U.S. patent application number 13/528593 was filed with the patent office on 2012-12-27 for microorganisms for producing 1,3-butanediol and methods related thereto.
This patent application is currently assigned to GENOMATICA, INC.. Invention is credited to ANTHONY P. BURGARD, MARK J. BURK, ROBIN E. OSTERHOUT, PRITI PHARKYA, JUN SUN.
Application Number | 20120329113 13/528593 |
Document ID | / |
Family ID | 47362202 |
Filed Date | 2012-12-27 |
View All Diagrams
United States Patent
Application |
20120329113 |
Kind Code |
A1 |
BURGARD; ANTHONY P. ; et
al. |
December 27, 2012 |
Microorganisms for Producing 1,3-Butanediol and Methods Related
Thereto
Abstract
Provided herein is a non-naturally occurring microbial organism
having a 1,3-butanediol (1,3-BDO) pathway and comprising at least
one exogenous nucleic acid encoding a 1,3-BDO pathway enzyme
expressed in a sufficient amount to produce 1,3-BDO. In some
embodiments, the pathway includes reducing equivalents from CO or
hydrogen. In certain embodiments, a 1,3-BDO pathway proceeds by way
of central metabolites pyruvate, succinate or alpha-ketoglutarate.
Also provided herein is a method for producing 1,3-BDO, includes
culturing such microbial organisms under conditions and for a
sufficient period of time to produce 1,3-BDO.
Inventors: |
BURGARD; ANTHONY P.;
(BELLEFONTE, PA) ; BURK; MARK J.; (SAN DIEGO,
CA) ; OSTERHOUT; ROBIN E.; (SAN DIEGO, CA) ;
SUN; JUN; (SAN DIEGO, CA) ; PHARKYA; PRITI;
(SAN DIEGO, CA) |
Assignee: |
GENOMATICA, INC.
SAN DIEGO
CA
|
Family ID: |
47362202 |
Appl. No.: |
13/528593 |
Filed: |
June 20, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61500131 |
Jun 22, 2011 |
|
|
|
61502702 |
Jun 29, 2011 |
|
|
|
Current U.S.
Class: |
435/158 ;
435/252.33 |
Current CPC
Class: |
C12N 15/52 20130101;
C12P 7/18 20130101 |
Class at
Publication: |
435/158 ;
435/252.33 |
International
Class: |
C12N 1/21 20060101
C12N001/21; C12P 7/18 20060101 C12P007/18 |
Claims
1. A non-naturally occurring microbial organism having a
1,3-butanediol pathway, wherein said microbial organism comprises
at least one exogenous nucleic acid encoding a 1,3-butanediol
pathway enzyme expressed in a sufficient amount to produce
1,3-butanediol; said non-naturally occurring microbial organism
further comprising: (i) a reductive TCA pathway, wherein said
microbial organism comprises at least one exogenous nucleic acid
encoding a reductive TCA pathway enzyme selected from the group
consisting of an ATP-citrate lyase, citrate lyase, a fumarate
reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase;
(ii) a reductive TCA pathway, wherein said microbial organism
comprises at least one exogenous nucleic acid encoding a reductive
TCA pathway enzyme selected from the group consisting of a
pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate
carboxylase, a phosphoenolpyruvate carboxykinase, a CO
dehydrogenase, and an H.sub.2 hydrogenase; or (iii) at least one
exogenous nucleic acid encoding an enzyme selected from the group
consisting of a CO dehydrogenase, an H.sub.2 hydrogenase, and
combinations thereof; wherein said 1,3-butanediol pathway comprises
a pathway selected from the group consisting of: (a) (1) a
2-amino-4-ketopentanoate (AKP) thiolase; (2) an AKP dehydrogenase;
(3) a 2-amino-4-hydroxypentanoate aminotransferase or
oxidoreductase (deaminating); (4) a 2-oxo-4-hydroxypentanoate
decarboxylase; and (5) a 3-hydroxybutyraldehyde reductase; (b) (1)
an AKP thiolase; (2) an AKP aminotransferase or oxidoreductase
(deaminating); (3) a 2,4-dioxopentanoate decarboxylase; (4) a
3-oxobutyraldehyde reductase (ketone reducing); and (5) a
3-hydroxybutyraldehyde reductase; (c) (1) an AKP thiolase; (2) an
AKP aminotransferase or oxidoreductase (deaminating); (3) a
2,4-dioxopentanoate decarboxylase; (4) a 3-oxobutyraldehyde
reductase (aldehyde reducing); and (5) a 4-hydroxy-2-butanone
reductase; (d) (1) an AKP thiolase; (2) an AKP decarboxylase; (3) a
4-aminobutan-2-one aminotransferase or oxidoreductase
(deaminating); (4) a 3-oxobutyraldehyde reductase (ketone
reducing); and (5) a 3-hydroxybutyraldehyde reductase; (e) (1) an
AKP thiolase; (2) an AKP decarboxylase; (3) a 4-aminobutan-2-one
aminotransferase or oxidoreductase (deaminating); (4) a
3-oxobutyraldehyde reductase (aldehyde reducing); and (5) a
4-hydroxy-2-butanone reductase; (f) (1) an AKP thiolase; (2) an AKP
decarboxylase; (3) a 4-aminobutan-2-one ammonia-lyase; (4) a
butenone hydratase; and (5) a 4-hydroxy-2-butanone reductase; (g)
(1) an AKP thiolase; (2) an AKP ammonia-lyase; (3) an
acetylacrylate decarboxylase; (4) a butenone hydratase; and (5) a
4-hydroxy-2-butanone reductase; (h) (1) an acetoacetyl-CoA
reductase (CoA-dependent, aldehyde forming); (2) a
3-oxobutyraldehyde reductase (ketone reducing); and (3) a
3-hydroxybutyraldehyde reductase; (i) an acetoacetyl-CoA reductase
(CoA dependent, alcohol forming) and (2) a 4-hydroxy-2-butanone
reductase; (j) (1) an acetoacetyl-CoA reductase (CoA-dependent,
aldehyde forming); (2) a 3-oxobutyraldehyde reductase (aldehyde
reducing); and (3) a 4-hydroxy-2-butanone reductase; (k) (1) an
acetoacetyl-CoA reductase (ketone reducing) and (2) a
3-hydroxybutyryl-CoA reductase (alcohol forming); (l) (1) an
acetoacetyl-CoA reductase (ketone reducing); (2) a
3-hydroxybutyryl-CoA reductase (aldehyde forming); and (3) a
3-hydroxybutyraldehyde reductase; (m) (1) a 4-hydroxybutyryl-CoA
dehydratase; (2) a crotonase; and (3) a 3-hydroxybutyryl-CoA
reductase (alcohol forming); and (n) (1) a 4-hydroxybutyryl-CoA
dehydratase; (2) a crotonase; (3) a 3-hydroxybutyryl-CoA reductase
(aldehyde forming); and (4) a 3-hydroxybutyraldehyde reductase; (o)
(1) a succinyl-CoA transferase, a succinyl-CoA synthetase or a
succinyl-CoA ligase, (2) a succinyl-CoA reductase (aldehyde
forming), (3) a 4-hydroxybutyrate dehydrogenase, (4) a
4-hydroxybutyrate kinase, (5) a phosphotrans-4-hydroxybutyrylase,
(6) a 4-hydroxybutyryl-CoA dehydratase, (7) a crotonase, (8) a
3-hydroxybutyryl-CoA reductase (aldehyde forming), and (9) a
3-hydroxybutanal reductase; (p) (1) (i) an alpha-ketoglutarate
decarboxylase, or (ii) (a) a glutamate dehydrogenase and/or a
glutamate transaminase, (b) a glutamate decarboxylase, and (c) a
4-aminobutyrate dehydrogenase and/or a 4-aminobutyrate
transaminase, (2) a 4-hydroxybutyrate dehydrogenase, (3) a
4-hydroxybutyrate kinase, (4) a phosphotrans-4-hydroxybutyrylase,
(5) a 4-hydroxybutyryl-CoA dehydratase; (6) a crotonase, (7) a
3-hydroxybutyryl-CoA reductase (aldehyde forming), and (8) a
3-hydroxybutanal reductase; (q) (1) (i) an alpha-ketoglutarate
decarboxylase, or (ii) (a) a glutamate dehydrogenase and/or a
glutamate transaminase, (b) a glutamate decarboxylase, and (c) a
4-aminobutyrate dehydrogenase and/or a 4-aminobutyrate
transaminase, (2) a 4-hydroxybutyrate dehydrogenase, (3) a
4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA
synthetase, (4) a 4-hydroxybutyryl-CoA dehydratase, (5) a
crotonase, (6) a 3-hydroxybutyryl-CoA reductase (aldehyde forming),
and (7) a 3-hydroxybutanal reductase; (r) (1) (i) an
alpha-ketoglutarate decarboxylase, or (ii) (a) a glutamate
dehydrogenase and/or a glutamate transaminase, (b) a glutamate
decarboxylase, and (c) a 4-aminobutyrate dehydrogenase and/or a
4-aminobutyrate transaminase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, and (7) a 3-hydroxybutyryl-CoA
reductase (alcohol forming); (s) (1) (i) an alpha-ketoglutarate
decarboxylase, or (ii) (a) a glutamate dehydrogenase and/or a
glutamate transaminase, (b) a glutamate decarboxylase, and (c) a
4-aminobutyrate dehydrogenase and/or a 4-aminobutyrate
transaminase, (2) a 4-hydroxybutyrate dehydrogenase, (3) a
4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA
synthetase, (4) a 4-hydroxybutyryl-CoA dehydratase, (5) a
crotonase, and (6) a 3-hydroxybutyryl-CoA reductase (alcohol
forming); (t) (1) (i) an alpha-ketoglutarate decarboxylase, or (ii)
(a) a glutamate dehydrogenase and/or a glutamate transaminase, (b)
a glutamate decarboxylase, and (c) a 4-aminobutyrate dehydrogenase
and/or a 4-aminobutyrate transaminase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA hydrolase,
transferase or synthetase, and (8) a 3-hydroxybutyrate reductase;
(u) (1) (i) an alpha-ketoglutarate decarboxylase, or (ii) (a) a
glutamate dehydrogenase and/or a glutamate transaminase, (b) a
glutamate decarboxylase, and (c) a 4-aminobutyrate dehydrogenase
and/or a 4-aminobutyrate transaminase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA
dehydratase, (5) a crotonase, (6) a 3-hydroxybutyryl-CoA hydrolase,
transferase or synthetase, and (7) a 3-hydroxybutyrate reductase.
(v) (1) a succinate reductase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA reductase
(aldehyde forming), and (8) a 3-hydroxybutanal reductase; (w) (1) a
succinate reductase, (2) a 4-hydroxybutyrate dehydrogenase, (3) a
4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA
synthetase, (4) a 4-hydroxybutyryl-CoA dehydratase, (5) a
crotonase, (6) a 3-hydroxybutyryl-CoA reductase (aldehyde forming),
(7) a 3-hydroxybutanal reductase; (x) (1) a succinate reductase,
(2) a 4-hydroxybutyrate dehydrogenase, (3) a 4-hydroxybutyrate
kinase, (4) a phosphotrans-4-hydroxybutyrylase, (5) a
4-hydroxybutyryl-CoA dehydratase, (6) a crotonase, and (7) a
3-hydroxybutyryl-CoA reductase (alcohol forming); (y) (1) a
succinate reductase, (2) a 4-hydroxybutyrate dehydrogenase, (3) a
4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA
synthetase, (4) a 4-hydroxybutyryl-CoA dehydratase, (5) a
crotonase, and (6) a 3-hydroxybutyryl-CoA reductase (alcohol
forming); (z) (1) a succinate reductase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA hydrolase,
transferase or synthetase, and (8) a 3-hydroxybutyrate reductase;
(aa) (1) a succinate reductase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA
dehydratase, (5) a crotonase, (6) a 3-hydroxybutyryl-CoA hydrolase,
transferase or synthetase, and (7) a 3-hydroxybutyrate reductase;
(bb) (1) a succinyl-CoA transferase, succinyl-CoA synthetase or
succinyl-CoA ligase, (2) a succinyl-CoA reductase (aldehyde
forming), (3) a 4-hydroxybutyrate dehydrogenase, (5) a
4-hydroxybutyrate kinase, (6) a pPhosphotrans-4-hydroxybutyrylase,
(7) a 4-hydroxybutyryl-CoA dehydratase, (8) a crotonase, and (9) a
3-hydroxybutyryl-CoA reductase (alcohol forming); (cc) (1) a
succinyl-CoA transferase, succinyl-CoA synthetase or succinyl-CoA
ligase, (2) a succinyl-CoA reductase (aldehyde forming), (3) a
4-hydroxybutyrate dehydrogenase, (4) a 4-hydroxybutyrate kinase,
(5) a phosphotrans-4-hydroxybutyrylase, (6) a 4-hydroxybutyryl-CoA
dehydratase, (7) a crotonase, (8) a 3-hydroxybutyryl-CoA hydrolase,
transferase or synthetase, and (9) a 3-hydroxybutyrate reductase;
(dd) (1) a succinyl-CoA transferase, succinyl-CoA synthetase or
succinyl-CoA ligase, (2) a succinyl-CoA reductase (alcohol
forming), (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA reductase
(aldehyde forming), and (8) a 3-hydroxybutanal reductase; (ee) (1)
a succinyl-CoA transferase, succinyl-CoA synthetase or succinyl-CoA
ligase, (2) a succinyl-CoA reductase (alcohol forming), (3) a
4-hydroxybutyrate kinase, (4) a phosphotrans-4-hydroxybutyrylase,
(5) a 4-hydroxybutyryl-CoA dehydratase, (6) a crotonase, and (7) a
3-hydroxybutyryl-CoA reductase (alcohol forming); (ff) (1) a
succinyl-CoA transferase, succinyl-CoA synthetase or succinyl-CoA
ligase, (2) a succinyl-CoA reductase (alcohol forming), (3) a
4-hydroxybutyrate kinase, (4) a phosphotrans-4-hydroxybutyrylase,
(5) a 4-hydroxybutyryl-CoA dehydratase, (6) a crotonase, (7) a
3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, and (8)
a 3-hydroxybutyrate reductase; (gg) (1) a succinyl-CoA transferase,
succinyl-CoA synthetase or succinyl-CoA ligase, (2) a succinyl-CoA
reductase (aldehyde forming), (3) a 4-hydroxybutyrate
dehydrogenase, (4) a 4-hydroxybutyryl-CoA transferase, or
4-hydroxybutyryl-CoA synthetase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA reductase
(aldehyde forming), and (8) a 3-hydroxybutanal reductase; (hh) (1)
a succinyl-CoA transferase, succinyl-CoA synthetase or succinyl-CoA
ligase, (2) a succinyl-CoA reductase (aldehyde forming), (3) a
4-hydroxybutyrate dehydrogenase, (4) a 4-hydroxybutyryl-CoA
transferase or 4-hydroxybutyryl-CoA synthetase, (5) a
4-hydroxybutyryl-CoA dehydratase, (6) a crotonase, and (7) a
3-hydroxybutyryl-CoA reductase (alcohol forming); (ii) (1) a
succinyl-CoA transferase, succinyl-CoA synthetase or succinyl-CoA
ligase, (2) a succinyl-CoA reductase (aldehyde forming), (3) a
4-hydroxybutyrate dehydrogenase, (4) a 4-hydroxybutyryl-CoA
transferase or 4-hydroxybutyryl-CoA synthetase, (5) a
4-hydroxybutyryl-CoA dehydratase, (6) a crotonase, (7) a
3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, (8) a
3-hydroxybutyrate reductase; (jj) (1) a succinyl-CoA transferase,
succinyl-CoA synthetase or succinyl-CoA ligase, (2) a succinyl-CoA
reductase (alcohol forming), (3) a 4-hydroxybutyryl-CoA transferase
or 4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA
dehydratase, (5) a crotonase, (6) a 3-hydroxybutyryl-CoA reductase
(aldehyde faulting), and (7) a 3-hydroxybutanal reductase; (kk) (1)
a succinyl-CoA transferase, succinyl-CoA synthetase or succinyl-CoA
ligase, (2) a succinyl-CoA reductase (alcohol forming), (3) a
4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA
synthetase, (4) a 4-hydroxybutyryl-CoA dehydratase, (5) a
crotonase, and (6) a 3-hydroxybutyryl-CoA reductase (alcohol
forming); and (ll) (1) a succinyl-CoA transferase, succinyl-CoA
synthetase or succinyl-CoA ligase, (2) a succinyl-CoA reductase
(alcohol forming), (3) a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA hydrolase,
transferase or synthetase, and (8) a 3-hydroxybutyrate
reductase.
2. The non-naturally occurring microbial organism of claim 1,
wherein said microbial organism further comprises an exogenous
nucleic acid encoding an enzyme selected from the group consisting
of 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, a ferredoxin, and combinations
thereof.
3. The non-naturally occurring microbial organism of claim 1,
wherein said microbial organism further comprises an exogenous
nucleic acid encoding an enzyme selected from the group consisting
of 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, seven, eight or nine exogenous nucleic acids, each encoding a
1,3-BDO pathway enzyme.
5. The non-naturally occurring microbial organism of claim 1,
wherein said microbial organism comprises exogenous nucleic acids
encoding each of the enzymes of at least one of the 1,3-butanediol
pathways selected from the group consisting of (a)-(ll).
6. The non-naturally occurring microbial organism of claim 1,
wherein said at least one exogenous nucleic acid is a heterologous
nucleic acid.
7. The non-naturally occurring microbial organism of claim 1,
wherein said non-naturally occurring microbial organism is in a
substantially anaerobic culture medium.
8. A method for producing 1,3-BDO, comprising culturing a
non-naturally occurring microbial organism according to claim 1,
under conditions and for a sufficient period of time to produce
1,3-BDO.
9. The method of claim 8, wherein said microbial organism further
comprises an exogenous nucleic acid encoding an enzyme selected
from the group consisting of 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.
10. The method of claim 8, wherein said microbial organism further
comprises an exogenous nucleic acid encoding an enzyme selected
from the group consisting of an aconitase, an isocitrate
dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA
transferase, a fumarase, a malate dehydrogenase, and combinations
thereof.
11. The method of claim 8, wherein said microbial organism
comprises two, three, four, five, six, seven, eight or nine
exogenous nucleic acids, each encoding a 1,3-BDO pathway
enzyme.
12. The method of claim 8, wherein said microbial organism
comprises exogenous nucleic acids encoding each of the enzymes of
at least one of the 1,3-butanediol pathways selected from the group
consisting of (a)-(ll).
13. The method of claim 8, wherein said at least one exogenous
nucleic acid is a heterologous nucleic acid.
14. The method of claim 8, wherein said non-naturally occurring
microbial organism is in a substantially anaerobic culture medium.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of priority to
U.S. Ser. Nos. 61/500,131, filed Jun. 22, 2011, and 61/502,702,
filed Jun. 29, 2011, the contents of which are herein incorporated
by reference in their entirety.
BACKGROUND
[0002] The present invention relates generally to biosynthetic
processes and organisms capable of producing organic compounds.
More specifically, the invention relates to non-naturally occurring
organisms that can produce the commodity chemical
1,3-butanediol.
[0003] 1,3-butanediol (1,3-BDO) is a four carbon diol traditionally
produced from acetylene via its hydration. The resulting
acetaldehyde is then converted to 3-hydroxybutyraldehdye which is
subsequently reduced to form 1,3-BDO. In more recent years,
acetylene has been replaced by the less expensive ethylene as a
source of acetaldehyde. 1,3-BDO is commonly used as an organic
solvent for food flavoring agents. It is also used as a co-monomer
for polyurethane and polyester resins and is widely employed as a
hypoglycaemic agent. Optically active 1,3-BDO is a useful starting
material for the synthesis of biologically active compounds and
liquid crystals. A substantial commercial use of 1,3-butanediol is
subsequent dehydration to afford 1,3-butadiene (Ichikawa et al., J.
of Molecular Catalysis A-Chemical, 256:106-112 (2006); Ichikawa et
al., J. of Molecular Catalysis A-Chemical, 231:181-189 (2005)), a
25 billion lb/yr petrochemical used to manufacture synthetic
rubbers (e.g., tires), latex, and resins. The reliance on petroleum
based feedstocks for either acetylene or ethylene warrants the
development of a renewable feedstock based route to 1,3-butanediol
and to butadiene.
[0004] Thus, there exists a need to develop microorganisms and
methods of their use to produce 1,3-BDO. The present invention
satisfies this need and provides related advantages as well.
SUMMARY
[0005] In some embodiments, the present invention is directed to a
non-naturally occurring microbial organism that includes a
microbial organism having a 1,3-butanediol (1,3-BDO) pathway having
at least one exogenous nucleic acid encoding a 1,3-BDO pathway
enzyme expressed in a sufficient amount to produce 1,3-BDO. The
1,3-BDO pathway includes an enzyme selected from the group
consisting of a 2-amino-4-ketopentanoate (AKP) thiolase, an AKP
dehydrogenase, a 2-amino-4-hydroxypentanoate aminotransferase, a
2-amino-4-hydroxypentanoate oxidoreductase (deaminating), a
2-oxo-4-hydroxypentanoate decarboxylase, a 3-hydroxybutyraldehyde
reductase, an AKP aminotransferase, an AKP oxidoreductase
(deaminating), a 2,4-dioxopentanoate decarboxylase, a
3-oxobutyraldehyde reductase (ketone reducing), a
3-oxobutyraldehyde reductase (aldehyde reducing), a
4-hydroxy-2-butanone reductase, an AKP decarboxylase, a
4-aminobutan-2-one aminotransferase, a 4-aminobutan-2-one
oxidoreductase (deaminating), a 4-aminobutan-2-one ammonia-lyase, a
butenone hydratase, an AKP ammonia-lyase, an acetylacrylate
decarboxylase, an acetoacetyl-CoA reductase (CoA-dependent,
aldehyde forming), an acetoacetyl-CoA reductase (CoA-dependent,
alcohol forming), an acetoacetyl-CoA reductase (ketone reducing), a
3-hydroxybutyryl-CoA reductase (aldehyde forming), a
3-hydroxybutyryl-CoA reductase (alcohol forming), a
4-hydroxybutyryl-CoA dehydratase, and a crotonase.
[0006] In some embodiments, the present invention is directed to a
method for producing 1,3-BDO that includes culturing such a
non-naturally occurring microbial organism, under conditions and
for a sufficient period of time to produce 1,3-BDO.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 shows pathways to 1,3-BDO from alanine. Enzymes are:
A) AKP thiolase, B) AKP aminotransferase or AKP oxidoreductase
(deaminating), C) 2,4-dioxopentanoate decarboxylase, D)
3-oxobutyraldehyde reductase (aldehyde reducing), E) AKP
decarboxylase, F) 4-aminobutan-2-one ammonia-lyase, G) Butenone
hydratase, H) 4-hydroxy,2-butanone reductase, I) AKP ammonia-lyase,
J) acetylacrylate decarboxylase, K) 4-aminobutan-2-one
aminotransferase or 4-aminobutan-2-one oxidoreductase
(deaminating), L) AKP dehydrogenase, M) 2-amino-4-hydroxypentanoate
aminotransferase or 2-amino-4-hydroxypentanoate oxidoreductase
(deaminating), N) 2-oxo-4-hydroxypentanoate decarboxylase, O)
3-oxobutyraldehyde reductase (ketone reducing), and P)
3-hydroxybutyraldehdye reductase.
[0008] FIG. 2 shows pathways from acetoacetyl-CoA to
1,3-butanediol. Enzymes are: A) acetoacetyl-CoA reductase
(CoA-dependent, aldehyde forming), B) 3-oxobutyraldehyde reductase
(ketone reducing), C) 3-hydroxybutyraldehyde reductase, D)
acetoacetyl-CoA reductase (CoA-dependent, alcohol forming), E)
3-oxobutyraldehyde reductase (aldehyde reducing), F)
4-hydroxy,2-butanone reductase, G) acetoacetyl-CoA reductase
(ketone reducing), H) 3-hydroxybutyryl-CoA reductase (aldehyde
forming), and I) 3-hydroxybutyryl-CoA reductase (alcohol
forming).
[0009] FIG. 3 shows pathways from 4-hydroxybutyryl-CoA to
1,3-butanediol. Enzymes are: A) 4-hydroxybutyryl-CoA dehydratase,
B) crotonase, C) 3-hydroxybutyryl-CoA reductase (aldehyde forming),
D) 3-hydroxybutyraldehyde reductase, and E) 3-hydroxybutyryl-CoA
reductase (alcohol forming).
[0010] FIG. 4 shows aldehyde dehydrogenases showing significant
activity on 3-hydroxybutyl-CoA.
[0011] FIG. 5 shows the specific activity of bld from Clostridium
saccharoperbutylacetonicum on 3-Hydroxybutyryl-CoA before and after
dialysis.
[0012] FIG. 6 shows 1,3-BDO concentrations when
3-hydroxybutyraldehyde was added as a substrate and in the control
samples with no substrate. The GI numbers for the alcohol
dehydrogenases are shown.
[0013] FIG. 7 shows 1,3-BDO concentrations when
3-hydroxybutyryl-CoA was added as a substrate and in the control
samples with no substrate. The GI numbers for the alcohol
dehydrogenases are shown. The GI number for the aldehyde
dehydrogenase tested in conjunction is 163762382.
[0014] FIG. 8A shows the pathways for fixation of CO.sub.2 to
pyruvate using the reductive TCA cycle.
[0015] FIG. 8B shows exemplary pathways for the biosynthesis of
1,3-butanediol from pyruvate; pyruvate is converted to alanine by
alanine dehydrogenase alanine aminotransferase; the remaining
enzymatic transformations shown are carried out by the following
enzymes: A) AKP thiolase, B) AKP aminotransferase or AKP
oxidoreductase (deaminating), C) 2,4-dioxopentanoate decarboxylase,
D) 3-oxobutyraldehyde reductase (aldehyde reducing), E) AKP
decarboxylase, F) 4-aminobutan-2-one ammonia-lyase, G) Butenone
hydratase, H) 4-hydroxy,2-butanone reductase, I) AKP ammonia-lyase,
J) acetylacrylate decarboxylase, K) 4-aminobutan-2-one
aminotransferase or 4-aminobutan-2-one oxidoreductase
(deaminating), L) AKP dehydrogenase, M) 2-amino-4-hydroxypentanoate
aminotransferase or 2-amino-4-hydroxypentanoate oxidoreductase
(deaminating), N) 2-oxo-4-hydroxypentanoate decarboxylase, O)
3-oxobutyraldehyde reductase (ketone reducing), and P)
3-hydroxybutyraldehdye reductase.
[0016] FIG. 9A shows the pathways for fixation of CO.sub.2 to
alpha-ketoglutarate, succinate and succinyl-CoA using the reductive
TCA cycle.
[0017] FIG. 9B shows exemplary pathways for the biosynthesis of
1,3-butanediol from alpha-ketoglutarate, succinate and
succinyl-CoA; the enzymatic transformations shown are carried out
by the following enzymes: A. Succinyl-CoA transferase, or
Succinyl-CoA synthetase (or succinyl-CoA ligase), B. Succinyl-CoA
reductase (aldehyde forming), C. 4-Hydroxybutyrate dehydrogenase,
D. 4-Hydroxybutyrate kinase, E. Phosphotrans-4-hydroxybutyrylase,
F. Succinate reductase, G. Succinyl-CoA reductase (alcohol
forming), H. 4-Hydroxybutyryl-CoA transferase, or
4-Hydroxybutyryl-CoA synthetase, or 4-Hydroxybutyryl-CoA ligase I.
Alpha-ketoglutarate decarboxylase, J. 4-hydroxybutyryl-CoA
dehydratase, K. crotonase, L. 3-hydroxybutyryl-CoA reductase
(aldehyde forming), M. 3-hydroxybutanal reductase, N.
3-hydroxybutyryl-CoA reductase (alcohol forming), O.
3-hydroxybutyryl-CoA hydrolase, transferase, or synthetase, P.
3-hydroxybutyrate reductase, Q. Glutamate dehydrogenase and/or R.
Glutamate transaminase; S. Glutamate decarboxylase; T.
4-aminobutyrate dehydrogenase and/or U. 4-aminobutyrate
transaminase and V. Alpha-ketoglutarate dehydrogenase.
[0018] FIG. 10 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.sub.--1202/1203) or Mtr (Moth.sub.--1197) proteins (50, 150,
250, 350, 450, 500, 750, 900, and 1000 ng).
[0019] FIG. 11 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.
[0020] FIG. 12A shows the nucleotide sequence (SEQ ID NO:1) of
carboxylic acid reductase from Nocardia iowensis (GNM.sub.--720),
and FIG. 12B shows the encoded amino acid sequence (SEQ ID
NO:2).
[0021] 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).
[0022] 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).
[0023] 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).
[0024] 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).
[0025] 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
[0026] This invention is directed, in part, to non-naturally
occurring microorganisms that express genes encoding enzymes that
catalyze 1,3-butanediol (1,3-BDO) production. Pathways for the
production of 1,3-butanediol disclosed herein are based on three
precursors: (i) D-alanine, (ii) acetoacetyl-CoA, and (iii)
4-hydroxybutyryl-CoA. Successfully engineering these pathways
entails identifying an appropriate set of enzymes with sufficient
activity and specificity, cloning their corresponding genes into a
production host, optimizing fermentation conditions, and assaying
for product formation following fermentation.
[0027] The conversion of alanine to 1,3-BDO can be accomplished by
a number of pathways in about five enzymatic steps as shown in FIG.
1. In the first step of all pathways (Step A), alanine and
acetyl-CoA are combined by 2-amino-4-ketopentanoate thiolase, a
highly selective enzyme. The product of this reaction,
2-amino-4-oxopentanoate (AKP) can then be transaminated, reduced,
decarboxylated or deaminated as shown in FIG. 1. Further synthetic
steps for the production of 1,3-BDO are discussed in detail below.
The theoretical yield of 1,3-BDO from each of these pathways is
calculated to be about 1.09 mole/mole of glucose consumed.
[0028] FIG. 2 outlines multiple routes for producing 1,3-BDO from
acetoacetyl-CoA. Each of these pathways from acetoacetyl-CoA to
1,3-BDO utilizes three reducing equivalents and provides a
theoretical yield of 1 mole of 1,3-BDO per mole of glucose
consumed. Other carbon substrates such as syngas can also be used
for the production of acetoacetyl-CoA. Gasification of glucose to
form syngas will result in the maximum theoretical yield of 1.09
moles of 1,3-BDO per mole of glucose consumed, assuming that 6
moles of CO and 6 moles of H.sub.2 are obtained from glucose
6CO+6H.sub.2.fwdarw.1.091C.sub.4H.sub.10O.sub.2+1.636CO.sub.2+0.545H.sub-
.2
[0029] 4-Hydroxybutyryl-CoA is an important starting metabolite
from which a number of industrially useful compounds can be made,
including 1,3-BDO as shown in FIG. 3. Although 4-hydroxybutyryl-CoA
is not a highly common central metabolite, methods for engineering
strains that synthesize 4-hydroxybutyryl-CoA have been described
previously by Applicants in U.S. Patent Application No.
2009/0075351. The 4-hydroxybutyryl-CoA to 1,3-butanediol pathway
has a theoretical yield of 1.09 mol/mol product yield assuming
glucose as the carbohydrate feedstock.
[0030] This invention is also directed, in part, to methods for
producing 1,3-BDO through culturing of these non-naturally
occurring microbial organisms. Dehydration of 1,3-BDO produced by
the organisms and methods described herein, provides an opportunity
to produce renewable butadiene in small end-use facilities
obviating the need to transport this flammable and reactive
chemical.
[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
1,3-butanediol 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 "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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] "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.
[0038] 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.
[0039] The non-naturally occurring microbal 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] Therefore, in identifying and constructing the non-naturally
occurring microbial organisms of the invention having 1,3-BDO
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.
[0046] 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.
[0047] 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.
[0048] In some embodiments, the present invention provides a
non-naturally occurring microbial organism that includes a
microbial organism having a 1,3-butanediol (1,3-BDO) pathway with
at least one exogenous nucleic acid encoding a 1,3-BDO pathway
enzyme expressed in a sufficient amount to produce 1,3-BDO. The
1,3-BDO pathway includes an enzyme selected from the group
consisting of a 2-amino-4-ketopentanoate (AKP) thiolase, an AKP
dehydrogenase, a 2-amino-4-hydroxypentanoate aminotransferase, a
2-amino-4-hydroxypentanoate oxidoreductase (deaminating), a
2-oxo-4-hydroxypentanoate decarboxylase, a 3-hydroxybutyraldehyde
reductase, an AKP aminotransferase, an AKP oxidoreductase
(deaminating), a 2,4-dioxopentanoate decarboxylase, a
3-oxobutyraldehyde reductase (ketone reducing), a
3-oxobutyraldehyde reductase (aldehyde reducing), a
4-hydroxy-2-butanone reductase, an AKP decarboxylase, a
4-aminobutan-2-one aminotransferase, a 4-aminobutan-2-one
oxidoreductase (deaminating), a 4-aminobutan-2-one ammonia-lyase, a
butenone hydratase, an AKP ammonia-lyase, an acetylacrylate
decarboxylase, an acetoacetyl-CoA reductase (CoA-dependent,
aldehyde forming), an acetoacetyl-CoA reductase (CoA-dependent,
alcohol forming), an acetoacetyl-CoA reductase (ketone reducing), a
3-hydroxybutyryl-CoA reductase (aldehyde forming), a
3-hydroxybutyryl-CoA reductase (alcohol forming), a
4-hydroxybutyryl-CoA dehydratase, and a crotonase.
[0049] Any combination and any number of the aforementioned enzymes
can be introduced into a host microbial organism to complete a
1,3-BDO pathway, as exemplified in FIGS. 1-3. For example, the
non-naturally occurring microbial organism can include one, two,
three, four, five, up to all of the nucleic acids in a 1,3-BDO
pathway, each nucleic acid encoding a 1,3-BDO pathway enzyme. Such
nucleic acids can include heterologous nucleic acids, additional
copies of existing genes, and gene regulatory elements, as
explained further below. The pathways of the non-naturally
occurring microbial organisms of the invention are also suitably
engineered to be cultured in a substantially anaerobic culture
medium.
[0050] In some embodiments, the non-naturally occurring microbial
organisms having a 1,3-BDO pathway include a set of 1,3-BDO pathway
enzymes. A set of 1,3-BDO pathway enzymes represents a group of
enzymes that can convert alanine, acetoacetyl-CoA, or
4-hydroxybutyryl-CoA to 1,3-BDO, as show in FIGS. 1-3. Exemplary
sets of 1,3-BDO pathway enzymes to convert alanine to 1,3-BDO,
according to FIG. 1 include (a) (1) a 2-amino-4-ketopentanoate
(AKP) thiolase; (2) an AKP dehydrogenase; (3) a
2-amino-4-hydroxypentanoate aminotransferase or oxidoreductase
(deaminating); (4) a 2-oxo-4-hydroxypentanoate decarboxylase; and
(5) a 3-hydroxybutyraldehyde reductase; (b) (1) a
2-amino-4-ketopentanoate (AKP) thiolase; (2) an AKP
aminotransferase or oxidoreductase (deaminating); (3) a
2,4-dioxopentanoate decarboxylase; (4) a 3-oxobutyraldehyde
reductase (ketone reducing); and (5) a 3-hydroxybutyraldehyde
reductase; (c) (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2)
an AKP aminotransferase or oxidoreductase (deaminating); (3) a
2,4-dioxopentanoate decarboxylase; (4) a 3-oxobutyraldehyde
reductase (aldehyde reducing); and (5) a 4-hydroxy-2-butanone
reductase; (d) (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2)
an AKP decarboxylase; (3) a 4-aminobutan-2-one aminotransferase or
oxidoreductase (deaminating); (4) a 3-oxobutyraldehyde reductase
(ketone reducing); and (5) a 3-hydroxybutyraldehyde reductase; (e)
(1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an AKP
decarboxylase; (3) a 4-aminobutan-2-one aminotransferase or
oxidoreductase (deaminating); (4) a 3-oxobutyraldehyde reductase
(aldehyde reducing); and (5) a 4-hydroxy-2-butanone reductase; (f)
(1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an AKP
decarboxylase; (3) a 4-aminobutan-2-one ammonia-lyase; (4) a
butenone hydratase; and (5) a 4-hydroxy-2-butanone reductase; and
(g) (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an AKP
ammonia-lyase; (3) an acetylacrylate decarboxylase; (4) a butenone
hydratase; and (5) a 4-hydroxy-2-butanone reductase;
[0051] Exemplary sets of 1,3-BDO pathway enzymes to convert
acetoacetyl-CoA to 1,3-BDO, according to FIG. 2 include (h) (1) an
acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming); (2) a
3-oxobutyraldehyde reductase (ketone reducing); and (3) a
3-hydroxybutyraldehyde reductase; (i) (1) an acetoacetyl-CoA
reductase (CoA dependent, alcohol forming) and (2) a
4-hydroxy-2-butanone reductase; (j) (1) an acetoacetyl-CoA
reductase (CoA-dependent, aldehyde forming); (2) a
3-oxobutyraldehyde reductase (aldehyde reducing); and (3) a
4-hydroxy-2-butanone reductase; (k) (1) an acetoacetyl-CoA
reductase (ketone reducing) and (2) a 3-hydroxybutyryl-CoA
reductase (alcohol forming); and (l) (1) an acetoacetyl-CoA
reductase (ketone reducing); (2) a 3-hydroxybutyryl-CoA reductase
(aldehyde forming); and (3) a 3-hydroxybutyraldehyde reductase;
[0052] Exemplary sets of 1,3-BDO pathway enzymes to convert
4-hydroxybutyryl-CoA to 1,3-BDO, according to FIG. 3 include (m)
(1) a 4-hydroxybutyryl-CoA dehydratase; (2) a crotonase; and (3) a
3-hydroxybutyryl-CoA reductase (alcohol forming); and (n) (1) a
4-hydroxybutyryl-CoA dehydratase; (2) a crotonase; (3) a
3-hydroxybutyryl-CoA reductase (aldehyde forming); and (4) a
3-hydroxybutyraldehyde reductase.
[0053] The conversion of alanine to 1,3-BDO can be accomplished by
a number of pathways involving about five enzymatic steps as shown
in FIG. 1. In the first step of all pathways (Step A), alanine and
acetyl-CoA are combined by 2-amino-4-ketopentanoate thiolase, a
highly selective enzyme. The product of this reaction,
2-amino-4-oxopentanoate (AKP) can then be transaminated, reduced,
decarboxylated or deaminated as shown in FIG. 1.
[0054] In one route, AKP converted to 2,4-dioxopentanoate, a 2-keto
acid similar in structure to alpha-ketoglutarate, by an
aminotransferase or deaminating oxidoreductase (Step B).
2,4-Dioxopentanoate is then converted to 3-oxobutyraldehyde by a
2-ketoacid decarboxylase (Step C). Reduction of the ketone and
aldehyde groups to their corresponding alcohols yields
1,3-butanediol. These reductions can occur in either order to form
the intermediates 3-hydroxybutyraldehyde (Steps O and P) or
4-hydroxy,2-butanone (Steps D and H).
[0055] In another route, the 4-oxo group of AKP is first reduced to
a secondary alcohol by AKP dehydrogenase (Step L). The product,
2-amino-4-hydroxypentanoate, is then converted to
2-oxo-4-hydroxypentanoate (Step M). The resulting 2-ketoacid is
decarboxylated to 3-hydroxybutyraldehyde (Step N). In the final
step of this route, the aldehyde of 3-hydroxybutyraldehyde is
reduced to a primary alcohol by 3-hydroxybutyraldehyde reductase,
forming 1,3-butanediol (Step P).
[0056] Yet another route involves decarboxylation of AKP by an
amino acid decarboxylase (Step E). The decarboxylation product,
4-aminobutan-2-one, can either be transaminated or oxidatively
deaminated to 3-oxobutyraldehyde (Step K) or deaminated to butenone
(Step F). When 3-oxobutyraldehyde is formed, two alcohol-forming
reduction steps are used to form 1,3-butanediol, as described
previously (Steps O and P, or Steps D and H). The deamination
product, butenone, is then hydrolyzed to 4-hydroxy,2-butanone (Step
G), which is reduced to 1,3-butanediol by 4-hydroxy-2-butanone
reductase (Step H).
[0057] Yet another route involves the deamination of AKP to
acetylacrylate (Step I). Acetylacrylate is decarboxylated to
butenone (Step J), which is then converted to 1,3-butandiol by
butenone hydratase (Step G) and 4-hydroxy,2-butanone reductase
(Step H).
[0058] Based on the routes described above for the production
1,3-BDO from alanine, in some embodiments, the non-naturally
occurring microbial organism has a set of 1,3-BDO pathway enzymes
that includes (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an
AKP dehydrogenase; (3) a 2-amino-4-hydroxypentanoate
aminotransferase or oxidoreductase (deaminating); (4) a
2-oxo-4-hydroxypentanoate decarboxylase; and (5) a
3-hydroxybutyraldehyde reductase. Any number of nucleic acids
encoding these enzymes can be introduced into a host microbial
organism including one, two, three, four, up to all five of the
nucleic acids that encode these enzymes. Where one, two, three, or
four exogenous nucleic acids are introduced, such nucleic acids can
be any permutation of the five nucleic acids.
[0059] In other embodiments non-naturally occurring microbial
organism has a set of 1,3-BDO pathway enzymes that includes (1) a
2-amino-4-ketopentanoate (AKP) thiolase; (2) an AKP
aminotransferase or oxidoreductase (deaminating); (3) a
2,4-dioxopentanoate decarboxylase; (4) a 3-oxobutyraldehyde
reductase (ketone reducing); and (5) a 3-hydroxybutyraldehyde
reductase. Any number of nucleic acids encoding these enzymes can
be introduced into a host microbial organism including one, two,
three, four, up to all five of the nucleic acids that encode these
enzymes. Where one, two, three, or four exogenous nucleic acids are
introduced, such nucleic acids can be any permutation of the five
nucleic acids.
[0060] In still other embodiments, the non-naturally occurring
microbial organism has a set of 1,3-BDO pathway enzymes that
includes (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an AKP
aminotransferase or oxidoreductase (deaminating); (3) a
2,4-dioxopentanoate decarboxylase; (4) a 3-oxobutyraldehyde
reductase (aldehyde reducing); and (5) a 4-hydroxy-2-butanone
reductase. Any number of nucleic acids encoding these enzymes can
be introduced into a host microbial organism including one, two,
three, four, up to all five of the nucleic acids that encode these
enzymes. Where one, two, three, or four exogenous nucleic acids are
introduced, such nucleic acids can be any permutation of the five
nucleic acids.
[0061] In yet further embodiments, the non-naturally occurring
microbial organism has a set of 1,3-BDO pathway enzymes that
includes (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an AKP
decarboxylase; (3) a 4-aminobutan-2-one aminotransferase or
oxidoreductase (deaminating); (4) a 3-oxobutyraldehyde reductase
(ketone reducing); and (5) a 3-hydroxybutyraldehyde reductase. Any
number of nucleic acids encoding these enzymes can be introduced
into a host microbial organism including one, two, three, four, up
to all five of the nucleic acids that encode these enzymes. Where
one, two, three, or four exogenous nucleic acids are introduced,
such nucleic acids can be any permutation of the five nucleic
acids.
[0062] In yet still further embodiments, the non-naturally
occurring microbial organism has a set of 1,3-BDO pathway enzymes
that includes (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an
AKP decarboxylase; (3) a 4-aminobutan-2-one aminotransferase or
oxidoreductase (deaminating); (4) a 3-oxobutyraldehyde reductase
(aldehyde reducing); and (5) a 4-hydroxy-2-butanone reductase. Any
number of nucleic acids encoding these enzymes can be introduced
into a host microbial organism including one, two, three, four, up
to all five of the nucleic acids that encode these enzymes. Where
one, two, three, or four exogenous nucleic acids are introduced,
such nucleic acids can be any permutation of the five nucleic
acids.
[0063] In still further embodiments, the non-naturally occurring
microbial organism has a set of 1,3-BDO pathway enzymes that
includes (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an AKP
decarboxylase; (3) a 4-aminobutan-2-one ammonia-lyase; (4) a
butenone hydratase; and (5) a 4-hydroxy-2-butanone reductase. Any
number of nucleic acids encoding these enzymes can be introduced
into a host microbial organism including one, two, three, four, up
to all five of the nucleic acids that encode these enzymes. Where
one, two, three, or four exogenous nucleic acids are introduced,
such nucleic acids can be any permutation of the five nucleic
acids.
[0064] In yet still further embodiments, the non-naturally
occurring microbial organism has a set of 1,3-BDO pathway enzymes
that includes (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an
AKP ammonia-lyase; (3) an acetylacrylate decarboxylase; (4) a
butenone hydratase; and (5) a 4-hydroxy-2-butanone reductase. Any
number of nucleic acids encoding these enzymes can be introduced
into a host microbial organism including one, two, three, four, up
to all five of the nucleic acids that encode these enzymes. Where
one, two, three, or four exogenous nucleic acids are introduced,
such nucleic acids can be any permutation of the five nucleic
acids.
[0065] FIG. 2 outlines multiple routes for producing 1,3-butanediol
from acetoacetyl-CoA. One route through steps A, B and C utilizes
(i) CoA-dependent, aldehyde forming acetoacetyl-CoA reductase to
convert acetoacetyl-CoA into 3-oxobutyraldehyde (FIG. 2, Step A),
(ii) 3-oxobutyraldehyde reductase to reduce 3-oxobutyraldehyde to
3-hydroxybutyraldehyde (FIG. 2, Step B), and (iii) finally,
3-hydroxybutyraldehyde reductase to foam 1,3-butanediol (FIG. 2,
Step C).
[0066] Alternatively, acetoacetyl-CoA can be reduced via the
aldehyde forming acetoacetyl-CoA reductase to form
4-hydroxy,2-butanone (FIG. 2, Step D). 4-hydroxy,2-butanone can
also be formed by the reduction of 3-oxobutyraldehyde by the
aldehyde reducing 3-oxobutyraldehyde reductase (FIG. 2, Step E).
Eventually, 4-hydroxy,2-butanone can be reduced to form 1,3-BDO by
4-hydroxy-2-butanone reductase (FIG. 2, Step F).
[0067] Yet another set of 1,3-BDO forming routes rely on the
reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA by the ketone
reducing acetoacetyl-CoA reductase (FIG. 2, Step G). This enzyme
reduces the ketone function in acetoacetyl-CoA to a hydroxyl group.
3-hydroxybutyryl-CoA can be reduced by the bifunctional
alcohol-forming 3-hydroxybutyryl-CoA reductase to form
1,3-butanediol (FIG. 2, Step I). Alternatively, it can first be
reduced to 3-hydroxybutyraldehyde via the aldehyde forming
3-hydroxybutyryl-CoA reductase (Step H) and 3-hydroxybutyraldehyde
can then be reduced as shown in Step C.
[0068] Based on the routes described above for the production
1,3-BDO from acetoacetyl-CoA, in some embodiments, the
non-naturally occurring microbial organism has a set of 1,3-BDO
pathway enzymes that includes (1) an acetoacetyl-CoA reductase
(CoA-dependent, aldehyde forming); (2) a 3-oxobutyraldehyde
reductase (ketone reducing); and (3) a 3-hydroxybutyraldehyde
reductase. Any number of nucleic acids encoding these enzymes can
be introduced into a host microbial organism including one, two up
to all three of the nucleic acids that encode these enzymes. Where
one or two exogenous nucleic acids are introduced, such nucleic
acids can be any permutation of the three nucleic acids.
[0069] In other embodiments, the non-naturally occurring microbial
organism has a set of 1,3-BDO pathway enzymes that includes (1) an
acetoacetyl-CoA reductase (CoA dependent, alcohol forming) and (2)
a 4-hydroxy-2-butanone reductase. Any number of nucleic acids
encoding these enzymes can be introduced into a host microbial
organism including one or both of the nucleic acids that encode
these enzymes. Where one exogenous nucleic acid is introduced, such
a nucleic acid can be either of the two nucleic acids.
[0070] In further embodiments, the non-naturally occurring
microbial organism has a set of 1,3-BDO pathway enzymes that
includes (1) an acetoacetyl-CoA reductase (CoA-dependent, aldehyde
forming); (2) a 3-oxobutyraldehyde reductase (aldehyde reducing);
and (3) a 4-hydroxy-2-butanone reductase. Any number of nucleic
acids encoding these enzymes can be introduced into a host
microbial organism including one, two up to all three of the
nucleic acids that encode these enzymes. Where one or two exogenous
nucleic acids are introduced, such nucleic acids can be any
permutation of the three nucleic acids.
[0071] In yet further embodiments, the non-naturally occurring
microbial organism has a set of 1,3-BDO pathway enzymes that
includes (1) an acetoacetyl-CoA reductase (ketone reducing) and (2)
a 3-hydroxybutyryl-CoA reductase (alcohol forming). Any number of
nucleic acids encoding these enzymes can be introduced into a host
microbial organism including one or both of the nucleic acids that
encode these enzymes. Where one exogenous nucleic acid is
introduced, such a nucleic acid can be either of the two nucleic
acids.
[0072] In still further embodiments, the non-naturally occurring
microbial organism has a set of 1,3-BDO pathway enzymes that
includes (1) an acetoacetyl-CoA reductase (ketone reducing); (2) a
3-hydroxybutyryl-CoA reductase (aldehyde forming); and (3) a
3-hydroxybutyraldehyde reductase. Any number of nucleic acids
encoding these enzymes can be introduced into a host microbial
organism including one, two up to all three of the nucleic acids
that encode these enzymes. Where one or two exogenous nucleic acids
are introduced, such nucleic acids can be any permutation of the
three nucleic acids.
[0073] 4-hydroxybutyryl-CoA is an important starting metabolite
from which a number of industrially useful compounds can be made.
Although 4-hydroxybutyryl-CoA is not a highly common central
metabolite, methods for engineering strains that synthesize
4-hydroxybutyryl-CoA have been described in Burk et al. (US
20090075351). An exemplary method involves synthesizing
4-hydroxybutyryl-CoA from succinyl-CoA by employing genes encoding
succinic semialdehyde dehydrogenase (CoA-dependent),
4-hydroxybutyrate dehydrogenase, 4-hydroxybutyrate kinase, and
phosphotransbutyrylase activities.
[0074] The first step in the pathway involves the dehydration of
4-hydroxybutyryl-CoA (Step A, FIG. 3) followed by the hydration of
crotonoyl-CoA to form 3-hydroxybutyryl-CoA (Step B).
3-hydroxybutyryl-CoA then undergoes two reduction steps to form
1,3-butanediol carried out by either two enzymes (Steps C and D) or
a single dual-function enzyme (Step E).
[0075] Thus, in some embodiments, the non-naturally occurring
microbial organism has a set of 1,3-BDO pathway enzymes that
includes (1) a 4-hydroxybutyryl-CoA dehydratase; (2) a crotonase;
and (3) a 3-hydroxybutyryl-CoA reductase (alcohol forming). Any
number of nucleic acids encoding these enzymes can be introduced
into a host microbial organism including one, two up to all three
of the nucleic acids that encode these enzymes. Where one or two
exogenous nucleic acids are introduced, such nucleic acids can be
any permutation of the three nucleic acids.
[0076] In other embodiments, the non-naturally occurring microbial
organism has a set of 1,3-BDO pathway enzymes that includes (1) a
4-hydroxybutyryl-CoA dehydratase; (2) a crotonase; (3) a
3-hydroxybutyryl-CoA reductase (aldehyde forming); and (4) a
3-hydroxybutyraldehyde reductase. Any number of nucleic acids
encoding these enzymes can be introduced into a host microbial
organism including one, two, three up to all four of the nucleic
acids that encode these enzymes. Where one, two, or three exogenous
nucleic acids are introduced, such nucleic acids can be any
permutation of the four nucleic acids.
[0077] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having a 1,3-BDO
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 alanine to 2-amino-4-oxopentanoate,
2-amino-4-oxopentanoate to 2-amino-4-hydroxypentanoate,
2-amino-4-hydroxypentanoate to 2-oxo-4-hydroxypentanoate,
2-oxo-4-hydroxypentanoate to 3-hydroxybutyraldehyde, and
3-hydroxybutyraldehyde to 1,3-BDO.
[0078] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having a 1,3-BDO
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 alanine to 2-amino-4-oxopentanoate,
2-amino-4-oxopentanoate to 2,4-dioxopentanoate, 2,4-dioxopentanoate
to 3-oxobutyraldehyde, 3-oxobutyraldehyde to
3-hydroxybutyraldehyde, and 3-hydroxybutyraldehyde to 1,3-BDO.
[0079] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having a 1,3-BDO
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 alanine to 2-amino-4-oxopentanoate,
2-amino-4-oxopentanoate to 2,4-dioxopentanoate, 2,4-dioxopentanoate
to 3-oxobutyraldehyde, 3-oxobutyraldehyde to 4-hydroxy-2-butanone,
and 4-hydroxy-2-butanone to 1,3-BDO.
[0080] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having a 1,3-BDO
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 alanine to 2-amino-4-oxopentanoate,
2-amino-4-oxopentanoate to 4-aminobutan-2-one, 4-aminobutan-2-one
to 3-oxobutyraldehyde, 3-oxobutyraldehyde to
3-hydroxybutyraldehyde, and 3-hydroxybutyraldehyde to 1,3-BDO.
[0081] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having a 1,3-BDO
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 alanine to 2-amino-4-oxopentanoate,
2-amino-4-oxopentanoate to 4-aminobutan-2-one, 4-aminobutan-2-one
to 3-oxobutyraldehyde, 3-oxobutyraldehyde to 4-hydroxy-2-butanone,
and 4-hydroxy-2-butanone to 1,3-BDO.
[0082] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having a 1,3-BDO
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 alanine to 2-amino-4-oxopentanoate,
2-amino-4-oxopentanoate to 4-aminobutan-2-one, 4-aminobutan-2-one
to butenone, butenone to 4-hydroxy-2-butanone, and
4-hydroxy-2-butanone to 1,3-BDO.
[0083] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having a 1,3-BDO
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 alanine to 2-amino-4-oxopentanoate,
2-amino-4-oxopentanoate to acetylacrylate, acetylacrylate to
butenone, butenone to 4-hydroxy-2-butanone, and
4-hydroxy-2-butanone to 1,3-BDO.
[0084] 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 1,3-BDO pathway converting alanine
to 1,3-BDO, as exemplified by the pathways shown in FIG. 1.
[0085] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having a 1,3-BDO
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 acetoacetyl-CoA to 4-hydroxy-2-butanone, and
4-hydroxy-2-butanone to 1,3-BDO.
[0086] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having a 1,3-BDO
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 acetoacetyl-CoA to 3-oxobutyraldehyde,
3-oxobutyraldehyde to 4-hydroxy-2-butanone, and
4-hydroxy-2-butanone to 1,3-BDO.
[0087] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having a 1,3 BDO
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 acetoacetyl-CoA to 3-oxobutyraldehyde,
3-oxobutyraldehyde to 3-hydroxybutryaldehyde, and
3-hydroxybutryaldehyde to 1,3-BDO.
[0088] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having a 1,3-BDO
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 acetoacetyl-CoA to 3-hydroxybutyryl-CoA,
3-hydroxybutyryl-CoA to 3-hydroxybutryaldehyde, and
3-hydroxybutryaldehyde to 1,3-BDO.
[0089] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having a 1,3-BDO
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 acetoacetyl-CoA to 3-hydroxybutyryl-CoA, and
3-hydroxybutyryl-CoA to 1,3-BDO.
[0090] 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 1,3-BDO pathway converting
acetoacetyl-CoA to 1,3-BDO, as exemplified by the pathways shown in
FIG. 2.
[0091] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having a 1,3-BDO
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 4-hydroxybutyryl-CoA to crotonoyl-CoA,
crotonoyl-CoA to 3-hydroxybutyryl-CoA, 3-hydroxybutyryl-CoA to
3-hydroxybutryaldehyde, and 3-hydroxybutryaldehyde to 1,3-BDO.
[0092] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having a 1,3-BDO
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 4-hydroxybutyryl-CoA to crotonoyl-CoA,
crotonoyl-CoA to 3-hydroxybutyryl-CoA, and 3-hydroxybutyryl-CoA to
1,3-BDO.
[0093] 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 1,3-BDO pathway, the pathway
converting 4-hydroxybutyryl-CoA to 1,3-BDO, as exemplified by the
pathways shown in FIG. 3.
[0094] This invention is also directed, in part to engineered
biosynthetic pathways to improve carbon flux through a central
metabolism intermediate en route to 1,3-butanediol. 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
1,3-butanediol. 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.
[0095] 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
1,3-butanediol by (i) enhancing carbon fixation via the reductive
TCA cycle, and/or (ii) accessing additional reducing equivalents
from gaseous carbon sources and/or syngas components such as CO,
CO.sub.2, and/or H.sub.2. In addition to syngas, other sources of
such gases include, but are not limited to, the atmosphere, either
as found in nature or generated.
[0096] 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 (FIGS. 8a and 9a). 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.
[0097] In some embodiments, the reductive TCA cycle, coupled with
carbon monoxide dehydrogenase and/or hydrogenase enzymes (FIGS. 8a
and 9a insert), 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.
[0098] 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 RICA 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 of NADH, 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.
[0099] In some embodiments, a non-naturally occurring microbial
organism has a 1,3-butanediol pathway and includes at least one
exogenous nucleic acid encoding a 1,3-butanediol pathway enzyme
expressed in a sufficient amount to produce 1,3-butanediol; wherein
the non-naturally occurring microbial organism further
includes:
[0100] A non-naturally occurring microbial organism having a
1,3-butanediol pathway, wherein said microbial organism comprises
at least one exogenous nucleic acid encoding a 1,3-butanediol
pathway enzyme expressed in a sufficient amount to produce
1,3-butanediol; said non-naturally occurring microbial organism
further comprising:
[0101] (i) a reductive TCA pathway, wherein said microbial organism
comprises at least one exogenous nucleic acid encoding a reductive
TCA pathway enzyme selected from the group consisting of an
ATP-citrate lyase, citrate lyase, a fumarate reductase, and an
alpha-ketoglutarate:ferredoxin oxidoreductase;
[0102] (ii) a reductive TCA pathway, wherein said microbial
organism comprises at least one exogenous nucleic acid encoding a
reductive TCA pathway enzyme selected from the group consisting of
a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate
carboxylase, a phosphoenolpyruvate carboxykinase, a CO
dehydrogenase, and an H.sub.2 hydrogenase; or
[0103] (iii) at least one exogenous nucleic acid encodes an enzyme
selected from the group consisting of a CO dehydrogenase, an
H.sub.2 hydrogenase, and combinations thereof;
[0104] wherein said 1,3-butanediol pathway comprises a pathway
selected from the group consisting of:
[0105] (a) (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an
AKP dehydrogenase; (3) a 2-amino-4-hydroxypentanoate
aminotransferase or oxidoreductase (deaminating); (4) a
2-oxo-4-hydroxypentanoate decarboxylase; and (5) a
3-hydroxybutyraldehyde reductase;
[0106] (b) (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an
AKP aminotransferase or oxidoreductase (deaminating); (3) a
2,4-dioxopentanoate decarboxylase; (4) a 3-oxobutyraldehyde
reductase (ketone reducing); and (5) a 3-hydroxybutyraldehyde
reductase;
[0107] (c) (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an
AKP aminotransferase or oxidoreductase (deaminating); (3) a
2,4-dioxopentanoate decarboxylase; (4) a 3-oxobutyraldehyde
reductase (aldehyde reducing); and (5) a 4-hydroxy-2-butanone
reductase;
[0108] (d) (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an
AKP decarboxylase; (3) a 4-aminobutan-2-one aminotransferase or
oxidoreductase (deaminating); (4) a 3-oxobutyraldehyde reductase
(ketone reducing); and (5) a 3-hydroxybutyraldehyde reductase;
[0109] (e) (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an
AKP decarboxylase; (3) a 4-aminobutan-2-one aminotransferase or
oxidoreductase (deaminating); (4) a 3-oxobutyraldehyde reductase
(aldehyde reducing); and (5) a 4-hydroxy-2-butanone reductase;
[0110] (f) (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an
AKP decarboxylase; (3) a 4-aminobutan-2-one ammonia-lyase; (4) a
butenone hydratase; and (5) a 4-hydroxy-2-butanone reductase;
[0111] (g) (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an
AKP ammonia-lyase; (3) an acetylacrylate decarboxylase; (4) a
butenone hydratase; and (5) a 4-hydroxy-2-butanone reductase;
[0112] (h) (1) an acetoacetyl-CoA reductase (CoA-dependent,
aldehyde forming); (2) a 3-oxobutyraldehyde reductase (ketone
reducing); and (3) a 3-hydroxybutyraldehyde reductase;
[0113] (i) (1) an acetoacetyl-CoA reductase (CoA dependent, alcohol
forming) and (2) a 4-hydroxy-2-butanone reductase;
[0114] (j) (1) an acetoacetyl-CoA reductase (CoA-dependent,
aldehyde forming); (2) a 3-oxobutyraldehyde reductase (aldehyde
reducing); and (3) a 4-hydroxy-2-butanone reductase;
[0115] (k) (1) an acetoacetyl-CoA reductase (ketone reducing) and
(2) a 3-hydroxybutyryl-CoA reductase (alcohol forming);
[0116] (l) (1) an acetoacetyl-CoA reductase (ketone reducing); (2)
a 3-hydroxybutyryl-CoA reductase (aldehyde forming); and (3) a
3-hydroxybutyraldehyde reductase;
[0117] (m) (1) a 4-hydroxybutyryl-CoA dehydratase; (2) a crotonase;
and (3) a 3-hydroxybutyryl-CoA reductase (alcohol forming); and
[0118] (n) (1) a 4-hydroxybutyryl-CoA dehydratase; (2) a crotonase;
(3) a 3-hydroxybutyryl-CoA reductase (aldehyde forming); and (4) a
3-hydroxybutyraldehyde reductase;
[0119] (o) (1) a succinyl-CoA transferase, a succinyl-CoA
synthetase or a succinyl-CoA ligase, (2) a succinyl-CoA reductase
(aldehyde forming), (3) a 4-hydroxybutyrate dehydrogenase, (4) a
4-hydroxybutyrate kinase, (5) a phosphotrans-4-hydroxybutyrylase,
(6) a 4-hydroxybutyryl-CoA dehydratase, (7) a crotonase, (8) a
3-hydroxybutyryl-CoA reductase (aldehyde forming), and (9) a
3-hydroxybutanal reductase;
[0120] (p) (1) (i) an alpha-ketoglutarate decarboxylase, or (ii)
(a) a glutamate dehydrogenase and/or a glutamate transaminase, (b)
a glutamate decarboxylase, and (c) a 4-aminobutyrate dehydrogenase
and/or a 4-aminobutyrate transaminase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase; (6) a crotonase, (7) a 3-hydroxybutyryl-CoA reductase
(aldehyde forming), and (8) a 3-hydroxybutanal reductase;
[0121] (q) (1) (i) an alpha-ketoglutarate decarboxylase, or (ii)
(a) a glutamate dehydrogenase and/or a glutamate transaminase, (b)
a glutamate decarboxylase, and (c) a 4-aminobutyrate dehydrogenase
and/or a 4-aminobutyrate transaminase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA
dehydratase, (5) a crotonase, (6) a 3-hydroxybutyryl-CoA reductase
(aldehyde forming), and (7) a 3-hydroxybutanal reductase;
[0122] (r) (1) (i) an alpha-ketoglutarate decarboxylase, or (ii)
(a) a glutamate dehydrogenase and/or a glutamate transaminase, (b)
a glutamate decarboxylase, and (c) a 4-aminobutyrate dehydrogenase
and/or a 4-aminobutyrate transaminase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, and (7) a 3-hydroxybutyryl-CoA
reductase (alcohol forming);
[0123] (s) (1) (i) an alpha-ketoglutarate decarboxylase, or (ii)
(a) a glutamate dehydrogenase and/or a glutamate transaminase, (b)
a glutamate decarboxylase, and (c) a 4-aminobutyrate dehydrogenase
and/or a 4-aminobutyrate transaminase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA
dehydratase, (5) a crotonase, and (6) a 3-hydroxybutyryl-CoA
reductase (alcohol forming);
[0124] (t) (1) (i) an alpha-ketoglutarate decarboxylase, or (ii)
(a) a glutamate dehydrogenase and/or a glutamate transaminase, (b)
a glutamate decarboxylase, and (c) a 4-aminobutyrate dehydrogenase
and/or a 4-aminobutyrate transaminase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA hydrolase,
transferase or synthetase, and (8) a 3-hydroxybutyrate
reductase;
[0125] (u) (1) (i) an alpha-ketoglutarate decarboxylase, or (ii)
(a) a glutamate dehydrogenase and/or a glutamate transaminase, (b)
a glutamate decarboxylase, and (c) a 4-aminobutyrate dehydrogenase
and/or a 4-aminobutyrate transaminase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA
dehydratase, (5) a crotonase, (6) a 3-hydroxybutyryl-CoA hydrolase,
transferase or synthetase, and (7) a 3-hydroxybutyrate
reductase.
[0126] (v) (1) a succinate reductase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA reductase
(aldehyde forming), and (8) a 3-hydroxybutanal reductase;
[0127] (w) (1) a succinate reductase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA
dehydratase, (5) a crotonase, (6) a 3-hydroxybutyryl-CoA reductase
(aldehyde forming), (7) a 3-hydroxybutanal reductase;
[0128] (x) (1) a succinate reductase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, and (7) a 3-hydroxybutyryl-CoA
reductase (alcohol forming);
[0129] (y) (1) a succinate reductase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA
dehydratase, (5) a crotonase, and (6) a 3-hydroxybutyryl-CoA
reductase (alcohol forming);
[0130] (z) (1) a succinate reductase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA hydrolase,
transferase or synthetase, and (8) a 3-hydroxybutyrate
reductase;
[0131] (aa) (1) a succinate reductase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA
dehydratase, (5) a crotonase, (6) a 3-hydroxybutyryl-CoA hydrolase,
transferase or synthetase, and (7) a 3-hydroxybutyrate
reductase;
[0132] (bb) (1) a succinyl-CoA transferase, succinyl-CoA synthetase
or succinyl-CoA ligase, (2) a succinyl-CoA reductase (aldehyde
forming), (3) a 4-hydroxybutyrate dehydrogenase, (5) a
4-hydroxybutyrate kinase, (6) a pPhosphotrans-4-hydroxybutyrylase,
(7) a 4-hydroxybutyryl-CoA dehydratase, (8) a crotonase, and (9) a
3-hydroxybutyryl-CoA reductase (alcohol forming);
[0133] (cc) (1) a succinyl-CoA transferase, succinyl-CoA synthetase
or succinyl-CoA ligase, (2) a succinyl-CoA reductase (aldehyde
forming), (3) a 4-hydroxybutyrate dehydrogenase, (4) a
4-hydroxybutyrate kinase, (5) a phosphotrans-4-hydroxybutyrylase,
(6) a 4-hydroxybutyryl-CoA dehydratase, (7) a crotonase, (8) a
3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, and (9)
a 3-hydroxybutyrate reductase;
[0134] (dd) (1) a succinyl-CoA transferase, succinyl-CoA synthetase
or succinyl-CoA ligase, (2) a succinyl-CoA reductase (alcohol
forming), (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA reductase
(aldehyde forming), and (8) a 3-hydroxybutanal reductase;
[0135] (ee) (1) a succinyl-CoA transferase, succinyl-CoA synthetase
or succinyl-CoA ligase, (2) a succinyl-CoA reductase (alcohol
forming), (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, and (7) a 3-hydroxybutyryl-CoA
reductase (alcohol forming);
[0136] (ff) (1) a succinyl-CoA transferase, succinyl-CoA synthetase
or succinyl-CoA ligase, (2) a succinyl-CoA reductase (alcohol
forming), (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA hydrolase,
transferase or synthetase, and (8) a 3-hydroxybutyrate
reductase;
[0137] (gg) (1) a succinyl-CoA transferase, succinyl-CoA synthetase
or succinyl-CoA ligase, (2) a succinyl-CoA reductase (aldehyde
forming), (3) a 4-hydroxybutyrate dehydrogenase, (4) a
4-hydroxybutyryl-CoA transferase, or 4-hydroxybutyryl-CoA
synthetase, (5) a 4-hydroxybutyryl-CoA dehydratase, (6) a
crotonase, (7) a 3-hydroxybutyryl-CoA reductase (aldehyde forming),
and (8) a 3-hydroxybutanal reductase;
[0138] (hh) (1) a succinyl-CoA transferase, succinyl-CoA synthetase
or succinyl-CoA ligase, (2) a succinyl-CoA reductase (aldehyde
limning), (3) a 4-hydroxybutyrate dehydrogenase, (4) a
4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA
synthetase, (5) a 4-hydroxybutyryl-CoA dehydratase, (6) a
crotonase, and (7) a 3-hydroxybutyryl-CoA reductase (alcohol
forming);
[0139] (ii) (1) a succinyl-CoA transferase, succinyl-CoA synthetase
or succinyl-CoA ligase, (2) a succinyl-CoA reductase (aldehyde
forming), (3) a 4-hydroxybutyrate dehydrogenase, (4) a
4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA
synthetase, (5) a 4-hydroxybutyryl-CoA dehydratase, (6) a
crotonase, (7) a 3-hydroxybutyryl-CoA hydrolase, transferase or
synthetase, (8) a 3-hydroxybutyrate reductase;
[0140] (jj) (1) a succinyl-CoA transferase, succinyl-CoA synthetase
or succinyl-CoA ligase, (2) a succinyl-CoA reductase (alcohol
forming), (3) a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA
dehydratase, (5) a crotonase, (6) a 3-hydroxybutyryl-CoA reductase
(aldehyde forming), and (7) a 3-hydroxybutanal reductase;
[0141] (kk) (1) a succinyl-CoA transferase, succinyl-CoA synthetase
or succinyl-CoA ligase, (2) a succinyl-CoA reductase (alcohol
forming), (3) a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA
dehydratase, (5) a crotonase, and (6) a 3-hydroxybutyryl-CoA
reductase (alcohol forming); and
[0142] (ll) (1) a succinyl-CoA transferase, succinyl-CoA synthetase
or succinyl-CoA ligase, (2) a succinyl-CoA reductase (alcohol
forming), (3) a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA hydrolase,
transferase or synthetase, and (8) a 3-hydroxybutyrate
reductase.
[0143] In some embodiments, the non-naturally occurring microbial
organism (e.g., having pathway (i)) further includes 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.
[0144] In some embodiments, the non-naturally occurring microbial
organism (e.g., having pathway (ii)) further includes 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.
[0145] In some embodiments, the non-naturally occurring microbial
organism includes two, three, four, five, six, seven, eight or nine
exogenous nucleic acids, each encoding a 1,3-BDO pathway
enzyme.
[0146] In some embodiments, the non-naturally occurring microbial
organism comprises exogenous nucleic acids encoding each of the
enzymes of at least one of the 1,3-butanediol pathways selected
from the group consisting of (a)-(ll).
[0147] In some embodiments, the non-naturally occurring microbial
organism has at least one exogenous nucleic acid is a heterologous
nucleic acid.
[0148] In some embodiments, the non-naturally occurring microbial
organism is in a substantially anaerobic culture medium.
[0149] 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)).
[0150] 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 during the conversion of
pyruvate to malate by malic enzyme and during the conversion of
pyruvate to oxaloacetate by pyruvate carboxylase.
[0151] 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.
[0152] 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 FIGS. 8A and 9A).
Enzymes and the corresponding genes required for these activities
are described herein below.
[0153] 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
1,3-butanediol 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.
[0154] In some embodiments, a 1,3-butanediol 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.
[0155] In some embodiments a non-naturally occurring microbial
organism having a 1,3-butanediol 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, an isocitrate
dehydrogenase, an aconitase, an isocitrate dehydrogenase, 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.
[0156] In some embodiments a method includes culturing a
non-naturally occurring microbial organism having a 1,3-butanediol
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, an isocitrate dehydrogenase, an aconitase, an
isocitrate dehydrogenase, 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.
[0157] In some embodiments a non-naturally occurring microbial
organism having a 1,3-butanediol 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, an
isocitrate dehydrogenase, an aconitase, an isocitrate
dehydrogenase, a pyruvate:ferredoxin oxidoreductase and an
alpha-ketoglutarate:ferredoxin oxidoreductase.
[0158] In some embodiments a non-naturally occurring microbial
organism having a 1,3-butanediol 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 1,3-butanediol.
[0159] In some embodiments, the non-naturally occurring microbial
organism having a 1,3-butanediol pathway includes two exogenous
nucleic acids each encoding a reductive TCA pathway enzyme. In some
embodiments, the non-naturally occurring microbial organism having
a 1,3-butanediol 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, any one of the three exogenous nucleic acids can
be an isocitrate dehydrogenase. 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 H2 hydrogenase. In some embodiments, the
non-naturally occurring microbial organism includes two exogenous
nucleic acids encoding a CO dehydrogenase and an H2
hydrogenase.
[0160] In some embodiments, the non-naturally occurring microbial
organisms having a 1,3-butanediol 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.
[0161] In some embodiments, the non-naturally occurring microbial
organism having a 1,3-butanediol 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.
[0162] In some embodiments, the non-naturally occurring microbial
organism having a 1,3-butanediol 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 1,3-butanediol pathway utilizes hydrogen for reducing
equivalents. In some embodiments, the non-naturally occurring
microbial organism having a 1,3-butanediol pathway utilizes CO for
reducing equivalents. In some embodiments, the non-naturally
occurring microbial organism having a 1,3-butanediol pathway
utilizes combinations of CO and hydrogen for reducing
equivalents.
[0163] In some embodiments, the non-naturally occurring microbial
organism having a 1,3-butanediol 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.
[0164] In some embodiments, the non-naturally occurring microbial
organism having a 1,3-butanediol 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.
[0165] In some embodiments, the non-naturally occurring microbial
organism having a 1,3-butanediol 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,
an acetyl-CoA transferase, and a ferredoxin.
[0166] 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.
[0167] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having a 1,3-butanediol
pathway, wherein the non-naturally occurring microbial organism
comprises at least one exogenous nucleic acid encoding an enzyme or
protein that converts a substrate to a product selected from the
group consisting of pyruvate to alanine, alanine to
2-amino-4-oxopentanoate, 2-amino-4-oxopentanoate to
2-amino-4-hydroxypentanoate, 2-amino-4-oxopentanoate to
2,4-dioxopentanoate, 2-amino-4-oxopentanoate to 4-aminobutan-2-one,
2-amino-4-oxopentanoate to acetylacrylate,
2-amino-4-hydroxypentanoate to 2-oxo-4-hydroxypentanoate,
2-oxo-4-hydroxypentanoate to 3-hydroxybutyraldehyde,
2,4-dioxopentanoate to 3-oxobutyraldehyde, 3-oxobutyraldehyde to
3-hydroxybutryaldehyde, 3-oxobutyraldehdye to 4-hydroxy-2-butanone,
4-aminobutan-2-one to 3-oxobutyraldehyde, 4-aminobutan-2-one to
butanone, butanone to 4-hydroxy-2-butanone and acetylacrylate to
butenone. 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 1,3-butanediol pathway, such as that shown in FIG.
8B.
[0168] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having a 1,3-butanediol
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 succinate to succinyl-CoA, succinate to
succinic semialdehyde, succinyl-CoA to succinic semialdehyde,
alpha-ketoglutarate to succinic semialdehyde, succinyl-CoA to
4-hydroxybutyrate, succinic semialdehyde to 4-hydroxybutyrate,
4-hydroxybutyrate to 4-hydroxybutyryl phosphate, 4-hydroxybutyrate
to 4-hydroxybutyryl-CoA, 4-hydroxybutyryl phosphate to
4-hydroxybutyryl CoA, 4-hydroxybutyryl-CoA to crotonyl-CoA,
crotonyl-CoA to 3-hydroxybutyryl-CoA, 3-hydroxybutyryl-CoA to
3-hydroxybutyrate, 3-hydroxybutyrate to 1,3-butanediol,
3-hydroxybutyryl-CoA to 1,3-butanediol, 3-hydroxybutyryl-CoA to
3-hydroxybutanal, 3-hydroxybutanal to 1,3-butanediol. 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 1,3-butanediol pathway, such as
that shown in FIG. 9B.
[0169] While generally described herein as a microbial organism
that contains a 1,3-butanediol pathway, it is understood that the
invention additionally provides a non-naturally occurring microbial
organism comprising at least one exogenous nucleic acid encoding a
1,3-butanediol pathway enzyme expressed in a sufficient amount to
produce an intermediate of a 1,3-butanediol pathway. For example,
as disclosed herein, a 1,3-butanediol pathway is exemplified in
FIGS. 1-3, 8A/B, and 9A/B. Therefore, in addition to a microbial
organism containing a 1,3-butanediol pathway that produces
1,3-butanediol, the invention additionally provides a non-naturally
occurring microbial organism comprising at least one exogenous
nucleic acid encoding a 1,3-butanediol pathway enzyme, where the
microbial organism produces a 1,3-butanediol pathway intermediate,
for example, alanine, 2-amino-4-pentanoate,
2-amino-4-hydroxypentanoate, 2-oxo-4-hydroxypentanoate,
3-hydroxybutyraldehyde, 2,4-dioxopentanoate, 3-oxobutyraldehyde,
4-aminobutan-2-one, acetylacrylate, butanone, 4-hydroxy-2-butanone,
succinic semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl
phosphate, 4-hydroxybutyryl-CoA, crotonyl-CoA,
3-hydroxybutyryl-CoA, 3-hydroxybutyrate, and 3-hydroxybutanal.
[0170] It is understood that any of the pathways disclosed herein,
as described in the Examples and exemplified in the Figures,
including the pathways of FIGS. 1-3, 8A/B, and 9A/B 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 1,3-butanediol pathway
intermediate can be utilized to produce the intermediate as a
desired product.
[0171] Successfully engineering any of these pathways entails
identifying an appropriate set of enzymes with sufficient activity
and specificity, cloning their corresponding genes into a
production host, optimizing fermentation conditions, and assaying
for product formation following fermentation. To engineer a
production host for the production of any of the aforementioned
products, one or more exogenous DNA sequence(s) can be expressed in
microorganisms. In addition, the microorganisms can have endogenous
gene(s) functionally deleted. These modifications will enable the
production of 1,3-BDO using renewable feedstocks.
[0172] Below, are described a number of biochemically characterized
genes capable of encoding enzymes that catalyze each of the steps
shown in FIGS. 1, 2 3, 8B, and 9B. Although we describe this method
for E. coli, one skilled in the art can apply these teachings to
essentially any other organism. Specifically, genes are listed that
are native to E. coli in addition to genes in other organisms that
can be applied to catalyze the appropriate transformations when
properly cloned and expressed.
[0173] 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.
[0174] As disclosed herein, intermediates en route to
1,3-butanediol can be carboxylic acids or CoA esters thereof, such
as 4-hydroxy butyrate, 3-hydroxybutyrate, their CoA esters, as well
as crotonyl-CoA. Any carboxylic acid intermediate 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
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
4-hydroxybutyrate, methyl-3-hydroxybutyrate, ethyl
4-hydroxybutyrate, ethyl 3-hydroxybutyrate, n-propyl
4-hydroxybutyrate, and n-propyl 3-hydroxybutyrate. 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.
[0175] All transformations depicted in FIGS. 1-3, 8B and 9B fall
into the 8 general categories of transformations shown in Table 1.
Below is described a number of biochemically characterized genes in
each category. Specifically listed are genes that can be applied to
catalyze the appropriate transformations in FIGS. 1-3, 8B, and 9B
when properly cloned and expressed. Exemplary genes for each of the
steps in FIGS. 1-3, 8B and 9B are provided further below in Tables
35-37.
[0176] Table 1 shows the enzyme types useful to convert common
central metabolic intermediates into 1,3-butanediol. The first
three digits of each label correspond to the first three Enzyme
Commission number digits which denote the general type of
transformation independent of substrate specificity.
TABLE-US-00001 TABLE 1 LABEL FUNCTION 1.1.1.a Oxidoreductase
(ketone to hydroxyl or aldehyde to alcohol) 1.1.1.c Oxidoreductase
(2 step, acyl-CoA to alcohol) 1.2.1.b Oxidoreductase (acyl-CoA to
aldehyde) 1.4.1.a Oxidoreductase (deaminating) 2.3.1.b
Acyltransferase 2.6.1.a Aminotransferase 4.1.1.a Carboxy-lyase
4.2.1.a Hydro-lyase 4.3.1.a Ammonia-lyase
[0177] Numerous transformation in FIGS. 1, 2 and 3 fall into the
category of oxidoreductases that reduce an aldehyde to alcohol. For
example, Steps D and P in FIG. 1 catalyzed by 3-oxobutyraldehyde
reductase (aldehyde reducing) and 3-hydroxybutyraldehyde reductase
respectively fall into this category. Similarly, Steps C and E in
FIG. 2 catalyzed by 3-hydroxybutyraldehyde reductase and
3-oxobutyraldehdye reductase (aldehyde reducing) respectively are
also oxidoreductases that convert the aldehyde functionality to
alcohol. Pathways in FIG. 3 involve oxidoreductases such as
3-hydroxybutyraldehdye reductase in Step D.
[0178] 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 C3
(Sulzenbacher et al., J. of Molecular Biology, 342:489-502 (2004)),
and bdh I and bdh II from C. acetobutylicum which converts
butyraldehyde into butanol (Walter et al., J. of Bacteriology,
174:7149-7158 (1992)). The gene product of yqhD catalyzes the
reduction of acetaldehyde, malondialdehyde, propionaldehyde,
butyraldehyde, and acrolein using NADPH as the cofactor (Perez et
al., J. Biol. Chem., 283:7346-7353 (2008)). 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)). Additional
aldehyde reductase candidates are encoded by bdh in C.
saccharoperbutylacetonicum and Cbei.sub.--1722, Cbei.sub.--2181 and
Cbei.sub.--2421 in C. beijerinckii.
[0179] Data related to the sequences for each of these exemplary
gene products can be found using the following GenBank accession
numbers shown in Table 2.
TABLE-US-00002 TABLE 2 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 bdh
BAF45463.1 124221917 Clostridium saccharoperbutyl- acetonicum
Cbei_1722 YP_001308850 150016596 Clostridium beijerinckii Cbei_2181
YP_001309304 150017050 Clostridium beijerinckii Cbei_2421
YP_001309535 150017281 Clostridium beijerinckii
[0180] Enzymes exhibiting 3-hydroxybutyraldehyde reductase activity
(EC 1.1.1.61) also fall into this category. Such enzymes have been
characterized in Ralstonia eutropha (Bravo 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)). Yet
another gene is the alcohol dehydrogenase adhI from Geobacillus
thermoglucosidasius (Jeon et al., J. Biotechnol., 135:127-133
(2008)). Data related to the sequences for each of these exemplary
gene products can be found using the following GenBank accession
numbers shown in Table 3.
TABLE-US-00003 TABLE 3 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 adhI AAR91477.1 40795502 Geobacillus
thermoglucosidasius M10EXG
[0181] Another exemplary enzyme is 3-hydroxyisobutyrate
dehydrogenase which catalyzes the reversible oxidation of
3-hydroxyisobutyrate to methylmalonate semialdehyde. This enzyme
participates in valine, leucine and isoleucine degradation and has
been identified in bacteria, eukaryotes, and mammals. The enzyme
encoded by P84067 from Thermus thermophilus HB8 has been
structurally characterized (Lokanath et al., J. Mol. Biol.,
352:905-917 (2005)). The reversibility of the human
3-hydroxyisobutyrate dehydrogenase was demonstrated using
isotopically-labeled substrate (Manning et al., Biochem J.,
231:481-484 (1985)). Additional genes encoding this enzyme include
3hidh in Homo sapiens (Hawes et al., Methods Enzymol, 324:218-228
(2000)) and Oryctolagus cuniculus (Hawes et al., supra; Chowdhury
et al., Biosci. Biotechnol Biochem., 60:2043-2047 (1996)), mmsB in
Pseudomonas aeruginosa and Pseudomonas putida (Liao et al., US
patent 20050221466), and dhat in Pseudomonas putida (Aberhart et
al., J. Chem. Soc., 6:1404-1406 (1979); Chowdhury et al., supra;
Chowdhury et al., Biosci. Biotechnol Biochem., 67:438-441 (2003)).
Data related to the sequences for each of these exemplary gene
products can be found using the following GenBank accession numbers
shown in Table 4.
TABLE-US-00004 TABLE 4 PROTEIN GENBANK ID GI NUMBER ORGANISM P84067
P84067 75345323 Thermus thermophilus 3hidh P31937.2 12643395 Homo
sapiens 3hidh P32185.1 416872 Oryctolagus cuniculus mmsB P28811.1
127211 Pseudomonas aeruginosa mmsB NP_746775.1 26991350 Pseudomonas
putida dhat Q59477.1 2842618 Pseudomonas putida
[0182] Oxidoreductases that convert a ketone functionality to the
corresponding hydroxyl group are also synthetic steps in the
disclosed pathways. Notably, Reactions L, O and H in FIG. 1
catalyzed by AKP dehydrogenase, 3-oxobutyraldehyde reductase
(ketone reducing), 4-hydroxy-2-butanone reductase respectively are
transformations of this category. The two latter transformations
are also encountered in Steps B and F respectively in FIG. 2. On
similar lines, the acetoacetyl-CoA reductase in Step G of FIG. 2
reduces acetoacetyl-CoA to 3-hydroxybutyryl-CoA.
[0183] The reduction of 4-oxo group of 2-amino-4-oxopentanoate
(AKP) by a dehydrogenase yields 2-amino-4-hydroxypentanoate (FIG.
1, step L). This reaction is very similar to the NAD(P)H-dependent
reduction of aspartate semialdehyde to homoserine catalyzed by
homoserine dehydrogenase (EC 1.1.13). 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
(1973)). 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, 152:205-112
(2006)) and Arabidopsis thaliana. The monofunctional homoserine
dehydrogenases encoded by hom6 in S. cerevisiae (Jacques et al.,
Biochem. Biophys. Acta, 1544:28-41 (2001)) and hom2 in
Lactobacillus plantarum (Cahyanto et al., supra) have been
functionally expressed and characterized in E. coli. Data related
to the sequences for each of these exemplary gene products can be
found using the following GenBank accession numbers shown in Table
5.
TABLE-US-00005 TABLE 5 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
[0184] Acetoacetyl-CoA reductase (Step G, FIG. 2) 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., 71C:403-411 (1981)). Yet other genes 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 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). 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.,
J. Biol. Chem., 207:631-638 (1954)). Data related to the sequences
for each of these exemplary gene products can be found using the
following GenBank accession numbers shown in Table 6.
TABLE-US-00006 TABLE 6 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 Clostridium
acetobutylicum HSD17B10 O02691.3 3183024 Bos Taurus phbB P23238.1
130017 Zoogloea ramigera phaB YP_353825.1 77464321 Rhodobacter
sphaeroides
[0185] A number of similar enzymes have been found in other species
of Clostridia and in Metallosphaera sedula (Berg et al., Archaea.
Science, 318:1782-1786 (2007)) as shown in Table 7.
TABLE-US-00007 TABLE 7 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
[0186] An exemplary alcohol dehydrogenase that converts a ketone to
a hydroxyl group is the secondary alcohol dehydrogenase that was
shown to convert acetone to isopropanol 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)). The gene product of adhA from
Pyrococcus furiosus, which exhibits maximum activity on 2-pentanol
and pyruvaldehyde, was shown to have very broad specificity which
includes isopropanol and acetone (Van der et al., Eur. J. Biochem.,
268:3062-3068 (2001)). Yet another secondary alcohol dehydrogenase
with activity on isopropanol and acetone is encoded by the gene
product of adh-A from Rhodococcus ruber (Edegger et al., Chem.
Commun. (Camb), 2402-2404 (2006); Kosjek et al., Biotechnol.
Bioeng., 86:55-62 (2004)). These genes along with others are listed
below in Table 8.
TABLE-US-00008 TABLE 8 Protein GenBank ID GI number Organism 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
[0187] Alternatively, 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 substrates of various chain lengths
such as lactate, 2-oxobutyrate, 2-oxopentanoate and 2-oxoglutarate
(Steinbuchel et al., Eur. J. Biochem., 130:329-334 (1983)).
Conversion of the oxo functionality to the hydroxyl group can also
be catalyzed by 2-keto-1,3-butanediol 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)). All of these enzymes can
provide a 3-oxobutyraldehyde reductase, and a 4-hydroxy-2-butanone
reductase. An additional enzyme for these steps 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. Data related to the sequences for
each of these exemplary gene products can be found using the
following GenBank accession numbers shown in Table 9.
TABLE-US-00009 TABLE 9 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
[0188] 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. (1995).
[0189] Several transformations in FIGS. 2 and 3 rely on the
two-step reduction of acyl-CoA to the corresponding alcohol. For
example, Steps D and I in FIG. 2, involving the acetoacetyl-CoA
reductase (CoA-dependent, alcohol forming) and 3-hydroxybutyryl-CoA
reductase (alcohol forming), and Step E in FIG. 3 involving
3-hydroxybutyryl-CoA reductase (alcohol forming), shows such a
transformation.
[0190] Exemplary two-step oxidoreductases that convert an acyl-CoA
to alcohol include those that transform substrates such as
acetyl-CoA to ethanol (e.g., adhE from E. coli (Kessler 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)). In addition to reducing acetyl-CoA to ethanol,
the enzyme encoded by adhE in Leuconostoc mesenteroides has been
shown to oxidize the branched chain compound isobutyraldehyde to
isobutyryl-CoA (Kazahaya et al., J. Gen. Appl. Microbiol., 18:43-55
(1972); Koo et al., Biotechnol. Lett., 27:505-510 (2005)). Data
related to the sequences for each of these exemplary gene products
can be found using the following GenBank accession numbers shown in
Table 10.
TABLE-US-00010 TABLE 10 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
[0191] 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., J. Bacteriol.,
184:2404-2410 (2002); Strauss et al., Eur. J. Biochem., 215:633-643
(1993)). This enzyme, with a mass of 300 kDa, is highly
substrate-specific and shows little sequence similarity to other
known oxidoreductases (Hugler et al., supra). 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)). Enzymes in other organisms including
Roseiflexus castenholzii, Erythrobacter sp. NAP1 and marine gamma
proteobacterium HTCC2080 can be inferred by sequence similarity.
Data related to the sequences for each of these exemplary gene
products can be found using the following GenBank accession numbers
shown in Table 11.
TABLE-US-00011 TABLE 11 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
[0192] Longer chain acyl-CoA molecules can be reduced by enzymes
such as the jojoba (Simmondsia chinensis) FAR which encodes an
alcohol-forming fatty acyl-CoA reductase. Its overexpression in E.
coli resulted in FAR activity and the accumulation of fatty alcohol
(Metz et al., Plant Physiology, 122:635-644 (2000)) (FAR,
AAD38039.1, 5020215, Simmondsia chinensis).
[0193] The pathways disclosed herein involve numerous
oxidoreductase-type transformations that convert an acyl-CoA to an
aldehyde. Specifically, Steps A and H in FIG. 2 catalyzed by
acetoacetyl-CoA reductase (aldehyde forming) and
3-hydroxybutyryl-CoA reductase (aldehyde forming), and Step C from
FIG. 3 showing the transformation catalyzed by 3-hydroxybutyryl-CoA
reductase.
[0194] Several acyl-CoA dehydrogenases are capable of reducing an
acyl-CoA to its corresponding aldehyde. Exemplary genes that encode
such enzymes include the Acinetobacter calcoaceticus acr1 encoding
a fatty acyl-CoA reductase (Reiser et al., J. of Bacteriology,
179:2969-2975 (1997)), the Acinetobacter sp. M-1 fatty acyl-CoA
reductase (Ishige et al., Appl. Environ. Microbiol., 68:1192-1195
(2002)), and a CoA- and NADP-dependent succinate semialdehyde
dehydrogenase encoded by the sucD gene in Clostridium kluyveri
(Sohling et al., J. Bacteriol., 178:871-880 (1996)). SucD of P.
gingivalis is another succinate semialdehyde dehydrogenase
(Takahashi et al., J. Bacteriol., 182:4704-4710 (2000)). The enzyme
acylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded by
bphG, is yet another enzyme demonstrated to oxidize and acylate
acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde and
formaldehyde (Powlowski et al., J. Bacteriol., 175:377-385 (1993)).
In addition to reducing acetyl-CoA to ethanol, the enzyme encoded
by adhE in Leuconostoc mesenteroides has been shown to oxidize the
branched chain compound isobutyraldehyde to isobutyryl-CoA
(Kazahaya et al., supra; Koo et al., supra). 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., 71:58-61 (2007)). Additional aldehyde
dehydrogenase enzyme candidates are found in Desulfatibacillum
alkenivorans, Citrobacter koseri, Salmonella enterica,
Lactobacillus brevis and Bacillus selenitireducens. Data related to
the sequences for each of these exemplary gene products can be
found using the following GenBank accession numbers shown in Table
12.
TABLE-US-00012 TABLE 12 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 ald ACL06658.1 218764192
Desulfatibacillum alkenivorans AK-01 ald YP_001452373 157145054
Citrobacter koseri ATCC BAA- 895 pduP NP_460996.1 16765381
Salmonella enterica Typhimurium pduP ABJ64680.1 116099531
Lactobacillus brevis ATCC 367 BselDRAFT_1651 ZP_02169447 163762382
Bacillus selenitireducens MLS10
[0195] An additional enzyme type that converts an acyl-CoA to its
corresponding aldehyde is malonyl-CoA reductase which transforms
malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is a key
enzyme in autotrophic carbon fixation via the 3-hydroxypropionate
cycle in thermoacidophilic archaeal bacteria (Berg et al., supra;
Thauer, R. K., Science, 318:1732-1733 (2007)). The enzyme utilizes
NADPH as a cofactor and has been characterized in Metallosphaera
and Sulfolobus spp (Alber et al., J. Bacteriol., 188:8551-8559
(2006); Hugler et al., supra). The enzyme is encoded by
Msed.sub.--0709 in Metallosphaera sedula (Alber et al., supra; Berg
et al., supra). A gene encoding a malonyl-CoA reductase from
Sulfolobus tokodaii was cloned and heterologously expressed in E.
coli (Alber et al., supra). This enzyme has also been shown to
catalyze the conversion of methylmalonyl-CoA to its corresponding
aldehyde (2007). Although the aldehyde dehydrogenase functionality
of these enzymes is similar to the bifunctional dehydrogenase from
Chloroflexus aurantiacus, there is little sequence similarity. Both
malonyl-CoA reductase enzymes have high sequence similarity to
aspartate-semialdehyde dehydrogenase, an enzyme catalyzing the
reduction and concurrent dephosphorylation of aspartyl-4-phosphate
to aspartate semialdehyde. Additional genes can be found by
sequence homology to proteins in other organisms including
Sulfolobus solfataricus and Sulfolobus acidocaldarius and have been
listed below. Yet another enzyme for CoA-acylating aldehyde
dehydrogenase is the ald gene from Clostridium beijerinckii (Toth
et al., Appl. Environ. Microbiol., 65:4973-4980 (1999)). This
enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to
their corresponding aldehydes. This gene is very similar to eutE
that encodes acetaldehyde dehydrogenase of Salmonella typhimurium
and E. coli (Toth et al., supra). Data related to the sequences for
each of these exemplary gene products can be found using the
following GenBank accession numbers shown in Table 13.
TABLE-US-00013 TABLE 13 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
[0196] The oxidative deamination of amino groups to their
corresponding oxo groups is catalyzed by deaminating
oxidoreductases in the EC class 1.4.1. Such enzymes utilize
NAD.sup.+, NADP.sup.+ or FAD.sup.+ as acceptor. Enzymes in this
class can convert 2-amino-4-oxopentanoate to 2,4-dioxopentanoate
(FIG. 1, Step B), 2-amino-4-hydroxypentanoate to
2-oxo-4-hydroxypentanoate (FIG. 1, Step M) and 4-aminobutan-2-one
to 3-oxobutyraldehyde (FIG. 1, Step K). Exemplary oxidoreductases
operating on similar substrates include glutamate dehydrogenase
(deaminating), encoded by gdhA, leucine dehydrogenase
(deaminating), encoded by ldh, and aspartate dehydrogenase
(deaminating), encoded by nadX. The gdhA gene product from
Escherichia coli (McPherson et al., Nucleic. Acids Res.
11:5257-5266 (1983); Korber et al., J. Mol. Biol. 234:1270-1273
(1993)), gdh from Thermotoga maritima (Kort et al., Extremophiles
1:52-60 (1997); Lebbink et al., J. Mol. Biol. 280:287-296 (1998);
Lebbink et al., J. Mol. Biol. 289:357-369 (1999)), and gdhA1 from
Halobacterium salinarum (Ingoldsby et al., Gene. 349:237-244
(2005)) catalyze the reversible interconversion of glutamate to
2-oxoglutarate and ammonia, while favoring NADP(H), NAD(H), or
both, respectively. Additional glutamate dehydrogenase gene
candidates are found in Bacillus subtilis (Khan et al., Biosci.
Biotechnol Biochem. 69:1861-1870 (2005)), Nicotiana tabacum
(Purnell et al., Planta 222:167-180 (2005)), Oryza sativa (Abiko et
al., Plant Cell Physiol 46:1724-1734 (2005)), Haloferax
mediterranei (Diaz et al., Extremophiles. 10:105-115 (2006)),
Halobactreium salinarum (Hayden et al., FEMS Microbiol Lett.
211:37-41 (2002)) and yeast (Roca et al., Appl Environ. Microbiol
69:4732-4736 (2003)). The Nicotiana tabacum enzyme is composed of
alpha and beta subunits encoded by gdh1 and gdh2 (Purnell et al.,
Planta 222:167-180 (2005)). The ldh gene of Bacillus cereus encodes
the LeuDH protein that accepts a wide of range of substrates
including leucine, isoleucine, valine, and 2-aminobutanoate (Stoyan
et al., J. Biotechnol 54:77-80 (1997); Ansorge et al., Biotechnol
Bioeng. 68:557-562 (2000)). The nadX gene from Thermotoga maritime
encoding for the aspartate dehydrogenase is involved in the
biosynthesis of NAD (Yang et al., J. Biol. Chem. 278:8804-8808
(2003)). Data related to the sequences for each of these exemplary
gene products can be found using the GenBank accession numbers
shown below in Table 14.
TABLE-US-00014 TABLE 14 Protein GenBank ID GI Number Organism gdhA
P00370 118547 Escherichia coli gdh P96110.4 6226595 Thermotoga
maritima gdhA1 NP_279651.1 15789827 Halobacterium salinarum rocG
NP_391659.1 16080831 Bacillus subtilis gdh1 AAR11534.1 38146335
Nicotiana tabacum gdh2 AAR11535.1 38146337 Nicotiana tabacum GDH
Q852M0 75243660 Oryza sativa GDH Q977U6 74499858 Haloferax
mediterranei GDH P29051 118549 Halobactreium salinarum GDH2
NP_010066.1 6319986 Saccharomyces cerevisiae ldh P0A393 61222614
Bacillus cereus nadX NP_229443.1 15644391 Thermotoga maritima
[0197] An enzyme with 4-aminobutan-2-one oxidoreductase
(deaminating) activity is required to convert 4-aminobutan-2-one to
its corresponding aldehyde (FIG. 1, Step K). Exemplary candidates
include 3,5-diaminohexanoate dehydrogenase (EC 1.4.1.11) and lysine
6-dehydrogenase (EC 1.4.1.18). 3,5-Diaminohexanoate dehydrogenase
interconverts 3-amino acids and 3-oxoacids and has been
characterized in organisms that ferment lysine. The gene encoding
3,5-diaminohexanoate dehydrogenase, kdd, was recently identified in
Fusobacterium nucleatum (Kreimeyer et al., J. Biol. Chem.
282:7191-7197 (2007)). The enzyme has been purified and
characterized in other organisms (Baker et al., J Biol. Chem.
247:7724-7734 (1972); Baker et al., Biochemistry 13:292-299 (1974))
but the genes associated with these enzymes are not known.
Candidates in other sequenced organisms can be inferred by sequence
homology. Lysine 6-dehydrogenase, encoded by the lysDH genes,
catalyzes the conversion of primary amines to their corresponding
aldehydes. This enzyme naturally catalyzes the reversible oxidative
deamination of the 6-amino group of L-lysine to form
2-aminoadipate-6-semialdehyde (Misono et al., J Bacteriol.
150:398-401 (1982)). Exemplary enzymes are found in Geobacillus
stearothermophilus (Heydari et al., Appl Environ. Microbiol
70:937-942 (2004)), Agrobacterium tumefaciens (Hashimoto et al., J
Biochem. 106:76-80 (1989); Misono and Nagasaki, J Bacteriol.
150:398-401 (1982)), and Achromobacter denitrificans
(Ruldeekulthamrong et al., BMB. Rep. 41:790-795 (2008)). Data
related to the sequences for each of these exemplary gene products
can be found using the following GenBank accession numbers shown in
Table 15.
TABLE-US-00015 TABLE 15 Protein GenBank ID GI Number Organism kdd
AAL93966.1 19713113 Fusobacterium nucleatum lysDH BAB39707 13429872
Geobacillus stearothermophilus lysDH NP_353966 15888285
Agrobacterium tumefaciens lysDH AAZ94428 74026644 Achromobacter
denitrificans
[0198] 2-Amino-4-oxopentanoate (AKP) thiolase or AKP thiolase
(AKPT) (Step 1, FIG. 1) is a pyridoxal phosphate-dependent enzyme
participating in ornithine degradation in Clostridium sticklandii
(Jeng et al., A. Biochemistry, 13:2898-2903 (1974); Kenklies et
al., Microbiology, 145:819-826 (1999)). A gene cluster encoding the
alpha and beta subunits of AKPT (or-2 (ortA) and or-3 (ortB)) was
recently identified and the biochemical properties of the enzyme
were characterized (Fonknechten et al., J. Bacteriol., In Press
(2009)). The enzyme is capable of operating in both directions and
reacts with the D-isomer of alanine. Enzyme engineering can be
performed to optimize function with L-alanine as a substrate. AKPT
from Clostridium sticklandii has been characterized but its protein
sequence has not yet been published. Enzymes with high sequence
homology are found in Clostridium difficile, Alkaliphilus
metalliredigenes QYF, Thermoanaerobacter sp. X514, and
Thermoanaerobacter tengcongensis MB4 (Fonknechten et al, supra).
Data related to the sequences for each of these exemplary gene
products can be found using the following GenBank accession numbers
shown in Table 16.
TABLE-US-00016 TABLE 16 Protein GenBank ID GI Number Organism ortA
(A) YP_001086914.1 126698017 Clostridium difficile 630 ortB
(.beta.) YP_001086915.1 126698018 Clostridium difficile 630
Amet_2368 YP_001320181.1 150390132 Alkaliphilus (.alpha.)
metalliredigenes QYF Amet_2369 YP_001320182.1 150390133
Alkaliphilus (.beta.) metalliredigenes QYF Teth514_1478
YP_001663101.1 167040116 Thermoanaerobacter (.alpha.) sp. X514
Teth514_1479 YP_001663102.1 167040117 Thermoanaerobacter (.beta.)
sp. X514 TTE1235 (.alpha.) NP_622858.1 20807687 Thermoanaerobacter
tengcongensis MB4 thrC (.beta.) NP_622859.1 20807688
Thermoanaerobacter tengcongensis MB4
[0199] The conversion of 2-amino-4-oxopentanoate (AKP) to
2,4-dioxopentanoate (Step B, FIG. 1) is accomplished by
2-amino-4-oxopentanoate aminotransferase or oxidoreductase
(deaminating). Selection of an appropriate enzyme for this
transformation is dependent on the stereochemistry of the
substrate. For example, if the substrate is in the D-configuration,
a D-amino acid aminotransferase (EC 2.6.1.21) can be utilized,
whereas the L-stereoisomer can utilize an L-aminotransferase such
as aspartate aminotransferase (EC 2.6.1.1).
[0200] Aspartate aminotransferase transfers an amino group from
aspartate to alpha-ketoglutarate, forming glutamate and
oxaloacetate. Aspartate is similar in structure to
2-amino-4-oxopentanoate. This conversion is catalyzed by, for
example, the gene products of aspC from Escherichia coli (Yagi et
al., FEBS Lett., 100:81-84 (1979); Yagi et al., Methods Enzymol.,
133:83-89 (1985)), AAT2 from Saccharomyces cerevisiae (Yagi et al.,
J. Biochem., 92:35-43 (1982)) and ASP5 from Arabidopsis thaliana
(Kwok et al., J. Exp. Bot., 55:595-604 (2004); De la et al., Plant
J., 46:414-425 (2006); Wilkie et al., Protein Expr. Purif.,
12:381-389 (1998)). The enzyme from Rattus norvegicus has been
shown to transaminate alternate substrates such as
2-aminohexanedioic acid and 2,4-diaminobutyric acid (Recasens et
al., Biochemistry, 19:4583-4589 (1980)). Aminotransferases that
work on other amino-acid-like substrates can also catalyze this
transformation. Valine aminotransferase catalyzes the conversion of
valine and pyruvate to 2-ketoisovalerate and alanine. The E. coli
gene, avtA, encodes one such enzyme (Whalen et al., J. Bacteriol.,
150:739-746 (1982)). This gene product also catalyzes the amination
of .alpha.-ketobutyrate to generate .alpha.-aminobutyrate, although
the amine donor in this reaction has not been identified (Whalen et
al., J. Bacteriol., 158:571-574 (1984)). An additional candidate is
alpha-aminoadipate transaminase (EC 2.6.1.39), an enzyme that
participates in lysine biosynthesis and degradation in some
organisms. The enzyme from Thermus thermophilus, encoded by lysN,
is active with several alternate substrates including oxaloacetate,
2-oxoisocaproate, 2-oxoisovalerate, and 2-oxo-3-methylvalerate
(Miyazaki et al., Microbiol. 150:2327-2334 (2004)). A similar
enzyme from Homo sapiens has been characterized (Okuno et al., Enz.
Prot. 47:136-148 (1993)). Data related to the sequences for each of
these exemplary gene products can be found using the following
GenBank accession numbers shown in Table 17.
TABLE-US-00017 TABLE 17 Protein GenBank ID GI Number Organism aspC
NP_415448.1 16128895 Escherichia coli AAT2 P23542.3 1703040
Saccharomyces cerevisiae ASP5 P46248.2 20532373 Arabidopsis
thaliana got2 P00507 112987 Rattus norvegicus avtA YP_026231.1
49176374 Escherichia coli lysN BAC76939.1 31096548 Thermus
thermophilus AadAT-II Q8N5Z0.2 46395904 Homo sapiens
[0201] When the substrate is present as the D-stereoisomer,
transamination can be catalyzed by D-aminotransferase (EC
2.6.1.21), also known as D-amino acid aminotransferase and
D-alanine aminotransferase (DAAT). This class of enzymes is noted
for its broad substrate specificity, which is species-specific. The
D-aminotransferase from Bacillus species YM-1, encoded by dat, has
been cloned, sequenced (Tanizawa et al., J. Biol. Chem.,
264:2450-2454 (1989)) and the crystal structure has been solved
(Peisach et al., Biochemistry, 37:4958-4967 (1998)). This enzyme
has also been the subject of protein engineering studies to alter
the substrate specificity (Gutierrez et al., Eur. J. Biochem,
267:7218-7223 (2000); Gutierrez et al., Protein Eng., 11:53-58
(1998)). Additional genes are found in Bacillus licheniformis ATCC
10716 (Taylor et al., Biochim. Biophys. Acta., 1350:38-40 (1997)),
Staphylococcus haemolyticus (Pucci et al., J. Bacteriol.,
177:336-342 (1995)) and Bacillus subtilis (Martinez-Carrion et al.,
J. Biol. Chem., 240:3538-3546 (1965)). Data related to the
sequences for each of these exemplary gene products can be found
using the following GenBank accession numbers shown in Table
18.
TABLE-US-00018 TABLE 18 Protein GenBank ID GI Number Organism dat
P19938 118222 Bacillus sp. YM-1 dat P54692 1706292 Bacillus
licheniformis ATCC 10716 dat P54694 1706294 Staphylococcus
haemolyticus dat O07597.1 3121979 Bacillus subtilis
[0202] In reaction K of FIG. 1, 4-aminobutan-2-one is transaminated
to form 3-oxobutanal. This transformation can likely be catalyzed
by an aminotransferase that interconverts terminal amines and
aldehydes. Exemplary candidate enzymes are
beta-alanine/alpha-ketoglutarate aminotransferase, GABA
aminotransferase, 3-amino-2-methylpropionate transaminase,
lysine-6-aminotransferase, 2,4-diaminobutanoate transaminase,
putrescine aminotransferase and diamine aminotransferase.
[0203] Cargill has developed and patented a
beta-alanine/alpha-ketoglutarate aminotransferase for producing
3-HP from beta-alanine via malonyl-semialdehyde (Chandra et al.,
ARch. Microbiol., 176:443-451 (2001)). The gene product of SkPYD4
in Saccharomyces kluyveri was also shown to preferentially use
beta-alanine as the amino group donor (Aberhart et al., J. Chem.
Soc. 6:1404-1406 (1979)). SkUGA1 encodes a homologue of
Saccharomyces cerevisiae GABA aminotransferase, UGA1 (Ichikawa et
al., J. Mol. Catalysis A-Chem., 256:106-112 (2006)), whereas SkPYD4
encodes an enzyme involved in both .beta.-alanine and GABA
transamination (Aberthart et al., Supra).
3-amino-2-methylpropionate transaminase catalyzes the
transformation from methylmalonate semialdehyde to
3-amino-2-methylpropionate. The enzyme has been characterized in
Rattus norvegicus and Sus scrofa and is encoded by Abat (Chopra et
al., Protein Expr. Purif, 25:533-540 (2002), Kuznetsova et al.,
FEMS Microbiol. Rev., 29:263-279 (2005)). Enzyme candidates in
other organisms with high sequence homology to
3-amino-2-methylpropionate transaminase include Gta-1 in C. elegans
and gabT in Bacillus subtilus. Additionally, one of the native GABA
aminotransferases in E. coli, encoded by gene gabT, has been shown
to have broad substrate specificity (Fontaine et al., J.
Bacteriol., 184:821-830 (2002), Kanamasa et al., Appl. Microbiol
Biotechnol., 80:223-229 (2008)). The gene puuE encodes the other
4-aminobutyrate transaminase in E. coli (Drummond et al., J. Biol.
Chem., 235:318-325 (1960)).
[0204] Lysine-6-aminotransferase converts lysine to
alpha-aminoadipate semialdehyde. Candidate enzymes have been
characterized in Candida utilis (Hammer et al., J Basic Microbiol
32:21-27 (1992)), Flavobacterium lutescens (Fujii et al., J
Biochem. 128:391-397 (2000)) and Streptomyces clavuligenus (Romero
et al., J Ind. Microbiol Biotechnol 18:241-246 (1997)). A
recombinant lysine-6-aminotransferase from S. clavuligenus was
functionally expressed in E. coli (Tobin et al., J Bacteriol.
173:6223-6229 (1991)). The F. lutescens enzyme is specific to
alpha-ketoglutarate as the amino acceptor (Soda et al.,
Biochemistry 7:4110-4119 (1968)). An enzyme with diaminobutanoate
transaminase activity is encoded by the dat gene product in
Acinetobacter baumanii (Ikai et al., J Bacteriol. 179:5118-5125
(1997)). In addition to its natural substrate, 2,4-diaminobutyrate,
DAT transaminates the terminal amines of lysine, 4-aminobutyrate
and ornithine. Candidate putrescine aminotransferase enzymes are
encoded by ygjG in E. coli and spuC of Pseudomonas aeruginosa (Lu
et al., J Bacteriol. 184:3765-3773 (2002)). The ygiG gene product
reacts with the alternate substrates cadaverine, spermidine and
1,7-diaminoheptanoate (Samsonova et al., BMC. Microbiol 3:2 (2003);
Kim, J Biol. Chem. 239:783-786 (1964)).
[0205] Data related to the sequences for each of these exemplary
gene products can be found using the following GenBank accession
numbers shown in Table 19.
TABLE-US-00019 TABLE 19 Protein GenBank ID GI Number Organism
SkyPYD4 ABF58893.1 98626772 Saccharomyces kluyveri SkUGA1
ABF58894.1 98626792 Saccharomyces kluyveri UGA1 NP_011533.1 6321456
Saccharomyces cerevisiae Abat P50554.3 122065191 Rattus norvegicus
Abat P80147.2 120968 Sus scrofa Gta-1 Q21217.1 6016091
Caenorhabditis elegans gabT P94427.1 6016090 Bacillus subtilis gabT
P22256.1 16130576 Escherichia coli K12 puuE NP_415818.1 16129263
Escherichia coli K12 lat BAB13756.1 10336502 Flavobacterium
lutescens lat AAA26777.1 153343 Streptomyces clavuligenus dat
P56744.1 6685373 Acinetobacter baumanii ygjG NP_417544 145698310
Escherichia coli spuC AAG03688 9946143 Pseudomonas aeruginosa
[0206] In FIG. 1, Step C, 2,4-dioxopentanoate is decarboxylated to
form 3-oxobutyraldehyde by 2,4-dioxopentanoate decarboxylase.
2,4-dioxopentanoate is similar to the native substrates of pyruvate
decarboxylase (EC 4.1.1.1) and benzoylformate decarboxylase (EC
4.1.1.7). Pyruvate decarboxylase (PDC), also termed keto-acid
decarboxylase, is a key enzyme in alcoholic fermentation,
catalyzing the decarboxylation of pyruvate to acetaldehyde. The
enzyme from Saccharomyces cerevisiae has a broad substrate range
for aliphatic 2-keto acids including 2-ketobutyrate,
2-ketovalerate, 3-hydroxypyruvate and 2-phenylpyruvate (Li et al.,
Biochemistry, 38:10004-10012 (1999)). This enzyme has been
extensively studied, engineered for altered activity, and
functionally expressed in E. coli (Killenberg-Jabs et al., Eur. J.
Biochem., 268:1698-1704 (2001); Li et al., supra; Schure et al.,
Appl. Environ. Microbiol., 64:1303-1307 (1998)). The PDC from
Zymomonas mobilus, encoded by pdc, also has a broad substrate range
and has been a subject of directed engineering studies to alter the
affinity for different substrates (Siegert et al., Protein Eng.
Des. Sel., 18:345-357 (2005)). The crystal structure of this enzyme
is available (Killenberg-Jabs, supra). Other well-characterized PDC
enzymes include the enzymes from Acetobacter pasteurians (Chandra
et al., Arch. Microbiol. 176:443-451 (2001)) and Kluyveromyces
lactis (Krieger et al., Eur. J. Biochem., 269:3256-3263 (2002)).
Data related to the sequences for each of these exemplary gene
products can be found using the following GenBank accession numbers
shown in Table 20.
TABLE-US-00020 TABLE 20 Protein GenBank ID GI Number Organism pdc
P06672.1 118391 Zymomonas mobilis pdc1 P06169 30923172
Saccharomyces cerevisiae pdc Q8L388 20385191 Acetobacter
pasteurians pdc1 Q12629 52788279 Kluyveromyces lactis
[0207] Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a
broad substrate range and has been the target of enzyme engineering
studies. The enzyme from Pseudomonas putida has been extensively
studied and crystal structures of this enzyme are available
(Polovnikova et al., Biochemistry 42:1820-1830 (2003); Hasson et
al., Biochemistry, 37:9918-9930 (1998)). Site-directed mutagenesis
of two residues in the active site of the Pseudomonas putida enzyme
altered the affinity (Km) of naturally and non-naturally occurring
substrates (Siegert et al., supra). The properties of this enzyme
have been further modified by directed engineering (Lingen et al.,
Chembiochem, 4:721-726 (2003); Lingen et al., Protein Eng.,
15:585-593 (2002)). The enzyme from Pseudomonas aeruginosa, encoded
by mdlC, has also been characterized experimentally (Barrowman et
al., FEMS Microbiology Letters, 34:57-60 (1986)). Additional genes
from Pseudomonas stutzeri, Pseudomonas fluorescens and other
organisms can be inferred by sequence homology or identified using
a growth selection system developed in Pseudomonas putida (Henning
et al., Appl. Environ. Microbiol., 72:7510-7517 (2006)). Data
related to the sequences for each of these exemplary gene products
can be found using the following GenBank accession numbers shown in
Table 21.
TABLE-US-00021 TABLE 21 Protein GenBank ID GI Number Organism mdlC
P20906.2 3915757 Pseudomonas putida mdlC Q9HUR2.1 81539678
Pseudomonas aeruginosa dpgB ABN80423.1 126202187 Pseudomonas
stutzeri ilvB-1 YP_260581.1 70730840 Pseudomonas fluorescens
[0208] A third enzyme capable of decarboxylating 2-oxoacids is
alpha-ketoglutarate decarboxylase (KGD). The substrate range of
this class of enzymes has not been studied to date. The KDC from
Mycobacterium tuberculosis (Tian et al., Proc. Natl. Acad. Sci.
USA, 102:10670-10675 (2005)) has been cloned and has been
functionally expressed in E. coli at Genomatica. KDC enzyme
activity has been detected in several species of Rhizobia including
Bradyrhizobium japonicum and Mesorhizobium loti (Green et al., J.
Bacteriol., 182:2838-2844 (2000)). Although the KDC-encoding
gene(s) have not been isolated in these organisms, the genome
sequences are available and several genes in each genome are
annotated as putative KDCs. A KDC from Euglena gracilis has also
been characterized but the gene associated with this activity has
not been identified to date (Shigeoka et al., Arch. Biochem.
Biophys., 288:22-28 (1991)). The first twenty amino acids starting
from the N-terminus were sequenced MTYKAPVKDVKFLLDKVFKV (Shigeoka
et al., supra). The gene can be identified by testing genes
containing this N-terminal sequence for KDC activity. Data related
to the sequences for each of these exemplary gene products can be
found using the following GenBank accession numbers shown in Table
22.
TABLE-US-00022 TABLE 22 Protein GenBank ID GI Number Organism kgd
O50463.4 160395583 Mycobacterium tuberculosis kgd NP_767092.1
27375563 Bradyrhizobium japonicum USDA110 kgd NP_105204.1 13473636
Mesorhizobium loti
[0209] A fourth enzyme for catalyzing this step is the branched
chain alpha-ketoacid decarboxylase (BCKA). This class of enzymes
has been shown to act on a variety of compounds varying in chain
length from 3 to 6 carbons (Oku et al., J. Biol. Chem.,
263:18386-18396 (1988); Smit et al., Appl. Environ. Microbiol.,
71:303-311 (2005)). The enzyme in Lactococcus lactis has been
characterized on a variety of branched and linear substrates
including 2-oxobutanoate, 2-oxohexanoate, 2-oxopentanoate,
3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate and isocaproate
(Smit et al., supra). The enzyme has been structurally
characterized (Berthold et al., D. Biol. Crystallogr., 63:1217-1224
(2007)). Sequence alignments between the Lactococcus lactis enzyme
and the pyruvate decarboxylase of Zymomonas mobilus indicate that
the catalytic and substrate recognition residues are nearly
identical (Siegert et al., supra), so this enzyme is readily
amenable to directed engineering. Additional BCKA genes can be
identified by homology to the Lactococcus lactis protein sequence
(kdcA, AAS49166.1, 44921617, Lactococcus lactis). Many of the
high-scoring BLASTp hits to this enzyme are annotated as
indolepyruvate decarboxylases (EC 4.1.1.74). Indolepyruvate
decarboxylase (IPDA) is an enzyme that catalyzes the
decarboxylation of indolepyruvate to indoleacetaldehyde in plants
and plant bacteria.
[0210] 2-amino-4-ketopentanoate is decarboxylated to form
4-aminobutan-2-one by AKP decarboxylase in Step E of FIG. 1. This
transformation can be catalyzed by an amino acid decarboxylase.
Selection of an appropriate decarboxylase depends on the
stereochemical configuration of 4-amino-4-oxopentanoate. When this
compound is in a D-configuration, a D-amino acid decarboxylase can
be utilized. One such D-amino acid decarboxylase is diaminopimelate
decarboxylase (DDC, EC 4.1.1.20). This enzyme decarboxylates the
D-stereocenter of meso-diaminopimelate, catalyzing the final step
of lysine biosynthesis. DDC has been studied in many organisms
including E. coli (Momany et al., D. Biol. Crystallogr., 58:549-552
(2002)), Mycobacterium tuberculosis (Kefala et al., Acta.
Crystallogr. Sect. F. Struct. Biol. Cryst. Commun., 61:782-784
(2005); Gokulan et al., J. Biol. Chem., 278:18588-18596 (2003);
Andersen et al., Gene, 124:105-109 (1993)), Methylophilus
methylotrophus (Tsujimoto et al., J. Biotechnol, 124:327-337
(2006)), and Helicobacter pylori (Hu et al., J. Biol. Chem.,
283:21284-21293 (2008)). Alternately, the ornithine decarboxylase
(EC 4.1.1.17) from Homo sapiens has a weak activity on the D-isomer
of ornithine (Qu et al., Biochem. J., 375:465-470 (2003);
Fitzgerald et al., DNA, 8:623-634 (1989)) and can be used for the
decarboxylation in step E. Data related to the sequences for each
of these exemplary gene products can be found using the following
GenBank accession numbers shown in Table 23.
TABLE-US-00023 TABLE 23 Protein GenBank ID GI Number Organism lysA
NP_417315.1 16130742 Escherichia coli lysA AAA25361.1 149964
Mycobacterium tuberculosis lysA BAC92756.1 37196770 Methylophilus
methylotrophus lysA ABW70801.1 158523325 Helicobacter pylori odc1
AA59969.1 386989 Homo sapiens
[0211] When 2-amino-4-ketopentanoate exhibits L-stereochemistry, an
amino acid decarboxylase such as aspartate decarboxylase (EC
4.1.1.11), ornithine decarboxylase (EC 4.1.1.17) or lysine
decarboxylase (EC 4.1.1.18) can be utilized. An exemplary enzyme is
aspartate decarboxylase (EC 4.1.1.11). 2-Amino-4-ketopentanoate
bears structural similarity to aspartate, the native substrate of
this enzyme. Aspartate decarboxylase participates in pantothenate
biosynthesis and is encoded by panD in Escherichia coli (Dusch et
al., Appl. Environ. Microbiol., 65:1530-1539 (1999); Ramjee et al.,
Biochem. J., 323:661-669 (1997); Merkel et al., FEMS Microbiol.
Lett., 143:247-252 (1996); Schmitzberger et al., EMBO J.,
22:6193-6204 (2003)). The enzymes from Mycobacterium tuberculosis
(Chopra et al., Protein Expr. Purif., 25:533-540 (2002)) and
Corynebacterium glutamicum (Dusch et al., supra) have been
expressed and characterized in E. coli. Lysine decarboxylase
enzymes are encoded in the E. coli genome by genes cadA and ldcC. A
lysine decarboxylase analogous to CadA was recently identified in
Vibrio parahaemolyticus (Tanaka et al., J. Appl. Microbiol.
104:1283-1293 (2008)). The lysine decarboxylase from Selenomonas
ruminantium, encoded by ldc, bears sequence similarity to
eukaryotic ornithine decarboxylases, and accepts both L-lysine and
L-ornithine as substrates (Takatsuka et al., Biosci. Biotechnol
Biochem. 63:1843-1846 (1999)). Ornithine decarboxylase enzyme
candidates are found in Nicotiana glutinosa (Lee et al., Biochem.
J. 360:657-665 (2001)), Lactobacillus sp. 30a (Guirard et al., J
Biol. Chem. 255:5960-5964 (1980)) and Vibrio vulnificus (Lee et
al., J Biol. Chem. 282:27115-27125 (2007)). The residues involved
in substrate specificity Vibrio vulnificus have been elucidated
(Lee et al., supra).
[0212] Data related to the sequences for each of these exemplary
gene products can be found using the following GenBank accession
numbers shown in Table 24.
TABLE-US-00024 TABLE 24 Protein GenBank ID GI Number Organism panD
P0A790 67470411 Escherichia coli panD Q9X4N0 18203593
Corynebacterium glutanicum panD P65660.1 54041701 Mycobacterium
tuberculosis cadA AAA23536. 145458 Escherichia coli ldcC AAC73297.1
1786384 Escherichia coli ldc O50657.1 13124043 Selenomonas
ruminantium cadA AB124819.1 44886078 Vibrio parahaemolyticus
AF323910.1:1..1299 AAG45222.1 12007488 Nicotiana glutinosa odc1
P43099.2 1169251 Lactobacillus sp. 30a VV2_1235 NP_763142.1
27367615 Vibrio vulnificus
[0213] In reaction J (FIG. 1), acetylacrylate is decarboxylated to
2-oxobutene by acetoacrylate decarboxylase. An enzyme catalyzing
this transformation has not been identified to date, but similar
reactions are catalyzed by the enzymes aconitate decarboxylase,
4-oxalocrotonate decarboxylase and cinnamate decarboxylase.
[0214] Aconitate decarboxylase catalyzes the final step in
itaconate biosynthesis in a strain of Candida and also in the
filamentous fungus Aspergillus terreus (Bonnarme et al., J.
Bacteriol., 177:3573-3578 (1995); Willke et al., Appl. Microbiol.
Biotechnol., 56:289-295 (2001)). A cis-aconitate decarboxylase
(CAD) (EC 4.1.16), encoded by ATEG.sub.--09971, has been identified
and extensively studied in Aspergillus terreus and other related
fungi. Recently, the gene has been cloned and functionally
characterized (Kanamasa et al., Appl. Biotechnol., 80:223-229
(2008)) and (WO/2009/014437).
[0215] 4-oxalocronate decarboxylase has been isolated from numerous
organisms and characterized. Genes encoding this enzyme include
dmpH and dmpE in Pseudomonas sp. (strain 600) (Shingler et al., J.
Bacteriol., 174:711-724 (1992)), xylII and xylIII from Pseudomonas
putida (Kato et al., Arch. Microbiol., 168:457-463 (1997); Stanley
et al., Biochemistry, 39:3514 (2000); Lian et al., J. Am. Chem.
Soc., 116:10403-10411 (1994)) and Reut_B5691 and Reut_B5692 from
Ralstonia eutropha JMP134 (Hughes et al., J. Bacteriol., 158:79-83
(1984)). The genes encoding the enzyme from Pseudomonas sp. (strain
600) have been cloned and expressed in E. coli (Shingler et al.,
supra). Data related to the sequences for each of these exemplary
gene products can be found using the following GenBank accession
numbers shown in Table 25.
TABLE-US-00025 TABLE 25 Protein GenBank ID GI Number Organism dmpH
CAA43228.1 45685 Pseudomonas sp. CF600 dmpE CAA43225.1 45682
Pseudomonas sp. CF600 xylII YP_709328.1 111116444 Pseudomonas
putida xylIII YP_709353.1 111116469 Pseudomonas putida Reut_B5691
YP_299880.1 73539513 Ralstonia eutropha JMP134 Reut_B5692
YP_299881.1 73539514 Ralstonia eutropha JMP134 ATEG_09971
EAU29420.1 114187720 Aspergillus terreus
[0216] An additional class of decarboxylases has been characterized
that catalyze the conversion of cinnamate (phenylacrylate) and
substituted cinnamate derivatives to the corresponding styrene
derivatives. These enzymes are common in a variety of organisms and
specific genes encoding these enzymes that have been cloned and
expressed in E. coli are: pad 1 from Saccharomyces cerevisae
(Clausen et al., Gene, 142:107-112 (1994)), pdc from Lactobacillus
plantarum (Barthelmebs et al., Appl. Environ. Microbiol.,
67:1063-1069 (2001); Rodriguez et al., J. Agric. Food Chem.,
56:3068-3072 (2008); Qi et al., Biochem. J., 375:465-470 (2007)),
pofK (pad) from Klebsiella oxytoca (Uchiyama et al., Biosci.
Biotechnol. Biochem., 72:116-123 (2008); Hashidoko et al., Biosci.
Biotech. Biochem., 58:217-218 (1994)), Pedicoccus pentosaceus
(Barthelmebs et al., supra) and padC from Bacillus subtilis and
Bacillus pumilus (Cavin et al., Appl. Environ. Microbiol.,
64:1466-1471 (1998)). A ferulic acid decarboxylase from Pseudomonas
fluorescens also has been purified and characterized (Huang et al.,
J. Bacteriol., 176:5912-5918 (1994)). Importantly, this class of
enzymes has been shown to be stable and does not require either
exogenous or internally bound co-factors, thus making these enzymes
ideally suitable for biotransformations (Sariaslani, F. S., Annu.
Rev. Microbiol., 61:51-69 (2007)). Data related to the sequences
for each of these exemplary gene products can be found using the
following GenBank accession numbers shown in Table 26.
TABLE-US-00026 TABLE 26 Protein GenBank ID GI Number Organism pad1
AAB64980.1 1165293 Saccharomyces cerevisae pdc AAC45282.1 1762616
Lactobacillus plantarum pad BAF65031.1 149941608 Klebsiella oxytoca
padC NP_391320.1 16080493 Bacillus subtilis pad YP_804027.1
116492292 Pedicoccus pentosaceus pad CAC18719.1 11691810 Bacillus
pumilus
[0217] An additional enzyme for decarboxylation is acetoacetate
decarboxylase (EC 4.1.1.4), an enzyme that decarboxylates
acetoacetate to acetone and has therefore been studied for its role
in bacterial solventogenesis. Exemplary bacterial enzymes have been
characterized from Clostridium acetobutylicum (Benner et al., J.
Am. Chem. So. 103:993-994 (1981); Hlghbarger et al., Biochemistry
35:41-46 (1996); Petersen et al., Appl. Environ. Microbiol.
56:3491-3498 (1990); Rozzel et al. J. Am. Chem. Soc. 106:4937-4941
(1984)) Clostridium saccharoperbutylacetonicum (Kosaka, et al.,
Biosci. Biotechnol Biochem. 71:58-68 (2007)) and Clostridium
beijerinckii (Ravagnani et al. Mol. Microbiol. 37:1172-1185
(2000)). Acetoacetate decarboxylase activity has also been
demonstrated in Pseudomonas putida and Bacillus polymyxa but genes
are not associated with this activity to date (Matiasek et al.,
Curr. Microbiol. 42: 276-281 (2001)). Bacterial genes in other
organisms such as Clostridium botulinum and Bacillus
amyloliquefaciens can be identified by sequence homology. In humans
and other mammals, acetoacetate decarboxylase catalyzes the final
step of the ketone-body pathway (Kalapos, Biochim. Biophys. Acta
1621:122-139 (2003)), but genes associated with this activity have
not been identified to date. Data related to the sequences for each
of these exemplary gene products can be found using the following
GenBank accession numbers shown in Table 27.
TABLE-US-00027 TABLE 27 Protein GenBank ID GI Number Organism adc
NP_149328.1 15004868 Clostridium acetobutylicum adc AAP42566.1
31075386 Clostridium saccharoperbutyl- acetonicum cbei_3835
YP_001310906.1 150018652 Clostridium beijerinckii CLL_A2135
YP_001886324.1 187933144 Clostridium botulinum RBAM_030030
YP_001422565.1 154687404 Bacillus amyloliquefaciens
[0218] All the aforementioned gene candidates can also be used to
catalyze the decarboxylation of 2-oxo-4-hydroxypentanoate to
3-hydroxybutyraldehyde in Step N of FIG. 1.
[0219] Butenone hydratase (Step G, FIG. 1), 4-hydroxybutyryl-CoA
dehydratase (Step A, FIG. 3) and crotonase (Step A, FIG. 3) are
hydrolyase-type transformations. Specifically, the hydration of
butenone to 4-hydroxy-2-butanone (Step G, FIG. 1) can be
accomplished by an enzyme in the hydratase family of enzymes.
Enzymes that can carry out this transformation include fumarate
hydratase (EC 4.2.1.2), 2-(hydroxymethyl)glutarate dehydratase (EC
4.2.1.-), dimethylmaleate hydratase (EC 4.2.1.85) and citramalate
hydrolyase (EC 4.2.1.34).
[0220] Fumarate hydratase enzymes naturally catalyze the reversible
hydration of fumarate to malate. Although the ability of fumarate
hydratase to react with butanone as a substrate has not been
described in the literature, a wealth of structural information is
available for this enzyme and other researchers have successfully
engineered the enzyme to alter activity, inhibition and
localization (Weaver, T., B. Biol. Crystallogr., 61:1395-1401
(2005)). E. coli has three fumarases: FumA, FumB, and FumC that are
regulated by growth conditions. FumB is oxygen sensitive and only
active under anaerobic conditions. FumA is active under
microanaerobic conditions, and FumC is the only active enzyme in
aerobic growth (Tseng et al., J. Bacteriol., 183:461-467 (2001);
Woods et al., Biochem. Biophys. Acta., 954:14-26 (1988); Guest et
al., J. Gen. Microbiol., 131:2971-2984 (1985)). Additional enzymes
are found in Campylobacter jejuni (Smith et al., Int. J. Biochem.
Cell Biol., 31:961-975 (1999)), Thermus thermophilus (Mizobata et
al., Arch. Biochem. Biophys., 355:49-55 (1998)) and Rattus
norvegicus (Kobayashi et al., J. Biochem., 89:1923-1931 (1981)).
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)). Data related to the
sequences for each of these exemplary gene products can be found
using the following GenBank accession numbers shown in Table
28.
TABLE-US-00028 TABLE 28 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 fumC
O69294 9789756 Campylobacter jejuni fumC P84127 75427690 Thermus
thermophilus fumH P14408 120605 Rattus norvegicus fum1 P93033
39931311 Arabidopsis thaliana fumC Q8NRN8 39931596 Corynebacterium
glutamicum MmcB YP_001211906 147677691 Pelotomaculum
thermopropionicum MmcC YP_001211907 147677692 Pelotomaculum
thermopropionicum
[0221] Two additional hydratase enzymes are
2-(hydroxymethyl)glutarate dehydratase and dimethylmaleate
hydratase, enzymes studied for their role in nicontinate catabolism
in Eubacterium barkeri (formerly Clostridium barkeri) (Alhapel et
al., Proc. Natl. Acad. Sci. USA, 103:12341-12346 (2006)).
2-(Hydroxymethyl)glutarate dehydratase is a [4Fe-4S]-containing
enzyme that dehydrates 2-(hydroxymethyl)glutarate to
2-methylene-glutarate. This enzyme is encoded by hmd in Eubacterium
barkeri (Alhapel et al., supra). Similar enzymes with high sequence
homology are found in Bacteroides capillosus, Anaerotruncus
colihominis, and Natranaerobius thermophilius. These enzymes are
homologous to the alpha and beta subunits of [4Fe-4S]-containing
bacterial serine dehydratases (e.g., E. coli enzymes encoded by
tdcG, sdhB, and sdaA). Dimethylmaleate hydratase (EC 4.2.1.85) is a
reversible Fe.sup.2+-dependent and oxygen-sensitive enzyme in the
aconitase family that hydrates dimethylmaeate to form
(2R,3S)-2,3-dimethylmalate. This enzyme is encoded by dmdAB in
Eubacterium barkeri (Alhapel, et al., supra; Kollmann-Koch et al.,
Physiol. Chem., 365:847-857 (1984)). Data related to the sequences
for each of these exemplary gene products can be found using the
following GenBank accession numbers shown in Table 29.
TABLE-US-00029 TABLE 29 Protein GenBank ID GI Number Organism hmd
ABC88407.1 86278275 Eubacterium barkeri BACCAP_02294 ZP_02036683.1
154498305 Bacteroides capillosus ATCC 29799 ANACOL_02527
ZP_02443222.1 167771169 Anaerotruncus colihominis DSM 17241
NtherDRAFT_2368 ZP_02852366.1 169192667 Natranaerobius thermophilus
JW/NM-WN-LF dmdA ABC88408 86278276 Eubacterium barkeri dmdB
ABC88409.1 86278277 Eubacterium barkeri
[0222] An additional enzyme is 2-methylmalate dehydratase, also
called citramalate hydrolyase, a reversible hydrolyase that
catalyzes the alpha, beta elimination of water from citramalate to
form mesaconate. This enzyme has been purified and characterized in
Clostridium tetanomorphum (Wang et al., J. Biol. Chem.,
244:2516-2526 (1969)). The activity of this enzyme has also been
detected in several bacteria in the genera Citrobacter and
Morganella in the context of the glutamate degradation VI pathway
(Kato et al., supra). Genes encoding this enzyme have not been
identified in any organism to date.
[0223] Hydration of crotonyl-CoA to form 3-hydroxybutyryl-CoA (Step
B, FIG. 3) is catalyzed by a crotonase (EC 4.2.1.55). These 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 (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., supra). Enoyl-CoA
hydratases, which are involved in fatty acid beta-oxidation and/or
the metabolism of various amino acids, can also catalyze the
hydration of crotonyl-CoA to form 3-hydroxybutyryl-CoA (Roberts et
al., Arch. Microbiol., 117:99-108 (1978); Agnihotri et al., Bioorg.
Med. Chem., 11:9-20 (2003); Conrad et al., J. Bacteriol.,
118:103-111 (1974)). An exemplary enoyl-CoA hydratase is the gene
product of ech from Pseudomonas putida (Roberts et al., supra). The
enoyl-CoA hydratases, phaA and phaB, of P. putida have been
indicated to carry out the hydroxylation of double bonds during
phenylacetate catabolism (Olivera et al., Proc. Natl. Acad. Sci
USA, 95:6419-6424 (1998)). The paaA and paaB from P. fluorescens
catalyze analogous transformations (Olivera et al., supra). 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; Park et
al., supra; Park et al., supra). Data related to the sequences for
each of these exemplary gene products can be found using the
following GenBank accession numbers shown in Table 30.
TABLE-US-00030 TABLE 30 Protein GenBank ID GI Number Organism crt
NP_349318.1 15895969 Clostridium acetobutylicum crt1 YP_001393856
153953091 Clostridium kluyveri DSM 555 ech NP_745498.1 26990073
Pseudomonas putida phaA ABF82233.1 26990002 Pseudomonas putida phaB
ABF82234.1 26990001 Pseudomonas putida paaA NP_745427.1 106636093
Pseudomonas fluorescens paaB NP_745426.1 106636094 Pseudomonas
fluorescens maoC NP_415905.1 16129348 Escherichia coli paaF
NP_415911.1 16129354 Escherichia coli paaG NP_415912.1 16129355
Escherichia coli
[0224] Alternatively, the E. coli gene products of fadA and fadB
encode a multienzyme complex involved in fatty acid oxidation that
exhibits enoyl-CoA hydratase activity (Haller et al., Biochemistry
39:4622-4629 (2000); Martinez-Carrion et al., J. Biol. Chem.
240:3538-3546 (1965); Matthies et al., Appl. Environ. Micriobiol.
58:1435-1439 (1992)). Knocking out a negative regulator encoded by
fadR can be utilized to activate the fadB gene product (Jeng et
al., A. Biochemistry 13:2898-2903 (1974)). The fadI and fadJ genes
encode similar functions and are naturally expressed under
anaerobic conditions (Atsumi et al., Nature 451:86-89 (2008)). Data
related to the sequences for each of these exemplary gene products
can be found using the following GenBank accession numbers shown in
Table 31.
TABLE-US-00031 TABLE 31 Protein GenBank ID GI Number Organism fadA
YP_026272.1 49176430 Escherichia coli fadB NP_418288.1 16131692
Escherichia coli fadI NP_416844.1 16130275 Escherichia coli fadJ
NP_416843.1 16130274 Escherichia coli fadR NP_415705.1 16129150
Escherichia coli
[0225] The reversible condensation of 4-hydroxybutyryl-CoA to
crotonyl-CoA (Step A, FIG. 3) is catalyzed by the bifunctional
enzyme 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA
.DELTA.-isomerase. This enzyme first dehydrates
4-hydroxybutyryl-CoA to vinylacetyl-CoA, which subsequently
rearranges to form crotonoyl-CoA. The enzymes from Clostridium
kluyveri and C. aminobutyrium have been purified, characterized,
and sequenced at the N-terminal domain (Scherf et al., Eur. J.
Biochem., 215:421-429 (1993); Scherf et al., Arch. Microbiol.,
161:239-245 (1994)). The abfD genes from C. aminobutyrium and C.
kluyveri match exactly with these N-terminal amino acid sequences,
and have been indicated to encode the 4-hydroxybutyrul-CoA
dehydratases/vinylacetyl-CoA A-isomerase activities. Similar genes
are identified through homology from genome projects, including
abfD from Porphyromonas gingivalis and Msed.sub.--1220 from
Metallosphaera sedula. Data related to the sequences for each of
these exemplary gene products can be found using the following
GenBank accession numbers shown in Table 32.
TABLE-US-00032 TABLE 32 Protein GenBank ID GI Number Organism abfD
YP_001396399.1 153955634 Clostridium kluyveri abfD P55792 84028213
Clostridium aminobutyricum abfD YP_001928843 188994591
Porphyromonas gingivalis Msed_1220 YP_001191305.1 146303989
Metallosphaera sedula
[0226] Deamination of 2-amino-4-ketopentanoate (FIG. 1, Reaction I)
and of 4-aminobutan-2-one (Step F, FIG. 1) can be accomplished by
AKP ammonia-lyase and 4-aminobutan-2-one ammonia-lyase
respectively. These deaminations are very similar to the
deamination of aspartate to fumarate by aspartase. The enzyme has
been extensively studied and several crystal structures are
available. The E. coli enzyme has been shown to react with
alternate substrates such as aspartatephenylmethylester,
asparagine, benzyl-aspartate and malate (Ma et al., Ann. N.Y. Acad.
Sci., 672:60-65 (1992). In a separate study, directed evolution has
been implemented on this enzyme to alter substrate specificity
(Asano et al., Biomol. Eng., 22:95-101 (2005)). Enzymes with
aspartase functionality have also been characterized in Haemophilus
influenzae (Sjostrom et al., Biochem. Biophys. Acta., 1324:182-190
(1997)), Pseudomonas fluorescens (Takagi et al., J. Biochem.,
96:545-552 (1984)), Bacillus subtilus (Sjostrom et al., supra) and
Serratia marcescens (Takagi et al., J. Bacteriol., 161:1-6 (1985)).
Data related to the sequences for each of these exemplary gene
products can be found using the following GenBank accession numbers
shown in Table 33.
TABLE-US-00033 TABLE 33 Protein GenBank ID GI Number Organism aspA
NP_418562 90111690 Escherichia coli aspA P44324.1 1168534
Haemophilus influenzae aspA P07346.1 114273 Pseudomonas fluorescens
ansB P26899.1 251757243 Bacillus subtilus aspA P33109.1 416661
Serratia marcescens
[0227] A similar ammonia lyase reaction is catalyzed by
methylaspartase (EC 4.3.1.2), an enzyme participating in the
glutamate fermentation route via mesaconate (Kato et al., supra).
This enzyme, also known as beta-methylaspartase and
3-methylaspartate ammonia-lyase, naturally catalyzes the
deamination of threo-3-methylasparatate to mesaconate. The
3-methylaspartase from Clostridium tetanomorphum has been cloned,
functionally expressed in E. coli, and crystallized (Asuncion et
al., 57:731-733 (2001); Asuncion et al., J Biol Chem. 277:8306-8311
(2002); Botting et al., 27:2953-2955 (1988); Goda et al.,
31:10747-10756 (1992)). In Citrobacter amalonaticus, this enzyme is
encoded by BAA28709 (Kato et al., Arch. Microbiol 168:457-463
(1997)). 3-Methylaspartase has also been crystallized from E. coli
YG1002 (Asano et al., FEMS Microbiol Lett. 118:255-258 (1994))
although the protein sequence is not listed in public databases
such as GenBank. Data related to the sequences for each of these
exemplary gene products can be found using the following GenBank
accession numbers shown in Table 34.
TABLE-US-00034 TABLE 34 Protein GenBank ID GI Number Organism mal
AAB24070.1 259429 Clostridium tetanomorphum BAA28709 BAA28709.1
3184397 Citrobacter amalonaticus
[0228] Referring now to FIG. 8B, gene candidates for alanine
dehydrogenase alanine aminotransferase are shown below which
convert pyruvate to alanine.
TABLE-US-00035 TABLE 35 glutamate-pyruvate Escherichia coli
Accession: AAC76384.1 aminotransferase (alaB) K-12 substr. GI:
1789759 MG1655 glutamate-pyruvate Escherichia coli Accession:
AAC75350.1 aminotransferase (alaA) K-12 substr. GI: 1788627 MG1655
glutamate-pyruvate Escherichia coli Accession: AAC75438.1
aminotransferase (alaC) K-12 substr. GI: 1788722 MG1655 alanine
transaminase Arabidopsis Accession: AEE30370.1 (AOAT1) thaliana col
GI: 332192249 alanine transaminase Arabidopsis Accession:
AEE35084.1 (AOAT2) thaliana col GI: 332196963 tryptophan
Arabidopsis Accession: aminotransferase (TAA1) thaliana col
NP_177213.1 GI: 15223183 alanine aminotransferase Homo sapiens
Accession: (GPT2) NP_597700.1 GI: 19263340 alanine aminotransferase
Homo sapiens Accession: AAB20194.1 (GPT) GI: 238134 alanine
aminotransferase Clostridium propionicum alanine dehydrogenase
Phormidium Accession: BAA24455.1 (ald) lapideum GI: 2804515 alanine
dehydrogenase Enterobacter Accession: BAA77513.1 (aladh) aerogenes
GI: 4803749 L-alanine dehydrogenase Bacillus cereus L-alanine
dehydrogenase Bacillus subtilis Accession: (ald) NP_391071.1 GI:
16080244 alanine dehydrogenase Bilophila Accession: AF269148.1
wadsworthia GI: 13661832 RZATAU
[0229] Five requisite pathways to achieve the biosynthesis of 4-HB
are exemplified herein and shown for purposes of illustration in
FIG. 9B. One requisite 4-HB biosynthetic pathway includes the
biosynthesis of 4-HB from succinate (the succinate pathway). The
enzymes participating in this 4-HB pathway include succinate
reductase, 4-hydroxybutanoate dehydrogenase (Steps F and C, FIG.
9B). Another requisite 4-HB biosynthetic pathway includes the
biosynthesis from succinate through succinyl-CoA (the succinyl-CoA
pathway). The enzymes participating in this 4-HB pathway include
succinyl-CoA synthetase, CoA-dependent succinic semialdehyde
dehydrogenase, and 4-hydroxybutanoate dehydrogenase (Steps A, I and
K, FIG. 9B). Three other requisite 4-HB biosynthetic pathways
include the biosynthesis of 4-HB from .alpha.-ketoglutarate (the
.alpha.-ketoglutarate pathways). Hence, a third requisite 4-HB
biosynthetic pathway is the biosynthesis of succinic semialdehyde
through succinyl-CoA (Steps V, B, and C, FIG. 9B). AKG can be
converted into succinyl-CoA by alpha-ketoglutarate dehydrogenase.
This is then transformed into succinate semialdehyde and 4-HB as
described earlier. Yet another pathway (Steps Q, R, S, T, U, FIG.
9B) for synthesizing 4-HB entails conversion of AKG into glutamate
via glutamate dehydrogenase or glutamate transaminase. Glutamate is
decarboxylated to form 4-aminobutyrate by glutamate decarboxylase
and then 4-aminobutyrate dehydrogenase or 4-aminobutyrate
transaminase can convert it into succinate semialdehyde. The final
4-HB biosynthetic pathway described here also includes the
biosynthesis of 4-HB from .alpha.-ketoglutarate (Steps I and C,
FIG. 9B), but utilizes .alpha.-ketoglutarate decarboxylase to
catalyze succinic semialdehyde synthesis. 4-hydroxybutanoate
dehydrogenase catalyzes the conversion of succinic semialdehyde to
4-HB. 4-hydroxybutanoate dehydrogenase catalyzes the conversion of
succinic semialdehyde to 4-HB. 4HB can further be converted into
4-HB-CoA by 4-HB-CoA ligase, 4-HB-CoA synthetase or 4-HB-CoA
transferase. Alternatively, the conversion of 4-HB to 4-HB CoA can
be carried out by 4-Hydroxybutyrate kinase and
Phosphotrans-4-hydroxybutyrylase (Steps D and E, FIG. 9B). Each of
these 4-HB and 4HB-CoA biosynthetic pathways, their substrates,
reactants and products are described further below in the
Examples.
[0230] The non-naturally occurring microbial organisms of the
invention can be produced by introducing expressible nucleic acids
encoding one or more of the enzymes participating in one or more
4-HB biosynthetic pathways. Depending on the host microbial
organism chosen for biosynthesis, nucleic acids for some or all of
a particular 4-HB biosynthetic pathway can be expressed. For
example, if a chosen host is deficient in both enzymes in the
succinate to 4-HB pathway and this pathway is selected for 4-HB
biosynthesis, then expressible nucleic acids for both succinate
reductase and 4-hydroxybutanoate dehydrogenase are introduced into
the host for subsequent exogenous expression. Alternatively, if the
chosen host exhibits endogenous succinate reductase, but is
deficient in 4-hydroxybutanoate dehydrogenase then an encoding
nucleic acid is needed for this enzyme to achieve 4-HB
biosynthesis.
[0231] In like fashion, where 4-HB biosynthesis is selected to
occur through the succinate to succinyl-CoA pathway (the
succinyl-CoA pathway), encoding nucleic acids for host deficiencies
in the enzymes succinyl-CoA synthetase, CoA-dependent succinic
semialdehyde dehydrogenase and/or 4-hydroxybutanoate dehydrogenase
are to be exogenously expressed in the recipient host. Selection of
4-HB biosynthesis through the .alpha.-ketoglutarate to succinic
semialdehyde pathway (the .alpha.-ketoglutarate pathway) can
utilize exogenous expression for host deficiencies in one or more
of the enzymes for glutamate dehydrogenase, glutamate transaminase,
glutamate decarboxylase, 4-aminobutyrate dehydrogenase,
4-aminobutyrate transaminase, and/or 4-hydroxybutanoate
dehydrogenase.
[0232] Depending on the 4-HB biosynthetic pathway constituents of a
selected host microbial organism, the non-naturally occurring
microbial 4-HB biocatalysts of the invention will include at least
one exogenously expressed 4-HB pathway-encoding nucleic acid and up
to all encoding nucleic acids for one or more 4-HB biosynthetic
pathways. For example, 4-HB-CoA biosynthesis can be established
from all five pathways in a host deficient in 4-hydroxybutanoate
dehydrogenase through exogenous expression of a 4-hydroxybutanoate
dehydrogenase encoding nucleic acid. In contrast, 4-HB biosynthesis
can be established from all five pathways in a host deficient in
all eleven enzymes through exogenous expression of all eight of
CoA-independent succinic semialdehyde dehydrogenase, succinyl-CoA
synthetase, CoA-dependent succinic semialdehyde dehydrogenase,
glutamate: succinic semialdehyde transaminase, glutamate
decarboxylase, .alpha.-ketoglutarate decarboxylase and
4-hydroxybutanoate dehydrogenase.
[0233] 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 4-HB 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
or seven or up to eleven nucleic acids encoding the above enzymes
constituting one or more 4-HB biosynthetic pathways. In some
embodiments, the non-naturally occurring microbial organisms also
can include other genetic modifications that facilitate or optimize
4-HB-CoA 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 4-HB-CoA pathway precursors such as succinate, succinyl-CoA
and/or .alpha.-ketoglutarate.
[0234] In some embodiments, a non-naturally occurring microbial
organism of the invention is generated from a host that contains
the enzymatic capability to synthesize 4-HB. In this specific
embodiment it can be useful to increase the synthesis or
accumulation of a 4-HB pathway product to, for example, drive 4-HB
pathway reactions toward 4-HB production. Increased synthesis or
accumulation can be accomplished by, for example, overexpression of
nucleic acids encoding one or more of the above-described 4-HB
pathway enzymes. Over expression of the 4-HB pathway enzyme or
enzymes 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 4-HB
producing microbial organisms of the invention through
overexpression of one, two, three, four, five, six or seven nucleic
or all eleven acids encoding 4-HB biosynthetic pathway enzymes. 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 4-HB biosynthetic pathway.
[0235] Non-naturally occurring microbial organisms also can be
generated which biosynthesize 1,3-BDO. As with the 4-HB producing
microbial organisms of the invention, the 1,3-BDO producing
microbial organisms also can produce intracellularly or secret the
1,3-BDO into the culture medium. Following the teachings and
guidance provided previously for the construction of microbial
organisms that synthesize 4-HB, additional 1,3-BDO pathways can be
incorporated into the 4-HB producing microbial organisms to
generate organisms that also synthesize 1,3-BDO and other 1,3-BDO
family compounds. The non-naturally occurring microbial organisms
of the invention capable of 1,3-BDO biosynthesis circumvent these
chemical synthesis using 4-HB as an entry point as illustrated in
FIG. 9B.
[0236] The additional 1,3-BDO pathways to introduce into 4-HB
producers include, for example, the exogenous expression in a host
deficient background or the overexpression of one or more of the
enzymes exemplified in FIG. 9B. An initial step in the entry
pathway to 1,3-BDO is the conversion of 4-hydroxybutyrate to
4-hydroxybutyryl-CoA using 4-hydroxybutyrate:CoA transferase or
4-hydroxybutyryl-CoA synthetase (or ligase) the combination of
butyrate kinase and phosphotransbutyrylase. Accordingly, the
additional initial 1,3-BDO pathways to introduce into 4-HB
producers to produce 4-hydroxybutyryl-CoA include, for example, the
exogenous expression in a host deficient background or the
overexpression of one or more of a 4-hydroxybutyrate:CoA
transferase, 4-hydroxybutyryl-CoA synthetase (or ligase), butyrate
kinase or phosphotransbutyrylase. In the absence of endogenous
acyl-CoA synthetase capable of modifying 4-HB, the non-naturally
occurring 1,3-BDO producing microbial organisms can further include
an exogenous acyl-CoA synthetase selective for 4-HB, or the
combination of multiple enzymes that have as a net reaction
conversion of 4-HB into 4-HB-CoA. As exemplified further below in
the Examples, butyrate kinase and phosphotransbutyrylase exhibit
1,3-BDO pathway activity and catalyze the conversions illustrated
in FIG. 9B with a 4-HB substrate. Therefore, these enzymes also can
be referred to herein as 4-hydroxybutyrate kinase and
phosphotranshydroxybutyrylase respectively. Once
4-hydroxybutyryl-CoA is generated it can then be utilized for the
biosynthesis of 1,3-BDO following the subsequent steps shown FIG.
9B.
[0237] Step A of FIG. 9B involves CoA synthetase or ligase
reactions for succinate as the substrate. Exemplary genes encoding
enzymes likely to carry out these transformations include the sucCD
genes of E. coli which naturally form a succinyl-CoA synthetase
complex. This enzyme complex naturally catalyzes the formation of
succinyl-CoA from succinate with the contaminant consumption of one
ATP, a reaction which is reversible in vivo (Buck et al., Biochem.
24:6245-6252 (1985)).
TABLE-US-00036 TABLE 36 Gene Accession No. GI No. Organism sucC
NP_415256.1 16128703 Escherichia coli sucD AAC73823.1 1786949
Escherichia coli
[0238] 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. 4-hydroxybutyryl-CoA synthetase activity has
been demonstrated in Metallosphaera sedula (Berg et al., Science
318:1782-1786 (2007)). This function has been tentatively assigned
to the Msed.sub.--1422 gene.
TABLE-US-00037 TABLE 37 Gene Accession No. GI No. 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 Msed_1422 YP_001191504 146304188 Metallosphaera sedula
[0239] ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is
another candidate enzyme that 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,
isobutyrate, 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).
TABLE-US-00038 TABLE 38 Gene Accession No. GI No. 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
[0240] The conversion of succinate to succinate semialdehyde (Step
F) can be catalyzed by a carboxylic acid reductase. 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. "Biocatalytic Reduction of
Carboxylic Acids: Mechanism and Applications" Chapter 15 in
Biocatalysis in the Pharmaceutical and Biotechnology Industires,
ed. R. N. Patel, CRC Press LLC, Boca Raton, Fla. (2006)).
Additional car and npt genes can be identified based on sequence
homology.
TABLE-US-00039 TABLE 39A Gene Accession No. GI No. Organism car
AAR91681.1 40796035 Nocardia iowensis (sp. NRRL 5646) npt
ABI83656.1 114848891 Nocardia iowensis (sp. NRRL 5646)
TABLE-US-00040 TABLE 39B 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 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
[0241] 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.sub.--665, an enzyme similar in sequence
to the Nocardia iowensis npt, may be beneficial.
TABLE-US-00041 TABLE 40 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
[0242] An enzyme with similar characteristics, alpha-aminoadipate
reductase (AAR, EC 1.2.1.31), participates in lysine biosynthesis
pathways in some fungal species. This enzyme naturally reduces
alpha-aminoadipate to alpha-aminoadipate semialdehyde. The carboxyl
group is first activated through the ATP-dependent formation of an
adenylate that is then reduced by NAD(P)H to yield the aldehyde and
AMP. Like CAR, this enzyme utilizes magnesium and requires
activation by a PPTase. Enzyme candidates for AAR and its
corresponding PPTase are found in Saccharomyces cerevisiae (Morris
et al., Gene 98:141-145 (1991)), Candida albicans (Guo et al., Mol.
Genet. Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe
(Ford et al., Curr. Genet. 28:131-137 (1995)). The AAR from S.
pombe exhibited significant activity when expressed in E. coli (Guo
et al., Yeast 21:1279-1288 (2004)). The AAR from Penicillium
chrysogenum accepts S-carboxymethyl-L-cysteine as an alternate
substrate, but did not react with adipate, L-glutamate or
diaminopimelate (Hijarrubia et al., J Biol. Chem 278:8250-8256
(2003)). The gene encoding the P. chrysogenum PPTase has not been
identified to date and no high-confidence hits were identified by
sequence comparison homology searching.
TABLE-US-00042 TABLE 41 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
[0243] The conversion of succinyl-CoA to succinate semialdehyde
(Step B, FIG. 9B) is catalyzed by an aldehyde forming succinyl-CoA
reductase. Several acyl-CoA dehydrogenases are capable of reducing
an acyl-CoA to its corresponding aldehyde. Exemplary genes that
encode such enzymes include the Acinetobacter calcoaceticus acr1
encoding a fatty acyl-CoA reductase (Reiser and Somerville, J.
Bacteriology 179:2969-2975 (1997)), the Acinetobacter sp. M-1 fatty
acyl-CoA reductase (Ishige et al. Appl. Environ. Microbiol.
68:1192-1195 (2002)), and a CoA- and NADP-dependent succinate
semialdehyde dehydrogenase encoded by the sucD gene in Clostridium
kluyveri (Sohling and Gottschalk J Bacteriol 178:871-80 (1996);
Sohling and Gottschalk J Bacteriol. 178:871-880 (1996)). SucD of P.
gingivalis is another aldehyde-forming succinyl-CoA reductase
(Takahashi et al. J. Bacteriol. 182:4704-4710 (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. J Bacteriol.
175:377-385 (1993)). In addition to reducing acetyl-CoA to ethanol,
the enzyme encoded by adhE in Leuconostoc mesenteroides has been
shown to oxidize the branched chain compound isobutyraldehyde to
isobutyryl-CoA (Koo et al., Biotechnol Lett. 27:505-510 (2005)).
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-00043 TABLE 42 Gene Accession No. GI No. 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 730847 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
[0244] 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 (2007); Thauer, R. K. Science 318:1732-1733 (2007)).
The enzyme utilizes NADPH as a cofactor and has been characterized
in Metallosphaera and Sulfolobus spp (Alber et al. J. Bacteriol.
188:8551-8559 (2006); Hugler et al. J. Bacteriol. 184:2404-2410
(2002)). The enzyme is encoded by Msed.sub.--0709 in Metallosphaera
sedula (Alber et al. J. Bacteriol. 188:8551-8559 (2006); Berg et
al. Science 318:1782-1786 (2007)). A gene encoding a malonyl-CoA
reductase from Sulfolobus tokodaii was cloned and heterologously
expressed in E. coli (Alber et al. J. Bacteriol. 188:8551-8559
(2006)). 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
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., Appl Environ. Microbiol 65:4973-4980
(1999)).
TABLE-US-00044 TABLE 43 Gene Accession No. GI No. 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
[0245] Glutamate dehydrogenase (Step Q, FIG. 9B) and
4-aminobutyrate dehydrogenase (Step T, FIG. 9B) can be catalyzed by
aminating oxidoreductases. Enzymes in this EC class (1.4.1.a)
catalyze the oxidative deamination of alpha-amino acids with
NAD.sup.+ or NADP.sup.+ as acceptor, and the reactions are
typically reversible. Exemplary oxidoreductases operating on amino
acids include glutamate dehydrogenase (deaminating), encoded by
gdhA, leucine dehydrogenase (deaminating), encoded by ldh, and
aspartate dehydrogenase (deaminating), encoded by nadX. The gdhA
gene product from Escherichia coli (Korber et al. J. Mol. Biol.
234:1270-1273 (1993); McPherson and Wootton Nucleic. Acids Res.
11:5257-5266 (1983)), gdh from Thermotoga maritima (Kort et al.
Extremophiles 1:52-60 (1997); Lebbink, et al. J. Mol. Biol.
280:287-296 (1998)); Lebbink et al. J. Mol. Biol. 289:357-369
(1999)), and gdhA1 from Halobacterium salinarum (Ingoldsby et al.
Gene 349:237-244 (2005)) catalyze the reversible interconversion of
glutamate to 2-oxoglutarate and ammonia, while favoring NADP(H),
NAD(H), or both, respectively. The ldh gene of Bacillus cereus
encodes the LeuDH protein that has a wide of range of substrates
including leucine, isoleucine, valine, and 2-aminobutanoate
(Ansorge and Kula Biotechnol Bioeng. 68:557-562 (2000); Stoyan et
al. J. Biotechnol 54:77-80 (1997)). The nadX gene from Thermotoga
maritime encoding for the aspartate dehydrogenase is involved in
the biosynthesis of NAD (Yang et al. J. Biol. Chem. 278:8804-8808
(2003)).
TABLE-US-00045 TABLE 44 Gene Accession No. GI No. Organism gdhA
P00370 118547 Escherichia coli gdh P96110.4 6226595 Thermotoga
maritima gdhA1 NP_279651.1 15789827 Halobacterium salinarum ldh
P0A393 61222614 Bacillus cereus nadX NP_229443.1 15644391
Thermotoga maritima
[0246] Additional glutamate dehydrogenase gene candidates are found
in Bacillus subtilis (Khan et al., Biosci. Biotechnol Biochem.
69:1861-1870 (2005)), Nicotiana tabacum (Purnell et al., Planta
222:167-180 (2005)), Oryza sativa (Abiko et al., Plant Cell Physiol
46:1724-1734 (2005)), Haloferax mediterranei (Diaz et al.,
Extremophiles. 10:105-115 (2006)) and Halobactreium salinarum
(Hayden et al., FEMS Microbiol Lett. 211:37-41 (2002)). The
Nicotiana tabacum enzyme is composed of alpha and beta subunits
encoded by gdh1 and gdh2 (Purnell et al., Planta 222:167-180
(2005)). Overexpression of the NADH-dependent glutamate
dehydrogenase was found to improve ethanol production in engineered
strains of S. cerevisiae (Roca et al., Appl Environ. Microbiol
69:4732-4736 (2003)).
TABLE-US-00046 TABLE 45 Gene Accession No. GI No. Organism rocG
NP_391659.1 16080831 Bacillus subtilis gdh1 AAR11534.1 38146335
Nicotiana tabacum gdh2 AAR11535.1 38146337 Nicotiana tabacum GDH
Q852M0 75243660 Oryza sativa GDH Q977U6 74499858 Haloferax
mediterranei GDH P29051 118549 Halobactreium salinarum GDH2
NP_010066.1 6319986 Saccharomyces cerevisiae
[0247] An exemplary enzyme for catalyzing the conversion of
aldehydes to their corresponding primary amines is lysine
6-dehydrogenase (EC 1.4.1.18), encoded by the lysDH genes. The
lysine 6-dehydrogenase (deaminating), encoded by lysDH gene,
catalyze the oxidative deamination of the .epsilon.-amino group of
L-lysine to form 2-aminoadipate-6-semialdehyde, which in turn
nonenzymatically cyclizes to form
.DELTA.1-piperideine-6-carboxylate (Misono and Nagasaki J.
Bacteriol. 150:398-401 (1982)). The lysDH gene from Geobacillus
stearothermophilus encodes a thermophilic NAD-dependent lysine
6-dehydrogenase (Heydari et al. Appl Environ. Microbiol 70:937-942
(2004)). The lysDH gene from Aeropyrum pernix K1 is identified
through homology from genome projects. Additional enzymes can be
found in Agrobacterium tumefaciens (Hashimoto et al., J Biochem.
106:76-80 (1989); Misono et al., J Bacteriol. 150:398-401 (1982))
and Achromobacter denitrificans (Ruldeekulthamrong et al., BMB.
Rep. 41:790-795 (2008)).
TABLE-US-00047 TABLE 46 Gene Accession No. GI No. Organism lysDH
BAB39707 13429872 Geobacillus stearothermophilus lysDH NP_147035.1
14602185 Aeropyrum pernix K1 lysDH NP_353966 15888285 Agrobacterium
tumefaciens lysDH AAZ94428 74026644 Achromobacter denitrificans
[0248] An enzyme that converts 3-oxoacids to 3-amino acids is
3,5-diaminohexanoate dehydrogenase (EC 1.4.1.11), an enzyme found
in organisms that ferment lysine. The gene encoding this enzyme,
kdd, was recently identified in Fusobacterium nucleatum (Kreimeyer
et al., 282:7191-7197 (2007)). The enzyme has been purified and
characterized in other organisms (Baker et al., 247:7724-7734
(1972); Baker et al., 13:292-299 (1974)) but the genes associated
with these enzymes are not known. Candidates in Myxococcus xanthus,
Porphyromonas gingivalis W83 and other sequenced organisms can be
inferred by sequence homology.
TABLE-US-00048 TABLE 47 Gene Accession No. GI No. Organism kdd
AAL93966.1 19713113 Fusobacterium nucleatum mxan_439 ABF87267.1
108462082 Myxococcus xanthus pg_1069 AAQ66183.1 34397119
Porphyromonas gingivalis
[0249] Steps R and U in FIG. 9B can be catalyzed by
aminotransferases that reversibly convert an aldehyde or ketone to
an amino group. Common amino donor/acceptor combinations include
glutamate/alpha-ketoglutarate, alanine/pyruvate, and
aspartate/oxaloacetate. Several enzymes have been shown to convert
aldehydes to primary amines, and vice versa.
Lysine-6-aminotransferase (EC 2.6.1.36) is one exemplary enzyme
capable of forming a primary amine. This enzyme function,
converting lysine to alpha-aminoadipate semialdehyde, has been
demonstrated in yeast and bacteria. Candidates from Candida utilis
(Hammer et al., J Basic Microbiol 32:21-27 (1992)), Flavobacterium
lutescens (Fujii et al., J Biochem. 128:391-397 (2000)) and
Streptomyces clavuligenus (Romero et al., J Ind. Microbiol
Biotechnol 18:241-246 (1997)) have been characterized. A
recombinant lysine-6-aminotransferase from S. clavuligenus was
functionally expressed in E. coli (Tobin et al., J Bacteriol.
173:6223-6229 (1991)). The F. lutescens enzyme is specific to
alpha-ketoglutarate as the amino acceptor (Soda et al., 7:4110-4119
(1968)). Other enzymes which convert aldehydes to terminal amines
include the dat gene product in Acinetobacter baumanii encoding
2,4-diaminobutanoate:2-ketoglutarate 4-transaminase (Ikai et al., J
Bacteriol. 179:5118-5125 (1997)). In addition to its natural
substrate, 2,4-diaminobutyrate, DAT transaminates the terminal
amines of lysine, 4-aminobutyrate and ornithine.
TABLE-US-00049 TABLE 48 Gene Accession No. GI No. Organism lat
BAB13756.1 10336502 Flavobacterium lutescens lat AAA26777.1 153343
Streptomyces clavuligenus dat P56744.1 6685373 Acinetobacter
baumanii
[0250] The conversion of an aldehyde to a terminal amine can also
be catalyzed by gamma-aminobutyrate transaminase (GABA transaminase
or 4-aminobutyrate transaminase). This enzyme naturally
interconverts succinic semialdehyde and glutamate to
4-aminobutyrate and alpha-ketoglutarate and is known to have a
broad substrate range (Schulz et al., 56:1-6 (1990); Liu et al.,
43:10896-10905 (2004)). The two GABA transaminases in E. coli are
encoded by gabT (Bartsch et al., J Bacteriol. 172:7035-7042 (1990))
and puuE (Kurihara et al., J. Biol. Chem. 280:4602-4608 (2005)).
GABA transaminases in Mus musculus, Pseudomonas fluorescens, and
Sus scrofa have been shown to react with a range of alternate
substrates including 6-aminocaproic acid (Cooper, 113:80-82 (1985);
SCOTT et al., 234:932-936 (1959)).
TABLE-US-00050 TABLE 49 Gene Accession No. GI No. Organism gabT
NP_417148.1 16130576 Escherichia coli puuE NP_415818.1 16129263
Escherichia coli Abat NP_766549.2 37202121 Mus musculus gabT
YP_257332.1 70733692 Pseudomonas fluorescens abat NP_999428.1
47523600 Sus scrofa
[0251] Additional enzyme candidates for interconverting aldehydes
and primary amines are putrescine transminases or other diamine
aminotransferases. The E. coli putrescine aminotransferase is
encoded by the ygjG gene and the purified enzyme also was able to
transaminate cadaverine and spermidine (Samsonova et al., BMC.
Microbiol 3:2 (2003)). In addition, activity of this enzyme on
1,7-diaminoheptane and with amino acceptors other than
2-oxoglutarate (e.g., pyruvate, 2-oxobutanoate) has been reported
(Samsonova et al., BMC. Microbiol 3:2 (2003); KIM, 239:783-786
(1964)). A putrescine aminotransferase with higher activity with
pyruvate as the amino acceptor than alpha-ketoglutarate is the spuC
gene of Pseudomonas aeruginosa (Lu et al., J Bacteriol.
184:3765-3773 (2002)).
TABLE-US-00051 TABLE 50 Gene Accession No. GI No. Organism ygjG
NP_417544 145698310 Escherichia coli spuC AAG03688 9946143
Pseudomonas aeruginosa
[0252] Enzymes that transaminate 3-oxoacids include GABA
aminotransferase (described above),
beta-alanine/alpha-ketoglutarate aminotransferase and
3-amino-2-methylpropionate aminotransferase.
Beta-alanine/alpha-ketoglutarate aminotransferase (WO08027742)
reacts with beta-alanine to form malonic semialdehyde, a 3-oxoacid.
The gene product of SkPYD4 in Saccharomyces kluyveri was shown to
preferentially use beta-alanine as the amino group donor (Andersen
et al., Gene 124:105-109 (1993)). SkUGA1 encodes a homologue of
Saccharomyces cerevisiae GABA aminotransferase, UGA1 (Ramos et al.,
Eur. J. Biochem. 149:401-404 (1985)), whereas SkPYD4 encodes an
enzyme involved in both beta-alanine and GABA transamination
(Andersen and Hansen, Gene 124:105-109 (1993)).
3-Amino-2-methylpropionate transaminase catalyzes the
transformation from methylmalonate semialdehyde to
3-amino-2-methylpropionate. The enzyme has been characterized in
Rattus norvegicus and Sus scrofa and is encoded by Abat (Tamaki et
al., 324:376-389 (2000); Kakimoto et al., 156:374-380 (1968)).
TABLE-US-00052 TABLE 51 Gene Accession No. GI No. Organism SkyPYD4
ABF58893.1 98626772 Lachancea kluyveri SkUGA1 ABF58894.1 98626792
Lachancea kluyveri UGA1 NP_011533.1 6321456 Saccharomyces
cerevisiae Abat P50554.3 122065191 Rattus norvegicus Abat P80147.2
120968 Sus scrofa
[0253] Several aminotransferases transaminate the amino groups of
amino acids to form 2-oxoacids. Aspartate aminotransferase is an
enzyme that naturally transfers an oxo group from oxaloacetate to
glutamate, forming alpha-ketoglutarate and aspartate. Aspartate is
similar in structure to OHED and 2-AHD. Aspartate aminotransferase
activity is catalyzed by, for example, the gene products of aspC
from Escherichia coli (Yagi et al., 100:81-84 (1979): Yagi et al.,
113:83-89 (1985)), AAT2 from Saccharomyces cerevisiae (Yagi et al.,
92:35-43 (1982)) and ASP5 from Arabidopsis thaliana (Kwok et al.,
55:595-604 (2004); de la et al., 46:414-425 (2006); Wilkie et al.,
Protein Expr. Purif. 12:381-389 (1998)). The enzyme from Rattus
norvegicus has been shown to transaminate alternate substrates such
as 2-aminohexanedioic acid and 2,4-diaminobutyric acid (Recasens et
al., 19:4583-4589 (1980)). Aminotransferases that work on other
amino-acid substrates may also be able to catalyze this
transformation. Valine aminotransferase catalyzes the conversion of
valine and pyruvate to 2-ketoisovalerate and alanine. The E. coli
gene, avtA, encodes one such enzyme (Whalen et al., J. Bacteriol.
150:739-746 (1982)). This gene product also catalyzes the
transamination of alpha-ketobutyrate to generate
.alpha.-aminobutyrate, although the amine donor in this reaction
has not been identified (Whalen et al., J. Bacteriol. 158:571-574
(1984)). The gene product of the E. coli serC catalyzes two
reactions, phosphoserine aminotransferase and
phosphohydroxythreonine aminotransferase (Lam et al., J. Bacteriol.
172:6518-6528 (1990)), and activity on nonphosphorylated substrates
could not be detected (Drewke et al., FEBS. Lett. 390:179-182
(1996)).
TABLE-US-00053 TABLE 52 Gene Accession No. GI No. Organism aspC
NP_415448.1 16128895 Escherichia coli AAT2 P23542.3 1703040
Saccharomyces cerevisiae ASP5 P46248.2 20532373 Arabidopsis
thaliana Got2 P00507 112987 Rattus norvegicus avtA YP_026231.1
49176374 Escherichia coli serC NP_415427.1 16128874 Escherichia
coli
[0254] Another enzyme candidate is alpha-aminoadipate
aminotransferase (EC 2.6.1.39), an enzyme that participates in
lysine biosynthesis and degradation in some organisms. This enzyme
interconverts 2-aminoadipate and 2-oxoadipate, using
alpha-ketoglutarate as the amino acceptor. Gene candidates are
found in Homo sapiens (Okuno et al., Enzyme Protein 47:136-148
(1993)) and Thermus thermophilus (Miyazaki et al., 150:2327-2334
(2004)). The Thermus thermophilus enzyme, encoded by lysN, is
active with several alternate substrates including oxaloacetate,
2-oxoisocaproate, 2-oxoisovalerate, and 2-oxo-3-methylvalerate.
TABLE-US-00054 TABLE 53 Gene Accession No. GI No. Organism lysN
BAC76939.1 31096548 Thermus thermophilus AadAT-II Q8N5Z0.2 46395904
Homo sapiens
[0255] Alpha-ketoglutarate decarboxylase (Step I, FIG. 9B) and
glutamate decarboxylase (Step S, FIG. 9B) all involve the
decarboxylation of an alpha-ketoacid. The decarboxylation of
keto-acids is catalyzed by a variety of enzymes with varied
substrate specificities, including pyruvate decarboxylase (EC
4.1.1.1), benzoylformate decarboxylase (EC 4.1.1.7),
alpha-ketoglutarate decarboxylase and branched-chain alpha-ketoacid
decarboxylase.
[0256] Pyruvate decarboxylase (PDC), also termed keto-acid
decarboxylase, is a key enzyme in alcoholic fermentation,
catalyzing the decarboxylation of pyruvate to acetaldehyde. The
enzyme from Saccharomyces cerevisiae has a broad substrate range
for aliphatic 2-keto acids including 2-ketobutyrate,
2-ketovalerate, 3-hydroxypyruvate and 2-phenylpyruvate. This enzyme
has been extensively studied, engineered for altered activity, and
functionally expressed in E. coli (Killenberg-Jabs et al.,
268:1698-1704 (2001); Li et al., Biochemistry. 38:10004-10012
(1999); ter Schure et al., Appl. Environ. Microbiol. 64:1303-1307
(1998)). The PDC from Zymomonas mobilus, encoded by pdc, also has a
broad substrate range and has been a subject of directed
engineering studies to alter the affinity for different substrates
(Siegert et al., 18:345-357 (2005)). The crystal structure of this
enzyme is available (Killenberg-Jabs et al., 268:1698-1704 (2001)).
Other well-characterized PDC candidates include the enzymes from
Acetobacter pasteurians (Chandra et al., 176:443-451 (2001)) and
Kluyveromyces lactis (Krieger et al., 269:3256-3263 (2002)).
TABLE-US-00055 TABLE 54 Gene Accession No. GI No. Organism pdc
P06672.1 118391 Zymomonas mobilus pdc1 P06169 30923172
Saccharomyces cerevisiae pdc AM21208 20385191 Acetobacter
pasteurians pdc1 Q12629 52788279 Kluyveromyces lactis
[0257] Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a
broad substrate range and has been the target of enzyme engineering
studies. The enzyme from Pseudomonas putida has been extensively
studied and crystal structures of this enzyme are available
(Polovnikova et al., 42:1820-1830 (2003); Hasson et al.,
37:9918-9930 (1998)). Site-directed mutagenesis of two residues in
the active site of the Pseudomonas putida enzyme altered the
affinity (Km) of naturally and non-naturally occurring substrates
(Siegert et al., 18:345-357 (2005)). The properties of this enzyme
have been further modified by directed engineering (Lingen et al.,
Chembiochem. 4:721-726 (2003); Lingen et al., Protein Eng
15:585-593 (2002)). The enzyme from Pseudomonas aeruginosa, encoded
by mdlC, has also been characterized experimentally (Barrowman et
al., 34:57-60 (1986)). Additional gene candidates from Pseudomonas
stutzeri, Pseudomonas fluorescens and other organisms can be
inferred by sequence homology or identified using a growth
selection system developed in Pseudomonas putida (Henning et al.,
Appl. Environ. Microbiol. 72:7510-7517 (2006)).
TABLE-US-00056 TABLE 55 Gene Accession No. GI No. Organism mdlC
P20906.2 3915757 Pseudomonas putida mdlC Q9HUR2.1 81539678
Pseudomonas aeruginosa dpgB ABN80423.1 126202187 Pseudomonas
stutzeri ilvB-1 YP_260581.1 70730840 Pseudomonas fluorescens
[0258] A third enzyme capable of decarboxylating 2-oxoacids is
alpha-ketoglutarate decarboxylase (KGD). The substrate range of
this class of enzymes has not been studied to date. The KDC from
Mycobacterium tuberculosis (Tian et al., 102:10670-10675 (2005))
has been cloned and functionally expressed in other internal
projects at Genomatica. However, it is not an ideal candidate for
strain engineering because it is large (.about.130 kDa) and
GC-rich. KDC enzyme activity has been detected in several species
of rhizobia including Bradyrhizobium japonicum and Mesorhizobium
loti (Green et al., 182:2838-2844 (2000)). Although the
KDC-encoding gene(s) have not been isolated in these organisms, the
genome sequences are available and several genes in each genome are
annotated as putative KDCs. A KDC from Euglena gracilis has also
been characterized but the gene associated with this activity has
not been identified to date (Shigeoka et al., 288:22-28 (1991)).
The first twenty amino acids starting from the N-terminus were
sequenced MTYKAPVKDVKFLLDKVFKV (Shigeoka and Nakano, 288:22-28
(1991)). The gene could be identified by testing candidate genes
containing this N-terminal sequence for KDC activity.
TABLE-US-00057 TABLE 56 Gene Accession No. GI No. Organism kgd
O50463.4 160395583 Mycobacterium tuberculosis kgd NP_767092.1
27375563 Bradyrhizobium japonicum kgd NP_105204.1 13473636
Mesorhizobium loti
[0259] A fourth candidate enzyme for catalyzing this reaction is
branched chain alpha-ketoacid decarboxylase (BCKA). This class of
enzyme has been shown to act on a variety of compounds varying in
chain length from 3 to 6 carbons (Oku et al., 263:18386-18396
(1988); Smit et al., 71:303-311 (2005)). The enzyme in Lactococcus
lactis has been characterized on a variety of branched and linear
substrates including 2-oxobutanoate, 2-oxohexanoate,
2-oxopentanoate, 3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate
and isocaproate (Smit et al., 71:303-311 (2005)). The enzyme has
been structurally characterized (Berg et al., 318:1782-1786
(2007)). Sequence alignments between the Lactococcus lactis enzyme
and the pyruvate decarboxylase of Zymomonas mobilus indicate that
the catalytic and substrate recognition residues are nearly
identical (Siegert et al., 18:345-357 (2005)), so this enzyme would
be a promising candidate for directed engineering. Decarboxylation
of alpha-ketoglutarate by a BCKA was detected in Bacillus subtilis;
however, this activity was low (5%) relative to activity on other
branched-chain substrates (Oku and Kaneda, 263:18386-18396 (1988))
and the gene encoding this enzyme has not been identified to date.
Additional BCKA gene candidates can be identified by homology to
the Lactococcus lactis protein sequence. Many of the high-scoring
BLASTp hits to this enzyme are annotated as indolepyruvate
decarboxylases (EC 4.1.1.74). Indolepyruvate decarboxylase (IPDA)
is an enzyme that catalyzes the decarboxylation of indolepyruvate
to indoleacetaldehyde in plants and plant bacteria.
TABLE-US-00058 Gene Accession No. GI No. Organism kdcA AAS49166.1
44921617 Lactococcus lactis
[0260] Recombinant branched chain alpha-keto acid decarboxylase
enzymes derived from the E1 subunits of the mitochondrial
branched-chain keto acid dehydrogenase complex from Homo sapiens
and Bos taurus have been cloned and functionally expressed in E.
coli (Davie et al., 267:16601-16606 (1992); Wynn et al.,
267:12400-12403 (1992); Wynn et al., 267:1881-1887 (1992)). In
these studies, the authors found that co-expression of chaperonins
GroEL and GroES enhanced the specific activity of the decarboxylase
by 500-fold (Wynn et al., 267:12400-12403 (1992)). These enzymes
are composed of two alpha and two beta subunits.
TABLE-US-00059 TABLE 57 Gene Accession No. GI No. Organism BCKDHB
NP_898871.1 34101272 Homo sapiens BCKDHA NP_000700.1 11386135 Homo
sapiens BCKDHB P21839 115502434 Bos taurus BCKDHA P11178 129030 Bos
taurus
[0261] Conversion of succinate semialdehyde to 4-hydroxybutyrate
(Step C, FIG. 9B) can be catalyzed by an oxidoreducatse that
converts an aldehyde to alcohol. Exemplary genes encoding enzymes
that catalyze the conversion of an aldehyde to alcohol, that is,
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 C3
(Sulzenbacher et al. Journal of Molecular Biology 342:489-502
(2004)), and bdh I and bdh II from C. acetobutylicum which converts
butyrylaldehyde into butanol (Walter et al. Journal of Bacteriology
174:7149-7158 (1992)). The protein sequences for each of these
exemplary gene products, if available, can be found using the
following GenBank accession numbers:
TABLE-US-00060 TABLE 58 Gene Accession No. GI No. 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
[0262] Enzymes exhibiting 4-hydroxybutyrate dehydrogenase activity
(EC 1.1.1.61) 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-00061 TABLE 59 Gene Accession No. GI No. Organism 4-HBd
YP_726053.1 113867564 Ralstonia eutropha H16 4-HBd EDK35022.1
146348486 Clostridium kluyveri DSM 555 4-HBd Q94B07 75249805
Arabidopsis thaliana
[0263] The adhl gene from Geobacillus thermoglucosidasius M10EXG
(Jeon et al., J Biotechnol 135:127-133 (2008)) has been indicated
to exhibit high activity on both 4-hydroxybutanal and butanal. Thus
this enzyme exhibits 1,4-butanediol dehydrogenase activity.
TABLE-US-00062 TABLE 60 Gene Accession No. GI No. Organism adh1
AAR91477.1 40795502 Geobacillus thermoglucosidasius M10EXG
[0264] Another exemplary enzyme is 3-hydroxyisobutyrate
dehydrogenase which catalyzes the reversible oxidation of
3-hydroxyisobutyrate to methylmalonate semialdehyde. This enzyme
participates in valine, leucine and isoleucine degradation and has
been identified in bacteria, eukaryotes, and mammals. The enzyme
encoded by P84067 from Thermus thermophilus HB8 has been
structurally characterized (Lokanath et al. J Mol Biol 352:905-17
(2005)). The reversibility of the human 3-hydroxyisobutyrate
dehydrogenase was demonstrated using isotopically-labeled substrate
(Manning et al. Biochem J 231:481-484 (1985)). Additional genes
encoding this enzyme include 3hidh in Homo sapiens (Hawes et al.
Methods Enzymol. 324:218-228 (2000)) and Oryctolagus cuniculus
(Chowdhury et al. Biosci. Biotechnol Biochem. 60:2043-2047 (1996);
Hawes et al. Methods Enzymol. 324:218-228 (2000)), mmsb in
Pseudomonas aeruginosa, and dhat in Pseudomonas putida (Aberhart et
al. J Chem. Soc. [Perkin 1] 6:1404-1406 (1979); Chowdhury et al.
Biosci. Biotechnol Biochem. 67:438-441 (2003); Chowdhury et al.
Biosci. Biotechnol Biochem. 60:2043-2047 (1996)).
TABLE-US-00063 TABLE 61 Gene Accession No. GI No. 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
[0265] Several 3-hydroxyisobutyrate dehydrogenase enzymes have also
been shown to convert malonic semialdehyde to 3-hydroxyproprionic
acid (3-HP). Three gene candidates exhibiting this activity are
mmsB from Pseudomonas aeruginosa PAO1(62), mmsB from Pseudomonas
putida KT2440 (Liao et al., US Publication 2005/0221466) and mmsB
from Pseudomonas putida E23 (Chowdhury et al., Biosci. Biotechnol.
Biochem. 60:2043-2047 (1996)). An enzyme with 3-hydroxybutyrate
dehydrogenase activity in Alcaligenes faecalis M3A has also been
identified (Gokam et al., U.S. Pat. No. 7,393,676; Liao et al., US
Publication No. 2005/0221466). Additional gene candidates from
other organisms including Rhodobacter spaeroides can be inferred by
sequence similarity.
TABLE-US-00064 TABLE 62 Gene Accession No. GI No. Organism mmsB
AAA25892.1 151363 Pseudomonas aeruginosa mmsB NP_252259.1 15598765
Pseudomonas aeruginosa PAO1 mmsB NP_746775.1 26991350 Pseudomonas
putida KT2440 mmsB JC7926 60729613 Pseudomonas putida E23 orfB1
AAL26884 16588720 Rhodobacter spaeroides
[0266] 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, E. R. 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. J. Bacteriol.
188:8551-8559 (2006)).
[0267] Step V in FIG. 9B depicts the conversion of
alpha-ketoglutarate to succinyl-CoA. This reaction is catalyzed by
alpha-ketoglutarate dehydrogenase, These enzymes are multi-enzyme
complexes that catalyze a series of partial reactions which result
in acylating oxidative decarboxylation of 2-keto-acids. This
2-keto-acid dehydrogenase complex occupies key positions in
intermediary metabolism, and enzyme activity is typically tightly
regulated (Fries et al. Biochemistry 42:6996-7002 (2003)). The
enzyme shares a complex but common structure composed of multiple
copies of three catalytic components: alpha-ketoacid decarboxylase
(E1), dihydrolipoamide acyltransferase (E2) and dihydrolipoamide
dehydrogenase (E3). The E3 component is shared among all
2-keto-acid dehydrogenase complexes in an organism, while the E1
and E2 components are encoded by different genes. The enzyme
components are present in numerous copies in the complex and
utilize multiple cofactors to catalyze a directed sequence of
reactions via substrate channeling. The overall size of these
dehydrogenase complexes is very large, with molecular masses
between 4 and 10 million Da (that is, larger than a ribosome).
[0268] Activity of enzymes in the 2-keto-acid dehydrogenase family
is normally low or limited under anaerobic conditions in E. coli.
Increased production of NADH (or NADPH) could lead to a
redox-imbalance, and NADH itself serves as an inhibitor to enzyme
function. Engineering efforts have increased the anaerobic activity
of the E. coli pyruvate dehydrogenase complex (Kim et al. Appl.
Environ. Microbiol. 73:1766-1771 (2007); Kim et al. J. Bacteriol.
190:3851-3858) 2008); Zhou et al. Biotechnol. Lett. 30:335-342
(2008)). For example, the inhibitory effect of NADH can be overcome
by engineering an H322Y mutation in the E3 component (Kim et al. J.
Bacteriol. 190:3851-3858 (2008)). Structural studies of individual
components and how they work together in complex provide insight
into the catalytic mechanisms and architecture of enzymes in this
family (Aevarsson et al. Nat. Struct. Biol. 6:785-792 (1999); Zhou
et al. Proc. Natl. Acad. Sci. U.S.A. 98:14802-14807 (2001)). The
substrate specificity of the dehydrogenase complexes varies in
different organisms, but generally branched-chain ketoacid
dehydrogenases have the broadest substrate range.
[0269] Alpha-ketoglutarate dehydrogenase (AKGD) converts
alpha-ketoglutarate to succinyl-CoA and is the primary site of
control of metabolic flux through the TCA cycle (Hansford, R. G.
Curr. Top. Bioenerg. 10:217-278 (1980)). Encoded by genes sucA,
sucB and lpd in E. coli, AKGD gene expression is downregulated
under anaerobic conditions and during growth on glucose (Park et
al. Mol. Microbiol. 15:473-482 (1995)). Structural studies of the
catalytic core of the E2 component pinpoint specific residues
responsible for substrate specificity (Knapp et al. J. Mol. Biol.
280:655-668 (1998)). The Bacillus subtilis AKGD, encoded by odhAB
(E1 and E2) and pdhD (E3, shared domain), is regulated at the
transcriptional level and is dependent on the carbon source and
growth phase of the organism (Resnekov et al. Mol. Gen. Genet.
234:285-296 (1992)). In yeast, the LPD1 gene encoding the E3
component is regulated at the transcriptional level by glucose (Roy
and Dawes J. Gen. Microbiol. 133:925-933 (1987)). The E1 component,
encoded by KGD1, is also regulated by glucose and activated by the
products of HAP2 and HAP3 (Repetto and Tzagoloff Mol. Cell Biol.
9:2695-2705 (1989)). The AKGD enzyme complex, inhibited by products
NADH and succinyl-CoA, is well-studied in mammalian systems, as
impaired function of has been linked to several neurological
diseases (Tretter and dam-Vizi Philos. Trans. R. Soc. Lond B Biol.
Sci. 360:2335-2345 (2005)).
TABLE-US-00065 TABLE 63 Gene Accession No. GI No. Organism sucA
NP_415254.1 16128701 Escherichia coli str. K12 sucB NP_415255.1
16128702 Escherichia coli str. K12 lpd NP_414658.1 16128109
Escherichia coli str. K12 odhA P23129.2 51704265 Bacillus subtilis
odhB P16263.1 129041 Bacillus subtilis pdhD P21880.1 118672
Bacillus subtilis KGD1 NP_012141.1 6322066 Saccharomyces cerevisiae
KGD2 NP_010432.1 6320352 Saccharomyces cerevisiae LPD1 NP_116635.1
14318501 Saccharomyces cerevisiae
[0270] Steps A and H in FIG. 9B involve CoA transferase activities.
The gene products of cat1, cat2, and cat3 of Clostridium kluyveri
have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and
butyryl-CoA acetyltransferase activities (Seedorf et al. Proc Natl
Acad Sci U.S.A. 105(6):2128-2133 (2008); Sohling and Gottschalk J
Bacteriol 178(3):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)).
TABLE-US-00066 TABLE 64 Gene Accession No. GI No. Organism cat1
P38946.1 729048 Clostridium kluyveri cat2 P38942.2 1705614
Clostridium kluyveri cat3 EDK35586.1 146349050 Clostridium kluyveri
TVAG_395550 XP_001330176 123975034 Trichomonas vaginalis G3
Tb11.02.0290 XP_828352 71754875 Trypanosoma brucei
[0271] An additionally useful enzyme for this type of
transformation is acyl-CoA:acetate-CoA transferase, also known as
acetate-CoA transferase (EC 2.8.3.8), which has been shown to
transfer the CoA moiety to acetate from a variety of branched and
linear acyl-CoA substrates, including isobutyrate (Matthies and
Schink Appl Environ Microbiol 58:1435-1439 (1992)), valerate
(Vanderwinkel et al. Biochem. Biophys. Res Commun. 33:902-908
(1968)) and butanoate (Vanderwinkel, supra (1968)). This enzyme is
encoded by atoA (alpha subunit) and atoD (beta subunit) in E. coli
sp. K12 (Korolev et al. Acta Crystallogr. D Biol Crystallogr.
58:2116-2121 (2002); Vanderwinkel, supra (1968)). Similar enzymes
exist in Corynebacterium glutamicum ATCC 13032 (Duncan et al.,
68:5186-5190 (2002)), Clostridium saccharoperbutylacetonicum
(Kosaka et al., Biosci. Biotechnol Biochem. 71:58-68 (2007)), and
Clostridium acetobutylicum (Cary et al., 56:1576-1583 (1990);
Wiesenbom et al., 55:323-329 (1989)).
TABLE-US-00067 TABLE 65 Gene Accession No. GI No. Organism atoA
P76459.1 2492994 Escherichia coli K12 atoD P76458.1 2492990
Escherichia coli K12 actA YP_226809.1 62391407 Corynebacterium
glutamicum cg0592 YP_224801.1 62389399 Corynebacterium glutamicum
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
[0272] 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 and Buckel 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 (Mac et al. Eur. J. Biochem. 226:41-51 (1994)).
TABLE-US-00068 TABLE 66 Gene Accession No. GI No. Organism gctA
CAA57199.1 559392 Acidaminococcus fermentans gctB CAA57200.1 559393
Acidaminococcus fermentans
[0273] Step H of FIG. 9B involves CoA synthetase or ligase
reactions for 4-HB as the substrate. 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. 4-hydroxybutyryl-CoA synthetase activity has
been demonstrated in Metallosphaera sedula (Berg et al., Science
318:1782-1786 (2007)). This function has been tentatively assigned
to the Msed.sub.--1422 gene.
TABLE-US-00069 TABLE 67 Gene Accession No. GI No. 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 Msed_1422 YP_001191504 146304188 Metallosphaera sedula
[0274] ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is
another candidate enzyme that 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,
isobutyrate, 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).
TABLE-US-00070 TABLE 68 Gene Accession No. GI No. 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
[0275] Exemplary phosphate transferring acyltransferases that
transform 4-hydroxybutyryl-CoA to 4-hydroxybutyryl-phosphate
include phosphotransacetylase, encoded by pta, and
phosphotransbutyrylase, encoded by ptb. The pta gene from E. coli
encodes an enzyme that can convert acetyl-CoA into
acetyl-phosphate, and vice versa (Suzuki, T. Biochim. Biophys. Acta
191:559-569 (1969)). This enzyme can also utilize propionyl-CoA
instead of acetyl-CoA forming propionate in the process (Hesslinger
et al. Mol. Microbiol 27:477-492 (1998)). Similarly, the ptb gene
from C. acetobutylicum encodes an enzyme that can convert
butyryl-CoA into butyryl-phosphate (Walter et al. Gene 134(1): p.
107-11 (1993)); Huang et al. J Mol Microbiol Biotechnol 2(1): p.
33-38 (2000). Additional ptb genes can be 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-00071 TABLE 69 Gene Accession No. GI No. Organism pta
NP_416800.1 16130232 Escherichia coli ptb NP_349676 15896327
Clostridium acetobutylicum ptb AAR19757.1 38425288
butyrate-producing bacterium L2-50 ptb CAC07932.1 10046659 Bacillus
megaterium
[0276] The conversion of 4-hydroxybutyryl-phosphate to
4-hydroxybutanal can be catalyzed by an oxidoreductase in the EC
class 1.2.1. Aspartate semialdehyde dehydrogenase (ASD, EC
1.2.1.11) catalyzes the NADPH-dependent reduction of 4-aspartyl
phosphate to aspartate-4-semialdehyde. ASD participates in amino
acid biosynthesis and recently has been studied as an antimicrobial
target (Hadfield et al., 40:14475-14483 (2001)). The E. coli ASD
structure has been solved (Hadfield et al., 289:991-1002 (1999))
and the enzyme has been shown to accept the alternate substrate
beta-3-methylaspartyl phosphate (Shames et al., 259:15331-15339
(1984)). The Haemophilus influenzae enzyme has been the subject of
enzyme engineering studies to alter substrate binding affinities at
the active site (Blanco et al., Acta Crystallogr. D. Biol.
Crystallogr. 60:1388-1395 (2004); Blanco et al., Acta Crystallogr.
D. Biol. Crystallogr. 60:1808-1815 (2004)). Other ASD candidates
are found in Mycobacterium tuberculosis (Shafiani et al., J Appl
Microbiol 98:832-838 (2005)), Methanococcus jannaschii (Faehnle et
al., 353:1055-1068 (2005)), and the infectious microorganisms
Vibrio cholera and Heliobacter pylori (Moore et al., Protein Expr.
Purif. 25:189-194 (2002)). A related enzyme candidate is
acetylglutamylphosphate reductase (EC 1.2.1.38), an enzyme that
naturally reduces acetylglutamylphosphate to
acetylglutamate-5-semialdehyde, found in S. cerevisiae (Pauwels et
al., Eur. J Biochem. 270:1014-1024 (2003)), B. subtilis (O'Reilly
et al., 140 (Pt 5):1023-1025 (1994)) and other organisms.
TABLE-US-00072 TABLE 70 Gene Accession No. GI No. Organism asd
NP_417891.1 16131307 Escherichia coli asd YP_248335.1 68249223
Haemophilus influenzae asd AAB49996 1899206 Mycobacterium
tuberculosis VC2036 NP_231670 15642038 Vibrio cholera asd
YP_002301787.1 210135348 Heliobacter pylori ARG5,6 NP_010992.1
6320913 Saccharomyces cerevisiae argC NP_389001.1 16078184 Bacillus
subtilis
[0277] Other exemplary enzymes in this class include glyceraldehyde
3-phosphate dehydrogenase which converts glyceraldehyde-3-phosphate
into D-glycerate 1,3-bisphosphate (e.g., E. coli gapA (Branlant et
al., Eur. J. Biochem. 150:61-66 (1985))),
N-acetyl-gamma-glutamyl-phosphate reductase which converts
N-acetyl-L-glutamate-5-semialdehyde into
N-acetyl-L-glutamyl-5-phosphate (e.g., E. coli argC (Parsot et al.,
Gene. 68:275-283 (1988))), and glutamate-5-semialdehyde
dehydrogenase which converts L-glutamate-5-semialdehyde into
L-glutamyl-5-phospate (e.g., E. coli proA (Smith et al., J.
Bacteriol. 157:545-551 (1984a))). Genes encoding
glutamate-5-semialdehyde dehydrogenase enzymes from Salmonella
typhimurium (Mahan et al., J Bacteriol. 156:1249-1262 (1983)) and
Campylobacter jejuni (Louie et al., 240:29-35 (1993)) were cloned
and expressed in E. coli.
TABLE-US-00073 TABLE 71 Gene Accession No. GI No. Organism gapA
P0A9B2.2 71159358 Escherichia coli argC NP_418393.1 16131796
Escherichia coli proA NP_414778.1 16128229 Escherichia coli proA
NP_459319.1 16763704 Salmonella typhimurium proA P53000.2 9087222
Campylobacter jejuni
[0278] In some embodiments, the 2-amino-4-ketopentanoate (AKP)
thiolase encoded by one or more genes selected from the group
consisting of ortA (.alpha.), ortB (.beta.), Amet.sub.--2368
(.alpha.), Amet.sub.--2369 (.beta.), Teth514.sub.--1478 (.alpha.),
Teth514.sub.--1479 (.beta.), TTE1235 (.alpha.), and thrC
(.beta.).
[0279] In some embodiments, the AKP dehydrogenase is encoded by one
or more genes selected from the group consisting of thrA, akthr2,
hom6, hom1, hom2, fadB, fadJ, Hbd2, Hbd1, hbd, HSD17B10, phbB,
phaB, Msed.sub.--1423, Msed.sub.--0399, Msed.sub.--0389,
Msed.sub.--1993, adh, adhA, adh-A, mdh, ldhA, ldh, and bdh.
[0280] In some embodiments, the 2-amino-4-hydroxypentanoate
aminotransferase is encoded by one or more genes selected from the
group consisting of aspC, AAT2, ASP5, got2, avtA, lysN, AadAT-II,
dat, lat, ygjG, spuC, SkyPYD4, SkUGA1, UGA1, Abat, Abat, Gta-1,
gabT, and puuE.
[0281] In some embodiments, the 2-amino-4-hydroxypentanoate
oxidoreductase (deaminating) is encoded by one or more genes
selected from the group consisting of gdhA, gdh, gdhA1, rocG, gdh1,
gdh2, GDH, GDH2, ldh and nadX.
[0282] In some embodiments, the 2-oxo-4-hydroxypentanoate
decarboxylase is encoded by one or more genes selected from the
group consisting of pdc, pdc1, mdlC, dpgB, ilvB-1, kgd, kdcA, lysA,
panD, cadA, ldc, ldcC, AF323910.1:1 . . . 1299, odc1,
VV2.sub.--1235, dmpH, dmpE, xylII, xylIII, Reut_B5691, Reut_B5692,
CAD, pad1, pofK (pad), padC, pad, adc, cbei.sub.--3835, CLL_A2135,
RBAM.sub.--030030,
[0283] In some embodiments, the 3-hydroxybutyraldehdye reductase is
encoded by one or more genes selected from the group consisting of
alrA, ADH2, yqhD, bdh I, bdh II, adhA, 4hbd, adhI, P84067, mmsb,
dhat, and 3hidh.
[0284] In some embodiments, the AKP aminotransferase is encoded by
one or more genes selected from the group consisting of aspC, AAT2,
ASP5, got2, avtA, lysN, AadAT-II, dat, lat, ygjG, spuC, SkyPYD4,
SkUGA1, UGA1, Abat, Gta-1, gabT, and puuE.
[0285] In some embodiments, the AKP oxidoreductase (deaminating) is
encoded by one or more genes selected from the group consisting of
gdhA, gdh, gdhA1, rocG, gdh1, gdh2, GDH, GDH2, ldh and nadX. In
some embodiments, the 2,4-dioxopentanoate decarboxylase is encoded
by one or more genes selected from the group consisting of pdc,
pdc1, mdlC, dpgB, ilvB-1, kgd, kdcA, lysA, panD, cadA, ldc, ldcC,
AF323910.1:1 . . . 1299, odc1, VV2.sub.--1235, dmpH, dmpE, xylII,
xylIII, Reut_B5691, Reut_B5692, CAD, pad1, padC, and pad, adc,
cbei.sub.--3835, CLL_A2135, RBAM.sub.--030030.
[0286] In some embodiments, the 3-oxobutyraldehyde reductase
(ketone reducing) is encoded by one or more genes selected from the
group consisting of thrA, akthr2, hom6, hom1, hom2, fadB, fadJ,
Hbd2, Hbd1, hbd, HSD17B10, phbB, phaB, Msed.sub.--1423,
Msed.sub.--0399, Msed.sub.--0389, Msed.sub.--1993, adh, adhA,
adh-A, mdh, ldhA, ldh, and bdh.
[0287] In some embodiments, the 3-oxobutyraldehyde reductase
(aldehyde reducing) is encoded by one or more genes selected from
the group consisting of alrA, ADH2, yqhD, bdh I, bdh II, adhA,
4hbd, adhI, P84067, mmsb, dhat, and 3hidh.
[0288] In some embodiments, the 4-hydroxy-2-butanone reductase is
encoded by one or more genes selected from the group consisting of
thrA, akthr2, hom6, hom1, hom2, fadB, fadJ, Hbd2, Hbd1, hbd,
HSD17B10, phbB, phaB, Msed.sub.--1423, Msed.sub.--0399,
Msed.sub.--0389, Msed.sub.--1993, adh, adhA, adh-A, mdh, ldhA, ldh,
and bdh.
[0289] In some embodiments, the AKP decarboxylase is encoded by one
or more genes selected from the group consisting of pdc, pdc1,
mdlC, dpgB, ilvB-1, kgd, kdcA, lysA, panD, cadA, ldc, ldcC,
AF323910.1:1 . . . 1299, odc1, VV2.sub.--1235, dmpH, dmpE, xylII,
xylIII, Reut_B5691, Reut_B5692, CAD, pad1, pofK(pad), padC,
pad.
[0290] In some embodiments, the 4-aminobutan-2-one aminotransferase
is encoded by one or more genes selected from the group consisting
of aspC, AAT2, ASP5, got2, avtA, lysN, AadAT-II, dat, lat, ygjG,
spuC, SkyPYD4, SkUGA1, UGA1, Abat, Gta-1, gabT, and puuE.
[0291] In some embodiments, the 4-aminobutan-2-one oxidoreductase
(deaminating) is encoded by one or more genes selected from the
group consisting of gdhA, gdh, gdhA1, rocG, gdh1, gdh2, GDH, GDH2,
ldh, nadX, kdd and lysDH.
[0292] In some embodiments, the 4-aminobutan-2-one ammonia-lyase is
encoded by one or more genes selected from the group consisting of
aspA, ansB, mal and BAA28709.
[0293] In some embodiments, the butenone hydratase is encoded by
one or more genes selected from the group consisting of fumA, fumB,
fumC, fumH, fum1, MmcB, MmcC, hmd, BACCAP.sub.--02294,
ANACOL.sub.--02527, NtherDRAFT.sub.--2368, dmdA, dmdB, crt, crt1,
ech paaA, paaB, phaA, phaB, maoC, paaF, paaG, abfD,
Msed.sub.--1220, fadA, fadB, fadI, fadJ, and fadR.
[0294] In some embodiments, the AKP ammonia-lyase is encoded by one
or more genes selected from the group consisting of aspA, ansB, mal
and BAA28709.
[0295] In some embodiments, the acetylacrylate decarboxylase is
encoded by one or more genes selected from the group consisting of
pdc, pdc1, mdlC, dpgB, ilvB-1, kgd, kdcA, lysA, panD, cadA, ldc,
ldcC, AF323910.1:1 . . . 1299, odc1, VV2.sub.--1235, dmpH, dmpE,
xylII, xylIII, Reut_B5691, Reut_B5692, CAD, pad1, pofK (pad), padC,
pad, adc, cbei.sub.--3835, CLL_A2135, RBAM.sub.--030030)
[0296] In some embodiments, the acetoacetyl-CoA reductase
(CoA-dependent, aldehyde forming) is encoded by one or more genes
selected from the group consisting of acr1, sucD, bphG, bld, adhE,
Msed.sub.--0709, mcr, asd-2, Saci.sub.--2370, Ald, and eutE.
[0297] In some embodiments, the acetoacetyl-CoA reductase
(CoA-dependent, alcohol forming) is encoded by one or more genes
selected from the group consisting of adhE, adhE2, mcr,
Rcas.sub.--2929, NAP1.sub.--02720, MGP2080.sub.--00535, and
FAR.
[0298] In some embodiments, the acetoacetyl-CoA reductase (ketone
reducing) is encoded by one or more genes selected from the group
consisting of thrA, akthr2, hom6, hom1, hom2, fadB, fadJ, Hbd2,
Hbd1, hbd, HSD17B10, phbB, phaB, Msed.sub.--1423, Msed.sub.--0399,
Msed.sub.--0389, Msed.sub.--1993, adh, adhA, adh-A, mdh, ldhA, ldh,
and bdh.
[0299] In some embodiments, the 3-hydroxybutyryl-CoA reductase
(aldehyde forming) is encoded by one or more genes selected from
the group consisting of acr1, sucD, bphG, bld, adhE,
Msed.sub.--0709, mcr, asd-2, SacI.sub.--2370, Ald, and eutE.
[0300] In some embodiments, the 3-hydroxybutyryl-CoA reductase
(alcohol forming) is encoded by one or more genes selected from the
group consisting of adhE, adhE2, mcr, Rcas.sub.--2929,
NAP1.sub.--02720, MGP2080.sub.--00535, and FAR.
[0301] In some embodiments, the 4-hydroxybutyryl-CoA dehydratase is
encoded by one or more genes selected from the group consisting of
fumA, fumB, fumC, fumH, fum1, MmcB, MmcC, hmd, BACCAP.sub.--02294,
ANACOL.sub.--02527, NtherDRAFT.sub.--2368, dmdA, dmdB, crt, crt1,
ech, paaA, paaB, phaA, phaB, maoC, paaF, paaG, abfD,
Msed.sub.--1220, fadA, fadB, fadI, fadJ, and fadR.
[0302] In some embodiments, the crotonase is encoded by one or more
genes selected from the group consisting of fumA, fumB, fumC, fumH,
fumI, MmcB, MmcC, hmd, BACCAP.sub.--02294, ANACOL.sub.--02527,
NtherDRAFT.sub.--2368, dmdA, dmdB, crt, crt1, ech paaA, paaB, phaA,
phaB, maoC, paaF, paaG, abfD, Msed.sub.--1220, fadA, fadB, fadI,
fadJ, and fadR.
[0303] In some embodiments, the 3-hydroxybutyryl-CoA hydrolase,
transferase, or synthetase is encoded by one or more genes selected
from a group consisting of acot12, ACH1, acot8, tesB, acot8, teas,
ybgC, paaI, pbdB, gctA, gctB, and hibCH.
[0304] In some embodiments, 3-hydroxybutyrate reductase is encoded
by one or more genes selected from a group consisting of car, npt,
fadD9, BCG.sub.--2812c, nfa20150, nfa40540, SGR.sub.--6790,
SGR.sub.--665, griC, griD.
[0305] 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 1,3-butanediol biosynthetic pathways. Depending on the
host microbial organism chosen for biosynthesis, nucleic acids for
some or all of a particular 1,3-butanediol 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 1,3-butanediol 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 1,3-butanediol.
[0306] Host microbial organisms can be selected from, and the
non-naturally occurring microbial organisms generated in, for
example, bacteria, yeast, fungus or any of a variety of other
microorganisms applicable to fermentation processes. Exemplary
bacteria include species selected from Escherichia coli, Klebsiella
oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus
succinogenes, Mannheimia succiniciproducens, Rhizobium etli,
Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter
oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus
plantarum, Streptomyces coelicolor, Clostridium acetobutylicum,
Pseudomonas fluorescens, and Pseudomonas putida. Exemplary yeasts
or fungi include species selected from Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces
marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris,
Rhizopus arrhizus, Rhizobus oryzae, 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.
[0307] Depending on the 1,3-butanediol 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 1,3-butanediol
pathway-encoding nucleic acid and up to all encoding nucleic acids
for one or more 1,3-butanediol biosynthetic pathways. For example,
1,3-butanediol 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 1,3-butanediol 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 1,3-butanediol can be included.
[0308] 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 1,3-butanediol 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, up to all nucleic acids encoding the enzymes or proteins
constituting a 1,3-butanediol biosynthetic pathway disclosed
herein. In some embodiments, the non-naturally occurring microbial
organisms also can include other genetic modifications that
facilitate or optimize 1,3-butanediol 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 1,3-butanediol pathway precursors
such as acetyl-CoA.
[0309] Generally, a host microbial organism is selected such that
it produces the precursor of a 1,3-butanediol 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 is 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 1,3-butanediol
pathway.
[0310] In some embodiments, a non-naturally occurring microbial
organism of the invention is generated from a host that contains
the enzymatic capability to synthesize 1,3-butanediol. In this
specific embodiment it can be useful to increase the synthesis or
accumulation of a 1,3-butanediol pathway product to, for example,
drive 1,3-butanediol pathway reactions toward 1,3-butanediol
production. Increased synthesis or accumulation can be accomplished
by, for example, overexpression of nucleic acids encoding one or
more of the above-described 1,3-butanediol pathway enzymes or
proteins. Overexpression of the enzyme or enzymes and/or protein or
proteins of the 1,3-butanediol 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 1,3-butanediol, through overexpression of
one, two, three, four, five, that is, up to all nucleic acids
encoding 1,3-butanediol 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 1,3-butanediol biosynthetic
pathway.
[0311] 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.
[0312] 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, 1,3-butanediol 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 1,3-butanediol biosynthetic capability. For
example, a non-naturally occurring microbial organism having
1,3-butanediol biosynthetic pathway can comprise at least two
exogenous nucleic acids encoding desired enzymes or proteins. For
example, the non-naturally occurring microbial organism can
comprise at least two exogenous nucleic acids encoding an
acetoacetyl-CoA reductase (CoA-dependent, alcohol forming) and a
4-hydroxy,2-butanone reductase (FIG. 2, steps D and F). 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 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. For example, the
non-naturally occurring microbial organism can comprise at least
three exogenous nucleic acids encoding an acetoacetyl-CoA reductase
(CoA-dependent, aldehyde forming), a 3-oxobutyraldehyde reductase
(ketone reducing), and a 3-hydroxybutyraldehyde reductase (FIG. 2,
steps A, B and C). Similarly, any combination of four, 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. For example, the
non-naturally occurring microbial organism can comprise at least
five exogenous nucleic acids encoding an AKP thiolase, an AKP
decarboxylase, a 4-aminobutan-2-one ammonia-lyase, a butenone
hydratase, and a 4-hydroxy,2-butanone reductase, (FIG. 8B, steps A,
E, F, G and H). Other individual pathways depicted in the figures
are also contemplated embodiments of the compositions and methods
provided herein.
[0313] In addition to the biosynthesis of 1,3-butanediol 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
1,3-butanediol other than use of the 1,3-butanediol producers is
through addition of another microbial organism capable of
converting 1,3-butanediol pathway intermediate to 1,3-butanediol.
One such procedure includes, for example, the fermentation of a
microbial organism that produces 1,3-butanediol pathway
intermediate. The 1,3-butanediol pathway intermediate can then be
used as a substrate for a second microbial organism that converts
the 1,3-butanediol pathway intermediate to 1,3-butanediol. The
1,3-butanediol pathway intermediate can be added directly to
another culture of the second organism or the original culture of
the 1,3-butanediol 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.
[0314] 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,
1,3-butanediol. 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 1,3-butanediol 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, 1,3-butanediol
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
1,3-butanediol intermediate and the second microbial organism
converts the intermediate to 1,3-butanediol.
[0315] 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 1,3-butanediol.
[0316] Sources of encoding nucleic acids for 1,3-butanediol 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, as well as
other exemplary species disclosed herein or available as source
organisms for corresponding genes. However, with the complete
genome sequence available for now more than 550 species (with more
than half of these available on public databases such as the NCBI),
including 395 microorganism genomes and a variety of yeast, fungi,
plant, and mammalian genomes, the identification of genes encoding
the requisite 1,3-butanediol 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
1,3-butanediol 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.
[0317] In some instances, such as when an alternative
1,3-butanediol biosynthetic pathway exists in an unrelated species,
1,3-butanediol 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 can 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 1,3-butanediol.
[0318] Host microbial organisms can be selected from, and the
non-naturally occurring microbial organisms generated in, for
example, bacteria, yeast, fungus or any of a variety of other
microorganisms applicable to fermentation processes. Exemplary
bacteria include species selected from Escherichia coli, Klebsiella
oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus
succinogenes, Mannheimia succiniciproducens, Rhizobium etli,
Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter
oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus
plantarum, Streptomyces coelicolor, Clostridium acetobutylicum,
Pseudomonas fluorescens, and Pseudomonas putida. Exemplary yeasts
or fungi include species selected from Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces
marxianus, Aspergillus terreus, Aspergillus niger and Pichia
pastoris. E. coli is a particularly useful host organism since it
is a well characterized microbial organism suitable for genetic
engineering. Other particularly useful host organisms include yeast
such as Saccharomyces cerevisiae.
[0319] Methods for constructing and testing the expression levels
of a non-naturally occurring 1,3-butanediol-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).
[0320] Exogenous nucleic acid sequences involved in a pathway for
production of 1,3-butanediol 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.
[0321] An expression vector or vectors can be constructed to
include one or more 1,3-butanediol 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.
[0322] The invention provides a method for producing 1,3-BDO that
includes culturing the non-naturally occurring microbial organism
disclosed herein, under conditions and for a sufficient period of
time to produce 1,3-BDO, including organisms that incorporate one,
two, three, four, five, up to all exogenous nucleic acids encoding
enzymes that complete a 1,3-BDO pathway. The 1,3-BDO pathways
include a set of 1,3-BDO pathway enzymes, where the set of 1,3-BDO
pathway enzymes are identified as above, namely: (a) (1) a
2-amino-4-ketopentanoate (AKP) thiolase; (2) an AKP dehydrogenase;
(3) a 2-amino-4-hydroxypentanoate aminotransferase or
oxidoreductase (deaminating); (4) a 2-oxo-4-hydroxypentanoate
decarboxylase; and (5) a 3-hydroxybutyraldehyde reductase; (b) (1)
a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an AKP
aminotransferase or oxidoreductase (deaminating); (3) a
2,4-dioxopentanoate decarboxylase; (4) a 3-oxobutyraldehyde
reductase (ketone reducing); and (5) a 3-hydroxybutyraldehyde
reductase; (c) (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2)
an AKP aminotransferase or oxidoreductase (deaminating); (3) a
2,4-dioxopentanoate decarboxylase; (4) a 3-oxobutyraldehyde
reductase (aldehyde reducing); and (5) a 4-hydroxy-2-butanone
reductase; (d) (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2)
an AKP decarboxylase; (3) a 4-aminobutan-2-one aminotransferase or
oxidoreductase (deaminating); (4) a 3-oxobutyraldehyde reductase
(ketone reducing); and (5) a 3-hydroxybutyraldehyde reductase; (e)
(1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an AKP
decarboxylase; (3) a 4-aminobutan-2-one aminotransferase or
oxidoreductase (deaminating); (4) a 3-oxobutyraldehyde reductase
(aldehyde reducing); and (5) a 4-hydroxy-2-butanone reductase; (f)
(1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an AKP
decarboxylase; (3) a 4-aminobutan-2-one ammonia-lyase; (4) a
butanone hydratase; and (5) a 4-hydroxy-2-butanone reductase; (g)
(1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an AKP
ammonia-lyase; (3) an acetylacrylate decarboxylase; (4) a butanone
hydratase; and (5) a 4-hydroxy-2-butanone reductase; (h) (1) an
acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming); (2) a
3-oxobutyraldehyde reductase (ketone reducing); and (3) a
3-hydroxybutyraldehyde reductase; (i) (1) an acetoacetyl-CoA
reductase (CoA dependent, alcohol faulting) and (2) a
4-hydroxy-2-butanone reductase; (j) (1) an acetoacetyl-CoA
reductase (CoA-dependent, aldehyde forming); (2) a
3-oxobutyraldehyde reductase (aldehyde reducing); and (3) a
4-hydroxy-2-butanone reductase; (k) (1) an acetoacetyl-CoA
reductase (ketone reducing) and (2) a 3-hydroxybutyryl-CoA
reductase (alcohol forming); (l) (1) an acetoacetyl-CoA reductase
(ketone reducing); (2) a 3-hydroxybutyryl-CoA reductase (aldehyde
forming); and (3) a 3-hydroxybutyraldehyde reductase; (m) (1) a
4-hydroxybutyryl-CoA dehydratase; (2) a crotonase; and (3) a
3-hydroxybutyryl-CoA reductase (alcohol forming); and (n) (1) a
4-hydroxybutyryl-CoA dehydratase; (2) a crotonase; (3) a
3-hydroxybutyryl-CoA reductase (aldehyde forming); and (4) a
3-hydroxybutyraldehyde reductase.
[0323] In some embodiments, a method for producing 1,3-BDO includes
culturing a non-naturally occurring microbial organism provided
herein under conditions and for a sufficient period of time to
produce 1,3-BDO. In some embodiments, the non-naturally occurring
microbial organism has a 1,3-butanediol pathway, wherein said
microbial organism comprises at least one exogenous nucleic acid
encoding a 1,3-butanediol pathway enzyme expressed in a sufficient
amount to produce 1,3-butanediol. In some embodiments, the
non-naturally occurring microbial organism includes at least one
of
[0324] (i) a reductive TCA pathway, wherein said microbial organism
comprises at least one exogenous nucleic acid encoding a reductive
TCA pathway enzyme selected from the group consisting of an
ATP-citrate lyase, citrate lyase, a fumarate reductase, and an
alpha-ketoglutarate:ferredoxin oxidoreductase;
[0325] (ii) a reductive TCA pathway, wherein said microbial
organism comprises at least one exogenous nucleic acid encoding a
reductive TCA pathway enzyme selected from the group consisting of
a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate
carboxylase, a phosphoenolpyruvate carboxykinase, a CO
dehydrogenase, and an H.sub.2 hydrogenase; or
[0326] (iii) at least one exogenous nucleic acid encodes an enzyme
selected from the group consisting of a CO dehydrogenase, an
H.sub.2 hydrogenase, and combinations thereof;
[0327] wherein said 1,3-butanediol pathway comprises a pathway
selected from the group consisting of:
[0328] (a) (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an
AKP dehydrogenase; (3) a 2-amino-4-hydroxypentanoate
aminotransferase or oxidoreductase (deaminating); (4) a
2-oxo-4-hydroxypentanoate decarboxylase; and (5) a
3-hydroxybutyraldehyde reductase;
[0329] (b) (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an
AKP aminotransferase or oxidoreductase (deaminating); (3) a
2,4-dioxopentanoate decarboxylase; (4) a 3-oxobutyraldehyde
reductase (ketone reducing); and (5) a 3-hydroxybutyraldehyde
reductase;
[0330] (c) (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an
AKP aminotransferase or oxidoreductase (deaminating); (3) a
2,4-dioxopentanoate decarboxylase; (4) a 3-oxobutyraldehyde
reductase (aldehyde reducing); and (5) a 4-hydroxy-2-butanone
reductase;
[0331] (d) (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an
AKP decarboxylase; (3) a 4-aminobutan-2-one aminotransferase or
oxidoreductase (deaminating); (4) a 3-oxobutyraldehyde reductase
(ketone reducing); and (5) a 3-hydroxybutyraldehyde reductase;
[0332] (e) (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an
AKP decarboxylase; (3) a 4-aminobutan-2-one aminotransferase or
oxidoreductase (deaminating); (4) a 3-oxobutyraldehyde reductase
(aldehyde reducing); and (5) a 4-hydroxy-2-butanone reductase;
[0333] (f) (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an
AKP decarboxylase; (3) a 4-aminobutan-2-one ammonia-lyase; (4) a
butenone hydratase; and (5) a 4-hydroxy-2-butanone reductase;
[0334] (g) (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an
AKP ammonia-lyase; (3) an acetylacrylate decarboxylase; (4) a
butenone hydratase; and (5) a 4-hydroxy-2-butanone reductase;
[0335] (h) (1) an acetoacetyl-CoA reductase (CoA-dependent,
aldehyde forming); (2) a 3-oxobutyraldehyde reductase (ketone
reducing); and (3) a 3-hydroxybutyraldehyde reductase;
[0336] (i) (1) an acetoacetyl-CoA reductase (CoA dependent, alcohol
forming) and (2) a 4-hydroxy-2-butanone reductase;
[0337] (j) (1) an acetoacetyl-CoA reductase (CoA-dependent,
aldehyde forming); (2) a 3-oxobutyraldehyde reductase (aldehyde
reducing); and (3) a 4-hydroxy-2-butanone reductase;
[0338] (k) (1) an acetoacetyl-CoA reductase (ketone reducing) and
(2) a 3-hydroxybutyryl-CoA reductase (alcohol forming);
[0339] (l) (1) an acetoacetyl-CoA reductase (ketone reducing); (2)
a 3-hydroxybutyryl-CoA reductase (aldehyde forming); and (3) a
3-hydroxybutyraldehyde reductase;
[0340] (m) (1) a 4-hydroxybutyryl-CoA dehydratase; (2) a crotonase;
and (3) a 3-hydroxybutyryl-CoA reductase (alcohol forming); and
[0341] (n) (1) a 4-hydroxybutyryl-CoA dehydratase; (2) a crotonase;
(3) a 3-hydroxybutyryl-CoA reductase (aldehyde forming); and (4) a
3-hydroxybutyraldehyde reductase;
[0342] (o) (1) a succinyl-CoA transferase, a succinyl-CoA
synthetase or a succinyl-CoA ligase, (2) a succinyl-CoA reductase
(aldehyde forming), (3) a 4-hydroxybutyrate dehydrogenase, (4) a
4-hydroxybutyrate kinase, (5) a phosphotrans-4-hydroxybutyrylase,
(6) a 4-hydroxybutyryl-CoA dehydratase, (7) a crotonase, (8) a
3-hydroxybutyryl-CoA reductase (aldehyde forming), and (9) a
3-hydroxybutanal reductase;
[0343] (p) (1) (i) an alpha-ketoglutarate decarboxylase, or (ii)
(a) a glutamate dehydrogenase and/or a glutamate transaminase, (b)
a glutamate decarboxylase, and (c) a 4-aminobutyrate dehydrogenase
and/or a 4-aminobutyrate transaminase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase; (6) a crotonase, (7) a 3-hydroxybutyryl-CoA reductase
(aldehyde forming), and (8) a 3-hydroxybutanal reductase;
[0344] (q) (1) (i) an alpha-ketoglutarate decarboxylase, or (ii)
(a) a glutamate dehydrogenase and/or a glutamate transaminase, (b)
a glutamate decarboxylase, and (c) a 4-aminobutyrate dehydrogenase
and/or a 4-aminobutyrate transaminase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA
dehydratase, (5) a crotonase, (6) a 3-hydroxybutyryl-CoA reductase
(aldehyde forming), and (7) a 3-hydroxybutanal reductase;
[0345] (r) (1) (i) an alpha-ketoglutarate decarboxylase, or (ii)
(a) a glutamate dehydrogenase and/or a glutamate transaminase, (b)
a glutamate decarboxylase, and (c) a 4-aminobutyrate dehydrogenase
and/or a 4-aminobutyrate transaminase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, and (7) a 3-hydroxybutyryl-CoA
reductase (alcohol forming);
[0346] (s) (1) (i) an alpha-ketoglutarate decarboxylase, or (ii)
(a) a glutamate dehydrogenase and/or a glutamate transaminase, (b)
a glutamate decarboxylase, and (c) a 4-aminobutyrate dehydrogenase
and/or a 4-aminobutyrate transaminase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA
dehydratase, (5) a crotonase, and (6) a 3-hydroxybutyryl-CoA
reductase (alcohol forming);
[0347] (t) (1) (i) an alpha-ketoglutarate decarboxylase, or (ii)
(a) a glutamate dehydrogenase and/or a glutamate transaminase, (b)
a glutamate decarboxylase, and (c) a 4-aminobutyrate dehydrogenase
and/or a 4-aminobutyrate transaminase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA hydrolase,
transferase or synthetase, and (8) a 3-hydroxybutyrate
reductase;
[0348] (u) (1) (i) an alpha-ketoglutarate decarboxylase, or (ii)
(a) a glutamate dehydrogenase and/or a glutamate transaminase, (b)
a glutamate decarboxylase, and (c) a 4-aminobutyrate dehydrogenase
and/or a 4-aminobutyrate transaminase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA
dehydratase, (5) a crotonase, (6) a 3-hydroxybutyryl-CoA hydrolase,
transferase or synthetase, and (7) a 3-hydroxybutyrate
reductase.
[0349] (v) (1) a succinate reductase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA reductase
(aldehyde forming), and (8) a 3-hydroxybutanal reductase;
[0350] (w) (1) a succinate reductase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA
dehydratase, (5) a crotonase, (6) a 3-hydroxybutyryl-CoA reductase
(aldehyde forming), (7) a 3-hydroxybutanal reductase;
[0351] (x) (1) a succinate reductase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, and (7) a 3-hydroxybutyryl-CoA
reductase (alcohol forming);
[0352] (y) (1) a succinate reductase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA
dehydratase, (5) a crotonase, and (6) a 3-hydroxybutyryl-CoA
reductase (alcohol forming);
[0353] (z) (1) a succinate reductase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA hydrolase,
transferase or synthetase, and (8) a 3-hydroxybutyrate
reductase;
[0354] (aa) (1) a succinate reductase, (2) a 4-hydroxybutyrate
dehydrogenase, (3) a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA
dehydratase, (5) a crotonase, (6) a 3-hydroxybutyryl-CoA hydrolase,
transferase or synthetase, and (7) a 3-hydroxybutyrate
reductase;
[0355] (bb) (1) a succinyl-CoA transferase, succinyl-CoA synthetase
or succinyl-CoA ligase, (2) a succinyl-CoA reductase (aldehyde
forming), (3) a 4-hydroxybutyrate dehydrogenase, (5) a
4-hydroxybutyrate kinase, (6) a pPhosphotrans-4-hydroxybutyrylase,
(7) a 4-hydroxybutyryl-CoA dehydratase, (8) a crotonase, and (9) a
3-hydroxybutyryl-CoA reductase (alcohol forming);
[0356] (cc) (1) a succinyl-CoA transferase, succinyl-CoA synthetase
or succinyl-CoA ligase, (2) a succinyl-CoA reductase (aldehyde
forming), (3) a 4-hydroxybutyrate dehydrogenase, (4) a
4-hydroxybutyrate kinase, (5) a phosphotrans-4-hydroxybutyrylase,
(6) a 4-hydroxybutyryl-CoA dehydratase, (7) a crotonase, (8) a
3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, and (9)
a 3-hydroxybutyrate reductase;
[0357] (dd) (1) a succinyl-CoA transferase, succinyl-CoA synthetase
or succinyl-CoA ligase, (2) a succinyl-CoA reductase (alcohol
forming), (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA reductase
(aldehyde forming), and (8) a 3-hydroxybutanal reductase;
[0358] (ee) (1) a succinyl-CoA transferase, succinyl-CoA synthetase
or succinyl-CoA ligase, (2) a succinyl-CoA reductase (alcohol
forming), (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, and (7) a 3-hydroxybutyryl-CoA
reductase (alcohol forming);
[0359] (ff) (1) a succinyl-CoA transferase, succinyl-CoA synthetase
or succinyl-CoA ligase, (2) a succinyl-CoA reductase (alcohol
forming), (3) a 4-hydroxybutyrate kinase, (4) a
phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA hydrolase,
transferase or synthetase, and (8) a 3-hydroxybutyrate
reductase;
[0360] (gg) (1) a succinyl-CoA transferase, succinyl-CoA synthetase
or succinyl-CoA ligase, (2) a succinyl-CoA reductase (aldehyde
forming), (3) a 4-hydroxybutyrate dehydrogenase, (4) a
4-hydroxybutyryl-CoA transferase, or 4-hydroxybutyryl-CoA
synthetase, (5) a 4-hydroxybutyryl-CoA dehydratase, (6) a
crotonase, (7) a 3-hydroxybutyryl-CoA reductase (aldehyde forming),
and (8) a 3-hydroxybutanal reductase;
[0361] (hh) (1) a succinyl-CoA transferase, succinyl-CoA synthetase
or succinyl-CoA ligase, (2) a succinyl-CoA reductase (aldehyde
forming), (3) a 4-hydroxybutyrate dehydrogenase, (4) a
4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA
synthetase, (5) a 4-hydroxybutyryl-CoA dehydratase, (6) a
crotonase, and (7) a 3-hydroxybutyryl-CoA reductase (alcohol
forming);
[0362] (ii) (1) a succinyl-CoA transferase, succinyl-CoA synthetase
or succinyl-CoA ligase, (2) a succinyl-CoA reductase (aldehyde
forming), (3) a 4-hydroxybutyrate dehydrogenase, (4) a
4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA
synthetase, (5) a 4-hydroxybutyryl-CoA dehydratase, (6) a
crotonase, (7) a 3-hydroxybutyryl-CoA hydrolase, transferase or
synthetase, (8) a 3-hydroxybutyrate reductase;
[0363] (jj) (1) a succinyl-CoA transferase, succinyl-CoA synthetase
or succinyl-CoA ligase, (2) a succinyl-CoA reductase (alcohol
forming), (3) a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA
dehydratase, (5) a crotonase, (6) a 3-hydroxybutyryl-CoA reductase
(aldehyde forming), and (7) a 3-hydroxybutanal reductase;
[0364] (kk) (1) a succinyl-CoA transferase, succinyl-CoA synthetase
or succinyl-CoA ligase, (2) a succinyl-CoA reductase (alcohol
forming), (3) a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA
dehydratase, (5) a crotonase, and (6) a 3-hydroxybutyryl-CoA
reductase (alcohol forming); and
[0365] (ll) (1) a succinyl-CoA transferase, succinyl-CoA synthetase
or succinyl-CoA ligase, (2) a succinyl-CoA reductase (alcohol
forming), (3) a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, (5) a 4-hydroxybutyryl-CoA
dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA hydrolase,
transferase or synthetase, and (8) a 3-hydroxybutyrate
reductase.
[0366] In some embodiments, the method includes a non-naturally
occurring microbial organism (e.g., having pathway (i)) that
further includes 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.
[0367] In some embodiments, the method includes a non-naturally
occurring microbial organism (e.g., having pathway (ii)) that
further includes 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.
[0368] In some embodiments, the method includes a microbial
organism that includes two, three, four, five, six, seven, eight or
nine exogenous nucleic acids, each encoding a 1,3-BDO pathway
enzyme.
[0369] In some embodiments, the method includes a microbial
organism that comprises exogenous nucleic acids encoding each of
the enzymes of at least one of the 1,3-butanediol pathways selected
from the group consisting of (a)-(ll).
[0370] In some embodiments, the method includes at least one
exogenous nucleic acid that is a heterologous nucleic acid.
[0371] In some embodiments, the method includes the non-naturally
occurring microbial organism in a substantially anaerobic culture
medium.
[0372] Suitable purification and/or assays to test for the
production of 1,3-butanediol 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 (see, for example, WO/2008/115840 and Hanai
et al., Appl. Environ. Microbiol. 73:7814-7818 (2007)).
[0373] The 1,3-butanediol 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.
[0374] 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
1,3-butanediol producers can be cultured for the biosynthetic
production of 1,3-butanediol.
[0375] For the production of 1,3-butanediol, 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
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 States Publication No. US-2009-0047719, filed Aug. 10, 2007.
Fermentations can be performed in a batch, fed-batch or continuous
manner, as disclosed herein.
[0376] 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.
[0377] In addition to renewable feedstocks such as those
exemplified above, the 1,3-butanediol 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 1,3-butanediol producing organisms to
provide a metabolic pathway for utilization of syngas or other
gaseous carbon source.
[0378] Organisms of the present invention can utilize, and 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 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 1,3-butanediol.
[0379] In addition to renewable feedstocks such as those
exemplified above, the 1,3-butanediol 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 1,3-butanediol producing organisms to
provide a metabolic pathway for utilization of syngas or other
gaseous carbon source.
[0380] 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.
[0381] The Wood-Ljungdahl pathway catalyzes the conversion of CO
and H.sub.2 to acetyl-CoA and other products such as acetate.
Organisms capable of utilizing CO and syngas also generally have
the capability of utilizing CO.sub.2 and CO.sub.2/H.sub.2 mixtures
through the same basic set of enzymes and transformations
encompassed by the Wood-Ljungdahl pathway. H.sub.2-dependent
conversion of CO.sub.2 to acetate by microorganisms was recognized
long before it was revealed that CO also could be used by the same
organisms and that the same pathways were involved. Many acetogens
have been shown to grow in the presence of CO.sub.2 and produce
compounds such as acetate as long as hydrogen is present to supply
the necessary reducing equivalents (see for example, Drake,
Acetogenesis, pp. 3-60 Chapman and Hall, New York, (1994)). This
can be summarized by the following equation:
2CO.sub.2+4H.sub.2+nADP+nPi.fwdarw.CH.sub.3COOH+2H.sub.2O+nATP
[0382] 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.
[0383] 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 1,3-butanediol 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.
[0384] 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 1,3-butanediol 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 1,3-butanediol 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.
[0385] 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, syngas, CO and/or CO2.
Such compounds include, for example, 1,3-butanediol and any of the
intermediate metabolites in the 1,3-butanediol 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 1,3-butanediol biosynthetic pathways. Accordingly, the
invention provides a non-naturally occurring microbial organism
that produces and/or secretes 1,3-butanediol when grown on a
carbohydrate or other carbon source and produces and/or secretes
any of the intermediate metabolites shown in the 1,3-butanediol
pathway when grown on a carbohydrate or other carbon source. The
1,3-butanediol producing microbial organisms of the invention can
initiate synthesis from an intermediate, for example,
acetyl-CoA.
[0386] 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 1,3-butanediol pathway enzyme or protein in sufficient
amounts to produce 1,3-butanediol. It is understood that the
microbial organisms of the invention are cultured under conditions
sufficient to produce 1,3-butanediol. Following the teachings and
guidance provided herein, the non-naturally occurring microbial
organisms of the invention can achieve biosynthesis of
1,3-butanediol resulting in intracellular concentrations between
about 0.1-200 mM or more. Generally, the intracellular
concentration of 1,3-butanediol is between about 3-150 mM,
particularly between about 5-125 mM and more particularly between
about 8-100 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, or
more. Intracellular concentrations between and above each of these
exemplary ranges also can be achieved from the non-naturally
occurring microbial organisms of the invention.
[0387] 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 1,3-butanediol producers can synthesize
1,3-butanediol 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, 1,3-butanediol producing microbial
organisms can produce 1,3-butanediol intracellularly and/or secrete
the product into the culture medium.
[0388] In addition to the culturing and fermentation conditions
disclosed herein, growth condition for achieving biosynthesis of
1,3-butanediol 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, dimethylsulfonioproprionate,
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.
[0389] 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 1,3-butanediol or any 1,3-butanediol 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 1,3-butanediol or
1,3-butanediol pathway intermediate including any 1,3-butanediol
impurities, or for side products generated in reactions diverging
away from a 1,3-butanediol pathway. Isotopic enrichment can be
achieved for any target atom including, for example, carbon,
hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or other
halogens.
[0390] 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.
[0391] 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 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 CO2, which can possess a larger amount of carbon-14
than its petroleum-derived counterpart.
[0392] 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".
[0393] 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.
[0394] 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.
[0395] 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 isotope with respect to another, for
example, the preferential uptake in biological systems of C.sup.12
over C.sup.13 over C.sup.14, and these corrections are reflected as
a Fm corrected for .delta..sup.13.
[0396] 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.
[0397] 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.
[0398] 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.
[0399] 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).
[0400] Accordingly, in some embodiments, the present invention
provides 1,3-butanediol or a 1,3-butanediol 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 1,3-butanediol or a
1,3-butanediol 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 1,3-butanediol
or a 1,3-butanediol intermediate that has a carbon-12, carbon-13,
and carbon-14 ratio that reflects petroleum-based carbon uptake
source. In this aspect, the 1,3-butanediol or a 1,3-butanediol
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 1,3-butanediol or a 1,3-butanediol 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.
[0401] Further, the present invention relates to the biologically
produced 1,3-butanediol or 1,3-butanediol intermediate as disclosed
herein, and to the products derived therefrom, wherein the
1,3-butanediol or a 1,3-butanediol 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 1,3-butanediol or a
bioderived 1,3-butanediol 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
1,3-butanediol or a bioderived 1,3-butanediol intermediate as
disclosed herein, wherein the bioderived product is chemically
modified to generate a final product. Methods of chemically
modifying a bioderived product of 1,3-butanediol, 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 organic solvents, polyurethane resins, polyester
resins, hypoglycaemic agents, butadiene and/or butadiene-based
products 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 organic solvents, polyurethane
resins, polyester resins, hypoglycaemic agents, butadiene and/or
butadiene-based products are generated directly from or in
combination with bioderived 1,3-butanediol or a bioderived
1,3-butanediol intermediate as disclosed herein.
[0402] 1,3-butanediol is a chemical used in commercial and
industrial applications and is also used as a raw material in the
production of a wide range of products. Non-limiting examples of
such applications and products include organic solvents,
polyurethane resins, polyester resins, hypoglycaemic agents,
butadiene and/or butadiene-based products organic solvents,
polyurethane resins, polyester resins, hypoglycaemic agents,
butadiene and/or butadiene-based products. Accordingly, in some
embodiments, the invention provides biobased used as a raw material
in the production of a wide range of products comprising one or
more bioderived 1,3-butanediol or bioderived 1,3-butanediol
intermediate produced by a non-naturally occurring microorganism of
the invention or produced using a method disclosed herein.
[0403] 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.
[0404] In some embodiments, the invention provides organic
solvents, polyurethane resins, polyester resins, hypoglycaemic
agents, butadiene and/or butadiene-based products comprising
bioderived 1,3-butanediol or bioderived 1,3-butanediol
intermediate, wherein the bioderived 1,3-butanediol or bioderived
1,3-butanediol intermediate includes all or part of the
1,3-butanediol or 1,3-butanediol intermediate used in the
production of organic solvents, polyurethane resins, polyester
resins, hypoglycaemic agents, butadiene and/or butadiene-based
products. Thus, in some aspects, the invention provides biobased
organic solvents, polyurethane resins, polyester resins,
hypoglycaemic agents, butadiene and/or butadiene-based products
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
1,3-butanediol or bioderived 1,3-butanediol intermediate as
disclosed herein. Additionally, in some aspects, the invention
provides biobased organic solvents, polyurethane resins, polyester
resins, hypoglycaemic agents, butadiene and/or butadiene-based
products wherein the 1,3-butanediol or 1,3-butanediol intermediate
used in its production is a combination of bioderived and petroleum
derived 1,3-butanediol or 1,3-butanediol intermediate. For example,
biobased organic solvents, polyurethane resins, polyester resins,
hypoglycaemic agents, butadiene and/or butadiene-based products can
be produced using 50% bioderived 1,3-butanediol and 50% petroleum
derived 1,3-butanediol 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 organic solvents,
polyurethane resins, polyester resins, hypoglycaemic agents,
butadiene and/or butadiene-based products using the bioderived
1,3-butanediol or bioderived 1,3-butanediol intermediate of the
invention are well known in the art.
[0405] 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 1,3-butanediol pathway enzyme or protein in sufficient
amounts to produce 1,3-butanediol. It is understood that the
microbial organisms of the invention are cultured under conditions
sufficient to produce 1,3-butanediol. Following the teachings and
guidance provided herein, the non-naturally occurring microbial
organisms of the invention can achieve biosynthesis of
1,3-butanediol resulting in intracellular concentrations between
about 0.1-2000 mM or more. Generally, the intracellular
concentration of 1,3-butanediol is between about 3-1800 mM,
particularly between about 5-1700 mM and more particularly between
about 8-1600 mM, including about 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.
[0406] 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. patent application No. US 2009/0047719, filed Aug. 10, 2007.
Any of these conditions can be employed with the non-naturally
occurring microbial organisms as well as other anaerobic conditions
well known in the art. Under such anaerobic conditions, the
1,3-butanediol producers can synthesize 1,3-butanediol 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, 1,3-butanediol producing microbial organisms can
produce 1,3-butanediol intracellularly and/or secrete the product
into the culture medium.
[0407] In addition to the culturing and fermentation conditions
disclosed herein, growth condition for achieving biosynthesis of
1,3-butanediol 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, dimethylsulfonioproprionate,
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.
[0408] 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.
[0409] As described herein, one exemplary growth condition for
achieving biosynthesis of 1,3-butanediol 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
N2/CO2 mixture or other suitable non-oxygen gas or gases.
[0410] The culture conditions described herein can be scaled up and
grown continuously for manufacturing of 1,3-butanediol. 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 1,3-butanediol. Generally, and as with non-continuous
culture procedures, the continuous and/or near-continuous
production of 1,3-butanediol will include culturing a non-naturally
occurring 1,3-butanediol 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 period 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.
[0411] Fermentation procedures are well known in the art. Briefly,
fermentation for the biosynthetic production of 1,3-butanediol 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.
[0412] In addition to the above fermentation procedures using the
1,3-butanediol producers of the invention for continuous production
of substantial quantities of 1,3-butanediol, the 1,3-butanediol
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.
For example, 1,3-butanediol can be dehydrated to provide
1,3-butadiene.
[0413] 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 1,3-butadiene.
[0414] In some embodiments, syngas can be used as a carbon
feedstock. Important process considerations for a syngas
fermentation are high biomass concentration and good gas-liquid
mass transfer (Bredwell et al., Biotechnol Prog., 15:834-844
(1999). The solubility of CO in water is somewhat less than that of
oxygen. Continuously gas-sparged fermentations can be performed in
controlled fermenters with constant off-gas analysis by mass
spectrometry and periodic liquid sampling and analysis by GC and
HPLC. The liquid phase can function in batch mode. Fermentation
products such as alcohols, organic acids, and residual glucose
along with residual methanol are quantified by HPLC (Shimadzu,
Columbia Md.), for example, using an Aminex.RTM. series of HPLC
columns (for example, HPX-87 series) (BioRad, Hercules Calif.),
using a refractive index detector for glucose and alcohols, and a
UV detector for organic acids. The growth rate is determined by
measuring optical density using a spectrophotometer (600 nm). All
piping in these systems is glass or metal to maintain anaerobic
conditions. The gas sparging is performed with glass fits to
decrease bubble size and improve mass transfer. Various sparging
rates are tested, ranging from about 0.1 to 1 vvm (vapor volumes
per minute). To obtain accurate measurements of gas uptake rates,
periodic challenges are performed in which the gas flow is
temporarily stopped, and the gas phase composition is monitored as
a function of time.
[0415] In order to achieve the overall target productivity, methods
of cell retention or recycle are employed. One method to increase
the microbial concentration is to recycle cells via a tangential
flow membrane from a sidestream. Repeated batch culture can also be
used, as previously described for production of acetate by Moorella
(Sakai et al., J Biosci. Bioeng, 99:252-258 (2005)). Various other
methods can also be used (Bredwell et al., Biotechnol Prog.,
15:834-844 (1999); Datar et al., Biotechnol Bioeng, 86:587-594
(2004)). Additional optimization can be tested such as overpressure
at 1.5 atm to improve mass transfer (Najafpour et al., Enzyme and
Microbial Technology, 38[1-2], 223-228 (2006)).
[0416] Once satisfactory performance is achieved using pure H2/CO
as the feed, synthetic gas mixtures are generated containing
inhibitors likely to be present in commercial syngas. For example,
a typical impurity profile is 4.5% CH4, 0.1% C2H2, 0.35% C2H6, 1.4%
C2H4, and 150 ppm nitric oxide (Datar et al., Biotechnol Bioeng,
86:587-594 (2004)). Tars, represented by compounds such as benzene,
toluene, ethylbenzene, p-xylene, o-xylene, and naphthalene, are
added at ppm levels to test for any effect on production. For
example, it has been shown that 40 ppm NO is inhibitory to C.
carboxidivorans (Ahmed et al., Biotechnol Bioeng, 97:1080-1086
(2007)). Cultures are tested in shake-flask cultures before moving
to a fermentor. Also, different levels of these potential
inhibitory compounds are tested to quantify the effect they have on
cell growth. This knowledge is used to develop specifications for
syngas purity, which is utilized for scale up studies and
production. If any particular component is found to be difficult to
decrease or remove from syngas used for scale up, an adaptive
evolution procedure is utilized to adapt cells to tolerate one or
more impurities.
[0417] Advances in the field of protein engineering make it
feasible to alter any of the enzymes disclosed herein to act
efficiently on substrates not known to be natural to them. Below
are several examples of broad-specificity enzymes from diverse
classes of interest and methods that have been used for evolving
such enzymes to act on non-natural substrates.
[0418] One class of enzymes in the pathways disclosed herein is the
oxidoreductases that interconvert ketones or aldehydes to alcohols
(1.1.1). Enzymes in this class that can operate on a wide range of
substrates. An alcohol dehydrogenase (1.1.1.1) purified from the
soil bacterium Brevibacterium sp KU 1309 (Hirano et al., J. Biosci.
Bioeng. 100:318-322 (2005)) was shown to operate on a plethora of
aliphatic as well as aromatic alcohols with high activities. Table
72 shows the activity of the enzyme and its Km on different
alcohols. The enzyme is reversible and has very high activity on
several aldehydes also as shown in Table 73.
TABLE-US-00074 TABLE 72 RELATIVE ACTIVITY K.sub.M SUBSTRATE (%)
(MM) 2-Phenylethanol 100 0.025 (S)-2-Phenylpropanol 156 0.157
(R)-2-Phenylpropanol 63 0.020 Benzyl alcohol 199 0.012
3-Phenylpropanol 135 0.033 Ethanol 76 1-Butanol 111 1-Octanol 101
1-Dodecanol 68 1-Phenylethanol 46 2-Propanol 54
[0419] In this Table, the activity of 2-phenylethanol,
corresponding to 19.2 U/mg, was taken as 100%.
TABLE-US-00075 TABLE 73 RELATIVE ACTIVITY .kappa..sub.M SUBSTRATE
(%) (MM) Phenylacetaldehyde 100 0.261 2-Phenylpropionaldehyde 188
0.864 1-Octylaldehyde 87 Acetophenone 0
[0420] Lactate dehydrogenase (1.1.1.27) from Ralstonia eutropha is
another enzyme that has been demonstrated to have high activities
on several 2-oxoacids such as 2-oxobutyrate, 2-oxopentanoate and
2-oxoglutarate (a C5 compound analogous to 2-oxoadipate)
(Steinbuchel et al., supra). Column 2 in Table 74 demonstrates the
activities of ldhA from R. eutropha (formerly A. eutrophus) on
different substrates (Steinbuchel et al., supra).
TABLE-US-00076 TABLE 74 Activity of L(+)- L(+)- D(-)- lactate
lactate dehydro- lactate dehydro-genase genase from dehydro-genase
from rabbit from Substrate A. eustrophus % muscle L. leischmanii
Glyoxylate 8.7 23.9 5.0 Pyruvate 100.0 100.0 100.0 2-Oxobutyrate
107.0 18.6 1.1 2-Oxovalerate 125.0 0.7 0.0 3-Methyl-2- 28.5 0.0 0.0
oxobutyrate 3-Methyl-2- 5.3 0.0 0.0 oxovalerate 4-Methyl-2- 39.0
1.4 1.1 oxopentanoate Oxaloacetate 0.0 33.1 23.1 2-Oxoglutarate
79.6 0.0 0.0 3-Fluoropyruvate 33.6 74.3 40.0
[0421] Oxidoreductases that can convert 2-oxoacids to their
acyl-CoA counterparts (1.2.1) have been shown to accept multiple
substrates as well. For example, branched-chain 2-keto-acid
dehydrogenase complex (BCKAD), also known as 2-oxoisovalerate
dehydrogenase (1.2.1.25), participates in branched-chain amino acid
degradation pathways, converting 2-keto acids derivatives of
valine, leucine and isoleucine to their acyl-CoA derivatives and
CO.sub.2. In some organisms including Rattus norvegicus (Paxton et
al., Biochem. J. 234:295-303 (1986)) and Saccharomyces cerevisiae
(Sinclair et al., Biochem. Mol. Biol. Int. 31:911-922 (1993)), this
complex has been shown to have a broad substrate range that
includes linear oxo-acids such as 2-oxobutanoate and
alpha-ketoglutarate, in addition to the branched-chain amino acid
precursors.
[0422] Members of yet another class of enzymes, namely
aminotransferases (2.6.1), have been reported to act on multiple
substrates. Aspartate aminotransferase (aspAT) from Pyrococcus
fursious has been identified, expressed in E. coli and the
recombinant protein characterized to demonstrate that the enzyme
has the highest activities towards aspartate and
alpha-ketoglutarate but lower, yet significant activities towards
alanine, glutamate and the aromatic amino acids (Ward et al.,
Archaea. 1:133-141 (2002)). In another instance, an
aminotransferase identified from Leishmania mexicana and expressed
in E. coli Vernal et al., FEMS Microbiol. Lett. 229:217-222 (2003))
was reported to have a broad substrate specificity towards tyrosine
(activity considered 100% on tyrosine), phenylalanine (90%),
tryptophan (85%), aspartate (30%), leucine (25%) and methionine
(25%) respectively (Vernal et al., Mol. Biochem. Parasitoi.
96:83-92 (1998)). Similar broad specificity has been reported for a
tyrosine aminotransferase from Trypanosoma cruzi, even though both
of these enzymes have a sequence homology of only 6%. Note that the
latter enzyme can accept leucine, methionine as well as tyrosine,
phenylalanine, tryptophan and alanine as efficient amino donors
(Nowicki et al., Biochim. Biophys. Acta 1546: 268-281 (2001)).
[0423] In contrast to these examples where the enzymes naturally
have broad substrate specificities, numerous enzymes have been
modified using directed evolution to broaden their specificity
towards their non-natural substrates. Alternatively, the substrate
preference of an enzyme has also been changed using directed
evolution. For example, it has been reported that the
enantioselectivity of a lipase from Pseudomonas aeruginosa was
improved significantly. This enzyme hydrolyzed p-nitrophenyl
2-methyldecanoate with only 2% enantiomeric excess (ee) in favor of
the (S)-acid. However, after four successive rounds of error-prone
mutagenesis and screening, a variant was produced that catalyzed
the requisite reaction with 81% ee Reetz et al., Angew. Chem. Int.
Ed Engl. 36:2830-2832 (1997)).
[0424] Directed evolution methods have made possible the
modification of an enzyme to function on an array of unnatural
substrates. The substrate specificity of the lipase in P.
aeruginosa was broadened by randomization of amino acid residues
near the active site. This allowed for the acceptance of
alpha-substituted carboxylic acid esters by this enzyme Reetz et
al., Angew. Chem. Int. Ed Engl. 44:4192-4196 (2005)). In another
successful attempt, DNA shuffling was employed to create an
Escherichia coli aminotransferase that accepted .beta.-branched
substrates, which were poorly accepted by the wild-type enzyme
(Yano et al., Proc. Natl. Acad. Sci. U.S.A 95:5511-5515 (1998)).
Specifically, at the end of four rounds of shuffling, the activity
of aspartate aminotransferase for valine and 2-oxovaline increased
by up to five orders of magnitude, while decreasing the activity
towards the natural substrate, aspartate, by up to 30-fold.
Recently, an algorithm was used to design a retro-aldolase that
could be used to catalyze the carbon-carbon bond cleavage in a
non-natural and non-biological substrate,
4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone. These algorithms
used different combinations of four different catalytic motifs to
design new enzymes and 20 of the selected designs for experimental
characterization had four-fold improved rates over the uncatalyzed
reaction (Jiang et al., Science 319:1387-1391 (2008)). Thus, not
only are these engineering approaches capable of expanding the
array of substrates on which an enzyme can act, but allow the
design and construction of very efficient enzymes. For example, a
method of DNA shuffling (random chimeragenesis on transient
templates or RACHITT) was reported to lead to an engineered
monooxygenase that had an improved rate of desulfurization on
complex substrates as well as 20-fold faster conversion of a
non-natural substrate (Coco et al. Nat. Biotechnol. 19:354-359
(2001)). Similarly, the specific activity of a sluggish mutant
triosephosphate isomerase enzyme was improved up to 19-fold from
1.3 fold (Hermes et al., Proc. Natl. Acad. Sci. U.S.A 87:696-700
(1990)). This enhancement in specific activity was accomplished by
using random mutagenesis over the whole length of the protein and
the improvement could be traced back to mutations in six amino acid
residues.
[0425] The effectiveness of protein engineering approaches to alter
the substrate specificity of an enzyme for a desired substrate has
also been demonstrated. Isopropylmalate dehydrogenase from Thermus
thermophilus was modified by changing residues close to the active
site so that it could now act on malate and D-lactate as substrates
(Fujita et al., Biosci. Biotechnol Biochem. 65:2695-2700 (2001)).
In this study as well as in others, it was pointed out that one or
a few residues could be modified to alter the substrate
specificity. A case in point is the dihydroflavonol 4-reductase for
which a single amino acid was changed in the presumed
substrate-binding region that could preferentially reduce
dihydrokaempferol Johnson et al., Plant J. 25:325-333 (2001)). The
substrate specificity of a very specific isocitrate dehydrogenase
from Escherichia coli was changed from isocitrate to
isopropylmalate by changing one residue in the active site (Doyle
et al., Biochemistry 40:4234-4241 (2001)). In a similar vein, the
cofactor specificity of a NAD+-dependent 1,5-hydroxyprostaglandin
dehydrogenase was altered to NADP+ by changing a few residues near
the N-terminal end Cho et al., Arch. Biochem. Biophys. 419:139-146
(2003)). Sequence analysis and molecular modeling analysis were
used to identify the key residues for modification, which were
further studied by site-directed mutagenesis.
[0426] A fucosidase was evolved from a galactosidase in E. coli by
DNA shuffling and screening (Zhang et al., Proc Natl Acad Sci US.A
94:4504-4509 (1997)). Similarly, aspartate aminotransferase from E.
coli was converted into a tyrosine aminotransferase using homology
modeling and site-directed mutagenesis (Onuffer et al., Protein
Sci. 4:1750-1757 (1995)). Site-directed mutagenesis of two residues
in the active site of benzoylformate decarboxylase from P. putida
reportedly altered the affinity (Km) towards natural and
non-natural substrates Siegert et al., Protein Eng Des Sel
18:345-357 (2005)). Cytochrome c peroxidase (CCP) from
Saccharomyces cerevisiae was subjected to directed molecular
evolution to generate mutants with increased activity against the
classical peroxidase substrate guaiacol, thus changing the
substrate specificity of CCP from the protein cytochrome c to a
small organic molecule. After three rounds of DNA shuffling and
screening, mutants were isolated which possessed a 300-fold
increased activity against guaiacol and up to 1000-fold increased
specificity for this substrate relative to that for the natural
substrate (Iffland et al., Biochemistry 39:10790-10798 (2000)).
[0427] In some cases, enzymes with different substrate preferences
than both the parent enzymes have been obtained. For example,
biphenyl-dioxygenase-mediated degradation of polychlorinated
biphenyls was improved by shuffling genes from two bacteria,
Pseudomonas pseudoalcaligens and Burkholderia cepacia (Kumamaru et
al., Nat. Biotechnol 16, 663-666 (1998)). The resulting chimeric
biphenyl oxygenases showed different substrate preferences than
both the parental enzymes and enhanced the degradation activity
towards related biphenyl compounds and single aromatic ring
hydrocarbons such as toluene and benzene which were originally poor
substrates for the enzyme.
[0428] It is not only possible to change the enzyme specificity but
also to enhance the activities on those substrates on which the
enzymes naturally have low activities. One study demonstrated that
amino acid racemase from P. putida that had broad substrate
specificity (on lysine, arginine, alanine, serine, methionine,
cysteine, leucine and histidine among others) but low activity
towards tryptophan could be improved significantly by random
mutagenesis Kino et al., Appl. Microbiol. Biotechnol. 73:1299-1305
(2007)). Similarly, the active site of the bovine BCKAD was
engineered to favor alternate substrate acetyl-CoA (Meng et al.
Biochemistry 33:12879-12885 (1994)). An interesting aspect of these
approaches is that even when random methods have been applied to
generate these mutated enzymes with efficacious activities, the
exact mutations or structural changes that confer the improvement
in activity can be identified. For example, in the aforementioned
study, the mutations that facilitated improved activity on
tryptophan could be traced back to two different positions.
[0429] Directed evolution has also been used to express proteins
that are difficult to express. For example, by subjecting the
horseradish peroxidase to random mutagenesis and gene
recombination, mutants could be extracted that had more than
14-fold activity than the wild type (Lin et al., Biotechnol. Frog.
15:467-471 (1999)).
[0430] A final example of directed evolution shows the extensive
modifications to which an enzyme can be subjected to achieve a
range of desired functions. The enzyme, lactate dehydrogenase from
Bacillus stearothermophilus was subjected to site-directed
mutagenesis, and three amino acid substitutions were made at sites
that were indicated to determine the specificity towards different
hydroxyacids (Clarke et al., Biochem. Biophys. Res. Commun.
148:15-23 (1987)). After these mutations, the specificity for
oxaloacetate over pyruvate was increased to 500 in contrast to the
wild type enzyme that had a catalytic specificity for pyruvate over
oxaloacetate of 1000. This enzyme was further engineered using
site-directed mutagenesis to have activity towards branched-chain
substituted pyruvates (Wilks et al., Biochemistry 29:8587-8591
(1990)). Specifically, the enzyme had a 55-fold improvement in Kcat
for alpha-ketoisocaproate. Three structural modifications were made
in the same enzyme to change its substrate specificity from lactate
to malate. The enzyme was highly active and specific towards malate
(Wilks et al., Science 242:1541-1544 (1988)). The same enzyme from
B. stearothermophilus was subsequently engineered to have high
catalytic activity towards alpha-keto acids with positively charged
side chains, such as those containing ammonium groups (Hogan et
al., Biochemistry 34:4225-4230 (1995)). Mutants with acidic amino
acids introduced at position 102 of the enzyme favored binding of
such side chain ammonium groups. The results obtained proved that
the mutants showed up to 25-fold improvements in kcat/Km values for
omega-amino-alpha-keto acid substrates. This enzyme was also
structurally modified to function as a phenyllactate dehydrogenase
instead of a lactate dehydrogenase (Wilks et al., Biochemistry
31:7802-7806 (1992)). Restriction sites were introduced into the
gene for the enzyme which allowed a region of the gene to be
excised. This region coded for a mobile surface loop of polypeptide
(residues 98-110) which normally seals the active site vacuole from
bulk solvent and is a major determinant of substrate specificity.
The variable length and sequence loops were inserted into the cut
gene and used to synthesize hydroxyacid dehydrogenases with altered
substrate specificities. With one longer loop construction,
activity with pyruvate was reduced one-million-fold but activity
with phenylpyruvate was largely unaltered. A switch in specificity
(kcat/Km) of 390,000-fold was achieved. The 1700:1 selectivity of
this enzyme for phenylpyruvate over pyruvate is that required in a
phenyllactate dehydrogenase.
[0431] As indicated above, directed evolution is a powerful
approach that involves the introduction of mutations targeted to a
specific gene in order to improve and/or alter the properties of an
enzyme. Improved and/or altered enzymes can be identified through
the development and implementation of sensitive high-throughput
screening assays that allow the automated screening of many enzyme
variants (e.g., >104). 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.
[0432] Numerous directed evolution technologies have been developed
(for reviews, see Hibbert, E. G., F. Baganz, H. C. Hailes, J. M.
Ward, G. J. Lye, J. M. Woodley, and P. A. Dalby, 2005, Directed
evolution of biocatalytic processes. Biomol. Eng 22:11-19; Huisman,
G. W. and J. J. Lalonde, 2007, Enzyme evolution for chemical
process applications, p. 717-742. In R. N. Patel (ed.),
Biocatalysis in the pharmaceutical and biotechnology industries.
CRC Press; Otten, L. G. and W. J. Quax. 2005. Directed evolution:
selecting today's biocatalysts. Biomol. Eng 22:1-9; and Sen, S., D.
Venkata, V, and B. Mandal, 2007, Developments in directed evolution
for improving enzyme functions. Appl Biochem. Biotechnol
143:212-223) 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.
[0433] 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 (Km)--broadens substrate binding to include
non-natural substrates; inhibition (Ki)--to remove inhibition by
products, substrates, or key intermediates; activity
(kcat)--increases enzymatic reaction rates to achieve desired flux;
expression levels--increases protein yields and overall pathway
flux; oxygen stability--for operation of air sensitive enzymes
under aerobic conditions; and anaerobic activity--for operation of
an aerobic enzyme in the absence of oxygen.
[0434] The following exemplary methods have been developed for the
mutagenesis and diversification of genes to target desired
properties of specific enzymes. Any of these can be used to
alter/optimize activity of a decarboxylase enzyme.
[0435] EpPCR (Pritchard, L., D. Come, D. Kell, J. Rowland, and M.
Winson, 2005, A general model of error-prone PCR. J Theor. Biol
234:497-509) introduces random point mutations by reducing the
fidelity of DNA polymerase in PCR reactions by the addition of Mn2+
ions, by biasing dNTP concentrations, or by other conditional
variations. The five step cloning process to confine the
mutagenesis to the target gene of interest involves: 1) error-prone
PCR amplification of the gene of interest; 2) restriction enzyme
digestion; 3) gel purification of the desired DNA fragment; 4)
ligation into a vector; 5) transformation of the gene variants into
a suitable host and screening of the library for improved
performance. This method can generate multiple mutations in a
single gene simultaneously, which can be useful. A high number of
mutants can be generated by EpPCR, so a high-throughput screening
assay or a selection method (especially using robotics) is useful
to identify those with desirable characteristics.
[0436] Error-prone Rolling Circle Amplification (epRCA) (Fujii, R.,
M. Kitaoka, and K. Hayashi, 2004, One-step random mutagenesis by
error-prone rolling circle amplification. Nucleic Acids Res
32:e145; and Fujii, R., M. Kitaoka, and K. Hayashi, 2006,
Error-prone rolling circle amplification: the simplest random
mutagenesis protocol. Nat. Protoc. 1:2493-2497) 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
Mn2+ concentration can vary the mutation rate somewhat. This
technique uses a simple error-prone, single-step method to create a
full copy of the plasmid with 3-4 mutations/kbp. No restriction
enzyme digestion or specific primers are required. Additionally,
this method is typically available as a kit.
[0437] DNA or Family Shuffling (Stemmer, W. P. 1994, DNA shuffling
by random fragmentation and reassembly: in vitro recombination for
molecular evolution. Proc Natl Acad Sci US.A 91:10747-10751; and
Stemmer, W. P. 1994. Rapid evolution of a protein in vitro by DNA
shuffling. Nature 370:389-391) typically involves digestion of 2 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 neutral mutations that might confer
antigenicity.
[0438] Staggered Extension (StEP) (Zhao, H., L. Giver, Z. Shao, J.
A. Affholter, and F. H. Arnold, 1998, Molecular evolution by
staggered extension process (StEP) in vitro recombination. Nat.
Biotechnol 16:258-261) 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.
[0439] In Random Priming Recombination (RPR) random sequence
primers are used to generate many short DNA fragments complementary
to different segments of the template. (Shao, Z., H. Zhao, L.
Giver, and F. H. Arnold, 1998, Random-priming in vitro
recombination: an effective tool for directed evolution. Nucleic
Acids Res 26:681-683.) 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.
[0440] In Heteroduplex Recombination linearized plasmid DNA is used
to form heteroduplexes that are repaired by mismatch repair.
(Volkov, A. A., Z. Shao, and F. H. Arnold. 1999. Recombination and
chimeragenesis by in vitro heteroduplex formation and in vivo
repair. Nucleic Acids Res 27:e18; and Volkov, A. A., Z. Shao, and
F. H. Arnold. 2000. Random chimeragenesis by heteroduplex
recombination. Methods Enzymol. 328:456-463.) 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.
[0441] Random Chimeragenesis on Transient Templates (RACHITT)
(Coco, W. M., W. E. Levinson, M. J. Crist, H. J. Hektor, A.
Darzins, P. T. Pienkos, C. H. Squires, and D. J. Monticello, 2001,
DNA shuffling method for generating highly recombined genes and
evolved enzymes. Nat. Biotechnol 19:354-359) employs Dnase I
fragmentation and size fractionation of ssDNA. Homologous fragments
are hybridized in the absence of polymerase to a complementary
ssDNA scaffold. Any overlapping unhybridized fragment ends are
trimmed down by an exonuclease. Gaps between fragments are filled
in, and then ligated to give a pool of full-length diverse strands
hybridized to the scaffold (that contains U to preclude
amplification). The scaffold then is destroyed and is replaced by a
new strand complementary to the diverse strand by PCR
amplification. The method involves one strand (scaffold) that is
from only one parent while the priming fragments derive from other
genes; the parent scaffold is selected against. Thus, no
reannealing with parental fragments occurs. Overlapping fragments
are trimmed with an exonuclease. Otherwise, this is conceptually
similar to DNA shuffling and StEP. Therefore, there should be no
siblings, few inactives, and no unshuffled parentals. This
technique has advantages in that few or no parental genes are
created and many more crossovers can result relative to standard
DNA shuffling.
[0442] 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, S. H., E. J. Ryu, M. J. Kang, E.-S. Wang, Z. C. Y.
Piao, K. J. J. Jung, and Y. Shin, 2003, A new approach to directed
gene evolution by recombined extension on truncated templates
(RETT). J. Molec. Catalysis 26:119-129.) No DNA endonucleases are
used. Unidirectional ssDNA is made by DNA polymerase with random
primers or serial deletion with exonuclease. Unidirectional ssDNA
are only templates and not primers. Random priming and exonucleases
don't introduce sequence bias as true of enzymatic cleavage of DNA
shuffling/RACHITT. RETT can be easier to optimize than StEP because
it uses normal PCR conditions instead of very short extensions.
Recombination occurs as a component of the PCR steps--no direct
shuffling. This method can also be more random than StEP due to the
absence of pauses.
[0443] In Degenerate Oligonucleotide Gene Shuffling (DOGS)
degenerate primers are used to control recombination between
molecules; (Bergquist, P. L. and M. D. Gibbs, 2007, Degenerate
oligonucleotide gene shuffling. Methods Mol. Biol. 352:191-204;
Bergquist, P. L., R. A. Reeves, and M. D. Gibbs, 2005, Degenerate
oligonucleotide gene shuffling (DOGS) and random drift mutagenesis
(RNDM): two complementary techniques for enzyme evolution. Biomol.
Eng 22:63-72; Gibbs, M. D., K. M. Nevalainen, and P. L. Bergquist,
2001, Degenerate oligonucleotide gene shuffling (DOGS): a method
for enhancing the frequency of recombination with family shuffling.
Gene 271:13-20) 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.
[0444] 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 US.A. 96:3562-3567 (1999); Ostermeier et al., 1999
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.
[0445] Thio-Incremental Truncation for the Creation of Hybrid
Enzymes (THIO-ITCHY) is almost the same as ITCHY except that
phosphothioate dNTPs are used to generate truncations. (Lutz, S.,
M. Ostermeier, and S. J. Benkovic, 2001, Rapid generation of
incremental truncation libraries for protein engineering using
alpha-phosphothioate nucleotides. Nucleic Acids Res 29:E16)
Relative to ITCHY, THIO-ITCHY can be easier to optimize, provide
more reproducibility, and adjustability.
[0446] SCRATCHY--ITCHY combined with DNA shuffling is a combination
of DNA shuffling and ITCHY; therefore, allowing multiple
crossovers. (Lutz et al., Proc Natl Acad Sci US.A. 98:11248-11253
(2001).) SCRATCHY combines the best features of ITCHY and DNA
shuffling. Computational predictions can be used in optimization.
SCRATCHY is more effective than DNA shuffling when sequence
identity is below 80%.
[0447] In Random Drift Mutagenesis (RNDM) mutations made via epPCR
followed by screening/selection for those retaining usable
activity. (Bergquist et al., Biomol. Eng. 22:63-72 (2005).) Then,
these are used in DOGS to generate recombinants with fusions
between multiple active mutants or between active mutants and some
other desirable parent. Designed to promote isolation of neutral
mutations; its purpose is to screen for retained catalytic activity
whether or not this activity is higher or lower than in the
original gene. RNDM is usable in high throughput assays when
screening is capable of detecting activity above background. RNDM
has been used as a front end to DOGS in generating diversity. The
technique imposes a requirement for activity prior to shuffling or
other subsequent steps; neutral drift libraries are indicated to
result in higher/quicker improvements in activity from smaller
libraries. Though published using epPCR, this could be applied to
other large-scale mutagenesis methods.
[0448] Sequence Saturation Mutagenesis (SeSaM) is a random
mutagenesis method that: 1) generates pool of random length
fragments using random incorporation of a phosphothioate nucleotide
and cleavage; this pool is used as a template to 2) extend in the
presence of "universal" bases such as inosine; 3) replication of a
inosine-containing complement gives random base incorporation and,
consequently, mutagenesis. (Wong et al., Biotechnol J. 3:74-82
(2008); Wong Nucleic Acids Res 32:e26; Wong et al. Anal. Biochem.
341:187-189 (2005).) Using this technique it can be possible to
generate a large library of mutants within 2-3 days using simple
methods. This is very non-directed compared to mutational bias of
DNA polymerases. Differences in this approach makes this technique
complementary (or alternative) to epPCR.
[0449] 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 more closely related sequences and it doesn't require
possessing the template genes physically.
[0450] 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. One can use other nucleotide analogs such as
8-oxo-guanine with this method. Additionally, the technique works
well with very short fragments (86 bp) and has a low error rate.
Chemical cleavage of DNA means very few unshuffled clones.
[0451] In Sequence Homology-Independent Protein Recombination
(SHIPREC) a linker is used to facilitate fusion between 2
distantly/unrelated genes; nuclease treatment is used to generate a
range of chimeras between the two. Result is a single crossover
library of these fusions. (Sieber, V., C. A. Martinez, and F. H.
Arnold. 2001. Libraries of hybrid proteins from distantly related
sequences. Nat. Biotechnol 19:456-460.) This produces a limited
type of shuffling; mutagenesis is a separate process. This
technique can create a library of chimeras with varying fractions
of each of 2 unrelated parent genes. No homology is needed. 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.
[0452] In Gene Site Saturation Mutagenesis (GSSM) the starting
materials are a supercoiled dsDNA plasmid with insert and 2 primers
degenerate at the desired site for mutations. (Kretz, K. A., T. H.
Richardson, K. A. Gray, D. E. Robertson, X. Tan, and J. M. Short,
2004, Gene site saturation mutagenesis: a comprehensive mutagenesis
approach. Methods Enzymol. 388:3-11.) Primers carry the mutation of
interest and anneal to the same sequence on opposite strands of
DNA; mutation in the middle of the primer and .about.20 nucleotides
of correct sequence flanking on each side. The sequence in the
primer is NNN or NNK (coding) and MNN (noncoding) (N=all 4, K=G, T,
M=A, C). After extension, DpnI is used to digest dam-methylated DNA
to eliminate the wild-type template. This technique explores all
possible amino acid substitutions at a given locus (i.e., one
codon). The technique facilitates the generation of all possible
replacements at one site with no nonsense codons and equal or
near-equal representation of most possible alleles. It does not
require prior knowledge of structure, mechanism, or domains of the
target enzyme. If followed by shuffling or Gene Reassembly, this
technology creates a diverse library of recombinants containing all
possible combinations of single-site up-mutations. The utility of
this technology combination has been demonstrated for the
successful evolution of over 50 different enzymes, and also for
more than one property in a given enzyme.
[0453] 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, J. F., J. U. Bowie, R. M. Breyer, J. C. Hu, K. L.
Knight, W. A. Lim, M. C. Mossing, D. A. Parsell, K. R. Shoemaker,
and R. T. Sauer, 1991, Random mutagenesis of protein sequences
using oligonucleotide cassettes. Methods Enzymol. 208:564-586; and
Reidhaar-Olson, J. F. and R. T. Sauer, 1988, Combinatorial cassette
mutagenesis as a probe of the informational content of protein
sequences. Science 241:53-57.) Simultaneous substitutions at 2 or 3
sites are possible using this technique. Additionally, the method
tests a large multiplicity of possible sequence changes at a
limited range of sites. It has been used to explore the information
content of lambda repressor DNA-binding domain.
[0454] Combinatorial Multiple Cassette Mutagenesis (CMCM) is
essentially similar to CCM except it is employed as part of a
larger program: 1) Use of epPCR at high mutation rate to 2) ID hot
spots and hot regions and then 3) extension by CMCM to cover a
defined region of protein sequence space. (Reetz, M. T., S.
Wilensek, D. Zha, and K. E. Jaeger, 2001, Directed Evolution of an
Enantioselective Enzyme through Combinatorial Multiple-Cassette
Mutagenesis. Angew. Chem. Int. Ed Engl. 40:3589-3591.) As with CCM,
this method can test virtually all possible alterations over a
target region. If used along with methods to create random
mutations and shuffled genes, it provides an excellent means of
generating diverse, shuffled proteins. This approach was successful
in increasing, by 51-fold, the enantioselectivity of an enzyme.
[0455] In the Mutator Strains technique conditional is mutator
plasmids allow increases of 20- to 4000-X in random and natural
mutation frequency during selection and to block accumulation of
deleterious mutations when selection is not required. (Selifonova,
0., F. Valle, and V. Schellenberger, 2001, Rapid evolution of novel
traits in microorganisms. Appl Environ Microbiol 67:3645-3649.)
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 of the strain that harbors the
plasmid. A broad-spectrum of base substitutions and frameshift
mutations occur. In order for effective use, the mutator plasmid
should be removed once the desired phenotype is achieved; this is
accomplished through a temperature sensitive origin of replication,
which allows plasmid curing at 41.degree. C. It should be noted
that mutator strains have been explored for quite some time (e.g.,
see Winter and coworkers, 1996, J. Mol. Biol. 260, 359-3680. 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.
[0456] "Look-Through Mutagenesis (LTM) is a multidimensional
mutagenesis method that assesses and optimizes combinatorial
mutations of selected amino acids." (Rajpal, A., N. Beyaz, L.
Haber, G. Cappuccilli, H. Yee, R. R. Bhatt, T. Takeuchi, R. A.
Lerner, and R. Crea, 2005, A general method for greatly improving
the affinity of antibodies by using combinatorial libraries. Proc
Natl Acad Sci US.A 102:8466-8471.) Rather than saturating each site
with all possible amino acid changes, a set of 9 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 should 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.
[0457] Gene Reassembly is a DNA shuffling method that can be
applied to multiple genes at one time or to creating a large
library of chimeras (multiple mutations) of a single gene. (on the
world-wide web at
www.verenium.com/Pages/Technology/EnzymeTech/TechEnzyTGR.html)
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,
a large range of mutations can be tested for improved activity. The
method allows "blending" and "fine tuning" of DNA shuffling, e.g.
codon usage can be optimized.
[0458] 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, R. J., J. Bentzien, M. L. Ary, M. Y. Hwang, J.
M. Jacinto, J. Vielmetter, A. Kundu, and B. I. Dahiyat, 2002,
Combining computational and experimental screening for rapid
optimization of protein properties. Proc Natl Acad Sci US.A
99:15926-15931.) This technology allows 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). Choice of sequence
variants to test is related to predictions based on most favorable
thermodynamics and 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.
[0459] Iterative Saturation Mutagenesis (ISM) involves 1) Use
knowledge of structure/function to choose a likely site for enzyme
improvement. 2) Saturation mutagenesis at chosen site using
Stratagene QuikChange (or other suitable means). 3) Screen/select
for desired properties. 4) With improved clone(s), start over at
another site and continue repeating. (Reetz, M. T. and J. D.
Carballeira, 2007, Iterative saturation mutagenesis (ISM) for rapid
directed evolution of functional enzymes. Nat. Protoc. 2:891-903;
and Reetz, M. T., J. D. Carballeira, and A. Vogel, 2006, Iterative
saturation mutagenesis on the basis of B factors as a strategy for
increasing protein thermostability. Angew. Chem. Int. Ed Engl.
45:7745-7751.) This is a proven methodology assures all possible
replacements at a given position are made for
screening/selection.
[0460] 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.
[0461] 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 1,3-butanediol.
[0462] 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 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.
[0463] 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 enable
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.
2002/0168654, WO 2002/055995, and U.S. 2009/0047719.
[0464] 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. 2003/0233218, filed Jun. 14, 2002, and in WO/2003/106998.
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.
[0465] 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.
[0466] 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.
[0467] 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.
[0468] 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.
[0469] 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..
[0470] 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.
[0471] 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)).
[0472] 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.
[0473] As disclosed herein, a nucleic acid encoding a desired
activity of a 1,3-butanediol pathway can be introduced into a host
organism. In some cases, it can be desirable to modify an activity
of a 1,3-butanediol pathway enzyme or protein to increase
production of 1,3-butanediol. 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.
[0474] 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.
[0475] A number of exemplary methods have been developed for the
mutagenesis and diversification of genes to target desired
properties of specific enzymes. Such methods are well known to
those skilled in the art. Any of these can be used to alter and/or
optimize the activity of a 1,3-butanediol pathway enzyme or
protein. Such methods include, but are not limited to EpPCR, which
introduces random point mutations by reducing the fidelity of DNA
polymerase in PCR reactions (Pritchard et al., J Theor. Biol.
234:497-509 (2005)); Error-prone Rolling Circle Amplification
(epRCA), which is similar to epPCR except a whole circular plasmid
is used as the template and random 6-mers with exonuclease
resistant thiophosphate linkages on the last 2 nucleotides are used
to amplify the plasmid followed by transformation into cells in
which the plasmid is re-circularized at tandem repeats (Fujii et
al., Nucleic Acids Res. 32:e145 (2004); and Fujii et al., Nat.
Protoc. 1:2493-2497 (2006)); DNA or Family Shuffling, which
typically involves digestion of two or more variant genes with
nucleases such as Dnase I or EndoV to generate a pool of random
fragments that are reassembled by cycles of annealing and extension
in the presence of DNA polymerase to create a library of chimeric
genes (Stemmer, Proc Natl Acad Sci USA 91:10747-10751 (1994); and
Stemmer, Nature 370:389-391 (1994)); Staggered Extension (StEP),
which entails template priming followed by repeated cycles of 2
step PCR with denaturation and very short duration of
annealing/extension (as short as 5 sec) (Zhao et al., Nat.
Biotechnol. 16:258-261 (1998)); Random Priming Recombination (RPR),
in which random sequence primers are used to generate many short
DNA fragments complementary to different segments of the template
(Shao et al., Nucleic Acids Res 26:681-683 (1998)).
[0476] Additional methods include Heteroduplex Recombination, in
which linearized plasmid DNA is used to form heteroduplexes that
are repaired by mismatch repair (Volkov et al, Nucleic Acids Res.
27:e18 (1999); and Volkov et al., Methods Enzymol. 328:456-463
(2000)); Random Chimeragenesis on Transient Templates (RACHITT),
which employs Dnase I fragmentation and size fractionation of
single stranded DNA (ssDNA) (Coco et al., Nat. Biotechnol.
19:354-359 (2001)); Recombined Extension on Truncated templates
(RETT), which entails template switching of unidirectionally
growing strands from primers in the presence of unidirectional
ssDNA fragments used as a pool of templates (Lee et al., J. Molec.
Catalysis 26:119-129 (2003)); Degenerate Oligonucleotide Gene
Shuffling (DOGS), in which degenerate primers are used to control
recombination between molecules; (Bergquist and Gibbs, Methods Mol.
Biol 352:191-204 (2007); Bergquist et al., Biomol. Eng 22:63-72
(2005); Gibbs et al., Gene 271:13-20 (2001)); Incremental
Truncation for the Creation of Hybrid Enzymes (ITCHY), which
creates a combinatorial library with 1 base pair deletions of a
gene or gene fragment of interest (Ostermeier et al., Proc. Natl.
Acad. Sci. USA 96:3562-3567 (1999); and Ostermeier et al., Nat.
Biotechnol. 17:1205-1209 (1999)); Thio-Incremental Truncation for
the Creation of Hybrid Enzymes (THIO-ITCHY), which is similar to
ITCHY except that phosphothioate dNTPs are used to generate
truncations (Lutz et al., Nucleic Acids Res 29:E16 (2001));
SCRATCHY, which combines two methods for recombining genes, ITCHY
and DNA shuffling (Lutz et al., Proc. Natl. Acad. Sci. USA
98:11248-11253 (2001)); Random Drift Mutagenesis (RNDM), in which
mutations made via epPCR are followed by screening/selection for
those retaining usable activity (Bergquist et al., Biomol. Eng.
22:63-72 (2005)); Sequence Saturation Mutagenesis (SeSaM), a random
mutagenesis method that generates a pool of random length fragments
using random incorporation of a phosphothioate nucleotide and
cleavage, which is used as a template to extend in the presence of
"universal" bases such as inosine, and replication of an
inosine-containing complement gives random base incorporation and,
consequently, mutagenesis (Wong et al., Biotechnol. J. 3:74-82
(2008); Wong et al., Nucleic Acids Res. 32:e26 (2004); and Wong et
al., Anal. Biochem. 341:187-189 (2005)); Synthetic Shuffling, which
uses overlapping oligonucleotides designed to encode "all genetic
diversity in targets" and allows a very high diversity for the
shuffled progeny (Ness et al., Nat. Biotechnol. 20:1251-1255
(2002)); Nucleotide Exchange and Excision Technology NexT, which
exploits a combination of dUTP incorporation followed by treatment
with uracil DNA glycosylase and then piperidine to perform endpoint
DNA fragmentation (Muller et al., Nucleic Acids Res. 33:e117
(2005)).
[0477] Further methods include Sequence Homology-Independent
Protein Recombination (SHIPREC), in which a linker is used to
facilitate fusion between two distantly related or unrelated genes,
and a range of chimeras is generated between the two genes,
resulting in libraries of single-crossover hybrids (Sieber et al.,
Nat. Biotechnol. 19:456-460 (2001)); Gene Site Saturation
Mutagenesis.TM. (GSSM.TM.), in which the starting materials include
a supercoiled double stranded DNA (dsDNA) plasmid containing an
insert and two primers which are degenerate at the desired site of
mutations (Kretz et al., Methods Enzymol. 388:3-11 (2004));
Combinatorial Cassette Mutagenesis (CCM), which involves the use of
short oligonucleotide cassettes to replace limited regions with a
large number of possible amino acid sequence alterations
(Reidhaar-Olson et al. Methods Enzymol. 208:564-586 (1991); and
Reidhaar-Olson et al. Science 241:53-57 (1988)); Combinatorial
Multiple Cassette Mutagenesis (CMCM), which is essentially similar
to CCM and uses epPCR at high mutation rate to identify hot spots
and hot regions and then extension by CMCM to cover a defined
region of protein sequence space (Reetz et al., Angew. Chem. Int.
Ed Engl. 40:3589-3591 (2001)); the Mutator Strains technique, in
which conditional is mutator plasmids, utilizing the mutD5 gene,
which encodes a mutant subunit of DNA polymerase III, to allow
increases of 20 to 4000-X in random and natural mutation frequency
during selection and block accumulation of deleterious mutations
when selection is not required (Selifonova et al., Appl. Environ.
Microbiol. 67:3645-3649 (2001)); Low et al., J. Mol. Biol.
260:359-3680 (1996)).
[0478] Additional exemplary methods include Look-Through
Mutagenesis (LTM), which is a multidimensional mutagenesis method
that assesses and optimizes combinatorial mutations of selected
amino acids (Rajpal et al., Proc. Natl. Acad. Sci. USA
102:8466-8471 (2005)); Gene Reassembly, which is a DNA shuffling
method that can be applied to multiple genes at one time or to
create a large library of chimeras (multiple mutations) of a single
gene (Tunable GeneReassembly.TM. (TGR.TM.) Technology supplied by
Verenium Corporation), in Silico Protein Design Automation (PDA),
which is an optimization algorithm that anchors the structurally
defined protein backbone possessing a particular fold, and searches
sequence space for amino acid substitutions that can stabilize the
fold and overall protein energetics, and generally works most
effectively on proteins with known three-dimensional structures
(Hayes et al., Proc. Natl. Acad. Sci. USA 99:15926-15931 (2002));
and Iterative Saturation Mutagenesis (ISM), which involves using
knowledge of structure/function to choose a likely site for enzyme
improvement, performing saturation mutagenesis at chosen site using
a mutagenesis method such as Stratagene QuikChange (Stratagene; San
Diego Calif.), screening/selecting for desired properties, and,
using improved clone(s), starting over at another site and continue
repeating until a desired activity is achieved (Reetz et al., Nat.
Protoc. 2:891-903 (2007); and Reetz et al., Angew. Chem. Int. Ed
Engl. 45:7745-7751 (2006)).
[0479] 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.
[0480] It is understood that modifications which do not
substantially affect the activity of the various embodiments of
this invention are also included within the definition of the
invention provided herein. Accordingly, the following examples are
intended to illustrate but not limit the present invention.
Example I
1,3-Butanediol Synthesis Via Alanine
[0481] This example describes the generation of a microbial
organism capable of producing 1,3-butanediol using the alanine
pathway in FIG. 1 via Steps A, B, C, D and H.
[0482] Escherichia coli is used as a target organism to engineer a
1,3-butanediol-producing pathway as shown in FIG. 1. E. coli
provides a good host for generating a non-naturally occurring
microorganism capable of producing 1,3-butanediol. E. coli is
amenable to genetic manipulation and is known to be capable of
producing various products, like ethanol, acetic acid, formic acid,
lactic acid, and succinic acid, effectively under anaerobic or
microaerobic conditions.
[0483] To generate an E. coli strain engineered to produce
1,3-butanediol, nucleic acids encoding the enzymes utilized in the
alanine pathway as described previously, are expressed in E. coli
using well known molecular biology techniques (see, for example,
Sambrook, supra, 2001; Ausubel supra, 1999; Roberts et al., supra,
1989).
[0484] In particular, the ortA (YP.sub.--001086914.1), ortB
(YP.sub.--001086915.1), dat (P19938), and pdc (P06672) genes
encoding the AKP thiolase, AKP aminotransferase and
2,4-dioxopentanoate decarboxylase activities, respectively, are
cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under
the PA1/lacO promoter. In addition, the yqhD (NP.sub.--417484.1)
and adh (AAA23199.2) genes encoding 3-oxobutyraldehdye reductase
(aldehyde reducing) and 4-hydroxy,2-butanone reductase,
respectively are cloned into the pZA33 vector (Expressys,
Ruelzheim, Germany) under the PA1/lacO promoter. The two sets of
plasmids are transformed into E. coli strain MG1655 to express the
proteins and enzymes required for 1,3-butanediol synthesis via the
alanine pathway. Note that E. coli possesses the ability to form
D-alanine.
[0485] The resulting genetically engineered organism is cultured in
glucose containing medium following procedures well known in the
art (see, for example, Sambrook et al., supra, 2001). The
expression of alanine pathway genes is corroborated using methods
well known in the art for determining polypeptide expression or
enzymatic activity, including for example, Northern blots, PCR
amplification of mRNA, immunoblotting. Enzymatic activities of the
expressed enzymes are confirmed using assays specific for the
individually activities. The ability of the engineered E. coli
strain to produce 1,3-butanediol is confirmed using HPLC, gas
chromatography-mass spectrometry (GCMS) or liquid
chromatography-mass spectrometry (LCMS).
[0486] Microbial strains engineered to have a functional
1,3-butanediol synthesis pathway are further augmented by
optimization for efficient utilization of the pathway. Briefly, the
engineered strain is assessed to determine whether any of the
exogenous genes are expressed at a rate limiting level. Expression
is increased for any enzymes expressed at low levels that can limit
the flux through the pathway by, for example, introduction of
additional gene copy numbers.
[0487] To generate better producers, metabolic modeling is utilized
to optimize growth conditions. Modeling is also 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 in 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 1,3-butanediol. One modeling method is the bilevel
optimization approach, OptKnock (Burgard et al., Biotechnol.
Bioengineer. 84:647-657 (2003)), which is applied to select gene
knockouts that collectively result in better production of
1,3-butanediol. Adaptive evolution also can be used to generate
better producers of, for example, alanine or
2-amino-4-oxopentanoate intermediates or the 1,3-butanediol
product. Adaptive evolution is performed to improve both growth and
production characteristics (Fong and Palsson, Nat. Genet.
36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)).
Based on the results, subsequent rounds of modeling, genetic
engineering and adaptive evolution can be applied to the
1,3-butanediol producer to further increase production.
[0488] For large-scale production of 1,3-butanediol, the above
alanine pathway-containing organism is cultured in a fermenter
using a medium known in the art to support growth of the organism
under anaerobic conditions. Fermentations are performed in either a
batch, fed-batch or continuous manner. Anaerobic conditions are
maintained by first sparging the medium with nitrogen and then
sealing culture vessel (e.g., flasks can be sealed with a septum
and crimp-cap). Microaerobic conditions also can be utilized by
providing a small hole for limited aeration. The pH of the medium
is maintained at a pH of 7 by addition of an acid, such as H2SO4.
The growth rate is determined by measuring optical density using a
spectrophotometer (600 nm), and the glucose uptake rate by
monitoring carbon source depletion over time. Byproducts such as
undesirable alcohols, organic acids, and residual glucose can be
quantified by HPLC (Shimadzu) with an HPX-087 column (BioRad),
using a refractive index detector for glucose and alcohols, and a
UV detector for organic acids, Lin et al., Biotechnol. Bioeng.,
775-779 (2005).
Example II
1,3-BDO Synthesis Using Acetoacetyl-CoA as the Intermediate
[0489] This Example describes the generation of a microbial
organism capable of producing 1,3-butanediol using acetoacetyl-CoA
as the precursor (Steps G, H and I in FIG. 2).
[0490] Escherichia coli is used as a target organism to engineer
the pathway through Steps G (conversion of acetoacetyl-CoA into
3-hydroxybutyryl-CoA), H (conversion of 3-hydroxybutyryl-CoA into
3-hydroxybutyraldehyde) and I (conversion of 3-hydroxybutyraldehyde
into 1,3-butanediol) in FIG. 2. E. coli provides a good host for
generating a non-naturally occurring microorganism capable of
producing 1,3-butanediol. E. coli is amenable to genetic
manipulation and is known to be capable of producing various
products, like ethanol, acetic acid, formic acid, lactic acid, and
succinic acid, effectively under anaerobic or microaerobic
conditions.
[0491] To generate an E. coli strain engineered to produce
1,3-butanediol, nucleic acids encoding the enzymes utilized in the
disclosed pathway (Steps G, H and I) as described previously, are
expressed in E. coli using well known molecular biology techniques
(see, for example, Sambrook, supra, 2001; Ausubel supra, 1999;
Roberts et al., supra, 1989). Note that E. coli has a native
thiolase encoded by atop (Accession number: NP.sub.--416728.1) that
condenses two molecules of acetyl-CoA to form acetoacetyl-CoA.
[0492] Further, hbd (NP.sub.--349314.1) encoding acetoacetyl-CoA
reductase (ketone reducing), is cloned into the pZE 13 vector
(Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. The
plasmid is transformed into E. coli strain MG1655 to express the
enzyme required for the formation of 3-hydroxybutyryl-CoA via
acetoacetyl-CoA. An aldehyde dehydrogenase (selected from Table 75
below) that converts 3-hydroxybutyryl-CoA into
3-hydroxybutyraldehyde, and an alcohol dehydrogenase (selected from
Table 76 below) that further reduces 3-hydroxybutyraldehyde into
1,3-BDO are also cloned into the pZE13 vector under the PA1/lacO
promoter.
[0493] The resulting genetically engineered organism is cultured in
glucose containing medium following procedures well known in the
art (see, for example, Sambrook et al., supra, 2001). The
expression of the pathway genes is corroborated using methods well
known in the art for determining polypeptide expression or
enzymatic activity, including, for example, Northern blots, PCR
amplification of mRNA, immunoblotting. Enzymatic activities of the
expressed enzymes are confirmed using assays specific for the
individually activities. The ability of the engineered E. coli
strain to produce 1,3-butanediol is confirmed using HPLC, gas
chromatography-mass spectrometry (GCMS) or liquid
chromatography-mass spectrometry (LCMS).
[0494] Microbial strains engineered to have a functional
1,3-butanediol synthesis pathway are further augmented by
optimization for efficient utilization of the pathway. Briefly, the
engineered strain is assessed to determine whether any of the
exogenous genes are expressed at a rate limiting level. Expression
is increased for any enzymes expressed at low levels that can limit
the flux through the pathway by, for example, introduction of
additional gene copy numbers.
[0495] To generate better producers, metabolic modeling is utilized
to optimize growth conditions. Modeling is also 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 in 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 1,3-butanediol. One modeling method is the bilevel
optimization approach, OptKnock (Burgard et al., Biotechnol.
Bioengineer. 84:647-657 (2003)), which is applied to select gene
knockouts that collectively result in better production of
1,3-butanediol. Adaptive evolution also can be used to generate
better producers of, for example, the acetyl-CoA intermediate or
the 1,3-butanediol product. Adaptive evolution is performed to
improve both growth and production characteristics (Fong and
Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science
314:1565-1568 (2006)). Based on the results, subsequent rounds of
modeling, genetic engineering and adaptive evolution can be applied
to the 1,3-butanediol producer to further increase production.
[0496] For large-scale production of 1,3-butanediol, the
recombinant organism is cultured in a fermenter using a medium
known in the art to support growth of the organism under anaerobic
conditions. Fermentations are performed in either a batch,
fed-batch or continuous manner. Anaerobic conditions are maintained
by first sparging the medium with nitrogen and then sealing culture
vessel (e.g., flasks can be sealed with a septum and crimp-cap).
Microaerobic conditions also can be utilized by providing a small
hole for limited aeration. The pH of the medium is maintained at a
pH of 7 by addition of an acid, such as H2SO4. The growth rate is
determined by measuring optical density using a spectrophotometer
(600 nm), and the glucose uptake rate by monitoring carbon source
depletion over time. Byproducts such as undesirable alcohols,
organic acids, and residual glucose can be quantified by HPLC
(Shimadzu) with an HPX-087 column (BioRad), using a refractive
index detector for glucose and alcohols, and a UV detector for
organic acids (Lin et al., Biotechnol. Bioeng., 90:775-779
(2005)).
[0497] Several aldehyde dehydrogenases were tested for activity on
3-hydroxybutyryl-CoA. Crude lysates of bacteria, each strain
carrying one out of six genes listed in Table 75 below encoding for
an aldehyde dehydrogenase was tested for activity on
3-hydroxybutyryl-CoA by measuring the release of CoA moiety. The
genes that were tested and were found to have significant activity
on 3-HBCoA encode the proteins with the following accession and GI
numbers:
TABLE-US-00077 TABLE 75 Protein GenBank ID GI Number Organism bld
AAP42563.1 31075383 Clostridium saccharoperbutyl- acetonicum ald
ACL06658.1 218764192 Desulfatibacillum alkenivorans AK-01 ald
YP_001452373 157145054 Citrobacter koseri ATCC BAA-895 pduP
NP_460996.1 16765381 Salmonella enterica Typhimurium pduP
ABJ64680.1 116099531 Lactobacillus brevis ATCC 367 BselDRAFT_1651
ZP_02169447 163762382 Bacillus selenitireducens MLS10
[0498] To correct for background activity in the lysate, measured
activities were compared to a negative control without ALD gene
(vector only, "Vo"). FIG. 4 shows the specific activity of each of
the tested genes on 3-hydroxybutyryl-CoA. The gene ids are shown on
the x-axis.
[0499] Further, bld (GenBank ID: AAP42563.1, GI number: 31075383)
was also tested for activity on 3-HBCoA. The following FIG. 5 shows
the activity of the gene on 3-hydroxybutyryl-CoA before and after
dialysis.
[0500] Alcohol dehydrogenases that were tested for activity on
3-hydroxybutyraldehyde and demonstrated to have significant
activity are listed below.
TABLE-US-00078 TABLE 76 Protein GenBank ID GI Number Organism Bdh
(Cbei_2181) YP_001309304 150017050 Clostridium beijerinckii Bdh
(Cbei_1722) YP_001309535.1 150016596 Clostridium beijerinckii Bdh
(Cbei_2421) YP_001309535.1 150017281 Clostridium beijerinckii
[0501] The following protocol was used to demonstrate alcohol
dehydrogenase activity (i.e., conversion of 3-hydroxybutyraldehyde
to 1,3-BDO) and combined aldehyde and alcohol dehydrogenase
activities (i.e., conversion of 3-hydroxybutyryl-CoA into
1,3-BDO).
[0502] Chemically competent cells were transformed with plasmids
containing either an aldehyde dehydrogenase or an alcohol
dehydrogenase (listed in Tables 75 and 76 above). Colonies from the
plates were picked and grown in LB plus 100 ug/ml carbenecillin
overnight, then 0.6 mL was used to inoculate 60 mL culture of each
alcohol dehydrogenase, or 1.5 mL was used to inoculate a 500 mL
culture of each aldehyde dehydrogenase. Cells were grown at
37.degree. C. to an O.D. of .about.0.7 and induced with IPTG. The
cultures were incubated at 30.degree. C. during protein expression
for 4 hours. The cell cultures were divided into 30 ml aliquots,
centrifuged and the cell pellets were stored at -80.degree. C. A
sample of the cell culture was used to estimate final cell
density.
[0503] Combinations of alcohol dehydrogenases and aldehyde
dehydrogenases were screened in a 96-well plate format with
3-hydroxybutyryl-CoA as a substrate plus a control (no substrate).
Alternatively, for testing the alcohol dehydrogenases activity,
only the alcohol dehydrogenases were added with and without the
substrate, 3-hydroxybutyraldehyde. Preparation of cell lysates was
performed on ice in the coldroom (4.degree. C.). Final cell density
was used to calculate the quantity of Bug Buster cell lysis reagent
for each cell pellet. Lysozyme (10 uL) and benzonase (10 uL) were
added to 35 ml bugbuster and gently inverted to mix. First, 50 tun
of dithiothreitol (100 mM stock) was added to the pellet, then 0.5
ml per O.D. of 1.0 (at 600 nm) of the Bug Buster plus enzyme
mixture was added to the cell pellet and gently mixed to
resuspend.
[0504] To each well, 50 ul of 1 M MOPS (pH=7.5), and 25 ul of
cofactor mixture (4 mM NADH and 4 mM NADPH), both 100 uL aldehyde
dehydrogenase cell lysate, 150 uL alcohol dehydrogenase cell lysate
or only 150 uL alcohol dehydrogenase cell lysate was added and
gently mixed. Then, the relevant substrate was added to the wells.
25 mg of 3-hydroxybutyryl CoA was resuspended in 250 uL water and 5
ul was added to each well testing for both alcohol and aldehyde
dehydrogenase activities for a final concentration of 1.8 mM. For
testing only the alcohol dehydrogenase activity, 50 uL of
3-hydroxybutyraldehyde (prepared by mixing 0.6 ml acetaldehyde in 5
ml water plus catalytic base (one pellet of NaOH) Guthrie, J. P.
(reference attached) was added to each well. The final
concentration of 3-hydroxybutyraldehyde in each well was
approximately 50 mM. The 96-deepwell plate was sealed with a
plastic PCR seal and incubated at 30.degree. C. shaking overnight
(18 hours total). Because protein and cell debris form precipitates
during the incubation period, the plates were centrifuged for 10
min at 4500.times.g, and the supernate was filtered through a
Whatman 96-well filter plate (0.45 .mu.m) prior to LC-MS analysis.
Samples were analyzed for 1,3-butanediol formation.
[0505] FIG. 6 shows 1,3-BDO concentrations when
3-hydroxybutyraldehyde was added as a substrate and in the control
samples with no substrate. The GI numbers for the alcohol
dehydrogenases are shown.
[0506] FIG. 7 shows 1,3-BDO concentrations when
3-hydroxybutyryl-CoA was added as a substrate and in the control
samples with no substrate. The GI numbers for the alcohol
dehydrogenases are shown. The GI number for the aldehyde
dehydrogenase tested in conjunction is 163762382.
Example III
1,3-BDO Synthesis Using 4-Hydroxybutyryl-CoA as the
Intermediate
[0507] This Example describes the generation of a microbial
organism capable of producing 1,3-butanediol using
4-hydroxybutyryl-CoA as the precursor (Steps A, B and E in FIG.
3).
[0508] Escherichia coli is used as a target organism to engineer
the pathway through Steps A, B and E in FIG. 3. E. coli provides a
good host for generating a non-naturally occurring microorganism
capable of producing 1,3-butanediol. E. coli is amenable to genetic
manipulation and is known to be capable of producing various
products, like ethanol, acetic acid, formic acid, lactic acid, and
succinic acid, effectively under anaerobic or microaerobic
conditions.
[0509] To generate an E. coli strain engineered to produce
1,3-butanediol, nucleic acids encoding the enzymes utilized in the
disclosed pathway (Steps A, B and E) as described previously, are
expressed in E. coli using well known molecular biology techniques
(see, for example, Sambrook, supra, 2001; Ausubel supra, 1999;
Roberts et al., supra, 1989). A recombinant strain that has ben
engineered to produce significant quantities of
4-hydroxybutyryl-CoA has been described by the applicants
previously (Burk et al. (US 20090075351) and will be used for
inserting the proposed pathway to 1,3-butanediol.
[0510] Further, abfD (YP.sub.--3001396399.1), crt
(NP.sub.--349318.1) and adhE2 (AAK09379.1) genes encoding
4-hydroxybutyryl-CoA dehydratase, crotonase and
3-hydroxybutyryl-CoA reductase (alcohol forming) activities
respectively, are cloned into the pZE 13 vector (Expressys,
Ruelzheim, Germany) under the PA1/lacO promoter. The plasmid is
transformed into the recombinant E. coli strain producing
4-hydroxybutyryl-CoA to express the proteins and enzymes required
for 1,3-butanediol synthesis from this metabolite.
[0511] The resulting genetically engineered organism is cultured in
glucose containing medium following procedures well known in the
art (see, for example, Sambrook et al., supra, 2001). The
expression of the pathway genes is corroborated using methods well
known in the art for determining polypeptide expression or
enzymatic activity, including, for example, Northern blots, PCR
amplification of mRNA, immunoblotting. Enzymatic activities of the
expressed enzymes are confirmed using assays specific for the
individually activities. The ability of the engineered E. coli
strain to produce 1,3-butanediol is confirmed using HPLC, gas
chromatography-mass spectrometry (GCMS) or liquid
chromatography-mass spectrometry (LCMS).
[0512] Microbial strains engineered to have a functional
1,3-butanediol synthesis pathway are further augmented by
optimization for efficient utilization of the pathway. Briefly, the
engineered strain is assessed to determine whether any of the
exogenous genes are expressed at a rate limiting level. Expression
is increased for any enzymes expressed at low levels that can limit
the flux through the pathway by, for example, introduction of
additional gene copy numbers.
[0513] To generate better producers, metabolic modeling is utilized
to optimize growth conditions. Modeling is also 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 in 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 1,3-butanediol. One modeling method is the bilevel
optimization approach, OptKnock (Burgard et al., Biotechnol.
Bioengineer. 84:647-657 (2003)), which is applied to select gene
knockouts that collectively result in better production of
1,3-butanediol. Adaptive evolution also can be used to generate
better producers of, for example, the acetyl-CoA intermediate or
the 1,3-butanediol product. Adaptive evolution is performed to
improve both growth and production characteristics (Fong and
Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science
314:1565-1568 (2006)). Based on the results, subsequent rounds of
modeling, genetic engineering and adaptive evolution can be applied
to the 1,3-butanediol producer to further increase production.
[0514] For large-scale production of 1,3-butanediol, the
recombinant organism is cultured in a fermenter using a medium
known in the art to support growth of the organism under anaerobic
conditions. Fermentations are performed in either a batch,
fed-batch or continuous manner. Anaerobic conditions are maintained
by first sparging the medium with nitrogen and then sealing culture
vessel (e.g., flasks can be sealed with a septum and crimp-cap).
Microaerobic conditions also can be utilized by providing a small
hole for limited aeration. The pH of the medium is maintained at a
pH of 7 by addition of an acid, such as H2SO4. The growth rate is
determined by measuring optical density using a spectrophotometer
(600 nm), and the glucose uptake rate by monitoring carbon source
depletion over time. Byproducts such as undesirable alcohols,
organic acids, and residual glucose can be quantified by HPLC
(Shimadzu) with an HPX-087 column (BioRad), using a refractive
index detector for glucose and alcohols, and a UV detector for
organic acids (Lin et al., Biotechnol Bioeng. 90:775-779
(2005)).
Example IV
Exemplary Hydrogenase and CO Dehydrogenase Enzymes for Extracting
Reducing Equivalents from Syngas and Exemplary Reductive TCA Cycle
Enzymes
[0515] 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.
[0516] 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 these enzymes is
tabulated below:
TABLE-US-00079 TABLE 77 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
[0517] 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. Micrbiol. 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.sub.--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-00080 TABLE 78 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
[0518] 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-00081 TABLE 79 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
[0519] 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. J. Biochem. Cell. Biol.
31:961-975 (1999)), Thermus thermophilus (Mizobata et al., Arch.
Biochem. Biophys. 355:49-55 (1998)) and Rattus norvegicus
(Kobayashi et al., J. Biochem. 89:1923-1931 (1981)). 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-00082 TABLE 80 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
[0520] 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-00083 TABLE 81 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
[0521] 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-00084 TABLE 82 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
[0522] 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 CO2 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 forDABGE, 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
CO2-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.sub.--0034 is predicted to function in the CO2-assimilating
direction. The genes associated with this enzyme, Moth.sub.--0034
have not been experimentally validated to date but can be inferred
by sequence similarity to known OFOR enzymes.
[0523] 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-00085 TABLE 83 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
[0524] 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-00086 TABLE 84 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
[0525] 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 denitrilicans and Thermocrinis
albus.
TABLE-US-00087 TABLE 85 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
[0526] 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-00088 TABLE 86 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
[0527] 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., fgrB 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-00089 TABLE 87 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
[0528] 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 pf1/3 (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)).
[0529] 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
phosphotransacetylyase are well-studied enzymes in several
Clostridia and Methanosarcina thermophila.
[0530] 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.
[0531] 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+ oxidoreductase (EC
1.18.1.3) and ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2).
Ferredoxin:NADP+ 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+ oxidoreductase enzyme is encoded in the E. coli
genome by fpr (Bianchi et al. 1993). Ferredoxin:NAD+ oxidoreductase
utilizes reduced ferredoxin to generate NADH from NAD+. In several
organisms, including E. coli, this enzyme is a component of
multifunctional dioxygenase enzyme complexes. The ferredoxin:NAD+
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). NADP oxidoreductase of
C. kluyveri, encoded by nfnAB, catalyzes the concomitant reduction
of ferredoxin and NAD+ with two equivalents of NADPH (Wang et al, J
Bacteriol 192: 5115-5123 (2010)). 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 and Clostridium ljungdahli.
TABLE-US-00090 TABLE 88 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
NfnA YP_001393861.1 153953096 Clostridium kluyveri NfnB
YP_001393862.1 153953097 Clostridium kluyveri 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 CLJU_c11410 ADK14209.1 300434442
Clostridium ljungdahli (RnfB) CLJU_c11400 ADK14208.1 300434441
Clostridium ljungdahli (RnfA) CLJU_c11390 ADK14207.1 300434440
Clostridium ljungdahli (RnfE) CLJU_c11380 ADK14206.1 300434439
Clostridium ljungdahli (RnfG) CLJU_c11370 ADK14205.1 300434438
Clostridium ljungdahli (RnfD) CLJU_c11360 ADK14204.1 300434437
Clostridium ljungdahli (RnfC)
[0532] 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+
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, Clostridium ljungdahli and
Rhodospirillum rubrum are predicted to encode several ferredoxins,
listed in the table below.
TABLE-US-00091 TABLE 89 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 CLJU_c00930 ADK13195.1 300433428
Clostridium ljungdahli CLJU_c00010 ADK13115.1 300433348 Clostridium
ljungdahli CLJU_c01820 ADK13272.1 300433505 Clostridium ljungdahli
CLJU_c17980 ADK14861.1 300435094 Clostridium ljungdahli CLJU_c17970
ADK14860.1 300435093 Clostridium ljungdahli CLJU_c22510 ADK15311.1
300435544 Clostridium ljungdahli CLJU_c26680 ADK15726.1 300435959
Clostridium ljungdahli CLJU_c29400 ADK15988.1 300436221 Clostridium
ljungdahli
[0533] 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.
[0534] 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-00092 TABLE 90 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
[0535] 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-00093 TABLE 91 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
[0536] 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
saccharoperhutylacetonicum (Kosaka et al., Biosci. Biotechnol
Biochem. 71:58-68 (2007)) are shown below.
TABLE-US-00094 TABLE 92 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
[0537] 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-00095 TABLE 93 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
[0538] 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-00096 TABLE 94 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
[0539] 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-00097 TABLE 95 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
[0540] 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., Microbioloy 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-00098 TABLE 96 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
[0541] The formation of acetyl-CoA from acetylphosphate is
catalyzed by phosphotransacetylase (EC 2.3.1.8). The pta 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-00099 TABLE 97 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
[0542] 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-00100 TABLE 98 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
[0543] The product yields per C-mol of substrate of microbial cells
synthesizing reduced fermentation products such as 1,3-butanediol,
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 H2 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, H2, 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.
[0544] Here, we show specific examples of how additional redox
availability from CO and/or H2 can improve the yields of reduced
products such as 1,3-butanediol. The maximum theoretical yield to
produce 1,3-butanediol from glucose is 1.09 mole 1,3-butanediol per
mole of glucose under aerobic conditions via the pathways shown in
FIG. 8B or 1.09 mole 1,3-butanediol per mole of glucose under
aerobic conditions via the pathways shown in FIG. 9B. Gasification
of glucose to form syngas will result in the maximum theoretical
yield of 1.09 moles of 1,3-BDO per mole of glucose consumed,
assuming that 6 moles of CO and 6 moles of H2 are obtained from
glucose.
6CO+6H.sub.2.fwdarw.1.091C.sub.4H.sub.10O.sub.2+1.636CO.sub.2+0.545H.sub-
.2
[0545] When additional reducing equivalents are provided, the yield
can be improved to 2 mol/mol glucose.
1C.sub.6H.sub.12O.sub.6+2CO+8H.sub.2.fwdarw.2C.sub.4H.sub.10O.sub.2+4H.s-
ub.2O
[0546] When both feedstocks of sugar and syngas are available, the
syngas components CO and H.sub.2 can be utilized together or
separately (from any source) to generate reducing equivalents by
employing the hydrogenase and/or CO dehydrogenase. The reducing
equivalents generated from CO and/or hydrogen will be utilized to
power the glucose to 1,3-butanediol production pathways.
Theoretically, all carbons in glucose will be conserved, thus
resulting in a maximal theoretical yield to produce 1,3-butanediol
from glucose.
[0547] 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 H2 and/or 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.
Such improvements provide environmental and economic benefits and
greatly enhance sustainable chemical production.
[0548] 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 H2 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 1,3-butanediol production from glucose or sugar, the
theoretical yields improve from 1.09 mol 1,3-butanediol per mol of
glucose to 2 mol 1,3-butanediol per mol of glucose. Such
improvements provide environmental and economic benefits and
greatly enhance sustainable chemical production.
[0549] Herein below the enzymes and the corresponding genes used
for extracting redox from syngas components are described. CODH is
a reversible enzyme that interconverts CO and CO2 at the expense or
gain of electrons. The natural physiological role of the CODH in
ACS/CODH complexes is to convert CO2 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).
[0550] 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.sub.--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-00101 TABLE 99 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 subsp. (CODH)
desulfuricans str. ATCC 27774 Ddes_0381 YP_002478972.1 220903660
Desulfovibrio desulfuricans subsp. (CooC) 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 CLJU_c09110 ADK13979.1 300434212
Clostridium ljungdahli CLJU_c09100 ADK13978.1 300434211 Clostridium
ljungdahli CLJU_c09090 ADK13977.1 300434210 Clostridium
ljungdahli
[0551] 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-00102 TABLE 100 GI Protein GenBank ID Number Organism
CODH-I (CooS-I) YP_360644 78043418 Carboxydothermus
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 (CooS) AAC45123
1498748 Rhodospirillum rubrum CooC AAC45124 1498749 Rhodospirillum
rubrum CooT AAC45125 1498750 Rhodospirillum rubrum CooJ AAC45126
1498751 Rhodospirillum rubrum
[0552] 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-00103 TABLE 101 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
TABLE-US-00104 TABLE 102 Protein GenBank ID GI Number Organism 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
[0553] 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-00105 TABLE 103 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
TABLE-US-00106 TABLE 104 Protein GenBank ID GI Number Organism 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
TABLE-US-00107 TABLE 105 Protein GenBank ID GI Number Organism 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
[0554] The M. thermoacetica hydrogenases are suitable for a host
that lacks sufficient endogenous hydrogenase activity. M.
thermoacetica can grow with CO2 as the exclusive carbon source
indicating that reducing equivalents are extracted from H2 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)). 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.
[0555] Proteins in M. thermoacetica whose genes are homologous to
the E. coli hyp genes are shown below.
TABLE-US-00108 TABLE 106 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
[0556] 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-00109 TABLE 107 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
[0557] In addition, several gene clusters encoding hydrogenase
functionality are present in M. thermoacetica and their
corresponding protein sequences are provided below.
TABLE-US-00110 TABLE 108A 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
[0558] Genes encoding hydrogenase enzymes from C. ljungdahli are
shown below.
TABLE-US-00111 TABLE 108B Protein GenBank ID GI Number Organism
CLJU_c20290 ADK15091.1 300435324 Clostridium ljungdahli CLJU_c07030
ADK13773.1 300434006 Clostridium ljungdahli CLJU_c07040 ADK13774.1
300434007 Clostridium ljungdahli CLJU_c07050 ADK13775.1 300434008
Clostridium ljungdahli CLJU_c07060 ADK13776.1 300434009 Clostridium
ljungdahli CLJU_c07070 ADK13777.1 300434010 Clostridium ljungdahli
CLJU_c07080 ADK13778.1 300434011 Clostridium ljungdahli CLJU_c14730
ADK14541.1 300434774 Clostridium ljungdahli CLJU_c14720 ADK14540.1
300434773 Clostridium ljungdahli CLJU_c14710 ADK14539.1 300434772
Clostridium ljungdahli CLJU_c14700 ADK14538.1 300434771 Clostridium
ljungdahli CLJU_c28670 ADK15915.1 300436148 Clostridium ljungdahli
CLJU_c28660 ADK15914.1 300436147 Clostridium ljungdahli CLJU_c28650
ADK15913.1 300436146 Clostridium ljungdahli CLJU_c28640 ADK15912.1
300436145 Clostridium ljungdahli
[0559] 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
O.sub.2-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-00112 TABLE 109 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
[0560] 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.
[0561] 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-00113 TABLE 110 Protein GenBank ID GI Number Organism Ppc
NP_418391 16131794 Escherichia coli ppcA AAB58883 28572162
Methylobacterium extorquens Ppc ABB53270 80973080 Corynebacterium
glutamicum
[0562] 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 CO2-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-00114 TABLE 111 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
[0563] 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-00115 TABLE 112 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
[0564] 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
.DELTA.pfl-.DELTA.ldhA 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-00116 TABLE 113 Protein GenBank ID GI Number Organism maeA
NP_415996 90111281 Escherichia coli maeB NP_416958 16130388
Escherichia coli NAD-ME P27443 126732 Ascaris suum
[0565] 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 above.
[0566] 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 H2, as disclosed herein, improve the yields of
1,3-butanediol when utilizing carbohydrate-based feedstock. For
example, 1,3-butanediol can be produced from succinyl-CoA via
succinate semialdehyde, 4-hydroxybutyrate,
4-hydroxybutyryl-phosphate, 4-hydroxybutyrl-CoA, crotonyl-CoA,
3-hydroxybutyryl-CoA, 3-hydroxybutanal, and/or 3-hydroxybutryate.
Exemplary enzymes for the conversion succinyl-CoA to 1,3-butanediol
include: A. Succinyl-CoA transferase, or Succinyl-CoA synthetase
(or succinyl-CoA ligase), B. Succinyl-CoA reductase (aldehyde
forming), C. 4-Hydroxybutyrate dehydrogenase, D. 4-Hydroxybutyrate
kinase, E. Phosphotrans-4-hydroxybutyrylase, F. Succinate
reductase, G. Succinyl-CoA reductase (alcohol forming), H.
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA
synthetase, I. Alpha-ketoglutarate decarboxylase or (Q. Glutamate
dehydrogenase and/or R. Glutamate transaminase; S. Glutamate
decarboxylase; T. 4-aminobutyrate dehydrogenase and/or U.
4-aminobutyrate transaminase), J. 4-hydroxybutyryl-CoA dehydratase,
K. crotonase, L. 3-hydroxybutyryl-CoA reductase (aldehyde forming),
M. 3-hydroxybutanal reductase, N. 3-hydroxybutyryl-CoA reductase
(alcohol forming), O. 3-hydroxybutyryl-CoA hydrolase, transferase,
or synthetase, P. 3-hydroxybutyrate reductase.
[0567] 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 H2, as described herein, improve the yields of all
these products on carbohydrates. For example, 1,3-butanediol can be
produced from the glycolysis intermediate, pyruvate. Exemplary
enzymes for the conversion of pyruvate to 1,3-butanediol include
alanine dehydrogenase alanine aminotransferase; the remaining
enzymatic transformations shown are carried out by the following
enzymes: A) AKP thiolase, B) AKP aminotransferase or AKP
oxidoreductase (deaminating), C) 2,4-dioxopentanoate decarboxylase,
D) 3-oxobutyraldehyde reductase (aldehyde reducing), E) AKP
decarboxylase, F) 4-aminobutan-2-one ammonia-lyase, G) Butenone
hydratase, H) 4-hydroxy,2-butanone reductase, I) AKP ammonia-lyase,
J) acetylacrylate decarboxylase, K) 4-aminobutan-2-one
aminotransferase or 4-aminobutan-2-one oxidoreductase
(deaminating), L) AKP dehydrogenase, M) 2-amino-4-hydroxypentanoate
aminotransferase or 2-amino-4-hydroxypentanoate oxidoreductase
(deaminating), N) 2-oxo-4-hydroxypentanoate decarboxylase, O)
3-oxobutyraldehyde reductase (ketone reducing), and P)
3-hydroxybutyraldehdye reductase.
Example V
Methods for Handling CO and Anaerobic Cultures
[0568] This example describes methods used in handling CO and
anaerobic cultures.
[0569] A. Handling of CO in small quantities for assays and small
cultures. 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.
[0570] 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.
[0571] B. Handling of CO in larger quantities fed to large-scale
cultures. Fermentation cultures are fed either CO or a mixture of
CO and H2 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.
[0572] 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.
[0573] C. Anaerobic chamber and conditions. Exemplary anaerobic
chambers are available commercially (see, for example, Vacuum
Atmospheres Company, Hawthorne Calif.; MBraun, Newburyport Mass.).
Conditions included an O2 concentration of 1 ppm or less and 1 atm
pure N2. In one example, 3 oxygen scrubbers/catalyst regenerators
were used, and the chamber included an O2 electrode (such as
Teledyne; City of Industry Calif.). 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 N2 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.
[0574] The anaerobic chambers achieved levels of O2 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 O2 monitoring, test
strips can be used instead.
[0575] D. Anaerobic microbiology. 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 B 12 (10
.quadrature.M cyanocobalamin), nickel chloride (NiCl2, 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 .quadrature.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.
[0576] 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 VI
CO Oxidation (CODH) Assay
[0577] This example describes assay methods for measuring CO
oxidation (CO dehydrogenase; CODH).
[0578] 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.
[0579] 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.
[0580] 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.
[0581] 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.60 U 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.
[0582] 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 mM with a 22 Ga. needle plus an exhaust needle. A
volume of 0.98 mL 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 (CH3 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 CH3 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 CH3 viologen stock to slightly reduce the CH3 viologen. The
temperature was equilibrated to 55.degree. C. in a heated Olis
spectrophotometer (Bogart GA). A blank reaction (CH3
viologen+buffer) was run first to measure the base rate of CH3
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 CH3 viologen turns purple.
The results of an assay are shown in Table 114.
TABLE-US-00117 TABLE 114 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
[0583] 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.
[0584] 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 (CH3 viologen reduction) seen in the
negative controls indicates that E. coli may have a limited ability
to reduce CH3 viologen.
[0585] 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.
[0586] 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. 11. 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
above. 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.
[0587] 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 VII
E. coli CO Tolerance Experiment and CO Concentration Assay
(Myoglobin Assay)
[0588] This example describes the tolerance of E. coli for high
concentrations of CO.
[0589] 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, NiCl2, Fe(II)NH4SO4, 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 N2 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.
[0590] 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.
[0591] 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. N2 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 115.
TABLE-US-00118 TABLE 115 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
[0592] The results shown in Table 115 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.
[0593] 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 VIII
Exemplary Carboxylic Acid Reductases
[0594] This example describes the use of carboxylic acid reductases
(CAR) to carry out the conversion of a carboxylic acid to an
aldehyde.
[0595] Any intermediate carboxylic acid in a 1,3-butanediol pathway
(or accessible carboxylic acid via its CoA derivative) can be
converted to an aldehyde, if so desired. 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-00119 TABLE 116 Gene Accession No. GI No. Organism car
AAR91681.1 40796035 Nocardia iowensis (sp. NRRL 5646) npt
ABI83656.1 114848891 Nocardia iowensis (sp. NRRL 5646)
[0596] Additional car and npt genes can be identified based on
sequence homology.
TABLE-US-00120 TABLE 117 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 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
[0597] 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.sub.--665, an enzyme similar in sequence
to the Nocardia iowensis npt, can be beneficial.
TABLE-US-00121 TABLE 118 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
[0598] 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-00122 TABLE 119 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
[0599] Cloning and Expression of Carboxylic Acid Reductase.
[0600] Escherichia coli is used as a target organism to engineer
the pathway for 1,3-butanediol. E. coli provides a good host for
generating a non-naturally occurring microorganism capable of
producing 1,3-butanedial. E. coli is amenable to genetic
manipulation and is known to be capable of producing various
intermediates and products effectively under various oxygenation
conditions.
[0601] To generate a microbial organism strain such as an E. coli
strain engineered to produce 1,3-butanedial, 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.
[0602] The car gene (GNM.sub.--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.sub.--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 1,3-butanedial production.
[0603] 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 891 GA 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; H15Q; D198E;
G446C; S392N; F699L; V8831; F467S; T987S; R12H; V295G; V295A;
V295S; V295T; V295C; V295V; V295L; V2951; 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.
[0604] The CAR variants were screened for activity, and numerous
CAR variants were found to exhibit CAR activity. This example
describes the use of CAR for converting carboxylic acids to
aldehydes.
TABLE-US-00123 TABLE 120 (Ref: FIG. 1) Step EC class Desired
substrate Desired product Enzyme name Gene name A 2.3.1.b D-alanine
2-amino-4- AKP Thiolase ortA oxopentanoate ortB Amet_2368 Amet_2369
Teth514_1478 Teth514_1479 B 2.6.1.a 2-amino-4- 2,4- 2-amino-4- aspC
oxopentanoate dioxopentanoate oxopentanoate aminotransferase or
oxidoreductase (deaminating) avtA AAT2 dat dat ldh nadX C 4.1.1.a
2,4-dioxopentanoate 3-oxobutyr- 2,4-dioxopentanoate pdc aldehyde
decarboxylase (3-oxobutanal) pdc1 mdlC kgd D 1.1.1.a
3-oxobutyraldehyde 4-hydroxy,2- 3-oxobutyraldehyde alrA butanone
reductase (aldehyde reducing) ADH2 yqhD bdh I bdh II 4hbd ADHI mmsb
P84067 E 4.1.1.a 2-amino-4- 4-aminobutan-2- 2-amino-4- lysA
oxopentanoate one oxopentanoate decarboxylase lysA lysA odc1 panD
panD panD F 4.3.1.a 4-aminobutan-2-one butenone 4-aminobutan-2-one
aspA ammonia lyase aspA aspA ansB aspA G 4.2.1.a butenone
4-hydroxy,2- butenone hydratase fumA butanone fumC fumC fumC fumH
hmd dmdA dmdB H 1.1.1.a 4-hydroxy,2- 1,3-butanediol 4-hydroxy,2-
bdh butanone butanone reductase adh adhA ldh adh I 4.3.1.a
2-amino-4- acetylacrylate 2-amino-4- aspA oxopentanoate
oxopentanoate ammonia lyase aspA aspA ansB aspA J 4.1.1.a
acetylacrylate butenone acetylacrylate xylII decarboxylase xylIII
dmpH dmpE pdc pad K 2.6.1.a 4-aminobutan-2-one 3-oxobutyr-
4-aminobutan-2-one SkyPYD4 aldehyde aminotransferase or
(3-oxobutanal) oxidoreductase (deaminating) gabT Abat UGA1 kdd
lysDH L 1.1.1.a 2-amino-4- 2-amino-4- 2-amino-4- thrA oxopentanoate
hydroxypentanoate oxopentanoate dehydrogenase hom6 hom2 akthr2 hom1
M 2.6.1.a 2-amino-4- 2-oxo-4- 2-amino-4- aspC hydroxypentanoate
hydroxypentanoate hydroxypentanoate aminotransferase or
oxidoreductase (deaminating) avtA AAT2 dat dat ldh nadX N 4.1.1.a
2-oxo-4- 3-hydroxybutyr- 2-oxo-4- pdc hydroxypentanoate aldehyde
hydroxypentanoate (3-hydroxybutanal) pdc1 mdlC kgd O 1.1.1.a
3-oxobutyraldehyde 3-hydroxybutyr- 3-oxobutyraldehyde bdh aldehyde
reductase (ketone reducing) adh adhA ldh adh P 1.1.1.a
3-hydroxybutyr- 1,3-butanediol 3-hydroxybutyr- alrA aldehyde
aldehyde reductase ADH2 yqhD bdh I bdh II 4hbd ADHI mmsb P84067
GenBank ID Step (if available) Organism Known Substrates A
YP_001086914.1 Clostridium D-alanine difficile 630 YP_001086915.1
Clostridium D-alanine difficile 630 YP_001320181.1 Alkaliphilus
D-alanine metalliredigenes QYF YP_001320182.1 Alkaliphilus
D-alanine metalliredigenes QYF YP_001663101.1 Thermoanaero-
D-alanine bacter sp. X514 YP_001663102.1 Thermoanaero- D-alanine
bacter sp. X514 B NP_415448.1 Escherichia coli L-aspartate
YP_026231.1 Escherichia coli L-alanine, L-valine P23542.3
Saccharomyces L-aspartate cerevisae P19938 Bacillus sp. YM-1
D-alanine, D-2- minobutanoate, D- aspartate O07597 Bacillus
subtilis D-alanine, D-2- aminobutanoate, D-aspartate P0A393
Bacillus cereus L-leucine, L-valine, 2-aminobutanoate, L-isoleucine
NP_229443.1 Thermotoga L-aspartate maritima C P06672.1 Zymomonas
2-ketobutyrate mobilus P06169 Saccharomyces 2-ketobutyrate, 3-
cerevisae hydroxypyruvate P20906.2 Pseudomonas 2-ketobutyrate
putdia O50463.4 Mycobacterium alpha-ketoglutarate tuberculosis D
BAB12273.1 Acinetobacter sp. C2-C14 aldehydes Strain M-1
NP_014032.1 Saccharymyces propionaldehyde, cerevisiae
isobutyraldehyde, butyraldehyde, 2- methylbutyraldehyde, 3-
methylbutyraldehyde, 2-phenylacetaldehyde NP_417484.1 Escherichia
coli acetaldehyde, malondialdehyde, propanaldehyde, butanaldehyde,
and acrolein NP_349892.1 Clostridium butyraldehyde acetobutylicum
NP_349891.1 Clostridium butyraldehyde acetobutylicum YP_726053.1
Ralstonia eutropha succinate H16 semialdehyde AAR91477.1
Geobacillus ethanol, 1-butanol, 1- thermo- pentanol, 1-heptanol,
glucosidasius 1-hexanol, 1-octanol, M10EXG 2-propanol P28811.1
Pseudomonas 3- aeruginosa hydroxybutyraldehyde, malonic
semialdehyde, methylmalonate semialdehyde P84067 Thermus
methylmalonate thermophilus semialdehyde E NP_417315.1 Escherichia
coli meso- diaminopimelate AAA25361.1 Mycobacterium meso-
tuberculosis diaminopimelate BAC92756.1 Methylophilus meso-
methylotrophus diaminopimelate AA59967.1 Homo sapiens D-ornithine
P0A790 Escherichia coli L-aspartate Q9X4N0 Corynebacterium
L-aspartate glutanicum P65660 Mycobacterium L-aspartate
tuberculosis F NP_418562 Escherichia coli L-aspartate K12 subsp.
MG1655 P44324.1 Haemophilus L-aspartate influenzae P07346.1
Pseudomonas L-aspartate fluorescens P26899.1 Bacillus subtilus
L-aspartate P33109.1 Serratia L-aspartate marcescens G P0AC33
Escherichia coli fumarate K12 P05042 Escherichia coli fumarate K12
O69294 Campylobacter fumarate jejuni P84127 Thermus fumarate
thermophilus P14408 Rattus norvegicus fumarate ABC88407.1
Eubacterium 2-methylene- barkeri glutarate ABC88408 Eubacterium
dimethylmaleate barkeri ABC88409.1 Eubacterium dimethylmaleate
barkeri H AAA58352.1 Homo sapiens 3-oxobutyrate AAA23199.2
Clostridium acetone beijerinckii NRRL B593 AAC25556 Pyrococuus
2-pentanaol, furiosus pyruvaldehyde YP_725182.1 Ralstonia eutropha
lactate, 2-
oxobutyrate, 2-oxopentaonotae, 2-oxoglutarate P14941.1
Thermoanaero- acetone bacter brockii HTD4 I NP_418562 Escherichia
coli L-aspartate K12 subsp. MG1655 P44324.1 Haemophilus L-aspartate
influenzae P07346.1 Pseudomonas L-aspartate fluorescens P26899.1
Bacillus subtilus L-aspartate P33109.1 Serratia L-aspartate
marcescens J YP_709328.1 Pseudomonas 4-oxalocrotonate putida
YP_709353.1 Pseudomonas 4-oxalocrotonate putida CAA43228.1
Pseudomonas sp. 4-oxalocrotonate CF600 CAA43225.1 Pseudomonas sp.
4-oxalocrotonate CF600 U63827 Lactobacillus cinnamate and plantarum
derivatives AB330293 Klebsiella oxytoca cinnamate and derivatives K
ABF58893 Saccharomyces beta-alanine kluyveri P22256 Escherichia
coli 4-aminobutyrate P50554 Rattus norvegicus 3-amino-2-
methylpropionate NP_011533 Saccharomyces 4-aminobutyrate cerevisae
AAL93966.1 Fusobacterium 3,5- nucleatum diaminohexanoate BAB39707
Geobacillus L-lysine stearothermophilus L AAC73113 Escherichia coli
aspartate semialdehyde CAA89671 Saccharomyces aspartate cerevisae
semialdehyde CAD63186 Lactobacillus aspartate plantarum
semialdehyde O81852 Arabidopsis aspartate thaliana semialdehyde
CAD64819 Lactobacillus aspartate plantarum semialdehyde M
NP_415448.1 Escherichia coli L-aspartate YP_026231.1 Escherichia
coli L-alanine, L-valine P23542.3 Saccharomyces L-aspartate
cerevisae Bacillus sp. YM-1 P19938 D-alanine, D-2- aminobutanoate,
D-aspartate Bacillus subtilis O07597 D-alanine, D-2-aminobutanoate,
D-aspartate P0A393 Bacillus cereus L-leucine, L-valine,
2-aminobutanoate, L- isoleucine NP_229443.1 Thermotoga L-aspartate
maritima N P06672.1 Zymomonas 2-ketobutyrate mobilus P06169
Saccharomyces 2-ketobutyrate, cerevisae 3-hydroxypyruvate P20906.2
Pseudomonas 2-ketobutyrate putdia O50463.4 Mycobacterium
alpha-ketoglutarate tuberculosis O AAA58352.1 Homo sapiens
3-oxobutyrate AAA23199.2 Clostridium acetone beijerinckii NRRL B593
AAC25556 Pyrococuus 2-pentanaol, furiosus pyruvaldehyde YP_725182.1
Ralstonia eutropha lactate, 2-oxobutyrate, 2-oxopentaonotae,
2-oxoglutarate P14941.1 Thermoanaero- acetone bacter brockii HTD4 P
BAB12273.1 Acinetobacter sp. C2-C14 aldehydes Strain M-1
NP_014032.1 Saccharymyces propionaldehyde, cerevisiae
isobutyraldehyde, butyraldehyde, 2-methylbutyr- aldehyde,
3-methylbutyr- aldehyde, 2- phenylacetaldehyde NP_417484.1
Escherichia coli acetaldehyde, malondialdehyde, propanaldehyde,
butanaldehyde, and acrolein NP_349892.1 Clostridium butyraldehyde
acetobutylicum NP_349891.1 Clostridium butyraldehyde acetobutylicum
YP_726053.1 Ralstonia eutropha succinate H16 semialdehyde
AAR91477.1 Geobacillus ethanol, 1-butanol, thermoglucosidasius
1-pentanol, M10EXG 1-heptanol, 1-hexanol, 1-octanol, 2-propanol
P28811.1 Pseudomonas 3-hydroxy- aeruginosa butyraldehyde, malonic
semialdehyde, methylmalonate semialdehyde P84067 Thermus
methylmalonate thermophilus semialdehyde
TABLE-US-00124 TABLE 121 (Ref: FIG. 2) EC GenBank ID (if Step class
Desired substrate Desired product Enzyme name Gene name available)
Organism Known Substrates A 1.2.1.b acetoacetyl-CoA
3-oxobutyraldehyde acetoacetyl- Ald AAT66436 Clostridium
butyryl-CoA CoA reductase beijerinckii (aldehdye forming) sucD
NP_904963.1 Porphyromonas succinyl-CoA gingivalis bphG BAA03892.1
Pseudomonas sp acetaldehyde, propionaldehyde, butyraldehyde,
isobutyraldehyde and formaldehyde Msed_0709 YP_001190808.1
Metallosphaera malonyl-CoA sedula mcr NP_378167 Sulfolobus
malonyl-CoA, tokodaii methylmalonyl- CoA B 1.1.1.a 3-oxobutyr- 3-
3-oxobutyr- bdh AAA58352.1 Homo sapiens 3-oxobutyrate aldehyde
hydroxybutyraldehyde aldehyde reductase (ketone- reducing) adh
AAA23199.2 Clostridium acetone beijerinckii NRRL B593 adhA AAC25556
Pyrococuus 2-pentanaol, furiosus pyruvaldehyde ldh YP_725182.1
Ralstonia lactate, 2- eutropha oxobutyrate, 2- oxopentaonotae,
2-oxoglutarate adh P14941.1 Thermoanaero- acetone bacter brockii
HTD4 C 1.1.1.a 3-hydroxybutyr- 1,3-butanediol 3-hydroxy- alrA
BAB12273.1 Acinetobacter sp. C2-C14 aldehyde butyraldehyde Strain
M-1 aldehydes reductase ADH2 NP_014032.1 Saccharymyces
propionaldehyde, cerevisiae isobutyraldehyde, butyraldehyde,
2-methylbutyr- aldehyde, 3-methylbutyr- aldehyde, 2-phenylacet-
aldehyde yqhD NP_417484.1 Escherichia coli acetaldehyde,
malondialdehyde, propanaldehyde, butanaldehyde, and acrolein bdh I
NP_349892.1 Clostridium butyraldehyde acetobutylicum bdh II
NP_349891.1 Clostridium butyraldehyde acetobutylicum 4hbd
YP_726053.1 Ralstonia succinate eutropha H16 semialdehyde ADHI
AAR91477.1 Geobacillus ethanol, thermoglucosidasius 1-butanol,
M10EXG 1-pentanol, 1-heptanol, 1-hexanol, 1-octanol, 2-propanol
mmsb P28811.1 Pseudomonas 3-hydroxybutyr- aeruginosa aldehyde,
malonic semialdehyde, methylmalonate semialdehyde P84067 P84067
Thermus methylmalonate thermophilus semialdehyde D 1.1.1.c
acetoacetyl-CoA 4-hydroxy,2-butanone acetoacetyl- adhE2 AAK09379.1
Clostridium butanoyl-CoA CoA reductase acetobutylicum (alcohol-
forming) mcr AAS20429.1 Chloroflexus malonyl-CoA aurantiacus FAR
AAD38039.1 Simmondsia long chain acyl- chinensis CoA E 1.1.1.a
3-oxobutyr- 4-hydroxy,2-butanone 3- alrA BAB12273.1 Acinetobacter
sp. C2-C14 aldehyde oxobutyraldeh Strain M-1 aldehydes dye
reductase (aldehyde reducing) ADH2 NP_014032.1 Saccharymyces
propionaldehyde, cerevisiae isobutyraldehyde, butyraldehyde, 2-
methyl- butyraldehyde, 3-methylbutyr- aldehyde, 2- phenyl-
acetaldehyde yqhD NP_417484.1 Escherichia coli acetaldehyde,
malondialdehyde, propanaldehyde, butanaldehyde, and acrolein bdh I
NP_349892.1 Clostridium butyraldehyde acetobutylicum bdh II
NP_349891.1 Clostridium butyraldehyde acetobutylicum 4hbd
YP_726053.1 Ralstonia succinate eutropha H16 semialdehyde ADHI
AAR91477.1 Geobacillus ethanol, thermoglucosidasius 1-butanol,
M10EXG 1-pentanol, 1-heptanol, 1-hexanol, 1-octanol, 2-propanol
mmsb P28811.1 Pseudomonas 3- aeruginosa hydroxy- butyraldehyde,
malonic semialdehyde, methylmalonate semialdehyde P84067 P84067
Thermus methylmalonate thermophilus semialdehyde F 1.1.1.a
4-hydroxy, 1,3-butanediol 4-hydroxy,2- bdh AAA58352.1 Homo sapiens
3-oxobutyrate 2-butanone butanone reductase adh AAA23199.2
Clostridium acetone beijerinckii NRRL B593 adhA AAC25556 Pyrococuus
2-pentanaol, furiosus pyruvaldehyde ldh YP_725182.1 Ralstonia
lactate, 2- eutropha oxobutyrate, 2- oxopentaonotae, 2-oxoglutarate
adh P14941.1 Thermoanaero- acetone bacter brockii HTD4 G 1.1.1.a
acetoacetyl-CoA 3-hydroxybutyryl-CoA acetaocetyl hbd NP_349314.1
Clostridium acetoacetyl-CoA CoA reductase acetobutylicum (ketone
reducing) hbd AAM14586.1 Clostridium acetoacetyl-CoA beijerinckii
Hbd2 EDK34807.1 Clostridium acetoacetyl-CoA kluyveri Hbd1
EDK32512.1 Clostridium acetoacetyl-CoA kluyveri Msed_1423
YP_001191505 Metallosphaera 3-hydroxybutyryl- sedula CoA
(suspected) Msed_0399 YP_001190500 Metallosphaera 3-hydroxybutyryl-
sedula CoA (suspected) Msed_0389 YP_001190490 Metallosphaera
3-hydroxybutyryl- sedula CoA (suspected) Msed_1993 YP_001192057
Metallosphaera 3-hydroxybutyryl- sedula CoA (suspected) fadB
P21177.2 Escherichia coli 3-oxoacyl-CoA fadJ P77399.1 Escherichia
coli 3-oxoacyl-CoA H 1.2.1.b 3-hydroxybutyryl- 3-hydroxybutyr-
3-hydroxy- Ald AAT66436 Clostridium butyryl-CoA CoA aldehyde
butyryl-CoA beijerinckii redcutase (aldehyde forming) sucD
NP_904963.1 Porphyromonas succinyl-CoA gingivalis bphG BAA03892.1
Pseudomonas sp acetaldehyde, propionaldehyde, butyraldehyde,
isobutyraldehyde and formaldehyde Msed_0709 YP_001190808.1
Metallosphaera malonyl-CoA sedula mcr NP_378167 Sulfolobus
malonyl-CoA, tokodaii methylmalonyl- CoA I 1.1.1.c
3-hydroxybutyryl- 1,3-butanediol 3-hydroxy- adhE2 AAK09379.1
Clostridium butanoyl-CoA CoA butyryl-CoA acetobutylicum reductase
(alcohol forming) mcr AAS20429.1 Chloroflexus malonyl-CoA
aurantiacus FAR AAD38039.1 Simmondsia long chain acyl- chinensis
CoA
TABLE-US-00125 TABLE 122 (Ref: FIG. 3) EC Gene GenBank ID (if Step
class Desired substrate Desired product Enzyme name name available)
Organism Known Substrates A 4.2.1.a 4-hydroxybutyryl- crotonyl-CoA
4-hydroxy- abfD YP_001396399.1 Clostridium 4-hydroxybutyryl- CoA
butyryl-CoA kluyveri DSM 555 CoA dehydratase abfD P55792
Clostridium 4-hydroxybutyryl- aminobutyricum CoA abfD YP_001928843
Porphyromonas 4-hydroxybutyryl- gingivalis ATCC CoA 33277 B 4.2.1.a
crotonyl-CoA 3-hydroxybutyryl- crotonase crt NP_349318.1
Clostridium 3-hydroxybutyryl- CoA acetobutylicum CoA crt1
YP_001393856 Clostridium 3-hydroxybutyryl- kluyveri DSM 555 CoA crt
YP_001929291.1 Porphyromonas example based on gingivalis ATCC
sequence similarity 33277 paaA NP_745427.1 Pseudomonas enoyl-CoA,
cis- putida dihydrodiol derivative of phenylacetyl-CoA paaB
NP_745426.1 Pseudomonas enoyl-CoA, cis- putida dihydrodiol
derivative of phenylacetyl-CoA phaA ABF82233.1 Pseudomonas
enoyl-CoA, cis- fluorescens dihydrodiol derivative of
phenylacetyl-CoA phaB ABF82234.1 Pseudomonas enoyl-CoA, cis-
fluorescens dihydrodiol derivative of phenylacetyl-CoA maoC
NP_415905.1 Escherichia coli enoyl-CoA, cis- dihydrodiol derivative
of phenylacetyl-CoA paaF NP_415911.1 Escherichia coli enoyl-CoA,
cis- dihydrodiol derivative of phenylacetyl-CoA paaG NP_415912.1
Escherichia coli enoyl-CoA, cis-dihydrodiol derivative of
phenylacetyl-CoA C 1.2.1.b 3-hydroxybutyryl- 3-hydroxy- 3-hydroxy-
Ald AAT66436 Clostridium butyryl-CoA CoA butyraldehyde butyryl-CoA
beijerinckii reductase (aldehyde forming) sucD NP_904963.1
Porphyromonas succinyl-CoA gingivalis bphG BAA03892.1 Pseudomonas
sp acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde
and formaldehyde Msed_0709 YP_001190808.1 Metallosphaera
malonyl-CoA sedula mcr NP_378167 Sulfolobus tokodaii malonyl-CoA,
methylmalonyl- CoA D 1.1.1.a 3-hydroxy- 1,3-butanediol 3-hydroxy-
alrA BAB12273.1 Acinetobacter sp. C2-C14 aldehydes butyraldehyde
butyraldehyde Strain M-1 reductase ADH2 NP_014032.1 Saccharymyces
propionaldehyde, cerevisiae isobutyraldehyde, butyraldehyde,
2-methylbutyr- aldehyde, 3-methylbutyr- aldehyde, 2-phenylacet-
aldehyde yqhD NP_417484.1 Escherichia coli acetaldehyde,
malondialdehyde, propanaldehyde, butanaldehyde, and acrolein bdh I
NP_349892.1 Clostridium butyraldehyde acetobutylicum bdh II
NP_349891.1 Clostridium butyraldehyde acetobutylicum 4hbd
YP_726053.1 Ralstonia eutropha succinate H16 semialdehyde ADHI
AAR91477.1 Geobacillus ethanol, 1-butanol, thermoglucosidasius
1-pentanol, M10EXG 1-heptanol, 1-hexanol, 1-octanol, 2-propanol
mmsb P28811.1 Pseudomonas 3-hydroxy- aeruginosa butyraldehyde,
malonic semialdehyde, methylmalonate semialdehyde P84067 P84067
Thermus methylmalonate thermophilus semialdehyde E 1.1.1.c
3-hydroxybutyryl- 1,3-butanediol 3-hydroxy- adhE2 AAK09379.1
Clostridium butanoyl-CoA CoA butyryl-CoA acetobutylicum reductase
(alcohol forming) mcr AAS20429.1 Chloroflexus malonyl-CoA
aurantiacus FAR AAD38039.1 Simmondsia long chain acyl- chinensis
CoA
Sequence Listing
[0605] The present specification is being filed with a computer
readable form (CRF) copy of the Sequence Listing. The CRF entitled
12956-143_SEQLIST.txt, which was created on Jun. 15, 2012 and is
77,805 bytes in size, is identical to the paper copy of the
Sequence Listing and is incorporated herein by reference in its
entirety.
[0606] 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 and embodiments 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
* * * * *
References