpyruvate carboxylase or a
phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2
hydrogenase; or a microbial organism comprising (c) can comprise
two exogenous nucleic acids encoding CO dehydrogenase and H2
hydrogenase. The invention further provides methods for producing
butadiene by culturing such non-naturally occurring microbial
organisms under conditions and for a sufficient period of time to
produce butadiene.
[0117] In some embodiments, the invention provides a method for
producing crotyl alcohol that includes culturing a non-naturally
occurring microbial organism as described herein, including a
microbial organism having a crotyl alcohol pathway comprising at
least one exogenous nucleic acid encoding a crotyl alcohol pathway
enzyme expressed in a sufficient amount to produce crotyl alcohol.
Such a microbial organism can further comprise (a) a reductive TCA
pathway comprising at least one exogenous nucleic acid encoding a
reductive TCA pathway enzyme, wherein the at least one exogenous
nucleic acid is selected from an ATP-citrate lyase, a citrate
lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate
reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase;
(b) a reductive TCA pathway comprising at least one exogenous
nucleic acid encoding a reductive TCA pathway enzyme, wherein the
at least one exogenous nucleic acid is selected from a
pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate
carboxylase, a phosphoenolpyruvate carboxykinase, a CO
dehydrogenase, and an H.sub.2 hydrogenase; or (c) at least one
exogenous nucleic acid encodes an enzyme selected from a CO
dehydrogenase, an H.sub.2 hydrogenase, and combinations
thereof.
[0118] In such a microbial organism used in a method for producing
crotyl alcohol, the crotyl alcohol pathway can be selected from any
of those disclosed herein and in the figures. For example, the
crtoyl alcohol pathway can be selected from (i) an
acetyl-CoA:acetyl-CoA acyltransferase; an acetoacetyl-CoA
reductase; a 3-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA
hydrolase, synthase, or transferase; a crotonate reductase; and a
crotonaldehyde reductase (alcohol forming); (ii) an
acetyl-CoA:acetyl-CoA acyltransferase; an acetoacetyl-CoA
reductase; a 3-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA
reductase (aldehyde forming); and a crotonaldehyde reductase
(alcohol forming); (iii) an acetyl-CoA:acetyl-CoA acyltransferase;
an acetoacetyl-CoA reductase; a 3-hydroxybutyryl-CoA dehydratase;
and a crotonyl-CoA reductase (alcohol forming); (iv) a
glutaconyl-CoA decarboxylase; a crotonyl-CoA hydrolase, synthase,
or transferase; a crotonate reductase; and a crotonaldehyde
reductase (alcohol forming); (v) a glutaconyl-CoA decarboxylase; a
crotonyl-CoA reductase (aldehyde forming); and a crotonaldehyde
reductase (alcohol forming); and (vi) a glutaconyl-CoA
decarboxylase; and a crotonyl-CoA reductase (alcohol forming).
(vii) a glutaryl-CoA dehydrogenase; a crotonyl-CoA hydrolase,
synthase, or transferase; a crotonate reductase; and a
crotonaldehyde reductase (alcohol forming); (viii) a glutaryl-CoA
dehydrogenase; a crotonyl-CoA reductase (aldehyde forming); and a
crotonaldehyde reductase (alcohol forming); (ix) a glutaryl-CoA
dehydrogenase; and a crotonyl-CoA reductase (alcohol forming); (x)
a 3-aminobutyryl-CoA deaminase; a crotonyl-CoA hydrolase, synthase,
or transferase; a crotonate reductase; and a crotonaldehyde
reductase (alcohol forming); (xi) a 3-aminobutyryl-CoA deaminase; a
crotonyl-CoA reductase (aldehyde forming); and a crotonaldehyde
reductase (alcohol forming); (xii) a 3-aminobutyryl-CoA deaminase;
and a crotonyl-CoA reductase (alcohol forming); (xiii) a
4-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA hydrolase,
synthase, or transferase; a crotonate reductase; and a
crotonaldehyde reductase (alcohol forming); (xiv) a
4-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA reductase
(aldehyde forming); and a crotonaldehyde reductase (alcohol
forming); and (xv) a 4-hydroxybutyryl-CoA dehydratase; and a
crotonyl-CoA reductase (alcohol forming).
[0119] In some aspects, the invention provides a method for
producing crotyl alcohol, where a microbial organism comprising (a)
can further comprise an exogenous nucleic acid encoding an enzyme
selected from a pyruvate:ferredoxin oxidoreductase, an aconitase,
an isocitrate dehydrogenase, a succinyl-CoA synthetase, a
succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an
acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase,
an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations
thereof. In some aspects, such a microbial organism used in a
method for producing crotyl alcohol include a microbial organism
comprising (b), which can further comprise an exogenous nucleic
acid encoding an enzyme selected from an aconitase, an isocitrate
dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA
transferase, a fumarase, a malate dehydrogenase, and combinations
thereof. Such a microbial organism can comprise two, three, four,
five, six or seven exogenous nucleic acids each encoding a crotyl
alcohol pathway enzyme.
[0120] For example, the microbial organism used in the methods for
producing croytal alcohol as disclosed herein can comprise
exogenous nucleic acids encoding each of the enzymes selected from
(i) an acetyl-CoA:acetyl-CoA acyltransferase; an acetoacetyl-CoA
reductase; a 3-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA
hydrolase, synthase, or transferase; a crotonate reductase; and a
crotonaldehyde reductase (alcohol forming); (ii) an
acetyl-CoA:acetyl-CoA acyltransferase; an acetoacetyl-CoA
reductase; a 3-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA
reductase (aldehyde forming); and a crotonaldehyde reductase
(alcohol forming); (iii) an acetyl-CoA:acetyl-CoA acyltransferase;
an acetoacetyl-CoA reductase; a 3-hydroxybutyryl-CoA dehydratase;
and a crotonyl-CoA reductase (alcohol forming); (iv) a
glutaconyl-CoA decarboxylase; a crotonyl-CoA hydrolase, synthase,
or transferase; a crotonate reductase; and a crotonaldehyde
reductase (alcohol forming); (v) a glutaconyl-CoA decarboxylase; a
crotonyl-CoA reductase (aldehyde forming); and a crotonaldehyde
reductase (alcohol forming); (vi) a glutaconyl-CoA decarboxylase;
and a crotonyl-CoA reductase (alcohol forming); (vii) a
glutaryl-CoA dehydrogenase; a crotonyl-CoA hydrolase, synthase, or
transferase; a crotonate reductase; and a crotonaldehyde reductase
(alcohol forming); (viii) a glutaryl-CoA dehydrogenase; a
crotonyl-CoA reductase (aldehyde forming); and a crotonaldehyde
reductase (alcohol forming); (ix) a glutaryl-CoA dehydrogenase; and
a crotonyl-CoA reductase (alcohol forming); (x) a
3-aminobutyryl-CoA deaminase; a crotonyl-CoA hydrolase, synthase,
or transferase; a crotonate reductase; and a crotonaldehyde
reductase (alcohol forming); (xi) a 3-aminobutyryl-CoA deaminase; a
crotonyl-CoA reductase (aldehyde forming); and a crotonaldehyde
reductase (alcohol forming); (xii) a 3-aminobutyryl-CoA deaminase;
and a crotonyl-CoA reductase (alcohol forming). (xiii) a
4-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA hydrolase,
synthase, or transferase; a crotonate reductase; and a
crotonaldehyde reductase (alcohol forming); (xiv) a
4-hydroxybutyryl-CoA dehydratase; a crotonyl-CoA reductase
(aldehyde forming); and a crotonaldehyde reductase (alcohol
forming); and (xv) a 4-hydroxybutyryl-CoA dehydratase; and a
crotonyl-CoA reductase (alcohol forming).
[0121] Such microbial organisms used in a method for producing
crotyl alcohol as disclosed herein can comprise two, three, four or
five exogenous nucleic acids each encoding enzymes of (a), (b) or
(c). For example, a microbial organism comprising (a) can comprise
three exogenous nucleic acids encoding ATP-citrate lyase or citrate
lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin
oxidoreductase; a microbial organism comprising (b) can comprise
four exogenous nucleic acids encoding a pyruvate:ferredoxin
oxidoreductase, a phosphoenolpyruvate carboxylase or a
phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an
H.sub.2 hydrogenase; or a microbial organism comprising (c) can
comprise two exogenous nucleic acids encoding a CO dehydrogenase
and an H.sub.2 hydrogenase.
[0122] Suitable purification and/or assays to test for the
production of butadiene can be performed using well known methods.
Suitable replicates such as triplicate cultures can be grown for
each engineered strain to be tested. For example, product and
byproduct formation in the engineered production host can be
monitored. The final product and intermediates, and other organic
compounds, can be analyzed by methods such as HPLC (High
Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass
Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy)
or other suitable analytical methods using routine procedures well
known in the art. The release of product in the fermentation broth
can also be tested with the culture supernatant. Byproducts and
residual glucose can be quantified by HPLC using, for example, a
refractive index detector for glucose and alcohols, and a UV
detector for organic acids (Lin et al., Biotechnol. Bioeng.
90:775-779 (2005)), or other suitable assay and detection methods
well known in the art. The individual enzyme or protein activities
from the exogenous DNA sequences can also be assayed using methods
well known in the art. For typical Assay Methods, see Manual on
Hydrocarbon Analysis (ASTM Manula Series, A. W. Drews, ed., 6th
edition, 1998, American Society for Testing and Materials,
Baltimore, Md.
[0123] The butadiene 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.
[0124] 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 butadiene
producers can be cultured for the biosynthetic production of
butadiene.
[0125] For the production of butadiene or crotyl alcohol, the
recombinant strains are cultured in a medium with carbon source and
other essential nutrients. It is sometimes desirable and can be
highly desirable to maintain anaerobic conditions in the fermenter
to reduce the cost of the overall process. Such conditions can be
obtained, for example, by first sparging the medium with nitrogen
and then sealing the flasks with a septum and crimp-cap. For
strains where growth is not observed anaerobically, microaerobic or
substantially anaerobic conditions can be applied by perforating
the septum with a small hole for limited aeration. Exemplary
anaerobic conditions have been described previously and are
well-known in the art. Exemplary aerobic and anaerobic conditions
are described, for example, in United State publication
2009/0047719, filed Aug. 10, 2007. Fermentations can be performed
in a batch, fed-batch or continuous manner, as disclosed
herein.
[0126] 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.
[0127] 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 butadiene or crotyl alcohol.
[0128] In addition to renewable feedstocks such as those
exemplified above, the butadiene or crotyl alcohol 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 butadiene or crotyl
alcohol producing organisms to provide a metabolic pathway for
utilization of syngas or other gaseous carbon source.
[0129] 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.
[0130] The Wood-Ljungdahl pathway catalyzes the conversion of CO
and H.sub.2 to acetyl-CoA and other products such as acetate.
Organisms capable of utilizing CO and syngas also generally have
the capability of utilizing CO.sub.2 and CO.sub.2/H.sub.2 mixtures
through the same basic set of enzymes and transformations
encompassed by the Wood-Ljungdahl pathway. H.sub.2-dependent
conversion of CO.sub.2 to acetate by microorganisms was recognized
long before it was revealed that CO also could be used by the same
organisms and that the same pathways were involved. Many acetogens
have been shown to grow in the presence of CO.sub.2 and produce
compounds such as acetate as long as hydrogen is present to supply
the necessary reducing equivalents (see for example, Drake,
Acetogenesis, pp. 3-60 Chapman and Hall, New York, (1994)). This
can be summarized by the following equation:
2CO.sub.2+4H.sub.2+n ADP+n Pi.fwdarw.CH.sub.3COOH+2H.sub.2O+n
ATP
[0131] 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.
[0132] The Wood-Ljungdahl pathway is well known in the art and
consists of 12 reactions which can be separated into two branches:
(1) methyl branch and (2) carbonyl branch. The methyl branch
converts syngas to methyl-tetrahydrofolate (methyl-THF) whereas the
carbonyl branch converts methyl-THF to acetyl-CoA. The reactions in
the methyl branch are catalyzed in order by the following enzymes
or proteins: ferredoxin oxidoreductase, formate dehydrogenase,
formyltetrahydrofolate synthetase, methenyltetrahydrofolate
cyclodehydratase, methylenetetrahydrofolate dehydrogenase and
methylenetetrahydrofolate reductase. The reactions in the carbonyl
branch are catalyzed in order by the following enzymes or proteins:
methyltetrahydrofolate:corrinoid protein methyltransferase (for
example, AcsE), corrinoid iron-sulfur protein, nickel-protein
assembly protein (for example, AcsF), ferredoxin, acetyl-CoA
synthase, carbon monoxide dehydrogenase and nickel-protein assembly
protein (for example, CooC). Following the teachings and guidance
provided herein for introducing a sufficient number of encoding
nucleic acids to generate a butadiene or crotyl alcohol 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.
[0133] Additionally, the reductive (reverse) tricarboxylic acid
cycle coupled with carbon monoxide dehydrogenase and/or hydrogenase
activities can also be used for the conversion of CO, CO.sub.2
and/or H.sub.2 to acetyl-CoA and other products such as acetate.
Organisms capable of fixing carbon via the reductive TCA pathway
can utilize one or more of the following enzymes: ATP
citrate-lyase, citrate lyase, aconitase, isocitrate dehydrogenase,
alpha-ketoglutarate:ferredoxin oxidoreductase, succinyl-CoA
synthetase, succinyl-CoA transferase, fumarate reductase, fumarase,
malate dehydrogenase, NAD(P)H:ferredoxin oxidoreductase, carbon
monoxide dehydrogenase, and hydrogenase. Specifically, the reducing
equivalents extracted from CO and/or H.sub.2 by carbon monoxide
dehydrogenase and hydrogenase are utilized to fix CO.sub.2 via the
reductive TCA cycle into acetyl-CoA or acetate. Acetate can be
converted to acetyl-CoA by enzymes such as acetyl-CoA transferase,
acetate kinase/phosphotransacetylase, and acetyl-CoA synthetase.
Acetyl-CoA can be converted to the butadiene or crotyl alcohol
precursors, glyceraldehyde-3-phosphate, phosphoenolpyruvate, and
pyruvate, by pyruvate:ferredoxin oxidoreductase and the enzymes of
gluconeogenesis. Following the teachings and guidance provided
herein for introducing a sufficient number of encoding nucleic
acids to generate a butadiene or a crotyl alcohol pathway, those
skilled in the art will understand that the same engineering design
also can be performed with respect to introducing at least the
nucleic acids encoding the reductive TCA pathway enzymes or
proteins absent in the host organism. Therefore, introduction of
one or more encoding nucleic acids into the microbial organisms of
the invention such that the modified organism contains a reductive
TCA pathway can confer syngas utilization ability.
[0134] 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, butadiene and any of the intermediate metabolites in
the butadiene 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 butadiene biosynthetic
pathways. Accordingly, the invention provides a non-naturally
occurring microbial organism that produces and/or secretes
butadiene when grown on a carbohydrate or other carbon source and
produces and/or secretes any of the intermediate metabolites shown
in the butadiene pathway when grown on a carbohydrate or other
carbon source. The butadiene producing microbial organisms of the
invention can initiate synthesis from an intermediate, for example,
acetoacetyl-CoA, 3-hydroxybutyryl-CoA, crotonyl-CoA,
crotonaldehyde, crotyl alcohol, 2-betenyl-phosphate,
2-butenyl-4-diphosphate, erythritol-4-phosphate, 4-(cytidine
5'-diphospho)-erythritol, 2-phospho-4-(cytidine
5'-diphospho)-erythritol, erythritol-2,4-cyclodiphosphate,
1-hydroxy-2-butenyl 4-diphosphate, butenyl 4-diphosphate, 2-butenyl
4-diphosphate, 3-oxoglutaryl-CoA, 3-hydroxyglutaryl-CoA,
3-hydroxy-5-oxopentanoate, 3,5-dihydroxy pentanoate,
3-hydroxy-5-phosphonatooxypentanoate,
3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate,
crotonate, erythrose, erythritol, 3,5-dioxopentanoate or
5-hydroxy-3-oxopentanoate.
[0135] The non-naturally occurring microbial organisms of the
invention are constructed using methods well known in the art as
exemplified herein to exogenously express at least one nucleic acid
encoding a butadiene or a crotyl alcohol pathway enzyme or protein
in sufficient amounts to produce butadiene or crotyl alcohol. It is
understood that the microbial organisms of the invention are
cultured under conditions sufficient to produce butadiene or crotyl
alcohol. Following the teachings and guidance provided herein, the
non-naturally occurring microbial organisms of the invention can
achieve biosynthesis of butadiene or crotyl alcohol resulting in
intracellular concentrations between about 0.001-2000 mM or more.
Generally, the intracellular concentration of butadiene or crotyl
alcohol is between about 3-1500 mM, particularly between about
5-1250 mM and more particularly between about 8-1000 mM, including
about 10 mM, 100 mM, 200 mM, 500 mM, 800 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.
[0136] In some embodiments, culture conditions include anaerobic or
substantially anaerobic growth or maintenance conditions. Exemplary
anaerobic conditions have been described previously and are well
known in the art. Exemplary anaerobic conditions for fermentation
processes are described herein and are described, for example, in
U.S. publication 2009/0047719, filed Aug. 10, 2007. Any of these
conditions can be employed with the non-naturally occurring
microbial organisms as well as other anaerobic conditions well
known in the art. Under such anaerobic or substantially anaerobic
conditions, the butadiene or crotyl alcohol producers can
synthesize butadiene or crotyl alcohol 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, butadiene or crotyl alcohol producing microbial
organisms can produce butadiene or crotyl alcohol intracellularly
and/or secrete the product into the culture medium.
[0137] In addition to the culturing and fermentation conditions
disclosed herein, growth condition for achieving biosynthesis of
butadiene or crotyl alcohol can include the addition of an
osmoprotectant to the culturing conditions. In certain embodiments,
the non-naturally occurring microbial organisms of the invention
can be sustained, cultured or fermented as described herein in the
presence of an osmoprotectant. Briefly, an osmoprotectant refers to
a compound that acts as an osmolyte and helps a microbial organism
as described herein survive osmotic stress. Osmoprotectants
include, but are not limited to, betaines, amino acids, and the
sugar trehalose. Non-limiting examples of such are glycine betaine,
praline betaine, dimethylthetin, dimethylslfonioproprionate,
3-dimethylsulfonio-2-methylproprionate, pipecolic acid,
dimethylsulfonioacetate, choline, L-carnitine and ectoine. In one
aspect, the osmoprotectant is glycine betaine. It is understood to
one of ordinary skill in the art that the amount and type of
osmoprotectant suitable for protecting a microbial organism
described herein from osmotic stress will depend on the microbial
organism used. The amount of osmoprotectant in the culturing
conditions can be, for example, no more than about 0.1 mM, no more
than about 0.5 mM, no more than about 1.0 mM, no more than about
1.5 mM, no more than about 2.0 mM, no more than about 2.5 mM, no
more than about 3.0 mM, no more than about 5.0 mM, no more than
about 7.0 mM, no more than about 10 mM, no more than about 50 mM,
no more than about 100 mM or no more than about 500 mM.
[0138] In some embodiments, the carbon feedstock and other cellular
uptake sources such as phosphate, ammonia, sulfate, chloride and
other halogens can be chosen to alter the isotopic distribution of
the atoms present in butadiene or crotyl alcohol or any butadiene
or crotyl alcohol pathway intermediate. The various carbon
feedstock and other uptake sources enumerated above will be
referred to herein, collectively, as "uptake sources." Uptake
sources can provide isotopic enrichment for any atom present in the
product butadiene or crotyl alcohol or butadiene or crotyl alcohol
pathway intermediate including any butadiene or crotyl alcohol
impurities generated in diverging away from the pathway at any
point. Isotopic enrichment can be achieved for any target atom
including, for example, carbon, hydrogen, oxygen, nitrogen, sulfur,
phosphorus, chloride or other halogens.
[0139] In some embodiments, the uptake sources can be selected to
alter the carbon-12, carbon-13, and carbon-14 ratios. In some
embodiments, the uptake sources can be selected to alter the
oxygen-16, oxygen-17, and oxygen-18 ratios. In some embodiments,
the uptake sources can be selected to alter the hydrogen,
deuterium, and tritium ratios. In some embodiments, the uptake
sources can be selected to alter the nitrogen-14 and nitrogen-15
ratios. In some embodiments, the uptake sources can be selected to
alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35 ratios. In
some embodiments, the uptake sources can be selected to alter the
phosphorus-31, phosphorus-32, and phosphorus-33 ratios. In some
embodiments, the uptake sources can be selected to alter the
chlorine-35, chlorine-36, and chlorine-37 ratios.
[0140] In some embodiments, the isotopic ratio of a target atom can
be varied to a desired ratio by selecting one or more uptake
sources. An uptake source can be derived from a natural source, as
found in nature, or from a man-made source, and one skilled in the
art can select a natural source, a man-made source, or a
combination thereof, to achieve a desired isotopic ratio of a
target atom. An example of a man-made uptake source includes, for
example, an uptake source that is at least partially derived from a
chemical synthetic reaction. Such isotopically enriched uptake
sources can be purchased commercially or prepared in the laboratory
and/or optionally mixed with a natural source of the uptake source
to achieve a desired isotopic ratio. In some embodiments, a target
atom isotopic ratio of an uptake source can be achieved by
selecting a desired origin of the uptake source as found in nature.
For example, as discussed herein, a natural source can be a
biobased derived from or synthesized by a biological organism or a
source such as petroleum-based products or the atmosphere. In some
such embodiments, a source of carbon, for example, can be selected
from a fossil fuel-derived carbon source, which can be relatively
depleted of carbon-14, or an environmental or atmospheric carbon
source, such as CO.sub.2, which can possess a larger amount of
carbon-14 than its petroleum-derived counterpart.
[0141] The unstable carbon isotope carbon-14 or radiocarbon makes
up for roughly 1 in 10.sup.12 carbon atoms in the earth's
atmosphere and has a half-life of about 5700 years. The stock of
carbon is replenished in the upper atmosphere by a nuclear reaction
involving cosmic rays and ordinary nitrogen (.sup.14N). Fossil
fuels contain no carbon-14, as it decayed long ago. Burning of
fossil fuels lowers the atmospheric carbon-14 fraction, the
so-called "Suess effect".
[0142] Methods of determining the isotopic ratios of atoms in a
compound are well known to those skilled in the art. Isotopic
enrichment is readily assessed by mass spectrometry using
techniques known in the art such as accelerated mass spectrometry
(AMS), Stable Isotope Ratio Mass Spectrometry (SIRMS) and
Site-Specific Natural Isotopic Fractionation by Nuclear Magnetic
Resonance (SNIF-NMR). Such mass spectral techniques can be
integrated with separation techniques such as liquid chromatography
(LC), high performance liquid chromatography (HPLC) and/or gas
chromatography, and the like.
[0143] In the case of carbon, ASTM D6866 was developed in the
United States as a standardized analytical method for determining
the biobased content of solid, liquid, and gaseous samples using
radiocarbon dating by the American Society for Testing and
Materials (ASTM) International. The standard is based on the use of
radiocarbon dating for the determination of a product's biobased
content. ASTM D6866 was first published in 2004, and the current
active version of the standard is ASTM D6866-11 (effective Apr. 1,
2011). Radiocarbon dating techniques are well known to those
skilled in the art, including those described herein.
[0144] The biobased content of a compound is estimated by the ratio
of carbon-14 (.sup.14C) to carbon-12 (.sup.12C). Specifically, the
Fraction Modern (Fm) is computed from the expression:
Fm=(S-B)/(M-B), where B, S and M represent the .sup.14C/.sup.12C
ratios of the blank, the sample and the modern reference,
respectively. Fraction Modern is a measurement of the deviation of
the .sup.14C/.sup.12C ratio of a sample from "Modern." Modern is
defined as 95% of the radiocarbon concentration (in AD 1950) of
National Bureau of Standards (NBS) Oxalic Acid I (i.e., standard
reference materials (SRM) 4990b) normalized to
.delta..sup.13C.sub.VPDB=-19 per mil (Olsson, The use of Oxalic
acid as a Standard. in, Radiocarbon Variations and Absolute
Chronology, Nobel Symposium, 12th Proc., John Wiley & Sons, New
York (1970)). Mass spectrometry results, for example, measured by
ASM, are calculated using the internationally agreed upon
definition of 0.95 times the specific activity of NBS Oxalic Acid I
(SRM 4990b) normalized to .delta..sup.13C.sub.VPDB=-19 per mil.
This is equivalent to an absolute (AD 1950).sup.14C/.sup.12C ratio
of 1.176.+-.0.010.times.10.sup.-12 (Karlen et al., Arkiv Geofysik,
4:465-471 (1968)). The standard calculations take into account the
differential uptake of one istope with respect to another, for
example, the preferential uptake in biological systems of C.sup.12
over C.sup.13 over C.sup.14, and these corrections are reflected as
a Fm corrected for .delta..sup.13.
[0145] An oxalic acid standard (SRM 4990b or HOx 1) was made from a
crop of 1955 sugar beet. Although there were 1000 lbs made, this
oxalic acid standard is no longer commercially available. The
Oxalic Acid II standard (HOx 2; N.I.S.T designation SRM 4990 C) was
made from a crop of 1977 French beet molasses. In the early 1980's,
a group of 12 laboratories measured the ratios of the two
standards. The ratio of the activity of Oxalic acid II to 1 is
1.2933.+-.0.001 (the weighted mean). The isotopic ratio of HOx II
is -17.8 per mille. ASTM D6866-11 suggests use of the available
Oxalic Acid II standard SRM 4990 C (Hox2) for the modern standard
(see discussion of original vs. currently available oxalic acid
standards in Mann, Radiocarbon, 25(2):519-527 (1983)). A Fm=0%
represents the entire lack of carbon-14 atoms in a material, thus
indicating a fossil (for example, petroleum based) carbon source. A
Fm=100%, after correction for the post-1950 injection of carbon-14
into the atmosphere from nuclear bomb testing, indicates an
entirely modern carbon source. As described herein, such a "modern"
source includes biobased sources.
[0146] As described in ASTM D6866, the percent modern carbon (pMC)
can be greater than 100% because of the continuing but diminishing
effects of the 1950s nuclear testing programs, which resulted in a
considerable enrichment of carbon-14 in the atmosphere as described
in ASTM D6866-11. Because all sample carbon-14 activities are
referenced to a "pre-bomb" standard, and because nearly all new
biobased products are produced in a post-bomb environment, all pMC
values (after correction for isotopic fraction) must be multiplied
by 0.95 (as of 2010) to better reflect the true biobased content of
the sample. A biobased content that is greater than 103% suggests
that either an analytical error has occurred, or that the source of
biobased carbon is more than several years old.
[0147] ASTM D6866 quantifies the biobased content relative to the
material's total organic content and does not consider the
inorganic carbon and other non-carbon containing substances
present. For example, a product that is 50% starch-based material
and 50% water would be considered to have a Biobased Content=100%
(50% organic content that is 100% biobased) based on ASTM D6866. In
another example, a product that is 50% starch-based material, 25%
petroleum-based, and 25% water would have a Biobased Content=66.7%
(75% organic content but only 50% of the product is biobased). In
another example, a product that is 50% organic carbon and is a
petroleum-based product would be considered to have a Biobased
Content=0% (50% organic carbon but from fossil sources). Thus,
based on the well known methods and known standards for determining
the biobased content of a compound or material, one skilled in the
art can readily determine the biobased content and/or prepared
downstream products that utilize of the invention having a desired
biobased content.
[0148] Applications of carbon-14 dating techniques to quantify
bio-based content of materials are known in the art (Currie et al.,
Nuclear Instruments and Methods in Physics Research B, 172:281-287
(2000)). For example, carbon-14 dating has been used to quantify
bio-based content in terephthalate-containing materials (Colonna et
al., Green Chemistry, 13:2543-2548 (2011)). Notably, polypropylene
terephthalate (PPT) polymers derived from renewable 1,3-propanediol
and petroleum-derived terephthalic acid resulted in Fm values near
30% (i.e., since 3/11 of the polymeric carbon derives from
renewable 1,3-propanediol and 8/11 from the fossil end member
terephthalic acid) (Currie et al., supra, 2000). In contrast,
polybutylene terephthalate polymer derived from both renewable
1,4-butanediol and renewable terephthalic acid resulted in
bio-based content exceeding 90% (Colonna et al., supra, 2011).
[0149] Accordingly, in some embodiments, the present invention
provides butadiene or crotyl alcohol or a butadiene or crotyl
alcohol intermediate that has a carbon-12, carbon-13, and carbon-14
ratio that reflects an atmospheric carbon, also referred to as
environmental carbon, uptake source. For example, in some aspects
the butadiene or crotyl alcohol or a butadiene or crotyl alcohol
intermediate can have an Fm value of at least 10%, at least 15%, at
least 20%, at least 25%, at least 30%, at least 35%, at least 40%,
at least 45%, at least 50%, at least 55%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 98% or as much as 100%. In some
such embodiments, the uptake source is CO.sub.2. In some
embodiments, the present invention provides butadiene or crotyl
alcohol or a butadiene or crotyl alcohol intermediate that has a
carbon-12, carbon-13, and carbon-14 ratio that reflects
petroleum-based carbon uptake source. In this aspect, the butadiene
or crotyl alcohol or a butadiene or crotyl alcohol intermediate can
have an Fm value of less than 95%, less than 90%, less than 85%,
less than 80%, less than 75%, less than 70%, less than 65%, less
than 60%, less than 55%, less than 50%, less than 45%, less than
40%, less than 35%, less than 30%, less than 25%, less than 20%,
less than 15%, less than 10%, less than 5%, less than 2% or less
than 1%. In some embodiments, the present invention provides
butadiene or crotyl alcohol or a butadiene or crotyl alcohol
intermediate that has a carbon-12, carbon-13, and carbon-14 ratio
that is obtained by a combination of an atmospheric carbon uptake
source with a petroleum-based uptake source. Using such a
combination of uptake sources is one way by which the carbon-12,
carbon-13, and carbon-14 ratio can be varied, and the respective
ratios would reflect the proportions of the uptake sources.
[0150] Further, the present invention relates to the biologically
produced butadiene or crotyl alcohol or butadiene or crotyl alcohol
intermediate as disclosed herein, and to the products derived
therefrom, wherein the butadiene or crotyl alcohol or a butadiene
or crotyl alcohol intermediate has a carbon-12, carbon-13, and
carbon-14 isotope ratio of about the same value as the CO.sub.2
that occurs in the environment. For example, in some aspects the
invention provides bioderived butadiene or crotyl alcohol or a
bioderived butadiene or crotyl alcohol intermediate having a
carbon-12 versus carbon-13 versus carbon-14 isotope ratio of about
the same value as the CO.sub.2 that occurs in the environment, or
any of the other ratios disclosed herein. It is understood, as
disclosed herein, that a product can have a carbon-12 versus
carbon-13 versus carbon-14 isotope ratio of about the same value as
the CO.sub.2 that occurs in the environment, or any of the ratios
disclosed herein, wherein the product is generated from bioderived
butadiene or crotyl alcohol or a bioderived butadiene or crotyl
alcohol intermediate as disclosed herein, wherein the bioderived
product is chemically modified to generate a final product. Methods
of chemically modifying a bioderived product of butadiene or crotyl
alcohol, or an intermediate thereof, to generate a desired product
are well known to those skilled in the art, as described herein.
The invention further provides a polymer, synthetic rubber, resin,
chemical, monomer, fine chemical, agricultural chemical, or
pharmaceutical having a carbon-12 versus carbon-13 versus carbon-14
isotope ratio of about the same value as the CO.sub.2 that occurs
in the environment, wherein the polymer, synthetic rubber, resin,
chemical, monomer, fine chemical, agricultural chemical, or
pharmaceutical is generated directly from or in combination with
bioderived butadiene or crotyl alcohol or a bioderived butadiene or
crotyl alcohol intermediate as disclosed herein.
[0151] Butadiene is a chemical commonly used in many commercial and
industrial applications. Non-limiting examples of such applications
include production of polymers, such as synthetic rubbers and ABS
resins, and chemicals, such as hexamethylenediamine and
1,4-butanediol. Accordingly, in some embodiments, the invention
provides a biobased polymer, synthetic rubber, resin, or chemical
comprising one or more bioderived butadiene or bioderived butadiene
intermediate produced by a non-naturally occurring microorganism of
the invention or produced using a method disclosed herein.
[0152] Crotyl alcohol is a chemical commonly used in many
commercial and industrial applications. Non-limiting examples of
such applications include production of crotyl halides, esters, and
ethers, which in turn are chemical are chemical intermediates in
the production of monomers, fine chemicals, such as sorbic acid,
trimethylhydroquinone, crotonic acid and 3-methoxybutanol,
agricultural chemicals, and pharmaceuticals. Crotyl alcohol can
also be used as a precursor in the production of 1,3-butadiene.
Accordingly, in some embodiments, the invention provides a biobased
monomer, fine chemical, agricultural chemical, or pharmaceutical
comprising one or more bioderived crotyl alcohol or bioderived
crotyl alcohol intermediate produced by a non-naturally occurring
microorganism of the invention or produced using a method disclosed
herein.
[0153] As used herein, the term "bioderived" means derived from or
synthesized by a biological organism and can be considered a
renewable resource since it can be generated by a biological
organism. Such a biological organism, in particular the microbial
organisms of the invention disclosed herein, can utilize feedstock
or biomass, such as, sugars or carbohydrates obtained from an
agricultural, plant, bacterial, or animal source. Alternatively,
the biological organism can utilize atmospheric carbon. As used
herein, the term "biobased" means a product as described above that
is composed, in whole or in part, of a bioderived compound of the
invention. A biobased or bioderived product is in contrast to a
petroleum derived product, wherein such a product is derived from
or synthesized from petroleum or a petrochemical feedstock.
[0154] In some embodiments, the invention provides a biobased
polymer, synthetic rubber, resin, or chemical comprising bioderived
butadiene or bioderived butadiene intermediate, wherein the
bioderived butadiene or bioderived butadiene intermediate includes
all or part of the butadiene or butadiene intermediate used in the
production of polymer, synthetic rubber, resin, or chemical. Thus,
in some aspects, the invention provides a biobased polymer,
synthetic rubber, resin, or chemical comprising at least 2%, at
least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, at least 50%,
at least 60%, at least 70%, at least 80%, at least 90%, at least
95%, at least 98% or 100% bioderived butadiene or bioderived
butadiene intermediate as disclosed herein. Additionally, in some
aspects, the invention provides a biobased polymer, synthetic
rubber, resin, or chemical wherein the butadiene or butadiene
intermediate used in its production is a combination of bioderived
and petroleum derived butadiene or butadiene intermediate. For
example, a biobased polymer, synthetic rubber, resin, or chemical
can be produced using 50% bioderived butadiene and 50% petroleum
derived butadiene or other desired ratios such as 60%/40%, 70%/30%,
80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%,
10%/90% of bioderived/petroleum derived precursors, so long as at
least a portion of the product comprises a bioderived product
produced by the microbial organisms disclosed herein. It is
understood that methods for producing polymer, synthetic rubber,
resin, or chemical using the bioderived butadiene or bioderived
butadiene intermediate of the invention are well known in the
art.
[0155] In some embodiments, the invention provides a biobased
monomer, fine chemical, agricultural chemical, or pharmaceutical
comprising bioderived crotyl alcohol or bioderived crotyl alcohol
intermediate, wherein the bioderived crotyl alcohol or bioderived
crotyl alcohol intermediate includes all or part of the crotyl
alcohol or crotyl alcohol intermediate used in the production of
monomer, fine chemical, agricultural chemical, or pharmaceutical.
Thus, in some aspects, the invention provides a biobased monomer,
fine chemical, agricultural chemical, or pharmaceutical comprising
at least 2%, at least 3%, at least 5%, at least 10%, at least 15%,
at least 20%, at least 25%, at least 30%, at least 35%, at least
40%, at least 50%, at least 60%, at least 70%, at least 80%, at
least 90%, at least 95%, at least 98% or 100% bioderived crotyl
alcohol or bioderived crotyl alcohol intermediate as disclosed
herein. Additionally, in some aspects, the invention provides a
biobased monomer, fine chemical, agricultural chemical, or
pharmaceutical wherein the crotyl alcohol or crotyl alcohol
intermediate used in its production is a combination of bioderived
and petroleum derived crotyl alcohol or crotyl alcohol
intermediate. For example, a biobased monomer, fine chemical,
agricultural chemical, or pharmaceutical can be produced using 50%
bioderived crotyl alcohol and 50% petroleum derived crotyl alcohol
or other desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%,
95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of
bioderived/petroleum derived precursors, so long as at least a
portion of the product comprises a bioderived product produced by
the microbial organisms disclosed herein. It is understood that
methods for producing monomer, fine chemical, agricultural
chemical, or pharmaceutical using the bioderived crotyl alcohol or
bioderived crotyl alcohol intermediate of the invention are well
known in the art.
[0156] 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.
[0157] As described herein, one exemplary growth condition for
achieving biosynthesis of butadiene or crotyl alcohol 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.
[0158] The culture conditions described herein can be scaled up and
grown continuously for manufacturing of butadiene or crotyl
alcohol. 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 butadiene or
crotyl alcohol. Generally, and as with non-continuous culture
procedures, the continuous and/or near-continuous production of
butadiene or crotyl alcohol will include culturing a non-naturally
occurring butadiene or crotyl alcohol producing organism of the
invention in sufficient nutrients and medium to sustain and/or
nearly sustain growth in an exponential phase. Continuous culture
under such conditions can include, for example, growth for 1 day,
2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous culture
can include longer time periods of 1 week, 2, 3, 4 or 5 or more
weeks and up to several months. Alternatively, organisms of the
invention can be cultured for hours, if suitable for a particular
application. It is to be understood that the continuous and/or
near-continuous culture conditions also can include all time
intervals in between these exemplary periods. It is further
understood that the time of culturing the microbial organism of the
invention is for a sufficient period of time to produce a
sufficient amount of product for a desired purpose.
[0159] Fermentation procedures are well known in the art. Briefly,
fermentation for the biosynthetic production of butadiene or crotyl
alcohol 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.
[0160] In addition to the above fermentation procedures using the
butadiene or crotyl alcohol producers of the invention for
continuous production of substantial quantities of butadiene or
crotyl alcohol, the butadiene or crotyl alcohol 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 or enzymatic conversion to convert the
product to other compounds, if desired.
[0161] 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 butadiene or crotyl alcohol.
[0162] 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.
[0163] 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.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] 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.
[0168] 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.
[0169] 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..
[0170] 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.
[0171] 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)).
[0172] 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.
[0173] As disclosed herein, a nucleic acid encoding a desired
activity of a butadiene or crotyl alcohol pathway can be introduced
into a host organism. In some cases, it can be desirable to modify
an activity of a butadiene or crotyl alcohol pathway enzyme or
protein to increase production of butadiene or crotyl alcohol. For
example, known mutations that increase the activity of a protein or
enzyme can be introduced into an encoding nucleic acid molecule.
Additionally, optimization methods can be applied to increase the
activity of an enzyme or protein and/or decrease an inhibitory
activity, for example, decrease the activity of a negative
regulator.
[0174] One such optimization method is directed evolution. Directed
evolution is a powerful approach that involves the introduction of
mutations targeted to a specific gene in order to improve and/or
alter the properties of an enzyme. Improved and/or altered enzymes
can be identified through the development and implementation of
sensitive high-throughput screening assays that allow the automated
screening of many enzyme variants (for example, >10.sup.4).
Iterative rounds of mutagenesis and screening typically are
performed to afford an enzyme with optimized properties.
Computational algorithms that can help to identify areas of the
gene for mutagenesis also have been developed and can significantly
reduce the number of enzyme variants that need to be generated and
screened. Numerous directed evolution technologies have been
developed (for reviews, see Hibbert et al., Biomol. Eng 22:11-19
(2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical
and biotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC
Press; Otten and Quax. Biomol. Eng 22:1-9 (2005); and Sen et al.,
Appl Biochem. Biotechnol 143:212-223 (2007)) to be effective at
creating diverse variant libraries, and these methods have been
successfully applied to the improvement of a wide range of
properties across many enzyme classes. Enzyme characteristics that
have been improved and/or altered by directed evolution
technologies include, for example: selectivity/specificity, for
conversion of non-natural substrates; temperature stability, for
robust high temperature processing; pH stability, for bioprocessing
under lower or higher pH conditions; substrate or product
tolerance, so that high product titers can be achieved; binding
(K.sub.m), including broadening substrate binding to include
non-natural substrates; inhibition (K.sub.i), to remove inhibition
by products, substrates, or key intermediates; activity (kcat), to
increases enzymatic reaction rates to achieve desired flux;
expression levels, to increase protein yields and overall pathway
flux; oxygen stability, for operation of air sensitive enzymes
under aerobic conditions; and anaerobic activity, for operation of
an aerobic enzyme in the absence of oxygen.
[0175] Described below in more detail are exemplary methods that
have been developed for the mutagenesis and diversification of
genes to target desired properties of specific enzymes. Such
methods are well known to those skilled in the art. Any of these
can be used to alter and/or optimize the activity of a butadiene or
crotyl alcohol pathway enzyme or protein.
[0176] 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 to screen a larger number of potential variants
having a desired activity. A high number of mutants can be
generated by EpPCR, so a high-throughput screening assay or a
selection method, for example, using robotics, is useful to
identify those with desirable characteristics.
[0177] 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 commercially available kit.
[0178] DNA or Family Shuffling (Stemmer, Proc Natl Acad Sci USA
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.
[0179] 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.
[0180] 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.
[0181] 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.
[0182] Random Chimeragenesis on Transient Templates (RACHITT) (Coco
et al., Nat. Biotechnol. 19:354-359 (2001)) employs Dnase I
fragmentation and size fractionation of single stranded DNA
(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, which
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.
[0183] 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 do not 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, that is, no direct shuffling. This method can also be
more random than StEP due to the absence of pauses.
[0184] 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.
[0185] 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. USA 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.
[0186] 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.
[0187] SCRATCHY combines two methods for recombining genes, ITCHY
and DNA shuffling (Lutz et al., Proc. Natl. Acad. Sci. USA
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%.
[0188] In Random Drift Mutagenesis (RNDM) mutations are 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.
[0189] Sequence Saturation Mutagenesis (SeSaM) is a random
mutagenesis method that: 1) generates a 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 an
inosine-containing complement gives random base incorporation and,
consequently, mutagenesis (Wong et al., Biotechnol. J. 3:74-82
(2008); Wong et al., Nucleic Acids Res. 32:e26 (2004); and Wong et
al., Anal. Biochem. 341:187-189 (2005)). Using this technique it
can be possible to generate a large library of mutants within 2 to
3 days using simple methods. This technique is non-directed in
comparison to the mutational bias of DNA polymerases. Differences
in this approach makes this technique complementary (or an
alternative) to epPCR.
[0190] 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.
[0191] 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.
[0192] In Sequence Homology-Independent Protein Recombination
(SHIPREC), a linker is used to facilitate fusion between two
distantly related or 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.
[0193] 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 approximately 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 (that is, 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 usefulness 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.
[0194] 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.
[0195] 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)
identify hot spots and hot regions and then 3) extension by CMCM to
cover a defined region of protein sequence space (Reetz et al.,
Angew. Chem. Int. Ed Engl. 40:3589-3591 (2001)). 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.
[0196] In the Mutator Strains technique, conditional ts mutator
plasmids allow increases of 20 to 4000-.times.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 (ts) 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 (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.
[0197] 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. USA 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.
[0198] Gene Reassembly is a DNA shuffling method that can be
applied to multiple genes at one time or to create a large library
of chimeras (multiple mutations) of a single gene (Tunable
GeneReassembly.TM. (TGR.TM.) Technology supplied by Verenium
Corporation). 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, for example, codon usage can be optimized.
[0199] 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. USA 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 (1050). 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.
[0200] Iterative Saturation Mutagenesis (ISM) involves: 1) using
knowledge of structure/function to choose a likely site for enzyme
improvement; 2) performing saturation mutagenesis at chosen site
using a mutagenesis method such as Stratagene QuikChange
(Stratagene; San Diego Calif.); 3) screening/selecting for desired
properties; and 4) using improved clone(s), start over at another
site and continue repeating until a desired activity is achieved
(Reetz et al., Nat. Protoc. 2:891-903 (2007); and Reetz et al.,
Angew. Chem. Int. Ed Engl. 45:7745-7751 (2006)). This is a proven
methodology, which assures all possible replacements at a given
position are made for screening/selection.
[0201] Any of the aforementioned methods for mutagenesis can be
used alone or in any combination. Additionally, any one or
combination of the directed evolution methods can be used in
conjunction with adaptive evolution techniques, as described
herein.
[0202] 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 Producing Butadiene
[0203] Disclosed herein are novel processes for the direct
production of butadiene using engineered non-natural microorganisms
that possess the enzymes necessary for conversion of common
metabolites into the four carbon diene, 1,3-butadiene. One novel
route to direct production of butadiene entails reduction of the
known butanol pathway metabolite crotonyl-CoA to crotyl alcohol via
reduction with aldehyde and alcohol dehydrogenases, followed by
phosphorylation with kinases to afford crotyl pyrophosphate and
subsequent conversion to butadiene using isoprene synthases or
variants thereof (see FIG. 2). Another route (FIG. 3) is a variant
of the well-characterized DXP pathway for isoprenoid biosynthesis.
In this route, the substrate lacks a 2-methyl group and provides
butadiene rather than isoprene via a butadiene synthase. Such a
butadiene synthase can be derived from an isoprene synthase using
methods, such as directed evolution, as described herein. Finally,
FIG. 4 shows a pathway to butadiene involving the substrate
3-hydroxyglutaryl-CoA, which serves as a surrogate for the natural
mevalonate pathway substrate 3-hydroxy-3-methyl-glutaryl-CoA (shown
in FIG. 1). Enzyme candidates for steps A-P of FIG. 2, steps A-K of
FIG. 3 and steps A-O of FIG. 4 are provided below.
Acetyl-CoA:Acetyl-CoA Acyltransferase (FIG. 2, Step A)
[0204] Acetoacetyl-CoA thiolase 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)).
TABLE-US-00001 Protein 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 Reductase (FIG. 2, Step B)
[0205] Acetoacetyl-CoA reductase catalyzing the reduction of
acetoacetyl-CoA to 3-hydroxybutyryl-CoA participates in the
acetyl-CoA fermentation pathway to butyrate in several species of
Clostridia and has been studied in detail (Jones et al., Microbiol
Rev. 50:484-524 (1986)). The enzyme from Clostridium
acetobutylicum, encoded by hbd, has been cloned and functionally
expressed in E. coli (Youngleson et al., J Bacteriol. 171:6800-6807
(1989)). Additionally, subunits of two fatty acid oxidation
complexes in E. coli, encoded by fadB and fadJ, function as
3-hydroxyacyl-CoA dehydrogenases (Binstock et al., Methods Enzymol.
71 Pt C:403-411 (1981)). Yet other gene candidates demonstrated to
reduce acetoacetyl-CoA to 3-hydroxybutyryl-CoA are phbB from
Zoogloea ramigera (Ploux et al., Eur. J Biochem. 174:177-182
(1988)) and phaB from Rhodobacter sphaeroides (Alber et al., Mol.
Microbiol 61:297-309 (2006)). The former gene candidate is
NADPH-dependent, its nucleotide sequence has been determined
(Peoples et al., Mol. Microbiol 3:349-357 (1989)) and the gene has
been expressed in E. coli. Substrate specificity studies on the
gene led to the conclusion that it could accept 3-oxopropionyl-CoA
as a substrate besides acetoacetyl-CoA (Ploux et al., supra,
(1988)). Additional gene candidates include Hbd1 (C-terminal
domain) and Hbd2 (N-terminal domain) in Clostridium kluyveri
(Hillmer and Gottschalk, Biochim. Biophys. Acta 3334:12-23 (1974))
and HSD17B10 in Bos taurus (WAKIL et al., J Biol. Chem. 207:631-638
(1954)).
TABLE-US-00002 Protein Genbank ID GI number Organism fadB P21177.2
119811 Escherichia coli fadJ P77399.1 3334437 Escherichia coli Hbd2
EDK34807.1 146348271 Clostridium kluyveri Hbd1 EDK32512.1 146345976
Clostridium kluyveri hbd P52041.2 18266893 Clostridium
acetobutylicum HSD17B10 O02691.3 3183024 Bos Taurus phbB P23238.1
130017 Zoogloea ramigera phaB YP_353825.1 77464321 Rhodobacter
sphaeroides
[0206] A number of similar enzymes have been found in other species
of Clostridia and in Metallosphaera sedula (Berg et al., Science.
318:1782-1786 (2007)).
TABLE-US-00003 Protein GenBank ID GI number Organism hbd
NP_349314.1 NP_349314.1 Clostridium acetobutylicum hbd AAM14586.1
AAM14586.1 Clostridium beijerinckii Msed_1423 YP_001191505
YP_001191505 Metallosphaera sedula Msed_0399 YP_001190500
YP_001190500 Metallosphaera sedula Msed_0389 YP_001190490
YP_001190490 Metallosphaera sedula Msed_1993 YP_001192057
YP_001192057 Metallosphaera sedula
3-Hydroxybutyryl-CoA Dehydratase (FIG. 2, Step C)
[0207] 3-Hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), also called
crotonase, is an enoyl-CoA hydratase that reversibly dehydrates
3-hydroxybutyryl-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);
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 (2007a)) 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); Park et al.,
Biotechnol Bioeng 86:681-686 (2004)) and paaG (Ismail et al.,
supra, (2003); Park and Lee, supra, (2004); Park and Yup, supra,
(2004)). These proteins are identified below.
TABLE-US-00004 Protein 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
Crotonyl-CoA Reductase (Aldehyde Forming) (FIG. 2, Step D)
[0208] Several acyl-CoA dehydrogenases are capable of reducing an
acyl-CoA to its corresponding aldehyde. Thus they can naturally
reduce crotonyl-CoA to crotonaldehyde or can be engineered to do
so. Exemplary genes that encode such enzymes include the
Acinetobacter calcoaceticus acr1 encoding a fatty acyl-CoA
reductase (Reiser et al., J. Bacteriol. 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 et al., J Bacteriol.
178:871-880 (1996); Sohling et al., J. Bacteriol. 178:871-80
(1996))). SucD of P. gingivalis is another succinate semialdehyde
dehydrogenase (Takahashi et al., J. Bacteriol. 182:4704-4710
(2000)). These succinate semialdehyde dehydrogenases were
specifically shown in ref. (Burk et al., WO/2008/115840: (2008)) to
convert 4-hydroxybutyryl-CoA to 4-hydroxybutanal as part of a
pathway to produce 1,4-butanediol. The enzyme acylating
acetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG, is
yet another capable enzyme as it has been demonstrated to oxidize
and acylate acetaldehyde, propionaldehyde, butyraldehyde,
isobutyraldehyde and formaldehyde (Powlowski et al., J. Bacteriol.
175:377-385 (1993)).
TABLE-US-00005 Protein 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
[0209] 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 archael bacteria (Berg et al., Science
318:1782-1786 (2007b); Thauer, 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); Hugler et al., J. Bacteriol. 184:2404-2410
(2002)). The enzyme is encoded by Msed_0709 in Metallosphaera
sedula (Alber et al., supra, (2006); Berg et al., supra, (2007b)).
A gene encoding a malonyl-CoA reductase from Sulfolobus tokodaii
was cloned and heterologously expressed in E. coli (Alber et al.,
supra, (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. 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 eutE that
encodes acetaldehyde dehydrogenase of Salmonella typhimurium and E.
coli (Toth, Appl. Environ. Microbiol. 65:4973-4980 (1999). These
proteins are identified below.
TABLE-US-00006 Protein 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
Crotonaldehyde Reductase (Alcohol Forming) (FIG. 2, Step E)
[0210] Enzymes exhibiting crotonaldehyde reductase (alcohol
forming) activity are capable of forming crotyl alcohol from
crotonaldehyde. The following enzymes can naturally possess this
activity or can be engineered to exhibit this activity. 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., J.
Mol. Biol. 342:489-502 (2004)), and bdh I and bdh II from C.
acetobutylicum which converts butyraldehyde into butanol (Walter et
al., J. Bacteriol. 174:7149-7158 (1992)). 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)). Cbei_2181 from Clostridium
beijerinckii NCIMB 8052 encodes yet another useful alcohol
dehydrogenase capable of converting crotonaldehyde to crotyl
alcohol.
TABLE-US-00007 Protein 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
Cbei_2181 YP_001309304.1 150017050 Clostridium beijerinckii NCIMB
8052
[0211] Enzymes exhibiting 4-hydroxybutyrate dehydrogenase activity
(EC 1.1.1.61) also fall into this category. Such enzymes have been
characterized in Ralstonia eutropha (Bravo et al., J. Forensic Sci.
49:379-387 (2004)), Clostridium kluyveri (Wolff et al., Protein
Expr. Purif 6:206-212 (1995)) and Arabidopsis thaliana (Breitkreuz
et al., J. Biol. Chem. 278:41552-41556 (2003)).
TABLE-US-00008 Protein 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
Crotyl Alcohol Kinase (FIG. 2, Step F)
[0212] Crotyl alcohol kinase enzymes catalyze the transfer of a
phosphate group to the hydroxyl group of crotyl alcohol. The
enzymes described below naturally possess such activity or can be
engineered to exhibit this activity. Kinases that catalyze transfer
of a phosphate group to an alcohol group are members of the EC
2.7.1 enzyme class. The table below lists several useful kinase
enzymes in the EC 2.7.1 enzyme class.
TABLE-US-00009 Enzyme Commission Number Enzyme Name 2.7.1.1
hexokinase 2.7.1.2 glucokinase 2.7.1.3 ketohexokinase 2.7.1.4
fructokinase 2.7.1.5 rhamnulokinase 2.7.1.6 galactokinase 2.7.1.7
mannokinase 2.7.1.8 glucosamine kinase 2.7.1.10 phosphoglucokinase
2.7.1.11 6-phosphofructokinase 2.7.1.12 gluconokinase 2.7.1.13
dehydrogluconokinase 2.7.1.14 sedoheptulokinase 2.7.1.15 ribokinase
2.7.1.16 ribulokinase 2.7.1.17 xylulokinase 2.7.1.18
phosphoribokinase 2.7.1.19 phosphoribulokinase 2.7.1.20 adenosine
kinase 2.7.1.21 thymidine kinase 2.7.1.22 ribosylnicotinamide
kinase 2.7.1.23 NAD+ kinase 2.7.1.24 dephospho-CoA kinase 2.7.1.25
adenylyl-sulfate kinase 2.7.1.26 riboflavin kinase 2.7.1.27
erythritol kinase 2.7.1.28 triokinase 2.7.1.29 glycerone kinase
2.7.1.30 glycerol kinase 2.7.1.31 glycerate kinase 2.7.1.32 choline
kinase 2.7.1.33 pantothenate kinase 2.7.1.34 pantetheine kinase
2.7.1.35 pyridoxal kinase 2.7.1.36 mevalonate kinase 2.7.1.39
homoserine kinase 2.7.1.40 pyruvate kinase 2.7.1.41
glucose-1-phosphate phosphodismutase 2.7.1.42 riboflavin
phosphotransferase 2.7.1.43 glucuronokinase 2.7.1.44
galacturonokinase 2.7.1.45 2-dehydro-3- deoxygluconokinase 2.7.1.46
L-arabinokinase 2.7.1.47 D-ribulokinase 2.7.1.48 uridine kinase
2.7.1.49 hydroxymethylpyrimidine kinase 2.7.1.50
hydroxyethylthiazole kinase 2.7.1.51 L-fuculokinase 2.7.1.52
fucokinase 2.7.1.53 L-xylulokinase 2.7.1.54 D-arabinokinase
2.7.1.55 allose kinase 2.7.1.56 1-phosphofructokinase 2.7.1.58
2-dehydro-3- deoxygalactonokinase 2.7.1.59 N-acetylglucosamine
kinase 2.7.1.60 N-acylmannosamine kinase 2.7.1.61
acyl-phosphate-hexose phosphotransferase 2.7.1.62
phosphoramidate-hexose phosphotransferase 2.7.1.63
polyphosphate-glucose phosphotransferase 2.7.1.64 inositol 3-kinase
2.7.1.65 scyllo-inosamine 4-kinase 2.7.1.66 undecaprenol kinase
2.7.1.67 1-phosphatidylinositol 4- kinase 2.7.1.68
1-phosphatidylinositol-4- phosphate 5-kinase 2.7.1.69 protein-Np-
phosphohistidine-sugar phosphotransferase 2.7.1.70 identical to EC
2.7.1.37. 2.7.1.71 shikimate kinase 2.7.1.72 streptomycin 6-kinase
2.7.1.73 inosine kinase 2.7.1.74 deoxycytidine kinase 2.7.1.76
deoxyadenosine kinase 2.7.1.77 nucleoside phosphotransferase
2.7.1.78 polynucleotide 5'-hydroxyl- kinase 2.7.1.79
diphosphate-glycerol phosphotransferase 2.7.1.80 diphosphate-serine
phosphotransferase 2.7.1.81 hydroxylysine kinase 2.7.1.82
ethanolamine kinase 2.7.1.83 pseudouridine kinase 2.7.1.84
alkylglycerone kinase 2.7.1.85 -glucoside kinase 2.7.1.86 NADH
kinase 2.7.1.87 streptomycin 3''-kinase 2.7.1.88
dihydrostreptomycin-6- phosphate 3'a-kinase 2.7.1.89 thiamine
kinase 2.7.1.90 diphosphate-fructose-6- phosphate 1-
phosphotransferase 2.7.1.91 sphinganine kinase 2.7.1.92
5-dehydro-2- deoxygluconokinase 2.7.1.93 alkylglycerol kinase
2.7.1.94 acylglycerol kinase 2.7.1.95 kanamycin kinase 2.7.1.100
S-methyl-5-thioribose kinase 2.7.1.101 tagatose kinase 2.7.1.102
hamamelose kinase 2.7.1.103 viomycin kinase 2.7.1.105
6-phosphofructo-2-kinase 2.7.1.106 glucose-1,6-bisphosphate
synthase 2.7.1.107 diacylglycerol kinase 2.7.1.108 dolichol kinase
2.7.1.113 deoxyguanosine kinase 2.7.1.114 AMP-thymidine kinase
2.7.1.118 ADP-thymidine kinase 2.7.1.119 hygromycin-B 7''-O-kinase
2.7.1.121 phosphoenolpyruvate- glycerone phosphotransferase
2.7.1.122 xylitol kinase 2.7.1.127 inositol-trisphosphate 3- kinase
2.7.1.130 tetraacyldisaccharide 4'- kinase 2.7.1.134
inositol-tetrakisphosphate 1-kinase 2.7.1.136 macrolide 2'-kinase
2.7.1.137 phosphatidylinositol 3- kinase 2.7.1.138 ceramide kinase
2.7.1.140 inositol-tetrakisphosphate 5-kinase 2.7.1.142
glycerol-3-phosphate- glucose phosphotransferase 2.7.1.143
diphosphate-purine nucleoside kinase 2.7.1.144 tagatose-6-phosphate
kinase 2.7.1.145 deoxynucleoside kinase 2.7.1.146 ADP-dependent
phosphofructokinase 2.7.1.147 ADP-dependent glucokinase 2.7.1.148
4-(cytidine 5'-diphospho)- 2-C-methyl-D-erythritol kinase 2.7.1.149
1-phosphatidylinositol-5- phosphate 4-kinase 2.7.1.150
1-phosphatidylinositol-3- phosphate 5-kinase 2.7.1.151
inositol-polyphosphate multikinase 2.7.1.153
phosphatidylinositol-4,5- bisphosphate 3-kinase 2.7.1.154
phosphatidylinositol-4- phosphate 3-kinase 2.7.1.156
adenosylcobinamide kinase 2.7.1.157 N-acetylgalactosamine kinase
2.7.1.158 inositol-pentakisphosphate 2-kinase 2.7.1.159
inositol-1,3,4-trisphosphate 5/6-kinase 2.7.1.160
2'-phosphotransferase 2.7.1.161 CTP-dependent riboflavin kinase
2.7.1.162 N-acetylhexosamine 1- kinase 2.7.1.163 hygromycin B
4-O-kinase 2.7.1.164 O-phosphoseryl-tRNASec kinase
[0213] A good candidate for this step is mevalonate kinase (EC
2.7.1.36) that phosphorylates the terminal hydroxyl group of the
methyl analog, mevalonate, of 3,5-dihydroxypentanote. Some gene
candidates for this step are erg12 from S. cerevisiae, mvk from
Methanocaldococcus jannaschi, MVK from Homo sapeins, and mvk from
Arabidopsis thaliana col.
TABLE-US-00010 Protein GenBank ID GI Number Organism erg12
CAA39359.1 3684 Sachharomyces cerevisiae mvk Q58487.1 2497517
Methanocaldococcus jannaschii mvk AAH16140.1 16359371 Homo sapiens
M\mvk NP_851084.1 30690651 Arabidopsis thaliana
[0214] Glycerol kinase also phosphorylates the terminal hydroxyl
group in glycerol to form glycerol-3-phosphate. This reaction
occurs in several species, including Escherichia coli,
Saccharomyces cerevisiae, and Thermotoga maritima. The E. coli
glycerol kinase has been shown to accept alternate substrates such
as dihydroxyacetone and glyceraldehyde (Hayashi et al., J Biol.
Chem. 242:1030-1035 (1967)). T, maritime has two glycerol kinases
(Nelson et al., Nature 399:323-329 (1999)). Glycerol kinases have
been shown to have a wide range of substrate specificity. Crans and
Whiteside studied glycerol kinases from four different organisms
(Escherichia coli, S. cerevisiae, Bacillus stearothermophilus, and
Candida mycoderma) (Crans et al., J. Am. Chem. Soc. 107:7008-7018
(2010); Nelson et al., supra, (1999)). They studied 66 different
analogs of glycerol and concluded that the enzyme could accept a
range of substituents in place of one terminal hydroxyl group and
that the hydrogen atom at C2 could be replaced by a methyl group.
Interestingly, the kinetic constants of the enzyme from all four
organisms were very similar. The gene candidates are:
TABLE-US-00011 Protein GenBank ID GI Number Organism glpK
AP_003883.1 89110103 Escherichia coli K12 glpK1 NP_228760.1
15642775 Thermotoga maritime MSB8 glpK2 NP_229230.1 15642775
Thermotoga maritime MSB8 Gut1 NP_011831.1 82795252 Saccharomyces
cerevisiae
[0215] Homoserine kinase is another possible candidate that can
lead to the phosphorylation of 3,5-dihydroxypentanoate. This enzyme
is also present in a number of organisms including E. coli,
Streptomyces sp, and S. cerevisiae. Homoserine kinase from E. coli
has been shown to have activity on numerous substrates, including,
L-2-amino,1,4-butanediol, aspartate semialdehyde, and
2-amino-5-hydroxyvalerate (Huo et al., Biochemistry 35:16180-16185
(1996); Huo et al., Arch. Biochem. Biophys. 330:373-379 (1996)).
This enzyme can act on substrates where the carboxyl group at the
alpha position has been replaced by an ester or by a hydroxymethyl
group. The gene candidates are:
TABLE-US-00012 Protein GenBank ID GI Number Organism thrB
BAB96580.2 85674277 Escherichia coli K12 SACT1DRAFT_4809
ZP_06280784.1 282871792 Streptomyces sp. ACT-1 Thr1 AAA35154.1
172978 Saccharomyces serevisiae
2-Butenyl-4-Phosphate Kinase (FIG. 2, Step G)
[0216] 2-Butenyl-4-phosphate kinase enzymes catalyze the transfer
of a phosphate group to the phosphate group of
2-butenyl-4-phosphate. The enzymes described below naturally
possess such activity or can be engineered to exhibit this
activity. Kinases that catalyze transfer of a phosphate group to
another phosphate group are members of the EC 2.7.4 enzyme class.
The table below lists several useful kinase enzymes in the EC 2.7.4
enzyme class.
TABLE-US-00013 Enzyme Commission Number Enzyme Name 2.7.4.1
polyphosphate kinase 2.7.4.2 phosphomevalonate kinase 2.7.4.3
adenylate kinase 2.7.4.4 nucleoside-phosphate kinase 2.7.4.6
nucleoside-diphosphate kinase 2.7.4.7 phosphomethylpyrimidine
kinase 2.7.4.8 guanylate kinase 2.7.4.9 dTMP kinase 2.7.4.10
nucleoside-triphosphate-adenylate kinase 2.7.4.11 (deoxy)adenylate
kinase 2.7.4.12 T2-induced deoxynucleotide kinase 2.7.4.13
(deoxy)nucleoside-phosphate kinase 2.7.4.14 cytidylate kinase
2.7.4.15 thiamine-diphosphate kinase 2.7.4.16 thiamine-phosphate
kinase 2.7.4.17 3-phosphoglyceroyl-phosphate-polyphosphate
phosphotransferase 2.7.4.18 farnesyl-diphosphate kinase 2.7.4.19
5-methyldeoxycytidine-5'-phosphate kinase 2.7.4.20
dolichyl-diphosphate-polyphosphate phosphotransferase 2.7.4.21
inositol-hexakisphosphate kinase 2.7.4.22 UMP kinase 2.7.4.23
ribose 1,5-bisphosphate phosphokinase 2.7.4.24
diphosphoinositol-pentakisphosphate kinase
[0217] Phosphomevalonate kinase enzymes are of particular interest.
Phosphomevalonate kinase (EC 2.7.4.2) catalyzes the analogous
transformation to 2-butenyl-4-phosphate kinase. This enzyme is
encoded by erg8 in Saccharomyces cerevisiae (Tsay et al., Mol. Cell
Biol. 11:620-631 (1991)) and mvaK2 in Streptococcus pneumoniae,
Staphylococcus aureus and Enterococcus faecalis (Doun et al.,
Protein Sci. 14:1134-1139 (2005); Wilding et al., J Bacteriol.
182:4319-4327 (2000)). The Streptococcus pneumoniae and
Enterococcus faecalis enzymes were cloned and characterized in E.
coli (Pilloff et al., J Biol. Chem. 278:4510-4515 (2003); Doun et
al., Protein Sci. 14:1134-1139 (2005)).
TABLE-US-00014 Protein GenBank ID GI Number Organism Erg8
AAA34596.1 171479 Saccharomyces cerevisiae mvaK2 AAG02426.1 9937366
Staphylococcus aureus mvaK2 AAG02457.1 9937409 Streptococcus
pneumoniae mvaK2 AAG02442.1 9937388 Enterococcus faecalis
Butadiene Synthase (FIG. 2, Step H)
[0218] Butadiene synthase catalyzes the conversion of
2-butenyl-4-diphosphate to 1,3-butadiene. The enzymes described
below naturally possess such activity or can be engineered to
exhibit this activity. Isoprene synthase naturally catalyzes the
conversion of dimethylallyl diphosphate to isoprene, but can also
catalyze the synthesis of 1,3-butadiene from
2-butenyl-4-diphosphate. Isoprene synthases can be found in several
organisms including Populus alba (Sasaki et al., FEBS Letters,
2005, 579 (11), 2514-2518), Pueraria montana (Lindberg et al.,
Metabolic Eng, 2010, 12 (1), 70-79; Sharkey et al., Plant Physiol.,
2005, 137 (2), 700-712), and Populus tremula.times.Populus alba
(Miller et al., Planta, 2001, 213 (3), 483-487). Additional
isoprene synthase enzymes are described in (Chotani et al.,
WO/2010/031079, Systems Using Cell Culture for Production of
Isoprene; Cervin et al., US Patent Application 20100003716,
Isoprene Synthase Variants for Improved Microbial Production of
Isoprene).
TABLE-US-00015 Protein GenBank ID GI Number Organism ispS
BAD98243.1 63108310 Populus alba ispS AAQ84170.1 35187004 Pueraria
montana ispS CAC35696.1 13539551 Populus tremula x Populus alba
Crotonyl-CoA Hydrolase, Synthetase, Transferase (FIG. 2, Step
I)
[0219] Crotonyl-CoA hydrolase catalyzes the conversion of
crotonyl-CoA to crotonate. The enzymes described below naturally
possess such activity or can be engineered to exhibit this
activity. 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 proteins are identified below.
TABLE-US-00016 Protein 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
[0220] 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 proteins are identified
below.
TABLE-US-00017 Protein GenBank ID GI Number Organism acot12
NP_570103.1 18543355 Rattus norvegicus ACH1 NP_009538 6319456
Saccharomyces cerevisiae
[0221] 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 proteins are identified below.
TABLE-US-00018 Protein 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
[0222] 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 can also serve as
candidates for this reaction step but would require certain
mutations to change their function. These proteins are identified
below.
TABLE-US-00019 Protein GenBank ID GI Number Organism gctA CAA57199
559392 Acidaminococcus fermentans gctB CAA57200 559393
Acidaminococcus fermentans
[0223] Crotonyl-CoA synthetase catalyzes the conversion of
crotonyl-CoA to crotonate. The enzymes described below naturally
possess such activity or can be engineered to exhibit this
activity. 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 proteins are identified
below.
TABLE-US-00020 Protein 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
[0224] 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 proteins are identified below.
TABLE-US-00021 Protein GenBank ID GI Number Organism sucC
NP_415256.1 16128703 Escherichia coli sucD AAC73823.1 1786949
Escherichia coli
[0225] 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 proteins are identified below.
TABLE-US-00022 Protein 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
[0226] Crotonyl-CoA transferase catalyzes the conversion of
crotonyl-CoA to crotonate. The enzymes described below naturally
possess such activity or can be engineered to exhibit this
activity. Many transferases have broad specificity and thus can
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. For example, an 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,
FEBSLetters, 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, Citrobacteryoungae ATCC 29220, Salmonella enterica
subsp. arizonae serovar, and Yersinia intermedia ATCC 29909. These
proteins are identified below.
TABLE-US-00023 Protein 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
[0227] 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 proteins are identified below.
TABLE-US-00024 Protein 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
[0228] 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 proteins are identified below.
TABLE-US-00025 Protein 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
[0229] The above enzymes can also exhibit the desired activities on
crotonyl-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 proteins are
identified below.
TABLE-US-00026 Protein 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
[0230] 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
proteins are identified below.
TABLE-US-00027 Protein GenBank ID GI Number Organism gctA
CAA57199.1 559392 Acidaminococcus fermentans gctB CAA57200.1 559393
Acidaminococcus fermentans
Crotonate Reductase (FIG. 2, Step J)
[0231] Crotonate reductase enzymes are capable of catalyzing the
conversion of crotonate to crotonaldehyde. The enzymes described
below naturally possess such activity or can be engineered to
exhibit this activity. Carboxylic acid reductase 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)). 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., in
Biocatalysis in the Pharmaceutical and Biotechnology Industires,
ed. R. N. Patel, Chapter 15, pp. 425-440, CRC Press LLC, Boca
Raton, Fla. (2006)).
TABLE-US-00028 Protein GenBank ID GI Number Organism Car AAR91681.1
40796035 Nocardia iowensis (sp. NRRL 5646) Npt ABI83656.1 114848891
Nocardia iowensis (sp. NRRL 5646)
[0232] Additional car and npt genes can be identified based on
sequence homology.
TABLE-US-00029 Protein 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 118473501 Mycobacterium smegmatis MC2 155 MSMEG_5739
YP_889972.1 118469671 Mycobacterium smegmatis MC2 155 MSMEG_2648
YP_886985.1 118471293 Mycobacterium smegmatis MC2 155 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
[0233] 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, can be beneficial.
TABLE-US-00030 Protein 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
[0234] 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.
TABLE-US-00031 Protein 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
Crotonyl-CoA Reductase (Alcohol Forming) (FIG. 2, Step K)
[0235] Crotonaldehyde reductase (alcohol forming) enzymes catalyze
the 2 reduction steps required to form crotyl alcohol from
crotonyl-CoA. Exemplary 2-step oxidoreductases that convert an
acyl-CoA to an alcohol are provided below. Such enzymes can
naturally convert crotonyl-CoA to crotyl alcohol or can be
engineered to do so. These enzymes 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))) and butyryl-CoA to
butanol (e.g. adhE2 from C. acetobutylicum (Fontaine et al., J.
Bacteriol. 184:821-830 (2002))). The adhE2 enzyme from C.
acetobutylicum was specifically shown in ref. (Burk et al., supra,
(2008)) to produce BDO from 4-hydroxybutyryl-CoA. In addition to
reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in
Leuconostoc mesenteroides has been shown to oxide the branched
chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al.,
J. Gen. Appl. Microbiol. 18:43-55 (1972); Koo et al., Biotechnol.
Lett. 27:505-510 (2005)).
TABLE-US-00032 Protein GenBank ID GI Number Organism adhE
NP_415757.1 16129202 Escherichia coli adhE2 AAK09379.1 12958626
Clostridium acetobutylicum adhE AAV66076.1 55818563 Leuconostoc
mesenteroides
[0236] 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 et al., supra, (2002); Strauss et
al., 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., supra, (2002)). No
enzymes in other organisms have been shown to catalyze this
specific reaction; however there is bioinformatic evidence that
other organisms can have similar pathways (Klatt et al., 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.
TABLE-US-00033 Protein 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
Glutaconyl-CoA Decarboxylase (FIG. 2, Step L)
[0237] Glutaconyl-CoA decarboxylase enzymes, characterized in
glutamate-fermenting anaerobic bacteria, are sodium-ion
translocating decarboxylases that utilize biotin as a cofactor and
are composed of four subunits (alpha, beta, gamma, and delta)
(Boiangiu et al., J Mol. Microbiol Biotechnol 10:105-119 (2005);
Buckel, Biochim Biophys Acta. 1505:15-27 (2001)). Such enzymes have
been characterized in Fusobacterium nucleatum (Beatrix et al., Arch
Microbiol. 154:362-369 (1990)) and Acidaminococcus fermentans
(Braune et al., Mol. Microbiol 31:473-487 (1999)). Analogs to the
F. nucleatum glutaconyl-CoA decarboxylase alpha, beta and delta
subunits are found in S. aciditrophicus. A gene annotated as an
enoyl-CoA dehydrogenase, syn_00480, another GCD, is located in a
predicted operon between a biotin-carboxyl carrier (syn_00479) and
a glutaconyl-CoA decarboxylase alpha subunit (syn_00481). The
protein sequences for exemplary gene products can be found using
the following GenBank accession numbers shown below.
TABLE-US-00034 Protein GenBank ID GI Number Organism gcdA CAA49210
49182 Acidaminococcus fermentans gcdC AAC69172 3777506
Acidaminococcus fermentans gcdD AAC69171 3777505 Acidaminococcus
fermentans gcdB AAC69173 3777507 Acidaminococcus fermentans FN0200
AAL94406 19713641 Fusobacterium nucleatum FN0201 AAL94407 19713642
Fusobacterium nucleatum FN0204 AAL94410 19713645 Fusobacterium
nucleatum syn_00479 YP_462066 85859864 Syntrophus aciditrophicus
syn_00481 YP_462068 85859866 Syntrophus aciditrophicus syn_01431
YP_460282 85858080 Syntrophus aciditrophicus syn_00480 ABC77899
85722956 Syntrophus aciditrophicus
Glutaryl-CoA Dehydrogenase (FIG. 2 Step M)
[0238] Glutaryl-CoA dehydrogenase (GCD, EC 1.3.99.7 and EC
4.1.1.70) is a bifunctional enzyme that catalyzes the oxidative
decarboxylation of glutaryl-CoA to crotonyl-CoA (FIG. 3, step 3).
Bifunctional GCD enzymes are homotetramers that utilize electron
transfer flavoprotein as an electron acceptor (Hartel et al., Arch
Microbiol. 159:174-181 (1993)). Such enzymes were first
characterized in cell extracts of Pseudomonas strains KB740 and
K172 during growth on aromatic compounds (Hartel et al., supra,
(1993)), but the associated genes in these organisms is unknown.
Genes encoding glutaryl-CoA dehydrogenase (gcdH) and its cognate
transcriptional regulator (gcdR) were identified in Azoarcus sp.
CIB (Blazquez et al., Environ Microbiol. 10:474-482 (2008)). An
Azoarcus strain deficient in gcdH activity was used to identify the
a heterologous gene gcdH from Pseudomonas putida (Blazquez et al.,
supra, (2008)). The cognate transcriptional regulator in
Pseudomonas putida has not been identified but the locus PP_0157
has a high sequence homology (>69% identity) to the Azoarcus
enzyme. Additional GCD enzymes are found in Pseudomonas fluorescens
and Paracoccus denitrificans (Husain et al., J Bacteriol.
163:709-715 (1985)). The human GCD has been extensively studied,
overexpressed in E. coli (Dwyer et al., Biochemistry 39:11488-11499
(2000)), crystallized, and the catalytic mechanism involving a
conserved glutamate residue in the active site has been described
(Fu et al., Biochemistry 43:9674-9684 (2004)). A GCD in Syntrophus
aciditrophicus operates in the CO.sub.2-assimilating direction
during growth on crotonate (Mouttaki et al., Appl Environ
Microbiol. 73:930-938 (2007))). Two GCD genes in S. aciditrophicus
were identified by protein sequence homology to the Azoarcus GcdH:
syn_00480 (31%) and syn_01146 (31%). No significant homology was
found to the Azoarcus GcdR regulatory protein. The protein
sequences for exemplary gene products can be found using the
following GenBank accession numbers shown below.
TABLE-US-00035 Protein GenBank ID GI Number Organism gcdH
ABM69268.1 123187384 Azoarcus sp. CIB gcdR ABM69269.1 123187385
Azoarcus sp. CIB gcdH AAN65791.1 24981507 Pseudomonas putida KT2440
PP_0157 (gcdR) AAN65790.1 24981506 Pseudomonas putida KT2440 gcdH
YP_257269.1 70733629 Pseudomonas fluorescens Pf-5 gcvA (gcdR)
YP_257268.1 70733628 Pseudomonas fluorescens Pf-5 gcd YP_918172.1
119387117 Paracoccus denitrificans gcdR YP_918173.1 119387118
Paracoccus denitrificans gcd AAH02579.1 12803505 Homo sapiens
syn_00480 ABC77899 85722956 Syntrophus aciditrophicus syn_01146
ABC76260 85721317 Syntrophus aciditrophicus
3-Aminobutyryl-CoA Deaminase (FIG. 2, Step N)
[0239] 3-aminobutyryl-CoA is an intermediate in lysine
fermentation. It also can be formed from acetoacetyl-CoA via a
transaminase or an aminating dehydrogenase. 3-aminobutyryl-CoA
deaminase (or 3-aminobutyryl-CoA ammonia lyase) catalyzes the
deamination of 3-aminobutyryl-CoA to form crotonyl-CoA. This
reversible enzyme is present in Fusobacterium nucleatum,
Porphyromonas gigivalis, Thermoanaerobacter tengcongensis, and
several other organisms and is co-localized with several genes
involved in lysine fermentation (Kreimeyer et al., J Biol Chem,
2007, 282(10) 7191-7197).
TABLE-US-00036 Protein GenBank ID GI Number Organism kal
NP_602669.1 19705174 Fusobacterium nucleatum subsp. nucleatum ATCC
25586 kal NP_905282.1 34540803 Porphyromonas gingivalis W83 kal
NP_622376.1 20807205 Thermoanaerobacter tengcongensis MB4
4-Hydroxybutyryl-CoA Dehydratase (FIG. 2, Step O)
[0240] Several enzymes naturally catalyze the dehydration of
4-hydroxybutyryl-CoA to crotonoyl-CoA. This transformation is
required for 4-aminobutyrate fermentation by Clostridium
aminobutyricum (Scherf et al., Eur. J Biochem. 215:421-429 (1993))
and succinate-ethanol fermentation by Clostridium kluyveri (Scherf
et al., Arch. Microbiol 161:239-245 (1994)). The transformation is
also a key step in Archaea, for example, Metallosphaera sedula, as
part of the 3-hydroxypropionate/4-hydroxybutyrate autotrophic
carbon dioxide assimilation pathway (Berg et al., supra, (2007)).
The reversibility of 4-hydroxybutyryl-CoA dehydratase is
well-documented (Muh et al., Biochemistry. 35:11710-11718 (1996);
Friedrich et al., Angew. Chem. Int. Ed. Engl. 47:3254-3257 (2008);
Muh et al., Eur. J. Biochem. 248:380-384 (1997)) and the
equilibrium constant has been reported to be about 4 on the side of
crotonoyl-CoA (Scherf and Buckel, supra, (1993)).
TABLE-US-00037 Protein GenBank ID GI Number Organism AbfD CAB60035
70910046 Clostridium aminobutyricum AbfD YP_001396399 153955634
Clostridium kluyveri Msed_1321 YP_001191403 146304087
Metallosphaera sedula Msed_1220 YP_001191305 146303989
Metallosphaera sedula
Crotyl Alcohol Diphosphokinase (FIG. 2, Step P)
[0241] Crotyl alcohol diphosphokinase enzymes catalyze the transfer
of a diphosphate group to the hydroxyl group of crotyl alcohol. The
enzymes described below naturally possess such activity or can be
engineered to exhibit this activity. Kinases that catalyze transfer
of a diphosphate group are members of the EC 2.7.6 enzyme class.
The table below lists several useful kinase enzymes in the EC 2.7.6
enzyme class.
TABLE-US-00038 Enzyme Commission Number Enzyme Name 2.7.6.1
ribose-phosphate diphosphokinase 2.7.6.2 thiamine diphosphokinase
2.7.6.3 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine
diphosphokinase 2.7.6.4 nucleotide diphosphokinase 2.7.6.5 GTP
diphosphokinase
[0242] Of particular interest are ribose-phosphate diphosphokinase
enzymes which have been identified in Escherichia coli (Hove-Jenson
et al., J Biol Chem, 1986, 261(15); 6765-71) and Mycoplasma
pneumoniae M129 (McElwain et al, International Journal of
Systematic Bacteriology, 1988, 38:417-423) as well as thiamine
diphosphokinase enzymes. Exemplary thiamine diphosphokinase enzymes
are found in Arabidopsis thaliana (Ajjawi, Plant Mol Biol, 2007,
65(1-2); 151-62).
TABLE-US-00039 Protein GenBank ID GI Number Organism prs
NP_415725.1 16129170 Escherichia coli prsA NP_109761.1 13507812
Mycoplasma pneumoniae M129 TPK1 BAH19964.1 222424006 Arabidopsis
thaliana col TPK2 BAH57065.1 227204427 Arabidopsis thaliana col
Erythrose-4-Phosphate Reductase (FIG. 3, Step A)
[0243] In Step A of the pathway, erythrose-4-phosphate is converted
to erythritol-4-phosphate by the erythrose-4-phosphate reductase or
erythritol-4-phosphate dehydrogenase. The reduction of
erythrose-4-phosphate was observed in Leuconostoc oenos during the
production of erythritol (Veiga-da-Cunha et al., J Bacteriol.
175:3941-3948 (1993)). NADPH was identified as the cofactor
(Veiga-da-Cunha et al., supra, (1993)). However, gene for
erythrose-4-phosphate was not identified. Thus, it is possible to
identify the erythrose-4-phosphate reductase gene from Leuconostoc
oenos and apply to this step. Additionally, enzymes catalyzing
similar reactions can be utilized for this step. An example of
these enzymes is 1-deoxy-D-xylulose-5-phosphate reductoisomerase
(EC 1.1.1.267) catalyzing the conversion of 1-deoxy-D-xylylose
5-phosphate to 2-C-methyl-D-erythritol-4-phosphate, which has one
additional methyl group comparing to Step A. The dxr or ispC genes
encode the 1-deoxy-D-xylulose-5-phosphate reductoisomerase have
been well studied: the Dxr proteins from Escherichia coli and
Mycobacterium tuberculosis were purified and their crystal
structures were determined (Yajima et al., Acta Crystallogr. Sect.
F. Struct. Biol. Cryst. Commun. 63:466-470 (2007); Mac et al., J
Mol. Biol. 345:115-127 (2005); Henriksson et al., Acta Crystallogr.
D. Biol. Crystallogr. 62:807-813 (2006); Henriksson et al., J Biol.
Chem. 282:19905-19916 (2007)); the Dxr protein from Synechocystis
sp was studied by site-directed mutagenesis with modified activity
and altered kinetics (Fernandes et al., Biochim. Biophys. Acta
1764:223-229 (2006); Fernandes et al., Arch. Biochem. Biophys.
444:159-164 (2005)). Furthermore, glyceraldehyde 3-phosphate
reductase YghZ from Escherichia coli catalyzes the conversion
between glyceraldehyde 3-phosphate and glycerol-3-phosphate (Desai
et al., Biochemistry 47:7983-7985 (2008)) can also be applied to
this step. The following genes can be used for Step A
conversion:
TABLE-US-00040 Protein GenBank ID GI Number Organism dxr P45568.2
2506592 Escherichia coli strain K12 dxr A5U6M4.1 166218269
Mycobacterium tuberculosis dxr Q55663.1 2496789 Synechocystis sp.
strain PCC6803 yghZ NP_417474.1 16130899 Escherichia coli strain
K12
Erythritol-4-Phospate Cytidylyltransferase (FIG. 3, Step B)
[0244] In Step B of the pathway, erythritol-4-phosphate is
converted to 4-(cytidine 5'-diphospho)-erythritol by the
erythritol-4-phospate cytidylyltransferase or 4-(cytidine
5'-diphospho)-erythritol synthase. The exact enzyme for this step
has not been identified. However, enzymes catalyzing similar
reactions can be applied to this step. An example is the
2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase or
4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol synthase (EC
2.7.7.60). The 2-C-methyl-D-erythritol 4-phospate
cytidylyltransferase is in the methylerythritol phosphate pathway
for the isoprenoid biosynthesis and catalyzes the conversion
between 2-C-methyl-D-erythritol 4-phospate and 4-(cytidine
5'-diphospho)-2-C-methyl-D-erythritol, with an extra methyl group
comparing to Step B conversion. The 2-C-methyl-D-erythritol
4-phosphate cytidylyltransferase is encoded by ispD gene and the
crystal structure of Escherichia coli IspD was determined (Kemp et
al., Acta Crystallogr. D. Biol. Crystallogr. 57:1189-1191 (2001);
Kemp et al., Acta Crystallogr. D. Biol. Crystallogr. 59:607-610
(2003); Richard et al., Nat. Struct. Biol. 8:641-648 (2001)). The
ispD gene from Mycobacterium tuberculosis H37Rv was cloned and
expressed in Escherichia coli, and the recombinant proteins were
purified with N-terminal His-tag (Shi et al., J Biochem. Mol. Biol.
40:911-920 (2007)). Additionally, the Streptomyces coelicolor ispD
gene was cloned and expressed in E. coli, and the recombinant
proteins were characterized physically and kinetically (Cane et
al., Bioorg. Med. Chem. 9:1467-1477 (2001)). The following genes
can be used for Step B conversion:
TABLE-US-00041 Protein GenBank ID GI Number Organism ispD Q46893.3
2833415 Escherichia coli strain K12 ispD A5U8Q7.1 166215456
Mycobacterium tuberculosis ispD Q9L0Q8.1 12230289 Streptomyces
coelicolor
4-(Cytidine 5'-Diphospho)-Erythritol Kinase (FIG. 3, Step C)
[0245] In Step C of the pathway, 4-(cytidine
5'-diphospho)-erythritol is converted to 2-phospho-4-(cytidine
5'-diphospho)-erythritol by the 4-(cytidine
5'-diphospho)-erythritol kinase. The exact enzyme for this step has
not been identified. However, enzymes catalyzing similar reactions
can be applied to this step. An example is the
4-diphosphocytidyl-2-C-methylerythritol kinase (EC 2.7.1.148). The
4-diphosphocytidyl-2-C-methylerythritol kinase is also in the
methylerythritol phosphate pathway for the isoprenoid biosynthesis
and catalyzes the conversion between 4-(cytidine
5'-diphospho)-2-C-methyl-D-erythritol and 2-phospho-4-(cytidine
5'-diphospho)-2-C-methyl-D-erythritol, with an extra methyl group
comparing to Step C conversion. The
4-diphosphocytidyl-2-C-methylerythritol kinase is encoded by ispE
gene and the crystal structures of Escherichia coli, Thermus
thermophilus HB8, and Aquifex aeolicus IspE were determined (Sgraja
et al., FEBS J 275:2779-2794 (2008); Miallau et al., Proc. Natl.
Acad. Sci. U.S.A 100:9173-9178 (2003); Wada et al., J Biol. Chem.
278:30022-30027 (2003)). The ispE genes from above organism were
cloned and expressed, and the recombinant proteins were purified
for crystallization. The following genes can be used for Step C
conversion:
TABLE-US-00042 Protein GenBank ID GI Number Organism ispE P62615.1
50402174 Escherichia coli strain K12 ispE P83700.1 51316201 Thermus
thermophilus HB8 ispE O67060.1 6919911 Aquifex aeolicus
Erythritol 2,4-Cyclodiphosphate Synthase (FIG. 3, Step D)
[0246] In Step D of the pathway, 2-phospho-4-(cytidine
5'-diphospho)-erythritol is converted to
erythritol-2,4-cyclodiphosphate by the Erythritol
2,4-cyclodiphosphate synthase. The exact enzyme for this step has
not been identified. However, enzymes catalyzing similar reactions
can be applied to this step. An example is the
2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (EC
4.6.1.12). The 2-C-methyl-D-erythritol 2,4-cyclodiphosphate
synthase is also in the methylerythritol phosphate pathway for the
isoprenoid biosynthesis and catalyzes the conversion between
2-phospho-4-(cytidine 5'diphospho)-2-C-methyl-D-erythritol and
2-C-methyl-D-erythritol-2,4-cyclodiphosphate, with an extra methyl
group comparing to step D conversion. The 2-C-methyl-D-erythritol
2,4-cyclodiphosphate synthase is encoded by ispF gene and the
crystal structures of Escherichia coli, Thermus thermophilus,
Haemophilus influenzae, and Campylobacter jejuni IspF were
determined (Richard et al., J Biol. Chem. 277:8667-8672 (2002);
Steinbacher et al., J Mol. Biol. 316:79-88 (2002); Lehmann et al.,
Proteins 49:135-138 (2002); Kishida et al., Acta Crystallogr. D.
Biol. Crystallogr. 59:23-31 (2003); Gabrielsen et al., J Biol.
Chem. 279:52753-52761 (2004)). The ispF genes from above organism
were cloned and expressed, and the recombinant proteins were
purified for crystallization. The following genes can be used for
Step D conversion:
TABLE-US-00043 Protein GenBank ID GI Number Organism ispF P62617.1
51317402 Escherichia coli strain K12 ispF Q8RQP5.1 51701599 Thermus
thermophilus HB8 ispF P44815.1 1176081 Haemophilus influenzae ispF
Q9PM68.1 12230305 Campylobacter jejuni
1-Hydroxy-2-Butenyl 4-Diphosphate Synthase (FIG. 3, Step E)
[0247] Step E of FIG. 3 entails conversion of
erythritol-2,4-cyclodiphosphate to 1-hydroxy-2-butenyl
4-diphosphate by 1-hydroxy-2-butenyl 4-diphosphate synthase. An
enzyme with this activity has not been characterized to date. This
transformation is analogous to the reduction of
2-C-methyl-D-erythritol-2,4-cyclodiphosphate to
1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate by
(E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase (EC
1.17.7.1). This enzyme is an iron-sulfur protein that participates
in the non-mevalonate pathway for isoprenoid biosynthesis found in
bacteria and plants. Most bacterial enzymes including the E. coli
enzyme, encoded by ispG, utilize reduced ferredoxin or flavodoxin
as an electron donor (Zepeck et al., J Org. Chem. 70:9168-9174
(2005)). An analogous enzyme from the thermophilic cyanobacterium
Thermosynechococcus elongatus BP-1, encoded by gcpE, was
heterologously expressed and characterized in E. coli (Okada et
al., J Biol. Chem. 280:20672-20679 (2005)). Additional enzyme
candidates from Thermus thermophilus and Arabidopsis thaliana have
been characterized and expressed in E. coli (Seemann et al., J
Biol. Inorg. Chem. 10:131-137 (2005); Kollas et al., FEBS Lett.
532:432-436 (2002)).
TABLE-US-00044 Protein GenBank ID GI Number Organism ispG
NP_417010.1 16130440 Escherichia coli gcpE NP_681786.1 22298539
Thermosynechococcus elongatus gcpE AAO21364.1 27802077 Thermus
thermophilus gcpE AAO15446.1 27462472 Arabidopsis thaliana
1-Hydroxy-2-Butenyl 4-Diphosphate Reductase (FIG. 3, Step F)
[0248] The concurrent dehydration and reduction of
1-hydroxy-2-butenyl 4-diphosphate is catalyzed by an enzyme with
1-hydroxy-2-butenyl 4-diphosphate reductase activity (FIG. 3, Step
F). Such an enzyme will form a mixture of products, butenyl
4-diphosphate or 2-butenyl 4-diphosphate. An analogous reaction is
catalyzed by 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC
1.17.1.2) in the non-mevalonate pathway for isoprenoid
biosynthesis. This enzyme is an iron-sulfur protein that utilizes
reduced ferredoxin or flavodoxin as an electron donor. Maximal
activity of 4-hydroxy-3-methylbut-2-enyl diphosphate reductase E.
coli, encoded by ispH, requires both flavodoxin and flavodoxin
reductase (Wolff et al., FEBS Lett. 541:115-120 (2003); Grawert et
al., J Am. Chem. Soc. 126:12847-12855 (2004)). In the characterized
catalytic system, reduced flavodoxin is regenerated by the
NAD(P)+-dependent flavodoxin reductase. The enzyme from Aquifex
aeolicus, encoded by lytB, was expressed as a His-tagged enzyme in
E. coli and characterized (Altincicek et al., FEBS Lett.
532:437-440 (2002)). An analogous enzyme in plants is encoded by
hdr of Arabidopsis thaliana (Botella-Pavia et al., Plant J
40:188-199 (2004)).
TABLE-US-00045 Protein GenBank ID GI Number Organism ispH
AAL38655.1 18652795 Escherichia coli lytB O67625.1 8928180 Aquifex
aeolicus hdr NP_567965.1 18418433 Arabidopsis thaliana
[0249] Altering the expression level of genes involved in
iron-sulfur cluster formation can have an advantageous effect on
the activities of iron-sulfur proteins in the proposed pathways
(for example, enzymes required in Steps E and F of FIG. 3). In E.
coli, it was demonstrated that overexpression of the iron-sulfur
containing protein IspH (analogous to Step F of FIG. 3) is enhanced
by coexpression of genes from the isc region involved in assembly
of iron-sulfur clusters (Grawert et al., J Am. Chem. Soc.
126:12847-12855 (2004)). The gene cluster is composed of the genes
icsS, icsU, icsA, hscB, hscA andfdx. Overexpression of these genes
was shown to improve the synthetic capability of the iron-sulfur
assembly pipeline, required for functional expression of
iron-sulfur proteins. A similar approach can be applicable in the
current application.
TABLE-US-00046 Protein GenBank ID GI Number Organism iscS
AAT48142.1 48994898 Escherichia coli iscU AAC75582.1 1788878
Escherichia coli iscA AAC75581.1 1788877 Escherichia coli hscB
AAC75580.1 1788876 Escherichia coli hscA AAC75579.1 1788875
Escherichia coli fdx AAC75578.1 1788874 Escherichia coli
Butenyl 4-Diphosphate Isomerase (FIG. 3, Step G)
[0250] Butenyl 4-diphosphate isomerase catalyzes the reversible
interconversion of 2-butenyl-4-diphosphate and
butenyl-4-diphosphate. The following enzymes can naturally possess
this activity or can be engineered to exhibit this activity. Useful
genes include those that encode enzymes that interconvert
isopenenyl diphosphate and dimethylallyl diphosphate. These include
isopentenyl diphosphate isomerase enzymes from Escherichia coli
(Rodriguez-Concepcion et al., FEBS Lett, 473(3):328-332),
Saccharomyces cerevisiae (Anderson et al., J Biol Chem, 1989,
264(32); 19169-75), and Sulfolobus shibatae (Yamashita et al, Eur J
Biochem, 2004, 271(6); 1087-93). The reaction mechanism of
isomerization, catalyzed by the Idi protein of E. coli, has been
characterized in mechanistic detail (de Ruyck et al., J Biol. Chem.
281:17864-17869 (2006)). Isopentenyl diphosphate isomerase enzymes
from Saccharomyces cerevisiae, Bacillus subtilis and Haematococcus
pluvialis have been heterologously expressed in E. coli (Laupitz et
al., Eur. J Biochem. 271:2658-2669 (2004); Kajiwara et al.,
Biochem. J 324 (Pt 2):421-426 (1997)).
TABLE-US-00047 Protein GenBank ID GI Number Organism Idi
NP_417365.1 16130791 Escherichia coli IDI1 NP_015208.1 6325140
Saccharomyces cerevisiae Idi BAC82424.1 34327946 Sulfolobus
shibatae Idi AAC32209.1 3421423 Haematococcus pluvialis Idi
BAB32625.1 12862826 Bacillus subtilis
Butadiene Synthase (FIG. 3, Step H)
[0251] Butadiene synthase catalyzes the conversion of
2-butenyl-4-diphosphate to 1,3-butadiene. The enzymes described
below naturally possess such activity or can be engineered to
exhibit this activity. Isoprene synthase naturally catalyzes the
conversion of dimethylallyl diphosphate to isoprene, but can also
catalyze the synthesis of 1,3-butadiene from
2-butenyl-4-diphosphate. Isoprene synthases can be found in several
organisms including Populus alba (Sasaki et al., FEBS Letters, 579
(11), 2514-2518 (2005)), Pueraria montana (Lindberg et al.,
Metabolic Eng, 12(1):70-79 (2010); Sharkey et al., Plant Physiol.,
137(2):700-712 (2005)), and Populus tremula.times.Populus alba
(Miller et al., Planta, 213(3):483-487 (2001)). Additional isoprene
synthase enzymes are described in (Chotani et al., WO/2010/031079,
Systems Using Cell Culture for Production of Isoprene; Cervin et
al., US Patent Application 20100003716, Isoprene Synthase Variants
for Improved Microbial Production of Isoprene).
TABLE-US-00048 Protein GenBank ID GI Number Organism ispS
BAD98243.1 63108310 Populus alba ispS AAQ84170.1 35187004 Pueraria
montana ispS CAC35696.1 13539551 Populus tremula .times. Populus
alba
Erythrose-4-Phosphate Kinase (FIG. 3, Step I)
[0252] In Step I of the pathway, erythrose-4-phosphate is converted
to erythrose by the erythrose-4-phosphate kinase. In industrial
fermentative production of erythritol by yeasts, glucose was
converted to erythrose-4-phosphate through the pentose phosphate
pathway and erythrose-4-phosphate was dephosphorylated and reduced
to produce erythritol (Moon et al., Appl. Microbiol Biotechnol.
86:1017-1025 (2010)). Thus, erythrose-4-phosphate kinase is present
in many of these erythritol-producing yeasts, including
Trichosporonoides megachiliensis SN-G42 (Sawada et al., J Biosci.
Bioeng. 108:385-390 (2009)), Candida magnolia (Kohl et al.,
Biotechnol. Lett. 25:2103-2105 (2003)), and Torula sp. (HAJNY et
al., Appl. Microbiol 12:240-246 (1964), Oh et al., J Ind. Microbiol
Biotechnol. 26:248-252 (2001)). However, the erythrose-4-phosphate
kinase genes were not identified yet. There are many polyol
phosphotransferases with wide substrate range that can be applied
to this step. An example is the triose kinase (EC 2.7.1.28)
catalyzing the reversible conversion between glyceraldehydes and
glyceraldehydes-3-phosphate, which are one carbon shorter comparing
to Step I. Other examples include the xylulokinase (EC 2.7.1.17) or
arabinokinase (EC 2.7.1.54) that catalyzes phosphorylation of 5 C
polyol aldehyde. The following genes can be used for Step I
conversion:
TABLE-US-00049 Protein GenBank ID GI Number Organism xylB P09099.1
139849 Escherichia coli strain K12 xks1 P42826.2 1723736
Saccharomyces cerevisiae xylB P29444.1 267426 Klebsiella pneumoniae
dak1 Q9HFC5 74624685 Zygosaccharomyces rouxii
Erythrose Reductase (FIG. 3, Step J)
[0253] In Step J of the pathway, erythrose is converted to
erythritol by the erythrose reductase. In industrial fermentative
production of erythritol by yeasts, glucose was converted to
erythrose-4-phosphate through the pentose phosphate pathway and
erythrose-4-phosphate was dephosphorylated and reduced to produce
erythritol (Moon et al., supra, (2010)). Thus, erythrose reductase
is present in many of these erythritol-producing yeasts, including
Trichosporonoides megachiliensis SN-G42 (Sawada et al., supra,
(2009)), Candida magnolia (Kohl et al., supra, (2003)), and Torula
sp. (HAJNY et al., supra, (1964); Oh et al., supra, (2001)).
Erythrose reductase was characterized and purified from Torula
corallina (Lee et al., Biotechnol. Prog. 19:495-500 (2003); Lee et
al., Appl. Environ. Microbiol 68:4534-4538 (2002)), Candida
magnolia (Lee et al., Appl. Environ. Microbiol 69:3710-3718 (2003))
and Trichosporonoides megachiliensis SN-G42 (Sawada et al., supra,
(2009)). Several erythrose reductase genes were cloned and can be
applied to Step J. The following genes can be used for Step J
conversion:
TABLE-US-00050 Protein GenBank ID GI Number Organism alr ACT78580.1
254679867 Candida magnoliae er1 BAD90687.1 60458781
Trichosporonoides megachiliensis er2 BAD90688.1 60458783
Trichosporonoides megachiliensis er3 BAD90689.1 60458785
Trichosporonoides megachiliensis
Erythritol Kinase (FIG. 3, Step K)
[0254] In Step K of the pathway, erythritol is converted to
erythritol-4-phosphate by the erythritol kinase. Erythritol kinase
(EC 2.7.1.27) catalyzes the phosphorylation of erythritol.
Erythritol kinase was characterized in erythritol utilizing
bacteria such as Brucella abortus (Sperry et al., J Bacteriol.
121:619-630 (1975)). The eryA gene of Brucella abortus has been
functionally expressed in Escherichia coli and the resultant EryA
was shown to catalyze the ATP-dependent conversion of erythritol to
erythritol-4-phosphate (Lillo et al., Bioorg. Med. Chem. Lett.
13:737-739 (2003)). The following genes can be used for Step K
conversion:
TABLE-US-00051 Protein GenBank ID GI Number Organism eryA Q8YCU8
81850596 Brucella melitensis eriA Q92NH0 81774560 Sinorhizobium
meliloti eryA YP_001108625.1 134102964 Saccharopolyspora erythraea
NRRL 2338
Malonyl-CoA:Acetyl-CoA Acyltransferase (FIG. 4, Step A)
[0255] In Step A of the pathway described in FIG. 4, malonyl-CoA
and acetyl-CoA are condensed to form 3-oxoglutaryl-CoA by
malonyl-CoA:acetyl-CoA acyl transferase, a beta-keothiolase.
Although no enzyme with activity on malonyl-CoA has been reported
to date, a good candidate for this transformation is
beta-ketoadipyl-CoA thiolase (EC 2.3.1.174), also called
3-oxoadipyl-CoA thiolase that converts beta-ketoadipyl-CoA to
succinyl-CoA and acetyl-CoA, and is a key enzyme of the
beta-ketoadipate pathway for aromatic compound degradation. The
enzyme is widespread in soil bacteria and fungi including
Pseudomonas putida (Harwood et al., J Bacteriol. 176:6479-6488
(1994)) and Acinetobacter calcoaceticus (Doten et al., J Bacteriol.
169:3168-3174 (1987)). The gene products encoded by pcaF in
Pseudomonas strain B13 (Kaschabek et al., J Bacteriol. 184:207-215
(2002)), phaD in Pseudomonas putida U (Olivera et al., supra,
(1998)), paaE in Pseudomonas fluorescens ST (Di Gennaro et al.,
Arch Microbiol. 88:117-125 (2007)), and paaJ from E. coli (Nogales
et al., Microbiology, 153:357-365 (2007)) also catalyze this
transformation. Several beta-ketothiolases exhibit significant and
selective activities in the oxoadipyl-CoA forming direction
including bkt from Pseudomonas putida, pcaF and bkt from
Pseudomonas aeruginosa PAO1, bkt from Burkholderia ambifaria AMMD,
paaJ from E. coli, and phaD from P. putida. These enzymes can also
be employed for the synthesis of 3-oxoglutaryl-CoA, a compound
structurally similar to 3-oxoadipyl-CoA.
TABLE-US-00052 Protein GenBank ID GI Number Organism paaJ
NP_415915.1 16129358 Escherichia coli pcaF AAL02407 17736947
Pseudomonas knackmussii (B13) phaD AAC24332.1 3253200 Pseudomonas
putida pcaF AAA85138.1 506695 Pseudomonas putida pcaF AAC37148.1
141777 Acinetobacter calcoaceticus paaE ABF82237.1 106636097
Pseudomonas fluorescens bkt YP_777652.1 115360515 Burkholderia
ambifaria AMMD bkt AAG06977.1 9949744 Pseudomonas aeruginosa PAO1
pcaF AAG03617.1 9946065 Pseudomonas aeruginosa PAO1
[0256] Another relevant beta-ketothiolase is
oxopimeloyl-CoA:glutaryl-CoA acyltransferase (EC 2.3.1.16) that
combines glutaryl-CoA and acetyl-CoA to form 3-oxopimeloyl-CoA. An
enzyme catalyzing this transformation is found in Ralstonia
eutropha (formerly known as Alcaligenes eutrophus), encoded by
genes bktB and bktC (Slater et al., J. Bacteriol. 180:1979-1987
(1998); Haywood et al., FEMS Microbiology Letters 52:91-96 (1988)).
The sequence of the BktB protein is known; however, the sequence of
the BktC protein has not been reported. The pim operon of
Rhodopseudomonas palustris also encodes a beta-ketothiolase,
encoded by pimB, predicted to catalyze this transformation in the
degradative direction during benzoyl-CoA degradation (Harrison et
al., Microbiology 151:727-736 (2005)). A beta-ketothiolase enzyme
candidate in S. aciditrophicus was identified by sequence homology
to bktB (43% identity, evalue=1e-93).
TABLE-US-00053 Protein GenBank ID GI Number Organism bktB YP_725948
11386745 Ralstonia eutropha pimB CAE29156 39650633 Rhodopseudomonas
palustris syn_02642 YP_462685.1 85860483 Syntrophus
aciditrophicus
[0257] Beta-ketothiolase enzymes catalyzing the formation of
beta-ketovaleryl-CoA from acetyl-CoA and propionyl-CoA can also be
able to catalyze the formation of 3-oxoglutaryl-CoA. Zoogloea
ramigera possesses two ketothiolases that can form
.beta.-ketovaleryl-CoA from propionyl-CoA and acetyl-CoA and R.
eutropha has a .beta.-oxidation ketothiolase that is also capable
of catalyzing this transformation (Slater et al., J. Bacteriol,
180:1979-1987 (1998)). The sequences of these genes or their
translated proteins have not been reported, but several candidates
in R. eutropha, Z. ramigera, or other organisms can be identified
based on sequence homology to bktB from R. eutropha. These
include:
TABLE-US-00054 Protein GenBank ID GI Number Organism phaA
YP_725941.1 113867452 Ralstonia eutropha h16_A1713 YP_726205.1
113867716 Ralstonia eutropha pcaF YP_728366.1 116694155 Ralstonia
eutropha h16_B1369 YP_840888.1 116695312 Ralstonia eutropha
h16_A0170 YP_724690.1 113866201 Ralstonia eutropha h16_A0462
YP_724980.1 113866491 Ralstonia eutropha h16_A1528 YP_726028.1
113867539 Ralstonia eutropha h16_B0381 YP_728545.1 116694334
Ralstonia eutropha h16_B0662 YP_728824.1 116694613 Ralstonia
eutropha h16_B0759 YP_728921.1 116694710 Ralstonia eutropha
h16_B0668 YP_728830.1 116694619 Ralstonia eutropha h16_A1720
YP_726212.1 113867723 Ralstonia eutropha h16_A1887 YP_726356.1
113867867 Ralstonia eutropha phbA P07097.4 135759 Zoogloea ramigera
bktB YP_002005382.1 194289475 Cupriavidus taiwanensis Rmet_1362
YP_583514.1 94310304 Ralstonia metallidurans Bphy_0975
YP_001857210.1 186475740 Burkholderia phymatum
[0258] Additional candidates include beta-ketothiolases that are
known to convert two molecules of acetyl-CoA into acetoacetyl-CoA
(EC 2.1.3.9). Exemplary acetoacetyl-CoA thiolase enzymes include
the gene products of atoB from E. coli (Martin et al., supra,
(2003)), thlA and thlB from C. acetobutylicum (Hanai et al., supra,
(2007); Winzer et al., supra, (2000)), and ERG10 from S. cerevisiae
(Hiser et al., supra, (1994)).
TABLE-US-00055 Protein GenBank ID GI Number Organism toB 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
3-Oxoglutaryl-CoA Reductase (Ketone-Reducing) (FIG. 4, Step B)
[0259] This enzyme catalyzes the reduction of the 3-oxo group in
3-oxoglutaryl-CoA to the 3-hydroxy group in Step B of the pathway
shown in FIG. 4.
[0260] 3-Oxoacyl-CoA dehydrogenase enzymes convert 3-oxoacyl-CoA
molecules into 3-hydroxyacyl-CoA molecules and are often involved
in fatty acid beta-oxidation or phenylacetate catabolism. For
example, subunits of two fatty acid oxidation complexes in E. coli,
encoded by fadB and fadJ, function as 3-hydroxyacyl-CoA
dehydrogenases (Binstock et al., Methods Enzymol. 71 Pt C:403-411
(1981)). Furthermore, the gene products encoded by phaC in
Pseudomonas putida U (Olivera et al., supra, (1998)) and paaC in
Pseudomonas fluorescens ST (Di et al., supra, (2007)) catalyze the
reversible oxidation of 3-hydroxyadipyl-CoA to form
3-oxoadipyl-CoA, during the catabolism of phenylacetate or styrene.
In addition, given the proximity in E. coli of paaH to other genes
in the phenylacetate degradation operon (Nogales et al., supra,
(2007)) and the fact that paaH mutants cannot grow on phenylacetate
(Ismail et al., supra, (2003)), it is expected that the E. coli
paaH gene encodes a 3-hydroxyacyl-CoA dehydrogenase.
TABLE-US-00056 Protein GenBank ID GI Number Organism fadB P21177.2
119811 Escherichia coli fadJ P77399.1 3334437 Escherichia coli paaH
NP_415913.1 16129356 Escherichia coli phaC NP_745425.1 26990000
Pseudomonas putida paaC ABF82235.1 106636095 Pseudomonas
fluorescens
[0261] 3-Hydroxybutyryl-CoA dehydrogenase, also called
acetoacetyl-CoA reductase, catalyzes the reversible
NAD(P)H-dependent conversion of acetoacetyl-CoA to
3-hydroxybutyryl-CoA. This enzyme participates in the acetyl-CoA
fermentation pathway to butyrate in several species of Clostridia
and has been studied in detail (Jones and Woods, supra, (1986)).
Enzyme candidates include hbd from C. acetobutylicum (Boynton et
al., J. Bacteriol. 178:3015-3024 (1996)), hbd from C. beijerinckii
(Colby et al., Appl Environ. Microbiol 58:3297-3302 (1992)), and a
number of similar enzymes from Metallosphaera sedula (Berg et al.,
supra, (2007)). The enzyme from Clostridium acetobutylicum, encoded
by hbd, has been cloned and functionally expressed in E. coli
(Youngleson et al., supra, (1989)). Yet other genes demonstrated to
reduce acetoacetyl-CoA to 3-hydroxybutyryl-CoA are phbB from
Zoogloea ramigera (Ploux et al., supra, (1988)) and phaB from
Rhodobacter sphaeroides (Alber et al., supra, (2006)). The former
gene is NADPH-dependent, its nucleotide sequence has been
determined (Peoples and Sinskey, supra, (1989)) and the gene has
been expressed in E. coli. Additional genes include hbd1
(C-terminal domain) and hbd2 (N-terminal domain) in Clostridium
kluyveri (Hillmer and Gottschalk, Biochim. Biophys. Acta 3334:12-23
(1974)) and HSD17B10 in Bos taurus (WAKIL et al., supra,
(1954)).
TABLE-US-00057 Protein GenBank ID GI Number Organism hbd
NP_349314.1 15895965 Clostridium acetobutylicum hbd AAM14586.1
20162442 Clostridium beijerinckii Msed_1423 YP_001191505 146304189
Metallosphaera sedula Msed_0399 YP_001190500 146303184
Metallosphaera sedula Msed_0389 YP_001190490 146303174
Metallosphaera sedula Msed_1993 YP_001192057 146304741
Metallosphaera sedula hbd2 EDK34807.1 146348271 Clostridium
kluyveri hbd1 EDK32512.1 146345976 Clostridium kluyveri HSD17B10
O02691.3 3183024 Bos taurus phaB YP_353825.1 77464321 Rhodobacter
sphaeroides phbB P23238.1 130017 Zoogloea ramigera
3-Hydroxyglutaryl-CoA Reductase (Aldehyde Forming) (FIG. 4, Step
C)
[0262] 3-hydroxyglutaryl-CoA reductase reduces
3-hydroxyglutaryl-CoA to 3-hydroxy-5-oxopentanoate. Several
acyl-CoA dehydrogenases reduce an acyl-CoA to its corresponding
aldehyde (EC 1.2.1). Exemplary genes that encode such enzymes
include the Acinetobacter calcoaceticus acr1 encoding a fatty
acyl-CoA reductase (Reiser and Somerville, supra, (1997)), the
Acinetobacter sp. M-1 fatty acyl-CoA reductase (Ishige et al.,
supra, (2002)), and a CoA- and NADP-dependent succinate
semialdehyde dehydrogenase encoded by the sucD gene in Clostridium
kluyveri (Sohling and Gottschalk, supra, (1996); Sohling and
Gottschalk, supra, (1996)). SucD of P. gingivalis is another
succinate semialdehyde dehydrogenase (Takahashi et al., supra,
(2000)). The enzyme acylating acetaldehyde dehydrogenase in
Pseudomonas sp, encoded by bphG, is yet another as it has been
demonstrated to oxidize and acylate acetaldehyde, propionaldehyde,
butyraldehyde, isobutyraldehyde and formaldehyde (Powlowski et al.,
supra, (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)).
Butyraldehyde dehydrogenase catalyzes a similar reaction,
conversion of butyryl-CoA to butyraldehyde, in solventogenic
organisms such as Clostridium saccharoperbutylacetonicum (Kosaka et
al., Biosci. Biotechnol Biochem. 71:58-68 (2007)).
TABLE-US-00058 Protein 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 bld AAP42563.1 31075383 Clostridium
saccharoperbutylacetonicum
[0263] 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 archael bacteria (Berg et al., supra,
(2007b); Thauer, supra, (2007)). The enzyme utilizes NADPH as a
cofactor and has been characterized in Metallosphaera and
Sulfolobus spp (Alber et al., supra, (2006); Hugler et al., supra,
(2002)). The enzyme is encoded by Msed_0709 in Metallosphaera
sedula (Alber et al., supra, (2006); Berg et al., supra, (2007b)).
A gene encoding a malonyl-CoA reductase from Sulfolobus tokodaii
was cloned and heterologously expressed in E. coli (Alber et al.,
supra, (2006)). This enzyme has also been shown to catalyze the
conversion of methylmalonyl-CoA to its corresponding aldehyde
(WO/2007/141208). 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. Yet another acyl-CoA reductase (aldehyde forming)
candidate 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. This gene is very similar to eutE that
encodes acetaldehyde dehydrogenase of Salmonella typhimurium and E.
coli (Toth et al., supra, (1999)).
TABLE-US-00059 Protein 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 9473535 Clostridium beijerinckii eutE
AAA80209 687645 Salmonella typhimurium eutE P77445 2498347
Escherichia coli
3-Hydroxy-5-Oxopentanoate Reductase (FIG. 4, Step D)
[0264] This enzyme reduces the terminal aldehyde group in
3-hydroxy-5-oxopentanote to the alcohol group. Exemplary genes
encoding enzymes that catalyze the conversion of an aldehyde to
alcohol (i.e., alcohol dehydrogenase or equivalently aldehyde
reductase, 1.1.1.a) include alrA encoding a medium-chain alcohol
dehydrogenase for C2-C14 (Tani et al., supra, (2000)), ADH2 from
Saccharomyces cerevisiae (Atsumi et al., supra, (2008)), yqhD from
E. coli which has preference for molecules longer than C(3)
(Sulzenbacher et al., supra, (2004)), and bdh I and bdh II from C.
acetobutylicum which converts butyryaldehyde into butanol (Walter
et al., supra, (1992)). The gene product of yqhD catalyzes the
reduction of acetaldehyde, malondialdehyde, propionaldehyde,
butyraldehyde, and acrolein using NADPH as the cofactor (Perez et
al., 283:7346-7353 (2008); 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)).
TABLE-US-00060 Protein 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
[0265] Enzymes exhibiting 4-hydroxybutyrate dehydrogenase activity
(EC 1.1.1.61) also fall into this category. Such enzymes have been
characterized in Ralstonia eutropha (Bravo et al., supra, (2004)),
Clostridium kluyveri (Wolff and Kenealy, supra, (1995)) and
Arabidopsis thaliana (Breitkreuz et al., supra, (2003)). The A.
thaliana enzyme was cloned and characterized in yeast [12882961].
Yet another gene is the alcohol dehydrogenase adhI from Geobacillus
thermoglucosidasius (Jeon et al., J Biotechnol 135:127-133
(2008)).
TABLE-US-00061 Protein 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
[0266] Another exemplary enzyme is 3-hydroxyisobutyrate
dehydrogenase (EC 1.1.1.31) 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
352:905-17 (2005)). The reversibility of the human
3-hydroxyisobutyrate dehydrogenase was demonstrated using
isotopically-labeled substrate (Manning et al., Biochem J 231:481-4
(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., supra, (2000); Chowdhury et
al., Biosci. Biotechnol Biochem. 60:2043-2047 (1996)), mmsb in
Pseudomonas aeruginosa, and dhat in Pseudomonas putida (Aberhart et
al., J Chem. Soc. [Perkin 1] 6:1404-1406 (1979); Chowdhury et al.,
supra, (1996); Chowdhury et al., Biosci. Biotechnol Biochem.
67:438-441 (2003)).
TABLE-US-00062 Protein 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
[0267] The conversion of malonic semialdehyde to 3-HP can also be
accomplished by two other enzymes: NADH-dependent
3-hydroxypropionate dehydrogenase and NADPH-dependent malonate
semialdehyde reductase. An NADH-dependent 3-hydroxypropionate
dehydrogenase is thought to participate in beta-alanine
biosynthesis pathways from propionate in bacteria and plants
(Rathinasabapathi B., Journal of Plant Pathology 159:671-674
(2002); Stadtman, 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 CO.sub.2-fixing bacteria. Although
the enzyme activity has been detected in Metallosphaera sedula, the
identity of the gene is not known (Alber et al., supra,
(2006)).
3,5-Dihydroxvpentanoate Kinase (FIG. 4, Step E)
[0268] This enzyme phosphorylates 3,5-dihydroxypentanotae in FIG. 4
(Step E) to form 3-hydroxy-5-phosphonatooxypentanoate (3H5PP). This
transformation can be catalyzed by enzymes in the EC class 2.7.1
that enable the ATP-dependent transfer of a phosphate group to an
alcohol.
[0269] A good candidate for this step is mevalonate kinase (EC
2.7.1.36) that phosphorylates the terminal hydroxyl group of the
methyl analog, mevalonate, of 3,5-dihydroxypentanote. Some gene
candidates for this step are erg12 from S. cerevisiae, mvk from
Methanocaldococcus jannaschi, MVK from Homo sapeins, and mvk from
Arabidopsis thaliana col.
TABLE-US-00063 Protein GenBank ID GI Number Organism erg12
CAA39359.1 3684 Sachharomyces cerevisiae mvk Q58487.1 2497517
Methanocaldococcus jannaschii mvk AAH16140.1 16359371 Homo sapiens
M\mvk NP_851084.1 30690651 Arabidopsis thaliana
[0270] Glycerol kinase also phosphorylates the terminal hydroxyl
group in glycerol to form glycerol-3-phosphate. This reaction
occurs in several species, including Escherichia coli,
Saccharomyces cerevisiae, and Thermotoga maritima. The E. coli
glycerol kinase has been shown to accept alternate substrates such
as dihydroxyacetone and glyceraldehyde (Hayashi and Lin, supra,
(1967)). T, maritime has two glycerol kinases (Nelson et al.,
supra, (1999)). Glycerol kinases have been shown to have a wide
range of substrate specificity. Crans and Whiteside studied
glycerol kinases from four different organisms (Escherichia coli,
S. cerevisiae, Bacillus stearothermophilus, and Candida mycoderma)
(Crans and Whitesides, supra, (2010); Nelson et al., supra,
(1999)). They studied 66 different analogs of glycerol and
concluded that the enzyme could accept a range of substituents in
place of one terminal hydroxyl group and that the hydrogen atom at
C2 could be replaced by a methyl group. Interestingly, the kinetic
constants of the enzyme from all four organisms were very similar.
The gene candidates are:
TABLE-US-00064 Protein GenBank ID GI Number Organism glpK
AP_003883.1 89110103 Escherichia coli K12 glpK1 NP_228760.1
15642775 Thermotoga maritime MSB8 glpK2 NP_229230.1 15642775
Thermotoga maritime MSB8 Gut1 NP_011831.1 82795252 Saccharomyces
cerevisiae
[0271] Homoserine kinase is another possible candidate that can
lead to the phosphorylation of 3,5-dihydroxypentanoate. This enzyme
is also present in a number of organisms including E. coli,
Streptomyces sp, and S. cerevisiae. Homoserine kinase from E. coli
has been shown to have activity on numerous substrates, including,
L-2-amino,1,4-butanediol, aspartate semialdehyde, and
2-amino-5-hydroxyvalerate (Huo and Viola, supra, (1996); Huo and
Viola, supra, (1996)). This enzyme can act on substrates where the
carboxyl group at the alpha position has been replaced by an ester
or by a hydroxymethyl group. The gene candidates are:
TABLE-US-00065 Protein GenBank ID GI Number Organism thrB
BAB96580.2 85674277 Escherichia coli K12 SACT1DRAFT_4809
ZP_06280784.1 282871792 Streptomyces sp. ACT-1 Thr1 AAA35154.1
172978 Saccharomyces serevisiae
3H.sub.5PP Kinase (FIG. 4, Step F)
[0272] Phosphorylation of 3H.sub.5PP to 3H.sub.5PDP is catalyzed by
3H.sub.5PP kinase (FIG. 4, Step F). Phosphomevalonate kinase (EC
2.7.4.2) catalyzes the analogous transformation in the mevalonate
pathway. This enzyme is encoded by erg8 in Saccharomyces cerevisiae
(Tsay et al., Mol. Cell Biol. 11:620-631 (1991)) and mvaK2 in
Streptococcus pneumoniae, Staphylococcus aureus and Enterococcus
faecalis (Doun et al., Protein Sci. 14:1134-1139 (2005); Wilding et
al., J Bacteriol. 182:4319-4327 (2000)). The Streptococcus
pneumoniae and Enterococcus faecalis enzymes were cloned and
characterized in E. coli (Pilloff et al., J Biol. Chem.
278:4510-4515 (2003); Doun et al., Protein Sci. 14:1134-1139
(2005)).
TABLE-US-00066 Protein GenBank ID GI Number Organism Erg8
AAA34596.1 171479 Saccharomyces cerevisiae mvaK2 AAG02426.1 9937366
Staphylococcus aureus mvaK2 AAG02457.1 9937409 Streptococcus
pneumoniae mvaK2 AAG02442.1 9937388 Enterococcus faecalis
3H.sub.5PDP Decarboxylase (FIG. 4, Step G)
[0273] Butenyl 4-diphosphate is formed from the ATP-dependent
decarboxylation of 3H5PDP by 3H5PDP decarboxylase (FIG. 4, Step G).
Although an enzyme with this activity has not been characterized to
date a similar reaction is catalyzed by mevalonate diphosphate
decarboxylase (EC 4.1.1.33), an enzyme participating in the
mevalonate pathway for isoprenoid biosynthesis. This reaction is
catalyzed by MVD1 in Saccharomyces cerevisiae, MVD in Homo sapiens
and MDD in Staphylococcus aureus and Trypsonoma brucei (Toth et
al., J Biol. Chem. 271:7895-7898 (1996); Byres et al., J Mol. Biol.
371:540-553 (2007)).
TABLE-US-00067 Protein GenBank ID GI Number Organism MVD1 P32377.2
1706682 Saccharomyces cerevisiae MVD NP_002452.1 4505289 Homo
sapiens MDD ABQ48418.1 147740120 Staphylococcus aureus MDD
EAN78728.1 70833224 Trypsonoma brucei
Butenyl 4-Diphosphate Isomerase (FIG. 4, Step H)
[0274] Butenyl 4-diphosphate isomerase catalyzes the reversible
interconversion of 2-butenyl-4-diphosphate and
butenyl-4-diphosphate. The following enzymes can naturally possess
this activity or can be engineered to exhibit this activity. Useful
genes include those that encode enzymes that interconvert
isopenenyl diphosphate and dimethylallyl diphosphate. These include
isopentenyl diphosphate isomerase enzymes from Escherichia coli
(Rodriguez-Concepcion et al., FEBS Lett, 473(3):328-332),
Saccharomyces cerevisiae (Anderson et al., J Biol Chem, 1989,
264(32); 19169-75), and Sulfolobus shibatae (Yamashita et al, Eur J
Biochem, 2004, 271(6); 1087-93). The reaction mechanism of
isomerization, catalyzed by the Idi protein of E. coli, has been
characterized in mechanistic detail (de Ruyck et al., J Biol. Chem.
281:17864-17869 (2006)). Isopentenyl diphosphate isomerase enzymes
from Saccharomyces cerevisiae, Bacillus subtilis and Haematococcus
pluvialis have been heterologously expressed in E. coli (Laupitz et
al., Eur. J Biochem. 271:2658-2669 (2004); Kajiwara et al.,
Biochem. J 324 (Pt 2):421-426 (1997)).
TABLE-US-00068 Protein GenBank ID GI Number Organism Idi
NP_417365.1 16130791 Escherichia coli IDI1 NP_015208.1 6325140
Saccharomyces cerevisiae Idi BAC82424.1 34327946 Sulfolobus
shibatae Idi AAC32209.1 3421423 Haematococcus pluvialis Idi
BAB32625.1 12862826 Bacillus subtilis
Butadiene Synthase (FIG. 4, Step I)
[0275] Butadiene synthase catalyzes the conversion of
2-butenyl-4-diphosphate to 1,3-butadiene. The enzymes described
below naturally possess such activity or can be engineered to
exhibit this activity. Isoprene synthase naturally catalyzes the
conversion of dimethylallyl diphosphate to isoprene, but can also
catalyze the synthesis of 1,3-butadiene from
2-butenyl-4-diphosphate. Isoprene synthases can be found in several
organisms including Populus alba (Sasaki et al., FEBS Letters,
2005, 579 (11), 2514-2518), Pueraria montana (Lindberg et al.,
Metabolic Eng, 12(1):70-79 (2010); Sharkey et al., Plant Physiol.,
137(2):700-712 (2005)), and Populus tremula.times.Populus alba
(Miller et al., Planta, 213(3):483-487 (2001)). Additional isoprene
synthase enzymes are described in (Chotani et al., WO/2010/031079,
Systems Using Cell Culture for Production of Isoprene; Cervin et
al., US Patent Application 20100003716, Isoprene Synthase Variants
for Improved Microbial Production of Isoprene).
TABLE-US-00069 Protein GenBank ID GI Number Organism ispS
BAD98243.1 63108310 Populus alba ispS AAQ84170.1 35187004 Pueraria
montana ispS CAC35696.1 13539551 Populus tremula .times. Populus
alba
3-Hydroxyglutaryl-CoA Reductase (Alcohol Forming) (FIG. 4, Step
J)
[0276] This step catalyzes the reduction of the acyl-CoA group in
3-hydroxyglutaryl-CoA to the alcohol group. Exemplary bifunctional
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 et al., supra, (1991)) and butyryl-CoA to
butanol (e.g. adhE2 from C. acetobutylicum (Fontaine et al., supra,
(2002)). In addition to reducing acetyl-CoA to ethanol, the enzyme
encoded by adhE in Leuconostoc mesenteroides has been shown to
oxide the branched chain compound isobutyraldehyde to
isobutyryl-CoA (Kazahaya et al., supra, (1972); Koo et al., supra,
(2005)).
[0277] Another exemplary enzyme can convert malonyl-CoA to 3-HP. An
NADPH-dependent enzyme with this activity has characterized in
Chloroflexus aurantiacus where it participates in the
3-hydroxypropionate cycle (Hugler et al., supra, (2002); Strauss
and Fuchs, supra, (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., supra, (2002)). No
enzymes in other organisms have been shown to catalyze this
specific reaction; however there is bioinformatic evidence that
other organisms can have similar pathways (Klatt et al., supra,
(2007)). Enzyme candidates in other organisms including Roseiflexus
castenholzii, Erythrobacter sp. NAP1 and marine gamma
proteobacterium HTCC2080 can be inferred by sequence
similarity.
TABLE-US-00070 Protein GenBank ID GI Number Organism adhE
NP_415757.1 16129202 Escherichia coli adhE2 AAK09379.1 12958626
Clostridium acetobutylicum adhE AAV66076.1 55818563 Leuconostoc
mesenteroides 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
[0278] Longer chain acyl-CoA molecules can be reduced to their
corresponding alcohols 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 et al., Plant
Physiology 122:635-644 (2000)).
TABLE-US-00071 Protein GenBank ID GI Number Organism FAR AAD38039.1
5020215 Simmondsia chinensis
[0279] Another candidate for catalyzing this step is
3-hydroxy-3-methylglutaryl-CoA reductase (or HMG-CoA reductase).
This enzyme reduces the CoA group in 3-hydroxy-3-methylglutaryl-CoA
to an alcohol forming mevalonate. Gene candidates for this step
include:
TABLE-US-00072 Protein GenBank ID GI Number Organism HMG1
CAA86503.1 587536 Saccharomyces cerevisiae HMG2 NP_013555 6323483
Saccharomyces cerevisiae HMG1 CAA70691.1 1694976 Arabidopsis
thaliana hmgA AAC45370.1 2130564 Sulfolobus solfataricus
[0280] The hmgA gene of Sulfolobus solfataricus, encoding
3-hydroxy-3-methylglutaryl-CoA reductase, has been cloned,
sequenced, and expressed in E. coli (Bochar et al., J Bacteriol.
179:3632-3638 (1997)). S. cerevisiae also has two HMG-CoA
reductases in it (Basson et al., Proc. Natl. Acad. Sci. U.S.A
83:5563-5567 (1986)). The gene has also been isolated from
Arabidopsis thaliana and has been shown to complement the HMG-COA
reductase activity in S. cerevisiae (Learned et al., Proc. Natl.
Acad. Sci. U.S.A 86:2779-2783 (1989)).
3-Oxoglutaryl-CoA Reductase (Aldehyde Forming) (FIG. 4, Step K)
[0281] Several acyl-CoA dehydrogenases are capable of reducing an
acyl-CoA to its corresponding aldehyde. Thus they can naturally
reduce 3-oxoglutaryl-CoA to 3,5-dioxopentanoate or can be
engineered to do so. Exemplary genes that encode such enzymes were
discussed in FIG. 4, Step C.
3,5-Dioxopentanoate Reductase (Ketone Reducing) (FIG. 4, Step
L)
[0282] There exist several exemplary alcohol dehydrogenases that
convert a ketone to a hydroxyl functional group. Two such enzymes
from E. coli are encoded by malate dehydrogenase (mdh) and lactate
dehydrogenase (ldhA). In addition, lactate dehydrogenase from
Ralstonia eutropha has been shown to demonstrate high activities on
2-ketoacids of various chain lengths including lactate,
2-oxobutyrate, 2-oxopentanoate and 2-oxoglutarate (Steinbuchel et
al., Eur. J. Biochem. 130:329-334 (1983)). Conversion of
alpha-ketoadipate into alpha-hydroxyadipate can be catalyzed by
2-ketoadipate reductase, an enzyme reported to be found in rat and
in human placenta (Suda et al., Arch. Biochem. Biophys. 176:610-620
(1976); Suda et al., Biochem. Biophys. Res. Commun. 77:586-591
(1977)). An additional candidate for this step is the mitochondrial
3-hydroxybutyrate dehydrogenase (bdh) from the human heart which
has been cloned and characterized (Marks et al., J. Biol. Chem.
267:15459-15463 (1992)). This enzyme is a dehydrogenase that
operates on a 3-hydroxyacid. Another exemplary alcohol
dehydrogenase converts acetone to isopropanol as was shown in C.
beijerinckii (Ismaiel et al., J. Bacteriol. 175:5097-5105 (1993))
and T. brockii (Lamed et al., Biochem. J. 195:183-190 (1981);
Peretz et al., Biochemistry. 28:6549-6555 (1989)). Methyl ethyl
ketone reductase, or alternatively, 2-butanol dehydrogenase,
catalyzes the reduction of MEK to form 2-butanol. Exemplary enzymes
can be found in Rhodococcus ruber (Kosjek et al., Biotechnol
Bioeng. 86:55-62 (2004)) and Pyrococcus furiosus (van der et al.,
Eur. J. Biochem. 268:3062-3068 (2001)).
TABLE-US-00073 Protein GenBank ID GI Number Organism mdh AAC76268.1
1789632 Escherichia coli ldhA NP_415898.1 16129341 Escherichia coli
ldh YP_725182.1 113866693 Ralstonia eutropha bdh AAA58352.1 177198
Homo sapiens adh AAA23199.2 60592974 Clostridium beijerinckii NRRL
B593 adh P14941.1 113443 Thermoanaerobacter brockii HTD4 adhA
AAC25556 3288810 Pyrococcus furiosus adh-A CAD36475 21615553
Rhodococcus ruber
[0283] A number of organisms can catalyze the reduction of
4-hydroxy-2-butanone to 1,3-butanediol, including those belonging
to the genus Bacillus, Brevibacterium, Candida, and Klebsiella
among others, as described by Matsuyama et al. U.S. Pat. No.
5,413,922. A mutated Rhodococcus phenylacetaldehyde reductase
(Sar268) and a Leifonia alcohol dehydrogenase have also been shown
to catalyze this transformation at high yields (Itoh et al., Appl.
Microbiol. Biotechnol. 75(6):1249-1256).
[0284] Homoserine dehydrogenase (EC 1.1.1.13) catalyzes the
NAD(P)H-dependent reduction of aspartate semialdehyde to
homoserine. In many organisms, including E. coli, homoserine
dehydrogenase is a bifunctional enzyme that also catalyzes the
ATP-dependent conversion of aspartate to aspartyl-4-phosphate
(Starnes et al., Biochemistry 11:677-687 (1972)). The functional
domains are catalytically independent and connected by a linker
region (Sibilli et al., J Biol Chem 256:10228-10230 (1981)) and
both domains are subject to allosteric inhibition by threonine. The
homoserine dehydrogenase domain of the E. coli enzyme, encoded by
thrA, was separated from the aspartate kinase domain,
characterized, and found to exhibit high catalytic activity and
reduced inhibition by threonine (James et al., Biochemistry
41:3720-3725 (2002)). This can be applied to other bifunctional
threonine kinases including, for example, hom1 of Lactobacillus
plantarum (Cahyanto et al., Microbiology 152:105-112 (2006)) and
Arabidopsis thaliana. The monofunctional homoserine dehydrogenases
encoded by hom6 in S. cerevisiae (Jacques et al., Biochim Biophys
Acta 1544:28-41 (2001)) and hom2 in Lactobacillus plantarum
(Cahyanto et al., supra, (2006)) have been functionally expressed
and characterized in E. coli.
TABLE-US-00074 Protein GenBank ID GI number Organism thrA
AAC73113.1 1786183 Escherichia coli K12 akthr2 O81852 75100442
Arabidopsis thaliana hom6 CAA89671 1015880 Saccharomyces cerevisiae
hom1 CAD64819 28271914 Lactobacillus plantarum hom2 CAD63186
28270285 Lactobacillus plantarum
3,5-Dioxopentanoate Reductase (Aldehyde Reducing) (FIG. 4, Step
M)
[0285] Several aldehyde reducing reductases are capable of reducing
an aldehyde to its corresponding alcohol. Thus they can naturally
reduce 3,5-dioxopentanoate to 5-hydroxy-3-oxopentanoate or can be
engineered to do so. Exemplary genes that encode such enzymes were
discussed in FIG. 4, Step D.
5-Hydroxy-3-Oxopentanoate Reductase (FIG. 4, Step N)
[0286] Several ketone reducing reductases are capable of reducing a
ketone to its corresponding hydroxyl group. Thus they can naturally
reduce 5-hydroxy-3-oxopentanoate to 3,5-dihydroxypentanoate or can
be engineered to do so. Exemplary genes that encode such enzymes
were discussed in FIG. 4, Step L.
3-Oxo-Glutaryl-CoA Reductase (CoA Reducing and Alcohol Forming)
(FIG. 4, Step O)
[0287] 3-oxo-glutaryl-CoA reductase (CoA reducing and alcohol
forming) enzymes catalyze the 2 reduction steps required to form
5-hydroxy-3-oxopentanoate from 3-oxo-glutaryl-CoA. Exemplary 2-step
oxidoreductases that convert an acyl-CoA to an alcohol were
provided for FIG. 4, Step J. Such enzymes can naturally convert
3-oxo-glutaryl-CoA to 5-hydroxy-3-oxopentanoate or can be
engineered to do so.
Example II
Exemplary Hydrogenase and CO Dehydrogenase Enzymes for Extracting
Reducing Equivalents from Syngas and Exemplary Reductive TCA Cycle
Enzymes
[0288] Enzymes of the reductive TCA cycle useful in the
non-naturally occurring microbial organisms of the present
invention include one or more of ATP-citrate lyase and three
CO.sub.2-fixing enzymes: isocitrate dehydrogenase,
alpha-ketoglutarate:ferredoxin oxidoreductase, pyruvate:ferredoxin
oxidoreductase. The presence of ATP-citrate lyase or citrate lyase
and alpha-ketoglutarate:ferredoxin oxidoreductase indicates the
presence of an active reductive TCA cycle in an organism. Enzymes
for each step of the reductive TCA cycle are shown below.
[0289] ATP-citrate lyase (ACL, EC 2.3.3.8), also called ATP citrate
synthase, catalyzes the ATP-dependent cleavage of citrate to
oxaloacetate and acetyl-CoA. ACL is an enzyme of the RTCA cycle
that has been studied in green sulfur bacteria Chlorobium limicola
and Chlorobium tepidum. The alpha(4)beta(4) heteromeric enzyme from
Chlorobium limicola was cloned and characterized in E. coli (Kanao
et al., Eur. J. Biochem. 269:3409-3416 (2002). The C. limicola
enzyme, encoded by aclAB, is irreversible and activity of the
enzyme is regulated by the ratio of ADP/ATP. A recombinant ACL from
Chlorobium tepidum was also expressed in E. coli and the holoenzyme
was reconstituted in vitro, in a study elucidating the role of the
alpha and beta subunits in the catalytic mechanism (Kim and Tabita,
J Bacteriol. 188:6544-6552 (2006). ACL enzymes have also been
identified in Balnearium lithotrophicum, Sulfurihydrogenibium
subterraneum and other members of the bacterial phylum Aquificae
(Hugler et al., Environ. Microbiol. 9:81-92 (2007)). This activity
has been reported in some fungi as well. Exemplary organisms
include Sordaria macrospora (Nowrousian et al., Curr. Genet.
37:189-93 (2000), Aspergillus nidulans, Yarrowia lipolytica (Hynes
and Murray, Eukaryotic Cell, July: 1039-1048, (2010) and
Aspergillus niger (Meijer et al. J. Ind. Microbiol. Biotechnol.
36:1275-1280 (2009). Other candidates can be found based on
sequence homology. Information related to hese enzymes is tabulated
below:
TABLE-US-00075 Protein GenBank ID GI Number Organism aclA
BAB21376.1 12407237 Chlorobium limicola aclB BAB21375.1 12407235
Chlorobium limicola aclA AAM72321.1 21647054 Chlorobium tepidum
aclB AAM72322.1 21647055 Chlorobium tepidum aclA ABI50076.1
114054981 Balnearium lithotrophicum aclB ABI50075.1 114054980
Balnearium lithotrophicum aclA ABI50085.1 114055040
Sulfurihydrogenibium subterraneum aclB ABI50084.1 114055039
Sulfurihydrogenibium subterraneum aclA AAX76834.1 62199504
Sulfurimonas denitrificans aclB AAX76835.1 62199506 Sulfurimonas
denitrificans acl1 XP_504787.1 50554757 Yarrowia lipolytica acl2
XP_503231.1 50551515 Yarrowia lipolytica SPBC1703.07 NP_596202.1
19112994 Schizosaccharomyces pombe SPAC22A12.16 NP_593246.1
19114158 Schizosaccharomyces pombe acl1 CAB76165.1 7160185 Sordaria
macrospora acl2 CAB76164.1 7160184 Sordaria macrospora aclA
CBF86850.1 259487849 Aspergillus nidulans aclB CBF86848 259487848
Aspergillus nidulans
[0290] In some organisms the conversion of citrate to oxaloacetate
and acetyl-CoA proceeds through a citryl-CoA intermediate and is
catalyzed by two separate enzymes, citryl-CoA synthetase (EC
6.2.1.18) and citryl-CoA lyase (EC 4.1.3.34) (Aoshima, M., Appl.
Microbiol. Biotechnol. 75:249-255 (2007). Citryl-CoA synthetase
catalyzes the activation of citrate to citryl-CoA. The
Hydrogenobacter thermophilus enzyme is composed of large and small
subunits encoded by ccsA and ccsB, respectively (Aoshima et al.,
Mol. Microbiol. 52:751-761 (2004)). The citryl-CoA synthetase of
Aquifex aeolicus is composed of alpha and beta subunits encoded by
sucC1 and sucD1 (Hugler et al., Environ. Microbiol. 9:81-92
(2007)). Citryl-CoA lyase splits citryl-CoA into oxaloacetate and
acetyl-CoA. This enzyme is a homotrimer encoded by ccl in
Hydrogenobacter thermophilus (Aoshima et al., Mol. Microbiol.
52:763-770 (2004)) and aq_150 in Aquifex aeolicus (Hugler et al.,
supra (2007)). The genes for this mechanism of converting citrate
to oxaloacetate and citryl-CoA have also been reported recently in
Chlorobium tepidum (Eisen et al., PNAS 99(14): 9509-14 (2002).
TABLE-US-00076 Protein GenBank ID GI Number Organism ccsA
BAD17844.1 46849514 Hydrogenobacter thermophilus ccsB BAD17846.1
46849517 Hydrogenobacter thermophilus sucC1 AAC07285 2983723
Aquifex aeolicus sucD1 AAC07686 2984152 Aquifex aeolicus ccl
BAD17841.1 46849510 Hydrogenobacter thermophilus aq_150 AAC06486
2982866 Aquifex aeolicus CT0380 NP_661284 21673219 Chlorobium
tepidum CT0269 NP_661173.1 21673108 Chlorobium tepidum CT1834
AAM73055.1 21647851 Chlorobium tepidum
[0291] Oxaloacetate is converted into malate by malate
dehydrogenase (EC 1.1.1.37), an enzyme which functions in both the
forward and reverse direction. S. cerevisiae possesses three copies
of malate dehydrogenase, MDH1 (McAlister-Henn and Thompson, J.
Bacteriol. 169:5157-5166 (1987), MDH2 (Minard and McAlister-Henn,
Mol. Cell. Biol. 11:370-380 (1991); Gibson and McAlister-Henn, J.
Biol. Chem. 278:25628-25636 (2003)), and MDH3 (Steffan and
McAlister-Henn, J. Biol. Chem. 267:24708-24715 (1992)), which
localize to the mitochondrion, cytosol, and peroxisome,
respectively. E. coli is known to have an active malate
dehydrogenase encoded by mdh.
TABLE-US-00077 Protein GenBank ID GI Number Organism MDH1 NP_012838
6322765 Saccharomyces cerevisiae MDH2 NP_014515 116006499
Saccharomyces cerevisiae MDH3 NP_010205 6320125 Saccharomyces
cerevisiae Mdh NP_417703.1 16131126 Escherichia coli
[0292] Fumarate hydratase (EC 4.2.1.2) catalyzes the reversible
hydration of fumarate to malate. The three fumarases of E. coli,
encoded by fumA, fumB and fumC, are regulated under different
conditions of oxygen availability. FumB is oxygen sensitive and is
active under anaerobic conditions. FumA is active under
microanaerobic conditions, and FumC is active under aerobic growth
conditions (Tseng et al., J. Bacteriol. 183:461-467 (2001); Woods
et al., Biochim. Biophys. Acta 954:14-26 (1988); Guest et al., J.
Gen. Microbiol. 131:2971-2984 (1985)). 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)). Additional fumarase enzymes are found in
Campylobacter jejuni (Smith et al., Int. I 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)). Similar
enzymes with high sequence homology include fum1 from Arabidopsis
thaliana and fumC from Corynebacterium glutamicum. The MmcBC
fumarase from Pelotomaculum thermopropionicum is another class of
fumarase with two subunits (Shimoyama et al., FEMS Microbiol. Lett.
270:207-213 (2007)).
TABLE-US-00078 Protein GenBank ID GI Number Organism fumA
NP_416129.1 16129570 Escherichia coli fumB NP_418546.1 16131948
Escherichia coli fumC NP_416128.1 16129569 Escherichia coli FUM1
NP_015061 6324993 Saccharomyces cerevisiae fumC Q8NRN8.1 39931596
Corynebacterium glutamicum 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
[0293] Fumarate reductase catalyzes the reduction of fumarate to
succinate. The fumarate reductase of E. coli, composed of four
subunits encoded by frdABCD, is membrane-bound and active under
anaerobic conditions. The electron donor for this reaction is
menaquinone and the two protons produced in this reaction do not
contribute to the proton gradient (Iverson et al., Science
284:1961-1966 (1999)). The yeast genome encodes two soluble
fumarate reductase isozymes encoded by 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 used during
anaerobic growth on glucose (Arikawa et al., FEMS Microbiol. Lett.
165:111-116 (1998)).
TABLE-US-00079 Protein GenBank ID GI Number Organism 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
[0294] The ATP-dependent acylation of succinate to succinyl-CoA is
catalyzed by succinyl-CoA synthetase (EC 6.2.1.5). 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 (Buck et al., Biochemistry 24:6245-6252 (1985)).
These proteins are identified below:
TABLE-US-00080 Protein 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
[0295] Alpha-ketoglutarate:ferredoxin oxidoreductase (EC 1.2.7.3),
also known as 2-oxoglutarate synthase or 2-oxoglutarate:ferredoxin
oxidoreductase (OFOR), forms alpha-ketoglutarate from CO.sub.2 and
succinyl-CoA with concurrent consumption of two reduced ferredoxin
equivalents. OFOR and pyruvate:ferredoxin oxidoreductase (PFOR) are
members of a diverse family of 2-oxoacid:ferredoxin (flavodoxin)
oxidoreductases which utilize thiamine pyrophosphate, CoA and
iron-sulfur clusters as cofactors and ferredoxin, flavodoxin and
FAD as electron carriers (Adams et al., Archaea. Adv. Protein Chem.
48:101-180 (1996)). Enzymes in this class are reversible and
function in the carboxylation direction in organisms that fix
carbon by the RTCA cycle such as Hydrogenobacter thermophilus,
Desulfobacter hydrogenophilus and Chlorobium species (Shiba et al.
1985; Evans et al., Proc. Natl. Acad. ScI. U.S.A. 55:92934 (1966);
Buchanan, 1971). The two-subunit enzyme from H. thermophilus,
encoded by korAB, has been cloned and expressed in E. coli (Yun et
al., Biochem. Biophys. Res. Commun. 282:589-594 (2001)). A five
subunit OFOR from the same organism with strict substrate
specificity for succinyl-CoA, encoded by for DABGE, was recently
identified and expressed in E. coli (Yun et al. 2002). The kinetics
of CO.sub.2 fixation of both H. thermophilus OFOR enzymes have been
characterized (Yamamoto et al., Extremophiles 14:79-85 (2010)). A
CO.sub.2-fixing OFOR from Chlorobium thiosulfatophilum has been
purified and characterized but the genes encoding this enzyme have
not been identified to date. Enzyme candidates in Chlorobium
species can be inferred by sequence similarity to the H.
thermophilus genes. For example, the Chlorobium limicola genome
encodes two similar proteins. Acetogenic bacteria such as Moorella
thermoacetica are predicted to encode two OFOR enzymes. The enzyme
encoded by Moth_0034 is predicted to function in the
CO.sub.2-assimilating direction. The genes associated with this
enzyme, Moth_0034 have not been experimentally validated to date
but can be inferred by sequence similarity to known OFOR
enzymes.
OFOR enzymes that function in the decarboxylation direction under
physiological conditions can also catalyze the reverse reaction.
The OFOR from the thermoacidophilic archaeon Sulfolobus sp. strain
7, encoded by ST2300, has been extensively studied (Zhang et al.
1996. A plasmid-based expression system has been developed for
efficiently expressing this protein in E. coli (Fukuda et al., Eur.
J. Biochem. 268:5639-5646 (2001)) and residues involved in
substrate specificity were determined (Fukuda and Wakagi, Biochim.
Biophys. Acta 1597:74-80 (2002)). The OFOR encoded by
Ape1472/Ape1473 from Aeropyrum pernix str. K1 was recently cloned
into E. coli, characterized, and found to react with 2-oxoglutarate
and a broad range of 2-oxoacids (Nishizawa et al., FEBS Lett.
579:2319-2322 (2005)). Another exemplary OFOR is encoded by oorDABC
in Helicobacter pylori (Hughes et al. 1998). An enzyme specific to
alpha-ketoglutarate has been reported in Thauera aromatica (Dorner
and Boll, J, Bacteriol. 184 (14), 3975-83 (2002). A similar enzyme
can be found in Rhodospirillum rubrum by sequence homology. A two
subunit enzyme has also been identified in Chlorobium tepidum
(Eisen et al., PNAS 99(14): 9509-14 (2002)).
TABLE-US-00081 Protein GenBank ID GI Number Organism korA BAB21494
12583691 Hydrogenobacter thermophilus korB BAB21495 12583692
Hydrogenobacter thermophilus forD BAB62132.1 14970994
Hydrogenobacter thermophilus forA BAB62133.1 14970995
Hydrogenobacter thermophilus forB BAB62134.1 14970996
Hydrogenobacter thermophilus forG BAB62135.1 14970997
Hydrogenobacter thermophilus forE BAB62136.1 14970998
Hydrogenobacter thermophilus Clim_0204 ACD89303.1 189339900
Chlorobium limicola Clim_0205 ACD89302.1 189339899 Chlorobium
limicola Clim_1123 ACD90192.1 189340789 Chlorobium limicola
Clim_1124 ACD90193.1 189340790 Chlorobium limicola Moth_1984
YP_430825.1 83590816 Moorella thermoacetica Moth_1985 YP_430826.1
83590817 Moorella thermoacetica Moth_0034 YP_428917.1 83588908
Moorella thermoacetica ST2300 NP_378302.1 15922633 Sulfolobus sp.
strain 7 Ape1472 BAA80470.1 5105156 Aeropyrum pernix Ape1473
BAA80471.2 116062794 Aeropyrum pernix oorD NP_207383.1 15645213
Helicobacter pylori oorA NP_207384.1 15645214 Helicobacter pylori
oorB NP_207385.1 15645215 Helicobacter pylori oorC NP_207386.1
15645216 Helicobacter pylori CT0163 NP_661069.1 21673004 Chlorobium
tepidum CT0162 NP_661068.1 21673003 Chlorobium tepidum korA
CAA12243.2 19571179 Thauera aromatica korB CAD27440.1 19571178
Thauera aromatica Rru_A2721 YP_427805.1 83594053 Rhodospirillum
rubrum Rru_A2722 YP_427806.1 83594054 Rhodospirillum rubrum
[0296] Isocitrate dehydrogenase catalyzes the reversible
decarboxylation of isocitrate to 2-oxoglutarate coupled to the
reduction of NAD(P).sup.+. IDH enzymes in Saccharomyces cerevisiae
and Escherichia coli are encoded by IDP1 and icd, respectively
(Haselbeck and McAlister-Henn, J. Biol. Chem. 266:2339-2345 (1991);
Nimmo, H. G., Biochem. J. 234:317-2332 (1986)). The reverse
reaction in the reductive TCA cycle, the reductive carboxylation of
2-oxoglutarate to isocitrate, is favored by the NADPH-dependent
CO.sub.2-fixing IDH from Chlorobium limicola and was functionally
expressed in E. coli (Kanao et al., Eur. J. Biochem. 269:1926-1931
(2002)). A similar enzyme with 95% sequence identity is found in
the C. tepidum genome in addition to some other candidates listed
below.
TABLE-US-00082 Protein GenBank ID GI Number Organism Icd ACI84720.1
209772816 Escherichia coli IDP1 AAA34703.1 171749 Saccharomyces
cerevisiae Idh BAC00856.1 21396513 Chlorobium limicola Icd
AAM71597.1 21646271 Chlorobium tepidum icd NP_952516.1 39996565
Geobacter sulfurreducens icd YP_393560. 78777245 Sulfurimonas
denitrificans
[0297] In H. thermophilus the reductive carboxylation of
2-oxoglutarate to isocitrate is catalyzed by two enzymes:
2-oxoglutarate carboxylase and oxalosuccinate reductase.
2-Oxoglutarate carboxylase (EC 6.4.1.7) catalyzes the ATP-dependent
carboxylation of alpha-ketoglutarate to oxalosuccinate (Aoshima and
Igarashi, Mol. Microbiol. 62:748-759 (2006)). This enzyme is a
large complex composed of two subunits. Biotinylation of the large
(A) subunit is required for enzyme function (Aoshima et al., Mol.
Microbiol. 51:791-798 (2004)). Oxalosuccinate reductase (EC
1.1.1.-) catalyzes the NAD-dependent conversion of oxalosuccinate
to D-threo-isocitrate. The enzyme is a homodimer encoded by icd in
H. thermophilus. The kinetic parameters of this enzyme indicate
that the enzyme only operates in the reductive carboxylation
direction in vivo, in contrast to isocitrate dehydrogenase enzymes
in other organisms (Aoshima and Igarashi, J. Bacteriol.
190:2050-2055 (2008)). Based on sequence homology, gene candidates
have also been found in Thiobacillus denitrificans and Thermocrinis
albus.
TABLE-US-00083 Protein GenBank ID GI Number Organism cfiA
BAF34932.1 116234991 Hydrogenobacter thermophilus cifB BAF34931.1
116234990 Hydrogenobacter thermophilus Icd BAD02487.1 38602676
Hydrogenobacter thermophilus Tbd_1556 YP_315314 74317574
Thiobacillus denitrificans Tbd_1555 YP_315313 74317573 Thiobacillus
denitrificans Tbd_0854 YP_314612 74316872 Thiobacillus
denitrificans Thal_0268 YP_003473030 289548042 Thermocrinis albus
Thal_0267 YP_003473029 289548041 Thermocrinis albus Thal_0646
YP_003473406 289548418 Thermocrinis albus
[0298] Aconitase (EC 4.2.1.3) is an iron-sulfur-containing protein
catalyzing the reversible isomerization of citrate and iso-citrate
via the intermediate cis-aconitate. Two aconitase enzymes are
encoded in the E. coli genome by acnA and acnB. AcnB is the main
catabolic enzyme, while AcnA is more stable and appears to be
active under conditions of oxidative or acid stress (Cunningham et
al., Microbiology 143 (Pt 12):3795-3805 (1997)). Two isozymes of
aconitase in Salmonella typhimurium are encoded by acnA and acnB
(Horswill and Escalante-Semerena, Biochemistry 40:4703-4713
(2001)). The S. cerevisiae aconitase, encoded by ACO1, is localized
to the mitochondria where it participates in the TCA cycle
(Gangloff et al., Mol. Cell. Biol. 10:3551-3561 (1990)) and the
cytosol where it participates in the glyoxylate shunt (Regev-Rudzki
et al., Mol. Biol. Cell. 16:4163-4171 (2005)).
TABLE-US-00084 Protein GenBank ID GI Number Organism acnA AAC7438.1
1787531 Escherichia coli acnB AAC73229.1 2367097 Escherichia coli
HP0779 NP_207572.1 15645398 Helicobacter pylori 26695 H16_B0568
CAJ95365.1 113529018 Ralstonia eutropha DesfrDRAFT_3783
ZP_07335307.1 303249064 Desulfovibrio fructosovorans JJ Suden_1040
ABB44318.1 78497778 Sulfurimonas denitrificans (acnB) Hydth_0755
ADO45152.1 308751669 Hydrogenobacter thermophilus CT0543 (acn)
AAM71785.1 21646475 Chlorobium tepidum Clim_2436 YP_001944436.1
189347907 Chlorobium limicola Clim_0515 ACD89607.1 189340204
Chlorobium limicola acnA NP_460671.1 16765056 Salmonella
typhimurium acnB NP_459163.1 16763548 Salmonella typhimurium ACO1
AAA34389.1 170982 Saccharomyces cerevisiae
[0299] Pyruvate:ferredoxin oxidoreductase (PFOR) catalyzes the
reversible 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. Two
cysteine residues in this enzyme form a disulfide bond that
protects it against inactivation in the form of oxygen. This
disulfide bond and the stability in the presence of oxygen has been
found in other Desulfovibrio species also (Vita et al.,
Biochemistry, 47: 957-64 (2008)). The M. thermoacetica PFOR is also
well characterized (Menon and Ragsdale, Biochemistry 36:8484-8494
(1997)) and was shown to have high activity in the direction of
pyruvate synthesis during autotrophic growth (Furdui and Ragsdale,
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., Eur. J. Biochem. 123:563-569 (1982)). PFORs
have also been described in other organisms, including Rhodobacter
capsulatas (Yakunin and Hallenbeck, Biochimica et Biophysica Acta
1409 (1998) 39-49 (1998)) and Choloboum tepidum (Eisen et al., PNAS
99(14): 9509-14 (2002)). The five subunit PFOR from H.
thermophilus, encoded by porEDABG, was cloned into E. coli and
shown to function in both the decarboxylating and
CO.sub.2-assimilating directions (Ikeda et al. 2006; Yamamoto et
al., Extremophiles 14:79-85 (2010)). Homologs also exist in C.
carboxidivorans P7. Several additional PFOR enzymes are described
in the following review (Ragsdale, S. W., 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 proteins are identified below.
TABLE-US-00085 Protein GenBank ID GI Number Organism
DesfrDRAFT_0121 ZP_07331646.1 303245362 Desulfovibrio
fructosovorans JJ Por CAA70873.1 1770208 Desulfovibrio africanus
por YP_012236.1 46581428 Desulfovibrio vulgaris str. Hildenborough
Dde_3237 ABB40031.1 78220682 DesulfoVibrio desulfuricans G20
Ddes_0298 YP_002478891.1 220903579 Desulfovibrio desulfuricans
subsp. desulfuricans str. ATCC 27774 Por YP_428946.1 83588937
Moorella thermoacetica YdbK NP_415896.1 16129339 Escherichia coli
nifJ (CT1628) NP_662511.1 21674446 Chlorobium tepidum CJE1649
YP_179630.1 57238499 Campylobacter jejuni nifJ ADE85473.1 294476085
Rhodobacter capsulatus porE BAA95603.1 7768912 Hydrogenobacter
thermophilus porD BAA95604.1 7768913 Hydrogenobacter thermophilus
porA BAA95605.1 7768914 Hydrogenobacter thermophilus porB
BAA95606.1 776891 Hydrogenobacter thermophilus porG BAA95607.1
7768916 Hydrogenobacter thermophilus FqrB YP_001482096.1 157414840
Campylobacter jejuni HP1164 NP_207955.1 15645778 Helicobacter
pylori 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
[0300] The conversion of pyruvate into acetyl-CoA can be catalyzed
by several other enzymes or their combinations thereof. For
example, pyruvate dehydrogenase can transform pyruvate into
acetyl-CoA with the concomitant reduction of a molecule of NAD into
NADH. It is a multi-enzyme complex that catalyzes a series of
partial reactions which results in acylating oxidative
decarboxylation of pyruvate. The enzyme comprises of three
subunits: the pyruvate decarboxylase (E1), dihydrolipoamide
acyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). This
enzyme is naturally present in several organisms, including E. coli
and S. cerevisiae. In the E. coli enzyme, specific residues in the
E1 component are responsible for substrate specificity (Bisswanger,
H., J. Biol. Chem. 256:815-82 (1981); Bremer, J., Eur. J. Biochem.
8:535-540 (1969); Gong et al., J. Biol. Chem. 275:13645-13653
(2000)). Enzyme engineering efforts have improved the E. coli PDH
enzyme activity under anaerobic conditions (Kim et al., J.
Bacteriol. 190:3851-3858 (2008); Kim et al., Appl. Environ.
Microbiol. 73:1766-1771 (2007); 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 (5). Crystal structures
of the enzyme complex from bovine kidney (18) and the E2 catalytic
domain from Azotobacter vinelandii are available (4). Yet another
enzyme that can catalyze this conversion is pyruvate formate lyase.
This enzyme 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
encoded by pflB (Knappe and Sawers, 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)). E. coli possesses an additional pyruvate formate lyase,
encoded by tdcE, that catalyzes the conversion of pyruvate or
2-oxobutanoate to acetyl-CoA or propionyl-CoA, respectively
(Hesslinger et al., Mol. Microbiol 27:477-492 (1998)). Both pflB
and tdcE from E. coli require the presence of pyruvate formate
lyase activating enzyme, encoded by pflA. Further, a short protein
encoded by yfiD in E. coli can associate with and restore activity
to oxygen-cleaved pyruvate formate lyase (Vey et al., Proc. Natl.
Acad. Sci. U.S.A. 105:16137-16141 (2008). Note that pflA and pflB
from E. coli were expressed in S. cerevisiae as a means to increase
cytosolic acetyl-CoA for butanol production as described in
WO/2008/080124]. Additional pyruvate formate lyase and activating
enzyme candidates, encoded by pfl and act, respectively, are found
in Clostridium pasteurianum (Weidner et al., J Bacteriol.
178:2440-2444 (1996)).
[0301] Further, different enzymes can be used in combination to
convert pyruvate into acetyl-CoA. For example, in S. cerevisiae,
acetyl-CoA is obtained in the cytosol by first decarboxylating
pyruvate to form acetaldehyde; the latter is oxidized to acetate by
acetaldehyde dehydrogenase and subsequently activated to form
acetyl-CoA by acetyl-CoA synthetase. Acetyl-CoA synthetase is a
native enzyme in several other organisms including E. coli (Kumari
et al., J. Bacteriol. 177:2878-2886 (1995)), Salmonella enterica
(Starai et al., Microbiology 151:3793-3801 (2005); Starai et al.,
J. Biol. Chem. 280:26200-26205 (2005)), and Moorella thermoacetica
(described already). Alternatively, acetate can be activated to
form acetyl-CoA by acetate kinase and phosphotransacetylase.
Acetate kinase first converts acetate into acetyl-phosphate with
the accompanying use of an ATP molecule. Acetyl-phosphate and CoA
are next converted into acetyl-CoA with the release of one
phosphate by phosphotransacetylase. Both acetate kinase and
phosphotransacetlyase are well-studied enzymes in several
Clostridia and Methanosarcina thermophila.
[0302] Yet another way of converting pyruvate to acetyl-CoA is via
pyruvate oxidase. Pyruvate oxidase converts pyruvate into acetate,
using ubiquione as the electron acceptor. In E. coli, this activity
is encoded by poxB. PoxB has similarity to pyruvate decarboxylase
of S. cerevisiae and Zymomonas mobilis. The enzyme has a thiamin
pyrophosphate cofactor (Koland and Gennis, Biochemistry
21:4438-4442 (1982)); O'Brien et al., Biochemistry 16:3105-3109
(1977); O'Brien and Gennis, J. Biol. Chem. 255:3302-3307 (1980))
and a flavin adenine dinucleotide (FAD) cofactor. Acetate can then
be converted into acetyl-CoA by either acetyl-CoA synthetase or by
acetate kinase and phosphotransacetylase, as described earlier.
Some of these enzymes can also catalyze the reverse reaction from
acetyl-CoA to pyruvate.
[0303] For enzymes that use reducing equivalents in the form of
NADH or NADPH, these reduced carriers can be generated by
transferring electrons from reduced ferredoxin. Two enzymes
catalyze the reversible transfer of electrons from reduced
ferredoxins to NAD(P).sup.+, ferredoxin:NAD.sup.+ oxidoreductase
(EC 1.18.1.3) and ferredoxin:NADP.sup.+ oxidoreductase (FNR, EC
1.18.1.2). Ferredoxin:NADP.sup.+ oxidoreductase (FNR, EC 1.18.1.2)
has a noncovalently bound FAD cofactor that facilitates the
reversible transfer of electrons from NADPH to low-potential
acceptors such as ferredoxins or flavodoxins (Blaschkowski et al.,
Eur. J Biochem. 123:563-569 (1982); Fujii et al., 1977). The
Helicobacter pylori FNR, encoded by HP1164 (fqrB), is coupled to
the activity of pyruvate:ferredoxin oxidoreductase (PFOR) resulting
in the pyruvate-dependent production of NADPH (St et al. 2007). An
analogous enzyme is found in Campylobacter jejuni (St et al. 2007).
A ferredoxin:NADP.sup.+ oxidoreductase enzyme is encoded in the E.
coli genome by fpr (Bianchi et al. 1993). Ferredoxin:NAD.sup.+
oxidoreductase utilizes reduced ferredoxin to generate NADH from
NAD.sup.+. In several organisms, including E. coli, this enzyme is
a component of multifunctional dioxygenase enzyme complexes. The
ferredoxin:NAD.sup.+ oxidoreductase of E. coli, encoded by hcaD, is
a component of the 3-phenylproppionate dioxygenase system involved
in involved in aromatic acid utilization (Diaz et al. 1998).
NADH:ferredoxin reductase activity was detected in cell extracts of
Hydrogenobacter thermophilus strain TK-6, although a gene with this
activity has not yet been indicated (Yoon et al. 2006). Finally,
the energy-conserving membrane-associated Rnf-type proteins
(Seedorf et al., Proc. Natl. Acad. Sci. U.S.A. 105:2128-2133
(2008); Herrmann et al., J. Bacteriol. 190:784-791 (2008)) provide
a means to generate NADH or NADPH from reduced ferredoxin.
Additional ferredoxin:NAD(P)+ oxidoreductases have been annotated
in Clostridium carboxydivorans P7.
TABLE-US-00086 Protein GenBank ID GI Number Organism HP1164
NP_207955.1 15645778 Helicobacter pylori RPA3954 CAE29395.1
39650872 Rhodopseudomonas palustris fpr BAH29712.1 225320633
Hydrogenobacter thermophilus yumC NP_391091.2 255767736 Bacillus
subtilis CJE0663 AAW35824.1 57167045 Campylobacter jejuni fpr
P28861.4 399486 Escherichia coli hcaD AAC75595.1 1788892
Escherichia coli LOC100282643 NP_001149023.1 226497434 Zea mays
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 CcarbDRAFT_2639 ZP_05392639.1
255525707 Clostridium carboxidivorans P7 CcarbDRAFT_2638
ZP_05392638.1 255525706 Clostridium carboxidivorans P7
CcarbDRAFT_2636 ZP_05392636.1 255525704 Clostridium carboxidivorans
P7 CcarbDRAFT_5060 ZP_05395060.1 255528241 Clostridium
carboxidivorans P7 CcarbDRAFT_2450 ZP_05392450.1 255525514
Clostridium carboxidivorans P7 CcarbDRAFT_1084 ZP_05391084.1
255524124 Clostridium carboxidivorans P7
[0304] Ferredoxins are small acidic proteins containing one or more
iron-sulfur clusters that function as intracellular electron
carriers with a low reduction potential. Reduced ferredoxins donate
electrons to Fe-dependent enzymes such as ferredoxin-NADP.sup.+
oxidoreductase, pyruvate:ferredoxin oxidoreductase (PFOR) and
2-oxoglutarate:ferredoxin oxidoreductase (OFOR). The H.
thermophilus gene fdx1 encodes a [4Fe-4S]-type ferredoxin that is
required for the reversible carboxylation of 2-oxoglutarate and
pyruvate by OFOR and PFOR, respectively (Yamamoto et al.,
Extremophiles 14:79-85 (2010)). The ferredoxin associated with the
Sulfolobus solfataricus 2-oxoacid:ferredoxin reductase is a
monomeric dicluster [3Fe-4S][4Fe-4S] type ferredoxin (Park et al.
2006). While the gene associated with this protein has not been
fully sequenced, the N-terminal domain shares 93% homology with the
zfx ferredoxin from S. acidocaldarius. The E. coli genome encodes a
soluble ferredoxin of unknown physiological function, fdx. Some
evidence indicates that this protein can function in iron-sulfur
cluster assembly (Takahashi and Nakamura, 1999). Additional
ferredoxin proteins have been characterized in Helicobacter pylori
(Mukhopadhyay et al. 2003) and Campylobacter jejuni (van Vliet et
al. 2001). A 2Fe-2S ferredoxin from Clostridium pasteurianum has
been cloned and expressed in E. coli (Fujinaga and Meyer,
Biochemical and Biophysical Research Communications, 192(3):
(1993)). Acetogenic bacteria such as Moorella thermoacetica,
Clostridium carboxidivorans P7 and Rhodospirillum rubrum are
predicted to encode several ferredoxins, listed in the table
below.
TABLE-US-00087 Protein GenBank ID GI Number Organism fdx1
BAE02673.1 68163284 Hydrogenobacter thermophilus M11214.1
AAA83524.1 144806 Clostridium pasteurianum Zfx AAY79867.1 68566938
Sulfolobus acidocalarius Fdx AAC75578.1 1788874 Escherichia coli
hp_0277 AAD07340.1 2313367 Helicobacter pylori fdxA CAL34484.1
112359698 Campylobacter jejuni Moth_0061 ABC18400.1 83571848
Moorella thermoacetica Moth_1200 ABC19514.1 83572962 Moorella
thermoacetica Moth_1888 ABC20188.1 83573636 Moorella thermoacetica
Moth_2112 ABC20404.1 83573852 Moorella thermoacetica Moth_1037
ABC19351.1 83572799 Moorella thermoacetica CcarbDRAFT_4383
ZP_05394383.1 255527515 Clostridium carboxidivorans P7
CcarbDRAFT_2958 ZP_05392958.1 255526034 Clostridium carboxidivorans
P7 CcarbDRAFT_2281 ZP_05392281.1 255525342 Clostridium
carboxidivorans P7 CcarbDRAFT_5296 ZP_05395295.1 255528511
Clostridium carboxidivorans P7 CcarbDRAFT_1615 ZP_05391615.1
255524662 Clostridium carboxidivorans P7 CcarbDRAFT_1304
ZP_05391304.1 255524347 Clostridium carboxidivorans P7 cooF
AAG29808.1 11095245 Carboxydothermus hydrogenoformans fdxN
CAA35699.1 46143 Rhodobacter capsulatus Rru_A2264 ABC23064.1
83576513 Rhodospirillum rubrum Rru_A1916 ABC22716.1 83576165
Rhodospirillum rubrum Rru_A2026 ABC22826.1 83576275 Rhodospirillum
rubrum cooF AAC45122.1 1498747 Rhodospirillum rubrum fdxN
AAA26460.1 152605 Rhodospirillum rubrum Alvin_2884 ADC63789.1
288897953 Allochromatium vinosum DSM 180 fdx YP_002801146.1
226946073 Azotobacter vinelandii DJ CKL_3790 YP_001397146.1
153956381 Clostridium kluyveri DSM 555 fer1 NP_949965.1 39937689
Rhodopseudomonas palustris CGA009 fdx CAA12251.1 3724172 Thauera
aromatica CHY_2405 YP_361202.1 78044690 Carboxydothermus
hydrogenoformans fer YP_359966.1 78045103 Carboxydothermus
hydrogenoformans fer AAC83945.1 1146198 Bacillus subtilis fdx1
NP_249053.1 15595559 Pseudomonas aeruginosa PA01 yfhL AP_003148.1
89109368 Escherichia coli K-12
[0305] 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
can utilize CoA acceptors as diverse as acetate, succinate,
propionate, butyrate, 2-methylacetoacetate, 3-ketohexanoate,
3-ketopentanoate, valerate, crotonate, 3-mercaptopropionate,
propionate, vinylacetate, and butyrate, among others.
[0306] The conversion of succinate to succinyl-CoA can be 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. 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 and
Gottschalk, J. Bacteriol. 178:871-880 (1996)). In addition, the
activity is present in Trichomonas vaginalis (van Grinsven et al.
2008) and Trypanosoma brucei (Riviere et al. 2004). The
succinyl-CoA:acetate CoA-transferase from Acetobacter aceti,
encoded by aarC, replaces succinyl-CoA synthetase in a variant TCA
cycle (Mullins et al. 2008). Similar succinyl-CoA transferase
activities are also present in Trichomonas vaginalis (van Grinsven
et al. 2008), Trypanosoma brucei (Riviere et al. 2004) and
Clostridium kluyveri (Sohling and Gottschalk, 1996c). The
beta-ketoadipate: succinyl-CoA transferase encoded by pcaI and pcaJ
in Pseudomonas putida is yet another candidate (Kaschabek et al.
2002). The aforementioned proteins are identified below.
TABLE-US-00088 Protein 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 pcaI AAN69545.1 24985644 Pseudomonas putida pcaJ
NP_746082.1 26990657 Pseudomonas putida aarC ACD85596.1 189233555
Acetobacter aceti
[0307] An additional exemplary transferase that converts succinate
to succinyl-CoA while converting a 3-ketoacyl-CoA to a 3-ketoacid
is succinyl-CoA:3:ketoacid-CoA transferase (EC 2.8.3.5). Exemplary
succinyl-CoA:3:ketoacid-CoA transferases are present in
Helicobacter pylori (Corthesy-Theulaz et al. 1997), Bacillus
subtilis, and Homo sapiens (Fukao et al. 2000; Tanaka et al. 2002).
The aforementioned proteins are identified below.
TABLE-US-00089 Protein 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
[0308] Converting succinate to succinyl-CoA by
succinyl-CoA:3:ketoacid-CoA transferase requires the simultaneous
conversion of a 3-ketoacyl-CoA such as acetoacetyl-CoA to a
3-ketoacid such as acetoacetate. Conversion of a 3-ketoacid back to
a 3-ketoacyl-CoA can be catalyzed by an acetoacetyl-CoA:
acetate:CoA transferase. Acetoacetyl-CoA: acetate:CoA transferase
converts acetoacetyl-CoA and acetate to acetoacetate and
acetyl-CoA, or vice versa. 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.
TABLE-US-00090 Protein 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
[0309] 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.
The aforementioned proteins are identified below.
TABLE-US-00091 Protein 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
[0310] Additionally, 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. The aforementioned
proteins are identified below.
TABLE-US-00092 Protein 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
[0311] Citrate lyase (EC 4.1.3.6) catalyzes a series of reactions
resulting in the cleavage of citrate to acetate and oxaloacetate.
The enzyme is active under anaerobic conditions and is composed of
three subunits: an acyl-carrier protein (ACP, gamma), an ACP
transferase (alpha), and a acyl lyase (beta). Enzyme activation
uses covalent binding and acetylation of an unusual prosthetic
group, 2'-(5''-phosphoribosyl)-3-'-dephospho-CoA, which is similar
in structure to acetyl-CoA. Acylation is catalyzed by CitC, a
citrate lyase synthetase. Two additional proteins, CitG and CitX,
are used to convert the apo enzyme into the active holo enzyme
(Schneider et al., Biochemistry 39:9438-9450 (2000)). Wild type E.
coli does not have citrate lyase activity; however, mutants
deficient in molybdenum cofactor synthesis have an active citrate
lyase (Clark, FEMS Microbiol. Lett. 55:245-249 (1990)). The E. coli
enzyme is encoded by citEFD and the citrate lyase synthetase is
encoded by citC (Nilekani and SivaRaman, Biochemistry 22:4657-4663
(1983)). The Leuconostoc mesenteroides citrate lyase has been
cloned, characterized and expressed in E. coli (Bekal et al., J
Bacteriol. 180:647-654 (1998)). Citrate lyase enzymes have also
been identified in enterobacteria that utilize citrate as a carbon
and energy source, including Salmonella typhimurium and Klebsiella
pneumoniae (Bott, Arch. Microbiol. 167: 78-88 (1997); Bott and
Dimroth, Mol. Microbiol. 14:347-356 (1994)). The aforementioned
proteins are tabulated below.
TABLE-US-00093 Protein GenBank ID GI Number Organism citF
AAC73716.1 1786832 Escherichia coli Cite AAC73717.2 87081764
Escherichia coli citD AAC73718.1 1786834 Escherichia coli citC
AAC73719.2 87081765 Escherichia coli citG AAC73714.1 1786830
Escherichia coli citX AAC73715.1 1786831 Escherichia coli citF
CAA71633.1 2842397 Leuconostoc mesenteroides Cite CAA71632.1
2842396 Leuconostoc mesenteroides citD CAA71635.1 2842395
Leuconostoc mesenteroides citC CAA71636.1 3413797 Leuconostoc
mesenteroides citG CAA71634.1 2842398 Leuconostoc mesenteroides
citX CAA71634.1 2842398 Leuconostoc mesenteroides citF NP_459613.1
16763998 Salmonella typhimurium cite AAL19573.1 16419133 Salmonella
typhimurium citD NP_459064.1 16763449 Salmonella typhimurium citC
NP_459616.1 16764001 Salmonella typhimurium citG NP_459611.1
16763996 Salmonella typhimurium citX NP_459612.1 16763997
Salmonella typhimurium citF CAA56217.1 565619 Klebsiella pneumoniae
cite CAA56216.1 565618 Klebsiella pneumoniae citD CAA56215.1 565617
Klebsiella pneumoniae citC BAH66541.1 238774045 Klebsiella
pneumoniae citG CAA56218.1 565620 Klebsiella pneumoniae citX
AAL60463.1 18140907 Klebsiella pneumoniae
[0312] Acetate kinase (EC 2.7.2.1) catalyzes the reversible
ATP-dependent phosphorylation of acetate to acetylphosphate.
Exemplary acetate kinase enzymes have been characterized in many
organisms including E. coli, Clostridium acetobutylicum and
Methanosarcina thermophila (Ingram-Smith et al., J. Bacteriol.
187:2386-2394 (2005); Fox and Roseman, J. Biol. Chem.
261:13487-13497 (1986); Winzer et al., Microbiology 143 (Pt
10):3279-3286 (1997)). Acetate kinase activity has also been
demonstrated in the gene product of E. coli purT (Marolewski et
al., Biochemistry 33:2531-2537 (1994). Some butyrate kinase enzymes
(EC 2.7.2.7), for example buk1 and buk2 from Clostridium
acetobutylicum, also accept acetate as a substrate (Hartmanis, M.
G., J. Biol. Chem. 262:617-621 (1987)).
TABLE-US-00094 Protein GenBank ID GI Number Organism ackA
NP_416799.1 16130231 Escherichia coli Ack AAB18301.1 1491790
Clostridium acetobutylicum Ack AAA72042.1 349834 Methanosarcina
thermophila purT AAC74919.1 1788155 Escherichia coli buk1 NP_349675
15896326 Clostridium acetobutylicum buk2 Q97II1 20137415
Clostridium acetobutylicum
[0313] The formation of acetyl-CoA from acetylphosphate is
catalyzed by phosphotransacetylase (EC 2.3.1.8). Thepta gene from
E. coli encodes an enzyme that reversibly converts acetyl-CoA into
acetyl-phosphate (Suzuki, T., Biochim. Biophys. Acta 191:559-569
(969)). Additional acetyltransferase enzymes have been
characterized in Bacillus subtilis (Rado and Hoch, Biochim.
Biophys. Acta 321:114-125 (1973), Clostridium kluyveri (Stadtman,
E., Methods Enzymol. 1:5896-599 (1955), and Thermotoga maritima
(Bock et al., J. Bacteriol. 181:1861-1867 (1999)). This reaction is
also catalyzed by some phosphotranbutyrylase enzymes (EC 2.3.1.19)
including the ptb gene products from Clostridium acetobutylicum
(Wiesenborn et al., App. Environ. Microbiol. 55:317-322 (1989);
Walter et al., Gene 134:107-111 (1993)). Additional ptb genes are
found in 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).
TABLE-US-00095 Protein GenBank ID GI Number Organism Pta
NP_416800.1 71152910 Escherichia coli Pta P39646 730415 Bacillus
subtilis Pta A5N801 146346896 Clostridium kluyveri Pta Q9X0L4
6685776 Thermotoga maritima Ptb NP_349676 34540484 Clostridium
acetobutylicum Ptb AAR19757.1 38425288 butyrate-producing bacterium
L2-50 Ptb CAC07932.1 10046659 Bacillus megaterium
[0314] The acylation of acetate to acetyl-CoA is catalyzed by
enzymes with acetyl-CoA synthetase activity. Two enzymes that
catalyze this reaction are AMP-forming acetyl-CoA synthetase (EC
6.2.1.1) and ADP-forming acetyl-CoA synthetase (EC 6.2.1.13).
AMP-forming acetyl-CoA synthetase (ACS) is the predominant enzyme
for activation of acetate to acetyl-CoA. Exemplary ACS enzymes are
found in E. coli (Brown et al., J. Gen. Microbiol. 102:327-336
(1977)), Ralstonia eutropha (Priefert and Steinbuchel, J.
Bacteriol. 174:6590-6599 (1992)), Methanothermobacter
thermautotrophicus (Ingram-Smith and Smith, Archaea 2:95-107
(2007)), Salmonella enterica (Gulick et al., Biochemistry
42:2866-2873 (2003)) and Saccharomyces cerevisiae (Jogl and Tong,
Biochemistry 43:1425-1431 (2004)). ADP-forming acetyl-CoA
synthetases are reversible enzymes with a generally broad substrate
range (Musfeldt and Schonheit, J. Bacteriol. 184:636-644 (2002)).
Two isozymes of ADP-forming acetyl-CoA synthetases are encoded in
the Archaeoglobus fulgidus genome by are encoded by AF1211 and
AF1983 (Musfeldt and Schonheit, supra (2002)). The enzyme from
Haloarcula marismortui (annotated as a succinyl-CoA synthetase)
also accepts acetate as a substrate and reversibility of the enzyme
was demonstrated (Brasen and Schonheit, 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
acetate, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA
(Brasen and Schonheit, supra (2004)). Directed evolution or
engineering can be used to modify 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 and
Schonheit, supra (2004); Musfeldt and Schonheit, supra (2002)).
Additional candidates include the succinyl-CoA synthetase encoded
by sucCD in E. coli (Buck et al., Biochemistry 24:6245-6252 (1985))
and the acyl-CoA ligase from Pseudomonas putida (Fernandez-Valverde
et al., Appl. Environ. Microbiol. 59:1149-1154 (1993)). The
aforementioned proteins are tabulated below.
TABLE-US-00096 Protein GenBank ID GI Number Organism acs AAC77039.1
1790505 Escherichia coli acoE AAA21945.1 141890 Ralstonia eutropha
acs1 ABC87079.1 86169671 Methanothermobacter thermautotrophicus
acs1 AAL23099.1 16422835 Salmonella enterica ACS1 Q01574.2
257050994 Saccharomyces cerevisiae AF1211 NP_070039.1 11498810
Archaeoglobus fulgidus AF1983 NP_070807.1 11499565 Archaeoglobus
fulgidus 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 paaF AAC24333.2 22711873 Pseudomonas putida
[0315] The product yields per C-mol of substrate of microbial cells
synthesizing reduced fermentation products such as butadiene or
crotyl alcohol, are limited by insufficient reducing equivalents in
the carbohydrate feedstock. Reducing equivalents, or electrons, can
be extracted from synthesis gas components such as CO and H.sub.2
using carbon monoxide dehydrogenase (CODH) and hydrogenase enzymes,
respectively. The reducing equivalents are then passed to acceptors
such as oxidized ferredoxins, oxidized quinones, oxidized
cytochromes, NAD(P)+, water, or hydrogen peroxide to form reduced
ferredoxin, reduced quinones, reduced cytochromes, NAD(P)H,
H.sub.2, or water, respectively. Reduced ferredoxin and NAD(P)H are
particularly useful as they can serve as redox carriers for various
Wood-Ljungdahl pathway and reductive TCA cycle enzymes.
[0316] Here, we show specific examples of how additional redox
availability from CO and/or H.sub.2 can improve the yields of
reduced products such as butadiene or crotyl alcohol.
[0317] The maximum theoretical yield to produce butadiene from
glucose is 1 mole/mole (0.3 g/g) based on the pathway described in
FIG. 2. For the pathway described in FIG. 4, the maximum
theoretical yield under aerobic conditions is 0.28 g/g. The maximum
theoretical yield based on stoichiometry is 1.09 mole/mole (0.33
g/g). Using rTCA and hydrogen, this yield can be improved to 2
mole/mole glucose (0.6 g/g). Similar yield improvements can be
attained for crotyl alcohol via the proposed routes.
[0318] When both feedstocks of sugar and syngas are available, the
syngas components CO and H.sub.2 can be utilized to generate
reducing equivalents by employing the hydrogenase and CO
dehydrogenase. The reducing equivalents generated from syngas
components will be utilized to power the glucose to butadiene or
crotyl alcohol production pathways.
[0319] As shown in above example, a combined feedstock strategy
where syngas is combined with a sugar-based feedstock or other
carbon substrate can greatly improve the theoretical yields. In
this co-feeding approach, syngas components H.sub.2 and CO can be
utilized by the hydrogenase and CO dehydrogenase to generate
reducing equivalents, that can be used to power chemical production
pathways in which the carbons from sugar or other carbon substrates
will be maximally conserved and the theoretical yields improved. In
case of butadiene or crotyl alcohol production from glucose or
sugar, the theoretical yields improve from 1.09 mol butadiene or
crotyl alcohol per mol of glucose to 2 mol butadiene or crotyl
alcohol per mol of glucose. Such improvements provide environmental
and economic benefits and greatly enhance sustainable chemical
production.
[0320] Herein below the enzymes and the corresponding genes used
for extracting redox from synags components are described. CODH is
a reversible enzyme that interconverts CO and CO.sub.2 at the
expense or gain of electrons. The natural physiological role of the
CODH in ACS/CODH complexes is to convert CO.sub.2 to CO for
incorporation into acetyl-CoA by acetyl-CoA synthase. Nevertheless,
such CODH enzymes are suitable for the extraction of reducing
equivalents from CO due to the reversible nature of such enzymes.
Expressing such CODH enzymes in the absence of ACS allows them to
operate in the direction opposite to their natural physiological
role (i.e., CO oxidation).
[0321] In M. thermoacetica, C. hydrogenoformans, C. carboxidivorans
P7, and several other organisms, additional CODH encoding genes are
located outside of the ACS/CODH operons. These enzymes provide a
means for extracting electrons (or reducing equivalents) from the
conversion of carbon monoxide to carbon dioxide. The M.
thermoacetica gene (Genbank Accession Number: YP_430813) is
expressed by itself in an operon and is believed to transfer
electrons from CO to an external mediator like ferredoxin in a
"Ping-pong" reaction. The reduced mediator then couples to other
reduced nicolinamide adenine dinucleotide phosphate (NAD(P)H)
carriers or ferredoxin-dependent cellular processes (Ragsdale,
Annals of the New York Academy of Sciences 1125: 129-136 (2008)).
The genes encoding the C. hydrogenoformans CODH-II and CooF, a
neighboring protein, were cloned and sequenced (Gonzalez and Robb,
FEMS Microbiol Lett. 191:243-247 (2000)). The resulting complex was
membrane-bound, although cytoplasmic fractions of CODH-II were
shown to catalyze the formation of NADPH suggesting an anabolic
role (Svetlitchnyi et al., J Bacteriol. 183:5134-5144 (2001)). The
crystal structure of the CODH-II is also available (Dobbek et al.,
Science 293:1281-1285 (2001)). Similar ACS-free CODH enzymes can be
found in a diverse array of organisms including Geobacter
metallireducens GS-15, Chlorobium phaeobacteroides DSM 266,
Clostridium cellulolyticum H10, Desulfovibrio desulfuricans subsp.
desulfuricans str. ATCC 27774, Pelobacter carbinolicus DSM 2380,
and Campylobacter curvus 525.92.
TABLE-US-00097 Protein GenBank ID GI Number Organism CODH
(putative) YP_430813 83590804 Moorella thermoacetica CODH-II
(CooS-II) YP_358957 78044574 Carboxydothermus hydrogenoformans CooF
YP_358958 78045112 Carboxydothermus hydrogenoformans CODH
(putative) ZP_05390164.1 255523193 Clostridium carboxidivorans P7
CcarbDRAFT_0341 ZP_05390341.1 255523371 Clostridium carboxidivorans
P7 CcarbDRAFT_1756 ZP_05391756.1 255524806 Clostridium
carboxidivorans P7 CcarbDRAFT_2944 ZP_05392944.1 255526020
Clostridium carboxidivorans P7 CODH YP_384856.1 78223109 Geobacter
metallireducens GS- 15 Cpha266_0148 YP_910642.1 119355998
Chlorobium (cytochrome c) phaeobacteroides DSM 266 Cpha266_0149
YP_910643.1 119355999 Chlorobium (CODH) phaeobacteroides DSM 266
Ccel_0438 YP_002504800.1 220927891 Clostridium cellulolyticum H10
Ddes_0382 YP_002478973.1 220903661 Desulfovibrio desulfuricans
(CODH) subsp. desulfuricans str. ATCC 27774 Ddes_0381 (CooC)
YP_002478972.1 220903660 Desulfovibrio desulfuricans subsp.
desulfuricans str. ATCC 27774 Pcar_0057 YP_355490.1 7791767
Pelobacter carbinolicus DSM (CODH) 2380 Pcar_0058 YP_355491.1
7791766 Pelobacter carbinolicus DSM (CooC) 2380 Pcar_0058
YP_355492.1 7791765 Pelobacter carbinolicus DSM (HypA) 2380 CooS
(CODH) YP_001407343.1 154175407 Campylobacter curvus 525.92
[0322] In some cases, hydrogenase encoding genes are located
adjacent to a CODH. In Rhodospirillum rubrum, the encoded
CODH/hydrogenase proteins form a membrane-bound enzyme complex that
has been indicated to be a site where energy, in the form of a
proton gradient, is generated from the conversion of CO and
H.sub.2O to CO.sub.2 and H.sub.2 (Fox et al., J Bacteriol.
178:6200-6208 (1996)). The CODH-I of C. hydrogenoformans and its
adjacent genes have been proposed to catalyze a similar functional
role based on their similarity to the R. rubrum CODH/hydrogenase
gene cluster (Wu et al., PLoS Genet. 1:e65 (2005)). The C.
hydrogenoformans CODH-I was also shown to exhibit intense CO
oxidation and CO.sub.2 reduction activities when linked to an
electrode (Parkin et al., J Am. Chem. Soc. 129:10328-10329 (2007)).
The protein sequences of exemplary CODH and hydrogenase genes can
be identified by the following GenBank accession numbers.
TABLE-US-00098 Protein GenBank ID GI Number Organism CODH-I
YP_360644 78043418 Carboxydothermus (CooS-I) hydrogenoformans CooF
YP_360645 78044791 Carboxydothermus hydrogenoformans HypA YP_360646
78044340 Carboxydothermus hydrogenoformans CooH YP_360647 78043871
Carboxydothermus hydrogenoformans CooU YP_360648 78044023
Carboxydothermus hydrogenoformans CooX YP_360649 78043124
Carboxydothermus hydrogenoformans CooL YP_360650 78043938
Carboxydothermus hydrogenoformans CooK YP_360651 78044700
Carboxydothermus hydrogenoformans CooM YP_360652 78043942
Carboxydothermus hydrogenoformans CooC YP_360654.1 78043296
Carboxydothermus hydrogenoformans CooA-1 YP_360655.1 78044021
Carboxydothermus hydrogenoformans CooL AAC45118 1515468
Rhodospirillum rubrum CooX AAC45119 1515469 Rhodospirillum rubrum
CooU AAC45120 1515470 Rhodospirillum rubrum CooH AAC45121 1498746
Rhodospirillum rubrum CooF AAC45122 1498747 Rhodospirillum rubrum
CODH AAC45123 1498748 Rhodospirillum rubrum (CooS) CooC AAC45124
1498749 Rhodospirillum rubrum CooT AAC45125 1498750 Rhodospirillum
rubrum CooJ AAC45126 1498751 Rhodospirillum rubrum
[0323] Native to E. coli and other enteric bacteria are multiple
genes encoding up to four hydrogenases (Sawers, G., Antonie Van
Leeuwenhoek 66:57-88 (1994); Sawers et al., J Bacteriol.
164:1324-1331 (1985); Sawers and Boxer, Eur. J Biochem. 156:265-275
(1986); Sawers et al., J Bacteriol. 168:398-404 (1986)). Given the
multiplicity of enzyme activities, E. coli or another host organism
can provide sufficient hydrogenase activity to split incoming
molecular hydrogen and reduce the corresponding acceptor. E. coli
possesses two uptake hydrogenases, Hyd-1 and Hyd-2, encoded by the
hyaABCDEF and hybOABCDEFG gene clusters, respectively (Lukey et
al., How E. coli is equipped to oxidize hydrogen under different
redox conditions, J Biol Chem published online Nov. 16, 2009).
Hyd-1 is oxygen-tolerant, irreversible, and is coupled to quinone
reduction via the hyaC cytochrome. Hyd-2 is sensitive to O.sub.2,
reversible, and transfers electrons to the periplasmic ferredoxin
hybA which, in turn, reduces a quinone via the hybB integral
membrane protein. Reduced quinones can serve as the source of
electrons for fumarate reductase in the reductive branch of the TCA
cycle. Reduced ferredoxins can be used by enzymes such as
NAD(P)H:ferredoxin oxidoreductases to generate NADPH or NADH. They
can alternatively be used as the electron donor for reactions such
as pyruvate ferredoxin oxidoreductase, AKG ferredoxin
oxidoreductase, and 5,10-methylene-H4folate reductase.
TABLE-US-00099 Protein GenBank ID GI Number Organism HyaA
AAC74057.1 1787206 Escherichia coli HyaB AAC74058.1 1787207
Escherichia coli HyaC AAC74059.1 1787208 Escherichia coli HyaD
AAC74060.1 1787209 Escherichia coli HyaE AAC74061.1 1787210
Escherichia coli HyaF AAC74062.1 1787211 Escherichia coli HybO
AAC76033.1 1789371 Escherichia coli HybA AAC76032.1 1789370
Escherichia coli HybB AAC76031.1 2367183 Escherichia coli HybC
AAC76030.1 1789368 Escherichia coli HybD AAC76029.1 1789367
Escherichia coli HybE AAC76028.1 1789366 Escherichia coli HybF
AAC76027.1 1789365 Escherichia coli HybG AAC76026.1 1789364
Escherichia coli
[0324] The hydrogen-lyase systems of E. coli include hydrogenase 3,
a membrane-bound enzyme complex using ferredoxin as an acceptor,
and hydrogenase 4 that also uses a ferredoxin acceptor. Hydrogenase
3 and 4 are encoded by the hyc and hyf gene clusters, respectively.
Hydrogenase 3 has been shown to be a reversible enzyme (Maeda et
al., Appl Microbiol Biotechnol 76(5): 1035-42 (2007)). Hydrogenase
activity in E. coli is also dependent upon the expression of the
hyp genes whose corresponding proteins are involved in the assembly
of the hydrogenase complexes (Jacobi et al., Arch. Microbiol
158:444-451 (1992); Rangarajan et al., J. Bacteriol. 190:1447-1458
(2008)).
TABLE-US-00100 Protein GenBank ID GI Number Organism HycA NP_417205
16130632 Escherichia coli HycB NP_417204 16130631 Escherichia coli
HycC NP_417203 16130630 Escherichia coli HycD NP_417202 16130629
Escherichia coli HycE NP_417201 16130628 Escherichia coli HycF
NP_417200 16130627 Escherichia coli HycG NP_417199 16130626
Escherichia coli HycH NP_417198 16130625 Escherichia coli HycI
NP_417197 16130624 Escherichia coli HyfA NP_416976 90111444
Escherichia coli HyfB NP_416977 16130407 Escherichia coli HyfC
NP_416978 90111445 Escherichia coli HyfD NP_416979 16130409
Escherichia coli HyfE NP_416980 16130410 Escherichia coli HyfF
NP_416981 16130411 Escherichia coli HyfG NP_416982 16130412
Escherichia coli HyfH NP_416983 16130413 Escherichia coli HyfI
NP_416984 16130414 Escherichia coli HyfJ NP_416985 90111446
Escherichia coli HyfR NP_416986 90111447 Escherichia coli HypA
NP_417206 16130633 Escherichia coli HypB NP_417207 16130634
Escherichia coli HypC NP_417208 16130635 Escherichia coli HypD
NP_417209 16130636 Escherichia coli HypE NP_417210 226524740
Escherichia coli HypF NP_417192 16130619 Escherichia coli
[0325] The M. thermoacetica hydrogenases are suitable for a host
that lacks sufficient endogenous hydrogenase activity. M.
thermoacetica can grow with CO.sub.2 as the exclusive carbon source
indicating that reducing equivalents are extracted from H.sub.2 to
enable acetyl-CoA synthesis via the Wood-Ljungdahl pathway (Drake,
H. L., J. Bacteriol. 150:702-709 (1982); Drake and Daniel, Res.
Microbiol. 155:869-883 (2004); Kellum and Drake, J. Bacteriol.
160:466-469 (1984)) (see FIG. 2A). M. thermoacetica has homologs to
several hyp, hyc, and hyf genes from E. coli. The protein sequences
encoded for by these genes are identified by the following GenBank
accession numbers.
[0326] Proteins in M. thermoacetica whose genes are homologous to
the E. coli hyp genes are shown below.
TABLE-US-00101 Protein GenBank ID GI Number Organism Moth_2175
YP_431007 83590998 Moorella thermoacetica Moth_2176 YP_431008
83590999 Moorella thermoacetica Moth_2177 YP_431009 83591000
Moorella thermoacetica Moth_2178 YP_431010 83591001 Moorella
thermoacetica Moth_2179 YP_431011 83591002 Moorella thermoacetica
Moth_2180 YP_431012 83591003 Moorella thermoacetica Moth_2181
YP_431013 83591004 Moorella thermoacetica
[0327] Proteins in M. thermoacetica that are homologous to the E.
coli Hydrogenase 3 and/or 4 proteins are listed in the following
table.
TABLE-US-00102 Protein GenBank ID GI Number Organism Moth_2182
YP_431014 83591005 Moorella thermoacetica Moth_2183 YP_431015
83591006 Moorella thermoacetica Moth_2184 YP_431016 83591007
Moorella thermoacetica Moth_2185 YP_431017 83591008 Moorella
thermoacetica Moth_2186 YP_431018 83591009 Moorella thermoacetica
Moth_2187 YP_431019 83591010 Moorella thermoacetica Moth_2188
YP_431020 83591011 Moorella thermoacetica Moth_2189 YP_431021
83591012 Moorella thermoacetica Moth_2190 YP_431022 83591013
Moorella thermoacetica Moth_2191 YP_431023 83591014 Moorella
thermoacetica Moth_2192 YP_431024 83591015 Moorella
thermoacetica
[0328] In addition, several gene clusters encoding hydrogenase
functionality are present in M. thermoacetica and their
corresponding protein sequences are provided below.
TABLE-US-00103 Protein GenBank ID GI Number Organism Moth_0439
YP_429313 83589304 Moorella thermoacetica Moth_0440 YP_429314
83589305 Moorella thermoacetica Moth_0441 YP_429315 83589306
Moorella thermoacetica Moth_0442 YP_429316 83589307 Moorella
thermoacetica Moth_0809 YP_429670 83589661 Moorella thermoacetica
Moth_0810 YP_429671 83589662 Moorella thermoacetica Moth_0811
YP_429672 83589663 Moorella thermoacetica Moth_0812 YP_429673
83589664 Moorella thermoacetica Moth_0814 YP_429674 83589665
Moorella thermoacetica Moth_0815 YP_429675 83589666 Moorella
thermoacetica Moth_0816 YP_429676 83589667 Moorella thermoacetica
Moth_1193 YP_430050 83590041 Moorella thermoacetica Moth_1194
YP_430051 83590042 Moorella thermoacetica Moth_1195 YP_430052
83590043 Moorella thermoacetica Moth_1196 YP_430053 83590044
Moorella thermoacetica Moth_1717 YP_430562 83590553 Moorella
thermoacetica Moth_1718 YP_430563 83590554 Moorella thermoacetica
Moth_1719 YP_430564 83590555 Moorella thermoacetica Moth_1883
YP_430726 83590717 Moorella thermoacetica Moth_1884 YP_430727
83590718 Moorella thermoacetica Moth_1885 YP_430728 83590719
Moorella thermoacetica Moth_1886 YP_430729 83590720 Moorella
thermoacetica Moth_1887 YP_430730 83590721 Moorella thermoacetica
Moth_1888 YP_430731 83590722 Moorella thermoacetica Moth_1452
YP_430305 83590296 Moorella thermoacetica Moth_1453 YP_430306
83590297 Moorella thermoacetica Moth_1454 YP_430307 83590298
Moorella thermoacetica
[0329] Ralstonia eutropha H16 uses hydrogen as an energy source
with oxygen as a terminal electron acceptor. Its membrane-bound
uptake [NiFe]-hydrogenase is an "O2-tolerant" hydrogenase
(Cracknell, et al. Proc Nat Acad Sci, 106(49) 20681-20686 (2009))
that is periplasmically-oriented and connected to the respiratory
chain via a b-type cytochrome (Schink and Schlegel, Biochim.
Biophys. Acta, 567, 315-324 (1979); Bernhard et al., Eur. J.
Biochem. 248, 179-186 (1997)). R. eutropha also contains an
02-tolerant soluble hydrogenase encoded by the Hox operon which is
cytoplasmic and directly reduces NAD+ at the expense of hydrogen
(Schneider and Schlegel, Biochim. Biophys. Acta 452, 66-80 (1976);
Burgdorf, J. Bact. 187(9) 3122-3132(2005)). Soluble hydrogenase
enzymes are additionally present in several other organisms
including Geobacter sulfurreducens (Coppi, Microbiology 151,
1239-1254 (2005)), Synechocystis str. PCC 6803 (Germer, J. Biol.
Chem., 284(52), 36462-36472 (2009)), and Thiocapsa roseopersicina
(Rakhely, Appl. Environ. Microbiol. 70(2) 722-728 (2004)). The
Synechocystis enzyme is capable of generating NADPH from hydrogen.
Overexpression of both the Hox operon from Synechocystis str. PCC
6803 and the accessory genes encoded by the Hyp operon from Nostoc
sp. PCC 7120 led to increased hydrogenase activity compared to
expression of the Hox genes alone (Germer, J. Biol. Chem. 284(52),
36462-36472 (2009)).
TABLE-US-00104 Protein GenBank ID GI Number Organism HoxF
NP_942727.1 38637753 Ralstonia eutropha H16 HoxU NP_942728.1
38637754 Ralstonia eutropha H16 HoxY NP_942729.1 38637755 Ralstonia
eutropha H16 HoxH NP_942730.1 38637756 Ralstonia eutropha H16 HoxW
NP_942731.1 38637757 Ralstonia eutropha H16 HoxI NP_942732.1
38637758 Ralstonia eutropha H16 HoxE NP_953767.1 39997816 Geobacter
sulfurreducens HoxF NP_953766.1 39997815 Geobacter sulfurreducens
HoxU NP_953765.1 39997814 Geobacter sulfurreducens HoxY NP_953764.1
39997813 Geobacter sulfurreducens HoxH NP_953763.1 39997812
Geobacter sulfurreducens GSU2717 NP_953762.1 39997811 Geobacter
sulfurreducens HoxE NP_441418.1 16330690 Synechocystis str. PCC
6803 HoxF NP_441417.1 16330689 Synechocystis str. PCC 6803 Unknown
NP_441416.1 16330688 Synechocystis str. PCC function 6803 HoxU
NP_441415.1 16330687 Synechocystis str. PCC 6803 HoxY NP_441414.1
16330686 Synechocystis str. PCC 6803 Unknown NP_441413.1 16330685
Synechocystis str. PCC function 6803 Unknown NP_441412.1 16330684
Synechocystis str. PCC function 6803 HoxH NP_441411.1 16330683
Synechocystis str. PCC 6803 HypF NP_484737.1 17228189 Nostoc sp.
PCC 7120 HypC NP_484738.1 17228190 Nostoc sp. PCC 7120 HypD
NP_484739.1 17228191 Nostoc sp. PCC 7120 Unknown NP_484740.1
17228192 Nostoc sp. PCC 7120 function HypE NP_484741.1 17228193
Nostoc sp. PCC 7120 HypA NP_484742.1 17228194 Nostoc sp. PCC 7120
HypB NP_484743.1 17228195 Nostoc sp. PCC 7120 Hox1E AAP50519.1
37787351 Thiocapsa roseopersicina Hox1F AAP50520.1 37787352
Thiocapsa roseopersicina Hox1U AAP50521.1 37787353 Thiocapsa
roseopersicina Hox1Y AAP50522.1 37787354 Thiocapsa roseopersicina
Hox1H AAP50523.1 37787355 Thiocapsa roseopersicina
[0330] Several enzymes and the corresponding genes used for fixing
carbon dioxide to either pyruvate or phosphoenolpyruvate to form
the TCA cycle intermediates, oxaloacetate or malate are described
below.
[0331] Carboxylation of phosphoenolpyruvate to oxaloacetate is
catalyzed by phosphoenolpyruvate carboxylase. Exemplary PEP
carboxylase enzymes are encoded by ppc in E. coli (Kai et al.,
Arch. Biochem. Biophys. 414:170-179 (2003), ppcA in
Methylobacterium extorquens AM1 (Arps et al., J. Bacteriol.
175:3776-3783 (1993), and ppc in Corynebacterium glutamicum
(Eikmanns et al., Mol. Gen. Genet. 218:330-339 (1989).
TABLE-US-00105 Protein GenBank ID GI Number Organism Ppc NP_418391
16131794 Escherichia coli ppcA AAB58883 28572162 Methylobacterium
extorquens Ppc ABB53270 80973080 Corynebacterium glutamicum
[0332] An alternative enzyme for converting phosphoenolpyruvate to
oxaloacetate is PEP carboxykinase, which simultaneously forms an
ATP while carboxylating PEP. In most organisms 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., J. Microbiol. Biotechnol. 16:1448-1452 (2006)). These
strains exhibited no growth defects and had increased succinate
production at high NaHCO.sub.3 concentrations. Mutant strains of E.
coli can adopt Pck as the dominant CO.sub.2-fixing enzyme following
adaptive evolution (Zhang et al. 2009). 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., 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.
supra). The PEP carboxykinase enzyme encoded by Haemophilus
influenza is effective at forming oxaloacetate from PEP.
TABLE-US-00106 Protein 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
[0333] Pyruvate carboxylase (EC 6.4.1.1) directly converts pyruvate
to oxaloacetate at the cost of one ATP. Pyruvate carboxylase
enzymes are encoded by PYC1 (Walker et al., Biochem. Biophys. Res.
Commun. 176:1210-1217 (1991) and PYC2 (Walker et al., supra) in
Saccharomyces cerevisiae, and pyc in Mycobacterium smegmatis
(Mukhopadhyay and Purwantini, Biochim. Biophys. Acta 1475:191-206
(2000)).
TABLE-US-00107 Protein GenBank ID GI Number Organism PYC1 NP_011453
6321376 Saccharomyces cerevisiae PYC2 NP_009777 6319695
Saccharomyces cerevisiae Pyc YP_890857.1 118470447 Mycobacterium
smegmatis
[0334] Malic enzyme can be applied to convert CO.sub.2 and pyruvate
to malate at the expense of one reducing equivalent. Malic enzymes
for this purpose can include, without limitation, malic enzyme
(NAD-dependent) and malic enzyme (NADP-dependent). For example, one
of the E. coli malic enzymes (Takeo, J. Biochem. 66:379-387 (1969))
or a similar enzyme with higher activity can be expressed to enable
the conversion of pyruvate and CO.sub.2 to malate. By fixing carbon
to pyruvate as opposed to PEP, malic enzyme allows the high-energy
phosphate bond from PEP to be conserved by pyruvate kinase whereby
ATP is generated in the formation of pyruvate or by the
phosphotransferase system for glucose transport. Although malic
enzyme is typically assumed to operate in the direction of pyruvate
formation from malate, overexpression of the NAD-dependent enzyme,
encoded by maeA, has been demonstrated to increase succinate
production in E. coli while restoring the lethal Apfl-AldhA
phenotype under anaerobic conditions by operating in the
carbon-fixing direction (Stols and Donnelly, Appl. Environ.
Microbiol. 63(7) 2695-2701 (1997)). A similar observation was made
upon overexpressing the malic enzyme from Ascaris suum in E. coli
(Stols et al., Appl. Biochem. Biotechnol. 63-65(1), 153-158
(1997)). The second E. coli malic enzyme, encoded by maeB, is
NADP-dependent and also decarboxylates oxaloacetate and other
alpha-keto acids (Iwakura et al., J. Biochem. 85(5):1355-65
(1979)).
TABLE-US-00108 Protein GenBank ID GI Number Organism maeA NP_415996
90111281 Escherichia coli maeB NP_416958 16130388 Escherichia coli
NAD-ME P27443 126732 Ascaris suum
[0335] The enzymes used for converting oxaloacetate (formed from,
for example, PEP carboxylase, PEP carboxykinase, or pyruvate
carboxylase) or malate (formed from, for example, malic enzyme or
malate dehydrogenase) to succinyl-CoA via the reductive branch of
the TCA cycle are malate dehydrogenase, fumarate dehydratase
(fumarase), fumarate reductase, and succinyl-CoA transferase. The
genes for each of the enzymes are described herein.
[0336] Enzymes, genes and methods for engineering pathways from
succinyl-CoA to various products into a microorganism are now known
in the art. The additional reducing equivalents obtained from CO
and/or H.sub.2, as disclosed herein, improve the yields of
butadiene or crotyl alcohol when utilizing carbohydrate-based
feedstock.
[0337] Enzymes, genes and methods for engineering pathways from
glycolysis intermediates to various products into a microorganism
are known in the art. The additional reducing equivalents obtained
from CO and H.sub.2, as described herein, improve the yields of all
these products, including butadiene and crotyl alcohol, on
carbohydrates.
Example III
Methods for Handling CO and Anaerobic Cultures
[0338] This example describes methods used in handling CO and
anaerobic cultures.
[0339] A. Handling of CO in Small Quantities for Assays and Small
Cultures.
[0340] CO is an odorless, colorless and tasteless gas that is a
poison. Therefore, cultures and assays that utilized CO required
special handling. Several assays, including CO oxidation,
acetyl-CoA synthesis, CO concentration using myoglobin, and CO
tolerance/utilization in small batch cultures, called for small
quantities of the CO gas that were dispensed and handled within a
fume hood. Biochemical assays called for saturating very small
quantities (<2 mL) of the biochemical assay medium or buffer
with CO and then performing the assay. All of the CO handling steps
were performed in a fume hood with the sash set at the proper
height and blower turned on; CO was dispensed from a compressed gas
cylinder and the regulator connected to a Schlenk line. The latter
ensures that equal concentrations of CO were dispensed to each of
several possible cuvettes or vials. The Schlenk line was set up
containing an oxygen scrubber on the input side and an oil pressure
release bubbler and vent on the other side. Assay cuvettes were
both anaerobic and CO-containing. Therefore, the assay cuvettes
were tightly sealed with a rubber stopper and reagents were added
or removed using gas-tight needles and syringes. Secondly, small
(.about.50 mL) cultures were grown with saturating CO in tightly
stoppered serum bottles. As with the biochemical assays, the
CO-saturated microbial cultures were equilibrated in the fume hood
using the Schlenk line setup. Both the biochemical assays and
microbial cultures were in portable, sealed containers and in small
volumes making for safe handling outside of the fume hood. The
compressed CO tank was adjacent to the fume hood.
[0341] Typically, a Schlenk line was used to dispense CO to
cuvettes, each vented. Rubber stoppers on the cuvettes were pierced
with 19 or 20 gage disposable syringe needles and were vented with
the same. An oil bubbler was used with a CO tank and oxygen
scrubber. The glass or quartz spectrophotometer cuvettes have a
circular hole on top into which a Kontes stopper sleeve, Sz7
774250-0007 was fitted. The CO detector unit was positioned
proximal to the fume hood.
[0342] B. Handling of CO in Larger Quantities Fed to Large-Scale
Cultures.
[0343] Fermentation cultures are fed either CO or a mixture of CO
and H.sub.2 to simulate syngas as a feedstock in fermentative
production. Therefore, quantities of cells ranging from 1 liter to
several liters can include the addition of CO gas to increase the
dissolved concentration of CO in the medium. In these
circumstances, fairly large and continuously administered
quantities of CO gas are added to the cultures. At different
points, the cultures are harvested or samples removed.
Alternatively, cells are harvested with an integrated continuous
flow centrifuge that is part of the fermenter.
[0344] The fermentative processes are carried out under anaerobic
conditions. In some cases, it is uneconomical to pump oxygen or air
into fermenters to ensure adequate oxygen saturation to provide a
respiratory environment. In addition, the reducing power generated
during anaerobic fermentation may be needed in product formation
rather than respiration. Furthermore, many of the enzymes for
various pathways are oxygen-sensitive to varying degrees. Classic
acetogens such as M. thermoacetica are obligate anaerobes and the
enzymes in the Wood-Ljungdahl pathway are highly sensitive to
irreversible inactivation by molecular oxygen. While there are
oxygen-tolerant acetogens, the repertoire of enzymes in the
Wood-Ljungdahl pathway might be incompatible in the presence of
oxygen because most are metallo-enzymes, key components are
ferredoxins, and regulation can divert metabolism away from the
Wood-Ljungdahl pathway to maximize energy acquisition. At the same
time, cells in culture act as oxygen scavengers that moderate the
need for extreme measures in the presence of large cell growth.
[0345] C. Anaerobic Chamber and Conditions.
[0346] Exemplary anaerobic chambers are available commercially
(see, for example, Vacuum Atmospheres Company, Hawthorne Calif.;
MBraun, Newburyport Mass.). Conditions included an O.sub.2
concentration of 1 ppm or less and 1 atm pure N.sub.2. In one
example, 3 oxygen scrubbers/catalyst regenerators were used, and
the chamber included an O.sub.2 electrode (such as Teledyne; City
of Industry CA). Nearly all items and reagents were cycled four
times in the airlock of the chamber prior to opening the inner
chamber door. Reagents with a volume>5 mL were sparged with pure
N.sub.2 prior to introduction into the chamber. Gloves are changed
twice/yr and the catalyst containers were regenerated periodically
when the chamber displays increasingly sluggish response to changes
in oxygen levels. The chamber's pressure was controlled through
one-way valves activated by solenoids. This feature allowed setting
the chamber pressure at a level higher than the surroundings to
allow transfer of very small tubes through the purge valve.
[0347] The anaerobic chambers achieved levels of O.sub.2 that were
consistently very low and were needed for highly oxygen sensitive
anaerobic conditions. However, growth and handling of cells does
not usually require such precautions. In an alternative anaerobic
chamber configuration, platinum or palladium can be used as a
catalyst that requires some hydrogen gas in the mix. Instead of
using solenoid valves, pressure release can be controlled by a
bubbler. Instead of using instrument-based O.sub.2 monitoring, test
strips can be used instead.
[0348] D. Anaerobic Microbiology.
[0349] Small cultures were handled as described above for CO
handling. In particular, serum or media bottles are fitted with
thick rubber stoppers and aluminum crimps are employed to seal the
bottle. Medium, such as Terrific Broth, is made in a conventional
manner and dispensed to an appropriately sized serum bottle. The
bottles are sparged with nitrogen for .about.30 min of moderate
bubbling. This removes most of the oxygen from the medium and,
after this step, each bottle is capped with a rubber stopper (such
as Bellco 20 mm septum stoppers; Bellco, Vineland, N.J.) and
crimp-sealed (Bellco 20 mm). Then the bottles of medium are
autoclaved using a slow (liquid) exhaust cycle. At least sometimes
a needle can be poked through the stopper to provide exhaust during
autoclaving; the needle needs to be removed immediately upon
removal from the autoclave. The sterile medium has the remaining
medium components, for example buffer or antibiotics, added via
syringe and needle. Prior to addition of reducing agents, the
bottles are equilibrated for 30-60 minutes with nitrogen (or CO
depending upon use). A reducing agent such as a 100.times.150 mM
sodium sulfide, 200 mM cysteine-HCl is added. This is made by
weighing the sodium sulfide into a dry beaker and the cysteine into
a serum bottle, bringing both into the anaerobic chamber,
dissolving the sodium sulfide into anaerobic water, then adding
this to the cysteine in the serum bottle. The bottle is stoppered
immediately as the sodium sulfide solution generates hydrogen
sulfide gas upon contact with the cysteine. When injecting into the
culture, a syringe filter is used to sterilize the solution. Other
components are added through syringe needles, such as B12 (10 .mu.M
cyanocobalamin), nickel chloride (NiCl.sub.2, 20 microM final
concentration from a 40 mM stock made in anaerobic water in the
chamber and sterilized by autoclaving or by using a syringe filter
upon injection into the culture), and ferrous ammonium sulfate
(final concentration needed is 100 .mu.M-made as 100-1000.times.
stock solution in anaerobic water in the chamber and sterilized by
autoclaving or by using a syringe filter upon injection into the
culture). To facilitate faster growth under anaerobic conditions,
the 1 liter bottles were inoculated with 50 mL of a preculture
grown anaerobically. Induction of the pA1-lacO1 promoter in the
vectors was performed by addition of isopropyl
.beta.-D-1-thiogalactopyranoside (IPTG) to a final concentration of
0.2 mM and was carried out for about 3 hrs.
[0350] Large cultures can be grown in larger bottles using
continuous gas addition while bubbling. A rubber stopper with a
metal bubbler is placed in the bottle after medium addition and
sparged with nitrogen for 30 minutes or more prior to setting up
the rest of the bottle. Each bottle is put together such that a
sterile filter will sterilize the gas bubbled in and the hoses on
the bottles are compressible with small C clamps. Medium and cells
are stirred with magnetic stir bars. Once all medium components and
cells are added, the bottles are incubated in an incubator in room
air but with continuous nitrogen sparging into the bottles.
Example IV
CO Oxidation (CODH) Assay
[0351] This example describes assay methods for measuring CO
oxidation (CO dehydrogenase; CODH).
[0352] The 7 gene CODH/ACS operon of Moorella thermoacetica was
cloned into E. coli expression vectors. The intact .about.10 kbp
DNA fragment was cloned, and it is likely that some of the genes in
this region are expressed from their own endogenous promoters and
all contain endogenous ribosomal binding sites. These clones were
assayed for CO oxidation, using an assay that quantitatively
measures CODH activity. Antisera to the M. thermoacetica gene
products was used for Western blots to estimate specific activity.
M. thermoacetica is Gram positive, and ribosome binding site
elements are expected to work well in E. coli. This activity,
described below in more detail, was estimated to be .about. 1/50th
of the M. thermoacetica specific activity. It is possible that CODH
activity of recombinant E. coli cells could be limited by the fact
that M. thermoacetica enzymes have temperature optima around
55.degree. C. Therefore, a mesophilic CODH/ACS pathway could be
advantageous such as the close relative of Moorella that is
mesophilic and does have an apparently intact CODH/ACS operon and a
Wood-Ljungdahl pathway, Desulfitobacterium hafniense. Acetogens as
potential host organisms include, but are not limited to,
Rhodospirillum rubrum, Moorella thermoacetica and
Desulfitobacterium hafniense.
[0353] CO oxidation is both the most sensitive and most robust of
the CODH/ACS assays. It is likely that an E. coli-based syngas
using system will ultimately need to be about as anaerobic as
Clostridial (i.e., Moorella) systems, especially for maximal
activity. Improvement in CODH should be possible but will
ultimately be limited by the solubility of CO gas in water.
[0354] Initially, each of the genes was cloned individually into
expression vectors. Combined expression units for multiple
subunits/1 complex were generated. Expression in E. coli at the
protein level was determined. Both combined M. thermoacetica
CODH/ACS operons and individual expression clones were made.
[0355] CO oxidation assay. This assay is one of the simpler,
reliable, and more versatile assays of enzymatic activities within
the Wood-Ljungdahl pathway and tests CODH (Seravalli et al.,
Biochemistry 43:3944-3955 (2004)). A typical activity of M.
thermoacetica CODH specific activity is 500 U at 55.degree. C. or
.about.60U at 25.degree. C. This assay employs reduction of methyl
viologen in the presence of CO. This is measured at 578 nm in
stoppered, anaerobic, glass cuvettes.
[0356] In more detail, glass rubber stoppered cuvettes were
prepared after first washing the cuvette four times in deionized
water and one time with acetone. A small amount of vacuum grease
was smeared on the top of the rubber gasket. The cuvette was gassed
with CO, dried 10 min with a 22 Ga. needle plus an exhaust needle.
A volume of 0.98 mlL of reaction buffer (50 mM Hepes, pH 8.5, 2 mM
dithiothreitol (DTT) was added using a 22 Ga. needle, with exhaust
needled, and 100% CO. Methyl viologen (CH.sub.3 viologen) stock was
1 M in water. Each assay used 20 microliters for 20 mM final
concentration. When methyl viologen was added, an 18 Ga needle
(partial) was used as a jacket to facilitate use of a Hamilton
syringe to withdraw the CH.sub.3 viologen. 4-5 aliquots were drawn
up and discarded to wash and gas equilibrate the syringe. A small
amount of sodium dithionite (0.1 M stock) was added when making up
the CH.sub.3 viologen stock to slightly reduce the CH.sub.3
viologen. The temperature was equilibrated to 55.degree. C. in a
heated Olis spectrophotometer (Bogart GA). A blank reaction
(CH.sub.3 viologen+buffer) was run first to measure the base rate
of CH.sub.3 viologen reduction. Crude E. coli cell extracts of
ACS90 and ACS91 (CODH-ACS operon of M. thermoacetica with and
without, respectively, the first cooC). 10 microliters of extract
were added at a time, mixed and assayed. Reduced CH.sub.3 viologen
turns purple. The results of an assay are shown in Table I.
TABLE-US-00109 TABLE I Crude extract CO Oxidation Activities. ACS90
7.7 mg/ml ACS91 11.8 mg/ml Mta98 9.8 mg/ml Mta99 11.2 mg/ml Extract
Vol OD/ U/ml U/mg ACS90 10 microliters 0.073 0.376 0.049 ACS91 10
microliters 0.096 0.494 0.042 Mta99 10 microliters 0.0031 0.016
0.0014 ACS90 10 microliters 0.099 0.51 0.066 Mta99 25 microliters
0.012 0.025 0.0022 ACS91 25 microliters 0.215 0.443 0.037 Mta98 25
microliters 0.019 0.039 0.004 ACS91 10 microliters 0.129 0.66 0.056
Averages ACS90 0.057 U/mg ACS91 0.045 U/mg Mta99 0.0018 U/mg
[0357] Mta98/Mta99 are E. coli MG1655 strains that express methanol
methyltransferase genes from M. thermoacetia and, therefore, are
negative controls for the ACS90 ACS91 E. coli strains that contain
M. thermoacetica CODH operons.
[0358] If .about.1% of the cellular protein is CODH, then these
figures would be approximately 100.times. less than the 500 U/mg
activity of pure M. thermoacetica CODH. Actual estimates based on
Western blots are 0.5% of the cellular protein, so the activity is
about 50.times. less than for M. thermoacetica CODH. Nevertheless,
this experiment demonstrates CO oxidation activity in recombinant
E. coli with a much smaller amount in the negative controls. The
small amount of CO oxidation (CH.sub.3 viologen reduction) seen in
the negative controls indicates that E. coli may have a limited
ability to reduce CH.sub.3 viologen.
[0359] To estimate the final concentrations of CODH and Mtr
proteins, SDS-PAGE followed by Western blot analyses were performed
on the same cell extracts used in the CO oxidation, ACS,
methyltransferase, and corrinoid Fe--S assays. The antisera used
were polyclonal to purified M. thermoacetica CODH-ACS and Mtr
proteins and were visualized using an alkaline phosphatase-linked
goat-anti-rabbit secondary antibody. The Westerns were performed
and results are shown in FIG. 9. The amounts of CODH in ACS90 and
ACS91 were estimated at 50 ng by comparison to the control lanes.
Expression of CODH-ACS operon genes including 2 CODH subunits and
the methyltransferase were confirmed via Western blot analysis.
Therefore, the recombinant E. coli cells express multiple
components of a 7 gene operon. In addition, both the
methyltransferase and corrinoid iron sulfur protein were active in
the same recombinant E. coli cells. These proteins are part of the
same operon cloned into the same cells.
[0360] The CO oxidation assays were repeated using extracts of
Moorella thermoacetica cells for the positive controls. Though CODH
activity in E. coli ACS90 and ACS91 was measurable, it was at about
130-150.times. lower than the M. thermoacetica control. The results
of the assay are shown in FIG. 10. Briefly, cells (M. thermoacetica
or E. coli with the CODH/ACS operon; ACS90 or ACS91 or empty
vector: pZA33S) were grown and extracts prepared as described
herein. Assays were performed as described above at 55.degree. C.
at various times on the day the extracts were prepared. Reduction
of methylviologen was followed at 578 nm over a 120 sec time
course.
[0361] These results describe the CO oxidation (CODH) assay and
results. Recombinant E. coli cells expressed CO oxidation activity
as measured by the methyl viologen reduction assay.
Example V
E. coli CO Tolerance Experiment and CO Concentration Assay
(Myoglobin Assay)
[0362] This example describes the tolerance of E. coli for high
concentrations of CO.
[0363] To test whether or not E. coli can grow anaerobically in the
presence of saturating amounts of CO, cultures were set up in 120
ml serum bottles with 50 ml of Terrific Broth medium (plus reducing
solution, NiCl.sub.2, Fe(II)NH.sub.4SO.sub.4, cyanocobalamin, IPTG,
and chloramphenicol) as described above for anaerobic microbiology
in small volumes. One half of these bottles were equilibrated with
nitrogen gas for 30 min. and one half was equilibrated with CO gas
for 30 min. An empty vector (pZA33) was used as a control, and
cultures containing the pZA33 empty vector as well as both ACS90
and ACS91 were tested with both N.sub.2 and CO. All were inoculated
and grown for 36 hrs with shaking (250 rpm) at 37.degree. C. At the
end of the 36 hour period, examination of the flasks showed high
amounts of growth in all. The bulk of the observed growth occurred
overnight with a long lag.
[0364] Given that all cultures appeared to grow well in the
presence of CO, the final CO concentrations were confirmed. This
was performed using an assay of the spectral shift of myoglobin
upon exposure to CO. Myoglobin reduced with sodium dithionite has
an absorbance peak at 435 nm; this peak is shifted to 423 nm with
CO. Due to the low wavelength and need to record a whole spectrum
from 300 nm on upwards, quartz cuvettes must be used. CO
concentration is measured against a standard curve and depends upon
the Henry's Law constant for CO of maximum water solubility=970
micromolar at 20.degree. C. and 1 atm.
[0365] For the myoglobin test of CO concentration, cuvettes were
washed 10.times. with water, 1.times. with acetone, and then
stoppered as with the CODH assay. N.sub.2 was blown into the
cuvettes for .about.10 min. A volume of 1 ml of anaerobic buffer
(HEPES, pH 8.0, 2 mM DTT) was added to the blank (not equilibrated
with CO) with a Hamilton syringe. A volume of 10 microliter
myoglobin (.about.1 mM--can be varied, just need a fairly large
amount) and 1 microliter dithionite (20 mM stock) were added. A CO
standard curve was made using CO saturated buffer added at 1
microliter increments. Peak height and shift was recorded for each
increment. The cultures tested were pZA33/CO, ACS90/CO, and
ACS91/CO. Each of these was added in 1 microliter increments to the
same cuvette. Midway through the experiment a second cuvette was
set up and used. The results are shown in Table II.
TABLE-US-00110 TABLE II Carbon Monoxide Concentrations, 36 hrs.
Strain and Growth Conditions Final CO concentration (micromolar)
pZA33-CO 930 ACS90-CO 638 494 734 883 ave 687 SD 164 ACS91-CO 728
812 760 611 ave. 728 SD 85
[0366] The results shown in Table II indicate that the cultures
grew whether or not a strain was cultured in the presence of CO or
not. These results indicate that E. coli can tolerate exposure to
CO under anaerobic conditions and that E. coli cells expressing the
CODH-ACS operon can metabolize some of the CO.
[0367] These results demonstrate that E. coli cells, whether
expressing CODH/ACS or not, were able to grow in the presence of
saturating amounts of CO. Furthermore, these grew equally well as
the controls in nitrogen in place of CO. This experiment
demonstrated that laboratory strains of E. coli are insensitive to
CO at the levels achievable in a syngas project performed at normal
atmospheric pressure. In addition, preliminary experiments
indicated that the recombinant E. coli cells expressing CODH/ACS
actually consumed some CO, probably by oxidation to carbon
dioxide.
Example VI
Exemplary Carboxylic Acid Reductases
[0368] This example describes the use of carboxylic acid reductases
to carry out the conversion of a carboxylic acid to an
aldehyde.
[0369] 1.2.1.e Acid Reductase.
[0370] The conversion of unactivated acids to aldehydes can be
carried out by an acid reductase. Examples of such conversions
include, but are not limited, the conversion of 4-hydroxybutyrate,
succinate, alpha-ketoglutarate, and 4-aminobutyrate to
4-hydroxybutanal, succinate semialdehyde, 2,5-dioxopentanoate, and
4-aminobutanal, respectively. One notable carboxylic acid reductase
can be found in Nocardia iowensis which 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)). This enzyme is encoded by the car gene and 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., in
Biocatalysis in the Pharmaceutical and Biotechnology Industires,
ed. R. N. Patel, Chapter 15, pp. 425-440, CRC Press LLC, Boca
Raton, Fla. (2006)).
TABLE-US-00111 Gene Accession No. GI No. Organism car AAR91681.1
40796035 Nocardia iowensis (sp. NRRL 5646) npt ABI83656.1 114848891
Nocardia iowensis (sp. NRRL 5646)
[0371] Additional car and npt genes can be identified based on
sequence homology.
TABLE-US-00112 Gene Accession No. GI No. 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 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
[0372] 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, can be beneficial.
TABLE-US-00113 Gene Accession No. GI No. Organism griC 182438036
YP_001825755.1 Streptomyces griseus subsp. griseus NBRC 13350 griD
182438037 YP_001825756.1 Streptomyces griseus subsp. griseus NBRC
13350 MSMEG_2956 YP_887275.1 YP_887275.1 Mycobacterium smegmatis
MC2 155 MSMEG_5739 YP_889972.1 118469671 Mycobacterium smegmatis
MC2 155 MSMEG_2648 YP_886985.1 118471293 Mycobacterium smegmatis
MC2 155 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
[0373] 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.
TABLE-US-00114 Gene Accession No. GI No. 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
[0374] Cloning and Expression of Carboxylic Acid Reductase.
[0375] Escherichia coli is used as a target organism to engineer
the pathway for butadiene or crotyl alcohol. E. coli provides a
good host for generating a non-naturally occurring microorganism
capable of producing butadiene or crotyl alcohol. E. coli is
amenable to genetic manipulation and is known to be capable of
producing various intermediates and products effectively under
various oxygenation conditions.
[0376] To generate a microbial organism strain such as an E. coli
strain engineered to produce butadiene or crotyl alcohol, nucleic
acids encoding a carboxylic acid reductase and phosphopantetheine
transferase are expressed in E. coli using well known molecular
biology techniques (see, for example, Sambrook, supra, 2001;
Ausubel supra, 1999). In particular, car genes from Nocardia
iowensis (designated 720), Mycobacterium smegmatis mc(2)155
(designated 890), Mycobacterium avium subspecies paratuberculosis
K-10 (designated 891) and Mycobacterium marinum M (designated 892)
were cloned into pZS*13 vectors (Expressys, Ruelzheim, Germany)
under control of PA1/lacO promoters. The npt (ABI83656.1) gene
(i.e., 721) was cloned into the pKJL33S vector, a derivative of the
original mini-F plasmid vector PML31 under control of promoters and
ribosomal binding sites similar to those used in pZS*13.
[0377] The car gene (GNM_720) was cloned by PCR from Nocardia
genomic DNA. Its nucleic acid and protein sequences are shown in
FIGS. 12A and 12B, respectively. A codon-optimized version of the
npt gene (GNM_721) was synthesized by GeneArt (Regensburg,
Germany). Its nucleic acid and protein sequences are shown in FIGS.
13A and 13B, respectively. The nucleic acid and protein sequences
for the Mycobacterium smegmatis mc(2)155 (designated 890),
Mycobacterium avium subspecies paratuberculosis K-10 (designated
891) and Mycobacterium marinum M (designated 892) genes and enzymes
can be found in FIGS. 14, 15, and 16, respectively. The plasmids
are transformed into a host cell to express the proteins and
enzymes required for butadiene or crotyl alcohol production or
intermediates thereof.
[0378] Additional CAR variants were generated. A codon optimized
version of CAR 891 was generated and designated 891GA. The nucleic
acid and amino acid sequences of CAR 891GA are shown in FIGS. 17A
and 17B, respectively. Over 2000 CAR variants were generated. In
particular, all 20 amino acid combinations were made at positions
V295, M296, G297, G391, G421, D413, G414, Y415, G416, and S417, and
additional variants were tested as well. Exemplary CAR variants
include: E16K; Q95L; L100M; A1011T; K823E; T941S; H.sub.15Q; D198E;
G446C; S392N; F699L; V883I; F467S; T987S; R12H; V295G; V295A;
V295S; V295T; V295C; V295V; V295L; V295I; V295M; V295P; V295F;
V295Y; V295W; V295D; V295E; V295N; V295Q; V295H; V295K; V295R;
M296G; M296A; M296S; M296T; M296C; M296V; M296L; M296I; M296M;
M296P; M296F; M296Y; M296W; M296D; M296E; M296N; M296Q; M296H;
M296K; M296R; G297G; G297A; G297S; G297T; G297C; G297V; G297L;
G297I; G297M; G297P; G297F; G297Y; G297W; G297D; G297E; G297N;
G297Q; G297H; G297K; G297R; G391G; G391A; G391S; G391T; G391C;
G391V; G391L; G391I; G391M; G391P; G391F; G391Y; G391W; G391D;
G391E; G391N; G391Q; G391H; G391K; G391R; G421G; G421A; G421S;
G421T; G421C; G421V; G421L; G421I G421M; G421P; G421F; G421Y;
G421W; G421D; G421E; G421N; G421Q; G421H; G421K; G421R; D413G;
D413A; D413S; D413T; D413C; D413V; D413L; D413I; D413M; D413P;
D413F; D413Y; D413W; D413D; D413E; D413N; D413Q; D413H; D413K;
D413R; G414G; G414A; G414S; G414T; G414C; G414V; G414L; G414I;
G414M; G414P; G414F; G414Y; G414W; G414D; G414E; G414N; G414Q;
G414H; G414K; G414R; Y415G; Y415A; Y415S; Y415T; Y415C; Y415V;
Y415L; Y415I; Y415M; Y415P; Y415F; Y415Y; Y415W; Y415D; Y415E;
Y415N; Y415Q; Y415H; Y415K; Y415R; G416G; G416A; G416S; G416T;
G416C; G416V; G416L; G416I; G416M; G416P; G416F; G416Y; G416W;
G416D; G416E; G416N; G416Q; G416H; G416K; G416R; S417G; S417A;
S417S; S417T; S417C; S417V S417L; S417I; S417M; S417P; S417F;
S417Y; S417W; S417D; S417E; S417N; S417Q; S417H; S417K; and
S417R.
[0379] The CAR variants were screened for activity, and numerous
CAR variants were found to exhibit CAR activity.
[0380] This example describes the use of CAR for converting
carboxylic acids to aldehydes.
[0381] 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
1213525DNANocardia iowensis 1atggcagtgg attcaccgga tgagcggcta
cagcgccgca ttgcacagtt gtttgcagaa 60gatgagcagg tcaaggccgc acgtccgctc
gaagcggtga gcgcggcggt gagcgcgccc 120ggtatgcggc tggcgcagat
cgccgccact gttatggcgg gttacgccga ccgcccggcc 180gccgggcagc
gtgcgttcga actgaacacc gacgacgcga cgggccgcac ctcgctgcgg
240ttacttcccc gattcgagac catcacctat cgcgaactgt ggcagcgagt
cggcgaggtt 300gccgcggcct ggcatcatga tcccgagaac cccttgcgcg
caggtgattt cgtcgccctg 360ctcggcttca ccagcatcga ctacgccacc
ctcgacctgg ccgatatcca cctcggcgcg 420gttaccgtgc cgttgcaggc
cagcgcggcg gtgtcccagc tgatcgctat cctcaccgag 480acttcgccgc
ggctgctcgc ctcgaccccg gagcacctcg atgcggcggt cgagtgccta
540ctcgcgggca ccacaccgga acgactggtg gtcttcgact accaccccga
ggacgacgac 600cagcgtgcgg ccttcgaatc cgcccgccgc cgccttgccg
acgcgggcag cttggtgatc 660gtcgaaacgc tcgatgccgt gcgtgcccgg
ggccgcgact taccggccgc gccactgttc 720gttcccgaca ccgacgacga
cccgctggcc ctgctgatct acacctccgg cagcaccgga 780acgccgaagg
gcgcgatgta caccaatcgg ttggccgcca cgatgtggca ggggaactcg
840atgctgcagg ggaactcgca acgggtcggg atcaatctca actacatgcc
gatgagccac 900atcgccggtc gcatatcgct gttcggcgtg ctcgctcgcg
gtggcaccgc atacttcgcg 960gccaagagcg acatgtcgac actgttcgaa
gacatcggct tggtacgtcc caccgagatc 1020ttcttcgtcc cgcgcgtgtg
cgacatggtc ttccagcgct atcagagcga gctggaccgg 1080cgctcggtgg
cgggcgccga cctggacacg ctcgatcggg aagtgaaagc cgacctccgg
1140cagaactacc tcggtgggcg cttcctggtg gcggtcgtcg gcagcgcgcc
gctggccgcg 1200gagatgaaga cgttcatgga gtccgtcctc gatctgccac
tgcacgacgg gtacgggtcg 1260accgaggcgg gcgcaagcgt gctgctcgac
aaccagatcc agcggccgcc ggtgctcgat 1320tacaagctcg tcgacgtgcc
cgaactgggt tacttccgca ccgaccggcc gcatccgcgc 1380ggtgagctgt
tgttgaaggc ggagaccacg attccgggct actacaagcg gcccgaggtc
1440accgcggaga tcttcgacga ggacggcttc tacaagaccg gcgatatcgt
ggccgagctc 1500gagcacgatc ggctggtcta tgtcgaccgt cgcaacaatg
tgctcaaact gtcgcagggc 1560gagttcgtga ccgtcgccca tctcgaggcc
gtgttcgcca gcagcccgct gatccggcag 1620atcttcatct acggcagcag
cgaacgttcc tatctgctcg cggtgatcgt ccccaccgac 1680gacgcgctgc
gcggccgcga caccgccacc ttgaaatcgg cactggccga atcgattcag
1740cgcatcgcca aggacgcgaa cctgcagccc tacgagattc cgcgcgattt
cctgatcgag 1800accgagccgt tcaccatcgc caacggactg ctctccggca
tcgcgaagct gctgcgcccc 1860aatctgaagg aacgctacgg cgctcagctg
gagcagatgt acaccgatct cgcgacaggc 1920caggccgatg agctgctcgc
cctgcgccgc gaagccgccg acctgccggt gctcgaaacc 1980gtcagccggg
cagcgaaagc gatgctcggc gtcgcctccg ccgatatgcg tcccgacgcg
2040cacttcaccg acctgggcgg cgattccctt tccgcgctgt cgttctcgaa
cctgctgcac 2100gagatcttcg gggtcgaggt gccggtgggt gtcgtcgtca
gcccggcgaa cgagctgcgc 2160gatctggcga attacattga ggcggaacgc
aactcgggcg cgaagcgtcc caccttcacc 2220tcggtgcacg gcggcggttc
cgagatccgc gccgccgatc tgaccctcga caagttcatc 2280gatgcccgca
ccctggccgc cgccgacagc attccgcacg cgccggtgcc agcgcagacg
2340gtgctgctga ccggcgcgaa cggctacctc ggccggttcc tgtgcctgga
atggctggag 2400cggctggaca agacgggtgg cacgctgatc tgcgtcgtgc
gcggtagtga cgcggccgcg 2460gcccgtaaac ggctggactc ggcgttcgac
agcggcgatc ccggcctgct cgagcactac 2520cagcaactgg ccgcacggac
cctggaagtc ctcgccggtg atatcggcga cccgaatctc 2580ggtctggacg
acgcgacttg gcagcggttg gccgaaaccg tcgacctgat cgtccatccc
2640gccgcgttgg tcaaccacgt ccttccctac acccagctgt tcggccccaa
tgtcgtcggc 2700accgccgaaa tcgtccggtt ggcgatcacg gcgcggcgca
agccggtcac ctacctgtcg 2760accgtcggag tggccgacca ggtcgacccg
gcggagtatc aggaggacag cgacgtccgc 2820gagatgagcg cggtgcgcgt
cgtgcgcgag agttacgcca acggctacgg caacagcaag 2880tgggcggggg
aggtcctgct gcgcgaagca cacgatctgt gtggcttgcc ggtcgcggtg
2940ttccgttcgg acatgatcct ggcgcacagc cggtacgcgg gtcagctcaa
cgtccaggac 3000gtgttcaccc ggctgatcct cagcctggtc gccaccggca
tcgcgccgta ctcgttctac 3060cgaaccgacg cggacggcaa ccggcagcgg
gcccactatg acggcttgcc ggcggacttc 3120acggcggcgg cgatcaccgc
gctcggcatc caagccaccg aaggcttccg gacctacgac 3180gtgctcaatc
cgtacgacga tggcatctcc ctcgatgaat tcgtcgactg gctcgtcgaa
3240tccggccacc cgatccagcg catcaccgac tacagcgact ggttccaccg
tttcgagacg 3300gcgatccgcg cgctgccgga aaagcaacgc caggcctcgg
tgctgccgtt gctggacgcc 3360taccgcaacc cctgcccggc ggtccgcggc
gcgatactcc cggccaagga gttccaagcg 3420gcggtgcaaa cagccaaaat
cggtccggaa caggacatcc cgcatttgtc cgcgccactg 3480atcgataagt
acgtcagcga tctggaactg cttcagctgc tctaa 352521174PRTNocardia
iowensis 2Met Ala Val Asp Ser Pro Asp Glu Arg Leu Gln Arg Arg Ile
Ala Gln1 5 10 15Leu Phe Ala Glu Asp Glu Gln Val Lys Ala Ala Arg Pro
Leu Glu Ala 20 25 30Val Ser Ala Ala Val Ser Ala Pro Gly Met Arg Leu
Ala Gln Ile Ala 35 40 45Ala Thr Val Met Ala Gly Tyr Ala Asp Arg Pro
Ala Ala Gly Gln Arg 50 55 60Ala Phe Glu Leu Asn Thr Asp Asp Ala Thr
Gly Arg Thr Ser Leu Arg65 70 75 80Leu Leu Pro Arg Phe Glu Thr Ile
Thr Tyr Arg Glu Leu Trp Gln Arg 85 90 95Val Gly Glu Val Ala Ala Ala
Trp His His Asp Pro Glu Asn Pro Leu 100 105 110Arg Ala Gly Asp Phe
Val Ala Leu Leu Gly Phe Thr Ser Ile Asp Tyr 115 120 125Ala Thr Leu
Asp Leu Ala Asp Ile His Leu Gly Ala Val Thr Val Pro 130 135 140Leu
Gln Ala Ser Ala Ala Val Ser Gln Leu Ile Ala Ile Leu Thr Glu145 150
155 160Thr Ser Pro Arg Leu Leu Ala Ser Thr Pro Glu His Leu Asp Ala
Ala 165 170 175Val Glu Cys Leu Leu Ala Gly Thr Thr Pro Glu Arg Leu
Val Val Phe 180 185 190Asp Tyr His Pro Glu Asp Asp Asp Gln Arg Ala
Ala Phe Glu Ser Ala 195 200 205Arg Arg Arg Leu Ala Asp Ala Gly Ser
Leu Val Ile Val Glu Thr Leu 210 215 220Asp Ala Val Arg Ala Arg Gly
Arg Asp Leu Pro Ala Ala Pro Leu Phe225 230 235 240Val Pro Asp Thr
Asp Asp Asp Pro Leu Ala Leu Leu Ile Tyr Thr Ser 245 250 255Gly Ser
Thr Gly Thr Pro Lys Gly Ala Met Tyr Thr Asn Arg Leu Ala 260 265
270Ala Thr Met Trp Gln Gly Asn Ser Met Leu Gln Gly Asn Ser Gln Arg
275 280 285Val Gly Ile Asn Leu Asn Tyr Met Pro Met Ser His Ile Ala
Gly Arg 290 295 300Ile Ser Leu Phe Gly Val Leu Ala Arg Gly Gly Thr
Ala Tyr Phe Ala305 310 315 320Ala Lys Ser Asp Met Ser Thr Leu Phe
Glu Asp Ile Gly Leu Val Arg 325 330 335Pro Thr Glu Ile Phe Phe Val
Pro Arg Val Cys Asp Met Val Phe Gln 340 345 350Arg Tyr Gln Ser Glu
Leu Asp Arg Arg Ser Val Ala Gly Ala Asp Leu 355 360 365Asp Thr Leu
Asp Arg Glu Val Lys Ala Asp Leu Arg Gln Asn Tyr Leu 370 375 380Gly
Gly Arg Phe Leu Val Ala Val Val Gly Ser Ala Pro Leu Ala Ala385 390
395 400Glu Met Lys Thr Phe Met Glu Ser Val Leu Asp Leu Pro Leu His
Asp 405 410 415Gly Tyr Gly Ser Thr Glu Ala Gly Ala Ser Val Leu Leu
Asp Asn Gln 420 425 430Ile Gln Arg Pro Pro Val Leu Asp Tyr Lys Leu
Val Asp Val Pro Glu 435 440 445Leu Gly Tyr Phe Arg Thr Asp Arg Pro
His Pro Arg Gly Glu Leu Leu 450 455 460Leu Lys Ala Glu Thr Thr Ile
Pro Gly Tyr Tyr Lys Arg Pro Glu Val465 470 475 480Thr Ala Glu Ile
Phe Asp Glu Asp Gly Phe Tyr Lys Thr Gly Asp Ile 485 490 495Val Ala
Glu Leu Glu His Asp Arg Leu Val Tyr Val Asp Arg Arg Asn 500 505
510Asn Val Leu Lys Leu Ser Gln Gly Glu Phe Val Thr Val Ala His Leu
515 520 525Glu Ala Val Phe Ala Ser Ser Pro Leu Ile Arg Gln Ile Phe
Ile Tyr 530 535 540Gly Ser Ser Glu Arg Ser Tyr Leu Leu Ala Val Ile
Val Pro Thr Asp545 550 555 560Asp Ala Leu Arg Gly Arg Asp Thr Ala
Thr Leu Lys Ser Ala Leu Ala 565 570 575Glu Ser Ile Gln Arg Ile Ala
Lys Asp Ala Asn Leu Gln Pro Tyr Glu 580 585 590Ile Pro Arg Asp Phe
Leu Ile Glu Thr Glu Pro Phe Thr Ile Ala Asn 595 600 605Gly Leu Leu
Ser Gly Ile Ala Lys Leu Leu Arg Pro Asn Leu Lys Glu 610 615 620Arg
Tyr Gly Ala Gln Leu Glu Gln Met Tyr Thr Asp Leu Ala Thr Gly625 630
635 640Gln Ala Asp Glu Leu Leu Ala Leu Arg Arg Glu Ala Ala Asp Leu
Pro 645 650 655Val Leu Glu Thr Val Ser Arg Ala Ala Lys Ala Met Leu
Gly Val Ala 660 665 670Ser Ala Asp Met Arg Pro Asp Ala His Phe Thr
Asp Leu Gly Gly Asp 675 680 685Ser Leu Ser Ala Leu Ser Phe Ser Asn
Leu Leu His Glu Ile Phe Gly 690 695 700Val Glu Val Pro Val Gly Val
Val Val Ser Pro Ala Asn Glu Leu Arg705 710 715 720Asp Leu Ala Asn
Tyr Ile Glu Ala Glu Arg Asn Ser Gly Ala Lys Arg 725 730 735Pro Thr
Phe Thr Ser Val His Gly Gly Gly Ser Glu Ile Arg Ala Ala 740 745
750Asp Leu Thr Leu Asp Lys Phe Ile Asp Ala Arg Thr Leu Ala Ala Ala
755 760 765Asp Ser Ile Pro His Ala Pro Val Pro Ala Gln Thr Val Leu
Leu Thr 770 775 780Gly Ala Asn Gly Tyr Leu Gly Arg Phe Leu Cys Leu
Glu Trp Leu Glu785 790 795 800Arg Leu Asp Lys Thr Gly Gly Thr Leu
Ile Cys Val Val Arg Gly Ser 805 810 815Asp Ala Ala Ala Ala Arg Lys
Arg Leu Asp Ser Ala Phe Asp Ser Gly 820 825 830Asp Pro Gly Leu Leu
Glu His Tyr Gln Gln Leu Ala Ala Arg Thr Leu 835 840 845Glu Val Leu
Ala Gly Asp Ile Gly Asp Pro Asn Leu Gly Leu Asp Asp 850 855 860Ala
Thr Trp Gln Arg Leu Ala Glu Thr Val Asp Leu Ile Val His Pro865 870
875 880Ala Ala Leu Val Asn His Val Leu Pro Tyr Thr Gln Leu Phe Gly
Pro 885 890 895Asn Val Val Gly Thr Ala Glu Ile Val Arg Leu Ala Ile
Thr Ala Arg 900 905 910Arg Lys Pro Val Thr Tyr Leu Ser Thr Val Gly
Val Ala Asp Gln Val 915 920 925Asp Pro Ala Glu Tyr Gln Glu Asp Ser
Asp Val Arg Glu Met Ser Ala 930 935 940Val Arg Val Val Arg Glu Ser
Tyr Ala Asn Gly Tyr Gly Asn Ser Lys945 950 955 960Trp Ala Gly Glu
Val Leu Leu Arg Glu Ala His Asp Leu Cys Gly Leu 965 970 975Pro Val
Ala Val Phe Arg Ser Asp Met Ile Leu Ala His Ser Arg Tyr 980 985
990Ala Gly Gln Leu Asn Val Gln Asp Val Phe Thr Arg Leu Ile Leu Ser
995 1000 1005Leu Val Ala Thr Gly Ile Ala Pro Tyr Ser Phe Tyr Arg
Thr Asp 1010 1015 1020Ala Asp Gly Asn Arg Gln Arg Ala His Tyr Asp
Gly Leu Pro Ala 1025 1030 1035Asp Phe Thr Ala Ala Ala Ile Thr Ala
Leu Gly Ile Gln Ala Thr 1040 1045 1050Glu Gly Phe Arg Thr Tyr Asp
Val Leu Asn Pro Tyr Asp Asp Gly 1055 1060 1065Ile Ser Leu Asp Glu
Phe Val Asp Trp Leu Val Glu Ser Gly His 1070 1075 1080Pro Ile Gln
Arg Ile Thr Asp Tyr Ser Asp Trp Phe His Arg Phe 1085 1090 1095Glu
Thr Ala Ile Arg Ala Leu Pro Glu Lys Gln Arg Gln Ala Ser 1100 1105
1110Val Leu Pro Leu Leu Asp Ala Tyr Arg Asn Pro Cys Pro Ala Val
1115 1120 1125Arg Gly Ala Ile Leu Pro Ala Lys Glu Phe Gln Ala Ala
Val Gln 1130 1135 1140Thr Ala Lys Ile Gly Pro Glu Gln Asp Ile Pro
His Leu Ser Ala 1145 1150 1155Pro Leu Ile Asp Lys Tyr Val Ser Asp
Leu Glu Leu Leu Gln Leu 1160 1165 1170Leu3669DNAArtificial
SequenceDescription of Artificial Sequence Synthetic codon
optimized phosphpantetheine transferase polynucleotide 3atgattgaaa
ccattctgcc tgcaggcgtt gaaagcgcag aactgctgga atatccggaa 60gatctgaaag
cacatccggc agaagaacat ctgattgcca aaagcgttga aaaacgtcgt
120cgtgatttta ttggtgcacg tcattgtgca cgtctggcac tggcagaact
gggtgaacct 180ccggttgcaa ttggtaaagg tgaacgtggt gcaccgattt
ggcctcgtgg tgttgttggt 240agcctgaccc attgtgatgg ttatcgtgca
gcagcagttg cacataaaat gcgctttcgc 300agcattggta ttgatgcaga
accgcatgca accctgccgg aaggtgttct ggatagcgtt 360agcctgccgc
cggaacgtga atggctgaaa accaccgata gcgcactgca tctggatcgt
420ctgctgtttt gtgcaaaaga agccacctat aaagcctggt ggccgctgac
agcacgttgg 480ctgggttttg aagaagccca tattaccttt gaaattgaag
atggtagcgc agatagcggt 540aatggcacct ttcatagcga actgctggtt
ccgggtcaga ccaatgatgg tggtacaccg 600ctgctgagct ttgatggtcg
ttggctgatt gcagatggtt ttattctgac cgcaattgcc 660tatgcctaa
6694222PRTArtificial SequenceDescription of Artificial Sequence
Synthetic codon optimized phosphpantetheine transferase polypeptide
4Met Ile Glu Thr Ile Leu Pro Ala Gly Val Glu Ser Ala Glu Leu Leu1 5
10 15Glu Tyr Pro Glu Asp Leu Lys Ala His Pro Ala Glu Glu His Leu
Ile 20 25 30Ala Lys Ser Val Glu Lys Arg Arg Arg Asp Phe Ile Gly Ala
Arg His 35 40 45Cys Ala Arg Leu Ala Leu Ala Glu Leu Gly Glu Pro Pro
Val Ala Ile 50 55 60Gly Lys Gly Glu Arg Gly Ala Pro Ile Trp Pro Arg
Gly Val Val Gly65 70 75 80Ser Leu Thr His Cys Asp Gly Tyr Arg Ala
Ala Ala Val Ala His Lys 85 90 95Met Arg Phe Arg Ser Ile Gly Ile Asp
Ala Glu Pro His Ala Thr Leu 100 105 110Pro Glu Gly Val Leu Asp Ser
Val Ser Leu Pro Pro Glu Arg Glu Trp 115 120 125Leu Lys Thr Thr Asp
Ser Ala Leu His Leu Asp Arg Leu Leu Phe Cys 130 135 140Ala Lys Glu
Ala Thr Tyr Lys Ala Trp Trp Pro Leu Thr Ala Arg Trp145 150 155
160Leu Gly Phe Glu Glu Ala His Ile Thr Phe Glu Ile Glu Asp Gly Ser
165 170 175Ala Asp Ser Gly Asn Gly Thr Phe His Ser Glu Leu Leu Val
Pro Gly 180 185 190Gln Thr Asn Asp Gly Gly Thr Pro Leu Leu Ser Phe
Asp Gly Arg Trp 195 200 205Leu Ile Ala Asp Gly Phe Ile Leu Thr Ala
Ile Ala Tyr Ala 210 215 22053522DNAMycobacterium smegmatis
5atgaccagcg atgttcacga cgccacagac ggcgtcaccg aaaccgcact cgacgacgag
60cagtcgaccc gccgcatcgc cgagctgtac gccaccgatc ccgagttcgc cgccgccgca
120ccgttgcccg ccgtggtcga cgcggcgcac aaacccgggc tgcggctggc
agagatcctg 180cagaccctgt tcaccggcta cggtgaccgc ccggcgctgg
gataccgcgc ccgtgaactg 240gccaccgacg agggcgggcg caccgtgacg
cgtctgctgc cgcggttcga caccctcacc 300tacgcccagg tgtggtcgcg
cgtgcaagcg gtcgccgcgg ccctgcgcca caacttcgcg 360cagccgatct
accccggcga cgccgtcgcg acgatcggtt tcgcgagtcc cgattacctg
420acgctggatc tcgtatgcgc ctacctgggc ctcgtgagtg ttccgctgca
gcacaacgca 480ccggtcagcc ggctcgcccc gatcctggcc gaggtcgaac
cgcggatcct caccgtgagc 540gccgaatacc tcgacctcgc agtcgaatcc
gtgcgggacg tcaactcggt gtcgcagctc 600gtggtgttcg accatcaccc
cgaggtcgac gaccaccgcg acgcactggc ccgcgcgcgt 660gaacaactcg
ccggcaaggg catcgccgtc accaccctgg acgcgatcgc cgacgagggc
720gccgggctgc cggccgaacc gatctacacc gccgaccatg atcagcgcct
cgcgatgatc 780ctgtacacct cgggttccac cggcgcaccc aagggtgcga
tgtacaccga ggcgatggtg 840gcgcggctgt ggaccatgtc gttcatcacg
ggtgacccca cgccggtcat caacgtcaac 900ttcatgccgc tcaaccacct
gggcgggcgc atccccattt ccaccgccgt gcagaacggt 960ggaaccagtt
acttcgtacc ggaatccgac atgtccacgc tgttcgagga tctcgcgctg
1020gtgcgcccga ccgaactcgg cctggttccg cgcgtcgccg acatgctcta
ccagcaccac 1080ctcgccaccg tcgaccgcct ggtcacgcag ggcgccgacg
aactgaccgc cgagaagcag 1140gccggtgccg aactgcgtga gcaggtgctc
ggcggacgcg tgatcaccgg attcgtcagc 1200accgcaccgc tggccgcgga
gatgagggcg ttcctcgaca tcaccctggg cgcacacatc 1260gtcgacggct
acgggctcac cgagaccggc gccgtgacac gcgacggtgt gatcgtgcgg
1320ccaccggtga tcgactacaa gctgatcgac gttcccgaac tcggctactt
cagcaccgac 1380aagccctacc cgcgtggcga actgctggtc aggtcgcaaa
cgctgactcc cgggtactac 1440aagcgccccg aggtcaccgc gagcgtcttc
gaccgggacg gctactacca caccggcgac 1500gtcatggccg agaccgcacc
cgaccacctg gtgtacgtgg accgtcgcaa caacgtcctc 1560aaactcgcgc
agggcgagtt cgtggcggtc gccaacctgg aggcggtgtt ctccggcgcg
1620gcgctggtgc gccagatctt cgtgtacggc aacagcgagc gcagtttcct
tctggccgtg 1680gtggtcccga cgccggaggc gctcgagcag tacgatccgg
ccgcgctcaa ggccgcgctg 1740gccgactcgc tgcagcgcac cgcacgcgac
gccgaactgc aatcctacga ggtgccggcc 1800gatttcatcg tcgagaccga
gccgttcagc gccgccaacg ggctgctgtc gggtgtcgga 1860aaactgctgc
ggcccaacct caaagaccgc tacgggcagc gcctggagca gatgtacgcc
1920gatatcgcgg ccacgcaggc caaccagttg cgcgaactgc ggcgcgcggc
cgccacacaa 1980ccggtgatcg acaccctcac ccaggccgct gccacgatcc
tcggcaccgg gagcgaggtg 2040gcatccgacg cccacttcac cgacctgggc
ggggattccc tgtcggcgct gacactttcg 2100aacctgctga gcgatttctt
cggtttcgaa gttcccgtcg gcaccatcgt gaacccggcc 2160accaacctcg
cccaactcgc ccagcacatc gaggcgcagc gcaccgcggg tgaccgcagg
2220ccgagtttca ccaccgtgca cggcgcggac gccaccgaga tccgggcgag
tgagctgacc 2280ctggacaagt tcatcgacgc cgaaacgctc cgggccgcac
cgggtctgcc caaggtcacc 2340accgagccac ggacggtgtt gctctcgggc
gccaacggct ggctgggccg gttcctcacg 2400ttgcagtggc tggaacgcct
ggcacctgtc ggcggcaccc tcatcacgat cgtgcggggc 2460cgcgacgacg
ccgcggcccg cgcacggctg acccaggcct acgacaccga tcccgagttg
2520tcccgccgct tcgccgagct ggccgaccgc cacctgcggg tggtcgccgg
tgacatcggc 2580gacccgaatc tgggcctcac acccgagatc tggcaccggc
tcgccgccga ggtcgacctg 2640gtggtgcatc cggcagcgct ggtcaaccac
gtgctcccct accggcagct gttcggcccc 2700aacgtcgtgg gcacggccga
ggtgatcaag ctggccctca ccgaacggat caagcccgtc 2760acgtacctgt
ccaccgtgtc ggtggccatg gggatccccg acttcgagga ggacggcgac
2820atccggaccg tgagcccggt gcgcccgctc gacggcggat acgccaacgg
ctacggcaac 2880agcaagtggg ccggcgaggt gctgctgcgg gaggcccacg
atctgtgcgg gctgcccgtg 2940gcgacgttcc gctcggacat gatcctggcg
catccgcgct accgcggtca ggtcaacgtg 3000ccagacatgt tcacgcgact
cctgttgagc ctcttgatca ccggcgtcgc gccgcggtcg 3060ttctacatcg
gagacggtga gcgcccgcgg gcgcactacc ccggcctgac ggtcgatttc
3120gtggccgagg cggtcacgac gctcggcgcg cagcagcgcg agggatacgt
gtcctacgac 3180gtgatgaacc cgcacgacga cgggatctcc ctggatgtgt
tcgtggactg gctgatccgg 3240gcgggccatc cgatcgaccg ggtcgacgac
tacgacgact gggtgcgtcg gttcgagacc 3300gcgttgaccg cgcttcccga
gaagcgccgc gcacagaccg tactgccgct gctgcacgcg 3360ttccgcgctc
cgcaggcacc gttgcgcggc gcacccgaac ccacggaggt gttccacgcc
3420gcggtgcgca ccgcgaaggt gggcccggga gacatcccgc acctcgacga
ggcgctgatc 3480gacaagtaca tacgcgatct gcgtgagttc ggtctgatct aa
352261173PRTMycobacterium smegmatis 6Met Thr Ser Asp Val His Asp
Ala Thr Asp Gly Val Thr Glu Thr Ala1 5 10 15Leu Asp Asp Glu Gln Ser
Thr Arg Arg Ile Ala Glu Leu Tyr Ala Thr 20 25 30Asp Pro Glu Phe Ala
Ala Ala Ala Pro Leu Pro Ala Val Val Asp Ala 35 40 45Ala His Lys Pro
Gly Leu Arg Leu Ala Glu Ile Leu Gln Thr Leu Phe 50 55 60Thr Gly Tyr
Gly Asp Arg Pro Ala Leu Gly Tyr Arg Ala Arg Glu Leu65 70 75 80Ala
Thr Asp Glu Gly Gly Arg Thr Val Thr Arg Leu Leu Pro Arg Phe 85 90
95Asp Thr Leu Thr Tyr Ala Gln Val Trp Ser Arg Val Gln Ala Val Ala
100 105 110Ala Ala Leu Arg His Asn Phe Ala Gln Pro Ile Tyr Pro Gly
Asp Ala 115 120 125Val Ala Thr Ile Gly Phe Ala Ser Pro Asp Tyr Leu
Thr Leu Asp Leu 130 135 140Val Cys Ala Tyr Leu Gly Leu Val Ser Val
Pro Leu Gln His Asn Ala145 150 155 160Pro Val Ser Arg Leu Ala Pro
Ile Leu Ala Glu Val Glu Pro Arg Ile 165 170 175Leu Thr Val Ser Ala
Glu Tyr Leu Asp Leu Ala Val Glu Ser Val Arg 180 185 190Asp Val Asn
Ser Val Ser Gln Leu Val Val Phe Asp His His Pro Glu 195 200 205Val
Asp Asp His Arg Asp Ala Leu Ala Arg Ala Arg Glu Gln Leu Ala 210 215
220Gly Lys Gly Ile Ala Val Thr Thr Leu Asp Ala Ile Ala Asp Glu
Gly225 230 235 240Ala Gly Leu Pro Ala Glu Pro Ile Tyr Thr Ala Asp
His Asp Gln Arg 245 250 255Leu Ala Met Ile Leu Tyr Thr Ser Gly Ser
Thr Gly Ala Pro Lys Gly 260 265 270Ala Met Tyr Thr Glu Ala Met Val
Ala Arg Leu Trp Thr Met Ser Phe 275 280 285Ile Thr Gly Asp Pro Thr
Pro Val Ile Asn Val Asn Phe Met Pro Leu 290 295 300Asn His Leu Gly
Gly Arg Ile Pro Ile Ser Thr Ala Val Gln Asn Gly305 310 315 320Gly
Thr Ser Tyr Phe Val Pro Glu Ser Asp Met Ser Thr Leu Phe Glu 325 330
335Asp Leu Ala Leu Val Arg Pro Thr Glu Leu Gly Leu Val Pro Arg Val
340 345 350Ala Asp Met Leu Tyr Gln His His Leu Ala Thr Val Asp Arg
Leu Val 355 360 365Thr Gln Gly Ala Asp Glu Leu Thr Ala Glu Lys Gln
Ala Gly Ala Glu 370 375 380Leu Arg Glu Gln Val Leu Gly Gly Arg Val
Ile Thr Gly Phe Val Ser385 390 395 400Thr Ala Pro Leu Ala Ala Glu
Met Arg Ala Phe Leu Asp Ile Thr Leu 405 410 415Gly Ala His Ile Val
Asp Gly Tyr Gly Leu Thr Glu Thr Gly Ala Val 420 425 430Thr Arg Asp
Gly Val Ile Val Arg Pro Pro Val Ile Asp Tyr Lys Leu 435 440 445Ile
Asp Val Pro Glu Leu Gly Tyr Phe Ser Thr Asp Lys Pro Tyr Pro 450 455
460Arg Gly Glu Leu Leu Val Arg Ser Gln Thr Leu Thr Pro Gly Tyr
Tyr465 470 475 480Lys Arg Pro Glu Val Thr Ala Ser Val Phe Asp Arg
Asp Gly Tyr Tyr 485 490 495His Thr Gly Asp Val Met Ala Glu Thr Ala
Pro Asp His Leu Val Tyr 500 505 510Val Asp Arg Arg Asn Asn Val Leu
Lys Leu Ala Gln Gly Glu Phe Val 515 520 525Ala Val Ala Asn Leu Glu
Ala Val Phe Ser Gly Ala Ala Leu Val Arg 530 535 540Gln Ile Phe Val
Tyr Gly Asn Ser Glu Arg Ser Phe Leu Leu Ala Val545 550 555 560Val
Val Pro Thr Pro Glu Ala Leu Glu Gln Tyr Asp Pro Ala Ala Leu 565 570
575Lys Ala Ala Leu Ala Asp Ser Leu Gln Arg Thr Ala Arg Asp Ala Glu
580 585 590Leu Gln Ser Tyr Glu Val Pro Ala Asp Phe Ile Val Glu Thr
Glu Pro 595 600 605Phe Ser Ala Ala Asn Gly Leu Leu Ser Gly Val Gly
Lys Leu Leu Arg 610 615 620Pro Asn Leu Lys Asp Arg Tyr Gly Gln Arg
Leu Glu Gln Met Tyr Ala625 630 635 640Asp Ile Ala Ala Thr Gln Ala
Asn Gln Leu Arg Glu Leu Arg Arg Ala 645 650 655Ala Ala Thr Gln Pro
Val Ile Asp Thr Leu Thr Gln Ala Ala Ala Thr 660 665 670Ile Leu Gly
Thr Gly Ser Glu Val Ala Ser Asp Ala His Phe Thr Asp 675 680 685Leu
Gly Gly Asp Ser Leu Ser Ala Leu Thr Leu Ser Asn Leu Leu Ser 690 695
700Asp Phe Phe Gly Phe Glu Val Pro Val Gly Thr Ile Val Asn Pro
Ala705 710 715 720Thr Asn Leu Ala Gln Leu Ala Gln His Ile Glu Ala
Gln Arg Thr Ala 725 730 735Gly Asp Arg Arg Pro Ser Phe Thr Thr Val
His Gly Ala Asp Ala Thr 740 745 750Glu Ile Arg Ala Ser Glu Leu Thr
Leu Asp Lys Phe Ile Asp Ala Glu 755 760 765Thr Leu Arg Ala Ala Pro
Gly Leu Pro Lys Val Thr Thr Glu Pro Arg 770 775 780Thr Val Leu Leu
Ser Gly Ala Asn Gly Trp Leu Gly Arg Phe Leu Thr785 790 795 800Leu
Gln Trp Leu Glu Arg Leu Ala Pro Val Gly Gly Thr Leu Ile Thr 805 810
815Ile Val Arg Gly Arg Asp Asp Ala Ala Ala Arg Ala Arg Leu Thr Gln
820 825 830Ala Tyr Asp Thr Asp Pro Glu Leu Ser Arg Arg Phe Ala Glu
Leu Ala 835 840 845Asp Arg His Leu Arg Val Val Ala Gly Asp Ile Gly
Asp Pro Asn Leu 850 855 860Gly Leu Thr Pro Glu Ile Trp His Arg Leu
Ala Ala Glu Val Asp Leu865 870 875 880Val Val His Pro Ala Ala Leu
Val Asn His Val Leu Pro Tyr Arg Gln 885 890 895Leu Phe Gly Pro Asn
Val Val Gly Thr Ala Glu Val Ile Lys Leu Ala 900 905 910Leu Thr Glu
Arg Ile Lys Pro Val Thr Tyr Leu Ser Thr Val Ser Val 915 920 925Ala
Met Gly Ile Pro Asp Phe Glu Glu Asp Gly Asp Ile Arg Thr Val 930 935
940Ser Pro Val Arg Pro Leu Asp Gly Gly Tyr Ala Asn Gly Tyr Gly
Asn945 950 955 960Ser Lys Trp Ala Gly Glu Val Leu Leu Arg Glu Ala
His Asp Leu Cys 965 970 975Gly Leu Pro Val Ala Thr Phe Arg Ser Asp
Met Ile Leu Ala His Pro 980 985 990Arg Tyr Arg Gly Gln Val Asn Val
Pro Asp Met Phe Thr Arg Leu Leu 995 1000 1005Leu Ser Leu Leu Ile
Thr Gly Val Ala Pro Arg Ser Phe Tyr Ile 1010 1015 1020Gly Asp Gly
Glu Arg Pro Arg Ala His Tyr Pro Gly Leu Thr Val 1025 1030 1035Asp
Phe Val Ala Glu Ala Val Thr Thr Leu Gly Ala Gln Gln Arg 1040 1045
1050Glu Gly Tyr Val Ser Tyr Asp Val Met Asn Pro His Asp Asp Gly
1055 1060 1065Ile Ser Leu Asp Val Phe Val Asp Trp Leu Ile Arg Ala
Gly His 1070 1075 1080Pro Ile Asp Arg Val Asp Asp Tyr Asp Asp Trp
Val Arg Arg Phe 1085 1090 1095Glu Thr Ala Leu Thr Ala Leu Pro Glu
Lys Arg Arg Ala Gln Thr 1100 1105 1110Val Leu Pro Leu Leu His Ala
Phe Arg Ala Pro Gln Ala Pro Leu 1115 1120 1125Arg Gly Ala Pro Glu
Pro Thr Glu Val Phe His Ala Ala Val Arg 1130 1135 1140Thr Ala Lys
Val Gly Pro Gly Asp Ile Pro His Leu Asp Glu Ala 1145 1150 1155Leu
Ile Asp Lys Tyr Ile Arg Asp Leu Arg Glu Phe Gly Leu Ile 1160 1165
117073522DNAMycobacterium avium 7atgtcgactg ccacccatga cgaacgactc
gaccgtcgcg tccacgaact catcgccacc 60gacccgcaat tcgccgccgc ccaacccgac
ccggcgatca ccgccgccct cgaacagccc 120gggctgcggc tgccgcagat
catccgcacc gtgctcgacg gctacgccga ccggccggcg 180ctgggacagc
gcgtggtgga gttcgtcacg gacgccaaga ccgggcgcac gtcggcgcag
240ctgctccccc gcttcgagac catcacgtac agcgaagtag cgcagcgtgt
ttcggcgctg 300ggccgcgccc tgtccgacga cgcggtgcac cccggcgacc
gggtgtgcgt gctgggcttc 360aacagcgtcg actacgccac catcgacatg
gcgctgggcg ccatcggcgc cgtctcggtg 420ccgctgcaga ccagcgcggc
aatcagctcg ctgcagccga tcgtggccga gaccgagccc 480accctgatcg
cgtccagcgt gaaccagctg tccgacgcgg tgcagctgat caccggcgcc
540gagcaggcgc ccacccggct ggtggtgttc gactaccacc cgcaggtcga
cgaccagcgc 600gaggccgtcc aggacgccgc ggcgcggctg tccagcaccg
gcgtggccgt ccagacgctg 660gccgagctgc tggagcgcgg caaggacctg
cccgccgtcg cggagccgcc cgccgacgag 720gactcgctgg ccctgctgat
ctacacctcc gggtccaccg gcgcccccaa gggcgcgatg 780tacccacaga
gcaacgtcgg caagatgtgg cgccgcggca gcaagaactg gttcggcgag
840agcgccgcgt cgatcaccct gaacttcatg ccgatgagcc acgtgatggg
ccgaagcatc 900ctctacggca cgctgggcaa cggcggcacc gcctacttcg
ccgcccgcag cgacctgtcc 960accctgcttg aggacctcga gctggtgcgg
cccaccgagc tcaacttcgt cccgcggatc 1020tgggagacgc tgtacggcga
attccagcgt caggtcgagc ggcggctctc cgaggccggg 1080gacgccggcg
aacgtcgcgc cgtcgaggcc gaggtgctgg ccgagcagcg ccagtacctg
1140ctgggcgggc ggttcacctt cgcgatgacg ggctcggcgc ccatctcgcc
ggagctgcgc 1200aactgggtcg agtcgctgct cgaaatgcac ctgatggacg
gctacggctc caccgaggcc 1260ggaatggtgt tgttcgacgg ggagattcag
cgcccgccgg tgatcgacta caagctggtc 1320gacgtgccgg acctgggcta
cttcagcacc gaccggccgc atccgcgcgg cgagctgctg 1380ctgcgcaccg
agaacatgtt cccgggctac tacaagcggg ccgaaaccac cgcgggcgtc
1440ttcgacgagg acggctacta ccgcaccggc gacgtgttcg ccgagatcgc
cccggaccgg 1500ctggtctacg tcgaccgccg caacaacgtg ctcaagctgg
cgcagggcga attcgtcacg 1560ctggccaagc tggaggcggt gttcggcaac
agcccgctga tccgccagat ctacgtctac 1620ggcaacagcg cccagcccta
cctgctggcg gtcgtggtgc ccaccgagga ggcgctggcc 1680tcgggtgacc
ccgagacgct caagcccaag atcgccgact cgctgcagca ggtcgccaag
1740gaggccggcc tgcagtccta cgaggtgccg cgcgacttca tcatcgagac
caccccgttc 1800agcctggaaa acggtctgct gaccgggatc cggaagctgg
cgtggccgaa actgaagcag 1860cactacgggg aacggctgga gcagatgtac
gccgacctgg ccgccggaca ggccaacgag 1920ctggccgagc tgcgccgcaa
cggtgcccag gcgccggtgt tgcagaccgt gagccgcgcc 1980gcgggcgcca
tgctgggttc ggccgcctcc gacctgtccc ccgacgccca cttcaccgat
2040ctgggcggag actcgttgtc ggcgttgaca ttcggcaacc tgctgcgcga
gatcttcgac 2100gtcgacgtgc cggtaggcgt gatcgtcagc ccggccaacg
acctggcggc catcgcgagc 2160tacatcgagg ccgagcggca gggcagcaag
cgcccgacgt tcgcctcggt gcacggccgg 2220gacgcgaccg tggtgcgcgc
cgccgacctg acgctggaca agttcctcga cgccgagacg 2280ctggccgccg
cgccgaacct gcccaagccg gccaccgagg tgcgcaccgt gctgctgacc
2340ggcgccaccg gcttcctggg ccgctacctg gccctggaat ggctggagcg
gatggacatg 2400gtggacggca aggtcatcgc cctggtccgg gcccgctccg
acgaggaggc acgcgcccgg 2460ctggacaaga ccttcgacag cggcgacccg
aaactgctcg cgcactacca gcagctggcc 2520gccgatcacc tggaggtcat
cgccggcgac aagggcgagg ccaatctggg cctgggccaa 2580gacgtttggc
aacgactggc cgacacggtc gacgtgatcg tcgaccccgc cgcgctggtc
2640aaccacgtgt tgccgtacag cgagctgttc gggcccaacg ccctgggcac
cgcggagctg 2700atccggctgg cgctgacgtc caagcagaag ccgtacacct
acgtgtccac catcggcgtg 2760ggcgaccaga tcgagccggg caagttcgtc
gagaacgccg acatccggca gatgagcgcc 2820acccgggcga tcaacgacag
ctacgccaac ggctatggca acagcaagtg ggccggcgag 2880gtgctgctgc
gcgaggcgca cgacctgtgc gggctgcccg tcgcggtgtt ccgctgcgac
2940atgatcctgg ccgacaccac gtatgccggg cagctcaacc tgccggacat
gttcacccgg 3000ctgatgctga gcctggtggc caccgggatc gcgcccggct
cgttctacga gctcgacgcc 3060gacggcaacc ggcagcgggc gcactacgac
ggcctgccgg tcgagttcat cgccgcggcg 3120atctcgacgc tgggttcgca
gatcaccgac agcgacaccg gcttccagac ctaccacgtg 3180atgaacccct
acgatgacgg cgtcggtctg gacgagtacg tcgattggct ggtggacgcc
3240ggctattcga tcgagcggat cgccgactac tccgaatggc tgcggcggtt
cgagacctcg 3300ctgcgggccc tgccggaccg gcagcgccag tactcgctgc
tgccgctgct gcacaactac 3360cgcacgccgg agaagccgat caacgggtcg
atagctccca ccgacgtgtt ccgggcagcg 3420gtgcaggagg cgaaaatcgg
ccccgacaaa gacattccgc acgtgtcgcc gccggtcatc 3480gtcaagtaca
tcaccgacct gcagctgctc gggctgctct aa 352281173PRTMycobacterium avium
8Met Ser Thr Ala Thr His Asp Glu Arg Leu Asp Arg Arg Val His Glu1 5
10 15Leu Ile Ala Thr Asp Pro Gln Phe Ala Ala Ala Gln Pro Asp Pro
Ala 20 25 30Ile Thr Ala Ala Leu Glu Gln Pro Gly Leu Arg Leu Pro Gln
Ile Ile 35 40 45Arg Thr Val Leu Asp Gly Tyr Ala Asp Arg Pro Ala Leu
Gly Gln Arg 50 55 60Val Val Glu Phe Val Thr Asp Ala Lys Thr Gly Arg
Thr Ser Ala Gln65 70 75 80Leu Leu Pro Arg Phe Glu Thr Ile Thr Tyr
Ser Glu Val Ala Gln Arg 85 90 95Val Ser Ala Leu Gly Arg Ala Leu Ser
Asp Asp Ala Val His Pro Gly 100 105 110Asp Arg Val Cys Val Leu Gly
Phe Asn Ser Val Asp Tyr Ala Thr Ile 115 120 125Asp Met Ala Leu Gly
Ala Ile Gly Ala Val Ser Val Pro Leu Gln Thr 130 135 140Ser Ala Ala
Ile Ser Ser Leu Gln Pro Ile Val Ala Glu Thr Glu Pro145 150 155
160Thr Leu Ile Ala Ser Ser Val Asn Gln Leu Ser Asp Ala Val Gln Leu
165 170 175Ile Thr Gly Ala Glu Gln Ala Pro Thr Arg Leu Val Val Phe
Asp Tyr 180 185 190His Pro Gln Val Asp Asp Gln Arg Glu Ala Val Gln
Asp Ala Ala Ala 195 200 205Arg Leu Ser Ser Thr Gly Val Ala Val Gln
Thr Leu Ala Glu Leu Leu 210 215 220Glu Arg Gly Lys Asp Leu Pro Ala
Val Ala Glu Pro Pro Ala Asp Glu225 230 235 240Asp Ser Leu Ala Leu
Leu Ile Tyr Thr Ser Gly Ser Thr Gly Ala Pro 245 250 255Lys Gly Ala
Met Tyr Pro Gln Ser Asn Val Gly Lys Met Trp Arg Arg 260 265 270Gly
Ser Lys Asn Trp Phe Gly Glu Ser Ala Ala Ser Ile Thr Leu Asn 275 280
285Phe Met Pro Met Ser His Val Met Gly Arg Ser Ile Leu Tyr Gly Thr
290 295 300Leu Gly Asn Gly Gly Thr Ala Tyr Phe Ala Ala Arg Ser Asp
Leu Ser305 310 315 320Thr Leu Leu Glu Asp Leu Glu Leu Val Arg Pro
Thr Glu Leu Asn Phe 325 330 335Val Pro Arg Ile Trp Glu Thr Leu Tyr
Gly Glu Phe Gln Arg Gln Val 340 345 350Glu Arg Arg Leu Ser Glu Ala
Gly Asp Ala Gly Glu Arg Arg Ala Val 355 360 365Glu Ala Glu Val Leu
Ala Glu Gln Arg Gln Tyr Leu Leu Gly Gly Arg 370 375 380Phe Thr Phe
Ala Met Thr Gly Ser Ala Pro Ile Ser Pro Glu Leu Arg385 390 395
400Asn Trp Val Glu Ser Leu Leu Glu Met His Leu Met Asp Gly Tyr Gly
405 410 415Ser Thr Glu Ala Gly Met Val Leu Phe Asp Gly Glu Ile Gln
Arg Pro 420 425 430Pro Val Ile Asp Tyr Lys Leu Val Asp Val Pro Asp
Leu Gly Tyr Phe 435
440 445Ser Thr Asp Arg Pro His Pro Arg Gly Glu Leu Leu Leu Arg Thr
Glu 450 455 460Asn Met Phe Pro Gly Tyr Tyr Lys Arg Ala Glu Thr Thr
Ala Gly Val465 470 475 480Phe Asp Glu Asp Gly Tyr Tyr Arg Thr Gly
Asp Val Phe Ala Glu Ile 485 490 495Ala Pro Asp Arg Leu Val Tyr Val
Asp Arg Arg Asn Asn Val Leu Lys 500 505 510Leu Ala Gln Gly Glu Phe
Val Thr Leu Ala Lys Leu Glu Ala Val Phe 515 520 525Gly Asn Ser Pro
Leu Ile Arg Gln Ile Tyr Val Tyr Gly Asn Ser Ala 530 535 540Gln Pro
Tyr Leu Leu Ala Val Val Val Pro Thr Glu Glu Ala Leu Ala545 550 555
560Ser Gly Asp Pro Glu Thr Leu Lys Pro Lys Ile Ala Asp Ser Leu Gln
565 570 575Gln Val Ala Lys Glu Ala Gly Leu Gln Ser Tyr Glu Val Pro
Arg Asp 580 585 590Phe Ile Ile Glu Thr Thr Pro Phe Ser Leu Glu Asn
Gly Leu Leu Thr 595 600 605Gly Ile Arg Lys Leu Ala Trp Pro Lys Leu
Lys Gln His Tyr Gly Glu 610 615 620Arg Leu Glu Gln Met Tyr Ala Asp
Leu Ala Ala Gly Gln Ala Asn Glu625 630 635 640Leu Ala Glu Leu Arg
Arg Asn Gly Ala Gln Ala Pro Val Leu Gln Thr 645 650 655Val Ser Arg
Ala Ala Gly Ala Met Leu Gly Ser Ala Ala Ser Asp Leu 660 665 670Ser
Pro Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala 675 680
685Leu Thr Phe Gly Asn Leu Leu Arg Glu Ile Phe Asp Val Asp Val Pro
690 695 700Val Gly Val Ile Val Ser Pro Ala Asn Asp Leu Ala Ala Ile
Ala Ser705 710 715 720Tyr Ile Glu Ala Glu Arg Gln Gly Ser Lys Arg
Pro Thr Phe Ala Ser 725 730 735Val His Gly Arg Asp Ala Thr Val Val
Arg Ala Ala Asp Leu Thr Leu 740 745 750Asp Lys Phe Leu Asp Ala Glu
Thr Leu Ala Ala Ala Pro Asn Leu Pro 755 760 765Lys Pro Ala Thr Glu
Val Arg Thr Val Leu Leu Thr Gly Ala Thr Gly 770 775 780Phe Leu Gly
Arg Tyr Leu Ala Leu Glu Trp Leu Glu Arg Met Asp Met785 790 795
800Val Asp Gly Lys Val Ile Ala Leu Val Arg Ala Arg Ser Asp Glu Glu
805 810 815Ala Arg Ala Arg Leu Asp Lys Thr Phe Asp Ser Gly Asp Pro
Lys Leu 820 825 830Leu Ala His Tyr Gln Gln Leu Ala Ala Asp His Leu
Glu Val Ile Ala 835 840 845Gly Asp Lys Gly Glu Ala Asn Leu Gly Leu
Gly Gln Asp Val Trp Gln 850 855 860Arg Leu Ala Asp Thr Val Asp Val
Ile Val Asp Pro Ala Ala Leu Val865 870 875 880Asn His Val Leu Pro
Tyr Ser Glu Leu Phe Gly Pro Asn Ala Leu Gly 885 890 895Thr Ala Glu
Leu Ile Arg Leu Ala Leu Thr Ser Lys Gln Lys Pro Tyr 900 905 910Thr
Tyr Val Ser Thr Ile Gly Val Gly Asp Gln Ile Glu Pro Gly Lys 915 920
925Phe Val Glu Asn Ala Asp Ile Arg Gln Met Ser Ala Thr Arg Ala Ile
930 935 940Asn Asp Ser Tyr Ala Asn Gly Tyr Gly Asn Ser Lys Trp Ala
Gly Glu945 950 955 960Val Leu Leu Arg Glu Ala His Asp Leu Cys Gly
Leu Pro Val Ala Val 965 970 975Phe Arg Cys Asp Met Ile Leu Ala Asp
Thr Thr Tyr Ala Gly Gln Leu 980 985 990Asn Leu Pro Asp Met Phe Thr
Arg Leu Met Leu Ser Leu Val Ala Thr 995 1000 1005Gly Ile Ala Pro
Gly Ser Phe Tyr Glu Leu Asp Ala Asp Gly Asn 1010 1015 1020Arg Gln
Arg Ala His Tyr Asp Gly Leu Pro Val Glu Phe Ile Ala 1025 1030
1035Ala Ala Ile Ser Thr Leu Gly Ser Gln Ile Thr Asp Ser Asp Thr
1040 1045 1050Gly Phe Gln Thr Tyr His Val Met Asn Pro Tyr Asp Asp
Gly Val 1055 1060 1065Gly Leu Asp Glu Tyr Val Asp Trp Leu Val Asp
Ala Gly Tyr Ser 1070 1075 1080Ile Glu Arg Ile Ala Asp Tyr Ser Glu
Trp Leu Arg Arg Phe Glu 1085 1090 1095Thr Ser Leu Arg Ala Leu Pro
Asp Arg Gln Arg Gln Tyr Ser Leu 1100 1105 1110Leu Pro Leu Leu His
Asn Tyr Arg Thr Pro Glu Lys Pro Ile Asn 1115 1120 1125Gly Ser Ile
Ala Pro Thr Asp Val Phe Arg Ala Ala Val Gln Glu 1130 1135 1140Ala
Lys Ile Gly Pro Asp Lys Asp Ile Pro His Val Ser Pro Pro 1145 1150
1155Val Ile Val Lys Tyr Ile Thr Asp Leu Gln Leu Leu Gly Leu Leu
1160 1165 117093525DNAMycobacterium marinum 9atgtcgccaa tcacgcgtga
agagcggctc gagcgccgca tccaggacct ctacgccaac 60gacccgcagt tcgccgccgc
caaacccgcc acggcgatca ccgcagcaat cgagcggccg 120ggtctaccgc
taccccagat catcgagacc gtcatgaccg gatacgccga tcggccggct
180ctcgctcagc gctcggtcga attcgtgacc gacgccggca ccggccacac
cacgctgcga 240ctgctccccc acttcgaaac catcagctac ggcgagcttt
gggaccgcat cagcgcactg 300gccgacgtgc tcagcaccga acagacggtg
aaaccgggcg accgggtctg cttgttgggc 360ttcaacagcg tcgactacgc
cacgatcgac atgactttgg cgcggctggg cgcggtggcc 420gtaccactgc
agaccagcgc ggcgataacc cagctgcagc cgatcgtcgc cgagacccag
480cccaccatga tcgcggccag cgtcgacgca ctcgctgacg ccaccgaatt
ggctctgtcc 540ggtcagaccg ctacccgagt cctggtgttc gaccaccacc
ggcaggttga cgcacaccgc 600gcagcggtcg aatccgcccg ggagcgcctg
gccggctcgg cggtcgtcga aaccctggcc 660gaggccatcg cgcgcggcga
cgtgccccgc ggtgcgtccg ccggctcggc gcccggcacc 720gatgtgtccg
acgactcgct cgcgctactg atctacacct cgggcagcac gggtgcgccc
780aagggcgcga tgtacccccg acgcaacgtt gcgaccttct ggcgcaagcg
cacctggttc 840gaaggcggct acgagccgtc gatcacgctg aacttcatgc
caatgagcca cgtcatgggc 900cgccaaatcc tgtacggcac gctgtgcaat
ggcggcaccg cctacttcgt ggcgaaaagc 960gatctctcca ccttgttcga
agacctggcg ctggtgcggc ccaccgagct gaccttcgtg 1020ccgcgcgtgt
gggacatggt gttcgacgag tttcagagtg aggtcgaccg ccgcctggtc
1080gacggcgccg accgggtcgc gctcgaagcc caggtcaagg ccgagatacg
caacgacgtg 1140ctcggtggac ggtataccag cgcactgacc ggctccgccc
ctatctccga cgagatgaag 1200gcgtgggtcg aggagctgct cgacatgcat
ctggtcgagg gctacggctc caccgaggcc 1260gggatgatcc tgatcgacgg
agccattcgg cgcccggcgg tactcgacta caagctggtc 1320gatgttcccg
acctgggtta cttcctgacc gaccggccac atccgcgggg cgagttgctg
1380gtcaagaccg atagtttgtt cccgggctac taccagcgag ccgaagtcac
cgccgacgtg 1440ttcgatgctg acggcttcta ccggaccggc gacatcatgg
ccgaggtcgg ccccgaacag 1500ttcgtgtacc tcgaccgccg caacaacgtg
ttgaagctgt cgcagggcga gttcgtcacc 1560gtctccaaac tcgaagcggt
gtttggcgac agcccactgg tacggcagat ctacatctac 1620ggcaacagcg
cccgtgccta cctgttggcg gtgatcgtcc ccacccagga ggcgctggac
1680gccgtgcctg tcgaggagct caaggcgcgg ctgggcgact cgctgcaaga
ggtcgcaaag 1740gccgccggcc tgcagtccta cgagatcccg cgcgacttca
tcatcgaaac aacaccatgg 1800acgctggaga acggcctgct caccggcatc
cgcaagttgg ccaggccgca gctgaaaaag 1860cattacggcg agcttctcga
gcagatctac acggacctgg cacacggcca ggccgacgaa 1920ctgcgctcgc
tgcgccaaag cggtgccgat gcgccggtgc tggtgacggt gtgccgtgcg
1980gcggccgcgc tgttgggcgg cagcgcctct gacgtccagc ccgatgcgca
cttcaccgat 2040ttgggcggcg actcgctgtc ggcgctgtcg ttcaccaacc
tgctgcacga gatcttcgac 2100atcgaagtgc cggtgggcgt catcgtcagc
cccgccaacg acttgcaggc cctggccgac 2160tacgtcgagg cggctcgcaa
acccggctcg tcacggccga ccttcgcctc ggtccacggc 2220gcctcgaatg
ggcaggtcac cgaggtgcat gccggtgacc tgtccctgga caaattcatc
2280gatgccgcaa ccctggccga agctccccgg ctgcccgccg caaacaccca
agtgcgcacc 2340gtgctgctga ccggcgccac cggcttcctc gggcgctacc
tggccctgga atggctggag 2400cggatggacc tggtcgacgg caaactgatc
tgcctggtcc gggccaagtc cgacaccgaa 2460gcacgggcgc ggctggacaa
gacgttcgac agcggcgacc ccgaactgct ggcccactac 2520cgcgcactgg
ccggcgacca cctcgaggtg ctcgccggtg acaagggcga agccgacctc
2580ggactggacc ggcagacctg gcaacgcctg gccgacacgg tcgacctgat
cgtcgacccc 2640gcggccctgg tcaaccacgt actgccatac agccagctgt
tcgggcccaa cgcgctgggc 2700accgccgagc tgctgcggct ggcgctcacc
tccaagatca agccctacag ctacacctcg 2760acaatcggtg tcgccgacca
gatcccgccg tcggcgttca ccgaggacgc cgacatccgg 2820gtcatcagcg
ccacccgcgc ggtcgacgac agctacgcca atggctactc gaacagcaag
2880tgggccggcg aggtgctgtt gcgcgaggcg catgacctgt gtggcctgcc
ggttgcggtg 2940ttccgctgcg acatgatcct ggccgacacc acatgggcgg
gacagctcaa tgtgccggac 3000atgttcaccc ggatgatcct gagcctggcg
gccaccggta tcgcgccggg ttcgttctat 3060gagcttgcgg ccgacggcgc
ccggcaacgc gcccactatg acggtctgcc cgtcgagttc 3120atcgccgagg
cgatttcgac tttgggtgcg cagagccagg atggtttcca cacgtatcac
3180gtgatgaacc cctacgacga cggcatcgga ctcgacgagt tcgtcgactg
gctcaacgag 3240tccggttgcc ccatccagcg catcgctgac tatggcgact
ggctgcagcg cttcgaaacc 3300gcactgcgcg cactgcccga tcggcagcgg
cacagctcac tgctgccgct gttgcacaac 3360tatcggcagc cggagcggcc
cgtccgcggg tcgatcgccc ctaccgatcg cttccgggca 3420gcggtgcaag
aggccaagat cggccccgac aaagacattc cgcacgtcgg cgcgccgatc
3480atcgtgaagt acgtcagcga cctgcgccta ctcggcctgc tctaa
3525101174PRTMycobacterium marinum 10Met Ser Pro Ile Thr Arg Glu
Glu Arg Leu Glu Arg Arg Ile Gln Asp1 5 10 15Leu Tyr Ala Asn Asp Pro
Gln Phe Ala Ala Ala Lys Pro Ala Thr Ala 20 25 30Ile Thr Ala Ala Ile
Glu Arg Pro Gly Leu Pro Leu Pro Gln Ile Ile 35 40 45Glu Thr Val Met
Thr Gly Tyr Ala Asp Arg Pro Ala Leu Ala Gln Arg 50 55 60Ser Val Glu
Phe Val Thr Asp Ala Gly Thr Gly His Thr Thr Leu Arg65 70 75 80Leu
Leu Pro His Phe Glu Thr Ile Ser Tyr Gly Glu Leu Trp Asp Arg 85 90
95Ile Ser Ala Leu Ala Asp Val Leu Ser Thr Glu Gln Thr Val Lys Pro
100 105 110Gly Asp Arg Val Cys Leu Leu Gly Phe Asn Ser Val Asp Tyr
Ala Thr 115 120 125Ile Asp Met Thr Leu Ala Arg Leu Gly Ala Val Ala
Val Pro Leu Gln 130 135 140Thr Ser Ala Ala Ile Thr Gln Leu Gln Pro
Ile Val Ala Glu Thr Gln145 150 155 160Pro Thr Met Ile Ala Ala Ser
Val Asp Ala Leu Ala Asp Ala Thr Glu 165 170 175Leu Ala Leu Ser Gly
Gln Thr Ala Thr Arg Val Leu Val Phe Asp His 180 185 190His Arg Gln
Val Asp Ala His Arg Ala Ala Val Glu Ser Ala Arg Glu 195 200 205Arg
Leu Ala Gly Ser Ala Val Val Glu Thr Leu Ala Glu Ala Ile Ala 210 215
220Arg Gly Asp Val Pro Arg Gly Ala Ser Ala Gly Ser Ala Pro Gly
Thr225 230 235 240Asp Val Ser Asp Asp Ser Leu Ala Leu Leu Ile Tyr
Thr Ser Gly Ser 245 250 255Thr Gly Ala Pro Lys Gly Ala Met Tyr Pro
Arg Arg Asn Val Ala Thr 260 265 270Phe Trp Arg Lys Arg Thr Trp Phe
Glu Gly Gly Tyr Glu Pro Ser Ile 275 280 285Thr Leu Asn Phe Met Pro
Met Ser His Val Met Gly Arg Gln Ile Leu 290 295 300Tyr Gly Thr Leu
Cys Asn Gly Gly Thr Ala Tyr Phe Val Ala Lys Ser305 310 315 320Asp
Leu Ser Thr Leu Phe Glu Asp Leu Ala Leu Val Arg Pro Thr Glu 325 330
335Leu Thr Phe Val Pro Arg Val Trp Asp Met Val Phe Asp Glu Phe Gln
340 345 350Ser Glu Val Asp Arg Arg Leu Val Asp Gly Ala Asp Arg Val
Ala Leu 355 360 365Glu Ala Gln Val Lys Ala Glu Ile Arg Asn Asp Val
Leu Gly Gly Arg 370 375 380Tyr Thr Ser Ala Leu Thr Gly Ser Ala Pro
Ile Ser Asp Glu Met Lys385 390 395 400Ala Trp Val Glu Glu Leu Leu
Asp Met His Leu Val Glu Gly Tyr Gly 405 410 415Ser Thr Glu Ala Gly
Met Ile Leu Ile Asp Gly Ala Ile Arg Arg Pro 420 425 430Ala Val Leu
Asp Tyr Lys Leu Val Asp Val Pro Asp Leu Gly Tyr Phe 435 440 445Leu
Thr Asp Arg Pro His Pro Arg Gly Glu Leu Leu Val Lys Thr Asp 450 455
460Ser Leu Phe Pro Gly Tyr Tyr Gln Arg Ala Glu Val Thr Ala Asp
Val465 470 475 480Phe Asp Ala Asp Gly Phe Tyr Arg Thr Gly Asp Ile
Met Ala Glu Val 485 490 495Gly Pro Glu Gln Phe Val Tyr Leu Asp Arg
Arg Asn Asn Val Leu Lys 500 505 510Leu Ser Gln Gly Glu Phe Val Thr
Val Ser Lys Leu Glu Ala Val Phe 515 520 525Gly Asp Ser Pro Leu Val
Arg Gln Ile Tyr Ile Tyr Gly Asn Ser Ala 530 535 540Arg Ala Tyr Leu
Leu Ala Val Ile Val Pro Thr Gln Glu Ala Leu Asp545 550 555 560Ala
Val Pro Val Glu Glu Leu Lys Ala Arg Leu Gly Asp Ser Leu Gln 565 570
575Glu Val Ala Lys Ala Ala Gly Leu Gln Ser Tyr Glu Ile Pro Arg Asp
580 585 590Phe Ile Ile Glu Thr Thr Pro Trp Thr Leu Glu Asn Gly Leu
Leu Thr 595 600 605Gly Ile Arg Lys Leu Ala Arg Pro Gln Leu Lys Lys
His Tyr Gly Glu 610 615 620Leu Leu Glu Gln Ile Tyr Thr Asp Leu Ala
His Gly Gln Ala Asp Glu625 630 635 640Leu Arg Ser Leu Arg Gln Ser
Gly Ala Asp Ala Pro Val Leu Val Thr 645 650 655Val Cys Arg Ala Ala
Ala Ala Leu Leu Gly Gly Ser Ala Ser Asp Val 660 665 670Gln Pro Asp
Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala 675 680 685Leu
Ser Phe Thr Asn Leu Leu His Glu Ile Phe Asp Ile Glu Val Pro 690 695
700Val Gly Val Ile Val Ser Pro Ala Asn Asp Leu Gln Ala Leu Ala
Asp705 710 715 720Tyr Val Glu Ala Ala Arg Lys Pro Gly Ser Ser Arg
Pro Thr Phe Ala 725 730 735Ser Val His Gly Ala Ser Asn Gly Gln Val
Thr Glu Val His Ala Gly 740 745 750Asp Leu Ser Leu Asp Lys Phe Ile
Asp Ala Ala Thr Leu Ala Glu Ala 755 760 765Pro Arg Leu Pro Ala Ala
Asn Thr Gln Val Arg Thr Val Leu Leu Thr 770 775 780Gly Ala Thr Gly
Phe Leu Gly Arg Tyr Leu Ala Leu Glu Trp Leu Glu785 790 795 800Arg
Met Asp Leu Val Asp Gly Lys Leu Ile Cys Leu Val Arg Ala Lys 805 810
815Ser Asp Thr Glu Ala Arg Ala Arg Leu Asp Lys Thr Phe Asp Ser Gly
820 825 830Asp Pro Glu Leu Leu Ala His Tyr Arg Ala Leu Ala Gly Asp
His Leu 835 840 845Glu Val Leu Ala Gly Asp Lys Gly Glu Ala Asp Leu
Gly Leu Asp Arg 850 855 860Gln Thr Trp Gln Arg Leu Ala Asp Thr Val
Asp Leu Ile Val Asp Pro865 870 875 880Ala Ala Leu Val Asn His Val
Leu Pro Tyr Ser Gln Leu Phe Gly Pro 885 890 895Asn Ala Leu Gly Thr
Ala Glu Leu Leu Arg Leu Ala Leu Thr Ser Lys 900 905 910Ile Lys Pro
Tyr Ser Tyr Thr Ser Thr Ile Gly Val Ala Asp Gln Ile 915 920 925Pro
Pro Ser Ala Phe Thr Glu Asp Ala Asp Ile Arg Val Ile Ser Ala 930 935
940Thr Arg Ala Val Asp Asp Ser Tyr Ala Asn Gly Tyr Ser Asn Ser
Lys945 950 955 960Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Asp
Leu Cys Gly Leu 965 970 975Pro Val Ala Val Phe Arg Cys Asp Met Ile
Leu Ala Asp Thr Thr Trp 980 985 990Ala Gly Gln Leu Asn Val Pro Asp
Met Phe Thr Arg Met Ile Leu Ser 995 1000 1005Leu Ala Ala Thr Gly
Ile Ala Pro Gly Ser Phe Tyr Glu Leu Ala 1010 1015 1020Ala Asp Gly
Ala Arg Gln Arg Ala His Tyr Asp Gly Leu Pro Val 1025 1030 1035Glu
Phe Ile Ala Glu Ala Ile Ser Thr Leu Gly Ala Gln Ser Gln 1040 1045
1050Asp Gly Phe His Thr Tyr His Val Met Asn Pro Tyr Asp Asp Gly
1055 1060 1065Ile Gly Leu Asp Glu Phe Val Asp Trp Leu Asn Glu Ser
Gly Cys 1070 1075 1080Pro Ile Gln Arg Ile Ala Asp Tyr Gly Asp Trp
Leu Gln Arg Phe 1085 1090 1095Glu Thr Ala Leu Arg Ala Leu Pro Asp
Arg Gln Arg His Ser Ser 1100 1105 1110Leu Leu Pro Leu Leu His Asn
Tyr Arg Gln Pro Glu Arg Pro Val 1115 1120 1125Arg Gly Ser Ile Ala
Pro Thr Asp Arg Phe Arg Ala Ala Val Gln 1130 1135 1140Glu Ala Lys
Ile Gly Pro Asp Lys Asp Ile
Pro His Val Gly Ala 1145 1150 1155Pro Ile Ile Val Lys Tyr Val Ser
Asp Leu Arg Leu Leu Gly Leu 1160 1165 1170Leu113522DNAArtificial
SequenceDescription of Artificial Sequence Synthetic carboxylic
acid reductase polynucleotide designated 891GA 11atgagcaccg
caacccatga tgaacgtctg gatcgtcgtg ttcatgaact gattgcaacc 60gatccgcagt
ttgcagcagc acagccggat cctgcaatta ccgcagcact ggaacagcct
120ggtctgcgtc tgccgcagat tattcgtacc gttctggatg gttatgcaga
tcgtccggca 180ctgggtcagc gtgttgttga atttgttacc gatgcaaaaa
ccggtcgtac cagcgcacag 240ctgctgcctc gttttgaaac cattacctat
agcgaagttg cacagcgtgt tagcgcactg 300ggtcgtgcac tgagtgatga
tgcagttcat ccgggtgatc gtgtttgtgt tctgggtttt 360aatagcgttg
attatgccac cattgatatg gcactgggtg caattggtgc agttagcgtt
420ccgctgcaga ccagcgcagc aattagcagc ctgcagccga ttgttgcaga
aaccgaaccg 480accctgattg caagcagcgt taatcagctg tcagatgcag
ttcagctgat taccggtgca 540gaacaggcac cgacccgtct ggttgttttt
gattatcatc cgcaggttga tgatcagcgt 600gaagcagttc aggatgcagc
agcacgtctg agcagcaccg gtgttgcagt tcagaccctg 660gcagaactgc
tggaacgtgg taaagatctg cctgcagttg cagaaccgcc tgcagatgaa
720gatagcctgg cactgctgat ttataccagc ggtagcacag gtgcaccgaa
aggtgcaatg 780tatccgcaga gcaatgttgg taaaatgtgg cgtcgtggta
gcaaaaattg gtttggtgaa 840agcgcagcaa gcattaccct gaatttcatg
ccgatgagcc atgttatggg tcgtagcatt 900ctgtatggca ccctgggtaa
tggtggcacc gcatattttg cagcacgtag cgatctgagc 960accctgctgg
aagatctgga actggttcgt ccgaccgaac tgaattttgt tccgcgtatt
1020tgggaaaccc tgtatggtga atttcagcgt caggttgaac gtcgtctgag
cgaagctggc 1080gatgccggtg aacgtcgtgc agttgaagca gaagttctgg
cagaacagcg tcagtatctg 1140ctgggtggtc gttttacctt tgcaatgacc
ggtagcgcac cgattagtcc ggaactgcgt 1200aattgggttg aaagcctgct
ggaaatgcat ctgatggatg gctatggtag caccgaagca 1260ggtatggttc
tgtttgatgg cgaaattcag cgtccgcctg tgattgatta taaactggtt
1320gatgttccgg atctgggtta ttttagcacc gatcgtccgc atccgcgtgg
tgaactgctg 1380ctgcgtaccg aaaatatgtt tccgggttat tataaacgtg
cagaaaccac cgcaggcgtt 1440tttgatgaag atggttatta tcgtaccggt
gatgtgtttg cagaaattgc accggatcgt 1500ctggtttatg ttgatcgtcg
taataatgtt ctgaaactgg cacagggtga atttgtgacc 1560ctggccaaac
tggaagcagt ttttggtaat agtccgctga ttcgtcagat ttatgtgtat
1620ggtaatagcg cacagccgta tctgctggca gttgttgttc cgaccgaaga
ggcactggca 1680agcggtgatc cggaaaccct gaaaccgaaa attgcagata
gcctgcagca ggttgcaaaa 1740gaagcaggtc tgcagagcta tgaagttccg
cgtgatttta ttattgaaac caccccgttt 1800agcctggaaa atggtctgct
gaccggtatt cgtaaactgg catggccgaa actgaaacag 1860cattatggtg
aacgcctgga acaaatgtat gcagatctgg cagcaggtca ggcaaatgaa
1920ctggccgaac tgcgtcgtaa tggtgcacag gcaccggttc tgcagaccgt
tagccgtgca 1980gccggtgcaa tgctgggtag cgcagccagc gatctgagtc
cggatgcaca ttttaccgat 2040ctgggtggtg atagcctgag cgcactgacc
tttggtaatc tgctgcgtga aatttttgat 2100gttgatgtgc cggttggtgt
tattgttagt ccggctaatg atctggcagc cattgcaagc 2160tatattgaag
cagaacgtca gggtagcaaa cgtccgacct ttgcaagcgt tcatggtcgt
2220gatgcaaccg ttgttcgtgc agcagatctg accctggata aatttctgga
tgcagaaacc 2280ctggcagcag caccgaatct gccgaaaccg gcaaccgaag
ttcgtaccgt gctgctgaca 2340ggtgcaaccg gttttctggg tcgttatctg
gcactggaat ggctggaacg tatggatatg 2400gttgatggta aagttattgc
actggttcgt gcccgtagtg atgaagaagc acgcgcacgt 2460ctggataaaa
cctttgatag tggtgatccg aaactgctgg cacattatca gcagctggct
2520gcagatcatc tggaagttat tgccggtgat aaaggtgaag caaatctggg
tctgggtcag 2580gatgtttggc agcgtctggc agataccgtt gatgttattg
tggatccggc agcactggtt 2640aatcatgttc tgccgtatag cgaactgttt
ggtccgaatg cactgggcac cgcagaactg 2700attcgtctgg cactgaccag
caaacagaaa ccgtatacct atgttagcac cattggtgtt 2760ggcgatcaga
ttgaaccggg taaatttgtt gaaaatgccg atattcgtca gatgagcgca
2820acccgtgcaa ttaatgatag ctatgcaaat ggctacggca atagcaaatg
ggcaggcgaa 2880gttctgctgc gcgaagcaca tgatctgtgt ggtctgccgg
ttgcagtttt tcgttgtgat 2940atgattctgg ccgataccac ctatgcaggt
cagctgaatc tgccggatat gtttacccgt 3000ctgatgctga gcctggttgc
aaccggtatt gcaccgggta gcttttatga actggatgca 3060gatggtaatc
gtcagcgtgc acattatgat ggcctgccgg ttgaatttat tgcagcagcc
3120attagcaccc tgggttcaca gattaccgat agcgataccg gttttcagac
ctatcatgtt 3180atgaacccgt atgatgatgg tgttggtctg gatgaatatg
ttgattggct ggttgatgcc 3240ggttatagca ttgaacgtat tgcagattat
agcgaatggc tgcgtcgctt tgaaacctca 3300ctgcgtgcac tgccggatcg
tcagcgccag tatagcctgc tgccgctgct gcacaattat 3360cgtacaccgg
aaaaaccgat taatggtagc attgcaccga ccgatgtttt tcgtgcagcc
3420gttcaagaag ccaaaattgg tccggataaa gatattccgc atgttagccc
tccggtgatt 3480gttaaatata ttaccgatct gcagctgctg ggtctgctgt aa
3522121173PRTArtificial SequenceDescription of Artificial Sequence
Synthetic carboxylic acid reductase polypeptide designated 891GA
12Met Ser Thr Ala Thr His Asp Glu Arg Leu Asp Arg Arg Val His Glu1
5 10 15Leu Ile Ala Thr Asp Pro Gln Phe Ala Ala Ala Gln Pro Asp Pro
Ala 20 25 30Ile Thr Ala Ala Leu Glu Gln Pro Gly Leu Arg Leu Pro Gln
Ile Ile 35 40 45Arg Thr Val Leu Asp Gly Tyr Ala Asp Arg Pro Ala Leu
Gly Gln Arg 50 55 60Val Val Glu Phe Val Thr Asp Ala Lys Thr Gly Arg
Thr Ser Ala Gln65 70 75 80Leu Leu Pro Arg Phe Glu Thr Ile Thr Tyr
Ser Glu Val Ala Gln Arg 85 90 95Val Ser Ala Leu Gly Arg Ala Leu Ser
Asp Asp Ala Val His Pro Gly 100 105 110Asp Arg Val Cys Val Leu Gly
Phe Asn Ser Val Asp Tyr Ala Thr Ile 115 120 125Asp Met Ala Leu Gly
Ala Ile Gly Ala Val Ser Val Pro Leu Gln Thr 130 135 140Ser Ala Ala
Ile Ser Ser Leu Gln Pro Ile Val Ala Glu Thr Glu Pro145 150 155
160Thr Leu Ile Ala Ser Ser Val Asn Gln Leu Ser Asp Ala Val Gln Leu
165 170 175Ile Thr Gly Ala Glu Gln Ala Pro Thr Arg Leu Val Val Phe
Asp Tyr 180 185 190His Pro Gln Val Asp Asp Gln Arg Glu Ala Val Gln
Asp Ala Ala Ala 195 200 205Arg Leu Ser Ser Thr Gly Val Ala Val Gln
Thr Leu Ala Glu Leu Leu 210 215 220Glu Arg Gly Lys Asp Leu Pro Ala
Val Ala Glu Pro Pro Ala Asp Glu225 230 235 240Asp Ser Leu Ala Leu
Leu Ile Tyr Thr Ser Gly Ser Thr Gly Ala Pro 245 250 255Lys Gly Ala
Met Tyr Pro Gln Ser Asn Val Gly Lys Met Trp Arg Arg 260 265 270Gly
Ser Lys Asn Trp Phe Gly Glu Ser Ala Ala Ser Ile Thr Leu Asn 275 280
285Phe Met Pro Met Ser His Val Met Gly Arg Ser Ile Leu Tyr Gly Thr
290 295 300Leu Gly Asn Gly Gly Thr Ala Tyr Phe Ala Ala Arg Ser Asp
Leu Ser305 310 315 320Thr Leu Leu Glu Asp Leu Glu Leu Val Arg Pro
Thr Glu Leu Asn Phe 325 330 335Val Pro Arg Ile Trp Glu Thr Leu Tyr
Gly Glu Phe Gln Arg Gln Val 340 345 350Glu Arg Arg Leu Ser Glu Ala
Gly Asp Ala Gly Glu Arg Arg Ala Val 355 360 365Glu Ala Glu Val Leu
Ala Glu Gln Arg Gln Tyr Leu Leu Gly Gly Arg 370 375 380Phe Thr Phe
Ala Met Thr Gly Ser Ala Pro Ile Ser Pro Glu Leu Arg385 390 395
400Asn Trp Val Glu Ser Leu Leu Glu Met His Leu Met Asp Gly Tyr Gly
405 410 415Ser Thr Glu Ala Gly Met Val Leu Phe Asp Gly Glu Ile Gln
Arg Pro 420 425 430Pro Val Ile Asp Tyr Lys Leu Val Asp Val Pro Asp
Leu Gly Tyr Phe 435 440 445Ser Thr Asp Arg Pro His Pro Arg Gly Glu
Leu Leu Leu Arg Thr Glu 450 455 460Asn Met Phe Pro Gly Tyr Tyr Lys
Arg Ala Glu Thr Thr Ala Gly Val465 470 475 480Phe Asp Glu Asp Gly
Tyr Tyr Arg Thr Gly Asp Val Phe Ala Glu Ile 485 490 495Ala Pro Asp
Arg Leu Val Tyr Val Asp Arg Arg Asn Asn Val Leu Lys 500 505 510Leu
Ala Gln Gly Glu Phe Val Thr Leu Ala Lys Leu Glu Ala Val Phe 515 520
525Gly Asn Ser Pro Leu Ile Arg Gln Ile Tyr Val Tyr Gly Asn Ser Ala
530 535 540Gln Pro Tyr Leu Leu Ala Val Val Val Pro Thr Glu Glu Ala
Leu Ala545 550 555 560Ser Gly Asp Pro Glu Thr Leu Lys Pro Lys Ile
Ala Asp Ser Leu Gln 565 570 575Gln Val Ala Lys Glu Ala Gly Leu Gln
Ser Tyr Glu Val Pro Arg Asp 580 585 590Phe Ile Ile Glu Thr Thr Pro
Phe Ser Leu Glu Asn Gly Leu Leu Thr 595 600 605Gly Ile Arg Lys Leu
Ala Trp Pro Lys Leu Lys Gln His Tyr Gly Glu 610 615 620Arg Leu Glu
Gln Met Tyr Ala Asp Leu Ala Ala Gly Gln Ala Asn Glu625 630 635
640Leu Ala Glu Leu Arg Arg Asn Gly Ala Gln Ala Pro Val Leu Gln Thr
645 650 655Val Ser Arg Ala Ala Gly Ala Met Leu Gly Ser Ala Ala Ser
Asp Leu 660 665 670Ser Pro Asp Ala His Phe Thr Asp Leu Gly Gly Asp
Ser Leu Ser Ala 675 680 685Leu Thr Phe Gly Asn Leu Leu Arg Glu Ile
Phe Asp Val Asp Val Pro 690 695 700Val Gly Val Ile Val Ser Pro Ala
Asn Asp Leu Ala Ala Ile Ala Ser705 710 715 720Tyr Ile Glu Ala Glu
Arg Gln Gly Ser Lys Arg Pro Thr Phe Ala Ser 725 730 735Val His Gly
Arg Asp Ala Thr Val Val Arg Ala Ala Asp Leu Thr Leu 740 745 750Asp
Lys Phe Leu Asp Ala Glu Thr Leu Ala Ala Ala Pro Asn Leu Pro 755 760
765Lys Pro Ala Thr Glu Val Arg Thr Val Leu Leu Thr Gly Ala Thr Gly
770 775 780Phe Leu Gly Arg Tyr Leu Ala Leu Glu Trp Leu Glu Arg Met
Asp Met785 790 795 800Val Asp Gly Lys Val Ile Ala Leu Val Arg Ala
Arg Ser Asp Glu Glu 805 810 815Ala Arg Ala Arg Leu Asp Lys Thr Phe
Asp Ser Gly Asp Pro Lys Leu 820 825 830Leu Ala His Tyr Gln Gln Leu
Ala Ala Asp His Leu Glu Val Ile Ala 835 840 845Gly Asp Lys Gly Glu
Ala Asn Leu Gly Leu Gly Gln Asp Val Trp Gln 850 855 860Arg Leu Ala
Asp Thr Val Asp Val Ile Val Asp Pro Ala Ala Leu Val865 870 875
880Asn His Val Leu Pro Tyr Ser Glu Leu Phe Gly Pro Asn Ala Leu Gly
885 890 895Thr Ala Glu Leu Ile Arg Leu Ala Leu Thr Ser Lys Gln Lys
Pro Tyr 900 905 910Thr Tyr Val Ser Thr Ile Gly Val Gly Asp Gln Ile
Glu Pro Gly Lys 915 920 925Phe Val Glu Asn Ala Asp Ile Arg Gln Met
Ser Ala Thr Arg Ala Ile 930 935 940Asn Asp Ser Tyr Ala Asn Gly Tyr
Gly Asn Ser Lys Trp Ala Gly Glu945 950 955 960Val Leu Leu Arg Glu
Ala His Asp Leu Cys Gly Leu Pro Val Ala Val 965 970 975Phe Arg Cys
Asp Met Ile Leu Ala Asp Thr Thr Tyr Ala Gly Gln Leu 980 985 990Asn
Leu Pro Asp Met Phe Thr Arg Leu Met Leu Ser Leu Val Ala Thr 995
1000 1005Gly Ile Ala Pro Gly Ser Phe Tyr Glu Leu Asp Ala Asp Gly
Asn 1010 1015 1020Arg Gln Arg Ala His Tyr Asp Gly Leu Pro Val Glu
Phe Ile Ala 1025 1030 1035Ala Ala Ile Ser Thr Leu Gly Ser Gln Ile
Thr Asp Ser Asp Thr 1040 1045 1050Gly Phe Gln Thr Tyr His Val Met
Asn Pro Tyr Asp Asp Gly Val 1055 1060 1065Gly Leu Asp Glu Tyr Val
Asp Trp Leu Val Asp Ala Gly Tyr Ser 1070 1075 1080Ile Glu Arg Ile
Ala Asp Tyr Ser Glu Trp Leu Arg Arg Phe Glu 1085 1090 1095Thr Ser
Leu Arg Ala Leu Pro Asp Arg Gln Arg Gln Tyr Ser Leu 1100 1105
1110Leu Pro Leu Leu His Asn Tyr Arg Thr Pro Glu Lys Pro Ile Asn
1115 1120 1125Gly Ser Ile Ala Pro Thr Asp Val Phe Arg Ala Ala Val
Gln Glu 1130 1135 1140Ala Lys Ile Gly Pro Asp Lys Asp Ile Pro His
Val Ser Pro Pro 1145 1150 1155Val Ile Val Lys Tyr Ile Thr Asp Leu
Gln Leu Leu Gly Leu Leu 1160 1165 1170
* * * * *