U.S. patent application number 13/530053 was filed with the patent office on 2013-02-07 for microorganisms for producing 1,4-butanediol and methods related thereto.
This patent application is currently assigned to Genomatica, Inc.. The applicant listed for this patent is Anthony P. BURGARD, Robin E. Osterhout, Priti Pharkya, Jun Sun. Invention is credited to Anthony P. BURGARD, Robin E. Osterhout, Priti Pharkya, Jun Sun.
Application Number | 20130034884 13/530053 |
Document ID | / |
Family ID | 47422957 |
Filed Date | 2013-02-07 |
United States Patent
Application |
20130034884 |
Kind Code |
A1 |
BURGARD; Anthony P. ; et
al. |
February 7, 2013 |
MICROORGANISMS FOR PRODUCING 1,4-BUTANEDIOL AND METHODS RELATED
THERETO
Abstract
The invention provides non-naturally occurring microbial
organisms comprising a 1,4-butanediol (BDO), 4-hydroxybutyryl-CoA,
4-hydroxybutanal or putrescine pathway comprising at least one
exogenous nucleic acid encoding a BDO, 4-hydroxybutyryl-CoA,
4-hydroxybutanal or putrescine pathway enzyme expressed in a
sufficient amount to produce BDO, 4-hydroxybutyryl-CoA,
4-hydroxybutanal or putrescine and further optimized for expression
of BDO. The invention additionally provides methods of using such
microbial organisms to produce BDO, 4-hydroxybutyryl-CoA,
4-hydroxybutanal or putrescine.
Inventors: |
BURGARD; Anthony P.;
(Bellefonte, PA) ; Osterhout; Robin E.; (San
Diego, CA) ; Sun; Jun; (San Diego, CA) ;
Pharkya; Priti; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BURGARD; Anthony P.
Osterhout; Robin E.
Sun; Jun
Pharkya; Priti |
Bellefonte
San Diego
San Diego
San Diego |
PA
CA
CA
CA |
US
US
US
US |
|
|
Assignee: |
Genomatica, Inc.
San Diego
CA
|
Family ID: |
47422957 |
Appl. No.: |
13/530053 |
Filed: |
June 21, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61500120 |
Jun 22, 2011 |
|
|
|
61502837 |
Jun 29, 2011 |
|
|
|
Current U.S.
Class: |
435/126 ;
435/146; 435/158; 435/189; 435/252.33; 435/254.2 |
Current CPC
Class: |
C12P 7/42 20130101; C12P
7/18 20130101; C12N 9/0008 20130101; C12Y 102/99006 20130101; C12P
7/16 20130101; Y02E 50/10 20130101; C12N 15/52 20130101 |
Class at
Publication: |
435/126 ;
435/158; 435/146; 435/252.33; 435/254.2; 435/189 |
International
Class: |
C12N 1/21 20060101
C12N001/21; C12N 9/02 20060101 C12N009/02; C12P 17/04 20060101
C12P017/04; C12N 1/19 20060101 C12N001/19; C12P 7/18 20060101
C12P007/18; C12P 7/42 20060101 C12P007/42 |
Claims
1. A non-naturally occurring microbial organism, comprising a
microbial organism having a 1,4-butanediol pathway comprising at
least one exogenous nucleic acid encoding a 1,4-butanediol pathway
enzyme expressed in a sufficient amount to produce 1,4-butanediol;
said non-naturally occurring microbial organism further comprising:
(i) a reductive TCA pathway comprising at least one exogenous
nucleic acid encoding a reductive TCA pathway enzyme, wherein said
at least one exogenous nucleic acid is selected from an ATP-citrate
lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA
lyase, a fumarate reductase, isocitrate dehydrogenase, and an
alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA
pathway comprising at least one exogenous nucleic acid encoding a
reductive TCA pathway enzyme, wherein said at least one exogenous
nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase,
a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate
carboxykinase, a CO dehydrogenase, and an H.sub.2 hydrogenase; or
(iii) at least one exogenous nucleic acid encodes an enzyme
selected from a CO dehydrogenase, an H.sub.2 hydrogenase, and
combinations thereof; wherein said 1,4-butanediol pathway comprises
a pathway selected from: (a) 4-hydroxybutanoate dehydrogenase,
succinyl-CoA synthetase, CoA-dependent succinic semialdehyde
dehydrogenase, and .alpha.-ketoglutarate decarboxylase; (b)
4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase,
CoA-dependent succinic semialdehyde dehydrogenase,
4-hydroxybutyrate:CoA transferase, 4-butyrate kinase,
phosphotransbutyrylase, .alpha.-ketoglutarate decarboxylase,
aldehyde dehydrogenase, alcohol dehydrogenase and an
aldehyde/alcohol dehydrogenase; (c) (i) an .alpha.-ketoglutarate
decarboxylase, or an .alpha.-ketoglutarate dehydrogenase and a
CoA-dependent succinic semialdehyde dehydrogenase, or a
glutamate:succinate semialdehyde transaminase and a glutamate
decarboxylase; (ii) a 4-hydroxybutanoate dehydrogenase; (iii) a
4-hydroxybutyryl-CoA:acetyl-CoA transferase, or a butyrate kinase
and a phosphotransbutyrylase; (iv) an aldehyde dehydrogenase; and
(v) an alcohol dehydrogenase; (d) 4-aminobutyrate CoA transferase,
4-aminobutyryl-CoA hydrolase, 4-aminobutyrate-CoA ligase,
4-aminobutyryl-CoA oxidoreductase (deaminating), 4-aminobutyryl-CoA
transaminase, and 4-hydroxybutyryl-CoA dehydrogenase; (e)
4-aminobutyrate CoA transferase, 4-aminobutyryl-CoA hydrolase,
4-aminobutyrate-CoA ligase, 4-aminobutyryl-CoA reductase (alcohol
forming), 4-aminobutyryl-CoA reductase, 4-aminobutan-1-ol
dehydrogenase, 4-aminobutan-1-ol oxidoreductase (deaminating) and
4-aminobutan-1-ol transaminase; (f) 4-aminobutyrate kinase,
4-aminobutyraldehyde dehydrogenase (phosphorylating),
4-aminobutan-1-ol dehydrogenase, 4-aminobutan-1-ol oxidoreductase
(deaminating), 4-aminobutan-1-ol transaminase,
[(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase
(deaminating), [(4-aminobutanolyl)oxy]phosphonic acid transaminase,
4-hydroxybutyryl-phosphate dehydrogenase, and
4-hydroxybutyraldehyde dehydrogenase (phosphorylating); (g)
alpha-ketoglutarate 5-kinase, 2,5-dioxopentanoic semialdehyde
dehydrogenase (phosphorylating), 2,5-dioxopentanoic acid reductase,
alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA
hydrolase, alpha-ketoglutaryl-CoA ligase, alpha-ketoglutaryl-CoA
reductase, 5-hydroxy-2-oxopentanoic acid dehydrogenase,
alpha-ketoglutaryl-CoA reductase (alcohol forming),
5-hydroxy-2-oxopentanoic acid decarboxylase, and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (h)
glutamate CoA transferase, glutamyl-CoA hydrolase, glutamyl-CoA
ligase, glutamate 5-kinase, glutamate-5-semialdehyde dehydrogenase
(phosphorylating), glutamyl-CoA reductase, glutamate-5-semialdehyde
reductase, glutamyl-CoA reductase (alcohol forming),
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating),
2-amino-5-hydroxypentanoic acid transaminase,
5-hydroxy-2-oxopentanoic acid decarboxylase,
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (i)
3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA
dehydratase, vinylacetyl-CoA .DELTA.-isomerase, or
4-hydroxybutyryl-CoA dehydratase; (j) homoserine deaminase,
homoserine CoA transferase, homoserine-CoA hydrolase,
homoserine-CoA ligase, homoserine-CoA deaminase,
4-hydroxybut-2-enoyl-CoA transferase, 4-hydroxybut-2-enoyl-CoA
hydrolase, 4-hydroxybut-2-enoyl-CoA ligase, 4-hydroxybut-2-enoate
reductase, 4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA
hydrolase, 4-hydroxybutyryl-CoA ligase, or 4-hydroxybut-2-enoyl-CoA
reductase; (k) succinyl-CoA reductase (alcohol forming),
4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or
4-hydroxybutanal dehydrogenase (phosphorylating); (l) glutamate
dehydrogenase, 4-aminobutyrate oxidoreductase (deaminating),
4-aminobutyrate transaminase, glutamate decarboxylase,
4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or
4-hydroxybutanal dehydrogenase (phosphorylating); (m)
4-aminobutyrate kinase; 4-aminobutyraldehyde dehydrogenase
(phosphorylating); 4-aminobutan-1-ol dehydrogenase; and
4-aminobutan-1-ol oxidoreductase (deaminating) or 4-aminobutan-1-ol
transaminase; (n) 4-aminobutyrate kinase;
[(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase (deaminating)
or [(4-aminobutanolyl)oxy]phosphonic acid transaminase;
4-hydroxybutyryl-phosphate dehydrogenase; and
4-hydroxybutyraldehyde dehydrogenase (phosphorylating); (o)
alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA
hydrolase, or alpha-ketoglutaryl-CoA ligase; alpha-ketoglutaryl-CoA
reductase (alcohol forming); and 5-hydroxy-2-oxopentanoic acid
decarboxylase; (p) alpha-ketoglutarate CoA transferase,
alpha-ketoglutaryl-CoA hydrolase, or alpha-ketoglutaryl-CoA ligase;
alpha-ketoglutaryl-CoA reductase (alcohol forming); and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (q)
alpha-ketoglutarate 5-kinase; 2,5-dioxopentanoic semialdehyde
dehydrogenase (phosphorylating); 2,5-dioxopentanoic acid reductase;
and 5-hydroxy-2-oxopentanoic acid decarboxylase; (r)
alpha-ketoglutarate 5-kinase; 2,5-dioxopentanoic semialdehyde
dehydrogenase (phosphorylating); 2,5-dioxopentanoic acid reductase;
and 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);
(s) alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA
hydrolase, or alpha-ketoglutaryl-CoA ligase; alpha-ketoglutaryl-CoA
reductase; 5-hydroxy-2-oxopentanoic acid dehydrogenase; and
5-hydroxy-2-oxopentanoic acid decarboxylase; (t)
alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA
hydrolase, or alpha-ketoglutaryl-CoA ligase; alpha-ketoglutaryl-CoA
reductase; 5-hydroxy-2-oxopentanoic acid dehydrogenase; and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (u)
glutamate CoA transferase, glutamyl-CoA hydrolase, or glutamyl-CoA
ligase; glutamyl-CoA reductase (alcohol forming);
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or
2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (v)
glutamate 5-kinase; glutamate-5-semialdehyde dehydrogenase
(phosphorylating); 2-amino-5-hydroxypentanoic acid oxidoreductase
(deaminating) or 2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (w)
glutamate CoA transferase, glutamyl-CoA hydrolase, or glutamyl-CoA
ligase; glutamyl-CoA reductase; glutamate-5-semialdehyde reductase;
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or
2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (x)
glutamate 5-kinase; glutamate-5-semialdehyde dehydrogenase
(phosphorylating); glutamate-5-semialdehyde reductase;
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or
2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (y)
homoserine deaminase; 4-hydroxybut-2-enoyl-CoA transferase,
4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA
ligase; 4-hydroxybut-2-enoyl-CoA reductase; (z) homoserine CoA
transferase, homoserine-CoA hydrolase, or homoserine-CoA ligase;
homoserine-CoA deaminase; and 4-hydroxybut-2-enoyl-CoA reductase;
(aa) homoserine deaminase; 4-hydroxybut-2-enoate reductase; and
4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA hydrolase,
or 4-hydroxybutyryl-CoA ligase; (bb) (i) alpha-ketoglutarate
decarboxylase; or alpha-ketoglutarate dehydrogenase and
CoA-dependent succinate semialdehyde dehydrogenase; or
glutamate:succinate semialdehyde transaminase and glutamate
decarboxylase; (ii) 4-hydroxybutyrate dehydrogenase; (iii)
4-hydroxybutyryl-CoA transferase; or 4-hydroxybutyrate kinase and
phosphotrans-4-hydroxybutyrylase; (iv) 4-hydroxybutyryl-CoA
reductase; and (v) 4-hydroxybutyraldehyde reductase; or
aldehyde/alcohol dehydrogenase; (cc) (i) alpha-ketoglutarate
decarboxylase; or succinyl-CoA synthetase and CoA-dependent
succinate semialdehyde dehydrogenase; (ii) 4-hydroxybutyrate
dehydrogenase; (iii) 4-hydroxybutyryl-CoA transferase; or
4-hydroxybutyrate kinase and phosphotrans-4-hydroxybutyrylase; and
(iv) aldehyde dehydrogenase; and alcohol dehydrogenase; or
aldehyde/alcohol dehydrogenase; (dd) (i) alpha-ketoglutarate
decarboxylase; or glutamate dehydrogenase; glutamate decarboxylase;
and deaminating 4-aminobutyrate oxidoreductase or 4-aminobutyrate
transaminase; or alpha-ketoglutarate dehydrogenase and
CoA-dependent succinate semialdehyde dehydrogenase; (ii)
4-hydroxybutyrate dehydrogenase; and (iii) 4-hydroxybutyrate
kinase; phosphotrans-4-hydroxybutyrylase; 4-hydroxybutyryl-CoA
reductase; and 4-hydroxybutyraldehyde reductase; or
4-hydroxybutyrate kinase; phosphorylating 4-hydroxybutanal
dehydrogenase; and 4-hydroxybutyraldehyde reductase; or
4-hydroxybutyrate kinase; phosphotrans-4-hydroxybutyrylase; and
alcohol forming 4-hydroxybutyryl-CoA reductase; or
4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA hydrolase
or 4-hydroxybutyryl-CoA ligase; 4-hydroxybutyryl-CoA reductase; and
4-hydroxybutyraldehyde reductase; or 4-hydroxybutyryl-CoA
transferase or 4-hydroxybutyryl-CoA hydrolase or
4-hydroxybutyryl-CoA ligase; and alcohol forming
4-hydroxybutyryl-CoA reductase; (ee) (i) glutamate CoA transferase
or glutamyl-CoA hydrolase or glutamyl-CoA ligase; glutamyl-CoA
reductase; and glutamate-5-semialdehyde reductase; or glutamate CoA
transferase or glutamyl-CoA hydrolase or glutamyl-CoA ligase; and
alcohol forming glutamyl-CoA reductase; or glutamate 5-kinase;
phosphorylating glutamate-5-semialdehyde dehydrogenase; and
glutamate-5-semialdehyde reductase; (ii) deaminating
2-amino-5-hydroxypentanoic acid oxidoreductase or
2-amino-5-hydroxypentanoic acid transaminase; and (iii)
5-hydroxy-2-oxopentanoic acid decarboxylase; and
4-hydroxybutyraldehyde reductase; or decarboxylating
5-hydroxy-2-oxopentanoic acid dehydrogenase; 4-hydroxybutyryl-CoA
reductase; and 4-hydroxybutyraldehyde reductase; or decarboxylating
5-hydroxy-2-oxopentanoic acid dehydrogenase and alcohol forming
4-hydroxybutyryl-CoA reductase; (ff) succinyl-CoA reductase
(aldehyde forming); 4-hydroxybutyrate dehydrogenase; and
4-hydroxybutyrate reductase; and optionally 1,4-butandiol
dehydrogenase; (gg) alpha-ketoglutarate decarboxylase;
4-hydroxybutyrate dehydrogenase; and 4-hydroxybutyrate reductase;
and optionally 1,4-butandiol dehydrogenase; (hh) succinate
reductase; 4-hydroxybutyrate dehydrogenase, and 4-hydroxybutyrate
reductase; and optionally 1,4-butandiol dehydrogenase; (ii)
alpha-ketoglutarate decarboxylase, or glutamate dehydrogenase or
glutamate transaminase and glutamate decarboxylase and
4-aminobutyrate dehydrogenase or 4-aminobutyrate transaminase;
4-hydroxybutyrate dehydrogenase; and 4-hydroxybutyrate reductase;
and optionally 1,4-butandiol dehydrogenase; (jj)
alpha-ketoglutarate reductase; 5-hydroxy-2-oxopentanoate
dehydrogenase; and 5-hydroxy-2-oxopentanoate decarboxylase; and
optionally 1,4-butandiol dehydrogenase; (kk) Acetoacetyl-CoA
thiolase or acetoacetyl-CoA synthase; 3-Hydroxybutyryl-CoA
dehydrogenase; Crotonase; Crotonyl-CoA hydratase; and
4-hydroxybutyryl-CoA reductase (alcohol forming); (ll)
Acetoacetyl-CoA thiolase or acetoacetyl-CoA synthase;
3-Hydroxybutyryl-CoA dehydrogenase; Crotonase; Crotonyl-CoA
hydratase; 4-hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase; (mm) Acetoacetyl-CoA thiolase or
acetoacetyl-CoA synthase; 3-Hydroxybutyryl-CoA dehydrogenase;
Crotonase; Crotonyl-CoA hydratase; 4-Hydroxybutyryl-CoA
transferase, 4-Hydroxybutyryl-CoA synthetase, 4-Hydroxybutyryl-CoA
hydrolase, or Phosphotrans-4-hydroxybutyrylase/4-Hydroxybutyrate
kinase; 4-Hydroxybutyrate reductase; and 1,4-butanediol
dehydrogenase; (nn) Succinate reductase; 4-Hydroxybutyrate
dehydrogenase; 4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylasel; 4-Hydroxybutyryl-CoA reductase
(aldehyde forming); and 1,4-butanediol dehydrogenase; (oo)
Succinate reductase; 4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase; 4-Hydroxybutyryl-phosphate reductase; and
1,4-butanediol dehydrogenase; (pp) Succinate reductase;
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyrate reductase; and
1,4-butanediol dehydrogenase; (qq) Succinate reductase;
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyryl-CoA transferase,
or 4-Hydroxybutyryl-CoA synthetase; and 4-Hydroxybutyryl-CoA
reductase (alcohol forming); (rr) Succinate reductase;
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyryl-CoA transferase,
or 4-Hydroxybutyryl-CoA synthetase; 4-Hydroxybutyryl-CoA reductase
(aldehyde forming); and 1,4-butanediol dehydrogenase; (ss)
Succinate reductase; 4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase; Phosphotrans-4-hydroxybutyrylase; and
4-Hydroxybutyryl-CoA reductase (alcohol forming); (tt) Succinyl-CoA
transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (aldehyde forming); 4-Hydroxybutyrate
dehydrogenase; 4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase; 4-Hydroxybutyryl-CoA reductase
(aldehyde forming); and 1,4-butanediol dehydrogenase; (uu)
Succinyl-CoA transferase, or Succinyl-CoA synthetase (or
succinyl-CoA ligase); Succinyl-CoA reductase (aldehyde forming);
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyrate kinase;
4-Hydroxybutyryl-phosphate reductase; and 1,4-butanediol
dehydrogenase; (vv) Succinyl-CoA transferase, or Succinyl-CoA
synthetase (or succinyl-CoA ligase); Succinyl-CoA reductase
(aldehyde forming); 4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate reductase; and 1,4-butanediol dehydrogenase; (ww)
Succinyl-CoA transferase, or Succinyl-CoA synthetase (or
succinyl-CoA ligase); Succinyl-CoA reductase (aldehyde forming);
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyryl-CoA transferase,
or 4-Hydroxybutyryl-CoA synthetase; and 4-Hydroxybutyryl-CoA
reductase (alcohol forming);
(xx) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or
succinyl-CoA ligase); Succinyl-CoA reductase (aldehyde forming);
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyryl-CoA transferase,
or 4-Hydroxybutyryl-CoA synthetase; 4-Hydroxybutyryl-CoA reductase
(aldehyde forming); and 1,4-butanediol dehydrogenase; (yy)
Succinyl-CoA transferase, or Succinyl-CoA synthetase (or
succinyl-CoA ligase); Succinyl-CoA reductase (aldehyde forming);
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase; and 4-Hydroxybutyryl-CoA
reductase (alcohol forming); (zz) Alpha-ketoglutarate decarboxylase
or (Glutamate dehydrogenase and/or Glutamate transaminase;
Glutamate decarboxylase; 4-aminobutyrate dehydrogenase and/or
4-aminobutyrate transaminase); 4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase; Phosphotrans-4-hydroxybutyrylase;
4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase; (aaa) Alpha-ketoglutarate
decarboxylase or (Glutamate dehydrogenase and/or Glutamate
transaminase; Glutamate decarboxylase; 4-aminobutyrate
dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyrate kinase;
4-Hydroxybutyryl-phosphate reductase; and 1,4-butanediol
dehydrogenase; (bbb) Alpha-ketoglutarate decarboxylase or
(Glutamate dehydrogenase and/or Glutamate transaminase; Glutamate
decarboxylase; 4-aminobutyrate dehydrogenase and/or 4-aminobutyrate
transaminase); 4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyrate
reductase; and 1,4-butanediol dehydrogenase; (ccc)
Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase
and/or Glutamate transaminase; Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyryl-CoA transferase,
or 4-Hydroxybutyryl-CoA synthetase; and 4-Hydroxybutyryl-CoA
reductase (alcohol forming); (ddd) Alpha-ketoglutarate
decarboxylase or (Glutamate dehydrogenase and/or Glutamate
transaminase; Glutamate decarboxylase; 4-aminobutyrate
dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyryl-CoA transferase,
or 4-Hydroxybutyryl-CoA synthetase; 4-Hydroxybutyryl-CoA reductase
(aldehyde forming); and 1,4-butanediol dehydrogenase; (eee)
Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase
and/or Glutamate transaminase; Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase; and 4-Hydroxybutyryl-CoA
reductase (alcohol forming); (fff) Succinyl-CoA transferase, or
Succinyl-CoA synthetase (or succinyl-CoA ligase); Succinyl-CoA
reductase (alcohol forming); 4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase; 4-Hydroxybutyryl-CoA reductase
(aldehyde forming); and 1,4-butanediol dehydrogenase; (ggg)
Succinyl-CoA transferase, or Succinyl-CoA synthetase (or
succinyl-CoA ligase); Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyrate kinase; 4-Hydroxybutyryl-phosphate reductase; and
1,4-butanediol dehydrogenase; (hhh) Succinyl-CoA transferase, or
Succinyl-CoA synthetase (or succinyl-CoA ligase); Succinyl-CoA
reductase (alcohol forming); 4-Hydroxybutyrate reductase; and
1,4-butanediol dehydrogenase; (iii) Succinyl-CoA transferase, or
Succinyl-CoA synthetase (or succinyl-CoA ligase); Succinyl-CoA
reductase (alcohol forming); 4-Hydroxybutyryl-CoA transferase, or
4-Hydroxybutyryl-CoA synthetase; and 4-Hydroxybutyryl-CoA reductase
(alcohol forming); (jjj) Succinyl-CoA transferase, or Succinyl-CoA
synthetase (or succinyl-CoA ligase); Succinyl-CoA reductase
(alcohol forming); 4-Hydroxybutyryl-CoA transferase, or
4-Hydroxybutyryl-CoA synthetase; 4-Hydroxybutyryl-CoA reductase
(aldehyde forming); and 1,4-butanediol dehydrogenase; (kkk)
Succinyl-CoA transferase, or Succinyl-CoA synthetase (or
succinyl-CoA ligase); Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyrate kinase; Phosphotrans-4-hydroxybutyrylase; and
4-Hydroxybutyryl-CoA reductase (alcohol forming); and (lll) any of
the pathways that produce 1,4-butanediol as shown in any of FIG. 1,
8-13, 58, 62, 63 or 72-74.
2. The non-naturally occurring microbial organism of claim 1,
wherein said microbial organism comprising (i) further comprises an
exogenous nucleic acid encoding an enzyme selected from a
pyruvate:ferredoxin oxidoreductase, an aconitase, a succinyl-CoA
synthetase, a succinyl-CoA transferase, a fumarase, a malate
dehydrogenase, an acetate kinase, a phosphotransacetylase, an
acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase,
ferredoxin, and combinations thereof.
3. The non-naturally occurring microbial organism of claim 1,
wherein said microbial organism comprising (ii) further comprises
an exogenous nucleic acid encoding an enzyme selected from an
aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase,
a succinyl-CoA transferase, a fumarase, a malate dehydrogenase,
NAD(P):ferredoxin oxidoreductase, ferredoxin, and combinations
thereof.
4. The non-naturally occurring microbial organism of claim 1,
wherein said microbial organism comprising (iii) further comprises
an exogenous nucleic acid encoding an enzyme selected from
NAD(P):ferredoxin oxidoreductase and ferredoxin.
5-6. (canceled)
7. The non-naturally occurring microbial organism of claim 1,
wherein said microbial organism: (i) comprising pathways (a), (b)
or (c) further comprises an enzyme selected from succinyl-CoA
synthetase, exogenous CoA-dependent succinic semialdehyde
dehydrogenase or exogenous succinyl-CoA synthetase and exogenous
CoA-dependent succinic semialdehyde dehydrogenase; (ii) comprising
pathway (d), (g), (h), (i), (j) further comprises an enzyme
selected from 4-hydroxybutyryl-CoA reductase (alcohol forming),
4-hydroxybutyryl-CoA reductase, or 1,4-butanediol dehydrogenase;
(iii) comprising pathway (e) or (f) further comprises
1,4-butanediol dehydrogenase; (iv) comprising pathway (k) further
comprises succinyl-CoA reductase, 4-hydroxybutyrate dehydrogenase,
4-hydroxybutyryl-CoA transferase, 4-hydroxybutyrate kinase,
phosphotrans-4-hydroxybutyrylase, 4-hydroxybutyryl-CoA reductase,
4-hydroxybutyryl-CoA reductase (alcohol forming), or 1,4-butanediol
dehydrogenase; (v) comprising pathway (1) further comprises
alpha-ketoglutarate decarboxylase, 4-hydroxybutyrate dehydrogenase,
4-hydroxybutyryl-CoA transferase, 4-hydroxybutyrate kinase,
phosphotrans-4-hydroxybutyrylase, 4-hydroxybutyryl-CoA reductase,
4-hydroxybutyryl-CoA reductase (alcohol forming), or 1,4-butanediol
dehydrogenase.
8. The non-naturally occurring microbial organism of claim 1,
wherein said microbial organism comprises two, three, four or five
exogenous nucleic acids each encoding enzymes of (i), (ii) or
(iii).
9. The non-naturally occurring microbial organism of claim 8,
wherein said microbial organism comprising (i) comprises three
exogenous nucleic acids encoding ATP-citrate lyase or citrate
lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin
oxidoreductase; wherein said microbial organism comprising (ii)
comprises five exogenous nucleic acids encoding pyruvate:ferredoxin
oxidoreductase, a phosphoenolpyruvate carboxylase, a
phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an
H.sub.2 hydrogenase; or wherein said microbial organism comprising
(iii) comprises two exogenous nucleic acids encoding CO
dehydrogenase and H.sub.2 hydrogenase.
10. (canceled)
11. A method for producing 1,4-butanediol, comprising culturing the
non-naturally occurring microbial organism of claim 1 under
conditions and for a sufficient period of time to produce
1,4-butanediol.
12. A non-naturally occurring microbial organism, comprising a
microbial organism having a 4-hydroxybutyrate pathway comprising at
least one exogenous nucleic acid encoding a 4-hydroxybutyrate
pathway enzyme 4-hydroxybutyrate expressed in a sufficient amount
to produce 4-hydroxybutyrate; said non-naturally occurring
microbial organism further comprising: (i) a reductive TCA pathway
comprising at least one exogenous nucleic acid encoding a reductive
TCA pathway enzyme, wherein said at least one exogenous nucleic
acid is selected from an ATP-citrate lyase, citrate lyase, a
citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase,
isocitrate dehydrogenase and an alpha-ketoglutarate:ferredoxin
oxidoreductase; (ii) a reductive TCA pathway comprising at least
one exogenous nucleic acid encoding a reductive TCA pathway enzyme,
wherein said at least one exogenous nucleic acid is selected from a
pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate
carboxylase, a phosphoenolpyruvate carboxykinase, a CO
dehydrogenase, and an H.sub.2 hydrogenase; or (iii) at least one
exogenous nucleic acid encodes an enzyme selected from a CO
dehydrogenase, an H.sub.2 hydrogenase, and combinations thereof;
(a) Acetoacetyl-CoA thiolase or acetoacetyl-CoA synthase;
3-Hydroxybutyryl-CoA dehydrogenase; Crotonase; Crotonyl-CoA
hydratase; and 4-Hydroxybutyryl-CoA transferase,
4-Hydroxybutyryl-CoA synthetase, 4-Hydroxybutyryl-CoA hydrolase, or
Phosphotrans-4-hydroxybutyrylase/4-Hydroxybutyrate kinase; (b)
Acetoacetyl-CoA thiolase or acetoacetyl-CoA synthase;
3-Hydroxybutyryl-CoA dehydrogenase; Crotonase; Crotonyl-CoA
hydratase; and 4-Hydroxybutyryl-CoA transferase, hydrolase or
synthetase; (c) Acetoacetyl-CoA thiolase or acetoacetyl-CoA
synthase; 3-Hydroxybutyryl-CoA dehydrogenase; Crotonase;
Crotonyl-CoA hydratase; Phosphotrans-4-hydroxybutyrylase; and
4-Hydroxybutyrate kinase; (d) Succinate reductase; and
4-Hydroxybutyrate dehydrogenase; (e) Succinyl-CoA transferase, or
Succinyl-CoA synthetase (or succinyl-CoA ligase); Succinyl-CoA
reductase (aldehyde forming); and 4-Hydroxybutyrate dehydrogenase;
(f) Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase
and/or Glutamate transaminase; Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase);
and 4-Hydroxybutyrate dehydrogenase; (g) Succinyl-CoA transferase,
or Succinyl-CoA synthetase (or succinyl-CoA ligase); and
Succinyl-CoA reductase (alcohol forming); (h) acetoacetyl-CoA
thiolase or acetoacetyl-CoA synthase, a 3-hydroxybutyryl-CoA
dehydrogenase, a crotonase, a crotonyl-CoA hydratase, a
4-hydroxybutyryl-CoA transferase, a
phosphotrans-4-hydroxybutyrylase, and a 4-hydroxybutyrate kinase;
(i) 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase,
CoA-dependent succinic semialdehyde dehydrogenase, and
.alpha.-ketoglutarate decarboxylase; (j) (i) an
.alpha.-ketoglutarate decarboxylase, or an .alpha.-ketoglutarate
dehydrogenase and a CoA-dependent succinic semialdehyde
dehydrogenase, or a glutamate:succinate semialdehyde transaminase
and a glutamate decarboxylase; (ii) a 4-hydroxybutanoate
dehydrogenase; (k) succinyl-CoA reductase (alcohol forming),
4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or
4-hydroxybutanal dehydrogenase (phosphorylating); (l) glutamate
dehydrogenase, 4-aminobutyrate oxidoreductase (deaminating),
4-aminobutyrate transaminase, glutamate decarboxylase,
4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or
4-hydroxybutanal dehydrogenase (phosphorylating); (m) homoserine
deaminase; 4-hydroxybut-2-enoyl-CoA transferase,
4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA
ligase; 4-hydroxybut-2-enoyl-CoA reductase; (n) homoserine CoA
transferase, homoserine-CoA hydrolase, or homoserine-CoA ligase;
homoserine-CoA deaminase; and 4-hydroxybut-2-enoyl-CoA reductase;
(o) homoserine deaminase; 4-hydroxybut-2-enoate reductase; and
4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA hydrolase,
or 4-hydroxybutyryl-CoA ligase; (p) succinyl-CoA reductase
(aldehyde forming); and 4-hydroxybutyrate dehydrogenase; (q)
alpha-ketoglutarate decarboxylase; and 4-hydroxybutyrate
dehydrogenase; (r) succinate reductase; and 4-hydroxybutyrate
dehydrogenase; (s) alpha-ketoglutarate decarboxylase, or glutamate
dehydrogenase or glutamate transaminase and glutamate decarboxylase
and 4-aminobutyrate dehydrogenase or 4-aminobutyrate transaminase;
and 4-hydroxybutyrate dehydrogenase; and (t) a 4-hydroxybutyrate
pathway selected from any of the pathways that produce
4-hydroxybutyrate as shown in any of FIG. 1, 8-13, 58, 62, 63 or
72-74.
13. The non-naturally occurring microbial organism of claim 12,
wherein said microbial organism comprising (i) further comprises an
exogenous nucleic acid encoding an enzyme selected from a
pyruvate:ferredoxin oxidoreductase, an aconitase, 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.
14. The non-naturally occurring microbial organism of claim 12,
wherein said microbial organism comprising (ii) further comprises
an exogenous nucleic acid encoding an enzyme selected from an
aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase,
a succinyl-CoA transferase, a fumarase, a malate dehydrogenase,
NAD(P):ferredoxin oxidoreductase, ferredoxin, and combinations
thereof.
15. The non-naturally occurring microbial organism of claim 12,
wherein said microbial organism comprising (iii) further comprises
an exogenous nucleic acid encoding an enzyme selected from
NAD(P):ferredoxin oxidoreductase and ferredoxin.
16-18. (canceled)
19. The non-naturally occurring microbial organism of claim 18,
wherein said microbial organism comprising (i) comprises three
exogenous nucleic acids encoding ATP-citrate lyase or citrate
lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin
oxidoreductase; wherein said microbial organism comprising (ii)
comprises five exogenous nucleic acids encoding pyruvate:ferredoxin
oxidoreductase, a phosphoenolpyruvate carboxylase, a
phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an
H.sub.2 hydrogenase; or wherein said microbial organism comprising
(iii) comprises two exogenous nucleic acids encoding CO
dehydrogenase and H.sub.2 hydrogenase.
20. (canceled)
21. A method for producing 4-hydroxybutyrate, comprising culturing
the non-naturally occurring microbial organism of claim 12 under
conditions and for a sufficient period of time to produce
4-hydroxybutyrate.
22. A non-naturally occurring microbial organism, comprising a
microbial organism having a gamma-butyrolactone pathway comprising
at least one exogenous nucleic acid encoding a gamma-butyrolactone
pathway enzyme expressed in a sufficient amount to produce
gamma-butyrolactone; said non-naturally occurring microbial
organism further comprising: (i) a reductive TCA pathway comprising
at least one exogenous nucleic acid encoding a reductive TCA
pathway enzyme, wherein said at least one exogenous nucleic acid is
selected from an ATP-citrate lyase, citrate lyase, a citryl-CoA
synthetase, a citryl-CoA lyase, a fumarate reductase, isocitrate
dehydrogenase and an alpha-ketoglutarate:ferredoxin oxidoreductase;
(ii) a reductive TCA pathway comprising at least one exogenous
nucleic acid encoding a reductive TCA pathway enzyme, wherein said
at least one exogenous nucleic acid is selected from a
pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate
carboxylase, a phosphoenolpyruvate carboxykinase, a CO
dehydrogenase, and an H.sub.2 hydrogenase; or (iii) at least one
exogenous nucleic acid encodes an enzyme selected from a CO
dehydrogenase, an H.sub.2 hydrogenase, and combinations thereof;
(a) Acetoacetyl-CoA thiolase or acetoacetyl-CoA synthase;
3-Hydroxybutyryl-CoA dehydrogenase; Crotonase; Crotonyl-CoA
hydratase; and spontaneous or enzyme catalyzed; (b) Acetoacetyl-CoA
thiolase or acetoacetyl-CoA synthase; 3-Hydroxybutyryl-CoA
dehydrogenase; Crotonase; Crotonyl-CoA hydratase;
Phosphotrans-4-hydroxybutyrylase; amd spontaneous or enzyme
catalyzed; (c) Succinate reductase; 4-Hydroxybutyrate
dehydrogenase; 4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase; and 4-Hydroxybutyryl-CoA
hydrolase or spontaneous; (d) Succinate reductase;
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyryl-CoA transferase,
or 4-Hydroxybutyryl-CoA synthetase; and 4-Hydroxybutyryl-CoA
hydrolase or spontaneous; (e) Succinyl-CoA transferase, or
Succinyl-CoA synthetase (or succinyl-CoA ligase); Succinyl-CoA
reductase (aldehyde forming); 4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase; Phosphotrans-4-hydroxybutyrylase; and
4-Hydroxybutyryl-CoA hydrolase or spontaneous; (f) Succinyl-CoA
transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (aldehyde forming); 4-Hydroxybutyrate
dehydrogenase; 4-Hydroxybutyryl-CoA transferase, or
4-Hydroxybutyryl-CoA synthetase; 4-Hydroxybutyryl-CoA hydrolase or
spontaneous; (g) Alpha-ketoglutarate decarboxylase or (Glutamate
dehydrogenase and/or Glutamate transaminase; Glutamate
decarboxylase; 4-aminobutyrate dehydrogenase and/or 4-aminobutyrate
transaminase); 4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyrate
kinase; Phosphotrans-4-hydroxybutyrylase; and 4-Hydroxybutyryl-CoA
hydrolase or spontaneous; (h) Alpha-ketoglutarate decarboxylase or
(Glutamate dehydrogenase and/or Glutamate transaminase; Glutamate
decarboxylase; 4-aminobutyrate dehydrogenase and/or 4-aminobutyrate
transaminase); 4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA
synthetase; and 4-Hydroxybutyryl-CoA hydrolase or spontaneous; (i)
Succinyl-CoA transferase, or Succinyl-CoA synthetase (or
succinyl-CoA ligase); Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyrate kinase; Phosphotrans-4-hydroxybutyrylase;
4-Hydroxybutyryl-CoA hydrolase or spontaneous; (j) Succinyl-CoA
transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (alcohol forming); 4-Hydroxybutyryl-CoA
transferase, or 4-Hydroxybutyryl-CoA synthetase; and
4-Hydroxybutyryl-CoA hydrolase or spontaneous; (k)
alpha-ketoglutarate reductase; 5-hydroxy-2-oxopentanoate
dehydrogenase; and 5-hydroxy-2-oxopentanoate dehydrogenase
(decarboxylation) (l) 4-hydroxybutanoate dehydrogenase,
succinyl-CoA synthetase, CoA-dependent succinic semialdehyde
dehydrogenase, and .alpha.-ketoglutarate decarboxylase; (m)
4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase,
CoA-dependent succinic semialdehyde dehydrogenase,
4-hydroxybutyrate:CoA transferase, 4-butyrate kinase,
phosphotransbutyrylase, .alpha.-ketoglutarate decarboxylase; (n)
(i) an .alpha.-ketoglutarate decarboxylase, or an
.alpha.-ketoglutarate dehydrogenase and a CoA-dependent succinic
semialdehyde dehydrogenase, or a glutamate:succinate semialdehyde
transaminase and a glutamate decarboxylase; (ii) a
4-hydroxybutanoate dehydrogenase; (iii) a
4-hydroxybutyryl-CoA:acetyl-CoA transferase, or a butyrate kinase
and a phosphotransbutyrylase; (o) 4-aminobutyrate CoA transferase,
4-aminobutyryl-CoA hydrolase, 4-aminobutyrate-CoA ligase,
4-aminobutyryl-CoA oxidoreductase (deaminating), 4-aminobutyryl-CoA
transaminase, and 4-hydroxybutyryl-CoA dehydrogenase; (p)
4-aminobutyrate CoA transferase, 4-aminobutyryl-CoA hydrolase,
4-aminobutyrate-CoA ligase, 4-aminobutyryl-CoA reductase (alcohol
forming), 4-aminobutyryl-CoA reductase, 4-aminobutan-1-ol
dehydrogenase, 4-aminobutan-1-ol oxidoreductase (deaminating) and
4-aminobutan-1-ol transaminase; (q) 4-aminobutyrate kinase,
4-aminobutyraldehyde dehydrogenase (phosphorylating),
4-aminobutan-1-ol dehydrogenase, 4-aminobutan-1-ol oxidoreductase
(deaminating), 4-aminobutan-1-ol transaminase,
[(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase
(deaminating), [(4-aminobutanolyl)oxy]phosphonic acid transaminase,
4-hydroxybutyryl-phosphate dehydrogenase, and
4-hydroxybutyraldehyde dehydrogenase (phosphorylating); (r)
alpha-ketoglutarate 5-kinase, 2,5-dioxopentanoic semialdehyde
dehydrogenase (phosphorylating), 2,5-dioxopentanoic acid reductase,
alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA
hydrolase, alpha-ketoglutaryl-CoA ligase, alpha-ketoglutaryl-CoA
reductase, 5-hydroxy-2-oxopentanoic acid dehydrogenase,
alpha-ketoglutaryl-CoA reductase (alcohol forming),
5-hydroxy-2-oxopentanoic acid decarboxylase, and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (s)
glutamate CoA transferase, glutamyl-CoA hydrolase, glutamyl-CoA
ligase, glutamate 5-kinase, glutamate-5-semialdehyde dehydrogenase
(phosphorylating), glutamyl-CoA reductase, glutamate-5-semialdehyde
reductase, glutamyl-CoA reductase (alcohol forming),
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating),
2-amino-5-hydroxypentanoic acid transaminase,
5-hydroxy-2-oxopentanoic acid decarboxylase,
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (t)
3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA
dehydratase, vinylacetyl-CoA .DELTA.-isomerase, or
4-hydroxybutyryl-CoA dehydratase; (u) homoserine deaminase,
homoserine CoA transferase, homoserine-CoA hydrolase,
homoserine-CoA ligase, homoserine-CoA deaminase,
4-hydroxybut-2-enoyl-CoA transferase, 4-hydroxybut-2-enoyl-CoA
hydrolase, 4-hydroxybut-2-enoyl-CoA ligase, 4-hydroxybut-2-enoate
reductase, 4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA
hydrolase, 4-hydroxybutyryl-CoA ligase, or 4-hydroxybut-2-enoyl-CoA
reductase; (v) succinyl-CoA reductase (alcohol forming),
4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or
4-hydroxybutanal dehydrogenase (phosphorylating); (w) glutamate
dehydrogenase, 4-aminobutyrate oxidoreductase (deaminating),
4-aminobutyrate transaminase, glutamate decarboxylase,
4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or
4-hydroxybutanal dehydrogenase (phosphorylating); (x)
4-aminobutyrate kinase; 4-aminobutyraldehyde dehydrogenase
(phosphorylating); 4-aminobutan-1-ol dehydrogenase; and
4-aminobutan-1-ol oxidoreductase (deaminating) or 4-aminobutan-1-ol
transaminase; (y) 4-aminobutyrate kinase;
[(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase (deaminating)
or [(4-aminobutanolyl)oxy]phosphonic acid transaminase;
4-hydroxybutyryl-phosphate dehydrogenase; and
4-hydroxybutyraldehyde dehydrogenase (phosphorylating); (z)
alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA
hydrolase, or alpha-ketoglutaryl-CoA ligase; alpha-ketoglutaryl-CoA
reductase (alcohol forming); and 5-hydroxy-2-oxopentanoic acid
decarboxylase; (aa) alpha-ketoglutarate CoA transferase,
alpha-ketoglutaryl-CoA hydrolase, or alpha-ketoglutaryl-CoA ligase;
alpha-ketoglutaryl-CoA reductase (alcohol forming); and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (bb)
alpha-ketoglutarate 5-kinase; 2,5-dioxopentanoic semialdehyde
dehydrogenase (phosphorylating); 2,5-dioxopentanoic acid reductase;
and 5-hydroxy-2-oxopentanoic acid decarboxylase; (cc)
alpha-ketoglutarate 5-kinase; 2,5-dioxopentanoic semialdehyde
dehydrogenase (phosphorylating); 2,5-dioxopentanoic acid reductase;
and 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);
(dd) alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA
hydrolase, or alpha-ketoglutaryl-CoA ligase; alpha-ketoglutaryl-CoA
reductase; 5-hydroxy-2-oxopentanoic acid dehydrogenase; and
5-hydroxy-2-oxopentanoic acid decarboxylase; (ee)
alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA
hydrolase, or alpha-ketoglutaryl-CoA ligase; alpha-ketoglutaryl-CoA
reductase; 5-hydroxy-2-oxopentanoic acid dehydrogenase; and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (ff)
glutamate CoA transferase, glutamyl-CoA hydrolase, or glutamyl-CoA
ligase; glutamyl-CoA reductase (alcohol forming);
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or
2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (gg)
glutamate 5-kinase; glutamate-5-semialdehyde dehydrogenase
(phosphorylating); 2-amino-5-hydroxypentanoic acid oxidoreductase
(deaminating) or 2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (hh)
glutamate CoA transferase, glutamyl-CoA hydrolase, or glutamyl-CoA
ligase; glutamyl-CoA reductase; glutamate-5-semialdehyde reductase;
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or
2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (ii)
glutamate 5-kinase; glutamate-5-semialdehyde dehydrogenase
(phosphorylating); glutamate-5-semialdehyde reductase;
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or
2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (jj)
homoserine deaminase; 4-hydroxybut-2-enoyl-CoA transferase,
4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA
ligase; 4-hydroxybut-2-enoyl-CoA reductase; (kk) homoserine CoA
transferase, homoserine-CoA hydrolase, or homoserine-CoA ligase;
homoserine-CoA deaminase; and 4-hydroxybut-2-enoyl-CoA reductase;
(ll) homoserine deaminase; 4-hydroxybut-2-enoate reductase; and
4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA hydrolase,
or 4-hydroxybutyryl-CoA ligase; (mm) (i) alpha-ketoglutarate
decarboxylase; or alpha-ketoglutarate dehydrogenase and
CoA-dependent succinate semialdehyde dehydrogenase; or
glutamate:succinate semialdehyde transaminase and glutamate
decarboxylase; (ii) 4-hydroxybutyrate dehydrogenase; (iii)
4-hydroxybutyryl-CoA transferase; or 4-hydroxybutyrate kinase and
phosphotrans-4-hydroxybutyrylase; and (iv) 4-hydroxybutyryl-CoA
reductase; (nn) (i) alpha-ketoglutarate decarboxylase; or
succinyl-CoA synthetase and CoA-dependent succinate semialdehyde
dehydrogenase; (ii) 4-hydroxybutyrate dehydrogenase; (iii)
4-hydroxybutyryl-CoA transferase; or 4-hydroxybutyrate kinase and
phosphotrans-4-hydroxybutyrylase; (oo) (i) alpha-ketoglutarate
decarboxylase; or glutamate dehydrogenase; glutamate decarboxylase;
and deaminating 4-aminobutyrate oxidoreductase or 4-aminobutyrate
transaminase; or alpha-ketoglutarate dehydrogenase and
CoA-dependent succinate semialdehyde dehydrogenase; (ii)
4-hydroxybutyrate dehydrogenase; and (iii) 4-hydroxybutyrate
kinase; phosphotrans-4-hydroxybutyrylase; 4-hydroxybutyryl-CoA
reductase; and 4-hydroxybutyraldehyde reductase; or
4-hydroxybutyrate kinase; phosphorylating 4-hydroxybutanal
dehydrogenase; and 4-hydroxybutyraldehyde reductase; or
4-hydroxybutyrate kinase; phosphotrans-4-hydroxybutyrylase; and
alcohol forming 4-hydroxybutyryl-CoA reductase; or
4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA hydrolase
or 4-hydroxybutyryl-CoA ligase; 4-hydroxybutyryl-CoA reductase; and
4-hydroxybutyraldehyde reductase; or 4-hydroxybutyryl-CoA
transferase or 4-hydroxybutyryl-CoA hydrolase or
4-hydroxybutyryl-CoA ligase; and alcohol forming
4-hydroxybutyryl-CoA reductase; (pp) (i) glutamate CoA transferase
or glutamyl-CoA hydrolase or glutamyl-CoA ligase; glutamyl-CoA
reductase; and glutamate-5-semialdehyde reductase; or glutamate CoA
transferase or glutamyl-CoA hydrolase or glutamyl-CoA ligase; and
alcohol forming glutamyl-CoA reductase; or glutamate 5-kinase;
phosphorylating glutamate-5-semialdehyde dehydrogenase; and
glutamate-5-semialdehyde reductase; (ii) deaminating
2-amino-5-hydroxypentanoic acid oxidoreductase or
2-amino-5-hydroxypentanoic acid transaminase; and (iii)
5-hydroxy-2-oxopentanoic acid decarboxylase; and
4-hydroxybutyraldehyde reductase; or decarboxylating
5-hydroxy-2-oxopentanoic acid dehydrogenase; 4-hydroxybutyryl-CoA
reductase; and 4-hydroxybutyraldehyde reductase; or decarboxylating
5-hydroxy-2-oxopentanoic acid dehydrogenase and alcohol forming
4-hydroxybutyryl-CoA reductase; (qq) a gamma-butyrolactone pathway
comprising a pathway selected from any of the pathways that produce
4-hydroxybutyryl-CoA or 4-hydroxybutyryl phosphate as shown in FIG.
1, 8-13, 58, 62-63 or 72-74, wherein gamma-butyrolactone is
produced enzymatically or by spontaneous chemical conversion.
23. The non-naturally occurring microbial organism of claim 22,
wherein said microbial organism comprising (i) further comprises an
exogenous nucleic acid encoding an enzyme selected from a
pyruvate:ferredoxin oxidoreductase, an aconitase, 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.
24. The non-naturally occurring microbial organism of claim 22,
wherein said microbial organism comprising (ii) further comprises
an exogenous nucleic acid encoding an enzyme selected from an
aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase,
a succinyl-CoA transferase, a fumarase, a malate dehydrogenase,
NAD(P):ferredoxin oxidoreductase, ferredoxin, and combinations
thereof.
25. The non-naturally occurring microbial organism of claim 22,
wherein said microbial organism comprising (iii) further comprises
an exogenous nucleic acid encoding an enzyme selected from
NAD(P):ferredoxin oxidoreductase and ferredoxin.
26-31. (canceled)
32. A method for producing gamma-butyrolactone, comprising
culturing the non-naturally occurring microbial organism of claim
22 under conditions and for a sufficient period of time to produce
gamma-butyrolactone.
33. A carboxylic acid reductase, comprising an amino acid sequence
having an amino acid substitution selected from E16K; Q95L; L100M;
A1011T; K823E; T941S; H15Q; D198E; G446C; S392N; F699L; V8831;
F467S; T987S; R12H; V295G; V295A; V295S; V295T; V295G; V295V;
V295L; V2951; V295M; V295P; V295F; V295Y; V295W; V295D; V295E;
V295N; V295Q; V295H; V295K; V295R; M296G; M296A; M296S; M296T;
M296C; M296V; M296L; M2961; M296M; M296P; M296F; M296Y; M296W;
M296D; M296E; M296N; M296Q; M296H; M296K; M296R; G297G; G297A;
G297S; G297T; G297C; G297V; G297L; G2971; 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; 5417A; S417S; S417T; S417C; S417V
S417L; S417I; S417M; S417P; S417F; S417Y; S417W; S417D; S417E;
S417N; S417Q; S417H; S417K; and S417R, or combinations thereof.
Description
[0001] This application claims the benefit of priority of U.S.
Provisional application Ser. No. 61/500,120, filed Jun. 22, 2011,
and U.S. Provisional application Ser. No. 61/502,837, filed Jun.
29, 2011, each of which the entire contents are incorporated herein
by reference.
[0002] Incorporated herein by reference is the Sequence Listing
being concurrently submitted via EFS-Web as an ASCII text file
named 12956-144-999 SEQLIST.TXT, created Jun. 21, 2012, and being
227,850 bytes in size.
[0003] This invention relates generally to in silico design of
organisms and engineering of organisms, more particularly to
organisms having 1,4-butanediol, 4-hydroxybutyryl-CoA,
4-hydroxybutanal or putrescine biosynthesis capability.
BACKGROUND OF THE INVENTION
[0004] The compound 4-hydroxybutanoic acid (4-hydroxybutanoate,
4-hydroxybutyrate, 4-HB) is a 4-carbon carboxylic acid that has
industrial potential as a building block for various commodity and
specialty chemicals. In particular, 4-HB has the potential to serve
as a new entry point into the 1,4-butanediol family of chemicals,
which includes solvents, resins, polymer precursors, and specialty
chemicals. 1,4-Butanediol (BDO) is a polymer intermediate and
industrial solvent. BDO is currently produced from petrochemical
precursors, primarily acetylene, maleic anhydride, and propylene
oxide.
[0005] For example, acetylene is reacted with 2 molecules of
formaldehyde in the Reppe synthesis reaction (Kroschwitz and Grant,
Encyclopedia of Chem. Tech., John Wiley and Sons, Inc., New York
(1999)), followed by catalytic hydrogenation to form
1,4-butanediol. It has been estimated that 90% of the acetylene
produced in the U.S. is consumed for butanediol production.
Alternatively, it can be formed by esterification and catalytic
hydrogenation of maleic anhydride, which is derived from butane.
Downstream, butanediol can be further transformed; for example, by
oxidation to .gamma.-butyrolactone, which can be further converted
to pyrrolidone and N-methyl-pyrrolidone, or hydrogenolysis to
tetrahydrofuran. These compounds have varied uses as polymer
intermediates, solvents, and additives, and have a combined market
of nearly 2 billion lb/year.
[0006] It is desirable to develop a method for production of these
chemicals by alternative means that not only substitute renewable
for petroleum-based feedstocks, and also use less energy- and
capital-intensive processes. The Department of Energy has proposed
1,4-diacids, and particularly succinic acid, as key
biologically-produced intermediates for the manufacture of the
butanediol family of products (DOE Report, "Top Value-Added
Chemicals from Biomass", 2004). However, succinic acid is costly to
isolate and purify and requires high temperatures and pressures for
catalytic reduction to butanediol.
[0007] Thus, there exists a need for alternative means for
effectively producing commercial quantities of 1,4-butanediol and
its chemical precursors. The present invention satisfies this need
and provides related advantages as well.
SUMMARY OF THE INVENTION
[0008] The invention provides non-naturally occurring microbial
organisms containing a 1,4-butanediol (BDO), 4-hydroxybutanal
(4-HBal), 4-hydroxybutyryl-CoA (4-HBCoA) and/or putrescine pathway
comprising at least one exogenous nucleic acid encoding a BDO,
4-HBal and/or putrescine pathway enzyme expressed in a sufficient
amount to produce BDO, 4-HBal, 4-HBCoA and/or putrescine. The
microbial organisms can be further optimized for expression of BDO,
4-HBal, 4-HBCoA and/or putrescine. The invention additionally
provides methods of using such microbial organisms to produce BDO,
4-HBal, 4-HBCoA and/or putrescine.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 is a schematic diagram showing biochemical pathways
to 4-hydroxybutyurate (4-HB) and to 1,4-butanediol production. The
first 5 steps are endogenous to E. coli, while the remainder can be
expressed heterologously. Enzymes catalyzing the biosynthetic
reactions are: (1) succinyl-CoA synthetase; (2) CoA-independent
succinic semialdehyde dehydrogenase or succinate reductase; (3)
.alpha.-ketoglutarate dehydrogenase; (4) glutamate:succinate
semialdehyde transaminase; (5) glutamate decarboxylase; (6)
CoA-dependent succinic semialdehyde dehydrogenase; (7)
4-hydroxybutanoate dehydrogenase; (8) .alpha.-ketoglutarate
decarboxylase; (9) 4-hydroxybutyryl CoA:acetyl-CoA transferase;
(10) butyrate kinase; (11) phosphotransbutyrylase; (12) aldehyde
dehydrogenase; (13) alcohol dehydrogenase.
[0010] FIG. 2 is a schematic diagram showing homoserine
biosynthesis in E. coli.
[0011] FIG. 3 shows the production of 4-HB in glucose minimal
medium using E. coli strains harboring plasmids expressing various
combinations of 4-HB pathway genes. (a) 4-HB concentration in
culture broth; (b) succinate concentration in culture broth; (c)
culture OD, measured at 600 nm. Clusters of bars represent the 24
hour, 48 hour, and 72 hour (if measured) timepoints. The codes
along the x-axis indicate the strain/plasmid combination used. The
first index refers to the host strain: 1, MG1655 lacI.sup.Q; 2,
MG1655 .DELTA.gabD lacI.sup.Q; 3, MG1655 AgabD .DELTA.aldA
lacI.sup.Q. The second index refers to the plasmid combination
used: 1, pZE13-0004-0035 and pZA33-0036; 2, pZE13-0004-0035 and
pZA33-0010n; 3, pZE13-0004-0008 and pZA33-0036; 4, pZE13-0004-0008
and pZA33-0010n; 5, Control vectors pZE13 and pZA33.
[0012] FIG. 4 shows the production of 4-HB from glucose in E. coli
strains expressing .alpha.-ketoglutarate decarboxylase from
Mycobacterium tuberculosis. Strains 1-3 contain pZE13-0032 and
pZA33-0036. Strain 4 expresses only the empty vectors pZE13 and
pZA33. Host strains are as follows: 1 and 4, MG1655 lacI.sup.Q; 2,
MG1655 AgabD lacI.sup.Q; 3, MG1655 .DELTA.gabD .DELTA.aldA
lacI.sup.Q. The bars refer to concentration at 24 and 48 hours.
[0013] FIG. 5 shows the production of BDO from 10 mM 4-HB in
recombinant E. coli strains. Numbered positions correspond to
experiments with MG1655 lacI.sup.Q containing pZA33-0024,
expressing cat2 from P. gingivalis, and the following genes
expressed on pZE13: 1, none (control); 2, 0002; 3, 0003; 4, 0003n;
5, 0011; 6, 0013; 7, 0023; 8, 0025; 9, 0008n; 10, 0035. Gene
numbers are defined in Table 6. For each position, the bars refer
to aerobic, microaerobic, and anaerobic conditions, respectively.
Microaerobic conditions were created by sealing the culture tubes
but not evacuating them.
[0014] FIG. 6 shows the mass spectrum of 4-HB and BDO produced by
MG1655 lacI.sup.Q pZE13-0004-0035-0002 pZA33-0034-0036 grown in M9
minimal medium supplemented with 4 g/L unlabeled glucose (a, c, e,
and g) uniformly labeled .sup.13C-glucose (b, d, f, and h). (a) and
(b), mass 116 characteristic fragment of derivatized BDO,
containing 2 carbon atoms; (c) and (d), mass 177 characteristic
fragment of derivatized BDO, containing 1 carbon atom; (e) and (f),
mass 117 characteristic fragment of derivatized 4-HB, containing 2
carbon atoms; (g) and (h), mass 233 characteristic fragment of
derivatized 4-HB, containing 4 carbon atoms.
[0015] FIG. 7 is a schematic process flow diagram of bioprocesses
for the production of .gamma.-butyrolactone. Panel (a) illustrates
fed-batch fermentation with batch separation and panel (b)
illustrates fed-batch fermentation with continuous separation.
[0016] FIGS. 8A and 8B show exemplary 1,4-butanediol (BDO)
pathways. FIG. 8A shows BDO pathways from succinyl-CoA. FIG. 8B
shows BDO pathways from alpha-ketoglutarate.
[0017] FIGS. 9A-9C show exemplary BDO pathways. FIGS. 9A and 9B
show pathways from 4-aminobutyrate. FIG. 9C shows a pathway from
acetoactyl-CoA to 4-aminobutyrate.
[0018] FIG. 10 shows exemplary BDO pathways from
alpha-ketoglutarate.
[0019] FIG. 11 shows exemplary BDO pathways from glutamate.
[0020] FIG. 12 shows exemplary BDO pathways from
acetoacetyl-CoA.
[0021] FIG. 13 shows exemplary BDO pathways from homoserine.
[0022] FIG. 14 shows the nucleotide and amino acid sequences of E.
coli succinyl-CoA synthetase. FIG. 14A shows the nucleotide
sequence (SEQ ID NO:46) of the E. coli sucCD operon. FIGS. 14B (SEQ
ID NO:47) and 14C (SEQ ID NO:48) show the amino acid sequences of
the succinyl-CoA synthetase subunits encoded by the sucCD
operon.
[0023] FIG. 15 shows the nucleotide and amino acid sequences of
Mycobacterium bovis alpha-ketoglutarate decarboxylase. FIG. 15A
shows the nucleotide sequence (SEQ ID NO:49) of Mycobacterium bovis
sucA gene. FIG. 15B shows the amino acid sequence (SEQ ID NO:50) of
M. bovis alpha-ketoglutarate decarboxylase.
[0024] FIG. 16 shows biosynthesis in E. coli of 4-hydroxybutyrate
from glucose in minimal medium via alpha-ketoglutarate under
anaerobic (microaerobic) conditions. The host strain is ECKh-401.
The experiments are labeled based on the upstream pathway genes
present on the plasmid pZA33 as follows: 1) 4hbd-sucA; 2)
sucCD-sucD-4hbd; 3) sucCD-sucD-4hbd-sucA.
[0025] FIG. 17 shows biosynthesis in E. coli of 4-hydroxybutyrate
from glucose in minimal medium via succinate and
alpha-ketoglutarate. The host strain is wild-type MG1655. The
experiments are labeled based on the genes present on the plasmids
pZE13 and pZA33 as follows: 1) empty control vectors 2) empty
pZE13, pZA33-4hbd; 3) pZE13-sucA, pZA33-4hbd.
[0026] FIG. 18A shows the nucleotide sequence (SEQ ID NO:51) of
CoA-dependent succinate semialdehyde dehydrogenase (sucD) from
Porphyromonas gingivalis, and FIG. 18B shows the encoded amino acid
sequence (SEQ ID NO:52).
[0027] FIG. 19A shows the nucleotide sequence (SEQ ID NO:53) of
4-hydroxybutyrate dehydrogenase (4-hbd) from Porphyromonas
gingivalis, and FIG. 19B shows the encoded amino acid sequence (SEQ
ID NO:54).
[0028] FIG. 20A shows the nucleotide sequence (SEQ ID NO:55) of
4-hydroxybutyrate CoA transferase (cat2) from Porphyromonas
gingivalis, and FIG. 20B shows the encoded amino acid sequence (SEQ
ID NO:56).
[0029] FIG. 21A shows the nucleotide sequence (SEQ ID NO:57) of
phosphotransbutyrylase (ptb) from Clostridium acetobutylicum, and
FIG. 21B shows the encoded amino acid sequence (SEQ ID NO:58).
[0030] FIG. 22A shows the nucleotide sequence (SEQ ID NO:59) of
butyrate kinase (buk1) from Clostridium acetobutylicum, and FIG.
22B shows the encoded amino acid sequence (SEQ ID NO:60).
[0031] FIG. 23 shows alternative nucleotide sequences for C.
acetobutylicum 020 (phosphtransbutyrylase) with altered codons for
more prevalent E. coli codons relative to the C. acetobutylicum
native sequence. FIGS. 23A-23D (020A-020D, SEQ ID NOS:61-64,
respectively) contain sequences with increasing numbers of rare E.
coli codons replaced by more prevalent codons
(A<B<C<D).
[0032] FIG. 24 shows alternative nucleotide sequences for C.
acetobutylicum 021 (butyrate kinase) with altered codons for more
prevalent E. coli codons relative to the C. acetobutylicum native
sequence. FIGS. 24A-24D (021A-021B, SEQ ID NOS:65-68, respectively)
contain sequences with increasing numbers of rare E. coli codons
replaced by more prevalent codons (A<B<C<D).
[0033] FIG. 25 shows improved expression of butyrate kinase (BK)
and phosphotransbutyrylase (PTB) with optimized codons for
expression in E. coli. FIG. 25A shows sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) stained for proteins
with Coomassie blue; lane 1, control vector with no insert; lane 2,
expression of C. acetobutylicum native sequences in E. coli; lane
3, expression of 020B-021B codon optimized PTB-BK; lane 4,
expression of 020C-021C codon optimized PTB-BK. The positions of BK
and PTB are shown. FIG. 25B shows the BK and PTB activities of
native C. acetobutylicum sequence (2021n) compared to codon
optimized 020B-021B (2021B) and 020C-021C (2021C).
[0034] FIG. 26 shows production of BDO and gamma-butyrylactone
(GBL) in various strains expressing BDO producing enzymes: Cat2
(034); 2021n; 2021B; 2021C.
[0035] FIG. 27A shows the nucleotide sequence (SEQ ID NO:69) of the
native Clostridium biejerinckii ald gene (025n), and FIG. 27B shows
the encoded amino acid sequence (SEQ ID NO:70).
[0036] FIGS. 28A-28D show alternative gene sequences for the
Clostridium beijerinckii ald gene (025A-025D, SEQ ID NOS:71-74,
respectively), in which increasing numbers of rare codons are
replaced by more prevalent codons (A<B<C<D).
[0037] FIG. 29 shows expression of native C. beijerinckii ald gene
and codon optimized variants; no ins (control with no insert),
025n, 025A, 025B, 025C, 025D.
[0038] FIG. 30 shows BDO or BDO and ethanol production in various
strains. FIG. 30 shows BDO production in strains containing the
native C. beijerinckii ald gene (025n) or variants with optimized
codons for expression in E. coli (025A-025D). FIG. 30B shows
production of ethanol and BDO in strains expressing the C.
acetobutylicum AdhE2 enzyme (002C) compared to the codon optimized
variant 025B. The third set shows expression of P. gingivalis sucD
(035). In all cases, P. gingivalis Cat2 (034) is also
expressed.
[0039] FIG. 31A shows the nucleotide sequence (SEQ ID NO:75) of the
adh1 gene from Geobacillus thermoglucosidasius, and FIG. 31B shows
the encoded amino acid sequence (SEQ ID NO:76).
[0040] FIG. 32A shows the expression of the Geobacillus
thermoglucosidasius adh1 gene in E. coli. Either whole cell lysates
or supernatants were analyzed by SDS-PAGE and stained with
Coomassie blue for plasmid with no insert, plasmid with 083
(Geotrichum capitatum N-benzyl-3-pyrrolidinol dehydrogenase) and
plasmid with 084 (Geobacillus thermoglucosidasius adh1) inserts.
FIG. 32B shows the activity of 084 with butyraldehyde (diamonds) or
4-hydroxybutyraldehyde (squares) as substrates.
[0041] FIG. 33 shows the production of BDO in various strains:
plasmid with no insert; 025B, 025B-026n; 025B-026A; 025B-026B;
025B-026C; 025B-050; 025B-052; 025B-053; 025B-055; 025B-057;
025B-058; 025B-071; 025B-083; 025B-084; PTSlacO-025B;
PTSlacO-025B-026n.
[0042] FIG. 34 shows a plasmid map for the vector pRE118-V2.
[0043] FIG. 35 shows the sequence (SEQ ID NO:77) of the ECKh-138
region encompassing the aceF and lpdA genes. The K. pneumonia lpdA
gene is underlined, and the codon changed in the Glu354Lys mutant
shaded.
[0044] FIG. 36 shows the protein sequence comparison of the native
E. coli lpdA (SEQ ID NO:78) and the mutant K. pneumonia lpdA (SEQ
ID NO:79).
[0045] FIG. 37 shows 4-hydroxybutyrate (left bars) and BDO (right
bars) production in the strains AB3, MG1655 .DELTA.ldhA and
ECKh-138. All strains expressed E. coli sucCD, P. gingivalis sucD,
P. gingivalis 4hbd on the medium copy plasmid pZA33, and P.
gingivalis Cat2, C. acetobutylicum AdhE2 on the high copy plasmid
pZE13.
[0046] FIG. 38 shows the nucleotide sequence (SEQ ID NO:80) of the
5' end of the aceE gene fused to the pflB-p6 promoter and ribosome
binding site (RBS). The 5' italicized sequence shows the start of
the aroP gene, which is transcribed in the opposite direction from
the pdh operon. The 3' italicized sequence shows the start of the
aceE gene. In upper case: pflB RBS. Underlined: FNR binding site.
In bold: pflB-p6 promoter sequence.
[0047] FIG. 39 shows the nucleotide sequence (SEQ ID NO:81) in the
aceF-lpdA region in the strain ECKh-456.
[0048] FIG. 40 shows the production of 4-hydroxybutyrate, BDO and
pyruvate (left to right bars, respectively) for each of strains
ECKh-439, ECKh-455 and ECKh-456.
[0049] FIG. 41A shows a schematic of the recombination sites for
deletion of the mdh gene. FIG. 41B shows the sequence (nucleotide
sequence, SEQ ID NO: 82; amino acid sequence, SEQ ID NO: 83) of the
PCR product of the amplification of chloramphenicol resistance gene
(CAT) flanked by FRT sites and homology regions from the mdh gene
from the plasmid pKD3.
[0050] FIG. 42 shows the sequence (SEQ ID NO:84) of the arcA
deleted region in strain ECKh-401.
[0051] FIG. 43 shows the sequence (SEQ ID NO:85) of the region
encompassing a mutated gltA gene of strain ECKh-422.
[0052] FIG. 44 shows the citrate synthase activity of wild type
gltA gene product and the R163L mutant. The assay was performed in
the absence (diamonds) or presence of 0.4 mM NADH (squares).
[0053] FIG. 45 shows the 4-hydroxybutyrate (left bars) and BDO
(right bars) production in strains ECKh-401 and ECKh-422, both
expressing genes for the complete BDO pathway on plasmids.
[0054] FIG. 46 shows central metabolic fluxes and associated 95%
confidence intervals from metabolic labeling experiments. Values
are molar fluxes normalized to a glucose uptake rate of 1 mmol/hr.
The result indicates that carbon flux is routed through citrate
synthase in the oxidative direction and that most of the carbon
enters the BDO pathway rather than completing the TCA cycle.
[0055] FIG. 47 shows extracellular product formation for strains
ECKh-138 and ECKh-422, both expressing the entire BDO pathway on
plasmids. The products measured were acetate (Ace), pyruvate (Pyr),
4-hydroxybutyrate (4HB), 1,4-butanediol (BDO), ethanol (EtOH), and
other products, which include gamma-butyrolactone (GBL), succinate,
and lactate.
[0056] FIG. 48 shows the sequence (SEQ ID NO:86) of the region
following replacement of PEP carboxylase (ppc) by H. influenzae
phosphoenolpyruvate carboxykinase (pepck). The pepck coding region
is underlined.
[0057] FIG. 49 shows growth of evolved pepCK strains grown in
minimal medium containing 50 mM NaHCO.sub.3.
[0058] FIG. 50 shows product formation in strain ECKh-453
expressing P. gingivalis Cat2 and C. beijerinckii Ald on the
plasmid pZS*13. The products measured were 1,4-butanediol (BDO),
pyruvate, 4-hydroxybutyrate (4HB), acetate, .gamma.-butyrolactone
(GBL) and ethanol.
[0059] FIG. 51 shows BDO production of two strains, ECKh-453 and
ECKh-432. Both contain the plasmid pZS*13 expressing P. gingivalis
Cat2 and C. beijerinckii Ald. The cultures were grown under
microaerobic conditions, with the vessels punctured with 27 or 18
gauge needles, as indicated.
[0060] FIG. 52 shows the nucleotide sequence (SEQ ID NO:87) of the
genomic DNA of strain ECKh-426 in the region of insertion of a
polycistronic DNA fragment containing a promoter, sucCD gene, sucD
gene, 4hbd gene and a terminator sequence.
[0061] FIG. 53 shows the nucleotide sequence (SEQ ID NO:88) of the
chromosomal region of strain ECKh-432 in the region of insertion of
a polycistronic sequence containing a promoter, sucA gene, C.
kluyveri 4hbd gene and a terminator sequence.
[0062] FIG. 54 shows BDO synthesis from glucose in minimal medium
in the ECKh-432 strain having upstream BDO pathway encoding genes
intergrated into the chromosome and containing a plasmid harboring
downstream BDO pathway genes.
[0063] FIG. 55 shows a PCR product (SEQ ID NO:89) containing the
non-phosphotransferase (non-PTS) sucrose utilization genes flanked
by regions of homology to the rrnC region.
[0064] FIG. 56 shows a schematic diagram of the integrations site
in the rrnC operon.
[0065] FIG. 57 shows average product concentration, normalized to
culture OD600, after 48 hours of growth of strain ECKh-432 grown on
glucose and strain ECKh-463 grown on sucrose. Both contain the
plasmid pZS*13 expressing P. gingivalis Cat2 and C. beijerinckii
Ald. The data is for 6 replicate cultures of each strain. The
products measured were 1,4-butanediol (BDO), 4-hydroxybutyrate
(4HB), .gamma.-butyrolactone (GBL), pyruvate (PYR) and acetate
(ACE) (left to right bars, respectively).
[0066] FIG. 58 shows exemplary pathways to 1,4-butanediol from
succcinyl-CoA and alpha-ketoglutarate. Abbreviations: A)
Succinyl-CoA reductase (aldehyde forming), B) Alpha-ketoglutarate
decarboxylase, C) 4-Hydroxybutyrate dehydrogenase, D)
4-Hydroxybutyrate reductase, E) 1,4-Butanediol dehydrogenase.
[0067] FIG. 59A shows the nucleotide sequence (SEQ ID NO:90) of
carboxylic acid reductase from Nocardia iowensis (GNM.sub.--720),
and FIG. 59B shows the encoded amino acid sequence (SEQ ID
NO:91).
[0068] FIG. 60A shows the nucleotide sequence (SEQ ID NO:92) of
phosphpantetheine transferase, which was codon optimized, and FIG.
60B shows the encoded amino acid sequence (SEQ ID NO:93).
[0069] FIG. 61 shows a plasmid map of plasmid pZS*-13S-720
721opt.
[0070] FIGS. 62A and 62B show pathways to 1,4-butanediol from
succinate, succcinyl-CoA, and alpha-ketoglutarate. Abbreviations:
A) Succinyl-CoA reductase (aldehyde forming), B)
Alpha-ketoglutarate decarboxylase, C) 4-Hydroxybutyrate
dehydrogenase, D) 4-Hydroxybutyrate reductase, E) 1,4-Butanediol
dehydrogenase, F) Succinate reductase, G) Succinyl-CoA transferase,
H) Succinyl-CoA hydrolase, I) Succinyl-CoA synthetase (or
Succinyl-CoA ligase), J) Glutamate dehydrogenase, K) Glutamate
transaminase, L) Glutamate decarboxylase, M) 4-aminobutyrate
dehydrogenase, N) 4-aminobutyrate transaminase, O)
4-Hydroxybutyrate kinase, P) Phosphotrans-4-hydroxybutyrylase, Q)
4-Hydroxybutyryl-CoA reductase (aldehyde forming), R)
4-hydroxybutyryl-phosphate reductase, S) Succinyl-CoA reductase
(alcohol forming), T) 4-Hydroxybutyryl-CoA transferase, U)
4-Hydroxybutyryl-CoA hydrolase, V) 4-Hydroxybutyryl-CoA synthetase
(or 4-Hydroxybutyryl-CoA ligase), W) 4-Hydroxybutyryl-CoA reductase
(alcohol forming), X) Alpha-ketoglutarate reductase, Y)
5-Hydroxy-2-oxopentanoate dehydrogenase, Z)
5-Hydroxy-2-oxopentanoate decarboxylase, AA)
5-hydroxy-2-oxopentanoate dehydrogenase (decarboxylation).
[0071] FIG. 63 shows pathways to putrescine from succinate,
succcinyl-CoA, and alpha-ketoglutarate. Abbreviations: A)
Succinyl-CoA reductase (aldehyde forming), B) Alpha-ketoglutarate
decarboxylase, C) 4-Aminobutyrate reductase, D) Putrescine
dehydrogenase, E) Putrescine transaminase, F) Succinate reductase,
G) Succinyl-CoA transferase, H) Succinyl-CoA hydrolase, I)
Succinyl-CoA synthetase (or Succinyl-CoA ligase), J) Glutamate
dehydrogenase, K) Glutamate transaminase, L) Glutamate
decarboxylase, M) 4-Aminobutyrate dehydrogenase, N) 4-Aminobutyrate
transaminase, O) Alpha-ketoglutarate reductase, P)
5-Amino-2-oxopentanoate dehydrogenase, Q) 5-Amino-2-oxopentanoate
transaminase, R) 5-Amino-2-oxopentanoate decarboxylase, S)
Ornithine dehydrogenase, T) Ornithine transaminase, U) Ornithine
decarboxylase.
[0072] FIG. 64A shows the nucleotide sequence (SEQ ID NO:94) of
carboxylic acid reductase from Mycobacterium smegmatis mc(2)155
(designated 890), and FIG. 64B shows the encoded amino acid
sequence (SEQ ID NO:95).
[0073] FIG. 65A shows the nucleotide sequence (SEQ ID NO:96) of
carboxylic acid reductase from Mycobacterium avium subspecies
paratuberculosis K-10 (designated 891), and FIG. 65B shows the
encoded amino acid sequence (SEQ ID NO:97).
[0074] FIG. 66A shows the nucleotide sequence (SEQ ID NO:98) of
carboxylic acid reductase from Mycobacterium marinum M (designated
892), and FIG. 66B shows the encoded amino acid sequence (SEQ ID
NO:99).
[0075] FIG. 67A shows the nucleotide sequence (SEQ ID NO:100) of
carboxylic acid reductase designated 891GA, and FIG. 67B shows the
encoded amino acid sequence (SEQ ID NO:101).
[0076] FIG. 68 shows the reverse TCA cycle for fixation of CO.sub.2
on carbohydrates as substrates. The enzymatic transformations are
carried out by the enzymes as shown.
[0077] FIG. 69 shows the pathway for the reverse TCA cycle coupled
with carbon monoxide dehydrogenase and hydrogenase for the
conversion of syngas to acetyl-CoA.
[0078] FIG. 70 shows Western blots of 10 micrograms ACS90 (lane 1),
ACS91 (lane 2), 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).
[0079] FIG. 71 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.
[0080] FIGS. 72A and 72B show exemplary pathways to 1,4-butanediol.
FIG. 72A shows the pathways for fixation of CO2 to acetyl-CoA using
the reductive TCA cycle. FIG. 72B shows exemplary pathways for the
biosynthesis of 1,4-butanediol and 4-hydroxybutyrate from
acetyl-CoA; the enzymatic transformations shown are carried out by
the following enzymes: 1) Acetoacetyl-CoA thiolase (AtoB), 2)
3-Hydroxybutyryl-CoA dehydrogenase (Hbd), 3) Crotonase (Crt), 4)
Crotonyl-CoA hydratase (4-Budh), 5) 4-hydroxybutyryl-CoA reductase
(alcohol forming), 6) 4-hydroxybutyryl-CoA reductase (aldehyde
forming), 7) 1,4-butanediol dehydrogenase, 8) 4-Hydroxybutyryl-CoA
transferase, 4-Hydroxybutyryl-CoA synthetase, 4-Hydroxybutyryl-CoA
hydrolase, or Phosphotrans-4-hydroxybutyrylase/4-Hydroxybutyrate
kinase, and 9) 4-Hydroxybutyrate reductase.
[0081] FIGS. 73A and 73B show exemplary pathways to
4-hydroxybutyrate and gamma-butyrolactone. FIG. 73A shows the
pathways for fixation of CO2 to acetyl-CoA using the reductive TCA
cycle. FIG. 73B shows exemplary pathways for the biosynthesis of
gamma-butyrolactone and 4-hydroxybutyrate from acetyl-CoA; the
enzymatic transformations shown are carried out by the following
enzymes: 1) Acetoacetyl-CoA thiolase (AtoB), 2)
3-Hydroxybutyryl-CoA dehydrogenase (Hbd), 3) Crotonase (Crt), 4)
Crotonyl-CoA hydratase (4-Budh), 5) 4-Hydroxybutyryl-CoA
transferase, hydrolase or synthetase, 6)
Phosphotrans-4-hydroxybutyrylase, 7) 4-Hydroxybutyrate kinase, 8)
spontaneous or enzyme catalyzed, and 9) spontaneous or enzyme
catalyzed.
[0082] FIGS. 74A and 74B show exemplary pathways to 1,4-butanediol
and gamma-butyrolactone. FIG. 74A shows the pathways for fixation
of CO2 to alpha-ketoglutarate, succinate and succinyl-CoA using the
reductive TCA cycle. FIG. 74B shows exemplary pathways for the
biosynthesis of 1,4-butanediol, 4-hydroxybutyrate and
gamma-butyrolactone 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. 4-Hydroxybutyryl-CoA reductase (aldehyde forming), G.
1,4-butanediol dehydrogenase, H. Succinate reductase, I.
Succinyl-CoA reductase (alcohol forming), J. 4-Hydroxybutyryl-CoA
transferase, or 4-Hydroxybutyryl-CoA synthetase, K.
4-Hydroxybutyrate reductase, L. 4-Hydroxybutyryl-phosphate
reductase, M. 4-Hydroxybutyryl-CoA reductase (alcohol forming), N.
Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase
and/or Glutamate transaminase; Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase),
O. 4-Hydroxybutyryl-CoA hydrolase or spontaneous.
DETAILED DESCRIPTION OF THE INVENTION
[0083] The present invention is directed to the design and
production of cells and organisms having biosynthetic production
capabilities for 4-hydroxybutanoic acid (4-HB),
.gamma.-butyrolactone, 1,4-butanediol (BDO), 4-hydroxybutanal
(4-HBal), 4-hydroxybutyryl-CoA (4-HBCoA) and/or putrescine. The
invention, in particular, relates to the design of microbial
organisms capable of producing BDO, 4-HBal, 4-HBCoA and/or
putrescine by introducing one or more nucleic acids encoding a BDO,
4-HBal, 4-HBCoA and/or putrescine pathway enzyme.
[0084] In one embodiment, the invention utilizes in silico
stoichiometric models of Escherichia coli metabolism that identify
metabolic designs for biosynthetic production of 4-hydroxybutanoic
acid (4-HB), 1,4-butanediol (BDO), 4-HBal, 4-HBCoA and/or
putrescine. The results described herein indicate that metabolic
pathways can be designed and recombinantly engineered to achieve
the biosynthesis of 4-HBal, 4-HBCoA or 4-HB and downstream products
such as 1,4-butanediol or putrescine in Escherichia coli and other
cells or organisms. Biosynthetic production of 4-HB, 4-HBal,
4-HBCoA, BDO and/or putrescine, for example, for the in silico
designs can be confirmed by construction of strains having the
designed metabolic genotype. These metabolically engineered cells
or organisms also can be subjected to adaptive evolution to further
augment 4-HB, 4-HBal, 4-HBCoA, BDO and/or putrescine biosynthesis,
including under conditions approaching theoretical maximum
growth.
[0085] In certain embodiments, the 4-HB, 4-HBal, 4-HBCoA, BDO
and/or putrescine biosynthesis characteristics of the designed
strains make them genetically stable and particularly useful in
continuous bioprocesses. Separate strain design strategies were
identified with incorporation of different non-native or
heterologous reaction capabilities into E. coli or other host
organisms leading to 4-HB and 1,4-butanediol producing metabolic
pathways from either CoA-independent succinic semialdehyde
dehydrogenase or succinate reductase, succinyl-CoA synthetase and
CoA-dependent succinic semialdehyde dehydrogenase, or
glutamate:succinic semialdehyde transaminase. In silico metabolic
designs were identified that resulted in the biosynthesis of 4-HB
in both E. coli and yeast species from each of these metabolic
pathways. The 1,4-butanediol intermediate .gamma.-butyrolactone can
be generated in culture by spontaneous cyclization under conditions
at pH<7.5, particularly under acidic conditions, such as below
pH 5.5, for example, pH<7, pH<6.5, pH<6, and particularly
at pH<5.5 or lower.
[0086] Strains identified via the computational component of the
platform can be put into actual production by genetically
engineering any of the predicted metabolic alterations which lead
to the biosynthetic production of 4-HB, 1,4-butanediol or other
intermediate and/or downstream products. In yet a further
embodiment, strains exhibiting biosynthetic production of these
compounds can be further subjected to adaptive evolution to further
augment product biosynthesis. The levels of product biosynthesis
yield following adaptive evolution also can be predicted by the
computational component of the system.
[0087] In other specific embodiments, microbial organisms were
constructed to express a 4-HB biosynthetic pathway encoding the
enzymatic steps from succinate to 4-HB and to 4-HB-CoA.
Co-expression of succinate coenzyme A transferase, CoA-dependent
succinic semialdehyde dehydrogenase, NAD-dependent
4-hydroxybutyrate dehydrogenase and 4-hydroxybutyrate coenzyme A
transferase in a host microbial organism resulted in significant
production of 4-HB compared to host microbial organisms lacking a
4-HB biosynthetic pathway. In a further specific embodiment,
4-HB-producing microbial organisms were generated that utilized
.alpha.-ketoglutarate as a substrate by introducing nucleic acids
encoding .alpha.-ketoglutarate decarboxylase and NAD-dependent
4-hydroxybutyrate dehydrogenase.
[0088] In another specific embodiment, microbial organisms
containing a 1,4-butanediol (BDO) biosynthetic pathway were
constructed that biosynthesized BDO when cultured in the presence
of 4-HB. The BDO biosynthetic pathway consisted of a nucleic acid
encoding either a multifunctional aldehyde/alcohol dehydrogenase or
nucleic acids encoding an aldehyde dehydrogenawse and an alcohol
dehydrogenase. To support growth on 4-HB substrates, these
BDO-producing microbial organisms also expressed 4-hydroxybutyrate
CoA transferase or 4-butyrate kinase in conjunction with
phosphotranshydroxybutyrlase. In yet a further specific embodiment,
microbial organisms were generated that synthesized BDO through
exogenous expression of nucleic acids encoding a functional 4-HB
biosynthetic pathway and a functional BDO biosynthetic pathway. The
4-HB biosynthetic pathway consisted of succinate coenzyme A
transferase, CoA-dependent succinic semialdehyde dehydrogenase,
NAD-dependent 4-hydroxybutyrate dehydrogenase and 4-hydroxybutyrate
coenzyme A transferase. The BDO pathway consisted of a
multifunctional aldehyde/alcohol dehydrogenase. Further described
herein are additional pathways for production of BDO (see FIGS.
8-13).
[0089] In a further embodiment, described herein is the cloning and
expression of a carboxylic acid reductase enzyme that functions in
a 4-hydroxybutanal, 4-hydroxybutyryl-CoA or 1,4-butanediol
metabolic pathway. Advantages of employing a carboxylic acid
reductase as opposed to an acyl-CoA reductase to form
4-hydroxybutyraldehyde (4-hydroxybutanal) include lower ethanol and
GBL byproduct formation accompanying the production of BDO. Also
disclosed herein is the application of carboxylic acid reductase as
part of additional numerous pathways to produce 1,4-butanediol and
putrescine from the tricarboxylic acid (TCA) cycle metabolites, for
example, succinate, succinyl-CoA, and/or alpha-ketoglutarate.
[0090] 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
biosynthetic pathway for a 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine
family of compounds.
[0091] 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.
[0092] 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.
[0093] 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.
[0094] As used herein, the term "4-hydroxybutanoic acid" is
intended to mean a 4-hydroxy derivative of butyric acid having the
chemical formula C.sub.4H.sub.8O.sub.3 and a molecular mass of
104.11 g/mol (126.09 g/mol for its sodium salt). The chemical
compound 4-hydroxybutanoic acid also is known in the art as 4-HB,
4-hydroxybutyrate, gamma-hydroxybutyric acid or GHB. The term as it
is used herein is intended to include any of the compound's various
salt forms and include, for example, 4-hydroxybutanoate and
4-hydroxybutyrate. Specific examples of salt forms for 4-HB include
sodium 4-HB and potassium 4-HB. Therefore, the terms
4-hydroxybutanoic acid, 4-HB, 4-hydroxybutyrate,
4-hydroxybutanoate, gamma-hydroxybutyric acid and GHB as well as
other art recognized names are used synonymously herein.
[0095] As used herein, the term "monomeric" when used in reference
to 4-HB is intended to mean 4-HB in a non-polymeric or
underivatized form. Specific examples of polymeric 4-HB include
poly-4-hydroxybutanoic acid and copolymers of, for example, 4-HB
and 3-HB. A specific example of a derivatized form of 4-HB is
4-HB-CoA. Other polymeric 4-HB forms and other derivatized forms of
4-HB also are known in the art.
[0096] As used herein, the term ".gamma.-butyrolactone" is intended
to mean a lactone having the chemical formula C.sub.4H.sub.6O.sub.2
and a molecular mass of 86.089 g/mol. The chemical compound
.gamma.-butyrolactone also is know in the art as GBL,
butyrolactone, 1,4-lactone, 4-butyrolactone, 4-hydroxybutyric acid
lactone, and gamma-hydroxybutyric acid lactone. The term as it is
used herein is intended to include any of the compound's various
salt forms.
[0097] As used herein, the term "1,4-butanediol" is intended to
mean an alcohol derivative of the alkane butane, carrying two
hydroxyl groups which has the chemical formula
C.sub.4H.sub.10O.sub.2 and a molecular mass of 90.12 g/mol. The
chemical compound 1,4-butanediol also is known in the art as BDO
and is a chemical intermediate or precursor for a family of
compounds referred to herein as BDO family of compounds.
[0098] As used herein, the term "4-hydroxybutanal" is intended to
mean an aldehyde having the chemical formula C.sub.4H.sub.8O.sub.2
and a molecular mass of 88.10512 g/mol. The chemical compound
4-hydroxybutanal (4-HBal) is also known in the art as
4-hydroxybutyraldehyde.
[0099] As used herein, the term "putrescine" is intended to mean a
diamine having the chemical formula C.sub.4H.sub.12N.sub.2 and a
molecular mass of 88.15148 g/mol. The chemical compound putrescine
is also known in the art as 1,4-butanediamine, 1,4-diaminobutane,
butylenediamine, tetramethylenediamine, tetramethyldiamine, and
1,4-butylenediamine.
[0100] As used herein, the term "tetrahydrofuran" is intended to
mean a heterocyclic organic compound corresponding to the fully
hydrogenated analog of the aromatic compound furan which has the
chemical formula C.sub.4H.sub.8O and a molecular mass of 72.11
g/mol. The chemical compound tetrahydrofuran also is known in the
art as THF, tetrahydrofuran, 1,4-epoxybutane, butylene oxide,
cyclotetramethylene oxide, oxacyclopentane, diethylene oxide,
oxolane, furanidine, hydrofuran, tetra-methylene oxide. The term as
it is used herein is intended to include any of the compound's
various salt forms.
[0101] 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.
[0102] 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.
[0103] The non-naturally occurring microbial organisms of the
invention can contain stable genetic alterations, which refers to
microorganisms that can be cultured for greater than five
generations without loss of the alteration. Generally, stable
genetic alterations include modifications that persist greater than
10 generations, particularly stable modifications will persist more
than about 25 generations, and more particularly, stable genetic
modifications will be greater than 50 generations, including
indefinitely.
[0104] Those skilled in the art will understand that the genetic
alterations, including metabolic modifications exemplified herein
are described with reference to a suitable source or host organism
such as E. coli, yeast, or other organisms disclosed herein and
their corresponding metabolic reactions or a suitable source
organism for desired genetic material such as genes encoding
enzymes for their corresponding metabolic reactions 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.
[0105] 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.
[0106] 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, including growth-coupled 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.
[0107] 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.
[0108] 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.
[0109] Therefore, in identifying and constructing the non-naturally
occurring microbial organisms of the invention having 4-HB, GBL,
4-HBal, 4-HBCoA, BDO and/or putrescine 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.
[0110] 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.
[0111] 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.
[0112] Disclosed herein are non-naturally occurring microbial
biocatalyst or microbial organisms including a microbial organism
having a 4-hydroxybutanoic acid (4-HB) biosynthetic pathway that
includes at least one exogenous nucleic acid encoding
4-hydroxybutanoate dehydrogenase, CoA-independent succinic
semialdehyde dehydrogenase, succinyl-CoA synthetase, CoA-dependent
succinic semialdehyde dehydrogenase, glutamate:succinic
semialdehyde transaminase, alpha-ketoglutarate decarboxylase, or
glutamate decarboxylase, wherein the exogenous nucleic acid is
expressed in sufficient amounts to produce monomeric
4-hydroxybutanoic acid (4-HB). 4-hydroxybutanoate dehydrogenase is
also referred to as 4-hydroxybutyrate dehydrogenase or 4-HB
dehydrogenase. Succinyl-CoA synthetase is also referred to as
succinyl-CoA synthase or succinyl-CoA ligase.
[0113] Also disclosed herein is a non-naturally occurring microbial
biocatalyst or microbial organism including a microbial organism
having a 4-hydroxybutanoic acid (4-HB) biosynthetic pathway having
at least one exogenous nucleic acid encoding 4-hydroxybutanoate
dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic
semialdehyde dehydrogenase, or .alpha.-ketoglutarate decarboxylase,
wherein the exogenous nucleic acid is expressed in sufficient
amounts to produce monomeric 4-hydroxybutanoic acid (4-HB).
[0114] The non-naturally occurring microbial biocatalysts or
microbial organisms can include microbial organisms that employ
combinations of metabolic reactions for biosynthetically producing
the compounds of the invention. The biosynthesized compounds can be
produced intracellularly and/or secreted into the culture medium.
Exemplary compounds produced by the non-naturally occurring
microorganisms include, for example, 4-hydroxybutanoic acid,
1,4-butanediol and .gamma.-butyrolactone.
[0115] In one embodiment, a non-naturally occurring microbial
organism is engineered to produce 4-HB. This compound is one useful
entry point into the 1,4-butanediol family of compounds. The
biochemical reactions for formation of 4-HB from succinate, from
succinate through succinyl-CoA or from .alpha.-ketoglutarate are
shown in steps 1-8 of FIG. 1.
[0116] It is understood that any combination of appropriate enzymes
of a BDO, 4-HBal, 4-HBCoA and/or putrescine pathway can be used so
long as conversion from a starting component to the BDO, 4-HBal,
4-HBCoA and/or putrescine product is achieved. Thus, it is
understood that any of the metabolic pathways disclosed herein can
be utilized and that it is well understood to those skilled in the
art how to select appropriate enzymes to achieve a desired pathway,
as disclosed herein.
[0117] In another embodiment, disclosed herein is a non-naturally
occurring microbial organism, comprising a microbial organism
having a 1,4-butanediol (BDO) pathway comprising at least one
exogenous nucleic acid encoding a BDO pathway enzyme expressed in a
sufficient amount to produce BDO, the BDO pathway comprising
4-aminobutyrate CoA transferase, 4-aminobutyryl-CoA hydrolase,
4-aminobutyrate-CoA ligase, 4-aminobutyryl-CoA oxidoreductase
(deaminating), 4-aminobutyryl-CoA transaminase, or
4-hydroxybutyryl-CoA dehydrogenase (see Example VII Table 17). The
BDO pathway further can comprise 4-hydroxybutyryl-CoA reductase
(alcohol forming), 4-hydroxybutyryl-CoA reductase, or
1,4-butanediol dehydrogenase.
[0118] It is understood by those skilled in the art that various
combinations of the pathways can be utilized, as disclosed herein.
For example, in a non-naturally occurring microbial organism, the
nucleic acids can encode 4-aminobutyrate CoA transferase,
4-aminobutyryl-CoA hydrolase, or 4-aminobutyrate-CoA ligase;
4-aminobutyryl-CoA oxidoreductase (deaminating) or
4-aminobutyryl-CoA transaminase; and 4-hydroxybutyryl-CoA
dehydrogenase. Other exemplary combinations are specifically
describe below and further can be found in FIGS. 8-13. For example,
the BDO pathway can further comprise 4-hydroxybutyryl-CoA reductase
(alcohol forming), 4-hydroxybutyryl-CoA reductase, or
1,4-butanediol dehydrogenase.
[0119] Additionally disclosed herein is a non-naturally occurring
microbial organism, comprising a microbial organism having a BDO
pathway comprising at least one exogenous nucleic acid encoding a
BDO pathway enzyme expressed in a sufficient amount to produce BDO,
the BDO pathway comprising 4-aminobutyrate CoA transferase,
4-aminobutyryl-CoA hydrolase, 4-aminobutyrate-CoA ligase,
4-aminobutyryl-CoA reductase (alcohol forming), 4-aminobutyryl-CoA
reductase, 4-aminobutan-1-ol dehydrogenase, 4-aminobutan-1-ol
oxidoreductase (deaminating) or 4-aminobutan-1-ol transaminase (see
Example VII and Table 18), and can further comprise 1,4-butanediol
dehydrogenase. For example, the exogenous nucleic acids can encode
4-aminobutyrate CoA transferase, 4-aminobutyryl-CoA hydrolase, or
4-aminobutyrate-CoA ligase; 4-aminobutyryl-CoA reductase (alcohol
forming); and 4-aminobutan-1-ol oxidoreductase (deaminating) or
4-aminobutan-1-ol transaminase. In addition, the nucleic acids can
encode. 4-aminobutyrate CoA transferase, 4-aminobutyryl-CoA
hydrolase, or 4-aminobutyrate-CoA ligase; 4-aminobutyryl-CoA
reductase; 4-aminobutan-1-ol dehydrogenase; and 4-aminobutan-1-ol
oxidoreductase (deaminating) or 4-aminobutan-1-ol transaminase.
[0120] Also disclosed herein is a non-naturally occurring microbial
organism, comprising a microbial organism having a BDO pathway
comprising at least one exogenous nucleic acid encoding a BDO
pathway enzyme expressed in a sufficient amount to produce BDO, the
BDO pathway comprising 4-aminobutyrate kinase, 4-aminobutyraldehyde
dehydrogenase (phosphorylating), 4-aminobutan-1-ol dehydrogenase,
4-aminobutan-1-ol oxidoreductase (deaminating), 4-aminobutan-1-ol
transaminase, [(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase
(deaminating), [(4-aminobutanolyl)oxy]phosphonic acid transaminase,
4-hydroxybutyryl-phosphate dehydrogenase, or 4-hydroxybutyraldehyde
dehydrogenase (phosphorylating) (see Example VII and Table 19). For
example, the exogenous nucleic acids can encode 4-aminobutyrate
kinase; 4-aminobutyraldehyde dehydrogenase (phosphorylating);
4-aminobutan-1-ol dehydrogenase; and 4-aminobutan-1-ol
oxidoreductase (deaminating) or 4-aminobutan-1-ol transaminase.
Alternatively, the exogenous nucleic acids can encode
4-aminobutyrate kinase; [(4-aminobutanolyl)oxy]phosphonic acid
oxidoreductase (deaminating) or [(4-aminobutanolyl)oxy]phosphonic
acid transaminase; 4-hydroxybutyryl-phosphate dehydrogenase; and
4-hydroxybutyraldehyde dehydrogenase (phosphorylating).
[0121] Additionally disclosed herein is a non-naturally occurring
microbial organism, comprising a microbial organism having a BDO
pathway comprising at least one exogenous nucleic acid encoding a
BDO pathway enzyme expressed in a sufficient amount to produce BDO,
the BDO pathway comprising alpha-ketoglutarate 5-kinase,
2,5-dioxopentanoic semialdehyde dehydrogenase (phosphorylating),
2,5-dioxopentanoic acid reductase, alpha-ketoglutarate CoA
transferase, alpha-ketoglutaryl-CoA hydrolase,
alpha-ketoglutaryl-CoA ligase, alpha-ketoglutaryl-CoA reductase,
5-hydroxy-2-oxopentanoic acid dehydrogenase, alpha-ketoglutaryl-CoA
reductase (alcohol forming), 5-hydroxy-2-oxopentanoic acid
decarboxylase, or 5-hydroxy-2-oxopentanoic acid dehydrogenase
(decarboxylation) (see Example VIII and Table 20). The BDO pathway
can further comprise 4-hydroxybutyryl-CoA reductase (alcohol
forming), 4-hydroxybutyryl-CoA reductase, or 1,4-butanediol
dehydrogenase. For example, the exogenous nucleic acids can encode
alpha-ketoglutarate 5-kinase; 2,5-dioxopentanoic semialdehyde
dehydrogenase (phosphorylating); 2,5-dioxopentanoic acid reductase;
and 5-hydroxy-2-oxopentanoic acid decarboxylase. Alternatively, the
exogenous nucleic acids can encode alpha-ketoglutarate 5-kinase;
2,5-dioxopentanoic semialdehyde dehydrogenase (phosphorylating);
2,5-dioxopentanoic acid reductase; and 5-hydroxy-2-oxopentanoic
acid dehydrogenase (decarboxylation). Alternatively, the exogenous
nucleic acids can encode alpha-ketoglutarate CoA transferase,
alpha-ketoglutaryl-CoA hydrolase, or alpha-ketoglutaryl-CoA ligase;
alpha-ketoglutaryl-CoA reductase, 5-hydroxy-2-oxopentanoic acid
dehydrogenase; and 5-hydroxy-2-oxopentanoic acid decarboxylase. In
another embodiment, the exogenous nucleic acids can encode
alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA
hydrolase, or alpha-ketoglutaryl-CoA ligase; alpha-ketoglutaryl-CoA
reductase, 5-hydroxy-2-oxopentanoic acid dehydrogenase, and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation).
Alternatively, the exogenous nucleic acids can encode
alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA
hydrolase, or alpha-ketoglutaryl-CoA ligase; alpha-ketoglutaryl-CoA
reductase (alcohol forming); and 5-hydroxy-2-oxopentanoic acid
decarboxylase. In yet another embodiment, the exogenous nucleic
acids can encode alpha-ketoglutarate CoA transferase,
alpha-ketoglutaryl-CoA hydrolase, or alpha-ketoglutaryl-CoA ligase;
alpha-ketoglutaryl-CoA reductase (alcohol forming); and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation).
[0122] Further disclosed herein is a non-naturally occurring
microbial organism, comprising a microbial organism having a BDO
pathway comprising at least one exogenous nucleic acid encoding a
BDO pathway enzyme expressed in a sufficient amount to produce BDO,
the BDO pathway comprising glutamate CoA transferase, glutamyl-CoA
hydrolase, glutamyl-CoA ligase, glutamate 5-kinase,
glutamate-5-semialdehyde dehydrogenase (phosphorylating),
glutamyl-CoA reductase, glutamate-5-semialdehyde reductase,
glutamyl-CoA reductase (alcohol forming),
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating),
2-amino-5-hydroxypentanoic acid transaminase,
5-hydroxy-2-oxopentanoic acid decarboxylase,
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation) (see
Example IX and Table 21). For example, the exogenous nucleic acids
can encode glutamate CoA transferase, glutamyl-CoA hydrolase, or
glutamyl-CoA ligase; glutamyl-CoA reductase;
glutamate-5-semialdehyde reductase; 2-amino-5-hydroxypentanoic acid
oxidoreductase (deaminating) or 2-amino-5-hydroxypentanoic acid
transaminase; and 5-hydroxy-2-oxopentanoic acid decarboxylase or
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation).
Alternatively, the exogenous nucleic acids can encode glutamate
5-kinase; glutamate-5-semialdehyde dehydrogenase (phosphorylating);
glutamate-5-semialdehyde reductase; 2-amino-5-hydroxypentanoic acid
oxidoreductase (deaminating) or 2-amino-5-hydroxypentanoic acid
transaminase; and 5-hydroxy-2-oxopentanoic acid decarboxylase or
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation). In
still another embodiment, the exogenous nucleic acids can encode
glutamate CoA transferase, glutamyl-CoA hydrolase, or glutamyl-CoA
ligase; glutamyl-CoA reductase (alcohol forming);
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or
2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation). In
yet another embodiment, the exogenous nucleic acids can encode
glutamate 5-kinase; glutamate-5-semialdehyde dehydrogenase
(phosphorylating); 2-amino-5-hydroxypentanoic acid oxidoreductase
(deaminating) or 2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation).
[0123] Also disclosed herein is a non-naturally occurring microbial
organism, comprising a microbial organism having a BDO pathway
comprising at least one exogenous nucleic acid encoding a BDO
pathway enzyme expressed in a sufficient amount to produce BDO, the
BDO pathway comprising 3-hydroxybutyryl-CoA dehydrogenase,
3-hydroxybutyryl-CoA dehydratase, vinylacetyl-CoA
.DELTA.-isomerase, or 4-hydroxybutyryl-CoA dehydratase (see Example
X and Table 22). For example, the exogenous nucleic acids can
encode 3-hydroxybutyryl-CoA dehydrogenase; 3-hydroxybutyryl-CoA
dehydratase; vinylacetyl-CoA A-isomerase; and 4-hydroxybutyryl-CoA
dehydratase.
[0124] Further disclosed herein is a non-naturally occurring
microbial organism, comprising a microbial organism having a BDO
pathway comprising at least one exogenous nucleic acid encoding a
BDO pathway enzyme expressed in a sufficient amount to produce BDO,
the BDO pathway comprising homoserine deaminase, homoserine CoA
transferase, homoserine-CoA hydrolase, homoserine-CoA ligase,
homoserine-CoA deaminase, 4-hydroxybut-2-enoyl-CoA transferase,
4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA
ligase, 4-hydroxybut-2-enoate reductase, 4-hydroxybutyryl-CoA
transferase, 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA
ligase, or 4-hydroxybut-2-enoyl-CoA reductase (see Example XI and
Table 23). For example, the exogenous nucleic acids can encode
homoserine deaminase; 4-hydroxybut-2-enoyl-CoA transferase,
4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA
ligase; 4-hydroxybut-2-enoyl-CoA reductase. Alternatively, the
exogenous nucleic acids can encode homoserine CoA transferase,
homoserine-CoA hydrolase, or homoserine-CoA ligase; homoserine-CoA
deaminase; and 4-hydroxybut-2-enoyl-CoA reductase. In a further
embodiment, the exogenous nucleic acids can encode homoserine
deaminase; 4-hydroxybut-2-enoate reductase; and
4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA hydrolase,
or 4-hydroxybutyryl-CoA ligase. Alternatively, the exogenous
nucleic acids can encode homoserine CoA transferase, homoserine-CoA
hydrolase, or homoserine-CoA ligase; homoserine-CoA deaminase; and
4-hydroxybut-2-enoyl-CoA reductase.
[0125] Further disclosed herein is a non-naturally occurring
microbial organism, comprising a microbial organism having a BDO
pathway comprising at least one exogenous nucleic acid encoding a
BDO pathway enzyme expressed in a sufficient amount to produce BDO,
the BDO pathway comprising succinyl-CoA reductase (alcohol
forming), 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA
ligase, 4-hydroxybutanal dehydrogenase (phosphorylating) (see Table
15). Such a BDO pathway can further comprise succinyl-CoA
reductase, 4-hydroxybutyrate dehydrogenase, 4-hydroxybutyryl-CoA
transferase, 4-hydroxybutyrate kinase,
phosphotrans-4-hydroxybutyrylase, 4-hydroxybutyryl-CoA reductase,
4-hydroxybutyryl-CoA reductase (alcohol forming), or 1,4-butanediol
dehydrogenase.
[0126] Additionally disclosed herein is a non-naturally occurring
microbial organism, comprising a microbial organism having a BDO
pathway comprising at least one exogenous nucleic acid encoding a
BDO pathway enzyme expressed in a sufficient amount to produce BDO,
the BDO pathway comprising glutamate dehydrogenase, glutamate
transaminase, 4-aminobutyrate oxidoreductase (deaminating),
4-aminobutyrate transaminase, glutamate decarboxylase,
4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase,
4-hydroxybutanal dehydrogenase (phosphorylating)(see Table 16).
Such a BDO pathway can further comprise alpha-ketoglutarate
decarboxylase, 4-hydroxybutyrate dehydrogenase,
4-hydroxybutyryl-CoA transferase, 4-hydroxybutyrate kinase,
phosphotrans-4-hydroxybutyrylase, 4-hydroxybutyryl-CoA reductase,
4-hydroxybutyryl-CoA reductase (alcohol forming), or 1,4-butanediol
dehydrogenase.
[0127] The pathways described above are merely exemplary. One
skilled in the art can readily select appropriate pathways from
those disclosed herein to obtain a suitable BDO pathway or other
metabolic pathway, as desired.
[0128] The invention provides genetically modified organisms that
allow improved production of a desired product such as BDO by
increasing the product or decreasing undesirable byproducts. As
disclosed herein, the invention provides a non-naturally occurring
microbial organism, comprising a microbial organism having a
1,4-butanediol (BDO) pathway comprising at least one exogenous
nucleic acid encoding a BDO pathway enzyme expressed in a
sufficient amount to produce BDO. In one embodiment, the microbial
organism is genetically modified to express exogenous succinyl-CoA
synthetase (see Example XII). For example, the succinyl-CoA
synthetase can be encoded by an Escherichia coli sucCD genes.
[0129] In another embodiment, the microbial organism is genetically
modified to express exogenous alpha-ketoglutarate decarboxylase
(see Example XIII). For example, the alpha-ketoglutarate
decarboxylase can be encoded by the Mycobacterium bovis sucA gene.
In still another embodiment, the microbial organism is genetically
modified to express exogenous succinate semialdehyde dehydrogenase
and 4-hydroxybutyrate dehydrogenase and optionally
4-hydroxybutyryl-CoA/acetyl-CoA transferase (see Example XIII). For
example, the succinate semialdehyde dehydrogenase (CoA-dependent),
4-hydroxybutyrate dehydrogenase and 4-hydroxybutyryl-CoA/acetyl-CoA
transferase can be encoded by Porphyromonas gingivalis W83 genes.
In an additional embodiment, the microbial organism is genetically
modified to express exogenous butyrate kinase and
phosphotransbutyrylase (see Example XIII). For example, the
butyrate kinase and phosphotransbutyrylase can be encoded by
Clostridium acetobutilicum buk1 and ptb genes.
[0130] In yet another embodiment, the microbial organism is
genetically modified to express exogenous 4-hydroxybutyryl-CoA
reductase (see Example XIII). For example, the 4-hydroxybutyryl-CoA
reductase can be encoded by Clostridium beijerinckii ald gene.
Additionally, in an embodiment of the invention, the microbial
organism is genetically modified to express exogenous
4-hydroxybutanal reductase (see Example XIII). For example, the
4-hydroxybutanal reductase can be encoded by Geobacillus
thermoglucosidasius adh1 gene. In another embodiment, the microbial
organism is genetically modified to express exogenous pyruvate
dehydrogenase subunits (see Example XIV). For example, the
exogenous pyruvate dehydrogenase can be NADH insensitive. The
pyruvate dehydrogenase subunit can be encoded by the Klebsiella
pneumonia lpdA gene. In a particular embodiment, the pyruvate
dehydrogenase subunit genes of the microbial organism can be under
the control of a pyruvate formate lyase promoter.
[0131] In still another embodiment, the microbial organism is
genetically modified to disrupt a gene encoding an aerobic
respiratory control regulatory system (see Example XV). For
example, the disruption can be of the arcA gene. Such an organism
can further comprise disruption of a gene encoding malate
dehydrogenase. In a further embodiment, the microbial organism is
genetically modified to express an exogenous NADH insensitive
citrate synthase (see Example XV). For example, the NADH
insensitive citrate synthase can be encoded by gltA, such as an
R163L mutant of gltA. In still another embodiment, the microbial
organism is genetically modified to express exogenous
phosphoenolpyruvate carboxykinase (see Example XVI). For example,
the phosphoenolpyruvate carboxykinase can be encoded by an
Haemophilus influenza phosphoenolpyruvate carboxykinase gene.
[0132] It is understood that any of a number of genetic
modifications, as disclosed herein, can be used alone or in various
combinations of one or more of the genetic modifications disclosed
herein to increase the production of BDO in a BDO producing
microbial organism. In a particular embodiment, the microbial
organism can be genetically modified to incorporate any and up to
all of the genetic modifications that lead to increased production
of BDO. In a particular embodiment, the microbial organism
containing a BDO pathway can be genetically modified to express
exogenous succinyl-CoA synthetase; to express exogenous
alpha-ketoglutarate decarboxylase; to express exogenous succinate
semialdehyde dehydrogenase and 4-hydroxybutyrate dehydrogenase and
optionally 4-hydroxybutyryl-CoA/acetyl-CoA transferase; to express
exogenous butyrate kinase and phosphotransbutyrylase; to express
exogenous 4-hydroxybutyryl-CoA reductase; and to express exogenous
4-hydroxybutanal reductase; to express exogenous pyruvate
dehydrogenase; to disrupt a gene encoding an aerobic respiratory
control regulatory system; to express an exogenous NADH insensitive
citrate synthase; and to express exogenous phosphoenolpyruvate
carboxykinase. Such strains for improved production are described
in Examples XII-XIX. It is thus understood that, in addition to the
modifications described above, such strains can additionally
include other modifications disclosed herein. Such modifications
include, but are not limited to, deletion of endogenous lactate
dehydrogenase (ldhA), alcohol dehydrogenase (adhE), and/or pyruvate
formate lyase (pflB)(see Examples XII-XIX and Table 28).
[0133] Additionally provided is a microbial organism in which one
or more genes encoding the exogenously expressed enzymes are
integrated into the fimD locus of the host organism (see Example
XVII). For example, one or more genes encoding a BDO pathway enzyme
can be integrated into the fimD locus for increased production of
BDO. Further provided is a microbial organism expressing a
non-phosphotransferase sucrose uptake system that increases
production of BDO.
[0134] Although the genetically modified microbial organisms
disclosed herein are exemplified with microbial organisms
containing particular BDO pathway enzymes, it is understood that
such modifications can be incorporated into any microbial organism
having a BDO, 4-HBal, 4-HBCoA and/or putrescine pathway suitable
for enhanced production in the presence of the genetic
modifications. The microbial organisms of the invention can thus
have any of the BDO, 4-HBal, 4-HBCoA and/or putrescine pathways
disclosed herein. For example, the BDO pathway can comprise
4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase,
CoA-dependent succinic semialdehyde dehydrogenase,
4-hydroxybutyrate:CoA transferase, 4-butyrate kinase,
phosphotransbutyrylase, alpha-ketoglutarate decarboxylase, aldehyde
dehydrogenase, alcohol dehydrogenase or an aldehyde/alcohol
dehydrogenase (see FIG. 1). Alternatively, the BDO pathway can
comprise 4-aminobutyrate CoA transferase, 4-aminobutyryl-CoA
hydrolase, 4-aminobutyrate-CoA ligase, 4-aminobutyryl-CoA
oxidoreductase (deaminating), 4-aminobutyryl-CoA transaminase, or
4-hydroxybutyryl-CoA dehydrogenase (see Table 17). Such a BDO
pathway can further comprise 4-hydroxybutyryl-CoA reductase
(alcohol forming), 4-hydroxybutyryl-CoA reductase, or
1,4-butanediol dehydrogenase.
[0135] Additionally, the BDO pathway can comprise 4-aminobutyrate
CoA transferase, 4-aminobutyryl-CoA hydrolase, 4-aminobutyrate-CoA
ligase, 4-aminobutyryl-CoA reductase (alcohol forming),
4-aminobutyryl-CoA reductase, 4-aminobutan-1-ol dehydrogenase,
4-aminobutan-1-ol oxidoreductase (deaminating) or 4-aminobutan-1-ol
transaminase (see Table 18). Also, the BDO pathway can comprise
4-aminobutyrate kinase, 4-aminobutyraldehyde dehydrogenase
(phosphorylating), 4-aminobutan-1-ol dehydrogenase,
4-aminobutan-1-ol oxidoreductase (deaminating), 4-aminobutan-1-ol
transaminase, [(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase
(deaminating), [(4-aminobutanolyl)oxy]phosphonic acid transaminase,
4-hydroxybutyryl-phosphate dehydrogenase, or 4-hydroxybutyraldehyde
dehydrogenase (phosphorylating) (see Table 19). Such a pathway can
further comprise 1,4-butanediol dehydrogenase.
[0136] The BDO pathway can also comprise alpha-ketoglutarate
5-kinase, 2,5-dioxopentanoic semialdehyde dehydrogenase
(phosphorylating), 2,5-dioxopentanoic acid reductase,
alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA
hydrolase, alpha-ketoglutaryl-CoA ligase, alpha-ketoglutaryl-CoA
reductase, 5-hydroxy-2-oxopentanoic acid dehydrogenase,
alpha-ketoglutaryl-CoA reductase (alcohol forming),
5-hydroxy-2-oxopentanoic acid decarboxylase, or
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation)(see
Table 20). Such a BDO pathway can further comprise
4-hydroxybutyryl-CoA reductase (alcohol forming),
4-hydroxybutyryl-CoA reductase, or 1,4-butanediol dehydrogenase.
Additionally, the BDO pathway can comprise glutamate CoA
transferase, glutamyl-CoA hydrolase, glutamyl-CoA ligase, glutamate
5-kinase, glutamate-5-semialdehyde dehydrogenase (phosphorylating),
glutamyl-CoA reductase, glutamate-5-semialdehyde reductase,
glutamyl-CoA reductase (alcohol forming),
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating),
2-amino-5-hydroxypentanoic acid transaminase,
5-hydroxy-2-oxopentanoic acid decarboxylase,
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation)(see
Table 21). Such a BDO pathway can further comprise
4-hydroxybutyryl-CoA reductase (alcohol forming),
4-hydroxybutyryl-CoA reductase, or 1,4-butanediol
dehydrogenase.
[0137] Additionally, the BDO pathway can comprise
3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA
dehydratase, vinylacetyl-CoA A-isomerase, or 4-hydroxybutyryl-CoA
dehydratase (see Table 22). Also, the BDO pathway can comprise
homoserine deaminase, homoserine CoA transferase, homoserine-CoA
hydrolase, homoserine-CoA ligase, homoserine-CoA deaminase,
4-hydroxybut-2-enoyl-CoA transferase, 4-hydroxybut-2-enoyl-CoA
hydrolase, 4-hydroxybut-2-enoyl-CoA ligase, 4-hydroxybut-2-enoate
reductase, 4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA
hydrolase, 4-hydroxybutyryl-CoA ligase, or 4-hydroxybut-2-enoyl-CoA
reductase (see Table 23). Such a BDO pathway can further comprise
4-hydroxybutyryl-CoA reductase (alcohol forming),
4-hydroxybutyryl-CoA reductase, or 1,4-butanediol
dehydrogenase.
[0138] The BDO pathway can additionally comprise succinyl-CoA
reductase (alcohol forming), 4-hydroxybutyryl-CoA hydrolase,
4-hydroxybutyryl-CoA ligase, or 4-hydroxybutanal dehydrogenase
(phosphorylating) (see Table 15). Such a pathway can further
comprise succinyl-CoA reductase, 4-hydroxybutyrate dehydrogenase,
4-hydroxybutyryl-CoA transferase, 4-hydroxybutyrate kinase,
phosphotrans-4-hydroxybutyrylase, 4-hydroxybutyryl-CoA reductase,
4-hydroxybutyryl-CoA reductase (alcohol forming), or 1,4-butanediol
dehydrogenase. Also, the BDO pathway can comprise glutamate
dehydrogenase, 4-aminobutyrate oxidoreductase (deaminating),
4-aminobutyrate transaminase, glutamate decarboxylase,
4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or
4-hydroxybutanal dehydrogenase (phosphorylating) (see Table 16).
Such a BDO pathway can further comprise alpha-ketoglutarate
decarboxylase, 4-hydroxybutyrate dehydrogenase,
4-hydroxybutyryl-CoA transferase, 4-hydroxybutyrate kinase,
phosphotrans-4-hydroxybutyrylase, 4-hydroxybutyryl-CoA reductase,
4-hydroxybutyryl-CoA reductase (alcohol forming), or 1,4-butanediol
dehydrogenase.
[0139] The invention additionally provides a non-naturally
occurring microbial organism, comprising a 4-hydroxybutanal pathway
comprising at least one exogenous nucleic acid encoding a
4-hydroxybutanal pathway enzyme expressed in a sufficient amount to
produce 4-hydroxybutanal, the 4-hydroxybutanal pathway comprising
succinyl-CoA reductase (aldehyde forming); 4-hydroxybutyrate
dehydrogenase; and 4-hydroxybutyrate reductase (see FIG. 58, steps
A-C-D). The invention also provides a non-naturally occurring
microbial organism, comprising a 4-hydroxybutanal pathway
comprising at least one exogenous nucleic acid encoding a
4-hydroxybutanal pathway enzyme expressed in a sufficient amount to
produce 4-hydroxybutanal, the 4-hydroxybutanal pathway comprising
alpha-ketoglutarate decarboxylase; 4-hydroxybutyrate dehydrogenase;
and 4-hydroxybutyrate reductase (FIG. 58, steps B-C-D).
[0140] The invention further provides a non-naturally occurring
microbial organism, comprising a 4-hydroxybutanal pathway
comprising at least one exogenous nucleic acid encoding a
4-hydroxybutanal pathway enzyme expressed in a sufficient amount to
produce 4-hydroxybutanal, the 4-hydroxybutanal pathway comprising
succinate reductase; 4-hydroxybutyrate dehydrogenase, and
4-hydroxybutyrate reductase (see FIG. 62, steps F-C-D). In yet
another embodiment, the invention provides a non-naturally
occurring microbial organism, comprising a 4-hydroxybutanal pathway
comprising at least one exogenous nucleic acid encoding a
4-hydroxybutanal pathway enzyme expressed in a sufficient amount to
produce 4-hydroxybutanal, the 4-hydroxybutanal pathway comprising
alpha-ketoglutarate decarboxylase, or glutamate dehydrogenase or
glutamate transaminase and glutamate decarboxylase and
4-aminobutyrate dehydrogenase or 4-aminobutyrate transaminase;
4-hydroxybutyrate dehydrogenase; and 4-hydroxybutyrate reductase
(see FIG. 62, steps B or ((J or K)-L-(M or N))-C-D).
[0141] The invention also provides a non-naturally occurring
microbial organism, comprising a 4-hydroxybutanal pathway
comprising at least one exogenous nucleic acid encoding a
4-hydroxybutanal pathway enzyme expressed in a sufficient amount to
produce 4-hydroxybutanal, the 4-hydroxybutanal pathway comprising
alpha-ketoglutarate reductase; 5-hydroxy-2-oxopentanoate
dehydrogenase; and 5-hydroxy-2-oxopentanoate decarboxylase (see
FIG. 62, steps X-Y-Z). In yet another embodiment, the invention
provides a non-naturally occurring microbial organism, comprising a
4-hydroxybutyryl-CoA pathway comprising at least one exogenous
nucleic acid encoding a 4-hydroxybutyryl-CoA pathway enzyme
expressed in a sufficient amount to produce 4-hydroxybutyryl-CoA,
the 4-hydroxybutyryl-CoA pathway comprising alpha-ketoglutarate
reductase; 5-hydroxy-2-oxopentanoate dehydrogenase; and
5-hydroxy-2-oxopentanoate dehydrogenase (decarboxylation) (see FIG.
62, steps X-Y-AA).
[0142] The invention additionally provides a non-naturally
occurring microbial organism, comprising a putrescine pathway
comprising at least one exogenous nucleic acid encoding a
putrescine pathway enzyme expressed in a sufficient amount to
produce putrescine, the putrescine pathway comprising succinate
reductase; 4-aminobutyrate dehydrogenase or 4-aminobutyrate
transaminase; 4-aminobutyrate reductase; and putrescine
dehydrogenase or putrescine transaminase (see FIG. 63, steps
F-M/N-C-D/E). In still another embodiment, the invention provides a
non-naturally occurring microbial organism, comprising a putrescine
pathway comprising at least one exogenous nucleic acid encoding a
putrescine pathway enzyme expressed in a sufficient amount to
produce putrescine, the putrescine pathway comprising
alpha-ketoglutarate decarboxylase; 4-aminobutyrate dehydrogenase or
4-aminobutyrate transaminase; 4-aminobutyrate reductase; and
putrescine dehydrogenase or putrescine transaminase (see FIG. 63,
steps B-M/N-C-D/E). The invention additionally provides a
non-naturally occurring microbial organism, comprising a putrescine
pathway comprising at least one exogenous nucleic acid encoding a
putrescine pathway enzyme expressed in a sufficient amount to
produce putrescine, the putrescine pathway comprising glutamate
dehydrogenase or glutamate transaminase; glutamate decarboxylase;
4-aminobutyrate reductase; and putrescine dehydrogenase or
putrescine transaminase (see FIG. 63, steps J/K-L-C-D/E).
[0143] The invention provides in another embodiment a non-naturally
occurring microbial organism, comprising a putrescine pathway
comprising at least one exogenous nucleic acid encoding a
putrescine pathway enzyme expressed in a sufficient amount to
produce putrescine, the putrescine pathway comprising
alpha-ketoglutarate reductase; 5-amino-2-oxopentanoate
dehydrogenase or 5-amino-2-oxopentanoate transaminase;
5-amino-2-oxopentanoate decarboxylase; and putrescine dehydrogenase
or putrescine transaminase (see FIG. 63, steps O-P/Q-R-D/E). Also
provided is a non-naturally occurring microbial organism,
comprising a putrescine pathway comprising at least one exogenous
nucleic acid encoding a putrescine pathway enzyme expressed in a
sufficient amount to produce putrescine, the putrescine pathway
comprising alpha-ketoglutarate reductase; 5-amino-2-oxopentanoate
dehydrogenase or 5-amino-2-oxopentanoate transaminase; ornithine
dehydrogenase or ornithine transaminase; and ornithine
decarboxylase (see FIG. 63, steps O-P/Q-S/T-U).
[0144] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having a 4-HB, 4-HBal,
4-HBCoA, BDO or putrescine pathway, wherein the non-naturally
occurring microbial organism comprises at least one exogenous
nucleic acid encoding an enzyme or protein that converts a
substrate of any of the pathways disclosed herein (see, for
example, the Examples and FIGS. 1, 8-13, 58, 62, 63 and 72-74). In
an exemplary embodiment for producing BDO, the microbial organism
can convert a substrate to a product selected from the group
consisting of succinate to succinyl-CoA; succinyl-CoA to succinic
semialdehyde; succinic semialdehyde to 4-hydroxybutrate;
4-hydroxybutyrate to 4-hydroxybutyryl-phosphate;
4-hydroxybutyryl-phosphate to 4-hydroxtbutyryl-CoA;
4-hydroxybutyryl-CoA to 4-hydroxybutanal; and 4-hydroxybutanal to
1,4-butanediol. In a pathway for producing 4-HBal, a microbial
organism can convert, for example, succinate to succinic
semialdehyde; succinic semialdehyde to 4-hydroxybutyrate; and
4-hydroxybutyrate to 4-hydroxybutanal. Such an organism can
additionally include activity to convert 4-hydroxybutanal to
1,4-butanediol in order to produce BDO. Yet another pathway for
producing 4-HBal can be, for example, alpha-ketoglutarate to
succinic semialdehyde; succinic semialdehyde to 4-hydroxybutyrate;
and 4-hydroxybutyrate to 4-hydroxybutanal. An alternative pathway
for producing 4-HBal can be, for example, alpha-ketoglutarate to
2,5-dioxopentanoic acid; 2,5-dioxopentanoic acid to
5-hydroxy-2-oxopentanooic acid; and 5-hydroxy-2-oxopentanoic acid
to 4-hydroxybutanal. An exemplary 4-hydroxybutyryl-CoA pathway can
be, for example, alpha-ketoglutarate to 2,5-dioxopentanoic acid;
2,5-dioxopentanoic acid to 5-hydroxy-2-oxopentanoic acid; and
5-hydroxy-2-oxopentanoic acid to 4-hydroxybutyryl-CoA. An exemplary
putrescine pathway can be, for example, succinate to succinyl-CoA;
succinyl-CoA to succinic semialdehyde; succinic semialdehyde to
4-aminobutyrate; 4-aminobutyrate to 4-aminobutanal; and
4-aminobutanal to putrescine. An alternative putrescine pathway can
be, for example, succinate to succinic semialdehyde; succinic
semialdehyde to 4-aminobutyrate; 4-aminobutyrate to 4-aminobutanal;
and 4-aminobutanal to putrescine. 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 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 pathway
(see FIGS. 1, 8-13, 58, 62, 63 and 72-74).
[0145] While generally described herein as a microbial organism
that contains a 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine pathway,
it is understood that the invention additionally provides a
non-naturally occurring microbial organism comprising at least one
exogenous nucleic acid encoding a 4-HB, 4-HBal, 4-HBCoA, BDO or
putrescine pathway enzyme or protein expressed in a sufficient
amount to produce an intermediate of a 4-HB, 4-HBal, 4-HBCoA, BDO
or putrescine pathway. For example, as disclosed herein, 4-HB,
4-HBal, 4-HBCoA, BDO and putrescine pathways are exemplified in
FIGS. 1, 8-13, 58, 62, 63 and 72-74). Therefore, in addition to a
microbial organism containing, for example, a BDO pathway that
produces BDO, the invention additionally provides a non-naturally
occurring microbial organism comprising at least one exogenous
nucleic acid encoding a BDO pathway enzyme, where the microbial
organism produces a BDO pathway intermediate as a product rather
than an intermediate of the pathway. In one exemplary embodiment as
shown in FIG. 62, for example, the invention provides a microbial
organism that produces succinyl-CoA, succinic semialdehyde,
4-hydroxybutyrate, 4-hydroxybutyryl-phosphate,
4-hydroxybutyryl-CoA, or 4-hydroxybutanal as a product rather than
an intermediate. Another exemplary embodiment includes, for
example, a microbial organism that produces alpha-ketoglutarate,
2,5-dioxopentanoic acid, 5-hydroxy-2-oxopentanoic acid, or
4-hydroxybutanal as a product rather than an intermediate. An
exemplary embodiment in a putrescine pathway includes, for example,
a microbial organism that produces glutamate, 4-aminobutyrate, or
4-aminobutanal as a product rather than an intermediate. An
alternative embodiment in a putrescine pathway can be, for example,
a microbial organism that produces 2,5-dioxopentanoate,
5-amino-2-oxopentanoate, or ornithine as a product rather than an
intermediate.
[0146] It is understood that any of the pathways disclosed herein,
as described in the Examples and exemplified in the Figures,
including the pathways of FIGS. 1, 8-13, 58, 62, 63 and 72-74), 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 4-HB, 4-HBal, 4-HBCoA,
BDO or putrescine pathway intermediate can be utilized to produce
the intermediate as a desired product.
[0147] This invention is also directed, in part to engineered
biosynthetic pathways to improve carbon flux through a central
metabolism intermediate en route to 1,4-butanediol,
4-hydroxybutyrate and/or gamma-butyrolactone or other products or
intermediates disclosed herein. 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 1,4-butanediol,
4-hydroxybutyrate and/or gamma-butyrolactone or other products or
intermediates disclosed herein. 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.
[0148] 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
1,4-butanediol, 4-hydroxybutyrate and/or gamma-butyrolactone 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, CO2, and/or H2. In
addition to syngas, other sources of such gases include, but are
not limited to, the atmosphere, either as found in nature or
generated.
[0149] The CO2-fixing reductive tricarboxylic acid (RTCA) cycle is
an endergenic anabolic pathway of CO2 assimilation which uses
reducing equivalents and ATP (FIG. 68). One turn of the RTCA cycle
assimilates two moles of CO2 into one mole of acetyl-CoA, or four
moles of CO2 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.
[0150] In some embodiments, the reductive TCA cycle, coupled with
carbon monoxide dehydrogenase and/or hydrogenase enzymes, can be
employed to allow syngas, CO2, CO, H2, and/or other gaseous carbon
source utilization by microorganisms. Synthesis gas (syngas), in
particular is a mixture of primarily H2 and CO, sometimes including
some amounts of CO2, 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 H2/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 CO2. 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.
[0151] The components of synthesis gas and/or other carbon sources
can provide sufficient CO2, reducing equivalents, and ATP for the
reductive TCA cycle to operate. One turn of the RTCA cycle
assimilates two moles of CO2 into one mole of acetyl-CoA and
requires 2 ATP and 4 reducing equivalents. CO and/or H2 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, reduced flavodoxins and reduced thioredoxins. 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.
[0152] 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)).
[0153] The key carbon-fixing enzymes of the reductive TCA cycle are
alpha-ketoglutarate:ferredoxin oxidoreductase, pyruvate:ferredoxin
oxidoreductase and isocitrate dehydrogenase. Additional carbon may
be fixed during the conversion of phosphoenolpyruvate to
oxaloacetate by phosphoenolpyruvate carboxylase or
phosphoenolpyruvate carboxykinase or by conversion of pyruvate to
malate by malic enzyme.
[0154] 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)+ dependent
decarboxylation of alpha-ketoglutarate by the alpha-ketoglutarate
dehydrogenase complex. The reverse reaction is catalyzed by
alpha-ketoglutarate:ferredoxin oxidoreductase.
[0155] An organism capable of utilizing the reverse tricarboxylic
acid cycle to enable production of acetyl-CoA-derived products on
1) CO, 2) CO.sub.2 and H.sub.2, 3) CO and CO.sub.2, 4) synthesis
gas comprising CO and H.sub.2, and 5) synthesis gas or other
gaseous carbon sources comprising CO, CO.sub.2, and H.sub.2 can
include any of the following enzyme activities: ATP-citrate lyase,
citrate lyase, aconitase, isocitrate dehydrogenase,
alpha-ketoglutarate:ferredoxin oxidoreductase, succinyl-CoA
synthetase, succinyl-CoA transferase, fumarate reductase, fumarase,
malate dehydrogenase, acetate kinase, phosphotransacetylase,
acetyl-CoA synthetase, acetyl-CoA transferase, pyruvate:ferredoxin
oxidoreductase, NAD(P)H:ferredoxin oxidoreductase, carbon monoxide
dehydrogenase, hydrogenase, and ferredoxin (see FIG. 69). Enzymes
and the corresponding genes required for these activities are
described herein.
[0156] 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
1,4-butanediol, 4-hydroxybutyrate and/or gamma-butyrolactone 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.
[0157] In some embodiments, a 1,4-butanediol, 4-hydroxybutyrate
and/or gamma-butyrolactone 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.
[0158] In some embodiments a non-naturally occurring microbial
organism having an 1,4-butanediol, 4-hydroxybutyrate and/or
gamma-butyrolactone 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 citryl-CoA synthetase, a citryl-CoA lyase, a
fumarate reductase, isocitrate dehydrogenase, aconitase, and an
alpha-ketoglutarate:ferredoxin oxidoreductase; and at least one
exogenous enzyme selected from a carbon monoxide dehydrogenase, a
hydrogenase, a NAD(P)H:ferredoxin oxidoreductase, and a ferredoxin,
expressed in a sufficient amount to allow the utilization of (1)
CO, (2) CO.sub.2, (3) H.sub.2, (4) CO.sub.2 and H.sub.2, (5) CO and
CO.sub.2, (6) CO and H.sub.2, or (7) CO, CO.sub.2, and H.sub.2.
[0159] In some embodiments a method includes culturing a
non-naturally occurring microbial organism having a 1,4-butanediol,
4-hydroxybutyrate and/or gamma-butyrolactone 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 citryl-CoA
synthetase, a citryl-CoA lyase, a fumarate reductase, isocitrate
dehydrogenase, aconitase, and an alpha-ketoglutarate:ferredoxin
oxidoreductase. Additionally, such an organism can also include at
least one exogenous enzyme selected from a carbon monoxide
dehydrogenase, a hydrogenase, a NAD(P)H:ferredoxin oxidoreductase,
and a ferredoxin, expressed in a sufficient amount to allow the
utilization of (1) CO, (2) CO.sub.2, (3) H.sub.2, (4) CO.sub.2 and
H.sub.2, (5) CO and CO.sub.2, (6) CO and H.sub.2, or (7) CO,
CO.sub.2, and H.sub.2 to produce a product.
[0160] In some embodiments a non-naturally occurring microbial
organism having an 1,4-butanediol, 4-hydroxybutyrate and/or
gamma-butyrolactone 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 citryl-CoA synthetase, a citryl-CoA lyase,
a fumarate reductase, a pyruvate:ferredoxin oxidoreductase,
isocitrate dehydrogenase, aconitase, and an
alpha-ketoglutarate:ferredoxin oxidoreductase.
[0161] In some embodiments a non-naturally occurring microbial
organism having an 1,4-butanediol, 4-hydroxybutyrate and/or
gamma-butyrolactone 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 1,4-butanediol, 4-hydroxybutyrate and/or
gamma-butyrolactone.
[0162] In some embodiments, the non-naturally occurring microbial
organism having an 1,4-butanediol, 4-hydroxybutyrate and/or
gamma-butyrolactone pathway includes two exogenous nucleic acids,
each encoding a reductive TCA pathway enzyme. In some embodiments,
the non-naturally occurring microbial organism having a
1,4-butanediol, 4-hydroxybutyrate and/or gamma-butyrolactone
pathway includes three exogenous nucleic acids each encoding a
reductive TCA pathway enzyme. In some embodiments, the
non-naturally occurring microbial organism includes three exogenous
nucleic acids encoding an ATP-citrate lyase, a fumarate reductase,
and an alpha-ketoglutarate:ferredoxin oxidoreductase. In some
embodiments, the non-naturally occurring microbial organism
includes three exogenous nucleic acids encoding a citrate lyase, or
a citryl-CoA synthetase or a citryl-CoA lyase, a fumarate
reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase. In
some embodiments, the non-naturally occurring microbial organism
includes four exogenous nucleic acids encoding a
pyruvate:ferredoxin oxidoreductase; a phosphoenolpyruvate
carboxylase or a phosphoenolpyruvate carboxykinase, a CO
dehydrogenase; and an H.sub.2 hydrogenase. In some embodiments, the
non-naturally occurring microbial organism includes two exogenous
nucleic acids encoding a CO dehydrogenase and an H.sub.2
hydrogenase.
[0163] In some embodiments, the non-naturally occurring microbial
organisms having a 1,4-butanediol, 4-hydroxybutyrate and/or
gamma-butyrolactone 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.
[0164] In some embodiments, the non-naturally occurring microbial
organism having a 1,4-butanediol, 4-hydroxybutyrate and/or
gamma-butyrolactone 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.
[0165] In some embodiments, the non-naturally occurring microbial
organism having a 1,4-butanediol, 4-hydroxybutyrate and/or
gamma-butyrolactone 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 1,4-butanediol,
4-hydroxybutyrate and/or gamma-butyrolactone pathway utilizes
hydrogen for reducing equivalents. In some embodiments, the
non-naturally occurring microbial organism having a 1,4-butanediol,
4-hydroxybutyrate and/or gamma-butyrolactone pathway utilizes CO
for reducing equivalents. In some embodiments, the non-naturally
occurring microbial organism having a 1,4-butanediol,
4-hydroxybutyrate and/or gamma-butyrolactone pathway utilizes
combinations of CO and hydrogen for reducing equivalents.
[0166] In some embodiments, the non-naturally occurring microbial
organism having a 1,4-butanediol, 4-hydroxybutyrate and/or
gamma-butyrolactone 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.
[0167] In some embodiments, the non-naturally occurring microbial
organism having a 1,4-butanediol, 4-hydroxybutyrate and/or
gamma-butyrolactone 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.
[0168] In some embodiments, the non-naturally occurring microbial
organism having a 1,4-butanediol, 4-hydroxybutyrate and/or
gamma-butyrolactone pathway further includes at least one exogenous
nucleic acid encoding a citrate lyase, an ATP-citrate lyase, a
citryl-CoA synthetase, a citryl-CoA lyase, an aconitase, an
isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA
transferase, a fumarase, a malate dehydrogenase, an acetate kinase,
a phosphotransacetylase, an acetyl-CoA synthetase, and a
ferredoxin.
[0169] 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.
[0170] 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.
[0171] In one embodiment, the invention provides a non-naturally
occurring microbial organism, comprising a microbial organism
having a 1,4-butanediol pathway comprising at least one exogenous
nucleic acid encoding a 1,4-butanediol pathway enzyme expressed in
a sufficient amount to produce 1,4-butanediol. Such a microbial
organism can further comprise (i) a reductive TCA pathway
comprising at least one exogenous nucleic acid encoding a reductive
TCA pathway enzyme, wherein the at least one exogenous nucleic acid
is selected from an ATP-citrate lyase, citrate lyase, a citryl-CoA
synthetase, a citryl-CoA lyase, a fumarate reductase, and an
alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA
pathway comprising at least one exogenous nucleic acid encoding a
reductive TCA pathway enzyme, wherein the at least one exogenous
nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase,
a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate
carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or (iii)
at least one exogenous nucleic acid encodes an enzyme selected from
a CO dehydrogenase, an H.sub.2 hydrogenase, and combinations
thereof.
[0172] In such microbial organisms, a 1,4-butanediol pathway can
comprise a pathway of any of those disclosed herein, including the
figures. For example, a 1,4-BDO pathway can be selected from (a)
4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase,
CoA-dependent succinic semialdehyde dehydrogenase, and
.alpha.-ketoglutarate decarboxylase; (b) 4-hydroxybutanoate
dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic
semialdehyde dehydrogenase, 4-hydroxybutyrate:CoA transferase,
4-butyrate kinase, phosphotransbutyrylase, .alpha.-ketoglutarate
decarboxylase, aldehyde dehydrogenase, alcohol dehydrogenase and an
aldehyde/alcohol dehydrogenase; (c) (i) an .alpha.-ketoglutarate
decarboxylase, or an .alpha.-ketoglutarate dehydrogenase and a
CoA-dependent succinic semialdehyde dehydrogenase, or a
glutamate:succinate semialdehyde transaminase and a glutamate
decarboxylase; (ii) a 4-hydroxybutanoate dehydrogenase; (iii) a
4-hydroxybutyryl-CoA:acetyl-CoA transferase, or a butyrate kinase
and a phosphotransbutyrylase; (iv) an aldehyde dehydrogenase; and
(v) an alcohol dehydrogenase; (d) 4-aminobutyrate CoA transferase,
4-aminobutyryl-CoA hydrolase, 4-aminobutyrate-CoA ligase,
4-aminobutyryl-CoA oxidoreductase (deaminating), 4-aminobutyryl-CoA
transaminase, and 4-hydroxybutyryl-CoA dehydrogenase; (e)
4-aminobutyrate CoA transferase, 4-aminobutyryl-CoA hydrolase,
4-aminobutyrate-CoA ligase, 4-aminobutyryl-CoA reductase (alcohol
forming), 4-aminobutyryl-CoA reductase, 4-aminobutan-1-ol
dehydrogenase, 4-aminobutan-1-ol oxidoreductase (deaminating) and
4-aminobutan-1-ol transaminase; (f) 4-aminobutyrate kinase,
4-aminobutyraldehyde dehydrogenase (phosphorylating),
4-aminobutan-1-ol dehydrogenase, 4-aminobutan-1-ol oxidoreductase
(deaminating), 4-aminobutan-1-ol transaminase,
[(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase
(deaminating), [(4-aminobutanolyl)oxy]phosphonic acid transaminase,
4-hydroxybutyryl-phosphate dehydrogenase, and
4-hydroxybutyraldehyde dehydrogenase (phosphorylating); (g)
alpha-ketoglutarate 5-kinase, 2,5-dioxopentanoic semialdehyde
dehydrogenase (phosphorylating), 2,5-dioxopentanoic acid reductase,
alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA
hydrolase, alpha-ketoglutaryl-CoA ligase, alpha-ketoglutaryl-CoA
reductase, 5-hydroxy-2-oxopentanoic acid dehydrogenase,
alpha-ketoglutaryl-CoA reductase (alcohol forming),
5-hydroxy-2-oxopentanoic acid decarboxylase, and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (h)
glutamate CoA transferase, glutamyl-CoA hydrolase, glutamyl-CoA
ligase, glutamate 5-kinase, glutamate-5-semialdehyde dehydrogenase
(phosphorylating), glutamyl-CoA reductase, glutamate-5-semialdehyde
reductase, glutamyl-CoA reductase (alcohol forming),
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating),
2-amino-5-hydroxypentanoic acid transaminase,
5-hydroxy-2-oxopentanoic acid decarboxylase,
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (i)
3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA
dehydratase, vinylacetyl-CoA A-isomerase, or 4-hydroxybutyryl-CoA
dehydratase; (j) homoserine deaminase, homoserine CoA transferase,
homoserine-CoA hydrolase, homoserine-CoA ligase, homoserine-CoA
deaminase, 4-hydroxybut-2-enoyl-CoA transferase,
4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA
ligase, 4-hydroxybut-2-enoate reductase, 4-hydroxybutyryl-CoA
transferase, 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA
ligase, or 4-hydroxybut-2-enoyl-CoA reductase; (k) succinyl-CoA
reductase (alcohol forming), 4-hydroxybutyryl-CoA hydrolase,
4-hydroxybutyryl-CoA ligase, or 4-hydroxybutanal dehydrogenase
(phosphorylating); (l) glutamate dehydrogenase, 4-aminobutyrate
oxidoreductase (deaminating), 4-aminobutyrate transaminase,
glutamate decarboxylase, 4-hydroxybutyryl-CoA hydrolase,
4-hydroxybutyryl-CoA ligase, or 4-hydroxybutanal dehydrogenase
(phosphorylating); (m) 4-aminobutyrate kinase; 4-aminobutyraldehyde
dehydrogenase (phosphorylating); 4-aminobutan-1-ol dehydrogenase;
and 4-aminobutan-1-ol oxidoreductase (deaminating) or
4-aminobutan-1-ol transaminase; (n) 4-aminobutyrate kinase;
[(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase (deaminating)
or [(4-aminobutanolyl)oxy]phosphonic acid transaminase;
4-hydroxybutyryl-phosphate dehydrogenase; and
4-hydroxybutyraldehyde dehydrogenase (phosphorylating); (o)
alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA
hydrolase, or alpha-ketoglutaryl-CoA ligase; alpha-ketoglutaryl-CoA
reductase (alcohol forming); and 5-hydroxy-2-oxopentanoic acid
decarboxylase; (p) alpha-ketoglutarate CoA transferase,
alpha-ketoglutaryl-CoA hydrolase, or alpha-ketoglutaryl-CoA ligase;
alpha-ketoglutaryl-CoA reductase (alcohol forming); and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (q)
alpha-ketoglutarate 5-kinase; 2,5-dioxopentanoic semialdehyde
dehydrogenase (phosphorylating); 2,5-dioxopentanoic acid reductase;
and 5-hydroxy-2-oxopentanoic acid decarboxylase; (r)
alpha-ketoglutarate 5-kinase; 2,5-dioxopentanoic semialdehyde
dehydrogenase (phosphorylating); 2,5-dioxopentanoic acid reductase;
and 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);
(s) alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA
hydrolase, or alpha-ketoglutaryl-CoA ligase; alpha-ketoglutaryl-CoA
reductase; 5-hydroxy-2-oxopentanoic acid dehydrogenase; and
5-hydroxy-2-oxopentanoic acid decarboxylase; (t)
alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA
hydrolase, or alpha-ketoglutaryl-CoA ligase; alpha-ketoglutaryl-CoA
reductase; 5-hydroxy-2-oxopentanoic acid dehydrogenase; and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (u)
glutamate CoA transferase, glutamyl-CoA hydrolase, or glutamyl-CoA
ligase; glutamyl-CoA reductase (alcohol forming);
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or
2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (v)
glutamate 5-kinase; glutamate-5-semialdehyde dehydrogenase
(phosphorylating); 2-amino-5-hydroxypentanoic acid oxidoreductase
(deaminating) or 2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (w)
glutamate CoA transferase, glutamyl-CoA hydrolase, or glutamyl-CoA
ligase; glutamyl-CoA reductase; glutamate-5-semialdehyde reductase;
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or
2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (x)
glutamate 5-kinase; glutamate-5-semialdehyde dehydrogenase
(phosphorylating); glutamate-5-semialdehyde reductase;
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or
2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (y)
homoserine deaminase; 4-hydroxybut-2-enoyl-CoA transferase,
4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA
ligase; 4-hydroxybut-2-enoyl-CoA reductase; (z) homoserine CoA
transferase, homoserine-CoA hydrolase, or homoserine-CoA ligase;
homoserine-CoA deaminase; and 4-hydroxybut-2-enoyl-CoA reductase;
(aa) homoserine deaminase; 4-hydroxybut-2-enoate reductase; and
4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA hydrolase,
or 4-hydroxybutyryl-CoA ligase; (bb) (i) alpha-ketoglutarate
decarboxylase; or alpha-ketoglutarate dehydrogenase and
CoA-dependent succinate semialdehyde dehydrogenase; or
glutamate:succinate semialdehyde transaminase and glutamate
decarboxylase; (ii) 4-hydroxybutyrate dehydrogenase; (iii)
4-hydroxybutyryl-CoA transferase; or 4-hydroxybutyrate kinase and
phosphotrans-4-hydroxybutyrylase; (iv) 4-hydroxybutyryl-CoA
reductase; and (v) 4-hydroxybutyraldehyde reductase; or
aldehyde/alcohol dehydrogenase; (cc) (i) alpha-ketoglutarate
decarboxylase; or succinyl-CoA synthetase and CoA-dependent
succinate semialdehyde dehydrogenase; (ii) 4-hydroxybutyrate
dehydrogenase; (iii) 4-hydroxybutyryl-CoA transferase; or
4-hydroxybutyrate kinase and phosphotrans-4-hydroxybutyrylase; and
(iv) aldehyde dehydrogenase; and alcohol dehydrogenase; or
aldehyde/alcohol dehydrogenase; (dd) (i) alpha-ketoglutarate
decarboxylase; or glutamate dehydrogenase; glutamate decarboxylase;
and deaminating 4-aminobutyrate oxidoreductase or 4-aminobutyrate
transaminase; or alpha-ketoglutarate dehydrogenase and
CoA-dependent succinate semialdehyde dehydrogenase; (ii)
4-hydroxybutyrate dehydrogenase; and (iii) 4-hydroxybutyrate
kinase; phosphotrans-4-hydroxybutyrylase; 4-hydroxybutyryl-CoA
reductase; and 4-hydroxybutyraldehyde reductase; or
4-hydroxybutyrate kinase; phosphorylating 4-hydroxybutanal
dehydrogenase; and 4-hydroxybutyraldehyde reductase; or
4-hydroxybutyrate kinase; phosphotrans-4-hydroxybutyrylase; and
alcohol forming 4-hydroxybutyryl-CoA reductase; or
4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA hydrolase
or 4-hydroxybutyryl-CoA ligase; 4-hydroxybutyryl-CoA reductase; and
4-hydroxybutyraldehyde reductase; or 4-hydroxybutyryl-CoA
transferase or 4-hydroxybutyryl-CoA hydrolase or
4-hydroxybutyryl-CoA ligase; and alcohol forming
4-hydroxybutyryl-CoA reductase; (ee) (i) glutamate CoA transferase
or glutamyl-CoA hydrolase or glutamyl-CoA ligase; glutamyl-CoA
reductase; and glutamate-5-semialdehyde reductase; or glutamate CoA
transferase or glutamyl-CoA hydrolase or glutamyl-CoA ligase; and
alcohol forming glutamyl-CoA reductase; or glutamate 5-kinase;
phosphorylating glutamate-5-semialdehyde dehydrogenase; and
glutamate-5-semialdehyde reductase; (ii) deaminating
2-amino-5-hydroxypentanoic acid oxidoreductase or
2-amino-5-hydroxypentanoic acid transaminase; and (iii)
5-hydroxy-2-oxopentanoic acid decarboxylase; and
4-hydroxybutyraldehyde reductase; or decarboxylating
5-hydroxy-2-oxopentanoic acid dehydrogenase; 4-hydroxybutyryl-CoA
reductase; and 4-hydroxybutyraldehyde reductase; or decarboxylating
5-hydroxy-2-oxopentanoic acid dehydrogenase and alcohol forming
4-hydroxybutyryl-CoA reductase; (ff) succinyl-CoA reductase
(aldehyde forming); 4-hydroxybutyrate dehydrogenase; and
4-hydroxybutyrate reductase; and optionally 1,4-butandiol
dehydrogenase; (gg) alpha-ketoglutarate decarboxylase;
4-hydroxybutyrate dehydrogenase; and 4-hydroxybutyrate reductase;
and optionally 1,4-butandiol dehydrogenase; (hh) succinate
reductase; 4-hydroxybutyrate dehydrogenase, and 4-hydroxybutyrate
reductase; and optionally 1,4-butandiol dehydrogenase; (ii)
alpha-ketoglutarate decarboxylase, or glutamate dehydrogenase or
glutamate transaminase and glutamate decarboxylase and
4-aminobutyrate dehydrogenase or 4-aminobutyrate transaminase;
4-hydroxybutyrate dehydrogenase; and 4-hydroxybutyrate reductase;
and optionally 1,4-butandiol dehydrogenase; (jj)
alpha-ketoglutarate reductase; 5-hydroxy-2-oxopentanoate
dehydrogenase; and 5-hydroxy-2-oxopentanoate decarboxylase; and
optionally 1,4-butandiol dehydrogenase; (kk) Acetoacetyl-CoA
thiolase or acetoacetyl-CoA synthase; 3-Hydroxybutyryl-CoA
dehydrogenase; Crotonase; Crotonyl-CoA hydratase; and
4-hydroxybutyryl-CoA reductase (alcohol forming) (see FIG. 72
reactions 1, 2, 3, 4 and 5); (ll) Acetoacetyl-CoA thiolase or
acetoacetyl-CoA synthase; 3-Hydroxybutyryl-CoA dehydrogenase;
Crotonase; Crotonyl-CoA hydratase; 4-hydroxybutyryl-CoA reductase
(aldehyde forming); and 1,4-butanediol dehydrogenase (see FIG. 72
reactions 1, 2, 3, 4, 6 and 7); (mm) Acetoacetyl-CoA thiolase or
acetoacetyl-CoA synthase; 3-Hydroxybutyryl-CoA dehydrogenase;
Crotonase; Crotonyl-CoA hydratase; 4-Hydroxybutyryl-CoA
transferase, 4-Hydroxybutyryl-CoA synthetase, 4-Hydroxybutyryl-CoA
hydrolase, or Phosphotrans-4-hydroxybutyrylase/4-Hydroxybutyrate
kinase; 4-Hydroxybutyrate reductase; and 1,4-butanediol
dehydrogenase (FIG. 72 reactions 1, 2, 3, 4, 8, 9 and 7); (nn)
Succinate reductase; 4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase; Phosphotrans-4-hydroxybutyrylasel;
4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase (FIG. 74 reactions H, C, D, E, F and
G); (oo) Succinate reductase; 4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase; 4-Hydroxybutyryl-phosphate reductase; and
1,4-butanediol dehydrogenase (FIG. 74 reactions H, C, D, L and G);
(pp) Succinate reductase; 4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate reductase; and 1,4-butanediol dehydrogenase (FIG.
74 reactions H, C, K +and G); (qq) Succinate reductase;
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyryl-CoA transferase,
or 4-Hydroxybutyryl-CoA synthetase; and 4-Hydroxybutyryl-CoA
reductase (alcohol forming) (FIG. 74 reactions H, C, J and M); (rr)
Succinate reductase; 4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA
synthetase; 4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase (FIG. 74 reactions H, C, J, F and G);
(ss) Succinate reductase; 4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase; Phosphotrans-4-hydroxybutyrylase; and
4-Hydroxybutyryl-CoA reductase (alcohol forming) (FIG. 74 reactions
H, C, D, E and M); (tt) Succinyl-CoA transferase, or Succinyl-CoA
synthetase (or succinyl-CoA ligase); Succinyl-CoA reductase
(aldehyde forming); 4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase; Phosphotrans-4-hydroxybutyrylase;
4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase (FIG. 74 reactions A, B, C, D, E, F
and G); (uu) Succinyl-CoA transferase, or Succinyl-CoA synthetase
(or succinyl-CoA ligase); Succinyl-CoA reductase (aldehyde
forming); 4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyrate
kinase; 4-Hydroxybutyryl-phosphate reductase; and 1,4-butanediol
dehydrogenase (FIG. 74 reactions A, B, C, D, L and G); (vv)
Succinyl-CoA transferase, or Succinyl-CoA synthetase (or
succinyl-CoA ligase); Succinyl-CoA reductase (aldehyde forming);
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyrate reductase; and
1,4-butanediol dehydrogenase (FIG. 74 reactions A, B, C, K and G);
(ww) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or
succinyl-CoA ligase); Succinyl-CoA reductase (aldehyde forming);
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyryl-CoA transferase,
or 4-Hydroxybutyryl-CoA synthetase; and 4-Hydroxybutyryl-CoA
reductase (alcohol forming) (FIG. 74 reactions A, B, C, J and M);
(xx) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or
succinyl-CoA ligase); Succinyl-CoA reductase (aldehyde forming);
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyryl-CoA transferase,
or 4-Hydroxybutyryl-CoA synthetase; 4-Hydroxybutyryl-CoA reductase
(aldehyde forming); and 1,4-butanediol dehydrogenase (FIG. 74
reactions A, B, C, J, F and G); (yy) Succinyl-CoA transferase, or
Succinyl-CoA synthetase (or succinyl-CoA ligase); Succinyl-CoA
reductase (aldehyde forming); 4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase; Phosphotrans-4-hydroxybutyrylase; and
4-Hydroxybutyryl-CoA reductase (alcohol forming) (
FIG. 74 reactions A, B, C, D, E and M); (zz) Alpha-ketoglutarate
decarboxylase or (Glutamate dehydrogenase and/or Glutamate
transaminase; Glutamate decarboxylase; 4-aminobutyrate
dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase; 4-Hydroxybutyryl-CoA reductase
(aldehyde forming); and 1,4-butanediol dehydrogenase (FIG. 74
reactions N, C, D, E, F and G); (aaa) Alpha-ketoglutarate
decarboxylase or (Glutamate dehydrogenase and/or Glutamate
transaminase; Glutamate decarboxylase; 4-aminobutyrate
dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyrate kinase;
4-Hydroxybutyryl-phosphate reductase; and 1,4-butanediol
dehydrogenase (FIG. 74 reactions N, C, D, L and G); (bbb)
Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase
and/or Glutamate transaminase; Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyrate reductase; and
1,4-butanediol dehydrogenase (FIG. 74 reactions N, C, K and G);
(ccc) Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase
and/or Glutamate transaminase; Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyryl-CoA transferase,
or 4-Hydroxybutyryl-CoA synthetase; and 4-Hydroxybutyryl-CoA
reductase (alcohol forming) (FIG. 74 reactions N, C, J and M);
[0173] (ddd) Alpha-ketoglutarate decarboxylase or (Glutamate
dehydrogenase and/or Glutamate transaminase; Glutamate
decarboxylase; 4-aminobutyrate dehydrogenase and/or 4-aminobutyrate
transaminase); 4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA
synthetase; 4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase (FIG. 74 reactions N, C, J, F and G);
(eee) Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase
and/or Glutamate transaminase; Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase; and 4-Hydroxybutyryl-CoA
reductase (alcohol forming) (FIG. 74 reactions N, C, D, E and M);
(fff) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or
succinyl-CoA ligase); Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyrate kinase; Phosphotrans-4-hydroxybutyrylase;
4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase (FIG. 74 reactions A, I, D, E, F and
G); (ggg) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or
succinyl-CoA ligase); Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyrate kinase; 4-Hydroxybutyryl-phosphate reductase; and
1,4-butanediol dehydrogenase (FIG. 74 reactions A, I, D, L and G);
(hhh) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or
succinyl-CoA ligase); Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyrate reductase; and 1,4-butanediol dehydrogenase (FIG.
74 reactions A, I, K and G); (iii) Succinyl-CoA transferase, or
Succinyl-CoA synthetase (or succinyl-CoA ligase); Succinyl-CoA
reductase (alcohol forming); 4-Hydroxybutyryl-CoA transferase, or
4-Hydroxybutyryl-CoA synthetase; and 4-Hydroxybutyryl-CoA reductase
(alcohol forming) (FIG. 74 reactions A, I, J and M); (jjj)
Succinyl-CoA transferase, or Succinyl-CoA synthetase (or
succinyl-CoA ligase); Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA
synthetase; 4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase (FIG. 74 reactions A, I, J, F and G);
(kkk) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or
succinyl-CoA ligase); Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyrate kinase; Phosphotrans-4-hydroxybutyrylase; and
4-Hydroxybutyryl-CoA reductase (alcohol forming) (FIG. 74 reactions
A, I, D, E and M); and (lll) any of the pathways that produce
1,4-butanediol as shown in any of FIG. 1, 8-13, 58, 62, 63 or
72-74.
[0174] In a further embodiment, such a microbial organism of the
invention comprising (i) can further comprise an exogenous nucleic
acid encoding an enzyme selected from a pyruvate:ferredoxin
oxidoreductase, an aconitase, an isocitrate dehydrogenase, a
succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a
malate dehydrogenase, an acetate kinase, a phosphotransacetylase,
an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase,
ferredoxin, and combinations thereof. In addition, a microbial
organism comprising (ii) can further comprise an exogenous nucleic
acid encoding an enzyme selected from an aconitase, an isocitrate
dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA
transferase, a fumarase, a malate dehydrogenase, and combinations
thereof.
[0175] In yet another embodiment, such a microbial organism can
comprise two, three, four, five, six or seven exogenous nucleic
acids each encoding a 1,4-butanediol pathway enzyme. For example,
such a microbial organism can comprise exogenous nucleic acids
encoding each of the enzymes of a pathway, for example, a
particular pathway as disclosed herein, including those shown in
FIGS. 1, 8-13, 58, 62, 63 and 72-74. A microbial organism can
comprise more than one pathway, if desired, which can be useful to
increase the yield of a desired product.
[0176] In a further embodiment, a microbial organism comprising
pathways (a), (b) or (c) further comprises an enzyme selected from
succinyl-CoA synthetase, exogenous CoA-dependent succinic
semialdehyde dehydrogenase or exogenous succinyl-CoA synthetase and
exogenous CoA-dependent succinic semialdehyde dehydrogenase. In
still a further embodiment, a microbial organism comprising pathway
(d), (g), (h), (i), (j) further comprises an enzyme selected from
4-hydroxybutyryl-CoA reductase (alcohol forming),
4-hydroxybutyryl-CoA reductase, or 1,4-butanediol dehydrogenase.
Additionally, a microbial organism comprising pathway (e) or (f)
can further comprise 1,4-butanediol dehydrogenase. In yet a further
embodiment, a microbial organism comprising pathway (k) can further
comprise succinyl-CoA reductase, 4-hydroxybutyrate dehydrogenase,
4-hydroxybutyryl-CoA transferase, 4-hydroxybutyrate kinase,
phosphotrans-4-hydroxybutyrylase, 4-hydroxybutyryl-CoA reductase,
4-hydroxybutyryl-CoA reductase (alcohol forming), or 1,4-butanediol
dehydrogenase. Still further, a microbial organism comprising
pathway (l) further comprises alpha-ketoglutarate decarboxylase,
4-hydroxybutyrate dehydrogenase, 4-hydroxybutyryl-CoA transferase,
4-hydroxybutyrate kinase, phosphotrans-4-hydroxybutyrylase,
4-hydroxybutyryl-CoA reductase, 4-hydroxybutyryl-CoA reductase
(alcohol forming), or 1,4-butanediol dehydrogenase. Such additional
pathway steps are disclosed herein.
[0177] In yet another embodiment of the invention, a microbial
organism can comprise two, three, four or five exogenous nucleic
acids each encoding enzymes of (i), (ii) or (iii). For example, a
microbial organism comprising (i) can comprise three exogenous
nucleic acids encoding ATP-citrate lyase or citrate lyase, a
fumarate reductase, and an alpha-ketoglutarate:ferredoxin
oxidoreductase; a microbial organism comprising (ii) can comprise
five exogenous nucleic acids encoding pyruvate:ferredoxin
oxidoreductase, a phosphoenolpyruvate carboxylase, a
phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an
H.sub.2 hydrogenase; or a microbial organism comprising (iii) can
comprise two exogenous nucleic acids encoding CO dehydrogenase and
H.sub.2 hydrogenase. The invention additionally provides methods
for producing 1,4-butanediol by culturing such non-naturally
occurring microbial organisms under conditions and for a sufficient
period of time to produce 1,4-butanediol.
[0178] The invention additionally provides a non-naturally
occurring microbial organism, comprising a microbial organism
having a 4-hydroxybutyrate pathway comprising at least one
exogenous nucleic acid encoding a 4-hydroxybutyrate pathway enzyme
4-hydroxybutyrate expressed in a sufficient amount to produce
4-hydroxybutyrate. Such a non-naturally occurring microbial
organism can further comprise (i) a reductive TCA pathway
comprising at least one exogenous nucleic acid encoding a reductive
TCA pathway enzyme, wherein said at least one exogenous nucleic
acid is selected from an ATP-citrate lyase, citrate lyase, a
citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase,
and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a
reductive TCA pathway comprising at least one exogenous nucleic
acid encoding a reductive TCA pathway enzyme, wherein said at least
one exogenous nucleic acid is selected from a pyruvate:ferredoxin
oxidoreductase, a phosphoenolpyruvate carboxylase, a
phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an
H.sub.2 hydrogenase; or (iii) at least one exogenous nucleic acid
encodes an enzyme selected from a CO dehydrogenase, an H.sub.2
hydrogenase, and combinations thereof. In such a microbial organism
the 4-hydroxybutyrate pathway can comprise a pathway from any of
those disclosed herein, including in the figures, for example, any
of FIG. 1, 8-13, 58, 62, 63 or 72-74.
[0179] In a particular embodiment, a microbial organism comprising
a 4-hydroxybutyrate pathway can be selected from (a)
Acetoacetyl-CoA thiolase or acetoacetyl-CoA synthase;
3-Hydroxybutyryl-CoA dehydrogenase; Crotonase; Crotonyl-CoA
hydratase; and 4-Hydroxybutyryl-CoA transferase,
4-Hydroxybutyryl-CoA synthetase, 4-Hydroxybutyryl-CoA hydrolase, or
Phosphotrans-4-hydroxybutyrylase/4-Hydroxybutyrate kinase (FIG. 72
reactions 1, 2, 3, 4 and 8); (b) Acetoacetyl-CoA thiolase or
acetoacetyl-CoA synthase; 3-Hydroxybutyryl-CoA dehydrogenase;
Crotonase; Crotonyl-CoA hydratase; and 4-Hydroxybutyryl-CoA
transferase, hydrolase or synthetase (FIG. 73 reactions 1, 2, 3, 4
and 5); (c) Acetoacetyl-CoA thiolase or acetoacetyl-CoA synthase;
3-Hydroxybutyryl-CoA dehydrogenase; Crotonase; Crotonyl-CoA
hydratase; Phosphotrans-4-hydroxybutyrylase; and 4-Hydroxybutyrate
kinase (FIG. 73 reactions 1, 2, 3, 4, 6 and 7); (d) Succinate
reductase; and 4-Hydroxybutyrate dehydrogenase (FIG. 74 reactions H
and C); (e) Succinyl-CoA transferase, or Succinyl-CoA synthetase
(or succinyl-CoA ligase); Succinyl-CoA reductase (aldehyde
forming); and 4-Hydroxybutyrate dehydrogenase (FIG. 74 reactions A,
B and C); (f) Alpha-ketoglutarate decarboxylase or (Glutamate
dehydrogenase and/or Glutamate transaminase; Glutamate
decarboxylase; 4-aminobutyrate dehydrogenase and/or 4-aminobutyrate
transaminase); and 4-Hydroxybutyrate dehydrogenase (FIG. 74
reactions N and C); (g) Succinyl-CoA transferase, or Succinyl-CoA
synthetase (or succinyl-CoA ligase); and Succinyl-CoA reductase
(alcohol forming) (FIG. 74 reactions A and I); (h) acetoacetyl-CoA
thiolase or acetoacetyl-CoA synthase, a 3-hydroxybutyryl-CoA
dehydrogenase, a crotonase, a crotonyl-CoA hydratase, a
4-hydroxybutyryl-CoA transferase, a
phosphotrans-4-hydroxybutyrylase, and a 4-hydroxybutyrate kinase;
(i) 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase,
CoA-dependent succinic semialdehyde dehydrogenase, and
.alpha.-ketoglutarate decarboxylase; (j) (i) an
.alpha.-ketoglutarate decarboxylase, or an .alpha.-ketoglutarate
dehydrogenase and a CoA-dependent succinic semialdehyde
dehydrogenase, or a glutamate:succinate semialdehyde transaminase
and a glutamate decarboxylase; (ii) a 4-hydroxybutanoate
dehydrogenase; (k) succinyl-CoA reductase (alcohol forming),
4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or
4-hydroxybutanal dehydrogenase (phosphorylating); (l) glutamate
dehydrogenase, 4-aminobutyrate oxidoreductase (deaminating),
4-aminobutyrate transaminase, glutamate decarboxylase,
4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or
4-hydroxybutanal dehydrogenase (phosphorylating); (m) homoserine
deaminase; 4-hydroxybut-2-enoyl-CoA transferase,
4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA
ligase; 4-hydroxybut-2-enoyl-CoA reductase; (n) homoserine CoA
transferase, homoserine-CoA hydrolase, or homoserine-CoA ligase;
homoserine-CoA deaminase; and 4-hydroxybut-2-enoyl-CoA reductase;
(o) homoserine deaminase; 4-hydroxybut-2-enoate reductase; and
4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA hydrolase,
or 4-hydroxybutyryl-CoA ligase; (p) succinyl-CoA reductase
(aldehyde forming); and 4-hydroxybutyrate dehydrogenase; (q)
alpha-ketoglutarate decarboxylase; and 4-hydroxybutyrate
dehydrogenase; (r) succinate reductase; and 4-hydroxybutyrate
dehydrogenase; (s) alpha-ketoglutarate decarboxylase, or glutamate
dehydrogenase or glutamate transaminase and glutamate decarboxylase
and 4-aminobutyrate dehydrogenase or 4-aminobutyrate transaminase;
and 4-hydroxybutyrate dehydrogenase; and (t) a 4-hydroxybutyrate
pathway selected from any of the pathways that produce
4-hydroxybutyrate as shown in any of FIG. 1, 8-13, 58, 62, 63 or
72-74.
[0180] Such a microbial organism comprising (i) can further
comprise an exogenous nucleic acid encoding an enzyme selected from
a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate
dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA
transferase, a fumarase, a malate dehydrogenase, an acetate kinase,
a phosphotransacetylase, an acetyl-CoA synthetase, an
NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations
thereof. In addition, a microbial organism comprising (ii) can
further comprise an exogenous nucleic acid encoding an enzyme
selected from an aconitase, an isocitrate dehydrogenase, a
succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a
malate dehydrogenase, and combinations thereof.
[0181] In a particular embodiment, the microbial organism can
comprise exogenous nucleic acids encoding each of the enzymes
selected from the pathway enzymes producing 4-hydroxybutyrate
pathway enzymes as shown in any of FIG. 1, 8-13, 58, 62, 63 or
72-74. Such microbial organisms can also comprise two, three, four
or five exogenous nucleic acids each encoding enzymes of (i), (ii)
or (iii). For example, a microbial organism comprising (i) can
comprise three exogenous nucleic acids encoding ATP-citrate lyase
or citrate lyase, a fumarate reductase, and an
alpha-ketoglutarate:ferredoxin oxidoreductase; a microbial organism
comprising (ii) can comprise five exogenous nucleic acids encoding
pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate
carboxylase, a phosphoenolpyruvate carboxykinase, a CO
dehydrogenase, and an H.sub.2 hydrogenase; or a microbial organism
comprising (iii) can comprise two exogenous nucleic acids encoding
CO dehydrogenase and H.sub.2 hydrogenase. The invention
additionally provides a method for producing 4-hydroxybutyrate, by
culturing the non-naturally occurring microbial organisms under
conditions and for a sufficient period of time to produce
4-hydroxybutyrate.
[0182] The invention also provides a non-naturally occurring
microbial organism, comprising a microbial organism having a
gamma-butyrolactone pathway comprising at least one exogenous
nucleic acid encoding a gamma-butyrolactone pathway enzyme
expressed in a sufficient amount to produce gamma-butyrolactone.
Such a microbial organism can further comprise (i) a reductive TCA
pathway comprising at least one exogenous nucleic acid encoding a
reductive TCA pathway enzyme, wherein said at least one exogenous
nucleic acid is selected from an ATP-citrate lyase, citrate lyase,
a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase,
and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a
reductive TCA pathway comprising at least one exogenous nucleic
acid encoding a reductive TCA pathway enzyme, wherein said at least
one exogenous nucleic acid is selected from a pyruvate:ferredoxin
oxidoreductase, a phosphoenolpyruvate carboxylase, a
phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an
H.sub.2 hydrogenase; or (iii) at least one exogenous nucleic acid
encodes an enzyme selected from a CO dehydrogenase, an H.sub.2
hydrogenase, and combinations thereof. Such microbial organisms can
comprise, for example, a pathway selected from any of the pathways
that produce gamma-butryolactone as shown in FIG. 1, 8-13, 58, 62,
63 or 72-74. As disclosed herein, both 4-hydroxybutyryl-CoA and
4-hydroxybutyryl phosphate can be enzymatically or can
spontaneously chemically convert to gamma-butyrolactone. Therefore,
it is understood that any of the pathways disclosed herein that
produce 4-hydroxybytyryl-CoA or 4-hydroxybutyryl phosphate can be
used to produce gamma-butyrolactone using enzymatic and/or chemical
conversion.
[0183] In such microbial organisms, a gamma-butyroloactone pathway
can comprise a pathway selected from, for example, (a)
Acetoacetyl-CoA thiolase or acetoacetyl-CoA synthase;
3-Hydroxybutyryl-CoA dehydrogenase; Crotonase; Crotonyl-CoA
hydratase; and spontaneous or enzyme catalyzed (FIG. 73 reactions
1, 2, 3, 4 and 8); (b) Acetoacetyl-CoA thiolase or acetoacetyl-CoA
synthase; 3-Hydroxybutyryl-CoA dehydrogenase; Crotonase;
Crotonyl-CoA hydratase; Phosphotrans-4-hydroxybutyrylase; amd
spontaneous or enzyme catalyzed (FIG. 73 reactions 1, 2, 3, 4, 6
and 9); (c) Succinate reductase; 4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase; Phosphotrans-4-hydroxybutyrylase; and
4-Hydroxybutyryl-CoA hydrolase or spontaneous (FIG. 74 reactions H,
C, D, E and O); (d) Succinate reductase; 4-Hydroxybutyrate
dehydrogenase; 4-Hydroxybutyryl-CoA transferase, or
4-Hydroxybutyryl-CoA synthetase; and 4-Hydroxybutyryl-CoA hydrolase
or spontaneous (FIG. 74 reactions H, C, J and O); (e) Succinyl-CoA
transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (aldehyde forming); 4-Hydroxybutyrate
dehydrogenase; 4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase; and 4-Hydroxybutyryl-CoA
hydrolase or spontaneous (FIG. 74 reactions A, B, C, D, E and O);
(f) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or
succinyl-CoA ligase); Succinyl-CoA reductase (aldehyde forming);
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyryl-CoA transferase,
or 4-Hydroxybutyryl-CoA synthetase; 4-Hydroxybutyryl-CoA hydrolase
or spontaneous (FIG. 74 reactions A, B, C, J and O); (g)
Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase
and/or Glutamate transaminase; Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase; and 4-Hydroxybutyryl-CoA
hydrolase or spontaneous (FIG. 74 reactions N, C, D, E and O); (h)
Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase
and/or Glutamate transaminase; Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase; 4-Hydroxybutyryl-CoA transferase,
or 4-Hydroxybutyryl-CoA synthetase; and 4-Hydroxybutyryl-CoA
hydrolase or spontaneous (FIG. 74 reactions N, C, J and O); (i)
Succinyl-CoA transferase, or Succinyl-CoA synthetase (or
succinyl-CoA ligase); Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyrate kinase; Phosphotrans-4-hydroxybutyrylase;
4-Hydroxybutyryl-CoA hydrolase or spontaneous (FIG. 74 reactions A,
I, D, E and O); (j) Succinyl-CoA transferase, or Succinyl-CoA
synthetase (or succinyl-CoA ligase); Succinyl-CoA reductase
(alcohol forming); 4-Hydroxybutyryl-CoA transferase, or
4-Hydroxybutyryl-CoA synthetase; and 4-Hydroxybutyryl-CoA hydrolase
or spontaneous (FIG. 74 reactions A, I, J and O); (k)
alpha-ketoglutarate reductase; 5-hydroxy-2-oxopentanoate
dehydrogenase; and 5-hydroxy-2-oxopentanoate dehydrogenase
(decarboxylation) (1) 4-hydroxybutanoate dehydrogenase,
succinyl-CoA synthetase, CoA-dependent succinic semialdehyde
dehydrogenase, and .alpha.-ketoglutarate decarboxylase; (m)
4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase,
CoA-dependent succinic semialdehyde dehydrogenase,
4-hydroxybutyrate:CoA transferase, 4-butyrate kinase,
phosphotransbutyrylase, .alpha.-ketoglutarate decarboxylase; (n)
(i) an .alpha.-ketoglutarate decarboxylase, or an
.alpha.-ketoglutarate dehydrogenase and a CoA-dependent succinic
semialdehyde dehydrogenase, or a glutamate:succinate semialdehyde
transaminase and a glutamate decarboxylase; (ii) a
4-hydroxybutanoate dehydrogenase; (iii) a
4-hydroxybutyryl-CoA:acetyl-CoA transferase, or a butyrate kinase
and a phosphotransbutyrylase; (o) 4-aminobutyrate CoA transferase,
4-aminobutyryl-CoA hydrolase, 4-aminobutyrate-CoA ligase,
4-aminobutyryl-CoA oxidoreductase (deaminating), 4-aminobutyryl-CoA
transaminase, and 4-hydroxybutyryl-CoA dehydrogenase; (p)
4-aminobutyrate CoA transferase, 4-aminobutyryl-CoA hydrolase,
4-aminobutyrate-CoA ligase, 4-aminobutyryl-CoA reductase (alcohol
forming), 4-aminobutyryl-CoA reductase, 4-aminobutan-1-ol
dehydrogenase, 4-aminobutan-1-ol oxidoreductase (deaminating) and
4-aminobutan-1-ol transaminase; (q) 4-aminobutyrate kinase,
4-aminobutyraldehyde dehydrogenase (phosphorylating),
4-aminobutan-1-ol dehydrogenase, 4-aminobutan-1-ol oxidoreductase
(deaminating), 4-aminobutan-1-ol transaminase,
[(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase
(deaminating), [(4-aminobutanolyl)oxy]phosphonic acid transaminase,
4-hydroxybutyryl-phosphate dehydrogenase, and
4-hydroxybutyraldehyde dehydrogenase (phosphorylating); (r)
alpha-ketoglutarate 5-kinase, 2,5-dioxopentanoic semialdehyde
dehydrogenase (phosphorylating), 2,5-dioxopentanoic acid reductase,
alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA
hydrolase, alpha-ketoglutaryl-CoA ligase, alpha-ketoglutaryl-CoA
reductase, 5-hydroxy-2-oxopentanoic acid dehydrogenase,
alpha-ketoglutaryl-CoA reductase (alcohol forming),
5-hydroxy-2-oxopentanoic acid decarboxylase, and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (s)
glutamate CoA transferase, glutamyl-CoA hydrolase, glutamyl-CoA
ligase, glutamate 5-kinase, glutamate-5-semialdehyde dehydrogenase
(phosphorylating), glutamyl-CoA reductase, glutamate-5-semialdehyde
reductase, glutamyl-CoA reductase (alcohol forming),
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating),
2-amino-5-hydroxypentanoic acid transaminase,
5-hydroxy-2-oxopentanoic acid decarboxylase,
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (t)
3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA
dehydratase, vinylacetyl-CoA A-isomerase, or 4-hydroxybutyryl-CoA
dehydratase; (u) homoserine deaminase, homoserine CoA transferase,
homoserine-CoA hydrolase, homoserine-CoA ligase, homoserine-CoA
deaminase, 4-hydroxybut-2-enoyl-CoA transferase,
4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA
ligase, 4-hydroxybut-2-enoate reductase, 4-hydroxybutyryl-CoA
transferase, 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA
ligase, or 4-hydroxybut-2-enoyl-CoA reductase; (v) succinyl-CoA
reductase (alcohol forming), 4-hydroxybutyryl-CoA hydrolase,
4-hydroxybutyryl-CoA ligase, or 4-hydroxybutanal dehydrogenase
(phosphorylating); (w) glutamate dehydrogenase, 4-aminobutyrate
oxidoreductase (deaminating), 4-aminobutyrate transaminase,
glutamate decarboxylase, 4-hydroxybutyryl-CoA hydrolase,
4-hydroxybutyryl-CoA ligase, or 4-hydroxybutanal dehydrogenase
(phosphorylating); (x) 4-aminobutyrate kinase; 4-aminobutyraldehyde
dehydrogenase (phosphorylating); 4-aminobutan-1-ol dehydrogenase;
and 4-aminobutan-1-ol oxidoreductase (deaminating) or
4-aminobutan-1-ol transaminase; (y) 4-aminobutyrate kinase;
[(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase (deaminating)
or [(4-aminobutanolyl)oxy]phosphonic acid transaminase;
4-hydroxybutyryl-phosphate dehydrogenase; and
4-hydroxybutyraldehyde dehydrogenase (phosphorylating); (z)
alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA
hydrolase, or alpha-ketoglutaryl-CoA ligase; alpha-ketoglutaryl-CoA
reductase (alcohol forming); and 5-hydroxy-2-oxopentanoic acid
decarboxylase; (aa) alpha-ketoglutarate CoA transferase,
alpha-ketoglutaryl-CoA hydrolase, or alpha-ketoglutaryl-CoA ligase;
alpha-ketoglutaryl-CoA reductase (alcohol forming); and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (bb)
alpha-ketoglutarate 5-kinase; 2,5-dioxopentanoic semialdehyde
dehydrogenase (phosphorylating); 2,5-dioxopentanoic acid reductase;
and 5-hydroxy-2-oxopentanoic acid decarboxylase; (cc)
alpha-ketoglutarate 5-kinase; 2,5-dioxopentanoic semialdehyde
dehydrogenase (phosphorylating); 2,5-dioxopentanoic acid reductase;
and 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);
(dd) alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA
hydrolase, or alpha-ketoglutaryl-CoA ligase; alpha-ketoglutaryl-CoA
reductase; 5-hydroxy-2-oxopentanoic acid dehydrogenase; and
5-hydroxy-2-oxopentanoic acid decarboxylase; (ee)
alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA
hydrolase, or alpha-ketoglutaryl-CoA ligase; alpha-ketoglutaryl-CoA
reductase; 5-hydroxy-2-oxopentanoic acid dehydrogenase; and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (ff)
glutamate CoA transferase, glutamyl-CoA hydrolase, or glutamyl-CoA
ligase; glutamyl-CoA reductase (alcohol forming);
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or
2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (gg)
glutamate 5-kinase; glutamate-5-semialdehyde dehydrogenase
(phosphorylating); 2-amino-5-hydroxypentanoic acid oxidoreductase
(deaminating) or 2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (hh)
glutamate CoA transferase, glutamyl-CoA hydrolase, or glutamyl-CoA
ligase; glutamyl-CoA reductase; glutamate-5-semialdehyde reductase;
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or
2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (ii)
glutamate 5-kinase; glutamate-5-semialdehyde dehydrogenase
(phosphorylating); glutamate-5-semialdehyde reductase;
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or
2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (jj)
homoserine deaminase; 4-hydroxybut-2-enoyl-CoA transferase,
4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA
ligase; 4-hydroxybut-2-enoyl-CoA reductase; (kk) homoserine CoA
transferase, homoserine-CoA hydrolase, or homoserine-CoA ligase;
homoserine-CoA deaminase; and 4-hydroxybut-2-enoyl-CoA reductase;
(ll) homoserine deaminase; 4-hydroxybut-2-enoate reductase; and
4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA hydrolase,
or 4-hydroxybutyryl-CoA ligase; (mm) (i) alpha-ketoglutarate
decarboxylase; or alpha-ketoglutarate dehydrogenase and
CoA-dependent succinate semialdehyde dehydrogenase; or
glutamate:succinate semialdehyde transaminase and glutamate
decarboxylase; (ii) 4-hydroxybutyrate dehydrogenase; (iii)
4-hydroxybutyryl-CoA transferase; or 4-hydroxybutyrate kinase and
phosphotrans-4-hydroxybutyrylase; and (iv) 4-hydroxybutyryl-CoA
reductase; (nn) (i) alpha-ketoglutarate decarboxylase; or
succinyl-CoA synthetase and CoA-dependent succinate semialdehyde
dehydrogenase; (ii) 4-hydroxybutyrate dehydrogenase; (iii)
4-hydroxybutyryl-CoA transferase; or 4-hydroxybutyrate kinase and
phosphotrans-4-hydroxybutyrylase; (oo) (i) alpha-ketoglutarate
decarboxylase; or glutamate dehydrogenase; glutamate decarboxylase;
and deaminating 4-aminobutyrate oxidoreductase or 4-aminobutyrate
transaminase; or alpha-ketoglutarate dehydrogenase and
CoA-dependent succinate semialdehyde dehydrogenase; (ii)
4-hydroxybutyrate dehydrogenase; and (iii) 4-hydroxybutyrate
kinase; phosphotrans-4-hydroxybutyrylase; 4-hydroxybutyryl-CoA
reductase; and 4-hydroxybutyraldehyde reductase; or
4-hydroxybutyrate kinase; phosphorylating 4-hydroxybutanal
dehydrogenase; and 4-hydroxybutyraldehyde reductase; or
4-hydroxybutyrate kinase; phosphotrans-4-hydroxybutyrylase; and
alcohol forming 4-hydroxybutyryl-CoA reductase; or
4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA hydrolase
or 4-hydroxybutyryl-CoA ligase; 4-hydroxybutyryl-CoA reductase; and
4-hydroxybutyraldehyde reductase; or 4-hydroxybutyryl-CoA
transferase or 4-hydroxybutyryl-CoA hydrolase or
4-hydroxybutyryl-CoA ligase; and alcohol forming
4-hydroxybutyryl-CoA reductase; (pp) (i) glutamate CoA transferase
or glutamyl-CoA hydrolase or glutamyl-CoA ligase; glutamyl-CoA
reductase; and glutamate-5-semialdehyde reductase; or glutamate CoA
transferase or glutamyl-CoA hydrolase or glutamyl-CoA ligase; and
alcohol forming glutamyl-CoA reductase; or glutamate 5-kinase;
phosphorylating glutamate-5-semialdehyde dehydrogenase; and
glutamate-5-semialdehyde reductase; (ii) deaminating
2-amino-5-hydroxypentanoic acid oxidoreductase or
2-amino-5-hydroxypentanoic acid transaminase; and (iii)
5-hydroxy-2-oxopentanoic acid decarboxylase; and
4-hydroxybutyraldehyde reductase; or decarboxylating
5-hydroxy-2-oxopentanoic acid dehydrogenase; 4-hydroxybutyryl-CoA
reductase; and 4-hydroxybutyraldehyde reductase; or decarboxylating
5-hydroxy-2-oxopentanoic acid dehydrogenase and alcohol forming
4-hydroxybutyryl-CoA reductase; (qq) a gamma-butyrolactone pathway
comprising a pathway selected from any of the pathways that produce
4-hydroxybutyryl-CoA or 4-hydroxybutyryl phosphate as shown in FIG.
1, 8-13, 58, 62-63 or 72-74, wherein gamma-butyrolactone is
produced enzymatically or by spontaneous chemical conversion.
[0184] In a particular embodiment, a microbial organism comprising
(i) can further comprise an exogenous nucleic acid encoding an
enzyme selected from a pyruvate:ferredoxin oxidoreductase, an
aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase,
a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an
acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase,
an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations
thereof. Additionally, a microbial organism comprising (ii) can
further comprise an exogenous nucleic acid encoding an enzyme
selected from an aconitase, an isocitrate dehydrogenase, a
succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a
malate dehydrogenase, and combinations thereof.
[0185] In a particular embodiment, a microbial organism can
comprise two, three, four, five, six or seven exogenous nucleic
acids each encoding a gamma-butyrolactone pathway enzyme. For
example, a microbial organism can comprise exogenous nucleic acids
encoding each of the enzymes selected from a gamma-butyrolactone
pathway shown in any of FIG. 1, 8-13, 58, 62, 63, or 72-74, in
particular pathways that produce 4-hydroxybutyryl-CoA and/or
4-hydroxybutyryl phosphate. Additionally, such a microbial organism
can comprise two, three, four or five exogenous nucleic acids each
encoding enzymes of (i), (ii) or (iii). For example, such a
microbial organism can comprising (i) can comprise three exogenous
nucleic acids encoding ATP-citrate lyase or citrate lyase, a
fumarate reductase, and an alpha-ketoglutarate:ferredoxin
oxidoreductase; a microbial organism comprising (ii) can comprise
five exogenous nucleic acids encoding pyruvate:ferredoxin
oxidoreductase, a phosphoenolpyruvate carboxylase, a
phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an
H.sub.2 hydrogenase; or a microbial organism comprising (iii) can
comprise two exogenous nucleic acids encoding CO dehydrogenase and
H.sub.2 hydrogenase. The invention additionally provides methods
for producing gamma-butyrolactone by culturing the non-naturally
occurring microbial organism under conditions and for a sufficient
period of time to produce gamma-butyrolactone.
[0186] 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 1,4-butanediol, 4-hydroxybutyrate and/or
gamma-butyrolactone or any 1,4-butanediol, 4-hydroxybutyrate and/or
gamma-butyrolactone 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 1,4-butanediol, 4-hydroxybutyrate and/or
gamma-butyrolactone or 1,4-butanediol, 4-hydroxybutyrate and/or
gamma-butyrolactone pathway intermediate, or for side products
generated in reactions diverging away from a 1,4-butanediol,
4-hydroxybutyrate and/or gamma-butyrolactone pathway. Isotopic
enrichment can be achieved for any target atom including, for
example, carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus,
chloride or other halogens.
[0187] 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 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.
[0188] 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
isotopic ratio of an uptake source can be obtained by selecting a
desired origin of the uptake source as found in nature For example,
as discussed herein, a natural source can be a biobased derived
from or synthesized by a biological organism or a source such as
petroleum-based products or the atmosphere. In some such
embodiments, a source of carbon, for example, can be selected from
a fossil fuel-derived carbon source, which can be relatively
depleted of carbon-14, or an environmental or atmospheric carbon
source, such as CO.sub.2, which can possess a larger amount of
carbon-14 than its petroleum-derived counterpart.
[0189] Isotopic enrichment is readily assessed by mass spectrometry
using techniques known in the art such as Stable Isotope Ratio Mass
Spectrometry (SIRMS) and Site-Specific Natural Isotopic
Fractionation by Nuclear Magnetic Resonance (SNIF-NMR). Such mass
spectral techniques can be integrated with separation techniques
such as liquid chromatography (LC) and/or high performance liquid
chromatography (HPLC).
[0190] 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".
[0191] 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.
[0192] 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.
[0193] 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.
[0194] 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.
[0195] 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.
[0196] 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.
[0197] 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).
[0198] Accordingly, in some embodiments, the present invention
provides 1,4-butanediol, 4-hydroxybutyrate, gamma-butyrolactone
and/or putrescine or a 1,4-butanediol, 4-hydroxybutyrate,
gamma-butyrolactone and/or putrescine pathway intermediate that has
a carbon-12, carbon-13, and carbon-14 ratio that reflects an
atmospheric carbon, also referred to as environmental carbon,
uptake source. For example, in some aspects the 1,4-butanediol,
4-hydroxybutyrate, gamma-butyrolactone and/or putrescine or a
1,4-butanediol, 4-hydroxybutyrate, gamma-butyrolactone and/or
putrescine 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 1,4-butanediol,
4-hydroxybutyrate, gamma-butyrolactone and/or putrescine or a
1,4-butanediol, 4-hydroxybutyrate, gamma-butyrolactone and/or
putrescine intermediate that has a carbon-12, carbon-13, and
carbon-14 ratio that reflects petroleum-based carbon uptake source.
In this aspect, the 1,4-butanediol, 4-hydroxybutyrate,
gamma-butyrolactone and/or putrescine or a 1,4-butanediol,
4-hydroxybutyrate, gamma-butyrolactone and/or putrescine
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 1,4-butanediol, 4-hydroxybutyrate, gamma-butyrolactone
and/or putrescine or a 1,4-butanediol, 4-hydroxybutyrate,
gamma-butyrolactone and/or putrescine 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.
[0199] Further, the present invention relates to biologically
produced 1,4-butanediol, 4-hydroxybutyrate, gamma-butyrolactone
and/or putrescine or 1,4-butanediol, 4-hydroxybutyrate,
gamma-butyrolactone and/or putrescine intermediate as disclosed
herein, and to the products derived therefrom, wherein the
1,4-butanediol, 4-hydroxybutyrate, gamma-butyrolactone and/or
putrescine or a 1,4-butanediol, 4-hydroxybutyrate,
gamma-butyrolactone and/or putrescine 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 1,4-butanediol,
4-hydroxybutyrate, gamma-butyrolactone and/or putrescine or a
bioderived 1,4-butanediol, 4-hydroxybutyrate, gamma-butyrolactone
and/or putrescine 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 1,4-butanediol,
4-hydroxybutyrate, gamma-butyrolactone and/or putrescine or a
bioderived 1,4-butanediol, 4-hydroxybutyrate, gamma-butyrolactone
and/or putrescine intermediate as disclosed herein, wherein the
bioderived product is chemically modified to generate a final
product. Methods of chemically modifying a bioderived product of
1,4-butanediol, 4-hydroxybutyrate, gamma-butyrolactone and/or
putrescine, 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 plastics, elastic fibers,
polyurethanes, polyesters, including polyhydroxyalkanoates such as
poly-4-hydroxybutyrate (P4HB) or co-polymers thereof,
poly(tetramethylene ether) glycol (PTMEG) (also referred to as
PTMO, polytetramethylene oxide) and polyurethane-polyurea
copolymers, referred to as spandex, elastane or Lycra.TM., nylons,
and the like, 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 plastics, elastic fibers,
polyurethanes, polyesters, including polyhydroxyalkanoates such as
poly-4-hydroxybutyrate (P4HB) or co-polymers thereof,
poly(tetramethylene ether) glycol (PTMEG) (also referred to as
PTMO, polytetramethylene oxide) and polyurethane-polyurea
copolymers, referred to as spandex, elastane or Lycra.TM., nylons,
and the like, are generated directly from or in combination with
bioderived 1,4-butanediol, 4-hydroxybutyrate, gamma-butyrolactone
and/or putrescine or a bioderived 1,4-butanediol,
4-hydroxybutyrate, gamma-butyrolactone and/or putrescine
intermediate as disclosed herein.
[0200] 1,4-butanediol, 4-hydroxybutyrate, gamma-butyrolactone
and/or putrescine are chemicals used in commercial and industrial
applications. Non-limiting examples of such applications include
production of plastics, elastic fibers, polyurethanes, polyesters,
including polyhydroxyalkanoates such as poly-4-hydroxybutyrate
(P4HB) or co-polymers thereof, poly(tetramethylene ether) glycol
(PTMEG) (also referred to as PTMO, polytetramethylene oxide) and
polyurethane-polyurea copolymers, referred to as spandex, elastane
or Lycra.TM., nylons, and the like. Moreover, 1,4-butanediol,
4-hydroxybutyrate, gamma-butyrolactone and/or putrescine are also
used as a raw material in the production of a wide range of
products including plastics, elastic fibers, polyurethanes,
polyesters, including polyhydroxyalkanoates such as
poly-4-hydroxybutyrate (P4HB) or co-polymers thereof,
poly(tetramethylene ether) glycol (PTMEG) (also referred to as
PTMO, polytetramethylene oxide) and polyurethane-polyurea
copolymers, referred to as spandex, elastane or Lycra.TM., nylons,
and the like. Accordingly, in some embodiments, the invention
provides biobased plastics, elastic fibers, polyurethanes,
polyesters, including polyhydroxyalkanoates such as
poly-4-hydroxybutyrate (P4HB) or co-polymers thereof,
poly(tetramethylene ether) glycol (PTMEG) (also referred to as
PTMO, polytetramethylene oxide) and polyurethane-polyurea
copolymers, referred to as spandex, elastane or Lycra.TM., nylons,
and the like, comprising one or more bioderived 1,4-butanediol,
4-hydroxybutyrate, gamma-butyrolactone and/or putrescine or
bioderived 1,4-butanediol, 4-hydroxybutyrate, gamma-butyrolactone
and/or putrescine intermediate produced by a non-naturally
occurring microorganism of the invention or produced using a method
disclosed herein.
[0201] 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.
[0202] In some embodiments, the invention provides plastics,
elastic fibers, polyurethanes, polyesters, including
polyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) or
co-polymers thereof, poly(tetramethylene ether) glycol (PTMEG)
(also referred to as PTMO, polytetramethylene oxide) and
polyurethane-polyurea copolymers, referred to as spandex, elastane
or Lycra.TM., nylons, and the like, comprising bioderived
1,4-butanediol, 4-hydroxybutyrate, gamma-butyrolactone and/or
putrescine or bioderived 1,4-butanediol, 4-hydroxybutyrate,
gamma-butyrolactone and/or putrescine intermediate, wherein the
bioderived 1,4-butanediol, 4-hydroxybutyrate, gamma-butyrolactone
and/or putrescine or bioderived 1,4-butanediol, 4-hydroxybutyrate,
gamma-butyrolactone and/or putrescine intermediate includes all or
part of the 1,4-butanediol, 4-hydroxybutyrate, gamma-butyrolactone
and/or putrescine or 1,4-butanediol, 4-hydroxybutyrate,
gamma-butyrolactone and/or putrescine intermediate used in the
production of plastics, elastic fibers, polyurethanes, polyesters,
including polyhydroxyalkanoates such as poly-4-hydroxybutyrate
(P4HB) or co-polymers thereof, poly(tetramethylene ether) glycol
(PTMEG) (also referred to as PTMO, polytetramethylene oxide) and
polyurethane-polyurea copolymers, referred to as spandex, elastane
or Lycra.TM., nylons, and the like. Thus, in some aspects, the
invention provides a biobased plastics, elastic fibers,
polyurethanes, polyesters, including polyhydroxyalkanoates such as
poly-4-hydroxybutyrate (P4HB) or co-polymers thereof,
poly(tetramethylene ether) glycol (PTMEG) (also referred to as
PTMO, polytetramethylene oxide) and polyurethane-polyurea
copolymers, referred to as spandex, elastane or Lycra.TM., nylons,
and the like, 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 1,4-butanediol, 4-hydroxybutyrate, gamma-butyrolactone
and/or putrescine or bioderived 1,4-butanediol, 4-hydroxybutyrate,
gamma-butyrolactone and/or putrescine intermediate as disclosed
herein. Additionally, in some aspects, the invention provides a
biobased plastics, elastic fibers, polyurethanes, polyesters,
including polyhydroxyalkanoates such as poly-4-hydroxybutyrate
(P4HB) or co-polymers thereof, poly(tetramethylene ether) glycol
(PTMEG) (also referred to as PTMO, polytetramethylene oxide) and
polyurethane-polyurea copolymers, referred to as spandex, elastane
or Lycra.TM., nylons, and the like, wherein the 1,4-butanediol,
4-hydroxybutyrate, gamma-butyrolactone and/or putrescine or
1,4-butanediol, 4-hydroxybutyrate, gamma-butyrolactone and/or
putrescine intermediate used in its production is a combination of
bioderived and petroleum derived 1,4-butanediol, 4-hydroxybutyrate,
gamma-butyrolactone and/or putrescine or 1,4-butanediol,
4-hydroxybutyrate, gamma-butyrolactone and/or putrescine
intermediate. For example, a biobased plastics, elastic fibers,
polyurethanes, polyesters, including polyhydroxyalkanoates such as
poly-4-hydroxybutyrate (P4HB) or co-polymers thereof,
poly(tetramethylene ether) glycol (PTMEG) (also referred to as
PTMO, polytetramethylene oxide) and polyurethane-polyurea
copolymers, referred to as spandex, elastane or Lycra.TM., nylons,
and the like, can be produced using 50% bioderived 1,4-butanediol,
4-hydroxybutyrate, gamma-butyrolactone and/or putrescine and 50%
petroleum derived 1,4-butanediol, 4-hydroxybutyrate,
gamma-butyrolactone and/or putrescine 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 plastics,
elastic fibers, polyurethanes, polyesters, including
polyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) or
co-polymers thereof, poly(tetramethylene ether) glycol (PTMEG)
(also referred to as PTMO, polytetramethylene oxide) and
polyurethane-polyurea copolymers, referred to as spandex, elastane
or Lycra.TM., nylons, and the like, using the bioderived
1,4-butanediol, 4-hydroxybutyrate, gamma-butyrolactone and/or
putrescine or bioderived 1,4-butanediol, 4-hydroxybutyrate,
gamma-butyrolactone and/or putrescine intermediate of the invention
are well known in the art.
[0203] 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 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 that reference to any of these metabolic constitutes
also references the gene or genes encoding the enzymes that
catalyze 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 as well as the reactants and products
of the reaction.
[0204] The production of 4-HB via biosynthetic modes using the
microbial organisms of the invention is particularly useful because
it can produce monomeric 4-HB. The non-naturally occurring
microbial organisms of the invention and their biosynthesis of 4-HB
and BDO family compounds also is particularly useful because the
4-HB product can be (1) secreted; (2) can be devoid of any
derivatizations such as Coenzyme A; (3) avoids thermodynamic
changes during biosynthesis; (4) allows direct biosynthesis of BDO,
and (5) allows for the spontaneous chemical conversion of 4-HB to
.gamma.-butyrolactone (GBL) in acidic pH medium. This latter
characteristic also is particularly useful for efficient chemical
synthesis or biosynthesis of BDO family compounds such as
1,4-butanediol and/or tetrahydrofuran (THF), for example.
[0205] Microbial organisms generally lack the capacity to
synthesize 4-HB and therefore any of the compounds disclosed herein
to be within the 1,4-butanediol family of compounds or known by
those in the art to be within the 1,4-butanediol family of
compounds. Moreover, organisms having all of the requisite
metabolic enzymatic capabilities are not known to produce 4-HB from
the enzymes described and biochemical pathways exemplified herein.
Rather, with the possible exception of a few anaerobic
microorganisms described further below, the microorganisms having
the enzymatic capability to use 4-HB as a substrate to produce, for
example, succinate. In contrast, the non-naturally occurring
microbial organisms of the invention can generate 4-HB, 4-HBal,
4-HBCoA, BDO and/or putrescine as a product. As described above,
the biosynthesis of 4-HB in its monomeric form is not only
particularly useful in chemical synthesis of BDO family of
compounds, it also allows for the further biosynthesis of BDO
family compounds and avoids altogether chemical synthesis
procedures.
[0206] The non-naturally occurring microbial organisms of the
invention that can produce 4-HB, 4-HBal, 4-HBCoA, BDO and/or
putrescine are produced by ensuring that a host microbial organism
includes functional capabilities for the complete biochemical
synthesis of at least one 4-HB, 4-HBal, 4-HBCoA, BDO and/or
putrscine biosynthetic pathway of the invention. Ensuring at least
one requisite 4-HB, 4-HBal, 4-HBCoA or BDO biosynthetic pathway
confers 4-HB biosynthesis capability onto the host microbial
organism.
[0207] Several 4-HB biosynthetic pathways are exemplified herein
and shown for purposes of illustration in FIG. 1. Additional 4-HB
and BDO pathways are described in FIGS. 8-13. One 4-HB biosynthetic
pathway includes the biosynthesis of 4-HB from succinate (the
succinate pathway). The enzymes participating in this 4-HB pathway
include CoA-independent succinic semialdehyde dehydrogenase and
4-hydroxybutanoate dehydrogenase. In this pathway, CoA-independent
succinic semialdehyde dehydrogenase (succinate reductase) catalyzes
the reverse reaction to the arrow shown in FIG. 1. Another 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. Three other 4-HB biosynthetic
pathways include the biosynthesis of 4-HB from
.alpha.-ketoglutarate (the .alpha.-ketoglutarate pathways). Hence,
a third 4-HB biosynthetic pathway is the biosynthesis of succinic
semialdehyde through glutamate:succinic semialdehyde transaminase,
glutamate decarboxylase and 4-hydroxybutanoate dehydrogenase. A
fourth 4-HB biosynthetic pathway also includes the biosynthesis of
4-HB from .alpha.-ketoglutarate, but utilizes .alpha.-ketoglutarate
decarboxylase to catalyze succinic semialdehyde synthesis.
4-hydroxybutanoate dehydrogenase catalyzes the conversion of
succinic semialdehyde to 4-HB. A fifth 4-HB biosynthetic pathway
includes the biosynthesis from .alpha.-ketoglutarate through
succinyl-CoA and utilizes .alpha.-ketoglutarate dehydrogenase to
produce succinyl-CoA, which funnels into the succinyl-CoA pathway
described above. Each of these 4-HB biosynthetic pathways, their
substrates, reactants and products are described further below in
the Examples. As described herein, 4-HB can further be
biosynthetically converted to BDO by inclusion of appropriate
enzymes to produce BDO (see Example). Thus, it is understood that a
4-HB pathway can be used with enzymes for converting 4-HB to BDO to
generate a BDO pathway.
[0208] As disclosed herein, the product 4-hydroxybutyrate, as well
as other intermediates and/or products, such as succinate, are
carboxylic acids, which can occur in various ionized forms,
including fully protonated, partially protonated, and fully
deprotonated forms. Accordingly, the suffix "-ate," or the acid
form, can be used interchangeably to describe both the free acid
form as well as any deprotonated form, in particular since the
ionized form is known to depend on the pH in which the compound is
found. It is understood that carboxylate products or intermediates
includes ester forms of carboxylate products or pathway
intermediates, such as O-carboxylate and S-carboxylate esters. O-
and S-carboxylates can include lower alkyl, that is C1 to C6,
branched or straight chain carboxylates. Some such O- or
S-carboxylates include, without limitation, methyl, ethyl,
n-propyl, n-butyl, i-propyl, sec-butyl, and tert-butyl, pentyl,
hexyl O- or S-carboxylates, any of which can further possess an
unsaturation, providing for example, propenyl, butenyl, pentyl, and
hexenyl O- or S-carboxylates. O-carboxylates can be the product of
a biosynthetic pathway. Exemplary O-carboxylates accessed via
biosynthetic pathways can include, without limitation, methyl
4-hydroxybutyrate, ethyl 4-hydroxybutyrate, and n-propyl
4-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.
[0209] 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, 4-HBal, 4-HBCoA, BDO or putrescine biosynthetic pathways.
Depending on the host microbial organism chosen for biosynthesis,
nucleic acids for some or all of a particular 4-HB, 4-HBal,
4-HBCoA, BDO or putrescine biosynthetic pathway can be expressed.
For example, if a chosen host is deficient in one or more enzymes
in a desired biosynthetic pathway, for example, the succinate to
4-HB pathway, then expressible nucleic acids for the deficient
enzyme(s), for example, both CoA-independent succinic semialdehyde
dehydrogenase and 4-hydroxybutanoate dehydrogenase in this example,
are introduced into the host for subsequent exogenous expression.
Alternatively, if the chosen host exhibits endogenous expression of
some pathway enzymes, but is deficient in others, then an encoding
nucleic acid is needed for the deficient enzyme(s) to achieve 4-HB,
4-HBal, 4-HBCoA, BDO or putrescine biosynthesis. For example, if
the chosen host exhibits endogenous CoA-independent succinic
semialdehyde dehydrogenase, but is deficient in 4-hydroxybutanoate
dehydrogenase, then an encoding nucleic acid is needed for this
enzyme to achieve 4-HB 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 4-HB, 4-HBal, 4-HBCoA,
BDO and/or putrescine.
[0210] 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:succinic semialdehyde transaminase,
glutamate decarboxylase and/or 4-hydroxybutanoate dehydrogenase, or
.alpha.-ketoglutarate decarboxylase and 4-hydroxybutanoate
dehydrogenase. One skilled in the art can readily determine pathway
enzymes for production of 4-HB or BDO, as disclosed herein.
[0211] Depending on the 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine
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 4-HB,
4-HB, 4-HBCoA, BDO or putrescine pathway-encoding nucleic acid and
up to all encoding nucleic acids for one or more 4-HB or BDO
biosynthetic pathways. For example, 4-HB, 4-HBal, 4-HBCoA, BDO or
putrescine 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 4-HB, 4-HB, 4-HBCoA, BDO or putrescine
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. If desired,
exogenous expression of all enzymes or proteins in a pathway for
production of 4-HB, 4-HB, 4-HBCoA, BDO or putrescine can be
included. For example, 4-HB 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 eight 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,
.alpha.-ketoglutarate dehydrogenase and 4-hydroxybutanoate
dehydrogenase.
[0212] 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, 4-HBal, 4-HBCoA, BDO or putrescine pathway
deficiencies of the selected host microbial organism. Therefore, a
non-naturally occurring microbial organism of the invention can
have one, two, three, four, five, six, seven, eight or up to all
nucleic acids encoding the enzymes disclosed herein constituting
one or more 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine biosynthetic
pathways. In some embodiments, the non-naturally occurring
microbial organisms also can include other genetic modifications
that facilitate or optimize 4-HB, 4-HBal, 4-HBCoA, BDO or
putrescine 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 pathway precursors such as succinate, succinyl-CoA,
.alpha.-ketoglutarate, 4-aminobutyrate, glutamate, acetoacetyl-CoA,
and/or homoserine.
[0213] Generally, a host microbial organism is selected such that
it produces the precursor of a 4-HB, 4-HBal, 4-HBCoA, BDO or
putrescine 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, succinyl-CoA,
.alpha.-ketoglutarate, 4-aminobutyrate, glutamate, acetoacetyl-CoA,
and homoserine are produced naturally in a host organism such as E.
coli. A host organism can be engineered to increase production of a
precursor, as disclosed herein. In addition, a microbial organism
that has been engineered to produce a desired precursor can be used
as a host organism and further engineered to express enzymes or
proteins of a 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine pathway.
[0214] 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, 4-HBal, 4-HBCoA, BDO
or putrescine. In this specific embodiment it can be useful to
increase the synthesis or accumulation of a 4-HB, 4-HBal, 4-HBCoA,
BDO or putrescine pathway product to, for example, drive 4-HB,
4-HBal, 4-HBCoA, BDO or putrescine pathway reactions toward 4-HB,
4-HBal, 4-HBCoA, BDO or putrescine production. Increased synthesis
or accumulation can be accomplished by, for example, overexpression
of nucleic acids encoding one or more of the 4-HB, 4-HBal, 4-HBCoA,
BDO or putrescine pathway enzymes disclosed herein. Over expression
of the 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine 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,
4-HBal, 4-HBCoA, BDO or putrescine producing microbial organisms of
the invention through overexpression of one, two, three, four,
five, six and so forth up to all nucleic acids encoding 4-HB,
4-HBal, 4-HBCoA, BDO or putrescine 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, 4-HBal, 4-HBCoA, BDO or
putrescine biosynthetic pathway.
[0215] 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 (see Examples).
[0216] "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.
[0217] 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.
[0218] Sources of encoding nucleic acids for a 4-HB, 4-HBal,
4-HBCoA, BDO or putrescine pathway enzyme 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,
Saccharomyces cerevisiae, Saccharomyces kluyveri, Clostridium
kluyveri, Clostridium acetobutylicum, Clostridium beijerinckii,
Clostridium saccharoperbutylacetonicum, Clostridium perfringens,
Clostridium difficile, Clostridium botulinum, Clostridium
tyrobutyricum, Clostridium tetanomorphum, Clostridium tetani,
Clostridium propionicum, Clostridium aminobutyricum, Clostridium
subterminale, Clostridium sticklandii, Ralstonia eutropha,
Mycobacterium bovis, Mycobacterium tuberculosis, Porphyromonas
gingivalis, Arabidopsis thaliana, Thermus thermophilus, Pseudomonas
species, including Pseudomonas aeruginosa, Pseudomonas putida,
Pseudomonas stutzeri, Pseudomonas fluorescens, Homo sapiens,
Oryctolagus cuniculus, Rhodobacter spaeroides, Thermoanaerobacter
brockii, Metallosphaera sedula, Leuconostoc mesenteroides,
Chloroflexus aurantiacus, Roseiflexus castenholzii, Erythrobacter,
Simmondsia chinensis, Acinetobacter species, including
Acinetobacter calcoaceticus and Acinetobacter baylyi, Porphyromonas
gingivalis, Sulfolobus tokodaii, Sulfolobus solfataricus,
Sulfolobus acidocaldarius, Bacillus subtilis, Bacillus cereus,
Bacillus megaterium, Bacillus brevis, Bacillus pumilus, Rattus
norvegicus, Klebsiella pneumonia, Klebsiella oxytoca, Euglena
gracilis, Treponema denticola, Moorella thermoacetica, Thermotoga
maritima, Halobacterium salinarum, Geobacillus stearothermophilus,
Aeropyrum pernix, Sus scrofa, Caenorhabditis elegans,
Corynebacterium glutamicum, Acidaminococcus fermentans, Lactococcus
lactis, Lactobacillus plantarum, Streptococcus thermophilus,
Enterobacter aerogenes, Candida, Aspergillus terreus, Pedicoccus
pentosaceus, Zymomonas mobilus, Acetobacter pasteurians,
Kluyveromyces lactis, Eubacterium barkeri, Bacteroides capillosus,
Anaerotruncus colihominis, Natranaerobius thermophilusm,
Campylobacter jejuni, Haemophilus influenzae, Serratia marcescens,
Citrobacter amalonaticus, Myxococcus xanthus, Fusobacterium
nuleatum, Penicillium chrysogenum, marine gamma proteobacterium,
butyrate producing bacterium, Nocardia iowensis, Nocardia
farcinica, Streptomyces griseus, Schizosaccharomyces pombe,
Geobacillus thermoglucosidasius, Salmonella typhimurium, Vibrio
cholera, Heliobacter pylori, Nicotiana tabacum, Oryza sativa,
Haloferax mediterranei, Agrobacterium tumefaciens, Achromobacter
denitrificans, Fusobacterium nucleatum, Streptomyces clavuligenus,
Acinetobacter baumanii, Mus musculus, Lachancea kluyveri,
Trichomonas vaginalis, Trypanosoma brucei, Pseudomonas stutzeri,
Bradyrhizobium japonicum, Mesorhizobium loti, Bos taurus, Nicotiana
glutinosa, Vibrio vulnificus, Selenomonas ruminantium, Vibrio
parahaemolyticus, Archaeoglobus fulgidus, Haloarcula marismortui,
Pyrobaculum aerophilum, Mycobacterium smegmatis MC2 155,
Mycobacterium avium subsp. paratuberculosis K-10, Mycobacterium
marinum M, Tsukamurella paurometabola DSM 20162, Cyanobium PCC7001,
Dictyostelium discoideum AX4, and others disclosed herein or
available as source organisms for corresponding genes (see
Examples). For example, microbial organisms having 4-HB, 4-HBal,
4-HBCoA, BDO or putrescine biosynthetic production are exemplified
herein with reference to E. coli and yeast hosts. 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 4-HB, 4-HBal, 4-HBCoA, BDO or
putrescine 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 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine
and other compounds of the invention described herein with
reference to a particular organism such as E. coli or yeast 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.
[0219] In some instances, such as when an alternative 4-HB, 4-HBal,
BDO or putrescine biosynthetic pathway exists in an unrelated
species, 4-HB, 4-HBal, BDO or putrescine biosynthesis can be
conferred onto the host species by, for example, exogenous
expression of a paralog or paralogs from the unrelated species that
catalyzes a similar, yet non-identical metabolic reaction to
replace the referenced reaction. Because certain differences among
metabolic networks exist between different organisms, those skilled
in the art will understand that the actual gene usage between
different organisms may differ. However, given the teachings and
guidance provided herein, those skilled in the art also will
understand that the teachings and methods of the invention can be
applied to all microbial organisms using the cognate metabolic
alterations to those exemplified herein to construct a microbial
organism in a species of interest that will synthesize 4-HB, such
as monomeric 4-HB, 4-HBal, BDO or putrescine.
[0220] Host microbial organisms can be selected from, and the
non-naturally occurring microbial organisms generated in, for
example, bacteria, yeast, fungus or any of a variety of other
microorganisms applicable to fermentation processes. Exemplary
bacteria include species selected from Escherichia coli, Klebsiella
oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus
succinogenes, Mannheimia succiniciproducens, Rhizobium etli,
Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter
oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus
plantarum, Streptomyces coelicolor, Clostridium acetobutylicum,
Pseudomonas fluorescens, and Pseudomonas putida. Exemplary yeasts
or fungi include species selected from Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces
marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris,
Rhizopus arrhizus, Rhizobus oryzae, and the like. E. coli is a
particularly useful host organisms since it is a well characterized
microbial organism suitable for genetic engineering. Other
particularly useful host organisms include yeast such as
Saccharomyces cerevisiae. It is understood that any suitable
microbial host organism can be used to introduce metabolic and/or
genetic modifications to produce a desired product.
[0221] Methods for constructing and testing the expression levels
of a non-naturally occurring 4-HB-, 4-HBal-, 4-HBCoA-, BDO-, or
putrescine-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); Ausubel et al., Current
Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.
(1999). 4-HB and GBL can be separated by, for example, HPLC using a
Spherisorb 5 ODS1 column and a mobile phase of 70% 10 mM phosphate
buffer (pH=7) and 30% methanol, and detected using a UV detector at
215 nm (Hennessy et al. 2004, J. Forensic Sci. 46(6):1-9). BDO is
detected by gas chromatography or by HPLC and refractive index
detector using an Aminex HPX-87H column and a mobile phase of 0.5
mM sulfuric acid (Gonzalez-Pajuelo et al., Met. Eng. 7:329-336
(2005)).
[0222] Exogenous nucleic acid sequences involved in a pathway for
production of 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine 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.
[0223] An expression vector or vectors can be constructed to harbor
one or more 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine biosynthetic
pathway and/or one or more biosynthetic 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.
[0224] 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 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine pathway enzyme
in sufficient amounts to produce 4-HB, such as monomeric 4-HB,
4-HBal, 4-HBCoA, BDO or putrescine. It is understood that the
microbial organisms of the invention are cultured under conditions
sufficient to produce 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine.
Exemplary levels of expression for 4-HB, 4-HBal, 4-HBCoA, BDO or
putrescine enzymes in each pathway are described further below in
the Examples. Following the teachings and guidance provided herein,
the non-naturally occurring microbial organisms of the invention
can achieve biosynthesis of 4-HB, such as monomeric 4-HB, 4-HBal,
4-HBCoA, BDO or putrescine resulting in intracellular
concentrations between about 0.1-200 mM or more, for example,
0.1-25 mM or more. Generally, the intracellular concentration of
4-HB, such as monomeric 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine is
between about 3-150 mM or more, particularly about 5-125 mM or
more, and more particularly between about 8-100 mM, for example,
about 3-20 mM, particularly between about 5-15 mM and more
particularly between about 8-12 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. In
particular embodiments, the microbial organisms of the invention,
particularly strains such as those disclosed herein (see Examples
XII-XIX and Table 28), can provide improved production of a desired
product such as 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine by
increasing the production of 4-HB, 4-HBal, 4-HBCoA, BDO or
putrescine and/or decreasing undesirable byproducts. Such
production levels include, but are not limited to, those disclosed
herein and including from about 1 gram to about 25 grams per liter,
for example about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, or even higher amounts of
product per liter.
[0225] In addition to the culturing and fermentation conditions
disclosed herein, growth condition for achieving biosynthesis of
BDO, 4-HB, 4-HBCoA, 4-HBal and/or putrescine can include the
addition of an osmoprotectant to the culturing conditions. In
certain embodiments, the non-naturally occurring microbial
organisms of the invention can be sustained, cultured or fermented
as described herein in the presence of an osmoprotectant. Briefly,
an osmoprotectant refers to a compound that acts as an osmolyte and
helps a microbial organism as described herein survive osmotic
stress. Osmoprotectants include, but are not limited to, betaines,
amino acids, and the sugar trehalose. Non-limiting examples of such
are glycine betaine, praline betaine, dimethylthetin,
dimethylslfonioproprionate, 3-dimethylsulfonio-2-methylproprionate,
pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine and
ectoine. In one aspect, the osmoprotectant is glycine betaine. It
is understood to one of ordinary skill in the art that the amount
and type of osmoprotectant suitable for protecting a microbial
organism described herein from osmotic stress will depend on the
microbial organism used. The amount of osmoprotectant in the
culturing conditions can be, for example, no more than about 0.1
mM, no more than about 0.5 mM, no more than about 1.0 mM, no more
than about 1.5 mM, no more than about 2.0 mM, no more than about
2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no
more than about 7.0 mM, no more than about 10 mM, no more than
about 50 mM, no more than about 100 mM or no more than about 500
mM.
[0226] In some embodiments, culture conditions include anaerobic or
substantially anaerobic growth or maintenance conditions. Exemplary
anaerobic conditions have been described previously and are well
known in the art. Exemplary anaerobic conditions for fermentation
processes are described herein and are described, for example, in
U.S. publication 2009/0047719, filed Aug. 10, 2007. Any of these
conditions can be employed with the non-naturally occurring
microbial organisms as well as other anaerobic conditions well
known in the art. Under such anaerobic conditions or substantially
anaerobic, the 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine producers
can synthesize 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine 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, 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine producing
microbial organisms can produce 4-HB, 4-HBal, 4-HBCoA, BDO or
putrescine intracellularly and/or secrete the product into the
culture medium.
[0227] 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.
[0228] As described herein, one exemplary growth condition for
achieving biosynthesis of 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine
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.
[0229] The invention also provides a non-naturally occurring
microbial biocatalyst including a microbial organism having
4-hydroxybutanoic acid (4-HB) and 1,4-butanediol (BDO) biosynthetic
pathways that include at least one exogenous nucleic acid encoding
4-hydroxybutanoate dehydrogenase, CoA-independent succinic
semialdehyde dehydrogenase, succinyl-CoA synthetase, CoA-dependent
succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:CoA
transferase, glutamate:succinic semialdehyde transaminase,
glutamate decarboxylase, CoA-independent aldehyde dehydrogenase,
CoA-dependent aldehyde dehydrogenase or alcohol dehydrogenase,
wherein the exogenous nucleic acid is expressed in sufficient
amounts to produce 1,4-butanediol (BDO). 4-Hydroxybutyrate:CoA
transferase also is known as 4-hydroxybutyryl CoA:acetyl-CoA
transferase. Additional 4-HB or BDO pathway enzymes are also
disclosed herein (see Examples and FIGS. 8-13).
[0230] The invention further provides non-naturally occurring
microbial biocatalyst including a microbial organism having
4-hydroxybutanoic acid (4-HB) and 1,4-butanediol (BDO) biosynthetic
pathways, the pathways include at least one exogenous nucleic acid
encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase,
CoA-dependent succinic semialdehyde dehydrogenase,
4-hydroxybutyrate:CoA transferase, 4-butyrate kinase,
phosphotransbutyrylase, .alpha.-ketoglutarate decarboxylase,
aldehyde dehydrogenase, alcohol dehydrogenase or an
aldehyde/alcohol dehydrogenase, wherein the exogenous nucleic acid
is expressed in sufficient amounts to produce 1,4-butanediol
(BDO).
[0231] Non-naturally occurring microbial organisms also can be
generated which biosynthesize BDO. As with the 4-HB producing
microbial organisms of the invention, the BDO producing microbial
organisms also can produce intracellularly or secret the BDO into
the culture medium. Following the teachings and guidance provided
previously for the construction of microbial organisms that
synthesize 4-HB, additional BDO pathways can be incorporated into
the 4-HB producing microbial organisms to generate organisms that
also synthesize BDO and other BDO family compounds. The chemical
synthesis of BDO and its downstream products are known. The
non-naturally occurring microbial organisms of the invention
capable of BDO biosynthesis circumvent these chemical synthesis
using 4-HB as an entry point as illustrated in FIG. 1. As described
further below, the 4-HB producers also can be used to chemically
convert 4-HB to GBL and then to BDO or THF, for example.
Alternatively, the 4-HB producers can be further modified to
include biosynthetic capabilities for conversion of 4-HB and/or GBL
to BDO.
[0232] The additional 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. 1 as steps 9-13. One such pathway includes, for
example, the enzyme activies necessary to carryout the reactions
shown as steps 9, 12 and 13 in FIG. 1, where the aldehyde and
alcohol dehydrogenases can be separate enzymes or a multifunctional
enzyme having both aldehyde and alcohol dehydrogenase activity.
Another such pathway includes, for example, the enzyme activities
necessary to carry out the reactions shown as steps 10, 11, 12 and
13 in FIG. 1, also where the aldehyde and alcohol dehydrogenases
can be separate enzymes or a multifunctional enzyme having both
aldehyde and alcohol dehydrogenase activity. Accordingly, the
additional 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 a
4-hydroxybutyrate:CoA transferase, butyrate kinase,
phosphotransbutyrylase, CoA-independent aldehyde dehydrogenase,
CoA-dependent aldehyde dehydrogenase or an alcohol dehydrogenase.
In the absence of endogenous acyl-CoA synthetase capable of
modifying 4-HB, the non-naturally occurring 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 BDO pathway activity and catalyze
the conversions illustrated in FIG. 1 with a 4-HB substrate.
Therefore, these enzymes also can be referred to herein as
4-hydroxybutyrate kinase and phosphotranshydroxybutyrylase
respectively.
[0233] Exemplary alcohol and aldehyde dehydrogenases that can be
used for these in vivo conversions from 4-HB to BDO are listed
below in Table 1.
TABLE-US-00001 TABLE1 Alcohol and Aldehyde Dehydrogenases for
Conversion of 4-HB to BDO. ALCOHOL DEHYDROGENASES ec:1.1.1.1
alcohol dehydrogenase ec:1.1.1.2 alcohol dehydrogenase (NADP+)
ec:1.1.1.4 (R,R)-butanediol dehydrogenase ec:1.1.1.5 acetoin
dehydrogenase ec:1.1.1.6 glycerol dehydrogenase ec:1.1.1.7
propanediol-phosphate dehydrogenase ec:1.1.1.8 glycerol-3-phosphate
dehydrogenase (NAD+) ec:1.1.1.11 D-arabinitol 4-dehydrogenase
ec:1.1.1.12 L-arabinitol 4-dehydrogenase ec:1.1.1.13 L-arabinitol
2-dehydrogenase ec:1.1.1.14 L-iditol 2-dehydrogenase ec:1.1.1.15
D-iditol 2-dehydrogenase ec:1.1.1.16 galactitol 2-dehydrogenase
ec:1.1.1.17 mannitol-l-phosphate 5- dehydrogenase ec:1.1.1.18
inositol 2-dehydrogenase ec:1.1.1.21 aldehyde reductase ec:1.1.1.23
histidinol dehydrogenase ec:1.1.1.26 glyoxylate reductase
ec:1.1.1.27 L-lactate dehydrogenase ec:1.1.1.28 D-lactate
dehydrogenase ec:1.1.1.29 glycerate dehydrogenase ec:1.1.1.30
3-hydroxybutyrate dehydrogenase ec:1.1.1.31 3-hydroxyisobutyrate
dehydrogenase ec:1.1.1.35 3-hydroxyacyl-CoA dehydrogenase
ec:1.1.1.36 acetoacetyl-CoA reductase ec:1.1.1.37 malate
dehydrogenase ec:1.1.1.38 malate dehydrogenase
(oxaloacetate-decarboxylating) ec:1.1.1.39 malate dehydrogenase
(decarboxylating) ec:1.1.1.40 malate dehydrogenase
(oxaloacetate-decarboxylating) (NADP+) ec:1.1.1.41 isocitrate
dehydrogenase (NAD+) ec:1.1.1.42 isocitrate dehydrogenase (NADP+)
ec:1.1.1.54 allyl-alcohol dehydrogenase ec:1.1.1.55 lactaldehyde
reductase (NADPH) ec:1.1.1.56 ribitol 2-dehydrogenase ec:1.1.1.59
3-hydroxypropionate dehydrogenase ec:1.1.1.60
2-hydroxy-3-oxopropionate reductase ec:1.1.1.61 4-hydroxybutyrate
dehydrogenase ec:1.1.1.66 omega-hydroxydecanoate dehydrogenase
ec:1.1.1.67 mannitol 2-dehydrogenase ec:1.1.1.71 alcohol
dehydrogenase [NAD(P)+] ec:1.1.1.72 glycerol dehydrogenase (NADP+)
ec:1.1.1.73 octanol dehydrogenase ec:1.1.1.75 (R)-aminopropanol
dehydrogenase ec:1.1.1.76 (S,S)-butanediol dehydrogenase
ec:1.1.1.77 lactaldehyde reductase ec:1.1.1.78 methylglyoxal
reductase (NADH- dependent) ec:1.1.1.79 glyoxylate reductase
(NADP+) ec:1.1.1.80 isopropanol dehydrogenase (NADP+) ec:1.1.1.81
hydroxypyruvate reductase ec:1.1.1.82 malate dehydrogenase (NADP+)
ec:1.1.1.83 D-malate dehydrogenase (decarboxylating) ec:1.1.1.84
dimethylmalate dehydrogenase ec:1.1.1.85 3-isopropylmalate
dehydrogenase ec:1.1.1.86 ketol-acid reductoisomerase ec:1.1.1.87
homoisocitrate dehydrogenase ec:1.1.1.88 hydroxymethylglutaryl-CoA
reductase ec:1.1.1.90 aryl-alcohol dehydrogenase ec:1.1.1.91
aryl-alcohol dehydrogenase (NADP+) ec:1.1.1.92 oxaloglycolate
reductase (decarboxylating) ec:1.1.1.94 glycerol-3-phosphate
dehydrogenase [NAD(P)+] ec:1.1.1.95 phosphoglycerate dehydrogenase
ec:1.1.1.97 3-hydroxybenzyl-alcohol dehydrogenase ec:1.1.1.101
acylglycerone-phosphate reductase ec:1.1.1.103 L-threonine
3-dehydrogenase ec:1.1.1.104 4-oxoproline reductase ec:1.1.1.105
retinol dehydrogenase ec:1.1.1.110 indolelactate dehydrogenase
ec:1.1.1.112 indanol dehydrogenase ec:1.1.1.113 L-xylose
1-dehydrogenase ec:1.1.1.129 L-threonate 3-dehydrogenase
ec:1.1.1.137 ribitol-5-phosphate 2- dehydrogenase ec:1.1.1.138
mannitol 2-dehydrogenase (NADP+) ec:1.1.1.140 sorbitol-6-phosphate
2- dehydrogenase ec:1.1.1.142 D-pinitol dehydrogenase ec:1.1.1.143
sequoyitol dehydrogenase ec:1.1.1.144 perillyl-alcohol
dehydrogenase ec:1.1.1.156 glycerol 2-dehydrogenase (NADP+)
ec:1.1.1.157 3-hydroxybutyryl-CoA dehydrogenase ec:1.1.1.163
cyclopentanol dehydrogenase ec:1.1.1.164 hexadecanol dehydrogenase
ec:1.1.1.165 2-alkyn-l-ol dehydrogenase ec:1.1.1.166
hydroxycyclohexanecarboxylate dehydrogenase ec:1.1.1.167
hydroxymalonate dehydrogenase ec:1.1.1.174 cyclohexane-1,2-diol
dehydrogenase ec:1.1.1.177 glycerol-3-phosphate 1- dehydrogenase
(NADP+) ec:1.1.1.178 3-hydroxy-2-methylbutyryl-CoA dehydrogenase
ec:1.1.1.185 L-glycol dehydrogenase ec:1.1.1.190
indole-3-acetaldehyde reductase (NADH) ec:1.1.1.191
indole-3-acetaldehyde reductase (NADPH) ec:1.1.1.192
long-chain-alcohol dehydrogenase ec:1.1.1.194 coniferyl-alcohol
dehydrogenase ec:1.1.1.195 cinnamyl-alcohol dehydrogenase
ec:1.1.1.198 (+)-borneol dehydrogenase ec:1.1.1.202 1,3-propanediol
dehydrogenase ec:1.1.1.207 (-)-menthol dehydrogenase ec:1.1.1.208
(+)-neomenthol dehydrogenase ec:1.1.1.216 farnesol dehydrogenase
ec:1.1.1.217 benzyl-2-methyl-hydroxybutyrate dehydrogenase
ec:1.1.1.222 (R)-4-hydroxyphenyllactate dehydrogenase ec:1.1.1.223
isopiperitenol dehydrogenase ec:1.1.1.226
4-hydroxycyclohexanecarboxylate dehydrogenase ec:1.1.1.229 diethyl
2-methyl-3-oxosuccinate reductase ec:1.1.1.237
hydroxyphenylpyruvate reductase ec:1.1.1.244 methanol dehydrogenase
ec:1.1.1.245 cyclohexanol dehydrogenase ec:1.1.1.250 D-arabinitol
2-dehydrogenase ec:1.1.1.251 galactitol 1-phosphate 5-
dehydrogenase ec:1.1.1.255 mannitol dehydrogenase ec:1.1.1.256
fluoren-9-ol dehydrogenase ec:1.1.1.257 4-
(hydroxymethyl)benzenesulfonate dehydrogenase ec:1.1.1.258
6-hydroxyhexanoate dehydrogenase ec:1.1.1.259 3-hydroxypimeloyl-CoA
dehydrogenase ec:1.1.1.261 glycerol-1 -phosphate dehydrogenase
[NAD(P)+] ec:1.1.1.265 3-methylbutanal reductase ec:1.1.1.283
methylglyoxal reductase (NADPH- dependent) ec:1.1.1.286
isocitrate-homoisocitrate dehydrogenase ec:1.1.1.287 D-arabinitol
dehydrogenase (NADP+) butanol dehydrogenase ALDEHYDE DEHYDROGENASES
ec:1.2.1.2 formate dehydrogenase ec:1.2.1.3 aldehyde dehydrogenase
(NAD+) ec:1.2.1.4 aldehyde dehydrogenase (NADP+) ec:1.2.1.5
aldehyde dehydrogenase [NAD(P)+] ec:1.2.1.7 benzaldehyde
dehydrogenase (NADP+) ec:1.2.1.8 betaine-aldehyde dehydrogenase
ec:1.2.1.9 glyceraldehyde-3-phosphate dehydrogenase (NADP+)
ec:1.2.1.10 acetaldehyde dehydrogenase (acetylating) ec:1.2.1.11
aspartate-semialdehyde dehydrogenase ec:1.2.1.12
glyceraldehyde-3-phosphate dehydrogenase (phosphorylating)
ec:1.2.1.13 glyceraldehyde-3-phosphate dehydrogenase (NADP+)
(phosphorylating) ec:1.2.1.15 malonate-semialdehyde dehydrogenase
ec:1.2.1.16 succinate-semialdehyde dehydrogenase [NAD(P)+]
ec:1.2.1.17 glyoxylate dehydrogenase (acylating) ec:1.2.1.18
malonate-semialdehyde dehydrogenase (acetylating) ec:1.2.1.19
aminobutyraldehyde dehydrogenase ec:1.2.1.20 glutarate-semialdehyde
dehydrogenase ec:1.2.1.21 glycolaldehyde dehydrogenase ec:1.2.1.22
lactaldehyde dehydrogenase ec:1.2.1.23 2-oxoaldehyde dehydrogenase
(NAD+) ec:1.2.1.24 succinate-semialdehyde dehydrogenase ec:1.2.1.25
2-oxoisovalerate dehydrogenase (acylating) ec: 1.2.1.26
2,5-dioxovalerate dehydrogenase ec:1.2.1.27
methylmalonate-semialdehyde dehydrogenase (acylating) ec:1.2.1.28
benzaldehyde dehydrogenase (NAD+) ec:1.2.1.29 aryl-aldehyde
dehydrogenase ec:1.2.1.30 aryl-aldehyde dehydrogenase (NADP+)
ec:1.2.1.31 L-aminoadipate-semialdehyde dehydrogenase ec:1.2.1.32
aminomuconate-semialdehyde dehydrogenase ec:1.2.1.36 retinal
dehydrogenase ec:1.2.1.39 phenylacetaldehyde dehydrogenase
ec:1.2.1.41 glutamate-5-semialdehyde dehydrogenase ec:1.2.1.42
hexadecanal dehydrogenase (acylating) ec:1.2.1.43 formate
dehydrogenase (NADP+) ec:1.2.1.45 4-carboxy-2-hydroxymuconate-6-
semialdehyde dehydrogenase ec:1.2.1.46 formaldehyde dehydrogenase
ec:1.2.1.47 4-trimethylammoniobutyraldehyde dehydrogenase
ec:1.2.1.48 long-chain-aldehyde dehydrogenase ec:1.2.1.49
2-oxoaldehyde dehydrogenase (NADP+) ec:1.2.1.51 pyruvate
dehydrogenase (NADP+) ec:1.2.1.52 oxoglutarate dehydrogenase
(NADP+) ec:1.2.1.53 4-hydroxyphenylacetaldehyde dehydrogenase
ec:1.2.1.57 butanal dehydrogenase ec:1.2.1.58 phenylglyoxylate
dehydrogenase (acylating) ec:1.2.1.59 glyceraldehyde-3-phosphate
dehydrogenase (NAD(P)+) (phosphorylating) ec:1.2.1.62
4-formylbenzenesulfonate dehydrogenase ec:1.2.1.63 6-oxohexanoate
dehydrogenase ec:1.2.1.64 4-hydroxybenzaldehyde dehydrogenase
ec:1.2.1.65 salicylaldehyde dehydrogenase ec:1.2.1.66
mycothiol-dependent formaldehyde dehydrogenase
ec:1.2.1.67 vanillin dehydrogenase ec:1.2.1.68 coniferyl-aldehyde
dehydrogenase ec:1.2.1.69 fluoroacetaldehyde dehydrogenase
ec:1.2.1.71 succinylglutamate-semialdehyde dehydrogenase
[0234] Other exemplary enzymes and pathways are disclosed herein
(see Examples). Furthermore, it is understood that enzymes can be
utilized for carry out reactions for which the substrate is not the
natural substrate. While the activity for the non-natural substrate
may be lower than the natural substrate, it is understood that such
enzymes can be utilized, either as naturally occurring or modified
using the directed evolution or adaptive evolution, as disclosed
herein (see also Examples).
[0235] BDO production through any of the pathways disclosed herein
are based, in part, on the identification of the appropriate
enzymes for conversion of precursors to BDO. A number of specific
enzymes for several of the reaction steps have been identified. For
those transformations where enzymes specific to the reaction
precursors have not been identified, enzyme candidates have been
identified that are best suited for catalyzing the reaction steps.
Enzymes have been shown to operate on a broad range of substrates,
as discussed below. In addition, advances in the field of protein
engineering also make it feasible to alter enzymes to act
efficiently on substrates, even if not a natural substrate.
Described below are several examples of broad-specificity enzymes
from diverse classes suitable for a BDO pathway as well as methods
that have been used for evolving enzymes to act on non-natural
substrates.
[0236] A key class of enzymes in BDO pathways is the
oxidoreductases that interconvert ketones or aldehydes to alcohols
(1.1.1). Numerous exemplary enzymes in this class 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. Biosc. Bioeng. 100:318-322 (2005)) was shown to operate
on a plethora of aliphatic as well as aromatic alcohols with high
activities. Table 2 shows the activity of the enzyme and its
K.sub.m on different alcohols. The enzyme is reversible and has
very high activity on several aldehydes also (Table 3).
TABLE-US-00002 TABLE 2 Relative activities of an alcohol
dehydrogenase from Brevibacterium sp KU to oxidize various
alcohols. Relative Activity K.sub.m Substrate (0%) (mM)
2-Phenylethanol 100* 0.025 (S)-2-Phenylpropanol 156 0.157
(R)-2-Phenylpropanol 63 0.020 Bynzyl 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 *The activity of
2-phenylethanol, corresponding to 19.2 U/mg, was taken as 100%.
TABLE-US-00003 TABLE 3 Relative activities of an alcohol
dehydrogenase from Brevibacterium sp KU 1309 to reduce various
carbonyl compounds. Relative Activity K.sub.m Substrate (%) (mM)
Phenylacetaldehyde 100 0.261 2-Phenylpropionaldehyde 188 0.864
1-Octylaldehyde 87 Acetophenone 0
[0237] 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 and Schlegel, Eur. J. Biochem. 130:329-334 (1983)).
Column 2 in Table 4 demonstrates the activities of ldhA from R.
eutropha (formerly A. eutrophus) on different substrates
(Steinbuchel and Schlegel, supra, 1983).
TABLE-US-00004 TABLE 4 The in vitro activity of R.eutropha IdhA
(Steinbuchel and Schlegel, supra, 1983) on different substrates and
compared with that on pyruvate. Activity (%) of L(+)-lactate
L(+)-lactate D(-)-lactate dehydrogenase dehydrogenase dehydrogenase
from A. from rabbit from L. Substrate eutrophus muscle leichmanii
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
[0238] 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
CO2. 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. 32:911-922 (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.
[0239] 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 133-141 (2002)). In another instance, an aminotransferase
indentified 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. Parasitol
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%. 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)).
[0240] CoA transferases (2.8.3) have been demonstrated to have the
ability to act on more than one substrate. Specifically, a CoA
transferase was purified from Clostridium acetobutylicum and was
reported to have the highest activities on acetate, propionate, and
butyrate. It also had significant activities with valerate,
isobutyrate, and crotonate (Wiesenborn et al., Appl. Environ.
Microbiol. 55:323-329 (1989)). In another study, the E. coli enzyme
acyl-CoA:acetate-CoA transferase, also known as acetate-CoA
transferase (EC 2.8.3.8), has been shown to transfer the CoA moiety
to acetate from a variety of branched and linear acyl-CoA
substrates, including isobutyrate (Matthies and Schink, App.
Environm. Microbiol. 58:1435-1439 (1992)), valerate (Vanderwinkel
et al., Biochem. Biophys. Res Commun. 33:902-908 (1968b)) and
butanoate (Vanderwinkel et al., Biochem. Biophys. Res Commun.
33:902-908 (1968a).
[0241] Other enzyme classes additionally support broad substrate
specificity for enzymes. Some isomerases (5.3.3) have also been
proven to operate on multiple substrates. For example, L-rhamnose
isomerase from Pseudomonas stutzeri catalyzes the isomerization
between various aldoalses and ketoses (Yoshida et al., J. Mol.
Biol. 365:1505-1516 (2007)). These include isomerization between
L-rhamnose and L-rhamnulose, L-mannose and L-fructose, L-xylose and
L-xylulose, D-ribose and D-ribulose, and D-allose and
D-psicose.
[0242] In yet another class of enzymes, the phosphotransferases
(2.7.1), the homoserine kinase (2.7.1.39) from E. coli that
converts L-homoserine to L-homoserine phosphate, was found to
phosphorylate numerous homoserine analogs. In these substrates, the
carboxyl functional group at the R-position had been replaced by an
ester or by a hydroxymethyl group (Huo and Viola, Biochemistry
35:16180-16185 (1996)). Table 5 demonstrates the broad substrate
specificity of this kinase.
TABLE-US-00005 TABLE 5 The substrate specificity of homoserine
kinase. Substrate k.sub.cat % k.sub.cat K.sub.m (mM)
k.sub.cat/K.sub.m L-homoserine 18.3 .+-. 0.1 100 0.14 .+-. 0.04 184
.+-. 17 D-homoserine 8.3 .+-. 1.1 32 31.8 .+-. 7.2 0.26 .+-. 0.03
L-aspartate .beta.- 2.1 .+-. 0.1 8.2 0.28 .+-. 0.02 7.5 .+-. 0.3
semialdehyde L-2-amino-1,4- 2.0 .+-. 0.5 7.9 11.6 .+-. 6.5 0.17
.+-. 0.06 butanediol L-2-amino-5- 2.5 .+-. 0.4 9.9 1.1 .+-. 0.5 2.3
.+-. 0.3 hydroxyvalerate L-homoserine methyl 14.7 .+-. 2.6 80 4.9
.+-. 2.0 3.0 .+-. 0.6 ester L-homoserine ethyl 13.6 .+-. 0.8 74 1.9
.+-. 0.5 7.2 .+-. 1.7 ester L-homoserine 13.6 .+-. 1.4 74 1.2 .+-.
0.5 11.3 .+-. 1.1 isopropyl ester L-homoserine n- 14.0 .+-. 0.4 76
3.5 .+-. 0.4 4.0 .+-. 1.2 propyl ester L-homoserine isobutyl 16.4
.+-. 0.8 84 6.9 .+-. 1.1 2.4 .+-. 0.3 ester L-homserine n-butyl
29.1 .+-. 1.2 160 5.8 .+-. 0.8 5.0 .+-. 0.5 ester
[0243] Another class of enzymes useful in BDO pathways is the
acid-thiol ligases (6.2.1). Like enzymes in other classes, certain
enzymes in this class have been determined to have broad substrate
specificity. For example, acyl CoA ligase from Pseudomonas putida
has been demonstrated to work on several aliphatic substrates
including acetic, propionic, butyric, valeric, hexanoic, heptanoic,
and octanoic acids and on aromatic compounds such as phenylacetic
and phenoxyacetic acids (Fernandez-Valverde et al., Appl. Environ.
Microbiol. 59:1149-1154 (1993)). A related enzyme, malonyl CoA
synthetase (6.3.4.9) from Rhizobium trifolii could convert several
diacids, namely, ethyl-, propyl-, allyl-, isopropyl-, dimethyl-,
cyclopropyl-, cyclopropylmethylene-, cyclobutyl-, and
benzyl-malonate into their corresponding monothioesters (Pohl et
al., J. Am. Chem. Soc. 123:5822-5823 (2001)). Similarly,
decarboxylases (4.1.1) have also been found with broad substrate
ranges. Pyruvate decarboxylase (PDC), also termed keto-acid
decarboxylase, is a key enzyme in alcoholic fermentation,
catalyzing the decarboxylation of pyruvate to acetaldehyde. The
enzyme isolated from Saccharomyces cerevisiae has a broad substrate
range for aliphatic 2-keto acids including 2-ketobutyrate,
2-ketovalerate, and 2-phenylpyruvate (Li and Jordan, Biochemistry
38:10004-10012 (1999)). Similarly, benzoylformate decarboxylase has
a broad substrate range and has been the target of enzyme
engineering studies. The enzyme from Pseudomonas putida has been
extensively studied and crystal structures of this enzyme are
available (Polovnikova et al., Biochemistry 42:1820-1830 (2003);
Hasson et al., Biochemistry 37:9918-9930 (1998)). Branched chain
alpha-ketoacid decarboxylase (BCKA) has been shown to act on a
variety of compounds varying in chain length from 3 to 6 carbons
(Oku and Kaneda, J. Biol. Chem. 263:18386-18396 (1998); Smit et
al., Appl. Environ. Microbiol. 71:303-311 (2005b)). The enzyme in
Lactococcus lactis has been characterized on a variety of branched
and linear substrates including 2-oxobutanoate, 2-oxohexanoate,
2-oxopentanoate, 3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate
and isocaproate (Smit et al., Appl. Environ. Microbiol. 71:303-311
(2005a).
[0244] Interestingly, enzymes known to have one dominant activity
have also been reported to catalyze a very different function. For
example, the cofactor-dependent phosphoglycerate mutase (5.4.2.1)
from Bacillus stearothermophilus and Bacillus subtilis is known to
function as a phosphatase as well (Rigden et al., Protein Sci.
10:1835-1846 (2001)). The enzyme from B. stearothermophilus is
known to have activity on several substrates, including
3-phosphoglycerate, alpha-napthylphosphate, p-nitrophenylphosphate,
AMP, fructose-6-phosphate, ribose-5-phosphate and CMP.
[0245] 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. Therefore, it is feasible to engineer a given enzyme for
efficient function on a natural, for example, improved efficiency,
or a non-natural substrate, for example, increased efficiency. For
example, it has been reported that the enantioselectivity of a
lipase from Pseudomonas aeruginosa was improved significantly
(Reetz et al., Agnew. Chem. Int. Ed Engl. 36:2830-2832 (1997)).
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., Agnew. Chem. Int. Ed Engl. 36:2830-2832
(1997)).
[0246] Directed evolution methods have been used to modify an
enzyme to function on an array of non-natural 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., Agnew. Chem. Int. Ed Engl.
44:4192-4196 (2005)). In another successful modification of an
enzyme, 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.
Nat. 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 (Jiang et al.,
Science 319:1387-1391 (2008)). These algorithms used different
combinations of four different catalytic motifs to design new
enzyme, 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 they 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. Nat. 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.
[0247] The effectiveness of protein engineering approaches to alter
the substrate specificity of an enzyme for a desired substrate has
also been demonstrated in several studies. 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. For example, the
dihydroflavonol 4-reductase for which a single amino acid was
changed in the presumed substrate-binding region 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 form
isocitrate to isopropylmalate by changing one residue in the active
site (Doyle et al., Biochemistry 40:4234-4241 (2001)). Similarly,
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.
[0248] Numerous examples exist spanning diverse classes of enzymes
where the function of enzyme was changed to favor one non-natural
substrate over the natural substrate of the enzyme. A fucosidase
was evolved from a galactosidase in E. coli by DNA shuffling and
screening (Zhang et al., Proc. Natl. Acad. Sci. U.S.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 and Kirsch, 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)).
[0249] In some cases, enzymes with different substrate preferences
than either of 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.
[0250] In addition to changing enzyme specificity, it is also
possible to enhance the activities on substrates for 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 and
Chuang, Biochemistry 33:12879-12885 (1994)). An interesting aspect
of these approaches is that even if 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 was traced back to two different
positions.
[0251] Directed evolution has also been used to express proteins
that are difficult to express. For example, by subjecting
horseradish peroxidase to random mutagenesis and gene
recombination, mutants were identified that had more than 14-fold
higher activity than the wild type (Lin et al., Biotechnol. Prog.
15:467-471 (1999)).
[0252] Another 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 believed 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. Interestingly, 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 the
polypeptide (residues 98-110) which normally seals the active site
from bulk solvent and is a major determinant of substrate
specificity. The variable length and sequence loops were inserted
so that hydroxyacid dehydrogenases with altered substrate
specificities were generated. 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. The studies described above indicate
that various approaches of enzyme engineering can be used to obtain
enzymes for the BDO pathways as disclosed herein.
[0253] As disclosed herein, biosynthetic pathways to 1,4-butanediol
from a number of central metabolic intermediates are can be
utilized, including acetyl-CoA, succinyl-CoA, alpha-ketoglutarate,
glutamate, 4-aminobutyrate, and homoserine. Acetyl-CoA,
succinyl-CoA and alpha-ketoglutarate are common intermediates of
the tricarboxylic acid (TCA) cycle, a series of reactions that is
present in its entirety in nearly all living cells that utilize
oxygen for cellular respiration and is present in truncated forms
in a number of anaerobic organisms. Glutamate is an amino acid that
is derived from alpha-ketoglutarate via glutamate dehydrogenase or
any of a number of transamination reactions (see FIG. 8B).
4-aminobutyrate can be formed by the decarboxylation of glutamate
(see FIG. 8B) or from acetoacetyl-CoA via the pathway disclosed in
FIG. 9C. Acetoacetyl-CoA is derived from the condensation of two
acetyl-CoA molecules by way of the enzyme, acetyl-coenzyme A
acetyltransferase, or equivalently, acetoacetyl-coenzyme A
thiolase. Homoserine is an intermediate in threonine and methionine
metabolism, formed from oxaloacetate via aspartate. The conversion
of oxaloacetate to homoserine requires one NADH, two NADPH, and one
ATP.
[0254] Pathways other than those exemplified above also can be
employed to generate the biosynthesis of BDO in non-naturally
occurring microbial organisms. In one embodiment, biosynthesis can
be achieved using a L-homoserine to BDO pathway (see FIG. 13). This
pathway has a molar yield of 0.90 mol/mol glucose, which appears
restricted by the availability of reducing equivalents. A second
pathway synthesizes BDO from acetoacetyl-CoA and is capable of
achieving the maximum theoretical yield of 1.091 mol/mol glucose
(see FIG. 9). Implementation of either pathway can be achieved by
introduction of two exogenous enzymes into a host organism such as
E. coli, and both pathways can additionally complement BDO
production via succinyl-CoA. Pathway enzymes, thermodynamics,
theoretical yields and overall feasibility are described further
below.
[0255] A homoserine pathway also can be engineered to generate
BDO-producing microbial organisms. Homoserine is an intermediate in
threonine and methionine metabolism, formed from oxaloacetate via
aspartate. The conversion of oxaloacetate to homoserine requires
one NADH, two NADPH, and one ATP (FIG. 2). Once formed, homoserine
feeds into biosynthetic pathways for both threonine and methionine.
In most organisms, high levels of threonine or methionine feedback
to repress the homoserine biosynthesis pathway (Caspi et al.,
Nucleic Acids Res. 34:D511-D516 (1990)).
[0256] The transformation of homoserine to 4-hydroxybutyrate (4-HB)
can be accomplished in two enzymatic steps as described herein. The
first step of this pathway is deamination of homoserine by a
putative ammonia lyase. In step 2, the product alkene,
4-hydroxybut-2-enoate is reduced to 4-HB by a putative reductase at
the cost of one NADH. 4-HB can then be converted to BDO.
[0257] Enzymes available for catalyzing the above transformations
are disclosed herein. For example, the ammonia lyase in step 1 of
the pathway closely resembles the chemistry of aspartate
ammonia-lyase (aspartase). Aspartase is a widespread enzyme in
microorganisms, and has been characterized extensively (Viola, R.
E., Mol. Biol. 74:295-341 (2008)). The crystal structure of the E.
coli aspartase has been solved (Shi et al., Biochemistry
36:9136-9144 (1997)), so it is therefore possible to directly
engineer mutations in the enzyme's active site that would alter its
substrate specificity to include homoserine. The oxidoreductase in
step 2 has chemistry similar to several well-characterized enzymes
including fumarate reductase in the E. coli TCA cycle. Since the
thermodynamics of this reaction are highly favorable, an endogenous
reductase with broad substrate specificity will likely be able to
reduce 4-hydroxybut-2-enoate. The yield of this pathway under
anaerobic conditions is 0.9 mol BDO per mol glucose.
[0258] The succinyl-CoA pathway was found to have a higher yield
due to the fact that it is more energetically efficient. The
conversion of one oxaloacetate molecule to BDO via the homoserine
pathway will require the expenditure of 2 ATP equivalents. Because
the conversion of glucose to two oxaloacetate molecules can
generate a maximum of 3 ATP molecules assuming PEP carboxykinase to
be reversible, the overall conversion of glucose to BDO via
homoserine has a negative energetic yield. As expected, if it is
assumed that energy can be generated via respiration, the maximum
yield of the homoserine pathway increases to 1.05 mol/mol glucose
which is 96% of the succinyl-CoA pathway yield. The succinyl-CoA
pathway can channel some of the carbon flux through pyruvate
dehydrogenase and the oxidative branch of the TCA cycle to generate
both reducing equivalents and succinyl-CoA without an energetic
expenditure. Thus, it does not encounter the same energetic
difficulties as the homoserine pathway because not all of the flux
is channeled through oxaloacetate to succinyl-CoA to BDO. Overall,
the homoserine pathway demonstrates a high-yielding route to
BDO.
[0259] An acetoacetate pathway also can be engineered to generate
BDO-producing microbial organisms. Acetoacetate can be formed from
acetyl-CoA by enzymes involved in fatty acid metabolism, including
acetyl-CoA acetyltransferase and acetoacetyl-CoA transferase.
Biosynthetic routes through acetoacetate are also particularly
useful in microbial organisms that can metabolize single carbon
compounds such as carbon monoxide, carbon dioxide or methanol to
form acetyl-CoA.
[0260] A three step route from acetoacetyl-CoA to 4-aminobutyrate
(see FIG. 9C) can be used to synthesize BDO through
acetoacetyl-CoA. 4-Aminobutyrate can be converted to succinic
semialdehyde as shown in FIG. 8B. Succinic semialdehyde, which is
one reduction step removed from succinyl-CoA or one decarboxylation
step removed from .alpha.-ketoglutarate, can be converted to BDO
following three reductions steps (FIG. 1). Briefly, step 1 of this
pathway involves the conversion of acetoacetyl-CoA to acetoacetate
by, for example, the E. coli acetoacetyl-CoA transferase encoded by
the atoA and atoD genes (Hanai et al., Appl. Environ. Microbiol.
73: 7814-7818 (2007)). Step 2 of the acetoacetyl-CoA biopathway
entails conversion of acetoacetate to 3-aminobutanoate by an
w-aminotransferase. The .omega.-amino acid:pyruvate
aminotransferase (.omega.-APT) from Alcaligens denitrificans was
overexpressed in E. coli and shown to have a high activity toward
3-aminobutanoate in vitro (Yun et al., Appl. Environ. Microbiol.
70:2529-2534 (2004)).
[0261] In step 2, a putative aminomutase shifts the amine group
from the 3- to the 4-position of the carbon backbone. An
aminomutase performing this function on 3-aminobutanoate has not
been characterized, but an enzyme from Clostridium sticklandii has
a very similar mechanism. The enzyme, D-lysine-5,6-aminomutase, is
involved in lysine biosynthesis.
[0262] The synthetic route to BDO from acetoacetyl-CoA passes
through 4-aminobutanoate, a metabolite in E. coli that is normally
formed from decarboxylation of glutamate. Once formed,
4-aminobutanoate can be converted to succinic semialdehyde by
4-aminobutanoate transaminase (2.6.1.19), an enzyme which has been
biochemically characterized.
[0263] One consideration for selecting candidate enzymes in this
pathway is the stereoselectivity of the enzymes involved in steps 2
and 3. The .omega.-ABT in Alcaligens denitrificans is specific to
the L-stereoisomer of 3-aminobutanoate, while
D-lysine-5,6-aminomutase likely requires the D-stereoisomer. If
enzymes with complementary stereoselectivity are not initially
found or engineered, a third enzyme can be added to the pathway
with racemase activity that can convert L-3-aminobutanoate to
D-3-aminobutanoate. While amino acid racemases are widespread,
whether these enzymes can function on w-amino acids is not
known.
[0264] The maximum theoretical molar yield of this pathway under
anaerobic conditions is 1.091 mol/mol glucose. In order to generate
flux from acetoacetyl-CoA to BDO it was necessary to assume that
acetyl-CoA:acetoacetyl-CoA transferase is reversible. The function
of this enzyme in E. coli is to metabolize short-chain fatty acids
by first converting them into thioesters.
[0265] While the operation of acetyl-CoA:acetoacetyl-CoA
transferase in the acetate-consuming direction has not been
demonstrated experimentally in E. coli, studies on similar enzymes
in other organisms support the assumption that this reaction is
reversible. The enzyme butyryl-CoA:acetate:CoA transferase in gut
microbes Roseburia sp. and F. prasnitzii operates in the acetate
utilizing direction to produce butyrate (Duncan et al., Appl.
Environ. Microbiol. 68:5186-5190 (2002)). Another very similar
enzyme, acetyl:succinate CoA-transferase in Trypanosoma brucei,
also operates in the acetate utilizing direction. This reaction has
a .DELTA.rxnG close to equilibrium, so high concentrations of
acetate can likely drive the reaction in the direction of interest.
At the maximum theoretical BDO production rate of 1.09 mol/mol
glucose simulations predict that E. coli can generate 1.098 mol ATP
per mol glucose with no fermentation byproducts. This ATP yield
should be sufficient for cell growth, maintenance, and production.
The acetoacetatyl-CoA biopathway is a high-yielding route to BDO
from acetyl-CoA.
[0266] Therefore, in addition to any of the various modifications
exemplified previously for establishing 4-HB biosynthesis in a
selected host, the BDO producing microbial organisms can include
any of the previous combinations and permutations of 4-HB pathway
metabolic modifications as well as any combination of expression
for CoA-independent aldehyde dehydrogenase, CoA-dependent aldehyde
dehydrogenase or an alcohol dehydrogenase or other enzymes
disclosed herein to generate biosynthetic pathways for GBL and/or
BDO. Therefore, the BDO producers of the invention can have
exogenous expression of, for example, one, two, three, four, five,
six, seven, eight, nine, or up to all enzymes corresponding to any
of the 4-HB pathway and/or any of the BDO pathway enzymes disclosed
herein.
[0267] Design and construction of the genetically modified
microbial organisms is carried out using methods well known in the
art to achieve sufficient amounts of expression to produce BDO. In
particular, the non-naturally occurring microbial organisms of the
invention can achieve biosynthesis of BDO resulting in
intracellular concentrations between about 0.1-200 mM or more, such
as about 0.1-25 mM or more, as discussed above. For example, the
intracellular concentration of BDO is between about 3-20 mM,
particularly between about 5-15 mM and more particularly between
about 8-12 mM, including about 10 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. As with the 4-HB producers, the BDO
producers also can be sustained, cultured or fermented under
anaerobic conditions.
[0268] The invention further provides a method for the production
of 4-HB. The method includes culturing a non-naturally occurring
microbial organism having a 4-hydroxybutanoic acid (4-HB)
biosynthetic pathway comprising at least one exogenous nucleic acid
encoding 4-hydroxybutanoate dehydrogenase, CoA-independent succinic
semialdehyde dehydrogenase, succinyl-CoA synthetase, CoA-dependent
succinic semialdehyde dehydrogenase, glutamate:succinic
semialdehyde transaminase, -ketoglutarate decarboxylase, or
glutamate decarboxylase under substantially anaerobic conditions
for a sufficient period of time to produce monomeric
4-hydroxybutanoic acid (4-HB). The method can additionally include
chemical conversion of 4-HB to GBL and to BDO or THF, for
example.
[0269] Additionally provided is a method for the production of
4-HB. The method includes culturing a non-naturally occurring
microbial organism having a 4-hydroxybutanoic acid (4-HB)
biosynthetic pathway including at least one exogenous nucleic acid
encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase,
CoA-dependent succinic semialdehyde dehydrogenase or
.alpha.-ketoglutarate decarboxylase under substantially anaerobic
conditions for a sufficient period of time to produce monomeric
4-hydroxybutanoic acid (4-HB). The 4-HB product can be secreted
into the culture medium.
[0270] Further provided is a method for the production of BDO. The
method includes culturing a non-naturally occurring microbial
biocatalyst or microbial organism, comprising a microbial organism
having 4-hydroxybutanoic acid (4-HB) and 1,4-butanediol (BDO)
biosynthetic pathways, the pathways including at least one
exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase,
succinyl-CoA synthetase, CoA-dependent succinic semialdehyde
dehydrogenase, 4-hydroxybutyrate:CoA transferase, 4-hydroxybutyrate
kinase, phosphotranshydroxybutyrylase, .alpha.-ketoglutarate
decarboxylase, aldehyde dehydrogenase, alcohol dehydrogenase or an
aldehyde/alcohol dehydrogenase for a sufficient period of time to
produce 1,4-butanediol (BDO). The BDO product can be secreted into
the culture medium.
[0271] Additionally provided are methods for producing BDO by
culturing a non-naturally occurring microbial organism having a BDO
pathway of the invention. The BDO pathway can comprise at least one
exogenous nucleic acid encoding a BDO pathway enzyme expressed in a
sufficient amount to produce BDO, under conditions and for a
sufficient period of time to produce BDO, the BDO pathway
comprising 4-aminobutyrate CoA transferase, 4-aminobutyryl-CoA
hydrolase, 4-aminobutyrate-CoA ligase, 4-aminobutyryl-CoA
oxidoreductase (deaminating), 4-aminobutyryl-CoA transaminase, or
4-hydroxybutyryl-CoA dehydrogenase (see Example VII and Table
17).
[0272] Alternatively, the BDO pathway can compare at least one
exogenous nucleic acid encoding a BDO pathway enzyme expressed in a
sufficient amount to produce BDO, under conditions and for a
sufficient period of time to produce BDO, the BDO pathway
comprising 4-aminobutyrate CoA transferase, 4-aminobutyryl-CoA
hydrolase, 4-aminobutyrate-CoA ligase, 4-aminobutyryl-CoA reductase
(alcohol forming), 4-aminobutyryl-CoA reductase, 4-aminobutan-1-ol
dehydrogenase, 4-aminobutan-1-ol oxidoreductase (deaminating) or
4-aminobutan-1-ol transaminase (see Example VII and Table 18).
[0273] In addition, the invention provides a method for producing
BDO, comprising culturing a non-naturally occurring microbial
organism having a BDO pathway, the pathway comprising at least one
exogenous nucleic acid encoding a BDO pathway enzyme expressed in a
sufficient amount to produce BDO, under conditions and for a
sufficient period of time to produce BDO, the BDO pathway
comprising 4-aminobutyrate kinase, 4-aminobutyraldehyde
dehydrogenase (phosphorylating), 4-aminobutan-1-ol dehydrogenase,
4-aminobutan-1-ol oxidoreductase (deaminating), 4-aminobutan-1-ol
transaminase, [(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase
(deaminating), [(4-aminobutanolyl)oxy]phosphonic acid transaminase,
4-hydroxybutyryl-phosphate dehydrogenase, or 4-hydroxybutyraldehyde
dehydrogenase (phosphorylating) (see Example VII and Table 19).
[0274] The invention further provides a method for producing BDO,
comprising culturing a non-naturally occurring microbial organism
having a BDO pathway, the pathway comprising at least one exogenous
nucleic acid encoding a BDO pathway enzyme expressed in a
sufficient amount to produce BDO, under conditions and for a
sufficient period of time to produce BDO, the BDO pathway
comprising alpha-ketoglutarate 5-kinase, 2,5-dioxopentanoic
semialdehyde dehydrogenase (phosphorylating), 2,5-dioxopentanoic
acid reductase, alpha-ketoglutarate CoA transferase,
alpha-ketoglutaryl-CoA hydrolase, alpha-ketoglutaryl-CoA ligase,
alpha-ketoglutaryl-CoA reductase, 5-hydroxy-2-oxopentanoic acid
dehydrogenase, alpha-ketoglutaryl-CoA reductase (alcohol forming),
5-hydroxy-2-oxopentanoic acid decarboxylase, or
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation)(see
Example VIII and Table 20).
[0275] The invention additionally provides a method for producing
BDO, comprising culturing a non-naturally occurring microbial
organism having a BDO pathway, the pathway comprising at least one
exogenous nucleic acid encoding a BDO pathway enzyme expressed in a
sufficient amount to produce BDO, under conditions and for a
sufficient period of time to produce BDO, the BDO pathway
comprising glutamate CoA transferase, glutamyl-CoA hydrolase,
glutamyl-CoA ligase, glutamate 5-kinase, glutamate-5-semialdehyde
dehydrogenase (phosphorylating), glutamyl-CoA reductase,
glutamate-5-semialdehyde reductase, glutamyl-CoA reductase (alcohol
forming), 2-amino-5-hydroxypentanoic acid oxidoreductase
(deaminating), 2-amino-5-hydroxypentanoic acid transaminase,
5-hydroxy-2-oxopentanoic acid decarboxylase,
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation)(see
Example IX and Table 21).
[0276] The invention additionally includes a method for producing
BDO, comprising culturing a non-naturally occurring microbial
organism having a BDO pathway, the pathway comprising at least one
exogenous nucleic acid encoding a BDO pathway enzyme expressed in a
sufficient amount to produce BDO, under conditions and for a
sufficient period of time to produce BDO, the BDO pathway
comprising 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA
dehydratase, vinylacetyl-CoA A-isomerase, or 4-hydroxybutyryl-CoA
dehydratase (see Example X and Table 22).
[0277] Also provided is a method for producing BDO, comprising
culturing a non-naturally occurring microbial organism having a BDO
pathway, the pathway comprising at least one exogenous nucleic acid
encoding a BDO pathway enzyme expressed in a sufficient amount to
produce BDO, under conditions and for a sufficient period of time
to produce BDO, the BDO pathway comprising homoserine deaminase,
homoserine CoA transferase, homoserine-CoA hydrolase,
homoserine-CoA ligase, homoserine-CoA deaminase,
4-hydroxybut-2-enoyl-CoA transferase, 4-hydroxybut-2-enoyl-CoA
hydrolase, 4-hydroxybut-2-enoyl-CoA ligase, 4-hydroxybut-2-enoate
reductase, 4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA
hydrolase, 4-hydroxybutyryl-CoA ligase, or 4-hydroxybut-2-enoyl-CoA
reductase (see Example XI and Table 23).
[0278] The invention additionally provides a method for producing
BDO, comprising culturing a non-naturally occurring microbial
organism having a BDO pathway, the pathway comprising at least one
exogenous nucleic acid encoding a BDO pathway enzyme expressed in a
sufficient amount to produce BDO, under conditions and for a
sufficient period of time to produce BDO, the BDO pathway
comprising succinyl-CoA reductase (alcohol forming),
4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase,
4-hydroxybutanal dehydrogenase (phosphorylating). Such a BDO
pathway can further comprise succinyl-CoA reductase,
4-hydroxybutyrate dehydrogenase, 4-hydroxybutyryl-CoA transferase,
4-hydroxybutyrate kinase, phosphotrans-4-hydroxybutyrylase,
4-hydroxybutyryl-CoA reductase, 4-hydroxybutyryl-CoA reductase
(alcohol forming), or 1,4-butanediol dehydrogenase.
[0279] Also provided is a method for producing BDO, comprising
culturing a non-naturally occurring microbial organism having a BDO
pathway, the pathway comprising at least one exogenous nucleic acid
encoding a BDO pathway enzyme expressed in a sufficient amount to
produce BDO, under conditions and for a sufficient period of time
to produce BDO, the BDO pathway comprising glutamate dehydrogenase,
4-aminobutyrate oxidoreductase (deaminating), 4-aminobutyrate
transaminase, glutamate decarboxylase, 4-hydroxybutyryl-CoA
hydrolase, 4-hydroxybutyryl-CoA ligase, 4-hydroxybutanal
dehydrogenase (phosphorylating).
[0280] The invention additionally provides methods of producing a
desired product using the genetically modified organisms disclosed
herein that allow improved production of a desired product such as
BDO by increasing the product or decreasing undesirable byproducts.
Thus, the invention provides a method for producing 1,4-butanediol
(BDO), comprising culturing the non-naturally occurring microbial
organisms disclosed herein under conditions and for a sufficient
period of time to produce BDO. In one embodiment, the invention
provides a method of producing BDO using a non-naturally occurring
microbial organism, comprising a microbial organism having a
1,4-butanediol (BDO) pathway comprising at least one exogenous
nucleic acid encoding a BDO pathway enzyme expressed in a
sufficient amount to produce BDO. In one embodiment, the microbial
organism is genetically modified to express exogenous succinyl-CoA
synthetase (see Example XII). For example, the succinyl-CoA
synthetase can be encoded by an Escherichia coli sucCD genes.
[0281] In another embodiment, the microbial organism is genetically
modified to express exogenous alpha-ketoglutarate decarboxylase
(see Example XIII). For example, the alpha-ketoglutarate
decarboxylase can be encoded by the Mycobacterium bovis sucA gene.
In still another embodiment, the microbial organism is genetically
modified to express exogenous succinate semialdehyde dehydrogenase
and 4-hydroxybutyrate dehydrogenase and optionally
4-hydroxybutyryl-CoA/acetyl-CoA transferase (see Example XIII). For
example, the succinate semialdehyde dehydrogenase (CoA-dependent),
4-hydroxybutyrate dehydrogenase and 4-hydroxybutyryl-CoA/acetyl-CoA
transferase can be encoded by Porphyromonas gingivalis W83 genes.
In an additional embodiment, the microbial organism is genetically
modified to express exogenous butyrate kinase and
phosphotransbutyrylase (see Example XIII). For example, the
butyrate kinase and phosphotransbutyrylase can be encoded by
Clostridium acetobutilicum buk1 and ptb genes.
[0282] In yet another embodiment, the microbial organism is
genetically modified to express exogenous 4-hydroxybutyryl-CoA
reductase (see Example XIII). For example, the 4-hydroxybutyryl-CoA
reductase can be encoded by Clostridium beijerinckii ald gene.
Additionally, in an embodiment of the invention, the microbial
organism is genetically modified to express exogenous
4-hydroxybutanal reductase (see Example XIII). For example, the
4-hydroxybutanal reductase can be encoded by Geobacillus
thermoglucosidasius adh1 gene. In another embodiment, the microbial
organism is genetically modified to express exogenous pyruvate
dehydrogenase subunits (see Example XIV). For example, the
exogenous pyruvate dehydrogenase can be NADH insensitive. The
pyruvate dehydrogenase subunit can be encoded by the Klebsiella
pneumonia lpdA gene. In a particular embodiment, the pyruvate
dehydrogenase subunit genes of the microbial organism can be under
the control of a pyruvate formate lyase promoter.
[0283] In still another embodiment, the microbial organism is
genetically modified to disrupt a gene encoding an aerobic
respiratory control regulatory system (see Example XV). For
example, the disruption can be of the arcA gene. Such an organism
can further comprise disruption of a gene encoding malate
dehydrogenase. In a further embodiment, the microbial organism is
genetically modified to express an exogenous NADH insensitive
citrate synthase (see Example XV). For example, the NADH
insensitive citrate synthase can be encoded by gltA, such as an
R163L mutant of gltA. In still another embodiment, the microbial
organism is genetically modified to express exogenous
phosphoenolpyruvate carboxykinase (see Example XVI). For example,
the phosphoenolpyruvate carboxykinase can be encoded by an
Haemophilus influenza phosphoenolpyruvate carboxykinase gene. It is
understood that strains exemplified herein for improved production
of BDO can similarly be used, with appropriate modifications, to
produce other desired products, for example, 4-hydroxybutyrate or
other desired products disclosed herein.
[0284] The invention additionally provides a method for producing
4-hydroxybutanal by culturing a non-naturally occurring microbial
organism, comprising a 4-hydroxybutanal pathway comprising at least
one exogenous nucleic acid encoding a 4-hydroxybutanal pathway
enzyme expressed in a sufficient amount to produce
4-hydroxybutanal, the 4-hydroxybutanal pathway comprising
succinyl-CoA reductase (aldehyde forming); 4-hydroxybutyrate
dehydrogenase; and 4-hydroxybutyrate reductase (see FIG. 58, steps
A-C-D). The invention also provides a method for producing
4-hydroxybutanal by culturing a non-naturally occurring microbial
organism, comprising a 4-hydroxybutanal pathway comprising at least
one exogenous nucleic acid encoding a 4-hydroxybutanal pathway
enzyme expressed in a sufficient amount to produce
4-hydroxybutanal, the 4-hydroxybutanal pathway comprising
alpha-ketoglutarate decarboxylase; 4-hydroxybutyrate dehydrogenase;
and 4-hydroxybutyrate reductase (FIG. 58, steps B-C-D).
[0285] The invention further provides a method for producing
4-hydroxybutanal by culturing a non-naturally occurring microbial
organism, comprising a 4-hydroxybutanal pathway comprising at least
one exogenous nucleic acid encoding a 4-hydroxybutanal pathway
enzyme expressed in a sufficient amount to produce
4-hydroxybutanal, the 4-hydroxybutanal pathway comprising succinate
reductase; 4-hydroxybutyrate dehydrogenase, and 4-hydroxybutyrate
reductase (see FIG. 62, steps F-C-D). In yet another embodiment,
the invention provides a method for producing 4-hydroxybutanal by
culturing a non-naturally occurring microbial organism, comprising
a 4-hydroxybutanal pathway comprising at least one exogenous
nucleic acid encoding a 4-hydroxybutanal pathway enzyme expressed
in a sufficient amount to produce 4-hydroxybutanal, the
4-hydroxybutanal pathway comprising alpha-ketoglutarate
decarboxylase, or glutamate dehydrogenase or glutamate transaminase
and glutamate decarboxylase and 4-aminobutyrate dehydrogenase or
4-aminobutyrate transaminase; 4-hydroxybutyrate dehydrogenase; and
4-hydroxybutyrate reductase (see FIG. 62, steps B or ((J or K)-L-(M
or N))-C-D).
[0286] The invention also provides a method for producing
4-hydroxybutanal by culturing a non-naturally occurring microbial
organism, comprising a 4-hydroxybutanal pathway comprising at least
one exogenous nucleic acid encoding a 4-hydroxybutanal pathway
enzyme expressed in a sufficient amount to produce
4-hydroxybutanal, the 4-hydroxybutanal pathway comprising
alpha-ketoglutarate reductase; 5-hydroxy-2-oxopentanoate
dehydrogenase; and 5-hydroxy-2-oxopentanoate decarboxylase (see
FIG. 62, steps X-Y-Z). The invention further provides a method for
producing 4-hydroxybutyryl-CoA by culturing a non-naturally
occurring microbial organism, comprising a 4-hydroxybutyryl-CoA
pathway comprising at least one exogenous nucleic acid encoding a
4-hydroxybutyryl-CoA pathway enzyme expressed in a sufficient
amount to produce 4-hydroxybutyryl-CoA, the 4-hydroxybutyryl-CoA
pathway comprising alpha-ketoglutarate reductase;
5-hydroxy-2-oxopentanoate dehydrogenase; and
5-hydroxy-2-oxopentanoate dehydrogenase (decarboxylation) (see FIG.
62, steps X-Y-AA).
[0287] The invention additionally provides a method for producing
putrescine by culturing a non-naturally occurring microbial
organism, comprising a putrescine pathway comprising at least one
exogenous nucleic acid encoding a putrescine pathway enzyme
expressed in a sufficient amount to produce putrescine, the
putrescine pathway comprising succinate reductase; 4-aminobutyrate
dehydrogenase or 4-aminobutyrate transaminase; 4-aminobutyrate
reductase; and putrescine dehydrogenase or putrescine transaminase
(see FIG. 63, steps F-M/N-C-D/E). In still another embodiment, the
invention provides a method for producing putrescine by culturing a
non-naturally occurring microbial organism, comprising a putrescine
pathway comprising at least one exogenous nucleic acid encoding a
putrescine pathway enzyme expressed in a sufficient amount to
produce putrescine, the putrescine pathway comprising
alpha-ketoglutarate decarboxylase; 4-aminobutyrate dehydrogenase or
4-aminobutyrate transaminase; 4-aminobutyrate reductase; and
putrescine dehydrogenase or putrescine transaminase (see FIG. 63,
steps B-M/N-C-D/E). The invention additionally provides a method
for producing putrescine by culturing a non-naturally occurring
microbial organism, comprising a putrescine pathway comprising at
least one exogenous nucleic acid encoding a putrescine pathway
enzyme expressed in a sufficient amount to produce putrescine, the
putrescine pathway comprising glutamate dehydrogenase or glutamate
transaminase; glutamate decarboxylase; 4-aminobutyrate reductase;
and putrescine dehydrogenase or putrescine transaminase (see FIG.
63, steps J/K-L-C-D/E).
[0288] The invention provides in another embodiment a method for
producing putrescine by culturing a non-naturally occurring
microbial organism, comprising a putrescine pathway comprising at
least one exogenous nucleic acid encoding a putrescine pathway
enzyme expressed in a sufficient amount to produce putrescine, the
putrescine pathway comprising alpha-ketoglutarate reductase;
5-amino-2-oxopentanoate dehydrogenase or 5-amino-2-oxopentanoate
transaminase; 5-amino-2-oxopentanoate decarboxylase; and putrescine
dehydrogenase or putrescine transaminase (see FIG. 63, steps
O-P/Q-R-D/E). Also provided is a method for producing putrescine by
culturing a non-naturally occurring microbial organism, comprising
a putrescine pathway comprising at least one exogenous nucleic acid
encoding a putrescine pathway enzyme expressed in a sufficient
amount to produce putrescine, the putrescine pathway comprising
alpha-ketoglutarate reductase; 5-amino-2-oxopentanoate
dehydrogenase or 5-amino-2-oxopentanoate transaminase; ornithine
dehydrogenase or ornithine transaminase; and ornithine
decarboxylase (see FIG. 63, steps O-P/Q-S/T-U). It is understood
that a microbial organism comprising any of the pathways disclosed
herein can be used to produce a a desired product or intermediate,
including 4-HB, 4-HBal, BDO or putrescine.
[0289] It is understood that, in methods of the invention, any of
the one or more exogenous nucleic acids can be introduced into a
microbial organism to produce a non-naturally occurring microbial
organism of the invention. The nucleic acids can be introduced so
as to confer, for example, a 4-HB, BDO, THF or GBL 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 4-HB, BDO, THF or GBL
biosynthetic capability. For example, a non-naturally occurring
microbial organism having a 4-HB biosynthetic pathway can comprise
at least two exogenous nucleic acids encoding desired enzymes, such
as the combination of 4-hydroxybutanoate dehydrogenase and
.alpha.-ketoglutarate decarboxylase; 4-hydroxybutanoate
dehydrogenase and CoA-independent succinic semialdehyde
dehydrogenase; 4-hydroxybutanoate dehydrogenase and CoA-dependent
succinic semialdehyde dehydrogenase; CoA-dependent succinic
semialdehyde dehydrogenase and succinyl-CoA synthetase;
succinyl-CoA synthetase and glutamate decarboxylase, and the like.
Thus, it is understood that any combination of two or more enzymes
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 of a
biosynthetic pathway can be included in a non-naturally occurring
microbial organism of the invention, for example,
4-hydroxybutanoate dehydrogenase, .alpha.-ketoglutarate
decarboxylase and CoA-dependent succinic semialdehyde
dehydrogenase; CoA-independent succinic semialdehyde dehydrogenase
and succinyl-CoA synthetase; 4-hydroxybutanoate dehydrogenase,
CoA-dependent succinic semialdehyde dehydrogenase and
glutamate:succinic semialdehyde transaminase, and so forth, as
desired, so long as the combination of enzymes of the desired
biosynthetic pathway results in production of the corresponding
desired product.
[0290] Similarly, for example, with respect to any one or more
exogenous nucleic acids introduced to confer BDO production, a
non-naturally occurring microbial organism having a BDO
biosynthetic pathway can comprise at least two exogenous nucleic
acids encoding desired enzymes, such as the combination of
4-hydroxybutanoate dehydrogenase and .alpha.-ketoglutarate
decarboxylase; 4-hydroxybutanoate dehydrogenase and
4-hydroxybutyryl CoA:acetyl-CoA transferase; 4-hydroxybutanoate
dehydrogenase and butyrate kinase; 4-hydroxybutanoate dehydrogenase
and phosphotransbutyrylase; 4-hydroxybutyryl CoA:acetyl-CoA
transferase and aldehyde dehydrogenase; 4-hydroxybutyryl
CoA:acetyl-CoA transferase and alcohol dehydrogenase;
4-hydroxybutyryl CoA:acetyl-CoA transferase and an aldehyde/alcohol
dehydrogenase, 4-aminobutyrate-CoA transferase and
4-aminobutyryl-CoA transaminase; 4-aminobutyrate kinase and
4-aminobutan-1-ol oxidoreductase (deaminating), and the like. Thus,
it is understood that any combination of two or more enzymes 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 of a biosynthetic
pathway can be included in a non-naturally occurring microbial
organism of the invention, for example, 4-hydroxybutanoate
dehydrogenase, .alpha.-ketoglutarate decarboxylase and
4-hydroxybutyryl CoA:acetyl-CoA transferase; 4-hydroxybutanoate
dehydrogenase, butyrate kinase and phosphotransbutyrylase;
4-hydroxybutanoate dehydrogenase, 4-hydroxybutyryl CoA:acetyl-CoA
transferase and aldehyde dehydrogenase; 4-hydroxybutyryl
CoA:acetyl-CoA transferase, aldehyde dehydrogenase and alcohol
dehydrogenase; butyrate kinase, phosphotransbutyrylase and an
aldehyde/alcohol dehydrogenase; 4-aminobutyryl-CoA hydrolase,
4-aminobutyryl-CoA reductase and 4-amino butan-1-ol transaminase;
3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA
dehydratase and 4-hydroxybutyryl-CoA dehydratase, and the like.
Similarly, any combination of four, five or more enzymes 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 of the desired
biosynthetic pathway results in production of the corresponding
desired product.
[0291] 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 4-HB
producers can be cultured for the biosynthetic production of 4-HB.
The 4-HB can be isolated or be treated as described below to
generate GBL, THF and/or BDO. Similarly, the BDO producers can be
cultured for the biosynthetic production of BDO. The BDO can be
isolated or subjected to further treatments for the chemical
synthesis of BDO family compounds, as disclosed herein.
[0292] 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, sucrose, 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, sucrose, 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 4-HB, 4-HBal, 4-HBCoA, BDO or
putrescine and other compounds of the invention.
[0293] Accordingly, given the teachings and guidance provided
herein, those skilled in the art will understand that a
non-naturally occurring microbial organism can be produced that
secretes the biosynthesized compounds of the invention when grown
on a carbon source such as a carbohydrate. Such compounds include,
for example, 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine and any of
the intermediates metabolites in the 4-HB, 4-HBal, 4-HBCoA, BDO or
putrescine pathways and/or the combined 4-HB, 4-HBal, 4-HBCoA, BDO
or putrescine pathways. All that is required is to engineer in one
or more of the enzyme activities shown in FIG. 1 to achieve
biosynthesis of the desired compound or intermediate including, for
example, inclusion of some or all of the 4-HB, 4-HBal, 4-HBCoA, BDO
or putrescine biosynthetic pathways. Accordingly, the invention
provides a non-naturally occurring microbial organism that secretes
4-HB when grown on a carbohydrate, secretes BDO when grown on a
carbohydrate and/or secretes any of the intermediate metabolites
shown in FIG. 1, 8-13, 58, 62, 63 or 72-74 when grown on a
carbohydrate. A BDO producing microbial organisms of the invention
can initiate synthesis from, for example, succinate, succinyl-CoA,
.alpha.-ketogluterate, succinic semialdehyde, 4-HB,
4-hydroxybutyrylphosphate, 4-hydroxybutyryl-CoA (4-HB-CoA) and/or
4-hydroxybutyraldehyde.
[0294] 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 below in the Examples. 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 4-HB,
4-HBal, 4-HBCoA, BDO or putrescine producers can synthesize
monomeric 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine, respectively,
at intracellular concentrations of 5-10 mM or more as well as all
other concentrations exemplified previously.
[0295] A number of downstream compounds also can be generated for
the 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine producing
non-naturally occurring microbial organisms of the invention. With
respect to the 4-HB producing microbial organisms of the invention,
monomeric 4-HB and GBL exist in equilibrium in the culture medium.
The conversion of 4-HB to GBL can be efficiently accomplished by,
for example, culturing the microbial organisms in acid pH medium. A
pH less than or equal to 7.5, in particular at or below pH 5.5,
spontaneously converts 4-HB to GBL.
[0296] The resultant GBL can be separated from 4-HB and other
components in the culture using a variety of methods well known in
the art. Such separation methods include, for example, the
extraction procedures exemplified in the Examples as well as
methods which 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. Separated GBL can be further purified by, for example,
distillation.
[0297] Another down stream compound that can be produced from the
4-HB producing non-naturally occurring microbial organisms of the
invention includes, for example, BDO. This compound can be
synthesized by, for example, chemical hydrogenation of GBL.
Chemical hydrogenation reactions are well known in the art. One
exemplary procedure includes the chemical reduction of 4-HB and/or
GBL or a mixture of these two components deriving from the culture
using a heterogeneous or homogeneous hydrogenation catalyst
together with hydrogen, or a hydride-based reducing agent used
stoichiometrically or catalytically, to produce 1,4-butanediol.
[0298] Other procedures well known in the art are equally
applicable for the above chemical reaction and include, for
example, WO No. 82/03854 (Bradley, et al.), which describes the
hydrogenolysis of gamma-butyrolactone in the vapor phase over a
copper oxide and zinc oxide catalyst. British Pat. No. 1,230,276,
which describes the hydrogenation of gamma-butyrolactone using a
copper oxide-chromium oxide catalyst. The hydrogenation is carried
out in the liquid phase. Batch reactions also are exemplified
having high total reactor pressures. Reactant and product partial
pressures in the reactors are well above the respective dew points.
British Pat. No. 1,314,126, which describes the hydrogenation of
gamma-butyrolactone in the liquid phase over a
nickel-cobalt-thorium oxide catalyst. Batch reactions are
exemplified as having high total pressures and component partial
pressures well above respective component dew points. British Pat.
No. 1,344,557, which describes the hydrogenation of
gamma-butyrolactone in the liquid phase over a copper
oxide-chromium oxide catalyst. A vapor phase or vapor-containing
mixed phase is indicated as suitable in some instances. A
continuous flow tubular reactor is exemplified using high total
reactor pressures. British Pat. No. 1,512,751, which describes the
hydrogenation of gamma-butyrolactone to 1,4-butanediol in the
liquid phase over a copper oxide-chromium oxide catalyst. Batch
reactions are exemplified with high total reactor pressures and,
where determinable, reactant and product partial pressures well
above the respective dew points. U.S. Pat. No. 4,301,077, which
describes the hydrogenation to 1,4-butanediol of
gamma-butyrolactone over a Ru--Ni--Co--Zn catalyst. The reaction
can be conducted in the liquid or gas phase or in a mixed
liquid-gas phase. Exemplified are continuous flow liquid phase
reactions at high total reactor pressures and relatively low
reactor productivities. U.S. Pat. No. 4,048,196, which describes
the production of 1,4-butanediol by the liquid phase hydrogenation
of gamma-butyrolactone over a copper oxide-zinc oxide catalyst.
Further exemplified is a continuous flow tubular reactor operating
at high total reactor pressures and high reactant and product
partial pressures. And U.S. Pat. No. 4,652,685, which describes the
hydrogenation of lactones to glycols.
[0299] A further downstream compound that can be produced form the
4-HB producing microbial organisms of the invention includes, for
example, THF. This compound can be synthesized by, for example,
chemical hydrogenation of GBL. One exemplary procedure well known
in the art applicable for the conversion of GBL to THF includes,
for example, chemical reduction of 4-HB and/or GBL or a mixture of
these two components deriving from the culture using a
heterogeneous or homogeneous hydrogenation catalyst together with
hydrogen, or a hydride-based reducing agent used stoichiometrically
or catalytically, to produce tetrahydrofuran. Other procedures well
know in the art are equally applicable for the above chemical
reaction and include, for example, U.S. Pat. No. 6,686,310, which
describes high surface area sol-gel route prepared hydrogenation
catalysts. Processes for the reduction of maleic acid to
tetrahydrofuran (THF) and 1,4-butanediol (BDO) and for the
reduction of gamma butyrolactone to tetrahydrofuran and
1,4-butanediol also are described.
[0300] The culture conditions can include, for example, liquid
culture procedures as well as fermentation and other large scale
culture procedures. As described further below in the Examples,
particularly useful yields of the biosynthetic products of the
invention can be obtained under anaerobic or substantially
anaerobic culture conditions.
[0301] Suitable purification and/or assays to test for the
production of 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine 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.
[0302] The 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine product 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.
[0303] The invention further provides a method of manufacturing
4-HB. The method includes fermenting a non-naturally occurring
microbial organism having a 4-hydroxybutanoic acid (4-HB)
biosynthetic pathway comprising at least one exogenous nucleic acid
encoding 4-hydroxybutanoate dehydrogenase, CoA-independent succinic
semialdehyde dehydrogenase, succinyl-CoA synthetase, CoA-dependent
succinic semialdehyde dehydrogenase, glutamate:succinic
semialdehyde transaminase, .alpha.-ketoglutarate decarboxylase, or
glutamate decarboxylase under substantially anaerobic conditions
for a sufficient period of time to produce monomeric
4-hydroxybutanoic acid (4-HB), the process comprising fed-batch
fermentation and batch separation; fed-batch fermentation and
continuous separation, or continuous fermentation and continuous
separation.
[0304] The culture and chemical hydrogenations described above also
can be scaled up and grown continuously for manufacturing of 4-HB,
4-HBal, 4-HBCoA, GBL, BDO and/or THF or putrescine. 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. Employing the 4-HB producers
allows for simultaneous 4-HB biosynthesis and chemical conversion
to GBL, BDO and/or THF by employing the above hydrogenation
procedures simultaneous with continuous cultures methods such as
fermentation. Other hydrogenation procedures also are well known in
the art and can be equally applied to the methods of the
invention.
[0305] Fermentation procedures are particularly useful for the
biosynthetic production of commercial quantities of 4-HB, 4-HBal,
4-HBCoA, BDO or putrescine. Generally, and as with non-continuous
culture procedures, the continuous and/or near-continuous
production of 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine will include
culturing a non-naturally occurring 4-HB, 4-HBal, 4-HBCoA, BDO or
putrescine producing organism of the invention in sufficient
nutrients and medium to sustain and/or nearly sustain growth in an
exponential phase. Continuous culture under such conditions can be
include, for example, 1 day, 2, 3, 4, 5, 6 or 7 days or more.
Additionally, continuous culture can include 1 week, 2, 3, 4 or 5
or more weeks and up to several months. Alternatively, organisms 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.
[0306] Fermentation procedures are well known in the art. Briefly,
fermentation for the biosynthetic production of 4-HB, 4-HBal,
4-HBCoA, BDO or putrescine or other 4-HB, 4-HBal, 4-HBCoA, BDO or
putrescine derived products, including intermediates, of the
invention 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 well known
in the art are exemplified further below in the Examples.
[0307] In addition to the above fermentation procedures using the
4-HB, 4-HBal, 4-HBCoA, BDO or putrescine producers of the invention
for continuous production of substantial quantities of 4-HB,
4-HBal, 4-HBCoA, BDO or putrescine, including monomeric 4-HB,
respectively, the 4-HB producers also can be, for example,
simultaneously subjected to chemical synthesis procedures to
convert the product to other compounds or the product as described
previously for the chemical conversion of monomeric 4-HB to, for
example, GBL, BDO and/or THF. The BDO producers can similarly be,
for example, simultaneously subjected to chemical synthesis
procedures as described previously for the chemical conversion of
BDO to, for example, THF, GBL, pyrrolidones and/or other BDO family
compounds. In addition, the products of the 4-HB, 4-HBal, 4-HBCoA,
BDO or putrescine producers can be separated from the fermentation
culture and sequentially subjected to chemical or enzymatic
conversion to convert the product to other compounds, if desired,
as disclosed herein.
[0308] Briefly, hydrogenation of GBL in the fermentation broth can
be performed as described by Frost et al., Biotechnology Progress
18: 201-211 (2002). Another procedure for hydrogenation during
fermentation include, for example, the methods described in, for
example, U.S. Pat. No. 5,478,952. This method is further
exemplified in the Examples below.
[0309] Therefore, the invention additionally provides a method of
manufacturing .gamma.-butyrolactone (GBL), tetrahydrofuran (THF) or
1,4-butanediol (BDO). The method includes fermenting a
non-naturally occurring microbial organism having 4-hydroxybutanoic
acid (4-HB) and/or 1,4-butanediol (BDO) biosynthetic pathways, the
pathways comprise at least one exogenous nucleic acid encoding
4-hydroxybutanoate dehydrogenase, CoA-independent succinic
semialdehyde dehydrogenase, succinyl-CoA synthetase, CoA-dependent
succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:CoA
transferase, glutamate:succinic semialdehyde transaminase,
alpha-ketoglutarate decarboxylase, glutamate decarboxylase,
4-hydroxybutanoate kinase, phosphotransbutyrylase, CoA-independent
1,4-butanediol semialdehyde dehydrogenase, CoA-dependent
1,4-butanediol semialdehyde dehydrogenase, CoA-independent
1,4-butanediol alcohol dehydrogenase or CoA-dependent
1,4-butanediol alcohol dehydrogenase, under substantially anaerobic
conditions for a sufficient period of time to produce
1,4-butanediol (BDO), GBL or THF, the fermenting comprising
fed-batch fermentation and batch separation; fed-batch fermentation
and continuous separation, or continuous fermentation and
continuous separation.
[0310] In addition to the biosynthesis of 4-HB, 4-HBal, 4-HBCoA,
BDO or putrescine and other products of the invention 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 BDO other than use of the
4-HB producers and chemical steps or other than use of the BDO
producer directly is through addition of another microbial organism
capable of converting 4-HB or a 4-HB product exemplified herein to
BDO.
[0311] One such procedure includes, for example, the fermentation
of a 4-HB producing microbial organism of the invention to produce
4-HB, as described above and below. The 4-HB can then be used as a
substrate for a second microbial organism that converts 4-HB to,
for example, BDO, GBL and/or THF. The 4-HB can be added directly to
another culture of the second organism or the original culture of
4-HB 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 utilized to produce
the final product without intermediate purification steps. One
exemplary second organism having the capacity to biochemically
utilize 4-HB as a substrate for conversion to BDO, for example, is
Clostridium acetobutylicum (see, for example, Jewell et al.,
Current Microbiology, 13:215-19 (1986)).
[0312] Thus, such a procedure includes, for example, the
fermentation of a microbial organism that produces a 4-HB, 4-HBal,
4-HBCoA, BDO or putrescine pathway intermediate. The 4-HB, 4-HBal,
4-HBCoA, BDO or putrescine pathway intermediate can then be used as
a substrate for a second microbial organism that converts the 4-HB,
4-HBal, 4-HBCoA, BDO or putrescine pathway intermediate to 4-HB,
4-HBal, 4-HBCoA, BDO or putrescine. The 4-HB, 4-HBal, 4-HBCoA, BDO
or putrescine pathway intermediate can be added directly to another
culture of the second organism or the original culture of the 4-HB,
4-HBal, 4-HBCoA BDO or putrescine 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.
[0313] 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,
4-HB and/or BDO as described. 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 BDO can be
accomplished as described previously by constructing a microbial
organism that contains biosynthetic pathways for conversion of one
pathway intermediate to another pathway intermediate or the
product, for example, a substrate such as endogenous succinate
through 4-HB to the final product BDO. Alternatively, BDO also can
be biosynthetically produced from microbial organisms through
co-culture or co-fermentation using two organisms in the same
vessel. A first microbial organism being a 4-HB producer with genes
to produce 4-HB from succinic acid, and a second microbial organism
being a BDO producer with genes to convert 4-HB to BDO. For
example, the biosynthesis of 4-HB, 4-HBal, 4-HBCoA, BDO or
putrescine 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, 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine also can be
biosynthetically produced from microbial organisms through
co-culture or co-fermentation using two organisms in the same
vessel, where the first microbial organism produces a 4-HB, 4-HBal,
4-HBCoA, BDO or putrescine intermediate and the second microbial
organism converts the intermediate to 4-HB, 4-HBal, 4-HBCoA, BDO or
putrescine.
[0314] 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 4-HB, BDO, GBL and THF products of
the invention.
[0315] It is understood that, in methods of the invention, any of
the one or more exogenous nucleic acids can be introduced into a
microbial organism to produce a non-naturally occurring microbial
organism of the invention. The nucleic acids can be introduced so
as to confer, for example, a 4-HB, 4-HBal, 4-HBCoA, BDO or
putrescine 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
4-HB, 4-HBal, 4-HBCoA, BDO or putrescine biosynthetic capability.
For example, a non-naturally occurring microbial organism having a
4-HB, 4-HBal, 4-HBCoA, BDO or putrescine biosynthetic pathway can
comprise at least two exogenous nucleic acids encoding desired
enzymes or proteins, such as the combination of enzymes as
disclosed herein (see Examples and FIG. 1, 8-13, 58, 62, 63 or
72-74), and the like. Thus, it is understood that any combination
of two or more enzymes or proteins of a biosynthetic pathway can be
included in a non-naturally occurring microbial organism of the
invention. Similarly, it is understood that any combination of
three or more enzymes or proteins of a biosynthetic pathway can be
included in a non-naturally occurring microbial organism of the
invention, for example, and so forth, as desired and disclosed
herein, so long as the combination of enzymes and/or proteins of
the desired biosynthetic pathway results in production of the
corresponding desired product. Similarly, any combination of four
or more enzymes or proteins of a biosynthetic pathway as disclosed
herein 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.
[0316] The invention additionally provides carboxylic acid
reductase variants. CAR variants were generated and tested for
activity. In a particular embodiment, a carboxylic acid reductase
can comprise an amino acid sequence having an amino acid
substitution selected from E16K; Q95L; L100M; A1011T; K823E; T941S;
H15Q; D198E; G446C; S392N; F699L; V883I; F467S; T987S; R12H; V295G;
V295A; V295S; V295T; V295C; V295V; V295L; V295I; V295M; V295P;
V295F; V295Y; V295W; V295D; V295E; V295N; V295Q; V295H; V295K;
V295R; M296G; M296A; M296S; M296T; M296C; M296V; M296L; M296I;
M296M; M296P; M296F; M296Y; M296W; M296D; M296E; M296N; M296Q;
M296H; M296K; M296R; G297G; G297A; G297S; G297T; G297C; G297V;
G297L; G297I; G297M; G297P; G297F; G297Y; G297W; G297D; G297E;
G297N; G297Q; G297H; G297K; G297R; G391G; G391A; G391S; G391T;
G391C; G391V; G391L; G391I; G391M; G391P; G391F; G391Y; G391W;
G391D; G391E; G391N; G391Q; G391H; G391K; G391R; G421G; G421A;
G421S; G421T; G421C; G421V; G421L; G421I; G421M; G421P; G421F;
G421Y; G421W; G421D; G421E; G421N; G421Q; G421H; G421K; G421R;
D413G; D413A; D413S; D413T; D413C; D413V; D413L; D413I; D413M;
D413P; D413F; D413Y; D413W; D413D; D413E; D413N; D413Q; D413H;
D413K; D413R; G414G; G414A; G414S; G414T; G414C; G414V; G414L;
G414I; G414M; G414P; G414F; G414Y; G414W; G414D; G414E; G414N;
G414Q; G414H; G414K; G414R; Y415G; Y415A; Y415S; Y415T; Y415C;
Y415V; Y415L; Y415I; Y415M; Y415P; Y415F; Y415Y; Y415W; Y415D;
Y415E; Y415N; Y415Q; Y415H; Y415K; Y415R; G416G; G416A; G416S;
G416T; G416C; G416V; G416L; G416I; G416M; G416P; G416F; G416Y;
G416W; G416D; G416E; G416N; G416Q; G416H; G416K; G416R; S417G;
S417A; S417S; S417T; S417C; S417V S417L; S417I; S417M; S417P;
S417F; S417Y; S417W; S417D; S417E; S417N; S417Q; S417H; S417K; and
S417R, or combinations thereof. The amino acid positions correspond
to amino acid positions of sequence of FIG. 67B, or equivalent
positions in a homologous CAR sequence. It is further understood
that a CAR variant includes a combination of one or more of the
amino acid substitutions, so long as the variant with multiple
amino acid substitutions exhibits measurable CAR activity, as
disclosed herein.
[0317] 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 4-HB, 4-HBal, 4-HBCoA, BDO or
putrescine.
[0318] One computational method for identifying and designing
metabolic alterations favoring biosynthesis of a desired product is
the OptKnock computational framework (Burgard et al., Biotechnol.
Bioeng. 84:647-657 (2003)). OptKnock is a metabolic modeling and
simulation program that suggests gene deletion or disruption
strategies that result in genetically stable microorganisms which
overproduce the target product. Specifically, the framework
examines the complete metabolic and/or biochemical network of a
microorganism in order to suggest genetic manipulations that force
the desired biochemical to become an obligatory byproduct of cell
growth. By coupling biochemical production with cell growth through
strategically placed gene deletions or other functional gene
disruption, the growth selection pressures imposed on the
engineered strains after long periods of time in a bioreactor lead
to improvements in performance as a result of the compulsory
growth-coupled biochemical production. Lastly, when gene deletions
are constructed there is a negligible possibility of the designed
strains reverting to their wild-type states because the genes
selected by OptKnock are to be completely removed from the genome.
Therefore, this computational methodology can be used to either
identify alternative pathways that lead to biosynthesis of a
desired product or used in connection with the non-naturally
occurring microbial organisms for further optimization of
biosynthesis of a desired product.
[0319] Briefly, OptKnock is a term used herein to refer to a
computational method and system for modeling cellular metabolism.
The OptKnock program relates to a framework of models and methods
that incorporate particular constraints into flux balance analysis
(FBA) models. These constraints include, for example, qualitative
kinetic information, qualitative regulatory information, and/or DNA
microarray experimental data. OptKnock also computes solutions to
various metabolic problems by, for example, tightening the flux
boundaries derived through flux balance models and subsequently
probing the performance limits of metabolic networks in the
presence of gene additions or deletions. OptKnock computational
framework allows the construction of model formulations that allow
an effective query of the performance limits of metabolic networks
and provides methods for solving the resulting mixed-integer linear
programming problems. The metabolic modeling and simulation methods
referred to herein as OptKnock are described in, for example, U.S.
publication 2002/0168654, filed Jan. 10, 2002, in International
Patent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S.
publication 2009/0047719, filed Aug. 10, 2007.
[0320] Another computational method for identifying and designing
metabolic alterations favoring biosynthetic production of a product
is a metabolic modeling and simulation system termed SimPheny.RTM..
This computational method and system is described in, for example,
U.S. publication 2003/0233218, filed Jun. 14, 2002, and in
International Patent Application No. PCT/US03/18838, filed Jun. 13,
2003. SimPheny.RTM. is a computational system that can be used to
produce a network model in silico and to simulate the flux of mass,
energy or charge through the chemical reactions of a biological
system to define a solution space that contains any and all
possible functionalities of the chemical reactions in the system,
thereby determining a range of allowed activities for the
biological system. This approach is referred to as
constraints-based modeling because the solution space is defined by
constraints such as the known stoichiometry of the included
reactions as well as reaction thermodynamic and capacity
constraints associated with maximum fluxes through reactions. The
space defined by these constraints can be interrogated to determine
the phenotypic capabilities and behavior of the biological system
or of its biochemical components.
[0321] 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.
[0322] 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.
[0323] 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.
[0324] 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.
[0325] 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..
[0326] 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.
[0327] 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)).
[0328] 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.
[0329] The methods exemplified above and further illustrated in the
Examples below allow the construction of cells and organisms that
biosynthetically produce, including obligatory couple production of
a target biochemical product to growth of the cell or organism
engineered to harbor the identified genetic alterations. In this
regard, metabolic alterations have been identified that result in
the biosynthesis of 4-HB and 1,4-butanediol. Microorganism strains
constructed with the identified metabolic alterations produce
elevated levels of 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine
compared to unmodified microbial organisms. These strains can be
beneficially used for the commercial production of 4-HB, BDO, THF,
GBL, 4-HBal, 4-HBCoA or putrescine, for example, in continuous
fermentation process without being subjected to the negative
selective pressures.
[0330] 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.
[0331] 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.
[0332] 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 4-HB,
4-HBal, 4-HBCoA, BDO or putrescine producers can be cultured for
the biosynthetic production of 4-HB, 4-HBal, 4-HBCoA, BDO or
putrescine.
[0333] For the production of 4-HB, 4-HBal, 4-HBCoA, BDO or
putrescine, the recombinant strains are cultured in a medium with
carbon source and other essential nutrients. It is highly desirable
to maintain anaerobic conditions in the fermenter to reduce the
cost of the overall process. Such conditions can be obtained, for
example, by first sparging the medium with nitrogen and then
sealing the flasks with a septum and crimp-cap. For strains where
growth is not observed anaerobically, microaerobic conditions can
be applied by perforating the septum with a small hole for limited
aeration. Exemplary anaerobic conditions have been described
previously and are well-known in the art. Exemplary aerobic and
anaerobic conditions are described, for example, in U.S.
publication 2009/0047719, filed Aug. 10, 2007. Fermentations can be
performed in a batch, fed-batch or continuous manner, as disclosed
herein.
[0334] 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.
[0335] In addition to renewable feedstocks such as those
exemplified above, the 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine
producing 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
4-HB, 4-HBal, 4-HBCoA, BDO or putrescine producing organisms to
provide a metabolic pathway for utilization of syngas or other
gaseous carbon source.
[0336] 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 H2 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 H2 and CO, syngas can also
include CO2 and other gases in smaller quantities. Thus, synthesis
gas provides a cost effective source of gaseous carbon such as CO
and, additionally, CO2.
[0337] The Wood-Ljungdahl pathway catalyzes the conversion of CO
and H2 to acetyl-CoA and other products such as acetate. Organisms
capable of utilizing CO and syngas also generally have the
capability of utilizing CO2 and CO2/H2 mixtures through the same
basic set of enzymes and transformations encompassed by the
Wood-Ljungdahl pathway. H2-dependent conversion of CO2 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 CO2 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:
2CO2+4H2+nADP+nPi.fwdarw.CH3COOH+2H2O+nATP
[0338] Hence, non-naturally occurring microorganisms possessing the
Wood-Ljungdahl pathway can utilize CO2 and H2 mixtures as well for
the production of acetyl-CoA and other desired products.
[0339] 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 4-HB, 4-HBal, 4-HBCoA, BDO or
putrescine 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.
[0340] Additionally, the reductive (reverse) tricarboxylic acid
cycle is and/or hydrogenase activities can also be used for the
conversion of CO, CO2 and/or H2 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 H2
by carbon monoxide dehydrogenase and hydrogenase are utilized to
fix CO2 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 4-HB,
4-HBal, 4-HBCoA, BDO or putrescine 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 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine
pathway, those skilled in the art will understand that the same
engineering design also can be performed with respect to
introducing at least the nucleic acids encoding the reductive TCA
pathway enzymes or proteins absent in the host organism. Therefore,
introduction of one or more encoding nucleic acids into the
microbial organisms of the invention such that the modified
organism contains the complete reductive TCA pathway will confer
syngas utilization ability.
[0341] Accordingly, given the teachings and guidance provided
herein, those skilled in the art will understand that a
non-naturally occurring microbial organism can be produced that
secretes the biosynthesized compounds of the invention when grown
on a carbon source such as a carbohydrate. Such compounds include,
for example, 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine and any of
the intermediate metabolites in the 4-HB, 4-HBal, 4-HBCoA, BDO or
putrescine 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 4-HB, 4-HBal, 4-HBCoA, BDO
or putrescine biosynthetic pathways. Accordingly, the invention
provides a non-naturally occurring microbial organism that produces
and/or secretes 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine when grown
on a carbohydrate or other carbon source and produces and/or
secretes any of the intermediate metabolites shown in the 4-HB,
4-HBal, 4-HBCoA, BDO or putrescine pathway when grown on a
carbohydrate or other carbon source. The 4-HB, 4-HBal, 4-HBCoA, BDO
or putrescine producing microbial organisms of the invention can
initiate synthesis from an intermediate in a 4-HB, 4-HBal, 4-HBCoA,
BDO or putrescine pathway, as disclosed herein.
[0342] 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 4-HB, 4-HBal, 4-HBCoA, BDO or
putrescine.
[0343] 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.
[0344] Briefly, OptKnock is a term used herein to refer to a
computational method and system for modeling cellular metabolism.
The OptKnock program relates to a framework of models and methods
that incorporate particular constraints into flux balance analysis
(FBA) models. These constraints include, for example, qualitative
kinetic information, qualitative regulatory information, and/or DNA
microarray experimental data. OptKnock also computes solutions to
various metabolic problems by, for example, tightening the flux
boundaries derived through flux balance models and subsequently
probing the performance limits of metabolic networks in the
presence of gene additions or deletions. OptKnock computational
framework allows the construction of model formulations that allow
an effective query of the performance limits of metabolic networks
and provides methods for solving the resulting mixed-integer linear
programming problems. The metabolic modeling and simulation methods
referred to herein as OptKnock are described in, for example, U.S.
publication 2002/0168654, filed Jan. 10, 2002, in International
Patent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S.
publication 2009/0047719, filed Aug. 10, 2007.
[0345] Another computational method for identifying and designing
metabolic alterations favoring biosynthetic production of a product
is a metabolic modeling and simulation system termed SimPheny.RTM..
This computational method and system is described in, for example,
U.S. publication 2003/0233218, filed Jun. 14, 2002, and in
International Patent Application No. PCT/US03/18838, filed Jun. 13,
2003. SimPheny.RTM. is a computational system that can be used to
produce a network model in silico and to simulate the flux of mass,
energy or charge through the chemical reactions of a biological
system to define a solution space that contains any and all
possible functionalities of the chemical reactions in the system,
thereby determining a range of allowed activities for the
biological system. This approach is referred to as
constraints-based modeling because the solution space is defined by
constraints such as the known stoichiometry of the included
reactions as well as reaction thermodynamic and capacity
constraints associated with maximum fluxes through reactions. The
space defined by these constraints can be interrogated to determine
the phenotypic capabilities and behavior of the biological system
or of its biochemical components.
[0346] 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.
[0347] 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.
[0348] 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.
[0349] 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.
[0350] 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..
[0351] 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.
[0352] 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)).
[0353] 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.
[0354] As disclosed herein, a nucleic acid encoding a desired
activity of a 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine pathway can
be introduced into a host organism. In some cases, it can be
desirable to modify an activity of a 4-HB, 4-HBal, 4-HBCoA BDO or
putrescine pathway enzyme or protein to increase production of
4-HB, 4-HBal, 4-HBCoA BDO or putrescine. 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.
[0355] 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, >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. 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
(Km), including broadening substrate binding to include non-natural
substrates; inhibition (Ki), 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.
[0356] 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 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine
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)).
[0357] 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)).
[0358] 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-.times. in random and natural mutation
frequency during selection and block accumulation of deleterious
mutations when selection is not required (Selifonova et al., Appl.
Environ. Microbiol. 67:3645-3649 (2001)); Low et al., J. Mol. Biol.
260:359-3680 (1996)).
[0359] 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)).
[0360] 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.
[0361] It is understood that modifications which do not
substantially affect the activity of the various embodiments of
this invention are also provided within the definition of the
invention provided herein. Accordingly, the following examples are
intended to illustrate but not limit the present invention.
Example I
Biosynthesis of 4-Hydroxybutanoic Acid
[0362] This example describes exemplary biochemical pathways for
4-HB production.
[0363] Previous reports of 4-HB synthesis in microbes have focused
on this compound as an intermediate in production of the
biodegradable plastic poly-hydroxyalkanoate (PHA) (U.S. Pat. No.
6,117,658). The use of 4-HB/3-HB copolymers over
poly-3-hydroxybutyrate polymer (PHB) can result in plastic that is
less brittle (Saito and Doi, Intl. J. Biol. Macromol. 16:99-104
(1994)). The production of monomeric 4-HB described herein is a
fundamentally distinct process for several reasons: (1) the product
is secreted, as opposed to PHA which is produced intracellularly
and remains in the cell; (2) for organisms that produce
hydroxybutanoate polymers, free 4-HB is not produced, but rather
the Coenzyme A derivative is used by the polyhydroxyalkanoate
synthase; (3) in the case of the polymer, formation of the granular
product changes thermodynamics; and (4) extracellular pH is not an
issue for production of the polymer, whereas it will affect whether
4-HB is present in the free acid or conjugate base state, and also
the equilibrium between 4-HB and GBL.
[0364] 4-HB can be produced in two enzymatic reduction steps from
succinate, a central metabolite of the TCA cycle, with succinic
semialdehyde as the intermediate (FIG. 1). The first of these
enzymes, succinic semialdehyde dehydrogenase, is native to many
organisms including E. coli, in which both NADH- and
NADPH-dependent enzymes have been found (Donnelly and Cooper, Eur.
J. Biochem. 113:555-561 (1981); Donnelly and Cooper, J. Bacteriol.
145:1425-1427 (1981); Marek and Henson, J. Bacteriol. 170:991-994
(1988)). There is also evidence supporting succinic semialdehyde
dehydrogenase activity in S. cerevisiae (Ramos et al., Eur. J.
Biochem. 149:401-404 (1985)), and a putative gene has been
identified by sequence homology. However, most reports indicate
that this enzyme proceeds in the direction of succinate synthesis,
as shown in FIG. 1 (Donnelly and Cooper, supra; Lutke-Eversloh and
Steinbuchel, FEMS Microbiol. Lett. 181:63-71 (1999)), participating
in the degradation pathway of 4-HB and gamma-aminobutyrate.
Succinic semialdehyde also is natively produced by certain
microbial organisms such as E. coli through the TCA cycle
intermediate .alpha.-ketogluterate via the action of two enzymes:
glutamate:succinic semialdehyde transaminase and glutamate
decarboxylase. An alternative pathway, used by the obligate
anaerobe Clostridium kluyveri to degrade succinate, activates
succinate to succinyl-CoA, then converts succinyl-CoA to succinic
semialdehyde using an alternative succinic semialdehyde
dehydrogenase which is known to function in this direction (Sohling
and Gottschalk, Eur. J. Biochem. 212:121-127 (1993)). However, this
route has the energetic cost of ATP required to convert succinate
to succinyl-CoA.
[0365] The second enzyme of the pathway, 4-hydroxybutanoate
dehydrogenase, is not native to E. coli or yeast but is found in
various bacteria such as C. kluyveri and Ralstonia eutropha
(Lutke-Eversloh and Steinbuchel, supra; Sohling and Gottschalk, J.
Bacteriol. 178:871-880 (1996); Valentin et al., Eur. J. Biochem.
227:43-60 (1995); Wolff and Kenealy, Protein Expr. Purif. 6:206-212
(1995)). These enzymes are known to be NADH-dependent, though
NADPH-dependent forms also exist. An additional pathway to 4-HB
from alpha-ketoglutarate was demonstrated in E. coli resulting in
the accumulation of poly(4-hydroxybutyric acid) (Song et al., Wei
Sheng Wu Xue. Bao. 45:382-386 (2005)). The recombinant strain
required the overexpression of three heterologous genes, PHA
synthase (R. eutropha), 4-hydroxybutyrate dehydrogenase (R.
eutropha) and 4-hydroxybutyrate:CoA transferase (C. kluyveri),
along with two native E. coli genes: glutamate:succinic
semialdehyde transaminase and glutamate decarboxylase. Steps 4 and
5 in FIG. 1 can alternatively be carried out by an
alpha-ketoglutarate decarboxylase such as the one identified in
Euglena gracilis (Shigeoka et al., Biochem. J. 282(Pt2):319-323
(1992); Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28
(1991); Shigeoka and Nakano, Biochem J. 292(Pt 2):463-467 (1993)).
However, this enzyme has not previously been applied to impact the
production of 4-HB or related polymers in any organism.
[0366] The microbial production capabilities of 4-hydroxybutyrate
were explored in two microbes, Escherichia coli and Saccharomyces
cerevisiae, using in silico metabolic models of each organism.
Potential pathways to 4-HB proceed via a succinate, succinyl-CoA,
or alpha-ketoglutarate intermediate as shown in FIG. 1.
[0367] A first step in the 4-HB production pathway from succinate
involves the conversion of succinate to succinic semialdehyde via
an NADH- or NADPH-dependant succinic semialdehyde dehydrogenase. In
E. coli, gabD is an NADP-dependant succinic semialdehyde
dehydrogenase and is part of a gene cluster involved in
4-aminobutyrate uptake and degradation (Niegemann et al., Arch.
Microbiol. 160:454-460 (1993); Schneider et al., J. Bacteriol.
184:6976-6986 (2002)). sad is believed to encode the enzyme for
NAD-dependant succinic semialdehyde dehydrogenase activity (Marek
and Henson, supra). S. cerevisiae contains only the NADPH-dependant
succinic semialdehyde dehydrogenase, putatively assigned to UGA2,
which localizes to the cytosol (Huh et al., Nature 425:686-691
(2003)). The maximum yield calculations assuming the succinate
pathway to 4-HB in both E. coli and S. cerevisiae require only the
assumption that a non-native 4-HB dehydrogenase has been added to
their metabolic networks.
[0368] The pathway from succinyl-CoA to 4-hydroxybutyrate was
described in U.S. Pat. No. 6,117,658 as part of a process for
making polyhydroxyalkanoates comprising 4-hydroxybutyrate monomer
units. Clostridium kluyveri is one example organism known to
possess CoA-dependant succinic semialdehyde dehydrogenase activity
(Sohling and Gottschalk, supra; Sohling and Gottschalk, supra). In
this study, it is assumed that this enzyme, from C. kluyveri or
another organism, is expressed in E. coli or S. cerevisiae along
with a non-native or heterologous 4-HB dehydrogenase to complete
the pathway from succinyl-CoA to 4-HB. The pathway from
alpha-ketoglutarate to 4-HB was demonstrated in E. coli resulting
in the accumulation of poly(4-hydroxybutyric acid) to 30% of dry
cell weight (Song et al., supra). As E. coli and S. cerevisiae
natively or endogenously possess both glutamate:succinic
semialdehyde transaminase and glutamate decarboxylase (Coleman et
al., J. Biol. Chem. 276:244-250 (2001)), the pathway from AKG to
4-HB can be completed in both organisms by assuming only that a
non-native 4-HB dehydrogenase is present.
Example II
Biosynthesis of 1,4-Butanediol from Succinate and
Alpha-Ketoglutarate
[0369] This example illustrates the construction and biosynthetic
production of 4-HB and BDO from microbial organisms. Pathways for
4-HB and BDO are disclosed herein.
[0370] There are several alternative enzymes that can be utilized
in the pathway described above. The native or endogenous enzyme for
conversion of succinate to succinyl-CoA (Step 1 in FIG. 1) can be
replaced by a CoA transferase such as that encoded by the cat1 gene
C. kluyveri (Sohling and Gottschalk, Eur. J Biochem. 212:121-127
(1993)), which functions in a similar manner to Step 9. However,
the production of acetate by this enzyme may not be optimal, as it
might be secreted rather than being converted back to acetyl-CoA.
In this respect, it also can be beneficial to eliminate acetate
formation in Step 9. As one alternative to this CoA transferase, a
mechanism can be employed in which the 4-HB is first phosphorylated
by ATP and then converted to the CoA derivative, similar to the
acetate kinase/phosphotransacetylase pathway in E. coli for the
conversion of acetate to acetyl-CoA. The net cost of this route is
one ATP, which is the same as is required to regenerate acetyl-CoA
from acetate. The enzymes phosphotransbutyrylase (ptb) and butyrate
kinase (bk) are known to carry out these steps on the
non-hydroxylated molecules for butyrate production in C.
acetobutylicum (Cary et al., Appl Environ Microbiol 56:1576-1583
(1990); Valentine, R. C. and R. S. Wolfe, J Biol Chem.
235:1948-1952 (1960)). These enzymes are reversible, allowing
synthesis to proceed in the direction of 4-HB.
[0371] BDO also can be produced via alpha-ketoglutarate in addition
to or instead of through succinate. A described previously, and
exemplified further below, one pathway to accomplish product
biosynthesis is with the production of succinic semialdehyde via
alpha-ketoglutarate using the endogenous enzymes (FIG. 1, Steps
4-5). An alternative is to use an alpha-ketoglutarate decarboxylase
that can perform this conversion in one step (FIG. 1, Step 8; Tian
et al., Proc Natl Acad Sci U.S.A 102:10670-10675 (2005)).
[0372] For the construction of different strains of BDO-producing
microbial organisms, a list of applicable genes was assembled for
corroboration. Briefly, one or more genes within the 4-HB and/or
BDO biosynthetic pathways were identified for each step of the
complete BDO-producing pathway shown in FIG. 1, using available
literature resources, the NCBI genetic database, and homology
searches. The genes cloned and assessed in this study are presented
below in in Table 6, along with the appropriate references and URL
citations to the polypeptide sequence. As discussed further below,
some genes were synthesized for codon optimization while others
were cloned via PCR from the genomic DNA of the native or wild-type
organism. For some genes both approaches were used, and in this
case the native genes are indicated by an "n" suffix to the gene
identification number when used in an experiment. Note that only
the DNA sequences differ; the proteins are identical.
TABLE-US-00006 TABLE 6 Genes expressed in host BDO-producting
microbial organisms. Reaction Gene ID number Gene number (FIG. 1)
name Source organism Enzyme name Link to protein sequence Reference
0001 9 Cat2 Clostridium kluyveri 4-hydroxybutyrate
ncbi.nlm.nih.gov/entrez/ 1 DSM 555 coenzyme A
viewer.fcgi?db=nuccore&id= transferase 1228100 0002 12/13 adhE
Clostridium acetobutylicum Aldehyde/alcohol
ncbi.nlm.nih.gov/entrez/ 2 ATCC 824 dehydrogenase
viewer.fcgi?db=protein&val= 15004739 0003 12/13 adhE2
Clostridium acetobutylicum Aldehyde/alcohol
ncbi.nlm.nih.gov/entrez/ 2 ATCC 824 dehydrogenase
viewer.fcgi?val=NP_149325.1 0004 1 Cat1 Clostridium kluyveri
Succinate ncbi.nlm.nih.gov/entrez/ 1 DSM 555 coenzyme A
viewer.fcgi?db=nuccore&id= transferase 1228100 0008 6 sucD
Clostridium kluyveri Succinic ncbi.nlm.nih.gov/entrez/ 1 DSM 555
semialdehyde viewer.fcgi?db=nuccore&id= dehydrogenase 1228100
(CoA-dependent) 0009 7 4-HBd Ralstonia eutropha 4-hydroxybutyrate
ncbi.nlm.nih.gov/entrez/ 2 H16 dehydrogenase
viewer.fcgi?val=YP_726053.1 (NAD-dependent) 0010 7 4-HBd
Clostridium kluyveri 4-hydroxybutyrate ncbi.nlm.nih.gov/entrez/ 1
DSM 555 dehydrogenase viewer.fcgi?db=nuccore&id=
(NAD-dependent) 1228100 0011 12/13 adhE E. coli Aldehyde/alcohol
shigen.nig.ac.jp/ecoli/pec/ dehydrogenase
genes.List.DetailAction.do? fromListFlag=true&featureType=
1&orfId=1219 0012 12/13 yqhD E. coli Aldehyde/alcohol
shigen.nig.ac.jp/ecoli/pec/ dehydrogenase
genes.List.DetailAction.do 0013 13 bdhB Clostridium acetobutylicum
Butanol ncbi.nlm.nih.gov/entrez/ 2 ATCC 824 dehydrogenase II
viewer.fcgi?val=NP_349891.1 0020 11 ptb Clostridium acetobutylicum
Phospho- ncbi.nlm.nih.gov/entrez/ 2 ATCC 824 transbutyrylase
viewer.fcgi?db=protein&id= 15896327 0021 10 buk1 Clostridium
acetobutylicum Butyrate kinase I ncbi.nlm.nih.gov/entrez/ 2 ATCC
824 viewer.fcgi?db=protein&id= 20137334 0022 10 buk2
Clostridium acetobutylicum Butyrate kinase II
ncbi.nlm.nih.gov/entrez/ 2 ATCC 824 viewer.fcgi?db=protein&id=
20137415 0023 13 adhEm isolated from metalibrary Alcohol (37)d} of
anaerobic sewage digester dehydrogenase microbial consortia 0024 13
adhE Clostridium thermocellum Alcohol genome.jp/dbget-
dehydrogenase bin/www_bget?cth:Cthe_0423 0025 13 ald Clostridium
beijerinckii Coenzyme A- ncbi.nlm.nih.gov/entrez/ (31)d} acylating
aldehyde viewer.fcgi?db=protein&id= dehydrogenase 49036681 0026
13 bdhA Clostridium acetobutylicum Butanol ncbi.nlm.nih.gov/entrez/
2 ATCC 824 dehydrogenase viewer.fcgi?val=NP_349892.1 0027 12 bld
Clostridium Butyraldehyde ncbi.nlm.nih.gov/entrez/ 4
saccharoperbutylacetonicum dehydrogenase
viewer.fcgi?db=protein&id= 31075383 0028 13 bdh Clostridium
Butanol ncbi.nlm.nih.gov/entrez/ 4 saccharoperbutylacetonicum
dehydrogenase viewer.fcgi?db=protein&id= 124221917 0029 12/13
adhE Clostridium tetani Aldehyde/alcohol genome.jp/dbget-
dehydrogenase bin/www_bget?ctc:CTC01366 0030 12/13 adhE Clostridium
perfringens Aldehyde/alcohol genome.jp/dbget- dehydrogenase
bin/www_bget?cpe:CPE2531 0031 12/13 adhE Clostridium difficile
Aldehyde/alcohol genome.jp/dbget- dehydrogenase
bin/www_bget?cdf:CD2966 0032 8 sucA Mycobacterium bovis
.alpha.-ketoglutarate ncbi.nlm.nih.gov/entrez/ 5 BCG, Pasteur
decarboxylase viewer.fcgi?val=YP_977400.1 0033 9 cat2 Clostridium
aminobutyricum 4-hydroxybutyrate ncbi.nlm.nih.gov/entrez/ coenzyme
A viewer.fcgi?db=protein&val= transferase 6249316 0034 9 cat2
Porphyromonas gingivalis 4-hydroxybutyrate ncbi.nlm.nih.gov/entrez/
W83 coenzyme A viewer.fcgi?db=protein&val= transferase 34541558
0035 6 sucD Porphyromonas gingivalis Succinic
ncbi.nlm.nih.gov/entrez/ W83 semialdehyde
viewer.fcgi?val=NP_904963.1 dehydrogenase (CoA-dependent) 0036 7
4-HBd Porphyromonas gingivalis NAD-dependent
ncbi.nlm.nih.gov/entrez/ W83 4-hydroxybutyrate
viewer.fcgi?val=NP_904964.1 dehydrogenase 0037 7 gbd Uncultured
bacterium 4-hydroxybutyrate ncbi.nlm.nih.gov/entrez/ 6
dehydrogenase viewer.fcgi?db=nuccore&id= 5916168 0038 1 sucCD
E. coli Succinyl-CoA shigen.nig.ac.jp/ecoli/pec/ synthetase
genes.List.DetailAction.do 1 Sohling and Gottschalk, Eur. J.
Biochem. 212: 121-127 (1993); Sohling and Gottschalk, J. Bacteriol.
178: 871-880 (1996) 2 Nolling et al., J., J. Bacteriol. 183:
4823-4838 (2001) 3 Pohlmann et al., Nat. Biotechnol. 24: 1257-1262
(2006) 4 Kosaka et al., Biosci. Biotechnol. Biochem. 71: 58-68
(2007) 5 Brosch et al., Proc. Natl. Acad. Sci. U.S.A. 104:
5596-5601 (2007) 6 Henne et al., Appl. Environ. Microbiol. 65:
3901-3907 (1999)
[0373] Expression Vector Construction for BDO Pathway.
[0374] Vector backbones and some strains were obtained from Dr.
Rolf Lutz of Expressys (expressys.de/). The vectors and strains are
based on the pZ Expression System developed by Dr. Rolf Lutz and
Prof Hermann Bujard (Lutz, R. and H. Bujard, Nucleic Acids Res
25:1203-1210 (1997)). Vectors obtained were pZE13luc, pZA33luc,
pZS*13luc and pZE22luc and contained the luciferase gene as a
stuffer fragment. To replace the luciferase stuffer fragment with a
lacZ-alpha fragment flanked by appropriate restriction enzyme
sites, the luciferase stuffer fragment was first removed from each
vector by digestion with EcoRI and XbaI. The lacZ-alpha fragment
was PCR amplified from pUC19 with the following primers:
TABLE-US-00007 lacZalpha-RI (SEQ ID NO: 1)
5'GACGAATTCGCTAGCAAGAGGAGAAGTCGACATGTCCAATTCACTGGC CGTCGTTTTAC3'
lacZalpha 3'BB (SEQ ID NO: 2)
5'-GACCCTAGGAAGCTTTCTAGAGTCGACCTATGCGGCATCAGAGCAG A-3'.
[0375] This generated a fragment with a 5' end of EcoRI site, NheI
site, a Ribosomal Binding Site, a SalI site and the start codon. On
the 3' end of the fragment contained the stop codon, XbaI, HindIII,
and AvrII sites. The PCR product was digested with EcoRI and AvrII
and ligated into the base vectors digested with EcoRI and XbaI
(XbaI and AvrII have compatible ends and generate a non-site).
Because NheI and XbaI restriction enzyme sites generate compatible
ends that can be ligated together (but generate a NheI/XbaI
non-site that is not digested by either enzyme), the genes cloned
into the vectors could be "Biobricked" together
(http://openwetware.org/wiki/Synthetic_Biology:BioBricks). Briefly,
this method allows joining an unlimited number of genes into the
vector using the same 2 restriction sites (as long as the sites do
not appear internal to the genes), because the sites between the
genes are destroyed after each addition.
[0376] All vectors have the pZ designation followed by letters and
numbers indication the origin of replication, antibiotic resistance
marker and promoter/regulatory unit. The origin of replication is
the second letter and is denoted by E for ColE1, A for p15A and S
for pSC101-based origins. The first number represents the
antibiotic resistance marker (1 for Ampicillin, 2 for Kanamycin, 3
for Chloramphenicol, 4 for Spectinomycin and 5 for Tetracycline).
The final number defines the promoter that regulated the gene of
interest (1 for PLtetO-1, 2 for PLlacO-1, 3 for PAllacO-1, and 4
for Plac/ara-1). The MCS and the gene of interest follows
immediately after. For the work discussed here we employed two base
vectors, pZA33 and pZE13, modified for the biobricks insertions as
discussed above. Once the gene(s) of interest have been cloned into
them, resulting plasmids are indicated using the four digit gene
codes given in Table 6; e.g., pZA33-XXXX-YYYY- . . . .
[0377] Host Strain Construction. The parent strain in all studies
described here is E. coli K-12 strain MG1655. Markerless deletion
strains in adhE, gabD, and aldA were constructed under service
contract by a third party using the redET method (Datsenko, K. A.
and B. L. Wanner, Proc Natl Acad Sci U.S.A. 97:6640-6645 (2000)).
Subsequent strains were constructed via bacteriophage P1 mediated
transduction (Miller, J. Experiments in Molecular Genetics, Cold
Spring Harbor Laboratories, New York (1973)). Strain C600Z1 (laciq,
PN25-tetR, SpR, lacY1, leuB6, mcrB+, supE44, thi-1, thr-1, tonA21)
was obtained from Expressys and was used as a source of a lacIq
allele for P1 transduction. Bacteriophage P1vir was grown on the
C600Z1 E. coli strain, which has the spectinomycin resistance gene
linked to the lacIq. The P1 lysate grown on C600Z1 was used to
infect MG1655 with selection for spectinomycin resistance. The
spectinomycin resistant colonies were then screened for the linked
lacIq by determining the ability of the transductants to repress
expression of a gene linked to a PA1lacO-1 promoter. The resulting
strain was designated MG1655 lacIq. A similar procedure was used to
introduce lacIQ into the deletion strains.
[0378] Production of 4-HB From Succinate. For construction of a
4-HB producer from succinate, genes encoding steps from succinate
to 4-HB and 4-HB-CoA (1, 6, 7, and 9 in FIG. 1) were assembled onto
the pZA33 and pZE13 vectors as described below. Various
combinations of genes were assessed, as well as constructs bearing
incomplete pathways as controls (Tables 7 and 8). The plasmids were
then transformed into host strains containing lacIQ, which allow
inducible expression by addition of isopropyl
.beta.-D-1-thiogalactopyranoside (IPTG). Both wild-type and hosts
with deletions in genes encoding the native succinic semialdehyde
dehydrogenase (step 2 in FIG. 1) were tested.
[0379] Activity of the heterologous enzymes were first tested in in
vitro assays, using strain MG1655 lacIQ as the host for the plasmid
constructs containing the pathway genes. Cells were grown
aerobically in LB media (Difco) containing the appropriate
antibiotics for each construct, and induced by addition of IPTG at
1 mM when the optical density (OD600) reached approximately 0.5.
Cells were harvested after 6 hours, and enzyme assays conducted as
discussed below.
[0380] In Vitro Enzyme Assays. To obtain crude extracts for
activity assays, cells were harvested by centrifugation at 4,500
rpm (Beckman-Coulter, Allegera X-15R) for 10 min. The pellets were
resuspended in 0.3 mL BugBuster (Novagen) reagent with benzonase
and lysozyme, and lysis proceeded for 15 minutes at room
temperature with gentle shaking. Cell-free lysate was obtained by
centrifugation at 14,000 rpm (Eppendorf centrifuge 5402) for 30 min
at 4.degree. C. Cell protein in the sample was determined using the
method of Bradford et al., Anal. Biochem. 72:248-254 (1976), and
specific enzyme assays conducted as described below. Activities are
reported in Units/mg protein, where a unit of activity is defined
as the amount of enzyme required to convert 1 .mu.mol of substrate
in 1 min. at room temperature. In general, reported values are
averages of at least 3 replicate assays.
[0381] Succinyl-CoA transferase (Cat1) activity was determined by
monitoring the formation of acetyl-CoA from succinyl-CoA and
acetate, following a previously described procedure Sohling and
Gottschalk, J. Bacteriol. 178:871-880 (1996). Succinyl-CoA
synthetase (SucCD) activity was determined by following the
formation of succinyl-CoA from succinate and CoA in the presence of
ATP. The experiment followed a procedure described by Cha and
Parks, J. Biol. Chem. 239:1961-1967 (1964). CoA-dependent succinate
semialdehyde dehydrogenase (SucD) activity was determined by
following the conversion of NAD to NADH at 340 nm in the presence
of succinate semialdehyde and CoA (Sohling and Gottschalk, Eur. J.
Biochem. 212:121-127 (1993)). 4-HB dehydrogenase (4-HBd) enzyme
activity was determined by monitoring the oxidation of NADH to NAD
at 340 nm in the presence of succinate semialdehyde. The experiment
followed a published procedure Gerhardt et al. Arch. Microbiol.
174:189-199 (2000). 4-HB CoA transferase (Cat2) activity was
determined using a modified procedure from Scherf and Buckel, Appl.
Environ. Microbiol. 57:2699-2702 (1991). The formation of 4-HB-CoA
or butyryl-CoA formation from acetyl-CoA and 4-HB or butyrate was
determined using HPLC.
[0382] Alcohol (ADH) and aldehyde (ALD) dehydrogenase was assayed
in the reductive direction using a procedure adapted from several
literature sources (Durre et al., FEMS Microbiol. Rev. 17:251-262
(1995); Palosaari and Rogers, J. Bacteriol. 170:2971-2976 (1988)
and Welch et al., Arch. Biochem. Biophys. 273:309-318 (1989). The
oxidation of NADH is followed by reading absorbance at 340 nM every
four seconds for a total of 240 seconds at room temperature. The
reductive assays were performed in 100 mM MOPS (adjusted to pH 7.5
with KOH), 0.4 mM NADH, and from 1 to 50 .mu.l of cell extract. The
reaction is started by adding the following reagents: 100 .mu.l of
100 mM acetaldehyde or butyraldehyde for ADH, or 100 .mu.l of 1 mM
acetyl-CoA or butyryl-CoA for ALD. The Spectrophotometer is quickly
blanked and then the kinetic read is started. The resulting slope
of the reduction in absorbance at 340 nM per minute, along with the
molar extinction coefficient of NAD(P)H at 340 nM (6000) and the
protein concentration of the extract, can be used to determine the
specific activity.
[0383] The enzyme activity of PTB is measured in the direction of
butyryl-CoA to butyryl-phosphate as described in Cary et al. J.
Bacteriol. 170:4613-4618 (1988). It provides inorganic phosphate
for the conversion, and follows the increase in free CoA with the
reagent 5,5'-dithiobis-(2-nitrobenzoic acid), or DTNB. DTNB rapidly
reacts with thiol groups such as free CoA to release the
yellow-colored 2-nitro-5-mercaptobenzoic acid (TNB), which absorbs
at 412 nm with a molar extinction coefficient of 14,140 M cm-1. The
assay buffer contained 150 mM potassium phosphate at pH 7.4, 0.1 mM
DTNB, and 0.2 mM butyryl-CoA, and the reaction was started by
addition of 2 to 50 .mu.L cell extract. The enzyme activity of BK
is measured in the direction of butyrate to butyryl-phosphate
formation at the expense of ATP. The procedure is similar to the
assay for acetate kinase previously described Rose et al., J. Biol.
Chem. 211:737-756 (1954). However we have found another acetate
kinase enzyme assay protocol provided by Sigma to be more useful
and sensitive. This assay links conversion of ATP to ADP by acetate
kinase to the linked conversion of ADP and phosphoenol pyruvate
(PEP) to ATP and pyruvate by pyruvate kinase, followed by the
conversion of pyruvate and NADH to lactate and NAD+ by lactate
dehydrogenase. Substituting butyrate for acetate is the only major
modification to allow the assay to follow BK enzyme activity. The
assay mixture contained 80 mM triethanolamine buffer at pH 7.6, 200
mM sodium butyrate, 10 mM MgCl2, 0.1 mM NADH, 6.6 mM ATP, 1.8 mM
phosphoenolpyruvate. Pyruvate kinase, lactate dehydrogenase, and
myokinase were added according to the manufacturer's instructions.
The reaction was started by adding 2 to 50 .mu.L cell extract, and
the reaction was monitored based on the decrease in absorbance at
340 nm indicating NADH oxidation.
[0384] Analysis of CoA Derivatives by HPLC. An HPLC based assay was
developed to monitor enzymatic reactions involving coenzyme A (CoA)
transfer. The developed method allowed enzyme activity
characterization by quantitative determination of CoA, acetyl CoA
(AcCoA), butyryl CoA (BuCoA) and 4-hydroxybutyrate CoA (4-HBCoA)
present in in-vitro reaction mixtures. Sensitivity down to low
.mu.M was achieved, as well as excellent resolution of all the CoA
derivatives of interest.
[0385] Chemical and sample preparation was performed as follows.
Briefly, CoA, AcCoA, BuCoA and all other chemicals, were obtained
from Sigma-Aldrich. The solvents, methanol and acetonitrile, were
of HPLC grade. Standard calibration curves exhibited excellent
linearity in the 0.01-1 mg/mL concentration range. Enzymatic
reaction mixtures contained 100 mM Tris HCl buffer (pH 7), aliquots
were taken at different time points, quenched with formic acid
(0.04% final concentration) and directly analyzed by HPLC.
[0386] HPLC analysis was performed using an Agilent 1100 HPLC
system equipped with a binary pump, degasser, thermostated
autosampler and column compartment, and diode array detector (DAD),
was used for the analysis. A reversed phase column, Kromasil 100
Sum C18, 4.6.times.150 mm (Peeke Scientific), was employed. 25 mM
potassium phosphate (pH 7) and methanol or acetonitrile, were used
as aqueous and organic solvents at 1 mL/min flow rate. Two methods
were developed: a short one with a faster gradient for the analysis
of well-resolved CoA, AcCoA and BuCoA, and a longer method for
distinguishing between closely eluting AcCoA and 4-HBCoA. Short
method employed acetonitrile gradient (0 min--5%, 6 min--30%, 6.5
min--5%, 10 min--5%) and resulted in the retention times 2.7, 4.1
and 5.5 min for CoA, AcCoA and BuCoA, respectively. In the long
method methanol was used with the following linear gradient: 0
min--5%, 20 min--35%, 20.5 min--5%, 25 min--5%. The retention times
for CoA, AcCoA, 4-HBCoA and BuCoA were 5.8, 8.4, 9.2 and 16.0 min,
respectively. The injection volume was 5 .mu.L, column temperature
30.degree. C., and UV absorbance was monitored at 260 nm.
[0387] The results demonstrated activity of each of the four
pathway steps (Table 7), though activity is clearly dependent on
the gene source, position of the gene in the vector, and the
context of other genes with which it is expressed. For example,
gene 0035 encodes a succinic semialdehyde dehydrogenase that is
more active than that encoded by 0008, and 0036 and 0010n are more
active 4-HB dehydrogenase genes than 0009. There also seems to be
better 4-HB dehydrogenase activity when there is another gene
preceding it on the same operon.
TABLE-US-00008 TABLE 7 In vitro enzyme activities in cell extracts
from MG1655 lacI.sup.Q containing the plasmids expressing genes in
the 4-HB-CoA pathway. Activities are reported in Units/mg protein,
where a unit of activity is defined as the amount of enzyme
required to convert 1 .mu.mol of substrate in 1 min. at room
temperature. Sample # pZE13 (a) pZA33 (b) OD600 Cell Prot (c) Cat1
SucD 4HBd Cat2 1 cat1 (0004) 2.71 6.43 1.232 0.00 2 cat1
(0004)-sucD (0035) 2.03 5.00 0.761 2.57 3 cat1 (0004)-sucD (0008)
1.04 3.01 0.783 0.01 4 sucD (0035) 2.31 6.94 2.32 5 sucD (0008)
1.10 4.16 0.05 6 4hbd (0009) 2.81 7.94 0.003 0.25 7 4hbd (0036)
2.63 7.84 3.31 8 4hbd (0010n) 2.00 5.08 2.57 9 cat1 (0004)-sucD
(0035) 4hbd (0009) 2.07 5.04 0.600 1.85 0.01 10 cat1 (0004)-sucD
(0035) 4hbd (0036) 2.08 5.40 0.694 1.73 0.41 11 cat1 (0004)-sucD
(0035) 4hbd (0010n) 2.44 4.73 0.679 2.28 0.37 12 cat1 (0004)-sucD
(0008) 4hbd (0009) 1.08 3.99 0.572 -0.01 0.02 13 cat1 (0004)-sucD
(0008) 4hbd (0036) 0.77 2.60 0.898 -0.01 0.04 14 cat1 (0004)-sucD
(0008) 4hbd (0010n) 0.63 2.47 0.776 0.00 0.00 15 cat2 (0034) 2.56
7.86 1.283 16 cat2(0034)-4hbd(0036) 3.13 8.04 24.86 0.993 17
cat2(0034)-4hbd(0010n) 2.38 7.03 7.45 0.675 18
4hbd(0036)-cat2(0034) 2.69 8.26 2.15 7.490 19
4hbd(0010n)-cat2(0034) 2.44 6.59 0.59 4.101 Genes expressed from
Plac on pZE13, a high-copy plasmid with colE1 origin and ampicillin
resistance. Gene identification numbers are as given in Table 6
Genes expressed from Plac on pZA33, a medium-copy plasmid with
pACYC origin and chloramphenicol resistance. (c) Cell protein given
as mg protein per mL extract.
[0388] Recombinant strains containing genes in the 4-HB pathway
were then evaluated for the ability to produce 4-HB in vivo from
central metabolic intermediates. Cells were grown anaerobically in
LB medium to OD600 of approximately 0.4, then induced with 1 mM
IPTG. One hour later, sodium succinate was added to 10 mM, and
samples taken for analysis following an additional 24 and 48 hours.
4-HB in the culture broth was analyzed by GC-MS as described below.
The results indicate that the recombinant strain can produce over 2
mM 4-HB after 24 hours, compared to essentially zero in the control
strain (Table 8).
TABLE-US-00009 TABLE 8 Production of 4-HB from succinate in E. coli
strains harboring plasmids expressing various combinations of 4-HB
pathway genes. Sample 24 Hours 48 Hours # Host Strain pZE13 pZA33
OD600 4HB, .mu.M 4HB norm. (a) OD600 4HB, .mu.M 4HB norm. (a) 1
MG1655 lacIq cat1 (0004)-sucD (0035) 4hbd (0009) 0.47 487 1036 1.04
1780 1711 2 MG1655 lacIq cat1 (0004)-sucD (0035) 4hbd (0027) 0.41
111 270 0.99 214 217 3 MG1655 lacIq cat1 (0004)-sucD (0035) 4hbd
(0036) 0.47 863 1835 0.48 2152 4484 4 MG1655 lacIq cat1 (0004)-sucD
(0035) 4hbd (0010n) 0.46 956 2078 0.49 2221 4533 5 MG1655 lacIq
cat1 (0004)-sucD (0008) 4hbd (0009) 0.38 493 1296 0.37 1338 3616 6
MG1655 lacIq cat1 (0004)-sucD (0008) 4hbd (0027) 0.32 26 81 0.27 87
323 7 MG1655 lacIq cat1 (0004)-sucD (0008) 4hbd (0036) 0.24 506
2108 0.31 1448 4672 8 MG1655 lacIq cat1 (0004)-sucD (0008) 4hbd
(0010n) 0.24 78 324 0.56 233 416 9 MG1655 lacIq gabD cat1
(0004)-sucD (0035) 4hbd (0009) 0.53 656 1237 1.03 1643 1595 10
MG1655 lacIq gabD cat1 (0004)-sucD (0035) 4hbd (0027) 0.44 92 209
0.98 214 218 11 MG1655 lacIq gabD cat1 (0004)-sucD (0035) 4hbd
(0036) 0.51 1072 2102 0.97 2358 2431 12 MG1655 lacIq gabD cat1
(0004)-sucD (0035) 4hbd (0010n) 0.51 981 1924 0.97 2121 2186 13
MG1655 lacIq gabD cat1 (0004)-sucD (0008) 4hbd (0009) 0.35 407 1162
0.77 1178 1530 14 MG1655 lacIq gabD cat1 (0004)-sucD (0008) 4hbd
(0027) 0.51 19 36 1.07 50 47 15 MG1655 lacIq gabD cat1 (0004)-sucD
(0008) 4hbd (0036) 0.35 584 1669 0.78 1350 1731 16 MG1655 lacIq
gabD cat1 (0004)-sucD (0008) 4hbd (0010n) 0.32 74 232 0.82 232 283
17 MG1655 lacIq vector only vector only 0.8 1 2 1.44 3 2 18 MG1655
lacIq gabD vector only vector only 0.89 1 2 1.41 7 5 (a) Normalized
4-HB concentration, .mu.M/OD600 units
[0389] An alternate to using a CoA transferase (cat1) to produce
succinyl-CoA from succinate is to use the native E. coli sucCD
genes, encoding succinyl-CoA synthetase. This gene cluster was
cloned onto pZE13 along with candidate genes for the remaining
steps to 4-HB to create pZE13-0038-0035-0036.
[0390] Production of 4-HB from Glucose. Although the above
experiments demonstrate a functional pathway to 4-HB from a central
metabolic intermediate (succinate), an industrial process would
require the production of chemicals from low-cost carbohydrate
feedstocks such as glucose or sucrose. Thus, the next set of
experiments was aimed to determine whether endogenous succinate
produced by the cells during growth on glucose could fuel the 4-HB
pathway. Cells were grown anaerobically in M9 minimal medium (6.78
g/L Na2HPO4, 3.0 g/L KH2PO4, 0.5 g/L NaCl, 1.0 g/L NH4Cl, 1 mM
MgSO4, 0.1 mM CaCl2) supplemented with 20 g/L glucose, 100 mM
3-(N-morpholino)propanesulfonic acid (MOPS) to improve the
buffering capacity, 10 .mu.g/mL thiamine, and the appropriate
antibiotics. 0.25 mM IPTG was added when OD600 reached
approximately 0.2, and samples taken for 4-HB analysis every 24
hours following induction. In all cases 4-HB plateaued after 24
hours, with a maximum of about 1 mM in the best strains (FIG. 3a),
while the succinate concentration continued to rise (FIG. 3b). This
indicates that the supply of succinate to the pathway is likely not
limiting, and that the bottleneck may be in the activity of the
enzymes themselves or in NADH availability. 0035 and 0036 are
clearly the best gene candidates for CoA-dependent succinic
semialdehyde dehydrogenase and 4-HB dehydrogenase, respectively.
The elimination of one or both of the genes encoding known (gabD)
or putative (aldA) native succinic semialdehyde dehydrogenases had
little effect on performance. Finally, it should be noted that the
cells grew to a much lower OD in the 4-HB-producing strains than in
the controls (FIG. 3c).
[0391] An alternate pathway for the production of 4-HB from glucose
is via .alpha.-ketoglutarate. We explored the use of an
.alpha.-ketoglutarate decarboxylase from Mycobacterium tuberculosis
Tian et al., Proc. Natl. Acad. Sci. USA 102:10670-10675 (2005) to
produce succinic semialdehyde directly from .alpha.-ketoglutarate
(step 8 in FIG. 1). To demonstrate that this gene (0032) was
functional in vivo, we expressed it on pZE13 in the same host as
4-HB dehydrogenase (gene 0036) on pZA33. This strain was capable of
producing over 1.0 mM 4-HB within 24 hours following induction with
1 mM IPTG (FIG. 4). Since this strain does not express a
CoA-dependent succinic semialdehyde dehydrogenase, the possibility
of succinic semialdehyde production via succinyl-CoA is eliminated.
It is also possible that the native genes responsible for producing
succinic semialdehyde could function in this pathway (steps 4 and 5
in FIG. 1); however, the amount of 4-HB produced when the
pZE13-0032 plasmid was left out of the host is the negligible.
[0392] Production of BDO from 4-HB. The production of BDO from 4-HB
required two reduction steps, catalyzed by dehydrogenases. Alcohol
and aldehyde dehydrogenases (ADH and ALD, respectively) are NAD+/H
and/or NADP+/H-dependent enzymes that together can reduce a
carboxylic acid group on a molecule to an alcohol group, or in
reverse, can perform the oxidation of an alcohol to a carboxylic
acid. This biotransformation has been demonstrated in wild-type
Clostridium acetobutylicum (Jewell et al., Current Microbiology,
13:215-19 (1986)), but neither the enzymes responsible nor the
genes responsible were identified. In addition, it is not known
whether activation to 4-HB-CoA is first required (step 9 in FIG.
1), or if the aldehyde dehydrogenase (step 12) can act directly on
4-HB. We developed a list of candidate enzymes from C.
acetobutylicum and related organisms based on known activity with
the non-hydroxylated analogues to 4-HB and pathway intermediates,
or by similarity to these characterized genes (Table 6). Since some
of the candidates are multifunctional dehydrogenases, they could
potentially catalyze both the NAD(P)H-dependent reduction of the
acid (or CoA-derivative) to the aldehyde, and of the aldehyde to
the alcohol. Before beginning work with these genes in E. coli, we
first validated the result referenced above using C. acetobutylicum
ATCC 824. Cells were grown in Schaedler broth (Accumedia, Lansing,
Mich.) supplemented with 10 mM 4-HB, in an anaerobic atmosphere of
10% CO2, 10% H2, and 80% N2 at 30.degree. C. Periodic culture
samples were taken, centrifuged, and the broth analyzed for BDO by
GC-MS as described below. BDO concentrations of 0.1 mM, 0.9 mM, and
1.5 mM were detected after 1 day, 2 days, and 7 days incubation,
respectively. No BDO was detected in culture grown without 4-HB
addition. To demonstrate that the BDO produced was derived from
glucose, we grew the best BDO producing strain MG1655 lacIQ
pZE13-0004-0035-0002 pZA33-0034-0036 in M9 minimal medium
supplemented with 4 g/L uniformly labeled 13C-glucose. Cells were
induced at OD of 0.67 with 1 mM IPTG, and a sample taken after 24
hours. Analysis of the culture supernatant was performed by mass
spectrometry.
[0393] Gene candidates for the 4-HB to BDO conversion pathway were
next tested for activity when expressed in the E. coli host MG1655
lacIQ. Recombinant strains containing each gene candidate expressed
on pZA33 were grown in the presence of 0.25 mM IPTG for four hours
at 37.degree. C. to fully induce expression of the enzyme. Four
hours after induction, cells were harvested and assayed for ADH and
ALD activity as described above. Since 4-HB-CoA and
4-hydroxybutyraldehyde are not available commercially, assays were
performed using the non-hydroxylated substrates (Table 9). The
ratio in activity between 4-carbon and 2-carbon substrates for C.
acetobutylicum adhE2 (0002) and E. coli adhE (0011) were similar to
those previously reported in the literature a Atsumi et al.,
Biochim. Biophys. Acta. 1207:1-11 (1994).
TABLE-US-00010 TABLE 9 In vitro enzyme activities in cell extracts
from MG1655 lacI.sup.Q containing pZA33 expressing gene candidates
for aldehyde and alcohol dehydrogenases. Activities are expressed
in .mu.mol min.sup.-1 mg cell protein.sup.-1. N.D., not determined.
Aldehyde dehydrogenase Alcohol dehydrogenase Gene Substrate
Butyryl-CoA Acetyl-CoA Butyraldehyde Acetaldehyde 0002 0.0076
0.0046 0.0264 0.0247 0003n 0.0060 0.0072 0.0080 0.0075 0011 0.0069
0.0095 0.0265 0.0093 0013 N.D. N.D. 0.0130 0.0142 0023 0.0089
0.0137 0.0178 0.0235 0025 0 0.0001 N.D. N.D. 0026 0 0.0005 0.0024
0.0008
[0394] For the BDO production experiments, cat2 from Porphyromonas
gingivalis W83 (gene 0034) was included on pZA33 for the conversion
of 4-HB to 4-HB-CoA, while the candidate dehydrogenase genes were
expressed on pZE13. The host strain was MG1655 lacIQ. Along with
the alcohol and aldehyde dehydrogenase candidates, we also tested
the ability of CoA-dependent succinic semialdehyde dehydrogenases
(sucD) to function in this step, due to the similarity of the
substrates. Cells were grown to an OD of about 0.5 in LB medium
supplemented with 10 mM 4-HB, induced with 1 mM IPTG, and culture
broth samples taken after 24 hours and analyzed for BDO as
described below. The best BDO production occurred using adhE2 from
C. acetobutylicum, sucD from C. kluyveri, or sucD from P.
gingivalis (FIG. 5). Interestingly, the absolute amount of BDO
produced was higher under aerobic conditions; however, this is
primarily due to the lower cell density achieved in anaerobic
cultures. When normalized to cell OD, the BDO production per unit
biomass is higher in anaerobic conditions (Table 10).
TABLE-US-00011 TABLE 10 Absolute and normalized BDO concentrations
from cultures of cells expressing adhE2 from C. acetobutylicum,
sucD from C. kluyveri, or sucD from P. gingivalis (data from
experiments 2, 9, and 10 in FIG. 3), as well as the negative
control (experiment 1). Gene BDO OD expressed Conditions (.mu.M)
(600 nm) BDO/OD none Aerobic 0 13.4 0 none Microaerobic 0.5 6.7
0.09 none Anaerobic 2.2 1.26 1.75 0002 Aerobic 138.3 9.12 15.2 0002
Microaerobic 48.2 5.52 8.73 0002 Anaerobic 54.7 1.35 40.5 0008n
Aerobic 255.8 5.37 47.6 0008n Microaerobic 127.9 3.05 41.9 0008n
Anaerobic 60.8 0.62 98.1 0035 Aerobic 21.3 14.0 1.52 0035
Microaerobic 13.1 4.14 3.16 0035 Anaerobic 21.3 1.06 20.1
[0395] As discussed above, it may be advantageous to use a route
for converting 4-HB to 4-HB-CoA that does not generate acetate as a
byproduct. To this aim, we tested the use of phosphotransbutyrylase
(ptb) and butyrate kinase (bk) from C. acetobutylicum to carry out
this conversion via steps 10 and 11 in FIG. 1. The native ptb/bk
operon from C. acetobutylicum (genes 0020 and 0021) was cloned and
expressed in pZA33. Extracts from cells containing the resulting
construct were taken and assayed for the two enzyme activities as
described herein. The specific activity of BK was approximately 65
U/mg, while the specific activity of PTB was approximately 5 U/mg.
One unit (U) of activity is defined as conversion of 1 .mu.M
substrate in 1 minute at room temperature. Finally, the construct
was tested for participation in the conversion of 4-HB to BDO. Host
strains were transformed with the pZA33-0020-0021 construct
described and pZE13-0002, and compared to use of cat2 in BDO
production using the aerobic procedure used above in FIG. 5. The
BK/PTB strain produced 1 mM BDO, compared to 2 mM when using cat2
(Table 11). Interestingly, the results were dependent on whether
the host strain contained a deletion in the native adhE gene.
TABLE-US-00012 TABLE 11 Absolute and normalized BDO concentrations
from cultures of cells expressing adhE2 from C. acetobutylicum in
pZE13 along with either cat2 from P. gingivalis (0034) or thePTB/BK
genes from C. acetobutylicum on pZA33. Host strains were either
MG1655 lacI.sup.Q or MG1655 .DELTA.adhE lacI.sup.Q. BDO OD Genes
Host Strain (.mu.M) (600 nm) BDO/OD 0034 MG1655 lacI.sup.Q 0.827
19.9 0.042 0020 + 00021 MG1655 lacI.sup.Q 0.007 9.8 0.0007 0034
MG1655 .DELTA.adhE 2.084 12.5 0.166 lacI.sup.Q 0020 + 0021 MG1655
.DELTA.adhE 0.975 18.8 0.052 lacI.sup.Q
[0396] Production of BDO from Glucose. The final step of pathway
corroboration is to express both the 4-HB and BDO segments of the
pathway in E. coli and demonstrate production of BDO in glucose
minimal medium. New plasmids were constructed so that all the
required genes fit on two plamids. In general, cat1, adhE, and sucD
genes were expressed from pZE13, and cat2 and 4-HBd were expressed
from pZA33. Various combinations of gene source and gene order were
tested in the MG1655 lacI.sup.Q background. Cells were grown
anaerobically in M9 minimal medium (6.78 g/L Na.sub.2HPO.sub.4, 3.0
g/L KH.sub.2PO.sub.4, 0.5 g/L NaCl, 1.0 g/L NH.sub.4Cl, 1 mM
MgSO.sub.4, 0.1 mM CaCl.sub.2) supplemented with 20 g/L glucose,
100 mM 3-(N-morpholino)propanesulfonic acid (MOPS) to improve the
buffering capacity, 10 .mu.g/mL thiamine, and the appropriate
antibiotics. 0.25 mM IPTG was added approximately 15 hours
following inoculation, and culture supernatant samples taken for
BDO, 4-HB, and succinate analysis 24 and 48 hours following
induction. The production of BDO appeared to show a dependency on
gene order (Table 12). The highest BDO production, over 0.5 mM, was
obtained with cat2 expressed first, followed by 4-HBd on pZA33, and
cat1 followed by P. gingivalis sucD on pZE13. The addition of C.
acetobutylicum adhE2 in the last position on pZE13 resulted in
slight improvement. 4-HB and succinate were also produced at higher
concentrations.
TABLE-US-00013 TABLE 12 Production of BDO, 4-HB, and succinate in
recombinant E. coli strains expressing combinations of BDO pathway
genes, grown in minimal medium supplemented with 20 g/L glucose.
Concentrations are given in mM. 24 Hours 48 Hours Induction OD600
OD600 Sample pZE13 pZA33 OD nm Su 4HB BDO nm Su 4HB BDO 1
cat1(0004)-sucD(0035) 4hbd (0036)-cat2(0034) 0.92 1.29 5.44 1.37
0.240 1.24 6.42 1.49 0.280 2 cat1(0004)-sucD(0008N) 4hbd
(0036)-cat2(0034) 0.36 1.11 6.90 1.24 0.011 1.06 7.63 1.33 0.011 3
adhE(0002)-cat1(0004)-sucD(0035) 4hbd (0036)-cat2(0034) 0.20 0.44
0.34 1.84 0.050 0.60 1.93 2.67 0.119 4
cat1(0004)-sucD(0035)-adhE(0002) 4hbd (0036)-cat2(0034) 1.31 1.90
9.02 0.73 0.073 1.95 9.73 0.82 0.077 5
adhE(0002)-cat1(0004)-sucD(0008N) 4hbd (0036)-cat2(0034) 0.17 0.45
1.04 1.04 0.008 0.94 7.13 1.02 0.017 6
cat1(0004)-sucD(0008N)-adhE(0002) 4hbd (0036)-cat2(0034) 1.30 1.77
10.47 0.25 0.004 1.80 11.49 0.28 0.003 7 cat1(0004)-sucD(0035)
cat2(0034)-4hbd(0036) 1.09 1.29 5.63 2.15 0.461 1.38 6.66 2.30
0.520 8 cat1(0004)-sucD(0008N) cat2(0034)-4hbd(0036) 1.81 2.01
11.28 0.02 0.000 2.24 11.13 0.02 0.000 9
adhE(0002)-cat1(0004)-sucD(0035) cat2(0034)-4hbd(0036) 0.24 1.99
2.02 2.32 0.106 0.89 4.85 2.41 0.186 10
cat1(0004)-sucD(0035)-adhE(0002) cat2(0034)-4hbd(0036) 0.98 1.17
5.30 2.08 0.569 1.33 6.15 2.14 0.640 11
adhE(0002)-cat1(0004)-sucD(0008N) cat2(0034)-4hbd(0036) 0.20 0.53
1.38 2.30 0.019 0.91 8.10 1.49 0.034 12
cat1(0004)-sucD(0008N)-adhE(0002) cat2(0034)-4hbd(0036) 2.14 2.73
12.07 0.16 0.000 3.10 11.79 0.17 0.002 13 vector only vector only
2.11 2.62 9.03 0.01 0.000 3.00 12.05 0.01 0.000
[0397] Analysis of BDO, 4-HB and succinate by GCMS. BDO, 4-HB and
succinate in fermentation and cell culture samples were derivatized
by silylation and quantitatively analyzed by GCMS using methods
adapted from literature reports ((Simonov et al., J. Anal Chem.
59:965-971 (2004)). The developed method demonstrated good
sensitivity down to 1 .mu.M, linearity up to at least 25 mM, as
well as excellent selectivity and reproducibility.
[0398] Sample preparation was performed as follows: 100 .mu.L
filtered (0.2 .mu.m or 0.45 .mu.m syringe filters) samples, e.g.
fermentation broth, cell culture or standard solutions, were dried
down in a Speed Vac Concentrator (Savant SVC-100H) for
approximately 1 hour at ambient temperature, followed by the
addition of 20 .mu.L 10 mM cyclohexanol solution, as an internal
standard, in dimethylformamide. The mixtures were vortexed and
sonicated in a water bath (Branson 3510) for 15 min to ensure
homogeneity. 100 .mu.L silylation derivatization reagent,
N,O-bis(trimethylsilyl)trifluoro-acetimide (BSTFA) with 1%
trimethylchlorosilane, was added, and the mixture was incubated at
70.degree. C. for 30 min. The derivatized samples were centrifuged
for 5 min, and the clear solutions were directly injected into
GCMS. All the chemicals and reagents were from Sigma-Aldrich, with
the exception of BDO which was purchased from J. T. Baker.
[0399] GCMS was performed on an Agilent gas chromatograph 6890N,
interfaced to a mass-selective detector (MSD) 5973N operated in
electron impact ionization (EI) mode has been used for the
analysis. A DB-5MS capillary column (J&W Scientific, Agilent
Technologies), 30 m.times.0.25 mm i.d..times.0.25 .mu.m film
thickness, was used. The GC was operated in a split injection mode
introducing 1 .mu.L of sample at 20:1 split ratio. The injection
port temperature was 250.degree. C. Helium was used as a carrier
gas, and the flow rate was maintained at 1.0 mL/min. A temperature
gradient program was optimized to ensure good resolution of the
analytes of interest and minimum matrix interference. The oven was
initially held at 80.degree. C. for 1 min, then ramped to
120.degree. C. at 2.degree. C./min, followed by fast ramping to
320.degree. C. at 100.degree. C./min and final hold for 6 min at
320.degree. C. The MS interface transfer line was maintained at
280.degree. C. The data were acquired using `lowmass` MS tune
settings and 30-400 m/z mass-range scan. The total analysis time
was 29 min including 3 min solvent delay. The retention times
corresponded to 5.2, 10.5, 14.0 and 18.2 min for BSTFA-derivatized
cyclohexanol, BDO, 4-HB and succinate, respectively. For
quantitative analysis, the following specific mass fragments were
selected (extracted ion chromatograms): m/z 157 for internal
standard cyclohexanol, 116 for BDO, and 147 for both 4-HB and
succinate. Standard calibration curves were constructed using
analyte solutions in the corresponding cell culture or fermentation
medium to match sample matrix as close as possible. GCMS data were
processed using Environmental Data Analysis ChemStation software
(Agilent Technologies).
[0400] The results indicated that most of the 4-HB and BDO produced
were labeled with 13C (FIG. 6, right-hand sides). Mass spectra from
a parallel culture grown in unlabeled glucose are shown for
comparison (FIG. 6, left-hand sides). Note that the peaks seen are
for fragments of the derivatized molecule containing different
numbers of carbon atoms from the metabolite. The derivatization
reagent also contributes some carbon and silicon atoms that
naturally-occurring label distribution, so the results are not
strictly quantitative.
[0401] Production of BDO from 4-HB using alternate pathways. The
various alternate pathways were also tested for BDO production.
This includes use of the native E. coli SucCD enzyme to convert
succinate to succinyl-CoA (Table 13, rows 2-3), use of
alpha-ketoglutarate decarboxylase in the alpha-ketoglutarate
pathway (Table 13, row 4), and use of PTB/BK as an alternate means
to generate the CoA-derivative of 4HB (Table 13, row 1). Strains
were constructed containing plasmids expressing the genes indicated
in Table 13, which encompass these variants. The results show that
in all cases, production of 4-HB and BDO occurred (Table 13).
TABLE-US-00014 TABLE 13 Production of BDO, 4-HB, and succinate in
recombinant E. coli strains genes for different BDO pathway
variants, grown anaerobically in minimal medium supplemented with
20 g/L glucose, and harvested 24 hours after induction with 0.1 mM
IPTG. Concentrations are given in mM. Genes on pZE13 Genes on pZA33
Succinate 4-HB BDO 0002 + 004 + 0035 0020n-0021n-0036 0.336 2.91
0.230 0038 + 0035 0034-0036 0.814 2.81 0.126 0038 + 0035 0036-0034
0.741 2.57 0.114 0035 + 0032 0034-0036 5.01 0.538 0.154
Example III
Biosynthesis of 4-Hydroxybutanoic Acid, .gamma.-Butyrolactone and
1,4-Butanediol
[0402] This Example describes the biosynthetic production of
4-hydroxybutanoic acid, .gamma.-butyrolactone and 1,4-butanediol
using fermentation and other bioprocesses.
[0403] Methods for the integration of the 4-HB fermentation step
into a complete process for the production of purified GBL,
1,4-butanediol (BDO) and tetrahydrofuran (THF) are described below.
Since 4-HB and GBL are in equilibrium, the fermentation broth will
contain both compounds. At low pH this equilibrium is shifted to
favor GBL. Therefore, the fermentation can operate at pH 7.5 or
less, generally pH 5.5 or less. After removal of biomass, the
product stream enters into a separation step in which GBL is
removed and the remaining stream enriched in 4-HB is recycled.
Finally, GBL is distilled to remove any impurities. The process
operates in one of three ways: 1) fed-batch fermentation and batch
separation; 2) fed-batch fermentation and continuous separation; 3)
continuous fermentation and continuous separation. The first two of
these modes are shown schematically in FIG. 7. The integrated
fermentation procedures described below also are used for the BDO
producing cells of the invention for biosynthesis of BDO and
subsequent BDO family products.
[0404] Fermentation protocol to produce 4-HB/GBL (batch): The
production organism is grown in a 10 L bioreactor sparged with an
N2/CO2 mixture, using 5 L broth containing 5 g/L potassium
phosphate, 2.5 g/L ammonium chloride, 0.5 g/L magnesium sulfate,
and 30 g/L corn steep liquor, and an initial glucose concentration
of 20 g/L. As the cells grow and utilize the glucose, additional
70% glucose is fed into the bioreactor at a rate approximately
balancing glucose consumption. The temperature of the bioreactor is
maintained at 30 degrees C. Growth continues for approximately 24
hours, until 4-HB reaches a concentration of between 20-200 g/L,
with the cell density being between 5 and 10 g/L. The pH is not
controlled, and will typically decrease to pH 3-6 by the end of the
run. Upon completion of the cultivation period, the fermenter
contents are passed through a cell separation unit (e.g.,
centrifuge) to remove cells and cell debris, and the fermentation
broth is transferred to a product separations unit. Isolation of
4-HB and/or GBL would take place by standard separations procedures
employed in the art to separate organic products from dilute
aqueous solutions, such as liquid-liquid extraction using a water
immiscible organic solvent (e.g., toluene) to provide an organic
solution of 4-HB/GBL. The resulting solution is then subjected to
standard distillation methods to remove and recycle the organic
solvent and to provide GBL (boiling point 204-205.degree. C.) which
is isolated as a purified liquid.
[0405] Fermentation protocol to produce 4-HB/GBL (fully
continuous): The production organism is first grown up in batch
mode using the apparatus and medium composition described above,
except that the initial glucose concentration is 30-50 g/L. When
glucose is exhausted, feed medium of the same composition is
supplied continuously at a rate between 0.5 L/hr and 1 L/hr, and
liquid is withdrawn at the same rate. The 4-HB concentration in the
bioreactor remains constant at 30-40 g/L, and the cell density
remains constant between 3-5 g/L. Temperature is maintained at 30
degrees C., and the pH is maintained at 4.5 using concentrated NaOH
and HCl, as required. The bioreactor is operated continuously for
one month, with samples taken every day to assure consistency of
4-HB concentration. In continuous mode, fermenter contents are
constantly removed as new feed medium is supplied. The exit stream,
containing cells, medium, and products 4-HB and/or GBL, is then
subjected to a continuous product separations procedure, with or
without removing cells and cell debris, and would take place by
standard continuous separations methods employed in the art to
separate organic products from dilute aqueous solutions, such as
continuous liquid-liquid extraction using a water immiscible
organic solvent (e.g., toluene) to provide an organic solution of
4-HB/GBL. The resulting solution is subsequently subjected to
standard continuous distillation methods to remove and recycle the
organic solvent and to provide GBL (boiling point 204-205.degree.
C.) which is isolated as a purified liquid.
[0406] GBL Reduction Protocol: Once GBL is isolated and purified as
described above, it will then be subjected to reduction protocols
such as those well known in the art (references cited) to produce
1,4-butanediol or tetrahydrofuran (THF) or a mixture thereof.
Heterogeneous or homogeneous hydrogenation catalysts combined with
GBL under hydrogen pressure are well known to provide the products
1,4-butanediol or tetrahydrofuran (THF) or a mixture thereof. It is
important to note that the 4-HB/GBL product mixture that is
separated from the fermentation broth, as described above, may be
subjected directly, prior to GBL isolation and purification, to
these same reduction protocols to provide the products
1,4-butanediol or tetrahydrofuran or a mixture thereof. The
resulting products, 1,4-butanediol and THF are then isolated and
purified by procedures well known in the art.
[0407] Fermentation and Hydrogenation Protocol to Produce BDO or
THF Directly (Batch):
[0408] Cells are grown in a 10 L bioreactor sparged with an N2/CO2
mixture, using 5 L broth containing 5 g/L potassium phosphate, 2.5
g/L ammonium chloride, 0.5 g/L magnesium sulfate, and 30 g/L corn
steep liquor, and an initial glucose concentration of 20 g/L. As
the cells grow and utilize the glucose, additional 70% glucose is
fed into the bioreactor at a rate approximately balancing glucose
consumption. The temperature of the bioreactor is maintained at 30
degrees C. Growth continues for approximately 24 hours, until 4-HB
reaches a concentration of between 20-200 g/L, with the cell
density being between 5 and 10 g/L. The pH is not controlled, and
will typically decrease to pH 3-6 by the end of the run. Upon
completion of the cultivation period, the fermenter contents are
passed through a cell separation unit (e.g., centrifuge) to remove
cells and cell debris, and the fermentation broth is transferred to
a reduction unit (e.g., hydrogenation vessel), where the mixture
4-HB/GBL is directly reduced to either 1,4-butanediol or THF or a
mixture thereof. Following completion of the reduction procedure,
the reactor contents are transferred to a product separations unit.
Isolation of 1,4-butanediol and/or THF would take place by standard
separations procedures employed in the art to separate organic
products from dilute aqueous solutions, such as liquid-liquid
extraction using a water immiscible organic solvent (e.g., toluene)
to provide an organic solution of 1,4-butanediol and/or THF. The
resulting solution is then subjected to standard distillation
methods to remove and recycle the organic solvent and to provide
1,4-butanediol and/or THF which are isolated as a purified
liquids.
[0409] Fermentation and hydrogenation protocol to produce BDO or
THF directly (fully continuous): The cells are first grown up in
batch mode using the apparatus and medium composition described
above, except that the initial glucose concentration is 30-50 g/L.
When glucose is exhausted, feed medium of the same composition is
supplied continuously at a rate between 0.5 L/hr and 1 L/hr, and
liquid is withdrawn at the same rate. The 4-HB concentration in the
bioreactor remains constant at 30-40 g/L, and the cell density
remains constant between 3-5 g/L. Temperature is maintained at 30
degrees C., and the pH is maintained at 4.5 using concentrated NaOH
and HCl, as required. The bioreactor is operated continuously for
one month, with samples taken every day to assure consistency of
4-HB concentration. In continuous mode, fermenter contents are
constantly removed as new feed medium is supplied. The exit stream,
containing cells, medium, and products 4-HB and/or GBL, is then
passed through a cell separation unit (e.g., centrifuge) to remove
cells and cell debris, and the fermentation broth is transferred to
a continuous reduction unit (e.g., hydrogenation vessel), where the
mixture 4-HB/GBL is directly reduced to either 1,4-butanediol or
THF or a mixture thereof. Following completion of the reduction
procedure, the reactor contents are transferred to a continuous
product separations unit. Isolation of 1,4-butanediol and/or THF
would take place by standard continuous separations procedures
employed in the art to separate organic products from dilute
aqueous solutions, such as liquid-liquid extraction using a water
immiscible organic solvent (e.g., toluene) to provide an organic
solution of 1,4-butanediol and/or THF. The resulting solution is
then subjected to standard continuous distillation methods to
remove and recycle the organic solvent and to provide
1,4-butanediol and/or THF which are isolated as a purified
liquids.
[0410] Fermentation protocol to produce BDO directly (batch): The
production organism is grown in a 10 L bioreactor sparged with an
N2/CO2 mixture, using 5 L broth containing 5 g/L potassium
phosphate, 2.5 g/L ammonium chloride, 0.5 g/L magnesium sulfate,
and 30 g/L corn steep liquor, and an initial glucose concentration
of 20 g/L. As the cells grow and utilize the glucose, additional
70% glucose is fed into the bioreactor at a rate approximately
balancing glucose consumption. The temperature of the bioreactor is
maintained at 30 degrees C. Growth continues for approximately 24
hours, until BDO reaches a concentration of between 20-200 g/L,
with the cell density generally being between 5 and 10 g/L. Upon
completion of the cultivation period, the fermenter contents are
passed through a cell separation unit (e.g., centrifuge) to remove
cells and cell debris, and the fermentation broth is transferred to
a product separations unit. Isolation of BDO would take place by
standard separations procedures employed in the art to separate
organic products from dilute aqueous solutions, such as
liquid-liquid extraction using a water immiscible organic solvent
(e.g., toluene) to provide an organic solution of BDO. The
resulting solution is then subjected to standard distillation
methods to remove and recycle the organic solvent and to provide
BDO (boiling point 228-229.degree. C.) which is isolated as a
purified liquid.
[0411] Fermentation protocol to produce BDO directly (fully
continuous): The production organism is first grown up in batch
mode using the apparatus and medium composition described above,
except that the initial glucose concentration is 30-50 g/L. When
glucose is exhausted, feed medium of the same composition is
supplied continuously at a rate between 0.5 L/hr and 1 L/hr, and
liquid is withdrawn at the same rate. The BDO concentration in the
bioreactor remains constant at 30-40 g/L, and the cell density
remains constant between 3-5 g/L. Temperature is maintained at 30
degrees C., and the pH is maintained at 4.5 using concentrated NaOH
and HCl, as required. The bioreactor is operated continuously for
one month, with samples taken every day to assure consistency of
BDO concentration. In continuous mode, fermenter contents are
constantly removed as new feed medium is supplied. The exit stream,
containing cells, medium, and the product BDO, is then subjected to
a continuous product separations procedure, with or without
removing cells and cell debris, and would take place by standard
continuous separations methods employed in the art to separate
organic products from dilute aqueous solutions, such as continuous
liquid-liquid extraction using a water immiscible organic solvent
(e.g., toluene) to provide an organic solution of BDO. The
resulting solution is subsequently subjected to standard continuous
distillation methods to remove and recycle the organic solvent and
to provide BDO (boiling point 228-229.degree. C.) which is isolated
as a purified liquid (mpt 20.degree. C.).
Example IV
Exemplary BDO Pathways
[0412] This example describes exemplary enzymes and corresponding
genes for 1,4-butandiol (BDO) synthetic pathways.
[0413] Exemplary BDO synthetic pathways are shown in FIGS. 8-13.
The pathways depicted in FIGS. 8-13 are from common central
metabolic intermediates to 1,4-butanediol. All transformations
depicted in FIGS. 8-13 fall into the 18 general categories of
transformations shown in Table 14. Below is described a number of
biochemically characterized candidate genes in each category.
Specifically listed are genes that can be applied to catalyze the
appropriate transformations in FIGS. 9-13 when cloned and expressed
in a host organism. The top three exemplary genes for each of the
key steps in FIGS. 9-13 are provided in Tables 15-23 (see below).
Exemplary genes were provided for the pathways depicted in FIG. 8
are described herein.
TABLE-US-00015 TABLE 14 Enzyme types required to convert common
central metabolic intermediates into 1,4-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. 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) 1.2.1.c Oxidoreductase (2-oxo
acid to acyl-CoA, decarboxylation) 1.2.1.d Oxidoreductase
(phosphorylating/dephosphorylating) 1.3.1.a Oxidoreductase
operating on CH--CH donors 1.4.1.a Oxidoreductase operating on
amino acids 2.3.1.a Acyltransferase (transferring phosphate group)
2.6.1.a Aminotransferase 2.7.2.a Phosphotransferase, carboxyl group
acceptor 2.8.3.a Coenzyme-A transferase 3.1.2.a Thiolester
hydrolase (CoA specific) 4.1.1.a Carboxy-lyase 4.2.1.a Hydro-lyase
4.3.1.a Ammonia-lyase 5.3.3.a Isomerase 5.4.3.a Aminomutase 6.2.1.a
Acid-thiol ligase
1.1.1.a--Oxidoreductase (Aldehyde to Alcohol or Ketone to
Hydroxyl)
[0414] 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
C(3) (Sulzenbacher et al. Journal of Molecular Biology 342:489-502
(2004)), and bdh I and bdh II from C. acetobutylicum which converts
butyryaldehyde 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-00016 Gene Accession No. GI No. Organism alrA BAB12273.1
9967138 Acinetobacter sp. Strain M-1 ADH2 NP_014032.1 6323961
Saccharymyces cerevisiae yqhD NP_417484.1 16130909 Escherichia coli
bdh I NP_349892.1 15896543 Clostridium acetobutylicum bdh II
NP_349891.1 15896542 Clostridium acetobutylicum
[0415] Enzymes exhibiting 4-hydroxybutyrate dehydrogenase activity
(EC 1.1.1.61) also fall into this category. Such enzymes have been
characterized in Ralstonia eutropha (Bravo et al. J. Forensic Sci.
49:379-387 (2004), Clostridium kluyveri (Wolff et al. Protein Expr.
Purif. 6:206-212 (1995)) and Arabidopsis thaliana (Breitkreuz et
al. J. Biol. Chem. 278:41552-41556 (2003)).
TABLE-US-00017 Gene Accession No. GI No. Organism 4hbd YP_726053.1
113867564 Ralstonia eutropha H16 4hbd L21902.1 146348486
Clostridium kluyveri DSM 555 4hbd Q94B07 75249805 Arabidopsis
thaliana
[0416] 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 J231:481-484 (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-00018 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
[0417] 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-00019 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
[0418] 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)).
[0419] Ketone to Hydroxyl.
[0420] 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 and Schlegel, Eur.
J. Biochem. 130:329-334 (1983)). Conversion of alpha-ketoadipate
into alpha-hydroxyadipate can be catalyzed by 2-ketoadipate
reductase, an enzyme reported to be found in rat and in human
placenta (Suda et al. Arch. Biochem. Biophys. 176:610-620 (1976);
Suda et al. Biochem. Biophys. Res. Commun. 77:586-591 (1977)). An
additional candidate for this step is the mitochondrial
3-hydroxybutyrate dehydrogenase (bdh) from the human heart which
has been cloned and characterized (Marks et al. J. Biol. Chem.
267:15459-15463 (1992)). This enzyme is a dehydrogenase that
operates on a 3-hydroxyacid. Another exemplary alcohol
dehydrogenase converts acetone to isopropanol as was shown in C.
beijerinckii (Ismaiel et al. J. Bacteriol. 175:5097-5105 (1993))
and T. brockii (Lamed et al. Biochem. J. 195:183-190 (1981); Peretz
and Burstein Biochemistry 28:6549-6555 (1989)).
TABLE-US-00020 Gene Accession No. GI No. Organism mdh AAC76268.1
1789632 Escherichia coli ldhA NP_415898.1 16129341 Escherichia coli
ldh YP_725182.1 113866693 Ralstonia eutropha bdh AAA58352.1 177198
Homo sapiens adh AAA23199.2 60592974 Clostridium beijerinckii NRRL
B593 adh P14941.1 113443 Thermoanaerobacter brockii HTD4
[0421] Exemplary 3-hydroxyacyl dehydrogenases which convert
acetoacetyl-CoA to 3-hydroxybutyryl-CoA include hbd from C.
acetobutylicum (Boynton et al. Journal of Bacteriology
178:3015-3024 (1996)), hbd from C. beijerinckii (Colby et al. Appl
Environ. Microbiol 58:3297-3302 (1992)), and a number of similar
enzymes from Metallosphaera sedula (Berg et al. Archaea. Science
318:1782-1786 (2007)).
TABLE-US-00021 Gene Accession No. GI No. Organism hbd NP_349314.1
15895965 Clostridium acetobutylicum hbd AAM14586.1 20162442
Clostridium beijerinckii Msed_1423 YP_001191505 146304189
Metallosphaera sedula Msed_0399 YP_001190500 146303184
Metallosphaera sedula Msed_0389 YP_001190490 146303174
Metallosphaera sedula Msed_1993 YP_001192057 146304741
Metallosphaera sedula
1.1.1.c--Oxidoredutase (2 Step, Acyl-CoA to Alcohol)
[0422] Exemplary 2-step oxidoreductases that convert an acyl-CoA to
alcohol include those that transform substrates such as acetyl-CoA
to ethanol (for example, adhE from E. coli (Kessler et al. FEBS.
Lett. 281:59-63 (1991)) and butyryl-CoA to butanol (for example,
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 oxide the branched chain compound isobutyraldehyde to
isobutyryl-CoA (Kazahaya et al. J. Gen. Appl. Microbiol. 18:43-55
(1972); Koo et al. Biotechnol Lett. 27:505-510 (2005)).
TABLE-US-00022 Gene Accession No. GI No. Organism adhE NP_415757.1
16129202 Escherichia coli adhE2 AAK09379.1 12958626 Clostridium
acetobutylicum adhE AAV66076.1 55818563 Leuconostoc
mesenteroides
[0423] 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 and Fuchs, Eur. J. Biochem.
215:633-643 (1993)). This enzyme, with a mass of 300 kDa, is highly
substrate-specific and shows little sequence similarity to other
known oxidoreductases (Hugler et al., J. Bacteriol. 184:2404-2410
(2002)). No enzymes in other organisms have been shown to catalyze
this specific reaction; however there is bioinformatic evidence
that other organisms may have similar pathways (Klatt et al.,
Environ. Microbiol. 9:2067-2078 (2007)). Enzyme candidates in other
organisms including Roseiflexus castenholzii, Erythrobacter sp.
NAP1 and marine gamma proteobacterium HTCC2080 can be inferred by
sequence similarity.
TABLE-US-00023 Gene Accession No. GI No. Organism mcr AAS20429.1
42561982 Chloroflexus aurantiacus Rcas_2929 YP_001433009.1
156742880 Roseiflexus castenholzii NAP1_02720 ZP_01039179.1
85708113 Erythrobacter sp. NAP 1 MGP2080_00535 ZP_01626393.1
119504313 marine gamma proteobacterium HTCC2080
[0424] 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)).
TABLE-US-00024 Gene Accession No. GI No. Organism FAR AAD38039.1
5020215 Simmondsia chinensis
1.2.1.b--Oxidoreductase (Acyl-CoA to Aldehyde)
[0425] 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 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 as it has been demonstrated to oxidize and acylate
acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde and
formaldehyde (Powlowski et al. J Bacteriol. 175:377-385
(1993)).
TABLE-US-00025 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
[0426] 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.
TABLE-US-00026 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
1.2.1.c--Oxidoreductase (2-Oxo Acid to Acyl-CoA,
Decarboxylation)
[0427] Enzymes in this family include 1) branched-chain 2-keto-acid
dehydrogenase, 2) alpha-ketoglutarate dehydrogenase, and 3) the
pyruvate dehydrogenase multienzyme complex (PDHC). These enzymes
are multi-enzyme complexes that catalyze a series of partial
reactions which result in acylating oxidative decarboxylation of
2-keto-acids. Each of the 2-keto-acid dehydrogenase complexes
occupies key positions in intermediary metabolism, and enzyme
activity is typically tightly regulated (Fries et al. Biochemistry
42:6996-7002 (2003)). The enzymes share 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).
[0428] 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 keto-acid
dehydrogenases have the broadest substrate range.
[0429] 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)). Although the substrate
range of AKGD is narrow, 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-00027 Gene Accession No. GI No. Organism sucA NP_415254.1
16128701 Escherichia coli str. K12 substr. MG1655 sucB NP_415255.1
16128702 Escherichia coli str. K12 substr. MG1655 lpd NP_414658.1
16128109 Escherichia coli str. K12 substr. MG1655 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
[0430] Branched-chain 2-keto-acid dehydrogenase complex (BCKAD),
also known as 2-oxoisovalerate dehydrogenase, 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. The complex has been studied in
many organisms including Bacillus subtilis (Wang et al. Eur. J.
Biochem. 213:1091-1099 (1993)), Rattus norvegicus (Namba et al. J.
Biol. Chem. 244:4437-4447 (1969)) and Pseudomonas putida (Sokatch
J. Bacteriol. 148:647-652 (1981)). In Bacillus subtilis the enzyme
is encoded by genes pdhD (E3 component), bfmBB (E2 component),
bfmBAA and bfmBAB (E1 component) (Wang et al. Eur. J. Biochem.
213:1091-1099 (1993)). In mammals, the complex is regulated by
phosphorylation by specific phosphatases and protein kinases. The
complex has been studied in rat hepatocites (Chicco et al. J. Biol.
Chem. 269:19427-19434 (1994)) and is encoded by genes Bckdha (E1
alpha), Bckdhb (E1 beta), Dbt (E2), and Dld (E3). The E1 and E3
components of the Pseudomonas putida BCKAD complex have been
crystallized (Aevarsson et al. Nat. Struct. Biol. 6:785-792 (1999);
Mattevi Science 255:1544-1550 (1992)) and the enzyme complex has
been studied (Sokatch et al. J. Bacteriol. 148:647-652 (1981)).
Transcription of the P. putida BCKAD genes is activated by the gene
product of bkdR (Hester et al. Eur. J. Biochem. 233:828-836
(1995)). 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. The active site of the bovine BCKAD was engineered to
favor alternate substrate acetyl-CoA (Meng and Chuang, Biochemistry
33:12879-12885 (1994)).
TABLE-US-00028 Gene Accession No. GI No. Organism bfmBB NP_390283.1
16079459 Bacillus subtilis bfmBAA NP_390285.1 16079461 Bacillus
subtilis bfmBAB NP_390284.1 16079460 Bacillus subtilis pdhD
P21880.1 118672 Bacillus subtilis lpdV P09063.1 118677 Pseudomonas
putida bkdB P09062.1 129044 Pseudomonas putida bkdA1 NP_746515.1
26991090 Pseudomonas putida bkdA2 NP_746516.1 26991091 Pseudomonas
putida Bckdha NP_036914.1 77736548 Rattus norvegicus Bckdhb
NP_062140.1 158749538 Rattus norvegicus Dbt NP_445764.1 158749632
Rattus norvegicus Dld NP_955417.1 40786469 Rattus norvegicus
[0431] The pyruvate dehydrogenase complex, catalyzing the
conversion of pyruvate to acetyl-CoA, has also been extensively
studied. In the E. coli enzyme, specific residues in the E1
component are responsible for substrate specificity (Bisswanger, H.
J Biol. Chem. 256:815-822 (1981); Bremer, J. Eur. J Biochem.
8:535-540 (1969); Gong et al. J Biol. Chem. 275:13645-13653
(2000)). As mentioned previously, enzyme engineering efforts have
improved the E. coli PDH enzyme activity under anaerobic conditions
(Kim et al. Appl. Environ. Microbiol. 73:1766-1771 (2007); Kim J.
Bacteriol. 190:3851-3858 (2008); 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 J. Bacteriol. 179:6749-6755 (1997)). The
Klebsiella pneumoniae PDH, characterized during growth on glycerol,
is also active under anaerobic conditions (Menzel et al. J.
Biotechnol. 56:135-142 (1997)). Crystal structures of the enzyme
complex from bovine kidney (Zhou et al. Proc. Natl. Acad. Sci.
U.S.A. 98:14802-14807 (2001)) and the E2 catalytic domain from
Azotobacter vinelandii are available (Mattevi et al. Science
255:1544-1550 (1992)). Some mammalian PDH enzymes complexes can
react on alternate substrates such as 2-oxobutanoate, although
comparative kinetics of Rattus norvegicus PDH and BCKAD indicate
that BCKAD has higher activity on 2-oxobutanoate as a substrate
(Paxton et al. Biochem. J. 234:295-303 (1986)).
TABLE-US-00029 Gene Accession No. GI No. Organism aceE NP_414656.1
16128107 Escherichia coli str. K12 substr. MG1655 aceF NP_414657.1
16128108 Escherichia coli str. K12 substr. MG1655 lpd NP_414658.1
16128109 Escherichia coli str. K12 substr. MG1655 pdhA P21881.1
3123238 Bacillus subtilis pdhB P21882.1 129068 Bacillus subtilis
pdhC P21883.2 129054 Bacillus subtilis pdhD P21880.1 118672
Bacillus subtilis aceE YP_001333808.1 152968699 Klebsiella
pneumonia MGH78578 aceF YP_001333809.1 152968700 Klebsiella
pneumonia MGH78578 lpdA YP_001333810.1 152968701 Klebsiella
pneumonia MGH78578 Pdha1 NP_001004072.2 124430510 Rattus norvegicus
Pdha2 NP_446446.1 16758900 Rattus norvegicus Dlat NP_112287.1
78365255 Rattus norvegicus Dld NP_955417.1 40786469 Rattus
norvegicus
[0432] As an alternative to the large multienzyme 2-keto-acid
dehydrogenase complexes described above, some anaerobic organisms
utilize enzymes in the 2-ketoacid oxidoreductase family (OFOR) to
catalyze acylating oxidative decarboxylation of 2-keto-acids.
Unlike the dehydrogenase complexes, these enzymes contain
iron-sulfur clusters, utilize different cofactors, and use
ferredoxin or flavodixin as electron acceptors in lieu of NAD(P)H.
While most enzymes in this family are specific to pyruvate as a
substrate (POR) some 2-keto-acid:ferredoxin oxidoreductases have
been shown to accept a broad range of 2-ketoacids as substrates
including alpha-ketoglutarate and 2-oxobutanoate (Fukuda and Wakagi
Biochim. Biophys. Acta 1597:74-80 (2002); Zhang et al. J. Biochem.
120:587-599 (1996)). One such enzyme is the OFOR from the
thermoacidophilic archaeon Sulfolobus tokodaii 7, which contains an
alpha and beta subunit encoded by gene ST2300 (Fukuda and Wakagi
Biochim. Biophys. Acta 1597:74-80 (2002); Zhang et al. J. Biochem.
120:587-599 (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)). Two OFORs from
Aeropyrum pernix str. K1 have also been recently cloned into E.
coli, characterized, and found to react with a broad range of
2-oxoacids (Nishizawa et al. FEBS Lett. 579:2319-2322 (2005)). The
gene sequences of these OFOR candidates are available, although
they do not have GenBank identifiers assigned to date. There is
bioinformatic evidence that similar enzymes are present in all
archaea, some anaerobic bacteria and amitochondrial eukarya (Fukuda
and Wakagi Biochim. Biophys. Acta 1597:74-80 (2005)). This class of
enzyme is also interesting from an energetic standpoint, as reduced
ferredoxin could be used to generate NADH by ferredoxin-NAD
reductase (Petitdemange et al. Biochim. Biophys. Acta 421:334-337
(1976)). Also, since most of the enzymes are designed to operate
under anaerobic conditions, less enzyme engineering may be required
relative to enzymes in the 2-keto-acid dehydrogenase complex family
for activity in an anaerobic environment.
TABLE-US-00030 Gene Accession No. GI No. Organism ST2300
NP_378302.1 15922633 Sulfolobus tokodaii 7
1.2.1.d--Oxidoreductase (Phosphorylating/Dephosphorylating)
[0433] Exemplary enzymes in this class include glyceraldehyde
3-phosphate dehydrogenase which converts glyceraldehyde-3-phosphate
into D-glycerate 1,3-bisphosphate (for example, E. coli gapA
(Branlant and Branlant Eur. J. Biochem. 150:61-66 (1985)),
aspartate-semialdehyde dehydrogenase which converts
L-aspartate-4-semialdehyde into L-4-aspartyl-phosphate (for
example, E. coli asd (Biellmann et al. Eur. J. Biochem. 104:53-58
(1980)), N-acetyl-gamma-glutamyl-phosphate reductase which converts
N-acetyl-L-glutamate-5-semialdehyde into
N-acetyl-L-glutamyl-5-phosphate (for example, 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 (for example, E. coli proA (Smith et al. J.
Bacteriol. 157:545-551 (1984)).
TABLE-US-00031 Gene Accession No. GI No. Organism gapA POA9B2.2
71159358 Escherichia coli asd NP_417891.1 16131307 Escherichia coli
argC NP_418393.1 16131796 Escherichia coli proA NP_414778.1
16128229 Escherichia coli
[0434] 1.3.1.a--Oxidoreductase Operating on CH--CH Donors
[0435] An exemplary enoyl-CoA reductase is the gene product of bcd
from C. acetobutylicum (Atsumi et al. Metab Eng (2007); Boynton et
al. Journal of Bacteriology 178:3015-3024 (1996), which naturally
catalyzes the reduction of crotonyl-CoA to butyryl-CoA. Activity of
this enzyme can be enhanced by expressing bcd in conjunction with
expression of the C. acetobutylicum etfAB genes, which encode an
electron transfer flavoprotein. An additional candidate for the
enoyl-CoA reductase step is the mitochondrial enoyl-CoA reductase
from E. gracilis (Hoffmeister et al. Journal of Biological
Chemistry 280:4329-4338 (2005)). A construct derived from this
sequence following the removal of its mitochondrial targeting
leader sequence was cloned in E. coli resulting in an active enzyme
(Hoffmeister et al., supra, (2005)). This approach is well known to
those skilled in the art of expressing eukarytotic genes,
particularly those with leader sequences that may target the gene
product to a specific intracellular compartment, in prokaryotic
organisms. A close homolog of this gene, TDE0597, from the
prokaryote Treponema denticola represents a third enoyl-CoA
reductase which has been cloned and expressed in E. coli (Tucci and
Martin FEBS Letters 581:1561-1566 (2007)).
TABLE-US-00032 Gene Accession No. GI No. Organism bcd NP_349317.1
15895968 Clostridium acetobutylicum etfA NP_349315.1 15895966
Clostridium acetobutylicum etfB NP_349316.1 15895967 Clostridium
acetobutylicum TER Q5EU90.1 62287512 Euglena gracilis TDE0597
NP_971211.1 42526113 Treponema denticola
[0436] Exemplary 2-enoate reductase (EC 1.3.1.31) enzymes are known
to catalyze the NADH-dependent reduction of a wide variety of
.alpha.,.beta.-unsaturated carboxylic acids and aldehydes (Rohdich
et al. J. Biol. Chem. 276:5779-5787 (2001)). 2-Enoate reductase is
encoded by enr in several species of Clostridia (Giesel and Simon
Arch Microbiol. 135(1): p. 51-57 (2001) including C. tyrobutyricum,
and C. thermoaceticum (now called Moorella thermoaceticum) (Rohdich
et al., supra, (2001)). In the recently published genome sequence
of C. kluyveri, 9 coding sequences for enoate reductases have been
reported, out of which one has been characterized (Seedorf et al.
Proc Natl Acad Sci U.S.A. 105(6):2128-33 (2008)). The enr genes
from both C. tyrobutyricum and C. thermoaceticum have been cloned
and sequenced and show 59% identity to each other. The former gene
is also found to have approximately 75% similarity to the
characterized gene in C. kluyveri (Giesel and Simon Arch Microbiol
135(1):51-57 (1983)). It has been reported based on these sequence
results that enr is very similar to the dienoyl CoA reductase in E.
coli (fadH) (163 Rohdich et al., supra (2001)). The C.
thermoaceticum enr gene has also been expressed in an enzymatically
active form in E. coli (163 Rohdich et al., supra (2001)).
TABLE-US-00033 Gene Accession No. GI No. Organism fadH NP_417552.1
16130976 Escherichia coli enr ACA54153.1 169405742 Clostridium
botulinum A3 str enr CAA71086.1 2765041 Clostridium tyrobutyricum
enr CAA76083.1 3402834 Clostridium kluyveri enr YP_430895.1
83590886 Moorella thermoacetica
1.4.1.a--Oxidoreductase Operating on Amino Acids
[0437] Most oxidoreductases operating on amino acids catalyze the
oxidative deamination of alpha-amino acids with NAD+ or NADP+ as
acceptor. 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-00034 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
[0438] 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)). In addition, the lysDH gene from Aeropyrum pernix K1 is
identified through homology from genome projects.
TABLE-US-00035 Gene Accession No. GI No. Organism lysDH AB052732
13429872 Geobacillus stearothermophilus lysDH NP_147035.1 14602185
Aeropyrum pernix K1 ldh P0A393 61222614 Bacillus cereus
2.3.1.a--Acyltransferase (Transferring Phosphate Group)
[0439] Exemplary phosphate transferring acyltransferases 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-00036 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
2.6.1.a--Aminotransferase
[0440] Aspartate aminotransferase transfers an amino group from
aspartate to alpha-ketoglutarate, forming glutamate and
oxaloacetate. 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. 113:83-89 (1985)),
AAT2 from Saccharomyces cerevisiae (Yagi et al. J Biochem. 92:35-43
(1982)) and ASPS from Arabidopsis thaliana (48, 108, 225 48. de la
et al. Plant J 46:414-425 (2006); Kwok and Hanson J Exp. Bot.
55:595-604 (2004); Wilkie and Warren Protein Expr. Purif.
12:381-389 (1998)). Valine aminotransferase catalyzes the
conversion of valine and pyruvate to 2-ketoisovalerate and alanine
The E. coli gene, avtA, encodes one such enzyme (Whalen and Berg 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 and Berg J. Bacteriol. 158:571-574
(1984)). The gene product of the E. coli serC catalyzes two
reactions, phosphoserine aminotransferase and
phosphohydroxythreonine aminotransferase (Lam and Winkler J.
Bacteriol. 172:6518-6528 (1990)), and activity on
non-phosphorylated substrates could not be detected (Drewke et al.
FEBS. Lett. 390:179-182 (1996)).
TABLE-US-00037 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 avtA
YP_026231.1 49176374 Escherichia coli serC NP_415427.1 16128874
Escherichia coli
[0441] Cargill has developed a beta-alanine/alpha-ketoglutarate
aminotransferase for producing 3-HP from beta-alanine via
malonyl-semialdehyde (PCT/US2007/076252 (Jessen et al)). The gene
product of SkPYD4 in Saccharomyces kluyveri was also shown to
preferentially use beta-alanine as the amino group donor (Andersen
et al. FEBS. J. 274:1804-1817 (2007)). 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 et al. FEBS. J. 274:1804-1817 (2007)).
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 (Kakimoto
et al. Biochim. Biophys. Acta 156:374-380 (1968); Tamaki et al.
Methods Enzymol. 324:376-389 (2000)). 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 (Liu et al. Biochemistry 43:10896-10905
(2004); Schulz et al. Appl Environ Microbiol 56:1-6 (1990)). The
gene product of puuE catalyzes the other 4-aminobutyrate
transaminase in E. coli (Kurihara et al. J. Biol. Chem.
280:4602-4608 (2005)).
TABLE-US-00038 Gene Accession No. GI No. 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 subtilus gabT
P22256.1 120779 Escherichia coli K12 puuE NP_415818.1 16129263
Escherichia coli K12
[0442] The X-ray crystal structures of E. coli 4-aminobutyrate
transaminase unbound and bound to the inhibitor were reported (Liu
et al. Biochemistry 43:10896-10905 (2004)). The substrates binding
and substrate specificities were studied and suggested. The roles
of active site residues were studied by site-directed mutagenesis
and X-ray crystallography (Liu et al. Biochemistry 44:2982-2992
(2005)). Based on the structural information, attempt was made to
engineer E. coli 4-aminobutyrate transaminase with novel enzymatic
activity. These studies provide a base for evolving transaminase
activity for BDO pathways.
2.7.2.a--Phosphotransferase, Carboxyl Group Acceptor
[0443] Exemplary kinases include the E. coli acetate kinase,
encoded by ackA (Skarstedt and Silverstein J. Biol. Chem.
251:6775-6783 (1976)), the C. acetobutylicum butyrate kinases,
encoded by buk1 and buk2 (Walter et al. Gene 134(1):107-111 (1993)
(Huang et al. J Mol Microbiol Biotechnol 2(1):33-38 (2000)), and
the E. coli gamma-glutamyl kinase, encoded by proB (Smith et al. J.
Bacteriol. 157:545-551 (1984)). These enzymes phosphorylate
acetate, butyrate, and glutamate, respectively. The ackA gene
product from E. coli also phosphorylates propionate (Hesslinger et
al. Mol. Microbiol 27:477-492 (1998)).
TABLE-US-00039 Gene Accession No. GI No. Organism ackA NP_416799.1
16130231 Escherichia coli buk1 NP_349675 15896326 Clostridium
acetobutylicum buk2 Q97II1 20137415 Clostridium acetobutylicum proB
NP_414777.1 16128228 Escherichia coli
2.8.3.a--Coenzyme-A Transferase
[0444] In the CoA-transferase family, E. coli enzyme
acyl-CoA:acetate-CoA transferase, also known as acetate-CoA
transferase (EC 2.8.3.8), has been shown to transfer the CoA moiety
to acetate from a variety of branched and linear acyl-CoA
substrates, including isobutyrate (Matthies 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)) and actA and cg0592 in Corynebacterium
glutamicum ATCC 13032 (Duncan et al. Appl Environ Microbiol
68:5186-5190 (2002)). Additional genes found by sequence homology
include atoD and atoA in Escherichia coli UT189.
TABLE-US-00040 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 ATCC 13032
cg0592 YP_224801.1 62389399 Corynebacterium glutamicum ATCC 13032
atoA ABE07971.1 91073090 Escherichia coli UT189 atoD ABE07970.1
91073089 Escherichia coli UT189
[0445] Similar transformations are catalyzed by the gene products
of cat1, cat2, and cat3 of Clostridium kluyveri which have been
shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and
butyryl-CoA acetyltransferase activity, respectively (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)).
TABLE-US-00041 Gene Accession No. GI No. Organism cat1 P38946.1
729048 Clostridium kluyveri cat2 P38942.2 1705614 Clostridium
kluyveri cat3 EDK35586.1 146349050 Clostridium kluyveri
[0446] 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-00042 Gene Accession No. GI No. Organism gctA CAA57199.1
559392 Acidaminococcus fermentans gctB CAA57200.1 559393
Acidaminococcus fermentans
3.1.1.a Hydroxyacylhydrolase
[0447] FIG. 64B is the transformation of 4-hydroxybutyrate to GBL.
This step can be catalyzed by enzymes in the 3.1.1 family that act
on carboxylic ester bonds molecules for the interconversion between
cyclic lactones and the open chain hydroxycarboxylic acids. The
1,4-lacton hydroxyacylhydrolase (EC 3.1.1.25), also known as
1,4-lactonase or gamma-lactonase, is specific for 1,4-lactones with
4-8 carbon atoms. It does not hydrolyze simple aliphatic esters,
acetylcholine, or sugar lactones. The gamma lactonase in human
blood and rat liver microsomes was purified (Fishbein et al., J
Biol Chem 241:4835-4841 (1966)) and the lactonase activity was
activated and stabilized by calcium ions (Fishbein et al., J Biol
Chem 241:4842-4847 (1966)). The optimal lactonase activities were
observed at pH 6.0, whereas high pH resulted in hydrolytic
activities (Fishbein and Bessman, J Biol Chem 241:4842-4847
(1966)). The following genes have been annotated as 1,4-lactonase
and can be utilized to catalyze the transformation of
4-hydroxybutyrate to GBL, including a lactonase from Fusarium
oxysporum (Zhang et al., Appl Microbiol Biotechnol 75:1087-1094
(2007)). The protein sequences for each of these exemplary gene
products, if available, can be found using the following GenBank
accession numbers shown below.
TABLE-US-00043 Gene Accession No. GI No. Organism xccb100_2516
YP_001903921.1 188991911 Xanthomonas campestris An16g06620
CAK46996.1 134083519 Aspergillus niger BAA34062 BAA34062.1 3810873
Fusarium oxysporum
[0448] Additionally, it has been reported that lipases such as
Candida antarctica lipase B can catalyze the lactonization of
4-hydroxybutyrate to GBL (Efe et al., Biotechnol Bioeng
99:1392-1406 (2008)). Therefore, the following genes coding for
lipases can also be utilized for Step AB in FIG. 1. The protein
sequences for each of these exemplary gene products, if available,
can be found using the following GenBank accession numbers shown
below.
TABLE-US-00044 Gene Accession No. GI No. Organism calB P41365.1
1170790 Candida antarctica lipB P41773.1 1170792 Pseudomonas
fluorescens estA P37957.1 7676155 Bacillus subtilis
3.1.2.a--Thiolester Hydrolase (CoA Specific)
[0449] In the CoA hydrolase family, the enzyme
3-hydroxyisobutyryl-CoA hydrolase is specific for 3-HIBCoA and has
been described to efficiently catalyze the desired transformation
during valine degradation (Shimomura et al. J Biol Chem
269:14248-14253 (1994)). Genes encoding this enzyme include hibch
of Rattus norvegicus (Shimomura et al., supra (1994); Shimomura et
al. Methods Enzymol. 324:229-240 (2000) and Homo sapiens (Shimomura
et al., supra, 2000). Candidate genes by sequence homology include
hibch of Saccharomyces cerevisiae and BC 2292 of Bacillus
cereus.
TABLE-US-00045 Gene Accession No. GI No. Organism hibch Q5XIE6.2
146324906 Rattus norvegicus hibch Q6NVY1.2 146324905 Homo sapiens
hibch P28817.2 2506374 Saccharomyces cerevisiae BC_2292 Q81DR3
81434808 Bacillus cereus
[0450] The conversion of adipyl-CoA to adipate can be carried out
by an acyl-CoA hydrolase or equivalently a thioesterase. The top E.
coli gene candidate is tesB (Naggert et al. J Biol. Chem.
266(17):11044-11050 (1991)) which shows high similarity to the
human acot8 which is a dicarboxylic acid acetyltransferase with
activity on adipyl-CoA (Westin et al. J Biol Chem 280(46):
38125-38132 (2005). This activity has also been characterized in
the rat liver (Deana, Biochem Int. 26(4): p. 767-773 (1992)).
TABLE-US-00046 Gene Accession No. GI No. Organism tesB NP_414986
16128437 Escherichia coli acot8 CAA15502 3191970 Homo sapiens acot8
NP_570112 51036669 Rattus norvegicus
[0451] Other potential E. coli thiolester hydrolases include the
gene products of tesA (Bonner and Bloch, J Biol Chem.
247(10):3123-3133 (1972)), ybgC (Kuznetsova et al., FEMS Microbiol
Rev. 29(2):263-279 (2005); Zhuang et al., FEBS Lett.
516(1-3):161-163 (2002)) paal (Song et al., J Biol Chem.
281(16):11028-11038 (2006)), and ybdB (Leduc et al., J Bacteriol.
189(19):7112-7126 (2007)).
TABLE-US-00047 Gene Accession No. GI No. Organism tesA NP_415027
16128478 Escherichia coli ybgC NP_415264 16128711 Escherichia coli
paaI NP_415914 16129357 Escherichia coli ybdB NP_415129 16128580
Escherichia coli
[0452] Several eukaryotic acetyl-CoA hydrolases (EC 3.1.2.1) have
broad substrate specificity. The enzyme from Rattus norvegicus
brain (Robinson et al. Biochem. Biophys. Res. Commun. 71:959-965
(1976)) can react with butyryl-CoA, hexanoyl-CoA and
malonyl-CoA.
TABLE-US-00048 Gene Accession No. GI No. Organism acotl2
NP_570103.1 18543355 Rattus norvegicus
4.1.1.a--Carboxy-Lyase
[0453] An exemplary carboxy-lyase is acetolactate decarboxylase
which participates in citrate catabolism and branched-chain amino
acid biosynthesis, converting 2-acetolactate to acetoin. In
Lactococcus lactis the enzyme is composed of six subunits, encoded
by gene aldB, and is activated by valine, leucine and isoleucine
(Goupil et al. Appl. Environ. Microbiol. 62:2636-2640 (1996);
Goupil-Feuillerat et al. J. Bacteriol. 182:5399-5408 (2000)). This
enzyme has been overexpressed and characterized in E. coli (Phalip
et al. FEBS Lett. 351:95-99 (1994)). In other organisms the enzyme
is a dimer, encoded by aldC in Streptococcus thermophilus (Monnet
et al. Lett. Appl. Microbiol. 36:399-405 (2003)), aldB in Bacillus
brevis (Diderichsen et al. J. Bacteriol. 172:4315-4321 (1990);
Najmudin et al. Acta Crystallogr. D. Biol. Crystallogr.
59:1073-1075 (2003)) and budA from Enterobacter aerogenes
(Diderichsen et al. J. Bacteriol. 172:4315-4321 (1990)). The enzyme
from Bacillus brevis was cloned and overexpressed in Bacillus
subtilis and characterized crystallographically (Najmudin et al.
Acta Crystallogr. D. Biol. Crystallogr. 59:1073-1075 (2003)).
Additionally, the enzyme from Leuconostoc lactis has been purified
and characterized but the gene has not been isolated (O'Sullivan et
al. FEMS Microbiol. Lett. 194:245-249 (2001)).
TABLE-US-00049 Gene Accession No. GI No. Organism aldB NP_267384.1
15673210 Lactococcus lactis aldC Q8L208 75401480 Streptococcus
thermophilus aldB P23616.1 113592 Bacillus brevis budA P05361.1
113593 Enterobacter aerogenes
[0454] 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 and Vorlop Appl Microbiol
Biotechnol 56:289-295 (2001)). Although itaconate is a compound of
biotechnological interest, the aconitate decarboxylase gene or
protein sequence has not been reported to date.
[0455] 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 and Asano Arch. Microbiol 168:457-463 (1997); Lian and
Whitman J. Am. Chem. Soc. 116:10403-10411 (1994); Stanley et al.
Biochemistry 39:3514 (2000)) 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. J
Bacteriol. 174:711-724 (1992)).
TABLE-US-00050 Gene Accession No. GI No. 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
[0456] 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); Qi et al. Metab Eng 9:268-276 (2007); Rodriguez et al. J.
Agric. Food Chem. 56:3068-3072 (2008)), pofK (pad) from Klebsiella
oxytoca (Hashidoko et al. Biosci. Biotech. Biochem. 58:217-218
(1994); Uchiyama et al. Biosci. Biotechnol. Biochem. 72:116-123
(2008)), Pedicoccus pentosaceus (Barthelmebs et al. Appl Environ
Microbiol 67:1063-1069 (2001)), and padC from Bacillus subtilis and
Bacillus pumilus (Lingen et al. Protein Eng 15:585-593 (2002)). 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 have been
shown to be stable and do not require either exogenous or
internally bound co-factors, thus making these enzymes ideally
suitable for biotransformations (Sariaslani, Annu. Rev. Microbiol.
61:51-69 (2007)).
TABLE-US-00051 Gene Accession No. GI No. Organism pad1 AB368798
188496948 Saccharomyces BAG32372.1 188496949 cerevisae pdc U63827
1762615, 1762616 Lactobacillus AAC45282.1 plantarum pofK (pad)
AB330293, 149941607, Klebsiella oxytoca BAF65031.1 149941608 padC
AF017117 2394281, 2394282 Bacillus subtilis AAC46254.1 pad AJ276891
11322456, 11322458 Pedicoccus CAC16794.1 pentosaceus pad AJ278683
11691809, 11691810 Bacillus pumilus CAC18719.1
[0457] Additional decarboxylase enzymes can form succinic
semialdehyde from alpha-ketoglutarate. These include the
alpha-ketoglutarate decarboxylase enzymes from Euglena gracilis
(Shigeoka et al. Biochem. J. 282(Pt 2):319-323 (1992); Shigeoka and
Nakano Arch. Biochem. Biophys. 288:22-28 (1991); Shigeoka and
Nakano Biochem. J. 292 (Pt 2):463-467 (1993)), whose corresponding
gene sequence has yet to be determined, and from Mycobacterium
tuberculosis (Tian et al. Proc Natl Acad Sci U.S.A. 102:10670-10675
(2005)). In addition, glutamate decarboxylase enzymes can convert
glutamate into 4-aminobutyrate such as the products of the E. coli
gadA and gadB genes (De Biase et al. Protein. Expr. Purif.
8:430-438 (1993)).
TABLE-US-00052 Gene Accession No. GI No. Organism kgd O50463.4
160395583 Mycobacterium tuberculosis gadA NP_417974 16131389
Escherichia coli gadB NP_416010 16129452 Escherichia coli
Keto-Acid Decarboxylases
[0458] Pyruvate decarboxylase (PDC, EC 4.1.1.1), also termed
keto-acid decarboxylase, is a key enzyme in alcoholic fermentation,
catalyzing the decarboxylation of pyruvate to acetaldehyde. This
enzyme has a broad substrate range for aliphatic 2-keto acids
including 2-ketobutyrate, 2-ketovalerate, 3-hydroxypyruvate and
2-phenylpyruvate (Berg et al. Science 318:1782-1786 (2007)). The
PDC from Zymomonas mobilus, encoded by pdc, has been a subject of
directed engineering studies that altered the affinity for
different substrates (Siegert et al. Protein Eng Des Sel 18:345-357
(2005)). The PDC from Saccharomyces cerevisiae has also been
extensively studied, engineered for altered activity, and
functionally expressed in E. coli (Killenberg-Jabs et al. Eur. J.
Biochem. 268:1698-1704 (2001); Li and Jordan Biochemistry
38:10004-10012 (1999); ter Schure et al. Appl. Environ. Microbiol.
64:1303-1307 (1998)). The crystal structure of this enzyme is
available (Killenberg-Jabs Eur. J. Biochem. 268:1698-1704 (2001)).
Other well-characterized PDC candidates include the enzymes from
Acetobacter pasteurians (Chandra et al. Arch. Microbiol.
176:443-451 (2001)) and Kluyveromyces lactis (Krieger et al. Eur.
J. Biochem. 269:3256-3263 (2002)).
TABLE-US-00053 Gene Accession No. GI No. Organism pdc P06672.1
118391 Zymomonas mobilus pdcl P06169 30923172 Saccharomyces
cerevisiae pdc Q8L388 75401616 Acetobacter pasteurians pdcl Q12629
52788279 Kluyveromyces lactis
[0459] 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 (Hasson
et al. Biochemistry 37:9918-9930 (1998); Polovnikova et al.
Biochemistry 42:1820-1830 (2003)). 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 Protein Eng Des Sel 18:345-357 (2005)). The
properties of this enzyme have been further modified by directed
engineering (Lingen et al. Protein Eng 15:585-593 (2002)); Lingen
Chembiochem 4:721-726 (2003)). The enzyme from Pseudomonas
aeruginosa, encoded by mdlC, has also been characterized
experimentally (Barrowman et al. FEMS Microbiology Letters 34:57-60
(1986)). Additional gene candidates from Pseudomonas stutzeri,
Pseudomonas fluorescens and other organisms can be inferred by
sequence homology or identified using a growth selection system
developed in Pseudomonas putida (Henning et al. Appl. Environ.
Microbiol. 72:7510-7517 (2006)).
TABLE-US-00054 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
4.2.1.a--Hydro-Lyase
[0460] The 2-(hydroxymethyl)glutarate dehydratase of Eubacterium
barkeri is an exemplary hydro-lyase. This enzyme has been studied
in the context of nicotinate catabolism and is encoded by hmd
(Alhapel et al. Proc Natl Acad Sci USA 103:12341-12346 (2006)).
Similar enzymes with high sequence homology are found in
Bacteroides capillosus, Anaerotruncus colihominis, and
Natranaerobius thermophilius.
TABLE-US-00055 Gene Accession No. GI No. 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
[0461] A second exemplary hydro-lyase is fumarate hydratase, an
enzyme catalyzing the dehydration of malate to fumarate. A wealth
of structural information is available for this enzyme and
researchers have successfully engineered the enzyme to alter
activity, inhibition and localization (Weaver, T. Acta Crystallogr.
D Biol Crystallogr. 61:1395-1401 (2005)). Additional fumarate
hydratases include those encoded by fumC from Escherichia coli
(Estevez et al. Protein Sci. 11:1552-1557 (2002); Hong and Lee
Biotechnol. Bioprocess Eng. 9:252-255 (2004); Rose and Weaver Proc
Natl Acad Sci U.S.A. 101:3393-3397 (2004)), Campylobacter jejuni
(Smith et al. Int. J Biochem. Cell Biol 31:961-975 (1999)) and
Thermus thermophilus (Mizobata et al. Arch. Biochem. Biophys.
355:49-55 (1998)), and fumH from 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.
TABLE-US-00056 Gene Accession No. GI No. Organism fumC P05042.1
120601 Escherichia coli K12 fumC O69294.1 9789756 Campylobacter
jejuni fumC P84127 75427690 Thermus thermophilus fumH P14408.1
120605 Rattus norvegicus fum1 P93033.2 39931311 Arabidopsis
thaliana fumC Q8NRN8.1 39931596 Corynebacterium glutamicum
[0462] Citramalate hydrolyase, also called 2-methylmalate
dehydratase, converts 2-methylmalate to mesaconate. 2-Methylmalate
dehydratase activity was detected in Clostridium tetanomorphum,
Morganella morganii, Citrobacter amalonaticus in the context of the
glutamate degradation VI pathway (Kato and Asano Arch. Microbiol
168:457-463 (1997)); however the genes encoding this enzyme have
not been sequenced to date.
[0463] The gene product of crt from C. acetobutylicum catalyzes the
dehydration of 3-hydroxybutyryl-CoA to crotonyl-CoA (Atsumi et al.
Metab Eng.; 29 (2007)); Boynton et al. Journal of Bacteriology
178:3015-3024 (1996)). The enoyl-CoA hydratases, phaA and phaB, of
P. putida are believed to carry out the hydroxylation of double
bonds during phenylacetate catabolism; (Olivera et al. Proc Natl
Acad Sci USA 95(11):6419-6424 (1998)). The paaA and paaB from P.
fluorescens catalyze analogous transformations (14 Olivera et al.,
supra, 1998). Lastly, a number of Escherichia coli genes have been
shown to demonstrate enoyl-CoA hydratase functionality including
maoC (Park and Lee J Bacteriol 185(18):5391-5397 (2003)), paaF
(Park and Lee Biotechnol Bioeng. 86(6):681-686 (2004a)); Park and
Lee Appl Biochem Biotechnol. 113-116: 335-346 (2004b)); Ismail et
al. Eur J Biochem 270(14):p. 3047-3054 (2003), and paaG (Park and
Lee, supra, 2004; Park and Lee supra, 2004b; Ismail et al., supra,
2003).
TABLE-US-00057 Gene Accession No. GI No. Organism maoC NP_415905.1
16129348 Escherichia coli paaF NP_415911.1 16129354 Escherichia
coli paaG NP_415912.1 16129355 Escherichia coli crt NP_349318.1
15895969 Clostridium acetobutylicum paaA NP_745427.1 26990002
Pseudomonas putida paaB NP_745426.1 26990001 Pseudomonas putida
phaA ABF82233.1 106636093 Pseudomonas fluorescens phaB ABF82234.1
106636094 Pseudomonas fluorescens
[0464] The E. coli genes fadA and fadB encode a multienzyme complex
that exhibits ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA
dehydrogenase, and enoyl-CoA hydratase activities (Yang et al.
Biochemistry 30(27): p. 6788-6795 (1991); Yang et al. J Biol Chem
265(18): p. 10424-10429 (1990); Yang et al. J Biol Chem 266(24): p.
16255 (1991); Nakahigashi and Inokuchi Nucleic Acids Res 18(16): p.
4937 (1990)). The fadI and fadJ genes encode similar functions and
are naturally expressed only anaerobically (Campbell et al. Mol
Microbiol 47(3): p. 793-805 (2003). A method for producing
poly[(R)-3-hydroxybutyrate] in E. coli that involves activating
fadB (by knocking out a negative regulator, fadR) and co-expressing
a non-native ketothiolase (phaA from Ralstonia eutropha) has been
described previously (Sato et al. J Biosci Bioeng 103(1): 38-44
(2007)). This work clearly demonstrates that a .beta.-oxidation
enzyme, in particular the gene product of fadB which encodes both
3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase activities,
can function as part of a pathway to produce longer chain molecules
from acetyl-CoA precursors.
TABLE-US-00058 Gene Accession No. GI No. 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
4.3.1.a--Ammonia-Lyase
[0465] Aspartase (EC 4.3.1.1), catalyzing the deamination of
aspartate to fumarate, is a widespread enzyme in microorganisms,
and has been characterized extensively (Viola, R. E. Adv. Enzymol.
Relat Areas Mol. Biol. 74:295-341 (2000)). The crystal structure of
the E. coli aspartase, encoded by aspA, has been solved (Shi et al.
Biochemistry 36:9136-9144 (1997)). The E. coli enzyme has also been
shown to react with alternate substrates
aspartatephenylmethylester, asparagine, benzyl-aspartate and malate
(Ma et al. Ann N.Y. Acad Sci 672:60-65 (1992)). In a separate
study, directed evolution was been employed 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. Biochim. Biophys. Acta
1324:182-190 (1997)), Pseudomonas fluorescens (Takagi et al. J.
Biochem. 96:545-552 (1984)), Bacillus subtilus (Sjostrom et al.
Biochim. Biophys. Acta 1324:182-190 (1997)) and Serratia marcescens
(Takagi and Kisumi J Bacteriol. 161:1-6 (1985)).
TABLE-US-00059 Gene Accession No. GI No. Organism aspA NP_418562
90111690 Escherichia coli K12 subsp. MG1655 aspA P44324.1 1168534
Haemophilus influenzae aspA P07346.1 114273 Pseudomonas fluorescens
ansB P26899.1 114271 Bacillus subtilus aspA P33109.1 416661
Serratia marcescens
[0466] 3-methylaspartase (EC 4.3.1.2), also known as
beta-methylaspartase or 3-methylaspartate ammonia-lyase, 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. Acta Crystallogr. D Biol Crystallogr. 57:731-733 (2001);
Asuncion et al. J Biol Chem. 277:8306-8311 (2002); Botting et al.
Biochemistry 27:2953-2955 (1988); Goda et al. Biochemistry
31:10747-10756 (1992). In Citrobacter amalonaticus, this enzyme is
encoded by BAA28709 (Kato and Asano Arch. Microbiol 168:457-463
(1997)). 3-Methylaspartase has also been crystallized from E. coli
YG1002 (Asano and Kato FEMS Microbiol Lett. 118:255-258 (1994))
although the protein sequence is not listed in public databases
such as GenBank. Sequence homology can be used to identify
additional candidate genes, including CTC 02563 in C. tetani and
ECs0761 in Escherichia coli O157:H7.
TABLE-US-00060 Gene Accession No. GI No. Organism MAL AAB24070.1
259429 Clostridium tetanomorphum BAA28709 BAA28709.1 3184397
Citrobacter amalonaticus CTC_02563 NP_783085.1 28212141 Clostridium
tetani ECs0761 BAB34184.1 13360220 Escherichia coli O157:H7 str.
Sakai
[0467] Ammonia-lyase enzyme candidates that form enoyl-CoA products
include beta-alanyl-CoA ammonia-lyase (EC 4.3.1.6), which
deaminates beta-alanyl-CoA, and 3-aminobutyryl-CoA ammonia-lyase
(EC 4.3.1.14). Two beta-alanyl-CoA ammonia lyases have been
identified and characterized in Clostridium propionicum (Herrmann
et al. FEBS J. 272:813-821 (2005)). No other beta-alanyl-CoA
ammonia lyases have been studied to date, but gene candidates can
be identified by sequence similarity. One such candidate is
MXAN.sub.--4385 in Myxococcus xanthus.
TABLE-US-00061 Gene Accession No. GI No. Organism ac12 CAG29275.1
47496504 Clostridium propionicum ac11 CAG29274.1 47496502
Clostridium propionicum MXAN_4385 YP_632558.1 108756898 Myxococcus
xanthus
5.3.3.a--Isomerase
[0468] The 4-hydroxybutyryl-CoA dehydratases from both Clostridium
aminobutyrium and C. kluyveri catalyze the reversible conversion of
4-hydroxybutyryl-CoA to crotonyl-CoA and posses an intrinsic
vinylacetyl-CoA A-isomerase activity (Scherf and Buckel Eur. J
Biochem. 215:421-429 (1993); Scherf et al. Arch. Microbiol
161:239-245 (1994)). Both native enzymes were purified and
characterized, including the N-terminal amino acid sequences
(Scherf and Buckel, supra, 1993; Scherf et al., supra, 1994). The
abfD genes from C. aminobutyrium and C. kluyveri match exactly with
these N-terminal amino acid sequences, thus are encoding the
4-hydroxybutyryl-CoA dehydratases/vinylacetyl-CoA
.DELTA.-isomerase. In addition, the abfD gene from Porphyromonas
gingivalis ATCC 33277 is identified through homology from genome
projects.
TABLE-US-00062 Gene Accession No. GI No. Organism abfD
YP_001396399.1 153955634 Clostridium kluyveri DSM 555 abfD P55792
84028213 Clostridium aminobutyricum abfD YP_001928843 188994591
Porphyromonas gingivalis ATCC 33277
5.4.3.a--Aminomutase
[0469] Lysine 2,3-aminomutase (EC 5.4.3.2) is an exemplary
aminomutase that converts lysine to (3S)-3,6-diaminohexanoate,
shifting an amine group from the 2- to the 3-position. The enzyme
is found in bacteria that ferment lysine to acetate and butyrate,
including as Fusobacterium nuleatum (kamA) (Barker et al. J.
Bacteriol. 152:201-207 (1982)) and Clostridium subterminale (kamA)
(Chirpich et al. J. Biol. Chem. 245:1778-1789 (1970)). The enzyme
from Clostridium subterminale has been crystallized (Lepore et al.
Proc. Natl. Acad. Sci. U.S.A 102:13819-13824 (2005)). An enzyme
encoding this function is also encoded by yodO in Bacillus subtilus
(Chen et al. Biochem. J. 348 Pt 3:539-549 (2000)). The enzyme
utilizes pyridoxal 5'-phosphate as a cofactor, requires activation
by S-Adenosylmethoionine, and is stereoselective, reacting with the
only with L-lysine. The enzyme has not been shown to react with
alternate substrates.
TABLE-US-00063 Gene Accession No. GI No. Organism yodO O34676.1
4033499 Bacillus subtilus kamA Q9XBQ8.1 75423266 Clostridium
subterminale kamA Q8RHX4 81485301 Fusobacterium nuleatum subsp.
nuleatum
[0470] A second aminomutase, beta-lysine 5,6-aminomutase (EC
5.4.3.3), catalyzes the next step of lysine fermentation to acetate
and butyrate, which transforms (3S)-3,6-diaminohexanoate to
(3S,5S)-3,5-diaminohexanoate, shifting a terminal amine group from
the 6- to the 5-position. This enzyme also catalyzes the conversion
of lysine to 2,5-diaminohexanoate and is also called
lysine-5,6-aminomutase (EC 5.4.3.4). The enzyme has been
crystallized in Clostridium sticklandii (kamD, kamE) (Berkovitch et
al. Proc. Natl. Acad. Sci. U.S.A 101:15870-15875 (2004)). The
enzyme from Porphyromonas gingivalis has also been characterized
(Tang et al. Biochemistry 41:8767-8776 (2002)).
TABLE-US-00064 Gene Accession No. GI No. Organism kamD AAC79717.1
3928904 Clostridium sticklandii kamE AAC79718.1 3928905 Clostridium
sticklandii kamD NC_002950.2 34539880, Porphyromonas gingivalis
34540809 W83 kamE NC_002950.2 34539880, Porphyromonas gingivalis
34540810 W83
[0471] Ornithine 4,5-aminomutase (EC 5.4.3.5) converts D-ornithine
to 2,4-diaminopentanoate, also shifting a terminal amine to the
adjacent carbon. The enzyme from Clostridium sticklandii is encoded
by two genes, oraE and oraS, and has been cloned, sequenced and
expressed in E. coli (Chen et al. J. Biol. Chem. 276:44744-44750
(2001)). This enzyme has not been characterized in other organisms
to date.
TABLE-US-00065 Gene Accession No. GI No. Organism oraE AAK72502
17223685 Clostridium sticklandii oraS AAK72501 17223684 Clostridium
sticklandii
[0472] Tyrosine 2,3-aminomutase (EC 5.4.3.6) participates in
tyrosine biosynthesis, reversibly converting tyrosine to
3-amino-3-(4-hydroxyphenyl)propanoate by shifting an amine from the
2- to the 3-position. In Streptomyces globisporus the enzyme has
also been shown to react with tyrosine derivatives (Christenson et
al. Biochemistry 42:12708-12718 (2003)). Sequence information is
not available.
[0473] Leucine 2,3-aminomutase (EC 5.4.3.7) converts L-leucine to
beta-leucine during leucine degradation and biosynthesis. An assay
for leucine 2,3-aminomutase detected activity in many organisms
(Poston, J. M. Methods Enzymol. 166:130-135 (1988)) but genes
encoding the enzyme have not been identified to date.
[0474] Cargill has developed a novel 2,3-aminomutase enzyme to
convert L-alanine to .beta.-alanine, thus creating a pathway from
pyruvate to 3-HP in four biochemical steps (Liao et al., U.S.
Publication No. 2005-0221466).
6.2.1.a--Acid-Thiol Ligase
[0475] An exemplary acid-thiol ligase is the gene products of sucCD
of E. coli which together catalyze the formation of succinyl-CoA
from succinate with the concaminant consumption of one ATP, a
reaction which is reversible in vivo (Buck et al. Biochemistry
24(22): p. 6245-6252 (1985)). Additional exemplary CoA-ligases
include the rat dicarboxylate-CoA ligase for which the sequence is
yet uncharacterized (Vamecq et al. Biochem J. 230(3): p. 683-693
(1985)), either of the two characterized phenylacetate-CoA ligases
from P. chrysogenum (Lamas-Maceiras et al. Biochem J395(1):147-155
(2006); Wang et al. Biochem Biophys Res Commun, 360(2):453-458
(2007)), the phenylacetate-CoA ligase from Pseudomonas putida
(Martinez-Blanco et al. J Biol Chem. 265(12):7084-7090 (1990)), and
the 6-carboxyhexanoate-CoA ligase from Bacillus subtilis (Bower et
al. J Bacteriol 178(14):4122-4130 (1996)).
TABLE-US-00066 Gene Accession No. GI No. Organism sucC NP_415256.1
16128703 Escherichia coli sucD AAC73823.1 1786949 Escherichia coli
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
Example V
Exemplary BDO Pathway from Succinyl-CoA
[0476] This example describes exemplary BDO pathways from
succinyl-CoA.
[0477] BDO pathways from succinyl-CoA are described herein and have
been described previously (see U.S. application Ser. No.
12/049,256, filed Mar. 14, 2008, and PCT application serial No.
US08/57168, filed Mar. 14, 2008, each of which is incorporated
herein by reference). Additional pathways are shown in FIG. 8A.
Enzymes of such exemplary BDO pathways are listed in Table 15,
along with exemplary genes encoding these enzymes.
[0478] Briefly, succinyl-CoA can be converted to succinic
semialdehyde by succinyl-CoA reductase (or succinate semialdehyde
dehydrogenase) (EC 1.2.1.b). Succinate semialdehyde can be
converted to 4-hydroxybutyrate by 4-hydroxybutyrate dehydrogenase
(EC 1.1.1.a), as previously described. Alternatively, succinyl-CoA
can be converted to 4-hydroxybutyrate by succinyl-CoA reductase
(alcohol forming) (EC 1.1.1.c). 4-Hydroxybutyrate can be converted
to 4-hydroxybutyryl-CoA by 4-hydroxybutyryl-CoA transferase (EC
2.8.3.a), as previously described, or by 4-hydroxybutyryl-CoA
hydrolase (EC 3.1.2.a) or 4-hydroxybutyryl-CoA ligase (or
4-hydroxybutyryl-CoA synthetase) (EC 6.2.1.a). Alternatively,
4-hydroxybutyrate can be converted to 4-hydroxybutyryl-phosphate by
4-hydroxybutyrate kinase (EC 2.7.2.a), as previously described.
4-Hydroxybutyryl-phosphate can be converted to 4-hydroxybutyryl-CoA
by phosphotrans-4-hydroxybutyrylase (EC 2.3.1.a), as previously
described. Alternatively, 4-hydroxybutyryl-phosphate can be
converted to 4-hydroxybutanal by 4-hydroxybutanal dehydrogenase
(phosphorylating) (EC 1.2.1.d). 4-Hydroxybutyryl-CoA can be
converted to 4-hydroxybutanal by 4-hydroxybutyryl-CoA reductase (or
4-hydroxybutanal dehydrogenase) (EC 1.2.1.b). Alternatively,
4-hydroxybutyryl-CoA can be converted to 1,4-butanediol by
4-hydroxybutyryl-CoA reductase (alcohol forming) (EC 1.1.1.c).
4-Hydroxybutanal can be converted to 1,4-butanediol by
1,4-butanediol dehydrogenase (EC 1.1.1.a), as previously
described.
TABLE-US-00067 TABLE 15 BDO pathway from succinyl-CoA. EC Desired
Desired Gene GenBank ID Known Figure class substrate product Enzyme
name name (if available) Organism Substrates 8A 1.2.1.b
succinyl-CoA succinic succinyl-CoA sucD P38947.1 Clostridium
succinyl-CoA semialdehyde reductase kluyveri (or succinate
semialdehyde dehydrogenase) sucD NP_904963.1 Porphyromonas
succinyl-CoA gingivalis Msed_0709 YP_001190808.1 Metallosphaera
malonyl-CoA sedula 8A 1.1.1.a succinate 4-hydroxy-
4-hydroxybutyrate 4hbd YP_726053.1 Ralstonia 4-hydroxy-
semialdehyde butyrate dehydrogenase eutropha H16 butyrate 4hbd
L21902.1 Clostridium 4-hydroxy- kluyveri DSM 555 butyrate 4hbd
Q94B07 Arabidopsis 4-hydroxy- thaliana butyrate 8A 1.1.1.c
succinyl-CoA 4-hydroxy- succinyl-CoA adhE2 AAK09379.1 Clostridium
butanoyl-CoA butyrate reductase acetobutylicum (alcohol forming)
mcr AAS20429.1 Chloroflexus malonyl-CoA aurantiacus FAR AAD38039.1
Simmondsia long chain chinensis acyl-CoA 8A 2.8.3.a 4-hydroxy-
4-hydroxy- 4-hydroxy- cat1, cat2, P38946.1, Clostridium succinate,
4- butyrate butyryl-CoA butyryl-CoA cat3 P38942.2, kluyveri
hydroxybutyrate, transferase EDK35586.1 butyrate gctA, gctB
CAA57199.1, Acidaminococcus glutarate CAA57200.1 fermentans atoA,
atoD P76459.1, Escherichia butanoate P76458.1 coli 8A 3.1.2.a
4-hydroxy- 4-hydroxy- 4-hydroxy- tesB NP_414986 Escherichia
adipyl-CoA butyrate butyryl-CoA butyryl-CoA coli hydrolase acot12
NP_570103.1 Rattus butyryl-CoA norvegicus hibch Q6NVY1.2 Homo
sapiens 3-hydroxy- propanoyl-CoA 8A 6.2.1.a 4-hydroxy- 4-hydroxy-
4-hydroxy- sucCD NP_415256.1, Escherichia succinate butyrate
butyryl-CoA butyryl-CoA AAC73823.1 coli ligase (or 4-
hydroxybutyryl- CoA synthetase) phl CAJ15517.1 Penicillium
phenylacetate chrysogenum bioW NP_390902.2 Bacillus 6-carboxy-
subtilis hexanoate 8A 2.7.2.a 4-hydroxy- 4-hydroxy- 4-hydroxy- ackA
NP_416799.1 Escherichia acetate, butyrate butyryl- butyrate coli
propionate phosphate kinase buk1 NP_349675 Clostridium butyrate
acetobutylicum buk2 Q97II1 Clostridium butyrate acetobutylicum 8A
2.3.1.a 4-hydroxy- 4-hydroxy- phosphotrans-4- ptb NP_349676
Clostridium butyryl- butyryl- butyryl-CoA hydroxybutyrylase
acetobutylicum phosphate phosphate ptb AAR19757.1 butyrate-
butyryl- producing phosphate bacterium L2-50 ptb CAC07932.1
Bacillus butyryl- megaterium phosphate 8A 1.2.1.d 4-hydroxy-
4-hydroxy- 4-hydroxybutanal asd NP_417891.1 Escherichia
L-4-aspartyl- butyryl- butanal dehydrogenase coli phosphate
phosphate (phosphorylating) proA NP_414778.1 Escherichia
L-glutamyl- coli 5-phospate gapA P0A9B2.2 Escherichia
Glyceraldehyde- coli 3-phosphate 8A 1.2.1.b 4-hydroxy- 4-hydroxy-
4-hydroxy- sucD P38947.1 Clostridium succinyl-CoA butyryl-CoA
butanal butyryl-CoA kluyveri reductase (or 4- hydroxybutanal
dehydrogenase) sucD NP_904963.1 Porphyromonas succinyl-CoA
gingivalis Msed_0709 YP_001190808.1 Metallosphaera malonyl-CoA
sedula 8A 1.1.1.c 4-hydroxy- 1,4- 4-hydroxy- adhE2 AAK09379.1
Clostridium butanoyl-CoA butyryl-CoA butanediol butyryl-CoA
acetobutylicum reductase (alcohol forming) mcr AAS20429.1
Chloroflexus malonyl-CoA aurantiacus FAR AAD38039.1 Simmondsia long
chain chinensis acyl-CoA 8A 1.1.1.a 4-hydroxy- 1,4- 1,4-butanediol
ADH2 NP_014032.1 Saccharymyces general butanal butanediol
dehydrogenase cerevisiae yqhD NP_417484.1 Escherichia >C3 coli
4hbd L21902.1 Clostridium Succinate kluyveri DSM 555
semialdehyde
Example VI
Additional Exemplary BDO Pathways from Alpha-Ketoglutarate
[0479] This example describes exemplary BDO pathways from
alpha-ketoglutarate.
[0480] BDO pathways from succinyl-CoA are described herein and have
been described previously (see U.S. application Ser. No.
12/049,256, filed Mar. 14, 2008, and PCT application serial No.
US08/57168, filed Mar. 14, 2008, each of which is incorporated
herein by reference). Additional pathways are shown in FIG. 8B.
Enzymes of such exemplary BDO pathways are listed in Table 16,
along with exemplary genes encoding these enzymes.
[0481] Briefly, alpha-ketoglutarate can be converted to succinic
semialdehyde by alpha-ketoglutarate decarboxylase (EC 4.1.1.a), as
previously described. Alternatively, alpha-ketoglutarate can be
converted to glutamate by glutamate dehydrogenase (EC 1.4.1.a).
4-Aminobutyrate can be converted to succinic semialdehyde by
4-aminobutyrate oxidoreductase (deaminating) (EC 1.4.1.a) or
4-aminobutyrate transaminase (EC 2.6.1.a). Glutamate can be
converted to 4-aminobutyrate by glutamate decarboxylase (EC
4.1.1.a). Succinate semialdehyde can be converted to
4-hydroxybutyrate by 4-hydroxybutyrate dehydrogenase (EC 1.1.1.a),
as previously described. 4-Hydroxybutyrate can be converted to
4-hydroxybutyryl-CoA by 4-hydroxybutyryl-CoA transferase (EC
2.8.3.a), as previously described, or by 4-hydroxybutyryl-CoA
hydrolase (EC 3.1.2.a), or 4-hydroxybutyryl-CoA ligase (or
4-hydroxybutyryl-CoA synthetase) (EC 6.2.1.a). 4-Hydroxybutyrate
can be converted to 4-hydroxybutyryl-phosphate by 4-hydroxybutyrate
kinase (EC 2.7.2.a). 4-Hydroxybutyryl-phosphate can be converted to
4-hydroxybutyryl-CoA by phosphotrans-4-hydroxybutyrylase (EC
2.3.1.a), as previously described. Alternatively,
4-hydroxybutyryl-phosphate can be converted to 4-hydroxybutanal by
4-hydroxybutanal dehydrogenase (phosphorylating) (EC 1.2.1.d).
4-Hydroxybutyryl-CoA can be converted to 4-hydroxybutanal by
4-hydroxybutyryl-CoA reductase (or 4-hydroxybutanal dehydrogenase)
(EC 1.2.1.b), as previously described. 4-Hydroxybutyryl-CoA can be
converted to 1,4-butanediol by 4-hydroxybutyryl-CoA reductase
(alcohol forming) (EC 1.1.1.c). 4-Hydroxybutanal can be converted
to 1,4-butanediol by 1,4-butanediol dehydrogenase (EC 1.1.1.a), as
previously described.
TABLE-US-00068 TABLE 16 BDO pathway from alpha-ketoglutarate. EC
Desired Desired Gene GenBank ID Known Figure class substrate
product Enzyme name name (if available) Organism Substrates 8B
4.1.1.a alpha-keto- succinic alpha-keto- kgd O50463.4 Mycobacterium
alpha-keto- glutarate semialdehyde glutarate tuberculosis glutarate
decarboxylase gadA NP_417974 Escherichia glutamate coli gadB
NP_416010 Escherichia glutamate coli 8B 1.4.1.a alpha-keto-
glutamate glutamate gdhA P00370 Escherichia glutamate glutarate
dehydrogenase coli gdh P96110.4 Thermotoga glutamate maritima gdhA1
NP_279651.1 Halobacterium glutamate salinarum 8B 1.4.1.a 4-amino-
succinic 4-aminobutyrate lysDH AB052732 Geobacillus lysine butyrate
semialdehyde oxidoreductase stearothermophilus (deaminating) lysDH
NP_147035.1 Aeropyrum lysine pernix K1 ldh P0A393 Bacillus leucine,
cereus isoleucine, valine, 2- aminobutanoate 8B 2.6.1.a 4-amino-
succinic 4-aminobutyrate gabT P22256.1 Escherichia 4-amino-
butyrate semialdehyde transaminase coli butyryate puuE NP_415818.1
Escherichia 4-amino- coli butyryate UGA1 NP_011533.1 Saccharomyces
4-amino- cerevisiae butyryate 8B 4.1.1.a glutamate 4-amino-
glutamate gadA NP_417974 Escherichia glutamate butyrate
decarboxylase coli gadB NP_416010 Escherichia glutamate coli kgd
O50463.4 Mycobacterium alpha-keto- tuberculosis glutarate 8B
1.1.1.a succinate 4-hydroxy- 4-hydroxybutyrate 4hbd YP_726053.1
Ralstonia 4-hydroxy- semialdehyde butyrate dehydrogenase eutropha
H16 butyrate 4hbd L21902.1 Clostridium 4-hydroxy- kluyveri DSM 555
butyrate 4hbd Q94B07 Arabidopsis 4-hydroxy- thaliana butyrate 8B
2.8.3.a 4-hydroxy- 4-hydroxy- 4-hydroxybutyryl- cat1, cat2,
P38946.1, Clostridium succinate, 4- butyrate butyryl-CoA CoA
transferase cat3 P38942.2, kluyveri hydroxybutyrate, EDK35586.1
butyrate gctA, gctB CAA57199.1, Acidaminococcus glutarate
CAA57200.1 fermentans atoA, atoD P76459.1, Escherichia butanoate
P76458.1 coli 8B 3.1.2.a 4-hydroxy- 4-hydroxy- 4-hydroxybutyryl-
tesB NP_414986 Escherichia adipyl-CoA butyrate butyryl-CoA CoA
hydrolase coli acot12 NP_570103.1 Rattus butyryl-CoA norvegicus
hibch Q6NVY1.2 Homo sapiens 3-hydroxy- propanoyl-CoA 8B 6.2.1.a
4-hydroxy- 4-hydroxy- 4-hydroxybutyryl- sucCD NP_415256.1,
Escherichia succinate butyrate butyryl-CoA CoA ligase (or 4-
AAC73823.1 coli hydroxybutyryl- CoA synthetase) phl CAJ15517.1
Penicillium phenylacetate chrysogenum bioW NP_390902.2 Bacillus
6-carboxy- subtilis hexanoate 8B 2.7.2.a 4-hydroxy- 4-hydroxy-
4-hydroxybutyrate ackA NP_416799.1 Escherichia acetate, butyrate
butyryl- kinase coli propionate phosphate buk1 NP_349675
Clostridium butyrate acetobutylicum buk2 Q97II1 Clostridium
butyrate acetobutylicum 8B 2.3.1.a 4-hydroxy- 4-hydroxy-
phosphotrans-4- ptb NP_349676 Clostridium butyryl- butyryl-
butyryl-CoA hydroxybutyrylase acetobutylicum phosphate phosphate
ptb AAR19757.1 butyrate- butyryl- producing phosphate bacterium
L2-50 ptb CAC07932.1 Bacillus butyryl- megaterium phosphate 8B
1.2.1.d 4-hydroxy- 4-hydroxy- 4-hydroxybutanal asd NP_417891.1
Escherichia L-4-aspartyl- butyryl- butanal dehydrogenase coli
phosphate phosphate (phosphorylating) proA NP_414778.1 Escherichia
L-glutamyl-5- coli phospate gapA P0A9B2.2 Escherichia
Glyceraldehyde- coli 3-phosphate 8B 1.2.1.b 4-hydroxy- 4-hydroxy-
4-hydroxybutyryl- sucD P38947.1 Clostridium succinyl-CoA
butyryl-CoA butanal CoA reductase (or kluyveri 4-hydroxybutanal
dehydrogenase) sucD NP_904963.1 Porphyromonas succinyl-CoA
gingivalis Msed_0709 YP_001190808.1 Metallosphaera malonyl-CoA
sedula 8B 1.1.1.c 4-hydroxy- 1,4- 4-hydroxybutyryl- adhE2
AAK09379.1 Clostridium butanoyl-CoA butyryl-CoA butanediol CoA
reductase acetobutylicum (alcohol forming) mcr AAS20429.1
Chloroflexus malonyl-CoA aurantiacus FAR AAD38039.1 Simmondsia long
chain chinensis acyl-CoA 8B 1.1.1.a 4-hydroxy- 1,4- 1,4-butanediol
ADH2 NP_014032.1 Saccharymyces general butanal butanediol
dehydrogenase cerevisiae yqhD NP_417484.1 Escherichia >C3 coli
4hbd L21902.1 Clostridium Succinate kluyveri DSM 555
semialdehyde
Example VII
BDO Pathways from 4-Aminobutyrate
[0482] This example describes exemplary BDO pathway from
4-aminobutyrate.
[0483] FIG. 9A depicts exemplary BDO pathways in which
4-aminobutyrate is converted to BDO. Enzymes of such an exemplary
BDO pathway are listed in Table 17, along with exemplary genes
encoding these enzymes.
[0484] Briefly, 4-aminobutyrate can be converted to
4-aminobutyryl-CoA by 4-aminobutyrate CoA transferase (EC 2.8.3.a),
4-aminobutyryl-CoA hydrolase (EC 3.1.2.a), or 4-aminobutyrate-CoA
ligase (or 4-aminobutyryl-CoA synthetase) (EC 6.2.1.a).
4-aminobutyryl-CoA can be converted to 4-oxobutyryl-CoA by
4-aminobutyryl-CoA oxidoreductase (deaminating) (EC 1.4.1.a) or
4-aminobutyryl-CoA transaminase (EC 2.6.1.a). 4-oxobutyryl-CoA can
be converted to 4-hydroxybutyryl-CoA by 4-hydroxybutyryl-CoA
dehydrogenase (EC 1.1.1.a). 4-hydroxybutyryl-CoA can be converted
to 1,4-butanediol by 4-hydroxybutyryl-CoA reductase (alcohol
forming) (EC 1.1.1.c). Alternatively, 4-hydroxybutyryl-CoA can be
converted to 4-hydroxybutanal by 4-hydroxybutyryl-CoA reductase (or
4-hydroxybutanal dehydrogenase) (EC 1.2.1.b). 4-hydroxybutanal can
be converted to 1,4-butanediol by 1,4-butanediol dehydrogenase (EC
1.1.1.a).
TABLE-US-00069 TABLE 17 BDO pathway from 4-aminobutyrate. EC
Desired Desired Gene GenBank ID Known Figure class substrate
product Enzyme name name (if available) Organism Substrates 9A
2.8.3.a 4-amino- 4-amino- 4-aminobutyrate cat1, cat2, P38946.1,
Clostridium succinate, 4- butyrate butyryl-CoA CoA transferase cat3
P38942.2, kluyveri hydroxybutyrate, EDK35586.1 butyrate gctA, gctB
CAA57199.1, Acidaminococcus glutarate CAA57200.1 fermentans atoA,
atoD P76459.1, Escherichia butanoate P76458.1 coli 9A 3.1.2.a
4-amino- 4-amino- 4-aminobutyryl- tesB NP_414986 Escherichia
adipyl-CoA butyrate butyryl-CoA CoA hydrolase coli acot12
NP_570103.1 Rattus butyryl-CoA norvegicus hibch Q6NVY1.2 Homo
sapiens 3-hydroxy- propanoyl-CoA 9A 6.2.1.a 4-amino- 4-amino-
4-aminobutyrate- sucCD NP_415256.1, Escherichia succinate butyrate
butyryl-CoA CoA ligase (or 4- AAC73823.1 coli aminobutyryl-CoA
synthetase) phl CAJ15517.1 Penicillium phenylacetate chrysogenum
bioW NP_390902.2 Bacillus 6-carboxy- subtilis hexanoate 9A 1.4.1.a
4-amino- 4-oxo- 4-aminobutyryl- lysDH AB052732 Geobacillus lysine
butyryl-CoA butyryl-CoA CoA oxidoreductase stearothermophilus
(deaminating) lysDH NP_147035.1 Aeropyrum lysine pernix K1 ldh
P0A393 Bacillus leucine, isoleucine, cereus valine, 2-
aminobutanoate 9A 2.6.1.a 4-amino- 4-oxo- 4-aminobutyryl- gabT
P22256.1 Escherichia 4-aminobutyryate butyryl-CoA butyryl-CoA CoA
transaminase coli abat P50554.3 Rattus 3-amino-2- norvegicus
methylpropionate SkyPYD4 ABF58893.1 Saccharomyces beta-alanine
kluyveri 9A 1.1.1.a 4-oxo- 4-hydroxy- 4-hydroxybutyryl- ADH2
NP_014032.1 Saccharymyces general butyryl-CoA butyryl-CoA CoA
dehydrogenase cerevisiae yqhD NP_417484.1 Escherichia >C3 coli
4hbd L21902.1 Clostridium Succinate kluyveri DSM 555 semialdehyde 8
1.1.1.c 4-hydroxy- 1,4- 4-hydroxybutyryl- adhE2 AAK09379.1
Clostridium butanoyl-CoA butyryl-CoA butanediol CoA reductase
acetobutylicum (alcohol forming) mcr AAS20429.1 Chloroflexus
malonyl-CoA aurantiacus FAR AAD38039.1 Simmondsia long chain
chinensis acyl-CoA 8 1.2.1.b 4-hydroxy- 4-hydroxy-
4-hydroxybutyryl- sucD P38947.1 Clostridium Succinyl-CoA
butyryl-CoA butanal CoA reductase (or kluyveri 4 -hydroxybutanal
dehydrogenase) sucD NP_904963.1 Porphyromonas Succinyl-CoA
gingivalis Msed_0709 YP_001190808.1 Metallosphaera Malonyl-CoA
sedula 8 1.1.1.a 4-hydroxy- 1,4- 1,4-butanediol ADH2 NP_014032.1
Saccharymyces general butanal butanediol dehydrogenase cerevisiae
yqhD NP_417484.1 Escherichia >C3 coli 4hbd L21902.1 Clostridium
Succinate kluyveri DSM 555 semialdehyde
[0485] Enzymes for another exemplary BDO pathway converting
4-aminobutyrate to BDO is shown in FIG. 9A. Enzymes of such an
exemplary BDO pathway are listed in Table 18, along with exemplary
genes encoding these enzymes.
[0486] Briefly, 4-aminobutyrate can be converted to
4-aminobutyryl-CoA by 4-aminobutyrate CoA transferase (EC 2.8.3.a),
4-aminobutyryl-CoA hydrolase (EC 3.1.2.a) or 4-aminobutyrate-CoA
ligase (or 4-aminobutyryl-CoA synthetase) (EC 6.2.1.a).
4-aminobutyryl-CoA can be converted to 4-aminobutan-1-ol by
4-aminobutyryl-CoA reductase (alcohol forming) (EC 1.1.1.c).
Alternatively, 4-aminobutyryl-CoA can be converted to
4-aminobutanal by 4-aminobutyryl-CoA reductase (or 4-aminobutanal
dehydrogenase) (EC 1.2.1.b), and 4-aminobutanal converted to
4-aminobutan-1-ol by 4-aminobutan-1-ol dehydrogenase (EC 1.1.1.a).
4-aminobutan-1-ol can be converted to 4-hydroxybutanal by
4-aminobutan-1-ol oxidoreductase (deaminating) (EC 1.4.1.a) or
4-aminobutan-1-ol transaminase (EC 2.6.1.a). 4-hydroxybutanal can
be converted to 1,4-butanediol by 1,4-butanediol dehydrogenase (EC
1.1.1.a).
TABLE-US-00070 TABLE 18 BDO pathway from 4-aminobutyrate. EC
Desired Desired Gene GenBank ID Known Figure class substrate
product Enzyme name name (if available) Organism Substrate 9A
2.8.3.a 4-amino- 4-amino- 4-aminobutyrate cat1, cat2, P38946.1,
Clostridium succinate, 4- butyrate butyryl-CoA CoA transferase cat3
P38942.2, kluyveri hydroxybutyrate, EDK35586.1 butyrate gctA, gctB
CAA57199.1, Acidaminococcus glutarate CAA57200.1 fermentans atoA,
atoD P76459.1, Escherichia butanoate P76458.1 coli 9A 3.1.2.a
4-amino- 4-amino- 4-aminobutyryl- tesB NP_414986 Escherichia
adipyl-CoA butyrate butyryl-CoA CoA hydrolase coli acot12
NP_570103.1 Rattus butyryl-CoA norvegicus hibch Q6NVY1.2 Homo
sapiens 3-hydroxy- propanoyl-CoA 9A 6.2.1.a 4-amino- 4-amino-
4-aminobutyrate- sucCD NP_415256.1, Escherichia succinate butyrate
butyryl-CoA CoA ligase (or 4- AAC73823.1 coli aminobutyryl-CoA
synthetase) phl CAJ15517.1 Penicillium phenylacetate chrysogenum
bioW NP_390902.2 Bacillus 6-carboxy- subtilis hexanoate 9A 1.1.1.c
4-amino- 4-amino- 4-aminobutyryl- adhE2 AAK09379.1 Clostridium
butanoyl-CoA butyryl-CoA butan-1-ol CoA reductase acetobutylicum
(alcohol forming) mcr AAS20429.1 Chloroflexus malonyl-CoA
aurantiacus FAR AAD38039.1 Simmondsia long chain chinensis acyl-CoA
9A 1.2.1.b 4-amino- 4-amino- 4-aminobutyryl- sucD P38947.1
Clostridium Succinyl-CoA butyryl-CoA butanal CoA reductase (or
kluyveri 4-aminobutanal dehydrogenase) sucD NP_904963.1
Porphyromonas Succinyl-CoA gingivalis Msed_0709 YP_001190808.1
Metallosphaera Malonyl-CoA sedula 9A 1.1.1.a 4-amino- 4-amino-
4-aminobutan-1-ol ADH2 NP_014032.1 Saccharymyces general butanal
butan-1-ol dehydrogenase cerevisiae yqhD NP_417484.1 Escherichia
>C3 coli 4hbd L21902.1 Clostridium Succinate kluyveri DSM 555
semialdehyde 9A 1.4.1.a 4-amino- 4-hydroxy- 4-aminobutan-1-ol lysDH
AB052732 Geobacillus lysine butan-1-ol butanal oxidoreductase
stearothermophilus (deaminating) lysDH NP_147035.1 Aeropyrum lysine
pernix K1 ldh P0A393 Bacillus leucine, cereus isoleucine, valine,
2- aminobutanoate 9A 2.6.1.a 4-amino- 4-hydroxy- 4-aminobutan-1-ol
gabT P22256.1 Escherichia 4-amino- butan-1-ol butanal transaminase
coli butyryate abat P50554.3 Rattus 3-amino-2- norvegicus
methylpropionate SkyPYD4 ABF58893.1 Saccharomyces beta-alanine
kluyveri 9A 1.1.1.a 4-hydroxy- 1,4- 1,4-butanediol ADH2 NP_014032.1
Saccharymyces general butanal butanediol dehydrogenase cerevisiae
yqhD NP_417484.1 Escherichia >C3 coli 4hbd L21902.1 Clostridium
Succinate kluyveri DSM 555 semialdehyde
[0487] FIG. 9B depicts exemplary BDO pathway in which
4-aminobutyrate is converted to BDO. Enzymes of such an exemplary
BDO pathway are listed in Table 19, along with exemplary genes
encoding these enzymes.
[0488] Briefly, 4-aminobutyrate can be converted to
[(4-aminobutanolyl)oxy] phosphonic acid by 4-aminobutyrate kinase
(EC 2.7.2.a). [(4-aminobutanolyl)oxy] phosphonic acid can be
converted to 4-aminobutanal by 4-aminobutyraldehyde dehydrogenase
(phosphorylating) (EC 1.2.1.d). 4-aminobutanal can be converted to
4-aminobutan-1-ol by 4-aminobutan-1-ol dehydrogenase (EC 1.1.1.a).
4-aminobutan-1-ol can be converted to 4-hydroxybutanal by
4-aminobutan-1-ol oxidoreductase (deaminating) (EC 1.4.1.a) or
4-aminobutan-1-ol transaminase (EC 2.6.1.a). Alternatively,
[(4-aminobutanolyl)oxy] phosphonic acid can be converted to
[(4-oxobutanolyl)oxy] phosphonic acid by
[(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase (deaminating)
(EC 1.4.1.a) or [(4-aminobutanolyl)oxy]phosphonic acid transaminase
(EC 2.6.1.a). [(4-oxobutanolyl)oxy] phosphonic acid can be
converted to 4-hydroxybutyryl-phosphate by
4-hydroxybutyryl-phosphate dehydrogenase (EC 1.1.1.a).
4-hydroxybutyryl-phosphate can be converted to 4-hydroxybutanal by
4-hydroxybutyraldehyde dehydrogenase (phosphorylating) (EC
1.2.1.d). 4-hydroxybutanal can be converted to 1,4-butanediol by
1,4-butanediol dehydrogenase (EC 1.1.1.a).
TABLE-US-00071 TABLE 19 BDO pathway from 4-aminobutyrate. EC
Desired Desired Gene GenBank ID Known Figure class substrate
product Enzyme name name (if available) Organism Substrate 9B
2.7.2.a 4-amino- [(4-amino- 4-amino- ackA NP_416799.1 Escherichia
acetate, butyrate butanolyl)oxy] butyrate kinase coli propionate
phosphonic acid buk1 NP_349675 Clostridium butyrate acetobutylicum
proB NP_414777.1 Escherichia glutamate coli 9B 1.2.1.d [(4-amino-
4-amino- 4-amino- asd NP_417891.1 Escherichia L-4-aspartyl-
butanolyl)oxy] butanal butyraldehyde coli phosphate phosphonic
dehydrogenase acid (phosphorylating) proA NP_414778.1 Escherichia
L-glutamyl- coli 5-phospate gapA P0A9B2.2 Escherichia
Glyceraldehyde- coli 3-phosphate 9B 1.1.1.a 4-amino- 4-amino-
4-aminobutan-1-ol ADH2 NP_014032.1 Saccharymyces general butanal
butan-1-ol dehydrogenase cerevisiae yqhD NP_417484.1 Escherichia
>C3 coli 4hbd L21902.1 Clostridium Succinate kluyveri DSM 555
semialdehyde 9B 1.4.1.a 4-amino- 4-hydroxy- 4-aminobutan-1-ol lysDH
AB052732 Geobacillus lysine butan-1-ol butanal oxidoreductase
stearothermophilus (deaminating) lysDH NP_147035.1 Aeropyrum lysine
pernix K1 ldh P0A393 Bacillus leucine, cereus isoleucine, valine,
2-aminobutanoate 9B 2.6.1.a 4-amino- 4-hydroxy- 4-aminobutan-1-ol
gabT P22256.1 Escherichia 4-aminobutyryate butan-1-ol butanal
transaminase coli abat P50554.3 Rattus 3-amino-2- norvegicus
methylpropionate SkyPYD4 ABF58893.1 Saccharomyces beta-alanine
kluyveri 9B 1.4.1.a [(4-amino- [(4-oxo- [(4-amino- lysDH AB052732
Geobacillus lysine butanolyl)oxy] butanolyl)oxy]
butanolyl)oxy]phos- stearothermophilus phosphonic phosphonic phonic
acid acid acid oxidoreductase (deaminating) lysDH NP_147035.1
Aeropyrum lysine pernix K1 ldh P0A393 Bacillus leucine, cereus
isoleucine, valine, 2-aminobutanoate 9B 2.6.1.a [(4-amino- [(4-oxo-
[(4-amino- gabT P22256.1 Escherichia 4-aminobutyryate
butanolyl)oxy] butanolyl)oxy] butanolyl)oxy]phos- coli phosphonic
phosphonic phonic acid acid acid transaminase SkyPYD4 ABF58893.1
Saccharomyces beta-alanine kluyveri serC NP_415427.1 Escherichia
phosphoserine, coli phospho- hydroxythreonine 9B 1.1.1.a [(4-oxo-
4-hydroxy- 4-hydroxy- ADH2 NP_014032.1 Saccharymyces general
butanolyl)oxy] butyryl- butyryl- cerevisiae phosphonic phosphate
phosphate acid dehydrogenase yqhD NP_417484.1 Escherichia >C3
coli 4hbd L21902.1 Clostridium Succinate kluyveri DSM 555
semialdehyde 9B 1.2.1.d 4-hydroxy- 4-hydroxy- 4-hydroxy- asd
NP_417891.1 Escherichia L-4-aspartyl- butyryl- butanal
butyraldehyde coli phosphate phosphate dehydrogenase
(phosphorylating) proA NP_414778.1 Escherichia L-glutamyl- coli
5-phospate gapA P0A9B2.2 Escherichia Glyceraldehyde- coli
3-phosphate 9B 1.1.1.a 4-hydroxy- 1,4- 1,4-butanediol ADH2
NP_014032.1 Saccharymyces general butanal butanediol dehydrogenase
cerevisiae yqhD NP_417484.1 Escherichia >C3 coli 4hbd L21902.1
Clostridium Succinate kluyveri DSM 555 semialdehyde
[0489] FIG. 9C shows an exemplary pathway through acetoacetate.
Example VIII
Exemplary BDO Pathways from Alpha-Ketoglutarate
[0490] This example describes exemplary BDO pathways from
alpha-ketoglutarate.
[0491] FIG. 10 depicts exemplary BDO pathways in which
alpha-ketoglutarate is converted to BDO. Enzymes of such an
exemplary BDO pathway are listed in Table 20, along with exemplary
genes encoding these enzymes.
[0492] Briefly, alpha-ketoglutarate can be converted to
alpha-ketoglutaryl-phosphate by alpha-ketoglutarate 5-kinase (EC
2.7.2.a). Alpha-ketoglutaryl-phosphate can be converted to
2,5-dioxopentanoic acid by 2,5-dioxopentanoic semialdehyde
dehydrogenase (phosphorylating) (EC 1.2.1.d). 2,5-dioxopentanoic
acid can be converted to 5-hydroxy-2-oxopentanoic acid by
2,5-dioxopentanoic acid reductase (EC 1.1.1.a). Alternatively,
alpha-ketoglutarate can be converted to alpha-ketoglutaryl-CoA by
alpha-ketoglutarate CoA transferase (EC 2.8.3.a),
alpha-ketoglutaryl-CoA hydrolase (EC 3.1.2.a) or
alpha-ketoglutaryl-CoA ligase (or alpha-ketoglutaryl-CoA
synthetase) (EC 6.2.1.a). Alpha-ketoglutaryl-CoA can be converted
to 2,5-dioxopentanoic acid by alpha-ketoglutaryl-CoA reductase (or
2,5-dioxopentanoic acid dehydrogenase) (EC 1.2.1.b).
2,5-Dioxopentanoic acid can be converted to
5-hydroxy-2-oxopentanoic acid by 5-hydroxy-2-oxopentanoic acid
dehydrogenase. Alternatively, alpha-ketoglutaryl-CoA can be
converted to 5-hydroxy-2-oxopentanoic acid by
alpha-ketoglutaryl-CoA reductase (alcohol forming) (EC 1.1.1.c).
5-hydroxy-2-oxopentanoic acid can be converted to 4-hydroxybutanal
by 5-hydroxy-2-oxopentanoic acid decarboxylase (EC 4.1.1.a).
4-hydroxybutanal can be converted to 1,4-butanediol by
1,4-butanediol dehydrogenase (EC 1.1.1.a). 5-hydroxy-2-oxopentanoic
acid can be converted to 4-hydroxybutyryl-CoA by
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation) (EC
1.2.1.c).
TABLE-US-00072 TABLE 20 BDO pathway from alpha-ketoglutarate. EC
Desired Desired Gene GenBank ID Known Figure class substrate
product Enzyme name name (if available) Organism Substrate 10
2.7.2.a alpha-keto- alpha-keto- alpha-keto- ackA NP_416799.1
Escherichia acetate, glutarate glutaryl- glutarate coli propionate
phosphate 5-kinase buk1 NP_349675 Clostridium butyrate
acetobutylicum proB NP_414777.1 Escherichia glutamate coli 10
1.2.1.d alpha-keto- 2,5-dioxo- 2,5- proA NP_414778.1 Escherichia
L-glutamyl- glutaryl- pentanoic acid dioxopentanoic coli 5-phospate
phosphate semialdehyde dehydrogenase (phosphorylating) asd
NP_417891.1 Escherichia L-4-aspartyl- coli phosphate gapA P0A9B2.2
Escherichia Glyceraldehyde- coli 3-phosphate 10 1.1.1.a 2,5-dioxo-
5-hydroxy-2- 2,5- ADH2 NP_014032.1 Saccharymyces general pentanoic
acid oxopentanoic dioxopentanoic cerevisiae acid acid reductase
yqhD NP_417484.1 Escherichia >C3 coli 4hbd L21902.1 Clostridium
Succinate kluyveri DSM 555 semialdehyde 10 2.8.3.a alpha-keto-
alpha-keto- alpha-keto- cat1, cat2, P38946.1, Clostridium
succinate, 4- glutarate glutaryl-CoA glutarate CoA cat3 P38942.2,
kluyveri hydroxybutyrate, transferase EDK35586.1 butyrate gctA,
gctB CAA57199.1, Acidaminococcus glutarate CAA57200.1 fermentans
atoA, atoD P76459.1, Escherichia butanoate P76458.1 coli 10 3.1.2.a
alpha-keto- alpha-keto- alpha-keto- tesB NP_414986 Escherichia
adipyl-CoA glutarate glutaryl-CoA glutaryl-CoA coli hydrolase
acot12 NP_570103.1 Rattus butyryl-CoA norvegicus hibch Q6NVY1.2
Homo sapiens 3-hydroxy- propanoyl-CoA 10 6.2.1.a alpha-keto-
alpha-keto- alpha- sucCD NP_415256.1, Escherichia succinate
glutarate glutaryl-CoA ketoglutaryl- AAC73823.1 coli CoA ligase (or
alpha- ketoglutaryl- CoA synthetase) phl CAJ15517.1 Penicillium
phenylacetate chrysogenum bioW NP_390902.2 Bacillus 6-carboxy-
subtilis hexanoate 10 1.2.1.b alpha-keto- 2,5-dioxo- alpha- sucD
P38947.1 Clostridium Succinyl-CoA glutaryl-CoA pentanoic acid
ketoglutaryl- kluyveri CoA reductase (or 2,5- dioxopentanoic acid
dehydrogenase) Msed_0709 YP_001190808.1 Metallosphaera Malonyl-CoA
sedula bphG BAA03892.1 Pseudomonas sp Acetaldehyde,
Propionaldehyde, Butyraldehyde, Isobutyraldehyde and Formaldehyde
10 1.1.1.a 2,5-dioxo- 5-hydroxy-2- 5-hydroxy-2- ADH2 NP_014032.1
Saccharymyces general pentanoic acid oxopentanoic oxopentanoic yqhD
NP_417484.1 cerevisiae >C3 acid acid 4hbd L21902.1 Escherichia
Succinate dehydrogenase coli semialdehyde Clostridium kluyveri DSM
555 10 1.1.1.c alpha-keto- 5-hydroxy-2- alpha- adhE2 AAK09379.1
Clostridium butanoyl-CoA glutaryl-CoA oxopentanoic ketoglutaryl-
acetobutylicum acid CoA reductase (alcohol forming) mcr AAS20429.1
Chloroflexus malonyl-CoA aurantiacus FAR AAD38039.1 Simmondsia long
chain chinensis acyl-CoA 10 4.1.1.a 5-hydroxy-2- 4-hydroxy-
5-hydroxy-2- pdc P06672.1 Zymomonas 2-oxopentanoic oxopentanoic
butanal oxopentanoic mobilus acid acid acid decarboxylase mdlC
P20906.2 Pseudomonas 2-oxopentanoic putida acid pdc1 P06169
Saccharomyces pyruvate cerevisiae 10 1.1.1.a 4-hydroxy- 1,4-
1,4-butanediol ADH2 NP_014032.1 Saccharymyces general butanal
butanediol dehydrogenase cerevisiae yqhD NP_417484.1 Escherichia
>C3 coli 4hbd L21902.1 Clostridium Succinate kluyveri DSM 555
semialdehyde 10 1.2.1.c 5-hydroxy-2- 4-hydroxy- 5-hydroxy-2- sucA,
sucB, NP_415254.1, Escherichia Alpha- oxopentanoic butyryl-CoA
oxopentanoic lpd NP_415255.1, coli ketoglutarate acid acid
NP_414658.1 dehydrogenase (decarboxylation) bfmBB, NP_390283.1,
Bacillus 2-keto acids bfmBAA, NP_390285.1, subtilis derivatives of
bfmBAB, NP_390284.1, valine, leucine bfmBAB, P21880.1 and
isoleucine pdhD Bckdha, NP_036914.1, Rattus 2-keto acids Bckdhb,
NP_062140.1, norvegicus derivatives of Dbt, Dld NP_445764.1,
valine, leucine NP_955417.1 and isoleucine
Example IX
Exemplary BDO Pathways from Glutamate
[0493] This example describes exemplary BDO pathways from
glutamate.
[0494] FIG. 11 depicts exemplary BDO pathways in which glutamate is
converted to BDO. Enzymes of such an exemplary BDO pathway are
listed in Table 21, along with exemplary genes encoding these
enzymes.
[0495] Briefly, glutamate can be converted to glutamyl-CoA by
glutamate CoA transferase (EC 2.8.3.a), glutamyl-CoA hydrolase (EC
3.1.2.a) or glutamyl-CoA ligase (or glutamyl-CoA synthetase) (EC
6.2.1.a). Alternatively, glutamate can be converted to
glutamate-5-phosphate by glutamate 5-kinase (EC 2.7.2.a).
Glutamate-5-phosphate can be converted to glutamate-5-semialdehyde
by glutamate-5-semialdehyde dehydrogenase (phosphorylating) (EC
1.2.1.d). Glutamyl-CoA can be converted to glutamate-5-semialdehyde
by glutamyl-CoA reductase (or glutamate-5-semialdehyde
dehydrogenase) (EC 1.2.1.b). Glutamate-5-semialdehyde can be
converted to 2-amino-5-hydroxypentanoic acid by
glutamate-5-semialdehyde reductase (EC 1.1.1.a). Alternatively,
glutamyl-CoA can be converted to 2-amino-5-hydroxypentanoic acid by
glutamyl-CoA reductase (alcohol forming) (EC 1.1.1.c).
2-Amino-5-hydroxypentanoic acid can be converted to
5-hydroxy-2-oxopentanoic acid by 2-amino-5-hydroxypentanoic acid
oxidoreductase (deaminating) (EC 1.4.1.a) or
2-amino-5-hydroxypentanoic acid transaminase (EC 2.6.1.a).
5-Hydroxy-2-oxopentanoic acid can be converted to 4-hydroxybutanal
by 5-hydroxy-2-oxopentanoic acid decarboxylase (EC 4.1.1.a).
4-Hydroxybutanal can be converted to 1,4-butanediol by
1,4-butanediol dehydrogenase (EC 1.1.1.a). Alternatively,
5-hydroxy-2-oxopentanoic acid can be converted to
4-hydroxybutyryl-CoA by 5-hydroxy-2-oxopentanoic acid dehydrogenase
(decarboxylation) (EC 1.2.1.c).
TABLE-US-00073 TABLE 21 BDO pathway from glutamate. EC Desired
Desired Gene GenBank ID Known Figure class substrate product Enzyme
name name (if available) Organism Substrate 11 2.8.3.a glutamate
glutamyl- glutamate CoA cat1, cat2, P38946.1, Clostridium
succinate, 4- CoA transferase cat3 P38942.2, kluyveri
hydroxybutyrate, EDK35586.1 butyrate gctA, gctB CAA57199.1,
Acidaminococcus glutarate CAA57200.1 fermentans atoA, atoD
P76459.1, Escherichia butanoate P76458.1 coli 11 3.1.2.a glutamate
glutamyl- glutamyl-CoA tesB NP_414986 Escherichia adipyl-CoA CoA
hydrolase coli acot12 NP_570103.1 Rattus butyryl-CoA norvegicus
hibch Q6NVY1.2 Homo sapiens 3-hydroxy- propanoyl-CoA 11 6.2.1.a
glutamate glutamyl- glutamyl-CoA sucCD NP_415256.1, Escherichia
succinate CoA ligase (or AAC73823.1 coli glutamyl- CoA synthetase)
phl CAJ15517.1 Penicillium phenylacetate chrysogenum bioW
NP_390902.2 Bacillus 6-carboxy- subtilis hexanoate 11 2.7.2.a
glutamate glutamate-5- glutamate ackA NP_416799.1 Escherichia
acetate, phosphate 5-kinase coli propionate buk1 NP_349675
Clostridium butyrate acetobutylicum proB NP_414777.1 Escherichia
glutamate coli 11 1.2.1.d glutamate-5- glutamate- glutamate-5- proA
NP_414778.1 Escherichia L-glutamyl-5- phosphate 5-semi-
semialdehyde coli phospate aldehyde dehydrogenase (phosphorylating)
asd NP_417891.1 Escherichia L-4-aspartyl- coli phosphate gapA
P0A9B2.2 Escherichia Glyceraldehyde- coli 3-phosphate 11 1.2.1.b
glutamyl- glutamate- glutamyl-CoA sucD P38947.1 Clostridium
Succinyl-CoA CoA 5-semi- reductase (or kluyveri aldehyde
glutamate-5- semialdehyde dehydrogenase) Msed_0709 YP_001190808.1
Metallosphaera Malonyl-CoA sedula bphG BAA03892.1 Pseudomonas sp
Acetaldehyde, Propionaldehyde, Butyraldehyde, Isobutyraldehyde and
Formaldehyde 11 1.1.1.a glutamate- 2-amino-5- glutamate-5- ADH2
NP_014032.1 Saccharymyces general 5-semi- hydroxy- semialdehyde
cerevisiae aldehyde pentanoic reductase acid yqhD NP_417484.1
Escherichia >C3 coli 4hbd L21902.1 Clostridium Succinate
kluyveri DSM 555 semialdehyde 11 1.1.1.c glutamyl- 2-amino-5-
glutamyl-CoA adhE2 AAK09379.1 Clostridium butanoyl-CoA CoA hydroxy-
reductase acetobutylicum pentanoic (alcohol forming) acid mcr
AAS20429.1 Chloroflexus malonyl-CoA aurantiacus FAR AAD38039.1
Simmondsia long chain chinensis acyl-CoA 11 1.4.1.a 2-amino-5-
5-hydroxy-2- 2-amino-5- gdhA P00370 Escherichia glutamate hydroxy-
oxopentanoic hydroxypentanoic coli pentanoic acid acid oxido- acid
reductase (deaminating) ldh P0A393 Bacillus leucine, cereus
isoleucine, valine, 2- aminobutanoate nadX NP_229443.1 Thermotoga
aspartate maritima 11 2.6.1.a 2-amino-5- 5-hydroxy-2- 2-amino-5-
aspC NP_415448.1 Escherichia aspartate hydroxy- oxopentanoic
hydroxy- coli pentanoic acid pentanoic acid acid transaminase AAT2
P23542.3 Saccharomyces aspartate cerevisiae avtA YP_026231.1
Escherichia valine, alpha- coli aminobutyrate 11 4.1.1.a
5-hydroxy-2- 4-hydroxy- 5-hydroxy-2- pdc P06672.1 Zymomonas
2-oxopentanoic oxopentanoic butanal oxopentanoic acid mobilus acid
acid decarboxylase mdlC P20906.2 Pseudomonas 2-oxopentanoic putida
acid pdc1 P06169 Saccharomyces pyruvate cerevisiae 11 1.1.1.a
4-hydroxy- 1,4- 1,4-butanediol ADH2 NP_014032.1 Saccharymyces
general butanal butanediol dehydrogenase cerevisiae yqhD
NP_417484.1 Escherichia >C3 coli 4hbd L21902.1 Clostridium
Succinate kluyveri DSM 555 semialdehyde 11 1.2.1.c 5-hydroxy-2-
4-hydroxy- 5-hydroxy-2- sucA, sucB, NP_415254.1, Escherichia
Alpha-keto- oxopentanoic butyryl-CoA oxopentanoic acid lpd
NP_415255.1, coli glutarate acid dehydrogenase NP_414658.1
(decarboxylation) bfmBB, NP_390283.1, Bacillus 2-keto acids bfmBAA,
NP_390285.1, subtilis derivatives of bfmBAB, NP_390284.1, valine,
leucine bfmBAB, pdhD P21880.1 and isoleucine Bckdha, NP_036914.1,
Rattus 2-keto acids Bckdhb, NP_062140.1, norvegicus derivatives of
Dbt, Dld NP_445764.1, valine, leucine NP_955417.1 and
isoleucine
Example X
Exemplary BDO from Acetoacetyl-CoA
[0496] This example describes an exemplary BDO pathway from
acetoacetyl-CoA.
[0497] FIG. 12 depicts exemplary BDO pathways in which
acetoacetyl-CoA is converted to BDO. Enzymes of such an exemplary
BDO pathway are listed in Table 22, along with exemplary genes
encoding these enzymes.
[0498] Briefly, acetoacetyl-CoA can be converted to
3-hydroxybutyryl-CoA by 3-hydroxybutyryl-CoA dehydrogenase (EC
1.1.1.a). 3-Hydroxybutyryl-CoA can be converted to crotonoyl-CoA by
3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.a). Crotonoyl-CoA can be
converted to vinylacetyl-CoA by vinylacetyl-CoA A-isomerase (EC
5.3.3.3). Vinylacetyl-CoA can be converted to 4-hydroxybutyryl-CoA
by 4-hydroxybutyryl-CoA dehydratase (EC 4.2.1.a).
4-Hydroxybutyryl-CoA can be converted to 1,4-butanediol by
4-hydroxybutyryl-CoA reductase (alcohol forming) (EC 1.1.1.c).
Alternatively, 4-hydroxybutyryl-CoA can be converted to
4-hydroxybutanal by 4-hydroxybutyryl-CoA reductase (or
4-hydroxybutanal dehydrogenase) (EC 1.2.1.b). 4-Hydroxybutanal can
be converted to 1,4-butanediol by 1,4-butanediol dehydrogenase (EC
1.1.1.a).
TABLE-US-00074 TABLE 22 BDO pathway from acetoacetyl-CoA. EC
Desired Desired Gene GenBank ID Known Figure class substrate
product Enzyme name name (if available) Organism Substrate 12
1.1.1.a acetoacetyl- 3-hydroxy- 3-hydroxy- hbd NP_349314.1
Clostridium 3-hydroxy- CoA butyryl-CoA butyryl-CoA acetobutylicum
butyryl-CoA dehydrogenase hbd AAM14586.1 Clostridium 3-hydroxy-
beijerinckii butyryl-CoA Msed_1423 YP_001191505 Metallosphaera
presumed 3- sedula hydroxy- butyryl-CoA 12 4.2.1.a 3-hydroxy-
crotonoyl- 3-hydroxy- crt NP_349318.1 Clostridium 3-hydroxy-
butyryl-CoA CoA butyryl-CoA acetobutylicum butyryl-CoA dehydratase
maoC NP_415905.1 Escherichia 3-hydroxy- coli butyryl-CoA paaF
NP_415911.1 Escherichia 3-hydroxy- coli adipyl-CoA 12 5.3.3.3
crotonoyl- vinylacetyl- vinylacetyl- abfD YP_001396399.1
Clostridium 4-hydroxy- CoA CoA CoA .DELTA.- kluyveri DSM 555
butyryl-CoA isomerase abfD P55792 Clostridium 4-hydroxy-
aminobutyricum butyryl-CoA abfD YP_001928843 Porphyromonas
4-hydroxy- gingivalis butyryl-CoA ATCC 33277 12 4.2.1.a
vinylacetyl- 4-hydroxy- 4-hydroxy- abfD YP_001396399.1 Clostridium
4-hydroxy- CoA butyryl-CoA butyryl-CoA kluyveri DSM 555 butyryl-CoA
dehydratase abfD P55792 Clostridium 4-hydroxy- aminobutyricum
butyryl-CoA abfD YP_001928843 Porphyromonas 4-hydroxy- gingivalis
butyryl-CoA ATCC 33277 12 1.1.1.c 4-hydroxy- 1,4- 4-hydroxy- adhE2
AAK09379.1 Clostridium butanoyl-CoA butyryl-CoA butanediol
butyryl-CoA acetobutylicum reductase (alcohol forming) mcr
AAS20429.1 Chloroflexus malonyl-CoA aurantiacus FAR AAD38039.1
Simmondsia long chain chinensis acyl-CoA 12 1.2.1.b 4-hydroxy-
4-hydroxy- 4-hydroxy- sucD P38947.1 Clostridium Succinyl-CoA
butyryl-CoA butanal butyryl-CoA kluyveri reductase (or 4-hydroxy-
butanal dehydrogenase) sucD NP_904963.1 Porphyromonas Succinyl-CoA
gingivalis Msed_0709 YP_001190808.1 Metallosphaera Malonyl-CoA
sedula 12 1.1.1.a 4-hydroxy- 1,4- 1,4- ADH2 NP_014032.1
Saccharymyces general butanal butanediol butanediol cerevisiae
dehydrogenase yqhD NP_417484.1 Escherichia >C3 coli 4hbd
L21902.1 Clostridium Succinate kluyveri DSM 555 semialdehyde
Example XI
Exemplary BDO Pathway from Homoserine
[0499] This example describes an exemplary BDO pathway from
homoserine.
[0500] FIG. 13 depicts exemplary BDO pathways in which homoserine
is converted to BDO. Enzymes of such an exemplary BDO pathway are
listed in Table 23, along with exemplary genes encoding these
enzymes.
[0501] Briefly, homoserine can be converted to
4-hydroxybut-2-enoate by homoserine deaminase (EC 4.3.1.a).
Alternatively, homoserine can be converted to homoserine-CoA by
homoserine CoA transferase (EC 2.8.3.a), homoserine-CoA hydrolase
(EC 3.1.2.a) or homoserine-CoA ligase (or homoserine-CoA
synthetase) (EC 6.2.1.a). Homoserine-CoA can be converted to
4-hydroxybut-2-enoyl-CoA by homoserine-CoA deaminase (EC 4.3.1.a).
4-Hydroxybut-2-enoate can be converted to 4-hydroxybut-2-enoyl-CoA
by 4-hydroxybut-2-enoyl-CoA transferase (EC 2.8.3.a),
4-hydroxybut-2-enoyl-CoA hydrolase (EC 3.1.2.a), or
4-hydroxybut-2-enoyl-CoA ligase (or 4-hydroxybut-2-enoyl-CoA
synthetase) (EC 6.2.1.a). Alternatively, 4-hydroxybut-2-enoate can
be converted to 4-hydroxybutyrate by 4-hydroxybut-2-enoate
reductase (EC 1.3.1.a). 4-Hydroxybutyrate can be converted to
4-hydroxybutyryl-coA by 4-hydroxybutyryl-CoA transferase (EC
2.8.3.a), 4-hydroxybutyryl-CoA hydrolase (EC 3.1.2.a), or
4-hydroxybutyryl-CoA ligase (or 4-hydroxybutyryl-CoA synthetase)
(EC 6.2.1.a). 4-Hydroxybut-2-enoyl-CoA can be converted to
4-hydroxybutyryl-CoA by 4-hydroxybut-2-enoyl-CoA reductase (EC
1.3.1.a). 4-Hydroxybutyryl-CoA can be converted to 1,4-butanediol
by 4-hydroxybutyryl-CoA reductase (alcohol forming) (EC 1.1.1.c).
Alternatively, 4-hydroxybutyryl-CoA can be converted to
4-hydroxybutanal by 4-hydroxybutyryl-CoA reductase (or
4-hydroxybutanal dehydrogenase) (EC 1.2.1.b). 4-Hydroxybutanal can
be converted to 1,4-butanediol by 1,4-butanediol dehydrogenase (EC
1.1.1.a).
TABLE-US-00075 TABLE 23 BDO pathway from homoserine. EC Desired
Desired Gene GenBank ID Known Figure class substrate product Enzyme
name name (if available) Organism Substrate 13 4.3.1.a homoserine
4-hydroxybut- homoserine aspA NP_418562 Escherichia aspartate
2-enoate deaminase coli aspA P44324.1 Haemophilus aspartate
influenzae aspA P07346 Pseudomonas aspartate fluorescens 13 2.8.3.a
homoserine homoserine- homoserine CoA cat1, cat2, P38946.1,
Clostridium succinate, 4- CoA transferase cat3 P38942.2, kluyveri
hydroxybutyrate, EDK35586.1 butyrate gctA, gctB CAA57199.1,
Acidaminococcus glutarate CAA57200.1 fermentans atoA, atoD
P76459.1, Escherichia butanoate P76458.1 coli 13 3.1.2.a homoserine
homoserine- homoserine-CoA tesB NP_414986 Escherichia adipyl-CoA
CoA hydrolase coli acot12 NP_570103.1 Rattus butyryl-CoA norvegicus
hibch Q6NVY1.2 Homo sapiens 3-hydroxy- propanoyl-CoA 13 6.2.1.a
homoserine homoserine- homoserine-CoA sucCD NP_415256.1,
Escherichia succinate CoA ligase (or AAC73823.1 coli homoserine-CoA
synthetase) phl CAJ15517.1 Penicillium phenylacetate chrysogenum
bioW NP_390902.2 Bacillus 6-carboxy- subtilis hexanoate 13 4.3.1.a
homoserine- 4-hydroxybut- homoserine-CoA acl1 CAG29274.1
Clostridium beta-alanyl- CoA 2-enoyl-CoA deaminase propionicum CoA
acl2 CAG29275.1 Clostridium beta-alanyl- propionicum CoA MXAN_4385
YP_632558.1 Myxococcus beta-alanyl- xanthus CoA 13 2.8.3.a
4-hydroxybut- 4-hydroxybut- 4-hydroxybut-2- cat1, cat2, P38946.1,
Clostridium succinate, 4- 2-enoate 2-enoyl-CoA enoyl-CoA cat3
P38942.2, kluyveri hydroxybutyrate, transferase EDK35586.1 butyrate
gctA, gctB CAA57199.1, Acidaminococcus glutarate CAA57200.1
fermentans atoA, atoD P76459.1, Escherichia butanoate P76458.1 coli
13 3.1.2.a 4-hydroxybut- 4-hydroxybut- 4-hydroxybut-2- tesB
NP_414986 Escherichia adipyl-CoA 2-enoate 2-enoyl-CoA enoyl-CoA
coli hydrolase acot12 NP_570103.1 Rattus butyryl-CoA norvegicus
hibch Q6NVY1.2 Homo sapiens 3-hydroxy- propanoyl-CoA 13 6.2.1.a
4-hydroxybut- 4-hydroxybut- 4-hydroxybut-2- sucCD NP_415256.1,
Escherichia succinate 2-enoate 2-enoyl-CoA enoyl-CoA AAC73823.1
coli ligase (or 4- hydroxybut-2- enoyl-CoA synthetase) phl
CAJ15517.1 Penicillium phenylacetate chrysogenum bioW NP_390902.2
Bacillus 6-carboxy- subtilis hexanoate 13 1.3.1.a 4-hydroxybut-
4-hydroxy- 4-hydroxybut-2- enr CAA71086.1 Clostridium 2-enoate
butyrate enoate reductase tyrobutyricum enr CAA76083.1 Clostridium
kluyveri enr YP_430895.1 Moorella thermoacetica 13 2.8.3.a
4-hydroxy- 4-hydroxy- 4-hydroxybutyryl- cat1, cat2, P38946.1,
Clostridium succinate, 4- butyrate butyryl-coA CoA transferase cat3
P38942.2, kluyveri hydroxybutyrate, EDK35586.1 butyrate gctA, gctB
CAA57199.1, Acidaminococcus glutarate CAA57200.1 fermentans atoA,
atoD P76459.1, Escherichia butanoate P76458.1 coli 13 3.1.2.a
4-hydroxy- 4-hydroxy- 4-hydroxybutyryl- tesB NP_414986 Escherichia
adipyl-CoA butyrate butyryl-coA CoA hydrolase coli acot12
NP_570103.1 Rattus butyryl-CoA norvegicus hibch Q6NVY1.2 Homo
sapiens 3-hydroxy- propanoyl-CoA 13 6.2.1.a 4-hydroxy- 4-hydroxy-
4-hydroxy- sucCD NP_415256.1, Escherichia succinate butyrate
butyryl-coA butyryl-CoA AAC73823.1 coli ligase (or 4-
hydroxybutyryl- CoA synthetase) phl CAJ15517.1 Penicillium
phenylacetate chrysogenum bioW NP_390902.2 Bacillus 6-carboxy-
subtilis hexanoate 13 1.3.1.a 4-hydroxybut- 4-hydroxy-
4-hydroxybut-2- bcd, etfA, NP_349317.1, Clostridium 2-enoyl-CoA
butyryl-CoA enoyl-CoA etfB NP_349315.1, acetobutylicum reductase
NP_349316.1 TER Q5EU90.1 Euglena gracilis TDE0597 NP_971211.1
Treponema denticola 8 1.1.1.c 4-hydroxy- 1,4- 4-hydroxybutyryl-
adhE2 AAK09379.1 Clostridium butanoyl-CoA butyryl-CoA butanediol
CoA reductase acetobutylicum (alcohol forming) mcr AAS20429.1
Chloroflexus malonyl-CoA aurantiacus FAR AAD38039.1 Simmondsia long
chain chinensis acyl-CoA 8 1.2.1.b 4-hydroxy- 4-hydroxy- 4-hydroxy-
sucD P38947.1 Clostridium Succinyl-CoA butyryl-CoA butanal
butyryl-CoA kluyveri reductase (or 4- hydroxybutanal dehydrogenase)
sucD NP_904963.1 Porphyromonas Succinyl-CoA gingivalis Msed_0709
YP_001190808.1 Metallosphaera Malonyl-CoA sedula 8 1.1.1.a
4-hydroxy- 1,4- 1,4-butanediol ADH2 NP_014032.1 Saccharymyces
general butanal butanediol dehydrogenase cerevisiae yqhD
NP_417484.1 Escherichia >C3 coli 4hbd L21902.1 Clostridium
Succinate kluyveri DSM 555 semialdehyde
Example XII
BDO Producing Strains Expressing Succinyl-CoA Synthetase
[0502] This example describes increased production of BDO in BDO
producing strains expressing succinyl-CoA synthetase.
[0503] As discussed above, succinate can be a precursor for
production of BDO by conversion to succinyl-CoA (see also
WO2008/115840, WO 2009/023493, U.S. publication 2009/0047719, U.S.
publication 2009/0075351). Therefore, the host strain was
genetically modified to overexpress the E. coli sucCD genes, which
encode succinyl-CoA synthetase. The nucleotide sequence of the E.
coli sucCD operon is shown in FIG. 14A, and the amino acid
sequences for the encoded succinyl-CoA synthetase subunits are
shown in FIGS. 14B and 14C. Briefly, the E. coli sucCD genes were
cloned by PCR from E. coli chromosomal DNA and introduced into
multicopy plasmids pZS*13, pZA13, and pZE33 behind the PA1lacO-1
promoter (Lutz and Bujard, Nucleic Acids Res. 25:1203-1210 (1997))
using standard molecular biology procedures.
[0504] The E. coli sucCD genes, which encode the succinyl-CoA
synthetase, were overexpressed. The results showed that introducing
into the strains sucCD to express succinyl-CoA synthetase improved
BDO production in various strains compared to either native levels
of expression or expression of cat1, which is a
succinyl-CoA/acetyl-CoA transferase. Thus, BDO production was
improved by overexpressing the native E. coli sucCD genes encoding
succinyl-CoA synthetase.
Example XIII
Expression of Heterologous Genes Encoding BDO Pathway Enzymes
[0505] This example describes the expression of various non-native
pathway enzymes to provide improved production of BDO.
[0506] Alpha-Ketoglutarate Decarboxylase.
[0507] The Mycobacterium bovis sucA gene encoding
alpha-ketoglutarate decarboxylase was expressed in host strains.
Overexpression of M. bovis sucA improved BDO production (see also
WO2008/115840, WO 2009/023493, U.S. publication 2009/0047719, U.S.
publication 2009/0075351). The nucleotide and amino acid sequences
of M. bovis sucA and the encoded alpha-ketoglutarate decarboxylase
are shown in FIG. 15.
[0508] To construct the M. bovis sucA expressing strains, fragments
of the sucA gene encoding the alpha-ketoglutarate decarboxylase
were amplified from the genomic DNA of Mycobacterium bovis BCG
(ATCC 19015; American Type Culture Collection, Manassas Va.) using
primers shown below. The full-length gene was assembled by ligation
reaction of the four amplified DNA fragments, and cloned into
expression vectors pZS*13 and pZE23 behind the P.sub.A1lacO-1
promoter (Lutz and Bujard, Nucleic Acids Res. 25:1203-1210 (1997)).
The nucleotide sequence of the assembled gene was verified by DNA
sequencing.
TABLE-US-00076 Primers for fragment 1: (SEQ ID NO: 3)
5'-ATGTACCGCAAGTTCCGC-3' (SEQ ID NO: 4) 5'-CAATTTGCCGATGCCCAG-3'
Primers for fragment 2: (SEQ ID NO: 5) 5'-GCTGACCACTGAAGACTTTG-3'
(SEQ ID NO: 6) 5'-GATCAGGGCTTCGGTGTAG-3' Primers for fragment 3:
(SEQ ID NO: 7) 5'-TTGGTGCGGGCCAAGCAGGATCTGCTC-3' (SEQ ID NO: 8)
5'-TCAGCCGAACGCCTCGTCGAGGATCTCCTG-3' Primers for fragment 4: (SEQ
ID NO: 9) 5'-TGGCCAACATAAGTTCACCATTCGGGCAAAAC-3' (SEQ ID NO: 10)
5'-TCTCTTCAACCAGCCATTCGTTTTGCCCG-3'
[0509] Functional expression of the alpha-ketoglutarate
decarboxylase was demonstrated using both in vitro and in vivo
assays. The SucA enzyme activity was measured by following a
previously reported method (Tian et al., Proc. Natl. Acad. Sci. USA
102:10670-10675 (2005)). The reaction mixture contained 50 mM
potassium phosphate buffer, pH 7.0, 0.2 mM thiamine pyrophosphate,
1 mM MgCl.sub.2, 0.8 mM ferricyanide, 1 mM alpha-ketoglutarate and
cell crude lysate. The enzyme activity was monitored by the
reduction of ferricyanide at 430 nm. The in vivo function of the
SucA enzyme was verified using E. coli whole-cell culture. Single
colonies of E. coli MG1655 lacI.sup.q transformed with plasmids
encoding the SucA enzyme and the 4-hydroxybutyrate dehydrogenase
(4Hbd) was inoculated into 5 mL of LB medium containing appropriate
antibiotics. The cells were cultured at 37.degree. C. overnight
aerobically. A 200 uL of this overnight culture was introduced into
8 mL of M9 minimal medium (6.78 g/L Na.sub.2HPO.sub.4, 3.0 g/L
KH.sub.2PO.sub.4, 0.5 g/L NaCl, 1.0 g/L NH.sub.4C1, 1 mM
MgSO.sub.4, 0.1 mM CaCl.sub.2) supplemented with 20 g/L glucose,
100 mM 3-(N-morpholino)propanesulfonic acid (MOPS) to improve the
buffering capacity, 10 .mu.g/mL thiamine, and the appropriate
antibiotics. Microaerobic conditions were established by initially
flushing capped anaerobic bottles with nitrogen for 5 minutes, then
piercing the septum with a 23 G needle following inoculation. The
needle was kept in the bottle during growth to allow a small amount
of air to enter the bottles. The protein expression was induced
with 0.2 mM isopropyl .beta.-D-1-thiogalactopyranoside (IPTG) when
the culture reached mid-log growth phase. As controls, E. coli
MG1655 lacIq strains transformed with only the plasmid encoding the
4-hydroxybutyrate dehydrogenase and only the empty vectors were
cultured under the same condition (see Table 23). The accumulation
of 4-hydroxybutyrate (4HB) in the culture medium was monitored
using LCMS method. Only the E. coli strain expressing the
Mycobacterium alpha-ketoglutarate decarboxylase produced
significant amount of 4HB (see FIG. 16).
TABLE-US-00077 TABLE 24 Three strains containing various plasmid
controls and encoding sucA and 4-hydroxybutyrate dehydrogenase.
Host pZE13 pZA33 1 MG1655 laclq vector vector 2 MG1655 laclq vector
4hbd 3 MG1655 laclq sucA 4hbd
[0510] A separate experiment demonstrated that the
alpha-ketoglutarate decarboxylase pathway functions independently
of the reductive TCA cycle. E. coli strain ECKh-401 (.DELTA.adhE
.DELTA.ldhA .DELTA.pflB .DELTA.lpdA::K.p.lpdA322 .DELTA.mdh
.DELTA.arcA) was used as the host strain. All the three constructs
contained the gene encoding 4HB dehydrogenase (4Hbd). Construct 1
also contained the gene encoding the alpha-ketoglutarate
decarboxylase (sucA). Construct 2 contained the genes encoding the
succinyl-CoA synthetase (sucCD) and the CoA-dependent succinate
semialdehyde dehydrogenase (sucD), which are required for the
synthesis of 4HB via the reductive TCA cycle. Construct 3 contains
all the genes from 1 and 2. The three E. coli strains were cultured
under the same conditions as described above except the second
culture was under the micro-aerobic condition. By expressing the
SucA enzyme, construct 3 produced more 4HB than construct 2, which
relies on the reductive TCA cycle for 4HB synthesis (see FIG.
17).
[0511] Further support for the contribution of alpha-ketoglutarate
decarboxylase to production of 4HB and BDO was provided by flux
analysis experiments. Cultures of ECKh-432, which contains both
sucCD-sucD and sucA on the chromosome, were grown in M9 minimal
medium containing a mixture of 1-13C-glucose (60%) and
U-13C-glucose (40%). The biomass was harvested, the protein
isolated and hydrolyzed to amino acids, and the label distribution
of the amino acids analyzed by gas chromatography-mass spectrometry
(GCMS) as described previously (Fischer and Sauer, Eur. J. Biochem.
270:880-891 (2003)). In addition, the label distribution of the
secreted 4HB and BDO was analyzed by GCMS as described in
WO2008115840 A2. This data was used to calculate the intracellular
flux distribution using established methods (Suthers et al., Metab.
Eng. 9:387-405 (2007)). The results indicated that between 56% and
84% of the alpha-ketoglutarate was channeled through
alpha-ketoglutarate decarboxylase into the BDO pathway. The
remainder was oxidized by alpha-ketoglutarate dehydrogenase, which
then entered BDO via the succinyl-CoA route.
[0512] These results demonstrate 4-hydroxybutyrate producing
strains that contain the sucA gene from Mycobacterium bovis BCG
expressed on a plasmid. When the plasmid encoding this gene is not
present, 4-hydroxybutyrate production is negligible when sucD
(CoA-dependant succinate semialdehyde dehydrogenase) is not
expressed. The M. bovis gene is a close homolog of the
Mycobacterium tuberculosis gene whose enzyme product has been
previously characterized (Tian et al., supra, 2005).
[0513] Succinate semialdehyde dehydrogenase (CoA-dependent),
4-hydroxybutyrate dehydrogenase, and
4-hydroxybutyryl-CoA/acetyl-CoA transferase. The genes from
Porphyromonas gingivalis W83 can be effective components of the
pathway for 1,4-butanediol production (see also WO2008/115840, WO
2009/023493, U.S. publication 2009/0047719, U.S. publication
2009/0075351). The nucleotide sequence of CoA-dependent succinate
semialdehyde dehydrogenase (sucD) from Porphyromonas gingivalis is
shown in FIG. 18A, and the encoded amino acid sequence is shown in
FIG. 18B. The nucleotide sequence of 4-hydroxybutyrate
dehydrogenase (4-hbd) from Porphymonas gingivalis is shown in FIG.
19A, and the encoded amino acid sequence is shown in FIG. 19B. The
nucleotide sequence of 4-hydroxybutyrate CoA transferase (cat2)
from Porphyromonas gingivalis is shown in FIG. 20A, and the encoded
amino acid sequence is shown in FIG. 20B.
[0514] Briefly, the genes from Porphyromonas gingivalis W83
encoding succinate semialdehyde dehydrogenase (CoA-dependent) and
4-hydroxybutyrate dehydrogenase, and in some cases additionally
4-hydroxybutyryl-CoA/acetyl-CoA, were cloned by PCR from P.
gingivalis chromosomal DNA and introduced into multicopy plasmids
pZS*13, pZA13, and pZE33 behind the PA1lacO-1 promoter (Lutz and
Bujard, Nucleic Acids Res. 25:1203-1210 (1997)) using standard
molecular biology procedures. These plasmids were then introduced
into host strains.
[0515] The Porphyromonas gingivalis W83 genes were introduced into
production strains as described above. Some strains included only
succinate semialdehyde dehydrogenase (CoA-dependant) and
4-hydroxybutyrate dehydrogenase without
4-hydroxybutyryl-CoA/acetyl-CoA transferase.
[0516] Butyrate Kinase and Phosphotransbutyrylase.
[0517] Butyrate kinase (BK) and phosphotransbutyrylase (PTB)
enzymes can be utlized to produce 4-hydroxybutyryl-CoA (see also
WO2008/115840, WO 2009/023493, U.S. publication 2009/0047719, U.S.
publication 2009/0075351). In particular, the Clostridium
acetobutylicum genes, buk1 and ptb, can be utilized as part of a
functional BDO pathway.
[0518] Initial experiments involved the cloning and expression of
the native C. acetobutylicum PTB (020) and BK (021) genes in E.
coli. Where required, the start codon and stop codon for each gene
were modified to "ATG" and "TAA," respectively, for more optimal
expression in E. coli. The C. acetobutylicum gene sequences (020N
and 021N) and their corresponding translated peptide sequences are
shown in FIGS. 21 and 22.
[0519] The PTB and BK genes exist in C. acetobutylicum as an
operon, with the PTB (020) gene expressed first. The two genes are
connected by the sequence "atta aagttaagtg gaggaatgtt aac" (SEQ ID
NO:11) that includes a re-initiation ribosomal binding site for the
downstream BK (021) gene. The two genes in this context were fused
to lac-controlled promoters in expression vectors for expression in
E. coli (Lutz and Bujard, Nucleic Acids Res. 25:1203-1210
(1997)).
[0520] Expression of the two proteins from these vector constructs
was found to be low in comparison with other exogenously expressed
genes due to the high incidence of codons in the C. acetobutylicum
genes that occur only rarely in E. coli. Therefore new 020 and 021
genes were predicted that changed rare codons for alternates that
are more highly represented in E. coli gene sequences. This method
of codon optimization followed algorithms described previously
(Sivaraman et al., Nucleic Acids Res. 36:e16 (2008)). This method
predicts codon replacements in context with their frequency of
occurrence when flanked by certain codons on either side.
Alternative gene sequences for 020 (FIG. 23) and 021 (FIG. 24) were
determined in which increasing numbers of rare codons were replaced
by more prevalent codons (A<B<C<D) based on their
incidence in the neighboring codon context. No changes in actual
peptide sequence compared to the native 020 and 021 peptide
sequences were introduced in these predicted sequences.
[0521] The improvement in expression of the BK and PTB proteins
resulting from codon optimization is shown in FIG. 25A. Expression
of the native gene sequences is shown in lane 2, while expression
of the 020B-021B and 020C-021C is shown in lanes 3 and 4,
respectively. Higher levels of protein expression in the
codon-optimized operons 020B-021B (2021B) and 020C-021C (2021C)
also resulted in increased activity compared to the native operon
(2021n) in equivalently-expressed E. coli crude extracts (FIG.
25B).
[0522] The codon optimized operons were expressed on a plasmid in
strain ECKh-432 (.DELTA.adhE .DELTA.ldhA .DELTA.pflB
.DELTA.lpdA::K.p.lpdA322 .DELTA.mdh .DELTA.arcA gltAR163L fimD::E.
coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd fimD:: M. bovis
sucA, C. kluyveri 4hbd) along with the C. acetobutylicum aldehyde
dehydrogenase to provide a complete BDO pathway. Cells were
cultured in M9 minimal medium containing 20 g/L glucose, using a 23
G needle to maintain microaerobic conditions as described above.
The resulting conversion of glucose to the final product BDO was
measured. Also measured was the accumulation of gamma-butyrylactone
(GBL), which is a spontaneously rearranged molecule derived from
4Hb-CoA, the immediate product of the PTB-BK enzyme pair. FIG. 26
shows that expression of the native 2021n operon resulted in
comparable BDO levels to an alternative enzyme function, Cat2
(034), that is capable of converting 4HB and free CoA to 4HB-CoA.
GBL levels of 034 were significantly higher than 2021n, suggesting
that the former enzyme has more activity than PTB-BK expressed from
the native genes. However levels of both BDO and GBL were higher
than either 034 or 2021n when the codon-optimized variants 2021B
and 2021C were expressed, indicating that codon optimization of the
genes for PTB and BK significantly increases their contributions to
BDO synthesis in E. coli.
[0523] These results demonstrate that butyrate kinase (BK) and
phosphotransbutyrylase (PTB) enzymes can be employed to convert
4-hydroxybutyrate to 4-hydroxybutyryl-CoA. This eliminates the need
for a transferase enzyme such as 4-hydroxybutyryl-CoA/Acetyl-CoA
transferase, which would generate one mole of acetate per mol of
4-hydroxybutyryl-CoA produced. The enzymes from Clostridium
acetobutylicum are present in a number of engineered strains for
BDO production.
[0524] 4-hydroxybutyryl-CoA Reductase.
[0525] The Clostridium beijerinckii ald gene can be utilized as
part of a functional BDO pathway (see also WO2008/115840, WO
2009/023493, U.S. publication 2009/0047719, U.S. publication
2009/0075351). The Clostridium beijerinckii ald can also be
utilized to lower ethanol production in BDO producing strains.
Additionally, a specific codon-optimized ald variant (GNM0025B) was
found to improve BDO production.
[0526] The native C. beijerinckii ald gene (025n) and the predicted
protein sequence of the enzyme are shown in FIG. 27. As was seen
for the Clostridium acetobutylicum PTB and BK genes, expression of
the native C. beijerinckii ald gene was very low in E. coli.
Therefore, four codon-optimized variants for this gene were
predicted. FIGS. 28A-28D show alternative gene sequences for 025,
in which increasing numbers of rare codons are replaced by more
prevalent codons (A<B<C<D) based on their incidence in the
neighboring codon context (25A, P=0.05; 25B, P=0.1; 25C, P=0.15;
25D, P=1). No changes in actual peptide sequence compared to the
native 025 peptide sequence were introduced in these predictions.
Codon optimization significantly increased expression of the C.
beijerinckii ald (see FIG. 29), which resulted in significantly
higher conversion of glucose to BDO in cells expressing the entire
BDO pathway (FIG. 30A).
[0527] The native and codon-optimized genes were expressed on a
plasmid along with P. gingivalis Cat2, in the host strain ECKh-432
(.DELTA.adhE .DELTA.ldhA .DELTA.pflB .DELTA.lpdA::K.p.lpdA322
.DELTA.mdh .DELTA.arcA gltAR163L .DELTA.ackA fimD::E. coli sucCD,
P. gingivalis sucD, P. gingivalis 4hbd fimD::M. bovis sucA, C.
kluyveri 4hbd), thus containing a complete BDO pathway. Cells were
cultured microaerobically in M9 minimal medium containing 20 g/L
glucose as described above. The relative production of BDO and
ethanol by the C. beijerinckii Ald enzyme (expressed from
codon-optimized variant gene 025B) was compared with the C.
acetobutylicum AdhE2 enzyme (see FIG. 30B). The C. acetobutylicum
AdhE2 enzyme (002C) produced nearly 4 times more ethanol than BDO.
In comparison, the C. beijerinckii Ald (025B) (in conjunction with
an endogenous ADH activity) produced equivalent amounts of BDO, yet
the ratio of BDO to ethanol production was reversed for this enzyme
compared to 002C. This suggests that the C. beijerinckii Ald is
more specific for 4HB-CoA over acetyl-coA than the C.
acetobutylicum AdhE2, and therefore the former is the preferred
enzyme for inclusion in the BDO pathway.
[0528] The Clostridium beijerinckii ald gene (Toth et al., Appi.
Environ. Microbiol. 65:4973-4980 (1999)) was tested as a candidate
for catalyzing the conversion of 4-hydroxybutyryl-CoA to
4-hydroxybutanal. Over fifty aldehyde dehydrogenases were screened
for their ability to catalyze the conversion of
4-hydroxybutyryl-CoA to 4-hydroxybutyraldehyde. The C. beijerinckii
ald gene was chosen for implementation into BDO-producing strains
due to the preference of this enzyme for 4-hydroxybutyryl-CoA as a
substrate as opposed to acetyl-CoA. This is important because most
other enzymes with aldehyde dehydrogenase functionality (for
example, adhE2 from C. acetobutylicum (Fontaine et al., J
Bacteriol. 184:821-830 (2002)) preferentially convert acetyl-CoA to
acetaldehyde, which in turn is converted to ethanol. Utilization of
the C. beijerinckii gene lowers the amount of ethanol produced as a
byproduct in BDO-producing organisms. Also, a codon-optimized
version of this gene expresses very well in E. coli (Sivaraman et
al., Nucleic Acids Res. 36:e16 (2008)).
[0529] 4-hydroxybutanal Reductase.
[0530] 4-hydroxybutanal reductase activity of adh1 from Geobacillus
thermoglucosidasius (M10EXG) was utilized. This led to improved BDO
production by increasing 4-hydroxybutanal reductase activity over
endogenous levels.
[0531] Multiple alcohol dehydrogenases were screened for their
ability to catalyze the reduction of 4-hydroxybutanal to BDO. Most
alcohol dehydrogenases with high activity on butyraldehyde
exhibited far lower activity on 4-hydroxybutyraldehyde. One notable
exception is the adh1 gene from Geobacillus thermoglucosidasius
M10EXG (Jeon et al., J. Biotechnol. 135:127-133 (2008)) (GNM0084),
which exhibits high activity on both 4-hydroxybutanal and
butanal.
[0532] The native gene sequence and encoded protein sequence if the
adh1 gene from Geobacillus thermoglucosidasius are shown in FIG.
31. The G. thermoglucosidasius ald1 gene was expressed in E.
coli.
[0533] The Adh1 enzyme (084) expressed very well from its native
gene in E. coli (see FIG. 32A). In ADH enzyme assays, the E. coli
expressed enzyme showed very high reductive activity when
butyraldehyde or 4HB-aldehyde were used as the substrates (see FIG.
32B). The Km values determined for these substrates were 1.2 mM and
4.0 mM, respectively. These activity values showed that the Adh1
enzyme was the most active on reduction of 4HB-aldehyde of all the
candidates tested.
[0534] The 084 enzyme was tested for its ability to boost BDO
production when coupled with the C. beijerinckii ald. The 084 gene
was inserted behind the C. beijerinckii ald variant 025B gene to
create a synthetic operon that results in coupled expression of
both genes. Similar constructs linked 025B with other ADH candidate
genes, and the effect of including each ADH with 025B on BDO
production was tested. The host strain used was ECKh-459
(.DELTA.adhE ldhA .DELTA.pflB .DELTA.lpdA::fnr-pflB6-K.p.lpdA322
.DELTA.mdh .DELTA.arcA gltAR163L fimD:: E. coli sucCD, P.
gingivalis sucD, P. gingivalis 4hbd fimD:: M. bovis sucA, C.
kluyveri 4hbd fimD:: C. acetobutylicum buk1, C. acetobutylicum
ptb), which contains the remainder of the BDO pathway on the
chromosome. The 084 ADH expressed in conjunction with 025B showed
the highest amount of BDO (right arrow in FIG. 33) when compared
with 025B only (left arrow in FIG. 33) and in conjunction with
endogenous ADH functions. It also produced more BDO than did other
ADH enzymes when paired with 025B, indicated as follows: 026A-C,
codon-optimized variants of Clostridium acetobutylicum butanol
dehydrogenase; 050, Zymomonas mobilis alcohol dehydrogenase I; 052,
Citrobacter freundii 1,3-propanediol dehydrogenase; 053,
Lactobacillus brevis 1,3-propanediol dehydrogenase; 057,
Bacteroides fragilis lactaldehyde reductase; 058, E. coli
1,3-propanediol dehydrogenase; 071, Bacillus subtilis 168
alpha-ketoglutarate semialdehyde dehydrogenase. The constructs
labeled "PT5lacO" are those in which the genes are driven by the
PT5lacO promoter. In all other cases, the PA1lacO-1 promoter was
used. This shows that inclusion of the 084 ADH in the BDO pathway
increased BDO production.
Example XIV
BDO Producing Strains Expressing Pyruvate Dehydrogenase
[0535] This example describes the utilization of pyruvate
dehydrogenase (PDH) to enhance BDO production. Heterologous
expression of the Klebsiella pneumonia lpdA gene was used to
enhance BDO production.
[0536] Computationally, the NADH-generating conversion of pyruvate
to acetyl-CoA is required to reach the maximum theoretical yield of
1,4-butanediol (see also WO2008/115840, WO 2009/023493, U.S.
publication 2009/0047719, U.S. publication 2009/0075351; WO
2008/018930; Kim et al., Appl. Environ. Microbiol. 73:1766-1771
(2007); Kim et al., J. Bacteriol. 190:3851-3858 (2008); Menzel et
al., J. Biotechnol. 56:135-142 (1997)). Lack of PDH activity was
shown to reduce the maximum anaerobic theoretical yield of BDO by
11% if phosphoenolpyruvate carboxykinase (PEPCK) activity cannot be
attained and by 3% if PEPCK activity can be attained. More
importantly, however, absence of PDH activity in the OptKnock
strain #439, described in WO 2009/023493 and U.S. publication
2009/0047719, which has the knockout of ADHEr, ASPT, LDH_D, MDH and
PFLi, would reduce the maximum anaerobic yield of BDO by 54% or by
43% if PEPCK activity is absent or present, respectively. In the
presence of an external electron acceptor, lack of PDH activity
would reduce the maximum yield of the knockout strain by 10% or by
3% assuming that PEPCK activity is absent or present,
respectively.
[0537] PDH is one of the most complicated enzymes of central
metabolism and is comprised of 24 copies of pyruvate decarboxylase
(E1) and 12 molecules of dihydrolipoyl dehydrogenase (E3), which
bind to the outside of the dihydrolipoyl transacetylase (E2) core.
PDH is inhibited by high NADH/NAD, ATP/ADP, and Acetyl-CoA/CoA
ratios. The enzyme naturally exhibits very low activity under
oxygen-limited or anaerobic conditions in organisms such as E. coli
due in large part to the NADH sensitivity of E3, encoded by lpdA.
To this end, an NADH-insensitive version of the lpdA gene from
Klebsiella pneumonia was cloned and expressed to increase the
activity of PDH under conditions where the NADH/NAD ratio is
expected to be high.
[0538] Replacement of the Native lpdA.
[0539] The pyruvate dehydrogenase operon of Klebsiella pneumoniae
is between 78 and 95% identical at the nucleotide level to the
equivalent operon of E. coli. It was shown previously that K.
pneumoniae has the ability to grow anaerobically in presence of
glycerol (Menzel et al., J. Biotechnol. 56:135-142 (1997); Menzel
et al., Biotechnol. Bioeng. 60:617-626 (1998)). It has also been
shown that two mutations in the lpdA gene of the operon of E. coli
would increase its ability to grow anaerobically (Kim et al. Appl.
Environ. Microbiol. 73:1766-1771 (2007); Kim et al., J. Bacteriol.
190:3851-3858 (2008)). The lpdA gene of K. pneumonia was amplified
by PCR using genomic DNA (ATCC700721D) as template and the primers
KP-lpdA-Bam (5'-acacgcggatccaacgtcccgg-3') (SEQ ID NO:12) and
KP-lpdA-Nhe (5'-agcggctccgctagccgcttatg-3') (SEQ ID NO:13). The
resulting fragment was cloned into the vector pCR-BluntII-TOPO
(Invitrogen; Carlsbad Calif.), leading to plasmid pCR-KP-lpdA.
[0540] The chromosomal gene replacement was performed using a
non-replicative plasmid and the sacB gene from Bacillus subtilis as
a means of counterselection (Gay et al., J. Bacteriol.
153:1424-1431 (1983)). The vector used is pREl 18 (ATCC87693)
deleted of the oriT and IS sequences, which is 3.6 kb in size and
carrying the kanamycin resistance gene. The sequence was confirmed,
and the vector was called pRE118-V2 (see FIG. 34).
[0541] The E. coli fragments flanking the lpdA gene were amplified
by PCR using the combination of primers: EC-aceF-Pst
(5'-aagccgttgctgcagctcttgagc-3') (SEQ ID NO:14)+EC-aceF-Bam2
(5'-atctccggcggtcggatccgtcg-3') (SEQ ID NO:15) and EC-yacH-Nhe
(5'-aaagcggctagccacgccgc-3') (SEQ ID NO:16)+EC-yacH-Kpn
(5'-attacacgaggtacccaacg-3') (SEQ ID NO:17). A BamHI-XbaI fragment
containing the lpdA gene of K. pneumonia was isolated from plasmid
pCR-KP-lpdA and was then ligated to the above E. coli fragments
digested with PstI+BamHI and NheI-KpnI respectively, and the
pRE118-V2 plasmid digested with KpnI and PstI. The resulting
plasmid (called pRE118-M2.1 lpdA yac) was subjected to Site
Directed Mutagenesis (SDM) using the combination of primers
KP-lpdA-HisTyr-F (5'-atgctggcgtacaaaggtgtcc-3') (SEQ ID NO:18) and
(5'-ggacacctttgtacgccagcat-3') (SEQ ID NO:19) for the mutation of
the His 322 residue to a Tyr residue or primers KP-lpdA-GluLys-F
(5'-atcgcctacactaaaccagaagtgg-3') (SEQ ID NO:20) and
KP-lpdA-GluLys-R (5'-ccacttctggtttagtgtaggcgat-3') (SEQ ID NO:21)
for the mutation of the residue Glu 354 to Lys residue. PCR was
performed with the Polymerase Pfu Turbo (Stratagene; San Diego
Calif.). The sequence of the entire fragment as well as the
presence of only the desired mutations was verified. The resulting
plasmid was introduced into electro competent cells of E. coli
.DELTA.adhE::Frt-.DELTA.ldhA::Frt by transformation. The first
integration event in the chromosome was selected on LB agar plates
containing Kanamycin (25 or 50 mg/L). Correct insertions were
verified by PCR using 2 primers, one located outside the region of
insertion and one in the kanamycin gene
(5'-aggcagttccataggatggc-3') (SEQ ID NO:22). Clones with the
correct insertion were selected for resolution. They were
sub-cultured twice in plain liquid LB at the desired temperature
and serial dilutions were plated on LB-no salt-sucrose 10% plates.
Clones that grew on sucrose containing plates were screened for the
loss of the kanamycin resistance gene on LB-low salt agar medium
and the lpdA gene replacement was verified by PCR and sequencing of
the encompassing region. Sequence of the insertion region was
verified, and is as described below. One clone (named 4-4-P1) with
mutation Glu354Lys was selected. This clone was then transduced
with P1 lysate of E. coli .DELTA.PflB::Frt leading to strain
ECKh-138 (.DELTA.adhE .DELTA.ldhA .DELTA.pflB
.DELTA.lpdA::K.p.lpdA322).
[0542] The sequence of the ECKh-138 region encompassing the aceF
and lpdA genes is shown in FIG. 35. The K. pneumonia lpdA gene is
underlined, and the codon changed in the Glu354Lys mutant shaded.
The protein sequence comparison of the native E. coli lpdA and the
mutant K. pneumonia lpdA is shown in FIG. 36.
[0543] To evaluate the benefit of using K. pneumoniae lpdA in a BDO
production strain, the host strains AB3 and ECKh-138 were
transformed with plasmids expressing the entire BDO pathway from
strong, inducible promoters. Specifically, E. coli sucCD, P.
gingivalis sucD, P. gingivalis 4hbd were expressed on the medium
copy plasmid pZA33, and P. gingivalis Cat2 and C. acetobutylicum
AdhE2 were expressed on the high copy plasmid pZE13. These plasmids
have been described in the literature (Lutz and H. Bujard, Nucleic
Acids Res 25:1203-1210 (1997)), and their use for BDO pathway
expression is described in Example XIII and WO2008/115840.
[0544] Cells were grown anaerobically at 37.degree. C. in M9
minimal medium (6.78 g/L Na.sub.2HPO.sub.4, 3.0 g/L
KH.sub.2PO.sub.4, 0.5 g/L NaCl, 1.0 g/L NH.sub.4Cl, 1 mM
MgSO.sub.4, 0.1 mM CaCl.sub.2) supplemented with 20 g/L glucose,
100 mM 3-(N-morpholino)propanesulfonic acid (MOPS) to improve the
buffering capacity, 10 .mu.g/mL thiamine, and the appropriate
antibiotics. Microaerobic conditions were established by initially
flushing capped anaerobic bottles with nitrogen for 5 minutes, then
piercing the septum with a 23 G needle following inoculation. The
needle was kept in the bottle during growth to allow a small amount
of air to enter the bottles. 0.25 mM IPTG was added when OD600
reached approximately 0.2 to induce the pathway genes, and samples
taken for analysis every 24 hours following induction. The culture
supernatants were analyzed for BDO, 4HB, and other by-products as
described in Example II and in WO2008/115840. BDO and 4HB
production in ECKh-138 was significantly higher after 48 hours than
in AB3 or the host used in previous work, MG1655 .DELTA.ldhA (FIG.
37).
[0545] PDH Promoter Replacement.
[0546] It was previously shown that the replacement of the pdhR
repressor by a transcriptional fusion containing the Fnr binding
site, one of the pflB promoters, and its ribosome binding site
(RBS), thus leading to expression of the aceEF-lpd operon by an
anaerobic promoter, should increase pdh activity anaerobically
(Zhou et al., Biotechnol. Lett. 30:335-342 (2008)). A fusion
containing the Fnr binding site, the pflB-p6 promoter and an RBS
binding site were constructed by overlapping PCR. Two fragments
were amplified, one using the primers aceE-upstream-RC
(5'-tgacatgtaacacctaccttctgtgcctgtgccagtggttgctgtgatatagaag-3')
(SEQ ID NO:23) and pflBp6-Up-Nde
(5'-ataataatacatatgaaccatgcgagttacgggcctataagccaggcg-3') (SEQ ID
NO:24) and the other using primers aceE-EcoRV-EC
(5'-agtttttcgatatctgcatcagacaccggcacattgaaacgg-3') (SEQ ID NO:25)
and aceE-upstream
(5'-ctggcacaggcacagaaggtaggtgttacatgtcagaacgtttacacaatgacgtggatc-3')
(SEQ ID NO:26). The tw fragments were assembled by overlapping PCR,
and the final DNA fragment was digested with the restriction
enzymes NdeI and BamHI. This fragment was subsequently introduced
upstream of the aceE gene of the E. coli operon using pRE118-V2 as
described above. The replacement was done in strains ECKh-138 and
ECKh-422. The nucleotide sequence encompassing the 5' region of the
aceE gene was verified and is shown in FIG. 37. FIG. 37 shows the
nucleotide sequence of 5' end of the aceE gene fused to the pflB-p6
promoter and ribosome binding site (RBS). The 5' italicized
sequence shows the start of the aroP gene, which is transcribed in
the opposite direction from the pdh operon. The 3' italicized
sequence shows the start of the aceE gene. In upper case: pflB RBS.
Underlined: FNR binding site. In bold: pflB-p6 promoter
sequence.
[0547] lpdA Promoter Replacement.
[0548] The promoter region containing the fnr binding site, the
pflB-p6 promoter and the RBS of the pflB gene was amplified by PCR
using chromosomal DNA template and primers aceF-pflBp6-fwd
(5'-agacaaatcggttgccgtttgttaagccaggcgagatatgatctatatc-3') (SEQ ID
NO:27) and lpdA-RBS-B-rev
(5'-gagttttgatttcagtactcatcatgtaacacctaccttcttgctgtgatatag-3') (SEQ
ID NO:28). Plasmid 2-4a was amplified by PCR using primers
B-RBS-lpdA fwd
(5'-ctatatcacagcaagaaggtaggtgttacatgatgagtactgaaatcaaaactc-3') (SEQ
ID NO:29) and pflBp6-aceF-rev
(5'-gatatagatcatatctcgcctggcttaacaaacggcaaccgatttgtct-3') (SEQ ID
NO:30). The two resulting fragments were assembled using the BPS
cloning kit (BPS Bioscience; San Diego Calif.). The resulting
construct was sequenced verified and introduced into strain
ECKh-439 using the pRE118-V2 method described above. The nucleotide
sequence encompassing the aceF-lpdA region in the resulting strain
ECKh-456 is shown in FIG. 39.
[0549] The host strain ECKh-439 (.DELTA.adhE .DELTA.ldhA
.DELTA.pflB .DELTA.lpdA::K.p.lpdA322 .DELTA.mdh .DELTA.arcA
gltAR163L ackA fimD:: E. coli sucCD, P. gingivalis sucD, P.
gingivalis 4hbd fimD:: M. bovis sucA, C. kluyveri 4hbd), the
construction of which is described below, and the pdhR and lpdA
promoter replacement derivatives ECKh-455 and ECKh-456, were tested
for BDO production. The strains were transformed with pZS*13
containing P. gingivalis Cat2 and C. beijerinckii Ald to provide a
complete BDO pathway. Cells were cultured in M9 minimal medium
supplemented with 20 g/L glucose as described above. 48 hours after
induction with 0.2 mM IPTG, the concentrations of BDO, 4HB, and
pyruvate were as shown in FIG. 40. The promoter replacement strains
produce slightly more BDO than the isogenic parent.
[0550] These results demonstrated that expression of pyruvate
dehydrogenase increased production of BDO in BDO producing
strains.
Example XV
BDO Producing Strains Expressing Citrate Synthase and Aconitase
[0551] This example describes increasing activity of citrate
synthase and aconitase to increase production of BDO. An R163L
mutation into gltA was found to improve BDO production.
Additionally, an arcA knockout was used to improve BDO
production.
[0552] Computationally, it was determined that flux through citrate
synthase (CS) and aconitase (ACONT) is required to reach the
maximum theoretical yield of 1,4-butanediol (see also
WO2008/115840, WO 2009/023493, U.S. publication 2009/0047719, U.S.
publication 2009/0075351). Lack of CS or ACONT activity would
reduce the maximum theoretical yield by 14% under anaerobic
conditions. In the presence of an external electron acceptor, the
maximum yield is reduced by 9% or by 6% without flux through CS or
ACONT assuming the absence or presence of PEPCK activity,
respectively. As with pyruvate dehydrogenase (PDH), the importance
of CS and ACONT is greatly amplified in the knockout strain
background in which ADHEr, ASPT, LDH_D, MDH and PFLi are knocked
out (design #439) (see WO 2009/023493 and U.S. publication
2009/0047719, which is incorporated herein by reference).
[0553] The minimal OptKnock strain design described in WO
2009/023493 and U.S. publication 2009/0047719 had one additional
deletion beyond ECKh-138, the mdh gene, encoding malate
dehydrogenase. Deletion of this gene is intended to prevent flux to
succinate via the reductive TCA cycle. The mdh deletion was
performed using the .lamda. red homologeous recombination method
(Datsenko and Wanner, Proc. Natl. Acad. Sci. USA 97:6640-6645
(2000)). The following oligonucleotides were used to PCR amplify
the chloramphenicol resistance gene (CAT) flanked by FRT sites from
pKD3:
TABLE-US-00078 S-mdh-Kan (SEQ ID NO: 31) 5'- TAT TGT GCA TAC AGA
TGA ATT TTT ATG CAA ACA GTC AGC CCT GAA GAA GGG TGT AGG CTG GAG CTG
CTT C -3' AS-mdh-Kan (SEQ ID NO: 32) 5'- CAA AAA ACC GGA GTC TGT
GCT CCG GTT TTT TAT TAT CCG CTA ATC AAT TAC ATA TGA ATA TCC TCC TTA
G -3'.
[0554] Underlined regions indicate homology to pKD3 plasmid and
bold sequence refers to sequence homology upstream and downstream
of the mdh ORF. After purification, the PCR product was
electroporated into ECKh-138 electrocompetent cells that had been
transformed with pRedET (tet) and prepared according to the
manufacturer's instructions (genebridges.com/gb/pdf/K001%20Q %20E
%20BAC%20Modification%20Kit-version2.6-2007-screen.pdf). The PCR
product was designed so that it integrated into the ECKh-138 genome
at a region upstream of the mdh gene, as shown in FIG. 41.
[0555] Recombinants were selected for chloramphenicol resistance
and streak purified. Loss of the mdh gene and insertion of CAT was
verified by diagnostic PCR. To remove the CAT gene, a temperature
sensitive plasmid pCP20 containing a FLP recombinase (Datsenko and
Wanner, Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)) was
transformed into the cell at 30.degree. C. and selected for
ampicillin resistance (AMP). Transformants were grown
nonselectively at 42.degree. C. overnight to thermally induce FLP
synthesis and to cause lose of the plasmid. The culture was then
streak purified, and individual colonies were tested for loss of
all antibiotic resistances. The majority lost the FRT-flanked
resistance gene and the FLP helper plasmid simultaneously. There
was also a "FRT" scar leftover. The resulting strain was named
ECKh-172.
[0556] CS and ACONT are not highly active or highly expressed under
anaerobic conditions. To this end, the arcA gene, which encodes for
a global regulator of the TCA cycle, was deleted. ArcA works during
microaerobic conditions to induce the expression of gene products
that allow the activity of central metabolism enzymes that are
sensitive to low oxygen levels, aceE, pflB and adhE. It was shown
that microaerobically, a deletion in arcA/arcB increases the
specific activities of ldh, icd, gltA, mdh, and gdh genes (Salmon
et al., J. Biol. Chem. 280:15084-15096 (2005); Shalel-Levanon et
al., Biotechnol. Bioeng. 92(2):147-159 (2005). The upstream and
downstream regions of the arcA gene of E. coli MG1655 were
amplified by PCR using primers ArcA-up-EcoRI
(5'-ataataatagaattcgtttgctacctaaattgccaactaaatcgaaacagg-3') (SEQ ID
NO:33) with ArcA-up-KpnI
(5'-tattattatggtaccaatatcatgcagcaaacggtgcaacattgccg-3') (SEQ ID
NO:34) and ArcA-down-EcoRI
(5'-tgatctggaagaattcatcggctttaccaccgtcaaaaaaaacggcg-3') (SEQ ID
NO:35) with ArcA-down-PstI
(5'-ataaaaccctgcagcggaaacgaagttttatccatttttggttacctg-3') (SEQ ID
NO:36), respectively. These fragments were subsequently digested
with the restriction enzymes EcoRI and KpnI (upstream fragment) and
EcoRI and PstI (downstream). They were then ligated into the
pRE118-V2 plasmid digested with PstI and KpnI, leading to plasmid
pRE118-.DELTA.arcA. The sequence of plasmid pRE118-.DELTA.arcA was
verified. pRE118-.DELTA.arcA was introduced into electro-competent
cells of E. coli strain ECKh-172 (.DELTA.adhE .DELTA.ldhA
.DELTA.pflB .DELTA.lpdA::K.p.lpdA322 .DELTA.mdh). After integration
and resolution on LB-no salt-sucrose plates as described above, the
deletion of the arcA gene in the chromosome of the resulting strain
ECKh-401 was verified by sequencing and is shown in FIG. 42.
[0557] The gltA gene of E. coli encodes for a citrate synthase. It
was previously shown that this gene is inhibited allosterically by
NADH, and the amino acids involved in this inhibition have been
identified (Pereira et al., J. Biol. Chem. 269(1):412-417 (1994);
Stokell et al., J. Biol. Chem. 278(37):35435-35443 (2003)). The
gltA gene of E. coli MG1655 was amplified by PCR using primers
gltA-up (5'-ggaagagaggctggtacccagaagccacagcagga-3') (SEQ ID NO:37)
and gltA-PstI (5'-gtaatcactgcgtaagcgccatgccccggcgttaattc-3') (SEQ
ID NO:38). The amplified fragment was cloned into pRE118-V2 after
digestion with KpnI and PstI. The resulting plasmid was called
pRE118-gltA. This plasmid was then subjected to site directed
mutagensis (SDM) using primers R163L-f
(5'-attgccgcgttcctcctgctgtcga-3') (SEQ ID NO:39) and R163L-r
(5'-cgacagcaggaggaacgcggcaat-3') (SEQ ID NO:40) to change the
residue Arg 163 to a Lys residue. The sequence of the entire
fragment was verified by sequencing. A variation of the .lamda. red
homologeous recombination method (Datsenko and Wanner, Proc. Natl.
Acad. Sci. USA 97:6640-6645 (2000)) was used to replace the native
gltA gene with the R163L mutant allele without leaving a Frt scar.
The general recombination procedure is the same as used to make the
mdh deletion described above. First, the strain ECKh-172 was made
streptomycin resistant by introducing an rpsL null mutation using
the .lamda. red homologeous recombination method. Next, a
recombination was done to replace the entire wild-type gltA coding
region in this strain with a cassette comprised of a kanamycin
resistance gene (kanR) and a wild-type copy of the E. coli rpsL
gene. When introduced into an E. coli strain harboring an rpsL null
mutation, the cassette causes the cells to change from resistance
to the drug streptomycin to streptomycin sensitivity. DNA fragments
were then introduced that included each of the mutant versions of
the gltA gene along with appropriate homologous ends, and resulting
colony growth was tested in the presence of streptomycin. This
selected for strains in which the kanR/rpsL cassette had been
replaced by the mutant gltA gene. Insertion of the mutant gene in
the correct locus was confirmed by PCR and DNA sequencing analyses.
The resulting strain was called ECKh-422, and has the genotype
.DELTA.adhE .DELTA.ldhA .DELTA.pflB .DELTA.lpdA::K.p.lpdA322
.DELTA.mdh .DELTA.arcA gltAR163L. The region encompassing the
mutated gltA gene of strain ECKh-422 was verified by sequencing, as
shown in FIG. 43.
[0558] Crude extracts of the strains ECKh-401 and the gltAR163L
mutant ECKh-422 were then evaluated for citrate synthase activity.
Cells were harvested by centrifugation at 4,500 rpm
(Beckman-Coulter, Allegera X-15R; Fullerton Calif.) for 10 min. The
pellets were resuspended in 0.3 mL BugBuster (Novagen/EMD; San
Diego Calif.) reagent with benzonase and lysozyme, and lysis
proceeded for 15 minutes at room temperature with gentle shaking
Cell-free lysate was obtained by centrifugation at 14,000 rpm
(Eppendorf centrifuge 5402; Hamburg Germany) for 30 min at
4.degree. C. Cell protein in the sample was determined using the
method of Bradford (Bradford, Anal. Biochem. 72:248-254
(1976)).
[0559] Citrate synthase activity was determined by following the
formation of free coenzyme A (HS-CoA), which is released from the
reaction of acetyl-CoA with oxaloacetate. The free thiol group of
HS-CoA reacts with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to
form 5-thio-2-nitrobenzoic acid (TNB). The concentration of TNB is
then monitored spectrophotometrically by measuring the absorbance
at 410 nm (maximum at 412 nm). The assay mixture contained 100 mM
Tris/HCl buffer (pH 7.5), 20 mM acetyl-CoA, 10 mM DTNB, and 20 mM
oxaloacetate. For the evaluation of NADH inhibition, 0.4 mM NADH
was also added to the reaction. The assay was started by adding 5
microliters of the cell extract, and the rate of reaction was
measured by following the absorbance change over time. A unit of
specific activity is defined as the .mu.mol of product converted
per minute per mg protein.
[0560] FIG. 44 shows the citrate synthase activity of wild type
gltA gene product and the R163L mutant. The assay was performed in
the absence or presence of 0.4 mM NADH.
[0561] Strains ECKh-401 and ECKh-422 were transformed with plasmids
expressing the entire BDO pathway. E. coli sucCD, P. gingivalis
sucD, P. gingivalis 4hbd, and M. bovis sucA were expressed on the
low copy plasmid pZS*13, and P. gingivalis Cat2 and C.
acetobutylicum AdhE2 were expressed on the medium copy plasmid
pZE23. Cultures of these strains were grown microaerobically in M9
minimal medium supplemented with 20 g/L glucose and the appropriate
antibiotics as described above. The 4HB and BDO concentrations at
48 hours post-induction averaged from duplicate cultures are shown
in FIG. 45. Both are higher in ECKh-422 than in ECKh-401,
demonstrating that the enhanced citrate synthase activity due to
the gltA mutation results in increased flux to the BDO pathway.
[0562] The host strain modifications described in this section were
intended to redirect carbon flux through the oxidative TCA cycle,
which is consistent with the OptKnock strain design described in WO
2009/023493 and U.S. publication 2009/0047719. To demonstrate that
flux was indeed routed through this pathway, .sup.13C flux analysis
was performed using the strain ECKh-432, which is a version of
ECKh-422 in which the upstream pathway is integrated into the
chromosome (as described in Example XVII). To complete the BDO
pathway, P. gingivalis Cat2 and C. beijerinckii Ald were expressed
from pZS*13. Four parallel cultures were grown in M9 minimal medium
(6.78 g/L Na.sub.2HPO.sub.4, 3.0 g/L KH.sub.2PO.sub.4, 0.5 g/L
NaCl, 1.0 g/L NH.sub.4C1, 1 mM MgSO.sub.4, 0.1 mM CaCl.sub.2)
containing 4 g/L total glucose of four different labeling ratios
(.sup.1-13C, only the first carbon atom in the glucose molecule is
labeled with .sup.13C; uniform-.sup.13C, all carbon atoms are
.sup.13C):
[0563] 1. 80 mol % unlabeled, 20 mol % uniform-.sup.13C
[0564] 2. 10 mol % unlabeled, 90 mol % uniform-.sup.13C
[0565] 3. 90 mol % .sup.1-13C, 10 mol % uniform-.sup.13C
[0566] 4. 40 mol % .sup.1-13C, 60 mol % uniform-.sup.13C
[0567] Parallel unlabeled cultures were grown in duplicate, from
which frequent samples were taken to evaluate growth rate, glucose
uptake rate, and product formation rates. In late exponential
phase, the labeled cultures were harvested, the protein isolated
and hydrolyzed to amino acids, and the label distribution of the
amino acids analyzed by gas chromatography-mass spectrometry (GCMS)
as described previously (Fischer and Sauer, Eur. J. Biochem.
270:880-891 (2003)). In addition, the label distribution of the
secreted 4HB and BDO in the broth from the labeled cultures was
analyzed by GCMS as described in WO2008115840. This data was
collectively used to calculate the intracellular flux distribution
using established methods (Suthers et al., Metab. Eng. 9:387-405
(2007)). The resulting central metabolic fluxes and associated 95%
confidence intervals are shown in FIG. 46. Values are molar fluxes
normalized to a glucose uptake rate of 1 mmol/hr. The result
indicates that carbon flux is routed through citrate synthase in
the oxidative direction, and that most of the carbon enters the BDO
pathway rather than completing the TCA cycle. Furthermore, it
confirms there is essentially no flux between malate and
oxaloacetate due to the mdh deletion in this strain.
[0568] The advantage of using a knockout strain such as strains
designed using OptKnock for BDO production (see WO 2009/023493 and
U.S. publication 2009/0047719) can be observed by comparing typical
fermentation profiles of ECKh-422 with that of the original strain
ECKh-138, in which BDO is produced from succinate via the reductive
TCA cycle (see FIG. 47). Fermentations were performed with 1 L
initial culture volume in 2 L Biostat B+ bioreactors (Sartorius;
Cedex France) using M9 minimal medium supplemented with 20 g/L
glucose. The temperature was controlled at 37.degree. C., and the
pH was controlled at 7.0 using 2 M NH.sub.4OH or Na.sub.2CO.sub.3.
Cells were grown aerobically to an OD600 of approximately 10, at
which time the cultures were induced with 0.2 mM IPTG. One hour
following induction, the air flow rate was reduced to 0.02 standard
liters per minute for microaerobic conditions. The agitation rate
was set at 700 rpm. Concentrated glucose was fed to maintain
glucose concentration in the vessel between 0.5 and 10 g/L. Both
strains were transformed with plasmids bearing the entire BDO
pathway, as in the examples above. In ECKh-138, acetate, pyruvate,
and 4HB dominate the fermentation, while with ECKh-422 BDO is the
major product.
Example XVI
BDO Strains Expression Phosphoenolpyruvate Carboxykinase
[0569] This example describes the utilization of
phosphoenolpyruvate carboxykinase (PEPCK) to enhance BDO
production. The Haemophilus influenza PEPCK gene was used for
heterologous expression.
[0570] Computationally, it was demonstrated that the ATP-generating
conversion of oxaloacetate to phosphoenolpyruvate is required to
reach the maximum theoretical yield of 1,4-butanediol (see also
WO2008/115840, WO 2009/023493, U.S. publication 2009/0047719, U.S.
publication 2009/0075351). Lack of PEPCK activity was shown to
reduce the maximum theoretical yield of BDO by 12% assuming
anaerobic conditions and by 3% assuming an external electron
acceptor such as nitrate or oxygen is present.
[0571] In organisms such as E. coli, PEPCK operates in the
gluconeogenic and ATP-consuming direction from oxaloacetate towards
phosphoenolpyruvate. It has been hypothesized that kinetic
limitations of PEPCK of E. coli prevent it from effectively
catalyzing the formation of oxaloacetate from PEP. PEP carboxylase
(PPC), which does not generate ATP but is required for efficient
growth, is naturally utilized by E. coli to form oxaloacetate from
phosphoenolpyruvate. Therefore, three non native PEPCK enzymes
(Table 25) were tested for their ability to complement growth of a
PPC mutant strain of E. coli in glucose minimal media.
TABLE-US-00079 TABLE 25 Sources of phosphoenolpyruvate
carboxykinase sequences. Accession Number, PEPCK Source Strain
GenBank Reference Sequence Haemophilus influenza NC_000907.1
Actinobacillus succinogenes YP_001343536.1 Mannheimia
succiniciproducens YP_089485.1
[0572] Growth complementation studies involved plasmid based
expression of the candidate genes in .DELTA.ppc mutant E. coli
JW3978 obtained from the Keio collection (Baba et al., Molecular
Systems Biology 2:2006.0008 (2006)). The genes were cloned behind
the PA1lacO-1 promoter in the expression vectors pZA23 (medium
copy) and pZE13 (high copy). These plasmids have been described
previously (Lutz and Bujard, Nucleic Acids Res. 25:1203-1210
(1997)), and their use in expression BDO pathway genes has been
described previously in WO2008115840.
[0573] Pre-cultures were grown aerobically in M9 minimal media with
4 g/L glucose. All pre-cultures were supplemented with aspartate (2
mM) to provide the .DELTA.ppc mutants with a source for generating
TCA cycle intermediates independent of PEPCK expression. M9 minimal
media was also used in the test conditions with 4 g/L glucose, but
no aspartate was added and IPTG was added to 0.5 mM. Table 26 shows
the results of the growth complementation studies.
TABLE-US-00080 TABLE 26 Complementation of .DELTA.ppc mutants with
PEPCK from H. influenzae, A. succinogenes and M. succinoproducens
when expressed from vectors pZA23 or pZE13. PEPCK Source Strain
Vector Time (h) OD.sub.600 H. influenzae pZA23BB 40 0.950
.DELTA.ppc Control pZA23BB 40 0.038 A. succinogenes pZA23BB 40
0.055 M. succinoproducens pZA23BB 40 0.214 A. succinogenes pZE13BB
40 0.041 M. succinoproducens pZE13BB 40 0.024 .DELTA.ppc Control
pZE13BB 40 0.042
[0574] Haemophilus influenza PEPCK was found to complement growth
in .DELTA.ppc mutant E. coli best among the genes that were tested
in the plasmid based screening. This gene was then integrated into
the PPC locus of wild-type E. coli (MG1655) using the SacB counter
selection method with pRE118-V2 discussed above (Gay et al., J.
Bacteriol. 153:1424-1431 (1983)). PEPCK was integrated retaining
the E. coli native PPC promoter, but utilizing the non-native PEPCK
terminator. The sequence of this region following replacement of
ppc by H. influenzae pepck is shown in FIG. 48. The pepck coding
region is underlined.
[0575] Techniques for adaptive evolution were applied to improve
the growth rate of the E. coli mutant (.DELTA.ppc::H. inf pepCK).
M9 minimal media with 4 g/L glucose and 50 mM sodium bicarbonate
was used to culture and evolve this strain in an anaerobic
environment. The high sodium bicarbonate concentration was used to
drive the equilibrium of the PEPCK reaction toward oxaloacetate
formation. To maintain exponential growth, the culture was diluted
2-fold whenever an OD600 of 0.5 was achieved. After about 100
generations over 3 weeks of adaptive evolution, anaerobic growth
rates improved from about 8 h to that of wild type, about 2 h.
Following evolution, individual colonies were isolated, and growth
in anaerobic bottles was compared to that of the initial mutant and
wild-type strain (see FIG. 49). M9 medium with 4 g/L glucose and 50
mM sodium bicarbonate was used.
[0576] The ppc/pepck gene replacement procedure described above was
then repeated, this time using the BDO-producing strains ECKh-432
(.DELTA.adhE .DELTA.ldhA .DELTA.pflB .DELTA.lpdA::K.p.lpdA322
.DELTA.mdh .DELTA.arcA gltAR163L .DELTA.ackA fimD:: E. coli sucCD,
P. gingivalis sucD, P. gingivalis 4hbd fimD:: M. bovis sucA, C.
kluyveri 4hbd) and ECKh-439 as the hosts. These strains contain the
TCA cycle enhancements discussed above as well as the upstream
pathway integrated in the chromosome. ECKh-439 is a derivative of
ECKh-432 that has the ackA gene deleted, which encodes acetate
kinase. This deletion was performed using the sacB counterselection
method described above.
[0577] The .DELTA.ppc::H. inf pepCK derivative of ECKh-439, called
ECKh-453, was run in a fermentation. The downstream BDO pathway was
supplied by pZS*13 containing P. gingivalis Cat2 and C.
beijerinckii Ald. This was performed with 1 L initial culture
volume in 2 L Biostat B+ bioreactors (Sartorius) using M9 minimal
medium supplemented with 20 g/L glucose and 50 mM NaHCO.sub.3. The
temperature was controlled at 37.degree. C., and the pH was
controlled at 7.0 using 2 M NH.sub.4OH or Na.sub.2CO.sub.3. Cells
were grown aerobically to an OD600 of approximately 2, at which
time the cultures were induced with 0.2 mM IPTG. One hour following
induction, the air flow rate was reduced to 0.01 standard liters
per minute for microaerobic conditions. The agitation rate was
initially set at 700 rpm. The aeration rate was gradually increased
throughout the fermentation as the culture density increased.
Concentrated glucose solution was fed to maintain glucose
concentration in the vessel between 0.5 and 10 g/L. The product
profile is shown in FIG. 50. The observed phenotype, in which BDO
and acetate are produced in approximately a one-to-one molar ratio,
is highly similar to that predicted in WO 2009/023493 for design
#439 (ADHEr, ASPT, LDH_D, MDH, PFLi). The deletion targeting the
ASPT reaction was deemed unnecessary as the natural flux through
aspartate ammonia-lyase is low.
[0578] A key feature of OptKnock strains is that production of the
metabolite of interest is generally coupled to growth, and further,
that, production should occur during exponential growth as well as
in stationary phase. The growth coupling potential of ECKh-432 and
ECKh-453 was evaluated by growth in microaerobic bottles with
frequent sampling during the exponential phase. M9 medium
containing 4 g/L glucose and either 10 mM NaHCO.sub.3 (for
ECKh-432) or 50 mM NaHCO.sub.3 (for ECKh-453) was used, and 0.2 mM
IPTG was included from inoculation. 18 G needles were used for
microaerobic growth of ECKh-432, while both 18 G and 27 G needles
were tested for ECKh-453. The higher gauge needles result in less
aeration. As shown in FIG. 51, ECKh-432 does not begin producing
BDO until 5 g/L glucose has been consumed, corresponding to the
onset of stationary phase. ECKh-453 produces BDO more evenly
throughout the experiment. In addition, growth coupling improves as
the aeration of the culture is reduced.
Example XVII
Integration of BDO Pathway Encoding Genes at Specific Integration
Sites
[0579] This example describes integration of various BDO pathway
genes into the fimD locus to provide more efficient expression and
stability.
[0580] The entire upstream BDO pathway, leading to 4HB, has been
integrated into the E. coli chromosome at the fimD locus. The
succinate branch of the upstream pathway was integrated into the E.
coli chromosome using the .lamda. red homologeous recombination
method (Datsenko and Wanner, Proc. Natl. Acad. Sci. USA
97:6640-6645 (2000)). The recipient E. coli strain was ECKh-422
(.DELTA.adhE .DELTA.ldhA .DELTA.pflB .DELTA.lpdA::K.p.lpdA322
.DELTA.mdh .DELTA.arcA gltAR163L). A polycistronic DNA fragment
containing a promoter, the sucCD gene, the sucD gene and the 4hbd
gene and a terminator sequence was inserted into the AflIII site of
the pKD3 plasmid. The following primers were used to amplify the
operon together with the chloramphenicol marker from the plasmid.
The underlined sequences are homologeous to the target insertion
site.
TABLE-US-00081 (SEQ ID NO: 41)
5'-GTTTGCACGCTATAGCTGAGGTTGTTGTCTTCCAGCAACGTACCGTA
TACAATAGGCGTATCACGAGGCCCTTTC-3' (SEQ ID NO: 42)
5'-GCTACAGCATGTCACACGATCTCAACGGTCGGATGACCAATCTGGCT
GGTATGGGAATTAGCCATGGTCC-3'
[0581] Following DpnI treatment and DNA electrophoresis, the
purified PCR product was used to transform E. coli strain harboring
plasmid pKD46. The candidate strain was selected on plates
containing chloramphenicol. Genomic DNA of the candidate strain was
purified. The insertion sequence was amplified and confirmed by DNA
sequencing. The chloramphenicol-resistant marker was removed from
chromosome by flipase. The nucleotide sequence of the region after
insertion and marker removal is shown in FIG. 52.
[0582] The alpha-ketoglutarate branch of the upstream pathway was
integrated into the chromosome by homologeous recombination. The
plasmid used in this modification was derived from vector
pRE118-V2, as referenced in Example XIV, which contains a
kanamycin-resistant gene, a gene encoding the levansucrase (sacB)
and a R6K conditional replication ori. The integration plasmid also
contained a polycistronic sequence with a promoter, the sucA gene,
the C. kluyveri 4hbd gene, and a terminator being inserted between
two 1.5-kb DNA fragments that are homologeous to the flanking
regions of the target insertion site. The resulting plasmid was
used to transform E. coli strain. The integration candidate was
selected on plates containing kanamycin. The correct integration
site was verified by PCR. To resolve the antibiotic marker from the
chromosome, the cells were selected for growth on medium containing
sucrose. The final strain was verified by PCR and DNA sequencing.
The nucleotide sequence of the chromosomal region after insertion
and marker removal is shown in FIG. 53.
[0583] The resulting upstream pathway integration strain ECKh-432
was transformed with a plasmid harboring the downstream pathway
genes. The construct was able to produce BDO from glucose in
minimal medium (see FIG. 54).
Example XVIII
Use of a Non-Phosphotransferase Sucrose Uptake System to Reduce
Pyruvate Byproduct Formation
[0584] This example describes the utilization of a
non-phosphotransferase (PTS) sucrose uptake system to reduce
pyruvate as a byproduct in the conversion of sucrose to BDO.
[0585] Strains engineered for the utilization of sucrose via a
phosphotransferase (PTS) system produce significant amounts of
pyruvate as a byproduct. Therefore, the use of a non-PTS sucrose
system can be used to decrease pyruvate formation because the
import of sucrose would not be accompanied by the conversion of
phosphoenolpyruvate (PEP) to pyruvate. This will increase the PEP
pool and the flux to oxaloacetate through PPC or PEPCK.
[0586] Insertion of a non-PTS sucrose operon into the rrnC region
was performed. To generate a PCR product containing the non-PTS
sucrose genes flanked by regions of homology to the rrnC region,
two oligos were used to PCR amplify the csc genes from Mach1.TM.
(Invitrogen, Carlsbad, Calif.). This strain is a descendent of W
strain which is an E. coli strain known to be able to catabolize
sucrose (Orencio-Trejo et al., Biotechnology Biofuels 1:8 (2008)).
The sequence was derived from E. coli W strain KO11 (accession
AY314757) (Shukla et al., Biotechnol. Lett. 26:689-693 (2004)) and
includes genes encoding a sucrose permease (cscB), D-fructokinase
(cscK), sucrose hydrolase (cscA), and a LacI-related
sucrose-specific repressor (cscR). The first 53 amino acids of cscR
was effectively removed by the placement of the AS primer. The
sequences of the oligos were: rrnC 23S del S-CSC 5'-TGT GAG TGA AAG
TCA CCT GCC TTA ATA TCT CAA AAC TCA TCT TCG GGT GAC GAA ATA TGG CGT
GAC TCG ATA C-3' (SEQ ID NO:43) and rrnC 23S del AS-CSC 5'-TCT GTA
TCA GGC TGA AAA TCT TCT CTC ATC CGC CAA AAC AGC TTC GGC GTT AAG ATG
CGC GCT CAA GGA C-3' (SEQ ID NO:44). Underlined regions indicate
homology to the csc operon, and bold sequence refers to sequence
homology upstream and downstream of the rrnC region. The sequence
of the entire PCR product is shown in FIG. 55.
[0587] After purification, the PCR product was electroporated into
MG1655 electrocompetent cells which had been transformed with
pRedET (tet) and prepared according to manufacturer's instructions
(genebridges.com/gb/pdf/K001%20Q%20E%20BAC%20Modification%20Kit-version2.-
6-2007-screen.pdf). The PCR product was designed so that it
integrated into genome into the rrnC region of the chromosome. It
effectively deleted 191 nucleotides upstream of rrlC (23S rRNA),
all of the rrlC rRNA gene and 3 nucleotides downstream of rrlC and
replaced it with the sucrose operon, as shown in FIG. 56.
[0588] Transformants were grown on M9 minimal salts medium with
0.4% sucrose and individual colonies tested for presence of the
sucrose operon by diagnostic PCR. The entire rrnC::crcAKB region
was transferred into the BDO host strain ECKh-432 by P1
transduction (Sambrook et al., Molecular Cloning: A Laboratory
Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001),
resulting in ECKh-463 (.DELTA.adhE .DELTA.ldhA .DELTA.pflB
.DELTA.lpdA::K.p.lpdA322 .DELTA.mdh .DELTA.arcA gltAR163L fimD:: E.
coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd fimD:: M. bovis
sucA, C. kluyveri 4hbd rrnC::cscAKB). Recombinants were selected by
growth on sucrose and verified by diagnostic PCR.
[0589] ECKh-463 was transformed with pZS*13 containing P.
gingivalis Cat2 and C. beijerinckii Ald to provide a complete BDO
pathway. Cells were cultured in M9 minimal medium (6.78 g/L
Na.sub.2HPO.sub.4, 3.0 g/L KH.sub.2PO.sub.4, 0.5 g/L NaCl, 1.0 g/L
NH.sub.4Cl, 1 mM MgSO.sub.4, 0.1 mM CaCl.sub.2) supplemented with
10 g/L sucrose. 0.2 mM IPTG was present in the culture from the
start. Anaerobic conditions were maintained using a bottle with 23
G needle. As a control, ECKh-432 containing the same plasmid was
cultured on the same medium, except with 10 g/L glucose instead of
sucrose. FIG. 57 shows average product concentration, normalized to
culture OD600, after 48 hours of growth. The data is for 6
replicate cultures of each strain. This demonstrates that BDO
production from ECKh-463 on sucrose is similar to that of the
parent strain on sucrose.
Example XIX
Summary of BDO Producing Strains
[0590] This example describes various BDO producing strains.
[0591] Table 27 summarizes various BDO producing strains disclosed
above in Examples XII-XVIII.
TABLE-US-00082 TABLE 27 Summary of various BDO production strains.
Strain # Host Strain # Host chromosome Host Description
Plasmid-based 1 .DELTA.ldhA Single deletion E. coli sucCD, P.
gingivalis derivative of E. coli sucD, P. gingivalis 4hbd, MG1655
P. gingivalis Cat2, C. acetobutylicum AdhE2 2 AB3 .DELTA.adhE
.DELTA.ldhA .DELTA.pflB Succinate producing E. coli sucCD, P.
gingivalis strain; derivative of sucD, P. gingivalis 4hbd, E. coli
MG1655 P. gingivalis Cat2, C. acetobutylicum AdhE2 3 ECKh-138
.DELTA.adhE .DELTA.ldhA .DELTA.pflB Improvement of E. coli sucCD,
P. gingivalis .DELTA.lpdA::K.p.lpdA322 lpdA to increase sucD, P.
gingivalis 4hbd, pyruvate P. gingivalis Cat2, dehydrogenase flux C.
acetobutylicum AdhE2 4 ECKh-138 .DELTA.adhE .DELTA.ldhA .DELTA.pflB
E. coli sucCD, P. gingivalis .DELTA.lpdA::K.p.lpdA322 sucD, P.
gingivalis 4hbd, C. acetobutylicum buk1, C. acetobutylicum ptb, C.
acetobutylicum AdhE2 5 ECKh-401 .DELTA.adhE .DELTA.ldhA .DELTA.pflB
Deletions in mdh and E. coli sucCD, P. gingivalis
.DELTA.lpdA::K.p.lpdA322 .DELTA.mdh .DELTA.arcA arcA to direct flux
sucD, P. gingivalis 4hbd, through oxidative P. gingivalis Cat2, TCA
cycle C. acetobutylicum AdhE2 6 ECKh-401 .DELTA.adhE .DELTA.ldhA
.DELTA.pflB M. bovis sucA, E. coli sucCD, .DELTA.lpdA::K.p.lpdA322
.DELTA.mdh .DELTA.arcA P. gingivalis sucD, P. gingivalis 4hbd, P.
gingivalis Cat2, C. acetobutylicum AdhE2 7 ECKh-422 .DELTA.adhE
.DELTA.ldhA .DELTA.pflB Mutation in citrate E. coli sucCD, P.
gingivalis .DELTA.lpdA::K.p.lpdA322 .DELTA.mdh .DELTA.arcA synthase
to improve sucD, P. gingivalis 4hbd, gltAR163L anaerobic activity
P. gingivalis Cat2, C. acetobutylicum AdhE2 8 ECKh-422 .DELTA.adhE
.DELTA.ldhA .DELTA.pflB M. bovis sucA, E. coli sucCD,
.DELTA.lpdA::K.p.lpdA322 .DELTA.mdh .DELTA.arcA P. gingivalis sucD,
gltAR163L P. gingivalis 4hbd, P. gingivalis Cat2, C. acetobutylicum
AdhE2 9 ECKh-422 .DELTA.adhE .DELTA.ldhA .DELTA.pflB M. bovis sucA,
E. coli sucCD, .DELTA.lpdA::K.p.lpdA322 .DELTA.mdh .DELTA.arcA P.
gingivalis sucD, gltAR163L P. gingivalis 4hbd, P. gingivalis Cat2,
C. beijerinckii Ald 10 ECKh-426 .DELTA.adhE .DELTA.ldhA .DELTA.pflB
Succinate branch of P. gingivalis Cat2, .DELTA.lpdA::K.p.lpdA322
.DELTA.mdh .DELTA.arcA upstream pathway C. beijerinckii Ald
gltAR163L fimD:: E. coli sucCD, integrated into P. gingivalis sucD,
P. gingivalis 4hbd ECKh-422 11 ECKh-432 .DELTA.adhE .DELTA.ldhA
.DELTA.pflB Succinate and alpha- P. gingivalis Cat2,
.DELTA.lpdA::K.p.lpdA322 .DELTA.mdh .DELTA.arcA ketoglutarate C.
beijerinckii Ald gltAR163L fimD:: E. coli sucCD, upstream pathway
P. gingivalis sucD, P. gingivalis 4hbd branches integrated fimD::
M. bovis sucA, C. kluyveri into ECKh-422 4hbd 12 ECKh-432
.DELTA.adhE .DELTA.ldhA .DELTA.pflB C. acetobutylicum buk1,
.DELTA.lpdA::K.p.lpdA322 .DELTA.mdh .DELTA.arcA C. acetobutylicum
ptb, gltAR163L fimD:: E. coli sucCD, C. beijerinckii Ald P.
gingivalis sucD, P. gingivalis 4hbd fimD:: M. bovis sucA, C.
kluyveri 4hbd 13 ECKh-439 .DELTA.adhE .DELTA.vldhA .DELTA.pflB
Acetate kinase P. gingivalis Cat2, .DELTA.lpdA::K.p.lpdA322
.DELTA.mdh .DELTA.arcA deletion of ECKh- C. beijerinckii Ald
gltAR163L .DELTA.ackA fimD:: E. coli 432 sucCD, P. gingivalis sucD,
P. gingivalis 4hbd fimD:: M. bovis sucA, C. kluyveri 4hbd 14
ECKh-453 .DELTA.adhE .DELTA.ldhA .DELTA.pflB Acetate kinase P.
gingivalis Cat2, .DELTA.lpdA::K.p.lpdA322 .DELTA.mdh .DELTA.arcA
deletion and C. beijerinckii Ald gltAR163L .DELTA.ackA
.DELTA.ppc::H.i.ppck PPC/PEPCK fimD:: E. coli sucCD, P. gingivalis
replacement of sucD, P. gingivalis 4hbd fimD:: ECKh-432 M. bovis
sucA, C. kluyveri 4hbd 15 ECKh-456 .DELTA.adhE .DELTA.ldhA
.DELTA.pflB .DELTA.lpdA::fnr-pflB6- Replacement of lpdA P.
gingivalis Cat2, K.p.lpdA322 .DELTA.mdh .DELTA.arcA gltAR163L
promoter with C. beijerinckii Ald fimD:: E. coli sucCD, P.
gingivalis anaerobic promoter sucD, P. gingivalis 4hbd fimD:: in
ECKh-432 M. bovis sucA, C. kluyveri 4hbd 16 ECKh-455 .DELTA.adhE
.DELTA.ldhA .DELTA.pflB .DELTA.lpdA:: Replacement of P. gingivalis
Cat2, K.p.lpdA322 .DELTA.pdhR:: fnr-pflB6 .DELTA.mdh pdhR and aceEF
C. beijerinckii Ald .DELTA.arcA gltAR163L fimD:: E. coli promoter
with sucCD, P. gingivalis sucD, anaerobic promoter P. gingivalis
4hbd fimD:: M. bovis sucA, in ECKh-432 C. kluyveri 4hbd 17 ECKh-459
.DELTA.adhE .DELTA.ldhA .DELTA.pflB .DELTA.lpdA:: Integration of C.
beijerinckii Ald K.p.lpdA322 .DELTA.mdh .DELTA.arcA gltAR163L
BK/PTB into ECKh- fimD:: E. coli sucCD, P. gingivalis 432 sucD, P.
gingivalis 4hbd fimD:: M. bovis sucA, C. kluyveri 4hbd fimD:: C.
acetobutylicum buk1, C. acetobutylicum ptb 18 ECKh-459 .DELTA.adhE
.DELTA.ldhA .DELTA.pflB .DELTA.lpdA:: C. beijerinckii Ald,
K.p.lpdA322 .DELTA.mdh .DELTA.arcA gltAR163L G. thermoglucosidasius
adh1 fimD:: E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd
fimD:: M. bovis sucA, C. kluyveri 4hbd fimD:: C. acetobutylicum
buk1, C. acetobutylicum ptb 19 ECKh-463 .DELTA.adhE .DELTA.ldhA
.DELTA.pflB Non-PTS sucrose P. gingivalis Cat2,
.DELTA.lpdA::K.p.lpdA322 .DELTA.mdh genes inserted into C.
beijerinckii Ald .DELTA.arcA gltAR163L fimD:: E. coli ECKh-432
sucCD, P. gingivalis sucD, P. gingivalis 4hbd fimD:: M. bovis sucA,
C. kluyveri 4hbd rrnC::cscAKB 20 ECKh-463 .DELTA.adhE .DELTA.ldhA
.DELTA.pflB C. acetobutylicum buk1, .DELTA.lpdA::K.p.lpdA322
.DELTA.mdh C. acetobutylicum ptb, .DELTA.arcA gltAR163L fimD:: E.
coli C. beijerinckii Ald sucCD, P. gingivalis sucD, P. gingivalis
4hbd fimD:: M. bovis sucA, C. kluyveri 4hbd rrnC::cscAKB
[0592] The strains summarized in Table 27 are as follows. Strain 1:
Single deletion derivative of E. coli MG1655, with deletion of
endogenous ldhA; plasmid expression of E. coli sucCD, P. gingivalis
sucD, P. gingivalis 4hbd, P. gingivalis Cat2, C. acetobutylicum
AdhE2. Strain 2: Host strain AB3, a succinate producing strain,
derivative of E. coli MG1655, with deletions of endogenous adhE
ldhA pflB; plasmid expression of E. coli sucCD, P. gingivalis sucD,
P. gingivalis 4hbd, P. gingivalis Cat2, C. acetobutylicum
AdhE2.
[0593] Strain 3: Host strain ECKh-138, deletion of endogenous adhE,
ldhA, pflB, deletion of endogenous lpdA and chromosomal insertion
of Klebsiella pneumoniae lpdA with a Glu354Lys mutation at the lpdA
locus; plasmid expression of E. coli sucCD, P. gingivalis sucD, P.
gingivalis 4hbd, P. gingivalis Cat2, C. acetobutylicum AdhE2;
strain provides improvement of lpdA to increase pyruvate
dehydrogenase flux. Strain 4: Host strain ECKh-138, deletion of
endogenous adhE, ldhA, pflB, and lpdA, chromosomal insertion of
Klebsiella pneumoniae lpdA with a Glu354Lys mutation; plasmid
expression E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd,
C. acetobutylicum buk1, C. acetobutylicum ptb, C. acetobutylicum
AdhE2.
[0594] Strain 5: Host strain ECKh-401, deletion of endogenous adhE,
ldhA, pflB, deletion of endogenous lpdA and chromosomal insertion
of Klebsiella pneumoniae lpdA with a Glu354Lys mutation at the lpdA
locus, deletion of endogenous mdh and arcA; plasmid expression of
E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd, P.
gingivalis Cat2, C. acetobutylicum AdhE2; strain has deletions in
mdh and arcA to direct flux through oxidative TCA cycle. Strain 6:
host strain ECKh-401, deletion of endogenous adhE, ldhA, pflB,
deletion of endogenous lpdA and chromosomal insertion of Klebsiella
pneumoniae lpdA with a Glu354Lys mutation at the lpdA locus,
deletion of endogenous mdh and arcA; plasmid expression of M. bovis
sucA, E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd, P.
gingivalis Cat2, C. acetobutylicum AdhE2.
[0595] Strain 7: Host strain ECKh-422, deletion of endogenous adhE,
ldhA, pflB, deletion of endogenous lpdA and chromosomal insertion
of Klebsiella pneumoniae lpdA with a Glu354Lys mutation at the lpdA
locus, deletion of endogenous mdh and arcA, chromosomal replacement
of gltA with gltA Arg163Leu mutant; plasmid expression of E. coli
sucCD, P. gingivalis sucD, P. gingivalis 4hbd, P. gingivalis Cat2,
C. acetobutylicum AdhE2; strain has mutation in citrate synthase to
improve anaerobic activity. Strain 8: strain ECKh-422, deletion of
endogenous adhE, ldhA, pflB, deletion of endogenous lpdA and
chromosomal insertion of Klebsiella pneumoniae lpdA with a
Glu354Lys mutation at the lpdA locus, deletion of endogenous mdh
and arcA, chromosomal replacement of gltA with gltA Arg163Leu
mutant; plasmid expression of M. bovis sucA, E. coli sucCD, P.
gingivalis sucD, P. gingivalis 4hbd, P. gingivalis Cat2, C.
acetobutylicum AdhE2. Strain 9: host strain ECKh-422, deletion of
endogenous adhE, ldhA, pflB, deletion of endogenous lpdA and
chromosomal insertion of Klebsiella pneumoniae lpdA with a
Glu354Lys mutation at the lpdA locus, deletion of endogenous mdh
and arcA, chromosomal replacement of gltA with gltA Arg163Leu
mutant; plasmid expression of M. bovis sucA, E. coli sucCD, P.
gingivalis sucD, P. gingivalis 4hbd, P. gingivalis Cat2, C.
beijerinckii Ald.
[0596] Strain 10: host strain ECKh-426, deletion of endogenous
adhE, ldhA, pflB, deletion of endogenous lpdA and chromosomal
insertion of Klebsiella pneumoniae lpdA with a Glu354Lys mutation
at the lpdA locus, deletion of endogenous mdh and arcA, chromosomal
replacement of gltA with gltA Arg163Leu mutant, chromosomal
insertion at the fimD locus of E. coli sucCD, P. gingivalis sucD,
P. gingivalis 4hbd; plasmid expression of P. gingivalis Cat2, C.
beijerinckii Ald; strain has succinate branch of upstream pathway
integrated into strain ECKh-422 at the fimD locus. Strain 11: host
strain ECKh-432, deletion of endogenous adhE, ldhA, pflB, deletion
of endogenous lpdA and chromosomal insertion of Klebsiella
pneumoniae lpdA with a Glu354Lys mutation at the lpdA locus,
deletion of endogenous mdh and arcA, chromosomal replacement of
gltA with gltA Arg163Leu mutant, chromosomal insertion at the fimD
locus of E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd,
chromosomal insertion at the fimD locus of M. bovis sucA, C.
kluyveri 4hbd; plasmid expression of P. gingivalis Cat2, C.
beijerinckii Ald; strain has succinate and alpha-ketoglutarate
upstream pathway branches integrated into ECKh-422. Strain 12: host
strain ECKh-432, deletion of endogenous adhE, ldhA, pflB, deletion
of endogenous lpdA and chromosomal insertion of Klebsiella
pneumoniae lpdA with a Glu354Lys mutation at the lpdA locus,
deletion of endogenous mdh and arcA, chromosomal replacement of
gltA with gltA Arg163Leu mutant, chromosomal insertion at the fimD
locus of E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd,
chromosomal insertion at the fimD locus of M. bovis sucA, C.
kluyveri 4hbd; plasmid expression of C. acetobutylicum buk1, C.
acetobutylicum ptb, C. beijerinckii Ald.
[0597] Strain 13: host strain ECKh-439, deletion of endogenous
adhE, ldhA, pflB, deletion of endogenous lpdA and chromosomal
insertion of Klebsiella pneumoniae lpdA with a Glu354Lys mutation
at the lpdA locus, deletion of endogenous mdh and arcA, chromosomal
replacement of gltA with gltA Arg163Leu mutant, deletion of
endogenous ackA, chromosomal insertion at the fimD locus of E. coli
sucCD, P. gingivalis sucD, P. gingivalis 4hbd, chromosomal
insertion at the fimD locus of M. bovis sucA, C. kluyveri 4hbd;
plasmid expression of P. gingivalis Cat2, C. beijerinckii Ald;
strain has acetate kinase deletion in strain ECKh-432. Strain 14:
host strain ECKh-453, deletion of endogenous adhE, ldhA, pflB,
deletion of endogenous lpdA and chromosomal insertion of Klebsiella
pneumoniae lpdA with a Glu354Lys mutation at the lpdA locus,
deletion of endogenous mdh and arcA, chromosomal replacement of
gltA with gltA Arg163Leu mutant, deletion of endogenous ackA,
deletion of endogenous ppc and insertion of Haemophilus influenza
ppck at the ppc locus, chromosomal insertion at the fimD locus of
E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd, chromosomal
insertion at the fimD locus of M. bovis sucA, C. kluyveri 4hbd;
plasmid expression of P. gingivalis Cat2, C. beijerinckii Ald;
strain has acetate kinase deletion and PPC/PEPCK replacement in
strain ECKh-432.
[0598] Strain 15: host strain ECKh-456, deletion of endogenous
adhE, ldhA, pflB, deletion of endogenous lpdA and chromosomal
insertion of Klebsiella pneumoniae lpdA with a Glu354Lys mutation
at the lpdA locus, deletion of endogenous mdh and arcA, chromosomal
replacement of gltA with gltA Arg163Leu mutant, chromosomal
insertion at the fimD locus of E. coli sucCD, P. gingivalis sucD,
P. gingivalis 4hbd, chromosomal insertion at the fimD locus of M.
bovis sucA, C. kluyveri 4hbd, replacement of lpdA promoter with fnr
binding site, pflB-p6 promoter and RBS of pflB; plasmid expression
of P. gingivalis Cat2, C. beijerinckii Ald; strain has replacement
of lpdA promoter with anaerobic promoter in strain ECKh-432. Strain
16: host strain ECKh-455, deletion of endogenous adhE, ldhA, pflB,
deletion of endogenous lpdA and chromosomal insertion of Klebsiella
pneumoniae lpdA with a Glu354Lys mutation at the lpdA locus,
deletion of endogenous mdh and arcA, chromosomal replacement of
gltA with gltA Arg163Leu mutant, chromosomal insertion at the fimD
locus of E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd,
chromosomal insertion at the fimD locus of M. bovis sucA, C.
kluyveri 4hbdI, replacement of pdhR and aceEF promoter with fnr
binding site, pflB-p6 promoter and RBS of pflB; plasmid expression
of P. gingivalis Cat2, C. beijerinckii Ald; strain has replacement
of pdhR and aceEF promoter with anaerobic promoter in ECKh-432.
[0599] Strain 17: host strain ECKh-459, deletion of endogenous
adhE, ldhA, pflB, deletion of endogenous lpdA and chromosomal
insertion of Klebsiella pneumoniae lpdA with a Glu354Lys mutation
at the lpdA locus, deletion of endogenous mdh and arcA, chromosomal
replacement of gltA with gltA Arg163Leu mutant, chromosomal
insertion at the fimD locus of E. coli sucCD, P. gingivalis sucD,
P. gingivalis 4hbd, chromosomal insertion at the fimD locus of M.
bovis sucA, C. kluyveri 4hbd, chromosomal insertion at the fimD
locus of C. acetobutylicum buk1, C. acetobutylicum ptb; plasmid
expression of C. beijerinckii Ald; strain has integration of BK/PTB
into strain ECKh-432. Strain 18: host strain ECKh-459, deletion of
endogenous adhE, ldhA, pflB, deletion of endogenous lpdA and
chromosomal insertion of Klebsiella pneumoniae lpdA with a
Glu354Lys mutation at the lpdA locus, deletion of endogenous mdh
and arcA, chromosomal replacement of gltA with gltA Arg163Leu
mutant, chromosomal insertion at the fimD locus of E. coli sucCD,
P. gingivalis sucD, P. gingivalis 4hbd, chromosomal insertion at
the fimD locus of M. bovis sucA, C. kluyveri 4hbd, chromosomal
insertion at the fimD locus of C. acetobutylicum buk1, C.
acetobutylicum ptb; plasmid expression of C. beijerinckii Ald, G.
thermoglucosidasius adh1.
[0600] Strain 19: host strain ECKh-463, deletion of endogenous
adhE, ldhA, pflB, deletion of endogenous lpdA and chromosomal
insertion of Klebsiella pneumoniae lpdA with a Glu354Lys mutation
at the lpdA locus, deletion of endogenous mdh and arcA, chromosomal
replacement of gltA with gltA Arg163Leu mutant, chromosomal
insertion at the fimD locus of E. coli sucCD, P. gingivalis sucD,
P. gingivalis 4hbd, chromosomal insertion at the fimD locus of M.
bovis sucA, C. kluyveri 4hbd, insertion at the rrnC locus of
non-PTS sucrose operon genes sucrose permease (cscB),
D-fructokinase (cscK), sucrose hydrolase (cscA), and a LacI-related
sucrose-specific repressor (cscR); plasmid expression of P.
gingivalis Cat2, C. beijerinckii Ald; strain has non-PTS sucrose
genes inserted into strain ECKh-432. Strain 20: host strain
ECKh-463 deletion of endogenous adhE, ldhA, pflB, deletion of
endogenous lpdA and chromosomal insertion of Klebsiella pneumoniae
lpdA with a Glu354Lys mutation at the lpdA locus, deletion of
endogenous mdh and arcA, chromosomal replacement of gltA with gltA
Arg163Leu mutant, chromosomal insertion at the fimD locus of E.
coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd, chromosomal
insertion at the fimD locus of M. bovis sucA, C. kluyveri 4hbd,
insertion at the rrnC locus of non-PTS sucrose operon; plasmid
expression of C. acetobutylicum buk1, C. acetobutylicum ptb, C.
beijerinckii Ald.
[0601] In addition to the BDO producing strains disclosed herein,
including those disclosed in Table 27, it is understood that
additional modifications can be incorporated that further increase
production of BDO and/or decrease undesirable byproducts. For
example, a BDO producing strain, or a strain of Table 27, can
incorporate additional knockouts to further increase the production
of BDO or decrease an undesirable byproduct. Exemplary knockouts
have been described previously (see U.S. publication 2009/0047719).
Such knockout strains include, but are not limited to, ADHEr,
NADH6; ADHEr, PPCK; ADHEr, SUCD4; ADHEr, ATPS4r; ADHEr, FUM; ADHEr,
MDH; ADHEr, PFLi, PPCK; ADHEr, PFLi, SUCD4; ADHEr, ACKr, NADH6;
ADHEr, NADH6, PFLi; ADHEr, ASPT, MDH; ADHEr, NADH6, PPCK; ADHEr,
PPCK, THD2; ADHEr, ATPS4r, PPCK; ADHEr, MDH, THD2; ADHEr, FUM,
PFLi; ADHEr, PPCK, SUCD4; ADHEr, GLCpts, PPCK; ADHEr, GLUDy, MDH;
ADHEr, GLUDy, PPCK; ADHEr, FUM, PPCK; ADHEr, MDH, PPCK; ADHEr, FUM,
GLUDy; ADHEr, FUM, HEX1; ADHEr, HEX1, PFLi; ADHEr, HEX1, THD2;
ADHEr, FRD2, LDH_D, MDH; ADHEr, FRD2, LDH_D, ME2; ADHEr, MDH, PGL,
THD2; ADHEr, G6PDHy, MDH, THD2; ADHEr, PFLi, PPCK, THD2; ADHEr,
ACKr, AKGD, ATPS4r; ADHEr, GLCpts, PFLi, PPCK; ADHEr, ACKr, ATPS4r,
SUCOAS; ADHEr, GLUDy, PFLi, PPCK; ADHEr, ME2, PFLi, SUCD4; ADHEr,
GLUDy, PFLi, SUCD4; ADHEr, ATPS4r, LDH_D, SUCD4; ADHEr, FUM, HEX1,
PFLi; ADHEr, MDH, NADH6, THD2; ADHEr, ATPS4r, MDH, NADH6; ADHEr,
ATPS4r, FUM, NADH6; ADHEr, ASPT, MDH, NADH6; ADHEr, ASPT, MDH,
THD2; ADHEr, ATPS4r, GLCpts, SUCD4; ADHEr, ATPS4r, GLUDy, MDH;
ADHEr, ATPS4r, MDH, PPCK; ADHEr, ATPS4r, FUM, PPCK; ADHEr, ASPT,
GLCpts, MDH; ADHEr, ASPT, GLUDy, MDH; ADHEr, ME2, SUCD4, THD2;
ADHEr, FUM, PPCK, THD2; ADHEr, MDH, PPCK, THD2; ADHEr, GLUDy, MDH,
THD2; ADHEr, HEX1, PFLi, THD2; ADHEr, ATPS4r, G6PDHy, MDH; ADHEr,
ATPS4r, MDH, PGL; ADHEr, ACKr, FRD2, LDH_D; ADHEr, ACKr, LDH_D,
SUCD4; ADHEr, ATPS4r, FUM, GLUDy; ADHEr, ATPS4r, FUM, HEX1; ADHEr,
ATPS4r, MDH, THD2; ADHEr, ATPS4r, FRD2, LDH_D; ADHEr, ATPS4r, MDH,
PGDH; ADHEr, GLCpts, PPCK, THD2; ADHEr, GLUDy, PPCK, THD2; ADHEr,
FUM, HEX1, THD2; ADHEr, ATPS4r, ME2, THD2; ADHEr, FUM, ME2, THD2;
ADHEr, GLCpts, GLUDy, PPCK; ADHEr, ME2, PGL, THD2; ADHEr, G6PDHy,
ME2, THD2; ADHEr, ATPS4r, FRD2, LDH_D, ME2; ADHEr, ATPS4r, FRD2,
LDH_D, MDH; ADHEr, ASPT, LDH_D, MDH, PFLi; ADHEr, ATPS4r, GLCpts,
NADH6, PFLi; ADHEr, ATPS4r, MDH, NADH6, PGL; ADHEr, ATPS4r, G6PDHy,
MDH, NADH6; ADHEr, ACKr, FUM, GLUDy, LDH_D; ADHEr, ACKr, GLUDy,
LDH_D, SUCD4; ADHEr, ATPS4r, G6PDHy, MDH, THD2; ADHEr, ATPS4r, MDH,
PGL, THD2; ADHEr, ASPT, G6PDHy, MDH, PYK; ADHEr, ASPT, MDH, PGL,
PYK; ADHEr, ASPT, LDH_D, MDH, SUCOAS; ADHEr, ASPT, FUM, LDH_D, MDH;
ADHEr, ASPT, LDH_D, MALS, MDH; ADHEr, ASPT, ICL, LDH_D, MDH; ADHEr,
FRD2, GLUDy, LDH_D, PPCK; ADHEr, FRD2, LDH_D, PPCK, THD2; ADHEr,
ACKr, ATPS4r, LDH_D, SUCD4; ADHEr, ACKr, ACS, PPC, PPCK; ADHEr,
GLUDy, LDH_D, PPC, PPCK; ADHEr, LDH_D, PPC, PPCK, THD2; ADHEr,
ASPT, ATPS4r, GLCpts, MDH; ADHEr, G6PDHy, MDH, NADH6, THD2; ADHEr,
MDH, NADH6, PGL, THD2; ADHEr, ATPS4r, G6PDHy, GLCpts, MDH; ADHEr,
ATPS4r, GLCpts, MDH, PGL; ADHEr, ACKr, LDH_D, MDH, SUCD4.
[0602] Table 28 shows the reactions of corresponding genes to be
knocked out of a host organism such as E. coli. The corresponding
metabolite corresponding to abbreviations in Table 28 are shown in
Table 29.
TABLE-US-00083 TABLE 28 Corresponding genes to be knocked out to
prevent a particular reaction from occurring in E. coli. Reaction
Genes Encoding the Enzyme(s) Abbreviation Reaction Stoichiometry*
Catalyzing Each Reaction& ACKr [c]: ac + atp <==> actp +
adp (b3115 or b2296 or b1849) ACS [c]: ac + atp + coa --> accoa
+ amp + ppi b4069 ACt6 ac[p] + h[p] <==> ac[c] + h[c]
Non-gene associated ADHEr [c]: etoh + nad <==> acald + h +
nadh (b0356 or b1478 or [c]: acald + coa + nad <==> accoa + h
+ nadh b1241) (b1241 or b0351) AKGD [c]: akg + coa + nad --> co2
+ nadh + succoa (b0116 and b0726 and b0727) ASNS2 [c]: asp-L + atp
+ nh4 --> amp + asn-L + h + ppi b3744 ASPT [c]: asp-L --> fum
+ nh4 b4139 ATPS4r adp[c] + (4) h[p] + pi[c] <==> atp[c] +
(3) h[c] + h2o[c] (((b3736 and b3737 and b3738) and (b3731 and
b3732 and b3733 and b3734 and b3735)) or ((b3736 and b3737 and
b3738) and (b3731 and b3732 and b3733 and b3734 and b3735) and
b3739)) CBMK2 [c]: atp + co2 + nh4 <==> adp + cbp + (2) h
(b0521 or b0323 or b2874) EDA [c]: 2ddg6p --> g3p + pyr b1850
ENO [c]: 2pg <==> h2o + pep b2779 FBA [c]: fdp <==>
dhap + g3p (b2097 or b2925 or b1773) FBP [c]: fdp + h2o --> f6p
+ pi (b4232 or b3925) FDH2 for[p] + (2) h[c] + q8[c] --> co2[c]
+ h[p] + q8h2[c] ((b3892 and b3893 for[p] + (2) h[c] + mqn8[c]
--> co2[c] + h[p] + mql8[c] and b3894) or (b1474 and b1475 and
b1476)) FRD2 [c]: fum + mql8 --> mqn8 + succ (b4151 and b4152
[c]: 2dmmql8 + fum --> 2dmmq8 + succ and b4153 and b4154) FTHFD
[c]: 10fthf + h2o --> for + h + thf b1232 FUM [c]: fum + h2o
<==> mal-L (b1612 or b4122 or b1611) G5SD [c]: glu5p + h +
nadph --> glu5sa + nadp + pi b0243 G6PDHy [c]: g6p + nadp
<==> 6pgl + h + nadph b1852 GLCpts glc-D[p] + pep[c] -->
g6p[c] + pyr[c] ((b2417 and b1101 and b2415 and b2416) or (b1817
and b1818 and b1819 and b2415 and b2416) or (b2417 and b1621 and
b2415 and b2416)) GLU5K [c]: atp + glu-L --> adp + glu5p b0242
GLUDy [c]: glu-L + h2o + nadp <==> akg + h + nadph + nh4
b1761 GLYCL [c]: gly + nad + thf --> co2 + mlthf + nadh + nh4
(b2904 and b2903 and b2905 and b0116) HEX1 [c]: atp + glc-D -->
adp + g6p + h b2388 ICL [c]: icit --> glx + succ b4015 LDH_D
[c]: lac-D + nad <==> h + nadh + pyr (b2133 or b1380) MALS
[c]: accoa + glx + h2o --> coa + h + mal-L (b4014 or b2976) MDH
[c]: mal-L + nad <==> h + nadh + oaa b3236 ME2 [c]: mal-L +
nadp --> co2 + nadph + pyr b2463 MTHFC [c]: h2o + methf
<==> 10fthf + h b0529 NADH12 [c]: h + mqn8 + nadh --> mql8
+ nad b1109 [c]: h + nadh + q8 --> nad + q8h2 [c]: 2dmmq8 + h +
nadh --> 2dmmql8 + nad NADH6 (4) h[c] + nadh[c] + q8[c] -->
(3) h[p] + nad[c] + q8h2[c] (b2276 and b2277 (4) h[c] + mqn8[c] +
nadh[c] --> (3) h[p] + mql8[c] + and b2278 and b2279 nad[c] and
b2280 and b2281 2dmmq8[c] + (4) h[c] + nadh[c] --> 2dmmql8[c] +
(3) and b2282 and b2283 h[p] + nad[c] and b2284 and b2285 and b2286
and b2287 and b2288) PFK [c]: atp + f6p --> adp + fdp + h (b3916
or b1723) PFLi [c]: coa + pyr --> accoa + for (((b0902 and
b0903) and b2579) or (b0902 and b0903) or (b0902 and b3114) or
(b3951 and b3952)) PGDH [c]: 6pgc + nadp --> co2 + nadph +
ru5p-D b2029 PGI [c]: g6p <==> f6p b4025 PGL [c]: 6pgl + h2o
--> 6pgc + h b0767 PGM [c]: 2pg <==> 3pg (b3612 or b4395
or b0755) PPC [c]: co2 + h2o + pep --> h + oaa + pi b3956 PPCK
[c]: atp + oaa --> adp + co2 + pep b3403 PRO1z [c]: fad + pro-L
--> 1pyr5c + fadh2 + h b1014 PYK [c]: adp + h + pep --> atp +
pyr b1854 or b1676) PYRt2 h[p] + pyr[p] <==> h[c] + pyr[c]
Non-gene associated RPE [c]: ru5p-D <==> xu5p-D (b4301 or
b3386) SO4t2 so4[e] <==> so4[p] (b0241 or b0929 or b1377 or
b2215) SUCD4 [c]: q8 + succ --> fum + q8h2 (b0721 and b0722 and
b0723 and b0724) SUCOAS [c]: atp + coa + succ <==> adp + pi +
succoa (b0728 and b0729) SULabc atp[c] + h2o[c] + so4[p] -->
adp[c] + h[c] + pi[c] + ((b2422 and b2425 so4[c] and b2424 and
b2423) or (b0763 and b0764 and b0765) or (b2422 and b2424 and b2423
and b3917)) TAL [c]: g3p + s7p <==> e4p + f6p (b2464 or
b0008) THD2 (2) h[p] + nadh[c] + nadp[c] --> (2) h[c] + nad[c] +
(b1602 and b1603) nadph[c] THD5 [c]: nad + nadph --> nadh + nadp
(b3962 or (b1602 and b1603)) TPI [c]: dhap <==> g3p b3919
TABLE-US-00084 TABLE 29 Metabolite names corresponding to
abbreviations used in Table 28. Metabolite Abbreviation Metabolite
Name 10fthf 10-Formyltetrahydrofolate 1pyr5c
1-Pyrroline-5-carboxylate 2ddg6p 2-Dehydro-3-deoxy-D-gluconate
6-phosphate 2dmmq8 2-Demethylmenaquinone 8 2dmmql8
2-Demethylmenaquinol 8 2pg D-Glycerate 2-phosphate 3pg
3-Phospho-D-glycerate 6pgc 6-Phospho-D-gluconate 6pgl
6-phospho-D-glucono-1,5-lactone ac Acetate acald Acetaldehyde accoa
Acetyl-CoA actp Acetyl phosphate adp ADP akg 2-Oxoglutarate amp AMP
asn-L L-Asparagine asp-L L-Aspartate atp ATP cbp Carbamoyl
phosphate co2 CO2 coa Coenzyme A dhap Dihydroxyacetone phosphate
e4p D-Erythrose 4-phosphate etoh Ethanol f6p D-Fructose 6-phosphate
fad Flavin adenine dinucleotide oxidized fadh2 Flavin adenine
dinucleotide reduced fdp D-Fructose 1,6-bisphosphate for Formate
fum Fumarate g3p Glyceraldehyde 3-phosphate g6p D-Glucose
6-phosphate glc-D D-Glucose glu5p L-Glutamate 5-phosphate glu5sa
L-Glutamate 5-semialdehyde glu-L L-Glutamate glx Glyoxylate gly
Glycine h H+ h2o H2O icit Isocitrate lac-D D-Lactate mal-L L-Malate
methf 5,10-Methenyltetrahydrofolate mlthf
5,10-Methylenetetrahydrofolate mql8 Menaquinol 8 mqn8 Menaquinone 8
nad Nicotinamide adenine dinucleotide nadh Nicotinamide adenine
dinucleotide - reduced nadp Nicotinamide adenine dinucleotide
phosphate nadph Nicotinamide adenine dinucleotide phosphate -
reduced nh4 Ammonium oaa Oxaloacetate pep Phosphoenolpyruvate pi
Phosphate ppi Diphosphate pro-L L-Proline pyr Pyruvate q8
Ubiquinone-8 q8h2 Ubiquinol-8 ru5p-D D-Ribulose 5-phosphate s7p
Sedoheptulose 7 -phosphate so4 Sulfate succ Succinate succoa
Succinyl-CoA thf 5,6,7,8-Tetrahydrofolate xu5p-D D-Xylulose
5-phosphate
Example XX
Exemplary Pathways for Producing BDO
[0603] This example describes exemplary pathways to produce
4-hydroxybutanal (4-HBal) and/or BDO using a carboxylic acid
reductase as a BDO pathway enzyme.
[0604] An exemplary pathway for production of BDO includes use of
an NAD+ or NADP+ aryl-aldehyde dehydrogenase (E.C.: 1.2.1.29 and
1.2.1.30) to convert 4-hydroxybutyrate to 4-hydroxybutanal and an
alcohol dehydrogenase to convert 4-hydroxybutanal to
1,4-butanediol. 4-Hydroxybutyrate can be derived from the
tricarboxylic acid cycle intermediates succinyl-CoA and/or
alpha-ketoglutarate as shown in FIG. 58.
[0605] Aryl-Aldehyde Dehydrogenase (or Carboxylic Acid
Reductase).
[0606] An aryl-aldehyde dehydrogenase, or equivalently a carboxylic
acid reductase, can be found in Nocardia iowensis. Carboxylic acid
reductase catalyzes the magnesium, ATP and NADPH-dependent
reduction of carboxylic acids to their corresponding aldehydes
(Venkitasubramanian et al., J. Biol. Chem. 282:478-485 (2007)) and
is capable of catalyzing the conversion of 4-hydroxybutyrate to
4-hydroxybutanal. This enzyme, encoded by car, was cloned and
functionally expressed in E. coli (Venkitasubramanian et al., J.
Biol. Chem. 282:478-485 (2007)). Expression of the npt gene product
improved activity of the enzyme via post-transcriptional
modification. The npt gene encodes a specific phosphopantetheine
transferase (PPTase) that converts the inactive apo-enzyme to the
active holo-enzyme. The natural substrate of this enzyme is
vanillic acid, and the enzyme exhibits broad acceptance of aromatic
and aliphatic substrates (Venkitasubramanian et al., 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-00085 Gene GenBank name GI No. Accession No. Organism car
40796035 AAR91681.1 Nocardia iowensis (sp. NRRL 5646) npt 114848891
ABI83656.1 Nocardia iowensis (sp. NRRL 5646)
[0607] Additional car and npt genes can be identified based on
sequence homology.
TABLE-US-00086 Gene GenBank name GI No. Accession No. Organism
fadD9 121638475 YP_978699.1 Mycobacterium bovis BCG BCG_2812c
121638674 YP_978898.1 Mycobacterium bovis BCG nfa20150 54023983
YP_118225.1 Nocardia farcinica IFM 10152 nfa40540 54026024
YP_120266.1 Nocardia farcinica IFM 10152 SGR_6790 182440583
YP_001828302.1 Streptomyces griseus subsp. griseus NBRC 13350
SGR_665 182434458 YP_001822177.1 Streptomyces griseuss 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 ZP_04026660.1 Tsukamurella
paurometabola DSM 20162 CPCC7001_1320 ZP_05045132.1 254431429
Cyanobium PCC7001 DDBDRAFT_0187729 XP_636931.1 66806417
Dictyostelium discoideum AX4
[0608] 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-00087 Gene GenBank name GI No. Accession No. Organism griC
182438036 YP_001825755.1 Streptomyces griseus subsp. griseus NBRC
13350 griD 182438037 YP_001825756.1 Streptomyces griseus subsp.
griseus NBRC 13350
[0609] 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-00088 Gene GenBank name GI No. Accession No. Organism LYS2
171867 AAA34747.1 Saccharomyces cerevisiae LYS5 1708896 P50113.1
Saccharomyces cerevisiae LYS2 2853226 AAC02241.1 Candida albicans
LYS5 28136195 AAO26020.1 Candida albicans Lys1p 13124791 P40976.3
Schizosaccharomyces pombe Lys7p 1723561 Q10474.1
Schizosaccharomyces pombe Lys2 3282044 CAA74300.1 Penicillium
chrysogenum
[0610] There are several advantages of using carboxylic acid
reductase for BDO production. There are at least two advantages of
forming 4-hydroxybutanal from 4-hydroxybutyrate via a carboxylic
acid reductase compared to forming 4-hydroxybutanal from an
activated version of 4-hydroxybutyrate (for example,
4-hydroxybutyryl-CoA, 4-hydroxybutyryl-Pi) via an acyl-CoA or
acyl-phosphate reductase. First, the formation of
gamma-butyrolactone (GBL) as a byproduct is greatly reduced. It is
believed that the activated versions of 4-hydroxybutyrate cyclize
to GBL more readily than unactivated 4-hydroxybutyrate. The use of
carboxylic acid reductase eliminates the need to pass through a
free activated 4-hydroxybutyrate intermediate, thus reducing the
formation of GBL as a byproduct accompanying BDO production.
Second, the formation of ethanol as a byproduct is greatly reduced.
Ethanol is often formed in varying amounts when an aldehyde- or an
alcohol-forming 4-hydroxybutyryl-CoA reductase is used to convert
4-hydroxybutyryl-CoA to 4-hydroxybutanal or 1,4-butanediol,
respectively. This is because most, if not all, aldehyde- or
alcohol-forming 4-hydroxybutyryl-CoA reductases can accept
acetyl-CoA as a substrate in addition to 4-hydroxybutyryl-CoA.
Aldehyde-forming enzymes, for example, often catalyze the
conversion of acetyl-CoA to acetaldehyde, which is subsequently
reduced to ethanol by native or non-native alcohol dehydrogenases.
Alcohol-forming 4-hydroxybutyryl-CoA reductases that accept
acetyl-CoA as a substrate will convert acetyl-CoA directly to
ethanol. It appears that carboxylic acid reductase enzymes have far
less activity on acetyl-CoA than aldehyde- or alcohol-forming
acyl-CoA reductase enzymes, and thus their application for BDO
production results in minimal ethanol byproduct formation (see
below).
Example XXI
Biosynthesis of 1,4-Butanediol Using a Carboxylic Acid Reductase
Enzyme
[0611] This example describes the generation of a microbial
organism that produces 1,4-butanediol using a carboxylic acid
reductase enzyme.
[0612] Escherichia coli is used as a target organism to engineer
the pathway for 1,4-butanediol synthesis described in FIG. 58. E.
coli provides a good host for generating a non-naturally occurring
microorganism capable of producing 1,4-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 various
oxygenation conditions.
[0613] Integration of 4-Hydroxybutyrate Pathway Genes into
Chromosome: Construction of ECKh-432.
[0614] The carboxylic acid reductase enzyme was expressed in a
strain of E. coli designated ECKh-432 whose construction is
described in Example XVII. This strain contained the components of
the BDO pathway, leading to 4HB, integrated into the chromosome of
E. coli at the fimD locus.
[0615] As described in Example XVII, the succinate branch of the
upstream pathway was integrated into the E. coli chromosome using
the .lamda. red homologeous recombination method (Datsenko and
Wanner, Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)). A
polycistronic DNA fragment containing a promoter, the sucCD gene of
Escherichia coli encoding succinyl-CoA ligase, the sucD gene of
Porphyromonas gingivalis encoding succinyl-CoA reductase (aldehyde
forming) (step A of FIG. 58), the 4hbd gene of Porphyromonas
gingivalis encoding 4-hydroxybutyrate dehydrogenase (step C of FIG.
58), and a terminator sequence was inserted into the AflIII site of
the pKD3 plasmid.
[0616] As described in Example XVII, the alpha-ketoglutarate branch
of the upstream pathway was integrated into the chromosome by
homologeous recombination. The plasmid used in this modification
was pRE118-V2 (pRE118 (ATCC87693) deleted of the oriT and IS
sequences), which contains a kanamycin-resistant gene, a gene
encoding the levansucrase (sacB) and a R6K conditional replication
ori. The integration plasmid also contained a polycistronic
sequence with a promoter, the sucA gene from Mycobacterium bovis
encoding alpha-ketoglutarate decarboxylase (step B of FIG. 58), the
Clostridium kluyveri 4hbd gene encoding 4-hydroxybutyrate
dehydrogenase (step C of FIG. 58), and a terminator being inserted
between two 1.5-kb DNA fragments that are homologous to the
flanking regions of the target insertion site. The resulting
plasmid was used to transform E. coli strain. The integration
candidate was selected on plates containing kanamycin. The correct
integration site was verified by PCR. To resolve the antibiotic
marker from the chromosome, the cells were selected for growth on
medium containing sucrose. The final strain was verified by PCR and
DNA sequencing.
[0617] The recipient E. coli strain was ECKh-422 (.DELTA.adhE
.DELTA.ldhA .DELTA.pflB .DELTA.lpdA::K.p.lpdA322 .DELTA.mdh
.DELTA.arcA gltAR163L) whose construction is described in Example
XV. ECKh-422 contains a mutation gltAR163L leading to
NADH-insensitivity of citrate synthase encoded by gltA. It further
contains an NADH-insensitive version of the lpdA gene from
Klebsiella pneumonia integrated into the chromosome as described
below.
[0618] Replacement of the native lpdA was replaced with a
NADH-insensitive lpdA from Klebsiella pneumonie, as described in
Example XIV. The resulting vector was designated pRE118-V2 (see
FIG. 34).
[0619] Cloning and Expression of Carboxylic Acid Reductase and
PPTase.
[0620] To generate an E. coli strain engineered to produce
1,4-butanediol, 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, the car
(AAR91681.1) and npt (ABI83656.1) genes were cloned into the pZS*13
vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter.
The car gene (GNM.sub.--720) was cloned by PCR from Nocardia
genomic DNA. Its nucleic acid and protein sequences are shown in
FIGS. 59A and 59B, respectively.
[0621] 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. 60A and 60B, respectively.
The resulting vector from cloning GNM.sub.--720 and GNM.sub.--721
into pZS*13 is shown in FIG. 61.
[0622] The plasmid was transformed into ECKh-432 to express the
proteins and enzymes required for 1,4-butanediol production.
Alternate versions of the plasmid containing only GNM.sub.--720 and
only GNM.sub.--721 were also constructed.
[0623] Demonstration of 1,4-BDO Production using Carboxylic Acid
Reductase.
[0624] Functional expression of the 1,4-butanediol pathway was
demonstrated using E. coli whole-cell culture. A single colony of
E. coli ECKh-432 transformed with the pZS*13 plasmid containing
both GNM.sub.--720 and GNM.sub.--721 was inoculated into 5 mL of LB
medium containing appropriate antibiotics. Similarly, single
colonies of E. coli ECKh-432 transformed with the pZS*13 plasmids
containing either GNM.sub.--720 or GNM.sub.--721 were inoculated
into additional 5 mL aliquots of LB medium containing appropriate
antibiotics. Ten mL micro-aerobic cultures were started by
inoculating fresh minimal in vivo conversion medium (see below)
containing the appropriate antibiotics with 1% of the first
cultures.
[0625] Recipe of the minimal in vivo conversion medium (for 1000
mL) is as follows:
TABLE-US-00089 final concentration 1M MOPS/KOH buffer 40 mM Glucose
(40%) 1% 10XM9 salts solution 1X MgSO4 (1M) 1 mM trace minerals
(x1000) 1X 1M NaHCO3 10 mM
[0626] Microaerobic conditions were established by initially
flushing capped anaerobic bottles with nitrogen for 5 minutes, then
piercing the septum with an 18 G needle following inoculation. The
needle was kept in the bottle during growth to allow a small amount
of air to enter the bottles. Protein expression was induced with
0.2 mM IPTG when the culture reached mid-log growth phase. This is
considered: time=0 hr. The culture supernatants were analyzed for
BDO, 4HB, and other by-products as described above and in
WO2008115840 (see Table 30).
TABLE-US-00090 TABLE 30 Production of BDO, 4-HB and other products
in various strains. mM Strain pZS*13S OD600 OD600 PA SA LA 4HB BDO
GBL ETOH.sub.Enz ECKh-432 720 0.420 2.221 6.36 0.00 0.10 7.71 3.03
0.07 >LLOQ ECKh-432 721 0.323 2.574 1.69 0.00 0.00 12.60 0.00
0.00 >LLOQ ECKh-432 720/721 0.378 2.469 1.70 0.00 0.01 4.23 9.16
0.24 1.52 PA = pyruvate, SA = succinate, LA = lactate, 4HB =
4-hydroxybutyrate, BDO = 1,4-butanediol, GBL = gamma-butyrolactone,
Etoh = ethanol, LLOQ = lower limit of quantification
[0627] These results demonstrate that the carboxylic acid reductase
gene, GNM.sub.--720, is required for BDO formation in ECKh-432 and
its effectiveness is increased when co-expressed with the PPTase,
GNM.sub.--721. GBL and ethanol were produced in far smaller
quantities than BDO in the strains expressing GNM.sub.--720 by
itself or in combination with GNM.sub.--721.
[0628] Additional Pathways to BDO Employing Carboxylic Acid
Reductase.
[0629] It is expected that carboxylic acid reductase can function
as a component of many pathways to 1,4-butanediol from the TCA
cycle metabolites: succinate, succinyl-CoA, and
alpha-ketoglutarate. Several of these pathways are disclosed in
FIG. 62. All routes can lead to theoretical BDO yields greater than
or equal to 1 mol/mol assuming glucose as the carbon source.
Similar high theoretical yields can be obtained from additional
substrates including sucrose, xylose, arabinose, synthesis gas,
among many others. It is expected that the expression of carboxylic
acid reductase alone or in combination with PPTase (that is, to
catalyze steps F and D of FIG. 62) is sufficient for 1,4-butanediol
production from succinate provided that sufficient endogenous
alcohol dehydrogenase activity is present to catalyze steps C and E
of FIG. 62. Candidate enzymes for steps A through Z of FIG. 62 are
described in section XXIII.
Example XXII
Pathways to Putrescine that Employ Carboxylic Acid Reductase
[0630] This example describes exemplary putrescine pathways
utilizing carboxylic acid reductase.
[0631] Putrescine, also known as 1,4-diaminobutane or
butanediamine, is an organic chemical compound of the formula
NH.sub.2(CH.sub.2).sub.4NH.sub.2. It can be reacted with adipic
acid to yield the polyamide Nylon-4,6, which is marketed by DSM
(Heerlen, Netherlands) under the trade name Stanyl.TM.. Putrescine
is naturally produced, for example, by the natural breakdown of
amino acids in living and dead organisms. E. coli has been
engineered to produce putrescine by overexpressing the native
ornithine biosynthetic machinery as well as an ornithine
decarboxylase (Qian, et al., Biotechnol. Bioeng. 104(4):651-662
(2009)).
[0632] FIG. 63 describes a number of additional biosynthetic
pathways leading to the production of putrescine from succinate,
succinyl-CoA, or alpha-ketoglutarate and employing a carboxylic
acid reductase. Note that none of these pathways require formation
of an activated version of 4-aminobutyrate such as
4-aminobutyryl-CoA, which can be reduced by an acyl-CoA reductase
to 4-aminobutanal but also can readily cyclize to its lactam,
2-pyrrolidinone (Ohsugi, et al., J. Biol. Chem. 256:7642-7651
(1981)). All routes can lead to theoretical putrescine yields
greater than or equal to 1 mol/mol assuming glucose as the carbon
source. Similar high theoretical yields can be obtained from
additional substrates including sucrose, xylose, arabinose,
synthesis gas, among many others. Candidate enzymes for steps A
through U of FIG. 63 are described in Example XXIII.
Example XXIII
Exemplary Enzymes for Production of C4 Compounds
[0633] This example describes exemplary enzymes for production of
C4 compounds such as 1,4-butanediol, 4-hydroxybutanal and
putrescine.
[0634] Enzyme Classes.
[0635] All transformations depicted in FIGS. 58, 62 and 63 fall
into the general categories of transformations shown in Table 31.
This example describes a number of biochemically characterized
genes in each category. Specifically listed are genes that can be
applied to catalyze the appropriate transformations in FIGS. 58, 62
and 63 when cloned and expressed. 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-00091 TABLE 31 Classes of Enzyme Transformations Depicted
in FIGS. 58, 62 and 63. LABEL FUNCTION 1.1.1.a Oxidoreductase (oxo
to alcohol) 1.1.1.c Oxidoreductase (2 step, acyl-CoA to alcohol)
1.2.1.b Oxidoreductase (acyl-CoA to aldehyde) 1.2.1.c
Oxidoreductase (2-oxo acid to acyl-CoA, decarboxylation) 1.2.1.d
Oxidoreductase (phosphonate reductase) 1.2.1.e Acid reductase
1.4.1.a Oxidoreductase (aminating) 2.3.1.a Acyltransferase
(transferring phosphate group to CoA) 2.6.1.a Aminotransferase
2.7.2.a Phosphotransferase (carboxy acceptor) 2.8.3.a CoA
transferase 3.1.2.a CoA hydrolase 4.1.1.a Carboxy-lyase 6.2.1.a CoA
synthetase
1.1.1.a Oxidoreductase (Oxo to Alcohol)
[0636] Aldehyde to Alcohol.
[0637] Three transformations described in FIGS. 58, 62 and 63
involve the conversion of an aldehyde to alcohol. These are
4-hydroxybutyrate dehydrogenase (step C, FIGS. 58 and 62),
1,4-butanediol dehydrogenase (step E, FIGS. 58 and 62), and
5-hydroxy-2-pentanoic acid (step Y, FIG. 62). 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. Appi. Environ. Microbiol.
66:5231-5235 (2000)), ADH2 from Saccharomyces cerevisiae (Atsumi et
al. Nature 451:86-89 (2008)), yqhD from E. coli, which has
preference for molecules longer than C(3) (Sulzenbacher et al. J.
Mol. Biol. 342:489-502 (2004)), and bdh I and bdh II from C.
acetobutylicum, which converts butyryaldehyde into butanol (Walter
et al. J. Bacteriol. 174:7149-7158 (1992)). The protein sequences
for each of exemplary gene products can be found using the
following GenBank accession numbers:
TABLE-US-00092 Gene Accession No. GI No. Organism alrA BAB12273.1
9967138 Acinetobacter sp. Strain M-1 ADH2 NP_014032.1 6323961
Saccharymyces cerevisiae yqhD NP_417484.1 16130909 Escherichia coli
bdh I NP_349892.1 15896543 Clostridium acetobutylicum bdh II
NP_349891.1 15896542 Clostridium acetobutylicum
[0638] Enzymes exhibiting 4-hydroxybutyrate dehydrogenase activity
(EC 1.1.1.61) also fall into this category. Such enzymes have been
characterized in Ralstonia eutropha (Bravo et al. J. Forensic Sci.
49:379-387 (2004)), Clostridium kluyveri (Wolff et al., Protein
Expr. Pur 6:206-212 (1995)) and Arabidopsis thaliana (Breitkreuz et
al. J. Biol. Chem. 278:41552-41556 (2003)).
TABLE-US-00093 Gene Accession No. GI No. Organism 4hbd YP_726053.1
113867564 Ralstonia eutropha H16 4hbd EDK35022.1 146348486
Clostridium kluyveri DSM 555 4hbd Q94B07 75249805 Arabidopsis
thaliana
[0639] The adh1 gene from Geobacillus thermoglucosidasius M10EXG
(Jeon et al., J. Biotechnol. 135:127-133 (2008)) was shown to
exhibit high activity on both 4-hydroxybutanal and butanal (see
above). Thus this enzyme exhibits 1,4-butanediol dehydrogenase
activity.
TABLE-US-00094 Gene Accession No. GI No. Organism adh1 AAR91477.1
40795502 Geobacillus thermoglucosidasius M10EXG
[0640] 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-00095 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
[0641] 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-00096 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
[0642] 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, J. Plant Pathol. 159:671-674 (2002); Stadtman,
J. Am. Chem. Soc. 77:5765-5766 (1955)). This enzyme has not been
associated with a gene in any organism to date. NADPH-dependent
malonate semialdehyde reductase catalyzes the reverse reaction in
autotrophic CO.sub.2-fixing bacteria. Although the enzyme activity
has been detected in Metallosphaera sedula, the identity of the
gene is not known (Alber et al. J. Bacteriol. 188:8551-8559
(2006)).
1.1.1.c Oxidoreductase (2 Step, Acyl-CoA to Alcohol).
[0643] Steps S and W of FIG. 62 depict bifunctional reductase
enzymes that can form 4-hydroxybutyrate and 1,4-butanediol,
respectively. Exemplary 2-step oxidoreductases that convert an
acyl-CoA to alcohol include those that transform substrates such as
acetyl-CoA to ethanol (for example, adhE from E. coli (Kessler et
al., FEBS. Lett. 281:59-63 (1991)) and butyryl-CoA to butanol (for
example, adhE2 from C. acetobutylicum (Fontaine et al., J.
Bacteriol. 184:821-830 (2002)). The C. acetobutylicum adhE2 gene
was shown to convert 4-hydroxybutyryl-CoA to 1,4-butanediol (see
above). 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)).
TABLE-US-00097 Gene Accession No. GI No. Organism adhE NP_415757.1
16129202 Escherichia coli adhE2 AAK09379.1 12958626 Clostridium
acetobutylicum adhE AAV66076.1 55818563 Leuconostoc
mesenteroides
[0644] 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 and Fuchs, Eur. J. Biochem.
215:633-643 (1993)). This enzyme, with a mass of 300 kDa, is highly
substrate-specific and shows little sequence similarity to other
known oxidoreductases (Hugler et al., J. Bacteriol. 184:2404-2410
(2002)). No enzymes in other organisms have been shown to catalyze
this specific reaction; however there is bioinformatic evidence
that other organisms may have similar pathways (Klatt et al.,
Environ. Microbiol. 9:2067-2078 (2007)). Enzyme candidates in other
organisms including Roseiflexus castenholzii, Erythrobacter sp.
NAP1 and marine gamma proteobacterium HTCC2080 can be inferred by
sequence similarity.
TABLE-US-00098 Gene Accession No. GI No. 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
[0645] 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 Physiol. 122:635-644 2000)).
TABLE-US-00099 Gene Accession No. GI No. Organism FAR AAD38039.1
5020215 Simmondsia chinensis
1.2.1.b Oxidoreductase (Acyl-CoA to Aldehyde).
[0646] Step A of FIGS. 58, 62 and 63 involves the conversion of
succinyl-CoA to succinate semialdehyde by an aldehyde forming
succinyl-CoA reductase. Step Q of FIG. 62 depicts the conversion of
4-hydroxybutyryl-CoA to 4-hydroxybutanal by an aldehyde-forming
4-hydroxybutyryl-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. Bacteriol. 179:2969-2975 (1997)), the Acinetobacter
sp. M-1 fatty acyl-CoA reductase (Ishige et al., Appl. Environ.
Microbiol. 68:1192-1195 (2002)), and a CoA- and NADP-dependent
succinate semialdehyde dehydrogenase encoded by the sucD gene in
Clostridium kluyveri (Sohling 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-00100 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
[0647] 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, 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-00101 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
1.2.1.c Oxidoreductase (2-Oxo Acid to Acyl-CoA,
Decarboxylation).
[0648] Step AA in FIG. 62 depicts the conversion of
5-hydroxy-2-oxopentanoic acid to 4-hydroxybutyryl-CoA. Candidate
enzymes for this transformation include 1) branched-chain
2-keto-acid dehydrogenase, 2) alpha-ketoglutarate dehydrogenase,
and 3) the pyruvate dehydrogenase multienzyme complex (PDHC). These
enzymes are multi-enzyme complexes that catalyze a series of
partial reactions which result in acylating oxidative
decarboxylation of 2-keto-acids. Each of the 2-keto-acid
dehydrogenase complexes occupies key positions in intermediary
metabolism, and enzyme activity is typically tightly regulated
(Fries et al. Biochemistry 42:6996-7002 (2003)). The enzymes share
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).
[0649] 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 keto-acid
dehydrogenases have the broadest substrate range.
[0650] 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)). Although the substrate
range of AKGD is narrow, 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-00102 Gene Accession No. GI No. Organism sucA NP_415254.1
16128701 Escherichia coli str. K12 substr. MG1655 sucB NP_415255.1
16128702 Escherichia coli str. K12 substr. MG1655 1pd NP_414658.1
16128109 Escherichia coli str. K12 substr. MG1655 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
[0651] Branched-chain 2-keto-acid dehydrogenase complex (BCKAD),
also known as 2-oxoisovalerate dehydrogenase, 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. The complex has been studied in
many organisms including Bacillus subtilis (Wang et al. Eur. J.
Biochem. 213:1091-1099 (1993)), Rattus norvegicus (Namba et al. J.
Biol. Chem. 244:4437-4447 (1969)) and Pseudomonas putida (Sokatch
J. Bacteriol. 148:647-652 (1981)). In Bacillus subtilis the enzyme
is encoded by genes pdhD (E3 component), bfmBB (E2 component),
bfmBAA and bfmBAB (E1 component) (Wang et al. Eur. J. Biochem.
213:1091-1099 (1993)). In mammals, the complex is regulated by
phosphorylation by specific phosphatases and protein kinases. The
complex has been studied in rat hepatocites (Chicco et al. J. Biol.
Chem. 269:19427-19434 (1994)) and is encoded by genes Bckdha (E1
alpha), Bckdhb (E1 beta), Dbt (E2), and Dld (E3). The E1 and E3
components of the Pseudomonas putida BCKAD complex have been
crystallized (Aevarsson et al. Nat. Struct. Biol. 6:785-792 (1999);
Mattevi Science 255:1544-1550 (1992)) and the enzyme complex has
been studied (Sokatch et al. J. Bacteriol. 148:647-652 (1981)).
Transcription of the P. putida BCKAD genes is activated by the gene
product of bkdR (Hester et al. Eur. J. Biochem. 233:828-836
(1995)). 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. The active site of the bovine BCKAD was engineered to
favor alternate substrate acetyl-CoA (Meng and Chuang, Biochemistry
33:12879-12885 (1994)).
TABLE-US-00103 Gene Accession No. GI No. Organism bfmBB NP_390283.1
16079459 Bacillus subtilis bfmBAA NP_390285.1 16079461 Bacillus
subtilis bfmBAB NP_390284.1 16079460 Bacillus subtilis pdhD
P21880.1 118672 Bacillus subtilis lpdV P09063.1 118677 Pseudomonas
putida bkdB P09062.1 129044 Pseudomonas putida bkdA1 NP_746515.1
26991090 Pseudomonas putida bkdA2 NP_746516.1 26991091 Pseudomonas
putida Bckdha NP_036914.1 77736548 Rattus norvegicus Bckdhb
NP_062140.1 158749538 Rattus norvegicus Dbt NP_445764.1 158749632
Rattus norvegicus Dld NP_955417.1 40786469 Rattus norvegicus
[0652] The pyruvate dehydrogenase complex, catalyzing the
conversion of pyruvate to acetyl-CoA, has also been extensively
studied. In the E. coli enzyme, specific residues in the E1
component are responsible for substrate specificity (Bisswanger, H.
J Biol. Chem. 256:815-822 (1981); Bremer, J. Eur. J Biochem.
8:535-540 (1969); Gong et al. J Biol. Chem. 275:13645-13653
(2000)). As mentioned previously, enzyme engineering efforts have
improved the E. coli PDH enzyme activity under anaerobic conditions
(Kim et al. Appl. Environ. Microbiol. 73:1766-1771 (2007); Kim J.
Bacteriol. 190:3851-3858 (2008); 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 J. Bacteriol. 179:6749-6755 (1997)). The
Klebsiella pneumoniae PDH, characterized during growth on glycerol,
is also active under anaerobic conditions (Menzel et al. J.
Biotechnol. 56:135-142 (1997)). Crystal structures of the enzyme
complex from bovine kidney (Zhou et al. Proc. Natl. Acad. Sci.
U.S.A. 98:14802-14807 (2001)) and the E2 catalytic domain from
Azotobacter vinelandii are available (Mattevi et al. Science
255:1544-1550 (1992)). Some mammalian PDH enzymes complexes can
react on alternate substrates such as 2-oxobutanoate, although
comparative kinetics of Rattus norvegicus PDH and BCKAD indicate
that BCKAD has higher activity on 2-oxobutanoate as a substrate
(Paxton et al. Biochem. J. 234:295-303 (1986)).
TABLE-US-00104 Gene Accession No. GI No. Organism aceE NP_414656.1
16128107 Escherichia coli str. K12 substr. MG1655 aceF NP_414657.1
16128108 Escherichia coli str. K12 substr. MG1655 lpd NP_414658.1
16128109 Escherichia coli str. K12 substr. MG1655 pdhA P21881.1
3123238 Bacillus subtilis pdhB P21882.1 129068 Bacillus subtilis
pdhC P21883.2 129054 Bacillus subtilis pdhD P21880.1 118672
Bacillus subtilis aceE YP_001333808.1 152968699 Klebsiella
pneumonia MGH78578 aceF YP_001333809.1 152968700 Klebsiella
pneumonia MGH78578 lpdA YP_001333810.1 152968701 Klebsiella
pneumonia MGH78578 Pdhal NP_001004072.2 124430510 Rattus norvegicus
Pdha2 NP_446446.1 16758900 Rattus norvegicus Dlat NP_112287.1
78365255 Rattus norvegicus Dld NP_955417.1 40786469 Rattus
norvegicus
[0653] As an alternative to the large multienzyme 2-keto-acid
dehydrogenase complexes described above, some anaerobic organisms
utilize enzymes in the 2-ketoacid oxidoreductase family (OFOR) to
catalyze acylating oxidative decarboxylation of 2-keto-acids.
Unlike the dehydrogenase complexes, these enzymes contain
iron-sulfur clusters, utilize different cofactors, and use
ferredoxin or flavodixin as electron acceptors in lieu of NAD(P)H.
While most enzymes in this family are specific to pyruvate as a
substrate (POR) some 2-keto-acid:ferredoxin oxidoreductases have
been shown to accept a broad range of 2-ketoacids as substrates
including alpha-ketoglutarate and 2-oxobutanoate (Fukuda and Wakagi
Biochim. Biophys. Acta 1597:74-80 (2002); Zhang et al. J. Biochem.
120:587-599 (1996)). One such enzyme is the OFOR from the
thermoacidophilic archaeon Sulfolobus tokodaii 7, which contains an
alpha and beta subunit encoded by gene ST2300 (Fukuda and Wakagi
Biochim. Biophys. Acta 1597:74-80 (2002); Zhang et al. J. Biochem.
120:587-599 (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)). Two OFORs from
Aeropyrum pernix str. K1 have also been recently cloned into E.
coli, characterized, and found to react with a broad range of
2-oxoacids (Nishizawa et al. FEBS Lett. 579:2319-2322 (2005)). The
gene sequences of these OFOR candidates are available, although
they do not have GenBank identifiers assigned to date. There is
bioinformatic evidence that similar enzymes are present in all
archaea, some anaerobic bacteria and amitochondrial eukarya (Fukuda
and Wakagi Biochim. Biophys. Acta 1597:74-80 (2005)). This class of
enzyme is also interesting from an energetic standpoint, as reduced
ferredoxin could be used to generate NADH by ferredoxin-NAD
reductase (Petitdemange et al. Biochim. Biophys. Acta 421:334-337
(1976)). Also, since most of the enzymes are designed to operate
under anaerobic conditions, less enzyme engineering may be required
relative to enzymes in the 2-keto-acid dehydrogenase complex family
for activity in an anaerobic environment.
TABLE-US-00105 Gene Accession No. GI No. Organism ST2300
NP_378302.1 15922633 Sulfolobus tokodaii 7
1.2.1.d Oxidoreductase (Phosphonate Reductase).
[0654] 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., Biochemistry 40:14475-14483 (2001). The E.
coli ASD structure has been solved (Hadfield et al., J. Mol. Biol.
289:991-1002 (1999)) and the enzyme has been shown to accept the
alternate substrate beta-3-methylaspartyl phosphate (Shames et al.,
J. Biol. Chem. 259:15331-15339 (1984)). The Haemophilus influenzae
enzyme has been the subject of enzyme engineering studies to alter
substrate binding affinities at the active site (Blanco et al.,
Acta Crystallogr. D. Biol. Crystallogr. 60:1388-1395 (2004); Blanco
et al., Acta Crystallogr. D. Biol. Crystallogr. 60:1808-1815
(2004)). Other ASD candidates are found in Mycobacterium
tuberculosis (Shafiani et al., J. Appl. Microbiol. 98:832-838
(2005), Methanococcus jannaschii (Faehnle et al., J. Mol. Biol.
353:1055-1068 (2005)), and the infectious microorganisms Vibrio
cholera and Heliobacter pylori (Moore et al., Protein Expr. Pur
25:189-194 (2002)). A related enzyme candidate is
acetylglutamylphosphate reductase (EC 1.2.1.38), an enzyme that
naturally reduces acetylglutamylphosphate to
acetylglutamate-5-semialdehyde, found in S. cerevisiae (Pauwels et
al., Eur. J. Biochem. 270:1014-1024 (2003), B. subtilis (O'Reilly
and Devine, Microbiology 140 (Pt 5):1023-1025 (1994)), and other
organisms.
TABLE-US-00106 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
[0655] Other exemplary enzymes in this class include glyceraldehyde
3-phosphate dehydrogenase which converts glyceraldehyde-3-phosphate
into D-glycerate 1,3-bisphosphate (for example, E. coli gapA
(Branlant and Branlant, 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 (for example, 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 (for example, E. coli proA (Smith et al., J.
Bacteriol. 157:545-551 (1984)). Genes encoding
glutamate-5-semialdehyde dehydrogenase enzymes from Salmonella
typhimurium (Mahan and Csonka, J. Bacteriol. 156:1249-1262 (1983))
and Campylobacter jejuni (Louie and Chan, Mol. Gen. Genet.
240:29-35 (1993)) were cloned and expressed in E. coli.
TABLE-US-00107 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
1.2.1.e Acid Reductase.
[0656] Several steps in FIGS. 58, 62 and 63 depict the conversion
of unactivated acids to aldehydes by an acid reductase. These
include 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-00108 Gene Accession No. GI No. Organism car AAR91681.1
40796035 Nocardia iowensis (sp. NRRL 5646) npt ABI83656.1 114848891
Nocardia iowensis (sp. NRRL 5646)
[0657] Additional car and npt genes can be identified based on
sequence homology.
TABLE-US-00109 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 Streptomyces griseus subsp. SGR_6790
YP_001828302.1 182440583 griseus NBRC 13350 Streptomyces griseus
subsp. SGR_665 YP_001822177.1 182434458 griseus NBRC 13350
[0658] 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-00110 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 Mycobacterium avium subsp. MAP_1040c NP_959974.1 41407138
paratuberculosis K-10 Mycobacterium avium subsp. MAP_2899c
NP_961833.1 41408997 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
[0659] 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-00111 Gene Accession No. GI No. Organism LYS2 AAA34747.1
171867 Saccharomyces cerevisiae LYS5 P50113.1 1708896 Saccharomyces
cerevisiae LYS2 AACO2241.1 2853226 Candida albicans LYS5 AA026020.1
28136195 Candida albicans Lys1p P40976.3 13124791
Schizosaccharomyces pombe Lys7p Q10474.1 1723561
Schizosaccharomyces pombe Lys2 CAA74300.1 3282044 Penicillium
chrysogenum
1.4.1.a Oxidoreductase (Aminating).
[0660] Glutamate dehydrogenase (Step J, FIGS. 62 and 63),
4-aminobutyrate dehydrogenase (Step M, FIGS. 62 and 63), putrescine
dehydrogenase (Step D, FIG. 63), 5-amino-2-oxopentanoate
dehydrogenase (Step P, FIG. 63), and ornithine dehydrogenase (Step
S, FIG. 63) can be catalyzed by aminating oxidoreductases. Enzymes
in this EC class catalyze the oxidative deamination of alpha-amino
acids with NAD+ or NADP+ 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-00112 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
[0661] 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-00113 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
[0662] 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 and Nagasaki, J.
Bacteriol. 150:398-401 (1982)) and Achromobacter denitrificans
(Ruldeekulthamrong et al., BMB. Rep. 41:790-795 (2008)).
TABLE-US-00114 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
[0663] 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., 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 and van der Drift,
Biochemistry 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-00115 Gene Accession No. GI No. Organism kdd AAL93966.1
19713113 Fusobacterium nucleatum mxan_4391 ABF87267.1 108462082
Myxococcus xanthus pg_1069 AAQ66183.1 34397119 Porphyromonas
gingivalis
2.3.1.a Acyltransferase (Transferring Phosphate Group to CoA).
[0664] Step P of FIG. 62 depicts the transformation of
4-hydroxybutyryl-CoA to 4-hydroxybutyryl-Pi. Exemplary phosphate
transferring acyltransferases 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, 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 134:107-111 (1993)); Huang et al., J Mol. Microbiol.
Biotechnol. 2:33-38 (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-00116 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
2.6.1. Aminotransferase.
[0665] Aminotransferases 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, such as
4-aminobutyrate, putrescine, and 5-amino-2-oxopentanoate. These
enzymes are particularly well suited to carry out the following
transformations: Step N in FIGS. 62 and 63, Steps E and Q in FIG.
63. 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 and Bode, 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 and Misono,
Biochemistry 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 and
Yamamoto, 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-00117 Gene Accession No. GI No. Organism lat BAB13756.1
10336502 Flavobacterium lutescens lat AAA26777.1 153343
Streptomyces clavuligenus dat P56744.1 6685373 Acinetobacter
baumanii
[0666] 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 (Liu et al., Biochemistry 43:10896-10905
2004); Schulz et al., Appl. Environ. Microbiol. 56:1-6 (1990)). 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, Methods Enzymol. 113:80-82 (1985);
Scott and Jakoby, J. Biol. Chem. 234:932-936 (1959)).
TABLE-US-00118 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
[0667] 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 (for example, pyruvate, 2-oxobutanoate) has been
reported (Kim, J. Biol. Chem. 239:783-786 (1964); Samsonova et al.,
BMC Microbiol. 3:2 (2003)). 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-00119 Gene Accession No. GI No. Organism ygjG NP_417544
145698310 Escherichia coli spuC AAG03688 9946143 Pseudomonas
aeruginosa
[0668] 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
and Hansen, 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 (Kakimoto
et al., Biochim. Biophys. Acta 156:374-380 (1968); Tamaki et al.,
Methods Enzymol. 324:376-389 (2000)).
TABLE-US-00120 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
[0669] 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., FEBS Lett. 100:81-84 (1979);
Yagi et al., Methods Enzymol. 113:83-89 (1985)), AAT2 from
Saccharomyces cerevisiae (Yagi et al., J. Biochem. 92:35-43 (1982))
and ASPS from Arabidopsis thaliana (de la Torre et al., Plant J.
46:414-425 (2006); Kwok and Hanson. J. Exp. Bot. 55:595-604 (2004);
Wilkie and Warren, Protein Expr. Pur 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 substrates can 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 and Berg, 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
and Berg, J. Bacteriol. 158:571-574 1984)). The gene product of the
E. coli serC catalyzes two reactions, phosphoserine
aminotransferase and phosphohydroxythreonine aminotransferase (Lam
and Winkler, J. Bacteriol. 172:6518-6528 (1990)), and activity on
non-phosphorylated substrates could not be detected (Drewke et al.,
FEBS Lett. 390:179-182 (1996)).
TABLE-US-00121 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
[0670] 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., Microbiology
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-00122 Gene Accession No. GI No. Organism lysN BAC76939.1
31096548 Thermus thermophilus AadAT- Q8N5Z0.2 46395904 Homo sapiens
II
2.7.2.a Phosphotransferase (Carboxy Acceptor).
[0671] Phosphotransferase enzymes in the EC class 2.7.2 transform
carboxylic acids to phosphonic acids with concurrent hydrolysis of
one ATP. Step O of FIG. 62 involves the conversion of
4-hydroxybutyrate to 4-hydroxybutyryl-phosphate by such an enzyme.
Butyrate kinase (EC 2.7.2.7) carries out the reversible conversion
of butyryl-phosphate to butyrate during acidogenesis in C.
acetobutylicum (Cary et al., Appl. Environ. Microbiol. 56:1576-1583
(1990)). This enzyme is encoded by either of the two buk gene
products (Huang et al., J. Mol. Microbiol. Biotechnol. 2:33-38
(2000)). Other butyrate kinase enzymes are found in C. butyricum
and C. tetanomorphum (Twarog and Wolfe, J. Bacteriol. 86:112-117
(1963)). Related enzyme isobutyrate kinase from Thermotoga maritima
has also been expressed in E. coli and crystallized (Diao et al.,
Acta Crystallogr. D. Biol. Crystallogr. 59:1100-1102 (2003); Diao
and Hasson, J. Bacteriol. 191:2521-2529 (2009)). Aspartokinase
catalyzes the ATP-dependent phosphorylation of aspartate and
participates in the synthesis of several amino acids. The
aspartokinase III enzyme in E. coli, encoded by lysC, has a broad
substrate range, and the catalytic residues involved in substrate
specificity have been elucidated (Keng and Viola, Arch. Biochem.
Biophys. 335:73-81 (1996)). Two additional kinases in E. coli are
also good candidates: acetate kinase and gamma-glutamyl kinase. The
E. coli acetate kinase, encoded by ackA (Skarstedt and Silverstein,
J. Biol. Chem. 251:6775-6783 (1976)), phosphorylates propionate in
addition to acetate (Hesslinger et al., Mol. Microbiol. 27:477-492
(1998)). The E. coli gamma-glutamyl kinase, encoded by proB (Smith
et al., J. Bacteriol. 157:545-551 (1984)), phosphorylates the gamma
carbonic acid group of glutamate.
TABLE-US-00123 Gene Accession No. GI No. Organism bukl NP_349675
15896326 Clostridium acetobutylicum buk2 Q97II1 20137415
Clostridium acetobutylicum buk2 Q9X278.1 6685256 Thermotoga
maritima lysC NP_418448.1 16131850 Escherichia coli ackA
NP_416799.1 16130231 Escherichia coli proB NP_414777.1 16128228
Escherichia coli
[0672] Acetylglutamate kinase phosphorylates acetylated glutamate
during arginine biosynthesis. This enzyme is not known to accept
alternate substrates; however, several residues of the E. coli
enzyme involved in substrate binding and phosphorylation have been
elucidated by site-directed mutagenesis (Marco-Marin et al., J.
Mol. Biol. 334:459-476 (2003); Ramon-Maiques et al., Structure
10:329-342 (2002)). The enzyme is encoded by argB in Bacillus
subtilis and E. coli (Parsot et al., Gene 68:275-283 (1988)), and
ARG5,6 in S. cerevisiae (Pauwels et al., Eur. J. Biochem.
270:1014-1024 (2003)). The ARG5,6 gene of S. cerevisiae encodes a
polyprotein precursor that is matured in the mitochondrial matrix
to become acetylglutamate kinase and acetylglutamylphosphate
reductase.
TABLE-US-00124 Gene Accession No. GI No. Organism argB NP_418394.3
145698337 Escherichia coli argB NP_389003.1 16078186 Bacillus
subtilis ARG5,6 NP_010992.1 6320913 Saccharomyces cerevisiae
2.8.3.a CoA Transferase.
[0673] The gene products of cat1, cat2, and cat3 of Clostridium
kluyveri have been shown to exhibit succinyl-CoA (Step G, FIGS. 62
and 63), 4-hydroxybutyryl-CoA (Step T, FIG. 62), and butyryl-CoA
acetyltransferase activity, respectively (Seedorf et al., Proc.
Natl. Acad. Sci. USA 105:2128-2133 (2008); Sohling and Gottschalk,
J Bacteriol 178:871-880 (1996)). Similar CoA transferase activities
are also present in Trichomonas vaginalis (van Grinsven et al., J.
Biol. Chem. 283:1411-1418 (2008)) and Trypanosoma brucei (Riviere
et al., J. Biol. Chem. 279:45337-45346 (2004)).
TABLE-US-00125 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
[0674] 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.,
Appl. Environ. Microbiol. 68:5186-5190 (2002)), Clostridium
acetobutylicum (Cary et al., Appl. Environ. Microbiol. 56:1576-1583
(1990); Wiesenborn et al., Appl. Environ. Microbiol. 55:323-329
(1989)), and Clostridium saccharoperbutylacetonicum (Kosaka et al.,
Biosci. Biotechnol. Biochem. 71:58-68 (2007)).
TABLE-US-00126 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
[0675] 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-00127 Gene Accession No. GI No. Organism gctA CAA57199.1
559392 Acidaminococcus fermentans gctB CAA57200.1 559393
Acidaminococcus fermentans
3.1.2.a CoA Hydrolase.
[0676] Enzymes in the 3.1.2 family hydrolyze acyl-CoA molecules to
their corresponding acids. However, such enzymes can be modified to
empart CoA-ligase or synthetase functionality if coupled to an
energy source such as a proton pump or direct ATP hydrolysis.
Several eukaryotic acetyl-CoA hydrolases (EC 3.1.2.1) have broad
substrate specificity. For example, the enzyme from Rattus
norvegicus brain (Robinson et al., Biochem. Biophys. Res. Commun.
71:959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA and
malonyl-CoA. Though its sequence has not been reported, the enzyme
from the mitochondrion of the pea leaf also has a broad substrate
specificity, with demonstrated activity on acetyl-CoA,
propionyl-CoA, butyryl-CoA, palmitoyl-CoA, oleoyl-CoA,
succinyl-CoA, and crotonyl-CoA (Zeiher and Randall, Plant. Physiol.
94:20-27 (1990)). The acetyl-CoA hydrolase, ACH1, from S.
cerevisiae represents another candidate hydrolase (Buu et al., J.
Biol. Chem. 278:17203-17209 (2003)).
TABLE-US-00128 Gene Accession No. GI No. Organism acot12
NP_570103.1 18543355 Rattus norvegicus ACH1 NP_009538 6319456
Saccharomyces cerevisiae
[0677] Another candidate hydrolase is the human dicarboxylic acid
thioesterase, acot8, which exhibits activity on glutaryl-CoA,
adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA (Westin
et al., J. Biol. Chem. 280:38125-38132 (2005)) and the closest E.
coli homolog, tesB, which can also hydrolyze a broad range of CoA
thioesters (Naggert et al., J. Biol. Chem. 266:11044-11050 (1991)).
A similar enzyme has also been characterized in the rat liver
(Deana, Biochem. Int. 26:767-773 (1992)). Other potential E. coli
thioester hydrolases include the gene products of tesA (Bonner and
Bloch, J. Biol. Chem. 247:3123-3133 (1972)), ybgC (Kuznetsova et
al., FEMS Microbiol. Rev. 29:263-279 (2005); Zhuang et al., FEBS
Lett. 516:161-163 (2002)), paaI (Song et al., J. Biol. Chem.
281:11028-11038 (2006)), and ybdB (Leduc et al., J. Bacteriol.
189:7112-7126 (2007)).
TABLE-US-00129 Gene Accession No. GI No. Organism acot8 CAA15502
3191970 Homo sapiens tesB NP_414986 16128437 Escherichia coli acot8
NP_570112 51036669 Rattus norvegicus tesA NP_415027 16128478
Escherichia coli ybgC NP_415264 16128711 Escherichia coli paaI
NP_415914 16129357 Escherichia coli ybdB NP_415129 16128580
Escherichia coli
[0678] Yet another candidate hydrolase is the glutaconate
CoA-transferase from Acidaminococcus fermentans. This enzyme was
transformed by site-directed mutagenesis into an acyl-CoA hydrolase
with activity on glutaryl-CoA, acetyl-CoA and 3-butenoyl-CoA (Mack
and Buckel, FEBS Lett. 405:209-212 (1997)). This indicates that the
enzymes encoding succinyl-CoA:3-ketoacid-CoA transferases and
acetoacetyl-CoA:acetyl-CoA transferases can also serve as
candidates for this reaction step but would likely require certain
mutations to change their function.
TABLE-US-00130 Gene Accession No. GI No. Organism gctA CAA57199.1
559392 Acidaminococcus fermentans gctB CAA57200.1 559393
Acidaminococcus fermentans
[0679] Additional hydrolase enzymes include 3-hydroxyisobutyryl-CoA
hydrolase which has been described to efficiently catalyze the
conversion of 3-hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate
during valine degradation (Shimomura et al., J. Biol. Chem.
269:14248-14253 (1994)). Genes encoding this enzyme include hibch
of Rattus norvegicus (Shimomura et al., supra (1994); Shimomura et
al., Methods Enzymol. 324:229-240 (2000)) and Homo sapiens
(Shimomura et al., supra (1994). Candidate genes by sequence
homology include hibch of Saccharomyces cerevisiae and
BC.sub.--2292 of Bacillus cereus.
TABLE-US-00131 Gene Accession No. GI No. Organism hibch Q5XIE6.2
146324906 Rattus norvegicus hibch Q6NVY1.2 146324905 Homo sapiens
hibch P28817.2 2506374 Saccharomyces cerevisiae BC_2292 AP09256
29895975 Bacillus cereus
4.1.1.a Carboxy-Lyase.
[0680] Decarboxylation of Alpha-Keto Acids.
[0681] Alpha-ketoglutarate decarboxylase (Step B, FIGS. 58, 62 and
63), 5-hydroxy-2-oxopentanoic acid decarboxylase (Step Z, FIG. 62),
and 5-amino-2-oxopentanoate decarboxylase (Step R, FIG. 63) 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.
[0682] 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 (Davie et
al., J. Biol. Chem. 267:16601-16606 (1992)). 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 and Jordan, 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., Protein Eng. Des. SeL 18:345-357
(2005)). The crystal structure of this enzyme is available
(Killenberg-Jabs et al., Eur. J. Biochem. 268:1698-1704 (2001)).
Other well-characterized PDC candidates 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)).
TABLE-US-00132 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
[0683] 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 (Hasson
et al., Biochemistry 37:9918-9930 (1998); Polovnikova et al.,
Biochemistry 42:1820-1830 (2003). Site-directed mutagenesis of two
residues in the active site of the Pseudomonas putida enzyme
altered the affinity (Km) of naturally and non-naturally occurring
substrates (Siegert et al., Protein Eng. Des. Sel. 18:345-357
(2005)). The properties of this enzyme have been further modified
by directed engineering (Lingen et al., Protein Eng. 15:585-593
(2002); Lingen et al., Chembiochem. 4:721-726 (2003)). The enzyme
from Pseudomonas aeruginosa, encoded by mdlC, has also been
characterized experimentally (Barrowman et al., FEMS Microbiol.
Lett. 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-00133 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
[0684] 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 functionally expressed.
However, it is not an ideal candidate for strain engineering
because it is large (.about.130 kD) and GC-rich. KDC enzyme
activity has been detected in several species of rhizobia including
Bradyrhizobium japonicum and Mesorhizobium loti (Green 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 and Nakano, Arch. Biochem.
Biophys. 288:22-28 (1991)). The first twenty amino acids starting
from the N-terminus were sequenced (MTYKAPVKDVKFLLDKVFKV; SEQ ID
NO:45) (Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28
(1991)). The gene can be identified by testing candidate genes
containing this N-terminal sequence for KDC activity.
TABLE-US-00134 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
[0685] 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 and Kaneda, J. Biol. Chem.
263:18386-18396 (1988); Smit et al., B. A., J. E. Hylckama Vlieg,
W. J. Engels, L. Meijer, J. T. Wouters, and G. Smit.
Identification, cloning, and characterization of a Lactococcus
lactis branched-chain alpha-keto acid decarboxylase involved in
flavor formation. Appl. Environ. Microbiol. 71:303-311 (2005)). The
enzyme in Lactococcus lactis has been characterized on a variety of
branched and linear substrates including 2-oxobutanoate,
2-oxohexanoate, 2-oxopentanoate, 3-methyl-2-oxobutanoate,
4-methyl-2-oxobutanoate and isocaproate (Smit et al., Appl.
Environ. Microbiol. 71:303-311 (2005)). The enzyme has been
structurally characterized (Berg et al., Science 318:1782-1786
(2007)). Sequence alignments between the Lactococcus lactis enzyme
and the pyruvate decarboxylase of Zymomonas mobilus indicate that
the catalytic and substrate recognition residues are nearly
identical (Siegert et al., Protein Eng. Des. Sel. 18:345-357
(2005)), so this enzyme would be a promising candidate for directed
engineering. 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. Biosynthesis of branched-chain fatty acids in Bacillus
subtilis. A decarboxylase is essential for branched-chain fatty
acid synthetase. J. Biol. Chem. 263:18386-18396 (1988)), and the
gene encoding this enzyme has not been identified to date.
Additional BCKA gene candidates can be identified by homology to
the Lactococcus lactis protein sequence. Many of the high-scoring
BLASTp hits to this enzyme are annotated as indolepyruvate
decarboxylases (EC 4.1.1.74). Indolepyruvate decarboxylase (IPDA)
is an enzyme that catalyzes the decarboxylation of indolepyruvate
to indoleacetaldehyde in plants and plant bacteria.
TABLE-US-00135 Gene Accession No. GI No. Organism kdcA AAS49166.1
44921617 Lactococcus lactis
[0686] Recombinant branched chain alpha-keto acid decarboxylase
enzymes derived from the E1 subunits of the mitochondrial
branched-chain keto acid dehydrogenase complex from Homo sapiens
and Bos taurus have been cloned and functionally expressed in E.
coli (Davie et al., J. Biol. Chem. 267:16601-16606 1992); Wynn et
al., J. Biol. Chem. 267:1881-1887 (1992); Wynn et al., J. Biol.
Chem. 267:12400-12403 (1992)). In these studies, the authors found
that co-expression of chaperonins GroEL and GroES enhanced the
specific activity of the decarboxylase by 500-fold (Wynn et al., J.
Biol. Chem. 267:12400-12403 (1992)). These enzymes are composed of
two alpha and two beta subunits.
TABLE-US-00136 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
[0687] Decarboxylation of Alpha-Keto Acids.
[0688] Several ornithine decarboxylase (Step U, FIG. 63) enzymes
also exhibit activity on lysine and other similar compounds. Such
enzymes are found in Nicotiana glutinosa (Lee and Cho, Biochem. J.
360:657-665 (2001)), Lactobacillus sp. 30a (Guirard and Snell, J.
Biol. Chem. 255:5960-5964 (1980)) and Vibrio vulnificus (Lee et
al., J. Biol. Chem. 282:27115-27125 (2007)). The enzymes from
Lactobacillus sp. 30a (Momany et al., J. Mol. Biol. 252:643-655
(1995)) and V. vulnificus have been crystallized. The V. vulnificus
enzyme efficiently catalyzes lysine decarboxylation, and the
residues involved in substrate specificity have been elucidated
(Lee et al., J. Biol. Chem. 282:27115-27125 (2007)). A similar
enzyme has been characterized in Trichomonas vaginalis, but the
gene encoding this enzyme is not known (Yarlett et al., Biochem. J.
293 (Pt 2):487-493 (1993)).
TABLE-US-00137 Gene Accession No. GI No. Organism AF323910.1:1 . .
1299 AAG45222.1 12007488 Nicotiana glutinosa odc1 P43099.2 1169251
Lactobacillus sp. 30a VV2_1235 NP_763142.1 27367615 Vibrio
vulnificus
[0689] Glutamate decarboxylase enzymes (Step L, FIGS. 62 and 63)
are also well-characterized. Exemplary glutamate decarboxylases can
be found in E. coli (De Biase et al., Protein Expr. Pur 8:430-438
(1996)), S. cerevisiae (Coleman et al., J. Biol. Chem. 276:244-250
(2001)), and Homo sapiens (Bu et al., Proc. Natl. Acad. Sci. USA
89:2115-2119 (1992); Bu and Tobin, Genomics 21:222-228 (1994)).
TABLE-US-00138 Gene Accession No. GI No. Organism GAD1 NP_000808
58331246 Homo sapiens GAD2 NP_001127838 197276620 Homo sapiens gadA
NP_417974 16131389 Escherichia coli gadB NP_416010 16129452
Escherichia coli GAD1 NP_013976 6323905 Saccharomyces
cerevisiae
[0690] Lysine decarboxylase (EC 4.1.1.18) catalyzes the
decarboxylation of lysine to cadaverine. Two isozymes of this
enzyme are encoded in the E. coli genome by genes cadA and ldcC.
CadA is involved in acid resistance and is subject to positive
regulation by the cadC gene product (Lemonnier and Lane,
Microbiology 144 (Pt 3):751-760 (1998)). CadC accepts hydroxylysine
and S-aminoethylcysteine as alternate substrates, and
2-Aminopimelate and 6-ACA act as competitive inhibitors to this
enzyme (Sabo et al., Biochemistry 13:662-670 (1974)). Directed
evolution or other enzyme engineering methods can be utilized to
increase the activity for this enzyme to decarboxylate
2-aminopimelate. The constitutively expressed ldc gene product is
less active than CadA (Lemonnier and Lane, Microbiology 144 (Pt
3):751-760 (1998)). 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)). Active site residues
were identified and engineered to alter the substrate specificity
of the enzyme (Takatsuka et al., J. Bacteriol. 182:6732-6741
(2000)).
TABLE-US-00139 Gene Accession No. GI No. Organism cadA AAA23536.1
145458 Escherichia coli ldcC AAC73297.1 1786384 Escherichia coli
ldc O50657.1 13124043 Selenomonas ruminantium cadA AB124819.1
44886078 Vibrio parahaemolyticus
6.2.1.a CoA Synthetase.
[0691] CoA synthetase or ligase reactions are required by Step I of
FIGS. 62 and 63, and Step V of FIG. 62. Succinate or
4-hydroxybutyrate are the required substrates. 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 concomitant
consumption of one ATP, a reaction which is reversible in vivo
(Buck et al., Biochem. 24:6245-6252 (1985)).
TABLE-US-00140 Gene Accession No. GI No. Organism sucC NP_415256.1
16128703 Escherichia coli sucD AAC73823.1 1786949 Escherichia
coli
[0692] Additional exemplary CoA-ligases include the rat
dicarboxylate-CoA ligase for which the sequence is yet
uncharacterized (Vamecq et al., Biochemical J. 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 Bacilis 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-00141 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
[0693] 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,
isobutyryate, isovalerate, succinate, fumarate, phenylacetate,
indoleacetate (Musfeldt et al., J. Bacteriol. 184:636-644 (2002)).
The enzyme from Haloarcula marismortui (annotated as a succinyl-CoA
synthetase) accepts propionate, butyrate, and branched-chain acids
(isovalerate and isobutyrate) as substrates, and was shown to
operate in the forward and reverse directions (Brasen et al., Arch.
Microbiol. 182:277-287 (2004)). The ACD encoded by PAE3250 from
hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the
broadest substrate range of all characterized ACDs, reacting with
acetyl-CoA, isobutyryl-CoA (preferred substrate) and
phenylacetyl-CoA (Brasen et al., supra (2004)). 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 (2004)).
TABLE-US-00142 Accession Gene 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
Example XXIII
Production of BDO Utilizing Carboxylic Acid Reductase
[0694] This example describes the generation of a microbial
organism that produces 1,4-butanediol using carboxylic acid
reductase enzymes.
[0695] Escherichia coli is used as a target organism to engineer
the pathway for 1,4-butanediol synthesis described in FIG. 58. E.
coli provides a good host for generating a non-naturally occurring
microorganism capable of producing 1,4-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 various
oxygenation conditions.
[0696] Integration of 4-Hydroxybutyrate Pathway Genes into
Chromosome: Construction of ECKh-432.
[0697] The carboxylic acid reductase enzymes were expressed in a
strain of E. coli designated ECKh-761 which is a descendent of
ECKh-432 with additional deletions of the sad and gabD genes
encoding succinate semialdehyde dehydrogenase enzymes. This strain
contained the components of the BDO pathway, leading to 4HB,
integrated into the chromosome of E. coli at the fimD locus as
described in Example XXI.
[0698] Cloning and Expression of Carboxylic Acid Reductase and
PPTase.
[0699] To generate an E. coli strain engineered to produce
1,4-butanediol, 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.
[0700] The car gene (GNM.sub.--720) was cloned by PCR from Nocardia
genomic DNA. Its nucleic acid and protein sequences are shown in
FIGS. 59A and 59B, 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.
60A and 60B, 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. 64, 65, and 66, respectively. The plasmids
were transformed into ECKh-761 to express the proteins and enzymes
required for 1,4-butanediol production.
[0701] Additional CAR variants were generated. A codon optimized
version of CAR 891 was generated and designated 891 GA. The nucleic
acid and amino acid sequences of CAR 891GA are shown in FIGS. 67A
and 67B, 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 5417 of
the CAR sequence, and additional variants were tested as well
(amino acid positions corresponding to amino acid positions of
sequence of FIG. 67B). Exemplary CAR variants include: E16K; Q95L;
L100M; A1011T; K823E; T941S; H15Q; D198E; G446C; S392N; F699L;
V883I; F467S; T987S; R12H; V295G; V295A; V295S; V295T; V295C;
V295V; V295L; V295I; V295M; V295P; V295F; V295Y; V295W; V295D;
V295E; V295N; V295Q; V295H; V295K; V295R; M296G; M296A; M296S;
M296T; M296C; M296V; M296L; M296I; M296M; M296P; M296F; M296Y;
M296W; M296D; M296E; M296N; M296Q; M296H; M296K; M296R; G297G;
G297A; G297S; G297T; G297C; G297V; G297L; G297I; G297M; G297P;
G297F; G297Y; G297W; G297D; G297E; G297N; G297Q; G297H; G297K;
G297R; G391G; G391A; G391S; G391T; G391C; G391V; G391L; G391I;
G391M; G391P; G391F; G391Y; G391W; G391D; G391E; G391N; G391Q;
G391H; G391K; G391R; G421G; G421A; G421S; G421T; G421C; G421V;
G421L; G421I; G421M; G421P; G421F; G421Y; G421W; G421D; G421E;
G421N; G421Q; G421H; G421K; G421R; D413G; D413A; D413S; D413T;
D413C; D413V; D413L; D413I; D413M; D413P; D413F; D413Y; D413W;
D413D; D413E; D413N; D413Q; D413H; D413K; D413R; G414G; G414A;
G414S; G414T; G414C; G414V; G414L; G414I; G414M; G414P; G414F;
G414Y; G414W; G414D; G414E; G414N; G414Q; G414H; G414K; G414R;
Y415G; Y415A; Y415S; Y415T; Y415C; Y415V; Y415L; Y415I; Y415M;
Y415P; Y415F; Y415Y; Y415W; Y415D; Y415E; Y415N; Y415Q; Y415H;
Y415K; Y415R; G416G; G416A; G416S; G416T; G416C; G416V; G416L;
G416I; G416M; G416P; G416F; G416Y; G416W; G416D; G416E; G416N;
G416Q; G416H; G416K; G416R; S417G; S417A; S417S; S417T; S417C;
S417V S417L; S417I; S417M; S417P; S417F; S417Y; S417W; S417D;
S417E; S417N; S417Q; S417H; S417K; and S417R.
[0702] The CAR variants were screened for activity, and numerous
CAR variants were found to exhibit CAR activity.
[0703] Demonstration of 1,4-BDO Production Using Carboxylic Acid
Reductase.
[0704] Functional expression of the 1,4-butanediol pathway was
demonstrated using E. coli whole-cell culture. Single colonies of
E. coli ECKh-761 transformed with the pZS*13 and pKJL33S plasmids
containing a car gene and GNM.sub.--721, respectively, were
inoculated into 5 mL of LB medium containing appropriate
antibiotics. Similarly, single colonies of E. coli ECKh-761
transformed with car-containing pZS*13 plasmids and pKJL33S
plasmids with no insert were inoculated into additional 5 mL
aliquots of LB medium containing appropriate antibiotics. Ten mL
micro-aerobic cultures were started by inoculating fresh minimal in
vivo conversion medium (see below) containing the appropriate
antibiotics with 1.5% of the first cultures.
[0705] Recipe of the minimal in vivo conversion medium (for 1000
mL) is as follows:
TABLE-US-00143 Final concentration 1M MOPS/KOH buffer 100 mM
Glucose (40%) 1% 10XM9 salts solution 1X MgSO4 (1 M) 1 mM trace
minerals (x1000) 1X 1M NaHCO3 10 mM
[0706] Microaerobic conditions were established by initially
flushing capped anaerobic bottles with nitrogen for 5 minutes, then
piercing the septum with an 18 G needle following inoculation. The
needle was kept in the bottle during growth to allow a small amount
of air to enter the bottles. Protein expression was induced with
0.2 mM IPTG when the culture reached mid-log growth phase. This is
considered: time=0 hr. The culture supernatants were analyzed for
BDO, 4HB, and other by-products as described above and in
WO2008115840 (see Table 30).
[0707] Table 32 shows the production of various products in the
strains expressing various carboxylic acid reductases, including
production of BDO.
TABLE-US-00144 TABLE 32 Production of various products in strains
expressing various carboxylic acid reductases. Cm10 Carb100 Carb100
0 h Strain pKLJ33S pZS*13S pZShc13S OD600 OD600 1 761 034rbs55 no
insert 0.54 2.13 5 761 721 720 0.48 1.88 7 761 721 890 0.45 1.63 8
761 721 891 0.48 1.65 9 761 721 892 0.45 1.31 12 761 no insert 720
0.50 1.72 14 761 no insert 890 0.51 1.96 15 761 no insert 891 0.19
2.36 16 761 no insert 892 0.05 1.40 48 h PA Su La 4HB BDO GBL
EtOH.sub.Enz 48 h, mM 1 10.60 0.00 0.20 8.08 2.40 2.97 0.65 5 3.41
0.00 0.02 6.93 8.53 0.24 1.82 7 0.00 0.00 0.00 6.26 12.30 0.47 5.85
8 2.16 0.00 0.00 7.61 9.08 0.46 2.84 9 0.36 0.00 0.00 5.89 7.83
0.15 2.89 12 8.30 0.00 0.13 9.91 1.99 0.14 0.64 14 2.57 0.00 0.01
9.77 3.53 0.14 1.44 15 1.73 0.00 0.00 9.71 2.68 0.10 0.79 16 0.02
0.00 0.00 10.80 1.30 0.07 0.55 48 h, mM/OD 1 4.98 0.00 0.09 3.80
1.13 1.40 0.31 5 1.81 0.00 0.01 3.69 4.54 0.13 0.97 7 0.00 0.00
0.00 3.84 7.55 0.29 3.59 8 1.31 0.00 0.00 4.61 5.50 0.28 1.72 9
0.27 0.00 0.00 4.50 5.99 0.12 2.21 12 4.83 0.00 0.07 5.76 1.16 0.08
0.37 14 1.31 0.00 0.01 4.99 1.80 0.07 0.74 15 0.73 0.00 0.00 4.11
1.13 0.04 0.33 16 0.01 0.00 0.00 7.71 0.93 0.05 0.39 PA = pyruvate,
SA = succinate, LA = lactate, 4HB = 4-hydroxybutyrate, BDO =
1,4-butanediol, GBL = gamma-butyrolactone, Etoh = ethanol, LLOQ =
lower limit of quantification
[0708] These results show that various carboxylic acid reductases
can function in a BDO pathway to produce BDO.
Example XXIV
4-Hydroxybutyrate and 1,4-Butanediol Synthesis Pathways
[0709] This example describes exemplary 4-hydroxybutyrate and
1,4-butanediol synthesis pathways, which have also been described
herein above.
[0710] Acetoacetyl-CoA thiolase converts two molecules of
acetyl-CoA into one molecule each of acetoacetyl-CoA and CoA.
Exemplary acetoacetyl-CoA thiolase enzymes include the gene
products of atoB from E. coli (Martin et al., Nat. Biotechnol.
21:796-802 (2003), thlA and thlB from C. acetobutylicum (Hanai et
al., Appl. Environ. Microbiol. 73:7814-7818 (2007); Winzer et al.,
J. Mol. Microbiol. Biotechnol. 2:531-541 (2000), and ERG10 from S.
cerevisiae (Hiser et al., J. Biol. Chem. 269:31383-31389 (1994).
The acetoacetyl-CoA thiolase from Zoogloea ramigera is irreversible
in the biosynthetic direction and a crystal structure is available
(Merilainen et al., Biochem. 48: 11011-11025 (2009)).
TABLE-US-00145 Protein GenBank ID GI number Organism AtoB NP_416728
16130161 Escherichia coli ThlA NP_349476.1 15896127 Clostridium
acetobutylicum ThlB NP_149242.1 15004782 Clostridium acetobutylicum
ERG10 NP_015297 6325229 Saccharomyces cerevisiae phbA P07097.4
135759 Zoogloea ramigera
[0711] Acetoacetyl-CoA can also be synthesized from acetyl-CoA and
malonyl-CoA by acetoacetyl-CoA synthase (EC 2.3.1.194). This enzyme
(FhsA) has been characterized in the soil bacterium Streptomyces
sp. CL 190 where it participates in mevalonate biosynthesis
(Okamura et al, PNAS USA 107:11265-11270 (2010)). As this enzyme
catalyzes an essentially irreversible reaction, it is particularly
useful for metabolic engineering applications for overproducing
metabolites, fuels or chemicals derived from acetoacetyl-CoA. For
example, the enzyme has been heterologously expressed in organisms
that biosynthesize butanol (Lan et al, PNAS USA (2012)) and
poly-(3-hydroxybutyrate) (Matsumoto et al, Biosci Biotech Biochem,
75:364-366 (2011). Other acetoacetyl-CoA synthase genes can be
identified by sequence homology to fhsA.
TABLE-US-00146 Protein GenBank ID GI Number Organism fhsA
BAJ83474.1 325302227 Streptomyces sp CL190 AB183750.1:11991 . . .
12971 BAD86806.1 57753876 Streptomyces sp. KO-3988 epzT ADQ43379.1
312190954 Streptomyces cinnamonensis ppzT CAX48662.1 238623523
Streptomyces anulatus O3I_22085 ZP_09840373.1 378817444 Nocardia
brasiliensis
[0712] Acetoacetyl-CoA can first be reduced to 3-hydroxybutyryl-CoA
by acetoacetyl-CoA reductase (ketone reducing). Acetoacetyl-CoA
reductase catalyzing the reduction of acetoacetyl-CoA to
3-hydroxybutyryl-CoA participates in the acetyl-CoA fermentation
pathway to butyrate in several species of Clostridia and has been
studied in detail (Jones and Woods, 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 and Schulz, Methods Enzymol. 71 Pt
C:403-411 (1981)). Yet other gene candidates demonstrated to reduce
acetoacetyl-CoA to 3-hydroxybutyryl-CoA are phbB from Zoogloea
ramigera (Ploux et al., Eur. J. Biochem. 174:177-182 (1988) and
phaB from Rhodobacter sphaeroides (Alber et al., Mol. Microbiol.
61:297-309 (2006). The former gene candidate is NADPH-dependent,
its nucleotide sequence has been determined (Peoples and Sinskey,
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., Eur. J. Biochem. 174:177-182
(1988)). Additional gene candidates include Hbd1 (C-terminal
domain) and Hbd2 (N-terminal domain) in Clostridium kluyveri
(Hillmer and Gottschalk, Biochim. Biophys. Acta 3334:12-23 (1974))
and HSD17B10 in Bos taurus (Wakil et al., J. Biol. Chem.
207:631-638 (1954)).
TABLE-US-00147 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
[0713] A number of similar enzymes have been found in other species
of Clostridia and in Metallosphaera sedula (Berg et al., Science
318:1782-1786 (2007).
TABLE-US-00148 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
[0714] This Example shows further enzymes that can be used in a
4-hydroxybutyrate pathway. The genes for the first enzyme,
acetoacetyl-CoA thiolase are described herein above.
[0715] Exemplary 3-hydroxyacyl dehydrogenases which convert
acetoacetyl-CoA to 3-hydroxybutyryl-CoA include hbd from C.
acetobutylicum (Boynton et al., J. Bacteriol. 178:3015-3024 (1996),
hbd from C. beijerinckii (Colby and Chen, Appl. Environ. Microbiol.
58:3297-3302 (1992), and a number of similar enzymes from
Metallosphaera sedula (Berg et al., Science 318:1782-1786
(2007).
TABLE-US-00149 Protein GenBank ID GI Number Organism hbd
NP_349314.1 15895965 Clostridium acetobutylicum hbd AAM14586.1
20162442 Clostridium beijerinckii Msed_1423 YP_001191505 146304189
Metallosphaera sedula Msed_0399 YP_001190500 146303184
Metallosphaera sedula Msed_0389 YP_001190490 146303174
Metallosphaera sedula Msed_1993 YP_001192057 146304741
Metallosphaera sedula
[0716] The gene product of crt from C. acetobutylicum catalyzes the
dehydration of 3-hydroxybutyryl-CoA to crotonyl-CoA (Boynton et
al., J. Bacteriol. 178:3015-3024 (1996); Atsumi et al., Metab. Eng.
(2007)). Further, enoyl-CoA hydratases are reversible enzymes and
thus suitable candidates for catalyzing the dehydration of
3-hydroxybutyryl-CoA to crotonyl-CoA. The enoyl-CoA hydratases,
phaA and phaB, of P. putida are believed to carry out the
hydroxylation of double bonds during phenylacetate catabolism
(Olivera et al., Proc. Nat. Acad. Sci. U.S.A. 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, paaF, and paaG (Park and
Lee, J. Bacteriol. 185:5391-5397 (2003); Park and Lee, Appl.
Biochem. Biotechnol. 113-116:335-346 (2004); Park and Yup,
Biotechnol. Bioeng. 86:681-686 (2004); Ismail et al., J. Bacteriol.
175:5097-5105 (2003)).
TABLE-US-00150 Protein GenBank ID GI Number Organism crt
NP_349318.1 15895969 Clostridium acetobutylicum paaA NP_745427.1
26990002 Pseudomonas putida paaB NP_745426.1 26990001 Pseudomonas
putida phaA ABF82233.1 106636093 Pseudomonas fluorescens phaB
ABF82234.1 106636094 Pseudomonas fluorescens maoC NP_415905.1
16129348 Escherichia coli paaF NP_415911.1 16129354 Escherichia
coli paaG NP_415912.1 16129355 Escherichia coli
[0717] Several enzymes that naturally catalyze the reverse reaction
(i.e., the dehydration of 4-hydroxybutyryl-CoA to crotonoyl-CoA) in
vivo have been identified in numerous species. This transformation
is required for 4-aminobutyrate fermentation by Clostridium
aminobutyricum (Scherf and Buckel, Eur. J. Biochem. 215:421-429
(1993) and succinate-ethanol fermentation by Clostridium kluyveri
(Scherf et al., Arch. Microbiol. 161:239-245 (1994)). The
transformation is also a key step in Archaea, for example,
Metallosphaera sedula, as part of the
3-hydroxypropionate/4-hydroxybutyrate autotrophic carbon dioxide
assimilation pathway (Berg et al., Science 318:1782-1786 (2007)).
This pathway requires the hydration of crotonoyl-CoA to form
4-hydroxybutyryl-CoA. The reversibility of 4-hydroxybutyryl-CoA
dehydratase is well-documented (Muh et al., Biochemistry
35:11710-11718 (1996); Friedrich et al., Agnew Chem. Int. Ed. Engl.
47:3254-3257 (2008); Muh et al., Eur. J. Biochem. 248:380-384
(1997) and the equilibrium constant has been reported to be about 4
on the side of crotonoyl-CoA (Scherf and Buckel, Eur. J. Biochem.
215:421-429 (1993). This implies that the downstream
4-hydroxybutyryl-CoA dehydrogenase must keep the
4-hydroxybutyryl-CoA concentration low so as to not create a
thermodynamic bottleneck at crotonyl-CoA. The reverse reaction of
4-hydroxybutyryl-CoA dehydratase is crotonyl-CoA hydratase.
TABLE-US-00151 Protein GenBank ID GI Number Organism AbfD CAB60035
70910046 Clostridium aminobutyricum AbfD YP_001396399 153955634
Clostridium kluyveri Msed_1321 YP_001191403 146304087
Metallosphaera sedula Msed_1220 YP_001191305 146303989
Metallosphaera sedula
[0718] Suitable acetoacetyl-CoA and 4-hydroxybutyryl-CoA
transferases are encoded by the gene products of cat1, cat2, and
cat3 of Clostridium kluyveri. These enzymes have been shown to
exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA
transferase activity, respectively (Seedorf et al., Proc. Natl.
Acad. Sci. USA 105:2128-2133 (2008); Sohling and Gottschalk, J
Bacteriol 178:871-880 (1996)). Similar CoA transferase activities
are also present in Trichomonas vaginalis (van Grinsven et al., J.
Biol. Chem. 283:1411-1418 (2008)) and Trypanosoma brucei (Riviere
et al., J. Biol. Chem. 279:45337-45346 (2004)). Yet another
transferase capable of the desired conversions is
butyryl-CoA:acetoacetate CoA-transferase. Exemplary enzymes can be
found in Fusobacterium nucleatum (Barker et al., J. Bacteriol.
152(1):201-7 (1982)), Clostridium SB4 (Barker et al., J. Biol.
Chem. 253(4):1219-25 (1978)), and Clostridium acetobutylicum
(Wiesenborn et al., Appl. Environ. Microbiol. 55(2):323-9 (1989)).
Although specific gene sequences were not provided for
butyryl-CoA:acetoacetate CoA-transferase in these references, the
genes FN0272 and FN0273 have been annotated as a
butyrate-acetoacetate CoA-transferase (Kapatral et al., J. Bact.
184(7) 2005-2018 (2002)). Homologs in Fusobacterium nucleatum such
as FN1857 and FN1856 also likely have the desired acetoacetyl-CoA
transferase activity. FN1857 and FN1856 are located adjacent to
many other genes involved in lysine fermentation and are thus very
likely to encode an acetoacetate:butyrate CoA transferase
(Kreimeyer, et al., J. Biol. Chem. 282 (10) 7191-7197 (2007)).
Additional candidates from Porphyrmonas gingivalis and
Thermoanaerobacter tengcongensis can be identified in a similar
fashion (Kreimeyer, et al., J. Biol. Chem. 282 (10) 7191-7197
(2007)). Information related to these proteins and genes is shown
below.
TABLE-US-00152 Protein GENBANK ID GI NUMBER 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 FN0272 NP_603179.1 19703617
Fusobacterium nucleatum FN0273 NP_603180.1 19703618 Fusobacterium
nucleatum FN1857 NP_602657.1 19705162 Fusobacterium nucleatum
FN1856 NP_602656.1 19705161 Fusobacterium nucleatum PG1066
NP_905281.1 34540802 Porphyromonas gingivalis W83 PG1075
NP_905290.1 34540811 Porphyromonas gingivalis W83 TTE0720
NP_622378.1 20807207 Thermoanaerobacter tengcongensis MB4 TTE0721
NP_622379.1 20807208 Thermoanaerobacter tengcongensis MB4
[0719] An alternative method for removing the CoA moiety from
acetoacetyl-CoA or 4-hydroxybutyryl-CoA is to apply a pair of
enzymes such as a phosphate-transferring acyltransferase and a
kinase to impart acetoacetyl-CoA or 4-hydroxybutyryl-CoA synthetase
activity. Exemplary names for these enzymes include
phosphotrans-4-hydroxybutyrylase/4-hydroxybutyrate kinase, which
can remove the CoA moiety from 4-hydroxybutyryl-CoA, and
phosphotransacetoacetylase/acetoacetate kinase which can remove the
CoA moiety from acetoacetyl-CoA. This general activity enables the
net hydrolysis of the CoA-ester of either molecule with the
simultaneous generation of ATP. For example, the butyrate kinase
(buk)/phosphotransbutyrylase (ptb) system from Clostridium
acetobutylicum has been successfully applied to remove the CoA
group from 3-hydroxybutyryl-CoA when functioning as part of a
pathway for 3-hydroxybutyrate synthesis (Tseng et al., Appl.
Environ. Microbiol. 75(10):3137-3145 (2009)). Specifically, the ptb
gene from C. acetobutylicum encodes an enzyme that can convert an
acyl-CoA into an acyl-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)). Additional exemplary
phosphate-transferring acyltransferases include
phosphotransacetylase, encoded by pta. 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)). Information related to
these proteins and genes is shown below.
TABLE-US-00153 Protein GENBANK ID GI NUMBER 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
[0720] Exemplary kinases include the E. coli acetate kinase,
encoded by ackA (Skarstedt and Silverstein J. Biol. Chem.
251:6775-6783 (1976)), the C. acetobutylicum butyrate kinases,
encoded by buk1 and buk2 ((Walter et al. Gene 134(1):107-111
(1993); Huang et al. J Mol Microbiol Biotechnol 2(1):33-38 (2000)),
and the E. coli gamma-glutamyl kinase, encoded by proB (Smith et
al. J. Bacteriol. 157:545-551 (1984)). These enzymes phosphorylate
acetate, butyrate, and glutamate, respectively. The ackA gene
product from E. coli also phosphorylates propionate (Hesslinger et
al. Mol. Microbiol 27:477-492 (1998)). Information related to these
proteins and genes is shown below:
TABLE-US-00154 Protein GENBANK ID GI NUMBER ORGANISM AckA
NP_416799.1 16130231 Escherichia coli Buk1 NP_349675 15896326
Clostridium acetobutylicum Buk2 Q97II1 20137415 Clostridium
acetobutylicum ProB NP_414777.1 16128228 Escherichia coli
[0721] Further enzymes that can be used in a 1,4-butanediol
pathway. The genes for acetoacetyl-CoA thiolase,
3-Hydroxybutyryl-CoA dehydrogenase (Hbd), Crotonase (Crt), and
Crotonyl-CoA hydratase (4-Budh) are described herein above.
Alcohol-forming 4-hydroxybutyryl-CoA reductase enzymes catalyze the
2 reduction steps required to form 1,4-butanediol from
4-hydroxybutyryl-CoA. Exemplary 2-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)). The adhE2 enzyme from C. acetobutylicum was specifically
shown in ref. (WO/2008/115840 (2008)) to produce BDO from
4-hydroxybutyryl-CoA. In addition to reducing acetyl-CoA to
ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides
has been shown to oxide the branched chain compound
isobutyraldehyde to isobutyryl-CoA (Kazahaya et al., J. Gen. Appl.
Microbiol. 18:43-55 (1972; Koo et al., Biotechnol. Lett. 27:505-510
(2005)).
TABLE-US-00155 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
[0722] 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 and Fuchs, Eur. J. Biochem.
215:633-643 (1993)). This enzyme, with a mass of 300 kDa, is highly
substrate-specific and shows little sequence similarity to other
known oxidoreductases (Hugler et al., J. Bacteriol. 184:2404-2410
(2002)). No enzymes in other organisms have been shown to catalyze
this specific reaction; however there is bioinformatic evidence
that other organisms may have similar pathways (Klatt et al.,
Environ. Microbiol. 9:2067-2078 (2007)). Enzyme candidates in other
organisms including Roseiflexus castenholzii, Erythrobacter sp.
NAP1 and marine gamma proteobacterium HTCC2080 can be inferred by
sequence similarity.
TABLE-US-00156 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
[0723] An alternative route to BDO from 4-hydroxybutyryl-CoA
involves first reducing this compound to 4-hydroxybutanal. 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. Bacteriol.
179:2969-2975 (1997), the Acinetobacter sp. M-1 fatty acyl-CoA
reductase (Ishige et al., Appl. Environ. Microbiol. 68:1192-1195
(2002), and a CoA- and NADP-- dependent succinate semialdehyde
dehydrogenase encoded by the sucD gene in Clostridium kluyveri
(Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996); Sohling
and Gottschalk, J. Bacteriol. 178:8710880 (1996)). SucD of P.
gingivalis is another succinate semialdehyde dehydrogenase
(Takahashi et al., J. Bacteriol. 182:4704-4710 (2000)). These
succinate semialdehyde dehydrogenases were specifically shown in
ref. (WO/2008/115840 (2008)) to convert 4-hydroxybutyryl-CoA to
4-hydroxybutanal as part of a pathway to produce 1,4-butanediol.
The enzyme acylating acetaldehyde dehydrogenase in Pseudomonas sp,
encoded by bphG, is yet another capable enzyme as it has been
demonstrated to oxidize and acylate acetaldehyde, propionaldehyde,
butyraldehyde, isobutyraldehyde and formaldehyde (Powlowski et al.,
J. Bacteriol. 175:377-385 (1993)).
TABLE-US-00157 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
[0724] These results show that various carboxylic acid reductases
can function in a BDO pathway to produce BDO. 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, 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. Mol. Microbiol. 61:297-309 (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,
Appl. Environ. Microbiol. 65:4973-4980 (1999). This enzyme has been
reported to reduce acetyl-CoA and butyryl-CoA to their
corresponding aldehydes. This gene is very similar to cutE that
encodes acetaldehyde dehydrogenase of Salmonella typhimurium and E.
coli (Toth, Appl. Environ. Microbiol. 65:4973-4980 (1999). These
proteins are identified below.
TABLE-US-00158 Protein GenBank ID GI Number Organism Msed_0709
YP_001190808.1 146303492 Metallosphaera sedula Mcr NP_378167.1
15922498 Sulfolobus tokodaii asd-2 NP_343563.1 15898958 Sulfolobus
solfataricus Saci_2370 YP_256941.1 70608071 Sulfolobus
acidocaldarius Ald AAT66436 49473535 Clostridium beijerinckii eutE
AAA80209 687645 Salmonella typhimurium eutE P77445 2498347
Escherichia coli
[0725] 4-Hydroxybutyryl-CoA can also be converted to
4-hydroxybutanal in several enzymatic steps, though the
intermediate 4-hydroxybutyrate. First, 4-hydroxybutyryl-CoA can be
converted to 4-hydroxybutyrate by a CoA transferase, hydrolase or
synthetase. Alternately, 4-hydroxybutyryl-CoA can be converted to
4-hydroxybutyrate via a phosphonated intermediate by enzymes with
phosphotrans-4-hydroxybutyrylase and 4-hydroxybutyrate kinase.
Exemplary candidates for these enzymes are described above.
[0726] Subsequent conversion of 4-hydroxybutyrate to
4-hydroxybutanal is catalyzed by an aryl-aldehyde dehydrogenase, or
equivalently a carboxylic acid reductase. Such an enzyme is found
in Nocardia iowensis. Carboxylic acid reductase catalyzes the
magnesium, ATP and NADPH-dependent reduction of carboxylic acids to
their corresponding aldehydes (Venkitasubramanian et al., J. Biol.
Chem. 282:478-485 (2007)) and is capable of catalyzing the
conversion of 4-hydroxybutyrate to 4-hydroxybutanal. This enzyme,
encoded by car, was cloned and functionally expressed in E. coli
(Venkitasubramanian et al., J. Biol. Chem. 282:478-485 (2007)).
Expression of the npt gene product improved activity of the enzyme
via post-transcriptional modification. The npt gene encodes a
specific phosphopantetheine transferase (PPTase) that converts the
inactive apo-enzyme to the active holo-enzyme. The natural
substrate of this enzyme is vanillic acid, and the enzyme exhibits
broad acceptance of aromatic and aliphatic substrates
(Venkitasubramanian et al., 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-00159 Gene name GI Number GenBank ID Organism Car 40796035
AAR91681.1 Nocardia iowensis (sp. NRRL 5646) Npt 114848891
ABI83656.1 Nocardia iowensis (sp. NRRL 5646)
[0727] Additional car and npt genes can be identified based on
sequence homology.
TABLE-US-00160 Gene name GI Number GenBank ID Organism fadD9
121638475 YP_978699.1 Mycobacterium bovis BCG BCG_2812c 121638674
YP_978898.1 Mycobacterium bovis BCG nfa20150 54023983 YP_118225.1
Nocardia farcinica IFM 10152 nfa40540 54026024 YP_120266.1 Nocardia
farcinica IFM 10152 SGR_6790 182440583 YP_001828302.1 Streptomyces
griseus subsp. griseus NBRC 13350 SGR_665 182434458 YP_001822177.1
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
ZP_04026660.1 Tsukamurella paurometabola DSM 20162 CPCC7001_1320
ZP_05045132.1 254431429 Cyanobium PCC7001 DDBDRAFT_0187729
XP_636931.1 66806417 Dictyostelium discoideum AX4
[0728] 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-00161 Gene name GI Number GenBank ID Organism griC
182438036 YP_001825755.1 Streptomyces griseus subsp. griseus NBRC
13350 Grid 182438037 YP_001825756.1 Streptomyces griseus subsp.
griseus NBRC 13350
[0729] 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-00162 Gene name GI Number GenBank ID Organism LYS2 171867
AAA34747.1 Saccharomyces cerevisiae LYS5 1708896 P50113.1
Saccharomyces cerevisiae LYS2 2853226 AAC02241.1 Candida albicans
LYS5 28136195 AAO26020.1 Candida albicans Lys1p 13124791 P40976.3
Schizosaccharomyces pombe Lys7p 1723561 Q10474.1
Schizosaccharomyces pombe Lys2 3282044 CAA74300.1 Penicillium
chrysogenum
[0730] Enzymes exhibiting 1,4-butanediol dehydrogenase activity are
capable of forming 1,4-butanediol from 4-hydroxybutanal. 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 C.sub.2-C.sub.14 (Tani Appl. Environ. Micro et
al. 66:5231-5235 (2000), ADH2 from Saccharomyces cerevisiae
(Aoshima et al., Mol. Microbiol. 51:791-798 (2004)), yqhD from E.
coli which has preference for molecules longer than C(3)
(Sulzenbacher et al., J. Mol. Biol. 342:489-502 (2004), and bdh I
and bdh II from C. acetobutylicum which converts butyraldehyde into
butanol (Walter et al., J. Bacteriol. 174:7149-7158 (1992)). ADH1
from Zymomonas mobilis has been demonstrated to have activity on a
number of aldehydes including formaldehyde, acetaldehyde,
propionaldehyde, butyraldehyde, and acrolein (Kinoshita, Appl.
Microbiol. Biotechnol. 22:249-254 (1985)).
TABLE-US-00163 Protein GenBank ID GI Number Organism alrA
BAB12273.1 9967138 Acinetobacter sp. Strain M-1 ADH2 NP_014032.1
6323961 Saccharymyces 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
[0731] Enzymes exhibiting 4-hydroxybutyrate dehydrogenase activity
(EC 1.1.1.61) also fall into this category. Such enzymes have been
characterized in Ralstonia eutropha (Bravo et al., J. Forensic Sci.
49:379-387 (2004), Clostridium kluyveri (Wolff and Kenealy, Protein
Expr. Purif. 6:206-212 (1995)) and Arabidopsis thaliana (Breitkreuz
et al., J. Biol. Chem. 278:41552-41556 (2003)).
TABLE-US-00164 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
Example XXV
Exemplary Hydrogenase and CO Dehydrogenase Enzymes for Extracting
Reducing Equivalents from Syngas and Exemplary Reductive TCA Cycle
Enzymes
[0732] 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
CO2-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.
[0733] 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-00165 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
[0734] 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-00166 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
[0735] 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-00167 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
[0736] 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-00168 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
[0737] 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-00169 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
[0738] 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-00170 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
[0739] 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. Biochem. Biophys.
Res. Commun. 292:280-286 (2002)). The kinetics of CO.sub.2 fixation
of both H. thermophilus OFOR enzymes have been characterized
(Yamamoto et al., Extremophiles 14:79-85 (2010)). A CO.sub.2-fixing
OFOR from Chlorobium thiosulfatophilum has been purified and
characterized but the genes encoding this enzyme have not been
identified to date. Enzyme candidates in Chlorobium species can be
inferred by sequence similarity to the H. thermophilus genes. For
example, the Chlorobium limicola genome encodes two similar
proteins. Acetogenic bacteria such as Moorella thermoacetica are
predicted to encode two OFOR enzymes. The enzyme encoded by
Moth.sub.--0034 is predicted to function in the
CO.sub.2-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.
[0740] 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., supra, 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., J. Bacteriol.
180:1119-1128 (1998)). An enzyme specific to alpha-ketoglutarate
has been reported in Thauera aromatics (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-00171 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 (AAC38210.1) (2935178) oorA NP_207384.1
15645214 Helicobacter pylori (AAC38211.1) (2935179) oorB
NP_207385.1 15645215 Helicobacter pylori (AAC38212.1) (2935180)
oorC NP_207386.1 15645216 Helicobacter pylori (AAC38213.1)
(2935181) 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
[0741] 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-00172 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
[0742] In H. thermophilus the reductive carboxylation of
2-oxoglutarate to isocitrate is catalyzed by two enzymes:
2-oxoglutarate carboxylase and oxalosuccinate reductase.
2-Oxoglutarate carboxylase (EC 6.4.1.7) catalyzes the ATP-dependent
carboxylation of alpha-ketoglutarate to oxalosuccinate (Aoshima and
Igarashi, Mol. Microbiol. 62:748-759 (2006)). This enzyme is a
large complex composed of two subunits. Biotinylation of the large
(A) subunit is required for enzyme function (Aoshima et al., Mol.
Microbiol. 51:791-798 (2004)). Oxalosuccinate reductase (EC
1.1.1.-) catalyzes the NAD-dependent conversion of oxalosuccinate
to D-threo-isocitrate. The enzyme is a homodimer encoded by icd in
H. thermophilus. The kinetic parameters of this enzyme indicate
that the enzyme only operates in the reductive carboxylation
direction in vivo, in contrast to isocitrate dehydrogenase enzymes
in other organisms (Aoshima and Igarashi, J. Bacteriol.
190:2050-2055 (2008)). Based on sequence homology, gene candidates
have also been found in Thiobacillus denitrificans and Thermocrinis
albus.
TABLE-US-00173 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
[0743] 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-00174 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 (acnB) denitrificans 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
[0744] 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
prtotects 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 CO2-assimilating
directions (Ikeda et al., Biochem Biophys Res Commun. 340:76-82
(2006); Yamamoto et al., Extremophiles 14:79-85 (2010)). Homologs
also exist in C. carboxidivorans P7. Several additional PFOR
enzymes are described in the following review (Ragsdale, S. W.,
Chem. Rev. 103:2333-2346 (2003)). Finally, flavodoxin reductases
(e.g., fqrB from Helicobacter pylori or Campylobacter jejuni) (St
Maurice et al., J. Bacteriol. 189:4764-4773 (2007)) or Rnf-type
proteins (Seedorf et al., Proc. Natl. Acad. Sci. U.S.A.
105:2128-2133 (2008); and Herrmann, J. Bacteriol 190:784-791
(2008)) provide a means to generate NADH or NADPH from the reduced
ferredoxin generated by PFOR. These proteins are identified
below.
TABLE-US-00175 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 stn. 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
thennophdus porD BAA95604.1 7768913 Hydrogenobacter thennophdus
porA BAA95605.1 7768914 Hydrogenobacter thennophdus porB BAA95606.1
776891 Hydrogenobacter thennophdus porG BAA95607.1 7768916
Hydrogenobacter thennophdus 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
[0745] The conversion of pyruvate into acetyl-CoA can be catalyzed
by several other enzymes or their combinations thereof. For
example, pyruvate dehydrogenase can transform pyruvate into
acetyl-CoA with the concomitant reduction of a molecule of NAD into
NADH. It is a multi-enzyme complex that catalyzes a series of
partial reactions which results in acylating oxidative
decarboxylation of pyruvate. The enzyme comprises of three
subunits: the pyruvate decarboxylase (E1), dihydrolipoamide
acyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). This
enzyme is naturally present in several organisms, including E. coli
and S. cerevisiae. In the E. coli enzyme, specific residues in the
E1 component are responsible for substrate specificity (Bisswanger,
H., J. Biol. Chem. 256:815-82 (1981); Bremer, J., Eur. J. Biochem.
8:535-540 (1969); Gong et al., J. Biol. Chem. 275:13645-13653
(2000)). Enzyme engineering efforts have improved the E. coli PDH
enzyme activity under anaerobic conditions (Kim et al., J.
Bacteriol. 190:3851-3858 (2008); Kim et al., Appl. Environ.
Microbiol. 73:1766-1771 (2007); Zhou et al., Biotechnol. Lett.
30:335-342 (2008)). In contrast to the E. coli PDH, the B. subtilis
complex is active and required for growth under anaerobic
conditions (Nakano et al., J. Bacteriol. 179:6749-6755 (1997)). The
Klebsiella pneumoniae PDH, characterized during growth on glycerol,
is also active under anaerobic conditions (5). Crystal structures
of the enzyme complex from bovine kidney (18) and the E2 catalytic
domain from Azotobacter vinelandii are available (4). Yet another
enzyme that can catalyze this conversion is pyruvate formate lyase.
This enzyme catalyzes the conversion of pyruvate and CoA into
acetyl-CoA and formate. Pyruvate formate lyase is a common enzyme
in prokaryotic organisms that is used to help modulate anaerobic
redox balance. Exemplary enzymes can be found in Escherichia coli
encoded by pflB (Knappe and Sawers, FEMS. Microbiol Rev. 6:383-398
(1990)), Lactococcus lactis (Melchiorsen et al., Appl Microbiol
Biotechnol 58:338-344 (2002)), and Streptococcus mutans
(Takahashi-Abbe et al., Oral. Microbiol Immunol. 18:293-297
(2003)). E. coli possesses an additional pyruvate formate lyase,
encoded by tdcE, that catalyzes the conversion of pyruvate or
2-oxobutanoate to acetyl-CoA or propionyl-CoA, respectively
(Hesslinger et al., Mol. Microbiol. 27:477-492 (1998)). Both pflB
and tdcE from E. coli require the presence of pyruvate formate
lyase activating enzyme, encoded by pflA. Further, a short protein
encoded by yfiD in E. coli can associate with and restore activity
to oxygen-cleaved pyruvate formate lyase (Vey et al., Proc. Natl.
Acad. Sci. U.S.A. 105:16137-16141 (2008). Note that pflA and pflB
from E. coli were expressed in S. cerevisiae as a means to increase
cytosolic acetyl-CoA for butanol production as described in
WO/2008/080124). Additional pyruvate formate lyase and activating
enzyme candidates, encoded by pfl and act, respectively, are found
in Clostridium pasteurianum (Weidner et al., J Bacteriol.
178:2440-2444 (1996)).
[0746] 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.
[0747] 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.
[0748] For enzymes that use reducing equivalents in the form of
NADH or NADPH, these reduced carriers can be generated by
transferring electrons from reduced ferredoxin. Two enzymes
catalyze the reversible transfer of electrons from reduced
ferredoxins to NAD(P).sup.+, ferredoxin:NAD.sup.+ oxidoreductase
(EC 1.18.1.3) and ferredoxin:NADP.sup.+ oxidoreductase (FNR, EC
1.18.1.2). Ferredoxin:NADP.sup.+ oxidoreductase (FNR, EC 1.18.1.2)
has a noncovalently bound FAD cofactor that facilitates the
reversible transfer of electrons from NADPH to low-potential
acceptors such as ferredoxins or flavodoxins (Blaschkowski et al.,
Eur. J. Biochem. 123:563-569 (1982); Fujii et al., 1977). The
Helicobacter pylori FNR, encoded by HP1164 (fqrB), is coupled to
the activity of pyruvate:ferredoxin oxidoreductase (PFOR) resulting
in the pyruvate-dependent production of NADPH (St Maurice et al., J
Bacteriol. 189(13):4764-4773 (2007)). An analogous enzyme is found
in Campylobacter jejuni (St et al., supra, 2007). A
ferredoxin:NADP.sup.+ oxidoreductase enzyme is encoded in the E.
coli genome by fpr (Bianchi et al., J. Bacteriol. 175:1590-1595
(1993)). Ferredoxin:NAD.sup.+ oxidoreductase utilizes reduced
ferredoxin to generate NADH from NAD.sup.+. In several organisms,
including E. coli, this enzyme is a component of multifunctional
dioxygenase enzyme complexes. The ferredoxin:NAD.sup.+
oxidoreductase of E. coli, encoded by hcaD, is a component of the
3-phenylproppionate dioxygenase system involved in involved in
aromatic acid utilization (Diaz et al., J Bacteriol. 180:2915-2923
(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-00176 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 (RnJB) ljungdahli CLJU_c11400 ADK14208.1 300434441
Clostridium (RnfA) ljungdahli CLJU_c11390 ADK14207.1 300434440
Clostridium (RnfE) ljungdahli CLJU_c11380 ADK14206.1 300434439
Clostridium (RnfG) ljungdahli CLJU_c11370 ADK14205.1 300434438
Clostridium (RnfD) ljungdahli CLJU_c11360 ADK14204.1 300434437
Clostridium (RnfC) ljungdahli
[0749] Ferredoxins are small acidic proteins containing one or more
iron-sulfur clusters that function as intracellular electron
carriers with a low reduction potential. Reduced ferredoxins donate
electrons to Fe-dependent enzymes such as ferredoxin-NADP.sup.+
oxidoreductase, pyruvate:ferredoxin oxidoreductase (PFOR) and
2-oxoglutarate:ferredoxin oxidoreductase (OFOR). The H.
thermophilus gene fdx1 encodes a [4Fe-4S]-type ferredoxin that is
required for the reversible carboxylation of 2-oxoglutarate and
pyruvate by OFOR and PFOR, respectively (Yamamoto et al.,
Extremophiles 14:79-85 (2010)). The ferredoxin associated with the
Sulfolobus solfataricus 2-oxoacid:ferredoxin reductase is a
monomeric dicluster [3Fe-4S][4Fe-4S] type ferredoxin (Park et al.,
J Biochem Mol. Biol. 39:46-54 (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, J Biochem. 126:917-926 (1999)). Additional
ferredoxin proteins have been characterized in Helicobacter pylori
(Mukhopadhyay et al., J Bacteriol. 185:2927-2935 (2003)) and
Campylobacter jejuni (van Vliet et al., FEMS Microbiol Lett.
196:189-193 (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):1115-1122 (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-00177 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
[0750] 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.
[0751] 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.,
J Biol. Chem. 283:1411-1418 (2008)) and Trypanosoma brucei (Riviere
et al., J Biol. Chem. 279(44):45337-45346 (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., J. Bacteriol. 190(14):4933-4940 (2008)).
Similar succinyl-CoA transferase activities are also present in
Trichomonas vaginalis (van Grinsven et al., supra 2008),
Trypanosoma brucei (Riviere et al., supra 2004) and Clostridium
kluyveri (Sohling and Gottschalk, J. Bacteriol. 178:871-880
(1996)). The beta-ketoadipate:succinyl-CoA transferase encoded by
pcaI and pcaJ in Pseudomonas putida is yet another candidate
(Kaschabek et al., J. Bacteriol. 184(1):207-215 (2002). The
aforementioned proteins are identified below.
TABLE-US-00178 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
[0752] 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., J. Biol. Chem.
272(41):25659-25667 (1997)), Bacillus subtilis, and Homo sapiens
(Fukao et al., Genomics 68(2):144-151 (2000); Tanaka et al., Mol
Hum Reprod. 8(1):16-23 (2002)). The aforementioned proteins are
identified below.
TABLE-US-00179 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
[0753] Converting succinate to succinyl-CoA by
succinyl-CoA:3:ketoacid-CoA transferase requires the simultaneous
conversion of a 3-ketoacyl-CoA such as acetoacetyl-CoA to a
3-ketoacid such as acetoacetate. Conversion of a 3-ketoacid back to
a 3-ketoacyl-CoA can be catalyzed by an acetoacetyl-CoA:acetate:CoA
transferase. Acetoacetyl-CoA:acetate:CoA transferase converts
acetoacetyl-CoA and acetate to acetoacetate and acetyl-CoA, or vice
versa. Exemplary enzymes include the gene products of atoAD from E.
coli (Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007),
ctfAB from C. acetobutylicum (Jojima et al., Appl Microbiol
Biotechnol 77:1219-1224 (2008), and ctfAB from Clostridium
saccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol
Biochem. 71:58-68 (2007)) are shown below.
TABLE-US-00180 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
[0754] 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 aromatics (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-00181 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
[0755] Additionally, yell encodes a propionyl CoA:succinate CoA
transferase in E. coli (Haller et al., Biochemistry, 39(16)
4622-4629). Close homologs can be found in, for example,
Citrobacter youngae ATCC 29220, Salmonella enterica subsp. arizonae
serovar, and Yersinia intermedia ATCC 29909. The aforementioned
proteins are identified below.
TABLE-US-00182 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
[0756] 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-00183 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
[0757] 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-00184 Protein GenBank ID GI Number Organism ackA
NP_416799.1 16130231 Escherichia coli Ack AAB18301.1 1491790
Clostridium acetobutylicum Ack AAA72042.1 349834 Methanosarcina
thermophile purT AAC74919.1 1788155 Escherichia coli buk1 NP_349675
15896326 Clostridium acetobutylicum buk2 Q97II1 20137415
Clostridium acetobutylicum
[0758] 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-00185 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
[0759] 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-00186 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
[0760] The product yields per C-mol of substrate of microbial cells
synthesizing reduced fermentation products such as 1,4-butanediol,
4-hydroxybutyrate and/or gamma-butyrolactone, are limited by
insufficient reducing equivalents in the carbohydrate feedstock.
Reducing equivalents, or electrons, can be extracted from synthesis
gas components such as CO and H.sub.2 using carbon monoxide
dehydrogenase (CODH) and hydrogenase enzymes, respectively. The
reducing equivalents are then passed to acceptors such as oxidized
ferredoxins, oxidized quinones, oxidized cytochromes, NAD(P)+,
water, or hydrogen peroxide to form reduced ferredoxin, reduced
quinones, reduced cytochromes, NAD(P)H, H.sub.2, or water,
respectively. Reduced ferredoxin and NAD(P)H are particularly
useful as they can serve as redox carriers for various
Wood-Ljungdahl pathway and reductive TCA cycle enzymes.
[0761] Here, we show specific examples of how additional redox
availability from CO and/or H.sub.2 can improve the yields of
reduced products such as 1,4-butanediol, 4-hydroxybutyrate and/or
gamma-butyrolactone. The maximum theoretical yield to produce
1,4-butanediol, 4-hydroxybutyrate and/or gamma-butyrolactone from
glucose is 1.09 mol BDO/mol glucose, 1.33 mol 4HB/mol glucose and
1.33 mol GBL/mol glucose under anaerobic conditions. Using reducing
equivalents from CO, H.sub.2 and their various combinations in
conjunction with carbohydrate feedstocks, such as glucose, yields
of all three products can be improved to 2 mole/mole glucose.
[0762] When both feedstocks of sugar and syngas are available, the
syngas components CO and H.sub.2 can be utilized to generate
reducing equivalents by employing the hydrogenase and CO
dehydrogenase. The reducing equivalents generated from syngas
components will be utilized to power the glucose to 1,4-butanediol,
4-hydroxybutyrate and/or gamma-butyrolactone production pathways.
Theoretically, all carbons in glucose will be conserved, thus
resulting in a maximal theoretical yield to produce 1,4-butanediol,
4-hydroxybutyrate and/or gamma-butyrolactone from glucose at 2 mole
of 1,4-butanediol, 4-hydroxybutyrate and/or gamma-butyrolactone per
mol of glucose under either aerobic or anaerobic conditions.
[0763] As shown in above example, a combined feedstock strategy
where syngas is combined with a sugar-based feedstock or other
carbon substrate can greatly improve the theoretical yields. In
this co-feeding approach, syngas components H.sub.2 and CO can be
utilized by the hydrogenase and CO dehydrogenase to generate
reducing equivalents, that can be used to power chemical production
pathways in which the carbons from sugar or other carbon substrates
will be maximally conserved and the theoretical yields improved.
For example, improved 1,4-butanediol, 4-hydroxybutyrate and/or
gamma-butyrolactone production from glucose or sugar can be
achieved. Such improvements provide environmental and economic
benefits and greatly enhance sustainable chemical production.
[0764] Herein below the enzymes and the corresponding genes used
for extracting redox from synags components are described. CODH is
a reversible enzyme that interconverts CO and CO.sub.2 at the
expense or gain of electrons. The natural physiological role of the
CODH in ACS/CODH complexes is to convert CO.sub.2 to CO for
incorporation into acetyl-CoA by acetyl-CoA synthase. Nevertheless,
such CODH enzymes are suitable for the extraction of reducing
equivalents from CO due to the reversible nature of such enzymes.
Expressing such CODH enzymes in the absence of ACS allows them to
operate in the direction opposite to their natural physiological
role (i.e., CO oxidation).
[0765] 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, C.
ljungdahli and Campylobacter curvus 525.92.
TABLE-US-00187 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
phaeobacteroides DSM 266 (cytochrome c) Cpha266_0149 YP_910643.1
119355999 Chlorobium phaeobacteroides DSM 266 (CODH) Ccel_0438
YP_002504800.1 220927891 Clostridium cellulolyticum H10 Ddes_0382
YP_002478973.1 220903661 Desulfovibrio desulfuricans (CODH) subsp.
desulfuricans str. ATCC 27774 Ddes_0381 YP_002478972.1 220903660
Desulfovibrio desulfuricans (CooC) subsp. desulfuricans str. ATCC
27774 Pcar_0057 YP_355490.1 7791767 Pelobacter carbinolicus DSM
2380 (CODH) Pcar_0058 YP_355491.1 7791766 Pelobacter carbinolicus
DSM 2380 (CooC) Pcar_0058 YP_355492.1 7791765 Pelobacter
carbinolicus DSM 2380 (HypA) 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
[0766] 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-00188 Protein GenBank ID GI Number Organism CODH-I
YP_360644 78043418 Carboxydothermus (CooS-I) hydrogenoformans CooF
YP_360645 78044791 Carboxydothermus hydrogenoformans HypA YP_360646
78044340 Carboxydothermus hydrogenoformans CooH YP_360647 78043871
Carboxydothermus hydrogenoformans CooU YP_360648 78044023
Carboxydothermus hydrogenoformans CooX YP_360649 78043124
Carboxydothermus hydrogenoformans CooL YP_360650 78043938
Carboxydothermus hydrogenoformans CooK YP_360651 78044700
Carboxydothermus hydrogenoformans CooM YP_360652 78043942
Carboxydothermus hydrogenoformans CooC YP_360654.1 78043296
Carboxydothermus hydrogenoformans CooA-1 YP_360655.1 78044021
Carboxydothermus hydrogenoformans CooL AAC45118 1515468
Rhodospirillum rubrum CooX AAC45119 1515469 Rhodospirillum rubrum
CooU AAC45120 1515470 Rhodospirillum rubrum CooH AAC45121 1498746
Rhodospirillum rubrum CooF AAC45122 1498747 Rhodospirillum rubrum
CODH AAC45123 1498748 Rhodospirillum rubrum (CooS) CooC AAC45124
1498749 Rhodospirillum rubrum CooT AAC45125 1498750 Rhodospirillum
rubrum CooJ AAC45126 1498751 Rhodospirillum rubrum
[0767] 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
(285(6):3928-3938 (2010)). 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-00189 Protein GenBank ID GI Number Organism HyaA
AAC74057.1 1787206 Escherichia coli HyaB AAC74058.1 1787207
Escherichia coli HyaC AAC74059.1 1787208 Escherichia coli HyaD
AAC74060.1 1787209 Escherichia coli HyaE AAC74061.1 1787210
Escherichia coli HyaF AAC74062.1 1787211 Escherichia coli HybO
AAC76033.1 1789371 Escherichia coli HybA AAC76032.1 1789370
Escherichia coli HybB AAC76031.1 2367183 Escherichia coli HybC
AAC76030.1 1789368 Escherichia coli HybD AAC76029.1 1789367
Escherichia coli HybE AAC76028.1 1789366 Escherichia coli HybF
AAC76027.1 1789365 Escherichia coli HybG AAC76026.1 1789364
Escherichia coli
[0768] 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-00190 Protein GenBank ID GI Number Organism HycA NP_417205
16130632 Escherichia coli HycB NP_417204 16130631 Escherichia coli
HycC NP_417203 16130630 Escherichia coli HycD NP_417202 16130629
Escherichia coli HycE NP_417201 16130628 Escherichia coli HycF
NP_417200 16130627 Escherichia coli HycG NP_417199 16130626
Escherichia coli HycH NP_417198 16130625 Escherichia coli HycI
NP_417197 16130624 Escherichia coli HyfA NP_416976 90111444
Escherichia coli HyfB NP_416977 16130407 Escherichia coli HyfC
NP_416978 90111445 Escherichia coli HyfD NP_416979 16130409
Escherichia coli HyfE NP_416980 16130410 Escherichia coli HyfF
NP_416981 16130411 Escherichia coli HyfG NP_416982 16130412
Escherichia coli HyfH NP_416983 16130413 Escherichia coli HyfI
NP_416984 16130414 Escherichia coli HyfJ NP_416985 90111446
Escherichia coli HyfR NP_416986 90111447 Escherichia coli HypA
NP_417206 16130633 Escherichia coli HypB NP_417207 16130634
Escherichia coli HypC NP_417208 16130635 Escherichia coli HypD
NP_417209 16130636 Escherichia coli HypE NP_417210 226524740
Escherichia coli HypF NP_417192 16130619 Escherichia coli
[0769] The M. thermoacetica hydrogenases are suitable for a host
that lacks sufficient endogenous hydrogenase activity. M.
thermoacetica can grow with CO.sub.2 as the exclusive carbon source
indicating that reducing equivalents are extracted from H.sub.2 to
enable acetyl-CoA synthesis via the Wood-Ljungdahl pathway (Drake,
H. L., J. Bacteriol. 150:702-709 (1982); Drake and Daniel, Res.
Microbiol. 155:869-883 (2004); Kellum and Drake, J. Bacteriol.
160:466-469 (1984)) (see FIG. 68). 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.
[0770] Proteins in M. thermoacetica whose genes are homologous to
the E. coli hyp genes are shown below.
TABLE-US-00191 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
[0771] 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-00192 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
In addition, several gene clusters encoding hydrogenase
functionality are present in M. thermoacetica and their
corresponding protein sequences are provided below.
TABLE-US-00193 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
[0772] 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-00194 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 6803 function 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 6803 function Unknown NP_441412.1 16330684
Synechocystis str. PCC 6803 function 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
[0773] Genes encoding hydrogenase enzymes from C. ljungdahli are
shown below.
TABLE-US-00195 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
[0774] 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.
[0775] 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-00196 Protein GenBank ID GI Number Organism Ppc NP_418391
16131794 Escherichia coli ppcA AAB58883 28572162 Methylobacterium
extorquens Ppc ABB53270 80973080 Corynebacterium glutamicum
[0776] 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., Appi. 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-00197 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
[0777] 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-00198 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
[0778] 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-00199 Protein GenBank ID GI Number Organism maeA NP_415996
90111281 Escherichia coli maeB NP_416958 16130388 Escherichia coli
NAD-ME P27443 126732 Ascaris suum
[0779] 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.
[0780] Enzymes, genes and methods for engineering pathways from
succinyl-CoA to various products into a microorganism are now known
in the art. The additional reducing equivalents obtained from CO
and/or H.sub.2, as disclosed herein, improve the yields of
1,4-butanediol, 4-hydroxybutyrate and/or gamma-butyrolactone when
utilizing carbohydrate-based feedstock. For example,
1,4-butanediol, 4-hydroxybutyrate and/or gamma-butyrolactone can be
produced as described herein, for example, produced from
succinyl-CoA via pathways shown in FIG. 64B and FIG. 8A. Exemplary
enzymes for the conversion succinyl-CoA to 1,4-butanediol,
4-hydroxybutyrate and/or gamma-butyrolactone include those
disclosed herein, including the figures.
[0781] Enzymes, genes and methods for engineering pathways from
glycolysis intermediates to various products into a microorganism
are known in the art. The additional reducing equivalents obtained
from CO and H.sub.2, as described herein, improve the yields of all
these products on carbohydrates. For example, 1,4-butanediol,
4-hydroxybutyrate and/or gamma-butyrolactone can be produced from
the glycolysis intermediate. Exemplary enzymes for the conversion
of to 1,4-butanediol, 4-hydroxybutyrate and/or gamma-butyrolactone
are described herein.
Example XXVI
Methods for Handling CO and Anaerobic Cultures
[0782] This example describes methods used in handling CO and
anaerobic cultures.
[0783] A. Handling of CO in Small Quantities for Assays and Small
Cultures.
[0784] 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.
[0785] 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.
[0786] B. Handling of CO in Larger Quantities Fed to Large-Scale
Cultures.
[0787] Fermentation cultures are fed either CO or a mixture of CO
and H.sub.2 to simulate syngas as a feedstock in fermentative
production. Therefore, quantities of cells ranging from 1 liter to
several liters can include the addition of CO gas to increase the
dissolved concentration of CO in the medium. In these
circumstances, fairly large and continuously administered
quantities of CO gas are added to the cultures. At different
points, the cultures are harvested or samples removed.
Alternatively, cells are harvested with an integrated continuous
flow centrifuge that is part of the fermenter.
[0788] 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.
[0789] C. Anaerobic Chamber and Conditions.
[0790] Exemplary anaerobic chambers are available commercially
(see, for example, Vacuum Atmospheres Company, Hawthorne Calif.;
MBraun, Newburyport Mass.). Conditions included an O.sub.2
concentration of 1 ppm or less and 1 atm pure N.sub.2. In one
example, 3 oxygen scrubbers/catalyst regenerators were used, and
the chamber included an O.sub.2 electrode (such as Teledyne; City
of Industry 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 N.sub.2 prior to introduction into the chamber. Gloves are
changed twice/yr and the catalyst containers were regenerated
periodically when the chamber displays increasingly sluggish
response to changes in oxygen levels. The chamber's pressure was
controlled through one-way valves activated by solenoids. This
feature allowed setting the chamber pressure at a level higher than
the surroundings to allow transfer of very small tubes through the
purge valve.
[0791] The anaerobic chambers achieved levels of O.sub.2 that were
consistently very low and were needed for highly oxygen sensitive
anaerobic conditions. However, growth and handling of cells does
not usually require such precautions. In an alternative anaerobic
chamber configuration, platinum or palladium can be used as a
catalyst that requires some hydrogen gas in the mix. Instead of
using solenoid valves, pressure release can be controlled by a
bubbler. Instead of using instrument-based O.sub.2 monitoring, test
strips can be used instead.
[0792] D. Anaerobic Microbiology.
[0793] Small cultures were handled as described above for CO
handling. In particular, serum or media bottles are fitted with
thick rubber stoppers and aluminum crimps are employed to seal the
bottle. Medium, such as Terrific Broth, is made in a conventional
manner and dispensed to an appropriately sized serum bottle. The
bottles are sparged with nitrogen for .about.30 min of moderate
bubbling. This removes most of the oxygen from the medium and,
after this step, each bottle is capped with a rubber stopper (such
as Bellco 20 mm septum stoppers; Bellco, Vineland, N.J.) and
crimp-sealed (Bellco 20 mm). Then the bottles of medium are
autoclaved using a slow (liquid) exhaust cycle. At least sometimes
a needle can be poked through the stopper to provide exhaust during
autoclaving; the needle needs to be removed immediately upon
removal from the autoclave. The sterile medium has the remaining
medium components, for example buffer or antibiotics, added via
syringe and needle. Prior to addition of reducing agents, the
bottles are equilibrated for 30-60 minutes with nitrogen (or CO
depending upon use). A reducing agent such as a 100.times.150 mM
sodium sulfide, 200 mM cysteine-HCl is added. This is made by
weighing the sodium sulfide into a dry beaker and the cysteine into
a serum bottle, bringing both into the anaerobic chamber,
dissolving the sodium sulfide into anaerobic water, then adding
this to the cysteine in the serum bottle. The bottle is stoppered
immediately as the sodium sulfide solution generates hydrogen
sulfide gas upon contact with the cysteine. When injecting into the
culture, a syringe filter is used to sterilize the solution. Other
components are added through syringe needles, such as B12 (10 .mu.M
cyanocobalamin), nickel chloride (NiCl.sub.2, 20 microM final
concentration from a 40 mM stock made in anaerobic water in the
chamber and sterilized by autoclaving or by using a syringe filter
upon injection into the culture), and ferrous ammonium sulfate
(final concentration needed is 100 .mu.M--made as 100-1000.times.
stock solution in anaerobic water in the chamber and sterilized by
autoclaving or by using a syringe filter upon injection into the
culture). To facilitate faster growth under anaerobic conditions,
the 1 liter bottles were inoculated with 50 mL of a preculture
grown anaerobically. Induction of the pA1-lacO1 promoter in the
vectors was performed by addition of isopropyl
.beta.-D-1-thiogalactopyranoside (IPTG) to a final concentration of
0.2 mM and was carried out for about 3 hrs.
[0794] 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 XXVII
CO Oxidation (CODH) Assay
[0795] This example describes assay methods for measuring CO
oxidation (CO dehydrogenase; CODH).
[0796] 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.
[0797] 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.
[0798] 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.
[0799] 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.
[0800] In more detail, glass rubber stoppered cuvettes were
prepared after first washing the cuvette four times in deionized
water and one time with acetone. A small amount of vacuum grease
was smeared on the top of the rubber gasket. The cuvette was gassed
with CO, dried 10 min with a 22 Ga. needle plus an exhaust needle.
A volume of 0.98 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 (CH.sub.3 viologen) stock was
1 M in water. Each assay used 20 microliters for 20 mM final
concentration. When methyl viologen was added, an 18 Ga needle
(partial) was used as a jacket to facilitate use of a Hamilton
syringe to withdraw the CH.sub.3 viologen. 4-5 aliquots were drawn
up and discarded to wash and gas equilibrate the syringe. A small
amount of sodium dithionite (0.1 M stock) was added when making up
the CH.sub.3 viologen stock to slightly reduce the CH.sub.3
viologen. The temperature was equilibrated to 55.degree. C. in a
heated Olis spectrophotometer (Bogart Ga.). A blank reaction
(CH.sub.3 viologen+buffer) was run first to measure the base rate
of CH.sub.3 viologen reduction. Crude E. coli cell extracts of
ACS90 and ACS91 (CODH-ACS operon of M. thermoacetica with and
without, respectively, the first cooC). 10 microliters of extract
were added at a time, mixed and assayed. Reduced CH.sub.3 viologen
turns purple. The results of an assay are shown in Table 33.
TABLE-US-00200 TABLE 33 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
[0801] 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.
[0802] If .about.1% of the cellular protein is CODH, then these
figures would be approximately 100.times. less than the 500 U/mg
activity of pure M. thermoacetica CODH. Actual estimates based on
Western blots are 0.5% of the cellular protein, so the activity is
about 50.times. less than for M. thermoacetica CODH. Nevertheless,
this experiment demonstrates CO oxidation activity in recombinant
E. coli with a much smaller amount in the negative controls. The
small amount of CO oxidation (CH.sub.3 viologen reduction) seen in
the negative controls indicates that E. coli may have a limited
ability to reduce CH.sub.3 viologen.
[0803] 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. 70. 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.
[0804] 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. 71. 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.
[0805] 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 XXVIII
E. coli CO Tolerance Experiment and CO Concentration Assay
(Myoglobin Assay)
[0806] This example describes the tolerance of E. coli for high
concentrations of CO.
[0807] To test whether or not E. coli can grow anaerobically in the
presence of saturating amounts of CO, cultures were set up in 120
ml serum bottles with 50 ml of Terrific Broth medium (plus reducing
solution, NiCl.sub.2, Fe(II)NH.sub.4SO.sub.4, cyanocobalamin, IPTG,
and chloramphenicol) as described above for anaerobic microbiology
in small volumes. One half of these bottles were equilibrated with
nitrogen gas for 30 min. and one half was equilibrated with CO gas
for 30 min. An empty vector (pZA33) was used as a control, and
cultures containing the pZA33 empty vector as well as both ACS90
and ACS91 were tested with both N.sub.2 and CO. All were inoculated
and grown for 36 hrs with shaking (250 rpm) at 37.degree. C. At the
end of the 36 hour period, examination of the flasks showed high
amounts of growth in all. The bulk of the observed growth occurred
overnight with a long lag.
[0808] 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.
[0809] For the myoglobin test of CO concentration, cuvettes were
washed 10.times. with water, 1.times. with acetone, and then
stoppered as with the CODH assay. N.sub.2 was blown into the
cuvettes for .about.10 min. A volume of 1 ml of anaerobic buffer
(HEPES, pH 8.0, 2 mM DTT) was added to the blank (not equilibrated
with CO) with a Hamilton syringe. A volume of 10 microliter
myoglobin (.about.1 mM--can be varied, just need a fairly large
amount) and 1 microliter dithionite (20 mM stock) were added. A CO
standard curve was made using CO saturated buffer added at 1
microliter increments. Peak height and shift was recorded for each
increment. The cultures tested were pZA33/CO, ACS90/CO, and
ACS91/CO. Each of these was added in 1 microliter increments to the
same cuvette. Midway through the experiment a second cuvette was
set up and used. The results are shown in Table 34.
TABLE-US-00201 TABLE 34 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
[0810] The results shown in Table 34 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.
[0811] 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.
[0812] Throughout this application various publications have been
referenced. The disclosures of these publications in their
entireties, including GenBank and GI number publications, are
hereby incorporated by reference in this application in order to
more fully describe the state of the art to which this invention
pertains. Although the invention has been described with reference
to the examples provided above, it should be understood that
various modifications can be made without departing from the spirit
of the invention.
Sequence CWU 1
1
101159DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1gacgaattcg ctagcaagag gagaagtcga catgtccaat
tcactggccg tcgttttac 59247DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 2gaccctagga agctttctag
agtcgaccta tgcggcatca gagcaga 47318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
3atgtaccgca agttccgc 18418DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 4caatttgccg atgcccag
18520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 5gctgaccact gaagactttg 20619DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
6gatcagggct tcggtgtag 19727DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 7ttggtgcggg ccaagcagga tctgctc
27830DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 8tcagccgaac gcctcgtcga ggatctcctg
30932DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 9tggccaacat aagttcacca ttcgggcaaa ac
321029DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 10tctcttcaac cagccattcg ttttgcccg
291127DNAClostridium acetobutylicum 11attaaagtta agtggaggaa tgttaac
271222DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 12acacgcggat ccaacgtccc gg 221323DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
13agcggctccg ctagccgctt atg 231424DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 14aagccgttgc tgcagctctt
gagc 241523DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 15atctccggcg gtcggatccg tcg 231620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
16aaagcggcta gccacgccgc 201720DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 17attacacgag gtacccaacg
201822DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 18atgctggcgt acaaaggtgt cc 221922DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
19ggacaccttt gtacgccagc at 222025DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 20atcgcctaca ctaaaccaga
agtgg 252125DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 21ccacttctgg tttagtgtag gcgat
252220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 22aggcagttcc ataggatggc 202355DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
23tgacatgtaa cacctacctt ctgtgcctgt gccagtggtt gctgtgatat agaag
552448DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 24ataataatac atatgaacca tgcgagttac gggcctataa
gccaggcg 482542DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 25agtttttcga tatctgcatc agacaccggc
acattgaaac gg 422660DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 26ctggcacagg cacagaaggt aggtgttaca
tgtcagaacg tttacacaat gacgtggatc 602749DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
27agacaaatcg gttgccgttt gttaagccag gcgagatatg atctatatc
492854DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 28gagttttgat ttcagtactc atcatgtaac acctaccttc
ttgctgtgat atag 542954DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 29ctatatcaca gcaagaaggt
aggtgttaca tgatgagtac tgaaatcaaa actc 543049DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
30gatatagatc atatctcgcc tggcttaaca aacggcaacc gatttgtct
493170DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 31tattgtgcat acagatgaat ttttatgcaa
acagtcagcc ctgaagaagg gtgtaggctg 60gagctgcttc 703270DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 32caaaaaaccg gagtctgtgc tccggttttt tattatccgc
taatcaatta catatgaata 60tcctccttag 703351DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
33ataataatag aattcgtttg ctacctaaat tgccaactaa atcgaaacag g
513447DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 34tattattatg gtaccaatat catgcagcaa acggtgcaac
attgccg 473547DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 35tgatctggaa gaattcatcg gctttaccac
cgtcaaaaaa aacggcg 473648DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 36ataaaaccct gcagcggaaa
cgaagtttta tccatttttg gttacctg 483735DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
37ggaagagagg ctggtaccca gaagccacag cagga 353838DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
38gtaatcactg cgtaagcgcc atgccccggc gttaattc 383925DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
39attgccgcgt tcctcctgct gtcga 254024DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
40cgacagcagg aggaacgcgg caat 244175DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
41gtttgcacgc tatagctgag gttgttgtct tccagcaacg taccgtatac aataggcgta
60tcacgaggcc ctttc 754270DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 42gctacagcat gtcacacgat
ctcaacggtc ggatgaccaa tctggctggt atgggaatta 60gccatggtcc
704373DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 43tgtgagtgaa agtcacctgc cttaatatct
caaaactcat cttcgggtga cgaaatatgg 60cgtgactcga tac
734470DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 44tctgtatcag gctgaaaatc ttctctcatc
cgccaaaaca gcttcggcgt taagatgcgc 60gctcaaggac 704520PRTEuglena
gracilis 45Met Thr Tyr Lys Ala Pro Val Lys Asp Val Lys Phe Leu Leu
Asp Lys1 5 10 15Val Phe Lys Val 20462036DNAEscherichia coli
46atgaacttac atgaatatca ggcaaaacaa ctttttgccc gctatggctt accagcaccg
60gtgggttatg cctgtactac tccgcgcgaa gcagaagaag ccgcttcaaa aatcggtgcc
120ggtccgtggg tagtgaaatg tcaggttcac gctggtggcc gcggtaaagc
gggcggtgtg 180aaagttgtaa acagcaaaga agacatccgt gcttttgcag
aaaactggct gggcaagcgt 240ctggtaacgt atcaaacaga tgccaatggc
caaccggtta accagattct ggttgaagca 300gcgaccgata tcgctaaaga
gctgtatctc ggtgccgttg ttgaccgtag ttcccgtcgt 360gtggtcttta
tggcctccac cgaaggcggc gtggaaatcg aaaaagtggc ggaagaaact
420ccgcacctga tccataaagt tgcgcttgat ccgctgactg gcccgatgcc
gtatcaggga 480cgcgagctgg cgttcaaact gggtctggaa ggtaaactgg
ttcagcagtt caccaaaatc 540ttcatgggcc tggcgaccat tttcctggag
cgcgacctgg cgttgatcga aatcaacccg 600ctggtcatca ccaaacaggg
cgatctgatt tgcctcgacg gcaaactggg cgctgacggc 660aacgcactgt
tccgccagcc tgatctgcgc gaaatgcgtg accagtcgca ggaagatccg
720cgtgaagcac aggctgcaca gtgggaactg aactacgttg cgctggacgg
taacatcggt 780tgtatggtta acggcgcagg tctggcgatg ggtacgatgg
acatcgttaa actgcacggc 840ggcgaaccgg ctaacttcct tgacgttggc
ggcggcgcaa ccaaagaacg tgtaaccgaa 900gcgttcaaaa tcatcctctc
tgacgacaaa gtgaaagccg ttctggttaa catcttcggc 960ggtatcgttc
gttgcgacct gatcgctgac ggtatcatcg gcgcggtagc agaagtgggt
1020gttaacgtac cggtcgtggt acgtctggaa ggtaacaacg ccgaactcgg
cgcgaagaaa 1080ctggctgaca gcggcctgaa tattattgca gcaaaaggtc
tgacggatgc agctcagcag 1140gttgttgccg cagtggaggg gaaataatgt
ccattttaat cgataaaaac accaaggtta 1200tctgccaggg ctttaccggt
agccagggga ctttccactc agaacaggcc attgcatacg 1260gcactaaaat
ggttggcggc gtaaccccag gtaaaggcgg caccacccac ctcggcctgc
1320cggtgttcaa caccgtgcgt gaagccgttg ctgccactgg cgctaccgct
tctgttatct 1380acgtaccagc accgttctgc aaagactcca ttctggaagc
catcgacgca ggcatcaaac 1440tgattatcac catcactgaa ggcatcccga
cgctggatat gctgaccgtg aaagtgaagc 1500tggatgaagc aggcgttcgt
atgatcggcc cgaactgccc aggcgttatc actccgggtg 1560aatgcaaaat
cggtatccag cctggtcaca ttcacaaacc gggtaaagtg ggtatcgttt
1620cccgttccgg tacactgacc tatgaagcgg ttaaacagac cacggattac
ggtttcggtc 1680agtcgacctg tgtcggtatc ggcggtgacc cgatcccggg
ctctaacttt atcgacattc 1740tcgaaatgtt cgaaaaagat ccgcagaccg
aagcgatcgt gatgatcggt gagatcggcg 1800gtagcgctga agaagaagca
gctgcgtaca tcaaagagca cgttaccaag ccagttgtgg 1860gttacatcgc
tggtgtgact gcgccgaaag gcaaacgtat gggccacgcg ggtgccatca
1920ttgccggtgg gaaagggact gcggatgaga aattcgctgc tctggaagcc
gcaggcgtga 1980aaaccgttcg cagcctggcg gatatcggtg aagcactgaa
aactgttctg aaataa 203647388PRTEscherichia coli 47Met Asn Leu His
Glu Tyr Gln Ala Lys Gln Leu Phe Ala Arg Tyr Gly1 5 10 15Leu Pro Ala
Pro Val Gly Tyr Ala Cys Thr Thr Pro Arg Glu Ala Glu 20 25 30Glu Ala
Ala Ser Lys Ile Gly Ala Gly Pro Trp Val Val Lys Cys Gln 35 40 45Val
His Ala Gly Gly Arg Gly Lys Ala Gly Gly Val Lys Val Val Asn 50 55
60Ser Lys Glu Asp Ile Arg Ala Phe Ala Glu Asn Trp Leu Gly Lys Arg65
70 75 80Leu Val Thr Tyr Gln Thr Asp Ala Asn Gly Gln Pro Val Asn Gln
Ile 85 90 95Leu Val Glu Ala Ala Thr Asp Ile Ala Lys Glu Leu Tyr Leu
Gly Ala 100 105 110Val Val Asp Arg Ser Ser Arg Arg Val Val Phe Met
Ala Ser Thr Glu 115 120 125Gly Gly Val Glu Ile Glu Lys Val Ala Glu
Glu Thr Pro His Leu Ile 130 135 140His Lys Val Ala Leu Asp Pro Leu
Thr Gly Pro Met Pro Tyr Gln Gly145 150 155 160Arg Glu Leu Ala Phe
Lys Leu Gly Leu Glu Gly Lys Leu Val Gln Gln 165 170 175Phe Thr Lys
Ile Phe Met Gly Leu Ala Thr Ile Phe Leu Glu Arg Asp 180 185 190Leu
Ala Leu Ile Glu Ile Asn Pro Leu Val Ile Thr Lys Gln Gly Asp 195 200
205Leu Ile Cys Leu Asp Gly Lys Leu Gly Ala Asp Gly Asn Ala Leu Phe
210 215 220Arg Gln Pro Asp Leu Arg Glu Met Arg Asp Gln Ser Gln Glu
Asp Pro225 230 235 240Arg Glu Ala Gln Ala Ala Gln Trp Glu Leu Asn
Tyr Val Ala Leu Asp 245 250 255Gly Asn Ile Gly Cys Met Val Asn Gly
Ala Gly Leu Ala Met Gly Thr 260 265 270Met Asp Ile Val Lys Leu His
Gly Gly Glu Pro Ala Asn Phe Leu Asp 275 280 285Val Gly Gly Gly Ala
Thr Lys Glu Arg Val Thr Glu Ala Phe Lys Ile 290 295 300Ile Leu Ser
Asp Asp Lys Val Lys Ala Val Leu Val Asn Ile Phe Gly305 310 315
320Gly Ile Val Arg Cys Asp Leu Ile Ala Asp Gly Ile Ile Gly Ala Val
325 330 335Ala Glu Val Gly Val Asn Val Pro Val Val Val Arg Leu Glu
Gly Asn 340 345 350Asn Ala Glu Leu Gly Ala Lys Lys Leu Ala Asp Ser
Gly Leu Asn Ile 355 360 365Ile Ala Ala Lys Gly Leu Thr Asp Ala Ala
Gln Gln Val Val Ala Ala 370 375 380Val Glu Gly
Lys38548289PRTEscherichia coli 48Met Ser Ile Leu Ile Asp Lys Asn
Thr Lys Val Ile Cys Gln Gly Phe1 5 10 15Thr Gly Ser Gln Gly Thr Phe
His Ser Glu Gln Ala Ile Ala Tyr Gly 20 25 30Thr Lys Met Val Gly Gly
Val Thr Pro Gly Lys Gly Gly Thr Thr His 35 40 45Leu Gly Leu Pro Val
Phe Asn Thr Val Arg Glu Ala Val Ala Ala Thr 50 55 60Gly Ala Thr Ala
Ser Val Ile Tyr Val Pro Ala Pro Phe Cys Lys Asp65 70 75 80Ser Ile
Leu Glu Ala Ile Asp Ala Gly Ile Lys Leu Ile Ile Thr Ile 85 90 95Thr
Glu Gly Ile Pro Thr Leu Asp Met Leu Thr Val Lys Val Lys Leu 100 105
110Asp Glu Ala Gly Val Arg Met Ile Gly Pro Asn Cys Pro Gly Val Ile
115 120 125Thr Pro Gly Glu Cys Lys Ile Gly Ile Gln Pro Gly His Ile
His Lys 130 135 140Pro Gly Lys Val Gly Ile Val Ser Arg Ser Gly Thr
Leu Thr Tyr Glu145 150 155 160Ala Val Lys Gln Thr Thr Asp Tyr Gly
Phe Gly Gln Ser Thr Cys Val 165 170 175Gly Ile Gly Gly Asp Pro Ile
Pro Gly Ser Asn Phe Ile Asp Ile Leu 180 185 190Glu Met Phe Glu Lys
Asp Pro Gln Thr Glu Ala Ile Val Met Ile Gly 195 200 205Glu Ile Gly
Gly Ser Ala Glu Glu Glu Ala Ala Ala Tyr Ile Lys Glu 210 215 220His
Val Thr Lys Pro Val Val Gly Tyr Ile Ala Gly Val Thr Ala Pro225 230
235 240Lys Gly Lys Arg Met Gly His Ala Gly Ala Ile Ile Ala Gly Gly
Lys 245 250 255Gly Thr Ala Asp Glu Lys Phe Ala Ala Leu Glu Ala Ala
Gly Val Lys 260 265 270Thr Val Arg Ser Leu Ala Asp Ile Gly Glu Ala
Leu Lys Thr Val Leu 275 280 285Lys 493696DNAMycobacterium bovis
49atggccaaca taagttcacc attcgggcaa aacgaatggc tggttgaaga gatgtaccgc
60aagttccgcg acgacccctc ctcggtcgat cccagctggc acgagttcct ggttgactac
120agccccgaac ccacctccca accagctgcc gaaccaaccc gggttacctc
gccactcgtt 180gccgagcggg ccgctgcggc cgccccgcag gcacccccca
agccggccga caccgcggcc 240gcgggcaacg gcgtggtcgc cgcactggcc
gccaaaactg ccgttccccc gccagccgaa 300ggtgacgagg tagcggtgct
gcgcggcgcc gccgcggccg tcgtcaagaa catgtccgcg 360tcgttggagg
tgccgacggc gaccagcgtc cgggcggtcc cggccaagct actgatcgac
420aaccggatcg tcatcaacaa ccagttgaag cggacccgcg gcggcaagat
ctcgttcacg 480catttgctgg gctacgccct ggtgcaggcg gtgaagaaat
tcccgaacat gaaccggcac 540tacaccgaag tcgacggcaa gcccaccgcg
gtcacgccgg cgcacaccaa tctcggcctg 600gcgatcgacc tgcaaggcaa
ggacgggaag cgttccctgg tggtggccgg catcaagcgg 660tgcgagacca
tgcgattcgc gcagttcgtc acggcctacg aagacatcgt acgccgggcc
720cgcgacggca agctgaccac tgaagacttt gccggcgtga cgatttcgct
gaccaatccc 780ggaaccatcg gcaccgtgca ttcggtgccg cggctgatgc
ccggccaggg cgccatcatc 840ggcgtgggcg ccatggaata ccccgccgag
tttcaaggcg ccagcgagga acgcatcgcc 900gagctgggca tcggcaaatt
gatcactttg acctccacct acgaccaccg catcatccag 960ggcgcggaat
cgggcgactt cctgcgcacc atccacgagt tgctgctctc ggatggcttc
1020tgggacgagg tcttccgcga actgagcatc ccatatctgc cggtgcgctg
gagcaccgac 1080aaccccgact cgatcgtcga caagaacgct cgcgtcatga
acttgatcgc ggcctaccgc 1140aaccgcggcc atctgatggc cgataccgac
ccgctgcggt tggacaaagc tcggttccgc 1200agtcaccccg acctcgaagt
gctgacccac ggcctgacgc tgtgggatct cgatcgggtg 1260ttcaaggtcg
acggctttgc cggtgcgcag tacaagaaac tgcgcgacgt gctgggcttg
1320ctgcgcgatg cctactgccg ccacatcggc gtggagtacg cccatatcct
cgaccccgaa 1380caaaaggagt ggctcgaaca acgggtcgag accaagcacg
tcaaacccac tgtggcccaa 1440cagaaataca tcctcagcaa gctcaacgcc
gccgaggcct ttgaaacgtt cctacagacc 1500aagtacgtcg gccagaagcg
gttctcgctg gaaggcgccg aaagcgtgat cccgatgatg 1560gacgcggcga
tcgaccagtg cgctgagcac ggcctcgacg aggtggtcat cgggatgccg
1620caccggggcc ggctcaacgt gctggccaac atcgtcggca agccgtactc
gcagatcttc 1680accgagttcg agggcaacct gaatccgtcg caggcgcacg
gctccggtga cgtcaagtac 1740cacctgggcg ccaccgggct gtacctgcag
atgttcggcg acaacgacat tcaggtgtcg 1800ctgaccgcca acccgtcgca
tctggaggcc gtcgacccgg tgctggaggg attggtgcgg 1860gccaagcagg
atctgctcga ccacggaagc atcgacagcg acggccaacg ggcgttctcg
1920gtggtgccgc tgatgttgca tggcgatgcc gcgttcgccg gtcagggtgt
ggtcgccgag 1980acgctgaacc tggcgaatct gccgggctac cgcgtcggcg
gcaccatcca catcatcgtc 2040aacaaccaga tcggcttcac caccgcgccc
gagtattcca ggtccagcga gtactgcacc 2100gacgtcgcaa agatgatcgg
ggcaccgatc tttcacgtca acggcgacga cccggaggcg 2160tgtgtctggg
tggcgcggtt ggcggtggac ttccgacaac ggttcaagaa ggacgtcgtc
2220atcgacatgc tgtgctaccg ccgccgcggg cacaacgagg gtgacgaccc
gtcgatgacc 2280aacccctaca tgtacgacgt cgtcgacacc aagcgcgggg
cccgcaaaag ctacaccgaa 2340gccctgatcg gacgtggcga catctcgatg
aaggaggccg aggacgcgct gcgcgactac
2400cagggccagc tggaacgggt gttcaacgaa gtgcgcgagc tggagaagca
cggtgtgcag 2460ccgagcgagt cggtcgagtc cgaccagatg attcccgcgg
ggctggccac tgcggtggac 2520aagtcgctgc tggcccggat cggcgatgcg
ttcctcgcct tgccgaacgg cttcaccgcg 2580cacccgcgag tccaaccggt
gctggagaag cgccgggaga tggcctatga aggcaagatc 2640gactgggcct
ttggcgagct gctggcgctg ggctcgctgg tggccgaagg caagctggtg
2700cgcttgtcgg ggcaggacag ccgccgcggc accttctccc agcggcattc
ggttctcatc 2760gaccgccaca ctggcgagga gttcacacca ctgcagctgc
tggcgaccaa ctccgacggc 2820agcccgaccg gcggaaagtt cctggtctac
gactcgccac tgtcggagta cgccgccgtc 2880ggcttcgagt acggctacac
tgtgggcaat ccggacgccg tggtgctctg ggaggcgcag 2940ttcggcgact
tcgtcaacgg cgcacagtcg atcatcgacg agttcatcag ctccggtgag
3000gccaagtggg gccaattgtc caacgtcgtg ctgctgttac cgcacgggca
cgaggggcag 3060ggacccgacc acacttctgc ccggatcgaa cgcttcttgc
agttgtgggc ggaaggttcg 3120atgaccatcg cgatgccgtc gactccgtcg
aactacttcc acctgctacg ccggcatgcc 3180ctggacggca tccaacgccc
gctgatcgtg ttcacgccca agtcgatgtt gcgtcacaag 3240gccgccgtca
gcgaaatcaa ggacttcacc gagatcaagt tccgctcagt gctggaggaa
3300cccacctatg aggacggcat cggagaccgc aacaaggtca gccggatcct
gctgaccagt 3360ggcaagctgt attacgagct ggccgcccgc aaggccaagg
acaaccgcaa tgacctcgcg 3420atcgtgcggc ttgaacagct cgccccgctg
cccaggcgtc gactgcgtga aacgctggac 3480cgctacgaga acgtcaagga
gttcttctgg gtccaagagg aaccggccaa ccagggtgcg 3540tggccgcgat
tcgggctcga actacccgag ctgctgcctg acaagttggc cgggatcaag
3600cgaatctcgc gccgggcgat gtcagccccg tcgtcaggct cgtcgaaggt
gcacgccgtc 3660gaacagcagg agatcctcga cgaggcgttc ggctaa
3696501231PRTMycobacterium bovis 50Met Ala Asn Ile Ser Ser Pro Phe
Gly Gln Asn Glu Trp Leu Val Glu1 5 10 15Glu Met Tyr Arg Lys Phe Arg
Asp Asp Pro Ser Ser Val Asp Pro Ser 20 25 30Trp His Glu Phe Leu Val
Asp Tyr Ser Pro Glu Pro Thr Ser Gln Pro 35 40 45Ala Ala Glu Pro Thr
Arg Val Thr Ser Pro Leu Val Ala Glu Arg Ala 50 55 60Ala Ala Ala Ala
Pro Gln Ala Pro Pro Lys Pro Ala Asp Thr Ala Ala65 70 75 80Ala Gly
Asn Gly Val Val Ala Ala Leu Ala Ala Lys Thr Ala Val Pro 85 90 95Pro
Pro Ala Glu Gly Asp Glu Val Ala Val Leu Arg Gly Ala Ala Ala 100 105
110Ala Val Val Lys Asn Met Ser Ala Ser Leu Glu Val Pro Thr Ala Thr
115 120 125Ser Val Arg Ala Val Pro Ala Lys Leu Leu Ile Asp Asn Arg
Ile Val 130 135 140Ile Asn Asn Gln Leu Lys Arg Thr Arg Gly Gly Lys
Ile Ser Phe Thr145 150 155 160His Leu Leu Gly Tyr Ala Leu Val Gln
Ala Val Lys Lys Phe Pro Asn 165 170 175Met Asn Arg His Tyr Thr Glu
Val Asp Gly Lys Pro Thr Ala Val Thr 180 185 190Pro Ala His Thr Asn
Leu Gly Leu Ala Ile Asp Leu Gln Gly Lys Asp 195 200 205Gly Lys Arg
Ser Leu Val Val Ala Gly Ile Lys Arg Cys Glu Thr Met 210 215 220Arg
Phe Ala Gln Phe Val Thr Ala Tyr Glu Asp Ile Val Arg Arg Ala225 230
235 240Arg Asp Gly Lys Leu Thr Thr Glu Asp Phe Ala Gly Val Thr Ile
Ser 245 250 255Leu Thr Asn Pro Gly Thr Ile Gly Thr Val His Ser Val
Pro Arg Leu 260 265 270Met Pro Gly Gln Gly Ala Ile Ile Gly Val Gly
Ala Met Glu Tyr Pro 275 280 285Ala Glu Phe Gln Gly Ala Ser Glu Glu
Arg Ile Ala Glu Leu Gly Ile 290 295 300Gly Lys Leu Ile Thr Leu Thr
Ser Thr Tyr Asp His Arg Ile Ile Gln305 310 315 320Gly Ala Glu Ser
Gly Asp Phe Leu Arg Thr Ile His Glu Leu Leu Leu 325 330 335Ser Asp
Gly Phe Trp Asp Glu Val Phe Arg Glu Leu Ser Ile Pro Tyr 340 345
350Leu Pro Val Arg Trp Ser Thr Asp Asn Pro Asp Ser Ile Val Asp Lys
355 360 365Asn Ala Arg Val Met Asn Leu Ile Ala Ala Tyr Arg Asn Arg
Gly His 370 375 380Leu Met Ala Asp Thr Asp Pro Leu Arg Leu Asp Lys
Ala Arg Phe Arg385 390 395 400Ser His Pro Asp Leu Glu Val Leu Thr
His Gly Leu Thr Leu Trp Asp 405 410 415Leu Asp Arg Val Phe Lys Val
Asp Gly Phe Ala Gly Ala Gln Tyr Lys 420 425 430Lys Leu Arg Asp Val
Leu Gly Leu Leu Arg Asp Ala Tyr Cys Arg His 435 440 445Ile Gly Val
Glu Tyr Ala His Ile Leu Asp Pro Glu Gln Lys Glu Trp 450 455 460Leu
Glu Gln Arg Val Glu Thr Lys His Val Lys Pro Thr Val Ala Gln465 470
475 480Gln Lys Tyr Ile Leu Ser Lys Leu Asn Ala Ala Glu Ala Phe Glu
Thr 485 490 495Phe Leu Gln Thr Lys Tyr Val Gly Gln Lys Arg Phe Ser
Leu Glu Gly 500 505 510Ala Glu Ser Val Ile Pro Met Met Asp Ala Ala
Ile Asp Gln Cys Ala 515 520 525Glu His Gly Leu Asp Glu Val Val Ile
Gly Met Pro His Arg Gly Arg 530 535 540Leu Asn Val Leu Ala Asn Ile
Val Gly Lys Pro Tyr Ser Gln Ile Phe545 550 555 560Thr Glu Phe Glu
Gly Asn Leu Asn Pro Ser Gln Ala His Gly Ser Gly 565 570 575Asp Val
Lys Tyr His Leu Gly Ala Thr Gly Leu Tyr Leu Gln Met Phe 580 585
590Gly Asp Asn Asp Ile Gln Val Ser Leu Thr Ala Asn Pro Ser His Leu
595 600 605Glu Ala Val Asp Pro Val Leu Glu Gly Leu Val Arg Ala Lys
Gln Asp 610 615 620Leu Leu Asp His Gly Ser Ile Asp Ser Asp Gly Gln
Arg Ala Phe Ser625 630 635 640Val Val Pro Leu Met Leu His Gly Asp
Ala Ala Phe Ala Gly Gln Gly 645 650 655Val Val Ala Glu Thr Leu Asn
Leu Ala Asn Leu Pro Gly Tyr Arg Val 660 665 670Gly Gly Thr Ile His
Ile Ile Val Asn Asn Gln Ile Gly Phe Thr Thr 675 680 685Ala Pro Glu
Tyr Ser Arg Ser Ser Glu Tyr Cys Thr Asp Val Ala Lys 690 695 700Met
Ile Gly Ala Pro Ile Phe His Val Asn Gly Asp Asp Pro Glu Ala705 710
715 720Cys Val Trp Val Ala Arg Leu Ala Val Asp Phe Arg Gln Arg Phe
Lys 725 730 735Lys Asp Val Val Ile Asp Met Leu Cys Tyr Arg Arg Arg
Gly His Asn 740 745 750Glu Gly Asp Asp Pro Ser Met Thr Asn Pro Tyr
Met Tyr Asp Val Val 755 760 765Asp Thr Lys Arg Gly Ala Arg Lys Ser
Tyr Thr Glu Ala Leu Ile Gly 770 775 780Arg Gly Asp Ile Ser Met Lys
Glu Ala Glu Asp Ala Leu Arg Asp Tyr785 790 795 800Gln Gly Gln Leu
Glu Arg Val Phe Asn Glu Val Arg Glu Leu Glu Lys 805 810 815His Gly
Val Gln Pro Ser Glu Ser Val Glu Ser Asp Gln Met Ile Pro 820 825
830Ala Gly Leu Ala Thr Ala Val Asp Lys Ser Leu Leu Ala Arg Ile Gly
835 840 845Asp Ala Phe Leu Ala Leu Pro Asn Gly Phe Thr Ala His Pro
Arg Val 850 855 860Gln Pro Val Leu Glu Lys Arg Arg Glu Met Ala Tyr
Glu Gly Lys Ile865 870 875 880Asp Trp Ala Phe Gly Glu Leu Leu Ala
Leu Gly Ser Leu Val Ala Glu 885 890 895Gly Lys Leu Val Arg Leu Ser
Gly Gln Asp Ser Arg Arg Gly Thr Phe 900 905 910Ser Gln Arg His Ser
Val Leu Ile Asp Arg His Thr Gly Glu Glu Phe 915 920 925Thr Pro Leu
Gln Leu Leu Ala Thr Asn Ser Asp Gly Ser Pro Thr Gly 930 935 940Gly
Lys Phe Leu Val Tyr Asp Ser Pro Leu Ser Glu Tyr Ala Ala Val945 950
955 960Gly Phe Glu Tyr Gly Tyr Thr Val Gly Asn Pro Asp Ala Val Val
Leu 965 970 975Trp Glu Ala Gln Phe Gly Asp Phe Val Asn Gly Ala Gln
Ser Ile Ile 980 985 990Asp Glu Phe Ile Ser Ser Gly Glu Ala Lys Trp
Gly Gln Leu Ser Asn 995 1000 1005Val Val Leu Leu Leu Pro His Gly
His Glu Gly Gln Gly Pro Asp 1010 1015 1020His Thr Ser Ala Arg Ile
Glu Arg Phe Leu Gln Leu Trp Ala Glu 1025 1030 1035Gly Ser Met Thr
Ile Ala Met Pro Ser Thr Pro Ser Asn Tyr Phe 1040 1045 1050His Leu
Leu Arg Arg His Ala Leu Asp Gly Ile Gln Arg Pro Leu 1055 1060
1065Ile Val Phe Thr Pro Lys Ser Met Leu Arg His Lys Ala Ala Val
1070 1075 1080Ser Glu Ile Lys Asp Phe Thr Glu Ile Lys Phe Arg Ser
Val Leu 1085 1090 1095Glu Glu Pro Thr Tyr Glu Asp Gly Ile Gly Asp
Arg Asn Lys Val 1100 1105 1110Ser Arg Ile Leu Leu Thr Ser Gly Lys
Leu Tyr Tyr Glu Leu Ala 1115 1120 1125Ala Arg Lys Ala Lys Asp Asn
Arg Asn Asp Leu Ala Ile Val Arg 1130 1135 1140Leu Glu Gln Leu Ala
Pro Leu Pro Arg Arg Arg Leu Arg Glu Thr 1145 1150 1155Leu Asp Arg
Tyr Glu Asn Val Lys Glu Phe Phe Trp Val Gln Glu 1160 1165 1170Glu
Pro Ala Asn Gln Gly Ala Trp Pro Arg Phe Gly Leu Glu Leu 1175 1180
1185Pro Glu Leu Leu Pro Asp Lys Leu Ala Gly Ile Lys Arg Ile Ser
1190 1195 1200Arg Arg Ala Met Ser Ala Pro Ser Ser Gly Ser Ser Lys
Val His 1205 1210 1215Ala Val Glu Gln Gln Glu Ile Leu Asp Glu Ala
Phe Gly 1220 1225 1230511356DNAPorphyromonas gingivalis
51atggaaatca aagaaatggt gagccttgca cgcaaggctc agaaggagta tcaagctacc
60cataaccaag aagcagttga caacatttgc cgagctgcag caaaagttat ttatgaaaat
120gcagctattc tggctcgcga agcagtagac gaaaccggca tgggcgttta
cgaacacaaa 180gtggccaaga atcaaggcaa atccaaaggt gtttggtaca
acctccacaa taaaaaatcg 240attggtatcc tcaatataga cgagcgtacc
ggtatgatcg agattgcaaa gcctatcgga 300gttgtaggag ccgtaacgcc
gacgaccaac ccgatcgtta ctccgatgag caatatcatc 360tttgctctta
agacctgcaa tgccatcatt attgcccccc accccagatc caaaaaatgc
420tctgcacacg cagttcgtct gatcaaagaa gctatcgctc cgttcaacgt
accggaaggt 480atggttcaga tcatcgaaga acccagcatc gagaagacgc
aggaactcat gggcgccgta 540gacgtagtag ttgctacggg tggtatgggc
atggtgaagt ctgcatattc ttcaggaaag 600ccttctttcg gtgttggagc
cggtaacgtt caggtgatcg tggatagcaa catcgatttc 660gaagctgctg
cagaaaaaat catcaccggt cgtgctttcg acaacggtat catctgctca
720ggcgaacaga gcatcatcta caacgaggct gacaaggaag cagttttcac
agcattccgc 780aaccacggtg catatttctg tgacgaagcc gaaggagatc
gggctcgtgc agctatcttc 840gaaaatggag ccatcgcgaa agatgtagta
ggtcagagcg ttgccttcat tgccaagaaa 900gcaaacatca atatccccga
gggtacccgt attctcgttg ttgaagctcg cggcgtagga 960gcagaagacg
ttatctgtaa ggaaaagatg tgtcccgtaa tgtgcgccct cagctacaag
1020cacttcgaag aaggtgtaga aatcgcacgt acgaacctcg ccaacgaagg
taacggccac 1080acctgtgcta tccactccaa caatcaggca cacatcatcc
tcgcaggatc agagctgacg 1140gtatctcgta tcgtagtgaa tgctccgagt
gccactacag caggcggtca catccaaaac 1200ggtcttgccg taaccaatac
gctcggatgc ggatcatggg gtaataactc tatctccgag 1260aacttcactt
acaagcacct cctcaacatt tcacgcatcg caccgttgaa ttcaagcatt
1320cacatccccg atgacaaaga aatctgggaa ctctaa
135652451PRTPorphyromonas gingivalis 52Met Glu Ile Lys Glu Met Val
Ser Leu Ala Arg Lys Ala Gln Lys Glu1 5 10 15Tyr Gln Ala Thr His Asn
Gln Glu Ala Val Asp Asn Ile Cys Arg Ala 20 25 30Ala Ala Lys Val Ile
Tyr Glu Asn Ala Ala Ile Leu Ala Arg Glu Ala 35 40 45Val Asp Glu Thr
Gly Met Gly Val Tyr Glu His Lys Val Ala Lys Asn 50 55 60Gln Gly Lys
Ser Lys Gly Val Trp Tyr Asn Leu His Asn Lys Lys Ser65 70 75 80Ile
Gly Ile Leu Asn Ile Asp Glu Arg Thr Gly Met Ile Glu Ile Ala 85 90
95Lys Pro Ile Gly Val Val Gly Ala Val Thr Pro Thr Thr Asn Pro Ile
100 105 110Val Thr Pro Met Ser Asn Ile Ile Phe Ala Leu Lys Thr Cys
Asn Ala 115 120 125Ile Ile Ile Ala Pro His Pro Arg Ser Lys Lys Cys
Ser Ala His Ala 130 135 140Val Arg Leu Ile Lys Glu Ala Ile Ala Pro
Phe Asn Val Pro Glu Gly145 150 155 160Met Val Gln Ile Ile Glu Glu
Pro Ser Ile Glu Lys Thr Gln Glu Leu 165 170 175Met Gly Ala Val Asp
Val Val Val Ala Thr Gly Gly Met Gly Met Val 180 185 190Lys Ser Ala
Tyr Ser Ser Gly Lys Pro Ser Phe Gly Val Gly Ala Gly 195 200 205Asn
Val Gln Val Ile Val Asp Ser Asn Ile Asp Phe Glu Ala Ala Ala 210 215
220Glu Lys Ile Ile Thr Gly Arg Ala Phe Asp Asn Gly Ile Ile Cys
Ser225 230 235 240Gly Glu Gln Ser Ile Ile Tyr Asn Glu Ala Asp Lys
Glu Ala Val Phe 245 250 255Thr Ala Phe Arg Asn His Gly Ala Tyr Phe
Cys Asp Glu Ala Glu Gly 260 265 270Asp Arg Ala Arg Ala Ala Ile Phe
Glu Asn Gly Ala Ile Ala Lys Asp 275 280 285Val Val Gly Gln Ser Val
Ala Phe Ile Ala Lys Lys Ala Asn Ile Asn 290 295 300Ile Pro Glu Gly
Thr Arg Ile Leu Val Val Glu Ala Arg Gly Val Gly305 310 315 320Ala
Glu Asp Val Ile Cys Lys Glu Lys Met Cys Pro Val Met Cys Ala 325 330
335Leu Ser Tyr Lys His Phe Glu Glu Gly Val Glu Ile Ala Arg Thr Asn
340 345 350Leu Ala Asn Glu Gly Asn Gly His Thr Cys Ala Ile His Ser
Asn Asn 355 360 365Gln Ala His Ile Ile Leu Ala Gly Ser Glu Leu Thr
Val Ser Arg Ile 370 375 380Val Val Asn Ala Pro Ser Ala Thr Thr Ala
Gly Gly His Ile Gln Asn385 390 395 400Gly Leu Ala Val Thr Asn Thr
Leu Gly Cys Gly Ser Trp Gly Asn Asn 405 410 415Ser Ile Ser Glu Asn
Phe Thr Tyr Lys His Leu Leu Asn Ile Ser Arg 420 425 430Ile Ala Pro
Leu Asn Ser Ser Ile His Ile Pro Asp Asp Lys Glu Ile 435 440 445Trp
Glu Leu 450531116DNAPorphyromonas gingivalis 53atgcaacttt
tcaaactcaa gagtgtaaca catcactttg acacttttgc agaatttgcc 60aaggaattct
gtcttggaga acgcgacttg gtaattacca acgagttcat ctatgaaccg
120tatatgaagg catgccagct cccctgccat tttgttatgc aggagaaata
tgggcaaggc 180gagccttctg acgaaatgat gaataacatc ttggcagaca
tccgtaatat ccagttcgac 240cgcgtaatcg gtatcggagg aggtacggtt
attgacatct ctaaactttt cgttctgaaa 300ggattaaatg atgtactcga
tgcattcgac cgcaaaatac ctcttatcaa agagaaagaa 360ctgatcattg
tgcccacaac atgcggaacg ggtagcgagg tgacgaacat ttctatcgca
420gaaatcaaaa gccgtcacac caaaatggga ttggctgacg atgccattgt
tgcagaccat 480gccatcatca tacctgaact tctgaagagc ttgcctttcc
acttctacgc atgcagtgca 540atcgatgctc ttatccatgc catcgagtca
tacgtatctc ctaaagccag tccatattct 600cgtctgttca gtgaggcggc
ttgggacatt atcctggaag tattcaagaa aatcgccgaa 660cacggccctg
aataccgctt cgaaaagctg ggagaaatga tcatggccag caactatgcc
720ggtatagcct tcggaaatgc aggagtagga gccgtccacg cactatccta
cccgttggga 780ggcaactatc acgtgccgca tggagaagca aactatcagt
tcttcacaga ggtattcaaa 840gtataccaaa agaagaatcc tttcggctat
atagtcgaac tcaactggaa gctctccaag 900atactgaact gccagcccga
atacgtatat ccgaagctgg atgaacttct cggatgcctt 960cttaccaaga
aacctttgca cgaatacggc atgaaggacg aagaggtaag aggctttgcg
1020gaatcagtgc ttaagacaca gcaaagattg ctcgccaaca actacgtaga
gcttactgta 1080gatgagatcg aaggtatcta cagaagactc tactaa
111654371PRTPorphyromonas gingivalis 54Met Gln Leu Phe Lys Leu Lys
Ser Val Thr His His Phe Asp Thr Phe1 5 10 15Ala Glu Phe Ala Lys Glu
Phe Cys Leu Gly Glu Arg Asp Leu Val Ile 20 25 30Thr Asn Glu Phe Ile
Tyr Glu Pro Tyr Met Lys Ala Cys Gln Leu Pro 35 40 45Cys His Phe Val
Met Gln Glu Lys Tyr Gly Gln Gly Glu Pro Ser Asp 50 55 60Glu Met Met
Asn Asn Ile Leu Ala Asp Ile Arg Asn Ile Gln Phe Asp65 70 75 80Arg
Val Ile Gly Ile Gly Gly Gly Thr Val Ile Asp Ile Ser Lys Leu 85 90
95Phe Val Leu Lys Gly Leu Asn Asp Val Leu Asp Ala Phe Asp Arg Lys
100 105 110Ile Pro Leu Ile Lys Glu Lys Glu Leu Ile Ile Val Pro Thr
Thr Cys 115 120 125Gly Thr Gly Ser Glu Val Thr
Asn Ile Ser Ile Ala Glu Ile Lys Ser 130 135 140Arg His Thr Lys Met
Gly Leu Ala Asp Asp Ala Ile Val Ala Asp His145 150 155 160Ala Ile
Ile Ile Pro Glu Leu Leu Lys Ser Leu Pro Phe His Phe Tyr 165 170
175Ala Cys Ser Ala Ile Asp Ala Leu Ile His Ala Ile Glu Ser Tyr Val
180 185 190Ser Pro Lys Ala Ser Pro Tyr Ser Arg Leu Phe Ser Glu Ala
Ala Trp 195 200 205Asp Ile Ile Leu Glu Val Phe Lys Lys Ile Ala Glu
His Gly Pro Glu 210 215 220Tyr Arg Phe Glu Lys Leu Gly Glu Met Ile
Met Ala Ser Asn Tyr Ala225 230 235 240Gly Ile Ala Phe Gly Asn Ala
Gly Val Gly Ala Val His Ala Leu Ser 245 250 255Tyr Pro Leu Gly Gly
Asn Tyr His Val Pro His Gly Glu Ala Asn Tyr 260 265 270Gln Phe Phe
Thr Glu Val Phe Lys Val Tyr Gln Lys Lys Asn Pro Phe 275 280 285Gly
Tyr Ile Val Glu Leu Asn Trp Lys Leu Ser Lys Ile Leu Asn Cys 290 295
300Gln Pro Glu Tyr Val Tyr Pro Lys Leu Asp Glu Leu Leu Gly Cys
Leu305 310 315 320Leu Thr Lys Lys Pro Leu His Glu Tyr Gly Met Lys
Asp Glu Glu Val 325 330 335Arg Gly Phe Ala Glu Ser Val Leu Lys Thr
Gln Gln Arg Leu Leu Ala 340 345 350Asn Asn Tyr Val Glu Leu Thr Val
Asp Glu Ile Glu Gly Ile Tyr Arg 355 360 365Arg Leu Tyr
370551296DNAPorphyromonas gingivalis 55atgaaagacg tattagcgga
atatgcctcc cgaattgttt cggccgaaga agccgtaaaa 60catatcaaaa atggagaacg
ggtagctttg tcacatgctg ccggagttcc tcagagttgt 120gttgatgcac
tggtacaaca ggccgacctt ttccagaatg tcgaaattta tcacatgctt
180tgtctcggcg aaggaaaata tatggcacct gaaatggccc ctcacttccg
acacataacc 240aattttgtag gtggtaattc tcgtaaagca gttgaggaaa
atagagccga cttcattccg 300gtattctttt atgaagtgcc atcaatgatt
cgcaaagaca tccttcacat agatgtcgcc 360atcgttcagc tttcaatgcc
tgatgagaat ggttactgta gttttggagt atcttgcgat 420tatagcaaac
cggcagcaga aagcgctcat ttagttatag gggaaatcaa ccgtcaaatg
480ccatatgtac atggcgacaa cttgattcac atatcgaagt tggattacat
cgtgatggca 540gactacccta tctattctct tgcaaagccc aaaatcggag
aagtagaaga agctatcggg 600cgtaattgtg ccgagcttat tgaagatggt
gccacactcc aactcggtat cggcgcgatt 660cctgatgcag ccctgttatt
cctcaaggac aaaaaagatc tggggatcca taccgagatg 720ttctccgatg
gtgttgtcga attagttcgc agtggagtaa ttacaggaaa gaaaaagaca
780cttcaccccg gaaagatggt cgcaaccttc ttaatgggaa gcgaagacgt
atatcatttc 840atcgacaaaa atcccgatgt agaactttat ccggtagatt
acgtcaatga tccgcgagta 900atcgctcaaa atgataatat ggtcagcatc
aatagctgta tcgaaatcga tcttatggga 960caagtcgtgt ccgaatgtat
aggaagcaag caattcagcg gaaccggcgg tcaagtagat 1020tatgttcgtg
gagcagcatg gtctaaaaac ggcaaaagca tcatggcaat tccctcaaca
1080gccaaaaacg gtactgcatc tcgaattgta cctataattg cagagggagc
tgctgtaaca 1140accctccgca acgaagtcga ttacgttgta accgaatacg
gtatagcaca actcaaagga 1200aagagtttgc gccagcgagc agaagctctt
attgccatag cccacccgga tttcagagag 1260gaactaacga aacatctccg
caaacgtttc ggataa 129656431PRTPorphyromonas gingivalis 56Met Lys
Asp Val Leu Ala Glu Tyr Ala Ser Arg Ile Val Ser Ala Glu1 5 10 15Glu
Ala Val Lys His Ile Lys Asn Gly Glu Arg Val Ala Leu Ser His 20 25
30Ala Ala Gly Val Pro Gln Ser Cys Val Asp Ala Leu Val Gln Gln Ala
35 40 45Asp Leu Phe Gln Asn Val Glu Ile Tyr His Met Leu Cys Leu Gly
Glu 50 55 60Gly Lys Tyr Met Ala Pro Glu Met Ala Pro His Phe Arg His
Ile Thr65 70 75 80Asn Phe Val Gly Gly Asn Ser Arg Lys Ala Val Glu
Glu Asn Arg Ala 85 90 95Asp Phe Ile Pro Val Phe Phe Tyr Glu Val Pro
Ser Met Ile Arg Lys 100 105 110Asp Ile Leu His Ile Asp Val Ala Ile
Val Gln Leu Ser Met Pro Asp 115 120 125Glu Asn Gly Tyr Cys Ser Phe
Gly Val Ser Cys Asp Tyr Ser Lys Pro 130 135 140Ala Ala Glu Ser Ala
His Leu Val Ile Gly Glu Ile Asn Arg Gln Met145 150 155 160Pro Tyr
Val His Gly Asp Asn Leu Ile His Ile Ser Lys Leu Asp Tyr 165 170
175Ile Val Met Ala Asp Tyr Pro Ile Tyr Ser Leu Ala Lys Pro Lys Ile
180 185 190Gly Glu Val Glu Glu Ala Ile Gly Arg Asn Cys Ala Glu Leu
Ile Glu 195 200 205Asp Gly Ala Thr Leu Gln Leu Gly Ile Gly Ala Ile
Pro Asp Ala Ala 210 215 220Leu Leu Phe Leu Lys Asp Lys Lys Asp Leu
Gly Ile His Thr Glu Met225 230 235 240Phe Ser Asp Gly Val Val Glu
Leu Val Arg Ser Gly Val Ile Thr Gly 245 250 255Lys Lys Lys Thr Leu
His Pro Gly Lys Met Val Ala Thr Phe Leu Met 260 265 270Gly Ser Glu
Asp Val Tyr His Phe Ile Asp Lys Asn Pro Asp Val Glu 275 280 285Leu
Tyr Pro Val Asp Tyr Val Asn Asp Pro Arg Val Ile Ala Gln Asn 290 295
300Asp Asn Met Val Ser Ile Asn Ser Cys Ile Glu Ile Asp Leu Met
Gly305 310 315 320Gln Val Val Ser Glu Cys Ile Gly Ser Lys Gln Phe
Ser Gly Thr Gly 325 330 335Gly Gln Val Asp Tyr Val Arg Gly Ala Ala
Trp Ser Lys Asn Gly Lys 340 345 350Ser Ile Met Ala Ile Pro Ser Thr
Ala Lys Asn Gly Thr Ala Ser Arg 355 360 365Ile Val Pro Ile Ile Ala
Glu Gly Ala Ala Val Thr Thr Leu Arg Asn 370 375 380Glu Val Asp Tyr
Val Val Thr Glu Tyr Gly Ile Ala Gln Leu Lys Gly385 390 395 400Lys
Ser Leu Arg Gln Arg Ala Glu Ala Leu Ile Ala Ile Ala His Pro 405 410
415Asp Phe Arg Glu Glu Leu Thr Lys His Leu Arg Lys Arg Phe Gly 420
425 43057906DNAClostridium acetobutylicum 57atgattaaga gttttaatga
aattatcatg aaggtaaaga gcaaagaaat gaaaaaagtt 60gctgttgctg tagcacaaga
cgagccagta cttgaagcag taagagatgc taagaaaaat 120ggtattgcag
atgctattct tgttggagac catgacgaaa tcgtgtcaat cgcgcttaaa
180ataggaatgg atgtaaatga ttttgaaata gtaaacgagc ctaacgttaa
gaaagctgct 240ttaaaggcag tagagcttgt atcaactgga aaagctgata
tggtaatgaa gggacttgta 300aatacagcaa ctttcttaag atctgtatta
aacaaagaag ttggacttag aacaggaaaa 360actatgtctc acgttgcagt
atttgaaact gagaaatttg atagactatt atttttaaca 420gatgttgctt
tcaatactta tcctgaatta aaggaaaaaa ttgatatagt aaacaattca
480gttaaggttg cacatgcaat aggaattgaa aatccaaagg ttgctccaat
ttgtgcagtt 540gaggttataa accctaaaat gccatcaaca cttgatgcag
caatgctttc aaaaatgagt 600gacagaggac aaattaaagg ttgtgtagtt
gacggacctt tagcacttga tatagcttta 660tcagaagaag cagcacatca
taagggagta acaggagaag ttgctggaaa agctgatatc 720ttcttaatgc
caaacataga aacaggaaat gtaatgtata agactttaac atatacaact
780gattcaaaaa atggaggaat cttagttgga acttctgcac cagttgtttt
aacttcaaga 840gctgacagcc atgaaacaaa aatgaactct atagcacttg
cagctttagt tgcaggcaat 900aaataa 90658301PRTClostridium
acetobutylicum 58Met Ile Lys Ser Phe Asn Glu Ile Ile Met Lys Val
Lys Ser Lys Glu1 5 10 15Met Lys Lys Val Ala Val Ala Val Ala Gln Asp
Glu Pro Val Leu Glu 20 25 30Ala Val Arg Asp Ala Lys Lys Asn Gly Ile
Ala Asp Ala Ile Leu Val 35 40 45Gly Asp His Asp Glu Ile Val Ser Ile
Ala Leu Lys Ile Gly Met Asp 50 55 60Val Asn Asp Phe Glu Ile Val Asn
Glu Pro Asn Val Lys Lys Ala Ala65 70 75 80Leu Lys Ala Val Glu Leu
Val Ser Thr Gly Lys Ala Asp Met Val Met 85 90 95Lys Gly Leu Val Asn
Thr Ala Thr Phe Leu Arg Ser Val Leu Asn Lys 100 105 110Glu Val Gly
Leu Arg Thr Gly Lys Thr Met Ser His Val Ala Val Phe 115 120 125Glu
Thr Glu Lys Phe Asp Arg Leu Leu Phe Leu Thr Asp Val Ala Phe 130 135
140Asn Thr Tyr Pro Glu Leu Lys Glu Lys Ile Asp Ile Val Asn Asn
Ser145 150 155 160Val Lys Val Ala His Ala Ile Gly Ile Glu Asn Pro
Lys Val Ala Pro 165 170 175Ile Cys Ala Val Glu Val Ile Asn Pro Lys
Met Pro Ser Thr Leu Asp 180 185 190Ala Ala Met Leu Ser Lys Met Ser
Asp Arg Gly Gln Ile Lys Gly Cys 195 200 205Val Val Asp Gly Pro Leu
Ala Leu Asp Ile Ala Leu Ser Glu Glu Ala 210 215 220Ala His His Lys
Gly Val Thr Gly Glu Val Ala Gly Lys Ala Asp Ile225 230 235 240Phe
Leu Met Pro Asn Ile Glu Thr Gly Asn Val Met Tyr Lys Thr Leu 245 250
255Thr Tyr Thr Thr Asp Ser Lys Asn Gly Gly Ile Leu Val Gly Thr Ser
260 265 270Ala Pro Val Val Leu Thr Ser Arg Ala Asp Ser His Glu Thr
Lys Met 275 280 285Asn Ser Ile Ala Leu Ala Ala Leu Val Ala Gly Asn
Lys 290 295 300591068DNAClostridium acetobutylicum 59atgtatagat
tactaataat caatcctggc tcgacctcaa ctaaaattgg tatttatgac 60gatgaaaaag
agatatttga gaagacttta agacattcag ctgaagagat agaaaaatat
120aacactatat ttgatcaatt tcaattcaga aagaatgtaa ttttagatgc
gttaaaagaa 180gcaaacatag aagtaagttc tttaaatgct gtagttggaa
gaggcggact cttaaagcca 240atagtaagtg gaacttatgc agtaaatcaa
aaaatgcttg aagaccttaa agtaggagtt 300caaggtcagc atgcgtcaaa
tcttggtgga attattgcaa atgaaatagc aaaagaaata 360aatgttccag
catacatagt tgatccagtt gttgtggatg agcttgatga agtttcaaga
420atatcaggaa tggctgacat tccaagaaaa agtatattcc atgcattaaa
tcaaaaagca 480gttgctagaa gatatgcaaa agaagttgga aaaaaatacg
aagatcttaa tttaatcgta 540gtccacatgg gtggaggtac ttcagtaggt
actcataaag atggtagagt aatagaagtt 600aataatacac ttgatggaga
aggtccattc tcaccagaaa gaagtggtgg agttccaata 660ggagatcttg
taagattgtg cttcagcaac aaatatactt atgaagaagt aatgaaaaag
720ataaacggca aaggcggagt tgttagttac ttaaatacta tcgattttaa
ggctgtagtt 780gataaagctc ttgaaggaga taagaaatgt gcacttatat
atgaagcttt cacattccag 840gtagcaaaag agataggaaa atgttcaacc
gttttaaaag gaaatgtaga tgcaataatc 900ttaacaggcg gaattgcgta
caacgagcat gtatgtaatg ccatagagga tagagtaaaa 960ttcatagcac
ctgtagttag atatggtgga gaagatgaac ttcttgcact tgcagaaggt
1020ggacttagag ttttaagagg agaagaaaaa gctaaggaat acaaataa
106860355PRTClostridium acetobutylicum 60Met Tyr Arg Leu Leu Ile
Ile Asn Pro Gly Ser Thr Ser Thr Lys Ile1 5 10 15Gly Ile Tyr Asp Asp
Glu Lys Glu Ile Phe Glu Lys Thr Leu Arg His 20 25 30Ser Ala Glu Glu
Ile Glu Lys Tyr Asn Thr Ile Phe Asp Gln Phe Gln 35 40 45Phe Arg Lys
Asn Val Ile Leu Asp Ala Leu Lys Glu Ala Asn Ile Glu 50 55 60Val Ser
Ser Leu Asn Ala Val Val Gly Arg Gly Gly Leu Leu Lys Pro65 70 75
80Ile Val Ser Gly Thr Tyr Ala Val Asn Gln Lys Met Leu Glu Asp Leu
85 90 95Lys Val Gly Val Gln Gly Gln His Ala Ser Asn Leu Gly Gly Ile
Ile 100 105 110Ala Asn Glu Ile Ala Lys Glu Ile Asn Val Pro Ala Tyr
Ile Val Asp 115 120 125Pro Val Val Val Asp Glu Leu Asp Glu Val Ser
Arg Ile Ser Gly Met 130 135 140Ala Asp Ile Pro Arg Lys Ser Ile Phe
His Ala Leu Asn Gln Lys Ala145 150 155 160Val Ala Arg Arg Tyr Ala
Lys Glu Val Gly Lys Lys Tyr Glu Asp Leu 165 170 175Asn Leu Ile Val
Val His Met Gly Gly Gly Thr Ser Val Gly Thr His 180 185 190Lys Asp
Gly Arg Val Ile Glu Val Asn Asn Thr Leu Asp Gly Glu Gly 195 200
205Pro Phe Ser Pro Glu Arg Ser Gly Gly Val Pro Ile Gly Asp Leu Val
210 215 220Arg Leu Cys Phe Ser Asn Lys Tyr Thr Tyr Glu Glu Val Met
Lys Lys225 230 235 240Ile Asn Gly Lys Gly Gly Val Val Ser Tyr Leu
Asn Thr Ile Asp Phe 245 250 255Lys Ala Val Val Asp Lys Ala Leu Glu
Gly Asp Lys Lys Cys Ala Leu 260 265 270Ile Tyr Glu Ala Phe Thr Phe
Gln Val Ala Lys Glu Ile Gly Lys Cys 275 280 285Ser Thr Val Leu Lys
Gly Asn Val Asp Ala Ile Ile Leu Thr Gly Gly 290 295 300Ile Ala Tyr
Asn Glu His Val Cys Asn Ala Ile Glu Asp Arg Val Lys305 310 315
320Phe Ile Ala Pro Val Val Arg Tyr Gly Gly Glu Asp Glu Leu Leu Ala
325 330 335Leu Ala Glu Gly Gly Leu Arg Val Leu Arg Gly Glu Glu Lys
Ala Lys 340 345 350Glu Tyr Lys 35561906DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
61atgattaaga gttttaatga aattatcatg aaggtaaaga gcaaagaaat gaaaaaagtt
60gctgttgctg tagcacaaga cgagccagta cttgaagcag tacgcgatgc taagaaaaat
120ggtattgcag atgctattct tgttggcgac catgacgaaa tcgtgtcaat
cgcgcttaaa 180ataggcatgg atgtaaatga ttttgaaata gtaaacgagc
ctaacgttaa gaaagctgct 240ttaaaggcag tagagctggt atcaactgga
aaagctgata tggtaatgaa gggacttgta 300aatacagcaa ctttcttacg
ctctgtatta aacaaagaag ttggactgag aacaggaaaa 360actatgtctc
acgttgcagt atttgaaact gagaaatttg atcgtctgtt atttttaaca
420gatgttgctt tcaatactta tcctgaatta aaggaaaaaa ttgatatcgt
aaacaattca 480gttaaggttg cacatgcaat aggtattgaa aatccaaagg
ttgctccaat ttgtgcagtt 540gaggttataa accctaaaat gccatcaaca
cttgatgcag caatgctttc aaaaatgagt 600gacagaggac aaattaaagg
ttgtgtagtt gacggaccgt tagcacttga tatcgcttta 660tcagaagaag
cagcacatca taagggcgta acaggagaag ttgctggaaa agctgatatc
720ttcttaatgc caaacattga aacaggaaat gtaatgtata agactttaac
atatacaact 780gatagcaaaa atggcggaat cttagttgga acttctgcac
cagttgtttt aacttcacgc 840gctgacagcc atgaaacaaa aatgaactct
attgcacttg cagctttagt tgcaggcaat 900aaataa 90662906DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
62atgattaaga gttttaatga aattatcatg aaggtaaaga gcaaagaaat gaaaaaagtt
60gctgttgctg tagcacaaga cgagccagta cttgaagcag tacgcgatgc taagaaaaat
120ggtattgccg atgctattct ggttggcgac catgacgaaa tcgtgtctat
cgcgctgaaa 180ataggcatgg atgtaaatga ttttgaaatt gttaacgagc
ctaacgttaa gaaagctgcg 240ttaaaggcag tagagctggt atcaactgga
aaagctgata tggtaatgaa gggactggta 300aataccgcaa ctttcttacg
ctctgtatta aacaaagaag ttggtctgcg tacaggaaaa 360accatgtctc
acgttgcagt atttgaaact gagaaatttg atcgtctgtt atttttaaca
420gatgttgctt tcaatactta tcctgaatta aaggaaaaaa ttgatatcgt
taacaatagc 480gttaaggttg cacatgccat tggtattgaa aatccaaagg
ttgctccaat ttgtgcagtt 540gaggttatta acccgaaaat gccatcaaca
cttgatgcag caatgctttc aaaaatgagt 600gaccgcggac aaattaaagg
ttgtgtagtt gacggaccgc tggcacttga tatcgcttta 660tcagaagaag
cagcacatca taaaggcgta acaggagaag ttgctggaaa agctgatatc
720ttcttaatgc caaacattga aacaggaaat gtaatgtata agacgttaac
ctataccact 780gatagcaaaa atggcggcat cctggttgga acttctgcac
cagttgtttt aacttcacgc 840gctgacagcc atgaaacaaa aatgaactct
attgcactgg cagcgctggt tgcaggcaat 900aaataa 90663906DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
63atgattaaga gttttaatga aattatcatg aaggtaaaga gcaaagaaat gaaaaaagtt
60gctgttgctg ttgcacaaga cgagccggta ctggaagcgg tacgcgatgc taagaaaaat
120ggtattgccg atgctattct ggttggcgac catgacgaaa tcgtctctat
cgcgctgaaa 180attggcatgg atgttaatga ttttgaaatt gttaacgagc
ctaacgttaa gaaagctgcg 240ctgaaggcgg tagagctggt ttccaccgga
aaagctgata tggtaatgaa agggctggtg 300aataccgcaa ctttcttacg
cagcgtactg aacaaagaag ttggtctgcg taccggaaaa 360accatgagtc
acgttgcggt atttgaaact gagaaatttg atcgtctgct gtttctgacc
420gatgttgctt tcaatactta tcctgaatta aaagaaaaaa ttgatatcgt
taacaatagc 480gttaaggttg cgcatgccat tggtattgaa aatccaaagg
ttgctccaat ttgtgcagtt 540gaggttatta acccgaaaat gccatcaaca
cttgatgccg caatgcttag caaaatgagt 600gaccgcggac aaattaaagg
ttgtgtggtt gacggcccgc tggcactgga tatcgcgtta 660agcgaagaag
cggcacatca taaaggcgta accggcgaag ttgctggaaa agctgatatc
720ttcctgatgc caaacattga aacaggcaat gtaatgtata aaacgttaac
ctataccact 780gatagcaaaa atggcggcat cctggttgga acttctgcac
cagttgtttt aacctcacgc 840gctgacagcc atgaaaccaa aatgaacagc
attgcactgg cagcgctggt tgcaggcaat 900aaataa 90664906DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
64atgattaaaa gttttaacga aattatcatg aaagtgaaaa gcaaagagat gaaaaaagtg
60gcggttgcgg ttgcgcagga tgaaccggtg ctggaagcgg tgcgcgatgc caaaaaaaac
120ggtattgccg atgccattct ggtgggcgat cacgatgaaa ttgtctctat
tgcgctgaaa 180attggcatgg atgttaacga ttttgaaatt
gttaatgaac cgaacgtgaa aaaagcggcg 240ctgaaagcgg ttgaactggt
ttccaccggt aaagccgata tggtgatgaa agggctggtg 300aataccgcaa
ccttcctgcg cagcgtgctg aataaagaag tgggtctgcg taccggtaaa
360accatgagtc atgttgcggt gtttgaaacc gaaaaatttg accgtctgct
gtttctgacc 420gatgttgcgt ttaataccta tccggaactg aaagagaaaa
ttgatatcgt taataacagc 480gtgaaagtgg cgcatgccat tggtattgaa
aacccgaaag tggcgccgat ttgcgcggtt 540gaagtgatta acccgaaaat
gccgtcaacg ctggatgccg cgatgctcag caaaatgagc 600gatcgcggtc
aaatcaaagg ctgtgtggtt gatggcccgc tggcgctgga tatcgcgctt
660agcgaagaag cggcgcatca taaaggcgtg accggcgaag tggccggtaa
agccgatatt 720ttcctgatgc cgaatattga aaccggcaac gtgatgtata
aaacgctgac ctataccacc 780gacagcaaaa acggcggcat tctggtgggt
accagcgcgc cggtggtgct gacctcgcgc 840gccgacagcc atgaaaccaa
aatgaacagc attgcgctgg cggcgctggt ggccggtaat 900aaataa
906651068DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 65atgtatcgtt tactgattat caatcctggc
tcgacctcaa ctaaaattgg tatttatgac 60gatgaaaaag agatatttga gaagacttta
cgtcattcag ctgaagagat agaaaaatat 120aacactatat ttgatcaatt
tcagttcaga aagaatgtaa ttctcgatgc gttaaaagaa 180gcaaacattg
aagtaagttc tttaaatgct gtagttggac gcggcggact gttaaagcca
240atagtaagtg gaacttatgc agtaaatcaa aaaatgcttg aagaccttaa
agtaggcgtt 300caaggtcagc atgcgtcaaa tcttggtgga attattgcaa
atgaaatagc aaaagaaata 360aatgttccag catacatcgt tgatccagtt
gttgtggatg agcttgatga agtttcacgt 420atatcaggaa tggctgacat
tccacgtaaa agtatattcc atgcattaaa tcaaaaagca 480gttgctagac
gctatgcaaa agaagttgga aaaaaatacg aagatcttaa tttaatcgtg
540gtccacatgg gtggcggtac ttcagtaggt actcataaag atggtagagt
aattgaagtt 600aataatacac ttgatggaga aggtccattc tcaccagaaa
gaagtggtgg cgttccaata 660ggcgatcttg tacgtttgtg cttcagcaac
aaatatactt atgaagaagt aatgaaaaag 720ataaacggca aaggcggcgt
tgttagttac ttaaatacta tcgattttaa ggctgtagtt 780gataaagctc
ttgaaggcga taagaaatgt gcacttatat atgaagcttt cacattccag
840gtagcaaaag agataggaaa atgttcaacc gttttaaaag gaaatgtaga
tgcaataatc 900ttaacaggcg gaattgcgta caacgagcat gtatgtaatg
ccatagagga tagagtaaaa 960ttcattgcac ctgtagttcg ttatggtgga
gaagatgaac ttcttgcact tgcagaaggt 1020ggactgcgcg ttttacgcgg
agaagaaaaa gctaaggaat acaaataa 1068661068DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
66atgtatcgtt tactgattat caatcctggc tcgacctcaa ctaaaattgg tatttatgac
60gatgaaaaag agatatttga gaagacgtta cgtcattcag ctgaagagat tgaaaaatat
120aacactatat ttgatcaatt tcagttccgc aagaatgtga ttctcgatgc
gttaaaagaa 180gcaaacattg aagtcagttc tttaaatgct gtagttggac
gcggcggact gttaaagcca 240attgtcagtg gaacttatgc agtaaatcaa
aaaatgcttg aagaccttaa agtgggcgtt 300caaggtcagc atgccagcaa
tcttggtggc attattgcca atgaaatcgc aaaagaaatc 360aatgttccag
catacatcgt tgatccggtt gttgtggatg agcttgatga agttagccgt
420ataagcggaa tggctgacat tccacgtaaa agtatattcc atgcattaaa
tcaaaaagca 480gttgctcgtc gctatgcaaa agaagttggt aaaaaatacg
aagatcttaa tttaatcgtg 540gtccacatgg gtggcggtac ttcagtaggt
actcataaag atggtcgcgt gattgaagtt 600aataatacac ttgatggcga
aggtccattc tcaccagaac gtagtggtgg cgttccaatt 660ggcgatctgg
tacgtttgtg cttcagcaac aaatatactt atgaagaagt gatgaaaaag
720ataaacggca aaggcggcgt tgttagttac ctgaatacta tcgattttaa
ggctgtagtt 780gataaagcgc ttgaaggcga taagaaatgt gcactgattt
atgaagcttt caccttccag 840gtagcaaaag agattggtaa atgttcaacc
gttttaaaag gaaatgttga tgccattatc 900ttaacaggcg gcattgctta
caacgagcat gtatgtaatg ccattgagga tcgcgtaaaa 960ttcattgcac
ctgtagttcg ttatggtggc gaagatgaac tgctggcact ggcagaaggt
1020ggactgcgcg ttttacgcgg cgaagaaaaa gcgaaggaat acaaataa
1068671068DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 67atgtatcgtc tgctgattat caatcctggc
tcgacctcaa ctaaaattgg tatttatgac 60gatgaaaaag agatatttga gaaaacgtta
cgtcatagcg ctgaagagat tgaaaaatat 120aacactattt ttgatcaatt
tcagttccgc aagaatgtga ttctcgatgc gctgaaagaa 180gcaaacattg
aagtcagttc gctgaatgcg gtagttggtc gcggcggtct gctgaagcca
240attgtcagcg gcacttatgc ggtaaatcaa aaaatgctgg aagacctgaa
agtgggcgtt 300caggggcagc atgccagcaa tcttggtggc attattgcca
atgaaatcgc caaagaaatc 360aatgttccgg catacatcgt tgatccggtt
gttgtggatg agctggatga agttagccgt 420atcagcggaa tggctgacat
tccacgtaaa agtattttcc atgcactgaa tcaaaaagcg 480gttgcgcgtc
gctatgcaaa agaagttggt aaaaaatacg aagatcttaa tctgatcgtg
540gtgcatatgg gtggcggtac tagcgtcggt actcataaag atggtcgcgt
gattgaagtt 600aataatacac ttgatggcga aggtccattc tcaccagaac
gtagcggtgg cgttccaatt 660ggcgatctgg tacgtttgtg cttcagcaac
aaatatacct atgaagaagt gatgaaaaag 720ataaacggca aaggcggcgt
tgttagttac ctgaatacta tcgattttaa ggcggtagtt 780gataaagcgc
tggaaggcga taagaaatgt gcactgattt atgaagcgtt caccttccag
840gtggcaaaag agattggtaa atgttcaacc gttctgaaag gcaatgttga
tgccattatc 900ctgaccggcg gcattgctta caacgagcat gtttgtaatg
ccattgagga tcgcgtaaaa 960ttcattgcac ctgtggttcg ttatggtggc
gaagatgaac tgctggcact ggcagaaggt 1020ggtctgcgcg ttttacgcgg
cgaagaaaaa gcgaaagaat acaaataa 1068681068DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
68atgtatcgtc tgctgattat caacccgggc agcacctcaa ccaaaattgg tatttacgac
60gatgaaaaag agatttttga aaaaacgctg cgtcacagcg cagaagagat tgaaaaatac
120aacaccattt tcgatcagtt ccagttccgc aaaaacgtga ttctcgatgc
gctgaaagaa 180gccaatattg aagtctcctc gctgaatgcg gtggtcggtc
gcggcggtct gctgaaaccg 240attgtcagcg gcacttatgc ggttaatcag
aaaatgctgg aagatctgaa agtgggcgtg 300caggggcagc atgccagcaa
tctcggcggc attatcgcca atgaaatcgc caaagagatc 360aacgtgccgg
cttatatcgt cgatccggtg gtggttgatg aactggatga agtcagccgt
420atcagcggca tggcggatat tccgcgtaaa agcattttcc atgcgctgaa
tcagaaagcg 480gttgcgcgtc gctatgccaa agaagtgggt aaaaaatatg
aagatctcaa tctgattgtg 540gtgcatatgg gcggcggcac cagcgtcggt
acgcataaag atggtcgcgt gattgaagtg 600aataacacgc tggatggcga
agggccgttc tcgccggaac gtagcggcgg cgtgccgatt 660ggcgatctgg
tgcgtctgtg tttcagcaat aaatacacct acgaagaagt gatgaaaaaa
720atcaacggca aaggcggcgt ggttagctat ctgaatacca tcgattttaa
agcggtggtt 780gataaagcgc tggaaggcga taaaaaatgc gcgctgattt
atgaagcgtt taccttccag 840gtggcgaaag agattggtaa atgttcaacc
gtgctgaaag gcaacgttga tgccattatt 900ctgaccggcg gcattgctta
taacgaacat gtttgtaatg ccattgaaga tcgcgtgaaa 960tttattgcgc
cggtggtgcg ttacggcggc gaagatgaac tgctggcgct ggcggaaggc
1020ggtctgcgcg tgctgcgcgg cgaagaaaaa gcgaaagagt acaaataa
1068691407DNAClostridium biejerinckii 69atgaataaag acacactaat
acctacaact aaagatttaa aagtaaaaac aaatggtgaa 60aacattaatt taaagaacta
caaggataat tcttcatgtt tcggagtatt cgaaaatgtt 120gaaaatgcta
taagcagcgc tgtacacgca caaaagatat tatcccttca ttatacaaaa
180gagcaaagag aaaaaatcat aactgagata agaaaggccg cattacaaaa
taaagaggtc 240ttggctacaa tgattctaga agaaacacat atgggaagat
atgaggataa aatattaaaa 300catgaattgg tagctaaata tactcctggt
acagaagatt taactactac tgcttggtca 360ggtgataatg gtcttacagt
tgtagaaatg tctccatatg gtgttatagg tgcaataact 420ccttctacga
atccaactga aactgtaata tgtaatagca taggcatgat agctgctgga
480aatgctgtag tatttaacgg acacccatgc gctaaaaaat gtgttgcctt
tgctgttgaa 540atgataaata aggcaattat ttcatgtggc ggtcctgaaa
atctagtaac aactataaaa 600aatccaacta tggagtctct agatgcaatt
attaagcatc cttcaataaa acttctttgc 660ggaactgggg gtccaggaat
ggtaaaaacc ctcttaaatt ctggtaagaa agctataggt 720gctggtgctg
gaaatccacc agttattgta gatgatactg ctgatataga aaaggctggt
780aggagcatca ttgaaggctg ttcttttgat aataatttac cttgtattgc
agaaaaagaa 840gtatttgttt ttgagaatgt tgcagatgat ttaatatcta
acatgctaaa aaataatgct 900gtaattataa atgaagatca agtatcaaaa
ttaatagatt tagtattaca aaaaaataat 960gaaactcaag aatactttat
aaacaaaaaa tgggtaggaa aagatgcaaa attattctta 1020gatgaaatag
atgttgagtc tccttcaaat gttaaatgca taatctgcga agtaaatgca
1080aatcatccat ttgttatgac agaactcatg atgccaatat tgccaattgt
aagagttaaa 1140gatatagatg aagctattaa atatgcaaag atagcagaac
aaaatagaaa acatagtgcc 1200tatatttatt ctaaaaatat agacaaccta
aatagatttg aaagagaaat agatactact 1260atttttgtaa agaatgctaa
atcttttgct ggtgttggtt atgaagcaga aggatttaca 1320actttcacta
ttgctggatc tactggtgag ggaataacct ctgcaaggaa ttttacaaga
1380caaagaagat gtgtacttgc cggctaa 140770468PRTClostridium
biejerinckii 70Met Asn Lys Asp Thr Leu Ile Pro Thr Thr Lys Asp Leu
Lys Val Lys1 5 10 15Thr Asn Gly Glu Asn Ile Asn Leu Lys Asn Tyr Lys
Asp Asn Ser Ser 20 25 30Cys Phe Gly Val Phe Glu Asn Val Glu Asn Ala
Ile Ser Ser Ala Val 35 40 45His Ala Gln Lys Ile Leu Ser Leu His Tyr
Thr Lys Glu Gln Arg Glu 50 55 60Lys Ile Ile Thr Glu Ile Arg Lys Ala
Ala Leu Gln Asn Lys Glu Val65 70 75 80Leu Ala Thr Met Ile Leu Glu
Glu Thr His Met Gly Arg Tyr Glu Asp 85 90 95Lys Ile Leu Lys His Glu
Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu 100 105 110Asp Leu Thr Thr
Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val 115 120 125Glu Met
Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn 130 135
140Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala
Gly145 150 155 160Asn Ala Val Val Phe Asn Gly His Pro Cys Ala Lys
Lys Cys Val Ala 165 170 175Phe Ala Val Glu Met Ile Asn Lys Ala Ile
Ile Ser Cys Gly Gly Pro 180 185 190Glu Asn Leu Val Thr Thr Ile Lys
Asn Pro Thr Met Glu Ser Leu Asp 195 200 205Ala Ile Ile Lys His Pro
Ser Ile Lys Leu Leu Cys Gly Thr Gly Gly 210 215 220Pro Gly Met Val
Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly225 230 235 240Ala
Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile 245 250
255Glu Lys Ala Gly Arg Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn
260 265 270Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn
Val Ala 275 280 285Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala
Val Ile Ile Asn 290 295 300Glu Asp Gln Val Ser Lys Leu Ile Asp Leu
Val Leu Gln Lys Asn Asn305 310 315 320Glu Thr Gln Glu Tyr Phe Ile
Asn Lys Lys Trp Val Gly Lys Asp Ala 325 330 335Lys Leu Phe Leu Asp
Glu Ile Asp Val Glu Ser Pro Ser Asn Val Lys 340 345 350Cys Ile Ile
Cys Glu Val Asn Ala Asn His Pro Phe Val Met Thr Glu 355 360 365Leu
Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu 370 375
380Ala Ile Lys Tyr Ala Lys Ile Ala Glu Gln Asn Arg Lys His Ser
Ala385 390 395 400Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg
Phe Glu Arg Glu 405 410 415Ile Asp Thr Thr Ile Phe Val Lys Asn Ala
Lys Ser Phe Ala Gly Val 420 425 430Gly Tyr Glu Ala Glu Gly Phe Thr
Thr Phe Thr Ile Ala Gly Ser Thr 435 440 445Gly Glu Gly Ile Thr Ser
Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys 450 455 460Val Leu Ala
Gly465711407DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 71atgaataaag acacactaat
acctacaact aaagatttaa aagtaaaaac aaatggtgaa 60aacattaatt taaagaacta
caaggataat tcttcatgtt tcggcgtatt cgaaaatgtt 120gaaaatgcta
taagcagcgc tgtacacgca caaaagatat tatcccttca ttatacaaaa
180gagcaacgtg aaaaaatcat aactgagata agaaaggccg cattacaaaa
taaagaggtc 240ttggctacaa tgattctgga agaaacacat atgggacgtt
atgaggataa aatattaaaa 300catgaattgg tagctaaata tactcctggt
acagaagatt taactactac tgcctggtca 360ggtgataatg gtctgacagt
tgtagaaatg tctccatatg gtgttattgg tgcaataact 420ccttctacga
atccaactga aactgtaata tgtaatagca taggcatgat tgctgctgga
480aatgctgtag tatttaacgg acacccatgc gctaaaaaat gtgttgcctt
tgctgttgaa 540atgataaata aggcaattat ttcatgtggc ggtcctgaaa
atctggtaac aactataaaa 600aatccaacca tggagtctct ggatgcaatt
attaagcatc cttcaataaa acttctttgc 660ggaactgggg gtccaggaat
ggtaaaaacc ctgttaaatt ctggtaagaa agctataggt 720gctggtgctg
gaaatccacc agttattgtc gatgatactg ctgatataga aaaggctggt
780cgtagcatca ttgaaggctg ttcttttgat aataatttac cttgtattgc
agaaaaagaa 840gtatttgttt ttgagaatgt tgcagatgat ttaatatcta
acatgctaaa aaataatgct 900gtaattataa atgaagatca agtatcaaaa
ttaatcgatt tagtattaca aaaaaataat 960gaaactcaag aatactttat
aaacaaaaaa tgggtaggaa aagatgcaaa attattcctc 1020gatgaaatag
atgttgagtc tccttcaaat gttaaatgca taatctgcga agtaaatgca
1080aatcatccat ttgttatgac agaactgatg atgccaatat tgccaattgt
acgcgttaaa 1140gatatcgatg aagctattaa atatgcaaag atagcagaac
aaaatagaaa acatagtgcc 1200tatatttatt ctaaaaatat cgacaacctg
aatcgctttg aacgtgaaat agatactact 1260atttttgtaa agaatgctaa
atcttttgct ggtgttggtt atgaagcaga aggatttaca 1320actttcacta
ttgctggatc tactggtgag ggaataacct ctgcacgtaa ttttacacgc
1380caacgtcgct gtgtacttgc cggctaa 1407721407DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
72atgaataaag acacactgat ccctacaact aaagatttaa aagtaaaaac aaatggtgaa
60aacattaatt taaagaacta caaagataat agcagttgtt tcggcgtatt cgaaaatgtt
120gaaaatgcta tcagcagcgc tgtacacgca caaaagatat tatcgctgca
ttatacaaaa 180gagcaacgtg aaaaaatcat cactgagata cgtaaggccg
cattacaaaa taaagaggtg 240ctggctacaa tgattctgga agaaacacat
atgggacgtt atgaggataa aatattaaaa 300catgaactgg tagctaaata
tactcctggt acagaagatt taactactac tgcctggagc 360ggtgataatg
gtctgacagt tgtagaaatg tctccatatg gtgttattgg tgcaataact
420ccttctacca atccaactga aactgtaatt tgtaatagca ttggcatgat
tgctgctgga 480aatgctgtag tatttaacgg acacccatgc gctaaaaaat
gtgttgcctt tgctgttgaa 540atgatcaata aggcaattat tagctgtggc
ggtccggaaa atctggtaac aactataaaa 600aatccaacca tggagtctct
ggatgccatt attaagcatc cttcaataaa actgctttgc 660ggaactggcg
gtccaggaat ggtaaaaacc ctgttaaatt ctggtaagaa agctattggt
720gctggtgctg gaaatccacc agttattgtc gatgatactg ctgatattga
aaaggctggt 780cgtagcatca ttgaaggctg ttcttttgat aataatttac
cttgtattgc agaaaaagaa 840gtatttgttt ttgagaatgt tgcagatgat
ttaatatcta acatgctgaa aaataatgct 900gtaattatca atgaagatca
ggtatcaaaa ttaatcgatt tagtattaca aaaaaataat 960gaaactcaag
aatactttat caacaaaaaa tgggtaggta aagatgcaaa attattcctc
1020gatgaaatcg atgttgagtc tccttcaaat gttaaatgca ttatctgcga
agtgaatgcc 1080aatcatccat ttgttatgac agaactgatg atgccaatat
tgccaattgt gcgcgttaaa 1140gatatcgatg aagctattaa atatgcaaag
attgcagaac aaaatagaaa acatagtgcc 1200tatatttata gcaaaaatat
cgacaacctg aatcgctttg aacgtgaaat cgatactact 1260atttttgtaa
agaatgctaa atcttttgct ggtgttggtt atgaagcaga aggatttacc
1320actttcacta ttgctggatc tactggtgag ggcataacct ctgcacgtaa
ttttacccgc 1380caacgtcgct gtgtactggc cggctaa
1407731407DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 73atgaataaag acacgctgat cccgacaact
aaagatctga aagtaaaaac caatggtgaa 60aacattaatc tgaagaacta caaagataat
agcagttgtt tcggcgtatt cgaaaatgtt 120gaaaatgcta tcagcagcgc
ggtacacgca caaaagatac tctcgctgca ttataccaaa 180gagcaacgtg
aaaaaatcat cactgagatc cgtaaggccg cattacaaaa taaagaggtg
240ctggcaacaa tgattctgga agaaacacat atgggacgtt atgaggataa
aatactgaaa 300catgaactgg tggcgaaata tacgcctggt actgaagatt
taaccaccac tgcctggagc 360ggtgataatg gtctgaccgt tgtggaaatg
tcgccttatg gtgttattgg tgcaattacg 420ccttcaacca atccaactga
aacggtaatt tgtaatagca ttggcatgat tgctgctgga 480aatgcggtag
tatttaacgg tcacccctgc gctaaaaaat gtgttgcctt tgctgttgaa
540atgatcaata aagcgattat tagctgtggc ggtccggaaa atctggtaac
cactataaaa 600aatccaacca tggagtcgct ggatgccatt attaagcatc
cttcaatcaa actgctgtgc 660ggcactggcg gtccaggaat ggtgaaaacc
ctgctgaata gcggtaagaa agcgattggt 720gctggtgctg gaaatccacc
agttattgtc gatgatactg ctgatattga aaaagcgggt 780cgtagcatca
ttgaaggctg ttcttttgat aataatttac cttgtattgc agaaaaagaa
840gtatttgttt ttgagaatgt tgccgatgat ctgatctcta acatgctgaa
aaataatgcg 900gtgattatca atgaagatca ggttagcaaa ctgatcgatc
tggtattaca aaaaaataat 960gaaactcaag aatactttat caacaaaaaa
tgggtaggta aagatgcaaa actgttcctc 1020gatgaaatcg atgttgagtc
gccttcaaat gttaaatgca ttatctgcga agtgaatgcc 1080aatcatccat
ttgtgatgac cgaactgatg atgccaattt tgccgattgt gcgcgttaaa
1140gatatcgatg aagcgattaa atatgcaaag attgcagaac aaaatcgtaa
acatagtgcc 1200tatatttata gcaaaaatat cgacaacctg aatcgctttg
aacgtgaaat cgataccact 1260atttttgtga agaatgctaa atcttttgct
ggtgttggtt atgaagcaga aggttttacc 1320actttcacta ttgctggaag
caccggtgaa ggcattacct ctgcacgtaa ttttacccgc 1380caacgtcgct
gtgtactggc cggctaa 1407741407DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 74atgaataaag
atacgctgat cccgaccacc aaagatctga aagtgaaaac caacggcgaa 60aatatcaacc
tgaaaaacta taaagataac agcagttgct ttggcgtgtt tgaaaacgtt
120gaaaacgcca tctccagcgc ggtgcatgcg caaaaaattc tctcgctgca
ttacaccaaa 180gagcagcgtg aaaaaattat caccgaaatc cgtaaagcgg
cgctgcaaaa caaagaagtg 240ctggcaacca tgatcctgga agaaacgcat
atggggcgtt atgaagataa aattctgaaa 300catgaactgg tggcgaaata
cacgccgggc actgaagatc tgaccaccac cgcctggagc 360ggcgataacg
gcctgaccgt ggtggagatg tcgccttatg gcgtgattgg cgcgattacg
420ccgtcaacca acccgaccga aacggtgatt tgtaacagca ttggcatgat
tgccgcgggt 480aatgcggtgg tgtttaacgg tcatccctgc gcgaaaaaat
gtgtggcgtt tgccgttgag 540atgatcaaca aagcgattat cagctgcggc
ggcccggaaa atctggtgac caccatcaaa 600aatccgacca tggaatcgct
ggatgccatt
atcaaacatc cttccatcaa actgctgtgc 660ggcaccggcg gcccgggcat
ggtgaaaacg ctgctgaaca gcggtaaaaa agcgattggc 720gcgggcgcgg
gtaacccgcc ggtgattgtc gatgacaccg ccgatattga aaaagcgggg
780cgtagcatta ttgaaggctg ttcttttgat aacaacctgc cctgcattgc
cgaaaaagaa 840gtgtttgtct ttgaaaacgt cgccgatgat ctgatcagca
atatgctgaa aaacaacgcg 900gtgattatca atgaagatca ggttagcaaa
ctgatcgatc tggtgctgca aaaaaacaac 960gaaacgcagg aatattttat
caacaaaaaa tgggttggta aagatgccaa actgtttctc 1020gatgaaatcg
atgttgaatc gccgtctaac gtgaaatgta ttatctgcga agtgaacgcc
1080aaccatccgt ttgtgatgac cgaactgatg atgccgattc tgccgattgt
gcgcgtgaaa 1140gatatcgatg aagcgattaa atatgccaaa attgccgaac
aaaaccgtaa acacagcgcc 1200tatatttaca gcaaaaatat cgataacctg
aaccgctttg aacgtgaaat cgataccacc 1260atttttgtga aaaatgccaa
aagttttgcc ggcgttggtt atgaagcgga aggttttacc 1320acctttacca
ttgccggtag caccggcgaa ggcattacca gcgcccgtaa ttttacccgc
1380cagcgtcgct gcgtgctggc gggctaa 1407751023DNAGeobacillus
thermoglucosidasius 75atgaaagctg cagtagtaga gcaatttaag gaaccattaa
aaattaaaga agtggaaaag 60ccatctattt catatggcga agtattagtc cgcattaaag
catgcggtgt atgccatacg 120gacttgcacg ccgctcatgg cgattggcca
gtaaaaccaa aacttccttt aatccctggc 180catgaaggag tcggaattgt
tgaagaagtc ggtccggggg taacccattt aaaagtggga 240gaccgcgttg
gaattccttg gttatattct gcgtgcggcc attgcgaata ttgtttaagc
300ggacaagaag cattatgtga acatcaacaa aacgccggct actcagtcga
cgggggttat 360gcagaatatt gcagagctgc gccagattat gtggtgaaaa
ttcctgacaa cttatcgttt 420gaagaagctg ctcctatttt ctgcgccgga
gttactactt ataaagcgtt aaaagtcaca 480ggtacaaaac cgggagaatg
ggtagcgatc tatggcatcg gcggccttgg acatgttgcc 540gtccagtatg
cgaaagcgat ggggcttcat gttgttgcag tggatatcgg cgatgagaaa
600ctggaacttg caaaagagct tggcgccgat cttgttgtaa atcctgcaaa
agaaaatgcg 660gcccaattta tgaaagagaa agtcggcgga gtacacgcgg
ctgttgtgac agctgtatct 720aaacctgctt ttcaatctgc gtacaattct
atccgcagag gcggcacgtg cgtgcttgtc 780ggattaccgc cggaagaaat
gcctattcca atctttgata cggtattaaa cggaattaaa 840attatcggtt
ccattgtcgg cacgcggaaa gacttgcaag aagcgcttca gttcgctgca
900gaaggtaaag taaaaaccat tattgaagtg caacctcttg aaaaaattaa
cgaagtattt 960gacagaatgc taaaaggaga aattaacgga cgggttgttt
taacgttaga aaataataat 1020taa 102376340PRTGeobacillus
thermoglucosidasius 76Met Lys Ala Ala Val Val Glu Gln Phe Lys Glu
Pro Leu Lys Ile Lys1 5 10 15Glu Val Glu Lys Pro Ser Ile Ser Tyr Gly
Glu Val Leu Val Arg Ile 20 25 30Lys Ala Cys Gly Val Cys His Thr Asp
Leu His Ala Ala His Gly Asp 35 40 45Trp Pro Val Lys Pro Lys Leu Pro
Leu Ile Pro Gly His Glu Gly Val 50 55 60Gly Ile Val Glu Glu Val Gly
Pro Gly Val Thr His Leu Lys Val Gly65 70 75 80Asp Arg Val Gly Ile
Pro Trp Leu Tyr Ser Ala Cys Gly His Cys Glu 85 90 95Tyr Cys Leu Ser
Gly Gln Glu Ala Leu Cys Glu His Gln Gln Asn Ala 100 105 110Gly Tyr
Ser Val Asp Gly Gly Tyr Ala Glu Tyr Cys Arg Ala Ala Pro 115 120
125Asp Tyr Val Val Lys Ile Pro Asp Asn Leu Ser Phe Glu Glu Ala Ala
130 135 140Pro Ile Phe Cys Ala Gly Val Thr Thr Tyr Lys Ala Leu Lys
Val Thr145 150 155 160Gly Thr Lys Pro Gly Glu Trp Val Ala Ile Tyr
Gly Ile Gly Gly Leu 165 170 175Gly His Val Ala Val Gln Tyr Ala Lys
Ala Met Gly Leu His Val Val 180 185 190Ala Val Asp Ile Gly Asp Glu
Lys Leu Glu Leu Ala Lys Glu Leu Gly 195 200 205Ala Asp Leu Val Val
Asn Pro Ala Lys Glu Asn Ala Ala Gln Phe Met 210 215 220Lys Glu Lys
Val Gly Gly Val His Ala Ala Val Val Thr Ala Val Ser225 230 235
240Lys Pro Ala Phe Gln Ser Ala Tyr Asn Ser Ile Arg Arg Gly Gly Thr
245 250 255Cys Val Leu Val Gly Leu Pro Pro Glu Glu Met Pro Ile Pro
Ile Phe 260 265 270Asp Thr Val Leu Asn Gly Ile Lys Ile Ile Gly Ser
Ile Val Gly Thr 275 280 285Arg Lys Asp Leu Gln Glu Ala Leu Gln Phe
Ala Ala Glu Gly Lys Val 290 295 300Lys Thr Ile Ile Glu Val Gln Pro
Leu Glu Lys Ile Asn Glu Val Phe305 310 315 320Asp Arg Met Leu Lys
Gly Glu Ile Asn Gly Arg Val Val Leu Thr Leu 325 330 335Glu Asn Asn
Asn 340774090DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 77atggctatcg aaatcaaagt
accggacatc ggggctgatg aagttgaaat caccgagatc 60ctggtcaaag tgggcgacaa
agttgaagcc gaacagtcgc tgatcaccgt agaaggcgac 120aaagcctcta
tggaagttcc gtctccgcag gcgggtatcg ttaaagagat caaagtctct
180gttggcgata aaacccagac cggcgcactg attatgattt tcgattccgc
cgacggtgca 240gcagacgctg cacctgctca ggcagaagag aagaaagaag
cagctccggc agcagcacca 300gcggctgcgg cggcaaaaga cgttaacgtt
ccggatatcg gcagcgacga agttgaagtg 360accgaaatcc tggtgaaagt
tggcgataaa gttgaagctg aacagtcgct gatcaccgta 420gaaggcgaca
aggcttctat ggaagttccg gctccgtttg ctggcaccgt gaaagagatc
480aaagtgaacg tgggtgacaa agtgtctacc ggctcgctga ttatggtctt
cgaagtcgcg 540ggtgaagcag gcgcggcagc tccggccgct aaacaggaag
cagctccggc agcggcccct 600gcaccagcgg ctggcgtgaa agaagttaac
gttccggata tcggcggtga cgaagttgaa 660gtgactgaag tgatggtgaa
agtgggcgac aaagttgccg ctgaacagtc actgatcacc 720gtagaaggcg
acaaagcttc tatggaagtt ccggcgccgt ttgcaggcgt cgtgaaggaa
780ctgaaagtca acgttggcga taaagtgaaa actggctcgc tgattatgat
cttcgaagtt 840gaaggcgcag cgcctgcggc agctcctgcg aaacaggaag
cggcagcgcc ggcaccggca 900gcaaaagctg aagccccggc agcagcacca
gctgcgaaag cggaaggcaa atctgaattt 960gctgaaaacg acgcttatgt
tcacgcgact ccgctgatcc gccgtctggc acgcgagttt 1020ggtgttaacc
ttgcgaaagt gaagggcact ggccgtaaag gtcgtatcct gcgcgaagac
1080gttcaggctt acgtgaaaga agctatcaaa cgtgcagaag cagctccggc
agcgactggc 1140ggtggtatcc ctggcatgct gccgtggccg aaggtggact
tcagcaagtt tggtgaaatc 1200gaagaagtgg aactgggccg catccagaaa
atctctggtg cgaacctgag ccgtaactgg 1260gtaatgatcc cgcatgttac
tcacttcgac aaaaccgata tcaccgagtt ggaagcgttc 1320cgtaaacagc
agaacgaaga agcggcgaaa cgtaagctgg atgtgaagat caccccggtt
1380gtcttcatca tgaaagccgt tgctgcagct cttgagcaga tgcctcgctt
caatagttcg 1440ctgtcggaag acggtcagcg tctgaccctg aagaaataca
tcaacatcgg tgtggcggtg 1500gataccccga acggtctggt tgttccggta
ttcaaagacg tcaacaagaa aggcatcatc 1560gagctgtctc gcgagctgat
gactatttct aagaaagcgc gtgacggtaa gctgactgcg 1620ggcgaaatgc
agggcggttg cttcaccatc tccagcatcg gcggcctggg tactacccac
1680ttcgcgccga ttgtgaacgc gccggaagtg gctatcctcg gcgtttccaa
gtccgcgatg 1740gagccggtgt ggaatggtaa agagttcgtg ccgcgtctga
tgctgccgat ttctctctcc 1800ttcgaccacc gcgtgatcga cggtgctgat
ggtgcccgtt tcattaccat cattaacaac 1860acgctgtctg acattcgccg
tctggtgatg taagtaaaag agccggccca acggccggct 1920tttttctggt
aatctcatga atgtattgag gttattagcg aatagacaaa tcggttgccg
1980tttgttgttt aaaaattgtt aacaattttg taaaataccg acggatagaa
cgacccggtg 2040gtggttaggg tattacttca cataccctat ggatttctgg
gtgcagcaag gtagcaagcg 2100ccagaatccc caggagctta cataagtaag
tgactggggt gagggcgtga agctaacgcc 2160gctgcggcct gaaagacgac
gggtatgacc gccggagata aatatataga ggtcatgatg 2220agtactgaaa
tcaaaactca ggtcgtggta cttggggcag gccccgcagg ttactccgct
2280gccttccgtt gcgctgattt aggtctggaa accgtaatcg tagaacgtta
caacaccctt 2340ggcggtgttt gtctgaacgt gggttgtatc ccttctaaag
cgctgctgca cgtggcaaaa 2400gttatcgaag aagcgaaagc gctggccgaa
cacggcatcg ttttcggcga accgaaaact 2460gacattgaca agatccgcac
ctggaaagaa aaagtcatca ctcagctgac cggtggtctg 2520gctggcatgg
ccaaaggtcg taaagtgaag gtggttaacg gtctgggtaa atttaccggc
2580gctaacaccc tggaagtgga aggcgaaaac ggcaaaaccg tgatcaactt
cgacaacgcc 2640atcatcgcgg cgggttcccg tccgattcag ctgccgttta
tcccgcatga agatccgcgc 2700gtatgggact ccaccgacgc gctggaactg
aaatctgtac cgaaacgcat gctggtgatg 2760ggcggcggta tcatcggtct
ggaaatgggt accgtatacc atgcgctggg ttcagagatt 2820gacgtggtgg
aaatgttcga ccaggttatc ccggctgccg acaaagacgt ggtgaaagtc
2880ttcaccaaac gcatcagcaa gaaatttaac ctgatgctgg aagccaaagt
gactgccgtt 2940gaagcgaaag aagacggtat ttacgtttcc atggaaggta
aaaaagcacc ggcggaagcg 3000cagcgttacg acgcagtgct ggtcgctatc
ggccgcgtac cgaatggtaa aaacctcgat 3060gcaggtaaag ctggcgtgga
agttgacgat cgcggcttca tccgcgttga caaacaaatg 3120cgcaccaacg
tgccgcacat ctttgctatc ggcgatatcg tcggtcagcc gatgctggcg
3180cacaaaggtg tccatgaagg ccacgttgcc gcagaagtta tctccggtct
gaaacactac 3240ttcgatccga aagtgatccc atccatcgcc tacactaaac
cagaagtggc atgggtcggt 3300ctgaccgaga aagaagcgaa agagaaaggc
atcagctacg aaaccgccac cttcccgtgg 3360gctgcttccg gccgtgctat
cgcttctgac tgcgcagatg gtatgaccaa actgatcttc 3420gacaaagaga
cccaccgtgt tatcggcggc gcgattgtcg gcaccaacgg cggcgagctg
3480ctgggtgaga tcggcctggc tatcgagatg ggctgtgacg ctgaagacat
cgccctgacc 3540atccacgctc acccgactct gcacgagtcc gttggcctgg
cggcggaagt gttcgaaggc 3600agcatcaccg acctgccaaa cgccaaagcg
aagaaaaagt aactttttct ttcaggaaaa 3660aagcataagc ggctccggga
gccgcttttt ttatgcctga tgtttagaac tatgtcactg 3720ttcataaacc
gctacacctc atacatactt taagggcgaa ttctgcagat atccatcaca
3780ctggcggccg ctcgagcatg catctagcac atccggcaat taaaaaagcg
gctaaccacg 3840ccgctttttt tacgtctgca atttaccttt ccagtcttct
tgctccacgt tcagagagac 3900gttcgcatac tgctgaccgt tgctcgttat
tcagcctgac agtatggtta ctgtcgttta 3960gacgttgtgg gcggctctcc
tgaactttct cccgaaaaac ctgacgttgt tcaggtgatg 4020ccgattgaac
acgctggcgg gcgttatcac gttgctgttg attcagtggg cgctgctgta
4080ctttttcctt 409078475PRTEscherichia coli 78Met Met Ser Thr Glu
Ile Lys Thr Gln Val Val Val Leu Gly Ala Gly1 5 10 15Pro Ala Gly Tyr
Ser Ala Ala Phe Arg Cys Ala Asp Leu Gly Leu Glu 20 25 30Thr Val Ile
Val Glu Arg Tyr Asn Thr Leu Gly Gly Val Cys Leu Asn 35 40 45Val Gly
Cys Ile Pro Ser Lys Ala Leu Leu His Val Ala Lys Val Ile 50 55 60Glu
Glu Ala Lys Ala Leu Ala Glu His Gly Ile Val Phe Gly Glu Pro65 70 75
80Lys Thr Asp Ile Asp Lys Ile Arg Thr Trp Lys Glu Lys Val Ile Asn
85 90 95Gln Leu Thr Gly Gly Leu Ala Gly Met Ala Lys Gly Arg Lys Val
Lys 100 105 110Val Val Asn Gly Leu Gly Lys Phe Thr Gly Ala Asn Thr
Leu Glu Val 115 120 125Glu Gly Glu Asn Gly Lys Thr Val Ile Asn Phe
Asp Asn Ala Ile Ile 130 135 140Ala Ala Gly Ser Arg Pro Ile Gln Leu
Pro Phe Ile Pro His Glu Asp145 150 155 160Pro Arg Ile Trp Asp Ser
Thr Asp Ala Leu Glu Leu Lys Glu Val Pro 165 170 175Glu Arg Leu Leu
Val Met Gly Gly Gly Ile Ile Gly Leu Glu Met Gly 180 185 190Thr Val
Tyr His Ala Leu Gly Ser Gln Ile Asp Val Val Glu Met Phe 195 200
205Asp Gln Val Ile Pro Ala Ala Asp Lys Asp Ile Val Lys Val Phe Thr
210 215 220Lys Arg Ile Ser Lys Lys Phe Asn Leu Met Leu Glu Thr Lys
Val Thr225 230 235 240Ala Val Glu Ala Lys Glu Asp Gly Ile Tyr Val
Thr Met Glu Gly Lys 245 250 255Lys Ala Pro Ala Glu Pro Gln Arg Tyr
Asp Ala Val Leu Val Ala Ile 260 265 270Gly Arg Val Pro Asn Gly Lys
Asn Leu Asp Ala Gly Lys Ala Gly Val 275 280 285Glu Val Asp Asp Arg
Gly Phe Ile Arg Val Asp Lys Gln Leu Arg Thr 290 295 300Asn Val Pro
His Ile Phe Ala Ile Gly Asp Ile Val Gly Gln Pro Met305 310 315
320Leu Ala His Lys Gly Val His Glu Gly His Val Ala Ala Glu Val Ile
325 330 335Ala Gly Lys Lys His Tyr Phe Asp Pro Lys Val Ile Pro Ser
Ile Ala 340 345 350Tyr Thr Glu Pro Glu Val Ala Trp Val Gly Leu Thr
Glu Lys Glu Ala 355 360 365Lys Glu Lys Gly Ile Ser Tyr Glu Thr Ala
Thr Phe Pro Trp Ala Ala 370 375 380Ser Gly Arg Ala Ile Ala Ser Asp
Cys Ala Asp Gly Met Thr Lys Leu385 390 395 400Ile Phe Asp Lys Glu
Ser His Arg Val Ile Gly Gly Ala Ile Val Gly 405 410 415Thr Asn Gly
Gly Glu Leu Leu Gly Glu Ile Gly Leu Ala Ile Glu Met 420 425 430Gly
Cys Asp Ala Glu Asp Ile Ala Leu Thr Ile His Ala His Pro Thr 435 440
445Leu His Glu Ser Val Gly Leu Ala Ala Glu Val Phe Glu Gly Ser Ile
450 455 460Thr Asp Leu Pro Asn Pro Lys Ala Lys Lys Lys465 470
47579475PRTKlebsiella pneumoniae 79Met Met Ser Thr Glu Ile Lys Thr
Gln Val Val Val Leu Gly Ala Gly1 5 10 15Pro Ala Gly Tyr Ser Ala Ala
Phe Arg Cys Ala Asp Leu Gly Leu Glu 20 25 30Thr Val Ile Val Glu Arg
Tyr Ser Thr Leu Gly Gly Val Cys Leu Asn 35 40 45Val Gly Cys Ile Pro
Ser Lys Ala Leu Leu His Val Ala Lys Val Ile 50 55 60Glu Glu Ala Lys
Ala Leu Ala Glu His Gly Ile Val Phe Gly Glu Pro65 70 75 80Lys Thr
Asp Ile Asp Lys Ile Arg Thr Trp Lys Glu Lys Val Ile Thr 85 90 95Gln
Leu Thr Gly Gly Leu Ala Gly Met Ala Lys Gly Arg Lys Val Lys 100 105
110Val Val Asn Gly Leu Gly Lys Phe Thr Gly Ala Asn Thr Leu Glu Val
115 120 125Glu Gly Glu Asn Gly Lys Thr Val Ile Asn Phe Asp Asn Ala
Ile Ile 130 135 140Ala Ala Gly Ser Arg Pro Ile Gln Leu Pro Phe Ile
Pro His Glu Asp145 150 155 160Pro Arg Val Trp Asp Ser Thr Asp Ala
Leu Glu Leu Lys Ser Val Pro 165 170 175Lys Arg Met Leu Val Met Gly
Gly Gly Ile Ile Gly Leu Glu Met Gly 180 185 190Thr Val Tyr His Ala
Leu Gly Ser Glu Ile Asp Val Val Glu Met Phe 195 200 205Asp Gln Val
Ile Pro Ala Ala Asp Lys Asp Val Val Lys Val Phe Thr 210 215 220Lys
Arg Ile Ser Lys Lys Phe Asn Leu Met Leu Glu Ala Lys Val Thr225 230
235 240Ala Val Glu Ala Lys Glu Asp Gly Ile Tyr Val Ser Met Glu Gly
Lys 245 250 255Lys Ala Pro Ala Glu Ala Gln Arg Tyr Asp Ala Val Leu
Val Ala Ile 260 265 270Gly Arg Val Pro Asn Gly Lys Asn Leu Asp Ala
Gly Lys Ala Gly Val 275 280 285Glu Val Asp Asp Arg Gly Phe Ile Arg
Val Asp Lys Gln Met Arg Thr 290 295 300Asn Val Pro His Ile Phe Ala
Ile Gly Asp Ile Val Gly Gln Pro Met305 310 315 320Leu Ala His Lys
Gly Val His Glu Gly His Val Ala Ala Glu Val Ile 325 330 335Ser Gly
Leu Lys His Tyr Phe Asp Pro Lys Val Ile Pro Ser Ile Ala 340 345
350Tyr Thr Lys Pro Glu Val Ala Trp Val Gly Leu Thr Glu Lys Glu Ala
355 360 365Lys Glu Lys Gly Ile Ser Tyr Glu Thr Ala Thr Phe Pro Trp
Ala Ala 370 375 380Ser Gly Arg Ala Ile Ala Ser Asp Cys Ala Asp Gly
Met Thr Lys Leu385 390 395 400Ile Phe Asp Lys Glu Thr His Arg Val
Ile Gly Gly Ala Ile Val Gly 405 410 415Thr Asn Gly Gly Glu Leu Leu
Gly Glu Ile Gly Leu Ala Ile Glu Met 420 425 430Gly Cys Asp Ala Glu
Asp Ile Ala Leu Thr Ile His Ala His Pro Thr 435 440 445Leu His Glu
Ser Val Gly Leu Ala Ala Glu Val Phe Glu Gly Ser Ile 450 455 460Thr
Asp Leu Pro Asn Ala Lys Ala Lys Lys Lys465 470
47580347DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 80ataataatac atatgaacca tgcgagttac
gggcctataa gccaggcgag atatgatcta 60tatcaatttc tcatctataa tgctttgtta
gtatctcgtc gccgacttaa taaagagaga 120gttagtgtga aagctgacaa
cccttttgat cttttacttc ctgctgcaat ggccaaagtg 180gccgaagagg
cgggtgtcta taaagcaacg aaacatccgc ttaagacttt ctatctggcg
240attaccgccg gtgttttcat ctcaatcgca ttcaccactg gcacaggcac
agaaggtagg 300tgttacatgt cagaacgttt acacaatgac gtggatccta ttattat
347814678DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 81aagaggtaaa agaataatgg ctatcgaaat
caaagtaccg gacatcgggg ctgatgaagt 60tgaaatcacc gagatcctgg tcaaagtggg
cgacaaagtt gaagccgaac agtcgctgat 120caccgtagaa ggcgacaaag
cctctatgga agttccgtct ccgcaggcgg gtatcgttaa 180agagatcaaa
gtctctgttg gcgataaaac ccagaccggc gcactgatta tgattttcga
240ttccgccgac ggtgcagcag acgctgcacc tgctcaggca gaagagaaga
aagaagcagc 300tccggcagca gcaccagcgg ctgcggcggc aaaagacgtt
aacgttccgg atatcggcag 360cgacgaagtt gaagtgaccg aaatcctggt
gaaagttggc gataaagttg aagctgaaca
420gtcgctgatc accgtagaag gcgacaaggc ttctatggaa gttccggctc
cgtttgctgg 480caccgtgaaa gagatcaaag tgaacgtggg tgacaaagtg
tctaccggct cgctgattat 540ggtcttcgaa gtcgcgggtg aagcaggcgc
ggcagctccg gccgctaaac aggaagcagc 600tccggcagcg gcccctgcac
cagcggctgg cgtgaaagaa gttaacgttc cggatatcgg 660cggtgacgaa
gttgaagtga ctgaagtgat ggtgaaagtg ggcgacaaag ttgccgctga
720acagtcactg atcaccgtag aaggcgacaa agcttctatg gaagttccgg
cgccgtttgc 780aggcgtcgtg aaggaactga aagtcaacgt tggcgataaa
gtgaaaactg gctcgctgat 840tatgatcttc gaagttgaag gcgcagcgcc
tgcggcagct cctgcgaaac aggaagcggc 900agcgccggca ccggcagcaa
aagctgaagc cccggcagca gcaccagctg cgaaagcgga 960aggcaaatct
gaatttgctg aaaacgacgc ttatgttcac gcgactccgc tgatccgccg
1020tctggcacgc gagtttggtg ttaaccttgc gaaagtgaag ggcactggcc
gtaaaggtcg 1080tatcctgcgc gaagacgttc aggcttacgt gaaagaagct
atcaaacgtg cagaagcagc 1140tccggcagcg actggcggtg gtatccctgg
catgctgccg tggccgaagg tggacttcag 1200caagtttggt gaaatcgaag
aagtggaact gggccgcatc cagaaaatct ctggtgcgaa 1260cctgagccgt
aactgggtaa tgatcccgca tgttactcac ttcgacaaaa ccgatatcac
1320cgagttggaa gcgttccgta aacagcagaa cgaagaagcg gcgaaacgta
agctggatgt 1380gaagatcacc ccggttgtct tcatcatgaa agccgttgct
gcagctcttg agcagatgcc 1440tcgcttcaat agttcgctgt cggaagacgg
tcagcgtctg accctgaaga aatacatcaa 1500catcggtgtg gcggtggata
ccccgaacgg tctggttgtt ccggtattca aagacgtcaa 1560caagaaaggc
atcatcgagc tgtctcgcga gctgatgact atttctaaga aagcgcgtga
1620cggtaagctg actgcgggcg aaatgcaggg cggttgcttc accatctcca
gcatcggcgg 1680cctgggtact acccacttcg cgccgattgt gaacgcgccg
gaagtggcta tcctcggcgt 1740ttccaagtcc gcgatggagc cggtgtggaa
tggtaaagag ttcgtgccgc gtctgatgct 1800gccgatttct ctctccttcg
accaccgcgt gatcgacggt gctgatggtg cccgtttcat 1860taccatcatt
aacaacacgc tgtctgacat tcgccgtctg gtgatgtaag taaaagagcc
1920ggcccaacgg ccggcttttt tctggtaatc tcatgaatgt attgaggtta
ttagcgaata 1980gacaaatcgg ttgccgtttg ttaagccagg cgagatatga
tctatatcaa tttctcatct 2040ataatgcttt gttagtatct cgtcgccgac
ttaataaaga gagagttagt cttctatatc 2100acagcaagaa ggtaggtgtt
acatgatgag tactgaaatc aaaactcagg tcgtggtact 2160tggggcaggc
cccgcaggtt actctgcagc cttccgttgc gctgatttag gtctggaaac
2220cgtcatcgta gaacgttaca gcaccctcgg tggtgtttgt ctgaacgtgg
gttgtatccc 2280ttctaaagcg ctgctgcacg tggcaaaagt tatcgaagaa
gcgaaagcgc tggccgaaca 2340cggcatcgtt ttcggcgaac cgaaaactga
cattgacaag atccgcacct ggaaagaaaa 2400agtcatcact cagctgaccg
gtggtctggc tggcatggcc aaaggtcgta aagtgaaggt 2460ggttaacggt
ctgggtaaat ttaccggcgc taacaccctg gaagtggaag gcgaaaacgg
2520caaaaccgtg atcaacttcg acaacgccat catcgcggcg ggttcccgtc
cgattcagct 2580gccgtttatc ccgcatgaag atccgcgcgt atgggactcc
accgacgcgc tggaactgaa 2640atctgtaccg aaacgcatgc tggtgatggg
cggcggtatc atcggtctgg aaatgggtac 2700cgtataccat gcgctgggtt
cagagattga cgtggtggaa atgttcgacc aggttatccc 2760ggctgccgac
aaagacgtgg tgaaagtctt caccaaacgc atcagcaaga aatttaacct
2820gatgctggaa gccaaagtga ctgccgttga agcgaaagaa gacggtattt
acgtttccat 2880ggaaggtaaa aaagcaccgg cggaagcgca gcgttacgac
gcagtgctgg tcgctatcgg 2940ccgcgtaccg aatggtaaaa acctcgatgc
aggtaaagct ggcgtggaag ttgacgatcg 3000cggcttcatc cgcgttgaca
aacaaatgcg caccaacgtg ccgcacatct ttgctatcgg 3060cgatatcgtc
ggtcagccga tgctggcgca caaaggtgtc catgaaggcc acgttgccgc
3120agaagttatc tccggtctga aacactactt cgatccgaaa gtgatcccat
ccatcgccta 3180cactaaacca gaagtggcat gggtcggtct gaccgagaaa
gaagcgaaag agaaaggcat 3240cagctacgaa accgccacct tcccgtgggc
tgcttccggc cgtgctatcg cttctgactg 3300cgcagatggt atgaccaaac
tgatcttcga caaagagacc caccgtgtta tcggcggcgc 3360gattgtcggc
accaacggcg gcgagctgct gggtgagatc ggcctggcta tcgagatggg
3420ctgtgacgct gaagacatcg ccctgaccat ccacgctcac ccgactctgc
acgagtccgt 3480tggcctggcg gcggaagtgt tcgaaggcag catcaccgac
ctgccaaacg ccaaagcgaa 3540gaaaaagtaa ctttttcttt caggaaaaaa
gcataagcgg ctccgggagc cgcttttttt 3600atgcctgatg tttagaacta
tgtcactgtt cataaaccgc tacacctcat acatacttta 3660agggcgaatt
ctgcagatat ccatcacact ggcggccgct cgagcatgca tctagcacat
3720ccggcaatta aaaaagcggc taaccacgcc gcttttttta cgtctgcaat
ttacctttcc 3780agtcttcttg ctccacgttc agagagacgt tcgcatactg
ctgaccgttg ctcgttattc 3840agcctgacag tatggttact gtcgtttaga
cgttgtgggc ggctctcctg aactttctcc 3900cgaaaaacct gacgttgttc
aggtgatgcc gattgaacac gctggcgggc gttatcacgt 3960tgctgttgat
tcagtgggcg ctgctgtact ttttccttaa acacctggcg ctgctctggt
4020gatgcggact gaatacgctc acgcgctgcg tctcttcgct gctggttctg
cgggttagtc 4080tgcattttct cgcgaaccgc ctggcgctgc tcaggcgagg
cggactgaat gcgctcacgc 4140gctgcctctc ttcgctgctg gatcttcggg
ttagtctgca ttctctcgcg aactgcctgg 4200cgctgctcag gcgaggcgga
ctgataacgc tgacgagcgg cgtccttttg ttgctgggtc 4260agtggttggc
gacggctgaa gtcgtggaag tcgtcatagc tcccatagtg ttcagcttca
4320ttaaaccgct gtgccgctgc ctgacgttgg gtacctcgtg taatgactgg
tgcggcgtgt 4380gttcgttgct gaaactgatt tgctgccgcc tgacgctggc
tgtcgcgcgt tggggcaggt 4440aattgcgtgg cgctcattcc gccgttgaca
tcggtttgat gaaaccgctt tgccatatcc 4500tgatcatgat agggcacacc
attacggtag tttggattgt gccgccatgc catattctta 4560tcagtaagat
gctcaccggt gatacggttg aaattgttga cgtcgatatt gatgttgtcg
4620ccgttgtgtt gccagccatt accgtcacga tgaccgccat cgtggtgatg ataatcat
4678821114DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 82caaaaaaccg gagtctgtgc tccggttttt
tattatccgc taatcaatta catatgaata 60tcctccttag ttcctattcc gaagttccta
ttctctagaa agtataggaa cttcggcgcg 120cctacctgtg acggaagatc
acttcgcaga ataaataaat cctggtgtcc ctgttgatac 180cgggaagccc
tgggccaact tttggcgaaa atgagacgtt gatcggcacg taagaggttc
240caactttcac cataatgaaa taagatcact accgggcgta ttttttgagt
tgtcgagatt 300ttcaggagct aaggaagcta aa atg gag aaa aaa atc act gga
tat acc acc 352 Met Glu Lys Lys Ile Thr Gly Tyr Thr Thr 1 5 10gtt
gat ata tcc caa tgg cat cgt aaa gaa cat ttt gag gca ttt cag 400Val
Asp Ile Ser Gln Trp His Arg Lys Glu His Phe Glu Ala Phe Gln 15 20
25tca gtt gct caa tgt acc tat aac cag acc gtt cag ctg gat att acg
448Ser Val Ala Gln Cys Thr Tyr Asn Gln Thr Val Gln Leu Asp Ile Thr
30 35 40gcc ttt tta aag acc gta aag aaa aat aag cac aag ttt tat ccg
gcc 496Ala Phe Leu Lys Thr Val Lys Lys Asn Lys His Lys Phe Tyr Pro
Ala 45 50 55ttt att cac att ctt gcc cgc ctg atg aat gct cat ccg gaa
tta cgt 544Phe Ile His Ile Leu Ala Arg Leu Met Asn Ala His Pro Glu
Leu Arg 60 65 70atg gca atg aaa gac ggt gag ctg gtg ata tgg gat agt
gtt cac cct 592Met Ala Met Lys Asp Gly Glu Leu Val Ile Trp Asp Ser
Val His Pro75 80 85 90tgt tac acc gtt ttc cat gag caa act gaa acg
ttt tca tcg ctc tgg 640Cys Tyr Thr Val Phe His Glu Gln Thr Glu Thr
Phe Ser Ser Leu Trp 95 100 105agt gaa tac cac gac gat ttc cgg cag
ttt cta cac ata tat tcg caa 688Ser Glu Tyr His Asp Asp Phe Arg Gln
Phe Leu His Ile Tyr Ser Gln 110 115 120gat gtg gcg tgt tac ggt gaa
aac ctg gcc tat ttc cct aaa ggg ttt 736Asp Val Ala Cys Tyr Gly Glu
Asn Leu Ala Tyr Phe Pro Lys Gly Phe 125 130 135att gag aat atg ttt
ttc gtc tca gcc aat ccc tgg gtg agt ttc acc 784Ile Glu Asn Met Phe
Phe Val Ser Ala Asn Pro Trp Val Ser Phe Thr 140 145 150agt ttt gat
tta aac gtg gcc aat atg gac aac ttc ttc gcc ccc gtt 832Ser Phe Asp
Leu Asn Val Ala Asn Met Asp Asn Phe Phe Ala Pro Val155 160 165
170ttc acc atg ggc aaa tat tat acg caa ggc gac aag gtg ctg atg ccg
880Phe Thr Met Gly Lys Tyr Tyr Thr Gln Gly Asp Lys Val Leu Met Pro
175 180 185ctg gcg att cag gtt cat cat gcc gtt tgt gat ggc ttc cat
gtc ggc 928Leu Ala Ile Gln Val His His Ala Val Cys Asp Gly Phe His
Val Gly 190 195 200aga tgc tta atg aat aca aca gta ctg cga
tgagtggcag ggcggggcgt 978Arg Cys Leu Met Asn Thr Thr Val Leu Arg
205 210aaggcgcgcc atttaaatga agttcctatt ccgaagttcc tattctctag
aaagtatagg 1038aacttcgaag cagctccagc ctacaccctt cttcagggct
gactgtttgc ataaaaattc 1098atctgtatgc acaata 111483212PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
83Met Glu Lys Lys Ile Thr Gly Tyr Thr Thr Val Asp Ile Ser Gln Trp1
5 10 15His Arg Lys Glu His Phe Glu Ala Phe Gln Ser Val Ala Gln Cys
Thr 20 25 30Tyr Asn Gln Thr Val Gln Leu Asp Ile Thr Ala Phe Leu Lys
Thr Val 35 40 45Lys Lys Asn Lys His Lys Phe Tyr Pro Ala Phe Ile His
Ile Leu Ala 50 55 60Arg Leu Met Asn Ala His Pro Glu Leu Arg Met Ala
Met Lys Asp Gly65 70 75 80Glu Leu Val Ile Trp Asp Ser Val His Pro
Cys Tyr Thr Val Phe His 85 90 95Glu Gln Thr Glu Thr Phe Ser Ser Leu
Trp Ser Glu Tyr His Asp Asp 100 105 110Phe Arg Gln Phe Leu His Ile
Tyr Ser Gln Asp Val Ala Cys Tyr Gly 115 120 125Glu Asn Leu Ala Tyr
Phe Pro Lys Gly Phe Ile Glu Asn Met Phe Phe 130 135 140Val Ser Ala
Asn Pro Trp Val Ser Phe Thr Ser Phe Asp Leu Asn Val145 150 155
160Ala Asn Met Asp Asn Phe Phe Ala Pro Val Phe Thr Met Gly Lys Tyr
165 170 175Tyr Thr Gln Gly Asp Lys Val Leu Met Pro Leu Ala Ile Gln
Val His 180 185 190His Ala Val Cys Asp Gly Phe His Val Gly Arg Cys
Leu Met Asn Thr 195 200 205Thr Val Leu Arg 210842521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
84ttatttggtg atattggtac caatatcatg cagcaaacgg tgcaacattg ccgtgtctcg
60ttgctctaaa agccccaggc gttgttgtaa ccagtcgacc agttttatgt catctgccac
120tgccagagtc gtcagcaatg tcatggctcg ttcgcgtaaa gcttgcagtt
gatgttggtc 180tgccgttgca tcacttttcg ccggttgttg tattaatgtt
gctaattgat agcaatagac 240catcaccgcc tgccccagat tgagcgaagg
ataatccgcc accatcggca caccagtaag 300aacgtcagcc aacgctaact
cttcgttagt caacccggaa tcttcgcgac caaacaccag 360cgcggcatgg
ctcatccatg aagatttttc ctctaacagc ggcaccagtt caactggcgt
420ggcgtagtaa tgatatttcg cccgactgcg cgcagtggtg gcgacagtga
aatcgacatc 480gtgtaacgat tcagccaatg tcgggaaaac tttaatatta
tcaataatat caccagatcc 540atgtgcgacc cagcgggtgg ctggctccag
gtgtgcctga ctatcgacaa tccgcagatc 600gctaaacccc atcgttttca
ttgcccgcgc cgctgcccca atattttctg ctctggcggg 660tgcgaccaga
ataatcgtta tacgcatatt gccactcttc ttgatcaaat aaccgcgaac
720cgggtgatca ctgtcaactt attacgcggt gcgaatttac aaattcttaa
cgtaagtcgc 780agaaaaagcc ctttacttag cttaaaaaag gctaaactat
ttcctgactg tactaacggt 840tgagttgtta aaaaatgcta catatccttc
tgtttactta ggataatttt ataaaaaata 900aatctcgaca attggattca
ccacgtttat tagttgtatg atgcaactag ttggattatt 960aaaataatgt
gacgaaagct agcatttaga tacgatgatt tcatcaaact gttaacgtgc
1020tacaattgaa cttgatatat gtcaacgaag cgtagtttta ttgggtgtcc
ggcccctctt 1080agcctgttat gttgctgtta aaatggttag gatgacagcc
gtttttgaca ctgtcgggtc 1140ctgagggaaa gtacccacga ccaagctaat
gatgttgttg acgttgatgg aaagtgcatc 1200aagaacgcaa ttacgtactt
tagtcatgtt acgccgatca tgttaatttg cagcatgcat 1260caggcaggtc
agggactttt gtacttcctg tttcgattta gttggcaatt taggtagcaa
1320acgaattcat cggctttacc accgtcaaaa aaaacggcgc tttttagcgc
cgtttttatt 1380tttcaacctt atttccagat acgtaactca tcgtccgttg
taacttcttt actggctttc 1440attttcggca gtgaaaacgc ataccagtcg
atattacggg tcacaaacat catgccggcc 1500agcgccacca ccagcacact
ggttcccaac aacagcgcgc tatcggcaga gttgagcagt 1560ccccacatca
caccatccag caacaacagc gcgagggtaa acaacatgct gttgcaccaa
1620cctttcaata ccgcttgcaa ataaataccg ttcattatcg ccccaatcag
actggcgatt 1680atccatgcca cggtaaaacc ggtatgttca gaaagcgcca
gcaagagcaa ataaaacatc 1740accaatgaaa gccccaccag caaatattgc
attgggtgta aacgttgcgc ggtgagcgtt 1800tcaaaaacaa agaacgccat
aaaagtcagt gcaatcagca gaatggcgta cttagtcgcc 1860cggtcagtta
attggtattg atcggctggc gtcgttactg cgacgctaaa cgccgggaag
1920ttttcccagc cggtatcatt gcctgaagca aaacgctcac cgagattatt
agcaaaccag 1980ctgctttgcc agtgcgcctg aaaacctgac tcgctaactt
cccgtttggc tggtagaaaa 2040tcacctaaaa aactgggatg cggccagttg
ctggttaagg tcatttcgct attacgcccg 2100ccaggcacca cagaaagatc
gccggtaccg cttaaattca gggccatatt cagcttcagg 2160ttctgcttcc
gccagtcccc ttcaggtaaa gggatatgca cgccctgccc gccttgctct
2220aacccggtgc cgggttcaat ggtcagcgcc gttccgttaa cttcaggcgc
tttcaccaca 2280ccaataccac gcgcatcccc gacgctaatc acaataaatg
gcttgcctaa ggtgatattt 2340ggcgcgttga gttcgctaag acgcgaaaca
tcgaaatcgg cttttaacgt taaatcactg 2400tgccagacct gaccggtata
aatccctatc ttgcgttctt ccacgttctg attgccatca 2460accatcaatg
actcaggtaa ccaaaaatgg ataaaacttc gtttccgctg cagggtttta 2520t
2521853010DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 85aagccacagc aggatgccca ctgcaacaaa
ggtgatcaca ccggaaacgc gatggagaat 60ggacgctatc gccgtgatgg ggaaccggat
ggtctgtagg tccagattaa caggtctttg 120ttttttcaca tttcttatca
tgaataacgc ccacatgctg ttcttattat tccctgggga 180ctacgggcac
agaggttaac tttctgttac ctggagacgt cgggatttcc ttcctccggt
240ctgcttgcgg gtcagacagc gtcctttcta taactgcgcg tcatgcaaaa
cactgcttcc 300agatgcgaaa acgacacgtt acaacgctgg gtggctcggg
attgcagggt gttccggaga 360cctggcggca gtataggctg ttcacaaaat
cattacaatt aacctacata tagtttgtcg 420ggttttatcc tgaacagtga
tccaggtcac gataacaaca tttatttaat ttttaatcat 480ctaatttgac
aatcattcaa caaagttgtt acaaacatta ccaggaaaag catataatgc
540gtaaaagtta tgaagtcggt atttcaccta agattaactt atgtaacagt
gtggaagtat 600tgaccaattc attcgggaca gttattagtg gtagacaagt
ttaataattc ggattgctaa 660gtacttgatt cgccatttat tcgtcatcaa
tggatccttt acctgcaagc gcccagagct 720ctgtacccag gttttcccct
ctttcacaga gcggcgagcc aaataaaaaa cgggtaaagc 780caggttgatg
tgcgaaggca aatttaagtt ccggcagtct tacgcaataa ggcgctaagg
840agaccttaaa tggctgatac aaaagcaaaa ctcaccctca acggggatac
agctgttgaa 900ctggatgtgc tgaaaggcac gctgggtcaa gatgttattg
atatccgtac tctcggttca 960aaaggtgtgt tcacctttga cccaggcttc
acttcaaccg catcctgcga atctaaaatt 1020acttttattg atggtgatga
aggtattttg ctgcaccgcg gtttcccgat cgatcagctg 1080gcgaccgatt
ctaactacct ggaagtttgt tacatcctgc tgaatggtga aaaaccgact
1140caggaacagt atgacgaatt taaaactacg gtgacccgtc ataccatgat
ccacgagcag 1200attacccgtc tgttccatgc tttccgtcgc gactcgcatc
caatggcagt catgtgtggt 1260attaccggcg cgctggcggc gttctatcac
gactcgctgg atgttaacaa tcctcgtcac 1320cgtgaaattg ccgcgttcct
cctgctgtcg aaaatgccga ccatggccgc gatgtgttac 1380aagtattcca
ttggtcagcc atttgtttac ccgcgcaacg atctctccta cgccggtaac
1440ttcctgaata tgatgttctc cacgccgtgc gaaccgtatg aagttaatcc
gattctggaa 1500cgtgctatgg accgtattct gatcctgcac gctgaccatg
aacagaacgc ctctacctcc 1560accgtgcgta ccgctggctc ttcgggtgcg
aacccgtttg cctgtatcgc agcaggtatt 1620gcttcactgt ggggacctgc
gcacggcggt gctaacgaag cggcgctgaa aatgctggaa 1680gaaatcagct
ccgttaaaca cattccggaa tttgttcgtc gtgcgaaaga caaaaatgat
1740tctttccgcc tgatgggctt cggtcaccgc gtgtacaaaa attacgaccc
gcgcgccacc 1800gtaatgcgtg aaacctgcca tgaagtgctg aaagagctgg
gcacgaagga tgacctgctg 1860gaagtggcta tggagctgga aaacatcgcg
ctgaacgacc cgtactttat cgagaagaaa 1920ctgtacccga acgtcgattt
ctactctggt atcatcctga aagcgatggg tattccgtct 1980tccatgttca
ccgtcatttt cgcaatggca cgtaccgttg gctggatcgc ccactggagc
2040gaaatgcaca gtgacggtat gaagattgcc cgtccgcgtc agctgtatac
aggatatgaa 2100aaacgcgact ttaaaagcga tatcaagcgt taatggttga
ttgctaagtt gtaaatattt 2160taacccgccg ttcatatggc gggttgattt
ttatatgcct aaacacaaaa aattgtaaaa 2220ataaaatcca ttaacagacc
tatatagata tttaaaaaga atagaacagc tcaaattatc 2280agcaacccaa
tactttcaat taaaaacttc atggtagtcg catttataac cctatgaaaa
2340tgacgtctat ctataccccc ctatatttta ttcatcatac aacaaattca
tgataccaat 2400aatttagttt tgcatttaat aaaactaaca atatttttaa
gcaaaactaa aaactagcaa 2460taatcaaata cgatattctg gcgtagctat
acccctattc tatatcctta aaggactctg 2520ttatgtttaa aggacaaaaa
acattggccg cactggccgt atctctgctg ttcactgcac 2580ctgtttatgc
tgctgatgaa ggttctggcg aaattcactt taagggggag gttattgaag
2640caccttgtga aattcatcca gaagatattg ataaaaacat agatcttgga
caagtcacga 2700caacccatat aaaccgggag catcatagca ataaagtggc
cgtcgacatt cgcttgatca 2760actgtgatct gcctgcttct gacaacggta
gcggaatgcc ggtatccaaa gttggcgtaa 2820ccttcgatag cacggctaag
acaactggtg ctacgccttt gttgagcaac accagtgcag 2880gcgaagcaac
tggggtcggt gtacgactga tggacaaaaa tgacggtaac atcgtattag
2940gttcagccgc gccagatctt gacctggatg caagctcatc agaacagacg
ctgaactttt 3000tcgcctggat 3010864180DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
86cgcgatgtcg acgtcacgaa actgaaaaaa ccgctctaca ttctggcgac tgctgatgaa
60gaaaccagta tggccggagc gcgttatttt gccgaaacta ccgccctgcg cccggattgc
120gccatcattg gcgaaccgac gtcactacaa ccggtacgcg cacataaagg
tcatatctct 180aacgccatcc gtattcaggg ccagtcgggg cactccagcg
atccagcacg cggagttaac 240gctatcgaac taatgcacga cgccatcggg
catattttgc aattgcgcga taacctgaaa 300gaacgttatc actacgaagc
gtttaccgtg ccatacccta cgctcaacct cgggcatatt 360cacggtggcg
acgcttctaa ccgtatttgc gcttgctgtg agttgcatat ggatattcgt
420ccgctgcctg gcatgacact caatgaactt aatggtttgc tcaacgatgc
attggctccg 480gtgagcgaac gctggccggg tcgtctgacg gtcgacgagc
tgcatccgcc gatccctggc 540tatgaatgcc caccgaatca tcaactggtt
gaagtggttg agaaattgct cggagcaaaa 600accgaagtgg tgaactactg
taccgaagcg ccgtttattc aaacgttatg cccgacgctg 660gtgttggggc
ctggctcaat taatcaggct catcaacctg atgaatatct ggaaacacgg
720tttatcaagc ccacccgcga actgataacc caggtaattc accatttttg
ctggcattaa 780aacgtaggcc ggataaggcg ctcgcgccgc
atccggcgct gttgccaaac tccagtgccg 840caataatgtc ggatgcgatg
cttgcgcatc ttatccgacc tacagtgact caaacgatgc 900ccaaccgtag
gccggataag gcgctcgcgc cgcatccggc actgttgcca aactccagtg
960ccgcaataat gtcggatgcg atacttgcgc atcttatccg accgacagtg
actcaaacga 1020tgcccaactg taggccggat aaggcgctcg cgccgcatcc
ggcactgttg ccaaactcca 1080gtgccgcaat aatgtcggat gcgatacttg
cgcatcttat ccgacctaca cctttggtgt 1140tacttggggc gattttttaa
catttccata agttacgctt atttaaagcg tcgtgaattt 1200aatgacgtaa
attcctgcta tttattcgtt tgctgaagcg atttcgcagc atttgacgtc
1260accgctttta cgtggcttta taaaagacga cgaaaagcaa agcccgagca
tattcgcgcc 1320aatgctagca agaggagaag tcgacatgac agacttaaat
aaagtggtaa aagaacttga 1380agctcttggt atttatgacg taaaagaagt
tgtttacaat ccaagctacg agcaattgtt 1440cgaagaagaa actaaaccag
gtttagaagg ctttgaaaaa ggtactttaa ctacgactgg 1500tgcagtggca
gtagatacag gtatcttcac aggtcgttct ccaaaagata aatatatcgt
1560gttagatgaa aaaaccaaag atactgtttg gtggacatct gaaacagcaa
aaaacgacaa 1620caagccaatg aaccaagcta catggcaaag cttaaaagac
ttggtaacca accagctttc 1680tcgtaaacgc ttatttgtag ttgatggttt
ctgtggtgcg agcgaacacg accgtattgc 1740agtacgtatt gtcactgaag
tagcgtggca agcacatttt gtaaaaaata tgtttattcg 1800cccaactgaa
gaacaactca aaaattttga accagatttc gttgtaatga atggttctaa
1860agtaaccaat ccaaactgga aagaacaagg tttaaattca gaaaactttg
ttgctttcaa 1920cttgactgaa cgcattcaat taatcggtgg tacttggtac
ggcggtgaaa tgaaaaaagg 1980tatgttctca atcatgaact acttcctacc
acttaaaggt gttggtgcaa tgcactgctc 2040agctaacgtt ggtaaagatg
gcgatgtagc aatcttcttc ggcttatctg gcacaggtaa 2100aacaaccctt
tcaacggatc caaaacgtga attaatcggt gacgatgaac acggctggga
2160tgatgtgggt atctttaact ttgaaggtgg ttgctatgcg aaaaccattc
acctttcaga 2220agaaaatgaa ccagatattt accgcgctat ccgtcgcgac
gcattattag aaaacgtggt 2280tgttcgtgca gatggttctg ttgatttcga
tgatggttca aaaacagaaa atactcgcgt 2340gtcttaccca atttatcaca
ttgataacat tgtaaaacca gtttctcgtg caggtcacgc 2400aactaaagtg
attttcttaa ctgcagatgc atttggcgta ttaccaccag tatctaaatt
2460gacaccagaa caaactaaat actacttctt atctggtttc acagcaaaat
tagcaggtac 2520tgaacgtggt attactgaac caactccaac tttctcagca
tgtttcggtg ctgcgttctt 2580aacccttcac ccaactcaat atgcagaagt
gttagtaaaa cgtatgcaag cagtgggtgc 2640tgaagcttac ttagtaaata
ctggttggaa tggcacaggc aaacgtatct caatcaaaga 2700tactcgcgga
atcattgatg caatcttaga tggctcaatt gaaaaagctg aaatgggcga
2760attaccaatc tttaacttag ccattcctaa agcattacca ggtgtagatt
ctgcaatctt 2820agatcctcgc gatacttacg cagataaagc acaatggcaa
tcaaaagctg aagacttagc 2880aggtcgtttt gtgaaaaact ttgttaaata
tgcaactaac gaagaaggca aagctttaat 2940tgcagctggt cctaaagctt
aatctagaaa gcttcctaga ggcatcaaat aaaacgaaag 3000gctcagtcga
aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
3060agtaggacga attcacttct gttctaacac cctcgttttc aatatatttc
tgtctgcatt 3120ttattcaaat tctgaatata ccttcagata tccttaagga
attgtcgtta cattcggcga 3180tattttttca agacaggttc ttactatgca
ttccacagaa gtccaggcta aacctctttt 3240tagctggaaa gccctgggtt
gggcactgct ctacttttgg tttttctcta ctctgctaca 3300ggccattatt
tacatcagtg gttatagtgg cactaacggc attcgcgact cgctgttatt
3360cagttcgctg tggttgatcc cggtattcct ctttccgaag cggattaaaa
ttattgccgc 3420agtaatcggc gtggtgctat gggcggcctc tctggcggcg
ctgtgctact acgtcatcta 3480cggtcaggag ttctcgcaga gcgttctgtt
tgtgatgttc gaaaccaaca ccaacgaagc 3540cagcgagtat ttaagccagt
atttcagcct gaaaattgtg cttatcgcgc tggcctatac 3600ggcggtggca
gttctgctgt ggacacgcct gcgcccggtc tatattccaa agccgtggcg
3660ttatgttgtc tcttttgccc tgctttatgg cttgattctg catccgatcg
ccatgaatac 3720gtttatcaaa aacaagccgt ttgagaaaac gttggataac
ctggcctcgc gtatggagcc 3780tgccgcaccg tggcaattcc tgaccggcta
ttatcagtat cgtcagcaac taaactcgct 3840aacaaagtta ctgaatgaaa
ataatgcctt gccgccactg gctaatttca aagatgaatc 3900gggtaacgaa
ccgcgcactt tagtgctggt gattggcgag tcgacccagc gcggacgcat
3960gagtctgtac ggttatccgc gtgaaaccac gccggagctg gatgcgctgc
ataaaaccga 4020tccgaatctg accgtgttta ataacgtagt tacgtctcgt
ccgtacacca ttgaaatcct 4080gcaacaggcg ctgacctttg ccaatgaaaa
gaacccggat ctgtatctga cgcagccgtc 4140gctgatgaac atgatgaaac
aggcgggtta taaaaccttc 4180874960DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 87aataggcgta
tcacgaggcc ctttcgtctt cacctcgaga attgtgagcg gataacaatt 60gacattgtga
gcggataaca agatactgag cacatcagca ggacgcactg accgaattca
120attaagctag caagaggaga agtcgagatg aacttacatg aatatcaggc
aaaacaactt 180tttgcccgct atggcttacc agcaccggtg ggttatgcct
gtactactcc gcgcgaagca 240gaagaagccg cttcaaaaat cggtgccggt
ccgtgggtag tgaaatgtca ggttcacgct 300ggtggccgcg gtaaagcggg
cggtgtgaaa gttgtaaaca gcaaagaaga catccgtgct 360tttgcagaaa
actggctggg caagcgtctg gtaacgtatc aaacagatgc caatggccaa
420ccggttaacc agattctggt tgaagcagcg accgatatcg ctaaagagct
gtatctcggt 480gccgttgttg accgtagttc ccgtcgtgtg gtctttatgg
cctccaccga aggcggcgtg 540gaaatcgaaa aagtggcgga agaaactccg
cacctgatcc ataaagttgc gcttgatccg 600ctgactggcc cgatgccgta
tcagggacgc gagctggcgt tcaaactggg tctggaaggt 660aaactggttc
agcagttcac caaaatcttc atgggcctgg cgaccatttt cctggagcgc
720gacctggcgt tgatcgaaat caacccgctg gtcatcacca aacagggcga
tctgatttgc 780ctcgacggca aactgggcgc tgacggcaac gcactgttcc
gccagcctga tctgcgcgaa 840atgcgtgacc agtcgcagga agatccgcgt
gaagcacagg ctgcacagtg ggaactgaac 900tacgttgcgc tggacggtaa
catcggttgt atggttaacg gcgcaggtct ggcgatgggt 960acgatggaca
tcgttaaact gcacggcggc gaaccggcta acttccttga cgttggcggc
1020ggcgcaacca aagaacgtgt aaccgaagcg ttcaaaatca tcctctctga
cgacaaagtg 1080aaagccgttc tggttaacat cttcggcggt atcgttcgtt
gcgacctgat cgctgacggt 1140atcatcggcg cggtagcaga agtgggtgtt
aacgtaccgg tcgtggtacg tctggaaggt 1200aacaacgccg aactcggcgc
gaagaaactg gctgacagcg gcctgaatat tattgcagca 1260aaaggtctga
cggatgcagc tcagcaggtt gttgccgcag tggaggggaa ataatgtcca
1320ttttaatcga taaaaacacc aaggttatct gccagggctt taccggtagc
caggggactt 1380tccactcaga acaggccatt gcatacggca ctaaaatggt
tggcggcgta accccaggta 1440aaggcggcac cacccacctc ggcctgccgg
tgttcaacac cgtgcgtgaa gccgttgctg 1500ccactggcgc taccgcttct
gttatctacg taccagcacc gttctgcaaa gactccattc 1560tggaagccat
cgacgcaggc atcaaactga ttatcaccat cactgaaggc atcccgacgc
1620tggatatgct gaccgtgaaa gtgaagctgg atgaagcagg cgttcgtatg
atcggcccga 1680actgcccagg cgttatcact ccgggtgaat gcaaaatcgg
tatccagcct ggtcacattc 1740acaaaccggg taaagtgggt atcgtttccc
gttccggtac actgacctat gaagcggtta 1800aacagaccac ggattacggt
ttcggtcagt cgacctgtgt cggtatcggc ggtgacccga 1860tcccgggctc
taactttatc gacattctcg aaatgttcga aaaagatccg cagaccgaag
1920cgatcgtgat gatcggtgag atcggcggta gcgctgaaga agaagcagct
gcgtacatca 1980aagagcacgt taccaagcca gttgtgggtt acatcgctgg
tgtgactgcg ccgaaaggca 2040aacgtatggg ccacgcgggt gccatcattg
ccggtgggaa agggactgcg gatgagaaat 2100tcgctgctct ggaagccgca
ggcgtgaaaa ccgttcgcag cctggcggat atcggtgaag 2160cactgaaaac
tgttctgaaa taatctagca agaggagaag tcgacatgga aatcaaagaa
2220atggtgagcc ttgcacgcaa ggctcagaag gagtatcaag ctacccataa
ccaagaagca 2280gttgacaaca tttgccgagc tgcagcaaaa gttatttatg
aaaatgcagc tattctggct 2340cgcgaagcag tagacgaaac cggcatgggc
gtttacgaac acaaagtggc caagaatcaa 2400ggcaaatcca aaggtgtttg
gtacaacctc cacaataaaa aatcgattgg tatcctcaat 2460atagacgagc
gtaccggtat gatcgagatt gcaaagccta tcggagttgt aggagccgta
2520acgccgacga ccaacccgat cgttactccg atgagcaata tcatctttgc
tcttaagacc 2580tgcaatgcca tcattattgc cccccacccc agatccaaaa
aatgctctgc acacgcagtt 2640cgtctgatca aagaagctat cgctccgttc
aacgtaccgg aaggtatggt tcagatcatc 2700gaagaaccca gcatcgagaa
gacgcaggaa ctcatgggcg ccgtagacgt agtagttgct 2760acgggtggta
tgggcatggt gaagtctgca tattcttcag gaaagccttc tttcggtgtt
2820ggagccggta acgttcaggt gatcgtggat agcaacatcg atttcgaagc
tgctgcagaa 2880aaaatcatca ccggtcgtgc tttcgacaac ggtatcatct
gctcaggcga acagagcatc 2940atctacaacg aggctgacaa ggaagcagtt
ttcacagcat tccgcaacca cggtgcatat 3000ttctgtgacg aagccgaagg
agatcgggct cgtgcagcta tcttcgaaaa tggagccatc 3060gcgaaagatg
tagtaggtca gagcgttgcc ttcattgcca agaaagcaaa catcaatatc
3120cccgagggta cccgtattct cgttgttgaa gctcgcggcg taggagcaga
agacgttatc 3180tgtaaggaaa agatgtgtcc cgtaatgtgc gccctcagct
acaagcactt cgaagaaggt 3240gtagaaatcg cacgtacgaa cctcgccaac
gaaggtaacg gccacacctg tgctatccac 3300tccaacaatc aggcacacat
catcctcgca ggatcagagc tgacggtatc tcgtatcgta 3360gtgaatgctc
cgagtgccac tacagcaggc ggtcacatcc aaaacggtct tgccgtaacc
3420aatacgctcg gatgcggatc atggggtaat aactctatct ccgagaactt
cacttacaag 3480cacctcctca acatttcacg catcgcaccg ttgaattcaa
gcattcacat ccccgatgac 3540aaagaaatct gggaactcta atctagcaag
aggagaagtc gacatgcaac ttttcaaact 3600caagagtgta acacatcact
ttgacacttt tgcagaattt gccaaggaat tctgtcttgg 3660agaacgcgac
ttggtaatta ccaacgagtt catctatgaa ccgtatatga aggcatgcca
3720gctcccctgc cattttgtta tgcaggagaa atatgggcaa ggcgagcctt
ctgacgaaat 3780gatgaataac atcttggcag acatccgtaa tatccagttc
gaccgcgtaa tcggtatcgg 3840aggaggtacg gttattgaca tctctaaact
tttcgttctg aaaggattaa atgatgtact 3900cgatgcattc gaccgcaaaa
tacctcttat caaagagaaa gaactgatca ttgtgcccac 3960aacatgcgga
acgggtagcg aggtgacgaa catttctatc gcagaaatca aaagccgtca
4020caccaaaatg ggattggctg acgatgccat tgttgcagac catgccatca
tcatacctga 4080acttctgaag agcttgcctt tccacttcta cgcatgcagt
gcaatcgatg ctcttatcca 4140tgccatcgag tcatacgtat ctcctaaagc
cagtccatat tctcgtctgt tcagtgaggc 4200ggcttgggac attatcctgg
aagtattcaa gaaaatcgcc gaacacggcc ctgaataccg 4260cttcgaaaag
ctgggagaaa tgatcatggc cagcaactat gccggtatag ccttcggaaa
4320tgcaggagta ggagccgtcc acgcactatc ctacccgttg ggaggcaact
atcacgtgcc 4380gcatggagaa gcaaactatc agttcttcac agaggtattc
aaagtatacc aaaagaagaa 4440tcctttcggc tatatagtcg aactcaactg
gaagctctcc aagatactga actgccagcc 4500cgaatacgta tatccgaagc
tggatgaact tctcggatgc cttcttacca agaaaccttt 4560gcacgaatac
ggcatgaagg acgaagaggt aagaggcttt gcggaatcag tgcttaagac
4620acagcaaaga ttgctcgcca acaactacgt agagcttact gtagatgaga
tcgaaggtat 4680ctacagaaga ctctactaat ctagaaagct tcctagaggc
atcaaataaa acgaaaggct 4740cagtcgaaag actgggcctt tcgttttatc
tgttgtttgt cggtgaacgc tctcctgagt 4800aggacaaatc cgccgcccta
gacctaggcg ttcggctgcg acacgtcttg agcgattgtg 4860taggctggag
ctgcttcgaa gttcctatac tttctagaga ataggaactt cggaatagga
4920actaaggagg atattcatat ggaccatggc taattcccat
4960885083DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 88tcgagaaatt tatcaaaaag agtgttgact
tgtgagcgga taacaatgat acttagattc 60aattgtgagc ggataacaat ttcacacaga
attcaattaa gctagcaaga ggagaagtcg 120acatggccaa cataagttca
ccattcgggc aaaacgaatg gctggttgaa gagatgtacc 180gcaagttccg
cgacgacccc tcctcggtcg atcccagctg gcacgagttc ctggttgact
240acagccccga acccacctcc caaccagctg ccgaaccaac ccgggttacc
tcgccactcg 300ttgccgagcg ggccgctgcg gccgccccgc aggcaccccc
caagccggcc gacaccgcgg 360ccgcgggcaa cggcgtggtc gccgcactgg
ccgccaaaac tgccgttccc ccgccagccg 420aaggtgacga ggtagcggtg
ctgcgcggcg ccgccgcggc cgtcgtcaag aacatgtccg 480cgtcgttgga
ggtgccgacg gcgaccagcg tccgggcggt cccggccaag ctactgatcg
540acaaccggat cgtcatcaac aaccagttga agcggacccg cggcggcaag
atctcgttca 600cgcatttgct gggctacgcc ctggtgcagg cggtgaagaa
attcccgaac atgaaccggc 660actacaccga agtcgacggc aagcccaccg
cggtcacgcc ggcgcacacc aatctcggcc 720tggcgatcga cctgcaaggc
aaggacggga agcgttccct ggtggtggcc ggcatcaagc 780ggtgcgagac
catgcgattc gcgcagttcg tcacggccta cgaagacatc gtacgccggg
840cccgcgacgg caagctgacc actgaagact ttgccggcgt gacgatttcg
ctgaccaatc 900ccggaaccat cggcaccgtg cattcggtgc cgcggctgat
gcccggccag ggcgccatca 960tcggcgtggg cgccatggaa taccccgccg
agtttcaagg cgccagcgag gaacgcatcg 1020ccgagctggg catcggcaaa
ttgatcactt tgacctccac ctacgaccac cgcatcatcc 1080agggcgcgga
atcgggcgac ttcctgcgca ccatccacga gttgctgctc tcggatggct
1140tctgggacga ggtcttccgc gaactgagca tcccatatct gccggtgcgc
tggagcaccg 1200acaaccccga ctcgatcgtc gacaagaacg ctcgcgtcat
gaacttgatc gcggcctacc 1260gcaaccgcgg ccatctgatg gccgataccg
acccgctgcg gttggacaaa gctcggttcc 1320gcagtcaccc cgacctcgaa
gtgctgaccc acggcctgac gctgtgggat ctcgatcggg 1380tgttcaaggt
cgacggcttt gccggtgcgc agtacaagaa actgcgcgac gtgctgggct
1440tgctgcgcga tgcctactgc cgccacatcg gcgtggagta cgcccatatc
ctcgaccccg 1500aacaaaagga gtggctcgaa caacgggtcg agaccaagca
cgtcaaaccc actgtggccc 1560aacagaaata catcctcagc aagctcaacg
ccgccgaggc ctttgaaacg ttcctacaga 1620ccaagtacgt cggccagaag
cggttctcgc tggaaggcgc cgaaagcgtg atcccgatga 1680tggacgcggc
gatcgaccag tgcgctgagc acggcctcga cgaggtggtc atcgggatgc
1740cgcaccgggg ccggctcaac gtgctggcca acatcgtcgg caagccgtac
tcgcagatct 1800tcaccgagtt cgagggcaac ctgaatccgt cgcaggcgca
cggctccggt gacgtcaagt 1860accacctggg cgccaccggg ctgtacctgc
agatgttcgg cgacaacgac attcaggtgt 1920cgctgaccgc caacccgtcg
catctggagg ccgtcgaccc ggtgctggag ggattggtgc 1980gggccaagca
ggatctgctc gaccacggaa gcatcgacag cgacggccaa cgggcgttct
2040cggtggtgcc gctgatgttg catggcgatg ccgcgttcgc cggtcagggt
gtggtcgccg 2100agacgctgaa cctggcgaat ctgccgggct accgcgtcgg
cggcaccatc cacatcatcg 2160tcaacaacca gatcggcttc accaccgcgc
ccgagtattc caggtccagc gagtactgca 2220ccgacgtcgc aaagatgatc
ggggcaccga tctttcacgt caacggcgac gacccggagg 2280cgtgtgtctg
ggtggcgcgg ttggcggtgg acttccgaca acggttcaag aaggacgtcg
2340tcatcgacat gctgtgctac cgccgccgcg ggcacaacga gggtgacgac
ccgtcgatga 2400ccaaccccta catgtacgac gtcgtcgaca ccaagcgcgg
ggcccgcaaa agctacaccg 2460aagccctgat cggacgtggc gacatctcga
tgaaggaggc cgaggacgcg ctgcgcgact 2520accagggcca gctggaacgg
gtgttcaacg aagtgcgcga gctggagaag cacggtgtgc 2580agccgagcga
gtcggtcgag tccgaccaga tgattcccgc ggggctggcc actgcggtgg
2640acaagtcgct gctggcccgg atcggcgatg cgttcctcgc cttgccgaac
ggcttcaccg 2700cgcacccgcg agtccaaccg gtgctggaga agcgccggga
gatggcctat gaaggcaaga 2760tcgactgggc ctttggcgag ctgctggcgc
tgggctcgct ggtggccgaa ggcaagctgg 2820tgcgcttgtc ggggcaggac
agccgccgcg gcaccttctc ccagcggcat tcggttctca 2880tcgaccgcca
cactggcgag gagttcacac cactgcagct gctggcgacc aactccgacg
2940gcagcccgac cggcggaaag ttcctggtct acgactcgcc actgtcggag
tacgccgccg 3000tcggcttcga gtacggctac actgtgggca atccggacgc
cgtggtgctc tgggaggcgc 3060agttcggcga cttcgtcaac ggcgcacagt
cgatcatcga cgagttcatc agctccggtg 3120aggccaagtg gggccaattg
tccaacgtcg tgctgctgtt accgcacggg cacgaggggc 3180agggacccga
ccacacttct gcccggatcg aacgcttctt gcagttgtgg gcggaaggtt
3240cgatgaccat cgcgatgccg tcgactccgt cgaactactt ccacctgcta
cgccggcatg 3300ccctggacgg catccaacgc ccgctgatcg tgttcacgcc
caagtcgatg ttgcgtcaca 3360aggccgccgt cagcgaaatc aaggacttca
ccgagatcaa gttccgctca gtgctggagg 3420aacccaccta tgaggacggc
atcggagacc gcaacaaggt cagccggatc ctgctgacca 3480gtggcaagct
gtattacgag ctggccgccc gcaaggccaa ggacaaccgc aatgacctcg
3540cgatcgtgcg gcttgaacag ctcgccccgc tgcccaggcg tcgactgcgt
gaaacgctgg 3600accgctacga gaacgtcaag gagttcttct gggtccaaga
ggaaccggcc aaccagggtg 3660cgtggccgcg attcgggctc gaactacccg
agctgctgcc tgacaagttg gccgggatca 3720agcgaatctc gcgccgggcg
atgtcagccc cgtcgtcagg ctcgtcgaag gtgcacgccg 3780tcgaacagca
ggagatcctc gacgaggcgt tcggctaatc tagcaagagg agaagtcgac
3840atgaagttat taaaattggc acctgatgtt tataaatttg atactgcaga
ggagtttatg 3900aaatacttta aggttggaaa aggtgacttt atacttacta
atgaattttt atataaacct 3960ttccttgaga aattcaatga tggtgcagat
gctgtatttc aggagaaata tggactcggt 4020gaaccttctg atgaaatgat
aaacaatata attaaggata ttggagataa acaatataat 4080agaattattg
ctgtaggggg aggatctgta atagatatag ccaaaatcct cagtcttaag
4140tatactgatg attcattgga tttgtttgag ggaaaagtac ctcttgtaaa
aaacaaagaa 4200ttaattatag ttccaactac atgtggaaca ggttcagaag
ttacaaatgt atcagttgca 4260gaattaaaga gaagacatac taaaaaagga
attgcttcag acgaattata tgcaacttat 4320gcagtacttg taccagaatt
tataaaagga cttccatata agttttttgt aaccagctcc 4380gtagatgcct
taatacatgc aacagaagct tatgtatctc caaatgcaaa tccttatact
4440gatatgttta gtgtaaaagc tatggagtta attttaaatg gatacatgca
aatggtagag 4500aaaggaaatg attacagagt tgaaataatt gaggattttg
ttataggcag caattatgca 4560ggtatagctt ttggaaatgc aggagtggga
gcggttcacg cactctcata tccaataggc 4620ggaaattatc atgtgcctca
tggagaagca aattatctgt tttttacaga aatatttaaa 4680acttattatg
agaaaaatcc aaatggcaag attaaagatg taaataaact attagcaggc
4740atactaaaat gtgatgaaag tgaagcttat gacagtttat cacaactttt
agataaatta 4800ttgtcaagaa aaccattaag agaatatgga atgaaagagg
aagaaattga aacttttgct 4860gattcagtaa tagaaggaca gcagagactg
ttggtaaaca attatgaacc tttttcaaga 4920gaagacatag taaacacata
taaaaagtta tattaatcta gaaagcttcc tagaggcatc 4980aaataaaacg
aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg
5040tgaacgctct cctgagtagg acaaatccgc cgccctagac cta
5083895097DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 89tctgtatcag gctgaaaatc ttctctcatc
cgccaaaaca gcttcggcgt taagatgcgc 60gctcaaggac gtaagccgtc gactctcgcc
gtgctggcgc aggacacggc taccactcct 120ttctctgttg atattctgct
tgccattgag caaaccgcca gcgagttcgg ctggaatagt 180tttttaatca
atattttttc tgaagatgac gctgcccgcg cggcacgtca gctgcttgcc
240caccgtccgg atggcattat ctatactaca atggggctgc gacatatcac
gctgcctgag 300tctctgtatg gtgaaaatat tgtattggcg aactgtgtgg
cggatgaccc agcgttaccc 360agttatatcc ctgatgatta cactgcacaa
tatgaatcaa cacagcattt gctcgcggcg 420ggctatcgtc aaccgttatg
cttctggcta ccggaaagtg cgttggcaac agggtatcgt 480cggcagggat
ttgagcaggc ctggcgtgat gctggacgag atctggctga ggtgaaacaa
540tttcacatgg caacaggtga tgatcactac accgatctcg caagtttact
caatgcccac 600ttcaaaccgg gcaaaccaga ttttgatgtt ctgatatgtg
gtaacgatcg cgcagccttt 660gtggcttatc aggttcttct ggcgaagggg
gtacgaatcc cgcaggatgt cgccgtaatg 720ggctttgata atctggttgg
cgtcgggcat ctgtttttac cgccgctgac cacaattcag 780cttccacatg
acattatcgg gcgggaagct gcattgcata ttattgaagg tcgtgaaggg
840ggaagagtga cgcggatccc ttgcccgctg ttgatccgtt gttccacctg
atattatgtt 900aacccagtag ccagagtgct ccatgttgca gcacagccac
tccgtgggag gcataaagcg 960acagttcccg ttcttctggc tgcggataga
ttcgactact catcaccgct tccccgtcgt 1020taataaatac ttccacggat
gatgtatcga taaatatcct tagggcgagc gtgtcacgct 1080gcgggagggg
aatactacgg tagccgtcta aattctcgtg tgggtaatac cgccacaaaa
1140caagtcgctc agattggtta tcaatataca gccgcattcc agtgccgagc
tgtaatccgt 1200aatgttcggc atcactgttc ttcagcgccc actgcaactg
aatctcaact gcttgcgcgt 1260tttcctgcaa aacatattta ttgctgattg
tgcggggaga gacagattga tgctgctggc
1320gtaacgactc agcttcgtgt accgggcgtt gtagaagttt gccattgctc
tctgatagct 1380cgcgcgccag cgtcatgcag cctgcccatc cttcacgttt
tgagggcatt ggcgattccc 1440acatatccat ccagccgata acaatacgcc
gaccatcctt cgctaaaaag ctttgtggtg 1500cataaaagtc atgcccgtta
tcaagttcag taaaatgccc ggattgtgca aaaagtcgtc 1560ctggcgacca
cattccgggt attacgccac tttgaaagcg atttcggtaa ctgtatccct
1620cggcattcat tccctgcggg gaaaacatca gataatgctg atcgccaagg
ctgaaaaagt 1680ccggacattc ccacatatag ctttcacccg catcagcgtg
ggccagtacg cgatcgaagg 1740tccattcacg caacgaactg ccgcgataaa
gcaggatctg ccccgtgttg cctggatctt 1800tcgccccgac taccatccac
catgtgtcgg cttcacgcca cactttagga tcgcggaagt 1860gcatgattcc
ttctggtgga gtgaggatca caccctgttt ctcgaaatga ataccatccc
1920gactggtagc cagacattgt acttcgcgaa ttgcatcgtc attacctgca
ccatcgagcc 1980agacgtgtcc ggtgtagata agtgagagga caccattgtc
atcgacagca ctacctgaaa 2040aacacccgtc tttgtcatta tcgtctcctg
gcgctagcgc aataggctca tgctgccagt 2100ggatcatatc gtcgctggtg
gcatgtcccc agtgcattgg cccccagtgt tcgctcatcg 2160gatgatgttg
ataaaacgcg tgataacgat cgttaaacca gatcaggccg tttggatcgt
2220tcatccaccc ggcaggaggc gcgaggtgaa aatggggata gaaagtgtta
ccccggtgct 2280catgaagttt tgctagggcg ttttgcgccg catgcaatcg
agattgcgtc attttaatca 2340tcctggttaa gcaaatttgg tgaattgtta
acgttaactt ttataaaaat aaagtccctt 2400actttcataa atgcgatgaa
tatcacaaat gttaacgtta actatgacgt tttgtgatcg 2460aatatgcatg
ttttagtaaa tccatgacga ttttgcgaaa aagaggttta tcactatgcg
2520taactcagat gaatttaagg gaaaaaaatg tcagccaaag tatgggtttt
aggggatgcg 2580gtcgtagatc tcttgccaga atcagacggg cgcctactgc
cttgtcctgg cggcgcgcca 2640gctaacgttg cggtgggaat cgccagatta
ggcggaacaa gtgggtttat aggtcgggtg 2700ggggatgatc cttttggtgc
gttaatgcaa agaacgctgc taactgaggg agtcgatatc 2760acgtatctga
agcaagatga atggcaccgg acatccacgg tgcttgtcga tctgaacgat
2820caaggggaac gttcatttac gtttatggtc cgccccagtg ccgatctttt
tttagagacg 2880acagacttgc cctgctggcg acatggcgaa tggttacatc
tctgttcaat tgcgttgtct 2940gccgagcctt cgcgtaccag cgcatttact
gcgatgacgg cgatccggca tgccggaggt 3000tttgtcagct tcgatcctaa
tattcgtgaa gatctatggc aagacgagca tttgctccgc 3060ttgtgtttgc
ggcaggcgct acaactggcg gatgtcgtca agctctcgga agaagaatgg
3120cgacttatca gtggaaaaac acagaacgat caggatatat gcgccctggc
aaaagagtat 3180gagatcgcca tgctgttggt gactaaaggt gcagaagggg
tggtggtctg ttatcgagga 3240caagttcacc attttgctgg aatgtctgtg
aattgtgtcg atagcacggg ggcgggagat 3300gcgttcgttg ccgggttact
cacaggtctg tcctctacgg gattatctac agatgagaga 3360gaaatgcgac
gaattatcga tctcgctcaa cgttgcggag cgcttgcagt aacggcgaaa
3420ggggcaatga cagcgctgcc atgtcgacaa gaactggaat agtgagaagt
aaacggcgaa 3480gtcgctctta tctctaaata ggacgtgaat tttttaacga
caggcaggta attatggcac 3540tgaatattcc attcagaaat gcgtactatc
gttttgcatc cagttactca tttctctttt 3600ttatttcctg gtcgctgtgg
tggtcgttat acgctatttg gctgaaagga catctagggt 3660tgacagggac
ggaattaggt acactttatt cggtcaacca gtttaccagc attctattta
3720tgatgttcta cggcatcgtt caggataaac tcggtctgaa gaaaccgctc
atctggtgta 3780tgagtttcat cctggtcttg accggaccgt ttatgattta
cgtttatgaa ccgttactgc 3840aaagcaattt ttctgtaggt ctaattctgg
gggcgctatt ttttggcttg gggtatctgg 3900cgggatgcgg tttgcttgat
agcttcaccg aaaaaatggc gcgaaatttt catttcgaat 3960atggaacagc
gcgcgcctgg ggatcttttg gctatgctat tggcgcgttc tttgccggca
4020tattttttag tatcagtccc catatcaact tctggttggt ctcgctattt
ggcgctgtat 4080ttatgatgat caacatgcgt tttaaagata aggatcacca
gtgcgtagcg gcagatgcgg 4140gaggggtaaa aaaagaggat tttatcgcag
ttttcaagga tcgaaacttc tgggttttcg 4200tcatatttat tgtggggacg
tggtctttct ataacatttt tgatcaacaa ctttttcctg 4260tcttttattc
aggtttattc gaatcacacg atgtaggaac gcgcctgtat ggttatctca
4320actcattcca ggtggtactc gaagcgctgt gcatggcgat tattcctttc
tttgtgaatc 4380gggtagggcc aaaaaatgca ttacttatcg gagttgtgat
tatggcgttg cgtatccttt 4440cctgcgcgct gttcgttaac ccctggatta
tttcattagt gaagttgtta catgccattg 4500aggttccact ttgtgtcata
tccgtcttca aatacagcgt ggcaaacttt gataagcgcc 4560tgtcgtcgac
gatctttctg attggttttc aaattgccag ttcgcttggg attgtgctgc
4620tttcaacgcc gactgggata ctctttgacc acgcaggcta ccagacagtt
ttcttcgcaa 4680tttcgggtat tgtctgcctg atgttgctat ttggcatttt
cttcttgagt aaaaaacgcg 4740agcaaatagt tatggaaacg cctgtacctt
cagcaatata gacgtaaact ttttccggtt 4800gttgtcgata gctctatatc
cctcaaccgg aaaataataa tagtaaaatg cttagccctg 4860ctaataatcg
cctaatccaa acgcctcatt catgttctgg tacagtcgct caaatgtact
4920tcagatgcgc ggttcgctga tttccaggac attgtcgtca ttcagtgacc
tgtcccgtgt 4980atcacggtcc tgcgaattca tcaaggaatg cattgcggag
tgaagtatcg agtcacgcca 5040tatttcgtca cccgaagatg agttttgaga
tattaaggca ggtgactttc actcaca 5097903525DNANocardia iowensis
90atggcagtgg 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
3525911174PRTNocardia iowensis 91Met 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
1170Leu92669DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 92atgattgaaa 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 66993222PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 93Met 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
220943522DNAMycobacterium smegmatis 94atgaccagcg 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
3522951173PRTMycobacterium smegmatis 95Met 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
1170963522DNAMycobacterium avium 96atgtcgactg 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 3522971173PRTMycobacterium avium 97Met 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 1170983525DNAMycobacterium marinum
98atgtcgccaa 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
3525991174PRTMycobacterium marinum 99Met 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 1170Leu1003522DNAArtificial
SequenceDescription of Artificial Sequence Synthetic carboxylic
acid reductase polynucleotide designated 891GA 100atgagcaccg
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
35221011173PRTArtificial SequenceDescription of Artificial Sequence
Synthetic carboxylic acid reductase polypeptide designated 891GA
101Met 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