U.S. patent application number 12/303161 was filed with the patent office on 2010-03-18 for microbiological production of 3-hydroxyisobutyric acid.
This patent application is currently assigned to Evonik Roehm GmbH. Invention is credited to Birgit Alber, Stefen Buchholz, Lothar Eggeling, Georg Fuchs, Achim Marx, Alexander May, Markus Poetter, Hermann Siegert.
Application Number | 20100068773 12/303161 |
Document ID | / |
Family ID | 38650473 |
Filed Date | 2010-03-18 |
United States Patent
Application |
20100068773 |
Kind Code |
A1 |
Marx; Achim ; et
al. |
March 18, 2010 |
MICROBIOLOGICAL PRODUCTION OF 3-HYDROXYISOBUTYRIC ACID
Abstract
The present invention relates to a cell which has been modified
in comparison with its wild type in such a way that it is capable
of forming more, by comparison with its wild, 3-hydroxyisobutyric
acid or poly-hydroxyalkanoates based on 3-hydroxyisobutyric acid
via methylmalonate-semialdehyde or 3-hydroxybutyryl-coenzyme A as
precursors. The invention also relates to a method of generating a
genetically modified cell, to the genetically modified cell
obtainable by these methods, to a method of producing
3-hydroxyisobutyric acid or polyhydroxyalkanoates based on
3-hydroxyisobutyric acid, to a method of producing methacrylic acid
or methacrylic esters, and to a method of producing polymethacrylic
acid or polymethacrylic esters. The present invention furthermore
relates to an isolated DNA, to a vector, to the use of this vector
for transforming a cell, to a transformed cell, and to a
polypeptide.
Inventors: |
Marx; Achim; (Gelnhausen,
DE) ; Poetter; Markus; (Muenster, DE) ;
Buchholz; Stefen; (Hanau, DE) ; May; Alexander;
(Darmstadt, DE) ; Siegert; Hermann;
(Seeheim-Jugenheim, DE) ; Fuchs; Georg;
(Heitersheim, DE) ; Alber; Birgit; (Stuttgart,
DE) ; Eggeling; Lothar; (Juelich, DE) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND MAIER & NEUSTADT, L.L.P.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
Evonik Roehm GmbH
Darmstadt
DE
|
Family ID: |
38650473 |
Appl. No.: |
12/303161 |
Filed: |
June 1, 2007 |
PCT Filed: |
June 1, 2007 |
PCT NO: |
PCT/EP2007/055394 |
371 Date: |
April 6, 2009 |
Current U.S.
Class: |
435/135 ;
435/146; 435/252.32; 435/320.1; 530/350; 536/23.1 |
Current CPC
Class: |
C12N 9/001 20130101;
C12P 7/52 20130101; C12N 9/1096 20130101; C12N 9/16 20130101; C12P
7/625 20130101; C12Y 103/99012 20130101; C12Y 102/04004 20130101;
C12N 9/0008 20130101; C08F 120/10 20130101; C12Y 402/01017
20130101; C12P 7/40 20130101; C12P 7/42 20130101; C12Y 206/01042
20130101; C12N 9/88 20130101; C12Y 301/02004 20130101; C08F 120/06
20130101 |
Class at
Publication: |
435/135 ;
435/252.32; 435/146; 536/23.1; 435/320.1; 530/350 |
International
Class: |
C12P 7/62 20060101
C12P007/62; C12N 1/21 20060101 C12N001/21; C12P 7/42 20060101
C12P007/42; C07H 21/04 20060101 C07H021/04; C12N 15/74 20060101
C12N015/74; C07K 14/00 20060101 C07K014/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 2, 2006 |
DE |
102006025821.5 |
Claims
1. A cell which has been genetically modified in comparison with
its wild type in such a way that it is capable of forming more
3-hydroxyisobutyric acid or polyhydroxyalkanoates based on
3-hydroxyisobutyric acid in comparison with its wild type.
2. The cell as claimed in claim 1, where the formation of
3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on
3-hydroxyisobutyric acid takes place via methylmalonate
semialdehyde as precursor.
3. The cell as claimed in claim 2, where the cell is capable of
forming 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on
3-hydroxyisobutyric acid via succinyl-coenzyme A as
intermediate.
4. The cell as claimed in claim 3, where the cell features an
activity of an enzyme E.sub.1, which catalyzes the conversion of
succinyl-coenzyme A into methylmalonyl-coenzyme A, which is
increased in comparison with its wild type.
5. The cell as claimed in claim 4, where the enzyme E.sub.1 is a
methylmalonyl-coenzyme A mutase (EC 5.4.99.2).
6. The cell as claimed in claim 3, where the cell features an
activity of at least one of the following enzymes E.sub.2 to
E.sub.4 which is increased in comparison with its wild type: of an
enzyme E.sub.2, which catalyzes the conversion of
methylmalonyl-coenzyme A into methyl malonate; of an enzyme
E.sub.3, which catalyzes the conversion of methyl malonate into
methylmalonate semialdehyde; of an enzyme E.sub.4 which catalyzes
the conversion of methylmalonate semialdehyde into
3-hydroxyisobutyrate.
7. The cell as claimed in claim 6, where the enzyme E.sub.2 is a
methylmalonyl-coenzyme A hydrolase (EC 3.1.2.17), E.sub.3 is an
aldehyde dehydrogenase (EC 1.2.1.3) or an aldehyde oxidase (EC
1.2.3.1) and E.sub.4 is a 3-hydroxyisobutyrate dehydrogenase (EC
1.1.1.31) or a 3-hydroxyacyl-coenzyme A dehydrogenase (EC
1.1.1.35).
8. The cell as claimed in claim 3, where the cell features an
activity of at least one of the following enzymes E.sub.4, E.sub.5,
E.sub.4 and E.sub.7 which is increased in comparison with its wild
type: of an enzyme E.sub.6, which catalyzes the conversion of (R)
methylmalonyl-coenzyme A into (S) methylmalonyl-coenzyme A; of an
enzyme E.sub.7, which catalyzes the conversion of (S)
methylmalonyl-coenzyme A into propionyl-coenzyme A; of an enzyme
E.sub.5, which catalyzes the conversion of propionyl-coenzyme A
into methylmalonate semialdehyde; of an enzyme E.sub.4, which
catalyzes the conversion of methylmalonate semialdehyde into
3-hydroxyisobutyric acid.
9. The cell as claimed in claim 8, where the enzyme E.sub.6 is a
methylmalonyl-coenzyme A epimerase (EC 5.1.99.1) E.sub.7 is a
methylmalonyl-coenzyme A decarboxylase (EC 4.1.1.41), E.sub.5 is a
methylmalonate-semialdehyde dehydrogenase (EC 1.2.1.27), and
E.sub.4 is a 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) or a
3-hydroxyacyl-coenzyme A dehydrogenase (EC 1.1.1.35).
10. The cell as claimed in claim 3, where the cell features an
activity of at least one of the following enzymes E.sub.4, E.sub.5
and E.sub.7 which is increased in comparison with its wild type: of
an enzyme E.sub.7, which catalyzes the conversion of
methylmalonyl-coenzyme A into propionyl-coenzyme A; of an enzyme
E.sub.5, which catalyzes the conversion of propionyl-coenzyme A
into methylmalonate semialdehyde; of an enzyme E.sub.4, which
catalyzes the conversion of methylmalonate semialdehyde into
3-hydroxyisobutyric acid.
11. The cell as claimed in claim 10, where the enzyme E.sub.7 is a
methylmalonyl-coenzyme A decarboxylase (EC 4.1.1.41), E.sub.5 is a
methylmalonate-semialdehyde dehydrogenase (EC 1.2.1.27), and
E.sub.4 is a 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) or a
3-hydroxyacyl-coenzyme A dehydrogenase (EC 1.1.1.35).
12. The cell as claimed in claim 3, where the cell features an
activity of at least one of the following enzymes E.sub.28 and
E.sub.46 which is increased in comparison with its wild type: of an
enzyme E.sub.46, which catalyzes the conversion of L-glutamate into
2-oxoglutarate; of an enzyme E.sub.28, which catalyzes the
conversion of 2-octoglutarate into succinyl-coenzyme A.
13. The cell as claimed in claim 12, where the enzyme E.sub.46 is a
glutamate synthase (EC 1.4.1.13 or EC 1.4.1.14), a glutamate
dehydrogenase (EC 1.4.1.2, EC 1.4.1.3 or EC 1.4.1.4) or an
aspartate transaminase (EC 2.6.1.1 or EC 2.6.1.2) and E.sub.28 is a
2-oxoglutarate synthase (EC 1.2.7.3).
14. The cell as claimed in claim 2, where the cell is capable of
forming 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on
3-hydroxyisobutyric acid via propionyl-coenzyme A as
intermediate.
15. The cell as claimed in claim 14, where the cell features an
activity of at least one of the following enzymes E.sub.4, E.sub.5
and E.sub.47 to E.sub.52 which is increased in comparison with its
wild type: of an enzyme E.sub.47, which catalyzes the conversion of
acetyl-coenzyme A into malonyl-coenzyme A; of an enzyme E.sub.48,
which catalyzes the conversion of malonyl-coenzyme A into malonate
semialdehyde; of an enzyme E.sub.49, which catalyzes the conversion
of malonate semialdehyde into 3-hydroxypropionate; of an enzyme
E.sub.50, which catalyzes the conversion of 3-hydroxypropionate
into 3-hydroxypropionyl-coenzyme A; of an enzyme E.sub.51, which
catalyzes the conversion of 3-hydroxypropionyl-coenzyme A into
acryloyl-coenzyme A; of an enzyme E.sub.52, which catalyzes the
conversion of acryloyl-coenzyme A into propionyl-coenzyme A; of an
enzyme E.sub.5, which catalyzes the conversion of
propionyl-coenzyme A into methylmalonate semialdehyde; of an enzyme
E.sub.4, which catalyzes the conversion of methylmalonate
semialdehyde into 3-hydroxyisobutyrate.
16. The cell as claimed in claim 15, where the enzyme E.sub.4 is a
3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) or a
3-hydroxyacyl-coenzyme A dehydrogenase (EC 1.1.1.35), E.sub.5 is a
methylmalonate-semialdehyde dehydrogenase (EC 1.2.1.27), E.sub.47
is a malonyl-coenzyme A decarboxylase (EC 4.1.1.9), a malonate
coenzyme A transferase (EC 2.8.3.3), a methylmalonyl-coenzyme A
carboxytransferase (EC 2.1.3.1) or an acetyl-coenzyme A carboxylase
(EC 6.4.1.2), E.sub.48 is a malonate-semialdehyde dehydrogenase (EC
1.2.1.18), E.sub.49 is a 3-hydroxypropionate dehydrogenase (EC
1.1.1.59), E.sub.50 is a 3-hydroxyisobutyryl-coenzyme A hydrolase
(EC 3.1.2.4), E.sub.51 is an enoyl-coenzyme A hydratase (EC
4.2.1.17) and E.sub.52 is an acyl-coenzyme A dehydrogenase (EC
1.3.99.3).
17. The cell as claimed in claim 2, where the cell is capable of
forming 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on
3-hydroxyisobutyric acid via acryloyl-coenzyme A as
intermediate.
18. The cell as claimed in claim 17, where the cell features an
activity of at least one of the following enzymes E.sub.10 to
E.sub.12, E.sub.56, E.sub.72 and E.sub.73 which is increased in
comparison with its wild type: of an enzyme E.sub.72, which
catalyzes the conversion of beta-alanine into beta-alanyl-coenzyme
A, of an enzyme E.sub.73, which catalyzes the conversion of
beta-alanyl-coenzyme A into acrylyl-coenzyme A, of an enzyme
E.sub.56, which catalyzes the conversion of acrylyl-coenzyme A into
methylmalonyl-coenzyme A, of an enzyme E.sub.10, which catalyzes
the conversion of methylmalonyl-coenzyme A into methyl malonate; of
an enzyme E.sub.11, which catalyzes the conversion of methyl
malonate into methylmalonate semialdehyde; of an enzyme E.sub.12,
which catalyzes the conversion of methylmalonate semialdehyde into
3-hydroxyisobutyric acid.
19. The cell as claimed in claim 18, where the enzyme E.sub.72 is a
coenzyme A transferase (EC 2.8.3.1) or coenzyme A synthetase,
preferably a coenzyme A transferase, E.sub.73 is a
beta-alanyl-coenzyme A ammonia-lyase (EC 4.3.1.6), E.sub.56 is a
crotonyl-coenzyme A decarboxylase E.sub.10 is a
methylmalonyl-coenzyme A hydrolase (EC 3.1.2.17), E.sub.11 is an
aldehyde dehydrogenase (EC 1.2.1.3) or an aldehyde oxidase (EC
1.2.3.1) and E.sub.12 is a 3-hydroxyisobutyrate dehydrogenase (EC
1.1.1.31) or a 3-hydroxyacyl-coenzyme A dehydrogenase (EC
1.1.1.35).
20. The cell as claimed in claim 1, where the formation of
3-hydroxyisobutyric acid or polyhydroxyalkanoates based on
3-hydroxyisobutyric acid takes place via 3-hydroxybutyryl-coenzyme
A as precursor.
21. The cell as claimed in claim 20, where the cell is capable of
forming 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on
3-hydroxyisobutyric acid via isobutyryl coenzyme A as
intermediate.
22. The cell as claimed in claim 21, where the cell features an
activity of at least one of the following enzymes E.sub.76 to
E.sub.79, E.sub.60, E.sub.61 and E.sub.8 which is increased in
comparison with its wild type: of an enzyme E.sub.76, which
catalyzes the conversion of pyruvate into 2-acetolactate; of an
enzyme E.sub.77, which catalyzes the conversion of 2-acetolactate
into 2,3-dihydroxyisovalerate; of an enzyme E.sub.78, which
catalyzes the conversion of 2,3-dihydroxyisolvalerate into
2-oxoisovalerate; of an enzyme E.sub.79, which catalyzes the
conversion of 2-oxoisovalerate into isobutyryl-coenzyme A; of an
enzyme E.sub.60, which catalyzes the conversion of
isobutyryl-coenzyme A into methacrylyl-coenzyme A; of an enzyme
E.sub.61, which catalyzes the conversion of methacrylyl-coenzyme A
into 3-hydroxyisobutyryl-coenzyme A; of an enzyme E.sub.8, which
catalyzes the conversion of 3-hydroxyisobutyryl-coenzyme A into
3-hydroxyisobutyrate.
23. The cell as claimed in claim 22, where the enzyme E.sub.8 is a
3-hydroxyisobutyryl-coenzyme A hydrolase (EC 3.1.2.4), E.sub.76 is
an acetolactate synthase (EC 2.2.1.6), E.sub.77 is a
dihydroxyisovalerate dehydrogenase (EC 1.1.1.86), E.sub.78 is a
2,3-dihydroxyisovalerate dehydratase (EC 4.2.1.9), E.sub.79 is a
2-oxoisovalerate dehydrogenase (EC 1.2.1.25 or EC 1.2.4.4),
E.sub.60 is an acyl-coenzyme A dehydrogenase (EC 1.3.99.3), a
butyryl-coenzyme A dehydrogenase (EC 1.3.99.2) or a
2-methylacyl-coenzyme A dehydrogenase (EC 1.3.99.12), and E.sub.61
is an enoyl-coenzyme A hydratase (EC 4.2.1.17).
24. The cell as claimed in claim 21, where the cell features an
activity of at least one of the following enzymes E.sub.8, E.sub.60
to E.sub.61 and E.sub.79 to E.sub.80 which is increased in
comparison with its wild type: of an enzyme E.sub.80, which
catalyzes the conversion of L-valine into 2-oxoisovalerate; of an
enzyme E.sub.79, which catalyzes the conversion of 2-oxoisovalerate
into isobutyryl-coenzyme A; of an enzyme E.sub.60, which catalyzes
the conversion of isobutyryl-coenzyme A into methacrylyl-coenzyme
A; of an enzyme E.sub.61, which catalyzes the conversion of
methacrylyl-coenzyme A into 3-hydroxyisobutyryl-coenzyme A; of an
enzyme E.sub.8, which catalyzes the conversion of
3-hydroxyisobutyryl-coenzyme A into 3-hydroxyisobutyrate.
25. The cell as claimed in claim 24, where the enzyme E.sub.8 is a
3-hydroxyisobutyryl-coenzyme A hydrolase (EC 3.1.2.4), E.sub.60 is
an enoyl-coenzyme A hydratase (EC 4.2.1.17), E.sub.61 is an
acyl-coenzyme A dehydrogenase (EC 1.3.99.3), a butyryl-coenzyme A
dehydrogenase (EC 1.3.99.2) or a 2-methylacyl-coenzyme A
dehydrogenase (EC 1.3.99.12), E.sub.79 is a 2-oxoisovalerate
dehydrogenase (EC 1.2.1.25 or EC 1.2.4.4), and E.sub.80 is an amino
acid transferase (EC 2.6.1.42).
26. The cell as claimed in claim 20, where the cell is capable of
forming 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on
3-hydroxyisobutyric acid via 3-hydroxybutyryl-coenzyme A as
intermediate.
27. The cell as claimed in claim 26, where the cell an activity of
at least one of the following enzymes E.sub.8, E.sub.53, E.sub.54
and E.sub.82 which is increased in comparison with its wild type:
of an enzyme E.sub.53, which catalyzes the conversion of
acetyl-coenzyme A into acetoacetyl-coenzyme A; of an enzyme
E.sub.54, which catalyzes the conversion of acetoacetyl-coenzyme A
into 3-hydroxybutyryl-coenzyme A; of an enzyme E.sub.81, which
catalyzes the conversion of 3-hydroxybutyryl-coenzyme A into
3-hydroxyisobutyryl-coenzyme A; of an enzyme E.sub.8, which
catalyzes the conversion of 3-hydroxyisobutyryl-coenzyme A into
3-hydroxyisobutyrate.
28. The cell as claimed in claim 27, where the enzyme E.sub.8 is a
3-hydroxyisobutyryl-coenzyme A hydrolase (EC 3.1.2.4) E.sub.53 is a
.beta.-kethothiolase (EC 2.3.1.9), E.sub.54 is an
acetoacetyl-coenzyme A reductase (EC 1.1.1.36), and E.sub.82 is an
isobutyryl-coenzyme mutase (EC 5.4.99.13).
29. A method of preparing a genetically modified cell which is
capable of forming 3-hydroxyisobutyric acid or
polyhydroxyalkanoates based on 3-hydroxyisobutyric acid via
methylmalonate semialdehyde or 3-hydroxybutyryl-coenzyme A, as
precursors, comprising the method step of increasing, in the cell,
the activity of at least one of the enzymes mentioned in claim
2.
30. A cell obtainable by a method as claimed in 29.
31. A method of producing 3-hydroxyisobutyric acid or
polyhydroxyalkanoates based on 3-hydroxyisobutyric acid, comprising
the method step of bringing a cell as claimed in claim 1 into
contact with a nutrient medium comprising, as carbon source,
carbohydrates, glycerol, carbon dioxide, methane, methanol,
L-valine or L-glutamate under conditions under which
3-hydroxyisobutyric acid or polyhydroxyalkanoates based on
3-hydroxyisobutyric acid are formed from the carbon source, and, if
appropriate, purification of the 3-hydroxyisobutyric acid from the
nutrient medium.
32. A method of preparing methacrylic acid or methacrylic esters,
comprising the method steps IA) preparation of 3-hydroxyisobutyric
acid by a method as claimed in claim 31 and, if appropriate,
neutralization of the 3-hydroxyisobutyric acid, IB) dehydration of
the 3-hydroxyisobutyric acid with formation of methacrylic acid
and, if appropriate, esterification methacrylic acid.
33. A method of preparing methacrylic acid or methacrylic esters,
comprising the method steps IIA) preparation of
polyhydroxyalkanoates based on 3-hydroxybutyric acid by a method as
claimed in claim 31, IIB) cleavage of the polyhydroxyalkanoates
based on 3-hydroxyisobutyric acid with formation of
3-hydroxyisobutyric acid and, if appropriate, neutralization of the
3-hydroxyisobutyric acid, IIC) dehydration of the
3-hydroxyisobutyric acid with formation of methacrylic acid and, if
appropriate, esterification of the methacrylic acid.
34. A method of preparing polymethacrylic acid or polymethacrylic
esters, comprising the method steps IIIA) preparation of
methacrylic acid by a method as claimed in claim 32, IIIB)
free-radical polymerization of the methacrylic acid, it being
possible, if appropriate, to esterify at least in part the carboxyl
groups of the methacrylic acid or the carboxylate group of the
methacrylate before or after the free-radical polymerization
reaction.
35. An isolated DNA, which is selected from the following
sequences: a) a sequence as shown in SEQ ID No 03, b) an
intron-free sequence which is derived from a sequence as specified
in a) and which codes for the same protein or peptide as the
sequence as shown in SEQ ID No 03, c) a sequence which codes for a
protein or peptide which comprises the amino acid sequence as shown
in SEQ ID No 04, d) a sequence with at least 80% identity with a
sequence as specified in a) to c), e) a sequence which hybridizes,
or, taking into consideration the degeneration of the genetic code,
would hybridize, with the counter strain of a sequence as specified
in any of groups a) to d), a derivative of a sequence as specified
in any of groups a) to e), obtained by substitution, addition,
inversion and/or deletion of one or more bases, and g) a sequence
which is complementary to a sequence as specified in any of groups
a) to f).
36. A vector, comprising a DNA sequence as specified in any of
groups a) to f), as defined in claim 35.
37. (canceled)
38. A transformed cell, obtainable by transformation with a vector
as claimed in claim 36.
39. An isolated polypeptide which features the amino acid sequence
with the SEQ ID No 04 or an amino acid sequence obtained when no
more than 10 amino acids in SEQ ID No 04 are deleted, inserted,
substituted or else added to the C and/or N terminus of the amino
acid sequence with the SEQ ID No 04.
40. A method of preparing polymethacrylic acid or polymethacrylic
esters, comprising the method steps IIIA) preparation of
methacrylic acid by a method as claimed in claim 33, IIIB)
free-radical polymerization of the methacrylic acid, it being
possible, if appropriate, to esterify at least in part the carboxyl
groups of the methacrylic acid or the carboxylate group of the
methacrylate before or after the free-radical polymerization
reaction.
41. The cell as claimed in claim 22, where the cell features an
activity of at least one of the following enzymes E.sub.8, E.sub.60
to E.sub.61 and E.sub.79 to E.sub.80 which is increased in
comparison with its wild type: of an enzyme E.sub.80, which
catalyzes the conversion of L-valine into 2-oxoisovalerate; of an
enzyme E.sub.79, which catalyzes the conversion of 2-oxoisovalerate
into isobutyryl-coenzyme A; of an enzyme E.sub.60, which catalyzes
the conversion of isobutyryl-coenzyme A into methacrylyl-coenzyme
A; of an enzyme E.sub.61, which catalyzes the conversion of
methacrylyl-coenzyme A into 3-hydroxyisobutyryl-coenzyme A; of an
enzyme E.sub.8, which catalyzes the conversion of
3-hydroxyisobutyryl-coenzyme A into 3-hydroxyisobutyrate.
42. A cell obtained by the method as claimed in 29.
Description
[0001] The present invention relates to cells which have been
genetically modified in comparison with their wild type, to methods
of generating a genetically modified cell, to the genetically
modified cells obtainable by these methods, to a process for the
preparation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates
based on 3-hydroxyisobutyric acid, to a process for the preparation
of methacrylic acid or methacrylic esters, and to a process for the
preparation of polymethacrylic acid or polymethacrylic esters. The
present invention furthermore relates to an isolated DNA, to a
vector, to the use of this vector for the transformation of a cell,
to a transformed cell, and to a polypeptide.
[0002] Methacrylic acid is an important intermediate which is
employed for the preparation of polymers, in particular in the form
of its alkyl esters. An example of a well-known methacrylic acid
derivative is the methyl ester of methacrylic acid. The current
global annual production of methyl methacrylate amounts to
approximately 1.5 million tonnes. The polymethacrylic esters are
raw materials in the plastics sector with a multiplicity of
uses.
[0003] Methacrylic acid is usually produced commercially by means
of the heterogeneous gas-phase oxidation of C.sub.4-carbon
compounds such as butylene, isobutylene, butane, isobutane, t-butyl
alcohol or methacrolein by two-step catalysis on solid multi-metal
oxide compositions as the catalyst. The resulting product gas
mixture, which, besides methacrylic acid, also comprises a large
number of secondary products, is subsequently either subjected to a
total condensation reaction, generating aqueous methacrylic acid
solution, or absorbed in a suitable solvent mixture. This is
usually followed by further purification of the resulting liquid
phases by means of distillation, crystallization, extraction, or a
combination of these measures. Besides the catalytic gas-phase
oxidation of C.sub.4-carbon compounds, methacrylic acid can also be
formed from isobutyric acid by catalytic oxidative dehydrogenation,
as is described for example in EP-A-0 356 315. A further
possibility for preparing methacrylic acid is what is known as the
"ACH process", in which acetone cyanohydrin and sulfuric acid are
reacted with the formation of methacrylamide as intermediate, which
then reacts further with water to give methacrylic acid. The
resulting methacrylic acid is subsequently purified by
distillation. This process is described for example in EP-A-1 359
137.
[0004] The disadvantage of these conventional processes for the
preparation of methacrylic acid is, inter alia, that during both
the preparation of the methacrylic acid itself and during the
subsequent steps, which involve purification by distillation, the
process steps, which cause thermal stress, result, owing to the
pronounced susceptibility of methacrylic acid to polymerization, in
the formation of dimers or oligomers; this not only entails
additional purification efforts, but also yield losses.
[0005] It was an object of the present invention to overcome the
disadvantages of the prior art.
[0006] In particular, it was an object of the present invention to
provide a process for the preparation of methacrylic acid which
generates methacrylic acid with a minimum of steps which involve
thermal stress.
[0007] Furthermore, it is intended that this process makes possible
the preparation of methacrylic acid from renewable resources, in
particular from carbohydrates and/or glycerol.
[0008] A contribution to achieving the abovementioned aims is
provided by a cell which has been genetically modified in
comparison with its wild type in such a way that it is capable of
forming more 3-hydroxyisobutyric acid, or polyhydroxyalkanoates
based on 3-hydroxyisobutyric acid, but preferably more
3-hydroxyisobutyric acid, in comparison with its wild type, this
formation preferably taking place via methylmalonate semialdehyde
or via 3-hydroxyisobutyryl-coenzyme A as precursor.
[0009] In the event that the formation of 3-hydroxyisobutyric acid
or of polyhydroxyalkanoates based on 3-hydroxy-isobutyric acid
takes place via methylmalonate semialdehyde as precursor, it is
furthermore preferred that the formation takes place via
succinyl-coenzyme A, propionyl-coenzyme A or acryloyl-coenzyme A,
especially preferably via succinyl-coenzyme A, as further
intermediate. In the event that the formation of
3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on
3-hydroxyisobutyric acid takes place via
3-hydroxyisobutyryl-coenzyme A as precursor, it is furthermore
preferred that the formation takes place via isobutyryl-coenzyme A
or via 3-hydroxybutyryl-coenzyme A, preferably via
3-hydroxybutyryl-coenzyme A, as further intermediate.
[0010] The term "precursor" as used in the present context defines
a chemical compound which can be converted enzymatically into
3-hydroxyisobutyric acid in just one reaction step, while the term
"intermediate" defines a chemical compound which cannot be
converted enzymatically into 3-hydroxyisobutyric acid in just one
reaction step.
[0011] The term "3-hydroxyisobutyric acid" as used in the present
context always describes the corresponding C.sub.4-carboxylic acid
in the form in which it is present as a function of the pH, after
having been formed by the microorganisms in question. As a
consequence, the term always comprises the pure acid form
(3-hydroxyisobutyric acid), the pure base form
(3-hydroxyisobutyrate) and mixtures of protonated and deprotonated
forms of the acid. Furthermore, the term "3-hydroxyisobutyric acid"
comprises, in principle, both the (R) and the (S) stereoisomer, the
(S) stereoisomer being especially preferred.
[0012] The wording "that it is capable of forming more
3-hydroxyisobutyric acid or polyhydroxyalkanoates based on
3-hydroxyisobutyric acid in comparison with its wild type" also
applies in the event that the wild type of the genetically modified
cell is not capable of forming any 3-hydroxyisobutyric acid or
polyhydroxyalkanoates based on 3-hydroxyisobutyric acid, but at
least no detectable amounts of these compounds, and that detectable
amounts of these components are only capable of being formed after
the genetic modification.
[0013] A "wild type" of a cell preferably refers to a cell whose
genome is present in a state as generated naturally as the result
of evolution. The term is used both for the entire cell and for
individual genes. As a consequence, the term "wild type" does not
cover in particular those cells, or those genes, whose gene
sequences have at least in part been modified by man by means of
recombinant methods.
[0014] The 3-hydroxyisobutyric acid can subsequently give rise to
methacrylic acid by subjecting it to a dehydration reaction under
mild conditions. In the case of polyhydroxyalkanoates based on
3-hydroxyisobutyric acid, the vesicles present in the cells, which
are filled with these polyhydroxyalkanoates, can be isolated and
the polymers can subsequently be cleaved to give
3-hydroxyisobutyric acid, which can then be dehydrated to give
methacrylic acid.
[0015] In this context, it is preferred according to the invention
that the genetically modified cell has been genetically modified in
such a way that it forms at least twice, especially preferably at
least 10 times, more preferably at least 100 times, even more
preferably at least 1000 times and most preferably at least 10 000
times more 3-hydroxyisobutyric acid or polyhydroxyalkanoates based
on 3-hydroxyisobutyric acid than the wild type of the cell within a
defined time interval, preferably within 2 hours, even more
preferably within 8 hours and most preferably within 24 hours. The
increase in the formation of product can be determined in this
context for example by growing the cell according to the invention
and the wild-type cell in each case separately, but under identical
conditions (identical cell density, identical nutrient medium,
identical culture conditions) for a particular time interval in a
suitable nutrient medium and subsequently determining the amount of
target product (3-hydroxyisobutyric acid or polyhydroxyalkanoates
based on 3-hydroxyisobutyric acid) in the nutrient medium.
[0016] The cells according to the invention may be prokaryotic or
eukaryotic cells. They may take the form of mammalian cells (such
as, for example, human cells), of plant cells or of microorganisms
such as yeasts, fungi or bacteria, with microorganisms being
especially preferred and bacteria and yeasts being most
preferred.
[0017] Suitable bacteria, yeasts or fungi are in particular those
bacteria, yeasts or fungi which have been deposited at the Deutsche
Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ),
Brunswick, Germany, as bacterial, yeast or fungal strains. Bacteria
which are suitable according to the invention belong to the genera
detailed under
http://www.dsmz.de/species/bacteria.htm, yeasts which are suitable
according to the invention belong to those genera which are
detailed under http://www.dsmz.de/species/yeasts.htm, and fungi
which are suitable according to the invention are those which are
detailed under http://www.dsmz.de/species/fungi.htm.
[0018] Cells which are especially preferred according to the
invention are those of the genera Corynebacterium, Brevibacterium,
Bacillus, Acinetobacter, Lactobacillus, Lactococcus, Candida,
Pichia, Kluveromyces, Saccharomyces, Escherichia, Zymomonas,
Yarrowia, Methylobacterium, Ralstonia, Pseudomonas, Burkholderia
and Clostridium, with Brevibacterium flavum, Brevibacterium
lactofermentum, Escherichia coli, Saccharomyces cerevisiae,
Kluveromyces lactis, Candida blankii, Candida rugosa,
Corynebacterium glutamicum, Corynebacterium efficiens, Zymonomas
mobilis, Yarrowia lipolytica, Methylobacterium extroquens,
Ralstonia eutropha, especially Ralstonia eutropha H16,
Rhodospirillum rubrum, Rhodobacter sphaeroides, Paracoccus
versutus, Pseudomonas aeroginosa, Acinetobacter calcoaceticus and
Pichia pastoris being especially preferred.
[0019] In accordance with a first variant of the cell according to
the invention, the formation of 3-hydroxyisobutyric acid or of
polyhydroxyalkanoates based on 3-hydroxyisobutyric acid takes place
via methylmalonate semialdehyde as precursor.
[0020] In accordance with a first special embodiment of this first
variant of the cell according to the invention, it is preferred
that the formation of 3-hydroxyisobutyric acid or of the
polyhydroxyalkanoate based on 3-hydroxyisobutyric acid
preferentially takes place via succinyl-coenzyme A as intermediate,
where the cell preferentially is capable of utilizing
carbohydrates, glycerol or glutamate as the carbon source.
[0021] Here, it may be advantageous in the context of the first
special embodiment of the first variant of the cell according to
the invention that the cell according to the invention features an
increased activity of an enzyme E.sub.1, which catalyzes the
conversion of succinyl-coenzyme A into methylmalonyl-coenzyme A, in
comparison with its wild type (see FIG. 1).
[0022] The term "increased activity of an enzyme" as used above in
connection with the enzyme E.sub.1 and in what follows in the
context of the enzymes E.sub.2 etc. is preferably to be understood
as increased intracellular activity.
[0023] What now follows on increasing the enzymatic activity in
cells applies both to increasing the activity of the enzyme E.sub.1
and to all enzymes mentioned thereafter, whose activity can, if
appropriate, be increased.
[0024] In principle, an increase in the enzymatic activity can be
achieved by increasing the copy number of the gene sequence(s)
which code for the enzyme, by using a strong promoter or by using a
gene or allele which codes for a corresponding enzyme with an
increased activity, and, if appropriate, combining these measures.
Cells which have been genetically modified in accordance with the
invention are generated for example by transformation,
transduction, conjugation or a combination of these methods with a
vector which comprises the desired gene, an allel of this gene or
parts thereof, and a vector which makes possible the expression of
the gene. The heterologous expression is achieved in particular by
integration of the gene, or of the alleles, into the chromosome of
the cell or into an extrachromosomally replicating vector.
[0025] An overview over the possibilities for increasing the
enzymatic activity in cells with pyruvate carboxylase by way of
example is found in DE-A-100 31 999, which is hereby incorporated
by reference and whose disclosure content regarding the
possibilities for increasing the enzymatic activity in cells forms
part of the disclosure of the present invention.
[0026] The expression of the enzymes or genes mentioned hereinabove
and in each case hereinbelow can be detected in the gel with the
aid of 1- and 2-dimensional protein gel separation and subsequent
visual identification of the protein concentration using suitable
evaluation software. When the increase in an enzymatic activity is
based exclusively on an increase in the expression of the gene in
question, the quantification of the increase in the enzymatic
activity can be determined in a simple manner by comparing the 1-
or 2-dimensional protein separations between the wild type and the
genetically modified cell. A conventional method of preparing the
protein gels in coryneform bacteria, and of identifying the
proteins, is the procedure described by Hermann et al.
(Electrophoresis, 22: 1712.23 (2001)). The protein concentration
can also be analyzed by Western blot hybridization using an
antibody which is specific for the protein to be detected (Sambrook
et al., Molecular Cloning: a laboratory manual, 2.sup.nd Ed. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. USA, 1989)
followed by visual evaluation with suitable software for
determining the concentration (Lohaus and Meyer (1989) Biospektrum,
5: 32-39; Lottspeich (1999), Angewandte Chemie 111: 2630-2647). The
activity of DNA-binding proteins can be measured by means of DNA
band shift assays (also referred to as gel retardation) (Wilson et
al. (2001) Journal of Bacteriology, 183: 2151-2155). The effect of
DNA-binding proteins on the expression of other genes can be
detected by various, extensively described methods of the reporter
gene assay (Sambrook et al., Molecular Cloning: a laboratory
manual, 2.sup.nd Ed. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y. USA, 1989). The intracellular enzymatic
activities can be detected by various methods which have been
described (Donahue et al. (2000) Journal of Bacteriology 182 (19):
5624-5627; Ray et al. (2000) Journal of Bacteriology 182 (8):
2277-2284; Freedberg et al. (1973) Journal of Bacteriology 115 (3):
816-823). In the event that no specific methods for determining the
activity of a particular enzyme are detailed in what follows, the
determination of the increase in the enzymatic activity, and also
the determination of the reduction in an enzymatic activity, is
preferably carried out by means of the methods described in Hermann
et al., Electrophoresis, 22: 1712-23 (2001), Lohaus et al.,
Biospektrum 5 32-39 (1998), Lottspeich, Angewandte Chemie 111:
2630-2647 (1999) and Wilson et al. (2001) Journal of Bacteriology,
183: 2151-2155 (2001).
[0027] If increasing the enzymatic activity is brought about by
mutating the endogenous gene, such mutations can be generated
either undirected, using traditional methods such as for example by
UV irradiation or by mutagenic chemicals, or directed by means of
recombinant methods such as deletion(s), insertion(s) and/or
nucleotide substitution(s). These mutations give rise to
genetically modified cells. Especially preferred mutants of enzymes
are in particular also those enzymes which are no longer capable of
being feedback-inhibited, or which are at least less capable of
being feedback-inhibited, in comparison with the wild-type
enzyme.
[0028] If increasing the enzymatic activity is brought about by
increasing the expression of an enzyme, then, for example, the copy
number of the respective genes are increased, or the promoter and
regulatory regions or the ribosomal binding site, which is located
upstream of the structural gene, are mutated. Expression cassettes
which are incorporated upstream of the structural gene act in the
same manner. By means of inducible promoters it is additionally
possible to increase the expression at any desired point in time.
Furthermore, the enzyme gene may also have assigned to it what are
known as enhancer sequences as regulatory sequences; these also
bring about an increased gene expression via an improved
interaction between RNA polymerase and DNA. Measures for extending
the life of the mRNA also improves expression. Furthermore,
preventing the degradation of the enzyme protein also enhances the
enzymatic activity. Here, the genes or gene constructs are either
present in plasmids in different copy numbers, or else they are
integrated and amplified in the chromosome. As an alternative,
overexpression of the genes in question may also be achieved by
modifying the media composition and the control of the culture.
[0029] Instructions for doing so can be found by the skilled worker
in Martin et al. (Bio/Technology 5, 137-146 (1987)) in Guerrero et
al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology
6, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in
EP-A-0 472 869, in U.S. Pat. No. 4,601,893, in Schwarzer and Puhler
((Bio/Technology 9, 84-87 (1991), in Reinscheid et al. (Applied and
Environmental Microbiology 60, 126-132 (1994)), in LaBarre et al.
(Journal of Bacteriology 175, 1001-1007 (1993)), in WO-A-96/15246,
in Malumbres et al. (Gene 134, 15-24 (1993), in JP-A-10-229891, in
Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195
(1998)), inter alia, and in known textbooks of genetics and
molecular biology. The above-described measures give rise to
genetically modified cells, as do the mutations.
[0030] Plasmids, for example episomal plasmids, are employed for
increasing the expression of the genes in question. Suitable
plasmids are in particular those which are replicated in coryneform
bacteria. A large number of known plasmid vectors such as, for
example, pZ1 (Menkel et al., Applied and Environmental Microbiology
64: 549-554 (1989)), pEKEx1 (Eikmanns et al., Gene 107: 69-74
(1991)) or pHS2-1 (Sonnen et al., Gene 107: 69-74 (1991)) are based
on the cryptic plasmids pHM1519, pBL1 or pGA1. Other plasmid
vectors such as, for example, those based on pCG4 (U.S. Pat. No.
4,489,160) or pNG2 {Serwold-Davis et al., FEMS Microbiology Letters
66: 119-124 (1990)} or pAG1 (U.S. Pat. No. 5,158,891), may be
employed in the same manner.
[0031] Others which are suitable are those plasmid vectors with the
aid of which the method of amplifying genes by integration into the
chromosome can be applied, as has been described for example by
Reinscheid et al. (Applied and Environmental Microbiology 60:
126-132 (1994)) for duplicating or amplifying the hom-thrB operon.
In this method, the entire gene is cloned into a plasmid vector
which is capable of replication in a host (typically Escherichia
coli), but not in Corynebacterium glutamicum. Suitable vectors are,
for example, pSUP301 (Simon et al, Bio/Technology 1: 784-791
(1983)), pK18mob or pK19mob (Schafer et al., Gene 145: 69-73
(1994)), pGEM-T (Promega Corporation, Madison, Wis., USA),
pCR2.1-TOPO (Shuman, Journal of Biological Chemistry 269: 32678-84
(1994)), pCR.RTM.Blunt (Invitrogen, Groningen, the Netherlands),
pEM1 (Schrumpf et al., Journal of Bacteriology 173: 4510-4516)) or
pBGS8 (Spratt et al., Gene 41: 337-342 (1986)). The plasmid vector,
which contains the gene to be amplified, is subsequently
transferred into the desired Corynebacterium glutamicum strain by
means of conjugation or transformation. The conjugation method is
described for example in Schafer et al., Applied and Environmental
Microbiology 60: 756-759 (1994). Transformation methods are
described for example in Thierbach et al., Applied Microbiology and
Biotechnology 29: 356-362 (1988), Dunican and Shivnan,
Bio/Technology 7: 1067-1070 (1989) and Tauch et al., FEMS
Microbiology Letters 123: 343-347 (1994). Following homologous
recombination by means of a cross-over event, the resulting strain
comprises at least two copies of the gene in question.
[0032] The wording "an activity of an enzyme E.sub.x which is
increased in comparison with its wild type" used hereinabove and in
what follows is preferably always understood as meaning an activity
of the respective enzyme E.sub.x which is increased by a factor of
at least 2, especially preferably of at least 10, more preferably
of at least 100, even more preferably of at least 1000 and most
preferably of at least 10 000. Furthermore, the cell according to
the invention which features "an activity of an enzyme E.sub.x
which is increased in comparison with its wild type", in particular
also a cell whose wild type features no, or at least no detectable,
activity of this enzyme E.sub.x and which only shows a detectable
activity of this enzyme E.sub.x after increasing the enzymatic
activity, for example by means of overexpression. In this context,
the term "overexpression", or the wording "increase in the
expression" used in what follows also comprises the case that a
starting cell, for example a wild-type cell, features no, or at
least no detectable, expression and detectable expression of the
enzyme E.sub.x is only induced by recombinant methods.
[0033] Accordingly, the wording "reduced activity of an enzyme
E.sub.x" used hereinbelow is understood as meaning an activity
which is preferably reduced by a factor of at least 0.5, especially
preferably of at least 0.1, more preferably of at least 0.01, even
more preferably of at least 0.001 and most preferably of at least
0.0001. The reduction in the activity of a specific enzyme can be
obtained for example by directed mutation, by the addition of
competitive or non-competitive inhibitors or by other measures for
reducing the expression of a specific enzyme which are known to the
skilled worker.
[0034] In the case of the enzyme E.sub.1, which catalyzes the
conversion of succinyl-coenzyme A into methylmalonyl-coenzyme A,
this preferably takes the form of a methylmalonyl-coenzyme A mutase
(EC 5.4.99.2). This enzyme is preferably encoded by the gene
selected from the group consisting of mut, mutA, mutB, sbm, sbmA,
sbmB, sbm5, bhbA, mcmA, mcmA1, mcmA2, mcmB, mcm1, mcm2, mcm3, icmA,
meaA1 and meaA2. The nucleotide sequence of these genes can be
found for example in the "Kyoto Encyclopedia of Genes and Genomes"
(KEGG database), the databases of the National Center for
Biotechnology Information (NCBI) of the National Library of
Medicine (Bethesda, Md., USA) or from the nucleotide sequence
database of the European Molecular Biologies Laboratories (EMBL,
Heidelberg, Germany and Cambridge, UK).
[0035] In accordance with an especially preferred embodiment of the
first variant of the cell according to the invention, the enzyme
E.sub.1 takes the form of the methylmalonyl-coenzyme A mutase from
Corynebacterium glutamicum ATCC 13032, which is encoded by a gene
with the DNA sequence as shown in SEQ ID No 01 and which has the
amino acid as shown in SEQ ID No 02.
[0036] Furthermore, it is preferred in accordance with a first
alternative of the cell according to the invention, where
succinyl-coenzyme A is formed as intermediate and methylmalonate
semialdehyde as precursor in the preparation of 3-hydroxyisobutyric
acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid,
that the cell, if appropriate in addition to the increased activity
of the enzyme E.sub.1, features an activity of at least one of the
following enzymes E.sub.2 to E.sub.4 which is increased in
comparison with its wild type (see FIG. 2): [0037] of an enzyme
E.sub.2, which catalyzes the conversion of methylmalonyl-coenzyme A
into methyl malonate; [0038] of an enzyme E.sub.3, which catalyzes
the conversion of methyl malonate into methylmalonate semialdehyde;
[0039] of an enzyme E.sub.4 which catalyzes the conversion of
methylmalonate semialdehyde into 3-hydroxyisobutyric acid.
[0040] In this context, cells which are especially preferred in
accordance with the invention are those in which the activity of
the following enzymes or enzyme combinations is increased: E.sub.2,
E.sub.3, E.sub.4, E.sub.2E.sub.3, E.sub.2E.sub.4, E.sub.3E.sub.4,
E.sub.2E.sub.3E.sub.4, where E.sub.2E.sub.3E.sub.4 is most
preferred. Furthermore, it is possible that an enzyme is also
capable of catalyzing at least two of the above-described reaction
steps. Thus, for example, it is possible to employ an enzyme which
features both the activity of enzyme E.sub.2 and that of enzyme
E.sub.3 (and which therefore catalyzes the conversion of
methylmalonyl-coenzyme A directly into methylmalonate semialdehyde)
such as, for example, the malonyl coenzyme A reductase from
Sulfolobus tokodaii, which is encoded by the DNA sequence with the
SEQ ID No 03 and which has the amino acid sequence as shown in SEQ
ID No 04, or else an enzyme which features all three enzymatic
activities E.sub.2, E.sub.3 and E.sub.4, such as the malonyl
coenzyme A reductase from Chloroflexus aurantiacus (Hugler et al.,
Journal of Bacteriology 184, pages 2404-2410, 2002).
[0041] In this context, it is especially preferred that the enzyme
[0042] E.sub.2 is a methylmalonyl-coenzyme A hydrolase (EC
3.1.2.17), [0043] E.sub.3 is an aldehyde dehydrogenase (EC 1.2.1.3)
or an aldehyde oxidase (EC 1.2.3.1) and [0044] E.sub.4 is a
3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) or a
3-hydroxyacyl-coenzyme A dehydrogenase (EC 1.1.1.35).
[0045] The enzyme E.sub.2 is preferably encoded by the aox1 gene.
The methylmalonyl-coenzyme A hydrolase from rat liver is described
for example in Kovachy et al., "Recognition, isolation, and
characterization of rat liver D-methylmalonyl coenzyme A
hydrolase", J. Biol. Chem. 258 (1983), pages 11415-11421.
[0046] The enzyme E.sub.3 is preferably encoded by genes selected
from the group consisting of aldh2, aldh3a1, aldh3a2, aldh1b1,
aldh9a1, aldh7a1, aldh1a4, aldh1a1, aldh1a2, mgc80785, mgc83352,
mgc89020, dmel-CG31075, cg3752, cg9629, alh-9, alh-1, alh-2,
f508.35, t7023.15, f15I1.19, tT17F15.130, ald1, ald2, ald4, ald5,
ald6, ac1044Wp, adr417wp, msc7, tb06.5F5.780, aldH, puuC, putA,
aldA, badH, alkH, pcD, rsp1591, rs01031, exaC, acoD, dhaL, pchA,
aldB, dhaS, betB, ywdH, ycbD, aldX, aldY, aldA1, aldA2, aldC, pcd,
cg10546, cg12668, cg12796, scg11A.05, sci30A.27c, sce9.27c,
sck13.05c, sc5H4.03, thcA, gabD2, alkH, aldH, aldH1, aldY1, aldY2,
aldY3, aldY4, aldY5, aldY6, aldY7 and aldhT.
[0047] Suitable genes for the enzyme E.sub.4 are selected from the
group consisting of hibadh, cg15093, cg15093, cg4747, mwL2.23,
t13k14.90, f19b15.150, hibA, ygbJ, mmsB, mmsB, garR, tsar, mmsB-1,
mmsB-2, yfjR, ykwC, ywjF, hibD, glxR, SCM1.40c, hibD, ehhahd,
hadh2, hadhsc, hsd17B4, loc488110, had, mgC81885, hadh2-prov,
cg3415, cg7113, ech-1, ech-8, ech-9, ard-1, yfcX, fadB, faoA,
fadB2x, hbd-1, hbd-2, hbd-3, hbd-4, hbd-5, hbd-6, hbd-7, hbd-8,
hbd-9, hbd-10, fadJ, rs04421, rs02946, rs05766, bbsD, bbsC, fadB1,
fadB2, fadB5, hbdA, pimF, fabJ-1, fabJ, scbac19f3.11, sci35.13,
scbac8d1.10c, sc5f2a.15, sc6a5.38, fadC2, fadC4, fadC5, fadC6, had
and paaH. Further suitable 3-hydroxyisobutyrate dehydrogenases are
described for example in Bannerjee et al. (1970), J. Biol. Chem.,
245, pages 1828 to 1835, Steele et al. (1992), J. Biol. Chem., 267,
pages 13585 to 13592, Harris et al. (1988), J. Biol. Chem., 263,
pages 327 to 331, Harris et al., Biochim. Biophys. Acta, 1645 (1),
pages 89 to 95, Hawes et al. (2000), Methods Enzymol., 324, pages
218 to 228, Harris et al., J. Biol. Chem., 275 (49), pages 38780 to
38786, Rougraff et al. (1988), J. Biol. Chem., 263(1), pages 327 to
331, Robinson et al., J. Biol. Chem., 225, pages 511 to 521, Hawes
et al. (1995), Biochemistry, 34, pages 4231 to 4237, Hasegawa J.
(1981), Agric. Biol. Chem., 45, pages 2805 to 2814, Hawes et al.
(1996), FEBS Lett., 389, pages 263 to 267, Hawes et al. (1996),
Enzymology and Molecular Biology of Carbonyl Metabolism, Plenum
Press, New York, pages 395 to 402, Adams et al. (1994), Structure,
2, pages 651 to 668, Zhang et et. (1999), Biochemistry, 38, pages
11231 to 11238, Mirny et al., (1999), J. Mol. Biol., 291, pages 177
to 196 and Lokanath et al. (2005), J Mol. Biol. The disclosure of
these publications is hereby incorporated by reference and forms
part of the disclosure of the present invention.
[0048] The nucleotide sequences of the abovementioned genes and of
further genes for the enzymes E.sub.2 to E.sub.4 can also be found
in the KEGG database, the NCBI database or the EMBL database, inter
alia.
[0049] In accordance with an especially preferred embodiment of
this alternative of the cell according to the invention, where
succinyl-coenzyme A is formed as intermediate and methylmalonate
semialdehyde as precursor in the preparation of 3-hydroxyisobutyric
acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid,
it is preferred that the malonyl coenzyme A reductase from
Sulfolobus tokodaii, which is encoded by the DNA sequence with the
SEQ ID No 03 and which has the amino acid sequence as shown in SEQ
ID No 04, is employed for the conversion of methylmalonyl-coenzyme
A into methylmalonate semialdehyde. In accordance with another
especially preferred embodiment of this variant, the malonyl
coenzyme A reductase from Chloroflexus aurantiacus (Huler et al.,
Journal of Bacteriology 184, pages 2404-2410, 2002) is employed for
the conversion of methylmalonyl-coenzyme A into 3-hydroxyisobutyric
acid.
[0050] Furthermore, it is preferred in the context of this first
alternative of the first special embodiment of the cell according
to the invention that the cell features an activity of an enzyme
E.sub.5, which features the conversion of methylmalonate
semialdehyde into propionyl-coenzyme A, which is reduced in
comparison with its wild type, this enzyme preferably taking the
form of a methylmalonate-semialdehyde dehydrogenase (EC
1.2.1.27).
[0051] In accordance with a second alternative of the cell
according to the invention, where succinyl-coenzyme A is formed as
intermediate and methylmalonate semialdehyde as precursor in the
preparation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates
based on 3-hydroxyisobutyric acid, it is preferred that the cell,
if appropriate in addition to the increased activity of the enzyme
E.sub.1, features an activity of at least one of the following
enzymes E.sub.4 to E.sub.7 which is increased in comparison with
its wild type (see FIG. 3): [0052] of an enzyme E.sub.6, which
catalyzes the conversion of (R) methylmalonyl-coenzyme A into (S)
methyl-malonyl-coenzyme A; [0053] of an enzyme E.sub.7, which
catalyzes the conversion of (S) methylmalonyl-coenzyme A into
propionyl-coenzyme A; [0054] of an enzyme E.sub.5, which catalyzes
the conversion of propionyl-coenzyme A into methylmalonate
semialdehyde; [0055] of an enzyme E.sub.4, which catalyzes the
conversion of methylmalonate semialdehyde into 3-hydroxyisobutyric
acid.
[0056] In this context, cells which are especially preferred in
accordance with the invention are those in which the activity of
the following enzymes or enzyme
[0057] combinations is increased: E.sub.4, E.sub.5, E.sub.6,
E.sub.7, E.sub.7, E.sub.4E.sub.5, E.sub.4E.sub.6, E.sub.4E.sub.7,
E.sub.5E.sub.6, E.sub.5E.sub.7, E.sub.6E.sub.7,
E.sub.4E.sub.5E.sub.6, E.sub.4E.sub.5E.sub.7,
E.sub.4E.sub.6E.sub.7, E.sub.5E.sub.6E.sub.7 and
E.sub.4E.sub.5E.sub.6E.sub.7, with E.sub.4E.sub.5E.sub.6E.sub.7
being most preferred.
[0058] In this context, it is especially preferred that the enzyme
[0059] E.sub.6 is a methylmalonyl-coenzyme A epimerase (EC
5.1.99.1) [0060] E.sub.7 is a methylmalonyl-coenzyme A
decarboxylase (EC 4.1.1.41), [0061] E.sub.5 is a
methylmalonate-semialdehyde dehydrogenase (EC 1.2.1.27), and [0062]
E.sub.4 is a 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) or a
3-hydroxyacyl-coenzyme A dehydrogenase (EC 1.1.1.35).
[0063] In this context, preferred enzymes E.sub.4 are those which
have already been mentioned above in the context of the first
variant of the first preferred embodiment of the cell according to
the invention.
[0064] The enzyme E.sub.6 is preferably encoded by the mcee gene. A
suitable methylmalonyl-coenzyme A decarboxylase (enzyme E.sub.7) is
described, for example, by Benning et al. in Biochemistry, Vol. 39
(2000), pages 4630-4639.
[0065] Suitable genes for the enzyme E.sub.5 are preferably
selected from the group consisting of aldh6a1, cg17896, t22c12.10,
ald6, putA1, mmsA, mmsA-1, mmsA-2, mmsA-3, mmsA-4, msdA, iolA and
iolAB.
[0066] Suitable genes for the enzyme E.sub.7 are preferably
selected from the group consisting of mmdA, bcc, oadB, oadB2,
oadB3, SC1C2.16, SC1G7.10, pccB1, accA2, mmdB, mmdC and ppcB.
[0067] The nucleotide sequences of the abovementioned genes for the
enzymes E.sub.5, E.sub.6 and E.sub.7 may, inter alia, also be found
in the KEGG database.
[0068] In accordance with a third alternative of the cell according
to the invention, where succinyl-coenzyme A is formed as
intermediate and methylmalonate semialdehyde as precursor in the
preparation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates
based on 3-hydroxyisobutyric acid, it is preferred that the cell,
if appropriate in addition to the increased activity of the enzyme
E.sub.1, features an activity of at least one of the following
enzymes E.sub.4, E.sub.5 and E.sub.7 which is increased in
comparison with its wild type (see FIG. 4): [0069] of an enzyme
E.sub.7, which catalyzes the conversion of methylmalonyl-coenzyme A
into propionyl-coenzyme A; [0070] of an enzyme E.sub.5, which
catalyzes the conversion of propionyl-coenzyme A into
methylmalonate-semialdehyde; [0071] of an enzyme E.sub.4, which
catalyzes the conversion of methylmalonate-semialdehyde into
3-hydroxyisobutyric acid.
[0072] This pathway corresponds essentially to the second variant
of the first preferred embodiment of the cell according to the
invention, but, as opposed to the second variant, propionyl-CoA is
prepared directly from methylmalonyl-coenzyme A. Preferred enzymes
and genes for the enzymes E.sub.4, E.sub.5 and E.sub.7 are those
genes or enzymes which have already been mentioned above in
connection with the second variant.
[0073] Furthermore, it may in accordance with the first special
embodiment of the cell according to the invention (and also in
accordance with all embodiments which are still to be described
hereinbelow) also be preferred that the cell is capable of
converting the formed 3-hydroxyisobutyric acid into a
polyhydroxy-alkanoate. Such polyhydroxydalkanoates are deposited
intracellularly by many microorganisms in the form of highly
refractive granula. In this context, it is especially preferred
that the cell according to the invention features an activity of at
least one of, preferably of the two, the following enzymes E.sub.9
and E.sub.10 which is increased in comparison with its wild type
(see FIG. 5): [0074] of an enzyme E.sub.8, which catalyzes the
conversion of 3-hydroxyisobutyric acid into
3-hydroxyisobutyryl-coenzyme A; [0075] of an enzyme E.sub.9, which
catalyzes the conversion of 3-hydroxyisobutyryl-coenzyme A to a
polyhydroxy-alkanoate based on 3-hydroxyisobutyric acid.
[0076] In this context, it is especially preferred that the enzyme
[0077] E.sub.8 is a 3-hydroxyisobutyryl CoA hydrolase (EC 3.1.2.4)
and [0078] E.sub.9 is a polyhydroxyalkanoate synthase.
[0079] As has already been explained above, the first preferred
embodiment of the cell according to the invention generates
3-hydroxyisobutyric acid or the polyhydroxyalkanoates based on
3-hydroxyisobutyric acid from succinyl coenzyme A as intermediate
and from methylmalonate semialdehyde as precursor. Here, it may
make sense in principle to influence not only one or more of the
abovementioned enzyme activities E.sub.1 to E.sub.9, but also those
enzyme activities which lead to an increased formation of
succinyl-coenzyme A in the cell.
[0080] In the event that, according to the first special embodiment
of the first variant of the cell according to the invention, the
formation of 3-hydroxyisobutyric acid or of the
polyhydroxyalkanoates based on 3-hydroxyisobutyric acid takes place
from carbohydrates or glycerol via succinyl-coenzyme A as
intermediate and methylmalonate semialdehyde as precursor, it is,
according to a special embodiment of the above-described first,
second or third alternative of the cell according to the invention,
preferred that the cell features an activity of at least one of
the, preferably of the two, following enzymes E.sub.10 and E.sub.11
which is increased in comparison with its wild type (see FIG. 6):
[0081] of an enzyme E.sub.10, which catalyzes the conversion of
phosphoenolpyruvate into oxaloacetate; [0082] of an enzyme
E.sub.11, which catalyzes the conversion of pyruvate into
oxaloacetate.
[0083] In this context, it is especially preferred that the enzyme
[0084] E.sub.10 is a phosphoenolpyruvate carboxylase (EC 4.1.1.31)
and [0085] E.sub.11 is a pyruvate carboxylase (EC 6.4.1.1).
[0086] The enzyme E.sub.10 is preferably encoded by the genes
selected from the group consisting of f12 m16.21, f14n22.13, k15
m2.8, ppc, clpA, pepC, capP, cgl1585, pepC, pck ppc and pccA, where
the ppc gene is especially preferred. Phosphoenolpyruvate
carboxylases which are preferred according to the invention are
also described in particular in U.S. Pat. No. 4,757,009, U.S. Pat.
No. 4,980,285, U.S. Pat. No. 5,573,945, U.S. Pat. No. 6,872,553 and
U.S. Pat. No. 6,599,732. As regards phosphoenolpyruvate
carboxylases, the disclosure content of these publications is
hereby incorporated by reference and forms part of the disclosure
of the present invention.
[0087] The enzyme E.sub.11 is preferably encoded by the genes
selected from the group consisting of pc, pcx, cg1516, cg1516,
pyc-1, pyc-2, aar162 Cp, pyr1, accC-2, pycA, pycA2, pca, cg10689,
pyc, pycB, accC, oadA, acc and accC1, where the pyc gene is
especially preferred. Pyruvate carboxylases which are preferred
according to the invention are also described in particular in U.S.
Pat. No. 6,455,284, U.S. Pat. No. 6,171,833, U.S. Pat. No.
6,884,606, U.S. Pat. No. 6,403,351, U.S. Pat. No. 6,852,516 and
U.S. Pat. No. 6,861,246. A further pyruvate carboxylase which is
especially preferred according to the invention is that mutant
which is described in "A novel methodology employing
Corynebacterium glutamicum genome information to generate a new
L-lysine-producing mutant", Ohnishi J et al., Applied Microbiology
and Biotechnology, Vol. 58 (2), pages 217-223 (2002).
[0088] The nucleotide sequences of suitable genes of the enzymes
E.sub.11 and E.sub.12 can be found in the KEGG database, the NCBI
database or the EMBL database.
[0089] Starting from the oxaloacetate intermediate stage, there are
several possibilities for arriving at succinyl-coenzyme A, which
can then be converted into 3-hydroxyisobutyric acid via
methylmalonyl-coenzyme A by means of the three variants mentioned
at the outset.
[0090] A first pathway leads via fumarate as intermediate. In this
case it is preferred in accordance with a first special embodiment
of the above-described first, second or third alternative of the
cell according to the invention, where methylmalonate-semialdehyde
is formed as precursor and succinyl-coenzyme A as intermediate,
that the cell, if appropriate additionally to an increased activity
of the enzyme E.sub.10 or E.sub.11, features an activity of at
least one of the following enzymes E.sub.12 to E.sub.15 which is
increased in comparison with its wild type (see FIG. 7): [0091] of
an enzyme E.sub.12, which catalyzes the conversion of oxaloacetate
into malate; [0092] of an enzyme E.sub.13, which catalyzes the
conversion of malate into fumarate; [0093] of an enzyme E.sub.14,
which catalyzes the conversion of fumarate into succinate; [0094]
of an enzyme E.sub.15, which catalyzes the conversion of succinate
into succinyl-coenzyme A.
[0095] In this context, cells which are especially preferred in
accordance with the invention are those in which the activity of
the following enzymes or enzyme combinations is increased:
E.sub.12, E.sub.13, E.sub.14, E.sub.15, E.sub.12E.sub.13,
E.sub.12E.sub.14, E.sub.12E.sub.15, E.sub.13E.sub.14,
E.sub.13E.sub.15, E.sub.14E.sub.15, E.sub.12E.sub.13E.sub.14,
E.sub.12E.sub.13E.sub.15, E.sub.12E.sub.14E.sub.15,
E.sub.13E.sub.14E.sub.15, E.sub.12E.sub.13E.sub.14E.sub.15, with
E.sub.12E.sub.13E.sub.14E.sub.15 being most preferred.
[0096] In this context, it is especially preferred that the enzyme
[0097] E.sub.12 is a malate dehydrogenase (EC 1.1.1.37) or a malate
quinone oxidoreductase (1.1.99.16), [0098] E.sub.13 is a fumarate
hydratase (EC 4.2.1.2), [0099] E.sub.14 is a succinate
dehydrogenase (EC 1.3.99.1 or EC 1.3.5.1) or a succinate quinone
oxidoreductase (1.3.5.1), and [0100] E.sub.15 is a succinate
coenzyme A ligase (EC 6.2.1.4 or EC 6.2.1.5).
[0101] The enzyme E.sub.12 is preferably encoded by genes selected
from the group consisting of mdh1, mdh2, mor1, cg10748, cg10749,
cg5362, mdh-1, f46e10.10, f19p19.13, f12 m16.14, t30120.4, k15
m2.16, f1p2.70, f17i14.150, mnl12.18, mik19.17, mdh3, adl164 cp,
adr152 cp, adr252wp, mdhA, mdhC, mdhB, ybiC, mdh, yiaK, ybiC, allD,
citH, yjmC, citH, cg12380, ldh, sqdB, mqo, yojH, mqoA, mqoB, mqo1,
mqo2, mqo3, mqo4 and cgl2001, where the mqo gene and the mdh gene
are especially preferred.
[0102] The enzyme E.sub.13 is preferably encoded by genes selected
from the group consisting of fh, fh1, sc4094, sc4095, t30b22.19,
k3k7.11, acr013/cp, fum1, fum2, fum3, fum4, fumH, fumA, fumB, fumC,
fumC1, fumC2, fum, ttdA, ttdB, fumB-alpha, fumB-beta, citG, citB,
fumX, fum-1 and fum-2, where the fum gene is especially
preferred.
[0103] The enzyme E.sub.14 is preferably encoded by genes selected
from the group consisting of sdh1, sdh2, sdh3, sdh4, sdh5, sdh6,
osm1, osm2, sdhA, sdhB, sdhC, sdhD, frdA, frdB, frdC, frdD, ifcA-1,
ifcA-2, sdhB-1, sdhB-2, frdC2, cgl0370, cgl0371, cgl0372,
scm10.10c, scm10.11c, scm10.12c, sc5g8.25c, sc5g8.26c,
scbac-31e11.02c, scbac31e11.02c, sc4b10.10c, sdhA2, sdhB2, sdhA1,
sdhB1, qcrB2, sdhA3, sdhB3, frdB1 and frdB2, where the genes sdhA,
sdhB and sdhC are especially preferred.
[0104] The enzyme E.sub.15 is preferably encoded by genes selected
from the group consisting of suclg1, suclg2, loc434885, cg10622,
dmel-CG6255, f11a3.3, f8115.30, mkd15.11, lsc1, lsc2, ael211wp,
afr134 cp, scsA, scsB, sucC and sucD.
[0105] Again, the nucleotide sequences of suitable genes of the
enzymes E.sub.12 to E.sub.15, can also be found in the KEGG
database, the NCBI database or the EMBL database.
[0106] In the event that the activity of one or more of the enzymes
E.sub.12 to E.sub.15 is increased, it may also prove advantageous
that the cell features an activity of one of the following enzymes
E.sub.16 to E.sub.23 which is reduced in comparison with its wild
type: [0107] of an enzyme E.sub.16, which catalyzes the conversion
of oxaloacetate into citrate; [0108] of an enzyme E.sub.17, which
catalyzes the conversion of malate into oxaloacetate; [0109] of an
enzyme E.sub.18, which catalyzes the conversion of
succinyl-coenzyme A into succinate, [0110] of an enzyme E.sub.19,
which catalyzes the conversion of oxaloacetate into
phosphoenolpyruvate, [0111] of an enzyme E.sub.20, which catalyzes
the conversion of oxaloacetate into pyruvate, [0112] of an enzyme
E.sub.21, which catalyzes the conversion of oxaloacetate into
aspartate, [0113] of an enzyme E.sub.22, which catalyzes the
conversion of malate into pyruvate, [0114] of an enzyme E.sub.23,
which catalyzes the conversion of pyruvate into acetate.
[0115] Cells which are especially preferred in accordance with the
invention are those in which the activity of the following enzymes
or enzyme combinations is reduced: E.sub.16, E.sub.17, E.sub.18,
E.sub.19, E.sub.20, E.sub.21, and
E.sub.16E.sub.17E.sub.18E.sub.19E.sub.20E.sub.21E.sub.22E.sub.23.
[0116] In this context, it is especially preferred that the enzyme
[0117] E.sub.16 is a citrate synthase (EC 2.3.3.1 or EC 2.3.3.8),
[0118] E.sub.17 is a malate oxidase (EC 1.1.3.3), [0119] E.sub.18
is a succinyl CoA hydrolase (EC 3.1.2.3), [0120] E.sub.19 is a
phosphoenolpyruvate carboxykinase (EC 4.1.1.49 or 4.1.1.32), [0121]
E.sub.20 is an oxaloacetate decarboxylase (EC 4.1.1.3), [0122]
E.sub.21 is an aspartate transaminase (EC 2.6.1.1), [0123] E.sub.22
is a malate dehydrogenase (EC 1.1.1.38, EC 1.1.1.39 or EC
1.1.1.40), [0124] E.sub.23 is a pyruvate dehydrogenase (EC
1.2.1.51).
[0125] The enzyme E.sub.16 is preferably encoded by genes selected
from the group consisting of glt, cs, csl, cg3861, cts-1, f7f19.21,
f4i1.16, t20n10.90, t20n10.100, t209.80, cit1, cit2, cit3, aar004
cp, agr002wp, cshA, gltA, citZ, cit, prpC, cisY, cis, mmgD, citA,
gltA1, gltA2, gltA3, cgl0829, prpC1, scd10.20, citA1, citA2, citA3,
acly, cg8322, f5e6.2, k7jJ8.14 and citE, where gltA is most
preferred.
[0126] The enzyme E.sub.19 is preferably encoded by genes selected
from the group consisting of pckA, pck1, pck2, cg10924, cg17725,
cg17725, pckG, ppcK, cgl2863, pck and 2sck36.02.
[0127] The enzyme E.sub.20 is preferably encoded by genes selected
from the group consisting of oadA, oadB, oadC, oadG, oag3, eda,
dcoA, oadA1, oadA2, pycB and mmdB.
[0128] The enzyme E.sub.21 is preferably encoded by genes selected
from the group consisting of myn8.7, glt1, adr290wp, gltB, gltD,
glt1, gls1, gltA, glt, glxD, gltD1, gltD2, gdh2, ag1040 Cp, gdhA1,
gdhA, gdhA2, gluD, gluD1, gluD2, rocG, ypcA, gudB, t11i18.2,
t2i1.150, mrg7.13, f19c24.7, gdh, gdh1, gdh2, gdh3, got1, got2,
cg4233, cg8430, f23n19.17, f13j11.16, t26c19.9, f7f1.18, F10N7.200,
t1611.170, f15n18.110, t20d1.70, aat1, aat2, ab1038wp, afr211 cp,
agx1, bn a4, aatA, aatB, ybdL, aspC, yfbQ, aat, avtA1, avtA2, tyrB,
avtA, avtB, argD1, argD2, aspB1, aspB2, aspB3, aspB, aspC1, aspC2,
aspC3, aspC4, RS05143, aspAT, ywfG, yhdR, argD, mtnV, alaT, hisC,
avtA1, avtA2, avtA3, cgl0240, cgl1103, cgl2599, cgl2844,
2sck36.07c, sc9e12.21, sc2h4.04c, tyrB, gtp, gtp1, gtp2, cg1640,
f20d23.34, f26f24.16, f24j13.15, t10d10.20 and agr085wp, where
aspC, aatA, gdh, gudB, gdhA, gltB and gltD are especially
preferred.
[0129] The enzyme E.sub.21 is preferably by genes selected from the
group consisting of myn8.7, glt1, adr290wp, gltB, gltD, glt1, gls1,
gltA, glt, glxD, gltD1, gltD2, gdh2, ag1040 Cp, gdhA1, gdhA, gdhA2,
gluD, gluD1, gluD2, rocG, ypcA,
[0130] The enzyme E.sub.22 is preferably encoded by genes selected
from the group consisting of me, me1, me2, me3, mae, mae1, mae2,
sfcA, sfcA1, maeA, maeB, tme, yqkJ, ywkA, yqkJ, malS, ytsJ, mleA,
mleS, mez, sce59.10c, 2sc7g11.23, malS1, malS2, dme, maeB1, maeB2,
mdh, mdh1, mdh2, dmel_cg10120, dmel_cg10120, dmel-cg5889,
f19k16.27, f6f22.7, t22p22.60, f18a17.1, mod1, tme, mao, cg13007,
malS and malE.
[0131] The enzyme E.sub.23 is preferably encoded by genes selected
from the group consisting of me, me1, me2, me3, mae, mae1, mae2,
sfcA, sfcA1, maeA, maeB, tme, yqkJ, ywkA, yqkJ, malS, ytsJ, mleA,
mleS, mez, sce59.10c, 2sc7g11.23, malS1, malS2, dme, maeB1, maeB2,
mdh, mdh1, mdh2, dmel_cg10120, dmel_cg10120, dmel-cg5889,
f19k16.27, f6f22.7, t22p22.60, f18a17.1, mod1, tme, mao, cgl3007,
malS and malE.
[0132] Furthermore, it is preferred in accordance with the
invention that, in the event where the increased provision of
succinyl-coenzyme A in the cell takes place by means of the
above-described pathway
(oxaloacetate.fwdarw.malate.fwdarw.fumarate.fwdarw.succinyl-coenzyme
A), the provision of reduction equivalents in the cell is also
increased in a targeted manner.
[0133] One possibility of increasing the reduction equivalents
consists in increasing the oxidative pentose phosphate pathway. In
this context, it is especially preferred that the activities of
glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and/or of
6-phospho-gluconate dehydrogenase (EC 1.1.1.44), which is
preferably encoded by the gnd gene, are increased while, if
appropriate, simultaneously inhibiting glycolysis, for example by
lowering the activity of glucose 6-phosphate isomerase, as
described in WO-A-01/07626. In addition to, or instead of, the
directed promotion of the pentose phosphate pathway, it may
furthermore be preferred to provide reduction equivalents by
supplying, to the cells, ethanol as the carbon source and by
promoting, in the cells, the conversion of the ethanol into
acetaldehyde by means of alcohol dehydrogenases (EC 1.1.1.1, EC
1.1.1.2, EC 1.1.1.71 or EC 1.1.99.8) and the further conversion of
the acetaldehyde into acetyl coenzyme A by means of acetaldehyde
dehydrogenases (EC 1.2.1.10). Again, suitable genes for alcohol
dehydrogenases and acetaldehyde dehydrogenases, can be found in
gene databases which are known to the skilled worker, such as, for
example, the KEGG database, the NCBI database or the EMBL
database.
[0134] A second pathway from oxaloacetate to succinyl-coenzyme A
leads via citrate as intermediate. In this case, it is preferred in
accordance with a second special embodiment of the above-described
first, second or third alternative of the cell according to the
invention that the cell, if appropriate in addition to an increased
activity of the enzyme E.sub.10 or E.sub.11, features an activity
of at least one of the following enzymes E.sub.13 to E.sub.16 and
E.sub.24 to E.sub.26 which is increased in comparison with its wild
type (see FIG. 8): [0135] of an enzyme E.sub.16, which catalyzes
the conversion of oxaloacetate into citrate; [0136] of an enzyme
E.sub.24, which catalyzes the conversion of citrate into
isocitrate; [0137] of an enzyme E.sub.25, which catalyzes the
conversion of isocitrate into glyoxalate and succinate; [0138] of
an enzyme E.sub.26, which catalyzes the conversion of glyoxalate
into malate; [0139] of an enzyme E.sub.13, which catalyzes the
conversion of malate into fumarate; [0140] of an enzyme E.sub.14,
which catalyzes the conversion of fumarate into succinate; [0141]
of an enzyme E.sub.15, which catalyzes the conversion of succinate
into succinyl-coenzyme A.
[0142] In this context, cells which are especially preferred in
accordance with the invention are those in which the activity of
the following enzymes or enzyme combinations is increased:
E.sub.13, E.sub.14, E.sub.15, E.sub.16, E.sub.24, E.sub.25,
E.sub.26, E.sub.13E.sub.14, E.sub.13E.sub.15, E.sub.13E.sub.16,
E.sub.13E.sub.24, E.sub.13E.sub.25, E.sub.13E.sub.26,
E.sub.14E.sub.15, E.sub.14E.sub.16, E.sub.14E.sub.24,
E.sub.14E.sub.25, E.sub.14E.sub.26, E.sub.15E.sub.16,
E.sub.15E.sub.24, E.sub.15E.sub.25, E.sub.15E.sub.26 and
E.sub.13E.sub.14E.sub.15E.sub.16E.sub.24E.sub.25E.sub.26, where
E.sub.13E.sub.14E.sub.15E.sub.16E.sub.24E.sub.25E.sub.26 is most
preferred.
[0143] In this context, it is especially preferred that the enzyme
[0144] E.sub.13 is a fumarate hydratase (EC 4.2.1.2), [0145]
E.sub.14 is a succinate dehydrogenase (EC 1.3.99.1 or EC 1.3.5.1)
or a succinate quinone oxidoreductase (1.3.5.1), [0146] E.sub.15 is
a succinate coenzyme A ligase (EC 6.2.1.4 or EC 6.2.1.5), [0147]
E.sub.16 is a citrate synthase (EC 2.3.3.1 or EC 2.3.3.8), [0148]
E.sub.24 is an aconitate hydratase (EC 4.2.1.3), [0149] E.sub.25 is
an isocitrate lyase (EC 4.1.3.1) and [0150] E.sub.26 is a malate
synthase (EC 2.3.3.9).
[0151] Preferred genes for the enzymes E.sub.13 to E.sub.16 are
those which have already been described above in connection with
the first pathway from oxaloacetate to succinyl-coenzyme A.
[0152] The enzyme E.sub.24 is preferably encoded by genes selected
from the group consisting of aco1, aco2, ratireb, dmel-CG4706,
dmel-CG4900, dmel-cg6342, cg9244, t3p4.5, f10 m23.310, f4b14.100,
adl032Wp, afr629wp, acnA, acnB, acnC, acnD, rpfA, acnA1, acnA2,
acnM, citB, leuC, cgl1540, sacA, can and aco, where acnA and acnB
are especially preferred.
[0153] The enzyme E.sub.25 is preferably encoded by genes selected
from the group consisting of msd21.4, icl1, icl2, adl066 cp,
agl057wp, aceA, icl, aceAa, aceAb, cgl0097 and cgl2331, where aceA
is especially preferred. In accordance with a particular
embodiment, genes which are preferred are those which code for an
isocitrate lyase which is deregulated at the gene level or protein
level.
[0154] The enzyme E.sub.26 is preferably encoded by genes selected
from the group consisting of med24.5, mlsS1, acr268 cp, masA, glcB,
aceB, mls, glcB-1, glcB-2, cgl2329, masZ, aceB1, aceB2 and mas,
where the aceB gene is especially preferred.
[0155] Again, the nucleotide sequences of suitable genes of the
enzymes E.sub.24 to E.sub.26 can be found in the KEGG database, the
NCBI database or the EMBL database.
[0156] When the provision of oxaloacetate from phosphoenol-pyruvate
or from pyruvate is promoted by increasing the activity of the
enzyme E.sub.10 or E.sub.11, the succinate which is formed, besides
glyoxalate, upon cleavage of the isocitrate by the isocitrate lyase
may also be utilized for the formation of succinyl-coenzyme A.
Furthermore, it may be advantageous in this second pathway from the
oxaloacetate to the succinate to reduce the activity of an enzyme
E.sub.27, which catalyzes the conversion of isocitrate into
2-oxoglutarate and which preferably takes the form of an isocitrate
dehydrogenase (EC 1.1.1.41 or EC 1.1.1.42). Preferably, the
isocitrate dehydrogenase takes the form of an enzyme which is
encoded by a gene selected from the group consisting of idh1, idh2,
cg7176, cg7176, cg7176, f20d21.16, f12p19.10, t15n1.80, idp1, idp2,
idp3, aal022Wp, aer061 Cp, idhC, idhM, icdA, icd, idh, icd1, icd2,
leuB, citC, citC, cgl0664, leuB2, idh3A, idg3B, idh3G, cg12233,
dmel-CG5028, dmel-CG6439, f6p23.14, f23e12.180, f8d20.160,
f12e4.20, adl223wp and afr137 cp, where icdA and citC are
especially preferred.
[0157] A third pathway from the oxaloacetate to the
succinyl-coenzyme A leads via 2-oxoglutarate as intermediate. In
this case, it is preferred in accordance with a third special
embodiment of the above-described first, second or third
alternative of the cell according to the invention that the cell
features an activity of at least one of the following enzymes
E.sub.16, E.sub.24, E.sub.27 and E.sub.28 which is increased in
comparison with its wild type, if appropriate in addition to an
increased activity of the enzyme E.sub.10 or E.sub.11 (see FIG. 9):
[0158] of an enzyme E.sub.16, which catalyzes the conversion of
oxaloacetate into citrate; [0159] of an enzyme E.sub.24, which
catalyzes the conversion of citrate into isocitrate; [0160] of an
enzyme E.sub.27, which catalyzes the conversion of isocitrate into
2-oxoglutarate; [0161] of an enzyme E.sub.28, which catalyzes the
conversion of 2-oxoglutarate into succinyl-coenzyme A.
[0162] In this context, cells which are especially preferred in
accordance with the invention are those in which the activity of
the following enzymes or enzyme combinations is increased:
E.sub.16, E.sub.24, E.sub.27, E.sub.28, E.sub.16E.sub.24,
E.sub.16E.sub.27, E.sub.16E.sub.28, E.sub.24E.sub.27,
E.sub.24E.sub.28, E.sub.27E.sub.28, E.sub.16E.sub.24E.sub.27,
E.sub.16E.sub.24E.sub.28, E.sub.24E.sub.27E.sub.28 and
E.sub.16E.sub.24E.sub.27E.sub.28, where
E.sub.16E.sub.24E.sub.27E.sub.28 is most preferred.
[0163] In this context, it is especially preferred that the enzyme
[0164] E.sub.16 is a citrate synthase (EC 2.3.3.1 or EC 2.3.3.8),
[0165] E.sub.24 is an aconitate hydratase (EC 4.2.1.3), [0166]
E.sub.27 is an isocitrate dehydrogenase (EC 1.1.1.41 or EC
1.1.1.42) and [0167] E.sub.28 is a 2-oxoglutarate synthase (EC
1.2.7.3).
[0168] Preferred genes for the enzymes E.sub.16, E.sub.24 and
E.sub.27 are those which have already been described above in
connection with the first and second pathway from the oxaloacetate
to the succinyl-coenzyme A.
[0169] The enzyme E.sub.28 is preferably encoded by genes selected
from the group consisting of korA, korB, kor D, korA1, korA2,
korB1, korB2, oorA, oorB, oorC, oorD, oforA, oforB, porA, porB,
porA1, porA2, porA3, porA4, porG, porG1, porG2, porB1, porB2,
porB3, SCD20.12c, SCD20.13c, SCAH10.34c, SCAH10.35c, korG, or A,
orB, korG1 and korG2. Furthermore, E.sub.28 may also take the form
of a dehydrogenase complex consisting of a plurality of subunits
which have different enzymatic activities. In particular, it may
take the form of a dehydrogenase complex comprising an oxoglutarate
dehydrogenase (EC 1.2.4.2), a dihydrolipoyl dehydrogenase (EC
1.8.1.4) and a dihydrolipoyllysine-residue succinyl transferase (EC
2.3.1.61). In this context, the oxoglutarate dehydrogenase (EC
1.2.4.2) is preferably encoded by genes selected from the group
consisting of ogdh, ghdhl, loc239017, mgc68800, mgc80496, cg11661,
t22e16.70, mpA24.10, kgd1, aer374 cp, sucA, odhA, kgdA and cgl1129,
where sucA and odhA are especially preferred. The dihydrolipoyl
dehydrogenase (EC 1.8.1.4) is preferably encoded by genes selected
from the group consisting of dld, dld-prov, dldh, cg7430, t2j15.6,
k14a17.6, at3g17240, mgd8.71pd1, afr512wp, dld1, lpd,
tb03.26j7.650, tb04.3 m17.450, tb927.8.7380, tb08.10 k10.200, lpdA,
lpdG, lpdV, lpd3, acoD, lpdA1, lpdA2, lpdA3, odhL, pdhD, pdhD1,
pdhD2, pdhD3, pdhD42, lpdAchl1, lpdAch2, lpdAc, acoL, bfmbC, bkdD,
cgl0366, cgl0688, scm1.17c, pdhL, sck13.11, lpdB2 and dld1, where
lpd is especially preferred. In this context, the
dihydrolipoyllysine-residue succinyl transferase (EC 2.3.1.61) is
preferably encoded by genes selected from the group consisting of
dlst, dlst-prov, mgc89125, dmel_CG5214, f10 m23.250, k13p22.8,
kgd2agl200wp, kgd2, odhB, sucB, aceF, kgdB, sucB1, sucB2, pdhC,
dlaT, kgd, sc5F7.20 and sc4B10.24c, where sucB and odhB are
especially preferred.
[0170] The nucleotide sequences of suitable genes of the enzyme
E.sub.28 or of the abovementioned subunits of the enzyme E.sub.28,
can, again, be found in the KEGG database, the NCBI database or the
EMBL database.
[0171] The above-described pathways from the oxaloacetate to the
succinyl-coenzyme A depart from phosphoenolpyruvate or from
pyruvate as substrate precursors. In this context, it may
furthermore be preferred to genetically modify the cells in such a
way that they are capable of providing especially large amounts of
pyruvate or phosphoenolpyruvate starting from carbohydrates and/or
from glycerol.
[0172] In the event that the cells are capable of utilizing
glycerol as nutrient source, it is preferred that the cell
according to the invention displays an activity of at least one,
preferably all, of the following enzymes E.sub.29 to E.sub.42 which
is increased in comparison with its wild type: [0173] of an enzyme
E.sub.29, which facilitates the diffusion of glycerol into the
cell, [0174] of an enzyme E.sub.30, which catalyzes the conversion
of glycerol into glycerol 3-phosphate, [0175] of an enzyme
E.sub.31, which catalyzes the conversion of glycerol 3-phosphate
into dihydroxyacetone phosphate, [0176] of an enzyme E.sub.32,
which catalyzes the transfer of sulfur to the sulfur acceptor
thioredoxin 1, [0177] of an enzyme E.sub.33, which catalyzes the
hydrolysis of phospholipids with formation of alcohols and
glycerol, [0178] of an enzyme E.sub.34, which catalyzes the
transport of glycerol 3-phosphate into the cell in exchange for
phosphate; [0179] of an enzyme E.sub.35, which catalyzes the
conversion of dihydroxyacetone phosphate into glyceraldehyde
3-phosphate, [0180] of an enzyme E.sub.36, which catalyzes the
conversion of glyceraldehyde 3-phosphate into
1,3-biphosphoglycerate, [0181] of an enzyme E.sub.37, which
catalyzes the conversion of 1,3-biphosphoglycerate into
3-phosphoglycerate, [0182] of an enzyme E.sub.38, which catalyzes
the conversion of 3-phosphoglycerate into 2-phosphoglycerate,
[0183] of an enzyme E.sub.39, which catalyzes the conversion of
2-phosphoglycerate into phosphoenolpyruvate, [0184] of an enzyme
E.sub.40, which catalyzes the conversion of phosphoenolpyruvate
into pyruvate, [0185] of an enzyme E.sub.41, which catalyzes the
conversion of glycerol into dihydroxyacetone, [0186] of an enzyme
E.sub.42, which catalyzes the conversion of dihydroxyacetone into
dihydroxyacetone phosphate.
[0187] In this context, cells which are especially preferred in
accordance with the invention are those in which the activity of
the following enzymes or enzyme combinations is reduced: E.sub.29,
E.sub.30, E.sub.31, E.sub.32, E.sub.33, E.sub.34, E.sub.35,
E.sub.36, E.sub.37, E.sub.38, E.sub.39, E.sub.40, E.sub.41,
E.sub.42, E.sub.29E.sub.30, E.sub.29E.sub.31, E.sub.29E.sub.32,
E.sub.29E.sub.33, E.sub.29E.sub.34, E.sub.29E.sub.35,
E.sub.29E.sub.36, E.sub.29E.sub.37, E.sub.29E.sub.38,
E.sub.29E.sub.39, E.sub.29E.sub.40, E.sub.29E.sub.41,
E.sub.29E.sub.42, E.sub.30E.sub.31, E.sub.30E.sub.32,
E.sub.30E.sub.33, E.sub.30E.sub.34, E.sub.30E.sub.35,
E.sub.30E.sub.36, E.sub.30E.sub.37, E.sub.30E.sub.38,
E.sub.30E.sub.39, E.sub.30E.sub.40, E.sub.30E.sub.41,
E.sub.30E.sub.42, E.sub.31E.sub.32, E.sub.31E.sub.33,
E.sub.31E.sub.34, E.sub.31E.sub.35, E.sub.31E.sub.36,
E.sub.31E.sub.37, E.sub.31E.sub.38, E.sub.31E.sub.39,
E.sub.31E.sub.40, E.sub.31E.sub.41, E.sub.31E.sub.42,
E.sub.32E.sub.33, E.sub.32E.sub.34, E.sub.32E.sub.35,
E.sub.32E.sub.36, E.sub.32E.sub.37, E.sub.32E.sub.38,
E.sub.32E.sub.39, E.sub.32E.sub.40, E.sub.32E.sub.41,
E.sub.32E.sub.42, E.sub.33E.sub.34, E.sub.33E.sub.35,
E.sub.33E.sub.36, E.sub.33E.sub.37, E.sub.33E.sub.38,
E.sub.33E.sub.39, E.sub.33E.sub.40, E.sub.34E.sub.41,
E.sub.33E.sub.42, E.sub.34E.sub.35, E.sub.34E.sub.36,
E.sub.34E.sub.37, E.sub.34E.sub.38, E.sub.34E.sub.39,
E.sub.34E.sub.40, E.sub.34E.sub.41, E.sub.34E.sub.42,
E.sub.35E.sub.36, E.sub.35E.sub.37, E.sub.35E.sub.38,
E.sub.35E.sub.39, E.sub.35E.sub.40, E.sub.35E.sub.41,
E.sub.35E.sub.42, E.sub.36E.sub.37, E.sub.36E.sub.38,
E.sub.36E.sub.39, E.sub.36E.sub.40, E.sub.36E.sub.41,
E.sub.36E.sub.42, E.sub.37E.sub.38, E.sub.37E.sub.39,
E.sub.37E.sub.40, E.sub.37E.sub.41, E.sub.37E.sub.42,
E.sub.38E.sub.39, E.sub.39E.sub.40, E.sub.39E.sub.41,
E.sub.39E.sub.42, E.sub.40E.sub.41, E.sub.40E.sub.42,
E.sub.41E.sub.42 and
E.sub.29E.sub.30E.sub.31E.sub.32E.sub.33E.sub.34E.sub.35E.sub.36E.sub.37E-
.sub.38E.sub.39-E.sub.40E.sub.41E.sub.42.
[0188] In this context, it is especially preferred that the enzyme
[0189] E.sub.29 is an aquaglyceroporin (glycerol facilitator) which
is preferably encoded by the glpF gene, [0190] E.sub.30 is a
glycerol kinase (EC 2.7.1.30) which is preferably encoded by the
glpK gene, [0191] E.sub.31 is a glycerol 3-phosphate dehydrogenase
(EC 1.1.99.5), preferably an FAD-dependent glycerol 3-phosphate
dehydrogenase, where the glycerol 3-phosphate dehydrogenase is
preferably encoded by the glpA gene, the glpB gene, the glpC gene
or the glpD gene, especially preferably by the glpD gene, [0192]
E.sub.32 is a sulfur transferase which is encoded by the glpE gene,
[0193] E.sub.33 is a glycerol phosphodiesterase (EC 3.1.4.46) which
is preferably encoded by the glpQ gene, [0194] E.sub.34 is a
glycerol 3-phosphate permease which is preferably encoded by the
glpT gene, [0195] E.sub.35 is a triose phosphate isomerase (EC
5.3.1.1), [0196] E.sub.36 is a glyceraldehyde 3-phosphate
dehydrogenase (EC 1.2.1.12), [0197] E.sub.37 is a phosphoglycerate
kinase (EC 2.7.2.3), [0198] E.sub.38 is a phosphoglycerate mutase
(EC 5.4.2.1), [0199] E.sub.39 is an enolase (EC 4.2.1.11), [0200]
E.sub.40 is a pyruvate kinase (EC 2.7.1.40), [0201] E.sub.41 is a
glycerol dehydrogenase (EC 1.1.1.6) which is preferably encoded by
the gldA gene, and [0202] E.sub.42 is a dihydroxyacetone kinase (EC
2.7.1.29) which is preferably encoded by the dhaK gene.
[0203] The gene sequences of the abovementioned enzymes can, again,
be found in the gene databases which are known to the skilled
worker, in particular the KEGG database, the NCBI database or the
EMBL database.
[0204] Furthermore, the gap gene, which codes for glyceraldehyde
3-phosphate dehydrogenase (Eikmanns (1992), Journal of Bacteriology
174: 6076-6086), the tpi gene, which codes for triose phosphate
isomerase (Eikmanns (1992), Journal of Bacteriology 174:
6076-6086), and the pgk gene, which codes for 3-phospho-glycerate
kinase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),
are also known from other sources.
[0205] Using the known genes of the enzymes E.sub.29 to E.sub.42,
it is possible to prepare genetically modified cells in which at
least one, preferably at least two, more preferably at least three
and most preferably all activities of the enzymes E.sub.29 to
E.sub.42 has been increased by means of the techniques (mutation of
the enzyme or increase in the expression of the enzyme) described
at the outset in connection with the enzyme E.sub.1. These cells
are capable of being cultured in the presence of glycerol as the
only carbon source (or else together with carbohydrates as further
carbon source).
[0206] In addition to increasing one or more of the enzymatic
activities E.sub.29 to E.sub.42, it may, in the event that the cell
is capable of utilizing glycerol as carbon source, also be
advantageous when the following genes are expressed, preferably
heterologously expressed, in the cells according to the invention:
[0207] the glpG gene or the 3925 gene, [0208] the glpX gene, [0209]
the dhaR gene, the ycgU gene or the b1201 gene, [0210] the fsa
gene, the mipB gene, the ybiZ gene or the B0825 gene, [0211] the
talC gene, the fsaB gene, the yijG gene or the b3946 gene.
[0212] Again, the nucleotide sequences of these genes can be found
in the KEGG database, the NCBI database or the EMBL database.
[0213] In the event that the cells are capable of utilizing
carbohydrates as nutrient source, it is preferred that the cell
according to the invention features an activity of at least one,
preferably of all, of the following enzymes E.sub.43 to E.sub.45
and E.sub.36 to E.sub.40 which is increased in comparison with its
wild type: [0214] of an enzyme E.sub.43, which catalyzes the
conversion of .alpha.-D-glucose 6-phosphate into .beta.-D-fructose
6-phosphate, [0215] of an enzyme E.sub.44, which catalyzes the
conversion of .beta.-D-fructose 6-phosphate into .beta.-D-fructose
1,6-biphosphate, [0216] of an enzyme E.sub.45, which catalyzes the
conversion of .beta.-D-fructose 1,6-biphosphate to glyceraldehyde
3-phosphate and dihydroxyacetone phosphate, [0217] of an enzyme
E.sub.36, which catalyzes the conversion of glyceraldehyde
3-phosphate into 1,3-biphospho-glycerate, [0218] of an enzyme
E.sub.37, which catalyzes the conversion of 1,3-biphosphoglycerate
into 3-phosphoglycerate, [0219] of an enzyme E.sub.38, which
catalyzes the conversion of 3-phosphoglycerate into
2-phosphoglycerate, [0220] of an enzyme E.sub.39, which catalyzes
the conversion of 2-phosphoglycerate into phosphoenolpyruvate, and
[0221] of an enzyme E.sub.40, which catalyzes the conversion of
phosphoenolpyruvate into pyruvate.
[0222] In this context genetically modified cells which are
especially preferred in accordance with the invention are those in
which the activity of the following enzymes or enzyme combinations
is increased: [0223] E.sub.36, E.sub.37, E.sub.38, E.sub.39,
E.sub.40, E.sub.43, E.sub.44, E.sub.45, E.sub.36E.sub.37,
E.sub.36E.sub.38, E.sub.36E.sub.39, E.sub.36E.sub.40,
E.sub.36E.sub.43, E.sub.36E.sub.44, E.sub.36E.sub.45,
E.sub.37E.sub.38, E.sub.37E.sub.39, E.sub.37E.sub.40,
E.sub.37E.sub.43, E.sub.37E.sub.44, E.sub.37E.sub.45,
E.sub.38E.sub.39, E.sub.38E.sub.40, E.sub.38E.sub.43,
E.sub.38E.sub.44, E.sub.38E.sub.45, E.sub.39E.sub.40,
E.sub.39E.sub.43, E.sub.39E.sub.44, E.sub.39E.sub.45,
E.sub.40E.sub.43, E.sub.40E.sub.44, E.sub.40E.sub.45,
E.sub.43E.sub.44, E.sub.43E.sub.45, E.sub.44E.sub.45 and
E.sub.36E.sub.37E.sub.38E.sub.39-E.sub.40E.sub.43E.sub.44E.sub.45.
[0224] In this context, it is especially preferred that the enzyme
[0225] E.sub.43 is a glucose 6-phosphate isomerase (EC 5.3.1.9),
[0226] E.sub.44 is a 6-phosphofructo kinase (EC 2.7.1.11), [0227]
E.sub.45 is a fructose bisphosphate aldolase (EC 4.1.2.13), [0228]
E.sub.36 is a glyceraldehyde 3-phosphate dehydrogenase (EC
1.2.1.12), [0229] E.sub.37 is a phosphoglycerate kinase (EC
2.7.2.3), [0230] E.sub.38 is a phosphoglycerate mutase (EC
5.4.2.1), [0231] E.sub.39 is an enolase (EC 4.2.1.11) and [0232]
E.sub.40 is a pyruvate kinase (EC 2.7.1.40).
[0233] Again, the nucleotide sequences of these genes can be found
be found in the KEGG database, the NCBI database or the EMBL
database.
[0234] In the event that the cell is capable of utilizing
carbohydrates as carbon source, it is furthermore preferred to
promote not only the abovementioned enzymes E.sub.43 to E.sub.45
and E.sub.36 to E.sub.40, but also the uptake of glucose into the
cells, for example by increasing the activity of enzymes of the
phosphotransferase system, in particular those enzymes which are
encoded by ptsI, ptsH and ptsM genes, or by enhancing glucokinase
(EC 2.7.1.2), which is preferably encoded by the glk gene. In this
context, reference is made in particular to U.S. Pat. No.
6,680,187, U.S. Pat. No. 6,818,432, U.S. Pat. No. 6,913,910 and
U.S. Pat. No. 6,884,614, whose disclosure content with regard to
the possibilities for overexpressing the ptsI, ptsH, ptsM and glk
genes is hereby incorporated by reference and forms part of the
disclosure of the present invention. In the event that
carbohydrates act as carbon source, it may also be advantageous to
promote the pentose phosphate pathway in a targeted manner, for
example by increasing the activity of glucose 6-phosphate
dehydrogenase (EC 1.1.1.49) and of 6-phosphogluconate dehydrogenase
(EC 1.1.1.44), which is preferably encoded by the gnd gene, while,
if appropriate, simultaneously inhibiting glycolysis, for example
by weakening the activity of glucose 6-phosphate isomerase, as is
described in WO-A-01/07626.
[0235] In the event that, according to the special embodiment of
the cell according to the invention where methylmalonate
semialdehyde is formed as precursor and succinyl-coenzyme A as
intermediate, the cells form 3-hydroxyisobutyric acid or
polyhydroxyalkanoates based on 3-hydroxyisobutyric acid via
oxaloacetate and pyruvate as intermediates, it may furthermore be
preferred to reduce the activity of at least one, preferably of
all, of the following enzymatic activities in the cell: [0236] of
an enzyme which catalyzes the conversion of oxaloacetate into
phosphoenolpyruvate, such as, for example, phosphoenylpyruvate
carboxykinase (EC 4.1.1.49) (see also DE-A-199 50 409), [0237] of
an enzyme which catalyzes the conversion of pyruvate into acetate
such as, for example, pyruvate oxidase (EC 1.2.2.2) (see also
DE-A-199 51 975), [0238] of an enzyme which catalyzes the
conversion of .alpha.-D-glucose 6-phosphate into .beta.-D-fructose
6-phosphate (see also U.S. Ser. No. 09/396,478), [0239] of an
enzyme which catalyzes the conversion of pyruvate into lactate such
as, for example, l-lactate dehydrogenase (EC 1.1.1.27) or
lactate-malate transhydrogenase (EC 1.1.99.7), [0240] of an enzyme
which catalyzes the conversion of pyruvate into acetyl-coenzyme A
such as, for example, pyruvate dehydrogenase (EC 1.2.1.51), [0241]
of an enzyme which catalyzes the conversion of pyruvate into acetyl
phosphate such as, for example, pyruvate oxidase (EC 1.2.3.3),
[0242] of an enzyme which catalyzes the conversion of pyruvate into
acetate, such as, for example, pyruvate dehydrogenase (EC 1.2.2.2),
[0243] of an enzyme which catalyzes the conversion of pyruvate into
phosphoenolpyruvate such as, for example, phosphoenolpyruvate
synthase (EC 2.7.9.2) or pyruvate, phosphate dikinase (EC 2.7.9.1),
[0244] of an enzyme which catalyzes the conversion of pyruvate into
alanine such as, for example, alanine transaminase (2.6.1.2) or
alanine-oxo-acid transaminase (EC 2.6.1.12), and/or [0245] of an
enzyme which converts pyruvate into acetolactate such as, for
example, acetohydroxy acid synthase (EC 2.2.1.6).
[0246] Cells which are especially preferred in accordance with the
invention and which are capable of forming 3-hydroxyisobutyric acid
or polyhydroxyalkanoates based on 3-hydroxybutyric acid from
carbohydrates as carbon source via succinyl-coenzyme A as
intermediate and in which one or more of the abovementioned
enzymatic activities, in particular one of the enzymatic activities
E.sub.1 to E.sub.45, more preferably the enzymatic activities
E.sub.1, E.sub.1E.sub.2E.sub.3E.sub.4,
E.sub.1E.sub.4E.sub.5E.sub.6E.sub.7 or and
E.sub.1E.sub.4E.sub.5E.sub.7, can be increased are those
microorganisms which have been described by Bennett et al., Metab.
Eng. (2005), 7 (3), pages 229 to 239, Bennett et al., Biotechnol.
Bioeng. (2005), 90 (6), pages 775 to 779, Bennett et al.,
Biotechnol. Prog. (2005), 21 (2), pages 358 to 365, Bennett et al.
(2005), Appl. Microbiol. Biotechnol., 67 (4), pages 515 to 523,
Vemuri et al. (2002), Applied and Environmental Microbiology 68
(4), pages 1715 to 1727 and in U.S. Pat. No. 6,455,284.
[0247] If, according to the first special embodiment of the cell
according to the invention, the formation of 3-hydroxyisobutyric
acid or of the polyhydroxy-alkanoates based on 3-hydroxyisobutyric
acid starting from L-glutamate as carbon source takes place via
succinyl-coenzyme A as intermediate, it is, in a further special
embodiment of the cell according to the invention, in which
methylmalonat semialdehyde is formed as precursor and
succinyl-coenzyme A as intermediate, furthermore preferred in
accordance with the invention that it features an activity of at
least one of the, preferably of the two, following enzymes E.sub.28
and E.sub.46 which is increased in comparison with its wild type
(see FIG. 10): [0248] of an enzyme E.sub.46, which catalyzes the
conversion of L-glutamate into 2-oxoglutarate; [0249] of an enzyme
E.sub.28, which catalyzes the conversion of 2-oxoglutarate into
succinyl-coenzyme A.
[0250] In this context, it is especially preferred that the enzyme
[0251] E.sub.46 is a glutamate synthase (EC 1.4.1.13 or EC
1.4.1.14), a glutamate dehydrogenase (EC 1.4.1.2, EC 1.4.1.3 or EC
1.4.1.4) or an aspartate transaminase (EC 2.6.1.1 or EC 2.6.1.2)
and [0252] E.sub.28 is a 2-oxoglutarate synthase (EC 1.2.7.3).
[0253] Preferred as enzyme E.sub.28 are those which have already
been mentioned at the outset as preferred enzymes E.sub.28.
[0254] The enzyme E.sub.46 is preferably encoded by the genes
selected from the group consisting of: myn8., glt1, adr290wp, gltB,
gltD, yeiT, aegA, ygfT, gltD-1, gltD-2, glt1, glt2, gls1, gltA,
glt, glxD, gltA, yerD, cgl0184, cgl0185, sc3c9.12, gdh1, gdh2,
agl140 cp, gdhA, gdhA1, gdhA2, gluD, rocG, ypcA, gudB, gluD, gdhA,
gdhA2, gdh, gdhA-1, gdhA2-2, gdhA-3, gluD1, gluD2, glud1-prov,
glud1a, t11I18.2, t2I1.150, mrg7.13, got1, got2, caspat, got2-prov,
xr406-prov, 406-prov, cg4233, cg4233, cg8430, cg8430, f23n19.17,
f13j11.16, t26c19.9, f7f1.18, f10n7.200, t1611.170, f15n18.110,
t20d1.70, aat, aat1, aat2, ab1038wp, afr211cp, agx1, bnA4, aatA,
aatB, ybdL, aspC, yfbQ, ydcR, avtA2, aspC-1, aspC-2, aspC-3,
aspC-4, aspB, aspB-1, aspB-2, aspB-3, aspB-4, argD1, argD2, aatAc,
ywfG, mtnV, alaT, avtA1, avtA2, avtA3, cgl0240, cgl1103, cgl2599,
cgl2844, dapC, 2sck36.07c, sc9e12.21, sc2h4.04c, aspB1, aspB2,
aspB3, tyrB, gpt, gpt1, gpt2, mgc82097, cg1640, c32f10.8,
f20d23.34, f26f24.16, f24j13.15, t10d10.20 or agrwp.
[0255] Again, the nucleotide sequences of these genes can be found
be found in the KEGG database, the NCBI database or the EMBL
database.
[0256] In accordance with a second special embodiment of the cell
according to the invention, where the formation of
3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on
3-hydroxyisobutyric acid takes place via methylmalonate
semialdehyde as precursor, it is preferred that the formation of
3-hydroxyisobutyric acid or of the polyhydroxyalkanoate based on
3-hydroxyisobutyric acid takes place via propionyl-coenzyme A as
intermediate, where the cell is capable of preferentially utilizing
carbohydrates, glycerol, methane or methanol as carbon source. In
this context, a variety of pathways exist for arriving at
3-hydroxy-isobutyric acid or polyhydroxyalkanoates based on
3-hydroxyisobutyric acid, departing from propionyl-coenzyme A.
[0257] In accordance with a first alternative of this second
special embodiment of the cell according to the invention, the
formation of intermediate propionyl-coenzyme A takes place via
acetyl-coenzyme A as further intermediate. In this context, it is
especially preferred that the cell features an activity of at least
one of the following enzymes E.sub.4, E.sub.5 and E.sub.47 to
E.sub.52 which is increased in comparison with its wild type (see
FIG. 11): [0258] of an enzyme E.sub.47, which catalyzes the
conversion of acetyl-coenzyme A into malonyl-coenzyme A; [0259] of
an enzyme E.sub.48, which catalyzes the conversion of
malonyl-coenzyme A into malonate semialdehyde; [0260] of an enzyme
E.sub.49, which catalyzes the conversion of malonate semialdehyde
into 3-hydroxypropionate; [0261] of an enzyme E.sub.50, which
catalyzes the conversion of 3-hydroxypropionate into
3-hydroxypropionyl-coenzyme A; [0262] of an enzyme E.sub.51, which
catalyzes the conversion of 3-hydroxypropionyl-coenzyme A into
acryloyl-coenzyme A; [0263] of an enzyme E.sub.52, which catalyzes
the conversion of acryloyl-coenzyme A into propionyl-coenzyme A;
[0264] of an enzyme E.sub.5, which catalyzes the conversion of
propionyl-coenzyme A into methylmalonate semialdehyde; [0265] of an
enzyme E.sub.4, which catalyzes the conversion of methylmalonate
semialdehyde into 3-hydroxy-isobutyrate.
[0266] In this context, genetically modified cells which are
especially preferred in accordance with the invention are those in
which the activity of the following enzymes or enzyme combinations
is increased: E.sub.47, E.sub.48, E.sub.49, E.sub.50, E.sub.51,
E.sub.52, E.sub.4, E.sub.5 and
E.sub.47E.sub.48E.sub.49E.sub.50E.sub.51E.sub.52E.sub.4E.sub.5.
[0267] Furthermore, it is particularly preferred in this context
that the enzyme [0268] E.sub.4 is a 3-hydroxyisobutyrate
dehydrogenase (EC 1.1.1.31) or a 3-hydroxyacyl-coenzyme A
dehydrogenase (EC 1.1.1.35), [0269] E.sub.5 is a
methylmalonate-semialdehyde dehydrogenase (EC 1.2.1.27), [0270]
E.sub.47 is a malonyl-coenzyme A decarboxylase (EC 4.1.1.9), a
malonate-coenzyme A transferase (EC 2.8.3.3), a
methylmalonyl-coenzyme A carboxy-transferase (EC 2.1.3.1) or an
acetyl-coenzyme A carboxylase (EC 6.4.1.2), [0271] E.sub.48 is a
malonate-semialdehyde dehydrogenase (EC 1.2.1.18), [0272] E.sub.49
is a 3-hydroxypropionate dehydrogenase (EC 1.1.1.59), [0273]
E.sub.50 is a 3-hydroxyisobutyryl-coenzyme A hydrolase (EC
3.1.2.4), [0274] E.sub.51 is an enoyl-coenzyme A hydratase (EC
4.2.1.17) and [0275] E.sub.52 is an acyl-coenzyme A dehydrogenase
(EC 1.3.99.3).
[0276] Preferred genes for the enzymes E.sub.4 and E.sub.5 are
those which have already been described above in connection with
the first special embodiment of the cell according to the
invention.
[0277] The enzyme E.sub.47 is preferably encoded by genes selected
from the group consisting of mlycd, t19b17.4, tb08.2904.110, matA,
acac, acaca, acacb, f5j5.21, f15c21.2, t8p21.5, acc1, aar071wp,
accA, accB, accC, accD, accC1, accC2, mmdA, fabG, accD1, accD2,
accD3, cgl0831, accBC, dtsR1, accDA, scc24.16c and cgl1327, where
accA, accC and accD are most preferred.
[0278] The enzyme E.sub.48 is preferably encoded by the iolD
gene.
[0279] The enzyme E.sub.51 is preferably encoded by genes selected
from the group consisting of echs1, ehhadh, hadha, echs1-prov,
cg4389, cg4389, cg6543, cg6984, cg8778, ech-1, ech-2, ech-3, ech-4,
ech-5, ech-6, ech-7, FCAALL.314, fcaall.21, fox2, eci1, eci2, paaF,
paaG, yfcX, fadB, faoA, rpfF, phaA, phaB, echA1, echA2, echA3,
echA4, echA5, echA6, echA7, echA8, echA9, echA9, echA10, echA11,
echA12, echA13, echA14, echA15, echA16, echA17, echA18, echA19,
echA20, echA21, fad-1, fad-2, fad-3, dcaE, hcaA, fadJ, rsp0671,
rsp0035, rsp0648, rsp0647, rs03234, rs03271, rs04421, rs04419,
rs02820, rs02946, paaG1, paaG2, paaG3, ech, pksH, ydbS, eccH1,
eccH2, pimF, fabJ1, fabJ2, caiD2, ysiB, yngF, yusL, fucA, cgl0919,
scf41.23, scd10.16, sck13.22, scp8.07c, stbac16h6.14, sc5f2a.15,
sc6a5.38, hbd-1, hbd-2, hdb-3, hdb-4, hdb-5, hdb-6, hdb-7, hdb-8,
hdb-9, hdb-10, fad-1, fad-2, fad-3, fad-4, fad-5, paaF-1, paaF-2,
paaF-3, paaF-4, paaF-5, paaF-6, paaF-7 and crt.
[0280] The enzyme E.sub.52 is preferably encoded by genes selected
from the group consisting of acadl, acadm, acad10, acad11,
acadm-prov, acadl-prov, mgc81873, cg12262, cg4703, cg4860, f3e22.5,
af1213wp, acdC, fadE13, acd-1, acd-2, acd-3, acd-4, acd-5, acd-6,
acd-7, acd-8, acd-9, acd-10, acd-11, acd-12, acd, fadE1, fadE2,
fadE3, fadE4, fadE5, fadE6, fadE7, fadE13, fadE14, fadE15, fadE16,
fadE17, fadE18, fadE19, fadE20, fadE21, fadE22, fadE23, fadE26,
fadE27, fadE30, fadE31, fadE33, fadE35, fadE38, fadE45, fadE, caiA,
aidB, RSp0036, RS03588, mmgC, acdA-3, bcd, acdA, acdH1, acdH2,
acdH3, aidB, acdI and acdH.
[0281] The nucleotide sequences of suitable genes for the enzymes
E.sub.47 to E.sub.52, in particular also of the enzymes E.sub.49
and E.sub.50, can be found in the KEGG database, the NCBI database
or the EMBL database.
[0282] According to a second alternative of this second special
embodiment of the cell according to the invention, the formation of
the intermediate propionyl-coenzyme A also takes place via
acetyl-coenzyme A as further intermediate, where, according to this
alternative, the propionyl-coenzyme A is not converted directly
into the methylmalonate semialdehyde, but via
methylmalonyl-coenzyme A. In this context, it is especially
preferred that the cell features an activity of at least one of the
following enzymes E.sub.2 to E.sub.4, E.sub.6, E.sub.7 and E.sub.47
to E.sub.52 which is increased in comparison with its wild type
(see FIG. 12): [0283] of an enzyme E.sub.47, which catalyzes the
conversion of acetyl-coenzyme A into malonyl-coenzyme A; [0284] of
an enzyme E.sub.48, which catalyzes the conversion of
malonyl-coenzyme A into malonate semialdehyde; [0285] of an enzyme
E.sub.49, which catalyzes the conversion of malonate semialdehyde
into 3-hydroxypropionate; [0286] of an enzyme E.sub.50, which
catalyzes the conversion of 3-hydroxypropionate into
3-hydroxypropionyl-coenzyme A; [0287] of an enzyme E.sub.51, which
catalyzes the conversion of 3-hydroxypropionyl-coenzyme A into
acryloyl-coenzyme A; [0288] of an enzyme E.sub.52, which catalyzes
the conversion of acryloyl-coenzyme A into propionyl-coenzyme A;
[0289] of an enzyme E.sub.7, which catalyzes the conversion of
propionyl-coenzyme A into (S)-methylmalonyl-coenzyme A; [0290] of
an enzyme E.sub.6, which catalyzes the conversion of
(S)-methylmalonyl-coenzyme A into (R)-methylmalonyl-coenzyme A;
[0291] of an enzyme E.sub.2, which catalyzes the conversion of
(R)-methylmalonyl-coenzyme A into methyl malonate; [0292] of an
enzyme E.sub.3, which catalyzes the conversion of methyl malonate
into methylmalonate semialdehyde; [0293] of an enzyme E.sub.4,
which catalyzes the conversion of methylmelonate-semialdehyde into
3-hydroxy-isobutyrate.
[0294] In this context, genetically modified cells which are
especially preferred according to the invention are those in which
the activity of the following enzymes or enzyme combinations is
increased: E.sub.2, E.sub.3, E.sub.4, E.sub.6, E.sub.7, E.sub.47,
E.sub.48, E.sub.49, E.sub.50, E.sub.51, E.sub.52 and
E.sub.2E.sub.3E.sub.4E.sub.6E.sub.7E.sub.47E.sub.48E.sub.49E.sub.50E.sub.-
51E.sub.52.
[0295] Preferred enzymes and genes of these enzymes are those genes
and enzymes which have already been mentioned above in connection
with the enzymes E.sub.2 to E.sub.4, E.sub.6, E.sub.7 and E.sub.47
to E.sub.52.
[0296] According to a third alternative of this first alternative
of the second special embodiment of the cell according to the
invention, the formation of the intermediate propionyl-coenzyme A
also takes place via acetyl-coenzyme A as further intermediate,
where, according to this alternative, the propionyl-coenzyme A is,
again, not converted directly into methylmalonate-semialdehyde, but
via (R)-methylmalonyl-coenzyme A (and not via
(S)-methylmalonyl-coenzyme A). In this context, it is especially
preferred that the cell features an activity of at least one of the
following enzymes E.sub.2 to E.sub.4, E.sub.7 and E.sub.47 to
E.sub.52 which is increased in comparison with its wild type (see
FIG. 13): [0297] of an enzyme E.sub.47, which catalyzes the
conversion of acetyl-coenzyme A into malonyl-coenzyme A; [0298] of
an enzyme E.sub.48, which catalyzes the conversion of
malonyl-coenzyme A into malonate semialdehyde; [0299] of an enzyme
E.sub.49, which catalyzes the conversion of malonate semialdehyde
into 3-hydroxypropionate; [0300] of an enzyme E.sub.50, which
catalyzes the conversion of 3-hydroxypropionate into
3-hydroxypropionyl-coenzyme A; [0301] of an enzyme E.sub.51, which
catalyzes the conversion of 3-hydroxypropionyl-coenzyme A into
acryloyl-coenzyme A; [0302] of an enzyme E.sub.52, which catalyzes
the conversion of acryloyl-coenzyme A into propionyl-coenzyme A;
[0303] of an enzyme E.sub.7, which catalyzes the conversion of
propionyl-coenzyme A into methylmalonyl-coenzyme A; [0304] of an
enzyme E.sub.2, which catalyzes the conversion of
methylmalonyl-coenzyme A into methylmalonate; [0305] of an enzyme
E.sub.3, which catalyzes the conversion of methyl malonate into
methylmalonate-semialdehyde; [0306] of an enzyme E.sub.4, which
catalyzes the conversion of methylmalonate-semialdehyde into
3-hydroxy-isobutyrate.
[0307] In this context, genetically modified cells which are
especially preferred according to the invention are those in which
the activity of the following enzymes or enzyme combinations is
increased: E.sub.2, E.sub.3, E.sub.4, E.sub.7, E.sub.47, E.sub.48,
E.sub.49, E.sub.50, E.sub.51, E.sub.52 and
E.sub.2E.sub.3E.sub.4E.sub.7E.sub.47E.sub.48E.sub.49E.sub.50E.sub.51E.sub-
.52.
[0308] Preferred enzymes and genes of these enzymes are, again,
those genes and enzymes which have already been mentioned above in
connection with the enzymes E.sub.2 to E.sub.4, E.sub.7 and
E.sub.47 to E.sub.52.
[0309] According to a fourth alternative of the second special
embodiment of the cell according to the invention, the formation of
the intermediate propionyl-coenzyme A also takes place via
acetyl-coenzyme A as further intermediate, where, according to this
alternative, acetoacetyl-coenzyme A is formed as intermediate. In
this context, it may be preferred that the cell features an
activity of at least one of the following enzymes E.sub.8 and
E.sub.53 to E.sub.61 which is increased in comparison with its wild
type: [0310] of an enzyme E.sub.53, which catalyzes the conversion
of acetyl-coenzyme A into acetoacetyl-coenzyme A; [0311] of an
enzyme E.sub.54, which catalyzes the conversion of
acetoacetyl-coenzyme A into 3-hydroxybutanoyl-coenzyme A; [0312] of
an enzyme E.sub.55, which catalyzes the conversion of
3-hydroxybutanoyl-coenzyme A into crotonyl-coenzyme A; [0313] of an
enzyme E.sub.56, which catalyzes the conversion of
crotonyl-coenzyme A into butyryl-coenzyme A; [0314] of an enzyme
E.sub.57, which catalyzes the conversion of butyryl-coenzyme A into
ethylmalonyl-coenzyme A; [0315] of an enzyme E.sub.58, which
catalyzes the conversion of ethylmalonyl-coenzyme A into
methylsuccinyl-coenzyme A; [0316] of an enzyme E.sub.59, which
catalyzes the conversion of methylsuccinyl-coenzyme A
isobutyryl-coenzyme A; [0317] of an enzyme E.sub.60, which
catalyzes the conversion of isobutyryl-coenzyme A into
methacrylyl-coenzyme A; [0318] of an enzyme E.sub.61, which
catalyzes the conversion of methacrylyl-coenzyme A into
3-hydroxyisobutyryl-coenzyme A; [0319] of an enzyme E.sub.8, which
catalyzes the conversion of 3-hydroxyisobutyryl-coenzyme A into
3-hydroxy-isobutyrate.
[0320] In this context, genetically modified cells which are
especially preferred according to the invention are those in which
the activity of the following enzymes or enzyme combinations is
increased: E.sub.8, E.sub.53, E.sub.54, E.sub.55, E.sub.56,
E.sub.57, E.sub.58, E.sub.59, E.sub.60, E.sub.61 and
E.sub.8E.sub.53E.sub.54E.sub.55E.sub.56E.sub.57E.sub.58E.sub.59E.sub.60E.-
sub.61.
[0321] This metabolic pathway and the enzymes which play a role in
this metabolic pathway are described, for example, in Korotkova et
al., Journal of Bacteriology (2002), pages 1750 to 1758.
[0322] According to a fifth alternative of the second special
embodiment of the cell according to the invention, the formation of
the intermediate propionyl-coenzyme A takes place via, again,
acetyl-coenzyme A as further intermediate, where, according to this
alternative, acetoacetyl-coenzyme A is formed as further
intermediate but where, in this case, ethylmalonyl-coenzyme A is
formed directly from crotonyl-coenzyme A. In this context, it may
be preferred that the cell features an activity of at least one of
the following enzymes E.sub.8 and E.sub.53 to E.sub.56 and E.sub.62
to E.sub.65 which is increased in comparison with its wild type
(see FIG. 14): [0323] of an enzyme E.sub.53, which catalyzes the
conversion of two acetyl-coenzyme A units into acetoacetyl-coenzyme
A; [0324] of an enzyme E.sub.54, which catalyzes the conversion of
acetoacetyl-coenzyme A into 3-hydroxybutyryl-coenzyme A; [0325] of
an enzyme E.sub.55, which catalyzes the conversion of
3-hydroxybutyryl-coenzyme A into crotonyl-coenzyme A; [0326] of an
enzyme E.sub.56, which catalyzes the conversion of
crotonyl-coenzyme A into ethylmalonyl-coenzyme A; [0327] of an
enzyme E.sub.62, which catalyzes the conversion of
ethylmalonyl-coenzyme A into methylsuccinyl-coenzyme A; [0328] of
an enzyme E.sub.63, which catalyzes the conversion of
methylsuccinyl-coenzyme A into mesaconyl-coenzyme A; [0329] of an
enzyme E.sub.64, which catalyzes the conversion of
mesaconyl-coenzyme A into .beta.-methylmalyl-coenzyme A; [0330] of
an enzyme E.sub.65, which catalyzes the conversion of
.beta.-methylmalyl-coenzyme A into glyoxylate and
propionyl-coenzyme A.
[0331] Then, from propionyl-coenzyme A can in the above-described
manner (increasing the activity of one or more of the enzymes
E.sub.7, E.sub.2, E.sub.3 and E.sub.4, increasing the activity of
one or more of the enzymes E.sub.7, E.sub.6, E.sub.2, E.sub.3 and
E.sub.4, or increasing the activity of one of the, or of both,
enzymes E.sub.4 and E.sub.5).
[0332] In this context, it is especially preferred that the enzyme
[0333] E.sub.53 is a .beta.-ketothiolase (EC 2.3.1.9), [0334]
E.sub.54 is an acetoacetyl-coenzyme A reductase (an EC 1.1.1.36),
[0335] E.sub.55 is an enoyl-coenzyme A hydratase (EC 4.2.1.17),
[0336] E.sub.56 is a crotonyl-coenzyme A decarboxylase, [0337]
E.sub.62 is an ethylmalonyl-coenzyme A mutase (EC 5.4.99.2), [0338]
E.sub.63 is a methylsuccinyl-coenzyme A dehydrogenase, [0339]
E.sub.64 is a mesaconyl-coenzyme A hydratase, and [0340] E.sub.65
is a .beta.-methylmalyl/L-malyl-coenzyme A lyase.
[0341] The enzyme E.sub.53 is preferably encoded by genes selected
from the group consisting of acat1, acat2, loc484063, loc489-421,
mgc69098, mgc81403, mgc81256, mgc83664, kat-1, erg10, ygeF, atoB,
fadAx, phbA-1, phbA-2, atoB-2, pcaF, pcaF-2, phb-A, bktB, phaA,
tioL, thlA, fadA, paaJ, phbAf, pimB, mmgA, yhfS, thl, vraB, th1,
mvaC, thiL, paaJ, fadA3, fadA4, fadA5, fadA6, cgl12392, catF,
sc8f4.03, thiL1, thiL2, acaB1, acaB2, acaB3, acaB4 or, where acat1,
acat2, atoB and phbA and the corresponding gene from Rhodobacter
sphaeroides are especially preferred.
[0342] The enzyme E.sub.54 is preferably encoded by genes selected
from the group consisting of phbB, fabG, phbN1, phbB2 or cgl12444,
where phbB is especially preferred and the corresponding gene from
Rhodobacter sphaeroides is especially preferred.
[0343] The enzyme E.sub.55 is preferably encoded by genes selected
from the group consisting of echS1, ehhadh, hadha, echs1-prov, Das
Enzym E.sub.55 wird vorzugsweise von Genen ausgewahlt aus der
Gruppe bestehend aus echS1, ehhadh, hadha, echs1-prov, cg4389,
cg4389, cg6543, cg6984, cg8778, ech-1, ech-2, ech-3, ech-4, ech-5,
ech-6, ech-7, FCAALL.314, fcaal1.21, fox2, eci1, eci2, paaF, paaG,
yfcX, fadB, faoA, rpfF, phaA, phaB, echA1, echA2, echA3, echA4,
echA5, echA6, echA7, echA8, echA9, echA9, echA10, echA11, echA12,
echA13, echA14, echA15, echA16, echA17, echA18, echA19, echA20,
echA21, fad-1, fad-2, fad-3, dcaE, hcaA, fadJ, rsp0671, rsp0035,
rsp0648, rsp0647, rs03234, rs03271, rs04421, rs04419, rs02820,
rs02946, paaG1, paaG2, paaG3, ech, pksH, ydbS, eccH1, ecCH2, pimF,
fabJ1, fabJ2, caiD2, ysiB, yngF, yusL, fucA, cgl0919, scf41.23,
scd10.16, sck13.22, scp8.07c, stbac16h6.14, sc5f2a.15, sc6a5.38,
hbd-1, hbd-2, hdb-3, hdb-4, hdb-5, hdb-6, hdb-7, hdb-8, hdb-9,
hdb-10, fad-1, fad-2, fad-3, fad-4, fad-5, paaF-1, paaF-2, paaF-3,
paaF-4, paaF-5, paaF-6, paaF-7 and crt
where the corresponding gene from Rhodobacter sphaeroides is
especially preferred.
[0344] The enzyme which is preferably employed as enzyme E.sub.56
is an enzyme from Rhodobacter sphaeroides which is encoded by the
DNA sequence with the SEQ ID No 05 and which has the amino acid
sequence as shown in SEQ ID No 06.
[0345] Suitable genes for the enzyme E.sub.62 are selected from the
group consisting of mut, mutA, mutB, sbm, sbmA, sbmB, sbm5, bhbA,
mcmA, mcmA1, mcmA2, mcmB, mcm1, mcm2, mcm3, icmA, meaA1 and meaA2,
where, again, the corresponding gene from Rhodobacter sphaeroides
is especially preferred.
[0346] Preferred genes for the enzymes E.sub.63, E.sub.64 and
E.sub.65 are, in particular, the genes for these enzymes from
Rhodobacter sphaeroides.
[0347] Further examples of nucleotide sequences of the
abovementioned genes can also be found in the KEGG database, the
NCBI database or the EMBL database, inter alia.
[0348] As has already been explained above, the first alternative
of the second preferred embodiment cell according to the invention
generates 3-hydroxyisobutyric acid or the polyhydroxyalkanoates
based on 3-hydroxyisobutyric acid via propionyl-coenzyme A and
acetyl-coenzyme A as intermediates. In this context, it may be
meaningful, in principle, to influence not only one or more of the
abovementioned enzymatic activities E.sub.2 to E.sub.8 and E.sub.47
to E.sub.65, but also those enzymatic activities which bring about
an increase in the formation of acetyl-coenzyme A in the cell.
[0349] In the event that 3-hydroxyisobutyric acid is formed from
carbohydrates or glycerol as carbon source, it may be preferred
that the cell features an increased activity in an enzyme E.sub.66,
which catalyzes the conversion of pyruvate into acetyl-coenzyme A.
This enzyme E.sub.66 preferably takes the form of a pyruvate
dehydrogenase (EC 1.2.1.51).
[0350] In the event that 3-hydroxyisobutyric acid is formed from
C.sub.1-carbon sources such as, for example, methane or methanol,
it may be preferred that the cell features an activity of at least
one of the enzymes E.sub.67 to E.sub.71 which is increased in
comparison with its wild type: [0351] of an enzyme E.sub.67, which
catalyzes the conversion of methane into methanol; [0352] of an
enzyme E.sub.68, which catalyzes the conversion of methanol into
formaldehyde; [0353] of an enzyme E.sub.69, which catalyzes the
conversion of formaldehyde into 5,10-methylenetetrahydrofolate;
[0354] of an enzyme E.sub.70, which catalyzes the conversion of
5,10-methylenetetrahydrofolate into 5-methyltetrahydrofolate;
[0355] of an enzyme E.sub.71, which catalyzes the conversion of
5-methyltetrahydrofolate into acetyl-coenzyme A.
[0356] In this context, it is especially preferred that the enzyme
[0357] E.sub.67 is a methane monooxygenase (EC 1.14.13.25), [0358]
E.sub.68 is a methanol dehydrogenase (EC 1.1.1.244), [0359]
E.sub.69 is a methylmalonate-semialdehyde dehydrogenase (EC
1.2.1.27), [0360] E.sub.70 is a methylenetetrahydrofolate reductase
(EC 1.5.1.20), [0361] E.sub.71 is a carbon monoxide dehydrogenase
(EC 1.2.99.2).
[0362] The nucleotide sequences of suitable genes for the enzymes
E.sub.63 to E.sub.67 can be found in the KEGG database, the NCBI
database or the EMBL database.
[0363] According to a third special embodiment of the cell
according to the invention, where the formation of
3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on
3-hydroxyisobutyric acid takes place via
methylmalonate-semialdehyde as precursor, it is preferred that the
formation of 3-hydroxyisobutyric acid or of the
polyhydroxyalkanoate based on 3-hydroxyisobutyric acid takes place
via acryloyl-coenzyme A as intermediate, where the cell is capable
of preferentially utilizing carbohydrates, glycerol or glutamate as
carbon source.
[0364] In connection with the third special embodiment of the cell
according to the invention, it is especially preferred when this
cell features an activity of at least one of the following enzymes
E.sub.10 to E.sub.12, E.sub.56, E.sub.72 and E.sub.73 which is
increased in comparison with its wild type (see FIG. 15): [0365] of
an enzyme E.sub.72, which catalyzes the conversion of beta-alanine
to beta-alanyl-coenzyme A, [0366] of an enzyme E.sub.73, which
catalyzes the conversion of beta-alanyl-coenzyme A into
acrylyl-coenzyme A, [0367] of an enzyme E.sub.56, which catalyzes
the conversion of acrylyl-coenzyme A into methylmalonyl-coenzyme A,
[0368] of an enzyme E.sub.10, which catalyzes the conversion of
methylmalonyl-coenzyme A into methyl malonate; [0369] of an enzyme
E.sub.11, which catalyzes the conversion of methyl malonate into
methylmalonate-semialdehyde; [0370] of an enzyme E.sub.12, which
catalyzes the conversion of methylmalonate-semialdehyde into
3-hydroxyisobutyric acid.
[0371] In this context, cells which are especially preferred
according to the invention are those in which the activity of the
following enzymes or enzyme combinations is increased:
E.sub.56E.sub.10, E.sub.56E.sub.11, E.sub.56E.sub.12,
E.sub.56E.sub.10E.sub.11 and
E.sub.72E.sub.73E.sub.56E.sub.10E.sub.11E.sub.12. In connection
with the fourth special embodiment, too, of the cell according to
the invention it may be advantageous to overexpress an enzyme which
is capable of catalyzing at least two of the above-described
reaction steps. Here too, it is possible for example to employ an
enzyme which features both the activity of the enzyme E.sub.10 and
the activity of the enzyme E.sub.11, such as, for example, the
malonyl-coenzyme A reductase from Sulfolobus tokodaii, which is
encoded by the DNA sequence with the SEQ ID No 03 and which
features the amino acid sequence as shown in SEQ ID No 04.
Furthermore, it is, in principle, also possible in the context of
the fourth special embodiment of the cell according to the
invention to employ a cell which is already capable of forming
especially large amounts of acrylyl-coenzyme A.
[0372] In this context, it is especially preferred that the enzyme
[0373] E.sub.72 is a coenzyme A transferase (EC 2.8.3.1) or a
coenzyme A synthetase, preferably a coenzyme A transferase, [0374]
E.sub.73 is a beta-alanyl-coenzyme A ammonia-lyase (EC 4.3.1.6),
[0375] E.sub.56 is a crotonyl-coenzyme A decarboxylase [0376]
E.sub.10 is a methylmalonyl-coenzyme A hydrolase (EC 3.1.2.17),
[0377] E.sub.11 is an aldehyde dehydrogenase (EC 1.2.1.3) or an
aldehyde oxidase (EC 1.2.3.1) and [0378] E.sub.12 is a
3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) or a
3-hydroxyacyl-coenzyme A dehydrogenase (EC 1.1.1.35).
[0379] Preferred enzymes E.sub.72 with a CoA transferase activity
are those from Megasphaera elsdenii, Clostridium propionicum,
Clostridium kluyveri and also from Escherichia coli. Examples which
may be mentioned at this point of a DNA sequence coding for a CoA
transferase is the sequence from Megasphaera elsdenii referred to
as SEQ ID No: 24 in WO-A-03/062173. Enzymes which are furthermore
preferred are those variants of the CoA transferase which are
described in WO-A-03/062173.
[0380] Suitable enzymes E.sub.73 with a beta-alanyl-coenzyme A
ammonia-lyase activity are, for example, those from Clostridium
propionicum. DNA sequences which code for such an enzyme can be
obtained for example from Clostridium propionicum as described in
Example 10 in WO-A-03/062173. The DNA sequence which codes for the
beta-alanyl-coenzyme A ammonia-lyase from Clostridium propionicum
is specified in WO-A-03/062173 as SEQ ID No: 22.
[0381] An enzyme E.sub.56 which is preferably employed is, again,
the crotonyl-coenzyme A decarboxylase from Rhodobacter sphaeroides,
which is encoded by the DNA sequence with the SEQ ID No 05 and
which features the amino acid sequence as shown in SEQ ID No 06.
This enzyme is not only capable of converting crotonyl-coenzyme A
into ethylmalonyl-coenzyme A, but also of converting
acrylyl-coenzyme A into methylmalonyl-coenzyme A.
[0382] Suitable genes for the enzymes E.sub.10 to E.sub.12 have
already been mentioned in connection with the first variant of the
cell according to the invention, where it is also preferred in
connection with the second variant, the above-described gene from
Sulfolobus tokodaii is especially preferred as gene for the enzyme
E.sub.11.
[0383] According to an especially preferred variant of the third
special embodiment of the cell according to the invention, this
cell features at least one activity of the enzyme E.sub.10 and
E.sub.56 or of the enzymes E.sub.10, E.sub.11 and E.sub.56 which is
increased in comparison with its wild type, where the E.sub.10 or
the enzymes E.sub.10 and E.sub.11 is encoded by a DNA sequence as
shown in SEQ ID No 03 and the enzyme E.sub.56 is encoded by a DNA
sequence as shown in SEQ ID No 05.
[0384] In this context, it is preferred when the increased activity
of these two enzymes is achieved by overexpressing, in the cell,
the polypeptides with SEQ ID No 04 and SEQ ID No 06 or else that
amino acid sequences with at least 50%, preferably at least 55%,
more preferably at least 60%, more preferably at least 65% and most
preferably at least 70% identity with the amino acid sequence as
shown in SEQ ID No 04 and SEQ ID No 06, respectively. In this
context, these two DNA sequences may be integrated into the genome
of the cell or else be present on a vector inside the cell.
[0385] In connection with the above-described third special
embodiment of the cell according to the invention, it may
furthermore be advantageous when the cell features not only an
increase in the activity of the enzyme E.sub.56 and/or of the
activity of the enzyme E.sub.10 or of the enzymes E.sub.10 and
E.sub.11, but at least one, preferably both, of the following
properties: [0386] an activity of an enzyme E.sub.n, which
catalyzes the conversion of pyruvate into oxaloacetate or of an
enzyme E.sub.74, which catalyzes the conversion of
phosphoenolpyruvate into oxaloacetate, but preferably of an enzyme
E.sub.11, which catalyzes the conversion of pyruvate into
oxaloacetate, which is increased in comparison with its wild type,
and [0387] an increased activity of an enzyme E.sub.75, which
catalyzes the conversion of aspartate into beta-alanine.
[0388] The enzyme E.sub.11 preferably takes the form of a
carboxylase, especially preferably of a pyruvate carboxylase (EC
number 6.4.1.1), which catalyzes the conversion of pyruvate into
oxaloacetate. A pyruvate carboxylase which is especially preferred
in this context is the mutant which is described in "A novel
methodology employing Corynebacterium glutamicum genome information
to generate a new L-lysine-producing mutant." Ohnishi J et al.,
Applied Microbiology and Biotechnology, Vol. 58 (2), pages 217-223
(2002). In this mutation, the amino acid proline at position 458
has been substituted by serine. The disclosure of this publication
with regard to the possibilities of preparing pyruvate carboxylate
mutants is hereby incorporated by referent and forms part of the
disclosure of the present invention.
[0389] The enzyme E.sub.75 preferably takes the form of a
decarboxylase, especially preferably of a glutamate decarboxylate
or of an aspartate decarboxylase, with a 1-aspartate
1-decarboxylase (EC number 4.1.1.11) which is encoded by the panD
gene being most preferred. Aspartate decarboxylase catalyzes the
conversion of aspartate into beta-alanine. Genes for aspartate
decarboxylase (panD genes) from, inter alia, Escherichia coli (FEMS
Microbiology Letters, 143, pages 247-252 (1996)), "Photorhabdus
luminescens subsp. Laumondii, Mycobacterium bovis subsp. Bovis")
and from a large number of other microorganisms have already been
cloned and sequenced. DE-A-198 55 313 describes in particular the
nucleotide sequence of the panD gene from Corynebacterium
glutamicum. In principle, it is possible to use panD genes of any
feasible origin, no matter whether from bacteria, yeasts or fungi.
Furthermore, it is possible to employ all alleles of the panD gene,
in particular also those which are the result of the degeneracy of
the genetic code or of function-neutral sense mutations. An
aspartate decarboxylase which is especially preferred according to
the invention, besides the aspartate decarboxylase from
Corynebacterium glutamicum, is the Escherichia coli mutant DV9
(Vallari and Rock, Journal of Bacteriology, 164, pages 136-142
(1985)). The disclosure of this publication with regard to the
abovementioned mutant is hereby incorporated by reference and forms
part of the disclosure of the present invention. The preparation of
recombinant cells in which both the activity of the pyruvate
carboxylase and the activity of the aspartate decarboxylase is
increased is described in DE-A-10 2005 048 818.
[0390] According to a second variant of the cell according to the
invention, the formation of 3-hydroxyisobutyric acid or of
polyhydroxyalkanoates based on 3-hydroxy-isobutyric acid takes
place via 3-hydroxyisobutyryl-coenzyme A as precursor.
[0391] In the event that, in the cell according to the invention,
the formation of 3-hydroxyisobutyric acid or of
polyhydroxyalkanoates based on 3-hydroxyisobutyric acid takes place
via 3-hydroxyisobutyryl-coenzyme A as precursor, as specified in
the second variant, it is preferred according to a first special
embodiment that the formation of 3-hydroxyisobutyric acid or of the
polyhydroxyalkanoate based on 3-hydroxyisobutyric acid takes place
via isobutyryl-coenzyme A as intermediate, where the cell is
capable of preferentially utilizing carbohydrates, glycerol or
L-valine as carbon source.
[0392] In the event that carbohydrates or glycerol act as the
carbon source, it is preferred, according to a first alternative of
this first special embodiment of the second variant of the cell
according to the invention that this cell features an activity of
at least one of the following enzymes E.sub.76 to E.sub.79,
E.sub.60, E.sub.61 and E.sub.8 which is increased in comparison
with its wild type (see FIG. 16): [0393] of an enzyme E.sub.76,
which catalyzes the conversion of pyruvate into 2-acetolactate;
[0394] of an enzyme E.sub.77, which catalyzes the conversion of
2-acetolactate into 2,3-dihydroxyisovalerate; [0395] of an enzyme
E.sub.78, which catalyzes the conversion of
2,3-dihydroxyisovalerate into 2-oxoisovalerate; [0396] of an enzyme
E.sub.79, which catalyzes the conversion of 2-oxoisovalerate into
isobutyryl-coenzyme A; [0397] of an enzyme E.sub.60, which
catalyzes the conversion of isobutyryl-coenzyme A into
methacrylyl-coenzyme A; [0398] of an enzyme E.sub.61, which
catalyzes the conversion of methacrylyl-coenzyme A into
3-hydroxyisobutyryl-coenzyme A; [0399] of an enzyme E.sub.8, which
catalyzes the conversion of 3-hydroxyisobutyryl-coenzyme A into
3-hydroxy-isobutyrate.
[0400] In this context, genetically modified cells which are
especially preferred in accordance with the invention are those in
which the activity of the following enzymes or enzyme combinations
is increased: E.sub.8, E.sub.60, E.sub.61, E.sub.76, E.sub.77,
E.sub.78, E.sub.79 and
E.sub.8E.sub.60E.sub.61E.sub.76E.sub.77E.sub.78E.sub.79.
[0401] In this context, it is especially preferred that the enzyme
[0402] E.sub.8 is a 3-hydroxyisobutyryl-coenzyme A hydrolase (EC
3.1.2.4), [0403] E.sub.76 is an acetolactate synthase (EC 2.2.1.6),
[0404] E.sub.77 is a dihydroxyisovalerate dehydrogenase (EC
1.1.1.86), [0405] E.sub.78 is a 2,3-dihydroxyisovalerate
dehydratase (EC 4.2.1.9), [0406] E.sub.79 is a 2-oxoisovalerate
dehydrogenase (EC 1.2.1.25 or EC 1.2.4.4), [0407] E.sub.60 is an
acyl-coenzyme A dehydrogenase (EC 1.3.99.3), a butyryl-coenzyme A
dehydrogenase (EC 1.3.99.2) or a 2-methylacyl-coenzyme A
dehydrogenase (EC 1.3.99.12), and [0408] E.sub.61 is an
enoyl-coenzyme A hydratase (EC 4.2.1.17).
[0409] Preferred enzymes E.sub.8, E.sub.60 and E.sub.61 are those
which have already been described above.
[0410] The enzyme E.sub.76 is preferably encoded by genes selected
from the group consisting of ilvbl, t8p19.70, ilv1, ilv2, ilv6,
aal021wp, ael305 cp, ilvI, ilvH, ilvN, ilvB, ilvM, ilvG, ilvN,
budB, ilvN-1, ilvN-2, atrC, ilvX, iolD, budB, alsS, ilvK, ilvB1,
ilvB2, ilvB3, ilvN1, ilvN2, cgl1271, cgl1272, iolD and
scc57A.40c.
[0411] The enzyme E.sub.77 is preferably encoded by genes selected
from the group consisting of f14p22.200, ilv5, ac1198Wp, ilvC,
ilvY, ilvC-1, ilvC-2, ilvC-3 and cgl1273, where the ilvC gene is
most preferred.
[0412] The enzyme E.sub.78 is preferably encoded by genes selected
from the group consisting of f14o13.18, ilv3, acl117wp, ilvD,
cgl1268, ilvD1 and ilvD2, where ilvD is most preferred.
[0413] In the event that L-valine acts as carbon source, it is
preferred according to a second modification of the first special
embodiment of the second alternative of the cell according to the
invention, where the formation of 3-hydroxyisobutyric acid or of
poly-hydroxyalkanoates based on 3-hydroxyisobutyric acid takes
place via 3-hydroxyisobutyryl-coenzyme A as precursor and
isobutyryl-coenzyme A as intermediate, that this cell features an
activity of at least one of the following enzymes E.sub.79,
E.sub.80, E.sub.60, E.sub.61 and E.sub.8 which is increased in
comparison with its wild type (see FIG. 17): [0414] of an enzyme
E.sub.80, which catalyzes the conversion of L-valine into
2-oxoisovalerate; [0415] of an enzyme E.sub.79, which catalyzes the
conversion of 2-oxoisovalerate into isobutyryl-coenzyme A; [0416]
of an enzyme E.sub.60, which catalyzes the conversion of
isobutyryl-coenzyme A into methacrylyl-coenzyme A; [0417] of an
enzyme E.sub.61, which catalyzes the conversion of
methacrylyl-coenzyme A into 3-hydroxyisobutyryl-coenzyme A; [0418]
of an enzyme E.sub.8, which catalyzes the conversion of
3-hydroxyisobutyryl-coenzyme A into 3-hydroxyisobutyrate.
[0419] In this context, genetically modified cells which are
especially preferred in accordance with the invention are those in
which the activity of the following enzymes or enzyme combinations
is increased: E.sub.8, E.sub.60, E.sub.61, E.sub.79, E.sub.80 and
E.sub.8E.sub.60E.sub.61E.sub.79E.sub.80.
[0420] In this context, it is especially preferred that the enzyme
[0421] E.sub.8 is a 3-hydroxyisobutyryl-coenzyme A hydrolase (EC
3.1.2.4), [0422] E.sub.60 is an acyl-coenzyme A dehydrogenase (EC
1.3.99.3), a butyryl-coenzyme A dehydrogenase (EC 1.3.99.2) or a
2-methylacyl-coenzyme A dehydrogenase (EC 1.3.99.12), [0423]
E.sub.61 is an enoyl-coenzyme A hydratase (EC 4.2.1.17), [0424]
E.sub.79 is a 2-oxoisovalerate dehydrogenase (EC 1.2.1.25 or EC
1.2.4.4), and [0425] E.sub.80 is an amino acid transferase (EC
2.6.1.42).
[0426] Preferred enzymes E.sub.8, E.sub.60, E.sub.61 and E.sub.79
are those which have already been described above.
[0427] The enzyme E.sub.80 is preferably encoded by genes selected
from the group consisting of bcat1, bcat2, t27I1.8, t27i1.9,
f2j10.5, f2j10.4, t12h1.16, mmb12.20, t9c5.3, mpa24.13, bat1, bat2,
ad1384wp, eca39, bcaA, ilvE, ilvE1, ilvE2, ilvE3, ywaA, ybgE, bcaT
and cgl2204, where ilvE is especially preferred.
[0428] The nucleotide sequences of suitable genes the enzyme
E.sub.80 can, again, be found in the KEGG database, the NCBI
database or the EMBL database.
[0429] In connection with this second alternative of the first
special embodiment of the second variant of the cell according to
the invention, it may furthermore be advantageous to reduce the
activity of an enzyme E.sub.4 which catalyzes the conversion of
methylmalonate-semialdehyde into 3-hydroxyisobutyric acid, where
this enzyme E.sub.4 preferably takes the form of a
3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) or of a
3-hydroxy-acyl-coenzyme A dehydrogenase (EC 1.1.1.35).
[0430] According to the second modification of the first special
embodiment of the second variant of the cell according to the
invention, where the formation of 3-hydroxyisobutyric acid or of
polyhydroxyalkanoates based on 3-hydroxyisobutyric acid takes place
via 3-hydroxyisobutyryl-coenzyme A as precursor and
isobutyryl-coenzyme A as intermediate and starting from L-valine as
carbon source, it may furthermore be preferred to employ those
cells which are already capable of forming large amounts of
L-valine. In this context, suitable cells are in particular those
which have been described by Blombach et al. in Applied
Environmental Microbiology, Vol. 73 (7) (2007), pages
2079-2084.
[0431] In the event that C.sub.1-compounds such as, for example,
methane or methanol act as carbon source, it is preferred in a
second special embodiment of the second variant of the cell
according to the invention, where the formation of
3-hydroxyisobutyric acid or of poly-hydroxyalkanoates based on
3-hydroxyisobutyric acid takes place via
3-hydroxyisobutyryl-coenzyme A as precursor, that the formation
takes place via 3-hydroxyisobutyryl-coenzyme A as intermediate. In
this context, it is preferred that the cell features an activity of
at least one of the following enzymes E.sub.8, E.sub.53, E.sub.54
and E.sub.81 which is increased in comparison with its wild type:
[0432] of an enzyme E.sub.53, which catalyzes the conversion of
acetyl-coenzyme A into acetoacetyl-coenzyme A; [0433] of an enzyme
E.sub.54, which catalyzes the conversion of acetoacetyl-coenzyme A
into 3-hydroxybutyryl-coenzyme A; [0434] of an enzyme E.sub.81,
which catalyzes the conversion of 3-hydroxybutyryl-coenzyme A into
3-hydroxy-isobutyryl-coenzyme A; [0435] of an enzyme E.sub.8, which
catalyzes the conversion of 3-hydroxyisobutyryl-coenzyme A into
3-hydroxyisobutyrate.
[0436] In this context, genetically modified cells which are
especially preferred in accordance with the invention are those in
which the activity of the following enzymes or enzyme combinations
is increased: E.sub.9, E.sub.53, E.sub.54, E.sub.81 and
E.sub.8E.sub.53E.sub.54E.sub.81.
[0437] In this context, it is especially preferred that the enzyme
[0438] E.sub.8 is a 3-hydroxyisobutyryl-coenzyme A hydrolase (EC
3.1.2.4), [0439] E.sub.53 is a .beta.-ketothiolase (EC 2.3.1.9),
[0440] E.sub.54 is an acetoacetyl-coenzyme A reductase (an EC
1.1.1.36), and [0441] E.sub.81 is an isobutyryl-coenzyme mutase (EC
5.4.99.13).
[0442] Preferred enzymes E.sub.8, E.sub.53 and E.sub.54 are those
which have already been described hereinabove. A preferred enzyme
E.sub.81 is the isobutyryl-coenzyme mutase from
.beta.-proteo-bacterium strain L108 which is described in Applied
And Environmental Microbiology, Vol. 72 (6), 2006, pages
4128-4135.
[0443] According to a special embodiment of the cell according to
the invention, it is furthermore preferred that this cell features
an expression of the glb0 gene which is increased in comparison
with its wild type. Furthermore, it may under certain circumstances
be preferred that the cell according to the invention features an
activity of the citrate transport protein which is encoded by the
dctA gene or the citP gene, which activity is reduced in comparison
with its wild type.
[0444] A contribution to the solution of the problems mentioned at
the outset is furthermore provided by a method of preparing a
genetically modified cell which is capable of forming
3-hydroxyisobutyric acid or polyhydroxyalkanoates based on
3-hydroxyisobutyric acid via methylmalonate-semialdehyde or
isobutyryl-coenzyme A, as precursors, comprising the method step of
increasing, in the cell, the activity of at least one of the
above-described enzymes, preferably of one or more of the enzymes
[0445] E.sub.1 to E.sub.4, [0446] E.sub.1, E.sub.4, E.sub.5,
E.sub.6 and E.sub.7, [0447] E.sub.1, E.sub.4, E.sub.5 and E.sub.7,
[0448] E.sub.4, E.sub.5 and E.sub.47 to E.sub.52, [0449] E.sub.2 to
E.sub.4, E.sub.6, E.sub.7 and E.sub.47 to E.sub.52, [0450] E.sub.2
to E.sub.4, E.sub.7 and E.sub.47 to E.sub.52, [0451] E.sub.8 and
E.sub.53 to E.sub.61, [0452] E.sub.8, E.sub.60, E.sub.61 and
E.sub.76 to E.sub.79, [0453] E.sub.8, E.sub.60, E.sub.61, E.sub.79
and E.sub.80, or [0454] E.sub.8, E.sub.53, E.sub.54 and E.sub.82 in
the cell, where increasing the enzymatic activity is preferably
carried out by the methods described at the outset.
[0455] Another contribution to the solution of the problems
mentioned at the outset is provided by the cells obtainable by the
above-described method.
[0456] Another contribution to the solution of the problems
mentioned at the outset is provided by a method of producing
3-hydroxyisobutyric acid or polyhydroxyalkanoates based on
3-hydroxyisobutyric acid, comprising the method step of bringing a
cell according to the invention into contact with a nutrient medium
comprising, as carbon source, carbohydrates, glycerol, carbon
dioxide, methane, methanol, L-valine or L-glutamate under
conditions under which 3-hydroxyisobutyric acid or
polyhydroxyalkanoates based on 3-hydroxyisobutyric acid are formed
from the carbon source, and, if appropriate, isolation of the
3-hydroxyisobutyric acid from the nutrient medium.
[0457] The genetically modified cells according to the invention
can be into contact with the nutrient medium, and thus cultured,
either continuously or batchwise in the batch method or in the
fed-batch method or in the repeated-fed-batch method in order to
produce 3-hydroxyisobutyrate or polyhydroxyalkanoates based on
3-hydroxyisobutyrate. A semicontinuous method as described in
GB-A-1009370 is also feasible. An overview over known culture
methods are described in the textbook by Chmiel ("Bioprozesstechnik
1. Einfuhrung in die Bioverfahrenstechnik" [Bioprocess technology
1. introduction to bioprocess technology] (Gustav Fischer Verlag,
Stuttgart, 1991)) or in the textbook by Storhas ("Bioreaktoren and
periphere Einrichtungen", [Bioreactors and peripheral equipment]
Vieweg Verlag, Braunschweig/Wiesbaden, 1994).
[0458] The culture medium to be used must suitably meet the
requirements of the strains in question. Descriptions of culture
media for various microorganisms can be found in the textbook
"Manual of Methods for General Bacteriology" of the American
Society for Bacteriology (Washington D.C., USA, 1981).
[0459] Carbon sources which may be used are carbohydrates such as,
for example, glucose, sucrose, lactose, fructose, maltose,
molasses, starch and cellulose, oils and fats such as, for example,
soy oil, sunflower oil, peanut oil and coconut fat, fatty acids
such as, for example, palmitic acid, stearic acid and linolic acid,
alcohols such as, for example, glycerol and methanol, hydrocarbons
such as methane, amino acids such as L-glutamate or L-valine, or
organic acids such as, for example, acetic acid. These substances
may be used singularly or as a mixture. It is especially preferred
to employ carbohydrates, in particular monosaccharides,
oligosaccharides or polysaccharides, as described in U.S. Pat. No.
601,494 and U.S. Pat. No. 6,136,576, or C.sub.5-sugars, or
glycerol.
[0460] Nitrogen sources which can be used are organic
nitrogen-comprising compounds such as peptones, yeast extract, meat
extract, malt extract, cornsteep liquor, soya mill and urea, or
inorganic compounds such as ammonium sulfate, ammonium chloride,
ammonium phosphate, ammonium carbonate and ammonium nitrate. The
nitrogen sources can be used singularly or as a mixture.
[0461] Phosphoric acid, potassium dihydrogen phosphate or
dipotassium hydrogen phosphate or the corresponding
sodium-comprising salts can be used as sources of phosphorus. The
culture medium must furthermore comprise salts of metals such as,
for example, magnesium sulfate or iron sulfate, which are required
for growth. Finally, essential growth factors such as amino acids
and vitamins may be employed in addition to the abovementioned
substances. Moreover, suitable precursors may be added to the
culture medium. The abovementioned input materials may be added to
the culture in the form of a single batch or else fed in a suitable
manner during culturing.
[0462] The pH for the culture can be controlled by employing, in an
appropriate manner, basic compounds such as sodium hydroxide,
potassium hydroxide, ammonia and aqueous ammonia, or acidic
compounds such as phosphoric acid or sulfuric acid. Foaming can be
controlled by employing antifoams such as, for example, fatty acid
polyglycol esters. To maintain the stability of plasmids, it is
possible to add to the medium suitable substances which have a
selective effect, such as, for example, antibiotics. Aerobic
conditions are maintained by introducing, into the culture, oxygen
or oxygen-containing gas mixtures such as, for example, ambient
air. The culture temperature is normally 20.degree. C. to
45.degree. C. and preferably 25.degree. C. to 40.degree. C. It may
be preferred to employ, as cells, those cells which are described
in U.S. Pat. No. 6,803,218, in particular when using cells which
are capable of converting glycerol as the substrate. In this case,
the cells can be cultured at temperatures in the range of from 40
to 100.degree. C.
[0463] The isolation of 3-hydroxyisobutyric acid from the nutrient
solution is preferably carried out continuously, it being
furthermore preferred in this context also to produce
3-hydroxyisobutyric acid by fermentation in a continuous manner, so
that the entire process from the production of 3-hydroxyisobutyric
acid up to its isolation from the fermentation liquor can be
carried out continuously. For the continuous isolation of the
production of 3-hydroxyisobutyric acid from the fermentation
liquor, the former is continuously passed over a device for
removing the microorganisms employed during fermentation,
preferably through a filter with an exclusion level in the range of
from 20 to 200 kDa, where a solid/liquid separation takes place. It
is also feasible to employ a centrifuge, a suitable sedimentation
device or a combination of these devices, it being especially
preferred to first separate at least part of the microorganisms by
sedimentation and subsequently to feed the fermentation liquor,
which has been freed from part of the microorganisms, to
ultrafiltration or to a centrifugation device.
[0464] After the microorganisms have been removed, the fermentation
product, which is enriched with regard to its 3-hydroxyisobutyric
acid fraction, is fed to a separation system, preferably a
multistep separation system. This separation system provides a
plurality of separation steps which are connected in series, from
which steps in each case return lines lead away and back to the
fermentation tank. Furthermore, exit pipes lead out of the
respective separation steps. The individual separation steps may
operate by the electrodialysis, the reverse osmosis, the
ultrafiltration or the nanofiltration principle. As a rule, these
are membrane separation devices in the individual separation steps.
The selection of the individual separation steps is a function of
the nature and the extent of the fermentation by-products and
substrate residues.
[0465] Besides the 3-hydroxyisobutyric acid being separated off by
means of electrodialysis, reverse osmosis, ultrafiltration or
nanofiltration, in the course of which an aqueous
3-hydroxyisobutyric acid solution is obtained as the end product,
the 3-hydroxyisobutyric acid can also be separated off by
extractive methods from the fermentation solution which has been
freed from microorganisms, in which case, finally, the pure
3-hydroxyisobutyric acid can be obtained. To separate the
3-hydroxyisobutryic acid by extraction, it is possible to add, to
the fermentation solution, for example ammonium compounds or amines
in order to form an ammonium salt of 3-hydroxyisobutyric acid. This
ammonium salt can then be separated from the fermentation solution
by adding an organic extractant and subsequently heating the
resulting mixture, whereby the ammonium salt is concentrated in the
organic phase. Then, the 3-hydroxyisobutyric acid can be isolated
from this phase for example by further extraction steps, giving the
pure 3-hydroxyisobutyric acid. More details regarding the
separation method can be found in WO-A-02/090312, whose disclosure
regarding the separation of hydroxycarboxylic acids from
fermentation solutions is hereby incorporated by reference and
forms part of the disclosure of the present application.
[0466] Depending on the way in which the 3-hydroxyisobutyric acid
is separated from the fermentation solution, either an aqueous
solution of 3-hydroxyisobutyric acid comprising 2 to 90% by weight,
preferably 7.5 to 50% by weight and especially preferably 10 to 25%
by weight of 3-hydroxyisobutyric acid, or else pure
3-hydroxyisobutyric acid is obtained.
[0467] Furthermore, the 3-hydroxyisobutyric acid prepared by the
method according to the invention can also be neutralized, either
before, during or after the purification, for which purpose bases
such as, for example, calcium hydroxide or sodium hydroxide can be
employed.
[0468] A contribution to solving the problems mentioned at the
outset is provided in particular also by a method of preparing
methacrylic acid or methacrylic esters, comprising the method steps
[0469] IA) preparation of 3-hydroxyisobutyric acid by the method
described above and, if appropriate, isolation and/or
neutralization of the 3-hydroxyisobutyric acid of the
3-hydroxyisobutyric acid, [0470] IB) dehydration of the
3-hydroxyisobutyric acid with formation of methacrylic acid and, if
appropriate, esterification of the methacrylate or of the
methacrylic acid.
[0471] According to method step IB), the 3-hydroxyisobutyric acid
is dehydrated with formation of methacrylic acid, for which purpose
it is possible either to employ the pure 3-hydroxyisobutyric acid
isolated from the fermentation solution or else the aqueous
solution of 3-hydroxyisobutryic acid, which has been isolated when
working up the fermentation solution, it also being possible to
concentrate the aqueous solution of 3-hydroxyisobutyric acid, if
appropriate, before the dehydration step, for example by means of
distillation, if appropriate in the presence of a suitable
entrainer.
[0472] The dehydration reaction can, in principle, be carried out
in liquid phase or in the gas phase. Furthermore, it is preferred
in accordance with the invention that the dehydration reaction is
carried out in the presence of a catalyst, with the nature of the
catalyst employed depending on whether a gas-phase or a
liquid-phase reaction is carried out.
[0473] Suitable dehydration catalysts are both acidic catalysts and
alkaline catalysts. Acidic catalysts are preferred, in particular
because they show less tendency to form oligomers. The dehydration
catalyst may be employed both as a homogeneous and as a
heterogeneous catalyst. If the dehydration catalyst is present in
the form of a heterogeneous catalyst, it is preferred that the
dehydration catalyst is in contact with a support x. Suitable
supports x are all solids believed by the skilled worker to be
suitable. In the present context, it is preferred that the solids
have suitable pore volumes which are suitable for good binding and
absorption of the dehydration catalyst. Furthermore, total pore
volumes as specified by DIN 66133 in a range of from 0.01 to 3 ml/g
are preferred, and total pore volumes in the range of from 0.1 to
1.5 ml/g are especially preferred. Moreover, it is preferred that
the solids which are suitable as support x have a surface area in
the range of from 0.001 to 1000 m.sup.2/g, preferably in the range
of from 0.005 to 450 m.sup.2/g and furthermore preferred in the
range of from 0.01 to 300 m.sup.2/g as determined by BET test as
specified in DIN 66131. A support which may be employed for the
dehydration catalyst can firstly be bulk material with a mean
particle diameter in the range of from 0.1 to 40 mm, preferably in
the range of from 1 to 10 mm, and furthermore preferably in the
range from 1.5 to 5 mm. The wall of the dehydration reactor may
furthermore act as support. Furthermore, the support may be acidic
or alkaline per se, or else an acidic or alkaline dehydration
catalyst may be applied to an inert support. Application techniques
which may be mentioned in particular are immersion or impregnation
or else incorporation into a support matrix.
[0474] Suitable supports x, which may also feature dehydration
catalyst properties, are, in particular, natural or synthetic
silicates such as, in particular, mordenite, montmorillonite,
acidic zeolites; supports which are coated with monobasic, dibasic
or polybasic inorganic acids, in particular phosphoric acid, or
with acidic salts of inorganic acids, such as substances of the
oxide or silicate type, for example Al.sub.2O.sub.3, TiO.sub.2;
oxides and mixed oxides such as, for example,
.gamma.-Al.sub.2O.sub.3 and ZnO--Al.sub.2O.sub.3 mixed oxides of
the heteropolyacids.
[0475] In accordance with an embodiment according to the invention,
the support x consists at least in part of a compound of the oxide
type. Such compounds of the oxide type should feature at least one
of the elements selected from among Si, Ti, Zr, Al, P or a
combination of at least two of these. Such supports may also act as
dehydration catalyst themselves, owing to their acidic or alkaline
properties. A preferred class of compounds, both as support by way
of x and by way of dehydration catalyst comprise
silicon/aluminum/phosphorus oxides. Preferred alkaline substances
which act both as dehydration catalyst and also as support x
comprise alkali, alkaline earth, lanthanum, lanthoids or a
combination of at least two of these in the form of their oxides.
Such acidic or alkaline dehydration catalysts are commercially
available both from Degussa AG and from Sudchemie AG. A further
class are ion exchangers. Again, these may be present both in
alkaline and in acidic form.
[0476] Suitable homogeneous dehydration catalysts are, in
particular, inorganic acids, preferably phosphorus-containing acids
and furthermore preferably phosphoric acid. These inorganic acids
can be immobilized on the support x by immersion or
impregnation.
[0477] The use of heterogeneous catalysts has proved particularly
advantageous in particular in the case of gas phase dehydration. In
the case of liquid-phase dehydration, however, both homogeneous and
heterogeneous dehydration catalysts are employed.
[0478] Furthermore, it is preferred that the method according to
the invention involves the use of a dehydration catalyst with an
H.sub.0 value in the range of from +1 to -10, preferably in the
range of from +2 to -8.2 and furthermore preferably, in the case of
liquid-phase dehydration, in the range of from +2 to -3 and in
gas-phase dehydration in the range of from -3 to -8.2. The H.sub.0
value corresponds to the acid function as defined by Hammert and
can be determined by what is known as amine titration and the use
of indicators, or by the absorption of a gaseous base (see "Studies
in Surface Science and Catalytics", vol. 51, 1989: "New solid Acids
and Bases, their catalytic Properties", K. Tannabe et al).
[0479] According to a special embodiment of the method according to
the invention, the acidic solid catalyst employed is a porous
support structure which has been brought into contact with an
inorganic acid, preferably with phosphoric acid or with superacids
such as, for example, sulfated or phosphated zirconium oxide and
which is based preferably on at least 90% by weight, furthermore
preferably at least 95% by weight and most preferably at least 99%
by weight of a silicon oxide, preferably an SiO.sub.2. The bringing
into contact of the porous support structure with the inorganic
acid is preferably carried out by impregnating the support
structure with the acid, with the latter preferably being brought
into contact with the former in an amount in a range of from 10 to
70% by weight, especially preferably in the range of from 20 to 60%
by weight and more preferably in a range of from 30 to 50% by
weight, based on the weight of the support structure, followed by
drying. After drying, the support structure is heated in order to
fix the inorganic acid, preferably at a temperature in a range of
from 300 to 600.degree. C., more preferably in a range of from 400
to 500.degree. C.
[0480] According to a special embodiment of the method according to
the invention, the dehydration reaction is carried out in the gas
phase. Here, it is possible to employ conventional apparatuses as
are known to the skilled worker in the field of gas phase reaction,
for example tubular reactors. It is especially preferred to employ
shell-and-tube heat exchangers and reactors which comprise
thermoplates as heat exchangers.
[0481] According to an embodiment of the gas-phase dehydration
reaction, pure 3-hydroxyisobutyric acid is introduced into a
reactor comprising one of the abovementioned fixed-bed catalysts.
According to another embodiment, the 3-hydroxyisobutyric acid is
introduced into the reactor in the form of an aqueous solution
comprising 2 to 80% by weight, especially preferably 5 to 50% by
weight and more preferably 10 to 25% by weight of
3-hydroxyisobutyric acid, in each case based on the total weight of
the aqueous solution. The pressure and temperature conditions
inside the reactor are chosen such that the 3-hydroxyisobutyric
acid, or the aqueous solution, is present in gaseous form when
entering the reactor. The dehydration in the gas phase is
preferably carried out in the temperature range of between 200 and
400.degree. C., especially preferably between 250 and 350.degree.
C. The pressure inside the reactor during the gas-phase dehydration
reaction is preferably in a range of from 0.1 to 50 bar, especially
preferably in a range of from 0.2 to 10 bar and most preferably in
a range of from 0.5 to 5 bar.
[0482] The amount of 3-hydroxyisobutyric acid introduced into the
reactor in the gas-phase dehydration reaction is preferably in a
range of from 10 to 100% by volume, especially preferably in a
range of from 20 to 100% by volume and most preferably in a range
of from 30 to 100% by volume.
[0483] According to another special embodiment of the method
according to the invention, the dehydration reaction is performed
in the liquid phase. The liquid-phase dehydration reaction can also
be carried out in all apparatuses which are known to the skilled
worker and in which a fluid can be heated to a desired reaction
temperature, during which process a pressure can be applied to the
apparatus which is sufficient for maintaining the reaction
components in the liquid state under the desired temperature
conditions.
[0484] According to a special embodiment of the method according to
the invention, the liquid-phase dehydration method comprises a
first method step, in which pure 3-hydroxyisobutyric acid or an
aqueous solution comprising 5 to 100% by weight, especially
preferably 20 to 100% by weight and most preferably 50 to 100% by
weight of 3-hydroxyisobutyric acid, based on the total weight of
the aqueous solution, is introduced into a reactor. The pressure
and temperature conditions inside the reactor are chosen such that
the 3-hydroxyisobutyric acid, or the aqueous solution, is present
in liquid form when entering the reactor. According to a special
embodiment of the method according to the invention in which the
dehydration reaction is carried out in the liquid phase, the
3-hydroxyisobutyric acid, or the aqueous solution, is passed in
such a way over a fixed catalyst bed inside the dehydration reactor
that the liquid phase trickles over the surface of the catalyst
particles. Such a procedure may be carried out for example in a
trickle-bed reactor.
[0485] The dehydration in the liquid phase is preferably carried
out in a temperature range of between 200 and 350.degree. C.,
especially preferably between 250 and 300.degree. C. The pressure
inside the reactor in the case of liquid-phase dehydration is
preferably in a range of from 1 to 50 bar, especially preferably in
a range of from 2 to 25 bar and most preferably in a range of from
3 to 10 bar.
[0486] The catalysis of the dehydration reaction may be homogeneous
or heterogeneous, both in the case of gas-phase dehydration and in
the case of liquid-phase dehydration.
[0487] In the case of homogeneous catalysis, the catalyst, which in
this case preferably takes the form of an inorganic acid such as,
for example, phosphoric acid or sulfuric acid, is first brought
into contact with the pure 3-hydroxyisobutyric acid or with the
aqueous solution comprising the 3-hydroxyisobutyric acid.
Thereafter, the resulting composition is introduced into the
reactor and converted into methacrylic acid under the desired
pressure and temperature conditions. It is also feasible to
introduce the inorganic acid independently of the
3-hydroxyisobutyric acid or the aqueous solution into the reactor.
In this case, the reactor features at least two feed lines, one for
the 3-hydroxyisobutyric acid, or the aqueous solution comprising
3-hydroxyisobutyric acid, and one for the catalyst. If the
dehydration reaction is carried out in liquid phase in a
trickle-bed reactor, it is preferred to introduce the catalyst
together with the 3-hydroxyisobutyric acid, or the aqueous solution
comprising the 3-hydroxyisobutyric acid, at the top of the
reactor.
[0488] In the case of heterogeneous catalysis, the catalyst is in
the form of a solid substrate located in the reaction space, for
example in the form of a fixed bed, in the form of catalyst-coated
plates, preferably thermoplates, which are arranged inside the
reactor, or else in the form of catalyst-coated reactor walls.
Reactors which are possible are described for example in DE-A-198
48 208, DE-A-100 19 381 and EP-A-I 234 612. In the case of
heterogeneous catalysis, preferred catalysts are support structures
which have been brought into contact with inorganic acids,
preferably impregnated porous support structures. The
3-hydroxyisobutyric acid, or the aqueous solution comprising the
3-hydroxyisobutyric acid, is then brought into contact with the
surface of the solid catalyst material in the form of a vapor, or
in liquid form.
[0489] According to an especially preferred embodiment of the
method according to the invention, the dehydration of the
3-hydroxyisobutyric acid is carried out in liquid phase at a
pressure in the range of from 200 to 500 mbar, at a temperature in
a range of from 200 to 230.degree. C. and in the presence of alkali
metal ions as the catalyst.
[0490] The reaction mixture which is obtained after the dehydration
reaction is either an aqueous methacrylic acid solution which does
not contain any catalyst components (such a solution is obtained in
the case of heterogeneously catalyzed dehydration) or else an
aqueous methacrylic acid solution which comprises catalysts (such a
solution is obtained in the case of homogeneously catalyzed
dehydration). Furthermore, the aqueous methacrylic acid solution
may be in liquid form (if the dehydration reaction has been
effected in the liquid phase) or in gaseous form (if the
dehydration reaction has been carried out in the gas phase).
[0491] If appropriate, the resulting methacrylic acid solution can,
according to a special embodiment of the method according to the
invention, be esterified without further processing. In such a
case, the methacrylic acid solution is brought into contact with
suitable alcohols such as, for example, methanol, ethanol,
1-propanol, 2-propanol or 1-butanol and suitable esterification
catalysts known to the skilled worker such as, for example,
concentrated acids, with heating, and the methacrylic acid is so
converted into the corresponding esters. However, it may be
advantageous additionally to purify the methacrylic acid before
esterification, it being possible to employ, in principle, any
purification method which is known to the skilled worker and which
is conventionally employed for the purification of contaminated
(meth)acrylic acid obtained by catalytic gas-phase oxidation of
propylene.
[0492] If the dehydration reaction has been carried out in the gas
phase, it is preferred that the methacrylic acid is first
condensed, generating an aqueous methacrylic acid solution. Here,
any condensation method known to the skilled worker may be employed
in principle, for example a fractional condensation as described in
WO-A-2004/035514, WO-A-03/014172 or EP-A-EP 1 163 201 or by total
condensation as described in EP-A-0 695 736. It is also feasible to
add additional solvents, in particular water, during the
condensation process in order to absorb the methacrylic acid as
completely as possible.
[0493] The aqueous methacrylic acid solution obtained after
condensation, or else the aqueous methacrylic acid solution
obtained in the event of liquid-phase dehydration, can then be
freed from water and other contaminants in further purification
steps. Here, it is possible first to remove the water by azeotrope
distillation in the presence of an entrainer as described, for
example, in DE-A-198 53 064. It is also feasible to employ
high-boiling organic solvents for absorbing the methacrylic acid,
as is disclosed for example in EP-A-0 974 574. In addition to these
distillation methods, membranes for dewatering may also be
employed, as proposed for example in DE-A-44 01 405. Employing
crystallization methods for purifying the aqueous methacrylic acid
solution, which has been generated in the case of liquid-phase
dehydration or which has been obtained by condensation, is
furthermore feasible.
[0494] The methacrylic acid obtained after dehydration can be
purified even further in further method steps. Thus, high-boiling
contaminants which are still present can be removed by further
distillation steps. However, it is especially preferred to further
purify the methacrylic acid obtained by dehydration using
crystallization methods as described for example in DE-A-101 49
353.
[0495] The resulting purified methacrylic acid can then be
esterified, if appropriate.
[0496] A contribution to solving the problems mentioned at the
outset is furthermore provided by a method of preparing methacrylic
acid or methacrylic esters, comprising the method steps [0497] IIA)
preparation of polyhydroxyalkanoates based on 3-hydroxyisobutyric
acid by the method described above, [0498] IB) cleavage of the
polyhydroxyalkanoates based on 3-hydroxyisobutyric acid with
formation of 3-hydroxyisobutyric acid and, if appropriate,
neutralization of the 3-hydroxyisobutyric acid and/or isolation of
the 3-hydroxyisobutyric acid, [0499] IIC) dehydration of the
3-hydroxyisobutyric acid with formation of methacrylic acid and, if
appropriate, esterification of the methacrylate or methacrylic
acid.
[0500] A contribution to solving the problems mentioned at the
outset is also provided by a method of preparing polymethacrylic
acid or polymethacrylic esters, comprising the method steps [0501]
IIIA) preparation of methacrylic acid by the method described
above, [0502] IIIB) free-radical polymerization of the methacrylic
acid, it being possible, if appropriate, to esterify at least in
part the carboxyl groups of the methacrylic acid before or after
the free-radical polymerization reaction.
[0503] A contribution to solving the problem mentioned at the
outset is furthermore provided by an isolated DNA, which is
selected from the following sequences: [0504] a) a sequence as
shown in SEQ ID No 03, [0505] b) an intron-free sequence which is
derived from a sequence as specified in a) and which codes for the
same protein or peptide as the sequence as shown in SEQ ID No 03,
[0506] c) a sequence which codes for a protein or peptide which
comprises the amino acid sequence as shown in SEQ ID No 04, [0507]
d) a sequence with at least 80%, especially preferably at least
90%, more preferably at least 95% and most preferably 99% identity
with a sequence as specified in one of groups a) to c), especially
preferably as specified in group a), this sequence preferably
coding for a protein or peptide which is capable of converting both
S- or R-methylmalonyl-coenzyme A and malonyl-coenzyme A into the
corresponding semialdehydes ((S)- or (R)-methylmalonate
semialdehyde and malonate semialdehyde, respectively), [0508] e) a
sequence which hybridizes, or, taking into consideration the
degeneration of the genetic code, would hybridize, with the counter
strain of a sequence as specified in any of groups a) to d),
especially preferably as specified in group a), this sequence
preferably coding for a protein or peptide which is capable of
converting both S- or R-methylmalonyl-coenzyme A and
malonyl-coenzyme A into the corresponding semialdehydes ((S)- or
(R)-methylmalonate semialdehyde and malonate semialdehyde,
respectively), [0509] f) a derivative of a sequence as specified in
any of groups a) to e), especially preferably as specified in group
a), this derivative preferably coding for a protein or peptide
which is capable of converting both S- or R-methylmalonyl-coenzyme
A and malonyl-coenzyme A into the corresponding semialdehydes ((S)-
or (R)-methylmalonate semialdehyde and malonate semialdehyde,
respectively), obtained by substitution, addition, inversion and/or
deletion of at least one base, preferably of at least 2 bases, more
preferably of at least 5 bases and most preferably at least 10
bases, but preferably of no more than 100 bases, especially
preferably of no more than 50 bases and most preferably of no more
than 25 bases, and [0510] g) a sequence which is complementary to a
sequence as specified in any of groups a) to f), especially
preferably as specified in group a).
[0511] Surprisingly, it has been found that a DNA which has been
isolated from bacteria of the strain Sulfolobus tokodaii (at the
Deutsche Sammlung von Mikroorganismen [German collection of
microorganisms], deposit number DSM 16993) and which has a DNA
sequence as shown in SEQ ID No 03 codes for a polypeptide (SEQ ID
No 04) which is capable even at temperatures of up to 75.degree. C.
of converting both S- or R-methylmalonyl-coenzyme A and
malonyl-coenzyme A into the corresponding semialdehydes ((S)- or
(R)-methylmalonate semialdehyde and malonate semialdehyde,
respectively). Since (S)- or (R)-methylmalonate semialdehyde and
malonate semialdehyde are natural metabolites which are formed for
example during the degradation of valine, of leucin or of
isoleucin, during the propanoate metabolism or during the pyruvate
metabolism, and because the formed semialdehydes are capable of
being reduced further in the course of the abovementioned metabolic
pathways to give the corresponding 3-hydroxyalkanoates, the
isolated DNA according to the invention can be utilized for
generating recombinant bacteria which are capable of directly
forming large amounts of 3-hydroxy-isobutyric acid (or
3-hydroxypropionic acid). If the cells are furthermore capable of
polymerizing the formed 3-hydroxyalkanoates with formation of
polyhydroxyalkanoates, this DNA would furthermore be suitable for
generating recombinant bacteria capable of producing
polyhydroxyalkanoates based on 3-hydroxyisobuytric acid (or on
3-hydroxypropionic acid).
[0512] The "nucleotide identity" in relation to SEQ ID No 03, which
is defined in alternative d), is determined with the aid of known
methods here. In general, specialist computer programs with
algorithms taking into consideration specific requirements are
used.
[0513] Preferred methods of determining the identity first generate
the maximum agreement between the sequences to be compared.
Computer programs for determining the identity comprise the GCG
program package, including but not limited thereto [0514] GAP
(Deveroy, J. et al., Nucleic Acid Research 12 (1984), page 387,
Genetics Computer Group University of Wisconsin, Medicine (Wi)),
and [0515] BLASTP, BLASTN and FASTA (Altschul. S. et al., Journal
of Molecular Biology 215 (1990), pages 403-410). The BLAST program
may be obtained from the Center For Biotechnology Information
(NCBI) and from other sources (BLAST Manual, Altschul S. et al.,
NCBI NLM NIH Bethesda Md. 22894; Altschul S. et al., above).
[0516] The Smith-Waterman algorithm, which is known, can also be
used for determining the nucleotide identity.
[0517] Preferred parameters for the nucleotide alignment comprise
the following: [0518] Algorithmus Needleman and Wunsch, Journal of
Molecular Biology 48 (1970), pages 443-453 [0519] alignment matrix
[0520] Matches=+10 [0521] Mismatches=0 [0522] Gap penalty=50 [0523]
Gap length penalty=3
[0524] The GAP program is also suitable for use with the above
parameters. The above parameters are the default parameters in the
nucleotide sequence alignment.
[0525] An identity of 80% according to the above algorithm means
80% identity in the context of the present invention. The same
applies to greater identities.
[0526] The feature "sequence which hybridizes, or, taking into
consideration the degeneracy of the genetic code, would hybridize,
with the counter strain of a sequence as specified in one of groups
a) to d), especially preferably as specified in group a),"
according to alternative e) indicates a sequence which hybridizes,
or would hybridize taking into consideration the degeneracy of the
genetic code, with the counter strand of a sequence as specified in
one of groups a) to d), especially preferably as specified in group
a), under preferably stringent conditions. For example, the
hybridization reactions can be carried out at 68.degree. C. in
2.times.SSC, or as described in the protocol of the dioxygenin
labeling kit from Boehringer (Mannheim). Examples of preferred
hybridization conditions are incubation overnight at 65.degree. C.
in 7% SDS, 1% BSA, 1 mM EDTA, 250 mM sodium phosphate buffer (pH
7.2), followed by washing at 65.degree. C. with 2.times.SSC; 0.1%
SDS.
[0527] The derivatives, of the isolated DNA according to the
invention, which can be obtained according to alternative f) by
substitution, addition, inversion and/or deletion of one or more
bases of a sequence as specified in any of groups a) to e) include
in particular those sequences which, in the protein which they
encode, lead to conservative amino acid substitutions such as, for
example, the substitution of glycine for alanine or of aspartate
for glutamic acid. Such function-neutral mutations are referred to
as sense mutations and do not lead to any principle modification of
the activity of the polypeptide. It is furthermore known that
modifications at the N and/or C terminus of a polypeptide do not
have a considerable adverse effect on its function; indeed, they
are even capable of stabilizing it, so that, as a consequence, the
present invention also comprises DNA sequences where bases are
added at the 3' terminus or at the 5' terminus of the sequence with
the SEQ ID No 03. The skilled worker will find information on this
subject in Ben Bassat et al. (Journal of Bacteriology 169:751-757
(1987)), in O'Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth
et al. (Protein Sciences 3:240-247 (1994)), in Hochuli et al.
(Bio/Technology 6: 1321-1325 (1988)) inter alia, and in known
textbooks of Genetics and Molecular Biology.
[0528] To isolate the DNA according to the invention, an
NADPH-dependant malonyl-coenzyme A reductase was first isolated
from a cell extract of Metallosphaera sedula and purified. The
first 20 amino acids of the N terminus of the polypeptide of the
resulting purified enzyme were sequenced. The gene for the
malonyl-coenzyme A reductase was subsequently determined in the
genome of Sulfolobus tokodaii, which has already been fully
sequenced (Kawarabayasi et al., "Complete genome sequence of an
aerobic thermoacidophilic crenarchaeon, Sulfolobus tokodaii
strain7.", DNA Research 8:123-40), by identifying the derived
protein sequence which is identical with the first 20 amino acids
of the polypeptide isolated from Metallosphaera sedula. The DNA
sequence according to the invention was then amplified by means of
PCR, using suitable primers (see example 2).
[0529] A contribution to solving the problems mentioned at the
outset is furthermore contributed by a vector, preferably an
expression vector, comprising a DNA with a sequence as specified in
one of groups a) to f) as defined above. Suitable vectors are all
vectors which are known to the skilled worker and which are
traditionally employed for introducing DNA into a host cell.
Preferred vectors are selected from the group consisting of
plasmids, such as, for example, the E. coli plasmids pTrc99A,
pBR345 and pBR322, viruses such as, for example, bacteriophages,
adenoviruses, vaccinia viruses, baculoviruses, measles viruses and
retroviruses, cosmids or YACs, with plasmids being most preferred
as vectors.
[0530] According to a preferred embodiment of the vector according
to the invention, the DNA with a sequence as specified in any of
groups a) to f) is under the control of a promoter capable of being
regulated, which promoter is suitable for expressing the
polypeptide encoded by these DNA sequences in the cell of a
microorganism, preferably in a bacterial cell, a yeast cell or a
fungal cell, especially preferably in a bacterial cell, most
preferably in an E. Coli cell. Examples of such promoters are the
trp promoter or the tac promoter.
[0531] Besides a promoter, the vector according to the invention
should preferably comprise a ribosomal binding site and terminator.
Here, it is especially preferred that the DNA according to the
invention is incorporated into an expression cassette of the vector
comprising the promoter, the ribosomal binding site and terminator.
Besides the abovementioned structural elements, the vector may
furthermore comprise selection genes known to the skilled
worker.
[0532] A contribution to solving the problems mentioned at the
outset is furthermore provided by the use of the above-described
vector for transforming a cell and by the cell obtained by
transformation of this vector. The cells which can be transformed
with the vector according to the invention may be prokaryotes or
eukaryotes. They may take the form of mammalian cells (such as, for
example, cells from humans), of plant cells or of microorganisms
such as yeasts, fungi or bacteria, with microorganisms being
especially preferred and bacteria and yeasts being most
preferred.
[0533] A contribution to solving the problems mentioned at the
outset is also provided by a polypeptide which features the amino
acid sequence with the SEQ ID No 04 or an amino acid sequence which
is obtained when no more than 40 amino acids, preferably no more
than 20 amino acids, even more preferably no more than 10 amino
acids and most preferably no more than 5 amino acids in SEQ ID No
04 are deleted, inserted, substituted or else added to the C and/or
N terminus of the amino acid sequence with the SEQ ID No 04. The
polypeptide takes the form of an enzyme which is capable of
catalyzing both the conversion of (S)- or
(R)-methylmalonyl-coenzyme A into (S)- or (R)-methylmalonate
semialdehyde and the conversion of malonyl-coenzyme A into malonate
semialdehyde. Such a polypeptide can be obtained for example via
the synthetic route, starting from the DNA sequence with the SEQ ID
No 03, or by transformation of a suitable cell with a suitable
vector comprising this nucleic acid sequence, expression, in the
cell, of the protein encoded by this nucleic acid sequence, lysis
of the cell, generating a cell extract, and subsequent purification
of the enzyme by means of purification techniques known to the
skilled worker, for example by means of HPLC or other
chromatographic methods. Besides chromatographic purification of
the polypeptide from cell extracts, one can also exploit the
advantage that the polypeptide with the amino acid sequence SEQ ID
No 04 is heat-resistant up to a temperature of at least 75.degree.
C. The cell extract can therefore be heated to a temperature of,
for example, 75.degree. C., which results in the coagulation, and
thus precipitation, in the cell extract of those polypeptides which
are not heat resistant. The polypeptide with the amino acid
sequence SEQ ID No 04 is retained in the cell extract in
nondenatured form.
[0534] The present invention will now be illustrated in greater
detail with reference to nonlimiting figures and examples.
[0535] FIG. 1 shows the conversion of succinyl-coenzyme A into
methylmalonyl-coenzyme A with catalysis by the enzyme E.sub.1.
[0536] FIG. 2 shows the conversion of methylmalonyl-coenzyme A into
3-hydroxyisobutyric acid with catalysis by the enzymes E.sub.2 to
E.sub.4 in accordance with the first alternative of the cell
according to the invention, where succinyl-coenzyme A is formed as
intermediate and methylmalonate semialdehyde as precursor in the
production of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates
based on 3-hydroxyisobutyric acid.
[0537] FIG. 3 shows the conversion of (R)-methylmalonyl-coenzyme A
into 3-hydroxyisobutyric acid with catalysis by the enzymes
E.sub.4, E.sub.6 and E.sub.7 in accordance with the second
alternative of the cell according to the invention, where
succinyl-coenzyme A is formed as intermediate and methylmalonate
semialdehyde as precursor in the production of 3-hydroxyisobutyric
acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric
acid.
[0538] FIG. 4 shows the conversion of methylmalonyl-coenzyme A into
3-hydroxyisobutyric acid with catalysis by the enzymes E.sub.4,
E.sub.5 and E.sub.7 in accordance with the third alternative of the
cell according to the invention, where succinyl-coenzyme A is
formed as intermediate and methylmalonate semialdehyde as precursor
in the production of 3-hydroxyisobutyric acid or of
polyhydroxyalkanoates based on 3-hydroxyisobutyric acid.
[0539] FIG. 5 shows the conversion of 3-hydroxyisobutyric acid into
a polyhydroxyalkanoate with catalysis by the enzymes E.sub.5 and
E.sub.9.
[0540] FIG. 6 shows the conversion of phosphoenolpyruvate or
pyruvate into oxalacetate with catalysis by the enzymes E.sub.10 or
E.sub.11 according to a special embodiment of the first, second or
third alternative of the cell according to the invention, where
succinyl-coenzyme A is formed as intermediate and methylmalonate
semialdehyde as precursor in the production of 3-hydroxyisobutyric
acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric
acid.
[0541] FIG. 7 shows the conversion of oxalacetate into
succinyl-coenzyme A with catalysis by the enzymes E.sub.12 to
E.sub.15 according to a first special embodiment of the first,
second or third alternative of the cell according to the invention,
where succinyl-coenzyme A is formed as intermediate and
methylmalonate semialdehyde as precursor in the production of
3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on
3-hydroxyisobutyric acid.
[0542] FIG. 8 shows the conversion of oxalacetate into
succinyl-coenzyme A with catalysis by the enzymes E.sub.13 to
E.sub.16 and E.sub.24 to E.sub.26 according to a second special
embodiment of the first, second or third alternative of the cell
according to the invention, where succinyl-coenzyme A is formed as
intermediate and methylmalonate semialdehyde as precursor in the
production of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates
based on 3-hydroxyisobutyric acid.
[0543] FIG. 9 shows the conversion of oxalacetate into
succinyl-coenzyme A with catalysis by the enzymes E.sub.16,
E.sub.24, E.sub.27 and E.sub.28 according to a third special
embodiment of the first, second or third alternative of the cell
according to the invention, where succinyl-coenzyme A is formed as
intermediate and methylmalonate semialdehyde as precursor in the
production of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates
based on 3-hydroxyisobutyric acid.
[0544] FIG. 10 shows the conversion of L-glutamate into
succinyl-coenzyme A with catalysis by the enzymes E.sub.46 and
E.sub.28 in accordance with a further special embodiment of the
first, second or third alternative of the cell according to the
invention, where succinyl-coenzyme A is formed as intermediate and
methylmalonate semialdehyde as precursor in the production of
3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on
3-hydroxyisobutyric acid.
[0545] FIG. 11 shows the conversion of acetyl-coenzyme A into
3-hydroxyisobutyric acid with catalysis by the enzymes E.sub.4,
E.sub.5 and E.sub.47 to E.sub.52 in accordance with a first
alternative of the second special embodiment of the cell according
to the invention, where propionyl-coenzyme A is formed as
intermediate and methylmalonate semialdehyde as precursor in the
production of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates
based on 3-hydroxyisobutyric acid.
[0546] FIG. 12 shows the conversion of propionyl-coenzyme A into
3-hydroxyisobutyric acid with catalysis by the enzymes E.sub.2 to
E.sub.4, E.sub.6, E.sub.7 and E.sub.47 to E.sub.52 in accordance
with a second alternative of the second special embodiment of the
cell according to the invention, where propionyl-coenzyme A is
formed as intermediate and methylmalonate semialdehyde as precursor
in the production of 3-hydroxyisobutyric acid or of
polyhydroxyalkanoates based on 3-hydroxyisobutyric acid.
[0547] FIG. 13 shows the conversion of propionyl-coenzyme A into
3-hydroxyisobutyric acid with catalysis by the enzymes E.sub.2 to
E.sub.4, E.sub.7 and E.sub.47 to E.sub.52 in accordance with a
third alternative of the second special embodiment of the cell
according to the invention, where propionyl-coenzyme A is formed as
intermediate and methylmalonate semialdehyde as precursor in the
production of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates
based on 3-hydroxyisobutyric acid.
[0548] FIG. 14 shows the conversion of propionyl-coenzyme A into
3-hydroxyisobutyric acid with catalysis by the enzymes E.sub.2 to
E.sub.4, E.sub.7 and E.sub.47 to E.sub.52 in accordance with a
fourth, fifth alternative of the second special embodiment of the
cell according to the invention, where propionyl-coenzyme A is
formed as intermediate and methylmalonate semialdehyde as precursor
in the production of 3-hydroxyisobutyric acid or of
polyhydroxyalkanoates based on 3-hydroxyisobutyric acid.
[0549] FIG. 15 shows the conversion of .beta.-alanine into
3-hydroxyisobutyric acid with catalysis by the enzymes E.sub.10 to
E.sub.12, E.sub.56, E.sub.72 and E.sub.73 according to a third
special embodiment of the cell according to the invention, where
acrylyl-coenzyme A is formed as intermediate and methylmalonate
semialdehyde as precursor in the production of 3-hydroxyisobutyric
acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric
acid.
[0550] FIG. 16 shows the conversion of pyruvate into
3-hydroxyisobuytric acid with catalysis by the enzymes E.sub.76 to
E.sub.79, E.sub.60, E.sub.61 and E.sub.8 according to a first
alternative of the first special embodiment of the second variant
of the cell according to the invention, where isobutyryl-coenzyme A
is formed as intermediate and 3-hydroxyisobutyryl-coenzyme A as
precursor in the production of 3-hydroxyisobutyric acid or of
polyhydroxyalkanoates based on 3-hydroxyisobutyric acid.
[0551] FIG. 17 shows the conversion of L-valine into
3-hydroxyisobutyric acid with catalysis by the enzymes E.sub.8,
E.sub.60, E.sub.61, E.sub.79 and E.sub.80 according to a second
alternative of the first special embodiment of the second variant
of the cell according to the invention, where isobutyryl-coenzyme A
is formed as intermediate and 3-hydroxyisobutyryl-coenzyme A as
precursor in the production of 3-hydroxyisobutyric acid or of
polyhydroxyalkanoates based on 3-hydroxyisobutyric acid.
EXAMPLES
Example 1
[0552] The present invention is now illustrated in Example 1 with
reference to a recombinant cell which is capable of producing
3-hydroxyisobutyric acid via 3-hydroxyisobutyryl-coenzyme A as
precursor and isobutyryl-coenzyme A as intermediate, starting from
L-valine as carbon source. To this end, the enzymes EC 2.6.1.42 and
EC 1.2.4.4 (in each case from Pseudomonas aeruginosa) and a cluster
comprising the three enzymes EC 1.3.99.12, EC 4.2.1.17 and EC
3.1.2.4 (from Acinetobacter calcoaceticus) were overexpressed in E.
coli BL21 (DE3).
[0553] Here, the enzyme EC 1.2.4.4 is encoded by a gene with the
DNA sequence as shown in SEQ ID No 07 and 08 (.alpha. and .beta.
subunit), while the enzyme EC 2.6.1.42 is encoded by a gene with
the DNA sequence as shown in SEQ ID No 09. The enzyme EC 1.3.99.12
is encoded by a gene with the DNA sequence with the SEQ ID No 10,
the enzyme EC 4.2.1.17 by a gene with the DNA sequence as shown in
SEQ ID No 11, and the enzyme EC 3.1.2.4 by a gene with the DNA
sequence as shown in SEQ ID No 12.
1. Organisms, Plasmids and Oligonucleotides
[0554] The following bacterial strains, vectors, genomic DNA and
oligonucleotides were used for preparing this recombinant cell:
TABLE-US-00001 [0554] TABLE 1 Bacterial strains used Reference
Strain (manufacturer) E. coli DH5 NEB E. coli BL21 (DE3)
Invitrogen
TABLE-US-00002 TABLE 2 Vectors used Reference Vector (manufacturer)
pCDFDuet-1 Novagen pET101/D-TOPO Invitrogen pCR2.1-TOPO
Invitrogen
TABLE-US-00003 TABLE 3 Genomic DNA used Strain Pseudomonas
aeruginosa PAO1 Acinetobacter calcoaceticus ADP1
TABLE-US-00004 TABLE 4 Oligonucleotides used Name Sequence
Aca_VClus_fw 5'-ATGCAATTTAATGAAGAACAGCTATTAATTC-3' (SEQ ID No. 13)
Aca_VClus_rev 5'-CAGTCTGAAATGACTAACCTAATTGGC-3' (SEQ ID No. 14)
Pae_26142_fw 5'-ACGGAATTCTGAAGGAGCTGGCAACTATG-3' (SEQ ID No. 15)
Pae_26142_rev 5'-TTGTCGACTTACTTGACCAGGGTACGCC-3' (SEQ ID No. 16)
Pae_1244_fw 5'-ACAGATCTGGAGGCCTGTCATGAGTGATTAC-3' (SEQ ID No. 17)
Pae_1244_rev 5'-ATGGGTACCCATTCAGACCTCCATC-3' (SEQ ID No. 18)
2. Amplification of the PCR Fragments 1.2.4.4 (2313 kb) and
2.6.1.42 (958 bp)
[0555] First, the fragments of 1.2.4.4 and 2.6.1.42 were amplified
by means of PCR starting from the total DNA from Pseudomonas
aeruginosa, using the primers as shown in SEQ ID No 15 to SEQ ID No
18, which are detailed in Table 4. 3. Digestion of the Vector
pCDF-Duet-1 and of the PCR Fragment 2.6.1.42 (958 bp) [0556] The
vector pCDFDuet-1 (featuring a streptomycin-/spectinomycin
resistance) is cleaved by means of EcoRI/SalI, as is the PCR
fragment 2.6.1.42, and the restrictions thus obtained are ligated
overnight with T4 ligase. This gives rise to the vector
pCDFDuet::2.6.1.42. 4. Cloning of the PCR Fragments into the Vector
pCR2.1-TOPO [0557] The preparation of a cloning vector comprising
the fragment 2.6.1.42 or the fragment 1.2.4.4, using the vector
pCR2.1-TOPO, was performed as specified in the manufacturer's
instructions. E. coli DH5.alpha. cells were transformed with the
resulting cloning vectors pCR2.1-TOPO::1.2.4.4 and
pCR2.1-TOPO::2.6.1.42. Since the pCR2.1-TOPO vectors feature a
kanamycin resistance and an ampicillin resistance, the
transformants were plated onto 2 AXI and KXI plates (20 and 40
.mu.l). The plasmids of the resulting clones were isolated and
digested:
TABLE-US-00005 [0557] pCR2.1-TOPO::1.2.4.4 BgIII + KpnI fragment
size 2313 bp pCR2.1-TOPO::2.6.1.42 EcoRI + SalI fragment size 958
bp
[0558] Each of the fragments was eluted from the gel and purified
with the QIAquick kit from Qiagen (following instructions). 5.
Preparation of the Vector pCDFDuet:2.6.1.42-1.2.4.4 [0559] The
vector pCDFDuet::2.6.1.42 and the vector pCR2.1-TOPO::1.2.4.4 are
digested with BgIII/KpnI. [0560] This is followed by the ligation
of pCDFDuet::2.6.1.42 (BgIII/KpnI) with pCR2.1-TOPO::1.2.4.4,
giving rise to the vector pCDFDuet::2.6.1.42-1.2.4.4. Again, E.
coli DH5.alpha. cells were transformed by means of this cloning
vector. The plasmids were isolated. The plasmid
pCDFDuet::2.6.1.42-1.2.4.4 features the DNA sequence as shown in
SEQ ID No 19. 6. Cloning the Valine Cluster from Acinetobacter
calcoaceticus (V-Clus.sub.Aca) [0561] Strain ATCC 33304
Acinetobacter calcoaceticus was cultured for the isolation of total
DNA (HH agar or medium). Total DNA was isolated by means of the
DNEasy kit from Qiagen (L1 and L2) and by a method comprising the
method steps i) centrifugation of 1 ml of culture, ii) addition of
200 .mu.l of H.sub.2O to the pellet, iii) heating for 10 min at
95.degree. C., iv) centrifugation (10 min, 13 000 rpm), and v)
removing the supernatant for a PCR. [0562] To amplify the valine
cluster from A. calcoaceticus, a PCR was carried out using the
primers as shown in SEQ ID No 13 and SEQ ID No 14, which have been
detailed in Table 4 (following the manufacturer's instructions
using the polymerases Pfu and Taq, respectively). [0563] The PCR
products were purified and, following the instructions, ligated to
the plasmid pET101/D-TOPO and transferred into E. coli DH5.alpha..
This gives rise to the plasmid pET101/D-TOPO::V-Cluster.sub.Aca.
Plasmid pET101/D-TOPO::V-Cluster.sub.Aca features the DNA sequence
as shown in SEQ ID No 20. 7. Preparation of a Recombinant Cell
which is Capable of Forming 3-Hydroxyisobutyric Acid from L-Valine
[0564] E. coli BL21 (DE3) was transformed with the plasmids
pET101/D-TOPO::V-Cluster.sub.Aca and pCDF-Duet::2.6.1.42-1.2.4.4
(plated onto LB spec./amp medium). The resulting cells were capable
of converting, in a nutrient medium comprising L-valine, the
L-valine into 3-hydroxyisobutyric acid. In contrast, the wild type
of the cells (E. coli BL21 (DE3)) was not capable of forming
detectable amounts of 3-hydroxyisobutyric acid in such a nutrient
medium.
Example 2
[0565] In this example, the DNA according to the invention is
isolated and the gene is overexpressed in E. coli.
1. Culturing and Harvesting Sulfolobus tokodaii [0566] Sulfolobus
tokodaii was grown in a small culture volume (40-200 ml) at
75.degree. C. and a pH of 3.0, with shaking (150 rpm). The growth
was monitored photometrically via measuring the optical density at
578 nm (OD.sub.578 nm). A modified Sulfolobus medium was used
(modified as described by Brock et al., Archives of Microbiology
84, pages 54-68, 1972; Suzuki et al., Extremophiles, 6, pages
39-44, 2002). The energy and carbohydrate source used were yeast
extract, casamino acids and glucose. The medium consisted of the
following components: basal medium, glucose stock solution, iron
stock solution and trace element stock solution. At an OD.sub.578
nm of 0.3-0.5 (exponential phase), the cells were harvested. The
centrifugation was carried out in a Sorvall centrifuge (SS34 rotor)
for 15 min at 9000 rpm. The cell pellet was employed directly for
the DNA extraction. [0567] Basal medium. KH.sub.2PO.sub.4 (0.28
g/l), (NH.sub.4).sub.2SO.sub.4 (1.3 g/l), MgSO.sub.4.times.7
H.sub.2O (0.25 g/l), CaCl.sub.2.times.6 H.sub.2O (0.07 g/l), yeast
extract (1g/l) and casamino acids (1g/l). Before autoclaving, the
pH was brought to 3.0 using H.sub.2SO.sub.4. [0568] Glucose stock
solution (100.times.). Glucose (100g/l). [0569] The solution was
filter-sterilized. [0570] Iron stock solution (1000.times.).
FeCl.sub.3.times.6 H.sub.2O (20g/l). The solution was
filter-sterilized. [0571] Trace element stock solution
(1000.times.). MnCl.sub.2.times.4 H.sub.2O (1.8 g/l),
Na.sub.2B.sub.4O.sub.7.times.10 H.sub.2O (4.5 g/l),
ZnSO.sub.4.times.7H.sub.2O (220 mg/l), CuCl.sub.2.times.2 H.sub.2O
(50 mg/l), Na.sub.2MoO.sub.4.times.2 H.sub.2O (30 mg/l),
VOSO.sub.4.times.5 H.sub.2O (30 mg/l), CoCl.sub.2.times.6 H.sub.2O
(8.4 mg/l). The individual components were dissolved in succession
in distilled H.sub.2O, the pH was brought to 3.0 using HCl, and the
solution was filter-sterilized. 2. Isolation of Genomic DNA from S.
tokodaii [0572] Genomic DNA was isolated by the method of Murray
and Thompson (Nucleic Acid Research, 8, pages 4321-4325, 1980). To
this end, 10-50 mg (fresh weight) of freshly harvested cells are
weighed into a 1.5 ml Eppendorf reaction vessel and resuspended in
570 ml of TE buffer (10 mM Tris/HCl (pH 8.0), 1 mM NaEDTA). 30
.mu.l of a 10% (w/v) SDS solution (sodium dodecyl sulfate solution)
and 3 .mu.l of Proteinase K (20 .mu.g/.mu.l) were added and the
mixture was incubated for 1 h at 52.degree. C. Thereafter, 100
.mu.l of 5 M NaCl solution and 80 .mu.l of pre-warmed 10% (w/v)
cetyltrimethylammonium bromide (CTAB) solution (10% (w/v) CTAB in
0.7 M NaCl) were added. After incubation for 10 min at 65.degree.
C., the complexes of CTAB, cell wall fragments and proteins were
extracted with 780 .mu.l of chloroform/iso-amyl alcohol (24:1
(v/v)) and spun down for 15 min at 14 000 rpm. The aqueous top
phase was transferred into a fresh Eppendorf reaction vessel and
the extraction was repeated. After the aqueous phase was free from
pigments, it was covered with a layer of 400 .mu.l of 100%
isopropanol. By carefully mixing the two phases, the chromosomal
DNA precipitated at the interface. Then, it was possible to fish
out the DNA with a drawn-out Pasteur pipette and washed in 200
.mu.l of 70% ethanol. After recentrifugation (5 min, 14 000 rpm),
the supernatant was pipetted off and the DNA was dried for 2 h at
room temperature and finally dissolved in 100 .mu.l of TE
buffer.
3. Amplification of the Malonyl-Coenzyme A Reductase Gene
[0572] [0573] The polymer chain reaction (PCR) (Mullis et al., Cold
Spring Harbor Symp. Quant. Biol., 51, pages 263-273, 1986) was
employed to amplify the malonyl-CoA reductase gene in a targeted
fashion, from the genomic Sulfolobus tokodaii DNA obtained in
Example 2. It was carried out in a thermocycler (Biometra,
Gottingen). [0574] A preparative PCR in which Pfu polymerase
(Pfunds, Genaxxon) was used, was employed. The Pfu polymerase
contains a 3'-5' exonuclease ("proofreading") function. [0575] The
following primers were used:
TABLE-US-00006 [0575] 5'-ATTATCCCATGGGGAGAACATTAAAAGC-3' ("forward
primer"; NcoI cleavage site is underlined; (SEQ ID No 21) and
5'-CGGGATCCTTACTTTTCAATATATCC-3' ("reverse primer"; BamHI cleavage
site is underlined; SEQ ID No 22)
[0576] The reaction mixture detailed in Table 1 hereinbelow was
employed for the PCR reactions. The PCR was carried out as a hot
start PCR, i.e. the reaction mixture was incubated for 2 min at
95.degree. C. before adding the Pfu polymerase. This was followed
by 30 cycles of in each case 1 minute at 95.degree. C., 1 minute at
45.degree. C. and 5 minutes at 72.degree. C., followed by a last
step of 30 seconds at 45.degree. C., 15 minutes at 72.degree. C.
and, finally, a pause at 6.degree. C.
TABLE-US-00007 [0576] TABLE 1 Standard reaction mixtures (50 .mu.l)
for proofreading PCR with Pfu polymerase Composition .mu.l/50 .mu.l
batch 10 .times. Pfu PCR 5 reaction buffer dNTP mix (2 mM per 5
nucleotide) Forward primer (2 .mu.M) 12.5 Reverse primer (2 .mu.M)
12.5 Chromosomal DNA 1 (10-50 ng) Pfu polymerase 2 (2.5 U/.mu.l)
dd-H.sub.2O 12
[0577] A gene fragment with a length of 1.1 kb was obtained.
4. Cloning the Malonyl-Coenzyme A Reductase Gene
[0577] [0578] To clone the malonyl-coenzyme A reductase gene from
Sulfolobus tokodaii, the gene amplified in Example 3 was cloned
unspecifically with the vector pCR T7/CT-Topo (Invitrogen,
Karlsruhe), using the "pCR T7 Topo TA Expression Kit" (Invitrogen,
Karlsruhe). This was done following the manufacturer's
instructions. [0579] To isolate the plasmid DNA, the plasmid DNA
was prepared using the "QIAprep Spin Plasmid Miniprep Kit" from
Qiagen (Hilden) following the manufacturer's instructions, starting
from 5 ml overnight cultures of transformed E. coli TOP10F'
cells.
5. Generation of an Expression Vector
[0579] [0580] To generate an expression vector comprising the
malonyl-coenzyme A reductase gene, the isolated cloning vector
obtained in Example 4 is subjected to restriction digestion with
the restriction enzymes NCoI and BamHI. To this end, 25-27 .mu.l of
plasmid DNA (expression vector pTrc99A and pCR T7/CT-Topo vector,
respectively, with the incorporated malonyl-coenzyme A reductase
gene) are mixed thoroughly with 5 .mu.l of a reaction buffer
(10.times.) and 2-3 .mu.l of restriction enzyme (10 U/.mu.l;
Fermentas, St. Leon-Rot). The reaction mixture was made up to 50
.mu.l with distilled H.sub.2O and incubated for 5 h at the
temperature specified by the manufacturer. An ethanol precipitation
was carried out before further use. To this end, the DNA was mixed
with 3 volumes of 100% ethanol and 0.1 volumes of 3 M sodium
acetate buffer (pH 5.3) and incubated for 2 h or overnight at
-80.degree. C. After a centrifugation step (20 min, 14 000 rpm,
4.degree. C., Eppendorf table-top centrifuge), the supernatant is
removed carefully, and the DNA was washed with 3 volumes of 70%
(v/v) ethanol. After 10 min incubation at room temperature, the
mixture was recentrifuged (10 min, 14 000 rpm, 4.degree. C.,
Eppendorf table-top centrifuge) and the supernatant was discarded.
The DNA was then dried for 1 hour at room temperature and
subsequently taken up in the desired volume of H.sub.2O or TE
buffer (10 mM Tris/HCl (pH 8.0), 1 mM NaEDTA). [0581] Then,
alkaline phosphatase is used for removing the 5'-phosphate groups
of the linearized double-stranded vector. In this manner, the
cloning efficiency is increased since religation of the vector is
prevented. Calf intestinal alkaline phosphatase was used for
dephosphorylating the digested vector. [0582] The dephosphorylation
was carried out in the same buffer as the restriction digestion. 50
.mu.l of restriction mixture were mixed with 1.5 .mu.l of CIAP
(Calf Intestine Alkaline Phosphatase (1 U/.mu.l; Fermentas, St.
Leon-Rot) and the mixture was incubated for 30 min at 37.degree. C.
Before further use of the cleaved and dephosphorylated vector, an
ethanol precipitation was carried out as described above. [0583] T4
DNA ligase was used the ligation of the insert DNA with the
expression vector, plasmid DNA and insert DNA being employed in a
molar ratio of from 1:3-1:6. [0584] Stock Solutions: [0585]
Ligation buffer (10.times.): 0.5 M Tris/HCl, pH 7.6 [0586] 100 mM
MgCl.sub.2 [0587] 0.5 mg/ml BSA [0588] filter-sterilized, storage
at room temperature [0589] 5 mM ATP (adenosine triphosphate) Always
make up freshly in sterile distilled H.sub.2O [0590] 50 mM DTE
(dithioerythritol) Always make up freshly in ligation buffer [0591]
The ligation mixtures had a volume of 50 .mu.l. Plasmid DNA (2-10
.mu.l), insert DNA (2-20 .mu.l), 5 .mu.l of ligation buffer with
DTE (50 mM) and the corresponding amount of sterile distilled
H.sub.2O were pipetted together, vortexed, spun down briefly and
subsequently incubated for 5 min at 45.degree. C. The mixture was
cooled on ice. 5 .mu.l of 5 mM ATP and 1.5 .mu.l of T4 DNA ligase
(1 U/.mu.l; Fermentas; St. Leon-Rot) were added, and everything was
mixed. Ligation was performed overnight at 16.degree. C. [0592] The
ligation mixture was employed directly for transforming chemically
competent cells. 6. Transformation of E. coli Cells with the
Expression Vector [0593] A 5 ml overnight culture was grown
starting from a single colony of E. coli Rosetta 2 cells. On the
next morning, 50 ml of LB medium (Sambrook et al., "Molecular
Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989) were inoculated with 0.5-1.0 ml of
this culture. After incubation for 1.5-2 h (37.degree. C., shaking
(180 rpm)), an OD.sub.578 of 0.6 was reached. The cells were cooled
on ice for 10 min and subsequently spun down for 5 min at 5000 rpm
and 4.degree. C. (GSA rotor, Sorvall centrifuge). The supernatant
was discarded and the cell pellet was resuspended in 2.7 ml of cold
0.1 M CaCl.sub.2 solution. After addition of 2.3 ml of sterile 50%
(v/v) glycerol, the cell suspension was divided into portions (in
each case 300 .mu.l) in 1.5-ml Eppendorf reaction vessels. The
competent cells were immediately frozen in liquid nitrogen and
subsequently stored at -80.degree. C.
[0594] To transform the cells, an aliquot of the chemically
competent cells (300 .mu.l) was defrosted on ice and treated with
25 .mu.l of a ligation mixture. Everything was mixed carefully and
incubated for 30 min on ice. After a heat shock (42.degree. C., 1
min) the mixture was reincubated on ice for 5 min. Thereafter, 800
.mu.l of LB medium (Sambrook et al., 1989) were added, and the
cells were shaken for 1 h at 37.degree. C. (Thermomixer, Eppendorf
5436). The mixture was concentrated and finally streaked onto LB
medium. To this end, the mixture was spun down for 1 min at 10 000
rpm, 750 .mu.m of the supernatant were discarded, and the cell
pellet was resuspended. 50 .mu.l, 100 .mu.l and 200 .mu.l of this
concentrated mixture were streaked onto LB plates (Sambrook et al.,
1989) supplemented with 100 .mu.g/ml ampicillin and incubated
overnight in the incubator at 37.degree. C. The plates were washed
with 1 ml LB medium. This cell suspension was used for subsequently
inoculating 150 ml LB medium (supplemented with 100 .mu.g/ml
ampicillin) in 500 ml Erlenmeyer flasks with baffles. The cultures
grew at 37.degree. C. and 180 rpm. Overexpression was performed by
inducing the promoter in pTrc99A by adding 0.5 M IPTG
(isopropyl-.beta.-D-thiogalactopyranoside) at an OD.sub.578nm of
0.6. The induced cultures were incubated for 3 h under the
abovementioned conditions and subsequently harvested at an
OD.sub.578nm=2.7.
7. Detection of the Enzymatic Activity
[0595] The E. coli strain obtained in Example 6 was disrupted by
means of a cell mill. The disrupted cells were heated for 15 min at
85.degree. C. During this heat precipitation, nonheat resistant
enzymes coagulate and are precipitated. Since the target protein is
heat resistant, it is retained in the supernatant. To measure the
malonyl-coenzyme A reductase activity, the supernatant was diluted
1:50 in TM buffer (50 mM Tris/Cl, 1 mM MgCl.sub.2, pH 8.1). 30
.mu.l of the diluted or undiluted (for detecting the
methylmalonyl-coenzyme A reductase activity) supernatant were
pipetted to 500 .mu.l of HIPS buffer (100 mM HEPES/NaOH, 5 mM
MgCl.sub.2, 1 mM dithioerythritol, containing 0.5 mM NADPH). [0596]
In a first batch, the reaction was started by adding
malonyl-coenzyme A, the final concentration being 0.5 mM. The drop
in the NADPH absorption at 365 nm was determined. The enzyme
activity determined was 15.5 .mu.mol/min/mg protein (15.5 U/mg).
[0597] In a second batch, the reaction was started by adding
methylmalonyl-coenzyme A (from Fluka, Article No.: 67767), the
final concentration being 2.0 mM. The drop in the NADPH absorption
at 365 nm was determined. The enzyme activity determined was 0.24
.mu.mol/min/mg protein (0.24 U/mg). [0598] It can be seen from
these results that the polypeptide which codes for the DNA sequence
with the SEQ ID No 03 catalyzes both the conversion of malonyl-CoA
and of methylmalonyl-coenzyme A. [0599] 1 mol of NADPH was oxidized
per mole of malonyl-CoA or methylmalonyl-Coa employed. From this it
can be concluded that the enzymatic reaction leads to the
corresponding semialdehyde.
Sequence CWU 1
1
2212214DNACorynebacterium glutamicum ATCC 13032 1atgacgtcga
tccctaattt ttcagacatc ccattgactg ctgagacacg tgcatcggag 60tcacacaacg
ttgacgccgg caaggtgtgg aacactcccg aaggcattga tgtcaagcgc
120gtattcacgc aggctgaccg cgacgaggcg caagcggcgg gacatccggt
ggattctttg 180ccaggtcaaa agccatttat gcgcgggccg tacccaacta
tgtacaccaa tcagccgtgg 240acgattcgcc agtacgcagg cttttcaacc
gccgcggaat ccaatgcgtt ttatcggagg 300aaccttgctg cgggtcaaaa
aggtttgtcg gttgcgttcg atctagcgac ccaccgcggt 360tatgactcgg
ataatgagcg cgtggtcggc gatgtgggta tggccggcgt ggcgattgat
420tcgattttgg atatgcgtca gctgtttgat ggcattgatt tgtccagcgt
gtcggtgtcg 480atgaccatga atggcgctgt gctgccgatt cttgcgttct
atatcgtggc ggctgaggaa 540caaggtgtgg gtccggagca gcttgcgggc
acgatccaga atgacatctt gaaagaattt 600atggtgcgca acacctatat
ttatccgccg aagccgtcga tgcgcatcat ttccaacatc 660tttgagtaca
cctccttgaa gatgccacgt tttaactcca tttcgatttc tggctatcac
720atccaggaag cgggagcgac tgccgatttg gagctggcct acactctggc
ggatggtatt 780gaatacatcc gtgcaggtaa agaggtaggc cttgacgtgg
ataagttcgc gcctcgtctg 840tccttcttct ggggtatttc tatgtacacc
ttcatggaga tcgcaaagct gcgtgcggga 900cgactgctgt ggagcgagtt
ggtggcaaaa ttcgatccga aaaacgccaa gtcccagtcg 960ctgcgcacgc
actcgcagac ctctggttgg tcgttgaccg cgcaggatgt gtacaacaac
1020gtcgcccgca ccgcgattga ggcgatggct gcaacccagg gccacaccca
gtcgctgcac 1080accaatgcac ttgatgaggc gttggcgctg cccaccgatt
tctctgctcg tatcgcccga 1140aacacccagc tgttgctgca gcaggaatct
ggcacggtgc gtccagttga tccatgggcg 1200ggctcctatt acgtggagtg
gttgaccaat gagctggcta accgcgcgcg caagcacatc 1260gatgaggtgg
aggaagccgg cggaatggcg caggccaccg cgcagggaat tcctaagctg
1320cgcattgagg aatcagcggc acgcacccag gctcgcattg attccggccg
ccaggcgctg 1380atcggcgtga atcgctacgt ggcggaagaa gatgaggaaa
ttgaagtcct caaggttgac 1440aacaccaagg ttcgcgcaga acagttggct
aaactcgcgc aactgaaagc agagcgcaac 1500gatgcggaag tcaaggctgc
gctggatgcg ttgacagctg ctgcccgcaa cgagcataaa 1560gagccagggg
atttggatca gaacctgctc aaacttgccg tcgatgctgc gcgcgcaaaa
1620gctaccattg gagagatctc cgatgctttg gaagttgtct ttggccgcca
cgaagcagaa 1680atcaggacgc tgtctggcgt gtacaaggat gaggttggaa
aggaaggcac agtgagcaac 1740gtcgaacgcg cgatcgccct ggctgacgcc
tttgaggctg aggaaggccg ccgcccacgt 1800atctttattg ccaagatggg
ccaggatgga catgaccgtg gacagaaggt tgtcgcgtct 1860gcctatgctg
acctgggcat ggacgtggat gttggaccgc tgtttcaaac tccagccgaa
1920gctgcccgcg ccgccgtgga cgccgatgtt cacgtggtgg gtatgtcttc
gctggcagca 1980ggccacctca ccttgctgcc cgagctgaag aaagaacttg
cagctcttgg ccgcgatgac 2040attctggtca ccgtgggcgg cgtcattccg
ccgggcgatt tccaggatct ctacgatatg 2100ggtgccgccg cgatttaccc
tccaggaacc gtcatcgcgg agtcggcgat cgatctgatc 2160acccgactcg
ccgcacacct gggctttgac ctggatgtgg atgtgaatga gtga
22142737PRTCorynebacterium glutamicum ATCC 13032 2Met Thr Ser Ile
Pro Asn Phe Ser Asp Ile Pro Leu Thr Ala Glu Thr1 5 10 15Arg Ala Ser
Glu Ser His Asn Val Asp Ala Gly Lys Val Trp Asn Thr 20 25 30Pro Glu
Gly Ile Asp Val Lys Arg Val Phe Thr Gln Ala Asp Arg Asp 35 40 45Glu
Ala Gln Ala Ala Gly His Pro Val Asp Ser Leu Pro Gly Gln Lys 50 55
60Pro Phe Met Arg Gly Pro Tyr Pro Thr Met Tyr Thr Asn Gln Pro Trp65
70 75 80Thr Ile Arg Gln Tyr Ala Gly Phe Ser Thr Ala Ala Glu Ser Asn
Ala 85 90 95Phe Tyr Arg Arg Asn Leu Ala Ala Gly Gln Lys Gly Leu Ser
Val Ala 100 105 110Phe Asp Leu Ala Thr His Arg Gly Tyr Asp Ser Asp
Asn Glu Arg Val 115 120 125Val Gly Asp Val Gly Met Ala Gly Val Ala
Ile Asp Ser Ile Leu Asp 130 135 140Met Arg Gln Leu Phe Asp Gly Ile
Asp Leu Ser Ser Val Ser Val Ser145 150 155 160Met Thr Met Asn Gly
Ala Val Leu Pro Ile Leu Ala Phe Tyr Ile Val 165 170 175Ala Ala Glu
Glu Gln Gly Val Gly Pro Glu Gln Leu Ala Gly Thr Ile 180 185 190Gln
Asn Asp Ile Leu Lys Glu Phe Met Val Arg Asn Thr Tyr Ile Tyr 195 200
205Pro Pro Lys Pro Ser Met Arg Ile Ile Ser Asn Ile Phe Glu Tyr Thr
210 215 220Ser Leu Lys Met Pro Arg Phe Asn Ser Ile Ser Ile Ser Gly
Tyr His225 230 235 240Ile Gln Glu Ala Gly Ala Thr Ala Asp Leu Glu
Leu Ala Tyr Thr Leu 245 250 255Ala Asp Gly Ile Glu Tyr Ile Arg Ala
Gly Lys Glu Val Gly Leu Asp 260 265 270Val Asp Lys Phe Ala Pro Arg
Leu Ser Phe Phe Trp Gly Ile Ser Met 275 280 285Tyr Thr Phe Met Glu
Ile Ala Lys Leu Arg Ala Gly Arg Leu Leu Trp 290 295 300Ser Glu Leu
Val Ala Lys Phe Asp Pro Lys Asn Ala Lys Ser Gln Ser305 310 315
320Leu Arg Thr His Ser Gln Thr Ser Gly Trp Ser Leu Thr Ala Gln Asp
325 330 335Val Tyr Asn Asn Val Ala Arg Thr Ala Ile Glu Ala Met Ala
Ala Thr 340 345 350Gln Gly His Thr Gln Ser Leu His Thr Asn Ala Leu
Asp Glu Ala Leu 355 360 365Ala Leu Pro Thr Asp Phe Ser Ala Arg Ile
Ala Arg Asn Thr Gln Leu 370 375 380Leu Leu Gln Gln Glu Ser Gly Thr
Val Arg Pro Val Asp Pro Trp Ala385 390 395 400Gly Ser Tyr Tyr Val
Glu Trp Leu Thr Asn Glu Leu Ala Asn Arg Ala 405 410 415Arg Lys His
Ile Asp Glu Val Glu Glu Ala Gly Gly Met Ala Gln Ala 420 425 430Thr
Ala Gln Gly Ile Pro Lys Leu Arg Ile Glu Glu Ser Ala Ala Arg 435 440
445Thr Gln Ala Arg Ile Asp Ser Gly Arg Gln Ala Leu Ile Gly Val Asn
450 455 460Arg Tyr Val Ala Glu Glu Asp Glu Glu Ile Glu Val Leu Lys
Val Asp465 470 475 480Asn Thr Lys Val Arg Ala Glu Gln Leu Ala Lys
Leu Ala Gln Leu Lys 485 490 495Ala Glu Arg Asn Asp Ala Glu Val Lys
Ala Ala Leu Asp Ala Leu Thr 500 505 510Ala Ala Ala Arg Asn Glu His
Lys Glu Pro Gly Asp Leu Asp Gln Asn 515 520 525Leu Leu Lys Leu Ala
Val Asp Ala Ala Arg Ala Lys Ala Thr Ile Gly 530 535 540Glu Ile Ser
Asp Ala Leu Glu Val Val Phe Gly Arg His Glu Ala Glu545 550 555
560Ile Arg Thr Leu Ser Gly Val Tyr Lys Asp Glu Val Gly Lys Glu Gly
565 570 575Thr Val Ser Asn Val Glu Arg Ala Ile Ala Leu Ala Asp Ala
Phe Glu 580 585 590Ala Glu Glu Gly Arg Arg Pro Arg Ile Phe Ile Ala
Lys Met Gly Gln 595 600 605Asp Gly His Asp Arg Gly Gln Lys Val Val
Ala Ser Ala Tyr Ala Asp 610 615 620Leu Gly Met Asp Val Asp Val Gly
Pro Leu Phe Gln Thr Pro Ala Glu625 630 635 640Ala Ala Arg Ala Ala
Val Asp Ala Asp Val His Val Val Gly Met Ser 645 650 655Ser Leu Ala
Ala Gly His Leu Thr Leu Leu Pro Glu Leu Lys Lys Glu 660 665 670Leu
Ala Ala Leu Gly Arg Asp Asp Ile Leu Val Thr Val Gly Gly Val 675 680
685Ile Pro Pro Gly Asp Phe Gln Asp Leu Tyr Asp Met Gly Ala Ala Ala
690 695 700Ile Tyr Pro Pro Gly Thr Val Ile Ala Glu Ser Ala Ile Asp
Leu Ile705 710 715 720Thr Arg Leu Ala Ala His Leu Gly Phe Asp Leu
Asp Val Asp Val Asn 725 730 735Glu31071DNASulfolobus tokodaii
3atgaggagaa cattaaaagc cgcaatatta ggtgctactg gtttagtagg aatcgaatac
60gtaagaatgc tatcaaatca tccttatatt aaaccagcat atttagctgg aaaaggttca
120gtgggtaaac cgtatggtga ggtagtaaga tggcaaacag taggacaagt
tcctaaggaa 180atagctgata tggaaataaa accaactgat cctaagttaa
tggatgatgt agacataata 240ttttctccat tacctcaagg tgctgctggc
ccagtagaag aacaatttgc aaaagaagga 300ttccctgtga ttagtaattc
accagatcat agatttgatc ctgatgttcc cttattggtt 360cctgaactaa
atcctcatac tattagctta attgatgagc aaagaaaaag aagagaatgg
420aaaggattta tagtaactac accactatgc acagcccagg gtgcagcaat
accattaggt 480gctatattta aagattataa gatggatgga gcatttataa
ctactattca atcgctatct 540ggtgccggtt atccaggaat accatcatta
gatgtagtag ataatatctt gcctttaggt 600gatggatacg atgccaagac
gataaaagag atcttcagaa ttttaagcga agttaagaga 660aatgtagatg
aacctaaatt agaagatgta agcttagcag caacaactca tagaatagct
720actatacatg gtcattatga agtactatat gtatcgttca aagaggaaac
tgctgctgaa 780aaagttaagg agactttaga aaactttaga ggggaaccac
aagatctaaa attaccaact 840gcaccttcaa agccaattat cgttatgaat
gaggatacaa gacctcaagt ctattttgat 900agatgggctg gggatattcc
aggaatgagt gtagttgtag gtagattaaa gcaagtgaat 960aagagaatga
taaggttagt atcattaatt cataacacgg tcagaggagc cgcaggagga
1020ggtatattag cagctgaatt acttgtcgaa aaaggatata ttgaaaagta a
10714356PRTSulfolobus tokodaii 4Met Arg Arg Thr Leu Lys Ala Ala Ile
Leu Gly Ala Thr Gly Leu Val1 5 10 15Gly Ile Glu Tyr Val Arg Met Leu
Ser Asn His Pro Tyr Ile Lys Pro 20 25 30Ala Tyr Leu Ala Gly Lys Gly
Ser Val Gly Lys Pro Tyr Gly Glu Val 35 40 45Val Arg Trp Gln Thr Val
Gly Gln Val Pro Lys Glu Ile Ala Asp Met 50 55 60Glu Ile Lys Pro Thr
Asp Pro Lys Leu Met Asp Asp Val Asp Ile Ile65 70 75 80Phe Ser Pro
Leu Pro Gln Gly Ala Ala Gly Pro Val Glu Glu Gln Phe 85 90 95Ala Lys
Glu Gly Phe Pro Val Ile Ser Asn Ser Pro Asp His Arg Phe 100 105
110Asp Pro Asp Val Pro Leu Leu Val Pro Glu Leu Asn Pro His Thr Ile
115 120 125Ser Leu Ile Asp Glu Gln Arg Lys Arg Arg Glu Trp Lys Gly
Phe Ile 130 135 140Val Thr Thr Pro Leu Cys Thr Ala Gln Gly Ala Ala
Ile Pro Leu Gly145 150 155 160Ala Ile Phe Lys Asp Tyr Lys Met Asp
Gly Ala Phe Ile Thr Thr Ile 165 170 175Gln Ser Leu Ser Gly Ala Gly
Tyr Pro Gly Ile Pro Ser Leu Asp Val 180 185 190Val Asp Asn Ile Leu
Pro Leu Gly Asp Gly Tyr Asp Ala Lys Thr Ile 195 200 205Lys Glu Ile
Phe Arg Ile Leu Ser Glu Val Lys Arg Asn Val Asp Glu 210 215 220Pro
Lys Leu Glu Asp Val Ser Leu Ala Ala Thr Thr His Arg Ile Ala225 230
235 240Thr Ile His Gly His Tyr Glu Val Leu Tyr Val Ser Phe Lys Glu
Glu 245 250 255Thr Ala Ala Glu Lys Val Lys Glu Thr Leu Glu Asn Phe
Arg Gly Glu 260 265 270Pro Gln Asp Leu Lys Leu Pro Thr Ala Pro Ser
Lys Pro Ile Ile Val 275 280 285Met Asn Glu Asp Thr Arg Pro Gln Val
Tyr Phe Asp Arg Trp Ala Gly 290 295 300Asp Ile Pro Gly Met Ser Val
Val Val Gly Arg Leu Lys Gln Val Asn305 310 315 320Lys Arg Met Ile
Arg Leu Val Ser Leu Ile His Asn Thr Val Arg Gly 325 330 335Ala Ala
Gly Gly Gly Ile Leu Ala Ala Glu Leu Leu Val Glu Lys Gly 340 345
350Tyr Ile Glu Lys 35551293DNARhodobacter sphaeroides 5atggccctcg
acgtgcagag cgatatcgtc gcctacgacg cgcccaagaa ggacctctac 60gagatcggcg
agatgccgcc tctcggccat gtgccgaagg agatgtatgc ttgggccatc
120cggcgcgagc gtcatggcga gccggatcag gccatgcaga tcgaggtggt
cgagacgccc 180tcgatcgaca gccacgaggt gctcgttctc gtgatggcgg
cgggcgtgaa ctacaacggc 240atctgggccg gcctcggcgt gcccgtctcg
ccgttcgacg gtcacaagca gccctatcac 300atcgcgggct ccgacgcgtc
gggcatcgtc tgggcggtgg gcgacaaggt caagcgctgg 360aaggtgggcg
acgaggtcgt gatccactgc aaccaggacg acggcgacga cgaggaatgc
420aacggcggcg acccgatgtt ctcgcccacc cagcggatct ggggctacga
gacgccggac 480ggctccttcg cccagttcac ccgcgtgcag gcgcagcagc
tgatgaagcg tccgaagcac 540ctgacctggg aagaggcggc ctgctacacg
ctgaccctcg ccaccgccta ccggatgctc 600ttcggccaca agccgcacga
cctgaagccg gggcagaacg tgctggtctg gggcgcctcg 660ggcggcctcg
gctcctacgc gatccagctc atcaacacgg cgggcgccaa tgccatcggc
720gtcatctcag aggaagacaa gcgcgacttc gtcatggggc tgggcgccaa
gggcgtcatc 780aaccgcaagg acttcaagtg ctggggccag ctgcccaagg
tgaactcgcc cgaatataac 840gagtggctga aggaggcgcg caagttcggc
aaggccatct gggacatcac cggcaagggc 900atcaacgtcg acatggtgtt
cgaacatccg ggcgaggcga ccttcccggt ctcgtcgctg 960gtggtgaaga
agggcggcat ggtcgtgatc tgcgcgggca ccaccggctt caactgcacc
1020ttcgacgtcc gctacatgtg gatgcaccag aagcgcctgc agggcagcca
tttcgccaac 1080ctcaagcagg cctccgcggc caaccagctg atgatcgagc
gccgcctcga tccctgcatg 1140tccgaggtct tcccctgggc cgagatcccg
gctgcccata cgaagatgta taagaaccag 1200cacaagcccg gcaacatggc
ggtgctggtg caggccccgc gcacggggtt gcgcaccttc 1260gccgacgtgc
tcgaggccgg ccgcaaggcc tga 12936430PRTRhodobacter sphaeroides 6Met
Ala Leu Asp Val Gln Ser Asp Ile Val Ala Tyr Asp Ala Pro Lys1 5 10
15Lys Asp Leu Tyr Glu Ile Gly Glu Met Pro Pro Leu Gly His Val Pro
20 25 30Lys Glu Met Tyr Ala Trp Ala Ile Arg Arg Glu Arg His Gly Glu
Pro 35 40 45Asp Gln Ala Met Gln Ile Glu Val Val Glu Thr Pro Ser Ile
Asp Ser 50 55 60His Glu Val Leu Val Leu Val Met Ala Ala Gly Val Asn
Tyr Asn Gly65 70 75 80Ile Trp Ala Gly Leu Gly Val Pro Val Ser Pro
Phe Asp Gly His Lys 85 90 95Gln Pro Tyr His Ile Ala Gly Ser Asp Ala
Ser Gly Ile Val Trp Ala 100 105 110Val Gly Asp Lys Val Lys Arg Trp
Lys Val Gly Asp Glu Val Val Ile 115 120 125His Cys Asn Gln Asp Asp
Gly Asp Asp Glu Glu Cys Asn Gly Gly Asp 130 135 140Pro Met Phe Ser
Pro Thr Gln Arg Ile Trp Gly Tyr Glu Thr Pro Asp145 150 155 160Gly
Ser Phe Ala Gln Phe Thr Arg Val Gln Ala Gln Gln Leu Met Lys 165 170
175Arg Pro Lys His Leu Thr Trp Glu Glu Ala Ala Cys Tyr Thr Leu Thr
180 185 190Leu Ala Thr Ala Tyr Arg Met Leu Phe Gly His Lys Pro His
Asp Leu 195 200 205Lys Pro Gly Gln Asn Val Leu Val Trp Gly Ala Ser
Gly Gly Leu Gly 210 215 220Ser Tyr Ala Ile Gln Leu Ile Asn Thr Ala
Gly Ala Asn Ala Ile Gly225 230 235 240Val Ile Ser Glu Glu Asp Lys
Arg Asp Phe Val Met Gly Leu Gly Ala 245 250 255Lys Gly Val Ile Asn
Arg Lys Asp Phe Lys Cys Trp Gly Gln Leu Pro 260 265 270Lys Val Asn
Ser Pro Glu Tyr Asn Glu Trp Leu Lys Glu Ala Arg Lys 275 280 285Phe
Gly Lys Ala Ile Trp Asp Ile Thr Gly Lys Gly Ile Asn Val Asp 290 295
300Met Val Phe Glu His Pro Gly Glu Ala Thr Phe Pro Val Ser Ser
Leu305 310 315 320Val Val Lys Lys Gly Gly Met Val Val Ile Cys Ala
Gly Thr Thr Gly 325 330 335Phe Asn Cys Thr Phe Asp Val Arg Tyr Met
Trp Met His Gln Lys Arg 340 345 350Leu Gln Gly Ser His Phe Ala Asn
Leu Lys Gln Ala Ser Ala Ala Asn 355 360 365Gln Leu Met Ile Glu Arg
Arg Leu Asp Pro Cys Met Ser Glu Val Phe 370 375 380Pro Trp Ala Glu
Ile Pro Ala Ala His Thr Lys Met Tyr Lys Asn Gln385 390 395 400His
Lys Pro Gly Asn Met Ala Val Leu Val Gln Ala Pro Arg Thr Gly 405 410
415Leu Arg Thr Phe Ala Asp Val Leu Glu Ala Gly Arg Lys Ala 420 425
43071233DNAPseudomonas aeruginosa 7atgagtgatt acgagccgtt gcgtctgcat
gtcccggagc ccaccgggcg tcctggctgc 60aagaccgact tttcctatct gcacctgtcc
cccgccggcg aggtacgcaa gccgccggtg 120gatgtcgagc ccgccgagac
cagcgacctg gcctacagcc tggtacgtgt gctcgacgac 180gacggccacg
ccgtcggtcc ctggaatccg cagctcagca acgaacaact gctgcgcggc
240atgcgggcga tgctcaagac ccgcctgttc gacgcgcgca tgctcaccgc
gcaacggcag 300aaaaagcttt ccttctatat gcaatgcctc ggcgaggaag
ccatcgccac cgcccacacc 360ctggccctgc gcgacggcga catgtgcttt
ccgacctatc gccagcaagg catcctgatc 420acccgcgaat acccgctggt
ggacatgatc tgccagcttc tctccaacga ggccgacccg 480ctcaagggcc
gccagctgcc gatcatgtac tcgagcaagg aggcaggttt cttctccatc
540tccggcaacc tcgccaccca gttcatccag gcggtcggct ggggcatggc
ctcggcgatc 600aagggcgaca cgcgcatcgc ctcggcctgg atcggcgacg
gcgccaccgc cgagtcggac 660ttccacaccg ccctcacctt cgcccatgtc
taccgcgcgc cggtaatcct caacgtggtc 720aacaaccagt gggcgatctc
caccttccag gccatcgccg gcggcgaagg caccaccttc 780gccaaccgtg
gcgtgggctg cgggatcgcc tcgctgcggg tcgacggcaa tgacttcctg
840gcggtctacg ccgcctccga gtgggccgcc gagcgcgccc ggcgcaacct
cgggccgagc 900ctgatcgaat gggtcaccta ccgcgccggc ccgcactcga
cttcggacga
cccgtccaag 960taccgccccg ccgacgactg gaccaacttc ccgctgggcg
acccgatcgc ccgcctgaag 1020cggcacatga tcggcctcgg catctggtcg
gaggaacagc acgaagccac ccacaaggcc 1080ctcgaagccg aagtactggc
cgcgcagaaa caggcggaga gccatggcac cctgatcgac 1140ggccgggtgc
cgagcgccgc cagcatgttc gaggacgtct atgcagaact gccggagcac
1200ctgcgccggc aacgccagga gctcggggta tga 123381049DNAPseudomonas
aeruginosa 8atgccatgaa cccgcaacac gagaacgccc agacggtcac cagcatgacc
atgatccagg 60cgctgcgctc ggcgatggac atcatgctcg agcgcgacga cgacgtggtg
gtattcggcc 120aggacgtcgg ctacttcggc ggcgtgttcc gctgcaccga
aggcctgcag aagaaatacg 180gcacctcgcg ggtgttcgat gcgccgatct
ccgagagcgg catcatcggc gccgcggtcg 240gcatgggtgc ctacggcctg
cgcccggtgg tggagatcca gttcgccgac tacgtctacc 300cggcctccga
ccagttgatc tccgaggcgg cgcgcctgcg ctatcgctcg gccggcgact
360tcatcgtgcc gatgaccgta cgcatgccct gtggcggcgg catctacggc
gggcaaacgc 420acagccagag cccggaggcg atgttcaccc aggtctgcgg
cctgcgcacg gtgatgccgt 480ccaaccccta cgacgccaag ggcctgctga
tcgcctgcat cgagaacgac gacccggtga 540tcttcctcga gcccaagcgc
ctctacaacg gcccgttcga tggccaccac gaccgcccgg 600tgacgccctg
gtccaagcat ccggccagcc aggtgccgga cggctactac aaggtgccgc
660tggacaaggc ggcgatcgtc cgccccggcg cggcgctgac cgtgctgacc
tacggcacca 720tggtctacgt ggcccaggcc gcggccgacg agaccggcct
ggacgccgag atcatcgacc 780tgcgcagcct ctggccgctg gacctggaaa
ccatcgtcgc ctcggtgaag aagaccggcc 840gctgcgtcat cgcccacgag
gcgacccgca cctgcgggtt cggcgccgag ctgatgtcgc 900tggtgcagga
gcactgcttc caccacctgg aggcgccgat cgagcgcgtc accggttggg
960acacccccta cccgcatgcc caggagtggg cgtatttccc cggccccgcg
cgcgtcggcg 1020cggcattcaa gcgtgtgatg gaggtctga
10499924DNAPseudomonas aeruginosa 9atgtcgatgg ccgatcgtga tggcgtgatc
tggtatgacg gtgaactggt gcagtggcgc 60gacgcgacca cgcacgtgct gacccatacc
ctgcactatg gaatgggcgt gttcgagggc 120gtgcgcgcct acgacacccc
gcagggcacg gcgatcttcc gcctgcaggc gcataccgac 180cggctgttcg
actccgcgca catcatgaac atgcagatcc cgtacagccg cgacgagatc
240aacgaggcga cccgcgccgc cgtgcgcgag aacaacctgg aaagcgccta
tatccgcccg 300atggtgttct acggaagcga aggcatgggc ctgcgcgcca
gcggcctgaa ggtccatgtg 360atcatcgccg cctggagctg gggcgcctac
atgggcgagg aagccctgca gcaaggcatc 420aaggtgcgca ccagttcctt
cacccgccac cacgtcaaca tctcgatgac ccgcgccaag 480tccaacggcg
cctacatcaa ctcgatgctg gccctccagg aagcgatctc cggcggcgcc
540gacgaggcca tgatgctcga tccggaaggc tacgtggccg aaggctccgg
cgagaacatc 600ttcatcatca aggatggcgt gatctacacc ccggaagtca
ccgcctgcct gaacggcatc 660actcgtaaca ctatcctgac cctggccgcc
gaacacggtt ttaaactggt cgagaagcgc 720atcacccgcg acgaggtgta
catcgccgac gaggccttct tcactggcac tgccgcggaa 780gtcacgccga
tccgcgaagt ggacggtcgc aagatcggcg ccggccgccg tggcccggtc
840accgaaaagc tgcagaaagc ctatttcgac ctggtcagcg gcaagaccga
ggcccacgcc 900gagtggcgta ccctggtcaa gtaa 924101128DNAAcinetobacter
calcoaceticus 10atgcaattta atgaagaaca gctattaatt caggatatgg
cgaaaagttt tgccaatgaa 60cagattaaat ctaatgcagc agaatgggat aagcatagca
tttttccaaa agacgttttg 120tcccaaatgg ggcaattggg ttttatggga
atgctggtga gtgagaaatg gggcggatca 180aatacaggaa atttagctta
tgtgctggca cttgaagaaa tcgctgccgc agatggtgcg 240acttcaacca
ttatgagtgt acataattct gttggctgtg tacccattgc taaatttggt
300acagaggagc aaaagcagaa atatctagtg cctttagcac aaggtgaaat
gatcggtgca 360tttgctttaa cggaaccaca tacaggttcc gatgccgcag
ccattaaaac ccgagcaatt 420aaacaaggtg atgaatggat tattaatggc
gctaaacaat ttataacatc aggtcataat 480gcgggcgtga ttattgtatt
tgctgtgaca gatccgaatg cagggaaaaa agggctgagt 540gcatttattg
tgccgcgtga aaccttgggt tatgaggtga ttcgcaccga agaaaaattg
600ggtttacatg cgtcagatac gtgccaaatt gctttaacgg atgttcgagt
acatcacagc 660ttaatgcttg gtcaggaagg tgagggacta aaaatagcat
tgtctaatct ggaaggtggc 720cgtattggga ttgcagcgca agccgttggt
ttggcacgtg ctgcactaga agaagcgaca 780aaatatgcca aagagcgtgt
gacctttgga aagcctattt ttgagcatca ggcgttagcc 840tttcgtttag
ccagtatggc cacagaaatt gaagcagcac gacaattggt tcattacgca
900gcgcggctta aagaagctgg aaaaccttgt ttaaatgaag catcaatggc
gaaattattt 960tcatctgaaa tggtcgaacg cgtatgttct gctgctttgc
aaatctttgg tggctatggc 1020tatttaaaag actttcccat cgagcgaatt
tatcgtgatg cacgtatttg ccagatttat 1080gaaggtacaa gtgatattca
gcgtttagtg atagcaagaa gcctataa 112811774DNAAcinetobacter
calcoaceticus 11atgacattcg caacaatttt attggaaaaa cgtaagggtg
tgggcttgat tacacttaac 60cgtccaaaag cattaaatgc tttaaactca gaattaattt
atgaaataaa tttagcctta 120gacgatttag aaaatgatca aacgattggt
tgtatcgtcc ttacaggttc agaaaaagcc 180tttgccgcag gtgcggatat
caaagaaatg gcagaattaa cttttccaaa tatttatttt 240gatgattttt
ttagtcttgc agatcgtatt gcacagcgtc gtaagccttt aattgccgca
300gtgagtggtt atgctttagg tggtggctgt gagttagcac tcatgtgtga
ctttatttat 360tgtgccgaca atgccaagtt tgcactacca gaagtaactt
taggtgtcat tcctggtatt 420ggtggaacac agcgtctaac gcttgcaata
ggcaaagcca aagccatgga aatgtgtttg 480actgcacggc aaatgcaggc
tgctgaggca gaacaaagtg gtttggtggc acgcgttttt 540agtaaagaag
aacttttaga acaaacctta caggctgccg aaaaaatagc ggaaaaatca
600cgggtatcta ccataatgat taaagagtca attaatcgag cttttgaagt
gagtttagca 660gagggtttac gttttgagcg ccgaatgttc cattcagttt
ttgcgacctt agatcagaaa 720gaaggcatgc aagcatttat tgataaacgt
ccagcccaat ttaaacatca ataa 774121029DNAAcinetobacter calcoaceticus
12atgactacta ctgacaatca tttactcatt gaacataaaa acgctttagg aacaattatt
60ttaaatcgtc cagcgagtct gaacgcgcta tctctagaaa tgattaatgc gattcgtcaa
120caagttgagg attggcaagg tgatgtaaat gttcaggcca tattaattaa
atcaaatagt 180cctaaagcat tttgtgcagg tggtgatatt cgctatcttt
atgaaagtta taaaagtgga 240tcagaagagt ataaagatta tttcattgct
gaatatgaga tgctcaatag cattcgaacg 300tctaaaaaaa cagtgattgt
tttattggat ggatatgtat tgggtggtgg ttttggttta 360gcacaggctt
gtcatatctt ggtgagtagt gaaaaatcac gattttcaat gccagaaaca
420gcaataggtt ttttcccaga tgttgcagcg acttatttct tatctcgttt
agatgatgtt 480ggggtatatt tggcactgac tggtgatcaa atcagtagta
gtgatgcatt gtatttagat 540ctgattgatt atcatgttcc gagtcagaat
tttgagcgac tagaaaatgc attcagccaa 600tcacagaact tagataaatt
tcatattcag aagattattt ctgcttatat ctccagccct 660gttcagagtg
aactcagtct atggcttgaa gccattcgtc agcattttgg tcttaaaaat
720gtgcaagata tcgaagaaag tttgaaaaat gaacaagatc ccaactatca
agtatggaca 780agtaaagtgt taaatacttt gcaacaacgt tcctctattg
caaaaaaaac cagtttaaag 840ttacagctgc tagggcgtgg atggtcatta
cagcaatgta tgcgtatcga gcgaaaatta 900caggatatct ggtttgaaca
tggtgatatg attgagggtg ttcgagcgtt gattattgat 960aaagataaac
aaccgcaatg gcagcagcat aatgcgactt tagataatat attaggccaa
1020ttaggttag 10291331DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 13atgcaattta atgaagaaca
gctattaatt c 311427DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 14cagtctgaaa tgactaacct aattggc
271529DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 15acggaattct gaaggagctg gcaactatg
291628DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 16ttgtcgactt acttgaccag ggtacgcc
281731DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 17acagatctgg aggcctgtca tgagtgatta c
311825DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 18atgggtaccc attcagacct ccatc
25196960DNAArtificial SequenceDescription of Artificial Sequence
Synthetic plasmid polynucleotide 19ggggaattgt gagcggataa caattcccct
gtagaaataa ttttgtttaa ctttaataag 60gagatatacc atgggcagca gccatcacca
tcatcaccac agccaggatc cgaattctga 120aggagctggc aactatgtcg
atggccgatc gtgatggcgt gatctggtat gacggtgaac 180tggtgcagtg
gcgcgacgcg accacgcacg tgctgaccca taccctgcac tatggaatgg
240gcgtgttcga gggcgtgcgc gcctacgaca ccccgcaggg cacggcgatc
ttccgcctgc 300aggcgcatac cgaccggctg ttcgactccg cgcacatcat
gaacatgcag atcccgtaca 360gccgcgacga gatcaacgag gcgacccgcg
ccgccgtgcg cgagaacaac ctggaaagcg 420cctatatccg cccgatggtg
ttctacggaa gcgaaggcat gggcctgcgc gccagcggcc 480tgaaggtcca
tgtgatcatc gccgcctgga gctggggcgc ctacatgggc gaggaagccc
540tgcagcaagg catcaaggtg cgcaccagtt ccttcacccg ccaccacgtc
aacatctcga 600tgacccgcgc caagtccaac ggcgcctaca tcaactcgat
gctggccctc caggaagcga 660tctccggcgg cgccgacgag gccatgatgc
tcgatccgga aggctacgtg gccgaaggct 720ccggcgagaa catcttcatc
atcaaggatg gcgtgatcta caccccggaa gtcaccgcct 780gcctgaacgg
catcactcgt aacactatcc tgaccctggc cgccgaacac ggttttaaac
840tggtcgagaa gcgcatcacc cgcgacgagg tgtacatcgc cgacgaggcc
ttcttcactg 900gcactgccgc ggaagtcacg ccgatccgcg aagtggacgg
tcgcaagatc ggcgccggcc 960gccgtggccc ggtcaccgaa aagctgcaga
aagcctattt cgacctggtc agcggcaaga 1020ccgaggccca cgccgagtgg
cgtaccctgg tcaagtaagt cgacaagctt gcggccgcat 1080aatgcttaag
tcgaacagaa agtaatcgta ttgtacacgg ccgcataatc gaaattaata
1140cgactcacta taggggaatt gtgagcggat aacaattccc catcttagta
tattagttaa 1200gtataagaag gagatataca tatggcagat ctggaggcct
gtcatgagtg attacgagcc 1260gttgcgtctg catgtcccgg agcccaccgg
gcgtcctggc tgcaagaccg acttttccta 1320tctgcacctg tcccccgccg
gcgaggtacg caagccgccg gtggatgtcg agcccgccga 1380gaccagcgac
ctggcctaca gcctggtacg tgtgctcgac gacgacggcc acgccgtcgg
1440tccctggaat ccgcagctca gcaacgaaca actgctgcgc ggcatgcggg
cgatgctcaa 1500gacccgcctg ttcgacgcgc gcatgctcac cgcgcaacgg
cagaaaaagc tttccttcta 1560tatgcaatgc ctcggcgagg aagccatcgc
caccgcccac accctggccc tgcgcgacgg 1620cgacatgtgc tttccgacct
atcgccagca aggcatcctg atcacccgcg aatacccgct 1680ggtggacatg
atctgccagc ttctctccaa cgaggccgac ccgctcaagg gccgccagct
1740gccgatcatg tactcgagca aggaggcagg tttcttctcc atctccggca
acctcgccac 1800ccagttcatc caggcggtcg gctggggcat ggcctcggcg
atcaagggcg acacgcgcat 1860cgcctcggcc tggatcggcg acggcgccac
cgccgagtcg gacttccaca ccgccctcac 1920cttcgcccat gtctaccgcg
cgccggtaat cctcaacgtg gtcaacaacc agtgggcgat 1980ctccaccttc
caggccatcg ccggcggcga aggcaccacc ttcgccaacc gtggcgtggg
2040ctgcgggatc gcctcgctgc gggtcgacgg caatgacttc ctggcggtct
acgccgcctc 2100cgagtgggcc gccgagcgcg cccggcgcaa cctcgggccg
agcctgatcg aatgggtcac 2160ctaccgcgcc ggcccgcact cgacttcgga
cgacccgtcc aagtaccgcc ccgccgacga 2220ctggaccaac ttcccgctgg
gcgacccgat cgcccgcctg aagcggcaca tgatcggcct 2280cggcatctgg
tcggaggaac agcacgaagc cacccacaag gccctcgaag ccgaagtact
2340ggccgcgcag aaacaggcgg agagccatgg caccctgatc gacggccggg
tgccgagcgc 2400cgccagcatg ttcgaggacg tctatgcaga actgccggag
cacctgcgcc ggcaacgcca 2460ggagctcggg gtatgaatgc catgaacccg
caacacgaga acgcccagac ggtcaccagc 2520atgaccatga tccaggcgct
gcgctcggcg atggacatca tgctcgagcg cgacgacgac 2580gtggtggtat
tcggccagga cgtcggctac ttcggcggcg tgttccgctg caccgaaggc
2640ctgcagaaga aatacggcac ctcgcgggtg ttcgatgcgc cgatctccga
gagcggcatc 2700atcggcgccg cggtcggcat gggtgcctac ggcctgcgcc
cggtggtgga gatccagttc 2760gccgactacg tctacccggc ctccgaccag
ttgatctccg aggcggcgcg cctgcgctat 2820cgctcggccg gcgacttcat
cgtgccgatg accgtacgca tgccctgtgg cggcggcatc 2880tacggcgggc
aaacgcacag ccagagcccg gaggcgatgt tcacccaggt ctgcggcctg
2940cgcacggtga tgccgtccaa cccctacgac gccaagggcc tgctgatcgc
ctgcatcgag 3000aacgacgacc cggtgatctt cctcgagccc aagcgcctct
acaacggccc gttcgatggc 3060caccacgacc gcccggtgac gccctggtcc
aagcatccgg ccagccaggt gccggacggc 3120tactacaagg tgccgctgga
caaggcggcg atcgtccgcc ccggcgcggc gctgaccgtg 3180ctgacctacg
gcaccatggt ctacgtggcc caggccgcgg ccgacgagac cggcctggac
3240gccgagatca tcgacctgcg cagcctctgg ccgctggacc tggaaaccat
cgtcgcctcg 3300gtgaagaaga ccggccgctg cgtcatcgcc cacgaggcga
cccgcacctg cgggttcggc 3360gccgagctga tgtcgctggt gcaggagcac
tgcttccacc acctggaggc gccgatcgag 3420cgcgtcaccg gttgggacac
cccctacccg catgcccagg agtgggcgta tttccccggc 3480cccgcgcgcg
tcggcgcggc attcaagcgt gtgatggagg tctgaatggg taccctcgag
3540tctggtaaag aaaccgctgc tgcgaaattt gaacgccagc acatggactc
gtctactagc 3600gcagcttaat taacctaggc tgctgccacc gctgagcaat
aactagcata accccttggg 3660gcctctaaac gggtcttgag gggttttttg
ctgaaacctc aggcatttga gaagcacacg 3720gtcacactgc ttccggtagt
caataaaccg gtaaaccagc aatagacata agcggctatt 3780taacgaccct
gccctgaacc gacgaccggg tcatcgtggc cggatcttgc ggcccctcgg
3840cttgaacgaa ttgttagaca ttatttgccg actaccttgg tgatctcgcc
tttcacgtag 3900tggacaaatt cttccaactg atctgcgcgc gaggccaagc
gatcttcttc ttgtccaaga 3960taagcctgtc tagcttcaag tatgacgggc
tgatactggg ccggcaggcg ctccattgcc 4020cagtcggcag cgacatcctt
cggcgcgatt ttgccggtta ctgcgctgta ccaaatgcgg 4080gacaacgtaa
gcactacatt tcgctcatcg ccagcccagt cgggcggcga gttccatagc
4140gttaaggttt catttagcgc ctcaaataga tcctgttcag gaaccggatc
aaagagttcc 4200tccgccgctg gacctaccaa ggcaacgcta tgttctcttg
cttttgtcag caagatagcc 4260agatcaatgt cgatcgtggc tggctcgaag
atacctgcaa gaatgtcatt gcgctgccat 4320tctccaaatt gcagttcgcg
cttagctgga taacgccacg gaatgatgtc gtcgtgcaca 4380acaatggtga
cttctacagc gcggagaatc tcgctctctc caggggaagc cgaagtttcc
4440aaaaggtcgt tgatcaaagc tcgccgcgtt gtttcatcaa gccttacggt
caccgtaacc 4500agcaaatcaa tatcactgtg tggcttcagg ccgccatcca
ctgcggagcc gtacaaatgt 4560acggccagca acgtcggttc gagatggcgc
tcgatgacgc caactacctc tgatagttga 4620gtcgatactt cggcgatcac
cgcttccctc atactcttcc tttttcaata ttattgaagc 4680atttatcagg
gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
4740caaatagcta gctcactcgg tcgctacgct ccgggcgtga gactgcggcg
ggcgctgcgg 4800acacatacaa agttacccac agattccgtg gataagcagg
ggactaacat gtgaggcaaa 4860acagcagggc cgcgccggtg gcgtttttcc
ataggctccg ccctcctgcc agagttcaca 4920taaacagacg cttttccggt
gcatctgtgg gagccgtgag gctcaaccat gaatctgaca 4980gtacgggcga
aacccgacag gacttaaaga tccccaccgt ttccggcggg tcgctccctc
5040ttgcgctctc ctgttccgac cctgccgttt accggatacc tgttccgcct
ttctccctta 5100cgggaagtgt ggcgctttct catagctcac acactggtat
ctcggctcgg tgtaggtcgt 5160tcgctccaag ctgggctgta agcaagaact
ccccgttcag cccgactgct gcgccttatc 5220cggtaactgt tcacttgagt
ccaacccgga aaagcacggt aaaacgccac tggcagcagc 5280cattggtaac
tgggagttcg cagaggattt gtttagctaa acacgcggtt gctcttgaag
5340tgtgcgccaa agtccggcta cactggaagg acagatttgg ttgctgtgct
ctgcgaaagc 5400cagttaccac ggttaagcag ttccccaact gacttaacct
tcgatcaaac cacctcccca 5460ggtggttttt tcgtttacag ggcaaaagat
tacgcgcaga aaaaaaggat ctcaagaaga 5520tcctttgatc ttttctactg
aaccgctcta gatttcagtg caatttatct cttcaaatgt 5580agcacctgaa
gtcagcccca tacgatataa gttgtaattc tcatgttagt catgccccgc
5640gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
agatcccggt 5700gcctaatgag tgagctaact tacattaatt gcgttgcgct
cactgcccgc tttccagtcg 5760ggaaacctgt cgtgccagct gcattaatga
atcggccaac gcgcggggag aggcggtttg 5820cgtattgggc gccagggtgg
tttttctttt caccagtgag acgggcaaca gctgattgcc 5880cttcaccgcc
tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
5940gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
cttcggtatc 6000gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc
ccggactcgg taatggcgcg 6060cattgcgccc agcgccatct gatcgttggc
aaccagcatc gcagtgggaa cgatgccctc 6120attcagcatt tgcatggttt
gttgaaaacc ggacatggca ctccagtcgc cttcccgttc 6180cgctatcggc
tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
6240cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
ccaatgcgac 6300cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
aaaataatac tgttgatggg 6360tgtctggtca gagacatcaa gaaataacgc
cggaacatta gtgcaggcag cttccacagc 6420aatggcatcc tggtcatcca
gcggatagtt aatgatcagc ccactgacgc gttgcgcgag 6480aagattgtgc
accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
6540cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
gcgacggcgc 6600gtgcagggcc agactggagg tggcaacgcc aatcagcaac
gactgtttgc ccgccagttg 6660ttgtgccacg cggttgggaa tgtaattcag
ctccgccatc gccgcttcca ctttttcccg 6720cgttttcgca gaaacgtggc
tggcctggtt caccacgcgg gaaacggtct gataagagac 6780accggcatac
tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
6840actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
cgatggtgtc 6900cgggatctcg acgctctccc ttatgcgact cctgcattag
gaaattaata cgactcacta 6960208757DNAArtificial SequenceDescription
of Artificial Sequence Synthetic plasmid polynucleotide
20caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
60acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
120ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
gtccggcgta 180gaggatcgag atctcgatcc cgcgaaatta atacgactca
ctatagggga attgtgagcg 240gataacaatt cccctctaga aataattttg
tttaacttta agaaggaatt caggagccct 300tatgcaattt aatgaagaac
agctattaat tcaggatatg gcgaaaagtt ttgccaatga 360acagattaaa
tctaatgcag cagaatggga taagcatagc atttttccaa aagacgtttt
420gtcccaaatg gggcaattgg gttttatggg aatgctggtg agtgagaaat
ggggcggatc 480aaatacagga aatttagctt atgtgctggc acttgaagaa
atcgctgccg cagatggtgc 540gacttcaacc attatgagtg tacataattc
tgttggctgt gtacccattg ctaaatttgg 600tacagaggag caaaagcaga
aatatctagt gcctttagca caaggtgaaa tgatcggtgc 660atttgcttta
acggaaccac atacaggttc cgatgccgca gccattaaaa cccgagcaat
720taaacaaggt gatgaatgga ttattaatgg cgctaaacaa tttataacat
caggtcataa 780tgcgggcgtg attattgtat ttgctgtgac agatccgaat
gcagggaaaa aagggctgag 840tgcatttatt gtgccgcgtg aaaccttggg
ttatgaggtg attcgcaccg aagaaaaatt 900gggtttacat gcgtcagata
cgtgccaaat tgctttaacg gatgttcgag tacatcacag 960cttaatgctt
ggtcaggaag gtgagggact aaaaatagca ttgtctaatc tggaaggtgg
1020ccgtattggg attgcagcgc aagccgttgg tttggcacgt gctgcactag
aagaagcgac 1080aaaatatgcc aaagagcgtg tgacctttgg aaagcctatt
tttgagcatc aggcgttagc 1140ctttcgttta gccagtatgg ccacagaaat
tgaagcagca cgacaattgg ttcattacgc 1200agcgcggctt aaagaagctg
gaaaaccttg tttaaatgaa gcatcaatgg cgaaattatt 1260ttcatctgaa
atggtcgaac gcgtatgttc tgctgctttg caaatctttg gtggctatgg
1320ctatttaaaa gactttccca tcgagcgaat ttatcgtgat gcacgtattt
gccagattta 1380tgaaggtaca agtgatattc agcgtttagt gatagcaaga
agcctataac tgacctttgc 1440tgctgtattt ttatcataaa attaagataa
ggattctaaa aatgacattc gcaacaattt 1500tattggaaaa acgtaagggt
gtgggcttga ttacacttaa ccgtccaaaa gcattaaatg 1560ctttaaactc
agaattaatt tatgaaataa
atttagcctt agacgattta gaaaatgatc 1620aaacgattgg ttgtatcgtc
cttacaggtt cagaaaaagc ctttgccgca ggtgcggata 1680tcaaagaaat
ggcagaatta acttttccaa atatttattt tgatgatttt tttagtcttg
1740cagatcgtat tgcacagcgt cgtaagcctt taattgccgc agtgagtggt
tatgctttag 1800gtggtggctg tgagttagca ctcatgtgtg actttattta
ttgtgccgac aatgccaagt 1860ttgcactacc agaagtaact ttaggtgtca
ttcctggtat tggtggaaca cagcgtctaa 1920cgcttgcaat aggcaaagcc
aaagccatgg aaatgtgttt gactgcacgg caaatgcagg 1980ctgctgaggc
agaacaaagt ggtttggtgg cacgcgtttt tagtaaagaa gaacttttag
2040aacaaacctt acaggctgcc gaaaaaatag cggaaaaatc acgggtatct
accataatga 2100ttaaagagtc aattaatcga gcttttgaag tgagtttagc
agagggttta cgttttgagc 2160gccgaatgtt ccattcagtt tttgcgacct
tagatcagaa agaaggcatg caagcattta 2220ttgataaacg tccagcccaa
tttaaacatc aataatagga tgaagcgatg actactactg 2280acaatcattt
actcattgaa cataaaaacg ctttaggaac aattatttta aatcgtccag
2340cgagtctgaa cgcgctatct ctagaaatga ttaatgcgat tcgtcaacaa
gttgaggatt 2400ggcaaggtga tgtaaatgtt caggccatat taattaaatc
aaatagtcct aaagcatttt 2460gtgcaggtgg tgatattcgc tatctttatg
aaagttataa aagtggatca gaagagtata 2520aagattattt cattgctgaa
tatgagatgc tcaatagcat tcgaacgtct aaaaaaacag 2580tgattgtttt
attggatgga tatgtattgg gtggtggttt tggtttagca caggcttgtc
2640atatcttggt gagtagtgaa aaatcacgat tttcaatgcc agaaacagca
ataggttttt 2700tcccagatgt tgcagcgact tatttcttat ctcgtttaga
tgatgttggg gtatatttgg 2760cactgactgg tgatcaaatc agtagtagtg
atgcattgta tttagatctg attgattatc 2820atgttccgag tcagaatttt
gagcgactag aaaatgcatt cagccaatca cagaacttag 2880ataaatttca
tattcagaag attatttctg cttatatctc cagccctgtt cagagtgaac
2940tcagtctatg gcttgaagcc attcgtcagc attttggtct taaaaatgtg
caagatatcg 3000aagaaagttt gaaaaatgaa caagatccca actatcaagt
atggacaagt aaagtgttaa 3060atactttgca acaacgttcc tctattgcaa
aaaaaaccag tttaaagtta cagctgctag 3120ggcgtggatg gtcattacag
caatgtatgc gtatcgagcg aaaattacag gatatctggt 3180ttgaacatgg
tgatatgatt gagggtgttc gagcgttgat tattgataaa gataaacaac
3240cgcaatggca gcagcataat gcgactttag ataatatatt aggccaatta
ggttagtcat 3300ttcagactga agggcgagct caattcgaag cttgaaggta
agcctatccc taaccctctc 3360ctcggtctcg attctacgcg taccggtcat
catcaccatc accattgagt ttgatccggc 3420tgctaacaaa gcccgaaagg
aagctgagtt ggctgctgcc accgctgagc aataactagc 3480ataacccctt
ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
3540atccggatat cccgcaagag gcccggcagt accggcataa ccaagcctat
gcctacagca 3600tccagggtga cggtgccgag gatgacgatg agcgcattgt
tagatttcat acacggtgcc 3660tgactgcgtt agcaatttaa ctgtgataaa
ctaccgcatt aaagcttatc gatgataagc 3720tgtcaaacat gagaattaat
tcttgaagac gaaagggcct cgtgatacgc ctatttttat 3780aggttaatgt
catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
3840tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
ccgctcatga 3900gacaataacc ctgataaatg cttcaataat attgaaaaag
gaagagtatg agtattcaac 3960atttccgtgt cgcccttatt cccttttttg
cggcattttg ccttcctgtt tttgctcacc 4020cagaaacgct ggtgaaagta
aaagatgctg aagatcagtt gggtgcacga gtgggttaca 4080tcgaactgga
tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
4140caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
gttgacgccg 4200ggcaagagca actcggtcgc cgcatacact attctcagaa
tgacttggtt gagtactcac 4260cagtcacaga aaagcatctt acggatggca
tgacagtaag agaattatgc agtgctgcca 4320taaccatgag tgataacact
gcggccaact tacttctgac aacgatcgga ggaccgaagg 4380agctaaccgc
ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
4440cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
gcagcaatgg 4500caacaacgtt gcgcaaacta ttaactggcg aactacttac
tctagcttcc cggcaacaat 4560taatagactg gatggaggcg gataaagttg
caggaccact tctgcgctcg gcccttccgg 4620ctggctggtt tattgctgat
aaatctggag ccggtgagcg tgggtctcgc ggtatcattg 4680cagcactggg
gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
4740aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
ctgattaagc 4800attggtaact gtcagaccaa gtttactcat atatacttta
gattgattta aaacttcatt 4860tttaatttaa aaggatctag gtgaagatcc
tttttgataa tctcatgacc aaaatccctt 4920aacgtgagtt ttcgttccac
tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 4980gagatccttt
ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
5040cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
actggcttca 5100gcagagcgca gataccaaat actgtccttc tagtgtagcc
gtagttaggc caccacttca 5160agaactctgt agcaccgcct acatacctcg
ctctgctaat cctgttacca gtggctgctg 5220ccagtggcga taagtcgtgt
cttaccgggt tggactcaag acgatagtta ccggataagg 5280cgcagcggtc
gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
5340acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
cccgaaggga 5400gaaaggcgga caggtatccg gtaagcggca gggtcggaac
aggagagcgc acgagggagc 5460ttccaggggg aaacgcctgg tatctttata
gtcctgtcgg gtttcgccac ctctgacttg 5520agcgtcgatt tttgtgatgc
tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 5580cggccttttt
acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
5640tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
accgctcgcc 5700gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
agcggaagag cgcctgatgc 5760ggtattttct ccttacgcat ctgtgcggta
tttcacaccg caatggtgca ctctcagtac 5820aatctgctct gatgccgcat
agttaagcca gtatacactc cgctatcgct acgtgactgg 5880gtcatggctg
cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
5940ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
gtgtcagagg 6000ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
aaagctcatc agcgtggtcg 6060tgaagcgatt cacagatgtc tgcctgttca
tccgcgtcca gctcgttgag tttctccaga 6120agcgttaatg tctggcttct
gataaagcgg gccatgttaa gggcggtttt ttcctgtttg 6180gtcactgatg
cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
6240cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
actggaacgt 6300tgtgagggta aacaactggc ggtatggatg cggcgggacc
agagaaaaat cactcagggt 6360caatgccagc gcttcgttaa tacagatgta
ggtgttccac agggtagcca gcagcatcct 6420gcgatgcaga tccggaacat
aatggtgcag ggcgctgact tccgcgtttc cagactttac 6480gaaacacgga
aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
6540cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
aaggcaaccc 6600cgccagccta gccgggtcct caacgacagg agcacgatca
tgcgcacccg tggccaggac 6660ccaacgctgc ccgagatgcg ccgcgtgcgg
ctgctggaga tggcggacgc gatggatatg 6720ttctgccaag ggttggtttg
cgcattcaca gttctccgca agaattgatt ggctccaatt 6780cttggagtgg
tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc gaggtggccc
6840ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg
gcgcctacaa 6900tccatgccaa cccgttccat gtgctcgccg aggcggcata
aatcgccgtg acgatcagcg 6960gtccaatgat cgaagttagg ctggtaagag
ccgcgagcga tccttgaagc tgtccctgat 7020ggtcgtcatc tacctgcctg
gacagcatgg cctgcaacgc gggcatcccg atgccgccgg 7080aagcgagaag
aatcataatg gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga
7140cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg
ccgaaacgtt 7200tggtggcggg accagtgacg aaggcttgag cgagggcgtg
caagattccg aataccgcaa 7260gcgacaggcc gatcatcgtc gcgctccagc
gaaagcggtc ctcgccgaaa atgacccaga 7320gcgctgccgg cacctgtcct
acgagttgca tgataaagaa gacagtcata agtgcggcga 7380cgatagtcat
gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
7440tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
ttgcgctcac 7500tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
ttaatgaatc ggccaacgcg 7560cggggagagg cggtttgcgt attgggcgcc
agggtggttt ttcttttcac cagtgagacg 7620ggcaacagct gattgccctt
caccgcctgg ccctgagaga gttgcagcaa gcggtccacg 7680ctggtttgcc
ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
7740gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
gcgcagcccg 7800gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
cgttggcaac cagcatcgca 7860gtgggaacga tgccctcatt cagcatttgc
atggtttgtt gaaaaccgga catggcactc 7920cagtcgcctt cccgttccgc
tatcggctga atttgattgc gagtgagata tttatgccag 7980ccagccagac
gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
8040tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
atgggagaaa 8100ataatactgt tgatgggtgt ctggtcagag acatcaagaa
ataacgccgg aacattagtg 8160caggcagctt ccacagcaat ggcatcctgg
tcatccagcg gatagttaat gatcagccca 8220ctgacgcgtt gcgcgagaag
attgtgcacc gccgctttac aggcttcgac gccgcttcgt 8280tctaccatcg
acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
8340acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
cagcaacgac 8400tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
aattcagctc cgccatcgcc 8460gcttccactt tttcccgcgt tttcgcagaa
acgtggctgg cctggttcac cacgcgggaa 8520acggtctgat aagagacacc
ggcatactct gcgacatcgt ataacgttac tggtttcaca 8580ttcaccaccc
tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
8640cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
gcattaggaa 8700gcagcccagt agtaggttga ggccgttgag caccgccgcc
gcaaggaatg gtgcatg 87572128DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 21attatcccat ggggagaaca
ttaaaagc 282226DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 22cgggatcctt acttttcaat atatcc 26
* * * * *
References