U.S. patent application number 15/182839 was filed with the patent office on 2017-03-09 for gene products of bacillus licheniformis which form odorous substances and improved biotechnological production methods based thereon.
The applicant listed for this patent is BASF SE. Invention is credited to Cornelius Bessler, Armin Ehrenreich, Stefan Evers, Jorg Feesche, Gerhard Gottschalk, Anke Henne, Christina Herzberg, Heiko Liesegang, Karl-Heinz Maurer, Birgit Veith.
Application Number | 20170067064 15/182839 |
Document ID | / |
Family ID | 34970441 |
Filed Date | 2017-03-09 |
United States Patent
Application |
20170067064 |
Kind Code |
A1 |
Bessler; Cornelius ; et
al. |
March 9, 2017 |
Gene Products of Bacillus Licheniformis Which Form Odorous
Substances and Improved Biotechnological Production Methods Based
Thereon
Abstract
The present invention relates to 25 hitherto undescribed genes
of B. licheniformis and gene products derived thereform and all
sufficiently homologous nucleic acids and proteins thereof. They
occur in five different metabolic pathways for the formation of
odorous substances. The metabolic pathways in question are for the
synthesis of: 1) isovalerian acid (as part of the catabolism of
leucine), 2) 2-methylbutyric acid and/or isobutyric acid (as part
of the catabolism of valine and/or isoleucine), 3) butanol and/or
butyric acid (as part of the metabolism of butyric acid), 4) propyl
acid (as part of the metabolism of propionate) and/or 5) cadaverine
and/or putrescine (as parts of the catabolism of lysine and/or
arginine). The identification of these genes allows
biotechnological production methods to be developed that are
improved to the extent that, to assist these nucleic acids, the
formation of the odorous substances synthesised via these metabolic
pathways can be reduced by deactivating the corresponding genes in
the micro-organism used for the biotechnological production. In
addition, these gene products are thus available for preparing
reactions or for methods according to their respective biochemical
properties.
Inventors: |
Bessler; Cornelius;
(Duesseldorf, DE) ; Feesche; Jorg; (Ekrath,
DE) ; Evers; Stefan; (Mettmann, DE) ; Maurer;
Karl-Heinz; (Erkrath, DE) ; Ehrenreich; Armin;
(Gottingen, DE) ; Veith; Birgit; (Gottingen,
DE) ; Liesegang; Heiko; (Hoxter, DE) ; Henne;
Anke; (Solingen, DE) ; Herzberg; Christina;
(Bilshausen, DE) ; Gottschalk; Gerhard;
(Norten-Hardenberg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BASF SE |
Ludwigshafen |
|
DE |
|
|
Family ID: |
34970441 |
Appl. No.: |
15/182839 |
Filed: |
June 15, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11616319 |
Dec 27, 2006 |
9394528 |
|
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15182839 |
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PCT/EP05/06540 |
Jun 17, 2005 |
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11616319 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/80 20130101; C12Y
602/01 20130101; C12N 15/52 20130101; C12P 7/40 20130101; C12Y
305/01011 20130101; C12N 9/88 20130101; C12N 9/1029 20130101; C12Y
206/01042 20130101; C12P 7/52 20130101; C12N 9/0006 20130101; C12N
15/75 20130101; Y02E 50/10 20130101; C12Y 207/02007 20130101; C12N
9/0016 20130101; C12N 9/93 20130101; C12P 7/42 20130101; C12N 9/78
20130101; C12Y 101/01157 20130101; C12Y 203/01019 20130101; C12Y
401/01018 20130101; C12P 13/001 20130101; C12Y 102/04002 20130101;
C12Y 402/01055 20130101; C12N 9/1096 20130101; C12N 9/16 20130101;
Y02P 20/52 20151101; C12N 9/0008 20130101; C12Y 305/01001 20130101;
C12P 7/16 20130101; C12Y 101/01 20130101; C12N 9/1217 20130101;
C12Y 402/01017 20130101; C12N 9/1025 20130101; C12Y 103/99
20130101; C12N 9/84 20130101; C12Y 401/01019 20130101; C12Y
602/01001 20130101; C12Y 101/01035 20130101; C12N 9/001
20130101 |
International
Class: |
C12N 15/52 20060101
C12N015/52; C12P 7/42 20060101 C12P007/42; C12P 7/52 20060101
C12P007/52; C12P 13/00 20060101 C12P013/00; C12N 15/75 20060101
C12N015/75; C12N 9/84 20060101 C12N009/84; C12N 9/88 20060101
C12N009/88; C12N 9/04 20060101 C12N009/04; C12N 9/02 20060101
C12N009/02; C12N 9/00 20060101 C12N009/00; C12N 9/12 20060101
C12N009/12; C12P 7/16 20060101 C12P007/16; C12N 9/10 20060101
C12N009/10 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 29, 2004 |
DE |
102004031177.3 |
Claims
1. A nucleic acid selected from the group consisting of: (a)
nucleic acids coding for a gene product (putative branched-chain
amino acid aminotransferase; E.C. 2.6.1.42) involved in the
synthesis of 2-methylbutyric acid and/or isobutyric acid and having
a nucleotide sequence which shows at least 67% identity to the
nucleotide sequence indicated in SEQ ID NO. 1; (b) nucleic acids
coding for a gene product (putative branched-chain amino acid
aminotransferase; E.C. 2.6.1.42) involved in the synthesis of
2-methylbutyric acid and/or isobutyric acid and having a nucleotide
sequence which shows at least 78% identity to the nucleotide
sequence indicated in SEQ ID NO. 3; (c) nucleic acids speA coding
for a gene product (lysine and/or arginine decarboxylase; E.C.
4.1.1.18 or 4.1.1.19) involved in the synthesis of cadaverine
and/or putrescine and having a nucleotide sequence which shows at
least 78% identity to the nucleotide sequence indicated in SEQ ID
No. 5; (d) nucleic acids yugJ coding for a gene product
(NADH-dependent butanol dehydrogenase A; E.C. 1.1.1.-) involved in
the synthesis of butanol and/or butyric acid and having a
nucleotide sequence which shows at least 81% identity to the
nucleotide sequence indicated in SEQ ID NO. 7; (e) nucleic acids
coding for a gene product (acyl-CoA dehydrogenase; E.C. 1.3.99.-)
involved in the synthesis of isovaleric acid, 2-methylbutyric acid,
isobutyric acid, butanol and/or butyric acid and having a
nucleotide sequence which shows at least 75% identity to the
nucleotide sequence indicated in SEQ ID NO. 9; (f) nucleic acids
coding for a gene product (acyl-CoA dehydrogenase; E.C. 1.3.99.-)
involved in the synthesis of isovaleric acid, 2-methylbutyric acid,
isobutyric acid, butanol and/or butyric acid and having a
nucleotide sequence which shows at least 64% identity to the
nucleotide sequence indicated in SEQ ID NO. 11; (g) nucleic acids
coding for a gene product (3-hydroxybutyryl-CoA dehydrogenase; E.C.
1.1.1.157) involved in the synthesis of butanol and/or butyric acid
and having a nucleotide sequence which shows at least 67% identity
to the nucleotide sequence indicated in SEQ ID NO. 13; (h) nucleic
acids coding for a gene product (putative enoyl-Coa hydratase
protein; E.C. 4.2.1.17) involved in the synthesis of isovaleric
acid, 2-methylbutyric acid and/or isobutyric acid and having a
nucleotide sequence which shows at least 65% identity to the
nucleotide sequence indicated in SEQ ID NO. 15; (i) nucleic acids
coding for a gene product (probable
enoyl-(3-hydroxyisobutyryl)-coenzyme A hydrolase protein) involved
in the synthesis of isovaleric acid, 2-methylbutyric acid and/or
isobutyric acid and having a nucleotide sequence which shows at
least 66% identity to the nucleotide sequence indicated in SEQ ID
NO. 17; (j) nucleic acids echA8 coding for a gene product (probable
enoyl-CoA hydratase; E.C. 4.2.1.17) involved in the synthesis of
isovaleric acid, 2-methylbutyric acid and/or isobutyric acid and
having a nucleotide sequence which shows at least 48% identity to
the nucleotide sequence indicated in SEQ ID NO. 19; (k) nucleic
acids coding for a gene product (acyl-CoA dehydrogenase; E.C.
1.3.99.-) involved in the synthesis of isovaleric acid,
2-methylbutyric acid and/or isobutyric acid and having a nucleotide
sequence which shows at least 54% identity to the nucleotide
sequence indicated in SEQ ID NO. 21; (l) nucleic acids acsA coding
for a gene product (acetyl-coenzyme A synthetase; E.C. 6.2.1.1)
involved in the synthesis of propionic acid and having a nucleotide
sequence which shows at least 67% identity to the nucleotide
sequence indicated in SEQ ID NO. 23; (m) nucleic acids yngF coding
for a gene product (3-hydroxybutyryl-CoA dehydratase; E.C.
4.2.1.55) involved in the synthesis of butanol and/or butyric acid
and having a nucleotide sequence which shows at least 68% identity
to the nucleotide sequence indicated in SEQ ID NO. 25; (n) nucleic
acids yusJ coding for a gene product (acyl-CoA dehydrogenase; E.C.
1.3.99.-) involved in the synthesis of isovaleric acid,
2-methylbutyric acid, isobutyric acid, butanol and/or butyric acid
and having a nucleotide sequence which shows at least 77% identity
to the nucleotide sequence indicated in SEQ ID NO. 27; (o) nucleic
acids ykwC coding for a gene product (hypothetical oxidoreductase;
E.C. 1.1.-.-) involved in the synthesis of 2-methylbutyric acid
and/or isobutyric acid and having a nucleotide sequence which shows
at least 77% identity to the nucleotide sequence indicated in SEQ
ID NO. 29; (p) nucleic acids coding for a gene product (probable
phosphate butyryltransferase; E.C. 2.3.1.19) involved in the
synthesis of butanol and/or butyric acid and having a nucleotide
sequence which shows at least 51% identity to the nucleotide
sequence indicated in SEQ ID NO. 31; (q) nucleic acids coding for a
gene product (probable butyrate kinase; E.C. 2.7.2.7) involved in
the synthesis of butanol and/or butyric acid and having a
nucleotide sequence which shows at least 77% identity to the
nucleotide sequence indicated in SEQ ID NO. 33; (r) nucleic acids
acsA coding for a gene product (acetyl-coenzyme A synthetase; E.C.
6.2.1.1) involved in the synthesis of propionic acid and having a
nucleotide sequence which shows at least 79% identity to the
nucleotide sequence indicated in SEQ ID NO. 35; (s) nucleic acids
ytcl coding for a gene product (acetate-CoA ligase; E.C. 6.2.1.1)
involved in the synthesis of propionic acid and having a nucleotide
sequence which shows at least 74% identity to the nucleotide
sequence indicated in SEQ ID NO. 37; (t) nucleic acids speA coding
for a gene product (lysine and/or arginine decarboxylase; E.C.
4.1.1.18 or E.C. 4.1.1.19) involved in the synthesis of cadaverine
and/or putrescine and having a nucleotide sequence which shows at
least 68% identity to the nucleotide sequence indicated in SEQ ID
NO. 39; (u) nucleic acids ysiB coding for a gene product (probable
Enoyl-CoA hydratase; E.C. 4.2.1.17) involved in the synthesis of
isovaleric acid, 2-methylbutyric acid and/or isobutyric acid and
having a nucleotide sequence which shows at least 75% identity to
the nucleotide sequence indicated in SEQ ID NO. 41; (v) nucleic
acids coding for a gene product (similar to 3-hydroxyacyl-CoA
dehydrogenase; E.C. 1.1.1.35) involved in the synthesis of
2-methylbutyric acid and having a nucleotide sequence which shows
at least 76% identity to the nucleotide sequence indicated in SEQ
ID NO. 43; (w) nucleic acids coding for a gene product
(2-oxoglutarate dehydrogenase E1 component; E.C. 1.2.4.2) involved
in the synthesis of isovaleric acid, 2-methylbutyric acid and/or
isobutyric acid and having a nucleotide sequence which shows at
least 80% identity to the nucleotide sequence indicated in SEQ ID
NO. 45; (x) nucleic acids yhfL coding for a gene product (probable
acid-CoA ligase; E.C. 6.2.1.-) involved in the synthesis of
propionic acid and having a nucleotide sequence which shows at
least 67% identity to the nucleotide sequence indicated in SEQ ID
NO. 47; and (y) nucleic acids ywhG coding for a gene product
(agmatinase; E.C. 3.5.1.11) involved in the synthesis of cadaverine
and/or putrescine and having a nucleotide sequence which shows at
least 85% identity to the nucleotide sequence indicated in SEQ ID
NO. 49.
2. (canceled)
3. A nucleic acid according to claim 1 coding for a gene product
involved in the synthesis of at least one compound selected from
the group consisting of isovaleric acid, 2-methylbutyric acid,
isobutyric acid, butanol, butyric acid, propionic acid, cadaverine
and putrescine, the nucleic acid being naturally present in a
microorganism.
4.-15. (canceled)
16. A process for fermenting a substrate using a microorganism, the
process comprising: (a) providing a microorganism, the
microorganism having at least one gene on a metabolic pathway for
synthesizing a predetermined product being functionally
inactivated, the functionally inactivated enzyme being selected
from the group consisting of: (1) functionally inactivated enzymes
of the metabolic pathway for synthesizing isovaleric acid (as part
of leucine catabolism) selected from the group consisting of: (i)
L-leucine dehydrogenase (E.C. 1.4.1.9), (ii)
3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate
dehydrogenase E1 (E.C. 1.2.4.2), (iii) enzyme for hydrolyzing
isovaleryl-CoA to isovaleric acid and coenzyme A, (iv) acyl-CoA
dehydrogenase (E.C. 1.3.99.-), (v) methylcrotonyl carboxylase, (vi)
3-methylglutaconyl-CoA hydratase, and (vii) enoyl-CoA hydratase
(protein) (E.C. 4.2.1.17); (2) functionally inactivated enzymes of
the metabolic pathway for synthesizing 2-methylbutyric acid and/or
isobutyric acid (as part of valine and/or isoleucine catabolism)
from the group consisting of: (i) branched-chain amino acid
aminotransferase (E.C. 2.6.1.42), (ii) 3-methyl-2-oxobutanoate
dehydrogenase or 2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2),
(iii) enzyme for hydrolyzing 2-methylbutyryl-CoA to 2-methylbutyric
acid or isobutyryl-CoA to isobutyric acid and coenzyme A, (iv)
acyl-CoA dehydrogenase (E.C. 1.3.99.-), (v) enoyl-CoA hydratase
(protein) (E.C. 4.2.1.17), (vi) 3-hydroxy-acyl-CoA dehydrogenase
(E.C. 1.1.1.35), (vii) acetyl-CoA acyltransferase, (viii)
enoyl-(3-hydroxyisobutyryl)-CoA hydrolase protein and (ix)
3-hydroxyisobutyrate dehydrogenase (E.C. 1.1.1.31) or
oxidoreductase (E.C.1.1.-.-); (3) functionally inactivated enzymes
of the metabolic pathway for synthesizing butanol and/or butyric
acid (as part of butyric acid metabolism) from the group consisting
of: (i) 3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157), (ii)
3-hydroxybutyryl-CoA dehydratase (E.C. 4.2.1.55), (iii) butyryl-CoA
dehydrogenase (E.C. 1.3.99.25), (iv) phosphate butyryltransferase
(E.C. 2.3.1.19), (v) butyrate kinase (E.C. 2.7.2.7), (vi)
butyraldehyde dehydrogenase and (vii) NADH-dependent butanol
dehydrogenase A (E.C. 1.1.1.-) (4) functionally inactivated enzymes
of the metabolic pathway for synthesizing propionic acid (as part
of propionate metabolism) from the group consisting of: (i)
3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157), (ii)
3-hydroxybutyryl-CoA dehydratase (E.C. 4.2.1.55), (iii) butyryl-CoA
dehydrogenase (E.C. 1.3.99.25), (iv) phosphate butyryltransferase
(E.C. 2.3.1.19), (v) butyrate kinase (E.C. 2.7.2.7), (vi)
butyraldehyde dehydrogenase, and (viii) NADH-dependent butanol
dehydrogenase A (E.C. 1.1.1.-) and (5) functionally inactivated
enzymes of metabolic pathway for synthesizing cadaverine and/or
putrescine (as parts of lysine and/or arginine catabolism) from the
group consisting of: (i) lysine decarboxylase (E.C. 4.1.1.18)
and/or arginine decarboxylase (E.C. 4.1.1.19), (ii) agmatinase
(E.C. 3.5.1.11) and (iii) ornithine decarboxylase (E.C. 4.1.1.17);
(b) providing a fermentable substrate; and (c) permitting the
microorganism to ferment the substrate.
17.-21. (canceled)
22. A microorganism in which at least one of the genes which
corresponds to the nucleic acid which codes for one predetermined
protein of B. licheniformis DSM 13 is functionally inactivated, the
predetermined protein being selected from the group consisting of:
putative branched-chain amino acid aminotransferase (E.C. 2.6.1.42)
defined by SEQ ID NO. 1, putative branched-chain amino acid
aminotransferase (E.C. 2.6.1.42) defined by SEQ ID NO. 3, lysine
and/or arginine decarboxylase (protein SpeA; E.C. 4.1.1.18 or E.C.
4.1.1.19) defined by SEQ ID NO. 5 (speA gene), NADH-dependent
butanol dehydrogenase A (protein YugJ; E.C. 1.1.1.-) defined by SEQ
ID NO. 7 (yugJ gene), butyryl-CoA dehydrogenase (E.C. 1.3.99.25) or
acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 9,
butyryl-CoA dehydrogenase (E.C. 1.3.99.25) or acyl-CoA
dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 11,
3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157) defined by SEQ
ID NO. 13, putative enoyl-CoA hydratase protein (E.C. 4.2.1.17)
defined by SEQ ID NO. 15, probable enoyl-(3-hydroxyisobutyryl)-CoA
hydrolase protein (E.C.4.2.1.17) defined by SEQ ID NO. 17, probable
enoyl-CoA hydratase (protein EchA8; E.C. 4.2.1.17) defined by SEQ
ID NO. 19 (echA8 gene), acyl-CoA dehydrogenase (E.C. 1.3.99.-)
defined by SEQ ID NO. 21, acetate-CoA ligase or propionate-CoA
ligase (or synthetase; protein AcsA; E.C. 6.2.1.1) defined by SEQ
ID NO. 23 (acsA gene), 3-hydroxybutyryl-CoA dehydratase (protein
YngF; E.C. 4.2.1.55) defined by SEQ ID NO. 25 (yngF gene),
butyryl-CoA dehydrogenase (protein YusJ; E.C. 1.3.99.25) or
acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 27
(yusJ gene), 3-hydroxyisobutyrate dehydrogenase (protein YkwC; E.C.
1.1.1.31) or oxidoreductase (E.C.1.1.-.-) defined by SEQ ID NO. 29
(ykwC gene), probable phosphate butyryltransferase (E.C. 2.3.1.19)
defined by SEQ ID NO. 31, probable butyrate kinase (E.C. 2.7.2.7)
defined by SEQ ID NO. 33, acetate-CoA ligase or synthetase or
propionate-CoA ligase or synthetase (protein AcsA; E.C. 6.2.1.1)
defined by SEQ ID NO. 35 (acsA gene), acetate-CoA ligase or
propionate-CoA ligase (protein Ytcl; E.C. 6.2.1.1) defined by SEQ
ID NO. 37 (ytcl gene), lysine and/or arginine decarboxylase
(protein speA; E.C. 4.1.1.18 or E.C. 4.1.1.19) defined by SEQ ID
NO. 39 (speA gene), probable enoyl-CoA hydratase (E.C. 4.2.1.17)
defined by SEQ ID NO. 41 (ysiB gene), similar to 3-hydroxyacyl-CoA
dehydrogenase (E.C. 1.1.1.35) defined by SEQ ID NO. 43,
3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate
dehydrogenase E1 (E.C. 1.2.4.2) defined by SEQ ID NO. 45, probable
acetate-CoA ligase or propionate-CoA ligase (protein YhfL; E.C.
6.2.1.1) or acid-CoA ligase (E.C. 6.2.1.-) defined by SEQ ID NO. 47
(yhfL gene) or agmatinase (E.C. 3.5.1.11) defined by SEQ ID NO. 49
(ywhG gene).
23. A microorganism according to claim 22 in which at least two of
the genes are functionally inactivated.
24. A microorganism according to claim 22, in which at least one of
the metabolic pathways selected from the group consisting of the
metabolic pathway (1.) for synthesizing isovaleric acid, (2.) for
synthesizing 2-methylbutyric acid and/or isobutyric acid, (3.) for
synthesizing butanol and/or butyric acid, (4.) for synthesizing
propionic acid and (5.) for synthesizing cadaverine and/or
putrescine, are blocked at least in part.
25. A microorganism according to claim 22, wherein the
microorganism is is a bacterium.
26. A microorganism according to claim 25, the bacterium being
selected from the group of Gram-negative bacteria consisting of the
genera Escherichia coli, Klebsiella, Pseudomonas and
Xanthomonas.
27. A microorganism according to claim 25, the bacterium being
selected from the group of Gram-negative bacteria strains
Escherichia coli BL21 (DE3), E. coli RV308, E. coli DH5.alpha., E.
coli JM109, E. coli XL-1 and Klebsiella planticola (Rf).
28. A microorganism according to claim 25, the bacterium being
selected from the group of Gram-positive genera Bacillus,
Staphylococcus and Corynebacterium.
29. A microorganism according to claim 25, the bacterium being
selected from the group of Gram-positive species Bacillus lentus,
B. licheniformis, B. amyloliquefaciens, B. subtilis, B. globigii or
B. alcalophilus, Staphylococcus carnosus and Corynebacterium
glutamicum
30. A microorganism according to claim 29 wherein the bacterium is
B. licheniformis DSM 13.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a Section 365(c) continuation of
International Application No. PCT/EP2005/006540 filed 17 Jun. 2005,
which in turn claims the priority of DE Application 10 2004 031
177.3 filed Jun. 29, 2004, each of which is incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to 25 not previously described
genes of B. licheniformis and gene products derived therefrom which
are involved in the formation of odorous substances in five
different metabolic pathways, and to biotechnological production
methods which are improved inasmuch as, on the basis of the
identification of these genes, the formation of these odorous
substances can be reduced.
[0003] The present invention is in the area of biotechnology, in
particular the preparation of valuable products by fermentation of
microorganisms able to form the valuable products of interest. This
includes for example the preparation of low molecular weight
compounds, for instance of dietary supplements or pharmaceutically
relevant compounds, or of proteins for which, because of their
diversity, there is in turn a large area of industrial use. In the
first case, the metabolic properties of the relevant microorganisms
are utilized and/or modified to prepare the valuable products; in
the second case, cells which express the genes of the proteins of
interest are employed. In both cases, genetically modified
organisms (GMO) are mostly involved.
[0004] There is an extensive prior art on the fermentation of
microorganisms, especially on the industrial scale; it extends from
the optimization of the relevant strains in relation to the
formation rate and the nutrient utilization via the technical
design of the fermenters and up to the isolation of the valuable
products from the relevant cells themselves and/or the fermentation
medium. Both genetic and microbiological, and process engineering
and biochemical approaches are applied thereto. The aim of the
present invention is to improve this process in relation to a
common property of the microorganisms employed, which impairs the
actual fermentation step, specifically at the level of the genetic
properties of the strains employed.
[0005] For industrial biotechnological production, the relevant
microorganisms are cultured in fermenters which are designed
appropriate for their metabolic properties. During the culturing,
they metabolize the provided substrate and normally form, besides
the actual product, a large number of other substances in which
there is usually no interest and/or which may lead to unwanted side
effects.
[0006] These include odorous and/or poisonous substances which are
a nuisance and/or harmful and are discharged even during the
fermentation via the exit air and/or are only incompletely removed
during the subsequent working up of the valuable product and thus
impair the quality of the product. The concomitant odorous and/or
poisonous substances are thus deleterious firstly for the
production process, meaning the staff involved and the surroundings
of the plant. Secondly, failure to reach a desired quality
(specification) of the product may lead to it being unavailable for
the intended area of use (for example food production), which means
a considerable economic disadvantage. Conversely, reducing the
formation of odorous and/or poisonous substances could increase
occupational and environmental safety and open up additional areas
of use and markets for sales of the product.
[0007] Odors frequently found during fermentation of microorganisms
are caused by small organic molecules from the classes of volatile,
branched and unbranched fatty acids, alcohols and diamines. These
include isovaleric acid, 2-methylbutyric acid, isobutyric acid from
the class of branched fatty acids, butyric acid, propionic acid
(unbranched fatty acids), butanol (alcohol), cadaverine and
putrescine (diamines).
[0008] Some of these volatile substances are additionally toxic for
humans and animals, for example cadaverine and putrescine, which
are also known as ptomaines. They can therefore be defined not only
as odorous substances but also, depending on the concentration and
the exposure time for the relevant organism, as poisonous
substances.
[0009] Efforts are being made even at present to remove such
compounds subsequently from fermentation products. For this
purpose, usual working up of the valuable products formed
comprises, besides steps to remove cell detritus and high molecular
weight compounds, also additional process steps which are referred
to as deodorizing. To these are ordinarily added filtrations,
precipitation steps and/or chromatography steps, each of which also
contribute to a certain extent to the deodorizing. Nevertheless,
all these steps carried out for removal lead to a purity which is
only inadequate according to the above-mentioned criteria.
[0010] The exit air from the fermenter is likewise checked in order
to minimize the pollution during the production process.
[0011] It would nevertheless be desirable to combat odors causally
where possible, i.e. to prevent the relevant substances being
produced at all. It would thus be possible firstly to keep the
number of subsequent purification and working-up steps small, which
appears to be advantageous because they represent in each case a
physicochemical stress on the desired product, and reduce the
yield. Overall, therefore, a better product quality would be
obtained. Secondly, the production conditions would be improved per
se, and the systems for filtering the fermenter exit air could be
kept simpler. Such a combating of odors causally would, if the
properties of the microorganism itself were to be changed thereby,
also increase its tolerability for further operations on this
microorganism.
SUMMARY OF THE INVENTION
[0012] The object was thus to reduce the formation of unpleasant
odors and/or poisonous compounds which occurs during the
fermentation of microorganisms, especially Gram-positive bacteria
of the species Bacillus, and is attributable to the same. It was
intended preferably that this take place at the genetic level in
order to obtain odorous and/or poisonous substance-depleted
microorganisms. In partial problems, this means identifying
metabolic pathways relevant thereto, finding genes which code for
proteins and/or enzymes which catalyze reactions lying on these
pathways and are suitable as possible starting points for solving
the problem, and, via identification of the relevant nucleotide
sequences, acquiring tools for the desired genetic modification and
providing corresponding applications.
[0013] To solve this problem, the following five metabolic pathways
have been identified:
(1) the metabolic pathway for synthesizing isovaleric acid (as part
of leucine catabolism), (2) the metabolic pathway for synthesizing
2-methylbutyric acid and/or isobutyric acid (as part of valine
and/or isoleucine catabolism), (3) the metabolic pathway for
synthesizing butanol and/or butyric acid (as part of butyric acid
metabolism), (4) the metabolic pathway for synthesizing propionic
acid (as part of propionate metabolism) and (5) the metabolic
pathway for synthesizing cadaverine and/or putrescine (as parts of
lysine and/or arginine catabolism).
[0014] The following genes which code for proteins and/or enzymes
which catalyze reactions lying on these pathways and are suitable
as starting points for biotechnological production processes of the
invention were then found; the non-consecutive numbering in some
cases is based in each case on the complete description hereinafter
of the respective metabolic pathways; in addition, some of them are
involved in more than one of these pathways: [0015] on the
metabolic pathway for synthesizing isovaleric acid and as part of
leucine catabolism: [0016] (1) L-leucine dehydrogenase (E.C.
1.4.1.9), [0017] (2) 3-methyl-2-oxobutanoate dehydrogenase or
2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2), [0018] (3) enzyme
for hydrolyzing isovaleryl-CoA to isovaleric acid and coenzyme A,
[0019] (4) acyl-CoA dehydrogenase (E.C. 1.3.99.-), [0020] (5)
methylcrotonyl carboxylase, [0021] (6) 3-methylglutaconyl-CoA
hydratase and [0022] (7) enoyl-CoA hydratase (E.C. 4.2.1.17);
[0023] on the metabolic pathway for synthesizing 2-methylbutyric
acid and/or isobutyric acid and as part of valine and/or isoleucine
catabolism: [0024] (1) branched-chain amino acid aminotransferase
(E.C. 2.6.1.42), [0025] (2) 3-methyl-2-oxobutanoate dehydrogenase
or 2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2), [0026] (3)
enzyme for hydrolyzing 2-methylbutyryl-CoA to 2-methylbutyric acid
or isobutyryl-CoA to isobutyric acid and coenzyme A, [0027] (4)
acyl-CoA dehydrogenase (E.C. 1.3.99.-), [0028] (5) enoyl-CoA
hydratase (protein) (E.C. 4.2.1.17), [0029] (6) 3-hydroxy-acyl-CoA
dehydrogenase (E.C. 1.1.1.35), [0030] (7) acetyl-CoA
acyltransferase, [0031] (8) enoyl-(3-hydroxyisobutyryl)-CoA
hydrolase protein and [0032] (9) 3-hydroxyisobutyrate dehydrogenase
(E.C. 1.1.1.31) or oxidoreductase (E.C. 1.1.-.-);
[0033] on the metabolic pathway for synthesizing butanol and/or
butyric acid and as part of butyric acid metabolism: [0034] (1)
3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157), [0035] (2)
3-hydroxybutyryl-CoA dehydratase (E.C. 4.2.1.55), [0036] (3)
butyryl-CoA dehydrogenase (E.C. 1.3.99.25), [0037] (4) phosphate
butyryltransferase (E.C. 2.3.1.19), [0038] (5) butyrate kinase
(E.C. 2.7.2.7), [0039] (6) butyraldehyde dehydrogenase and [0040]
(8) NADH-dependent butanol dehydrogenase A (E.C. 1.1.1.-);
[0041] on the metabolic pathway for synthesizing propionic acid and
as part of propionate metabolism: [0042] (1) succinate-propionate
CoA-transferase, [0043] (2) acetate-CoA ligase or synthetase or
propionate-CoA ligase or synthetase (E.C. 6.2.1.1) and [0044] (3)
acetate-CoA ligase or synthetase or propionate-CoA ligase or
synthetase (E.C. 6.2.1.1); and
[0045] on the metabolic pathway for synthesizing cadaverine and/or
putrescine and as parts of lysine and/or arginine catabolism:
[0046] (1) lysine decarboxylase (E.C. 4.1.1.18) and/or arginine
decarboxylase (E.C. 4.1.1.19), [0047] (2) agmatinase (E.C.
3.5.1.11) and [0048] (3) ornithine decarboxylase (E.C.
4.1.1.17).
[0049] Finally, nucleotide and amino acid sequences coding for
these proteins/enzymes were completely determined by sequencing
relevant genes in B. licheniformis DSM 13, and thus made available
for the desired modification of the microorganisms of interest.
They are compiled in the sequence listing for the present
application. These involve the following nucleic acids (odd
numbers) and amino acid sequences derived therefrom for enzymes or
proteins as parts of those enzymes which consist of a plurality of
subunits (even numbers below in each case):
[0050] putative branched-chain amino acid aminotransferase (E.C.
2.6.1.42), defined by SEQ ID NO. 1 and 2,
[0051] putative branched-chain amino acid aminotransferase (E.C.
2.6.1.42) defined by SEQ ID NO. 3 and 4,
[0052] lysine and/or arginine decarboxylase (protein SpeA; E.C.
4.1.1.18 or E.C. 4.1.1.19) defined by SEQ ID NO. 5 (speA gene) and
6,
[0053] NADH-dependent butanol dehydrogenase A (protein YugJ; E.C.
1.1.1.-) defined by SEQ ID NO. 7 (yugJ gene) and 8,
[0054] butyryl-CoA dehydrogenase (E.C. 1.3.99.25) or acyl-CoA
dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 9 and 10,
[0055] butyryl-CoA dehydrogenase (E.C. 1.3.99.25) or acyl-CoA
dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 11 and 12,
[0056] 3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157) defined
by SEQ ID NO. 13 and 14,
[0057] putative enoyl-CoA hydratase protein (E.C. 4.2.1.17) defined
by SEQ ID NO. 15 and 16,
[0058] probable enoyl-(3-hydroxyisobutyryl)-CoA hydrolase protein
defined by SEQ ID NO. 17 and 18,
[0059] probable enoyl-CoA hydratase (protein EchA8; E.C. 4.2.1.17)
defined by SEQ ID NO. 19 (echA8 gene) and 20,
[0060] acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO.
21 and 22,
[0061] acetate-CoA ligase or propionate-CoA ligase (or synthetase;
protein AcsA; E.C. 6.2.1.1) defined by SEQ ID NO. 23 (acsA gene)
and 24,
[0062] 3-hydroxybutyryl-CoA dehydratase (protein YngF; E.C.
4.2.1.55) defined by SEQ ID No. 25 (yngF gene) and 26,
[0063] butyryl-CoA dehydrogenase (protein YusJ; E.C. 1.3.99.25) or
acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 27
(yusJ gene) and 28,
[0064] 3-hydroxyisobutyrate dehydrogenase (protein YkwC; E.C.
1.1.1.31) or oxidoreductase (E.C. 1.1.-.-) defined by SEQ ID NO. 29
(ykwC gene) and 30,
[0065] probable phosphate butyryltransferase (E.C. 2.3.1.19)
defined by SEQ ID NO. 31 and 32,
[0066] probable butyrate kinase (E.C. 2.7.2.7) defined by SEQ ID
NO. 33 and 34,
[0067] acetate-CoA ligase or synthetase or propionate-CoA ligase or
synthetase (protein AcsA; E.C. 6.2.1.1) defined by SEQ ID NO. 35
(acsA gene) and 36,
[0068] acetate-CoA ligase or propionate-CoA ligase (protein Ytcl;
E.C. 6.2.1.1) defined by SEQ ID NO. 37 (ytcl gene) and 38,
[0069] lysine and/or arginine decarboxylase (protein speA; E.C.
4.1.1.18 or E.C. 4.1.1.19) defined by SEQ ID NO. 39 (speA gene) and
40,
[0070] probable enoyl-CoA hydratase (E.C. 4.2.1.17) defined by SEQ
ID NO. 41 (ysiB gene) and 42,
[0071] similar to 3-hydroxy-acyl-CoA dehydrogenase (E.C. 1.1.1.35)
defined by SEQ ID NO. 43 and 44,
[0072] 3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate
dehydrogenase E1 (E.C. 1.2.4.2) defined by SEQ ID NO. 45 and
46,
[0073] probable acetate-CoA ligase or propionate-CoA ligase
(protein YhfL; E.C. 6.2.1.1) or acid-CoA ligase (E.C. 6.2.1.-)
defined by SEQ ID NO. 47 (yhfL gene) and 48 or
[0074] agmatinase (E.C. 3.5.1.11) defined by SEQ ID NO. 49 (ywhG
gene) and 50.
[0075] All of them are made available by the present
application.
[0076] The stated problem is thus solved in the same way in
principle by all 25 nucleic acids of SEQ ID NO. 1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,
47 and 49 which are indicated in the sequence listing and are
obtainable from B. licheniformis DSM 13, including an in each case
corresponding homology region which is defined hereinafter and
which effects a delimitation from the sequences described in the
prior art. It is likewise solved by the gene products derived
therefrom of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,
26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50, once again
including a corresponding homology region defined hereinafter. The
respective most similar nucleic acid and amino acid sequences
described in the prior art are compiled in Example 2 (Table 1) with
reference to the relevant database entries. The homology regions
claimed in each case have been defined on the basis of this
information. Solutions according to the invention of the stated
problem are preferably in each case those nucleic acids and
proteins which actually originate from microorganisms.
[0077] Which of these genes is preferred must be ascertained
experimentally taking account of the individual strain to be
cultured (and possibly different gene activities) and the
respective metabolic situation (for example (over)supply of certain
C or N sources) in the individual case. For this it is necessary
for a series of several mutants, which are to be produced in the
same way in principle, of the various relevant genes to be
generated in parallel and cultured under conditions which are
otherwise identical.
[0078] Further solutions are represented by fermentation processes
in which one or more of the metabolic pathways for synthesizing (1)
isovaleric acid (as part of leucine catabolism), (2)
2-methylbutyric acid and/or isobutyric acid (as part of valine
and/or isoleucine catabolism), (3) butanol and/or butyric acid (as
part of butyric acid metabolism), (4) propionic acid (as part of
propionate metabolism) and/or (5) cadaverine and/or putrescine (as
parts of lysine and/or arginine catabolism) are functionally
inactivated, preferably via the abovementioned enzymes/proteins
which are active on these pathways, and particularly preferably via
the nucleotide sequences provided according to the invention. The
latter can be used in a manner known per se and established in the
prior art, for example for producing knock-out constructs and for
introducing them via vectors in the host cells so that gene
disruption takes place.
[0079] Further solutions are represented by appropriately modified
microorganisms in particular relevant to industrial production, all
fermentation processes in which these are employed, and among these
especially those used to produce valuable products.
[0080] In addition, these gene products are available on the basis
of the present invention for reaction mixtures or processes
according to their respective biochemical properties, by which is
meant in particular the synthesis of (1) isovaleric acid, (2)
2-methylbutyric acid and/or isobutyric acid, (3) butanol and/or
butyric acid, (4) propionic acid and/or (5) cadaverine and/or
putrescine.
[0081] The present invention enables, at least as far as these
important metabolic pathways are concerned, causal combating of
odors. This is because it is possible by switching off the
identified metabolic pathways via the proteins involved with the
aid of the nucleic acids coding for these proteins to substantially
prevent the relevant substances being produced at all. It is thus
possible firstly to keep the number of subsequent purification and
working-up steps small, which is advantageous because they
represent in each case a physicochemical stress on the desired
product and reduce the yield; the product quality is thus overall
improved. Secondly, the production conditions are improved per se,
and the systems for filtration of the fermenter exit air can be
kept simpler. This causal combating of odors acts, because it
operates at the genetic level, on the properties of the respective
microorganism itself, thus increasing its tolerability of further
operations on this microorganism.
[0082] In particular, industrial fermentation is improved thereby,
which ought also to lead to a reduction of the costs of the
fermentation products.
[0083] In addition, the identified genes and gene products are thus
available for diverse applications, for example for chemical and/or
at least partly biocatalyzed synthesis of the relevant
compounds.
[0084] As described in the examples of the present application, it
was possible by sequencing the genomic DNA of the B. licheniformis
DSM 13, the reference strain obtainable from the Deutschen Sammlung
von Mikroorganismen and Zellkulturen GmbH, Mascheroder Weg 1b,
38124 Brunswick (http://www.dsmz.de), to identify said 25 novel
genes for this species. These are ones which code for enzymes or
enzyme subunits which are involved in the reactions described
herein for synthesizing odorous substances.
[0085] The most similar genes and relevant proteins in each case
which are known in this connection in the prior art show the
sequence homologies indicated in Example 2 (Table 1) of the present
application. The range of protection covered in each case by the
present application is defined thereby. Accordingly, all the
following nucleic acids and proteins represent in principle
equivalent embodiments of the present invention:
[0086] nucleic acid coding for a gene product (putative
branched-chain amino acid aminotransferase; E.C. 2.6.1.42) involved
in the synthesis of 2-methylbutyric acid and/or isobutyric acid and
having a nucleotide sequence which shows at least 67% identity and
with increasing preference at least 70%, 75%, 80%, 85%, 90%, 92%,
94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity
to the nucleotide sequence indicated in SEQ ID NO. 1,
[0087] gene product (putative branched-chain amino acid
aminotransferase; E.C. 2.6.1.42) involved in the synthesis of
2-methylbutyric acid and/or isobutyric acid and having an amino
acid sequence which shows at least 73% identity and with increasing
preference at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%,
99% and particularly preferably 100% identity to the amino acid
sequence indicated in SEQ ID NO. 2;
[0088] nucleic acid coding for a gene product (putative
branched-chain amino acid aminotransferase; E.C. 2.6.1.42) involved
in the synthesis of 2-methylbutyric acid and/or isobutyric acid and
having a nucleotide sequence which shows at least 78% identity and
with increasing preference at least 80%, 85%, 90%, 92%, 94%, 95%,
96%, 97%, 98%, 99% and particularly preferably 100% identity to the
nucleotide sequence indicated in SEQ ID NO. 3;
[0089] gene product (putative branched-chain amino acid
aminotransferase; E.C. 2.6.1.42) involved in the synthesis of
2-methylbutyric acid and/or isobutyric acid and having an amino
acid sequence which shows at least 83% identity and with increasing
preference at least 85%, 87.5% 90%, 92%, 94%, 96%, 97%, 98%, 99%
and particularly preferably 100% identity to the amino acid
sequence indicated in SEQ ID NO. 4;
[0090] nucleic acid speA coding for a gene product (lysine and/or
arginine decarboxylase; E.C. 4.1.1.18 or 4.1.1.19) involved in the
synthesis of cadaverine and/or putrescine and having a nucleotide
sequence which shows at least 78% identity and with increasing
preference at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%,
99% and particularly preferably 100% identity to the nucleotide
sequence indicated in SEQ ID NO. 5;
[0091] gene product SpeA (lysine and/or arginine decarboxylase;
E.C. 4.1.1.18 or E.C. 4.1.1.19) involved in the synthesis of
cadaverine and/or putrescine and having an amino acid sequence
which shows at least 89% identity and with increasing preference at
least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% and particularly
preferably 100% identity to the amino acid sequence indicated in
SEQ ID NO. 6;
[0092] nucleic acid yugJ coding for a gene product (NADH-dependent
butanol dehydrogenase A; E.C. 1.1.1.-) involved in the synthesis of
butanol and/or butyric acid and having a nucleotide sequence which
shows at least 81% identity and with increasing preference at least
85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and particularly
preferably 100% identity to the nucleotide sequence indicated in
SEQ ID NO. 7;
[0093] gene product YugJ (NADH-dependent butanol dehydrogenase A;
E.C. 1.1.1.-) involved in the synthesis of butanol and/or butyric
acid and having an amino acid sequence which shows at least 93%
identity and with increasing preference at least 94%, 95%, 96%,
97%, 98%, 98.5%, 99%, 99.5% and particularly preferably 100%
identity to the amino acid sequence indicated in SEQ ID NO. 8;
[0094] nucleic acid coding for a gene product (acyl-CoA
dehydrogenase; E.C. 1.3.99.-) involved in the synthesis of
isovaleric acid, 2-methylbutyric acid, isobutyric acid, butanol
and/or butyric acid and having a nucleotide sequence which shows at
least 79% identity and with increasing preference at least 75%,
80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly
preferably 100% identity to the nucleotide sequence indicated in
SEQ ID NO. 9;
[0095] gene product (acyl-CoA dehydrogenase; E.C. 1.3.99.-)
involved in the synthesis of isovaleric acid, 2-methylbutyric acid,
isobutyric acid or butanol and/or butyric acid and having an amino
acid sequence which shows at least 86% identity and with increasing
preference at least 87.5%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% and particularly preferably 100% identity to the amino acid
sequence indicated in SEQ ID NO. 10;
[0096] nucleic acid coding for a gene product (acyl-CoA
dehydrogenase; E.C. 1.3.99.-) involved in the synthesis of
isovaleric acid, 2-methylbutyric acid, isobutyric acid, butanol
and/or butyric acid and having a nucleotide sequence which shows at
least 64% identity and with increasing preference at least 65%,
70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and
particularly preferably 100% identity to the nucleotide sequence
indicated in SEQ ID NO. 11;
[0097] gene product (acyl-CoA dehydrogenase; E.C. 1.3.99.-)
involved in the synthesis of isovaleric acid, 2-methylbutyric acid,
isobutyric acid, butanol and/or butyric acid and having an amino
acid sequence which shows at least 67% identity and with increasing
preference at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92%, 94%, 95%,
96%, 97%, 98%, 99% and particularly preferably 100% identity to the
amino acid sequence indicated in SEQ ID NO. 12;
[0098] nucleic acid coding for a gene product (3-hydroxybutyryl-CoA
dehydrogenase; E.C. 1.1.1.157) involved in the synthesis of butanol
and/or butyric acid and having a nucleotide sequence which shows at
least 67% identity and with increasing preference at least 70%,
75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly
preferably 100% identity to the nucleotide sequence indicated in
SEQ ID NO. 13;
[0099] gene product (3-hydroxybutyryl-CoA dehydrogenase; E.C.
1.1.1.157) involved in the synthesis of butanol and/or butyric acid
and having an amino acid sequence which shows at least 69% identity
and with increasing preference at least 70%, 75%, 80%, 85%, 90%,
92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100%
identity to the amino acid sequence indicated in SEQ ID NO. 14;
[0100] nucleic acid coding for a gene product (putative enoyl-CoA
hydratase protein; E.C. 4.2.1.17) involved in the synthesis of
isovaleric acid, 2-methylbutyric acid and/or isobutyric acid and
having a nucleotide sequence which shows at least 65% identity and
with increasing preference at least 70%, 75%, 80%, 85%, 90%, 92%,
94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity
to the nucleotide sequence indicated in SEQ ID NO. 15;
[0101] gene product (putative enoyl-CoA hydratase protein; E.C.
4.2.1.17) involved in the synthesis of isovaleric acid,
2-methylbutyric acid and/or isobutyric acid and having an amino
acid sequence which shows at least 62% identity and with increasing
preference at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%,
97%, 98%, 99% and particularly preferably 100% identity to the
amino acid sequence indicated in SEQ ID NO. 16;
[0102] nucleic acid coding for a gene product (probable
enoyl-(3-hydroxyisobutyryl)-coenzyme A hydrolase protein) involved
in the synthesis of isovaleric acid, 2-methylbutyric acid and/or
isobutyric acid and having a nucleotide sequence which shows at
least 66% identity and with increasing preference at least 70%,
75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly
preferably 100% identity to the nucleotide sequence indicated in
SEQ ID NO. 17;
[0103] gene product (probable enoyl-(3-hydroxyisobutyryl)-coenzyme
A hydrolase protein) involved in the synthesis of isovaleric acid,
2-methylbutyric acid and/or isobutyric acid and having an amino
acid sequence which shows at least 66% identity and with increasing
preference at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92%, 94%, 96%,
97%, 98%, 99% and particularly preferably 100% identity to the
amino acid sequence indicated in SEQ ID NO. 18;
[0104] nucleic acid echA8 coding for a gene product (probable
enoyl-CoA hydratase; E.C. 4.2.1.17) involved in the synthesis of
isovaleric acid, 2-methylbutyric acid and/or isobutyric acid and
having a nucleotide sequence which shows at least 48% identity and
with increasing preference at least 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly
preferably 100% identity to the nucleotide sequence indicated in
SEQ ID NO. 19;
[0105] gene product EchA8 (probable enoyl-CoA hydratase; E.C.
4.2.1.17) involved in the sythesis of isovaleric acid,
2-methylbutyric acid and/or isobutyric acid and having an amino
acid sequence which shows at least 52% identity and with increasing
preference at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 94%,
96%, 97%, 98%, 99% and particularly preferably 100% identity to the
amino acid sequence indicated in SEQ ID NO. 20;
[0106] nucleic acid coding for a gene product (acyl-CoA
dehydrogenase; E.C. 1.3.99.-) involved in the synthesis of
isovaleric acid, 2-methylbutyric acid and/or isobutyric acid and
having a nucleotide sequence which shows at least 54% identity and
with increasing preference at least 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably
100% identity to the nucleotide sequence indicated in SEQ ID NO.
21;
[0107] gene product (acyl-CoA dehydrogenase) involved in the
synthesis of isovaleric acid, 2-methylbutyric acid and/or
isobutyric acid and having an amino acid sequence which shows at
least 65% identity and with increasing preference at least 70%,
75%, 80%, 85%, 87.5%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and
particularly preferably 100% identity to the amino acid sequence
indicated in SEQ ID NO. 22;
[0108] nucleic acid acsA coding for a gene product (acetyl-coenzyme
A synthetase; E.C. 6.2.1.1) involved in the synthesis of propionic
acid and having a nucleotide sequence which shows at least 67%
identity and with increasing preference at least 70%, 75%, 80%,
85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably
100% identity to the nucleotide sequence indicated in SEQ ID NO.
23;
[0109] gene product AscA (acetyl-coenzyme A synthetase; E.C.
6.2.1.1) involved in the synthesis of propionic acid and having an
amino acid sequence which shows at least 65% identity and with
increasing preference at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92%,
94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity
to the amino acid sequence indicated in SEQ ID NO. 24;
[0110] nucleic acid yngF coding for a gene product
(3-hydroxybutyryl-CoA dehydratase; E.C. 4.2.1.55) involved in the
synthesis of butanol and/or butyric acid and having a nucleotide
sequence which shows at least 68% identity and with increasing
preference at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%,
98%, 99% and particularly preferably 100% identity to the
nucleotide sequence indicated in SEQ ID NO. 25;
[0111] gene product YngF (3-hydroxybutyryl-CoA dehydratase; E.C.
4.2.1.55) involved in the synthesis of butanol and/or butyric acid
and having an amino acid sequence which shows at least 69% identity
and with increasing preference at least 70%, 75%, 80%, 85%, 87.5%,
90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100%
identity to the amino acid sequence indicated in SEQ ID NO. 26;
[0112] nucleic acid yusJ coding for a gene product (acyl-CoA
dehydrogenase; E.C. 1.3.99.-) involved in the synthesis of
isovaleric acid, 2-methylbutyric acid, isobutyric acid, butanol
and/or butyric acid and having a nucleotide sequence which shows at
least 77% identity and with increasing preference at least 80%,
85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% and particularly
preferably 100% identity to the nucleotide sequence indicated in
SEQ ID NO. 27;
[0113] gene product YusJ (acyl-CoA dehydrogenase; E.C. 1.3.99.-)
involved in the synthesis of isovaleric acid, 2-methylbutyric acid,
isobutyric acid, butanol and/or butyric acid and having an amino
acid sequence which shows at least 86% identity and with increasing
preference at least 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% and particularly preferably 100% identity to the amino
acid sequence indicated in SEQ ID NO. 28;
[0114] nucleic acid ykwC coding for a gene product (hypothetical
oxidoreductase; E.C. 1.1.-.-) involved in the synthesis of
2-methylbutyric acid and/or isobutyric acid and having a nucleotide
sequence which shows at least 77% identity and with increasing
preference at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%,
99% and particularly preferably 100% identity to the nucleotide
sequence indicated in SEQ ID NO. 29;
[0115] gene product YkwC (hypothetical oxidoreductase; E.C.
1.1.-.-) involved in the synthesis of 2-methylbutyric acid and/or
isobutyric acid and having an amino acid sequence which shows at
least 85% identity and with increasing preference at least 87.5%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and particularly
preferably 100% identity to the amino acid sequence indicated in
SEQ ID NO. 30;
[0116] nucleic acid coding for a gene product (probable phosphate
butyryltransferase; E.C. 2.3.1.19) involved in the synthesis of
butanol and/or butyric acid and having a nucleotide sequence which
shows at least 51% identity and with increasing preference at least
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%,
99% and particularly preferably 100% identity to the nucleotide
sequence indicated in SEQ ID NO. 31;
[0117] gene product (probable phosphate butyryltransferase; E.C.
2.3.1.19) involved in the synthesis of butanol and/or butyric acid
and having an amino acid sequence which shows at least 69% identity
and with increasing preference at least 70%, 75%, 80%, 85%, 90%,
92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100%
identity to the amino acid sequence indicated in SEQ ID NO. 32;
[0118] nucleic acid coding for a gene product (probable butyrate
kinase; E.C. 2.7.2.7) involved in the synthesis of butanol and/or
butyric acid and having a nucleotide sequence which shows at least
77% identity and with increasing preference at least 80%, 85%, 90%,
92%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100%
identity to the nucleotide sequence indicated in SEQ ID NO. 33;
[0119] gene product (probable butyrate kinase; E.C. 2.7.2.7)
involved in the synthesis of butanol and/or butyric acid and having
an amino acid sequence which shows at least 84% identity and with
increasing preference at least 85%, 87.5%, 90%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% and particularly preferably 100% identity to the
amino acid sequence indicated in SEQ ID NO. 34;
[0120] nucleic acid acsA coding for a gene product (acetyl-coenzyme
A synthetase; E.C. 6.2.1.1) involved in the synthesis of propionic
acid and having a nucleotide sequence which shows at least 79%
identity and with increasing preference at least 80%, 85%, 90%,
92%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100%
identity to the nucleotide sequence indicated in SEQ ID NO. 35;
[0121] gene product AcsA (acetyl-coenzyme A synthetase: E.C.
6.2.1.1) involved in the synthesis of propionic acid and having an
amino acid sequence which shows at least 85% identity and with
increasing preference at least 87.5%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% and particularly preferably 100% identity to the
amino acid sequence indicated in SEQ ID NO. 36;
[0122] nucleic acid ytcl coding for a gene product (acetate-CoA
ligase; E.C. 6.2.1.1) involved in the synthesis of propionic acid
and having a nucleotide sequence which shows at least 74% identity
and with increasing preference at least 75%, 80%, 85%, 90%, 92%,
94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity
to the nucleotide sequence indicated in SEQ ID NO. 37;
[0123] gene product Ytcl (acetate-CoA ligase; E.C. 6.2.1.1)
involved in the synthesis of propionic acid and having an amino
acid sequence which shows at least 77% identity and with increasing
preference at least 80%, 85%, 87.5%, 90%, 92%, 94%, 96%, 97%, 98%,
99% and particularly preferably 100% identity to the amino acid
sequence indicated in SEQ ID NO. 38;
[0124] nucleic acid speA coding for a gene product (lysine and/or
arginine decarboxylase; E.C. 4.1.1.18 or E.C. 4.1.1.19) involved in
the synthesis of cadaverine and/or putrescine and having a
nucleotide sequence which shows at least 68% identity and with
increasing preference at least 75%, 80%, 85%, 90%, 92%, 94%, 96%,
97%, 98%, 99% and particularly preferably 100% identity to the
nucleotide sequence indicated in SEQ ID NO. 39;
[0125] gene product SpeA (lysine and/or arginine decarboxylase;
E.C. 4.1.1.18 or E.C. 4.1.1.19) involved in the synthesis of
cadaverine and/or putrescine and having an amino acid sequence
which shows at least 66% identity and with increasing preference at
least 70%, 75%, 80%, 85%, 87.5%, 90%, 92%, 94%, 96%, 97%, 98%, 99%
and particularly preferably 100% identity to the amino acid
sequence indicated in SEQ ID NO. 40;
[0126] nucleic acid ysiB coding for a gene product (probable
enoyl-CoA hydratrase; E.C. 4.2.1.17) involved in the synthesis of
isovaleric acid, 2-methylbutyric acid and/or isobutyric acid and
having a nucleotide sequence which shows at least 75% identity and
with increasing preference at least 80%, 85%, 90%, 92%, 94%, 95%,
96%, 97%, 98%, 99% and particularly preferably 100% identity to the
nucleotide sequence indicated in SEQ ID NO. 41;
[0127] gene product YsiB (probable enoyl-CoA hydratrase; E.C.
4.2.1.17) involved in the synthesis of isovaleric acid,
2-methylbutyric acid and/or isobutyric acid and having an amino
acid sequence which shows at least 77% identity and with increasing
preference at least 80%, 85%, 87.5%, 90%, 92%, 94%, 96%, 97%, 98%,
99% and particularly preferably 100% identity to the amino acid
sequence indicated in SEQ ID NO. 42;
[0128] nucleic acid coding for a gene product (similar to
3-hydroxyacyl-CoA dehydrogenase; E.C. 1.1.1.35) involved in the
synthesis of 2-methylbutyric acid and having a nucleotide sequence
which shows at least 76% identity and with increasing preference at
least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% and
particularly preferably 100% identity to the nucleotide sequence
indicated in SEQ ID NO. 43;
[0129] gene product (similar to 3-hydroxyacyl-CoA dehydrogenase)
involved in the synthesis of 2-methylbutyric acid and having an
amino acid sequence which shows at least 80% identity and with
increasing preference at least 85%, 87.5%, 90%, 92%, 94%, 95%, 96%,
97%, 98%, 99% and particularly preferably 100% identity to the
amino acid sequence indicated in SEQ ID NO. 44;
[0130] nucleic acid coding for a gene product (2-oxoglutarate
dehydrogenase E1 component; E.C. 1.2.4.2) involved in the synthesis
of isovaleric acid, 2-methylbutyric acid and/or isobutyric acid and
having a nucleotide sequence which shows at least 80% identity and
with increasing preference at least 80%, 85%, 90%, 92%, 94%, 95%,
96%, 97%, 98%, 99% and particularly preferably 100% identity to the
nucleotide sequence indicated in SEQ ID NO. 45;
[0131] gene product (2-oxoglutarate dehydrogenase E1 component;
E.C. 1.2.4.2) involved in the synthesis of isovaleric acid,
2-methylbutyric acid and/or isobutyric acid and having an amino
acid sequence which shows at least 82% identity and with increasing
preference at least 85%, 87.5%, 90%, 92%, 94%, 95%, 96%, 97%, 98%,
99% and particularly preferably 100% identity to the amino acid
sequence indicated in SEQ ID NO. 46;
[0132] nucleic acid yhfL coding for a gene product (probable
acid-CoA ligase; E.C. 6.2.1.-) involved in the synthesis of
propionic acid and having a nucleotide sequence which shows at
least 67% identity and with increasing preference at least 70%,
75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly
preferably 100% identity to the nucleotide sequence indicated in
SEQ ID NO. 47;
[0133] gene product YhfL (probable acid-CoA ligase; E.C. 6.2.1.-)
involved in the synthesis of propionic acid and having an amino
acid sequence which shows at least 76% identity and with increasing
preference at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%,
99% and particularly preferably 100% identity to the amino acid
sequence indicated in SEQ ID NO. 48;
[0134] nucleic acid ywhG coding for a gene product (agmatinase;
E.C. 3.5.1.11) involved in the synthesis of cadaverine and/or
putrescine and having a nucleotide sequence which shows at least
85% identity and with increasing preference at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100%
identity to the nucleotide sequence indicated in SEQ ID NO. 49;
[0135] gene product YwhG (agmatinase; E.C. 3.5.1.11) involved in
the synthesis of cadaverine and/or putrescine and having an amino
acid sequence which shows at least 97% identity and with increasing
preference at least 97.5%, 98%, 98.5%, 99%, 99.5% and particularly
preferably 100% identity to the amino acid sequence indicated in
SEQ ID NO. 50.
[0136] In connection with the present application, an expression of
the form "at least X %" means "X % to 100%, including the extreme
values X and 100 and all integral and non-integral percentages in
between".
BRIEF DESCRIPTION OF THE FIGURES
[0137] FIG. 1: Metabolic pathway for the formation of isovaleric
acid. Explanations: see text
[0138] FIG. 2: Metabolic pathway for the formation of
2-methylbutyric acid and/or isobutyric acid; aspect of the
formation of 2-methylbutyric acid. Explanations: see text
[0139] FIG. 3: Metabolic pathway for the formation of
2-methylbutyric acid and/or isobutyric acid; aspect of the
formation of isobutyric acid. Explanations: see text
[0140] FIG. 4: Metabolic pathway for the formation of butanol
and/or butyric acid. Explanations: see text
[0141] FIG. 5: Metabolic pathway for the formation of propionic
acid. Explanations: see text
[0142] FIG. 6: Metabolic pathway for the formation of cadaverine
and/or putrescine; aspect of the formation of cadaverine.
Explanations: see text
[0143] FIG. 7: Metabolic pathway for the formation of cadaverine
and/or putrescine; aspect of the formation of putrescine.
Explanations: see text
DETAILED DESCRIPTION
[0144] The designations of the respective enzymes are governed by
the specific reactions catalyzed by them, as are depicted for
example in FIGS. 1 to 7. (Detailed explanations of the figures and
of the relevant metabolic pathways following hereinafter.) Thus, it
is also possible for a single enzyme to be able to catalyze two
reactions which are chemically virtually identical but are assigned
to different pathways on the basis of the respective substrate.
This may also be associated with a different enzyme classification
(E.C. numbers) according to IUBMB. The enzyme designation is
governed according to the invention according to the respective
specific reaction. This is because the specific function which is
implemented in the course of the present invention or is to be
switched off where appropriate is also associated therewith.
[0145] For illustration, reference may be made by way of example to
the enzyme which is indicated in SEQ ID NO. 18 and with which such
a deviation is in fact located on the same metabolic pathway
defined according to the invention. According to the relevant
statement in SEQ ID NO. 17, this is a "probable
enoyl-(3-hydroxyisobutyryl)-coenzyme A hydrolase protein". At the
time of the application, the IUBMB has not yet allocated an E.C.
number for this reaction, which is why reference can be made for
definition of the relevant enzymic activity only to reaction (6.)
in FIG. 3. On the same metabolic pathway for synthesizing
2-methylbutyric acid and/or isobutyric acid (as part of valine
and/or isoleucine catabolism) there is also a reaction which is
catalyzed by an enoyl-CoA hydratase, reaction (3.) in FIG. 3; the
situation is likewise for reaction (7.) in FIG. 1. A plurality of
enzymes with E.C. class 4.2.1.17 are in each case suitable for
this, for example those shown in SEQ ID NO. 16, 20 and 42 (see
below), but also the enzyme according to SEQ ID NO. 18. In the
course of this specific reaction, the enzyme according to SEQ ID
NO. 18 is thus to be regarded as enoyl-CoA hydratase and assigned
to E.C. class 4.2.1.17.
[0146] These genes and gene products can now be synthesized
artificially by methods known per se, and without the need to
reproduce the sequencing described in Example 1, in a targeted
manner on the basis of these sequences.
[0147] As a further alternative thereto, it is possible to obtain
the relevant genes from a Bacillus strain, in particular the strain
B. licheniformis DSM 13 which is obtainable from the DSMZ, via PCR,
it being possible to use the respective border sequences listed in
the sequence listing for synthesizing primers. On use of other
strains, the genes homologous thereto are obtained in each case,
and the success of the PCR should increase with the closeness of
the relationship of the selected strains to B. licheniformis DSM
13, because an increasing agreement in sequence also within the
primer binding regions should be associated therewith.
[0148] As an alternative thereto, the nucleic acids indicated in
the sequence listing can also be employed as DNA probes in order to
detect the respective homologous genes in preparations of genomic
DNA from other species. The procedure for this is known per se; as
is the isolation of the genes obtained in this way, their cloning,
their expression and obtaining of the relevant proteins.
Consideration is given in this connection in particular to
operating steps like those described for B. licheniformis itself in
Example 1.
[0149] The existence of the relevant proteins in a strain of
interest is detected in the first place by a chemical detection of
whether the relevant odorous substances are formed. It is then
possible for the enzymic activities presumed therefor to be
ascertained in suitable detection reactions. This takes place for
example by the starting compound relevant to the reaction in
question being incubated with a cell extract. When the relevant
enzymic activity is present, the products following in the relevant
metabolic pathway should accumulate and, if all the subsequent
enzymes are present, result in the odorous substance.
[0150] As detection at the level of molecular biology it is
possible to synthesize proteins on the basis of the amino acid
sequences shown in the present sequence listing, and to form
antibodies against them. These can then be used for example in
Western blots for detecting the homologous protein in cell extracts
of the host cells of interest.
[0151] Among the nucleic acids mentioned herein and coding for a
gene product of the invention involved in the synthesis of
isovaleric acid, 2-methyl-butyric acid, isobutyric acid, butanol,
butyric acid, propionic acid, cadaverine and/or putrescine and
defined as above on the basis of SEQ ID NO. 1, 3, 5, 7, 9, 11, 13,
15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47
or 49, preference is given in each case to that present naturally
in a microorganism, preferably a bacterium, particularly preferably
a Gram-positive bacterium, among these preferably one of the genus
Bacillus, among these particularly preferably one of the species B.
licheniformis and among these very particularly preferably B.
licheniformis DSM13.
[0152] It is thus possible as just described comparatively easy in
relation to neosynthesis for the relevant nucleic acids to be
obtained from natural species, especially microorganisms. Among
these, increasing preference is given in view of the stated problem
to those which can be fermented and which can in fact be employed
in industrial fermentations. These include in particular
representatives of the genera Staphylococcus, Corynebacterium and
Bacillus. Mention should be made among these for example of S.
carnosus and C. glutamicum, and B. subtilis, B. licheniformis, B.
amyloliquefaciens, B. agaradherens, B. lentus, B. globigii and B.
alkalophilus. Most preference is given to B. licheniformis DSM 13
because it was possible to obtain therefrom exactly the sequences
listed in the sequence listing.
[0153] These explanations apply in the same way to the relevant
proteins.
[0154] Thus, among the gene products mentioned herein and involved
in the synthesis of isovaleric acid, 2-methylbutyric acid,
isobutyric acid, butanol, butyric acid, propionic acid, cadaverine
and/or putrescine and defined on the basis of SEQ ID NO. 2, 4, 6,
8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,
42, 44, 46, 48 or 50, preference is given in each case to those
naturally formed by a microorganism, preferably by a bacterium,
particularly preferably by a Gram-positive bacterium, among these
preferably by one of the genus Bacillus, among these particularly
preferably by one of the species B. licheniforms and among these
very particularly preferably by B. licheniforms DSM 13.
[0155] The metabolic pathway utilized in Gram-positive bacteria of
the genus Bacillus for synthesizing isovaleric acid as part of
leucine catabolism is depicted in FIG. 1. It ultimately represents
an interface between the citrate cycle and/or fatty acid metabolism
and pyruvate metabolism as far as the synthesis of leucine.
[0156] The enzymes involved in the reactions shown in FIG. 1 are,
as mentioned above, the following, where the relevant number
designates the respective reaction step indicated in the figure:
[0157] (1.) L-leucine dehydrogenase (E.C. 1.4.1.9), [0158] (2.)
3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate
dehydrogenase E1 (E.C. 1.2.4.2), [0159] (3.) enzyme for hydrolyzing
isovaleryl-CoA to isovaleric acid and coenzyme A (where
non-enzymatic hydrolysis is also possible), [0160] (4.) acyl-CoA
dehydrogenase (E.C. 1.3.99.-), [0161] (5.) methylcrotonyl
carboxylase, [0162] (6.) 3-methylglutaconyl-CoA hydratase and
[0163] (7.) enoyl-CoA hydratase (E.C. 4.2.1.17).
[0164] Solutions of the stated problem and thus independent
embodiments of the present invention are thus represented by all
processes for fermenting a microorganism in which at least one of
the genes on a metabolic pathway for synthesizing isovaleric acid
(as part of leucine catabolism) is functionally inactivated.
[0165] The advantages previously explained are associated with this
solution.
[0166] Preference is given in this connection to any process of
this type in which the microorganism now forms only 50% of the
amount naturally formed under the same conditions, preferably now
only 10%, particularly preferably no isovaleric acid.
[0167] These percentages (and all subsequent corresponding data for
the further metabolic pathways) mean, in analogy to the statement
made above for the sequence homology, once again all intermediate
integral or fractional percentages in correspondingly preferred
gradation. To determine these values, cells of an untreated strain
and of a treated strain are fermented under conditions which are
otherwise identical and, during the fermentation, the rate of
formation of the unwanted odorous substance is suitably ascertained
in a manner known per se. Since the strains are otherwise
identical, the differences in the formation of this substance are
attributable to the different gene activities. In this connection,
any reduction in the formation of the odorous substance is desired
according to the invention. Values comparable in percentage terms
are obtained by taking samples (for instance from the exit air)
from both fermentations and determining the content of the
respective substance by analytical methods known per se. It is
preferred to determine this value at the transition to the
stationary phase of growth, because this time can usually be
identified unambiguously and, at the same time, is normally
associated with the highest metabolic rate.
[0168] Account is taken thereby of the generally high flexibility
of microorganisms in relation to their metabolism. Thus, it is
conceivable for inactivation of one gene to be partly compensated
by enhancement of the activity of another gene and/or protein which
is possibly not quite as effective in vivo. However, increasing
preference is given to inactivation of the said pathway as
extensively as possible. It is possible for this to test in the
individual case the inactivation of various genes for the
effectiveness according to the invention thereof and to select
those with the strongest effect. It is additionally possible to
combine a plurality of inactivations together.
[0169] Preference is given to a process according to the invention
in which at least one of the following enzymes is functionally
inactivated: [0170] (1.) L-leucine dehydrogenase (E.C. 1.4.1.9),
[0171] (2.) 3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate
dehydrogenase E1 (E.C. 1.2.4.2), [0172] (3.) enzyme for hydrolyzing
isovaleryl-CoA to isovaleric acid and coenzyme A, [0173] (4.)
acyl-CoA dehydrogenase (E.C. 1.3.99.-), [0174] (5.) methylcrotonyl
carboxylase, [0175] (6.) 3-methylglutaconyl-CoA hydratase and
[0176] (7.) enoyl-CoA hydratase (protein) (E.C. 4.2.1.17).
[0177] This is because, as depicted in FIG. 1, these activities may
be connected with the metabolic pathway under consideration
here.
[0178] As already stated above and described in the examples of the
present application, it was possible by sequencing the genomic DNA
of B. licheniformis DSM 13 to identify several of the genes coding
for enzymes located on this pathway, or for subunits thereof. The
genes involved are the following (the preceding number designates
in each case the reaction in which the relevant enzyme is
involved): [0179] (2.) 3-methyl-2-oxobutanoate dehydrogenase or
2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2) defined by SEQ ID
NO. 45, [0180] (4.) a subunit of acyl-CoA dehydrogenase (E.C.
1.3.99.-) defined by SEQ ID NO. 9, 11, 21 or 27 (yusJ gene), and
[0181] (7.) enoyl-CoA hydratase (protein) (E.C. 4.2.1.17) defined
by SEQ ID NO. 15, 17, 19 (echA8 gene) or 41 (ysiB gene).
[0182] The amino acid sequences derived therefrom are indicated in
SEQ ID NO. 46, 10, 12, 22, 28, 16, 18, 20 and 42, respectively. It
was thus possible to identify these specific gene products in the
course of the present invention as involved in this metabolic
pathway for synthesizing isovaleric acid (as part of leucine
catabolism).
[0183] A process of the invention which is therefore preferred is
one where the functionally inactivated enzyme is the homolog, which
is naturally active in the relevant microorganism, to one of the
following proteins from B. licheniformis DSM 13: [0184] (2.)
3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate
dehydrogenase E1 (E.C. 1.2.4.2) defined by SEQ ID NO. 46, [0185]
(4.) a subunit of acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by
SEQ ID NO. 10, 12, 22 or 28, and [0186] (7.) enoyl-CoA hydratase
(protein) (E.C. 4.2.1.17) defined by SEQ ID NO. 16, 18, 20 or
42.
[0187] A preferred process of the invention is one where the enzyme
is functionally inactivated at the genetic level, preferably by
inactivation of a gene which corresponds to the nucleic acid which
codes for one of the following proteins from B. licheniformis DSM
13: [0188] (2.) 3-methyl-2-oxobutanoate dehydrogenase or
2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2) defined by SEQ ID
NO. 45, [0189] (4.) a subunit of acyl-CoA dehydrogenase (E.C.
1.3.99.-) defined by SEQ ID NO. 9, 11, 21 or 27 (yusJ gene), and
[0190] (7.) enoyl-CoA hydratase (protein) (E.C. 4.2.1.17) defined
by SEQ ID NO. 15, 17, 19 (echA8 gene) or 41 (ysiB gene).
[0191] This is because, in accordance with the stated problem, it
was preferably intended to find a causal solution, meaning one
applying at the level of molecular biology. This is available with
the stated nucleotide sequences. Example 3 explains how
corresponding deletions can be undertaken; further statements
concerning this are given hereinafter because they apply in
principle to all described metabolic pathways.
[0192] A preferred process of the invention is thus one where, for
inactivation at the genetic level, one of the nucleic acids of the
invention within the region designated above homologous to [0193]
(2.) SEQ ID NO. 45, [0194] (4.) 9, 11, 21 or 27 and [0195] (7.) 15,
17, 19 or 41 has been used, preferably one, particularly preferably
two parts in each case one of these sequences which in each case
comprise at least 70 connected positions.
[0196] This can be detected for example by a molecular biological
investigation (such as, for example, restriction, sequencing) of
the gene region modified by the mutagenesis.
[0197] A further embodiment of the present invention is represented
by the use of a gene which corresponds to the nucleic acid which
codes for one of the following proteins of B. licheniformis DSM 13
for functional inactivation of a metabolic pathway for synthesizing
isovaleric acid (as part of leucine catabolism) at the genetic
level in a microorganism: [0198] (2.) 3-methyl-2-oxobutanoate
dehydrogenase or 2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2)
defined by SEQ ID NO. 45, [0199] (4.) a subunit of acyl-CoA
dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 9, 11, 21 or 27
(yusJ gene), and [0200] (7.) enoyl-CoA hydratase (protein) (E.C.
4.2.1.17) defined by SEQ ID NO. 15, 17, 19 (echA8 gene) or 41 (ysiB
gene).
[0201] The same statements as previously made about the
corresponding processes apply in principle to such uses.
[0202] Accordingly, a preferred use according to the invention of
nucleic acids of the invention is within the region of homology
designated above to
(2.) SEQ ID NO. 45,
[0203] (4.) 9, 11, 21 or 27 and (7.) 15, 17, 19 or 41 for
functional inactivation, preferably of one, particularly preferably
of two parts in each case of one of these sequences, where these
parts in each case comprise at least 70 connected positions.
[0204] Further embodiments based on these fermentation processes
and uses are detailed hereinafter because they can be applied in
principle to all the metabolic pathways described within the scope
of the present invention.
[0205] The metabolic pathway utilized in Gram-positive bacteria of
the genus Bacillus for synthesizing 2-methylbutyric acid as part of
isoleucine catabolism is depicted in FIG. 2; the corresponding
pathway proceeding via the same enzymes in principle for
synthesizing isobutyric acid as part of valine catabolism is
evident from FIG. 3. This aspect, which is regarded in connection
with the present application as a single pathway, of bacterial
metabolism ultimately represents, like the pathway considered
previously too, an interface between the citrate cycle and/or fatty
acid metabolism and pyruvate metabolism as far as the synthesis of
the two amino acids isoleucine and valine.
[0206] As already mentioned, the following enzymes are involved in
the reactions shown in FIGS. 2 and 3, in each case the relevant
numbers of the reaction steps indicated in the figures being
indicated: [0207] (1.) branched-chain amino acid aminotransferase
(E.C. 2.6.1.42; reaction 1 in FIGS. 2 and 3), [0208] (2.)
3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate
dehydrogenase E1 (E.C. 1.2.4.2; reaction 2 in FIGS. 2 and 3),
[0209] (3.) enzyme for hydrolyzing 2-methylbutyryl-CoA to
2-methylbutyric acid (reaction 3 in FIG. 2) or isobutyryl-CoA to
isobutyric acid and coenzyme A (reaction 3 in FIG. 3; a
non-enzymatic hydrolysis also being possible in both cases), [0210]
(4.) acyl-CoA dehydrogenase (E.C. 1.3.99.-; reaction 4 in FIGS. 2
and 3), [0211] (5.) enoyl-CoA hydratase (protein) (E.C. 4.2.1.17;
reaction 5 in FIGS. 2 and 3), [0212] (6.) 3-hydroxy-acyl-CoA
dehydrogenase (E.C. 1.1.1.35) (reaction 6 in FIG. 2), [0213] (7.)
acetyl-CoA acyltransferase (reaction step 7 in FIG. 2), [0214] (8.)
enoyl-(3-hydroxyisobutyryl)-CoA hydrolase protein (step 6 in FIG.
3) and [0215] (9.) 3-hydroxyisobutyrate dehydrogenase (E.C.
1.1.1.31) or oxidoreductase (E.C. 1.1.-.-; step 7 in FIG. 3).
[0216] Solutions of the stated problem and thus independent
embodiments of the present invention are thus represented by all
processes for fermenting a microorganism in which at least one of
the genes on a metabolic pathway for synthesizing 2-methylbutyric
acid and/or isobutyric acid (as part of valine and/or isoleucine
catabolism) is functionally inactivated.
[0217] The advantages already explained are associated with this
solution.
[0218] Preference is given in this connection to any process of
this type in which the microorganism now forms only 50% of the
amount formed naturally under the same conditions, preferably now
only 10%, particularly preferably no 2-methylbutyric acid and/or
isobutyric acid.
[0219] Account is thereby taken, as explained above for the first
metabolic pathway described, of the generally high flexibility of
microorganisms in relation to their metabolism.
[0220] A preferred process of the invention is one in which at
least one of the following enzymes is functionally inactivated:
[0221] (1.) branched-chain amino acid aminotransferase (E.C.
2.6.1.42), [0222] (2.) 3-methyl-2-oxobutanoate dehydrogenase or
2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2), [0223] (3.) enzyme
for hydrolyzing 2-methylbutyryl-CoA to 2-methylbutyric acid or
isobutyryl-CoA to isobutyric acid and coenzyme A, [0224] (4.)
acyl-CoA dehydrogenase (E.C. 1.3.99.-), [0225] (5.) enoyl-CoA
hydratase (protein) (E.C. 4.2.1.17), [0226] (6.) 3-hydroxy-acyl-CoA
dehydrogenase (E.C. 1.1.1.35), [0227] (7.) acetyl-CoA
acyltransferase, [0228] (8.) enoyl-(3-hydroxyisobutyryl)-CoA
hydrolase protein and [0229] (9.) 3-hydroxyisobutyrate
dehydrogenase (E.C. 1.1.1.31) or oxidoreductase (E.C. 1.1.-.-).
[0230] This is because, as depicted in FIGS. 2 and 3, these
activities may be associated with the metabolic pathway
considered.
[0231] As stated previously and described in the examples of the
present application, it was possible by sequencing the genomic DNA
of B. licheniformis DSM 13 to identify several of the genes which
code for enzymes located on this pathway, or for subunits thereof.
These involve the following genes (the preceding number designates
in each case the reaction in which the relevant enzyme is
involved): [0232] (1.) branched-chain amino acid aminotransferase
(E.C. 2.6.1.42) defined by SEQ ID NO. 1 or 3, [0233] (2.)
3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate
dehydrogenase E1 (E.C. 1.2.4.2) defined by SEQ ID NO. 45, [0234]
(4.) acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO.
9, 11, 21 or 27 (yusJ gene), [0235] (5.) enoyl-CoA hydratase
(protein) (E.C. 4.2.1.17) defined by SEQ ID NO. 15, 17, 19 (echA8
gene) or 41 (ysiB gene), [0236] (6.) 3-hydroxy-acyl-CoA
dehydrogenase (E.C. 1.1.1.35) defined by SEQ ID NO. 43, [0237] (8.)
enoyl-(3-hydroxyisobutyryl)-CoA hydrolase protein defined by SEQ ID
NO. 17 and [0238] (9.) 3-hydroxyisobutyrate dehydrogenase (E.C.
1.1.1.31) or oxidoreductase (E.C. 1.1.-.-) defined by SEQ ID NO. 29
(ykwC gene).
[0239] The amino acid sequences derived therefrom are indicated in
SEQ ID NO. 2, 4, 46, 10, 12, 22, 28, 16, 18, 20, 42, 44, 18 and 30.
It was thus possible in the course of the present invention to
identify the specific gene products as involved in this metabolic
pathway for synthesizing 2-methyl-butyric acid and/or isobutyric
acid (as part of valine and/or isoleucine catabolism).
[0240] A preferred process of the invention is therefore one where
the functionally inactivated enzyme is the homolog, naturally
active in the relevant microorganism, to one of the following
proteins from B. licheniformis DSM 13: [0241] (1.) branched-chain
amino acid aminotransferase (E.C. 2.6.1.42) defined by SEQ ID NO. 2
or 4, [0242] (2.) 3-methyl-2-oxobutanoate dehydrogenase or
2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2) defined by SEQ ID
NO. 46, [0243] (4.) acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined
by SEQ ID NO. 10, 12, 22 or 28, [0244] (5.) enoyl-CoA hydratase
(protein) (E.C. 4.2.1.17) defined by SEQ ID NO. 16, 18, 20 or 42,
[0245] (6.) 3-hydroxy-acyl-CoA dehydrogenase (E.C. 1.1.1.35)
defined by SEQ ID NO. 44, [0246] (8.)
enoyl-(3-hydroxyisobutyryl)-CoA hydrolase protein defined by SEQ ID
NO. 18 and [0247] (9.) 3-hydroxyisobutyrate dehydrogenase (E.C.
1.1.1.31) or oxidoreductase (E.C. 1.1.-.-) defined by SEQ ID NO.
30.
[0248] A preferred process of the invention is one where the enzyme
is functionally inactivated at the genetic level, preferably by
inactivation of a gene which corresponds to the nucleic acid which
codes for one of the following proteins of B. licheniformis DSM 13:
[0249] (1.) branched-chain amino acid aminotransferase (E.C.
2.6.1.42) defined by SEQ ID NO. 1 or 3, [0250] (2.)
3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate
dehydrogenase E1 (E.C. 1.2.4.2) defined by SEQ ID NO. 45, [0251]
(4.) acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO.
9, 11, 21 or 27 (yusJ gene), [0252] (5.) enoyl-CoA hydratase
(protein) (E.C. 4.2.1.17) defined by SEQ ID NO. 15, 17, 19 (echA8
gene) or 41 (ysiB gene), [0253] (6.) 3-hydroxy-acyl-CoA
dehydrogenase (E.C. 1.1.1.35) defined by SEQ ID NO. 43, [0254] (8.)
enoyl-(3-hydroxyisobutyryl)-CoA hydrolase protein defined by SEQ ID
NO. 17 and [0255] (9.) 3-hydroxyisobutyrate dehydrogenase (E.C.
1.1.1.31) or oxidoreductase (E.C. 1.1.-.-) defined by SEQ ID NO. 29
(ykwC gene).
[0256] This is because, in accordance with the stated problem, the
intention was preferably to find a causal solution, meaning one
applying at the level of molecular biology. Example 3 explains how
corresponding deletions can be undertaken; further statements
concerning this are given hereinafter.
[0257] A preferred process of the invention is thus one where, for
inactivation at the genetic level, one of the nucleic acids of the
invention within the region designated above and homologous to
[0258] (1.) SEQ ID NO. 1 or 3, [0259] (2.) 45, [0260] (4.) 9, 11,
21 or 27, [0261] (5.) 15, 17, 19 or 41, [0262] (6.) 43, [0263] (8.)
17 and [0264] (9.) 29 has been used, preferably one, particularly
preferably two parts in each case of one of these sequences which
in each case comprise at least 70 connected positions.
[0265] A further embodiment of the present invention is represented
by the use of a gene which corresponds to the nucleic acid which
codes for one of the following proteins of B. licheniformis DSM 13
for functional inactivation of a metabolic pathway for synthesizing
isovaleric acid (as part of leucine catabolism) at the genetic
level in a microorganism: [0266] (1.) branched-chain amino acid
aminotransferase (E.C. 2.6.1.42) defined by SEQ ID NO. 1 or 3,
[0267] (2.) 3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate
dehydrogenase E1 (E.C. 1.2.4.2) defined by SEQ ID NO. 45, [0268]
(4.) acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO.
9, 11, 21 or 27 (yusJ gene), [0269] (5.) enoyl-CoA hydratase
(protein) (E.C. 4.2.1.17) defined by SEQ ID NO. 15, 17, 19 (echA8
gene) or 41 (ysiB gene), [0270] (6.) 3-hydroxy-acyl-CoA
dehydrogenase (E.C. 1.1.1.35) defined by SEQ ID NO. 43, [0271] (8.)
enoyl-(3-hydroxyisobutyryl)-CoA hydrolase protein defined by SEQ ID
NO. 17 and [0272] (9.) 3-hydroxyisobutyrate dehydrogenase (E.C.
1.1.1.31) or oxidoreductase (E.C. 1.1.-.-) defined by SEQ ID NO. 29
(ykwC gene).
[0273] The same as previously stated concerning the corresponding
processes applies in principle to such uses.
[0274] Accordingly, a preferred use according to the invention is
of nucleic acids of the invention within the regions designated
above and homologous to [0275] (1.) SEQ ID NO. 1 or 3, [0276] (2.)
45, [0277] (4.) 9, 11, 21 or 27, [0278] (5.) 15, 17, 19 or 41,
[0279] (6.) 43, [0280] (8.) 17 and [0281] (9.) 29 for functional
inactivation, preferably of one, particularly preferably of two
parts in each case of one of these sequences, where these parts
comprise in each case at least 70 connected positions.
[0282] Further embodiments based on these fermentation processes
and uses are detailed hereinafter.
[0283] The metabolic pathway utilized in Gram-positive bacteria of
the genus Bacillus for synthesizing butanol and/or butyric acid as
part of butyric acid metabolism is depicted in FIG. 4. This
metabolic pathway is ultimately derived from fatty acid
metabolism.
[0284] As previously mentioned, the following enzymes are involved
in the reactions shown in FIG. 4, the relevant number designating
the respective reaction step indicated in the figure: [0285] (1.)
3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157), [0286] (2.)
3-hydroxybutyryl-CoA dehydrogenase (E.C. 4.2.1.55), [0287] (3.)
butyryl-CoA dehydrogenase (E.C. 1.3.99.25), [0288] (4.) phosphate
butyryltransferase (E.C. 2.3.1.19), [0289] (5.) butyrate kinase
(E.C. 2.7.2.7), [0290] (6.) butyraldehyde dehydrogenase and [0291]
(8.) NADH-dependent butanol dehydrogenase A (E.C. 1.1.1.-).
[0292] Reaction (7.) normally takes place by non-enzymatic
oxidation by atmospheric oxygen.
[0293] Solutions of the stated problem and thus independent
embodiments of the present invention are thus represented by all
processes for fermenting a microorganism in which at least one of
the genes on a metabolic pathway for synthesizing butanol and/or
butyric acid (as part of butyric acid metabolism) is functionally
inactivated.
[0294] The advantages already explained are associated with this
solution.
[0295] Preference is given in this connection to any process of
this type in which the microorganism now forms only 50% of the
amount naturally formed under the same conditions, preferably now
only 10%, particularly preferably no butanol or no butyric
acid.
[0296] This takes account, as explained above for the first
metabolic pathway described, of the generally high flexibility of
microorganisms in relation to their metabolism.
[0297] A preferred process of the invention is one in which at
least one of the following enzymes is functionally inactivated:
[0298] (1.) 3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157),
[0299] (2.) 3-hydroxybutyryl-CoA dehydratase (E.C. 4.2.1.55),
[0300] (3.) butyryl-CoA dehydrogenase (E.C. 1.3.99.25), [0301] (4.)
phosphate butyryltransferase (E.C. 2.3.1.19), [0302] (5.) butyrate
kinase (E.C. 2.7.2.7), [0303] (6.) butyraldehyde dehydrogenase and
[0304] (8.) NADH-dependent butanol dehydrogenase A (E.C.
1.1.1.-).
[0305] This is because, as depicted in FIG. 4, these activities can
be associated with the metabolic pathway under consideration
here.
[0306] As stated above and described in the examples of the present
application, it was possible by sequencing the genomic DNA of B.
licheniformis DSM 13 to identify several of the genes which code
for enzymes located on this pathway, or for subunits thereof. These
are the following genes (the preceding number designates in each
case the reaction in which the relevant enzyme is involved): [0307]
(1.) 3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157) defined by
SEQ ID NO. 13, [0308] (2.) 3-hydroxybutyryl-CoA dehydratase (E.C.
4.2.1.55) defined by SEQ ID NO. 25 (yngF gene), [0309] (3.)
butyryl-CoA dehydrogenase (E.C. 1.3.99.25) defined by SEQ ID NO. 9,
11 or 27 (yusJ gene), [0310] (4.) phosphate butyryltransferase
(E.C. 2.3.1.19) defined by SEQ ID NO. 31, [0311] (5.) butyrate
kinase (E.C. 2.7.2.7) defined by SEQ ID NO. 33 and [0312] (8.)
NADH-dependent butanol dehydrogenase A (E.C. 1.1.1.-) defined by
SEQ ID NO. 7 (yugJ gene).
[0313] The amino acid sequences derived therefrom are indicated in
SEQ ID NO. 14, 26, 10, 12, 28, 32, 34 and 8. It was thus possible
in the course of the present invention to identify these specific
gene products as involved in this metabolic pathway for
synthesizing butanol and/or butyric acid (as part of butyric acid
metabolism).
[0314] A preferred process of the invention is therefore one where
the functionally inactivated enzyme is the homolog, which is
naturally active in the relevant microorganism, to one of the
following proteins from B. licheniformis DSM 13: [0315] (1.)
3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157) defined by SEQ
ID NO. 14, [0316] (2.) 3-hydroxybutyryl-CoA dehydratase (E.C.
4.2.1.55) defined by SEQ ID NO. 26, [0317] (3.) butyryl-CoA
dehydrogenase (E.C. 1.3.99.25) defined by SEQ ID NO. 10, 12 or 28,
[0318] (4.) phosphate butyryltransferase (E.C. 2.3.1.19) defined by
SEQ ID NO. 32, [0319] (5.) butyrate kinase (E.C. 2.7.2.7) defined
by SEQ ID NO. 34 and [0320] (8.) NADH-dependent butanol
dehydrogenase A (E.C. 1.1.1.-) defined by SEQ ID NO. 8.
[0321] The preferred process according to the invention is one
where the enzyme is functionally inactivated at the genetic level,
preferably by inactivation of a gene which corresponds to the
nucleic acid which codes for one of the following proteins of B.
licheniformis DSM 13: [0322] (1.) 3-hydroxybutyryl-CoA
dehydrogenase (E.C. 1.1.1.157) defined by SEQ ID NO. 13, [0323]
(2.) 3-hydroxybutyryl-CoA dehydratase (E.C. 4.2.1.55) defined by
SEQ ID NO. 25 (yngF gene), [0324] (3.) butyryl-CoA dehydrogenase
(E.C. 1.3.99.25) defined by SEQ ID NO. 9, 11 or 27 (yusJ gene),
[0325] (4.) phosphate butyryltransferase (E.C. 2.3.1.19) defined by
SEQ ID NO. 31, [0326] (5.) butyrate kinase (E.C. 2.7.2.7) defined
by SEQ ID NO. 33 and [0327] (8.) NADH-dependent butanol
dehydrogenase A (E.C. 1.1.1.-) defined by SEQ ID NO. 7 (yugJ
gene).
[0328] This is because, in accordance with the stated problem, it
was intended preferably to find a causal solution, meaning one
applying at the level of molecular biology. Example 3 explains how
corresponding deletions can be undertaken; further statements
concerning this are given hereinafter.
[0329] Thus, a preferred process of the invention is one where, for
inactivation at the genetic level, one of the nucleic acids of the
invention within the region designated above and homologous to
[0330] (1.) SEQ ID NO. 13, [0331] (2.) 25, [0332] (3.) 9, 11 or 27,
[0333] (4.) 31, [0334] (5.) 33 and [0335] (6.) 7 has been used,
preferably one, particularly preferably two parts in each case of
one of these sequences, which in each case comprise at least 70
connected positions.
[0336] A further embodiment of the present invention is represented
by the use of a gene which corresponds to the nucleic acid which
codes for one of the following proteins of B. licheniformis DSM 13
for functional inactivation of a metabolic pathway for synthesizing
butanol and/or butyric acid (as part of butyric acid metabolism) at
the genetic level in a microorganism: [0337] (1.)
3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157) defined by SEQ
ID NO. 13, [0338] (2.) 3-hydroxybutyryl-CoA dehydratase (E.C.
4.2.1.55) defined by SEQ ID NO. 25 (yngF gene), [0339] (3.)
butyryl-CoA dehydrogenase (E.C. 1.3.99.25) defined by SEQ ID NO. 9,
11 or 27 (yusJ gene), [0340] (4.) phosphate butyryltransferase
(E.C. 2.3.1.19) defined by SEQ ID NO. 31, [0341] (5.) butyrate
kinase (E.C. 2.7.2.7) defined by SEQ ID NO. 33 and [0342] (8.)
NADH-dependent butanol dehydrogenase A (E.C. 1.1.1.-) defined by
SEQ ID NO. 7 (yugJ gene).
[0343] The same as has previously been stated concerning the
corresponding processes applies in principle to such uses.
[0344] Accordingly, a preferred use according to the invention is
of nucleic acids of the invention within the region designated
above and homologous to [0345] (1.) SEQ ID NO. 13, [0346] (2.) 25,
[0347] (3.) 9, 11 or 27, [0348] (4.) 31, [0349] (5.) 33 and [0350]
(8.) 7 for functional inactivation, preferably of one, particularly
preferably of two parts in each case of one of these sequences,
where these parts comprise in each case at least 70 connected
positions.
[0351] Further embodiments based on these fermentation processes
and uses are detailed hereinafter.
[0352] The metabolic pathway utilized in Gram-positive bacteria of
the genus Bacillus for synthesizing propionic acid (as part of
propionate metabolism) is depicted in FIG. 5. This metabolic
pathway ultimately represents an interface between the citrate
cycle and fatty acid metabolism.
[0353] As already mentioned, the following enzymes are involved in
the reactions shown in FIG. 5, where the relevant number designates
the respective reaction step indicated in the figure: [0354] (1.)
succinate-propionate CoA-transferase, [0355] (2.) acetate-CoA
ligase or synthetase or propionate-CoA ligase or synthetase (E.C.
6.2.1.1) and [0356] (3.) acetate-CoA ligase or synthetase or
propionate-CoA ligase or synthetase (E.C. 6.2.1.1).
[0357] Solutions of the stated problem and thus independent
embodiments of the present invention are thus represented by all
processes for fermenting a microorganism in which at least one of
the genes on a metabolic pathway for synthesizing propionic acids
(as part of propionate metabolism) is functionally inactivated.
[0358] The previously explained advantages are associated with this
solution.
[0359] Preference is given in this connection to any process of
this type in which the microorganism now forms only 50% of the
amount naturally formed under the same conditions, preferably now
only 10%, particularly preferably no propionic acid.
[0360] This takes account, as explained above for the first
metabolic pathway described, of the generally high flexibility of
microorganisms in relation to their metabolism.
[0361] A preferred process of the invention is one in which at
least one of the following enzymes is functionally inactivated:
[0362] (1.) succinate-propionate CoA-transferase, [0363] (2.)
acetate-CoA ligase or synthetase or propionate-CoA ligase or
synthetase (E.C. 6.2.1.1) and [0364] (3.) acetate-CoA ligase or
synthetase or propionate-CoA ligase or synthetase (E.C.
6.2.1.1).
[0365] This is because, as depicted in FIG. 5, these activities can
be connected with the metabolic pathway under consideration
herein.
[0366] As already stated above and described in the examples in the
present application, it was possible to identify by sequencing the
genomic DNA of B. licheniformis DSM 13 several of the genes which
code for enzymes located on this pathway, or for subunits thereof.
These are the following genes (the preceding number designates in
each case the reaction in which the relevant enzyme is
involved):
acetate-CoA ligase or synthetase or propionate-CoA ligase or
synthetase (E.C. 6.2.1.1) defined by SEQ ID NO. 35 (acsA gene), 37
(ytcl gene), 47 (yhfL gene) or 23 (acsA gene).
[0367] The amino acid sequences derived therefrom are indicated in
SEQ ID NO. 36, 38, 48 and 24. It was thus possible to identify the
specific gene products in the course of the present invention as
involved in this metabolic pathway for synthesizing propionic acid
(as part of propionate metabolism).
[0368] A preferred process of the invention is therefore one where
the functionally inactivated enzyme is the homolog, which is
naturally active in the relevant microorganism, to one of the
following proteins from B. licheniformis DSM 13: acetate-CoA ligase
or synthetase or propionate-CoA ligase or synthetase (E.C. 6.2.1.1)
defined by SEQ ID NO. 36, 38, 48 or 24.
[0369] A preferred process of the invention is one where the enzyme
is functionally inactivated at the genetic level, preferably by
inactivation of a gene which corresponds to the nucleic acid which
codes for one of the following proteins of B. licheniformis DSM 13:
acetate-CoA ligase or synthetase or propionate-CoA ligase or
synthetase (E.C. 6.2.1.1) defined by SEQ ID NO. 35 (acsA gene), 37
(ytcl gene), 47 (yhfL gene) or 23 (acsA gene).
[0370] This is because, in accordance with the stated problem, the
intention was preferably to find a causal solution, meaning one
applying at the level of molecular biology. Example 3 explains how
corresponding deletions can be undertaken; further statements
concerning this are given hereinafter.
[0371] A preferred process of the invention is thus one where, for
the inactivation at the genetic level, one of the nucleic acids of
the invention has been used within the region designated above and
homologous to SEQ ID NO. 35, 37, 47 or 23, preferably one,
particularly preferably two parts in each case of one of these
sequences which comprise in each case at least 70 connected
positions.
[0372] A further embodiment of the present invention is represented
by the use of a gene which corresponds to the nucleic acid which
codes for one of the following proteins of B. licheniformis DSM 13
for the functional inactivation of a metabolic pathway for
synthesizing propionic acid (as part of propionate metabolism) at
the genetic level in a microorganism: acetate-CoA ligase or
synthetase or propionate-CoA ligase or synthetase (E.C. 6.2.1.1)
defined by SEQ ID NO. 35 (acsA gene), 37 (ytcl gene), 47 (yhfL
gene) or 23 (acsA gene).
[0373] The same as previously stated concerning the corresponding
processes applies in principle to uses of this type.
[0374] Accordingly, preference is given to such a use according to
the invention of nucleic acids of the invention within the region
designated above and homologous to SEQ ID NO. 35, 37, 47 or 23 for
functional inactivation, preferably of one, particularly preferably
of two parts in each case of one of these sequences, where these
parts comprise in each case at least 70 connected positions.
[0375] Further embodiments based on these fermentation processes
and uses are detailed hereinafter.
[0376] The metabolic pathway utilized in Gram-positive bacteria of
the genus Bacillus for synthesizing cadaverine and/or putrescine
(as parts of lysine and/or arginine catabolism) is depicted in FIG.
6 (for lysine and the cadaverine derived therefrom) and 7 (for
arginine and the putrescine derived therefrom). This aspect, which
is designated as a single pathway in the present application, of
the bacterial metabolism is ultimately derived as side pathway from
amino acid metabolism and in the second case additionally from the
urea cycle.
[0377] As already mentioned, the following enzymes are involved in
the reactions shown in FIGS. 6 and 7, where the relevant number
designates the respective reaction step indicated in the figures:
[0378] (1.) lysine decarboxylase (E.C. 4.1.1.18) and/or arginine
decarboxylase (E.C. 4.1.1.19) (single demonstrated reaction in FIG.
6; step 1 in FIG. 7; the case where the same enzyme is able to
catalyze both reactions also applies here), [0379] (2.) agmatinase
(E.C. 3.5.1.11); step 2 in FIG. 7) and [0380] (3.) ornithine
decarboxylase (E.C. 4.1.1.17; step 3 in FIG. 7).
[0381] Solutions of the stated problem and thus independent
embodiments of the present invention are thus represented by all
processes for fermenting a microorganism in which at least one of
the genes on a metabolic pathway for synthesizing cadaverine and/or
putrescine (as parts of lysine and/or arginine catabolism) is
functionally inactivated.
[0382] The previously explained advantages are associated with this
solution.
[0383] Preference is given in this connection to any process of
this type in which the microorganism now forms only 50% of the
amount naturally formed under the same conditions, preferably now
only 10%, particularly preferably no cadaverine and/or no
putrescine.
[0384] This takes account, as explained above for the first
metabolic pathway described, of the generally high flexibility of
microorganisms in relation to their metabolism.
[0385] A preferred process of the invention is one where at least
one of the following enzymes is functionally inactivated:
(1.) lysine decarboxylase (E.C. 4.1.1.18) and/or arginine
decarboxylase (E.C. 4.1.1.19), (2.) agmatinase (E.C. 3.5.1.11) and
(3.) ornithine decarboxylase (E.C. 4.1.1.17).
[0386] This is because, as depicted in FIGS. 6 and 7, these
activities can be associated with the metabolic pathway under
consideration here.
[0387] As stated above and described in the examples of the present
application, it was possible by sequencing the genomic DNA of B.
licheniformis DSM 13 to identify several of the genes coding for
enzymes located on this pathway, or for subunits thereof. These are
the following genes (the preceding number designates in each case
the reaction in which the relevant enzyme is involved): [0388] (1.)
lysine and/or arginine decarboxylase (E.C. 4.1.1.18 or E.C.
4.1.1.19) defined by SEQ ID NO. 5 (speA gene) or 39 (speA gene) and
[0389] (2.) agmatinase (E.C. 3.5.1.11) defined by SEQ ID NO. 49
(ywhG gene).
[0390] The amino acid sequences derived therefrom are indicated in
SEQ ID NO. 6, 40 and 50. It was thus possible in the course of the
present invention to identify these specific gene products as
involved in this metabolic pathway for synthesizing cadaverine
and/or putrescine (as parts of lysine and/or arginine
catabolism).
[0391] A preferred process of the invention is therefore one where
the functionally inactivated enzyme is the homolog, which is
naturally active in the relevant microorganism, to one of the
following proteins from licheniformis DSM 13: [0392] (1.) lysine
and/or arginine decarboxylase (E.C. 4.1.1.18 or E.C. 4.1.1.19)
defined by SEQ ID NO. 6 or 40 and [0393] (2.) agmatinase (E.C.
3.5.1.11) defined by SEQ ID NO. 50.
[0394] A preferred process of the invention is one where the enzyme
is functionally inactivated at the genetic level, preferably by
inactivation of a gene which corresponds to the nucleic acid which
codes for one of the following proteins of B. licheniformis DSM 13:
[0395] (1.) lysine and/or arginine decarboxylase (E.C. 4.1.1.18 or
E.C. 4.1.1.19) defined by SEQ ID NO. 5 (speA gene) or 39 (speA
gene) and [0396] (2.) agmatinase (E.C. 3.5.1.11) defined by SEQ ID
NO. 49 (ywhG gene).
[0397] This is because, in accordance with the stated problem, the
intention was preferably to find a causal solution, meaning one
applying at the level of molecular biology. Example 3 explains how
corresponding deletions can be undertaken; further statements
concerning this are given hereinafter.
[0398] Thus, preference is given to a process of the invention
where for the inactivation at the genetic level one of the nucleic
acids of the invention within the region designated above and
homologous to [0399] (1.) SEQ ID NO. 5 or 39 and [0400] (2.) 49 has
been used, preferably one, particularly preferably two parts in
each case of one of these sequences which in each case comprise at
least 70 connected positions.
[0401] A further embodiment of the present invention is represented
by the use of a gene which corresponds to the nucleic acid which
codes for one of the following proteins of B. licheniformis DSM 13
for functional inactivation of a metabolic pathway for synthesizing
cadaverine and/or putrescine (as parts of lysine and/or arginine
catabolism) at the genetic level in a microorganism: [0402] (1.)
lysine and/or arginine decarboxylase (E.C. 4.1.1.18 or E.C.
4.1.1.19) defined by SEQ ID NO. 5 (speA gene) or 39 (speA gene) and
[0403] (2.) agmatinase (E.C. 3.5.1.11) defined by SEQ ID NO. 49
(ywhG gene).
[0404] The same as has previously been stated concerning
corresponding processes applies in principle to such uses.
[0405] Accordingly, preference is given to a use according to the
invention of nucleic acids of the invention within the region
designated above and homologous to [0406] (1.) SEQ ID NO. 5 or 39
and [0407] (2.) 49 for functional inactivation, preferably of one,
particularly preferably of two parts in each case of one of these
sequences, where these parts comprise in each case at least 70
connected positions.
[0408] Further embodiments based on these fermentation processes
and uses are detailed hereinafter.
[0409] Embodiments which are preferred in each case of the uses
described above according to the invention of the genes and/or
nucleic acids on each of the described five metabolic pathways are
those where the functional inactivation takes place during the
fermentation of the microorganism.
[0410] This is because in accordance with the stated problem the
intention was to improve the fermentation at the genetic level. On
fermentation of microorganisms which have been correspondingly
modified via these genes and/or nucleic acids is to be expected
that the amount of the odorous and/or poisonous substances is less
than with unmodified strains. This advantage, which emerges during
the fermentation, is preferred according to the invention because
it has advantageous effects both on the production process, meaning
the fermentation process, and on the subsequent working up.
[0411] Among these, preference is given to any use of this type
where (if present) with increasing preference 2, 3 or 4 of the
genes mentioned for each metabolic pathway ((1.) for synthesizing
isovaleric acid, (2.) for synthesizing 2-methylbutyric acid and/or
isobutyric acid, (3.) for synthesizing butanol and/or butyric acid,
(4) for synthesizing propionic acid and/or (5.) for synthesizing
cadaverine and/or putrescine) are inactivated.
[0412] This is because, as already explained, microorganisms may in
individual cases escape inactivation by activating an alternative
pathway or at least enzymes with comparable reactions and thus
continuing to form the relevant odorous and/or poisonous substance.
This problem can be solved in particular by blocking a plurality of
single reactions.
[0413] Preference is further given to any use of this type where
(if present in the relevant microorganism) with increasing
preference 2, 3, 4 or 5 of the metabolic pathways (1.) for
synthesizing isovaleric acid, (2.) for synthesizing 2-methylbutyric
acid and/or isobutyric acid, (3.) for synthesizing butanol and/or
butyric acid, (4.) for synthesizing propionic acid and/or (5.) for
synthesizing cadaverine and/or putrescine are blocked at least in
part.
[0414] This is because firstly the inactivation of a single
reaction may block a plurality of said pathways. This applies for
example to butyryl-CoA dehydrogenase (E.C. 1.3.99.25) defined by
SEQ ID NO. 9, 11 or 27 (yusJ gene) which occurs on the first three
metabolic pathways mentioned; or to the three following enzymes or
groups of enzymes which are equally involved in the two pathways
mentioned first: 3-methyl-2-oxobutanoate dehydrogenase or
2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2) defined by SEQ ID
NO. 46, a subunit of acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined
by SEQ ID NO. 10, 12, 22 or 28, and enoyl-CoA hydratase (protein)
(E.C. 4.2.1.17) defined by SEQ ID NO. 16, 18, 20 or 42. In these
cases too, the enzymic activities are defined with reference to the
reactions described above and indicated in the figures.
[0415] Secondly, it is possible by generally known methods of
molecular biology to inactivate a plurality of genes in parallel,
so that in principle all these pathways can be switched off and
thus correspondingly favorable fermentation processes can be
obtained.
[0416] In one alternative, all these uses of genes and/or of the
described nucleic acids of the invention are ones where in each
case a nucleic acid coding for an inactive protein and having a
point mutation is employed.
[0417] Nucleic acids of this type can be generated by methods of
point mutagenesis known per se. Such methods are described for
example in relevant handbooks such as that of Fritsch, Sambrook and
Maniatis "Molecular cloning: a laboratory manual", Cold Spring
Harbour Laboratory Press, New York, 1989. In addition, numerous
commercial construction kits are now available therefor, for
instance the QuickChange.RTM. kit from Stratagene, La Jolla, USA.
The principle thereof is for oligonucleotides having single
exchanges (mismatch primers) to be synthesized and hybridized with
the gene in single-stranded form; subsequent DNA polymerization
then affords corresponding point mutants. It is possible to use for
this purpose the respective species-specific sequences of these
genes. Owing to the high homologies, it is possible and
particularly advantageous according to the invention to carry out
this reaction on the basis of the nucleotide sequences provided in
the sequence listing. These sequences can also serve to design
appropriate mismatch primers for related species.
[0418] In one alternative, all these uses of genes and/or of the
described nucleic acids of the invention are ones where in each
case a nucleic acid with a deletion mutation or insertion mutation
is employed, preferably comprising the border sequences, in each
case comprising at least 70 to 150 nucleic acid positions, of the
region coding for the protein.
[0419] These methods are also familiar per se to the skilled
worker. It is thus possible to prevent the formation of one or more
of the described gene products by the host cell by cutting out part
of the relevant gene on an appropriate transformation vector via
restriction endonucleases, and subsequently transforming the vector
into the host of interest, where the active gene is replaced by the
inactive copy via the homologous recombination which is still
possible until then. In the embodiment of insertion mutation it is
possible merely to introduce the intact gene interruptingly or,
instead of a gene portion, another gene, for example a selection
marker. Phenotypical checking of the mutation event is possible
thereby in a manner known per se.
[0420] In order to enable these recombination events which are
necessary in each case between the defective gene introduced into
the cell and the intact gene copy which is endogenously present for
example on the chromosome, it is necessary according to the current
state of knowledge that in each case there is agreement in at least
70 to 150 connected nucleic acid positions, in each case in the two
border sequences to the non-agreeing part, with the part lying
between being immaterial. Accordingly, preferred embodiments are
those including only two flanking regions with at least one of
these sizes.
[0421] In an alternative embodiment of this use, nucleic acids
having a total of two nucleic acid segments which in each case
comprise at least 70 to 150 nucleic acid positions, and thus flank
at least partly, preferably completely, the region coding for the
protein, are employed. The flanking regions can in this connection
be ascertained starting from the known sequences by methods known
per se, for example with the aid of outwardly directed PCR primers
and a preparation of genomic DNA as template (anchored PCR). This
is because it is not obligatory for the segments to be
protein-encoding in order to make it possible to exchange the two
gene copies by homologous recombination. According to the present
invention it is possible to design the primers required for this on
the basis of the nucleotide sequences indicated in the sequence
listing also for other species of Gram-positive bacteria and, among
these, in particular for those of the genus Bacillus. As an
alternative to this experimental approach it is possible to take
such regions which are at least in part non-coding for many of the
genes from related species, for example from B. subtilis database
entries, for example the SubtiList database of the Institute
Pasteur, Paris, France
(http://genolist.pasteur.fr/SubtiList/genome.cgi) or the databases
specified in Example 2.
[0422] The present invention is aimed in particular at providing
genetically improved microorganisms for biotechnological
production. Thus, every microorganism in which at least one of the
genes which corresponds to the nucleic acid which codes for one of
the following proteins of B. licheniformis DSM 13 is functionally
inactivated represents an embodiment of the present invention:
[0423] putative branched-chain amino acid aminotransferase (E.C.
2.6.1.42) defined by SEQ ID NO. 1,
[0424] putative branched-chain amino acid aminotransferase (E.C.
2.6.1.42) defined by SEQ ID NO. 3,
[0425] lysine and/or arginine decarboxylase (protein SpeA; E.C.
4.1.1.18 or E.C. 4.1.1.19) defined by SEQ ID NO. 5 (speA gene),
[0426] NADH-dependent butanol dehydrogenase A (protein YugJ; E.C.
1.1.1.-) defined by SEQ ID NO. 7 (yugJ gene),
[0427] butyryl-CoA dehydrogenase (E.C. 1.3.99.25) or acyl-CoA
dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 9,
[0428] butyryl-CoA dehydrogenase (E.C. 1.3.99.25) or acyl-CoA
dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 11,
[0429] 3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157) defined
by SEQ ID NO. 13,
[0430] putative enoyl-CoA hydratase protein (E.C. 4.2.1.17) defined
by SEQ ID NO. 15,
[0431] probable enoyl-(3-hydroxyisobutyryl)-CoA hydrolase protein
defined by SEQ ID NO. 17,
[0432] probable enoyl-CoA hydratase (protein EchA8; E.C. 4.2.1.17)
defined by SEQ ID NO. 19 (echA8 gene),
[0433] acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO.
21,
[0434] acetate-CoA ligase or propionate-CoA ligase (or synthetase;
protein AcsA; E.C. 6.2.1.1) defined by SEQ ID NO. 23 (acsA
gene),
[0435] 3-hydroxybutyryl-CoA dehydratase (protein YngF; E.C.
4.2.1.55) defined by SEQ ID NO. 25 (yngF gene),
[0436] butyryl-CoA dehydrogenase (protein YusJ; E.C. 1.3.99.25) or
acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 27
(yusJ gene),
[0437] 3-hydroxyisobutyrate dehydrogenase (protein YkwC; E.C.
1.1.1.31) or oxidoreductase (E.C. 1.1.-.-) defined by SEQ ID NO. 29
(ykwC gene),
[0438] probable phosphate butyryltransferase (E.C. 2.3.1.19)
defined by SEQ ID NO. 31,
[0439] probable butyrate kinase (E.C. 2.7.2.7) defined by SEQ ID
NO. 33,
[0440] acetate-CoA ligase or synthetase or propionate-CoA ligase or
synthetase (protein AcsA; E.C. 6.2.1.1) defined by SEQ ID NO. 35
(acsA gene),
[0441] acetate-CoA ligase or propionate-CoA ligase (protein Ytcl;
E.C. 6.2.1.1) defined by SEQ ID NO. 37 (ytcl gene),
[0442] lysine and/or arginine decarboxylase (protein speA; E.C.
4.1.1.18 or E.C. 4.1.1.19) defined by SEQ ID NO. 39 (speA
gene),
[0443] probable enoyl-CoA hydratase (E.C. 4.2.1.17) defined by SEQ
ID NO. 41 (ysiB gene),
[0444] similar to 3-hydroxyacyl-CoA dehydrogenase (E.C. 1.1.1.35)
defined by SEQ ID NO. 43,
[0445] 3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate
dehydrogenase E1 (E.C. 1.2.4.2) defined by SEQ ID NO. 45,
[0446] probable acetate-CoA ligase or propionate-CoA ligase
(protein YhfL; E.C. 6.2.1.1) or acid-CoA ligase (E.C. 6.2.1.-)
defined by SEQ ID NO. 47 (yhfL gene) or
[0447] agmatinase (E.C. 3.5.1.11) defined by SEQ ID NO. 49 (ywhG
gene).
[0448] "Corresponds" means in this connection in each case a gene
of the organism under consideration which codes for a gene product
having the same biochemical activity as defined above in connection
with the respective metabolic pathways. This is generally at the
same time the gene of all those of this organism which are
translated in vivo which shows the greatest homology in each case
to the stated gene from B. licheniformis (usually more than 40%
identity, as can be found by an alignment of the two sequences as
carried out in Example 2).
[0449] Among these, in accordance with the above statements, there
is increasing preference in each case for a microorganism in which
2, 3 or 4 of the genes mentioned for each metabolic pathway ((1.)
for synthesizing isovaleric acid, (2.) for synthesizing
2-methylbutyric acid and/or isobutyric acid, (3.) for synthesizing
butanol and/or butyric acid, (4.) for synthesizing propionic acid
and/or (5.) for synthesizing cadaverine and/or putrescine) are
inactivated.
[0450] In addition, in accordance with the above statements, there
is increasing preference in each case for a microorganism in which
2, 3, 4 or 5 of the metabolic pathways (1.) for synthesizing
isovaleric acid, (2.) for synthesizing 2-methylbutyric acid and/or
isobutyric acid, (3.) for synthesizing butanol and/or butyric acid,
(4.) for synthesizing propionic acid and/or (5.) for synthesizing
cadaverine and/or putrescine are blocked at least in part.
[0451] In addition, among these in each case a microorganism which
is a bacterium is preferred.
[0452] This is because they have particular importance for
biotechnological production. On the other hand, the relevant
pathways have been described for microorganisms of the genus
Bacillus.
[0453] A microorganism which is preferred among these is in each
case a Gram-negative bacterium, in particular one of the genera
Escherichia coli, Klebsiella, Pseudomonas or Xanthomonas, in
particular strains of E. coli K12, E. coli B or Klebsiella
planticola, and very especially derivatives of the strains
Escherichia coli BL21 (DE3), E. coli RV308, E. coli DH5.alpha., E.
coli JM109, E. coli XL-1 or Klebsiella planticola (Rf).
[0454] This is because these are important strains for molecular
biological operations on genes, for instance for cloning (see
examples), and additionally important producer strains.
[0455] As alternative thereto, in each case a microorganism which
is a Gram-positive bacterium is preferred, in particular one of the
genera Bacillus, Staphylococcus or Corynebacterium, very especially
of the species Bacillus lentus, B. licheniformis, B.
amyloliquefaciens, B. subtilis, B. globigii or B. alcalophilus,
Staphylococcus carnosus or Corynebacterium glutamicum, and among
these very particularly preferably B. licheniformis DSM 13.
[0456] This is because these are particularly important for the
biotechnological production of valuable products and proteins
because they are naturally able to secrete them into the
surrounding medium. On the other hand, they are increasingly
related to the B. licheniformis employed for the present
application, so that the working steps described and derived from
the sequences disclosed in each case should proceed more
successfully as the extent of relationship to B. licheniformis DSM
13 increases. It is thus to be assumed for example that a gene
indicated in the sequence listing can, after point mutation, be
used in a related species directly for a deletion mutation without
the need to isolate the homologous gene from the strain itself for
this purpose.
[0457] The present invention is aimed in particular at improving
fermentation processes. Thus, every process for fermenting a
microorganism of the invention described above represents an
embodiment of the present invention.
[0458] These processes and the processes described above in each
case in connection with an influence on one of the five metabolic
pathways described are in particular processes where a valuable
product is produced, in particular a low molecular weight compound
or a protein.
[0459] This is because these are the essential areas of use of
biotechnological production by fermentation of microorganisms.
[0460] Among these, preference is given in each case to a process
where the low molecular weight compound is a natural product, a
dietary supplement or a pharmaceutically relevant compound.
[0461] This is because they are important product groups for
biotechnological production by fermentation of microorganisms.
[0462] Among such biotechnological processes for producing proteins
by fermentation of microorganisms, preference is given in each case
to a process where the protein is an enzyme, in particular one from
the group of .alpha.-amylases, proteases, cellulases, lipases,
oxidoreductases, peroxidases, laccases, oxidases and
hemicellulases.
[0463] This is because these are important enzymes produced on the
industrial scale, for example for incorporation in detergent or
cleaning compositions.
[0464] In addition, the gene products provided according to the
invention are available for further applications. Thus, the present
invention is also implemented by any use of any gene product of the
invention in a reaction mixture or process appropriate for its
biochemical properties, which is defined as described above with
reference to SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,
26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50.
[0465] Among these are preferably included uses (1.) for
synthesizing isovaleric acid, (2.) for synthesizing 2-methylbutyric
acid and/or isobutyric acid, (3.) for synthesizing butanol and/or
butyric acid, (4.) for synthesizing propionic acid and/or (5.) for
synthesizing cadaverine and/or putrescine, where appropriate in
suitable combination with further enzymes.
[0466] Thus, the products of the metabolic pathways described are
simple organic chemical compounds for which there is certainly a
need in chemistry, for example to employ them as starting materials
for more complex syntheses. Preparation thereof can be considerably
simplified, especially when stereochemical reactions are involved,
by the use of appropriate enzymes, because they in most cases
specifically form one enantiomer. The term used when such synthetic
routes are undertaken in at least one reaction step by biological
catalysts is biotransformation. All gene products of the invention
are suitable in principle therefor.
[0467] The following examples illustrate the present invention
further.
EXAMPLES
[0468] All molecular biological working steps follow standard
methods as indicated for example in the handbook by Fritsch,
Sambrook and Maniatis "Molecular cloning: a laboratory manual",
Cold Spring Harbour Laboratory Press, New York, 1989, or comparable
relevant works. Enzymes and construction kits are employed in
accordance with the respective manufacturer's instructions.
Example 1
Identification of the Nucleic Acids Shown in SEQ ID NO. 1, 3, 5, 7,
9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,
43, 45, 47 and 49 from B. licheniformis DSM 13
[0469] The genomic DNA was prepared by standard methods from the
strain B. licheniformis DSM 13, which is available to anyone from
the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH,
Mascheroder Weg 1 b, 38124 Brunswick (http://www.dsmz.de),
mechanically fractionated and fractionated by electrophoresis in a
0.8% agarose gel. For a shotgun cloning of the smaller fragments,
the fragments 2 to 2.5 kb in size were eluted from the agarose gel,
dephosphorylated and ligated as blunt-ended fragments into the Smal
restriction cleavage site of the vector pTZ19R-Cm. This is a
derivative which confers chloramphenicol resistance of the plasmid
pTZ19R which is obtainable from Fermentas (St. Leon-Rot). A gene
library of the smaller fragments was obtained thereby. As second
shotgun cloning, the genomic fragments obtained by a partial
restriction with the enzyme Saulllal were ligated into the SuperCos
1 vector system ("Cosmid Vector Kit") from Stratagene, La Jolla,
USA, resulting in a gene library over the predominantly larger
fragments.
[0470] The relevant recombinant plasmids were isolated and
sequenced from the bacteria E. coli DH5.alpha. (D. Hannahan (1983):
"Studies on transformation on Escherichia coli"; J. Mol.
Microbiol., volume 166, pages 557-580) obtainable by transformation
with the relevant gene libraries. The dye termination method (dye
terminator chemistry) was employed in this case, carried out by the
automatic sequencers MegaBACE 1000/4000 (Amersham Bioscience,
Piscataway, USA) and ABI Prism 377 (Applied Biosystems, Foster
City, USA).
[0471] In this way, inter alia, the sequences SEQ ID NO. 1, 3, 5,
7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,
41, 43, 45, 47 and 49 indicated in the sequence listing of the
present application were obtained. The amino acid sequences derived
therefrom are indicated--the relevant ones under the higher number
in each case--under SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,
22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50.
Example 2
Sequence Homologies
[0472] After ascertaining the DNA and amino acid sequences as in
Example 1, in each case the most similar homologs disclosed to date
were ascertained by searching the databases GenBank (National
Center for Biotechnology Information NCBI, National Institute of
Health, Bethesda, Md., USA), EMBL European Bioinformatics Institute
(EBI) in Cambridge, Great Britain (http://www.ebi.ac.uk),
Swiss-Prot (Geneva Bioinformatics (GeneBio) S.A., Geneva,
Switzerland; http://www.genebio.com/sprot.html) and PIR (Protein
Information Resource, National Biomedical Research Foundation,
Georgetown University Medical Center, Washington, D.C., USA;
http://www.pir.georgetown.edu). The nr (nonredundant) option was
chosen in this connection.
[0473] The ascertained DNA and amino acid sequences were compared
with one another via alignments in order to determine the degree of
homology; the computer program used for this was Vector NTI.RTM.
Suite Version 7, which is obtainable from Informax Inc., Bethesda,
USA. In this case, the standard parameters of this program were
used, meaning for comparison of the DNA sequences: K-tuple size: 2;
Number of best Diagonals: 4; Window size: 4; Gap penalty: 5; Gap
opening penalty: 15 and Gap extension penalty: 6.66. The following
standard parameters applied to the comparison of the amino acid
sequences: K-tuple size: 1; Number of best Diagonals: 5; Window
size: 5; Gap penalty: 3; Gap opening penalty: 10 and Gap extension
penalty: 0.1. The results of these sequence comparisons are
compiled in Table 1 below, together with an indication of the
respective enzyme names, meaning functions, E.C. numbers and the
relevant metabolic pathways. The numbering of enzymes known in the
prior art is the consistent nomenclature of the abovementioned
databases.
TABLE-US-00001 TABLE 1 Genes and proteins of most similarity to the
genes and proteins respectively ascertained in Example 1. The
meanings therein are: ID the SEQ ID NO. indicated in the sequence
listing in the present application; E.C. No. the number according
to the international enzyme classification (Enzyme Nomenclature of
the IUBMB). Name of the enzyme (where possible of Identity to the
Identity to the the gene) and additional most closely most closely
information where Metabolic related at the related at the ID
appropriate E.C. No. pathway DNA level % protein level % 1, 2
putative branched- 2.6.1.42 valine/isoleucine 62.40% to
gb|AE017003.1|, 69% to chain amino acid catabolism B. cereus ATCC
aminotransferase aminotransferase 14579, section 6 IV from B. of 18
of the anthracis Ames complete genome (NP_655296.1) 3, 4 putative
branched- 2.6.1.42 valine/isoleucine 73.80% to 79% to chain amino
acid catabolism emb|Z49992.1|BS branched-chain aminotransferase
CELABCD, amino acid B. subtilis genes aminotransferase celA, celB,
celC, from B. subtilis celD and ywaA 168 (NP_391734.1) 5, 6 lysine
and/or 4.1.1.18 cadaverine 74.00% to 85% to lysine arginine or
and/or emb|X58433.1|BS decarboxylase decarboxylase 4.1.1.19
putrescine CADDNA from B. subtilis (speA) synthesis (lysine B.
subtilis, cad (NP_389346; and/or arginine gene for lysine A54546)
catabolism) decarboxylase 7, 8 NADH-dependent 1.1.1.- butyric acid
76.30% to 89% to NADH- butanol metabolism emb|Z93934.1|BS dependent
dehydrogenase A Z93934, butanol (yugJ) B. subtilis, dehydrogenase
genomic DNA from B. subtilis fragment from 168 patB to yugK
(NP_391015.1) 9, 10 acyl-CoA 1.3.99.-/ leucine 74.70% to 82% to
short- dehydrogenase 1.3.99.25 catabolism emb|Z49782.1|BS chain
specific (sic, i.e. more valine/ DNA320D, acyl-CoA generally
indicated isoleucine B. subtilis, dehydrogenase in the sequence
catabolism, chromosomal from B. cereus listing)/butyryl- butyric
acid DNA (region 320- ATCC 14579 CoA dehydrogenase metabolism 321
degrees) (NP_835003.1) 11, 12 acyl-CoA 1.3.99.- leucine 59.30% to
63% to C- dehydrogenase catabolism emb|Z49782.1|BS terminal domain
(sic, i.e. more DNA320D, of acyl-CoA generally indicated B.
subtilis, dehydrogenase in the sequence chromosomal from B.
anthracis listing)/butyryl- DNA (region 320- Ames CoA
dehydrogenase. 321 degrees) (NP_653803.1) The first codon ought to
be translated in vivo as methionine 13, 14 3-hydroxyburyryl-
1.1.1.157 butyric cid 62.40% to 65% to the NAD- CoA dehydrogenase
metabolism gb|AE017015.1|, binding domain B. cereus ATCC of
3-hydroxyacyl- 14579, section 18 CoA of 18 of the dehydrogenase,
complete genome from B. anthracis Ames (NP_653804.1) 15, 16
putative enoyl- 4.2.1.17 leucine 61.00% to 58% to YhaR CoA
hydratase catabolism, emb|Y14078.1|BS from B. subtilis protein
valine/ Y14078, 168 isoleucine B. subtilis, 8.7 Kb (CAB12828.2)
catabolism chromosomal DNA: downstream of the glyB-prsA region 17,
18 probable enoyl- not yet allo- leucine 61.90% to 62% to 3-
(3-hydroxy- cated catabolism, gb|AE017031.1|, hydroxy- isobutyryl)-
valine/ B. anthracis isobutyryl- coenzyme A isoleucine Ames,
section 8 coenzyme A hydrolase protein catabolism of 18 of the
hydrolase from complete genome B. cereus ATCC 14579 (NP_832055.1;
AAP09256) 19, 20 probable enoyl-CoA 4.2.1.17 leucine 43.50% to 48%
to 3-hydroxy- hydratase (echA8). catabolism, gb|AC084761.2|,
butyryl-CoA The first codon valine/ Callus gallus, dehydratase from
ought to be isoleucine clone WAG-69H2, B subtilis 168 translated in
vivo catabolism complete (NP_390732.1) as methionine sequence 21,
22 actyl-CoA 1.3.99.- leucine 49.90% to 61% to acyl-CoA
dehydrogenase catabolism, gb|AE015940.1|, dehydrogenase valine/
Clostridium tetani from B. cereus isoleucine E88, section 5 of ATCC
14579 catabolism 10 of the (NP_832051.1) complete genome 23, 24
acetyl-coenzyme 6.2.1.1 propionate 63.00% to 61% to acetyl- A
synthetase metabolism dbj|AP001511.1|, CoA synthetase (indicated
thus in B. halodurans, from B. the sequence genomic DNA, halodurans
listing) or section 5/14 (NP_242003.1) propionate-CoA ligase (acsA)
25, 26 3-hydroxybutyryl- 4.2.1.55 butyric acid 63.80% to 65% to CoA
dehydratase metabolism emb|Y13917.1|BS hydroxybutyryl (yngF)
Y13917, dehydratase B. subtilis, genes from ppsE, yngL, B. subtilis
yngK, yotB, yngJ, (AAF32340.1) yngI, yngH, yngG and yngF and
partial genes ppsD and yngE 27, 28 acyl-CoA 1.3.99./ leucine 72.90%
to 82% of butyryl- dehydrogenase 1.3.99.25 catabolism,
emb|Y13917.1|BS CoA (sic, i.e. more valine/ Y13917, dehydrogenase
generally, isoleucine B. subtilis, genes from B. subtilis indicated
in the catabolism, ppsE, yngL, 168 sequence listing)/ butyric acid
yngK, yotB, yngJ, (NP_389708.1) butyryl-CoA metabolism yngI, yngH,
yngG dehydrogenase and yngF and (yusJ) partial genes ppsD and yngE
29, 30 3-hydroxy- 1.1.1.31 or 1.1.-.- valine 72.80% to 81% to 3-
isobutyrate catabolism emb|AJ222587.1| hydroxy dehydrogenase/
BS16829KB, isobutyrate hypothetical B. subtilis, 29 kB
dehydrogenase oxidoreductase DNA fragment from B. subtilis (sic,
i.e. more from the gene 168 generally, ykwC to the gene
(NP_389279.1) indicated in the cse15 sequence listing) (ykwC) 31,
32 probable phosphate 2.3.1.19 butyric acid 46.30% to 65% to
butyryl-transferase metabolism gb|S81735.1|S81 phosphate 735,
leucine butyryl- dehydrogenase transferase from B. subtilis 168
(NP_390289.1) 33, 34 probable butyrate 2.7.2.7 butyric acid 72.50%
to 80% to kinase metabolism emb|Z99116.2|BS branched-chain UB0013,
fatty acid kinase B. subtilis, from B. subtilis complete genome 168
(section 13 of 21): (NP_390287.1) from 2409151 to 2613687 35, 36
acetyl-coenzyme A 6.2.1.1 propionate 74.90% to 81% to acetyl-
synthetase metabolism emb|Z99119.2|BS CoA synthetase (indicated
thus in UB0016, from B. subtilis the sequence B. subtilis, 168
listing) or complete genome (NP_390846.1) propionate-CoA (section
16 of 21): and to acetate- ligase (acsA) from 3013458 to CoA ligase
from 3213379 B. subtilis (P39062, S39646) 37, 38 acetate-CoA ligase
6.2.1.1 propionate 70% to 73% to acetate- (indicated thus in
metabolism emb|Z99119.2|BS CoA ligase from the sequence UB0016, B.
subtilis 168 listing) or B. subtilis, (NP_390834.1, propionate-CoA
complete genome E69989) ligase (ytcI). (section 16 of 21): The
first codon from 3013458 to ought to be 3213379 translated in vivo
as methionine 39, 40 lysine and/or 4.1.1.18 cadaverine 63.40% to
62% to lysine arginine or and/or emb|Z99104.2|BS decarboxylase
decarboxylase 4.1.1.19 putrescine UB0001, from B. subtilis (speA)
synthesis B. subtilis, 168 (lysine and/or complete genome
(NP_387908.1) arginine (section 1 of 21): and B. perfrigens
catabolism) from 1 to 213080 (NP_976355) 41, 42 probable enoyl-CoA
4.2.1.17 leucine 70.30% to 73% to 3- hydratase (ysiB) catabolism,
emb|Z75208.1|BS hydroxybutyryl- valine/ Z75208, CoA dehydratase
isoleucine B. subtilis, genomic from B. subtilis catabolism
sequence, 168 89009bp (NP_390732.1) 43, 44 similar to 1.1.1.35
isoleucine 71.60% to 76% to 3- 3-hydroxyacyl-CoA catabolism
emb|Z99120.2|BS hydroxyacyl-CoA dehydrogenase UB0017, dehydrogenase
B. subtilis, from B. subtilis complete genome 168 (section 17 of
21): (NP_391163.1) from 3213330 to 3414388 45, 46 3-methyl-2-
1.2.4.2 leucine 75.70% to 78% to E1 oxobutanoate catabolism,
emb|X54805.1|BS subunit of dehydrogenase/ valine/ ODHA, B.
subtilis, 2-oxoglutarate 2-oxoglutarate isoleucine odhA gene for
dehydrogenase dehydrogenase E1 catabolism 2-oxoglutarate from B.
subtilis component (sic, dehydrogenase (CAB13829.2) i.e. indicated
more generally in the sequence listing) 47, 48 probable acid-CoA
6.2.1.- propionate 62.20% to 72% to long- ligase (yhfL) metabolism
gb|AE017001.1|, chain fatty acid- B. cereus ATCC CoA ligase from
14579, section 4 B. subtilis 168 of 18 of the (NP_388908.1)
complete genome 49, 50 agmatinase (ywhG) 3.5.1.11 cadaverine 80.9%
to 95% to and/or B. subtilis, gene agmatinase putrescine BSUB0020
(agmatine synthesis (lysine (Genebank, ureohydrolase) and/or
arginine complete from B. subtilis catabolism) genome) 168
(P70999)
[0474] It is evident that the genes found and the gene products
derived therefrom are respectively novel genes and proteins with a
clear distance from the prior art disclosed to date.
Example 3
Functional Inactivation of One or More of the Genes Shown in SEQ ID
NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,
35, 37, 39, 41, 43, 45, 47 and 49 in B. Licheniformis
Principle of the Preparation of a Deletion Vector
[0475] Each of these genes can be functionally inactivated for
example by means of a so-called deletion vector. This procedure is
described per se for example by J. Vehmaanpera et al. (1991) in the
publication "Genetic manipulation of Bacillus amyloliquefaciens";
J. Biotechnol., volume 19, pages 221-240.
[0476] A suitable vector for this is pE194 which is characterized
in the publication "Replication and incompatibility properties of
plasmid pE194 in Bacillus subtilis" by T. J. Gryczan et al. (1982),
J. Bacteriol., volume 152, pages 722-735. The advantage of this
deletion vector is that it possesses a temperature-dependent origin
of replication. pE194 is able to replicate in the transformed cell
at 33.degree. C., so that initial selection for successful
transformation takes place at this temperature. Subsequently, the
cells comprising the vector are incubated at 42.degree. C. The
deletion vector no longer replicates at this temperature, and a
selection pressure is exerted on the integration of the plasmid via
a previously selected homologous region into the chromosome. A
second homologous recombination via a second homologous region then
leads to excision of the vector together with the intact gene copy
from the chromosome and thus to deletion of the gene which is
located in the chromosome in vivo. Another possibility as second
recombination would be the reverse reaction to integration, meaning
recombination of the vector out of the chromosome, so that the
chromosomal gene would remain intact. The gene deletion must
therefore be detected by methods known per se, for instance in a
southern blot after restriction of the chromosomal DNA with
suitable enzymes or with the aid of the PCR technique on the basis
of the size of the amplified region.
[0477] It is thus necessary to select two homologous regions of the
gene to be deleted, each of which should include at least 70 base
pairs in each case, for example the 5' region and the 3' region of
the selected gene. These are cloned into the vector in such a way
that they flank a part coding for an inactive protein, or are in
direct succession, omitting the region in between. The deletion
vector is obtained thereby.
Deletion of the Genes Considered Here
[0478] A deletion vector of the invention is constructed by PCR
amplification of the 5' and 3' regions of one of these genes of
interest in each case. The sequences SEQ ID NO. 1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,
47 and 49 indicated in the sequence listing are available for
designing suitable primers and originate from B. licheniformis, but
ought also to be suitable, because of the homologies to be
expected, for other species, especially of the genus Bacillus.
[0479] The two amplified regions suitably undergo intermediate
cloning in direct succession on a vector useful for these
operations, for example on the vector pUC18 which is suitable for
cloning steps in E. coli.
[0480] The next step is a subcloning into the vector pE194 selected
for deletion, and transformation thereof into B. subtilis DB104,
for instance by the method of protoplast transformation according
to Chang & Cohen (1979; "High Frequency Transformation of
Bacillus subtilis Protoplasts by Plasmid DNA"; Molec. Gen. Genet.
(1979), volume 168, pages 111-115). All working steps must be
carried out at 33.degree. C. in order to ensure replication of the
vector.
[0481] In a next step, the vector which has undergone intermediate
cloning is likewise transformed by the method of protoplast
transformation into the desired host strain, in this case B.
licheniformis. The transformants obtained in this way and
identified as positive by conventional methods (selection via the
resistance marker of the plasmid; check by plasmid preparation and
PCR for the insert) are subsequently cultured at 42.degree. C.
under selection pressure for presence of the plasmid through
addition of erythromycin. The deletion vector is unable to
replicate at this temperature, and the only cells to survive are
those in which the vector is integrated into the chromosome, and
this integration most probably takes place in homologous or
identical regions. Excision of the deletion vector can then be
induced subsequently by culturing at 33.degree. C. without
erythromycin selection pressure, the chromosomally encoded gene
being completely deleted from the chromosome. The success of the
deletion is subsequently checked by southern blotting after
restriction of the chromosomal DNA with suitable
[0482] Such transformants in which the relevant gene is deleted are
normally additionally distinguished by a limitation on the
formation of the odorous or poisonous substance resulting from the
relevant metabolic pathway. In the cases where the cell has no
substitute pathway for synthesizing the relevant compound, the
relevant metabolic pathway is completely blocked so that this
compound is no longer formed at all, and the strain modified in
this way no longer has the relevant odorous component.
Sequence CWU 1
1
501918DNABacillus licheniformis DSM 13gene(1)..(918)Putative
branched-chain amino acid aminotransferase (E.C. 2.6.1.42) 1atg ggg
gac cag aaa gac cag tgg atc ttc cta aac gac aaa ctc gtt 48Met Gly
Asp Gln Lys Asp Gln Trp Ile Phe Leu Asn Asp Lys Leu Val 1 5 10 15
aaa aaa gaa gac gct aaa ata tca gtc tat gat cac gga ttt tta tac
96Lys Lys Glu Asp Ala Lys Ile Ser Val Tyr Asp His Gly Phe Leu Tyr
20 25 30 ggg gac ggc gtg ttt gaa ggg atc agg gta tac gac ggc aac
atc ttc 144Gly Asp Gly Val Phe Glu Gly Ile Arg Val Tyr Asp Gly Asn
Ile Phe 35 40 45 aga atg caa gag cac atg gac cgc ctc tac gat tct
gcg aga tcg atc 192Arg Met Gln Glu His Met Asp Arg Leu Tyr Asp Ser
Ala Arg Ser Ile 50 55 60 atg ctg gag att cca tat cca cag gaa gaa
ctg aca cag cac gta ctc 240Met Leu Glu Ile Pro Tyr Pro Gln Glu Glu
Leu Thr Gln His Val Leu 65 70 75 80 aaa aca gtc gaa aaa aac ggg ctg
aaa gac gct tac atc cgc ttg gtc 288Lys Thr Val Glu Lys Asn Gly Leu
Lys Asp Ala Tyr Ile Arg Leu Val 85 90 95 gtt tca aga gga gca ggt
gac ctc gga ctc gat cca aac aat tgt tca 336Val Ser Arg Gly Ala Gly
Asp Leu Gly Leu Asp Pro Asn Asn Cys Ser 100 105 110 aac ccg agt gtc
atc ata att gtc gaa cca ttg gca ata ttc ccg aaa 384Asn Pro Ser Val
Ile Ile Ile Val Glu Pro Leu Ala Ile Phe Pro Lys 115 120 125 cat tta
tat gaa acg ggg att gac att gtt acg gtt ccg aca aga agg 432His Leu
Tyr Glu Thr Gly Ile Asp Ile Val Thr Val Pro Thr Arg Arg 130 135 140
aac aga ccc gat gtg ctg agc cct aaa gta aaa tcg ctg aac tac tta
480Asn Arg Pro Asp Val Leu Ser Pro Lys Val Lys Ser Leu Asn Tyr Leu
145 150 155 160 aac aat att ctt gtc cgg atc gag gcg cat atg gcg ggt
gtg acg gaa 528Asn Asn Ile Leu Val Arg Ile Glu Ala His Met Ala Gly
Val Thr Glu 165 170 175 gcg ctc atg ctc aat gat caa ggc tat gtc gcc
gaa ggg tct gcg gat 576Ala Leu Met Leu Asn Asp Gln Gly Tyr Val Ala
Glu Gly Ser Ala Asp 180 185 190 aac gta ttt att tat aaa aac gga aag
ctc ttg acg cct ccg ggc tat 624Asn Val Phe Ile Tyr Lys Asn Gly Lys
Leu Leu Thr Pro Pro Gly Tyr 195 200 205 atc gga gcg ctt gaa gga atc
acc cgg aat gcc atc atc gaa ata gcg 672Ile Gly Ala Leu Glu Gly Ile
Thr Arg Asn Ala Ile Ile Glu Ile Ala 210 215 220 cga gag ctc ggc tat
gaa gtg aaa gaa gag ccg ttt acc cgc cat gac 720Arg Glu Leu Gly Tyr
Glu Val Lys Glu Glu Pro Phe Thr Arg His Asp 225 230 235 240 gta tac
aca gcc gag gaa gtg ttt tta acc gga acg gct gca gaa gtc 768Val Tyr
Thr Ala Glu Glu Val Phe Leu Thr Gly Thr Ala Ala Glu Val 245 250 255
atc gcg gtc gta aaa gtt gac ggc cgc aag atc ggg gac ggc aaa ccg
816Ile Ala Val Val Lys Val Asp Gly Arg Lys Ile Gly Asp Gly Lys Pro
260 265 270 gga gtc cac aca aac cgg atg ctt gaa aag ttc cgc gag cgc
gtc gtc 864Gly Val His Thr Asn Arg Met Leu Glu Lys Phe Arg Glu Arg
Val Val 275 280 285 cgt gaa ggg tta aaa gtc agc ctc aaa gat caa agc
tta agt gtc agc 912Arg Glu Gly Leu Lys Val Ser Leu Lys Asp Gln Ser
Leu Ser Val Ser 290 295 300 tga ata 9182304PRTBacillus
licheniformis DSM 13 2Met Gly Asp Gln Lys Asp Gln Trp Ile Phe Leu
Asn Asp Lys Leu Val 1 5 10 15 Lys Lys Glu Asp Ala Lys Ile Ser Val
Tyr Asp His Gly Phe Leu Tyr 20 25 30 Gly Asp Gly Val Phe Glu Gly
Ile Arg Val Tyr Asp Gly Asn Ile Phe 35 40 45 Arg Met Gln Glu His
Met Asp Arg Leu Tyr Asp Ser Ala Arg Ser Ile 50 55 60 Met Leu Glu
Ile Pro Tyr Pro Gln Glu Glu Leu Thr Gln His Val Leu 65 70 75 80 Lys
Thr Val Glu Lys Asn Gly Leu Lys Asp Ala Tyr Ile Arg Leu Val 85 90
95 Val Ser Arg Gly Ala Gly Asp Leu Gly Leu Asp Pro Asn Asn Cys Ser
100 105 110 Asn Pro Ser Val Ile Ile Ile Val Glu Pro Leu Ala Ile Phe
Pro Lys 115 120 125 His Leu Tyr Glu Thr Gly Ile Asp Ile Val Thr Val
Pro Thr Arg Arg 130 135 140 Asn Arg Pro Asp Val Leu Ser Pro Lys Val
Lys Ser Leu Asn Tyr Leu 145 150 155 160 Asn Asn Ile Leu Val Arg Ile
Glu Ala His Met Ala Gly Val Thr Glu 165 170 175 Ala Leu Met Leu Asn
Asp Gln Gly Tyr Val Ala Glu Gly Ser Ala Asp 180 185 190 Asn Val Phe
Ile Tyr Lys Asn Gly Lys Leu Leu Thr Pro Pro Gly Tyr 195 200 205 Ile
Gly Ala Leu Glu Gly Ile Thr Arg Asn Ala Ile Ile Glu Ile Ala 210 215
220 Arg Glu Leu Gly Tyr Glu Val Lys Glu Glu Pro Phe Thr Arg His Asp
225 230 235 240 Val Tyr Thr Ala Glu Glu Val Phe Leu Thr Gly Thr Ala
Ala Glu Val 245 250 255 Ile Ala Val Val Lys Val Asp Gly Arg Lys Ile
Gly Asp Gly Lys Pro 260 265 270 Gly Val His Thr Asn Arg Met Leu Glu
Lys Phe Arg Glu Arg Val Val 275 280 285 Arg Glu Gly Leu Lys Val Ser
Leu Lys Asp Gln Ser Leu Ser Val Ser 290 295 300 31095DNABacillus
licheniformis DSM 13gene(1)..(1095)Putative branched-chain amino
acid aminotransferase (E.C. 2.6.1.42) 3atg aca aaa caa acg atc agc
gta cag ctc agt aca gca aaa aag caa 48Met Thr Lys Gln Thr Ile Ser
Val Gln Leu Ser Thr Ala Lys Lys Gln 1 5 10 15 aag cca gag gct gac
aag ctc gaa ttc ggc cgg acc ttt acc gac cac 96Lys Pro Glu Ala Asp
Lys Leu Glu Phe Gly Arg Thr Phe Thr Asp His 20 25 30 atg ttt atc
atg gac tat acg gct gaa aac ggc tgg cac gat ccg aga 144Met Phe Ile
Met Asp Tyr Thr Ala Glu Asn Gly Trp His Asp Pro Arg 35 40 45 atc
gtt cct tac cag ccg att gaa atg gac ccg gct gca atg gtt tac 192Ile
Val Pro Tyr Gln Pro Ile Glu Met Asp Pro Ala Ala Met Val Tyr 50 55
60 cac tac gga caa tcc gtt ttt gaa gga tta aaa gct tat tta tca agc
240His Tyr Gly Gln Ser Val Phe Glu Gly Leu Lys Ala Tyr Leu Ser Ser
65 70 75 80 gaa ggc aga gtt ctt ctg ttc aga cct gaa aaa aac ttc gag
aga ctc 288Glu Gly Arg Val Leu Leu Phe Arg Pro Glu Lys Asn Phe Glu
Arg Leu 85 90 95 aat aaa tcc aac gac cgc ctc tgc att ccc cgg gtt
gat cct gaa atc 336Asn Lys Ser Asn Asp Arg Leu Cys Ile Pro Arg Val
Asp Pro Glu Ile 100 105 110 gtt ctg gaa ggg ctg aag cag ctg gtt cag
atc gac aag gaa tgg att 384Val Leu Glu Gly Leu Lys Gln Leu Val Gln
Ile Asp Lys Glu Trp Ile 115 120 125 cct caa gct gag ggg aca tcc ctt
tat atc cgt ccg ttc att att tca 432Pro Gln Ala Glu Gly Thr Ser Leu
Tyr Ile Arg Pro Phe Ile Ile Ser 130 135 140 aca gaa ccg tac ctt ggc
gtc gcc cca tcc aat atg tat aaa atg ctg 480Thr Glu Pro Tyr Leu Gly
Val Ala Pro Ser Asn Met Tyr Lys Met Leu 145 150 155 160 atc att tta
tcg ccg gtc gga tct tat tac aaa gaa ggc atc cgc cct 528Ile Ile Leu
Ser Pro Val Gly Ser Tyr Tyr Lys Glu Gly Ile Arg Pro 165 170 175 gtg
aaa att gct gtt gaa agc gaa ttt gtc cgt gct gtg gca ggc ggt 576Val
Lys Ile Ala Val Glu Ser Glu Phe Val Arg Ala Val Ala Gly Gly 180 185
190 aca ggc aat gca aaa acg gcc gga aac tac gct gcg agc ctg aag gct
624Thr Gly Asn Ala Lys Thr Ala Gly Asn Tyr Ala Ala Ser Leu Lys Ala
195 200 205 cag gaa gtt gcg gaa agc aaa ggc ttc tca caa gtg ctg tgg
ctt gac 672Gln Glu Val Ala Glu Ser Lys Gly Phe Ser Gln Val Leu Trp
Leu Asp 210 215 220 gga gtt gaa aag aaa tac att gaa gaa gta ggc agc
atg aac atc ttc 720Gly Val Glu Lys Lys Tyr Ile Glu Glu Val Gly Ser
Met Asn Ile Phe 225 230 235 240 ttc aaa atc agc ggt gaa att gtc act
ccg gct cta aac gga agc att 768Phe Lys Ile Ser Gly Glu Ile Val Thr
Pro Ala Leu Asn Gly Ser Ile 245 250 255 ttg gaa ggc att acg aga aac
tcc gtc atc cat ctc tta aaa caa tgg 816Leu Glu Gly Ile Thr Arg Asn
Ser Val Ile His Leu Leu Lys Gln Trp 260 265 270 gga ctc tcc gta acc
gaa aga aaa att tca gtt gat gaa ctg gtt cag 864Gly Leu Ser Val Thr
Glu Arg Lys Ile Ser Val Asp Glu Leu Val Gln 275 280 285 gct cac aaa
gac ggc ctg ctt gag gaa gca ttc ggc acc gga acc gcc 912Ala His Lys
Asp Gly Leu Leu Glu Glu Ala Phe Gly Thr Gly Thr Ala 290 295 300 gct
gtc att tcc ccc gtc ggc gag ctg atc tgg aaa gac gaa agc ctt 960Ala
Val Ile Ser Pro Val Gly Glu Leu Ile Trp Lys Asp Glu Ser Leu 305 310
315 320 gtg atc aac aac ggt caa act gga gaa atc gcc aaa agg ctc tac
caa 1008Val Ile Asn Asn Gly Gln Thr Gly Glu Ile Ala Lys Arg Leu Tyr
Gln 325 330 335 acg atc acc ggt att caa aaa ggc gct ttg cct gac aca
ttc ggc tgg 1056Thr Ile Thr Gly Ile Gln Lys Gly Ala Leu Pro Asp Thr
Phe Gly Trp 340 345 350 aca gtt gaa gtt gat aaa gta agc cag tcc tgc
taa gcg 1095Thr Val Glu Val Asp Lys Val Ser Gln Ser Cys 355 360
4363PRTBacillus licheniformis DSM 13 4Met Thr Lys Gln Thr Ile Ser
Val Gln Leu Ser Thr Ala Lys Lys Gln 1 5 10 15 Lys Pro Glu Ala Asp
Lys Leu Glu Phe Gly Arg Thr Phe Thr Asp His 20 25 30 Met Phe Ile
Met Asp Tyr Thr Ala Glu Asn Gly Trp His Asp Pro Arg 35 40 45 Ile
Val Pro Tyr Gln Pro Ile Glu Met Asp Pro Ala Ala Met Val Tyr 50 55
60 His Tyr Gly Gln Ser Val Phe Glu Gly Leu Lys Ala Tyr Leu Ser Ser
65 70 75 80 Glu Gly Arg Val Leu Leu Phe Arg Pro Glu Lys Asn Phe Glu
Arg Leu 85 90 95 Asn Lys Ser Asn Asp Arg Leu Cys Ile Pro Arg Val
Asp Pro Glu Ile 100 105 110 Val Leu Glu Gly Leu Lys Gln Leu Val Gln
Ile Asp Lys Glu Trp Ile 115 120 125 Pro Gln Ala Glu Gly Thr Ser Leu
Tyr Ile Arg Pro Phe Ile Ile Ser 130 135 140 Thr Glu Pro Tyr Leu Gly
Val Ala Pro Ser Asn Met Tyr Lys Met Leu 145 150 155 160 Ile Ile Leu
Ser Pro Val Gly Ser Tyr Tyr Lys Glu Gly Ile Arg Pro 165 170 175 Val
Lys Ile Ala Val Glu Ser Glu Phe Val Arg Ala Val Ala Gly Gly 180 185
190 Thr Gly Asn Ala Lys Thr Ala Gly Asn Tyr Ala Ala Ser Leu Lys Ala
195 200 205 Gln Glu Val Ala Glu Ser Lys Gly Phe Ser Gln Val Leu Trp
Leu Asp 210 215 220 Gly Val Glu Lys Lys Tyr Ile Glu Glu Val Gly Ser
Met Asn Ile Phe 225 230 235 240 Phe Lys Ile Ser Gly Glu Ile Val Thr
Pro Ala Leu Asn Gly Ser Ile 245 250 255 Leu Glu Gly Ile Thr Arg Asn
Ser Val Ile His Leu Leu Lys Gln Trp 260 265 270 Gly Leu Ser Val Thr
Glu Arg Lys Ile Ser Val Asp Glu Leu Val Gln 275 280 285 Ala His Lys
Asp Gly Leu Leu Glu Glu Ala Phe Gly Thr Gly Thr Ala 290 295 300 Ala
Val Ile Ser Pro Val Gly Glu Leu Ile Trp Lys Asp Glu Ser Leu 305 310
315 320 Val Ile Asn Asn Gly Gln Thr Gly Glu Ile Ala Lys Arg Leu Tyr
Gln 325 330 335 Thr Ile Thr Gly Ile Gln Lys Gly Ala Leu Pro Asp Thr
Phe Gly Trp 340 345 350 Thr Val Glu Val Asp Lys Val Ser Gln Ser Cys
355 360 51494DNABacillus licheniformis DSM 13gene(1)..(1494)Lysine
and/or Arginine decarboxylase (E.C. 4.1.1.18 or E.C. 4.1.1.19,
respectively), speA 5atg aag gtg gga aaa caa ttg ttg cag cac gaa
aca ccc cta tat acc 48Met Lys Val Gly Lys Gln Leu Leu Gln His Glu
Thr Pro Leu Tyr Thr 1 5 10 15 gga tta aaa aaa cac gcc ggc aaa aac
ccg atc cag ttc cat ata ccg 96Gly Leu Lys Lys His Ala Gly Lys Asn
Pro Ile Gln Phe His Ile Pro 20 25 30 ggg cac aaa aaa ggc tct ggg
atg gac cct gaa ttc agg gag ttt atc 144Gly His Lys Lys Gly Ser Gly
Met Asp Pro Glu Phe Arg Glu Phe Ile 35 40 45 gga gaa aat gca tta
agc ata gat tta atc aac atc gag cct ctc gac 192Gly Glu Asn Ala Leu
Ser Ile Asp Leu Ile Asn Ile Glu Pro Leu Asp 50 55 60 gat ctg cac
gcg ccg aaa gga ata atc aaa cag gcg cag gat ctg gcg 240Asp Leu His
Ala Pro Lys Gly Ile Ile Lys Gln Ala Gln Asp Leu Ala 65 70 75 80 gct
gaa gca ttc gga gcc gac tac acg ttc ttt tcc gtc cag gga acg 288Ala
Glu Ala Phe Gly Ala Asp Tyr Thr Phe Phe Ser Val Gln Gly Thr 85 90
95 agc ggc gcc atc atg acg atg gtc atg gcc gta tgc gga ccc gga gac
336Ser Gly Ala Ile Met Thr Met Val Met Ala Val Cys Gly Pro Gly Asp
100 105 110 aaa atc atc gtc ccg aga aat gtt cat aaa tcg gtg atg tca
gcg atc 384Lys Ile Ile Val Pro Arg Asn Val His Lys Ser Val Met Ser
Ala Ile 115 120 125 gtc ttc tca ggc gcc gta ccg atc ttc atc cat ccg
gaa atc gac gat 432Val Phe Ser Gly Ala Val Pro Ile Phe Ile His Pro
Glu Ile Asp Asp 130 135 140 gag ctg ggg att tca cac ggg atc acc cct
gaa tca gcg aaa aaa gcg 480Glu Leu Gly Ile Ser His Gly Ile Thr Pro
Glu Ser Ala Lys Lys Ala 145 150 155 160 ctg ctt gag cat cct gac gca
aaa gga ctg ctc gtc atc aac ccg act 528Leu Leu Glu His Pro Asp Ala
Lys Gly Leu Leu Val Ile Asn Pro Thr 165 170 175 tat ttc ggc ata gct
gca gac tta aaa agc atc gtc gag ctg gct cat 576Tyr Phe Gly Ile Ala
Ala Asp Leu Lys Ser Ile Val Glu Leu Ala His 180 185 190 tct ttt cat
gtc ccg gtg cta gtt gac gag gcg cac ggc gtt cat atc 624Ser Phe His
Val Pro Val Leu Val Asp Glu Ala His Gly Val His Ile 195 200 205 cac
ttc cat gaa gat ctg cct ctt tcg gca atg cag gcc gga gcg gat 672His
Phe His Glu Asp Leu Pro Leu Ser Ala Met Gln Ala Gly Ala Asp 210 215
220 atg gcg gcg acg agc gtt cat aag ctc gga gga tcc ctt aca caa agt
720Met Ala Ala Thr Ser Val His Lys Leu Gly Gly Ser Leu Thr Gln Ser
225 230 235 240 tcg att ctc aat atg aaa gaa ggt ctg gtt tca aag gaa
agg gtg caa
768Ser Ile Leu Asn Met Lys Glu Gly Leu Val Ser Lys Glu Arg Val Gln
245 250 255 tcg att tta agc atg ctg acg acg aca tcg acc tcc tat cta
ttg ctc 816Ser Ile Leu Ser Met Leu Thr Thr Thr Ser Thr Ser Tyr Leu
Leu Leu 260 265 270 gct tcg ctt gat gtc gcc aga aaa cgc tta gcc aca
gag gga cac gaa 864Ala Ser Leu Asp Val Ala Arg Lys Arg Leu Ala Thr
Glu Gly His Glu 275 280 285 ctg atc gaa caa acg att aag ctc gcc aac
gaa aca agg gaa cgc atc 912Leu Ile Glu Gln Thr Ile Lys Leu Ala Asn
Glu Thr Arg Glu Arg Ile 290 295 300 aat aat atc aac ggg att tca tgc
gtc ggg agg gaa atc ctc ggc tcc 960Asn Asn Ile Asn Gly Ile Ser Cys
Val Gly Arg Glu Ile Leu Gly Ser 305 310 315 320 aaa gcg gct ttt gac
tat gat ccg acc aaa ttg atc ata tct gtg aaa 1008Lys Ala Ala Phe Asp
Tyr Asp Pro Thr Lys Leu Ile Ile Ser Val Lys 325 330 335 gac ctc ggc
ctg acc ggt cat gat gtg gaa aaa tgg ctg cgc gag tca 1056Asp Leu Gly
Leu Thr Gly His Asp Val Glu Lys Trp Leu Arg Glu Ser 340 345 350 tgc
caa atc gaa gtg gag ctt tct gac tta tac aac atc ctg tgc att 1104Cys
Gln Ile Glu Val Glu Leu Ser Asp Leu Tyr Asn Ile Leu Cys Ile 355 360
365 ttt aca ccg gga gac cgg aaa gag gat gca gat gcg tta att aaa gga
1152Phe Thr Pro Gly Asp Arg Lys Glu Asp Ala Asp Ala Leu Ile Lys Gly
370 375 380 tta acc gag att gct caa cag gcc gct tca tct gcg gaa aac
aga cgc 1200Leu Thr Glu Ile Ala Gln Gln Ala Ala Ser Ser Ala Glu Asn
Arg Arg 385 390 395 400 aag cct gaa gtg ctt ctg cca aac att ccg gcg
ctt gcg atg aca ccg 1248Lys Pro Glu Val Leu Leu Pro Asn Ile Pro Ala
Leu Ala Met Thr Pro 405 410 415 cgt gac gct ttt tac gca aac acg gag
atc att ccg ttc aaa aaa gca 1296Arg Asp Ala Phe Tyr Ala Asn Thr Glu
Ile Ile Pro Phe Lys Lys Ala 420 425 430 gct ggc aga atg att gcc gag
ttt gtt atg gtt tat ccg cca ggg ata 1344Ala Gly Arg Met Ile Ala Glu
Phe Val Met Val Tyr Pro Pro Gly Ile 435 440 445 ccg atc ttc att ccg
ggc gag atc att acc gag gat aat atc aac tac 1392Pro Ile Phe Ile Pro
Gly Glu Ile Ile Thr Glu Asp Asn Ile Asn Tyr 450 455 460 atc gaa aag
aac ctg gaa gct ggg ctg cct gtt cag gga ccg gaa gat 1440Ile Glu Lys
Asn Leu Glu Ala Gly Leu Pro Val Gln Gly Pro Glu Asp 465 470 475 480
gac acc ctc cat atg atc cgc gtt att aaa gaa cag cag gca atc ctg
1488Asp Thr Leu His Met Ile Arg Val Ile Lys Glu Gln Gln Ala Ile Leu
485 490 495 taa aaa 14946496PRTBacillus licheniformis DSM 13 6Met
Lys Val Gly Lys Gln Leu Leu Gln His Glu Thr Pro Leu Tyr Thr 1 5 10
15 Gly Leu Lys Lys His Ala Gly Lys Asn Pro Ile Gln Phe His Ile Pro
20 25 30 Gly His Lys Lys Gly Ser Gly Met Asp Pro Glu Phe Arg Glu
Phe Ile 35 40 45 Gly Glu Asn Ala Leu Ser Ile Asp Leu Ile Asn Ile
Glu Pro Leu Asp 50 55 60 Asp Leu His Ala Pro Lys Gly Ile Ile Lys
Gln Ala Gln Asp Leu Ala 65 70 75 80 Ala Glu Ala Phe Gly Ala Asp Tyr
Thr Phe Phe Ser Val Gln Gly Thr 85 90 95 Ser Gly Ala Ile Met Thr
Met Val Met Ala Val Cys Gly Pro Gly Asp 100 105 110 Lys Ile Ile Val
Pro Arg Asn Val His Lys Ser Val Met Ser Ala Ile 115 120 125 Val Phe
Ser Gly Ala Val Pro Ile Phe Ile His Pro Glu Ile Asp Asp 130 135 140
Glu Leu Gly Ile Ser His Gly Ile Thr Pro Glu Ser Ala Lys Lys Ala 145
150 155 160 Leu Leu Glu His Pro Asp Ala Lys Gly Leu Leu Val Ile Asn
Pro Thr 165 170 175 Tyr Phe Gly Ile Ala Ala Asp Leu Lys Ser Ile Val
Glu Leu Ala His 180 185 190 Ser Phe His Val Pro Val Leu Val Asp Glu
Ala His Gly Val His Ile 195 200 205 His Phe His Glu Asp Leu Pro Leu
Ser Ala Met Gln Ala Gly Ala Asp 210 215 220 Met Ala Ala Thr Ser Val
His Lys Leu Gly Gly Ser Leu Thr Gln Ser 225 230 235 240 Ser Ile Leu
Asn Met Lys Glu Gly Leu Val Ser Lys Glu Arg Val Gln 245 250 255 Ser
Ile Leu Ser Met Leu Thr Thr Thr Ser Thr Ser Tyr Leu Leu Leu 260 265
270 Ala Ser Leu Asp Val Ala Arg Lys Arg Leu Ala Thr Glu Gly His Glu
275 280 285 Leu Ile Glu Gln Thr Ile Lys Leu Ala Asn Glu Thr Arg Glu
Arg Ile 290 295 300 Asn Asn Ile Asn Gly Ile Ser Cys Val Gly Arg Glu
Ile Leu Gly Ser 305 310 315 320 Lys Ala Ala Phe Asp Tyr Asp Pro Thr
Lys Leu Ile Ile Ser Val Lys 325 330 335 Asp Leu Gly Leu Thr Gly His
Asp Val Glu Lys Trp Leu Arg Glu Ser 340 345 350 Cys Gln Ile Glu Val
Glu Leu Ser Asp Leu Tyr Asn Ile Leu Cys Ile 355 360 365 Phe Thr Pro
Gly Asp Arg Lys Glu Asp Ala Asp Ala Leu Ile Lys Gly 370 375 380 Leu
Thr Glu Ile Ala Gln Gln Ala Ala Ser Ser Ala Glu Asn Arg Arg 385 390
395 400 Lys Pro Glu Val Leu Leu Pro Asn Ile Pro Ala Leu Ala Met Thr
Pro 405 410 415 Arg Asp Ala Phe Tyr Ala Asn Thr Glu Ile Ile Pro Phe
Lys Lys Ala 420 425 430 Ala Gly Arg Met Ile Ala Glu Phe Val Met Val
Tyr Pro Pro Gly Ile 435 440 445 Pro Ile Phe Ile Pro Gly Glu Ile Ile
Thr Glu Asp Asn Ile Asn Tyr 450 455 460 Ile Glu Lys Asn Leu Glu Ala
Gly Leu Pro Val Gln Gly Pro Glu Asp 465 470 475 480 Asp Thr Leu His
Met Ile Arg Val Ile Lys Glu Gln Gln Ala Ile Leu 485 490 495
71167DNABacillus licheniformis DSM 13gene(1)..(1167)NADH-dependent
butanol dehydrogenase A (E.C. 1.1.1.-), yugJ 7atg gat aac ttt aca
tat tgg aat ccc aca aag ctg ata ttc ggc cgg 48Met Asp Asn Phe Thr
Tyr Trp Asn Pro Thr Lys Leu Ile Phe Gly Arg 1 5 10 15 gga gaa gtt
gaa aag ctg gca gaa gag gtg aaa caa tac ggc cgc aat 96Gly Glu Val
Glu Lys Leu Ala Glu Glu Val Lys Gln Tyr Gly Arg Asn 20 25 30 gtc
ctg ctc gta tac ggc gga ggc agc att aag cga aac ggt tta tac 144Val
Leu Leu Val Tyr Gly Gly Gly Ser Ile Lys Arg Asn Gly Leu Tyr 35 40
45 gat caa gtc att tca atc ctt gaa aag gcg ggc gcg acc gtc cat gaa
192Asp Gln Val Ile Ser Ile Leu Glu Lys Ala Gly Ala Thr Val His Glu
50 55 60 ctg ccc ggc gtc gaa ccg aat ccg cgt gtt gcc act gtg aat
aaa gga 240Leu Pro Gly Val Glu Pro Asn Pro Arg Val Ala Thr Val Asn
Lys Gly 65 70 75 80 gtt gcg atc tgc aaa gag aac gat att gac ttt ctt
ttg gca gtc ggc 288Val Ala Ile Cys Lys Glu Asn Asp Ile Asp Phe Leu
Leu Ala Val Gly 85 90 95 ggc gga agc gtc att gat tgt acg aaa gca
att gct gcc gga gcg aaa 336Gly Gly Ser Val Ile Asp Cys Thr Lys Ala
Ile Ala Ala Gly Ala Lys 100 105 110 tac gac ggc gat gcg tgg gat att
gtg acg aaa aaa cat att ccg gct 384Tyr Asp Gly Asp Ala Trp Asp Ile
Val Thr Lys Lys His Ile Pro Ala 115 120 125 gat gcg ctg ccg ttt gga
aca gtt tta acg tta gca gca aca ggc tct 432Asp Ala Leu Pro Phe Gly
Thr Val Leu Thr Leu Ala Ala Thr Gly Ser 130 135 140 gaa atg aac tcg
gga tct gtg atc aca aat tgg gaa acc aat gaa aaa 480Glu Met Asn Ser
Gly Ser Val Ile Thr Asn Trp Glu Thr Asn Glu Lys 145 150 155 160 tac
ggc tgg gga agc ccg ctc gta ttc cct aaa ttt tca att ctt gat 528Tyr
Gly Trp Gly Ser Pro Leu Val Phe Pro Lys Phe Ser Ile Leu Asp 165 170
175 ccg gtc aac acg ttt acc gtc ccg aaa gac cac acg att tac ggc att
576Pro Val Asn Thr Phe Thr Val Pro Lys Asp His Thr Ile Tyr Gly Ile
180 185 190 gtc gac atg atg tcc cac gtg ttt gag caa tat ttt cac cat
acc gaa 624Val Asp Met Met Ser His Val Phe Glu Gln Tyr Phe His His
Thr Glu 195 200 205 aat acc cct tat cag gac cgg atg tgc gaa tcc ctg
ctt aaa acg gta 672Asn Thr Pro Tyr Gln Asp Arg Met Cys Glu Ser Leu
Leu Lys Thr Val 210 215 220 att gaa aca gct cct aag ctc att gaa gac
cta gaa aac tat gag ctg 720Ile Glu Thr Ala Pro Lys Leu Ile Glu Asp
Leu Glu Asn Tyr Glu Leu 225 230 235 240 cgt gaa acg att ctg tat aca
ggc acc att gcg ctg aac ggc atg cta 768Arg Glu Thr Ile Leu Tyr Thr
Gly Thr Ile Ala Leu Asn Gly Met Leu 245 250 255 tca atg ggc gca cgc
gga gac tgg gca acg cac aat atc gag cac gct 816Ser Met Gly Ala Arg
Gly Asp Trp Ala Thr His Asn Ile Glu His Ala 260 265 270 gtt tca gcc
gta tac gat att ccg cat gcg gga ggg ctt gcg att ctg 864Val Ser Ala
Val Tyr Asp Ile Pro His Ala Gly Gly Leu Ala Ile Leu 275 280 285 ttc
ccg aat tgg atg aag cac acg ctt tcc gag aac gtc ggc cgc ttt 912Phe
Pro Asn Trp Met Lys His Thr Leu Ser Glu Asn Val Gly Arg Phe 290 295
300 aaa cag ctt gcc gtc cgt gtt ttt gac gta gat gaa aca gga aaa acc
960Lys Gln Leu Ala Val Arg Val Phe Asp Val Asp Glu Thr Gly Lys Thr
305 310 315 320 gat cgc gaa gtg gcg ctt gtt gga atc gag aaa ctg tct
gaa ttc tgg 1008Asp Arg Glu Val Ala Leu Val Gly Ile Glu Lys Leu Ser
Glu Phe Trp 325 330 335 acc agc ctt ggc gcg cca aat cgt ctt gcc gat
tat gac att aca gat 1056Thr Ser Leu Gly Ala Pro Asn Arg Leu Ala Asp
Tyr Asp Ile Thr Asp 340 345 350 gag aag ctt gat ctc att gcc gac aaa
gcg atg gca aac ggc gaa ttc 1104Glu Lys Leu Asp Leu Ile Ala Asp Lys
Ala Met Ala Asn Gly Glu Phe 355 360 365 ggc cgc ttt aaa acg ctg aat
aaa gac gat gtt ctg tct att ttg aag 1152Gly Arg Phe Lys Thr Leu Asn
Lys Asp Asp Val Leu Ser Ile Leu Lys 370 375 380 gct tct tta taa gat
1167Ala Ser Leu 385 8387PRTBacillus licheniformis DSM 13 8Met Asp
Asn Phe Thr Tyr Trp Asn Pro Thr Lys Leu Ile Phe Gly Arg 1 5 10 15
Gly Glu Val Glu Lys Leu Ala Glu Glu Val Lys Gln Tyr Gly Arg Asn 20
25 30 Val Leu Leu Val Tyr Gly Gly Gly Ser Ile Lys Arg Asn Gly Leu
Tyr 35 40 45 Asp Gln Val Ile Ser Ile Leu Glu Lys Ala Gly Ala Thr
Val His Glu 50 55 60 Leu Pro Gly Val Glu Pro Asn Pro Arg Val Ala
Thr Val Asn Lys Gly 65 70 75 80 Val Ala Ile Cys Lys Glu Asn Asp Ile
Asp Phe Leu Leu Ala Val Gly 85 90 95 Gly Gly Ser Val Ile Asp Cys
Thr Lys Ala Ile Ala Ala Gly Ala Lys 100 105 110 Tyr Asp Gly Asp Ala
Trp Asp Ile Val Thr Lys Lys His Ile Pro Ala 115 120 125 Asp Ala Leu
Pro Phe Gly Thr Val Leu Thr Leu Ala Ala Thr Gly Ser 130 135 140 Glu
Met Asn Ser Gly Ser Val Ile Thr Asn Trp Glu Thr Asn Glu Lys 145 150
155 160 Tyr Gly Trp Gly Ser Pro Leu Val Phe Pro Lys Phe Ser Ile Leu
Asp 165 170 175 Pro Val Asn Thr Phe Thr Val Pro Lys Asp His Thr Ile
Tyr Gly Ile 180 185 190 Val Asp Met Met Ser His Val Phe Glu Gln Tyr
Phe His His Thr Glu 195 200 205 Asn Thr Pro Tyr Gln Asp Arg Met Cys
Glu Ser Leu Leu Lys Thr Val 210 215 220 Ile Glu Thr Ala Pro Lys Leu
Ile Glu Asp Leu Glu Asn Tyr Glu Leu 225 230 235 240 Arg Glu Thr Ile
Leu Tyr Thr Gly Thr Ile Ala Leu Asn Gly Met Leu 245 250 255 Ser Met
Gly Ala Arg Gly Asp Trp Ala Thr His Asn Ile Glu His Ala 260 265 270
Val Ser Ala Val Tyr Asp Ile Pro His Ala Gly Gly Leu Ala Ile Leu 275
280 285 Phe Pro Asn Trp Met Lys His Thr Leu Ser Glu Asn Val Gly Arg
Phe 290 295 300 Lys Gln Leu Ala Val Arg Val Phe Asp Val Asp Glu Thr
Gly Lys Thr 305 310 315 320 Asp Arg Glu Val Ala Leu Val Gly Ile Glu
Lys Leu Ser Glu Phe Trp 325 330 335 Thr Ser Leu Gly Ala Pro Asn Arg
Leu Ala Asp Tyr Asp Ile Thr Asp 340 345 350 Glu Lys Leu Asp Leu Ile
Ala Asp Lys Ala Met Ala Asn Gly Glu Phe 355 360 365 Gly Arg Phe Lys
Thr Leu Asn Lys Asp Asp Val Leu Ser Ile Leu Lys 370 375 380 Ala Ser
Leu 385 91143DNABacillus licheniformis DSM
13gene(1)..(1143)Acyl-CoA dehydrogenase (E.C. 1.3.99.-) 9atg atc
ttt aag ctg agc gaa gag cac caa atg ata cag aag atg gtg 48Met Ile
Phe Lys Leu Ser Glu Glu His Gln Met Ile Gln Lys Met Val 1 5 10 15
cgc gac ttt gcc cat cac gag gtg gag ccg act gcg aaa gag cgg gat
96Arg Asp Phe Ala His His Glu Val Glu Pro Thr Ala Lys Glu Arg Asp
20 25 30 gag gaa gag cgg ttt gac atg gaa ctg ttt gca aaa atg gcg
gaa ttg 144Glu Glu Glu Arg Phe Asp Met Glu Leu Phe Ala Lys Met Ala
Glu Leu 35 40 45 ggg ctc acg ggt ata ccg tgg cca gag gaa tac ggg
gga atc ggc agc 192Gly Leu Thr Gly Ile Pro Trp Pro Glu Glu Tyr Gly
Gly Ile Gly Ser 50 55 60 gac tat ctc gcc tac gtc atc gcg gtt gaa
gag ctt tcg aaa gtc tgc 240Asp Tyr Leu Ala Tyr Val Ile Ala Val Glu
Glu Leu Ser Lys Val Cys 65 70 75 80 gcc tca acg ggt gtc acc ttg tct
gcc cat aca tcg ctt gcg ggc tgg 288Ala Ser Thr Gly Val Thr Leu Ser
Ala His Thr Ser Leu Ala Gly Trp 85 90 95 ccg att tat gca ttt gga
aca gaa gaa cag aaa cag gaa tat tta aag 336Pro Ile Tyr Ala Phe Gly
Thr Glu Glu Gln Lys Gln Glu Tyr Leu Lys 100 105 110 ccg atg gcc cgc
ggc gaa aag ata gga gcc tac gga ttg acg gag ccc 384Pro Met Ala Arg
Gly Glu Lys Ile Gly Ala Tyr Gly Leu Thr Glu Pro 115 120 125 ggt tca
ggc tcc gat gcc ggc ggc atg aaa acg acc gca gaa aaa aaa 432Gly Ser
Gly Ser Asp Ala Gly Gly Met Lys Thr Thr Ala Glu Lys Lys 130 135 140
ggc gat gaa tat att ttg aac ggg acg aag atc ttt atc aca aac ggc
480Gly Asp Glu Tyr Ile Leu Asn Gly Thr Lys Ile Phe Ile Thr Asn Gly
145 150 155 160 gga atc gcg gac ttc tac atc gtg ttt gcc aac ctt gcc
ccg gaa cag 528Gly Ile Ala Asp Phe Tyr Ile Val Phe Ala Asn Leu Ala
Pro Glu Gln 165 170 175 aaa cac aaa ggc aca acc gcc ttt atc gtt gaa
aag gac ttc ccc ggc 576Lys His Lys Gly Thr Thr Ala Phe Ile Val Glu
Lys Asp Phe Pro Gly 180 185 190 ttt tct gtc gga aaa aaa gag agg aaa
ctg ggc atc cgt tca tca ccg 624Phe Ser Val Gly Lys Lys Glu Arg Lys
Leu Gly Ile Arg Ser Ser Pro 195 200 205 aca acc gaa atc atc ttt cag
gac tgc cgc gtg cct tta aaa aac cgc 672Thr Thr Glu Ile Ile Phe Gln
Asp Cys Arg Val Pro Leu Lys Asn Arg 210 215 220 ctt ggc ggg gaa ggt
gaa ggc ttt aaa atc gcg atg aag acg ctc gat 720Leu Gly Gly Glu Gly
Glu Gly Phe Lys Ile Ala Met Lys Thr Leu Asp 225 230 235 240 gga ggc
aga aac gga att gcc gct caa gcc gtc ggg att gcc cag ggc 768Gly Gly
Arg Asn Gly Ile Ala Ala Gln Ala Val Gly Ile Ala Gln Gly 245 250 255
gca ttt gag gcg gcg aag gcg tat gcg aaa gag cgg aag caa ttc ggc
816Ala Phe Glu Ala Ala Lys Ala Tyr Ala Lys Glu Arg Lys Gln Phe Gly
260 265 270 agg ccg atc gcc gag cag cag ggc atc gct ttt aaa ctg gct
gat atg 864Arg Pro Ile Ala Glu Gln Gln Gly Ile Ala Phe Lys Leu Ala
Asp Met 275 280 285 gcg acg gag att gaa gct tca agg ctt tta acc tac
cag gcg gca tgg 912Ala Thr Glu Ile Glu Ala Ser Arg Leu Leu Thr Tyr
Gln Ala Ala Trp 290 295 300 ctg gaa tca gaa gga ctg cct tat gga aag
gct tct gcc atg tca aag 960Leu Glu Ser Glu Gly Leu Pro Tyr Gly Lys
Ala Ser Ala Met Ser Lys 305 310 315 320 ctt tac gca ggg gat acg gcc
atg aaa gtg acg acg gag gcc gtg caa 1008Leu Tyr Ala Gly Asp Thr Ala
Met Lys Val Thr Thr Glu Ala Val Gln 325 330 335 ata ttc ggc ggg tac
ggc tac aca aag gat tat ccg gtc gag cgc ttt 1056Ile Phe Gly Gly Tyr
Gly Tyr Thr Lys Asp Tyr Pro Val Glu Arg Phe 340 345 350 atg cgc gat
gca aaa atc aca cag atc tat gaa ggt acg cag gaa att 1104Met Arg Asp
Ala Lys Ile Thr Gln Ile Tyr Glu Gly Thr Gln Glu Ile 355 360 365 cag
aag ctc gtc att tcg aga atg ctg atg aaa taa ggg 1143Gln Lys Leu Val
Ile Ser Arg Met Leu Met Lys 370 375 10379PRTBacillus licheniformis
DSM 13 10Met Ile Phe Lys Leu Ser Glu Glu His Gln Met Ile Gln Lys
Met Val 1 5 10 15 Arg Asp Phe Ala His His Glu Val Glu Pro Thr Ala
Lys Glu Arg Asp 20 25 30 Glu Glu Glu Arg Phe Asp Met Glu Leu Phe
Ala Lys Met Ala Glu Leu 35 40 45 Gly Leu Thr Gly Ile Pro Trp Pro
Glu Glu Tyr Gly Gly Ile Gly Ser 50 55 60 Asp Tyr Leu Ala Tyr Val
Ile Ala Val Glu Glu Leu Ser Lys Val Cys 65 70 75 80 Ala Ser Thr Gly
Val Thr Leu Ser Ala His Thr Ser Leu Ala Gly Trp 85 90 95 Pro Ile
Tyr Ala Phe Gly Thr Glu Glu Gln Lys Gln Glu Tyr Leu Lys 100 105 110
Pro Met Ala Arg Gly Glu Lys Ile Gly Ala Tyr Gly Leu Thr Glu Pro 115
120 125 Gly Ser Gly Ser Asp Ala Gly Gly Met Lys Thr Thr Ala Glu Lys
Lys 130 135 140 Gly Asp Glu Tyr Ile Leu Asn Gly Thr Lys Ile Phe Ile
Thr Asn Gly 145 150 155 160 Gly Ile Ala Asp Phe Tyr Ile Val Phe Ala
Asn Leu Ala Pro Glu Gln 165 170 175 Lys His Lys Gly Thr Thr Ala Phe
Ile Val Glu Lys Asp Phe Pro Gly 180 185 190 Phe Ser Val Gly Lys Lys
Glu Arg Lys Leu Gly Ile Arg Ser Ser Pro 195 200 205 Thr Thr Glu Ile
Ile Phe Gln Asp Cys Arg Val Pro Leu Lys Asn Arg 210 215 220 Leu Gly
Gly Glu Gly Glu Gly Phe Lys Ile Ala Met Lys Thr Leu Asp 225 230 235
240 Gly Gly Arg Asn Gly Ile Ala Ala Gln Ala Val Gly Ile Ala Gln Gly
245 250 255 Ala Phe Glu Ala Ala Lys Ala Tyr Ala Lys Glu Arg Lys Gln
Phe Gly 260 265 270 Arg Pro Ile Ala Glu Gln Gln Gly Ile Ala Phe Lys
Leu Ala Asp Met 275 280 285 Ala Thr Glu Ile Glu Ala Ser Arg Leu Leu
Thr Tyr Gln Ala Ala Trp 290 295 300 Leu Glu Ser Glu Gly Leu Pro Tyr
Gly Lys Ala Ser Ala Met Ser Lys 305 310 315 320 Leu Tyr Ala Gly Asp
Thr Ala Met Lys Val Thr Thr Glu Ala Val Gln 325 330 335 Ile Phe Gly
Gly Tyr Gly Tyr Thr Lys Asp Tyr Pro Val Glu Arg Phe 340 345 350 Met
Arg Asp Ala Lys Ile Thr Gln Ile Tyr Glu Gly Thr Gln Glu Ile 355 360
365 Gln Lys Leu Val Ile Ser Arg Met Leu Met Lys 370 375
111134DNABacillus licheniformis DSM 13gene(1)..(1134)Acyl-CoA
dehydrogenase (E.C. 1.3.99.-) 11ttg aat ttt cag ttt aca gaa gag cag
acc cgc atg cag aag gtt gta 48Leu Asn Phe Gln Phe Thr Glu Glu Gln
Thr Arg Met Gln Lys Val Val 1 5 10 15 agg gat ttt gtc aaa cag gag
gtc gct cct ttt gta cct gaa atg gaa 96Arg Asp Phe Val Lys Gln Glu
Val Ala Pro Phe Val Pro Glu Met Glu 20 25 30 aaa ggc cgt ttt cca
acc tct ctt ttg aag aaa atg gcg gag cgc ggc 144Lys Gly Arg Phe Pro
Thr Ser Leu Leu Lys Lys Met Ala Glu Arg Gly 35 40 45 tgg atg ggg
ctt ccg ata ccg gct aag tac aac ggg gcc ggt cat gac 192Trp Met Gly
Leu Pro Ile Pro Ala Lys Tyr Asn Gly Ala Gly His Asp 50 55 60 ttt
atc acc tat atg atg acc ata cat gag ctg tca aag aaa agc gcc 240Phe
Ile Thr Tyr Met Met Thr Ile His Glu Leu Ser Lys Lys Ser Ala 65 70
75 80 gtt ctt ggt gcg gtt cta tct gta cac acg tca att gtc acc att
ccc 288Val Leu Gly Ala Val Leu Ser Val His Thr Ser Ile Val Thr Ile
Pro 85 90 95 att ctt tta aac ggt aat gag cgg caa aaa gag cac tac
gtt aaa aag 336Ile Leu Leu Asn Gly Asn Glu Arg Gln Lys Glu His Tyr
Val Lys Lys 100 105 110 ctg gca gca ggg cag tac ttg gga gct ttt tgt
tta acc gaa ccg agt 384Leu Ala Ala Gly Gln Tyr Leu Gly Ala Phe Cys
Leu Thr Glu Pro Ser 115 120 125 gcc ggt tcc gat gcg ggc agt cta aaa
acc agg gcc gaa aag cgc ggc 432Ala Gly Ser Asp Ala Gly Ser Leu Lys
Thr Arg Ala Glu Lys Arg Gly 130 135 140 gat acc tat gtg ctg aac gga
acg aaa gtt ttt att aca aac ggc gga 480Asp Thr Tyr Val Leu Asn Gly
Thr Lys Val Phe Ile Thr Asn Gly Gly 145 150 155 160 gcc gcg gac att
tat ctt gtt ttc gct tca acc gat ccg gag gcg gga 528Ala Ala Asp Ile
Tyr Leu Val Phe Ala Ser Thr Asp Pro Glu Ala Gly 165 170 175 acg gga
ggc att tcc gct ttt atc gtg gag aag ggc acg ccg ggc ttt 576Thr Gly
Gly Ile Ser Ala Phe Ile Val Glu Lys Gly Thr Pro Gly Phe 180 185 190
ttc atc gga aaa aat gaa gag aaa atg ggg ctc cac ggt tcg cta acg
624Phe Ile Gly Lys Asn Glu Glu Lys Met Gly Leu His Gly Ser Leu Thr
195 200 205 gtc act tta aat ttt gat aac gct gtc att ccc gcc cgg cag
ctg ctg 672Val Thr Leu Asn Phe Asp Asn Ala Val Ile Pro Ala Arg Gln
Leu Leu 210 215 220 ggg gag gaa gga atg gga ttc aaa atg gcc ctg tcc
aac ctg gat acc 720Gly Glu Glu Gly Met Gly Phe Lys Met Ala Leu Ser
Asn Leu Asp Thr 225 230 235 240 ggc cgg atc gga att gcg gcg cag gct
ctg ggt atc gcc gag gga gcg 768Gly Arg Ile Gly Ile Ala Ala Gln Ala
Leu Gly Ile Ala Glu Gly Ala 245 250 255 ctt tct gaa gcc gtt caa ttt
cta aaa aaa cgg tat ccg gac gga gag 816Leu Ser Glu Ala Val Gln Phe
Leu Lys Lys Arg Tyr Pro Asp Gly Glu 260 265 270 ctg tat aag aac ggc
caa gcg ctt gcc ttt aag ctg gcc gac atg gcg 864Leu Tyr Lys Asn Gly
Gln Ala Leu Ala Phe Lys Leu Ala Asp Met Ala 275 280 285 gcg agg aca
gag gcg gcc agg ctt ctc gtc tac cag gcc gcc tcg ttg 912Ala Arg Thr
Glu Ala Ala Arg Leu Leu Val Tyr Gln Ala Ala Ser Leu 290 295 300 aaa
cag caa ggc atg cag acg ggg aaa gct gct tca atg gcc aaa ttg 960Lys
Gln Gln Gly Met Gln Thr Gly Lys Ala Ala Ser Met Ala Lys Leu 305 310
315 320 ttc gct tcg gaa acg gcg atg tat gtc gcc gga gag gcc gtg cag
ctg 1008Phe Ala Ser Glu Thr Ala Met Tyr Val Ala Gly Glu Ala Val Gln
Leu 325 330 335 ctc gga gat ttc gga tac aca aag gat ttc agc gct gaa
cga tat ttc 1056Leu Gly Asp Phe Gly Tyr Thr Lys Asp Phe Ser Ala Glu
Arg Tyr Phe 340 345 350 agg gat gcg aaa gtg tgt gaa att tat gag ggc
acg agc gag atc cag 1104Arg Asp Ala Lys Val Cys Glu Ile Tyr Glu Gly
Thr Ser Glu Ile Gln 355 360 365 cgg atc gtc atc ggt aaa cat tta taa
ggt 1134Arg Ile Val Ile Gly Lys His Leu 370 375 12376PRTBacillus
licheniformis DSM 13 12Leu Asn Phe Gln Phe Thr Glu Glu Gln Thr Arg
Met Gln Lys Val Val 1 5 10 15 Arg Asp Phe Val Lys Gln Glu Val Ala
Pro Phe Val Pro Glu Met Glu 20 25 30 Lys Gly Arg Phe Pro Thr Ser
Leu Leu Lys Lys Met Ala Glu Arg Gly 35 40 45 Trp Met Gly Leu Pro
Ile Pro Ala Lys Tyr Asn Gly Ala Gly His Asp 50 55 60 Phe Ile Thr
Tyr Met Met Thr Ile His Glu Leu Ser Lys Lys Ser Ala 65 70 75 80 Val
Leu Gly Ala Val Leu Ser Val His Thr Ser Ile Val Thr Ile Pro 85 90
95 Ile Leu Leu Asn Gly Asn Glu Arg Gln Lys Glu His Tyr Val Lys Lys
100 105 110 Leu Ala Ala Gly Gln Tyr Leu Gly Ala Phe Cys Leu Thr Glu
Pro Ser 115 120 125 Ala Gly Ser Asp Ala Gly Ser Leu Lys Thr Arg Ala
Glu Lys Arg Gly 130 135 140 Asp Thr Tyr Val Leu Asn Gly Thr Lys Val
Phe Ile Thr Asn Gly Gly 145 150 155 160 Ala Ala Asp Ile Tyr Leu Val
Phe Ala Ser Thr Asp Pro Glu Ala Gly 165 170 175 Thr Gly Gly Ile Ser
Ala Phe Ile Val Glu Lys Gly Thr Pro Gly Phe 180 185 190 Phe Ile Gly
Lys Asn Glu Glu Lys Met Gly Leu His Gly Ser Leu Thr 195 200 205 Val
Thr Leu Asn Phe Asp Asn Ala Val Ile Pro Ala Arg Gln Leu Leu 210 215
220 Gly Glu Glu Gly Met Gly Phe Lys Met Ala Leu Ser Asn Leu Asp Thr
225 230 235 240 Gly Arg Ile Gly Ile Ala Ala Gln Ala Leu Gly Ile Ala
Glu Gly Ala 245 250 255 Leu Ser Glu Ala Val Gln Phe Leu Lys Lys Arg
Tyr Pro Asp Gly Glu 260 265 270 Leu Tyr Lys Asn Gly Gln Ala Leu Ala
Phe Lys Leu Ala Asp Met Ala 275 280 285 Ala Arg Thr Glu Ala Ala Arg
Leu Leu Val Tyr Gln Ala Ala Ser Leu 290 295 300 Lys Gln Gln Gly Met
Gln Thr Gly Lys Ala Ala Ser Met Ala Lys Leu 305 310 315 320 Phe Ala
Ser Glu Thr Ala Met Tyr Val Ala Gly Glu Ala Val Gln Leu 325 330 335
Leu Gly Asp Phe Gly Tyr Thr Lys Asp Phe Ser Ala Glu Arg Tyr Phe 340
345 350 Arg Asp Ala Lys Val Cys Glu Ile Tyr Glu Gly Thr Ser Glu Ile
Gln 355 360 365 Arg Ile Val Ile Gly Lys His Leu 370 375
13867DNABacillus licheniformis DSM
13gene(1)..(867)3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157)
13atg aaa aaa gac acg atc atg gtc atc gga gcc ggc cag atg ggg tcc
48Met Lys Lys Asp Thr Ile Met Val Ile Gly Ala Gly Gln Met Gly Ser 1
5 10 15 ggg atc gct cag gtc tct gcc cag gcc gga tac aat gtt tac atg
tat 96Gly Ile Ala Gln Val Ser Ala Gln Ala Gly Tyr Asn Val Tyr Met
Tyr 20 25 30 gac gta tca cct gaa caa att gaa aaa gga atg aag cgc
att tcc ggc 144Asp Val Ser Pro Glu Gln Ile Glu Lys Gly Met Lys Arg
Ile Ser Gly 35 40 45 cag ctt ttc aga cag gcg gaa aaa ggc aag ctg
ccg cat gaa gat gtg 192Gln Leu Phe Arg Gln Ala Glu Lys Gly Lys Leu
Pro His Glu Asp Val 50 55 60 aag caa atc tac cag cgc ctt tct ccg
tcg gct gcg ctc gac gaa gcg 240Lys Gln Ile Tyr Gln Arg Leu Ser Pro
Ser Ala Ala Leu Asp Glu Ala 65 70 75 80 cgg gaa gct ttt ctc atc att
gaa gcg gct gtt gaa caa atg gac gta 288Arg Glu Ala Phe Leu Ile Ile
Glu Ala Ala Val Glu Gln Met Asp Val 85 90 95 aaa aaa gac att ttc
acg cgg ctt gat gaa gtg acc gaa gac tca gcg 336Lys Lys Asp Ile Phe
Thr Arg Leu Asp Glu Val Thr Glu Asp Ser Ala 100 105 110 ata ttg gca
tca aat aca tcg tcc ctg tcg att acg gaa ctt gct gcc 384Ile Leu Ala
Ser Asn Thr Ser Ser Leu Ser Ile Thr Glu Leu Ala Ala 115 120 125 gtc
aca aaa aaa cct caa aac gtc atc ggc atg cat ttt atg aac ccg 432Val
Thr Lys Lys Pro Gln Asn Val Ile Gly Met His Phe Met Asn Pro 130 135
140 gtt ccc gtc atg cag ctt gtc gaa gtc atc agg ggg ctt gag acg agc
480Val Pro Val Met Gln Leu Val Glu Val Ile Arg Gly Leu Glu Thr Ser
145 150 155 160 ggc gaa aca tat gaa acc gtc gtg gct gcg gcg gag cgg
atg aac aag 528Gly Glu Thr Tyr Glu Thr Val Val Ala Ala Ala Glu Arg
Met Asn Lys 165 170 175 gtt ccg ata gag gtg cgg gat ttc ccg gga ttt
atc tcc aac cgc atc 576Val Pro Ile Glu Val Arg Asp Phe Pro Gly Phe
Ile Ser Asn Arg Ile 180 185 190 tta atg ccg atg att aat gag gcg gtt
ttt gca ctt tat gaa ggc atc 624Leu Met Pro Met Ile Asn Glu Ala Val
Phe Ala Leu Tyr Glu Gly Ile 195 200 205 gcc gag aaa gaa agc ata gac
gga atc atg aag ctt ggc atg aac cat 672Ala Glu Lys Glu Ser Ile Asp
Gly Ile Met Lys Leu Gly Met Asn His 210 215 220 ccg atg ggc ccg ttg
gct ctc gcc gat ctg atc ggt ctg gat acg tgt 720Pro Met Gly Pro Leu
Ala Leu Ala Asp Leu Ile Gly Leu Asp Thr Cys 225 230 235 240 ctg tat
att atg gag acg ctg cat gaa ggc ttt ggc gac gat aag tac 768Leu Tyr
Ile Met Glu Thr Leu His Glu Gly Phe Gly Asp Asp Lys Tyr 245 250 255
agg cct tgt ccg ctc ctt aaa caa tat gtc agc gca gga cga ctc gga
816Arg Pro Cys Pro Leu Leu Lys Gln Tyr Val Ser Ala Gly Arg Leu Gly
260 265 270 aag aaa acg ggc aga ggg ttt tat acg tat gaa aag cag ccg
aca taa 864Lys Lys Thr Gly Arg Gly Phe Tyr Thr Tyr Glu Lys Gln Pro
Thr 275 280 285 gga 867 14287PRTBacillus licheniformis DSM 13 14Met
Lys Lys Asp Thr Ile Met Val Ile Gly Ala Gly Gln Met Gly Ser 1 5 10
15 Gly Ile Ala Gln Val Ser Ala Gln Ala Gly Tyr Asn Val Tyr Met Tyr
20 25 30 Asp Val Ser Pro Glu Gln Ile Glu Lys Gly Met Lys Arg Ile
Ser Gly 35 40 45 Gln Leu Phe Arg Gln Ala Glu Lys Gly Lys Leu Pro
His Glu Asp Val 50 55 60 Lys Gln Ile Tyr Gln Arg Leu Ser Pro Ser
Ala Ala Leu Asp Glu Ala 65 70 75 80 Arg Glu Ala Phe Leu Ile Ile Glu
Ala Ala Val Glu Gln Met Asp Val 85 90 95 Lys Lys Asp Ile Phe Thr
Arg Leu Asp Glu Val Thr Glu Asp Ser Ala 100 105 110 Ile Leu Ala Ser
Asn Thr Ser Ser Leu Ser Ile Thr Glu Leu Ala Ala 115 120 125 Val Thr
Lys Lys Pro Gln Asn Val Ile Gly Met His Phe Met Asn Pro 130 135 140
Val Pro Val Met Gln Leu Val Glu Val Ile Arg Gly Leu Glu Thr Ser 145
150 155 160 Gly Glu Thr Tyr Glu Thr Val Val Ala Ala Ala Glu Arg Met
Asn Lys 165 170 175 Val Pro Ile Glu Val Arg Asp Phe Pro Gly Phe Ile
Ser Asn Arg Ile 180 185 190 Leu Met Pro Met Ile Asn Glu Ala Val Phe
Ala Leu Tyr Glu Gly Ile 195 200 205 Ala Glu Lys Glu Ser Ile Asp Gly
Ile Met Lys Leu Gly Met Asn His 210 215 220 Pro Met Gly Pro Leu Ala
Leu Ala Asp Leu Ile Gly Leu Asp Thr Cys 225 230 235 240 Leu Tyr Ile
Met Glu Thr Leu His Glu Gly Phe Gly Asp Asp Lys Tyr 245 250 255 Arg
Pro Cys Pro Leu Leu Lys Gln Tyr Val Ser Ala Gly Arg Leu Gly 260 265
270 Lys Lys Thr Gly Arg Gly Phe Tyr Thr Tyr Glu Lys Gln Pro Thr 275
280 285 15780DNABacillus licheniformis DSM 13gene(1)..(780)Putative
enoyl-CoA hydratase protein (E.C. 4.2.1.17) 15atg gaa tat atc aaa
tgg aaa gac gaa gac ggg att ttt gaa att gta 48Met Glu Tyr Ile Lys
Trp Lys Asp Glu Asp Gly Ile Phe Glu Ile Val 1 5 10 15 ctg aat cgc
tct gag gcg tac aat gcc ttg aat gaa aaa atg ctt gaa 96Leu Asn Arg
Ser Glu Ala Tyr Asn Ala Leu Asn Glu Lys Met Leu Glu 20 25 30 gaa
ctg aat gaa gct ttg cgt gtt gca gag gaa agc gaa tcg ctg ctt 144Glu
Leu Asn Glu Ala Leu Arg Val Ala Glu Glu Ser Glu Ser Leu Leu 35 40
45 ctc ctt gtg aga ggc agc ggc aaa gga ttt tcg gca ggc gga gac att
192Leu Leu Val Arg Gly Ser Gly Lys Gly Phe Ser Ala Gly Gly Asp Ile
50 55 60 aag atg atg ctg tct tcc gga gat caa gac agc tcc gcc cgc
gtc att 240Lys Met Met Leu Ser Ser Gly Asp Gln Asp Ser Ser Ala Arg
Val Ile 65 70 75 80 gac aca att tct gaa att gcg gtg aag ctt tac agc
atg ccg aag atg 288Asp Thr Ile Ser Glu Ile Ala Val Lys Leu Tyr Ser
Met Pro Lys Met 85 90 95 acg aca gcg gct gtc cac ggt gcg gct gcg
ggt ctg gga ttg agc ctc 336Thr Thr Ala Ala Val His Gly Ala Ala Ala
Gly Leu Gly Leu Ser Leu 100 105 110 gcg ctc agc tgc gac cat gta ctg
gtc gaa aaa gag gcg aag ctg gcg 384Ala Leu Ser Cys Asp His Val Leu
Val Glu Lys Glu Ala Lys Leu Ala 115 120 125 atg aat ttt atc ggc atc
ggg ctt gtt ccc gac gga ggc gga cac ttc 432Met Asn Phe Ile Gly Ile
Gly Leu Val Pro Asp Gly Gly Gly His Phe 130 135 140 ttt tta gag cgg
aga atc ggt gaa act gcg gcc aaa gaa ttg att tgg 480Phe Leu Glu Arg
Arg Ile Gly Glu Thr Ala Ala Lys Glu Leu Ile Trp 145 150 155 160 agc
ggg aaa aaa ttg acg ggg gcc gaa gcg cac gag ctt cgg atc gca 528Ser
Gly Lys Lys Leu Thr Gly Ala Glu Ala His Glu Leu Arg Ile Ala 165 170
175 gac gcc gta ttc agc ggg gac tcc ggc cgt ttt gcg cgc atc tat ctt
576Asp Ala Val Phe Ser Gly Asp Ser Gly Arg Phe Ala Arg Ile Tyr Leu
180 185 190 gaa aag ctt ctg cac gct ccg ctg gca gcg atg att gag aca
aaa aag 624Glu Lys Leu Leu His Ala Pro Leu Ala Ala Met Ile Glu Thr
Lys Lys 195 200 205 atc tat cag gcg ttg aat gga ggc agg ctg cag aaa
acg ctt gaa ctc 672Ile Tyr Gln Ala Leu Asn Gly Gly Arg Leu Gln Lys
Thr Leu Glu Leu 210 215 220 gag aaa acg gcc cag atg aaa atg agg ctg
aca agc gac cat cag gaa 720Glu Lys Thr Ala Gln Met Lys Met Arg Leu
Thr Ser Asp His Gln Glu 225 230 235 240 ggg atc cgc gca ttt tta gaa
aag cgc cag ccg caa ttt aac cgt cag 768Gly Ile Arg Ala Phe Leu Glu
Lys Arg Gln Pro Gln Phe Asn Arg Gln 245 250 255 caa gta taa caa
780Gln Val 16258PRTBacillus licheniformis DSM 13 16Met Glu Tyr Ile
Lys Trp Lys Asp Glu Asp Gly Ile Phe Glu Ile Val 1 5 10 15 Leu Asn
Arg Ser Glu Ala Tyr Asn Ala Leu Asn Glu Lys Met Leu Glu 20 25 30
Glu Leu Asn Glu Ala Leu Arg Val Ala Glu Glu Ser Glu Ser Leu Leu 35
40 45 Leu Leu Val Arg Gly Ser Gly Lys Gly Phe Ser Ala Gly Gly Asp
Ile 50 55 60 Lys Met Met Leu Ser Ser Gly Asp Gln Asp Ser Ser Ala
Arg Val Ile 65 70 75 80 Asp Thr Ile Ser Glu Ile Ala Val Lys Leu Tyr
Ser Met Pro Lys Met 85 90 95 Thr Thr Ala Ala Val His Gly Ala Ala
Ala Gly Leu Gly Leu Ser Leu 100 105 110 Ala Leu Ser Cys Asp His Val
Leu Val Glu Lys Glu Ala Lys Leu Ala 115 120 125 Met Asn Phe Ile Gly
Ile Gly Leu Val Pro Asp Gly Gly Gly His Phe 130 135 140 Phe Leu Glu
Arg Arg Ile Gly Glu Thr Ala Ala Lys Glu Leu Ile Trp 145 150 155 160
Ser Gly Lys Lys Leu Thr Gly Ala Glu Ala His Glu Leu Arg Ile Ala 165
170 175 Asp Ala Val Phe Ser Gly Asp Ser Gly Arg Phe Ala Arg Ile Tyr
Leu 180 185 190 Glu Lys Leu Leu His Ala Pro Leu Ala Ala Met Ile Glu
Thr Lys Lys 195 200 205 Ile Tyr Gln Ala Leu Asn Gly Gly Arg Leu Gln
Lys Thr Leu Glu Leu 210 215 220 Glu Lys Thr Ala Gln Met Lys Met Arg
Leu Thr Ser Asp His Gln Glu 225 230 235 240 Gly Ile Arg Ala Phe Leu
Glu Lys Arg Gln Pro Gln Phe Asn Arg Gln 245 250 255 Gln Val
171065DNABacillus licheniformis DSM 13gene(1)..(1065)Probable
Enoyl(3-hydroxyisobutyryl)-coenzyme A hydrolase protein 17atg tcc
gat gac gtg ctg ttt tcc gtc aat caa aac ggc gcc gca gcg 48Met Ser
Asp Asp Val Leu Phe Ser Val Asn Gln Asn Gly Ala Ala Ala 1 5 10 15
att gtt ctg aat cgc ccg aaa gcg ctc aac tca ctc aca tac gac atg
96Ile Val Leu Asn Arg Pro Lys Ala Leu Asn Ser Leu Thr Tyr Asp Met
20 25 30 gtc cgt ctg att ggt gaa aag tta aac gag tgg gag aca gat
caa aac 144Val Arg Leu Ile Gly Glu Lys Leu Asn Glu Trp Glu Thr Asp
Gln Asn 35 40 45 gtt tct atc gtg gtc atc aaa ggt gca gga cca aaa
gga cta tgt gcc 192Val Ser Ile Val Val Ile Lys Gly Ala Gly Pro Lys
Gly Leu Cys Ala 50 55 60 gga ggg gat att aag gca ctc tat gaa gct
cgt tcg tca aaa cag gcc 240Gly Gly Asp Ile Lys Ala Leu Tyr Glu Ala
Arg Ser Ser Lys Gln Ala 65 70 75 80 ctg caa gat gcc gag cgc ttt ttt
gaa aca gag tac gaa gtc gat atg 288Leu Gln Asp Ala Glu Arg Phe Phe
Glu Thr Glu Tyr Glu Val Asp Met 85 90 95 gca gtc cat cga ttt tcg
aaa ccg atc atc gcc tgc ttg gac ggg atc 336Ala Val His Arg Phe Ser
Lys Pro Ile Ile Ala Cys Leu Asp Gly Ile 100 105 110 gtc atg ggg gga
ggc gtc ggc ctg acg tac ggg gcc agc cac cgg atc 384Val Met Gly Gly
Gly Val Gly Leu Thr Tyr Gly Ala Ser His Arg Ile 115 120 125 gtc acg
gag agg aca aaa tgg gcg atg ccc gaa atg aat atc ggc ttc 432Val Thr
Glu Arg Thr Lys Trp Ala Met Pro Glu Met Asn Ile Gly Phe 130 135 140
ttt ccg gat gtc ggg gca gcc tat ttt tta aac aaa gcc ccg ggc cgc
480Phe Pro Asp Val Gly Ala Ala Tyr Phe Leu Asn Lys Ala Pro Gly Arg
145 150 155 160 tta ggg cgg tat ctt gga tta acg gcg tct gtc atc cat
gca gcc gac 528Leu Gly Arg Tyr Leu Gly Leu Thr Ala Ser Val Ile His
Ala Ala Asp 165 170 175 gtg ctg tat atc aat ggg gca gac gcc tac atg
gag agc ggc gct tta 576Val Leu Tyr Ile Asn Gly Ala Asp Ala Tyr Met
Glu Ser Gly Ala Leu 180 185 190 gaa cga ttg ctt caa gca gtg gaa caa
acc gat tgg cgc ctt gca agc 624Glu Arg Leu Leu Gln Ala Val Glu Gln
Thr Asp Trp Arg Leu Ala Ser 195 200 205 gtt gaa gaa aag ctc gat cag
ctg atc cgc gaa tcg aaa acg gag ccc 672Val Glu Glu Lys Leu Asp Gln
Leu Ile Arg Glu Ser Lys Thr Glu Pro 210 215 220 tcc cag gag agc acg
ctc gcc cgt gat cag caa gcg att gac cgt cat 720Ser Gln Glu Ser Thr
Leu Ala Arg Asp Gln Gln Ala Ile Asp Arg His 225 230 235 240 ttt aag
tat gat aag ctg gaa gag atc ctt caa tcg ctc gaa agc gag 768Phe Lys
Tyr Asp Lys Leu Glu Glu Ile Leu Gln Ser Leu Glu Ser Glu 245 250 255
gga agc acc ttt agc tcg aat gtg aaa aaa aca atg ctt tcc aaa tcg
816Gly Ser Thr Phe Ser Ser Asn Val Lys Lys Thr Met Leu Ser Lys Ser
260 265 270 cca ttt tca tta aaa atc aca ttg aaa cag ctg gcg gac gga
cgt caa 864Pro Phe Ser Leu Lys Ile Thr Leu Lys Gln Leu Ala Asp Gly
Arg Gln 275 280 285 aaa aca ctg gaa gaa tgc ttt gcc acg gat ctg gtg
ctg gca aag aac 912Lys Thr Leu Glu Glu Cys Phe Ala Thr Asp Leu Val
Leu Ala Lys Asn 290 295 300 ttt ttg aag cac aat gat ttc ttc gaa ggc
gtc agg tcc gtc ctg atc 960Phe Leu Lys His Asn Asp Phe Phe Glu Gly
Val Arg Ser Val Leu Ile 305 310 315 320 gac cgt gat caa tca ccg aac
tac aag tac cgg aac gtt tca gat gta 1008Asp Arg Asp Gln Ser Pro Asn
Tyr Lys Tyr Arg Asn Val Ser Asp Val 325 330 335 acc gat gaa gcg gtg
gac cgg ttt ttc caa ccc tct gaa tct gtc cgg 1056Thr Asp Glu Ala Val
Asp Arg Phe Phe Gln Pro Ser Glu Ser Val Arg 340 345 350 ttt taa aag
1065Phe 18353PRTBacillus licheniformis DSM 13 18Met Ser Asp Asp Val
Leu Phe Ser Val Asn Gln Asn Gly Ala Ala Ala 1 5 10 15 Ile Val Leu
Asn Arg Pro Lys Ala Leu Asn Ser Leu Thr Tyr Asp Met 20 25 30 Val
Arg Leu Ile Gly Glu Lys Leu Asn Glu Trp Glu Thr Asp Gln Asn 35 40
45 Val Ser Ile Val Val Ile Lys Gly Ala Gly Pro Lys Gly Leu Cys Ala
50 55 60 Gly Gly Asp Ile Lys Ala Leu Tyr Glu Ala Arg Ser Ser Lys
Gln Ala 65 70 75 80 Leu Gln Asp Ala Glu Arg Phe Phe Glu Thr Glu Tyr
Glu Val Asp Met 85 90 95 Ala Val His Arg Phe Ser Lys Pro Ile Ile
Ala Cys Leu Asp Gly Ile 100 105 110 Val Met Gly Gly Gly Val Gly Leu
Thr Tyr Gly Ala Ser His Arg Ile 115 120 125 Val Thr Glu Arg Thr Lys
Trp Ala Met Pro Glu Met Asn Ile Gly Phe 130 135 140 Phe Pro Asp Val
Gly Ala Ala Tyr Phe Leu Asn Lys Ala Pro Gly Arg 145 150 155 160 Leu
Gly Arg Tyr Leu Gly Leu Thr Ala Ser Val Ile His Ala Ala Asp 165 170
175 Val Leu Tyr Ile Asn Gly Ala Asp Ala Tyr Met Glu Ser Gly Ala Leu
180 185 190 Glu Arg Leu Leu Gln Ala Val Glu Gln Thr Asp Trp Arg Leu
Ala Ser 195 200 205 Val Glu Glu Lys Leu Asp Gln Leu Ile Arg Glu Ser
Lys Thr Glu Pro 210 215 220 Ser Gln Glu Ser Thr Leu Ala Arg Asp Gln
Gln Ala Ile Asp Arg His 225 230 235 240 Phe Lys Tyr Asp Lys Leu Glu
Glu Ile Leu Gln Ser Leu Glu Ser Glu 245 250 255 Gly Ser Thr Phe Ser
Ser Asn Val Lys Lys Thr Met Leu Ser Lys Ser 260 265 270 Pro Phe Ser
Leu Lys Ile Thr Leu Lys Gln Leu Ala Asp Gly Arg Gln 275 280 285 Lys
Thr Leu Glu Glu Cys Phe Ala Thr Asp Leu Val Leu Ala Lys Asn 290 295
300 Phe Leu Lys His Asn Asp Phe Phe Glu Gly Val Arg Ser Val Leu Ile
305 310 315 320 Asp Arg Asp Gln Ser Pro Asn Tyr Lys Tyr Arg Asn Val
Ser Asp Val 325 330 335 Thr Asp Glu Ala Val Asp Arg Phe Phe Gln Pro
Ser Glu Ser Val Arg 340 345 350 Phe 19789DNABacillus licheniformis
DSM 13gene(1)..(789)Probable enoyl-CoA hydratase (E.C. 4.2.1.17),
echA8 19ttg aac ttg aaa tat gcc aca ctt aaa aca gaa cat ggc atc aca
acc 48Leu Asn Leu Lys Tyr Ala Thr Leu Lys Thr Glu His Gly Ile Thr
Thr 1 5 10 15 gtg acg ttg aac aat ccg ccg gca aat aca ctt tct tct
tcc tgt atc 96Val Thr Leu Asn Asn Pro Pro Ala Asn Thr Leu Ser Ser
Ser Cys Ile 20 25 30 gcc gaa ttg cgc tct ctt ttt cgg gaa ttg gcc
cgc gac gag gag aca 144Ala Glu Leu Arg Ser Leu Phe Arg Glu Leu Ala
Arg Asp Glu Glu Thr 35 40 45 aaa gca atc atc att act gga gaa ggc
cgc ttt ttt gtg gcg gga gcg 192Lys Ala Ile Ile Ile Thr Gly Glu Gly
Arg Phe Phe Val Ala Gly Ala 50 55 60 gat ata aaa gaa ttc gtt tca
aaa ctt ggg gac caa aaa caa gga ttg 240Asp Ile Lys Glu Phe Val Ser
Lys Leu Gly Asp Gln Lys Gln Gly Leu 65 70 75 80 gcg ctc gca caa ggg
ggc cag gcg ctc tgc gat gaa atc gaa gct tcc 288Ala Leu Ala Gln Gly
Gly Gln Ala Leu Cys Asp Glu Ile Glu Ala Ser 85 90 95 aaa aaa ccc
gtc att gcg gcg ata aac gga ccg gct ctt ggc gga ggc 336Lys Lys Pro
Val Ile Ala Ala Ile Asn Gly Pro Ala Leu Gly Gly Gly 100 105 110 ctg
gaa ctc gcg atg agc tgc cac ttc aga atc gta tca gac gat gca 384Leu
Glu Leu Ala Met Ser Cys His Phe Arg Ile Val Ser Asp Asp Ala 115 120
125
aca gtc ggt ctt ccc gaa tta aag ctc ggc ttg att cct gca ttt ggt
432Thr Val Gly Leu Pro Glu Leu Lys Leu Gly Leu Ile Pro Ala Phe Gly
130 135 140 ggt aca cag cgg ctt cgc aac ata acg gac aca gcg aca gca
ctc gac 480Gly Thr Gln Arg Leu Arg Asn Ile Thr Asp Thr Ala Thr Ala
Leu Asp 145 150 155 160 ctt atc ctg acg ggc cga tcg ctt tca gct caa
gag gcg gta gag ctg 528Leu Ile Leu Thr Gly Arg Ser Leu Ser Ala Gln
Glu Ala Val Glu Leu 165 170 175 aaa att gca cag atg gct gta aag gga
gag gaa ctg atg aag acg gca 576Lys Ile Ala Gln Met Ala Val Lys Gly
Glu Glu Leu Met Lys Thr Ala 180 185 190 gct gct gtc gcg tcg tct ttt
atc gaa gga aaa agc atg acc agc gtg 624Ala Ala Val Ala Ser Ser Phe
Ile Glu Gly Lys Ser Met Thr Ser Val 195 200 205 agg cgc gcc gtc gaa
tgt gtc gta cag ggc gcc agc gaa agc atg gaa 672Arg Arg Ala Val Glu
Cys Val Val Gln Gly Ala Ser Glu Ser Met Glu 210 215 220 caa gca ctg
gag agg gag cga aac aga ttc gcc gag ctg ttt gtc act 720Gln Ala Leu
Glu Arg Glu Arg Asn Arg Phe Ala Glu Leu Phe Val Thr 225 230 235 240
tca gat gcc aaa gaa gga att cac gca ttt gtc gaa aag cgc aaa cca
768Ser Asp Ala Lys Glu Gly Ile His Ala Phe Val Glu Lys Arg Lys Pro
245 250 255 aac ttt cat cat tca taa aag 789Asn Phe His His Ser 260
20261PRTBacillus licheniformis DSM 13 20Leu Asn Leu Lys Tyr Ala Thr
Leu Lys Thr Glu His Gly Ile Thr Thr 1 5 10 15 Val Thr Leu Asn Asn
Pro Pro Ala Asn Thr Leu Ser Ser Ser Cys Ile 20 25 30 Ala Glu Leu
Arg Ser Leu Phe Arg Glu Leu Ala Arg Asp Glu Glu Thr 35 40 45 Lys
Ala Ile Ile Ile Thr Gly Glu Gly Arg Phe Phe Val Ala Gly Ala 50 55
60 Asp Ile Lys Glu Phe Val Ser Lys Leu Gly Asp Gln Lys Gln Gly Leu
65 70 75 80 Ala Leu Ala Gln Gly Gly Gln Ala Leu Cys Asp Glu Ile Glu
Ala Ser 85 90 95 Lys Lys Pro Val Ile Ala Ala Ile Asn Gly Pro Ala
Leu Gly Gly Gly 100 105 110 Leu Glu Leu Ala Met Ser Cys His Phe Arg
Ile Val Ser Asp Asp Ala 115 120 125 Thr Val Gly Leu Pro Glu Leu Lys
Leu Gly Leu Ile Pro Ala Phe Gly 130 135 140 Gly Thr Gln Arg Leu Arg
Asn Ile Thr Asp Thr Ala Thr Ala Leu Asp 145 150 155 160 Leu Ile Leu
Thr Gly Arg Ser Leu Ser Ala Gln Glu Ala Val Glu Leu 165 170 175 Lys
Ile Ala Gln Met Ala Val Lys Gly Glu Glu Leu Met Lys Thr Ala 180 185
190 Ala Ala Val Ala Ser Ser Phe Ile Glu Gly Lys Ser Met Thr Ser Val
195 200 205 Arg Arg Ala Val Glu Cys Val Val Gln Gly Ala Ser Glu Ser
Met Glu 210 215 220 Gln Ala Leu Glu Arg Glu Arg Asn Arg Phe Ala Glu
Leu Phe Val Thr 225 230 235 240 Ser Asp Ala Lys Glu Gly Ile His Ala
Phe Val Glu Lys Arg Lys Pro 245 250 255 Asn Phe His His Ser 260
211719DNABacillus licheniformis DSM 13gene(1)..(1719)Acyl-CoA
dehydrogenase (E.C. 1.3.99.-) 21atg ggg aaa gca aaa ctt cga tgg aat
gaa ccc ttg att tct caa cac 48Met Gly Lys Ala Lys Leu Arg Trp Asn
Glu Pro Leu Ile Ser Gln His 1 5 10 15 gaa tcg gct gct gaa gga ttt
aca ccg gaa gat ttc aca gag gaa gat 96Glu Ser Ala Ala Glu Gly Phe
Thr Pro Glu Asp Phe Thr Glu Glu Asp 20 25 30 cga ctg att tca aaa
acg aca gaa tca ttt gtc aaa aac gaa gtc atg 144Arg Leu Ile Ser Lys
Thr Thr Glu Ser Phe Val Lys Asn Glu Val Met 35 40 45 ccc ctt ctt
gaa tcg att gat cag cag gat cac gaa agc gtg aaa aaa 192Pro Leu Leu
Glu Ser Ile Asp Gln Gln Asp His Glu Ser Val Lys Lys 50 55 60 ttg
ttt caa aaa gca gga gag ctc ggt ttg ctc agt atc gaa gtt ccg 240Leu
Phe Gln Lys Ala Gly Glu Leu Gly Leu Leu Ser Ile Glu Val Pro 65 70
75 80 gag gat tgc ggc ggc ctt tca ctc agc aag aag ctt tcc ggg ttg
gtg 288Glu Asp Cys Gly Gly Leu Ser Leu Ser Lys Lys Leu Ser Gly Leu
Val 85 90 95 gca gag aaa atg gga gcc ggc gga tcg ttc agc gtc tcc
ttt aat att 336Ala Glu Lys Met Gly Ala Gly Gly Ser Phe Ser Val Ser
Phe Asn Ile 100 105 110 cat gcg gga gtc ggg aca ctg ccg tat att tat
tat gga aca gag gaa 384His Ala Gly Val Gly Thr Leu Pro Tyr Ile Tyr
Tyr Gly Thr Glu Glu 115 120 125 caa aaa caa aaa tac ctt cca aaa ctg
gca tcg ggc gaa tgg atc gga 432Gln Lys Gln Lys Tyr Leu Pro Lys Leu
Ala Ser Gly Glu Trp Ile Gly 130 135 140 gca tat gct ctg aca gag ccg
ggc gca gga tcg gat gct tta aac gca 480Ala Tyr Ala Leu Thr Glu Pro
Gly Ala Gly Ser Asp Ala Leu Asn Ala 145 150 155 160 aaa acg aca gcc
gtc ttg aat agg gaa ggg aca gcc tgg att tta aat 528Lys Thr Thr Ala
Val Leu Asn Arg Glu Gly Thr Ala Trp Ile Leu Asn 165 170 175 ggg gaa
aag cag tgg att acg aac gcg caa gta gct gat gta tat gtt 576Gly Glu
Lys Gln Trp Ile Thr Asn Ala Gln Val Ala Asp Val Tyr Val 180 185 190
gtc ttt gca aaa acg gcg gaa ggc atg aca gca ttt atc gtc gaa cgc
624Val Phe Ala Lys Thr Ala Glu Gly Met Thr Ala Phe Ile Val Glu Arg
195 200 205 tcg ttt aaa ggt gtt tcc atc gga cct gaa gag aag aag atg
gga atc 672Ser Phe Lys Gly Val Ser Ile Gly Pro Glu Glu Lys Lys Met
Gly Ile 210 215 220 aaa ggg tct tcg aca gca acc tta atc ttg gag gaa
gtc gag gtg cca 720Lys Gly Ser Ser Thr Ala Thr Leu Ile Leu Glu Glu
Val Glu Val Pro 225 230 235 240 agc gac aat gtt cta ggt cat atc ggg
aaa ggt cat cac gtc gct ttg 768Ser Asp Asn Val Leu Gly His Ile Gly
Lys Gly His His Val Ala Leu 245 250 255 aac att tta aac atg gcc cgc
tta aag ctc gcg ttt tcg aac att gga 816Asn Ile Leu Asn Met Ala Arg
Leu Lys Leu Ala Phe Ser Asn Ile Gly 260 265 270 acg gca aaa caa gca
ttg aac ctt gct gtt agc tac gcc aaa cag cga 864Thr Ala Lys Gln Ala
Leu Asn Leu Ala Val Ser Tyr Ala Lys Gln Arg 275 280 285 aag caa ttt
aac aag ccg atc atc ggt ttt tca atg att caa gaa aag 912Lys Gln Phe
Asn Lys Pro Ile Ile Gly Phe Ser Met Ile Gln Glu Lys 290 295 300 att
gcc gac atg gcg gtc tcg att ttc ggc gcg gaa agc gct gct tac 960Ile
Ala Asp Met Ala Val Ser Ile Phe Gly Ala Glu Ser Ala Ala Tyr 305 310
315 320 aga acg gca gat tgc ttg gac aat gtt tta gat tcg gct ctc cca
tta 1008Arg Thr Ala Asp Cys Leu Asp Asn Val Leu Asp Ser Ala Leu Pro
Leu 325 330 335 gac gat aga ctg aga aaa ctc aca aac tat gca tcc gaa
tgt gcg atc 1056Asp Asp Arg Leu Arg Lys Leu Thr Asn Tyr Ala Ser Glu
Cys Ala Ile 340 345 350 aat aaa gtt tac tgc tcc gaa atc ctc ggc cgg
atc gca gac gaa gcg 1104Asn Lys Val Tyr Cys Ser Glu Ile Leu Gly Arg
Ile Ala Asp Glu Ala 355 360 365 gtg cag att cat ggc ggc tac gga tac
atg cag gag tac gaa gtc gaa 1152Val Gln Ile His Gly Gly Tyr Gly Tyr
Met Gln Glu Tyr Glu Val Glu 370 375 380 cga ttg tac cgg gac gcg agg
atc agc cgg att ttc gag ggg aca aat 1200Arg Leu Tyr Arg Asp Ala Arg
Ile Ser Arg Ile Phe Glu Gly Thr Asn 385 390 395 400 gaa ata aac cgc
tta acg atc gcc aaa ctg ctc atg aaa gaa gtg cag 1248Glu Ile Asn Arg
Leu Thr Ile Ala Lys Leu Leu Met Lys Glu Val Gln 405 410 415 caa aac
ggc atc tca gag ccg gaa gct caa cta ggc agc gag gga aat 1296Gln Asn
Gly Ile Ser Glu Pro Glu Ala Gln Leu Gly Ser Glu Gly Asn 420 425 430
cga aac cgg cga ttt att cag ctg tcc aac agg ctt ttc ggc aag aca
1344Arg Asn Arg Arg Phe Ile Gln Leu Ser Asn Arg Leu Phe Gly Lys Thr
435 440 445 ctg aaa gcg ctt atc cgc tct cgc gtg aac act caa gaa gat
cag gaa 1392Leu Lys Ala Leu Ile Arg Ser Arg Val Asn Thr Gln Glu Asp
Gln Glu 450 455 460 tac gca cgg ctt ctc gcc gac atg aaa aaa gaa atc
tat gtg atg gaa 1440Tyr Ala Arg Leu Leu Ala Asp Met Lys Lys Glu Ile
Tyr Val Met Glu 465 470 475 480 tcc gcc gcc cgc cga acc gaa aaa gct
agg caa ata tac ggt ggt gaa 1488Ser Ala Ala Arg Arg Thr Glu Lys Ala
Arg Gln Ile Tyr Gly Gly Glu 485 490 495 aaa gca cgg ttg aaa gaa atg
atg acg aat gtc att tgt gaa gaa ggc 1536Lys Ala Arg Leu Lys Glu Met
Met Thr Asn Val Ile Cys Glu Glu Gly 500 505 510 tac cgc aga atc gaa
gag atg gcg gtg acc gcc tta tca agc ata gca 1584Tyr Arg Arg Ile Glu
Glu Met Ala Val Thr Ala Leu Ser Ser Ile Ala 515 520 525 tct gat gaa
gcc gaa aga aaa ctc gca ttt gaa gag gct cgc agc gtt 1632Ser Asp Glu
Ala Glu Arg Lys Leu Ala Phe Glu Glu Ala Arg Ser Val 530 535 540 tct
ttg cct ctt ttc agc aat ctg ttt act caa aaa cgc gaa atc gca 1680Ser
Leu Pro Leu Phe Ser Asn Leu Phe Thr Gln Lys Arg Glu Ile Ala 545 550
555 560 gaa aaa ata gcc gct cat gaa aaa tat acg gtg tga tcg 1719Glu
Lys Ile Ala Ala His Glu Lys Tyr Thr Val 565 570 22571PRTBacillus
licheniformis DSM 13 22Met Gly Lys Ala Lys Leu Arg Trp Asn Glu Pro
Leu Ile Ser Gln His 1 5 10 15 Glu Ser Ala Ala Glu Gly Phe Thr Pro
Glu Asp Phe Thr Glu Glu Asp 20 25 30 Arg Leu Ile Ser Lys Thr Thr
Glu Ser Phe Val Lys Asn Glu Val Met 35 40 45 Pro Leu Leu Glu Ser
Ile Asp Gln Gln Asp His Glu Ser Val Lys Lys 50 55 60 Leu Phe Gln
Lys Ala Gly Glu Leu Gly Leu Leu Ser Ile Glu Val Pro 65 70 75 80 Glu
Asp Cys Gly Gly Leu Ser Leu Ser Lys Lys Leu Ser Gly Leu Val 85 90
95 Ala Glu Lys Met Gly Ala Gly Gly Ser Phe Ser Val Ser Phe Asn Ile
100 105 110 His Ala Gly Val Gly Thr Leu Pro Tyr Ile Tyr Tyr Gly Thr
Glu Glu 115 120 125 Gln Lys Gln Lys Tyr Leu Pro Lys Leu Ala Ser Gly
Glu Trp Ile Gly 130 135 140 Ala Tyr Ala Leu Thr Glu Pro Gly Ala Gly
Ser Asp Ala Leu Asn Ala 145 150 155 160 Lys Thr Thr Ala Val Leu Asn
Arg Glu Gly Thr Ala Trp Ile Leu Asn 165 170 175 Gly Glu Lys Gln Trp
Ile Thr Asn Ala Gln Val Ala Asp Val Tyr Val 180 185 190 Val Phe Ala
Lys Thr Ala Glu Gly Met Thr Ala Phe Ile Val Glu Arg 195 200 205 Ser
Phe Lys Gly Val Ser Ile Gly Pro Glu Glu Lys Lys Met Gly Ile 210 215
220 Lys Gly Ser Ser Thr Ala Thr Leu Ile Leu Glu Glu Val Glu Val Pro
225 230 235 240 Ser Asp Asn Val Leu Gly His Ile Gly Lys Gly His His
Val Ala Leu 245 250 255 Asn Ile Leu Asn Met Ala Arg Leu Lys Leu Ala
Phe Ser Asn Ile Gly 260 265 270 Thr Ala Lys Gln Ala Leu Asn Leu Ala
Val Ser Tyr Ala Lys Gln Arg 275 280 285 Lys Gln Phe Asn Lys Pro Ile
Ile Gly Phe Ser Met Ile Gln Glu Lys 290 295 300 Ile Ala Asp Met Ala
Val Ser Ile Phe Gly Ala Glu Ser Ala Ala Tyr 305 310 315 320 Arg Thr
Ala Asp Cys Leu Asp Asn Val Leu Asp Ser Ala Leu Pro Leu 325 330 335
Asp Asp Arg Leu Arg Lys Leu Thr Asn Tyr Ala Ser Glu Cys Ala Ile 340
345 350 Asn Lys Val Tyr Cys Ser Glu Ile Leu Gly Arg Ile Ala Asp Glu
Ala 355 360 365 Val Gln Ile His Gly Gly Tyr Gly Tyr Met Gln Glu Tyr
Glu Val Glu 370 375 380 Arg Leu Tyr Arg Asp Ala Arg Ile Ser Arg Ile
Phe Glu Gly Thr Asn 385 390 395 400 Glu Ile Asn Arg Leu Thr Ile Ala
Lys Leu Leu Met Lys Glu Val Gln 405 410 415 Gln Asn Gly Ile Ser Glu
Pro Glu Ala Gln Leu Gly Ser Glu Gly Asn 420 425 430 Arg Asn Arg Arg
Phe Ile Gln Leu Ser Asn Arg Leu Phe Gly Lys Thr 435 440 445 Leu Lys
Ala Leu Ile Arg Ser Arg Val Asn Thr Gln Glu Asp Gln Glu 450 455 460
Tyr Ala Arg Leu Leu Ala Asp Met Lys Lys Glu Ile Tyr Val Met Glu 465
470 475 480 Ser Ala Ala Arg Arg Thr Glu Lys Ala Arg Gln Ile Tyr Gly
Gly Glu 485 490 495 Lys Ala Arg Leu Lys Glu Met Met Thr Asn Val Ile
Cys Glu Glu Gly 500 505 510 Tyr Arg Arg Ile Glu Glu Met Ala Val Thr
Ala Leu Ser Ser Ile Ala 515 520 525 Ser Asp Glu Ala Glu Arg Lys Leu
Ala Phe Glu Glu Ala Arg Ser Val 530 535 540 Ser Leu Pro Leu Phe Ser
Asn Leu Phe Thr Gln Lys Arg Glu Ile Ala 545 550 555 560 Glu Lys Ile
Ala Ala His Glu Lys Tyr Thr Val 565 570 231956DNABacillus
licheniformis DSM 13gene(1)..(1956)Acetyl-coenzyme A synthetase
(E.C. 6.2.1.1), acsA 23atg ggc gaa aaa gcg gtt tgg cag cct gat ccg
gaa ttt gta aaa aca 48Met Gly Glu Lys Ala Val Trp Gln Pro Asp Pro
Glu Phe Val Lys Thr 1 5 10 15 acc cgg ctg ttt caa tgg atg aca gcc
ctc ggt ttt tcc gac tat gat 96Thr Arg Leu Phe Gln Trp Met Thr Ala
Leu Gly Phe Ser Asp Tyr Asp 20 25 30 gat ttt ttg aaa gca agt aca
aac gat atc gcc tgg ttc tgg gaa gag 144Asp Phe Leu Lys Ala Ser Thr
Asn Asp Ile Ala Trp Phe Trp Glu Glu 35 40 45 gcc gaa aaa gcg ctc
ggg atc agc tgg tac aag cgg tac agc caa aca 192Ala Glu Lys Ala Leu
Gly Ile Ser Trp Tyr Lys Arg Tyr Ser Gln Thr 50 55 60 ttg aat ctc
gac aaa ggc ata aaa tgg ccg caa tgg ttt acc ggc ggc 240Leu Asn Leu
Asp Lys Gly Ile Lys Trp Pro Gln Trp Phe Thr Gly Gly 65 70 75 80 cgc
tta aat gcc gtt tac aac gcc gtg gaa aaa tgg gcc cgc cgg cct 288Arg
Leu Asn Ala Val Tyr Asn Ala Val Glu Lys Trp Ala Arg Arg Pro 85 90
95 gat acg gcc ggc agg acg gca ctc atc tgg gaa agt gaa gac gga aaa
336Asp Thr Ala Gly Arg Thr Ala Leu Ile Trp Glu Ser Glu Asp Gly Lys
100 105 110 aca gaa cag atc acc tat tct tct tta cac caa caa gtc gcc
cgt gcg
384Thr Glu Gln Ile Thr Tyr Ser Ser Leu His Gln Gln Val Ala Arg Ala
115 120 125 gcg gca ggc ttt aaa aag caa ggc atc tca aaa ggg gat gtc
att gcg 432Ala Ala Gly Phe Lys Lys Gln Gly Ile Ser Lys Gly Asp Val
Ile Ala 130 135 140 att tac atg ccg atg atc ccc gaa acg gtc atc gcc
atg ctg gcc gcc 480Ile Tyr Met Pro Met Ile Pro Glu Thr Val Ile Ala
Met Leu Ala Ala 145 150 155 160 gct aaa atc gga gcg gta ttc tca ccg
gtt ttt tca ggc tac ggc gcc 528Ala Lys Ile Gly Ala Val Phe Ser Pro
Val Phe Ser Gly Tyr Gly Ala 165 170 175 cat gca gca gcg gcg aga ctt
acc gct gcc gga gcg aaa atc ctt gtc 576His Ala Ala Ala Ala Arg Leu
Thr Ala Ala Gly Ala Lys Ile Leu Val 180 185 190 aca gca gat gcc ttt
ttg cga agg gga aag aag gtc tgc atg aag aaa 624Thr Ala Asp Ala Phe
Leu Arg Arg Gly Lys Lys Val Cys Met Lys Lys 195 200 205 gaa gct gac
aaa gcc gcg gac cgt tcc ccc act gtt caa aaa gtc gtc 672Glu Ala Asp
Lys Ala Ala Asp Arg Ser Pro Thr Val Gln Lys Val Val 210 215 220 gtc
tgc aag ctt cac ggc ggc gat caa gat tgg aat tat aag aga gat 720Val
Cys Lys Leu His Gly Gly Asp Gln Asp Trp Asn Tyr Lys Arg Asp 225 230
235 240 atc gac tgg aat gaa ttg atg aaa aac gag ccc atg caa aac acc
gaa 768Ile Asp Trp Asn Glu Leu Met Lys Asn Glu Pro Met Gln Asn Thr
Glu 245 250 255 gaa atg gac agt tca gat ccg ctc atg ctg cta tac aca
tca ggg acg 816Glu Met Asp Ser Ser Asp Pro Leu Met Leu Leu Tyr Thr
Ser Gly Thr 260 265 270 aca gga cag tcg aag gga gcg gtt cat acc cat
gcc ggt ttt ccg ctg 864Thr Gly Gln Ser Lys Gly Ala Val His Thr His
Ala Gly Phe Pro Leu 275 280 285 aaa gct gca ttt gat gcg gga ttc ggg
atg gat gtc aaa caa ggg gac 912Lys Ala Ala Phe Asp Ala Gly Phe Gly
Met Asp Val Lys Gln Gly Asp 290 295 300 aca ttt ttc tgg ttt aca gac
atg ggc tgg atg atg ggg ccg ttt tta 960Thr Phe Phe Trp Phe Thr Asp
Met Gly Trp Met Met Gly Pro Phe Leu 305 310 315 320 ata ttc ggg ggc
ctc ata aac gga gcg gct gtt ttg ctg ttt gac gga 1008Ile Phe Gly Gly
Leu Ile Asn Gly Ala Ala Val Leu Leu Phe Asp Gly 325 330 335 gca ccg
gac tac ccg gcc ccg gat cgg ctg tgg gag ctt gtc agc aga 1056Ala Pro
Asp Tyr Pro Ala Pro Asp Arg Leu Trp Glu Leu Val Ser Arg 340 345 350
cac cgg gtg acg cat ctc ggc gtc tct ccg acg ctc att cgc tcg ctg
1104His Arg Val Thr His Leu Gly Val Ser Pro Thr Leu Ile Arg Ser Leu
355 360 365 atg cag cac ggc gaa gat ttt ctc tat caa tac aat ctg aac
agt ctg 1152Met Gln His Gly Glu Asp Phe Leu Tyr Gln Tyr Asn Leu Asn
Ser Leu 370 375 380 aag gca atc ggc tca acg ggc gaa cca tgg aat tat
gag ccg tgg atg 1200Lys Ala Ile Gly Ser Thr Gly Glu Pro Trp Asn Tyr
Glu Pro Trp Met 385 390 395 400 tgg ctg ttc cgc cat gtt gga aaa gaa
cgg att cct ata ttt aat tat 1248Trp Leu Phe Arg His Val Gly Lys Glu
Arg Ile Pro Ile Phe Asn Tyr 405 410 415 tca gga gga aca gag atc tca
ggc gga att tta ggc aat gtg ctc ctg 1296Ser Gly Gly Thr Glu Ile Ser
Gly Gly Ile Leu Gly Asn Val Leu Leu 420 425 430 cgg ccg atc acg ccg
atg acg ttt aat tcg cct ctt ccc ggc atg gcg 1344Arg Pro Ile Thr Pro
Met Thr Phe Asn Ser Pro Leu Pro Gly Met Ala 435 440 445 gcc aat gtc
ttc aat gaa aaa gga gag gaa gtc gtc aat gaa gtc gga 1392Ala Asn Val
Phe Asn Glu Lys Gly Glu Glu Val Val Asn Glu Val Gly 450 455 460 gag
ctt gtc ctg aca aag ccc tgg gtc ggc atg acg aac ggt ttt tgg 1440Glu
Leu Val Leu Thr Lys Pro Trp Val Gly Met Thr Asn Gly Phe Trp 465 470
475 480 aag gag ccg tca aga tac gaa gaa gca tat tgg agc cgc tgg acc
gac 1488Lys Glu Pro Ser Arg Tyr Glu Glu Ala Tyr Trp Ser Arg Trp Thr
Asp 485 490 495 gtc tgg gtg cac ggc gat tgg gca aaa cgg gat gaa aac
ggc tac tgg 1536Val Trp Val His Gly Asp Trp Ala Lys Arg Asp Glu Asn
Gly Tyr Trp 500 505 510 acg atc agc gga cgc tct gat gat gtg atc aat
gct gcc ggg aaa agg 1584Thr Ile Ser Gly Arg Ser Asp Asp Val Ile Asn
Ala Ala Gly Lys Arg 515 520 525 atc ggc ccc gcc gaa ata gag tcc gtt
ctg gtc ggc cat cca gcg gtg 1632Ile Gly Pro Ala Glu Ile Glu Ser Val
Leu Val Gly His Pro Ala Val 530 535 540 gcg gaa gca ggc gtc atc ggc
gtt ccg gat aag ctc aaa ggc cag gct 1680Ala Glu Ala Gly Val Ile Gly
Val Pro Asp Lys Leu Lys Gly Gln Ala 545 550 555 560 gcc gtc tgc ttc
gtc gtc ctc aga cag tcg gaa aag ccg tcg gaa gaa 1728Ala Val Cys Phe
Val Val Leu Arg Gln Ser Glu Lys Pro Ser Glu Glu 565 570 575 tta aaa
gat gat ttg ctg aac ctt gca tct gat gcg atc ggt aaa gcg 1776Leu Lys
Asp Asp Leu Leu Asn Leu Ala Ser Asp Ala Ile Gly Lys Ala 580 585 590
gtc aag ccc aaa gcg gtt tat ttt gtc agc ggt ttg ccg aag acg aga
1824Val Lys Pro Lys Ala Val Tyr Phe Val Ser Gly Leu Pro Lys Thr Arg
595 600 605 aat gca aaa gtg atg aga cgg ctg atc aga gct gcc tat atg
aac gag 1872Asn Ala Lys Val Met Arg Arg Leu Ile Arg Ala Ala Tyr Met
Asn Glu 610 615 620 ccc gca ggc gat ttg tca act ttg gaa aac cgc gaa
aca tat gat gaa 1920Pro Ala Gly Asp Leu Ser Thr Leu Glu Asn Arg Glu
Thr Tyr Asp Glu 625 630 635 640 att gcc ggt ctt tca gtg cga aaa aat
ctg tag tat 1956Ile Ala Gly Leu Ser Val Arg Lys Asn Leu 645 650
24650PRTBacillus licheniformis DSM 13 24Met Gly Glu Lys Ala Val Trp
Gln Pro Asp Pro Glu Phe Val Lys Thr 1 5 10 15 Thr Arg Leu Phe Gln
Trp Met Thr Ala Leu Gly Phe Ser Asp Tyr Asp 20 25 30 Asp Phe Leu
Lys Ala Ser Thr Asn Asp Ile Ala Trp Phe Trp Glu Glu 35 40 45 Ala
Glu Lys Ala Leu Gly Ile Ser Trp Tyr Lys Arg Tyr Ser Gln Thr 50 55
60 Leu Asn Leu Asp Lys Gly Ile Lys Trp Pro Gln Trp Phe Thr Gly Gly
65 70 75 80 Arg Leu Asn Ala Val Tyr Asn Ala Val Glu Lys Trp Ala Arg
Arg Pro 85 90 95 Asp Thr Ala Gly Arg Thr Ala Leu Ile Trp Glu Ser
Glu Asp Gly Lys 100 105 110 Thr Glu Gln Ile Thr Tyr Ser Ser Leu His
Gln Gln Val Ala Arg Ala 115 120 125 Ala Ala Gly Phe Lys Lys Gln Gly
Ile Ser Lys Gly Asp Val Ile Ala 130 135 140 Ile Tyr Met Pro Met Ile
Pro Glu Thr Val Ile Ala Met Leu Ala Ala 145 150 155 160 Ala Lys Ile
Gly Ala Val Phe Ser Pro Val Phe Ser Gly Tyr Gly Ala 165 170 175 His
Ala Ala Ala Ala Arg Leu Thr Ala Ala Gly Ala Lys Ile Leu Val 180 185
190 Thr Ala Asp Ala Phe Leu Arg Arg Gly Lys Lys Val Cys Met Lys Lys
195 200 205 Glu Ala Asp Lys Ala Ala Asp Arg Ser Pro Thr Val Gln Lys
Val Val 210 215 220 Val Cys Lys Leu His Gly Gly Asp Gln Asp Trp Asn
Tyr Lys Arg Asp 225 230 235 240 Ile Asp Trp Asn Glu Leu Met Lys Asn
Glu Pro Met Gln Asn Thr Glu 245 250 255 Glu Met Asp Ser Ser Asp Pro
Leu Met Leu Leu Tyr Thr Ser Gly Thr 260 265 270 Thr Gly Gln Ser Lys
Gly Ala Val His Thr His Ala Gly Phe Pro Leu 275 280 285 Lys Ala Ala
Phe Asp Ala Gly Phe Gly Met Asp Val Lys Gln Gly Asp 290 295 300 Thr
Phe Phe Trp Phe Thr Asp Met Gly Trp Met Met Gly Pro Phe Leu 305 310
315 320 Ile Phe Gly Gly Leu Ile Asn Gly Ala Ala Val Leu Leu Phe Asp
Gly 325 330 335 Ala Pro Asp Tyr Pro Ala Pro Asp Arg Leu Trp Glu Leu
Val Ser Arg 340 345 350 His Arg Val Thr His Leu Gly Val Ser Pro Thr
Leu Ile Arg Ser Leu 355 360 365 Met Gln His Gly Glu Asp Phe Leu Tyr
Gln Tyr Asn Leu Asn Ser Leu 370 375 380 Lys Ala Ile Gly Ser Thr Gly
Glu Pro Trp Asn Tyr Glu Pro Trp Met 385 390 395 400 Trp Leu Phe Arg
His Val Gly Lys Glu Arg Ile Pro Ile Phe Asn Tyr 405 410 415 Ser Gly
Gly Thr Glu Ile Ser Gly Gly Ile Leu Gly Asn Val Leu Leu 420 425 430
Arg Pro Ile Thr Pro Met Thr Phe Asn Ser Pro Leu Pro Gly Met Ala 435
440 445 Ala Asn Val Phe Asn Glu Lys Gly Glu Glu Val Val Asn Glu Val
Gly 450 455 460 Glu Leu Val Leu Thr Lys Pro Trp Val Gly Met Thr Asn
Gly Phe Trp 465 470 475 480 Lys Glu Pro Ser Arg Tyr Glu Glu Ala Tyr
Trp Ser Arg Trp Thr Asp 485 490 495 Val Trp Val His Gly Asp Trp Ala
Lys Arg Asp Glu Asn Gly Tyr Trp 500 505 510 Thr Ile Ser Gly Arg Ser
Asp Asp Val Ile Asn Ala Ala Gly Lys Arg 515 520 525 Ile Gly Pro Ala
Glu Ile Glu Ser Val Leu Val Gly His Pro Ala Val 530 535 540 Ala Glu
Ala Gly Val Ile Gly Val Pro Asp Lys Leu Lys Gly Gln Ala 545 550 555
560 Ala Val Cys Phe Val Val Leu Arg Gln Ser Glu Lys Pro Ser Glu Glu
565 570 575 Leu Lys Asp Asp Leu Leu Asn Leu Ala Ser Asp Ala Ile Gly
Lys Ala 580 585 590 Val Lys Pro Lys Ala Val Tyr Phe Val Ser Gly Leu
Pro Lys Thr Arg 595 600 605 Asn Ala Lys Val Met Arg Arg Leu Ile Arg
Ala Ala Tyr Met Asn Glu 610 615 620 Pro Ala Gly Asp Leu Ser Thr Leu
Glu Asn Arg Glu Thr Tyr Asp Glu 625 630 635 640 Ile Ala Gly Leu Ser
Val Arg Lys Asn Leu 645 650 25786DNABacillus licheniformis DSM
13gene(1)..(786)3-hydroxybutyryl-CoA dehydratase (E.C. 4.2.1.55),
yngF 25atg gag ccc aat gtt ctt tat tcc ata aac gag cag tcg gtc gcc
gtg 48Met Glu Pro Asn Val Leu Tyr Ser Ile Asn Glu Gln Ser Val Ala
Val 1 5 10 15 ttg acg ctg aac agg ccg cag gct gca aat gcc ctc tcg
ctt ggg ctt 96Leu Thr Leu Asn Arg Pro Gln Ala Ala Asn Ala Leu Ser
Leu Gly Leu 20 25 30 ctc gac gac ttt cag cgc atc ctt cga gat att
cgt tca aac ccg gcc 144Leu Asp Asp Phe Gln Arg Ile Leu Arg Asp Ile
Arg Ser Asn Pro Ala 35 40 45 gtc cgc tgt gtc atc ata acg gga aaa
ggg gac agg acg ttt tgt gca 192Val Arg Cys Val Ile Ile Thr Gly Lys
Gly Asp Arg Thr Phe Cys Ala 50 55 60 ggc gcc gat tta aag gaa aga
gcc cgc atg agc caa aca gaa gcg aag 240Gly Ala Asp Leu Lys Glu Arg
Ala Arg Met Ser Gln Thr Glu Ala Lys 65 70 75 80 cag gct gtt tcc ctg
att caa cgc gtg gtc agc gaa acg gaa aaa ctg 288Gln Ala Val Ser Leu
Ile Gln Arg Val Val Ser Glu Thr Glu Lys Leu 85 90 95 ccg cag ccc
gtc atc gct tca tta aac gga agc gct tta gga ggg ggg 336Pro Gln Pro
Val Ile Ala Ser Leu Asn Gly Ser Ala Leu Gly Gly Gly 100 105 110 ctg
gag ctt gca ttg gcg tgc gac atc agg atc gca gcc gaa cat att 384Leu
Glu Leu Ala Leu Ala Cys Asp Ile Arg Ile Ala Ala Glu His Ile 115 120
125 gaa ctc ggc ctc ccc gaa aca acg ctc gca atc att cca ggg gca gga
432Glu Leu Gly Leu Pro Glu Thr Thr Leu Ala Ile Ile Pro Gly Ala Gly
130 135 140 ggg aca cag cgg ctg ccc cgc ttg atc ggc agg gga aag gcg
aaa gaa 480Gly Thr Gln Arg Leu Pro Arg Leu Ile Gly Arg Gly Lys Ala
Lys Glu 145 150 155 160 atg atc ttt acc ggc tgc cgc atc agc gcc gaa
gaa gcg caa aag atc 528Met Ile Phe Thr Gly Cys Arg Ile Ser Ala Glu
Glu Ala Gln Lys Ile 165 170 175 agc ctg gtt gaa cat gtc gtt ccg ctt
tcg aag tta aag gaa gcg agt 576Ser Leu Val Glu His Val Val Pro Leu
Ser Lys Leu Lys Glu Ala Ser 180 185 190 gaa agc atc gcg gcg aac atc
gcg gcg aac gga ccg gta gcc gtc aga 624Glu Ser Ile Ala Ala Asn Ile
Ala Ala Asn Gly Pro Val Ala Val Arg 195 200 205 caa gcg aag ttt gcc
atc aat caa ggc ctt gag aca gct atc gaa aca 672Gln Ala Lys Phe Ala
Ile Asn Gln Gly Leu Glu Thr Ala Ile Glu Thr 210 215 220 ggg ctt gcc
att gaa caa aaa gcc tat gaa ctg acg att ccg acg aaa 720Gly Leu Ala
Ile Glu Gln Lys Ala Tyr Glu Leu Thr Ile Pro Thr Lys 225 230 235 240
gac agg aca gaa ggg ctg aaa gct ttt gca gaa aag cgg aag ccg gat
768Asp Arg Thr Glu Gly Leu Lys Ala Phe Ala Glu Lys Arg Lys Pro Asp
245 250 255 tat acg gga gaa taa aac 786Tyr Thr Gly Glu 260
26260PRTBacillus licheniformis DSM 13 26Met Glu Pro Asn Val Leu Tyr
Ser Ile Asn Glu Gln Ser Val Ala Val 1 5 10 15 Leu Thr Leu Asn Arg
Pro Gln Ala Ala Asn Ala Leu Ser Leu Gly Leu 20 25 30 Leu Asp Asp
Phe Gln Arg Ile Leu Arg Asp Ile Arg Ser Asn Pro Ala 35 40 45 Val
Arg Cys Val Ile Ile Thr Gly Lys Gly Asp Arg Thr Phe Cys Ala 50 55
60 Gly Ala Asp Leu Lys Glu Arg Ala Arg Met Ser Gln Thr Glu Ala Lys
65 70 75 80 Gln Ala Val Ser Leu Ile Gln Arg Val Val Ser Glu Thr Glu
Lys Leu 85 90 95 Pro Gln Pro Val Ile Ala Ser Leu Asn Gly Ser Ala
Leu Gly Gly Gly 100 105 110 Leu Glu Leu Ala Leu Ala Cys Asp Ile Arg
Ile Ala Ala Glu His Ile 115 120 125 Glu Leu Gly Leu Pro Glu Thr Thr
Leu Ala Ile Ile Pro Gly Ala Gly 130 135 140 Gly Thr Gln Arg Leu Pro
Arg Leu Ile Gly Arg Gly Lys Ala Lys Glu 145 150 155 160 Met Ile Phe
Thr Gly Cys Arg Ile Ser Ala Glu Glu Ala Gln Lys Ile 165 170 175 Ser
Leu Val Glu His Val Val Pro Leu Ser Lys Leu Lys Glu Ala Ser 180 185
190 Glu Ser Ile Ala Ala Asn Ile Ala Ala Asn Gly Pro Val Ala Val Arg
195 200 205 Gln Ala Lys Phe Ala Ile Asn Gln Gly Leu Glu Thr Ala Ile
Glu Thr 210 215 220 Gly Leu Ala Ile Glu Gln Lys Ala Tyr Glu Leu Thr
Ile Pro Thr Lys 225
230 235 240 Asp Arg Thr Glu Gly Leu Lys Ala Phe Ala Glu Lys Arg Lys
Pro Asp 245 250 255 Tyr Thr Gly Glu 260 271164DNABacillus
licheniformis DSM 13gene(1)..(1164)Acyl-CoA dehydrogenase (E.C.
1.3.99.-), yusJ 27atg aat ttt gaa cta acc aga gaa cag caa atg att
cgt gaa ctc gca 48Met Asn Phe Glu Leu Thr Arg Glu Gln Gln Met Ile
Arg Glu Leu Ala 1 5 10 15 aga gat ttt gcg aaa cag gaa att gca ccg
cac gcc gaa cat gtt gac 96Arg Asp Phe Ala Lys Gln Glu Ile Ala Pro
His Ala Glu His Val Asp 20 25 30 agg acg gga gaa ttt ccg att gag
aca ttt aaa aaa atg ggg gag ctc 144Arg Thr Gly Glu Phe Pro Ile Glu
Thr Phe Lys Lys Met Gly Glu Leu 35 40 45 ggc ctc ttg ggg att ccg
ttt cct gaa agc tac ggc ggt tca ggc gga 192Gly Leu Leu Gly Ile Pro
Phe Pro Glu Ser Tyr Gly Gly Ser Gly Gly 50 55 60 gat acg att tcc
tat gca ctt agc gtc gaa gaa atc ggc aaa gcg tgc 240Asp Thr Ile Ser
Tyr Ala Leu Ser Val Glu Glu Ile Gly Lys Ala Cys 65 70 75 80 gga agc
acc ggt ctt agc tat gct gcg gct gta tcg ctc ggg gct gcg 288Gly Ser
Thr Gly Leu Ser Tyr Ala Ala Ala Val Ser Leu Gly Ala Ala 85 90 95
ccg att tat tat ttc ggc act gaa gaa caa aaa caa gaa tat ctc gtc
336Pro Ile Tyr Tyr Phe Gly Thr Glu Glu Gln Lys Gln Glu Tyr Leu Val
100 105 110 ccg ctt gcg acg ggc cgg gcg ctc gga gca ttt ggg ctg acc
gaa ccg 384Pro Leu Ala Thr Gly Arg Ala Leu Gly Ala Phe Gly Leu Thr
Glu Pro 115 120 125 aat gca ggt tcc gat gcg ggc ggc acc cgg aca aaa
gcc cgc tcg gaa 432Asn Ala Gly Ser Asp Ala Gly Gly Thr Arg Thr Lys
Ala Arg Ser Glu 130 135 140 ggg gac agc tat gtg atc agc ggt gag aaa
tgc tgg atc aca aat gca 480Gly Asp Ser Tyr Val Ile Ser Gly Glu Lys
Cys Trp Ile Thr Asn Ala 145 150 155 160 gga ttt gcc agg acc gtc atc
gtc acc gcc gtc acc gga ata gat gac 528Gly Phe Ala Arg Thr Val Ile
Val Thr Ala Val Thr Gly Ile Asp Asp 165 170 175 aac gga aaa aac atc
att tcc gcc atc atc gtt ccg aca gat tcg gag 576Asn Gly Lys Asn Ile
Ile Ser Ala Ile Ile Val Pro Thr Asp Ser Glu 180 185 190 ggc ttc acc
att aaa agc gaa tat gac aaa atg ggt gtc cgc ggc tcc 624Gly Phe Thr
Ile Lys Ser Glu Tyr Asp Lys Met Gly Val Arg Gly Ser 195 200 205 aat
aca tca cag ctc ata ttg gac aat gtc cgc gta cca aaa caa aat 672Asn
Thr Ser Gln Leu Ile Leu Asp Asn Val Arg Val Pro Lys Gln Asn 210 215
220 cta ttg gga agc ccg gaa aaa ggg ttt aaa caa ttt ctc aat aca ctt
720Leu Leu Gly Ser Pro Glu Lys Gly Phe Lys Gln Phe Leu Asn Thr Leu
225 230 235 240 gac ggc ggc aga att tcg atc gca gcg ctg gct gtc ggt
att gcc caa 768Asp Gly Gly Arg Ile Ser Ile Ala Ala Leu Ala Val Gly
Ile Ala Gln 245 250 255 ggc gca ttt gag gcg gcg ctc aca tac gcg cgc
gaa cga aaa caa ttc 816Gly Ala Phe Glu Ala Ala Leu Thr Tyr Ala Arg
Glu Arg Lys Gln Phe 260 265 270 ggc cga ccg atc tct tat ttc cag gcg
att cag ttc aag ctt gcc gac 864Gly Arg Pro Ile Ser Tyr Phe Gln Ala
Ile Gln Phe Lys Leu Ala Asp 275 280 285 atg gcc atg gaa att gag ctc
gcc cgc aat atg gtg ctg aag gcc gcc 912Met Ala Met Glu Ile Glu Leu
Ala Arg Asn Met Val Leu Lys Ala Ala 290 295 300 tgg ctg aaa gat caa
gga cgt ccg ttt aca aaa gaa gcg gct ttt gcc 960Trp Leu Lys Asp Gln
Gly Arg Pro Phe Thr Lys Glu Ala Ala Phe Ala 305 310 315 320 aag ctt
tat gcc tca gaa atg gcg ttc agg aca tgc aat cag tcc att 1008Lys Leu
Tyr Ala Ser Glu Met Ala Phe Arg Thr Cys Asn Gln Ser Ile 325 330 335
caa ata cac gga gga tac ggg tat atg aaa gag tat gga gtg gag cgc
1056Gln Ile His Gly Gly Tyr Gly Tyr Met Lys Glu Tyr Gly Val Glu Arg
340 345 350 atg ctg cgg gac gca aaa tta atg gaa atc ggt gaa ggc act
tca gaa 1104Met Leu Arg Asp Ala Lys Leu Met Glu Ile Gly Glu Gly Thr
Ser Glu 355 360 365 att caa cgg ctc gtc atc gca agg cag ctc ggc atc
ggc aaa caa gcg 1152Ile Gln Arg Leu Val Ile Ala Arg Gln Leu Gly Ile
Gly Lys Gln Ala 370 375 380 ctg aaa tga aaa 1164Leu Lys 385
28386PRTBacillus licheniformis DSM 13 28Met Asn Phe Glu Leu Thr Arg
Glu Gln Gln Met Ile Arg Glu Leu Ala 1 5 10 15 Arg Asp Phe Ala Lys
Gln Glu Ile Ala Pro His Ala Glu His Val Asp 20 25 30 Arg Thr Gly
Glu Phe Pro Ile Glu Thr Phe Lys Lys Met Gly Glu Leu 35 40 45 Gly
Leu Leu Gly Ile Pro Phe Pro Glu Ser Tyr Gly Gly Ser Gly Gly 50 55
60 Asp Thr Ile Ser Tyr Ala Leu Ser Val Glu Glu Ile Gly Lys Ala Cys
65 70 75 80 Gly Ser Thr Gly Leu Ser Tyr Ala Ala Ala Val Ser Leu Gly
Ala Ala 85 90 95 Pro Ile Tyr Tyr Phe Gly Thr Glu Glu Gln Lys Gln
Glu Tyr Leu Val 100 105 110 Pro Leu Ala Thr Gly Arg Ala Leu Gly Ala
Phe Gly Leu Thr Glu Pro 115 120 125 Asn Ala Gly Ser Asp Ala Gly Gly
Thr Arg Thr Lys Ala Arg Ser Glu 130 135 140 Gly Asp Ser Tyr Val Ile
Ser Gly Glu Lys Cys Trp Ile Thr Asn Ala 145 150 155 160 Gly Phe Ala
Arg Thr Val Ile Val Thr Ala Val Thr Gly Ile Asp Asp 165 170 175 Asn
Gly Lys Asn Ile Ile Ser Ala Ile Ile Val Pro Thr Asp Ser Glu 180 185
190 Gly Phe Thr Ile Lys Ser Glu Tyr Asp Lys Met Gly Val Arg Gly Ser
195 200 205 Asn Thr Ser Gln Leu Ile Leu Asp Asn Val Arg Val Pro Lys
Gln Asn 210 215 220 Leu Leu Gly Ser Pro Glu Lys Gly Phe Lys Gln Phe
Leu Asn Thr Leu 225 230 235 240 Asp Gly Gly Arg Ile Ser Ile Ala Ala
Leu Ala Val Gly Ile Ala Gln 245 250 255 Gly Ala Phe Glu Ala Ala Leu
Thr Tyr Ala Arg Glu Arg Lys Gln Phe 260 265 270 Gly Arg Pro Ile Ser
Tyr Phe Gln Ala Ile Gln Phe Lys Leu Ala Asp 275 280 285 Met Ala Met
Glu Ile Glu Leu Ala Arg Asn Met Val Leu Lys Ala Ala 290 295 300 Trp
Leu Lys Asp Gln Gly Arg Pro Phe Thr Lys Glu Ala Ala Phe Ala 305 310
315 320 Lys Leu Tyr Ala Ser Glu Met Ala Phe Arg Thr Cys Asn Gln Ser
Ile 325 330 335 Gln Ile His Gly Gly Tyr Gly Tyr Met Lys Glu Tyr Gly
Val Glu Arg 340 345 350 Met Leu Arg Asp Ala Lys Leu Met Glu Ile Gly
Glu Gly Thr Ser Glu 355 360 365 Ile Gln Arg Leu Val Ile Ala Arg Gln
Leu Gly Ile Gly Lys Gln Ala 370 375 380 Leu Lys 385
29876DNABacillus licheniformis DSM 13gene(1)..(876)Hypothetical
oxidoreductase (E.C. 1.1.-.-), ykwC 29atg aaa aaa acg gtc gga ttt
atc gga ctc ggt gta atg gga aac agc 48Met Lys Lys Thr Val Gly Phe
Ile Gly Leu Gly Val Met Gly Asn Ser 1 5 10 15 atg gcc tcg cac att
tta gcc gcg ggc tat ccc gtc agc gcc tat acg 96Met Ala Ser His Ile
Leu Ala Ala Gly Tyr Pro Val Ser Ala Tyr Thr 20 25 30 aga acg aaa
cac aaa gcg gac agc ctc gtg gaa aaa ggg gcg gaa tgg 144Arg Thr Lys
His Lys Ala Asp Ser Leu Val Glu Lys Gly Ala Glu Trp 35 40 45 aaa
tca tcc gtc aaa gca ctg gcg cag tcg tct gat gtg atc atc aca 192Lys
Ser Ser Val Lys Ala Leu Ala Gln Ser Ser Asp Val Ile Ile Thr 50 55
60 atg gtc ggc tac cca aaa gac gtt gaa gag gtt tac ttt gga agc gaa
240Met Val Gly Tyr Pro Lys Asp Val Glu Glu Val Tyr Phe Gly Ser Glu
65 70 75 80 ggc att att gaa aat gcc aaa aaa ggt tcc tac ctt atc gat
atg acg 288Gly Ile Ile Glu Asn Ala Lys Lys Gly Ser Tyr Leu Ile Asp
Met Thr 85 90 95 act tcc aaa cct tcg ctt gcc aaa caa atc gag act
gcc gca aaa gag 336Thr Ser Lys Pro Ser Leu Ala Lys Gln Ile Glu Thr
Ala Ala Lys Glu 100 105 110 aaa gga ctt tac gct ttg gat gct ccg gtt
tca ggc ggg gac gtc ggc 384Lys Gly Leu Tyr Ala Leu Asp Ala Pro Val
Ser Gly Gly Asp Val Gly 115 120 125 gcg agg aac ggc acg ctc gct atc
atg gtc gga gga gaa cgg aaa gct 432Ala Arg Asn Gly Thr Leu Ala Ile
Met Val Gly Gly Glu Arg Lys Ala 130 135 140 tat gat gaa tgc tac ccg
ctc ttt tcg atc atg ggt gaa aac atc cag 480Tyr Asp Glu Cys Tyr Pro
Leu Phe Ser Ile Met Gly Glu Asn Ile Gln 145 150 155 160 tat cag ggg
ccg gcc gga agc ggc cag cat acg aaa atg tgc aac cag 528Tyr Gln Gly
Pro Ala Gly Ser Gly Gln His Thr Lys Met Cys Asn Gln 165 170 175 att
gcg att gcc gca ggg atg atc ggc gtc gca gaa gcg atg gcc tac 576Ile
Ala Ile Ala Ala Gly Met Ile Gly Val Ala Glu Ala Met Ala Tyr 180 185
190 gcc gaa aaa tcc gga ctc gat ccc gac aac gtg ctg aaa agc att acg
624Ala Glu Lys Ser Gly Leu Asp Pro Asp Asn Val Leu Lys Ser Ile Thr
195 200 205 acc ggc gct gcg gga agc tgg tcg ctc tca aat cta gcg cct
aga atg 672Thr Gly Ala Ala Gly Ser Trp Ser Leu Ser Asn Leu Ala Pro
Arg Met 210 215 220 ctg aaa ggc gac ttt gaa ccg ggt ttt tac gtc aaa
cac ttt gtt aaa 720Leu Lys Gly Asp Phe Glu Pro Gly Phe Tyr Val Lys
His Phe Val Lys 225 230 235 240 gac atg ggc atc gcg ctt gaa gag gcg
gag ctg atg ggc gag aaa atg 768Asp Met Gly Ile Ala Leu Glu Glu Ala
Glu Leu Met Gly Glu Lys Met 245 250 255 ccg ggg ctc gag ctt gcg aaa
agc ctt tac gac acc ctt gtt gaa aaa 816Pro Gly Leu Glu Leu Ala Lys
Ser Leu Tyr Asp Thr Leu Val Glu Lys 260 265 270 ggc gaa gaa aac agc
ggc acc caa agt ctg tac aag ctt tgg aca gaa 864Gly Glu Glu Asn Ser
Gly Thr Gln Ser Leu Tyr Lys Leu Trp Thr Glu 275 280 285 tac aaa taa
cga 876Tyr Lys 290 30290PRTBacillus licheniformis DSM 13 30Met Lys
Lys Thr Val Gly Phe Ile Gly Leu Gly Val Met Gly Asn Ser 1 5 10 15
Met Ala Ser His Ile Leu Ala Ala Gly Tyr Pro Val Ser Ala Tyr Thr 20
25 30 Arg Thr Lys His Lys Ala Asp Ser Leu Val Glu Lys Gly Ala Glu
Trp 35 40 45 Lys Ser Ser Val Lys Ala Leu Ala Gln Ser Ser Asp Val
Ile Ile Thr 50 55 60 Met Val Gly Tyr Pro Lys Asp Val Glu Glu Val
Tyr Phe Gly Ser Glu 65 70 75 80 Gly Ile Ile Glu Asn Ala Lys Lys Gly
Ser Tyr Leu Ile Asp Met Thr 85 90 95 Thr Ser Lys Pro Ser Leu Ala
Lys Gln Ile Glu Thr Ala Ala Lys Glu 100 105 110 Lys Gly Leu Tyr Ala
Leu Asp Ala Pro Val Ser Gly Gly Asp Val Gly 115 120 125 Ala Arg Asn
Gly Thr Leu Ala Ile Met Val Gly Gly Glu Arg Lys Ala 130 135 140 Tyr
Asp Glu Cys Tyr Pro Leu Phe Ser Ile Met Gly Glu Asn Ile Gln 145 150
155 160 Tyr Gln Gly Pro Ala Gly Ser Gly Gln His Thr Lys Met Cys Asn
Gln 165 170 175 Ile Ala Ile Ala Ala Gly Met Ile Gly Val Ala Glu Ala
Met Ala Tyr 180 185 190 Ala Glu Lys Ser Gly Leu Asp Pro Asp Asn Val
Leu Lys Ser Ile Thr 195 200 205 Thr Gly Ala Ala Gly Ser Trp Ser Leu
Ser Asn Leu Ala Pro Arg Met 210 215 220 Leu Lys Gly Asp Phe Glu Pro
Gly Phe Tyr Val Lys His Phe Val Lys 225 230 235 240 Asp Met Gly Ile
Ala Leu Glu Glu Ala Glu Leu Met Gly Glu Lys Met 245 250 255 Pro Gly
Leu Glu Leu Ala Lys Ser Leu Tyr Asp Thr Leu Val Glu Lys 260 265 270
Gly Glu Glu Asn Ser Gly Thr Gln Ser Leu Tyr Lys Leu Trp Thr Glu 275
280 285 Tyr Lys 290 31915DNABacillus licheniformis DSM
13gene(1)..(915)Probable phosphate butyryltransferase (E.C.
2.3.1.19) 31atg aag ctg aaa caa cta ttg caa aaa gcg gcg gag ctt gac
aat aaa 48Met Lys Leu Lys Gln Leu Leu Gln Lys Ala Ala Glu Leu Asp
Asn Lys 1 5 10 15 acg gtc gcc gtc gca cat gcg gaa gat gac gaa gtg
ctg caa gcg gtc 96Thr Val Ala Val Ala His Ala Glu Asp Asp Glu Val
Leu Gln Ala Val 20 25 30 aaa ctt gcg gtc gac aaa caa ttt gcc cgg
ttc ttg ctg atc ggg cac 144Lys Leu Ala Val Asp Lys Gln Phe Ala Arg
Phe Leu Leu Ile Gly His 35 40 45 aga gaa aaa ctt aga cat atg atg
aca gag cag aat att tca aaa cgg 192Arg Glu Lys Leu Arg His Met Met
Thr Glu Gln Asn Ile Ser Lys Arg 50 55 60 cac gtg gat atc att cat
tcg gaa tcg ccg gcg gat tct gcg aga att 240His Val Asp Ile Ile His
Ser Glu Ser Pro Ala Asp Ser Ala Arg Ile 65 70 75 80 gcc gtt caa gct
gtc aaa agc ggc aat gcg gat gtt ctt atg aag ggg 288Ala Val Gln Ala
Val Lys Ser Gly Asn Ala Asp Val Leu Met Lys Gly 85 90 95 aat gtc
ccg aca gct gtg cta tta aaa gcc gtt ttg aat aaa gag tac 336Asn Val
Pro Thr Ala Val Leu Leu Lys Ala Val Leu Asn Lys Glu Tyr 100 105 110
ggg ctt cgt tcc tcg cac gtg ctg tca cat gta gca gca ttt gaa gtc
384Gly Leu Arg Ser Ser His Val Leu Ser His Val Ala Ala Phe Glu Val
115 120 125 agc ggg ttt gag agg ctg att tat gta aca gat gcg gcg atg
aat atc 432Ser Gly Phe Glu Arg Leu Ile Tyr Val Thr Asp Ala Ala Met
Asn Ile 130 135 140 agc ccc aag ctt gat gag ctg aag cag att tta gaa
aac gca gtc ggc 480Ser Pro Lys Leu Asp Glu Leu Lys Gln Ile Leu Glu
Asn Ala Val Gly 145 150 155 160 gtg gcg agg tcg gtc ggc gtg caa atg
ccg aaa gtc gct tgt ctt gcc 528Val Ala Arg Ser Val Gly Val Gln Met
Pro Lys Val Ala Cys Leu Ala 165 170 175 gca gtg gaa aca gtg aat ccc
gcg atg gaa gcg aca ttg aat gca gct 576Ala Val Glu Thr Val Asn Pro
Ala Met Glu Ala Thr Leu Asn Ala Ala 180 185 190 gcc ttg acg cag atg
aat cat cgg ggc caa atc aaa aac tgc gtt gtt 624Ala Leu Thr Gln Met
Asn His Arg Gly Gln Ile Lys Asn
Cys Val Val 195 200 205 gac ggg cct ctt gca ttg gat aac gcg ata tcg
ccg ctt gcc gcc cgg 672Asp Gly Pro Leu Ala Leu Asp Asn Ala Ile Ser
Pro Leu Ala Ala Arg 210 215 220 cat aaa aac att tcc ggg atc gta gca
ggc gag gcc gat atc ctg ctt 720His Lys Asn Ile Ser Gly Ile Val Ala
Gly Glu Ala Asp Ile Leu Leu 225 230 235 240 gtt cct tca att gaa aca
ggc aat gtc ctt tat aaa tca ttg att cat 768Val Pro Ser Ile Glu Thr
Gly Asn Val Leu Tyr Lys Ser Leu Ile His 245 250 255 ttt gcg ggt gca
aaa gtg gga gcc att tta gca ggg gca aaa gca ccc 816Phe Ala Gly Ala
Lys Val Gly Ala Ile Leu Ala Gly Ala Lys Ala Pro 260 265 270 atc gcc
ttg aca agc agg gcc gat tcc gca gaa aac aag ttg tat tcg 864Ile Ala
Leu Thr Ser Arg Ala Asp Ser Ala Glu Asn Lys Leu Tyr Ser 275 280 285
att gct ttg gcg ctg tgt acg tct gaa gca cga cat gag gag gaa taa
912Ile Ala Leu Ala Leu Cys Thr Ser Glu Ala Arg His Glu Glu Glu 290
295 300 aaa 91532303PRTBacillus licheniformis DSM 13 32Met Lys Leu
Lys Gln Leu Leu Gln Lys Ala Ala Glu Leu Asp Asn Lys 1 5 10 15 Thr
Val Ala Val Ala His Ala Glu Asp Asp Glu Val Leu Gln Ala Val 20 25
30 Lys Leu Ala Val Asp Lys Gln Phe Ala Arg Phe Leu Leu Ile Gly His
35 40 45 Arg Glu Lys Leu Arg His Met Met Thr Glu Gln Asn Ile Ser
Lys Arg 50 55 60 His Val Asp Ile Ile His Ser Glu Ser Pro Ala Asp
Ser Ala Arg Ile 65 70 75 80 Ala Val Gln Ala Val Lys Ser Gly Asn Ala
Asp Val Leu Met Lys Gly 85 90 95 Asn Val Pro Thr Ala Val Leu Leu
Lys Ala Val Leu Asn Lys Glu Tyr 100 105 110 Gly Leu Arg Ser Ser His
Val Leu Ser His Val Ala Ala Phe Glu Val 115 120 125 Ser Gly Phe Glu
Arg Leu Ile Tyr Val Thr Asp Ala Ala Met Asn Ile 130 135 140 Ser Pro
Lys Leu Asp Glu Leu Lys Gln Ile Leu Glu Asn Ala Val Gly 145 150 155
160 Val Ala Arg Ser Val Gly Val Gln Met Pro Lys Val Ala Cys Leu Ala
165 170 175 Ala Val Glu Thr Val Asn Pro Ala Met Glu Ala Thr Leu Asn
Ala Ala 180 185 190 Ala Leu Thr Gln Met Asn His Arg Gly Gln Ile Lys
Asn Cys Val Val 195 200 205 Asp Gly Pro Leu Ala Leu Asp Asn Ala Ile
Ser Pro Leu Ala Ala Arg 210 215 220 His Lys Asn Ile Ser Gly Ile Val
Ala Gly Glu Ala Asp Ile Leu Leu 225 230 235 240 Val Pro Ser Ile Glu
Thr Gly Asn Val Leu Tyr Lys Ser Leu Ile His 245 250 255 Phe Ala Gly
Ala Lys Val Gly Ala Ile Leu Ala Gly Ala Lys Ala Pro 260 265 270 Ile
Ala Leu Thr Ser Arg Ala Asp Ser Ala Glu Asn Lys Leu Tyr Ser 275 280
285 Ile Ala Leu Ala Leu Cys Thr Ser Glu Ala Arg His Glu Glu Glu 290
295 300 331110DNABacillus licheniformis DSM
13gene(1)..(1110)Probable butyrate kinase (E.C. 2.7.2.7) 33atg cag
gta cag gaa aaa cgt att ctc gtc atc aat ccg gga tct aca 48Met Gln
Val Gln Glu Lys Arg Ile Leu Val Ile Asn Pro Gly Ser Thr 1 5 10 15
tct aca aag atc ggc gtt ttt cat gat gac cgt tcg att ttc gaa aaa
96Ser Thr Lys Ile Gly Val Phe His Asp Asp Arg Ser Ile Phe Glu Lys
20 25 30 tca atc cgt cat gac gag gct gag cta cag caa tat cag acc
att att 144Ser Ile Arg His Asp Glu Ala Glu Leu Gln Gln Tyr Gln Thr
Ile Ile 35 40 45 gat caa tat tcg ttc aga aaa cag gcg ata ctc gaa
acc ctg cat gaa 192Asp Gln Tyr Ser Phe Arg Lys Gln Ala Ile Leu Glu
Thr Leu His Glu 50 55 60 cag gga atc aat att tct aaa ttg gat gcc
gtt tgc gcc agg gga ggg 240Gln Gly Ile Asn Ile Ser Lys Leu Asp Ala
Val Cys Ala Arg Gly Gly 65 70 75 80 ctg ctt cgg ccg att gaa ggc ggc
act tac gaa gtc aat gat gcg atg 288Leu Leu Arg Pro Ile Glu Gly Gly
Thr Tyr Glu Val Asn Asp Ala Met 85 90 95 att gtc gat ttg aaa aac
ggc tat gcg ggg cag cat gca tca aat ctc 336Ile Val Asp Leu Lys Asn
Gly Tyr Ala Gly Gln His Ala Ser Asn Leu 100 105 110 ggg ggc atc atc
gcc agg gag att gcc gac ggg tta aat att ccc gct 384Gly Gly Ile Ile
Ala Arg Glu Ile Ala Asp Gly Leu Asn Ile Pro Ala 115 120 125 ttt atc
gtc gac ccc gtt gtt gtg gat gaa atg gct cct atc gca aaa 432Phe Ile
Val Asp Pro Val Val Val Asp Glu Met Ala Pro Ile Ala Lys 130 135 140
att tcc ggc acc ccg gct att gaa agg cgc agc att ttt cac gcg ctc
480Ile Ser Gly Thr Pro Ala Ile Glu Arg Arg Ser Ile Phe His Ala Leu
145 150 155 160 aac caa aaa gca gtt gca agg aaa gcg gct tgg cag ttt
ggg aag cgt 528Asn Gln Lys Ala Val Ala Arg Lys Ala Ala Trp Gln Phe
Gly Lys Arg 165 170 175 tat gaa gat atg aaa atg atc atc acc cac atg
gga ggc ggc att acg 576Tyr Glu Asp Met Lys Met Ile Ile Thr His Met
Gly Gly Gly Ile Thr 180 185 190 atc ggc gtc cat tgc cgc ggc cgg gtg
atc gac gtc aac aac ggc ctc 624Ile Gly Val His Cys Arg Gly Arg Val
Ile Asp Val Asn Asn Gly Leu 195 200 205 cac ggg gaa ggt ccg ctc agt
cca gag cgg gcc gga acc att cct gcg 672His Gly Glu Gly Pro Leu Ser
Pro Glu Arg Ala Gly Thr Ile Pro Ala 210 215 220 ggt gat ctg atc gat
atg tgc ttt tcc ggc gaa tat acg aaa gac gag 720Gly Asp Leu Ile Asp
Met Cys Phe Ser Gly Glu Tyr Thr Lys Asp Glu 225 230 235 240 ctg atg
aaa atg ctt gtc ggc ggc gga ggg ctt gcc ggc tat ctc ggc 768Leu Met
Lys Met Leu Val Gly Gly Gly Gly Leu Ala Gly Tyr Leu Gly 245 250 255
acg acg gat gcg gta aaa gtt gag aaa atg atc aag gaa ggc gat caa
816Thr Thr Asp Ala Val Lys Val Glu Lys Met Ile Lys Glu Gly Asp Gln
260 265 270 aaa gct gcg ctc atc tat gaa gcg atg gct tat caa atc gcc
aaa gaa 864Lys Ala Ala Leu Ile Tyr Glu Ala Met Ala Tyr Gln Ile Ala
Lys Glu 275 280 285 atc ggg gcg gcc agc gcc gtc tta aaa ggc gaa gtc
gat gtc att att 912Ile Gly Ala Ala Ser Ala Val Leu Lys Gly Glu Val
Asp Val Ile Ile 290 295 300 ttg aca gga gga ctg gca tat gga aaa tcg
ttt att tcc tcg atc aga 960Leu Thr Gly Gly Leu Ala Tyr Gly Lys Ser
Phe Ile Ser Ser Ile Arg 305 310 315 320 caa tac ata gac tgg att tcg
gat gtc gtc gtc ttt cca gga gaa aat 1008Gln Tyr Ile Asp Trp Ile Ser
Asp Val Val Val Phe Pro Gly Glu Asn 325 330 335 gaa ctt caa gca ttg
gct gaa ggt gca ttt cgc gta ttg aac ggc gaa 1056Glu Leu Gln Ala Leu
Ala Glu Gly Ala Phe Arg Val Leu Asn Gly Glu 340 345 350 gaa gag gca
aaa cag tat ccg aac cag agg agg gaa agt cat ggc aac 1104Glu Glu Ala
Lys Gln Tyr Pro Asn Gln Arg Arg Glu Ser His Gly Asn 355 360 365 tga
ata 1110 34368PRTBacillus licheniformis DSM 13 34Met Gln Val Gln
Glu Lys Arg Ile Leu Val Ile Asn Pro Gly Ser Thr 1 5 10 15 Ser Thr
Lys Ile Gly Val Phe His Asp Asp Arg Ser Ile Phe Glu Lys 20 25 30
Ser Ile Arg His Asp Glu Ala Glu Leu Gln Gln Tyr Gln Thr Ile Ile 35
40 45 Asp Gln Tyr Ser Phe Arg Lys Gln Ala Ile Leu Glu Thr Leu His
Glu 50 55 60 Gln Gly Ile Asn Ile Ser Lys Leu Asp Ala Val Cys Ala
Arg Gly Gly 65 70 75 80 Leu Leu Arg Pro Ile Glu Gly Gly Thr Tyr Glu
Val Asn Asp Ala Met 85 90 95 Ile Val Asp Leu Lys Asn Gly Tyr Ala
Gly Gln His Ala Ser Asn Leu 100 105 110 Gly Gly Ile Ile Ala Arg Glu
Ile Ala Asp Gly Leu Asn Ile Pro Ala 115 120 125 Phe Ile Val Asp Pro
Val Val Val Asp Glu Met Ala Pro Ile Ala Lys 130 135 140 Ile Ser Gly
Thr Pro Ala Ile Glu Arg Arg Ser Ile Phe His Ala Leu 145 150 155 160
Asn Gln Lys Ala Val Ala Arg Lys Ala Ala Trp Gln Phe Gly Lys Arg 165
170 175 Tyr Glu Asp Met Lys Met Ile Ile Thr His Met Gly Gly Gly Ile
Thr 180 185 190 Ile Gly Val His Cys Arg Gly Arg Val Ile Asp Val Asn
Asn Gly Leu 195 200 205 His Gly Glu Gly Pro Leu Ser Pro Glu Arg Ala
Gly Thr Ile Pro Ala 210 215 220 Gly Asp Leu Ile Asp Met Cys Phe Ser
Gly Glu Tyr Thr Lys Asp Glu 225 230 235 240 Leu Met Lys Met Leu Val
Gly Gly Gly Gly Leu Ala Gly Tyr Leu Gly 245 250 255 Thr Thr Asp Ala
Val Lys Val Glu Lys Met Ile Lys Glu Gly Asp Gln 260 265 270 Lys Ala
Ala Leu Ile Tyr Glu Ala Met Ala Tyr Gln Ile Ala Lys Glu 275 280 285
Ile Gly Ala Ala Ser Ala Val Leu Lys Gly Glu Val Asp Val Ile Ile 290
295 300 Leu Thr Gly Gly Leu Ala Tyr Gly Lys Ser Phe Ile Ser Ser Ile
Arg 305 310 315 320 Gln Tyr Ile Asp Trp Ile Ser Asp Val Val Val Phe
Pro Gly Glu Asn 325 330 335 Glu Leu Gln Ala Leu Ala Glu Gly Ala Phe
Arg Val Leu Asn Gly Glu 340 345 350 Glu Glu Ala Lys Gln Tyr Pro Asn
Gln Arg Arg Glu Ser His Gly Asn 355 360 365 351722DNABacillus
licheniformis DSM 13gene(1)..(1722)Acetyl-coenzyme A synthetase
(E.C. 6.2.1.1), acsA 35atg aaa ttg aaa gcg ctg cca gca gaa aag gga
aat tac aac ttg aaa 48Met Lys Leu Lys Ala Leu Pro Ala Glu Lys Gly
Asn Tyr Asn Leu Lys 1 5 10 15 gac tat gat gaa aca tac cgg aca ttt
gac tgg aag gat gcc gaa aag 96Asp Tyr Asp Glu Thr Tyr Arg Thr Phe
Asp Trp Lys Asp Ala Glu Lys 20 25 30 cat ttt tca tgg cac aaa acg
gga aaa atc aat gca gct tat gaa gct 144His Phe Ser Trp His Lys Thr
Gly Lys Ile Asn Ala Ala Tyr Glu Ala 35 40 45 atc gac cgc cac gct
gag tca aat ttg aaa aac aaa gtg gca ttt tac 192Ile Asp Arg His Ala
Glu Ser Asn Leu Lys Asn Lys Val Ala Phe Tyr 50 55 60 tac aaa gat
ccg gtc cgc gaa gaa aag tac act ttc aga gag atg aaa 240Tyr Lys Asp
Pro Val Arg Glu Glu Lys Tyr Thr Phe Arg Glu Met Lys 65 70 75 80 aat
gaa acc aac aaa gcc ggg aat gtc tta aag cag cat gcc gat gtg 288Asn
Glu Thr Asn Lys Ala Gly Asn Val Leu Lys Gln His Ala Asp Val 85 90
95 gga aag gga gac cgt gtg ttt gtt ttt atg ccg aga tcg ccc gag ctt
336Gly Lys Gly Asp Arg Val Phe Val Phe Met Pro Arg Ser Pro Glu Leu
100 105 110 tat ttt att ctt ctc ggc gcc atc aaa ttg gga gcg atc gtc
ggg ccg 384Tyr Phe Ile Leu Leu Gly Ala Ile Lys Leu Gly Ala Ile Val
Gly Pro 115 120 125 tta ttt gaa gcg ttt atg gaa ggt gcc gtc aaa gac
agg ctt gca aac 432Leu Phe Glu Ala Phe Met Glu Gly Ala Val Lys Asp
Arg Leu Ala Asn 130 135 140 agc gga gcg aag gtc atc gtg acg acg ccg
gaa ttg ctt gaa cgg gtg 480Ser Gly Ala Lys Val Ile Val Thr Thr Pro
Glu Leu Leu Glu Arg Val 145 150 155 160 ccg gcc gat gaa ctt ccg gat
ctt gaa tca atc att gtc gtt gga gaa 528Pro Ala Asp Glu Leu Pro Asp
Leu Glu Ser Ile Ile Val Val Gly Glu 165 170 175 ggc gta aag gaa gaa
gga cct gtc att gat tat tac gcg aaa gcg gcg 576Gly Val Lys Glu Glu
Gly Pro Val Ile Asp Tyr Tyr Ala Lys Ala Ala 180 185 190 gaa gca ggc
act gat ctt gag att gaa tgg gtg gat cag gaa gac ggg 624Glu Ala Gly
Thr Asp Leu Glu Ile Glu Trp Val Asp Gln Glu Asp Gly 195 200 205 atg
ctg ctt cac tat acg tcg ggt tcg acc ggc gcg cca aaa ggg gtt 672Met
Leu Leu His Tyr Thr Ser Gly Ser Thr Gly Ala Pro Lys Gly Val 210 215
220 ctc cac gtc cat aaa gca atg atc cag cat tat caa aca gcc aaa tgg
720Leu His Val His Lys Ala Met Ile Gln His Tyr Gln Thr Ala Lys Trp
225 230 235 240 gtt ctt gat ctg cat gac gat gac atc tat tgg tgc acc
gct gat ccc 768Val Leu Asp Leu His Asp Asp Asp Ile Tyr Trp Cys Thr
Ala Asp Pro 245 250 255 ggc tgg gtc acc gga acg gtt tac ggg att ttc
ggt ccg tgg ctg aat 816Gly Trp Val Thr Gly Thr Val Tyr Gly Ile Phe
Gly Pro Trp Leu Asn 260 265 270 gga gct acg aat gtt gtc gta ggc ggc
aga ttc agt cct gag gca tgg 864Gly Ala Thr Asn Val Val Val Gly Gly
Arg Phe Ser Pro Glu Ala Trp 275 280 285 tac gaa acg att gaa aaa atg
gaa gtg acg gta tgg tac agc gcg cca 912Tyr Glu Thr Ile Glu Lys Met
Glu Val Thr Val Trp Tyr Ser Ala Pro 290 295 300 acg gct ttc cgg atg
ctg atg ggt gca ggc gac gat ctt gtg aat aaa 960Thr Ala Phe Arg Met
Leu Met Gly Ala Gly Asp Asp Leu Val Asn Lys 305 310 315 320 tat aat
cta agc tcc ttg cgg cat att tta agc gta ggg gag ccg tta 1008Tyr Asn
Leu Ser Ser Leu Arg His Ile Leu Ser Val Gly Glu Pro Leu 325 330 335
aat ccc gaa gtc atc agg tgg ggg cat aaa gtc ttc ggc aac cgg att
1056Asn Pro Glu Val Ile Arg Trp Gly His Lys Val Phe Gly Asn Arg Ile
340 345 350 cat gat act tgg tgg atg act gaa aca gga tcg cag ctc atc
tgc aat 1104His Asp Thr Trp Trp Met Thr Glu Thr Gly Ser Gln Leu Ile
Cys Asn 355 360 365 tac ccg tgc atg gaa att aaa ccg gga tca atg ggc
aag ccg att ccc 1152Tyr Pro Cys Met Glu Ile Lys Pro Gly Ser Met Gly
Lys Pro Ile Pro 370 375 380 ggt gta gag gct gca atc gtc gac aac cag
gga aat gaa ctg cct cct 1200Gly Val Glu Ala Ala Ile Val Asp Asn Gln
Gly Asn Glu Leu Pro Pro 385 390 395 400 tac aga atg gga aat ctc gcc
att aaa aaa ggc tgg ccg tcg atg atg 1248Tyr Arg Met Gly Asn Leu Ala
Ile Lys Lys Gly Trp Pro Ser Met Met 405 410 415 cat tcg atc tgg aac
aat cct gaa aaa tat agc tcc tat ttt atg ccg 1296His Ser Ile Trp Asn
Asn Pro Glu Lys Tyr Ser Ser Tyr Phe Met Pro 420 425 430 ggc gat tgg
tat gtg tca gga gat tcc gcc tac atg gat gaa gac ggg 1344Gly Asp Trp
Tyr Val Ser Gly Asp Ser Ala Tyr Met Asp Glu Asp Gly 435 440 445
tac ttc tgg ttc cag gga cgg atc gac gat gtc atc atg aca tcg ggc
1392Tyr Phe Trp Phe Gln Gly Arg Ile Asp Asp Val Ile Met Thr Ser Gly
450 455 460 gaa cgc gtc ggc ccg ttt gaa gtc gag agc aag ctt gtt gag
cat cag 1440Glu Arg Val Gly Pro Phe Glu Val Glu Ser Lys Leu Val Glu
His Gln 465 470 475 480 gcc gtc gct gaa gca ggc gtc atc ggc aaa ccg
gat ccc gtc cgg ggt 1488Ala Val Ala Glu Ala Gly Val Ile Gly Lys Pro
Asp Pro Val Arg Gly 485 490 495 gaa att att aaa gcg ttc atc gcc ttg
agg gac ggt tat gaa ccg tca 1536Glu Ile Ile Lys Ala Phe Ile Ala Leu
Arg Asp Gly Tyr Glu Pro Ser 500 505 510 gat gcg tta aaa gaa gaa atc
agg cag ttc gtg aaa caa ggc ttg gcc 1584Asp Ala Leu Lys Glu Glu Ile
Arg Gln Phe Val Lys Gln Gly Leu Ala 515 520 525 gca cat gcc gcg cca
agg gaa atc gaa ttt aaa gat aaa ctg ccg aag 1632Ala His Ala Ala Pro
Arg Glu Ile Glu Phe Lys Asp Lys Leu Pro Lys 530 535 540 aca aga agc
gga aag atc atg aga cgc gtc ctg aaa gca tgg gag ctg 1680Thr Arg Ser
Gly Lys Ile Met Arg Arg Val Leu Lys Ala Trp Glu Leu 545 550 555 560
aac ctt ccg gca ggc gat ctc tca tcg atg gaa gac tga tgt 1722Asn Leu
Pro Ala Gly Asp Leu Ser Ser Met Glu Asp 565 570 36572PRTBacillus
licheniformis DSM 13 36Met Lys Leu Lys Ala Leu Pro Ala Glu Lys Gly
Asn Tyr Asn Leu Lys 1 5 10 15 Asp Tyr Asp Glu Thr Tyr Arg Thr Phe
Asp Trp Lys Asp Ala Glu Lys 20 25 30 His Phe Ser Trp His Lys Thr
Gly Lys Ile Asn Ala Ala Tyr Glu Ala 35 40 45 Ile Asp Arg His Ala
Glu Ser Asn Leu Lys Asn Lys Val Ala Phe Tyr 50 55 60 Tyr Lys Asp
Pro Val Arg Glu Glu Lys Tyr Thr Phe Arg Glu Met Lys 65 70 75 80 Asn
Glu Thr Asn Lys Ala Gly Asn Val Leu Lys Gln His Ala Asp Val 85 90
95 Gly Lys Gly Asp Arg Val Phe Val Phe Met Pro Arg Ser Pro Glu Leu
100 105 110 Tyr Phe Ile Leu Leu Gly Ala Ile Lys Leu Gly Ala Ile Val
Gly Pro 115 120 125 Leu Phe Glu Ala Phe Met Glu Gly Ala Val Lys Asp
Arg Leu Ala Asn 130 135 140 Ser Gly Ala Lys Val Ile Val Thr Thr Pro
Glu Leu Leu Glu Arg Val 145 150 155 160 Pro Ala Asp Glu Leu Pro Asp
Leu Glu Ser Ile Ile Val Val Gly Glu 165 170 175 Gly Val Lys Glu Glu
Gly Pro Val Ile Asp Tyr Tyr Ala Lys Ala Ala 180 185 190 Glu Ala Gly
Thr Asp Leu Glu Ile Glu Trp Val Asp Gln Glu Asp Gly 195 200 205 Met
Leu Leu His Tyr Thr Ser Gly Ser Thr Gly Ala Pro Lys Gly Val 210 215
220 Leu His Val His Lys Ala Met Ile Gln His Tyr Gln Thr Ala Lys Trp
225 230 235 240 Val Leu Asp Leu His Asp Asp Asp Ile Tyr Trp Cys Thr
Ala Asp Pro 245 250 255 Gly Trp Val Thr Gly Thr Val Tyr Gly Ile Phe
Gly Pro Trp Leu Asn 260 265 270 Gly Ala Thr Asn Val Val Val Gly Gly
Arg Phe Ser Pro Glu Ala Trp 275 280 285 Tyr Glu Thr Ile Glu Lys Met
Glu Val Thr Val Trp Tyr Ser Ala Pro 290 295 300 Thr Ala Phe Arg Met
Leu Met Gly Ala Gly Asp Asp Leu Val Asn Lys 305 310 315 320 Tyr Asn
Leu Ser Ser Leu Arg His Ile Leu Ser Val Gly Glu Pro Leu 325 330 335
Asn Pro Glu Val Ile Arg Trp Gly His Lys Val Phe Gly Asn Arg Ile 340
345 350 His Asp Thr Trp Trp Met Thr Glu Thr Gly Ser Gln Leu Ile Cys
Asn 355 360 365 Tyr Pro Cys Met Glu Ile Lys Pro Gly Ser Met Gly Lys
Pro Ile Pro 370 375 380 Gly Val Glu Ala Ala Ile Val Asp Asn Gln Gly
Asn Glu Leu Pro Pro 385 390 395 400 Tyr Arg Met Gly Asn Leu Ala Ile
Lys Lys Gly Trp Pro Ser Met Met 405 410 415 His Ser Ile Trp Asn Asn
Pro Glu Lys Tyr Ser Ser Tyr Phe Met Pro 420 425 430 Gly Asp Trp Tyr
Val Ser Gly Asp Ser Ala Tyr Met Asp Glu Asp Gly 435 440 445 Tyr Phe
Trp Phe Gln Gly Arg Ile Asp Asp Val Ile Met Thr Ser Gly 450 455 460
Glu Arg Val Gly Pro Phe Glu Val Glu Ser Lys Leu Val Glu His Gln 465
470 475 480 Ala Val Ala Glu Ala Gly Val Ile Gly Lys Pro Asp Pro Val
Arg Gly 485 490 495 Glu Ile Ile Lys Ala Phe Ile Ala Leu Arg Asp Gly
Tyr Glu Pro Ser 500 505 510 Asp Ala Leu Lys Glu Glu Ile Arg Gln Phe
Val Lys Gln Gly Leu Ala 515 520 525 Ala His Ala Ala Pro Arg Glu Ile
Glu Phe Lys Asp Lys Leu Pro Lys 530 535 540 Thr Arg Ser Gly Lys Ile
Met Arg Arg Val Leu Lys Ala Trp Glu Leu 545 550 555 560 Asn Leu Pro
Ala Gly Asp Leu Ser Ser Met Glu Asp 565 570 371593DNABacillus
licheniformis DSM 13gene(1)..(1593)acetate-CoA ligase (E.C.
6.2.1.1), ytcI 37ttg aga aga gaa gat ttg att gcg ccg gag aag tat
aat gcg gtt gat 48Leu Arg Arg Glu Asp Leu Ile Ala Pro Glu Lys Tyr
Asn Ala Val Asp 1 5 10 15 gaa att gaa aaa ttt aaa tct tcc cgc gat
aag acc gca ttg atc tgg 96Glu Ile Glu Lys Phe Lys Ser Ser Arg Asp
Lys Thr Ala Leu Ile Trp 20 25 30 gaa gat gaa tca ggg cgt caa gtg
tca tgg tcc tat gaa aaa ttg att 144Glu Asp Glu Ser Gly Arg Gln Val
Ser Trp Ser Tyr Glu Lys Leu Ile 35 40 45 gaa aag gct tac aaa atc
ggc agc ata ttg acc cgt tct gga ctg aaa 192Glu Lys Ala Tyr Lys Ile
Gly Ser Ile Leu Thr Arg Ser Gly Leu Lys 50 55 60 aaa ggt gac aag
ctt atc gtg atg atg ccg cgg ata ccg gaa acg tat 240Lys Gly Asp Lys
Leu Ile Val Met Met Pro Arg Ile Pro Glu Thr Tyr 65 70 75 80 gcc gtg
tac atg gcc att tta aaa gct gga atg gtg gtc atc cca tgt 288Ala Val
Tyr Met Ala Ile Leu Lys Ala Gly Met Val Val Ile Pro Cys 85 90 95
tcc gaa atg ctt cgg gcg aaa gac ttg gat tac agg atc aag cat gca
336Ser Glu Met Leu Arg Ala Lys Asp Leu Asp Tyr Arg Ile Lys His Ala
100 105 110 ggc gtc aaa gga gcc gtc gta tat tca gca ttt ctt gat gct
ttt cta 384Gly Val Lys Gly Ala Val Val Tyr Ser Ala Phe Leu Asp Ala
Phe Leu 115 120 125 gat gtt cgt tca aaa gag gca ctg tcg tta ttt gcc
gtc gga gaa agc 432Asp Val Arg Ser Lys Glu Ala Leu Ser Leu Phe Ala
Val Gly Glu Ser 130 135 140 agc gaa ggg tgg atc aat ctg ctc gaa aaa
atg aat cag gcc att gca 480Ser Glu Gly Trp Ile Asn Leu Leu Glu Lys
Met Asn Gln Ala Ile Ala 145 150 155 160 gcg gat ttt caa gcg gcg gat
acc tct cgc gat gac atc gca ttt tta 528Ala Asp Phe Gln Ala Ala Asp
Thr Ser Arg Asp Asp Ile Ala Phe Leu 165 170 175 tct tac aca tcc ggc
acg acc ggt cag cca aaa ggg gtt gta cat aca 576Ser Tyr Thr Ser Gly
Thr Thr Gly Gln Pro Lys Gly Val Val His Thr 180 185 190 cac ggc tgg
gct tat gct cac tta aga acg aca gct tcc gca tgg ctt 624His Gly Trp
Ala Tyr Ala His Leu Arg Thr Thr Ala Ser Ala Trp Leu 195 200 205 gac
att tcg gaa ggg gat ctc gtc tgg gcg aca gca ggg ccg ggc tgg 672Asp
Ile Ser Glu Gly Asp Leu Val Trp Ala Thr Ala Gly Pro Gly Trp 210 215
220 caa aaa tgg gta tgg agc ccg ttt tta gca gtg ctc ggt agc ggt gca
720Gln Lys Trp Val Trp Ser Pro Phe Leu Ala Val Leu Gly Ser Gly Ala
225 230 235 240 acc ggc ttt atc tat cat gga aaa ttc acg ccg gaa acg
tat ttg cgg 768Thr Gly Phe Ile Tyr His Gly Lys Phe Thr Pro Glu Thr
Tyr Leu Arg 245 250 255 ctg att gag cgc cat caa gtc aat gtg ctg tgc
tgt acg ccg aca gag 816Leu Ile Glu Arg His Gln Val Asn Val Leu Cys
Cys Thr Pro Thr Glu 260 265 270 tac cgg ttc atg gcg aaa gtc aat gat
ttg tcc cga ttt gac ctg tct 864Tyr Arg Phe Met Ala Lys Val Asn Asp
Leu Ser Arg Phe Asp Leu Ser 275 280 285 tct ctg cac agc gct gtt tcg
gcc gga gag ccg ttg aac agg gaa gtc 912Ser Leu His Ser Ala Val Ser
Ala Gly Glu Pro Leu Asn Arg Glu Val 290 295 300 atc gat aca ttt aaa
aag cat ttt cat att gct gtg cgg gac gga tac 960Ile Asp Thr Phe Lys
Lys His Phe His Ile Ala Val Arg Asp Gly Tyr 305 310 315 320 gga caa
acg gag agc acg ctg ttg gtg ggg att tta aaa ggt atg aaa 1008Gly Gln
Thr Glu Ser Thr Leu Leu Val Gly Ile Leu Lys Gly Met Lys 325 330 335
atc aag cct gga agc atg gga aaa ccg acg cct gga aac ttg gtt gat
1056Ile Lys Pro Gly Ser Met Gly Lys Pro Thr Pro Gly Asn Leu Val Asp
340 345 350 att att gac ggg aat gga aag agc tgt ccc ccg ggc gaa aca
ggc gat 1104Ile Ile Asp Gly Asn Gly Lys Ser Cys Pro Pro Gly Glu Thr
Gly Asp 355 360 365 att gcc gtt cac tta agc acg ccg gct ctt ttt aaa
gaa tat tac aaa 1152Ile Ala Val His Leu Ser Thr Pro Ala Leu Phe Lys
Glu Tyr Tyr Lys 370 375 380 gat caa gaa cga acg ctc cga caa aga aga
ggg gat tac ttt ata aca 1200Asp Gln Glu Arg Thr Leu Arg Gln Arg Arg
Gly Asp Tyr Phe Ile Thr 385 390 395 400 ggg gat aag gcg cga aag gat
gaa gac ggc tat ttt tgg ttt gaa agc 1248Gly Asp Lys Ala Arg Lys Asp
Glu Asp Gly Tyr Phe Trp Phe Glu Ser 405 410 415 cgc aat gac gat gtg
atc atc agc tca gga tat aca atc ggc ccg ttt 1296Arg Asn Asp Asp Val
Ile Ile Ser Ser Gly Tyr Thr Ile Gly Pro Phe 420 425 430 gaa gtt gaa
gat gcg ctg atc aag cat cct gag gtc aaa gaa tgt gct 1344Glu Val Glu
Asp Ala Leu Ile Lys His Pro Glu Val Lys Glu Cys Ala 435 440 445 gtt
gtg gca agc cct gat gaa atc agg gga tcg atc gtg aaa gca tac 1392Val
Val Ala Ser Pro Asp Glu Ile Arg Gly Ser Ile Val Lys Ala Tyr 450 455
460 gtt gtc tta aag gac cca tcc cgc gga aat gaa cac ctg aca aag gaa
1440Val Val Leu Lys Asp Pro Ser Arg Gly Asn Glu His Leu Thr Lys Glu
465 470 475 480 tta caa gac cat gta aaa gcc atg acg gcc cct tat aaa
tac ccc cgg 1488Leu Gln Asp His Val Lys Ala Met Thr Ala Pro Tyr Lys
Tyr Pro Arg 485 490 495 gag ata gaa ttc atc gaa gaa ctg ccg aaa acg
cct tcg gcg aaa atc 1536Glu Ile Glu Phe Ile Glu Glu Leu Pro Lys Thr
Pro Ser Ala Lys Ile 500 505 510 aag cgc ttt gaa cta aga caa cgg gaa
atc gca ctt aaa acg aaa gct 1584Lys Arg Phe Glu Leu Arg Gln Arg Glu
Ile Ala Leu Lys Thr Lys Ala 515 520 525 gaa taa cca 1593Glu
38529PRTBacillus licheniformis DSM 13 38Leu Arg Arg Glu Asp Leu Ile
Ala Pro Glu Lys Tyr Asn Ala Val Asp 1 5 10 15 Glu Ile Glu Lys Phe
Lys Ser Ser Arg Asp Lys Thr Ala Leu Ile Trp 20 25 30 Glu Asp Glu
Ser Gly Arg Gln Val Ser Trp Ser Tyr Glu Lys Leu Ile 35 40 45 Glu
Lys Ala Tyr Lys Ile Gly Ser Ile Leu Thr Arg Ser Gly Leu Lys 50 55
60 Lys Gly Asp Lys Leu Ile Val Met Met Pro Arg Ile Pro Glu Thr Tyr
65 70 75 80 Ala Val Tyr Met Ala Ile Leu Lys Ala Gly Met Val Val Ile
Pro Cys 85 90 95 Ser Glu Met Leu Arg Ala Lys Asp Leu Asp Tyr Arg
Ile Lys His Ala 100 105 110 Gly Val Lys Gly Ala Val Val Tyr Ser Ala
Phe Leu Asp Ala Phe Leu 115 120 125 Asp Val Arg Ser Lys Glu Ala Leu
Ser Leu Phe Ala Val Gly Glu Ser 130 135 140 Ser Glu Gly Trp Ile Asn
Leu Leu Glu Lys Met Asn Gln Ala Ile Ala 145 150 155 160 Ala Asp Phe
Gln Ala Ala Asp Thr Ser Arg Asp Asp Ile Ala Phe Leu 165 170 175 Ser
Tyr Thr Ser Gly Thr Thr Gly Gln Pro Lys Gly Val Val His Thr 180 185
190 His Gly Trp Ala Tyr Ala His Leu Arg Thr Thr Ala Ser Ala Trp Leu
195 200 205 Asp Ile Ser Glu Gly Asp Leu Val Trp Ala Thr Ala Gly Pro
Gly Trp 210 215 220 Gln Lys Trp Val Trp Ser Pro Phe Leu Ala Val Leu
Gly Ser Gly Ala 225 230 235 240 Thr Gly Phe Ile Tyr His Gly Lys Phe
Thr Pro Glu Thr Tyr Leu Arg 245 250 255 Leu Ile Glu Arg His Gln Val
Asn Val Leu Cys Cys Thr Pro Thr Glu 260 265 270 Tyr Arg Phe Met Ala
Lys Val Asn Asp Leu Ser Arg Phe Asp Leu Ser 275 280 285 Ser Leu His
Ser Ala Val Ser Ala Gly Glu Pro Leu Asn Arg Glu Val 290 295 300 Ile
Asp Thr Phe Lys Lys His Phe His Ile Ala Val Arg Asp Gly Tyr 305 310
315 320 Gly Gln Thr Glu Ser Thr Leu Leu Val Gly Ile Leu Lys Gly Met
Lys 325 330 335 Ile Lys Pro Gly Ser Met Gly Lys Pro Thr Pro Gly Asn
Leu Val Asp 340 345 350 Ile Ile Asp Gly Asn Gly Lys Ser Cys Pro Pro
Gly Glu Thr Gly Asp 355 360 365 Ile Ala Val His Leu Ser Thr Pro Ala
Leu Phe Lys Glu Tyr Tyr Lys 370 375 380 Asp Gln Glu Arg Thr Leu Arg
Gln Arg Arg Gly Asp Tyr Phe Ile Thr 385 390 395 400 Gly Asp Lys Ala
Arg Lys Asp Glu Asp Gly Tyr Phe Trp Phe Glu Ser 405 410 415 Arg Asn
Asp Asp Val Ile Ile Ser Ser Gly Tyr Thr Ile Gly Pro Phe 420 425 430
Glu Val Glu Asp Ala Leu Ile Lys His Pro Glu Val Lys Glu Cys Ala 435
440 445 Val Val Ala Ser Pro Asp Glu Ile Arg Gly Ser Ile Val Lys Ala
Tyr 450 455 460 Val Val Leu Lys Asp Pro Ser Arg Gly Asn Glu His Leu
Thr Lys Glu 465 470 475 480 Leu Gln Asp His Val Lys Ala Met Thr Ala
Pro Tyr Lys Tyr Pro Arg 485 490 495 Glu Ile Glu Phe Ile Glu Glu Leu
Pro Lys Thr Pro Ser Ala Lys Ile 500 505 510 Lys Arg Phe Glu Leu Arg
Gln Arg Glu Ile Ala Leu Lys Thr Lys Ala 515 520 525 Glu
391524DNABacillus licheniformis DSM 13gene(1)..(1524)Lysine and/or
arginine decarboxylase (E.C. 4.1.1.18 or E.C. 4.1.1 .19,
respectively), speA 39atg cag aat gtc ata tcg gca tcc ggc agc gcc
tgg atg ccg aag aaa 48Met Gln Asn Val Ile Ser Ala Ser Gly Ser Ala
Trp Met
Pro Lys Lys 1 5 10 15 ata gag aag atg act aaa gga tcg gtt tat atg
aag aca cct tta tat 96Ile Glu Lys Met Thr Lys Gly Ser Val Tyr Met
Lys Thr Pro Leu Tyr 20 25 30 aca gca ttg gtg aac cat gct gaa gga
cat cat tat tct ttt cac gtt 144Thr Ala Leu Val Asn His Ala Glu Gly
His His Tyr Ser Phe His Val 35 40 45 ccg gga cat cac aat gga gat
gtc ttt ttt gat gaa gca aaa act ttt 192Pro Gly His His Asn Gly Asp
Val Phe Phe Asp Glu Ala Lys Thr Phe 50 55 60 ttt gaa acg att ctc
aaa gtg gat cta act gaa ctg aca gga ctg gat 240Phe Glu Thr Ile Leu
Lys Val Asp Leu Thr Glu Leu Thr Gly Leu Asp 65 70 75 80 gat tta cat
gag cca tct ggc gtc atc aag gaa gca cag gat tta gtt 288Asp Leu His
Glu Pro Ser Gly Val Ile Lys Glu Ala Gln Asp Leu Val 85 90 95 tcg
cgg ctt tac gga gcg gaa gaa agc ttt ttt ctc gtc aat ggt tcg 336Ser
Arg Leu Tyr Gly Ala Glu Glu Ser Phe Phe Leu Val Asn Gly Ser 100 105
110 acc gtt ggg aat ctg gct atg atc ctg gct gtt tgt cag ccg ggt gat
384Thr Val Gly Asn Leu Ala Met Ile Leu Ala Val Cys Gln Pro Gly Asp
115 120 125 acg ata ctc gtt cag cgt aac tgc cat aag tct gta ttt cat
gcg att 432Thr Ile Leu Val Gln Arg Asn Cys His Lys Ser Val Phe His
Ala Ile 130 135 140 gaa ctt tca ggc gcc cat ccg gtt ttt ttg aca ccg
gaa att gat gag 480Glu Leu Ser Gly Ala His Pro Val Phe Leu Thr Pro
Glu Ile Asp Glu 145 150 155 160 gcg atg gct gtt ccc aca cat ata ctg
tac gaa aca gtt gaa gat gca 528Ala Met Ala Val Pro Thr His Ile Leu
Tyr Glu Thr Val Glu Asp Ala 165 170 175 att tca caa tat ccg cac gca
aaa ggg atc gtg ttg aca tat cca aat 576Ile Ser Gln Tyr Pro His Ala
Lys Gly Ile Val Leu Thr Tyr Pro Asn 180 185 190 tac tac ggc cat gca
gtt gat ctg aag ccg atc ata gaa aag gcc cac 624Tyr Tyr Gly His Ala
Val Asp Leu Lys Pro Ile Ile Glu Lys Ala His 195 200 205 caa cac gat
att tcc gtt tta gtc gac gag gct cac ggc gcc cat ttt 672Gln His Asp
Ile Ser Val Leu Val Asp Glu Ala His Gly Ala His Phe 210 215 220 gtg
ctg ggc cat cca ttt ccc cag tcg tcg ttg aaa gcg ggc gct gat 720Val
Leu Gly His Pro Phe Pro Gln Ser Ser Leu Lys Ala Gly Ala Asp 225 230
235 240 gcc gtc gtc cag tcc gcg cat aaa acg ctc ccg gcg atg acg atg
ggg 768Ala Val Val Gln Ser Ala His Lys Thr Leu Pro Ala Met Thr Met
Gly 245 250 255 tcc tat ctg cat ctt aac agc ggc agg atc aac agg gac
aga ctg gca 816Ser Tyr Leu His Leu Asn Ser Gly Arg Ile Asn Arg Asp
Arg Leu Ala 260 265 270 tac tat ctg tcc gtg ctt caa agc agc agt cct
tcc tac cca att atg 864Tyr Tyr Leu Ser Val Leu Gln Ser Ser Ser Pro
Ser Tyr Pro Ile Met 275 280 285 gca tct ttg gat ata gct agg gca tac
gcc gaa gat atc ttg aaa aca 912Ala Ser Leu Asp Ile Ala Arg Ala Tyr
Ala Glu Asp Ile Leu Lys Thr 290 295 300 aat cga aca gcc gat att gag
aaa gag ctc atc aat atg aga gaa gtc 960Asn Arg Thr Ala Asp Ile Glu
Lys Glu Leu Ile Asn Met Arg Glu Val 305 310 315 320 ttt tct caa ata
aac gga gct gat att gtc gag ccg gct gat gcc cgt 1008Phe Ser Gln Ile
Asn Gly Ala Asp Ile Val Glu Pro Ala Asp Ala Arg 325 330 335 atc cgg
caa gat ccg ctt aag ctg tgt atc aga tca gcg tac ggc cac 1056Ile Arg
Gln Asp Pro Leu Lys Leu Cys Ile Arg Ser Ala Tyr Gly His 340 345 350
tcc ggt ttt gaa ctg aaa tcc ata ttt gaa gca aac ggc atc cat cct
1104Ser Gly Phe Glu Leu Lys Ser Ile Phe Glu Ala Asn Gly Ile His Pro
355 360 365 gaa ttg gca gat gaa agg caa gtc ctg ttg atc ctg ccg ctt
gaa gga 1152Glu Leu Ala Asp Glu Arg Gln Val Leu Leu Ile Leu Pro Leu
Glu Gly 370 375 380 aaa aat atg ccc gcg ccg gaa ctg atc agc aca atc
agt aaa gac atg 1200Lys Asn Met Pro Ala Pro Glu Leu Ile Ser Thr Ile
Ser Lys Asp Met 385 390 395 400 aaa gat aca gca gtt cga aat gat ctt
cct gcc gga atc gga atc cct 1248Lys Asp Thr Ala Val Arg Asn Asp Leu
Pro Ala Gly Ile Gly Ile Pro 405 410 415 tct gaa aaa gtc acg gct ttg
cct tac cgg aaa agc aag ttg tct gcc 1296Ser Glu Lys Val Thr Ala Leu
Pro Tyr Arg Lys Ser Lys Leu Ser Ala 420 425 430 ttc aaa aaa gaa tcg
gtg cct ttt acc gaa gca gcg ggc aga ata agc 1344Phe Lys Lys Glu Ser
Val Pro Phe Thr Glu Ala Ala Gly Arg Ile Ser 435 440 445 gcc gaa tcg
gtc aca cct tat cct ccg ggc att ccg ttg atc atg gcg 1392Ala Glu Ser
Val Thr Pro Tyr Pro Pro Gly Ile Pro Leu Ile Met Ala 450 455 460 ggt
gag aga ata aca aaa gag acg atc agc cgg ctg acg cgg ctc gtc 1440Gly
Glu Arg Ile Thr Lys Glu Thr Ile Ser Arg Leu Thr Arg Leu Val 465 470
475 480 gat ctg aat gta cac att cag ggc agc aat cag ctc aaa caa aag
caa 1488Asp Leu Asn Val His Ile Gln Gly Ser Asn Gln Leu Lys Gln Lys
Gln 485 490 495 tta act gta tat ata gaa gag gag aaa tca tga acg
1524Leu Thr Val Tyr Ile Glu Glu Glu Lys Ser 500 505
40506PRTBacillus licheniformis DSM 13 40Met Gln Asn Val Ile Ser Ala
Ser Gly Ser Ala Trp Met Pro Lys Lys 1 5 10 15 Ile Glu Lys Met Thr
Lys Gly Ser Val Tyr Met Lys Thr Pro Leu Tyr 20 25 30 Thr Ala Leu
Val Asn His Ala Glu Gly His His Tyr Ser Phe His Val 35 40 45 Pro
Gly His His Asn Gly Asp Val Phe Phe Asp Glu Ala Lys Thr Phe 50 55
60 Phe Glu Thr Ile Leu Lys Val Asp Leu Thr Glu Leu Thr Gly Leu Asp
65 70 75 80 Asp Leu His Glu Pro Ser Gly Val Ile Lys Glu Ala Gln Asp
Leu Val 85 90 95 Ser Arg Leu Tyr Gly Ala Glu Glu Ser Phe Phe Leu
Val Asn Gly Ser 100 105 110 Thr Val Gly Asn Leu Ala Met Ile Leu Ala
Val Cys Gln Pro Gly Asp 115 120 125 Thr Ile Leu Val Gln Arg Asn Cys
His Lys Ser Val Phe His Ala Ile 130 135 140 Glu Leu Ser Gly Ala His
Pro Val Phe Leu Thr Pro Glu Ile Asp Glu 145 150 155 160 Ala Met Ala
Val Pro Thr His Ile Leu Tyr Glu Thr Val Glu Asp Ala 165 170 175 Ile
Ser Gln Tyr Pro His Ala Lys Gly Ile Val Leu Thr Tyr Pro Asn 180 185
190 Tyr Tyr Gly His Ala Val Asp Leu Lys Pro Ile Ile Glu Lys Ala His
195 200 205 Gln His Asp Ile Ser Val Leu Val Asp Glu Ala His Gly Ala
His Phe 210 215 220 Val Leu Gly His Pro Phe Pro Gln Ser Ser Leu Lys
Ala Gly Ala Asp 225 230 235 240 Ala Val Val Gln Ser Ala His Lys Thr
Leu Pro Ala Met Thr Met Gly 245 250 255 Ser Tyr Leu His Leu Asn Ser
Gly Arg Ile Asn Arg Asp Arg Leu Ala 260 265 270 Tyr Tyr Leu Ser Val
Leu Gln Ser Ser Ser Pro Ser Tyr Pro Ile Met 275 280 285 Ala Ser Leu
Asp Ile Ala Arg Ala Tyr Ala Glu Asp Ile Leu Lys Thr 290 295 300 Asn
Arg Thr Ala Asp Ile Glu Lys Glu Leu Ile Asn Met Arg Glu Val 305 310
315 320 Phe Ser Gln Ile Asn Gly Ala Asp Ile Val Glu Pro Ala Asp Ala
Arg 325 330 335 Ile Arg Gln Asp Pro Leu Lys Leu Cys Ile Arg Ser Ala
Tyr Gly His 340 345 350 Ser Gly Phe Glu Leu Lys Ser Ile Phe Glu Ala
Asn Gly Ile His Pro 355 360 365 Glu Leu Ala Asp Glu Arg Gln Val Leu
Leu Ile Leu Pro Leu Glu Gly 370 375 380 Lys Asn Met Pro Ala Pro Glu
Leu Ile Ser Thr Ile Ser Lys Asp Met 385 390 395 400 Lys Asp Thr Ala
Val Arg Asn Asp Leu Pro Ala Gly Ile Gly Ile Pro 405 410 415 Ser Glu
Lys Val Thr Ala Leu Pro Tyr Arg Lys Ser Lys Leu Ser Ala 420 425 430
Phe Lys Lys Glu Ser Val Pro Phe Thr Glu Ala Ala Gly Arg Ile Ser 435
440 445 Ala Glu Ser Val Thr Pro Tyr Pro Pro Gly Ile Pro Leu Ile Met
Ala 450 455 460 Gly Glu Arg Ile Thr Lys Glu Thr Ile Ser Arg Leu Thr
Arg Leu Val 465 470 475 480 Asp Leu Asn Val His Ile Gln Gly Ser Asn
Gln Leu Lys Gln Lys Gln 485 490 495 Leu Thr Val Tyr Ile Glu Glu Glu
Lys Ser 500 505 41780DNABacillus licheniformis DSM
13gene(1)..(780)probable enoyl-CoA hydratase (E.C. 4.2.1.17), ysiB
41atg gca gca tta tca tac gcc gta gac ggc cat gtt gcg acg att acg
48Met Ala Ala Leu Ser Tyr Ala Val Asp Gly His Val Ala Thr Ile Thr 1
5 10 15 att cag cac ccg ccg gca aat gcg cta tcg acg caa gtg ctt gaa
gat 96Ile Gln His Pro Pro Ala Asn Ala Leu Ser Thr Gln Val Leu Glu
Asp 20 25 30 ctt tcg gca tgc ctt gat gaa ctt tca gaa cgt cag gat
gtc aga agc 144Leu Ser Ala Cys Leu Asp Glu Leu Ser Glu Arg Gln Asp
Val Arg Ser 35 40 45 gtc gtt att cac ggg gaa gga aga ttt ttc tcg
gca ggc gct gat att 192Val Val Ile His Gly Glu Gly Arg Phe Phe Ser
Ala Gly Ala Asp Ile 50 55 60 aaa gag ttt aca tca ttg atg gac ggg
tct gat tat gca aat ttg gct 240Lys Glu Phe Thr Ser Leu Met Asp Gly
Ser Asp Tyr Ala Asn Leu Ala 65 70 75 80 gat aag ggc cag cag att ttc
gaa aaa gta gaa tct ttt cca aaa ccg 288Asp Lys Gly Gln Gln Ile Phe
Glu Lys Val Glu Ser Phe Pro Lys Pro 85 90 95 gtg atc gcc gcg att
cac ggg gcc gct ctt gga ggc ggt ctt gag cta 336Val Ile Ala Ala Ile
His Gly Ala Ala Leu Gly Gly Gly Leu Glu Leu 100 105 110 gcg atg gca
tgc cac atc cgg att gcc gag gaa agc gca aag ctc ggc 384Ala Met Ala
Cys His Ile Arg Ile Ala Glu Glu Ser Ala Lys Leu Gly 115 120 125 ctt
ccc gaa ctg aat ctc gga atc att ccc ggc ttt gcg gga acg cag 432Leu
Pro Glu Leu Asn Leu Gly Ile Ile Pro Gly Phe Ala Gly Thr Gln 130 135
140 cgc ctt ccc aag tat gtg ggc acc gcc aaa gcg ctt gaa atg atc ggg
480Arg Leu Pro Lys Tyr Val Gly Thr Ala Lys Ala Leu Glu Met Ile Gly
145 150 155 160 aca tca gag ccg ata tcc ggc aag gaa gct ttt gaa tac
ggt ctt gtc 528Thr Ser Glu Pro Ile Ser Gly Lys Glu Ala Phe Glu Tyr
Gly Leu Val 165 170 175 acg att ttg gcg gcg aat gaa gaa gaa gtg ctg
caa aag gca aaa gag 576Thr Ile Leu Ala Ala Asn Glu Glu Glu Val Leu
Gln Lys Ala Lys Glu 180 185 190 ctc gca caa aaa ttc gct gaa aaa agt
ccg cag acg atg gct tat gtc 624Leu Ala Gln Lys Phe Ala Glu Lys Ser
Pro Gln Thr Met Ala Tyr Val 195 200 205 att gaa ctc ctg aat tcg agc
aaa gtg tat tcg tat gaa gga ggc ctc 672Ile Glu Leu Leu Asn Ser Ser
Lys Val Tyr Ser Tyr Glu Gly Gly Leu 210 215 220 aag cta gaa ggg aaa
tac ttc ggc gaa gtg ttc caa tcc gag gat gcg 720Lys Leu Glu Gly Lys
Tyr Phe Gly Glu Val Phe Gln Ser Glu Asp Ala 225 230 235 240 aag gaa
ggg att cag gca ttt ctt gaa aag cgg aag ccg cat ttt aaa 768Lys Glu
Gly Ile Gln Ala Phe Leu Glu Lys Arg Lys Pro His Phe Lys 245 250 255
ggg aaa taa caa 780Gly Lys 42258PRTBacillus licheniformis DSM 13 42
Met Ala Ala Leu Ser Tyr Ala Val Asp Gly His Val Ala Thr Ile Thr 1 5
10 15 Ile Gln His Pro Pro Ala Asn Ala Leu Ser Thr Gln Val Leu Glu
Asp 20 25 30 Leu Ser Ala Cys Leu Asp Glu Leu Ser Glu Arg Gln Asp
Val Arg Ser 35 40 45 Val Val Ile His Gly Glu Gly Arg Phe Phe Ser
Ala Gly Ala Asp Ile 50 55 60 Lys Glu Phe Thr Ser Leu Met Asp Gly
Ser Asp Tyr Ala Asn Leu Ala 65 70 75 80 Asp Lys Gly Gln Gln Ile Phe
Glu Lys Val Glu Ser Phe Pro Lys Pro 85 90 95 Val Ile Ala Ala Ile
His Gly Ala Ala Leu Gly Gly Gly Leu Glu Leu 100 105 110 Ala Met Ala
Cys His Ile Arg Ile Ala Glu Glu Ser Ala Lys Leu Gly 115 120 125 Leu
Pro Glu Leu Asn Leu Gly Ile Ile Pro Gly Phe Ala Gly Thr Gln 130 135
140 Arg Leu Pro Lys Tyr Val Gly Thr Ala Lys Ala Leu Glu Met Ile Gly
145 150 155 160 Thr Ser Glu Pro Ile Ser Gly Lys Glu Ala Phe Glu Tyr
Gly Leu Val 165 170 175 Thr Ile Leu Ala Ala Asn Glu Glu Glu Val Leu
Gln Lys Ala Lys Glu 180 185 190 Leu Ala Gln Lys Phe Ala Glu Lys Ser
Pro Gln Thr Met Ala Tyr Val 195 200 205 Ile Glu Leu Leu Asn Ser Ser
Lys Val Tyr Ser Tyr Glu Gly Gly Leu 210 215 220 Lys Leu Glu Gly Lys
Tyr Phe Gly Glu Val Phe Gln Ser Glu Asp Ala 225 230 235 240 Lys Glu
Gly Ile Gln Ala Phe Leu Glu Lys Arg Lys Pro His Phe Lys 245 250 255
Gly Lys 432394DNABacillus licheniformis DSM
13gene(1)..(2394)similar to 3-hydroxyacyl-CoA dehydrogenase (E.C.
1.1.1.35) 43atg gtc aag cat att cga aaa gcc gcc gtt atc ggt tcc ggt
gtt atg 48Met Val Lys His Ile Arg Lys Ala Ala Val Ile Gly Ser Gly
Val Met 1 5 10 15 ggt tcc ggc atc gcg gct cac tta gcc aat atc ggg
att cct gta tta 96Gly Ser Gly Ile Ala Ala His Leu Ala Asn Ile Gly
Ile Pro Val Leu 20 25 30 ctg ctc gat atg gtg ccg cat gaa tta aca
gat gag gaa agg aaa aaa 144Leu Leu Asp Met Val Pro His Glu Leu Thr
Asp Glu Glu Arg Lys Lys 35 40 45 ggc cgc acc ctt gaa gac ccg gat
gtg cgc aat cgg ctg gca cgg aca 192Gly Arg Thr Leu Glu Asp Pro Asp
Val Arg Asn Arg Leu Ala Arg Thr 50 55 60 gcc gta caa aag ctg cta
aag caa aaa ccg gct ccg ctg act tca aag 240Ala Val Gln Lys Leu Leu
Lys Gln Lys Pro Ala Pro Leu Thr Ser Lys 65 70 75 80 aag aat atc agc
cgt atc agc aca ggg aac atg gaa gac gat ttt gaa 288Lys Asn Ile Ser
Arg Ile Ser Thr Gly Asn Met Glu Asp Asp Phe Glu 85 90 95 aag att
aaa gat gcc gac tgg att att gaa gtt gtt gtc gaa aac ctg 336Lys Ile
Lys Asp Ala Asp Trp Ile Ile Glu Val Val Val
Glu Asn Leu 100 105 110 gag atc aaa aag caa gtg ttt tca aag gtc gat
caa tac aga aaa caa 384Glu Ile Lys Lys Gln Val Phe Ser Lys Val Asp
Gln Tyr Arg Lys Gln 115 120 125 gga agc atc gtc agc agc aac aca tca
ggc atc tcc gtc agg cag atg 432Gly Ser Ile Val Ser Ser Asn Thr Ser
Gly Ile Ser Val Arg Gln Met 130 135 140 gct gag gga agg tcc gct gat
ttt aaa aag cac ttt tta gga acg cac 480Ala Glu Gly Arg Ser Ala Asp
Phe Lys Lys His Phe Leu Gly Thr His 145 150 155 160 ttc ttc aat ccg
gcc cgc tat tta aag ctc ctt gag gtg att ccg att 528Phe Phe Asn Pro
Ala Arg Tyr Leu Lys Leu Leu Glu Val Ile Pro Ile 165 170 175 gac gaa
acg gag ccc gaa gtg ctg tcc ttt atg aag aaa ttc ggc gaa 576Asp Glu
Thr Glu Pro Glu Val Leu Ser Phe Met Lys Lys Phe Gly Glu 180 185 190
gac gtt ctt ggc aaa ggg gtt gtt gaa gcg aaa gac acg cca aac ttt
624Asp Val Leu Gly Lys Gly Val Val Glu Ala Lys Asp Thr Pro Asn Phe
195 200 205 atc gcc aac cgc atc gga acc tac ggc ttg ctt gtg acg gtt
cag gaa 672Ile Ala Asn Arg Ile Gly Thr Tyr Gly Leu Leu Val Thr Val
Gln Glu 210 215 220 atg ctg aaa ggc ggc tac acg ata ggc gaa gtc gat
tcg att aca ggt 720Met Leu Lys Gly Gly Tyr Thr Ile Gly Glu Val Asp
Ser Ile Thr Gly 225 230 235 240 ccg ctg atc gga cgc ccg aaa agc gcg
acc ttc aga acg ctc gat gtc 768Pro Leu Ile Gly Arg Pro Lys Ser Ala
Thr Phe Arg Thr Leu Asp Val 245 250 255 gtc ggc ctt gat aca ttt tca
cac gtt gcc aaa aat gtc tat aac caa 816Val Gly Leu Asp Thr Phe Ser
His Val Ala Lys Asn Val Tyr Asn Gln 260 265 270 gtc acg ggc gaa gaa
aaa aac gtt ttc cgg ctc cct gaa ttt att gaa 864Val Thr Gly Glu Glu
Lys Asn Val Phe Arg Leu Pro Glu Phe Ile Glu 275 280 285 caa atg ctc
gaa aaa ggc tgg atc ggg agc aaa gcg aaa caa ggt ttt 912Gln Met Leu
Glu Lys Gly Trp Ile Gly Ser Lys Ala Lys Gln Gly Phe 290 295 300 tac
aaa aaa gaa gga aaa gac atc ctt gag ctc gat ccg atg acg atg 960Tyr
Lys Lys Glu Gly Lys Asp Ile Leu Glu Leu Asp Pro Met Thr Met 305 310
315 320 acc tac agc gcg cgt caa aca tta aaa aca tcg gga ctg gaa gcg
gcc 1008Thr Tyr Ser Ala Arg Gln Thr Leu Lys Thr Ser Gly Leu Glu Ala
Ala 325 330 335 aag caa atg aaa ggc gca gga gcg aga ttg aaa gcg ctt
gtt tat tcc 1056Lys Gln Met Lys Gly Ala Gly Ala Arg Leu Lys Ala Leu
Val Tyr Ser 340 345 350 gat gac agg gcg gga agc ctc ctt tgg aag att
aca gct ccg acg ctt 1104Asp Asp Arg Ala Gly Ser Leu Leu Trp Lys Ile
Thr Ala Pro Thr Leu 355 360 365 gta tat tcg gcg gag ctg aca ggg gaa
atc gcc gac agc ata acg gcg 1152Val Tyr Ser Ala Glu Leu Thr Gly Glu
Ile Ala Asp Ser Ile Thr Ala 370 375 380 gtt gat cag gcg atg aaa tgg
gga ttc ggc tgg tcg gaa ggg ccg ttt 1200Val Asp Gln Ala Met Lys Trp
Gly Phe Gly Trp Ser Glu Gly Pro Phe 385 390 395 400 gaa atg tgg gac
agc atc ggt gtg aaa agc tcg gtg caa aag ctt gaa 1248Glu Met Trp Asp
Ser Ile Gly Val Lys Ser Ser Val Gln Lys Leu Glu 405 410 415 gca gag
ggc tgg aag gtc gcg gac tgg gtg aag gaa atg ctc gca aaa 1296Ala Glu
Gly Trp Lys Val Ala Asp Trp Val Lys Glu Met Leu Ala Lys 420 425 430
gga cat gaa acc ttc tac ctt aag gaa aac gga aag acg ttc tac tac
1344Gly His Glu Thr Phe Tyr Leu Lys Glu Asn Gly Lys Thr Phe Tyr Tyr
435 440 445 tgt ttt gaa agc ggc gaa tac agg gcg ctt caa gaa aac aag
aaa acg 1392Cys Phe Glu Ser Gly Glu Tyr Arg Ala Leu Gln Glu Asn Lys
Lys Thr 450 455 460 atc cat ttg tcg tca ctc aaa gaa aca aaa ggc gtc
atc aag aaa aac 1440Ile His Leu Ser Ser Leu Lys Glu Thr Lys Gly Val
Ile Lys Lys Asn 465 470 475 480 agc ggg gca agt ttg atc gat tta ggc
gat gat gtc gca ctg ctc gaa 1488Ser Gly Ala Ser Leu Ile Asp Leu Gly
Asp Asp Val Ala Leu Leu Glu 485 490 495 ttt cac tcg aaa agc aac gca
atc ggc ctg gac atc att caa atg ctg 1536Phe His Ser Lys Ser Asn Ala
Ile Gly Leu Asp Ile Ile Gln Met Leu 500 505 510 aaa ttt gct ctc gag
gaa gta gac gcc aac tat aaa ggg ctt gtg atc 1584Lys Phe Ala Leu Glu
Glu Val Asp Ala Asn Tyr Lys Gly Leu Val Ile 515 520 525 ggc aac cag
ggc aaa aat ttc tgc gtc gga gcc aat ctc gcg atg atg 1632Gly Asn Gln
Gly Lys Asn Phe Cys Val Gly Ala Asn Leu Ala Met Met 530 535 540 ctg
atg gaa gct caa gac gac aac tat atg gaa att gac atg atc atc 1680Leu
Met Glu Ala Gln Asp Asp Asn Tyr Met Glu Ile Asp Met Ile Ile 545 550
555 560 cgt cag ttc caa gag acg atg atg aaa gtg aaa tac agt tcc aag
ccg 1728Arg Gln Phe Gln Glu Thr Met Met Lys Val Lys Tyr Ser Ser Lys
Pro 565 570 575 gtc gtc gct gct ccg ttc ggc atg acg ctt ggc ggg ggg
act gag cta 1776Val Val Ala Ala Pro Phe Gly Met Thr Leu Gly Gly Gly
Thr Glu Leu 580 585 590 tgc ctg cct gct gcc cgt gtt caa gcc tca agc
gaa tca tac atg ggc 1824Cys Leu Pro Ala Ala Arg Val Gln Ala Ser Ser
Glu Ser Tyr Met Gly 595 600 605 ctc gtt gaa aca ggc gtc ggt ctg att
ccg ggc ggc gga ggc aat aaa 1872Leu Val Glu Thr Gly Val Gly Leu Ile
Pro Gly Gly Gly Gly Asn Lys 610 615 620 gag ctt tat tta aat tac ttg
aaa ggg ctg cct gaa ggc gtt aaa ccg 1920Glu Leu Tyr Leu Asn Tyr Leu
Lys Gly Leu Pro Glu Gly Val Lys Pro 625 630 635 640 gat atc cag gag
gcc gcc atc aag acg ttt gag aca att gcc ctt gca 1968Asp Ile Gln Glu
Ala Ala Ile Lys Thr Phe Glu Thr Ile Ala Leu Ala 645 650 655 aaa acg
tcg act tcg gct caa gaa gcg aaa gaa ctg aac atc tta acc 2016Lys Thr
Ser Thr Ser Ala Gln Glu Ala Lys Glu Leu Asn Ile Leu Thr 660 665 670
gca gag gat caa atc agc atc aat cag gat cac ttg tta tac gat gcc
2064Ala Glu Asp Gln Ile Ser Ile Asn Gln Asp His Leu Leu Tyr Asp Ala
675 680 685 aaa aaa ctc gtc ctg aca ctt tcc gaa agc ggc tac cgt ccg
ccg gtg 2112Lys Lys Leu Val Leu Thr Leu Ser Glu Ser Gly Tyr Arg Pro
Pro Val 690 695 700 aaa gag aaa gtt ccg gtg acg ggc gaa acc ggc tat
gcg gcg ctc ctg 2160Lys Glu Lys Val Pro Val Thr Gly Glu Thr Gly Tyr
Ala Ala Leu Leu 705 710 715 720 ctc ggt gct gaa tcg tta aaa ctt tcc
ggc gcc atc tct gaa cac gac 2208Leu Gly Ala Glu Ser Leu Lys Leu Ser
Gly Ala Ile Ser Glu His Asp 725 730 735 atg aag att gcg aaa aaa ctg
gcg ttt gtc atc gcg ggc ggc cga gtt 2256Met Lys Ile Ala Lys Lys Leu
Ala Phe Val Ile Ala Gly Gly Arg Val 740 745 750 cca ttc ggc gcc gaa
gta acg gaa gaa tac ttg ctc aat ttg gaa aga 2304Pro Phe Gly Ala Glu
Val Thr Glu Glu Tyr Leu Leu Asn Leu Glu Arg 755 760 765 gaa gcg ttt
ctg agc ctt gtg agc gaa ccg aaa tcg cag gcg aga atg 2352Glu Ala Phe
Leu Ser Leu Val Ser Glu Pro Lys Ser Gln Ala Arg Met 770 775 780 cag
cat atg ctt gtg aaa ggc aaa cct ttg cgt aat tag gag 2394Gln His Met
Leu Val Lys Gly Lys Pro Leu Arg Asn 785 790 795 44796PRTBacillus
licheniformis DSM 13 44Met Val Lys His Ile Arg Lys Ala Ala Val Ile
Gly Ser Gly Val Met 1 5 10 15 Gly Ser Gly Ile Ala Ala His Leu Ala
Asn Ile Gly Ile Pro Val Leu 20 25 30 Leu Leu Asp Met Val Pro His
Glu Leu Thr Asp Glu Glu Arg Lys Lys 35 40 45 Gly Arg Thr Leu Glu
Asp Pro Asp Val Arg Asn Arg Leu Ala Arg Thr 50 55 60 Ala Val Gln
Lys Leu Leu Lys Gln Lys Pro Ala Pro Leu Thr Ser Lys 65 70 75 80 Lys
Asn Ile Ser Arg Ile Ser Thr Gly Asn Met Glu Asp Asp Phe Glu 85 90
95 Lys Ile Lys Asp Ala Asp Trp Ile Ile Glu Val Val Val Glu Asn Leu
100 105 110 Glu Ile Lys Lys Gln Val Phe Ser Lys Val Asp Gln Tyr Arg
Lys Gln 115 120 125 Gly Ser Ile Val Ser Ser Asn Thr Ser Gly Ile Ser
Val Arg Gln Met 130 135 140 Ala Glu Gly Arg Ser Ala Asp Phe Lys Lys
His Phe Leu Gly Thr His 145 150 155 160 Phe Phe Asn Pro Ala Arg Tyr
Leu Lys Leu Leu Glu Val Ile Pro Ile 165 170 175 Asp Glu Thr Glu Pro
Glu Val Leu Ser Phe Met Lys Lys Phe Gly Glu 180 185 190 Asp Val Leu
Gly Lys Gly Val Val Glu Ala Lys Asp Thr Pro Asn Phe 195 200 205 Ile
Ala Asn Arg Ile Gly Thr Tyr Gly Leu Leu Val Thr Val Gln Glu 210 215
220 Met Leu Lys Gly Gly Tyr Thr Ile Gly Glu Val Asp Ser Ile Thr Gly
225 230 235 240 Pro Leu Ile Gly Arg Pro Lys Ser Ala Thr Phe Arg Thr
Leu Asp Val 245 250 255 Val Gly Leu Asp Thr Phe Ser His Val Ala Lys
Asn Val Tyr Asn Gln 260 265 270 Val Thr Gly Glu Glu Lys Asn Val Phe
Arg Leu Pro Glu Phe Ile Glu 275 280 285 Gln Met Leu Glu Lys Gly Trp
Ile Gly Ser Lys Ala Lys Gln Gly Phe 290 295 300 Tyr Lys Lys Glu Gly
Lys Asp Ile Leu Glu Leu Asp Pro Met Thr Met 305 310 315 320 Thr Tyr
Ser Ala Arg Gln Thr Leu Lys Thr Ser Gly Leu Glu Ala Ala 325 330 335
Lys Gln Met Lys Gly Ala Gly Ala Arg Leu Lys Ala Leu Val Tyr Ser 340
345 350 Asp Asp Arg Ala Gly Ser Leu Leu Trp Lys Ile Thr Ala Pro Thr
Leu 355 360 365 Val Tyr Ser Ala Glu Leu Thr Gly Glu Ile Ala Asp Ser
Ile Thr Ala 370 375 380 Val Asp Gln Ala Met Lys Trp Gly Phe Gly Trp
Ser Glu Gly Pro Phe 385 390 395 400 Glu Met Trp Asp Ser Ile Gly Val
Lys Ser Ser Val Gln Lys Leu Glu 405 410 415 Ala Glu Gly Trp Lys Val
Ala Asp Trp Val Lys Glu Met Leu Ala Lys 420 425 430 Gly His Glu Thr
Phe Tyr Leu Lys Glu Asn Gly Lys Thr Phe Tyr Tyr 435 440 445 Cys Phe
Glu Ser Gly Glu Tyr Arg Ala Leu Gln Glu Asn Lys Lys Thr 450 455 460
Ile His Leu Ser Ser Leu Lys Glu Thr Lys Gly Val Ile Lys Lys Asn 465
470 475 480 Ser Gly Ala Ser Leu Ile Asp Leu Gly Asp Asp Val Ala Leu
Leu Glu 485 490 495 Phe His Ser Lys Ser Asn Ala Ile Gly Leu Asp Ile
Ile Gln Met Leu 500 505 510 Lys Phe Ala Leu Glu Glu Val Asp Ala Asn
Tyr Lys Gly Leu Val Ile 515 520 525 Gly Asn Gln Gly Lys Asn Phe Cys
Val Gly Ala Asn Leu Ala Met Met 530 535 540 Leu Met Glu Ala Gln Asp
Asp Asn Tyr Met Glu Ile Asp Met Ile Ile 545 550 555 560 Arg Gln Phe
Gln Glu Thr Met Met Lys Val Lys Tyr Ser Ser Lys Pro 565 570 575 Val
Val Ala Ala Pro Phe Gly Met Thr Leu Gly Gly Gly Thr Glu Leu 580 585
590 Cys Leu Pro Ala Ala Arg Val Gln Ala Ser Ser Glu Ser Tyr Met Gly
595 600 605 Leu Val Glu Thr Gly Val Gly Leu Ile Pro Gly Gly Gly Gly
Asn Lys 610 615 620 Glu Leu Tyr Leu Asn Tyr Leu Lys Gly Leu Pro Glu
Gly Val Lys Pro 625 630 635 640 Asp Ile Gln Glu Ala Ala Ile Lys Thr
Phe Glu Thr Ile Ala Leu Ala 645 650 655 Lys Thr Ser Thr Ser Ala Gln
Glu Ala Lys Glu Leu Asn Ile Leu Thr 660 665 670 Ala Glu Asp Gln Ile
Ser Ile Asn Gln Asp His Leu Leu Tyr Asp Ala 675 680 685 Lys Lys Leu
Val Leu Thr Leu Ser Glu Ser Gly Tyr Arg Pro Pro Val 690 695 700 Lys
Glu Lys Val Pro Val Thr Gly Glu Thr Gly Tyr Ala Ala Leu Leu 705 710
715 720 Leu Gly Ala Glu Ser Leu Lys Leu Ser Gly Ala Ile Ser Glu His
Asp 725 730 735 Met Lys Ile Ala Lys Lys Leu Ala Phe Val Ile Ala Gly
Gly Arg Val 740 745 750 Pro Phe Gly Ala Glu Val Thr Glu Glu Tyr Leu
Leu Asn Leu Glu Arg 755 760 765 Glu Ala Phe Leu Ser Leu Val Ser Glu
Pro Lys Ser Gln Ala Arg Met 770 775 780 Gln His Met Leu Val Lys Gly
Lys Pro Leu Arg Asn 785 790 795 452838DNABacillus licheniformis DSM
13gene(1)..(2838)2-oxoglutarate dehydrogenase E1 component (E.C.
1.2.4.2) 45atg ttt caa aat agt atg aaa caa aga atg act tgg gaa gaa
ttt cac 48Met Phe Gln Asn Ser Met Lys Gln Arg Met Thr Trp Glu Glu
Phe His 1 5 10 15 ggt ccg aac ctc ggc tat gtg ctg gag ctt tac gat
cag tac gtc aag 96Gly Pro Asn Leu Gly Tyr Val Leu Glu Leu Tyr Asp
Gln Tyr Val Lys 20 25 30 gat cca gaa agc ttg gat gct gat tta aaa
gag atg ttt gac gaa ctt 144Asp Pro Glu Ser Leu Asp Ala Asp Leu Lys
Glu Met Phe Asp Glu Leu 35 40 45 gga gct ccc ccg ggc gat att agg
gcc gcc tct caa aaa aac gaa gag 192Gly Ala Pro Pro Gly Asp Ile Arg
Ala Ala Ser Gln Lys Asn Glu Glu 50 55 60 gca gat ttc acg gct gga
tct att caa aaa atc gca tca gcg gta aaa 240Ala Asp Phe Thr Ala Gly
Ser Ile Gln Lys Ile Ala Ser Ala Val Lys 65 70 75 80 ctt gca gaa gat
att aga acc tat ggc cat tta aac gct tcc gtc aat 288Leu Ala Glu Asp
Ile Arg Thr Tyr Gly His Leu Asn Ala Ser Val Asn 85 90 95 cca ctg
aga aaa aca caa gag aaa cag gag ctt ttt cct ctt gct gag 336Pro Leu
Arg Lys Thr Gln Glu Lys Gln Glu Leu Phe Pro Leu Ala Glu 100 105 110
tac ggg tta act gag cag gat gtg aaa aaa atc ccg gcg tct gtc ata
384Tyr Gly Leu Thr Glu Gln Asp Val Lys Lys Ile Pro Ala Ser Val Ile
115 120 125 tgc aaa gat gcc cct aaa gaa gta acg aac ggt tta gaa gcc
atc cag 432Cys Lys Asp Ala Pro Lys Glu Val Thr Asn Gly Leu Glu Ala
Ile Gln 130 135 140 tac tta aga aac aca tac aaa aaa tcg att tct ttt
gaa ttt gac cat 480Tyr Leu Arg Asn Thr Tyr Lys Lys Ser Ile Ser Phe
Glu Phe Asp His 145 150 155 160 gtg cac att ttt gaa gag cgc aac tgg
ctg atg aaa aag atc gaa tcc 528Val His Ile Phe Glu Glu Arg Asn Trp
Leu Met Lys Lys Ile Glu Ser
165 170 175 ggg gaa tta ttc acc ccg aaa tcg aaa gaa aaa ctg gta gaa
gtt tta 576Gly Glu Leu Phe Thr Pro Lys Ser Lys Glu Lys Leu Val Glu
Val Leu 180 185 190 aga agg ctt aca gaa gtg gaa agc ctt gaa cag ttt
ctc cac aaa acc 624Arg Arg Leu Thr Glu Val Glu Ser Leu Glu Gln Phe
Leu His Lys Thr 195 200 205 ttt gtc ggg caa aaa cgc ttt tca ata gaa
gga ctt gat gcg ctt gtg 672Phe Val Gly Gln Lys Arg Phe Ser Ile Glu
Gly Leu Asp Ala Leu Val 210 215 220 ccc atg ctg gat gat att atc gcc
aag tcc gtt tcg gca ggt acg aca 720Pro Met Leu Asp Asp Ile Ile Ala
Lys Ser Val Ser Ala Gly Thr Thr 225 230 235 240 aac gtc aat atc gga
atg gcg cac agg ggc cgc ctg aat gtt ctt gcg 768Asn Val Asn Ile Gly
Met Ala His Arg Gly Arg Leu Asn Val Leu Ala 245 250 255 cat gtg ctc
gga aaa cct tat gaa atc att ttt tct gaa ttc cag cat 816His Val Leu
Gly Lys Pro Tyr Glu Ile Ile Phe Ser Glu Phe Gln His 260 265 270 gcg
ccg aac aaa gat ctc gtt ccg tcg gaa ggt tcg acc ggg atc aat 864Ala
Pro Asn Lys Asp Leu Val Pro Ser Glu Gly Ser Thr Gly Ile Asn 275 280
285 tac ggc tgg acg ggc gac gta aaa tac cac ctc ggc gcc aac cgc cag
912Tyr Gly Trp Thr Gly Asp Val Lys Tyr His Leu Gly Ala Asn Arg Gln
290 295 300 att cag gat gag cat acg aaa acg gcg cgc att gcg ctc gcg
aac aat 960Ile Gln Asp Glu His Thr Lys Thr Ala Arg Ile Ala Leu Ala
Asn Asn 305 310 315 320 ccg agc cac ctt gag ttc atc gat ccg atc gtt
gag gga tcg aca aga 1008Pro Ser His Leu Glu Phe Ile Asp Pro Ile Val
Glu Gly Ser Thr Arg 325 330 335 gcc gcc cag gaa acg aga acg gag agc
ggc tat ccg gtt caa gac gtc 1056Ala Ala Gln Glu Thr Arg Thr Glu Ser
Gly Tyr Pro Val Gln Asp Val 340 345 350 aaa aaa tcg atg gcg att ctg
att cac ggc gat gcg gca ttc cca ggg 1104Lys Lys Ser Met Ala Ile Leu
Ile His Gly Asp Ala Ala Phe Pro Gly 355 360 365 gaa ggc att gtc gcg
gaa acg ctg aat tta agc cag ctt aaa ggg tat 1152Glu Gly Ile Val Ala
Glu Thr Leu Asn Leu Ser Gln Leu Lys Gly Tyr 370 375 380 caa gtg ggc
gga gcg att cac att atc gcc aat aac atg atc ggc ttt 1200Gln Val Gly
Gly Ala Ile His Ile Ile Ala Asn Asn Met Ile Gly Phe 385 390 395 400
acg acg gaa agc aat gag tca aga tcg acg aaa tat gca agc gac ctt
1248Thr Thr Glu Ser Asn Glu Ser Arg Ser Thr Lys Tyr Ala Ser Asp Leu
405 410 415 gcg aaa ggt ttt gaa att ccg atc gtc cac gtc aat gct gat
gat ccc 1296Ala Lys Gly Phe Glu Ile Pro Ile Val His Val Asn Ala Asp
Asp Pro 420 425 430 gaa gca tgt ctt tca gcg gtt cag ctc gct gtt gaa
tac cgc atg act 1344Glu Ala Cys Leu Ser Ala Val Gln Leu Ala Val Glu
Tyr Arg Met Thr 435 440 445 ttc aac aaa gac ttt ttg atc gat ctg atc
ggc tac cgc cgt ttt ggc 1392Phe Asn Lys Asp Phe Leu Ile Asp Leu Ile
Gly Tyr Arg Arg Phe Gly 450 455 460 cac aat gaa atg gat gag ccg tcc
gca acg cag ccg atg ctg tat gat 1440His Asn Glu Met Asp Glu Pro Ser
Ala Thr Gln Pro Met Leu Tyr Asp 465 470 475 480 gcg gtc aga aag cat
ccg acc gtc aaa aac atc ttt gct gaa aag ctg 1488Ala Val Arg Lys His
Pro Thr Val Lys Asn Ile Phe Ala Glu Lys Leu 485 490 495 att cat aaa
ggg atc gtc gat aaa gaa acc gtc ggc aaa atc aag gac 1536Ile His Lys
Gly Ile Val Asp Lys Glu Thr Val Gly Lys Ile Lys Asp 500 505 510 gct
gtc cag aag cgt tta gaa gaa gcc tat cgc aaa gtg ccg gcc aaa 1584Ala
Val Gln Lys Arg Leu Glu Glu Ala Tyr Arg Lys Val Pro Ala Lys 515 520
525 aag gaa gac atg acg cat gaa atc gta ctt cca gag ccg gtc tcc aac
1632Lys Glu Asp Met Thr His Glu Ile Val Leu Pro Glu Pro Val Ser Asn
530 535 540 ggt ttt cct gat gtt gac aca tcg gtt gat ttt gaa act ttg
cgc aaa 1680Gly Phe Pro Asp Val Asp Thr Ser Val Asp Phe Glu Thr Leu
Arg Lys 545 550 555 560 atc aat cag gag ctt gtt tca tgg ccg gaa aac
ttc aac gtt ttc gat 1728Ile Asn Gln Glu Leu Val Ser Trp Pro Glu Asn
Phe Asn Val Phe Asp 565 570 575 aag cta aaa cga atc ctt gaa agg cgc
gcc aaa gct ttc gaa gat gac 1776Lys Leu Lys Arg Ile Leu Glu Arg Arg
Ala Lys Ala Phe Glu Asp Asp 580 585 590 cga aaa gtc gac tgg tcg ctt
gca gag gcg atg gcg ttt gcg tcg att 1824Arg Lys Val Asp Trp Ser Leu
Ala Glu Ala Met Ala Phe Ala Ser Ile 595 600 605 ttg aaa gac ggt acg
ccg cta agg ctg acc ggg cag gat tca gaa cgc 1872Leu Lys Asp Gly Thr
Pro Leu Arg Leu Thr Gly Gln Asp Ser Glu Arg 610 615 620 ggc aca ttc
gca cac cgc aac ctt gtc ctt cac gac agc aag aca ggg 1920Gly Thr Phe
Ala His Arg Asn Leu Val Leu His Asp Ser Lys Thr Gly 625 630 635 640
gac gaa ttc atc gcg ctg cat cac ctt gcc gat acg aaa gcg tca ttt
1968Asp Glu Phe Ile Ala Leu His His Leu Ala Asp Thr Lys Ala Ser Phe
645 650 655 gcg gtt cac aac agc ccg ctt tct gaa ggg tcc gtc ctc ggc
ttc gaa 2016Ala Val His Asn Ser Pro Leu Ser Glu Gly Ser Val Leu Gly
Phe Glu 660 665 670 tac ggc tat aac gtg tct tcg ccg gaa acg atg gtg
atc tgg gaa gcg 2064Tyr Gly Tyr Asn Val Ser Ser Pro Glu Thr Met Val
Ile Trp Glu Ala 675 680 685 cag ttt gga gat ttt gca aat gcg gcg caa
gtt tac ttt gac cag ttc 2112Gln Phe Gly Asp Phe Ala Asn Ala Ala Gln
Val Tyr Phe Asp Gln Phe 690 695 700 att tct gca gga aga gcg aag tgg
ggt caa aaa tca ggg ctg gtt gtt 2160Ile Ser Ala Gly Arg Ala Lys Trp
Gly Gln Lys Ser Gly Leu Val Val 705 710 715 720 ctc ttg ccg cac ggc
tat gaa ggg cag ggg cct gag cat tca agc gga 2208Leu Leu Pro His Gly
Tyr Glu Gly Gln Gly Pro Glu His Ser Ser Gly 725 730 735 aga aca gag
cga ttc ctt caa ttg gcg gcg gaa aac aac tgg act gtc 2256Arg Thr Glu
Arg Phe Leu Gln Leu Ala Ala Glu Asn Asn Trp Thr Val 740 745 750 gcc
aac ctg acg agc gct gcc caa tac ttt cat att tta aga agg cag 2304Ala
Asn Leu Thr Ser Ala Ala Gln Tyr Phe His Ile Leu Arg Arg Gln 755 760
765 gcg aag atg ctc ctt cgc gag gag atc cgc ccg ctg atc atc atg acg
2352Ala Lys Met Leu Leu Arg Glu Glu Ile Arg Pro Leu Ile Ile Met Thr
770 775 780 ccg aaa agc ctg ctg aga aat ccg aat acc gtg tca gaa gtg
cag gag 2400Pro Lys Ser Leu Leu Arg Asn Pro Asn Thr Val Ser Glu Val
Gln Glu 785 790 795 800 ctc agt aac agc agc ttt aag ccg gtc tat gaa
atg tca gga ctt tcc 2448Leu Ser Asn Ser Ser Phe Lys Pro Val Tyr Glu
Met Ser Gly Leu Ser 805 810 815 cat caa tat gac aaa gtg acg cgc ctc
gtc ctt tca agc ggc aaa gtt 2496His Gln Tyr Asp Lys Val Thr Arg Leu
Val Leu Ser Ser Gly Lys Val 820 825 830 tcg att gac atc agc gac cat
ttc aat aaa atg gaa ggt gaa aag gat 2544Ser Ile Asp Ile Ser Asp His
Phe Asn Lys Met Glu Gly Glu Lys Asp 835 840 845 tgg ctg cac att gca
cgg gtt gaa gag ctg tat cct ttc cct gca aag 2592Trp Leu His Ile Ala
Arg Val Glu Glu Leu Tyr Pro Phe Pro Ala Lys 850 855 860 cat att aaa
gcg atc ttc agc aaa ctt ccg aat ttg gag gag atc gtc 2640His Ile Lys
Ala Ile Phe Ser Lys Leu Pro Asn Leu Glu Glu Ile Val 865 870 875 880
tgg gta cag gaa gaa ccg caa aat atg ggc gca tgg aac tat atc gag
2688Trp Val Gln Glu Glu Pro Gln Asn Met Gly Ala Trp Asn Tyr Ile Glu
885 890 895 cct tat tta aga gag gta gct cca aag gac gtg aag gtc cgc
tat att 2736Pro Tyr Leu Arg Glu Val Ala Pro Lys Asp Val Lys Val Arg
Tyr Ile 900 905 910 ggc aga aga aga cgt tca agt ccg gca gaa ggg gat
ccg acg gtt cat 2784Gly Arg Arg Arg Arg Ser Ser Pro Ala Glu Gly Asp
Pro Thr Val His 915 920 925 aaa aag gaa cag gaa cgc att gta tct gat
agc ttg act cgc aaa aat 2832Lys Lys Glu Gln Glu Arg Ile Val Ser Asp
Ser Leu Thr Arg Lys Asn 930 935 940 taa ggg 283846944PRTBacillus
licheniformis DSM 13 46Met Phe Gln Asn Ser Met Lys Gln Arg Met Thr
Trp Glu Glu Phe His 1 5 10 15 Gly Pro Asn Leu Gly Tyr Val Leu Glu
Leu Tyr Asp Gln Tyr Val Lys 20 25 30 Asp Pro Glu Ser Leu Asp Ala
Asp Leu Lys Glu Met Phe Asp Glu Leu 35 40 45 Gly Ala Pro Pro Gly
Asp Ile Arg Ala Ala Ser Gln Lys Asn Glu Glu 50 55 60 Ala Asp Phe
Thr Ala Gly Ser Ile Gln Lys Ile Ala Ser Ala Val Lys 65 70 75 80 Leu
Ala Glu Asp Ile Arg Thr Tyr Gly His Leu Asn Ala Ser Val Asn 85 90
95 Pro Leu Arg Lys Thr Gln Glu Lys Gln Glu Leu Phe Pro Leu Ala Glu
100 105 110 Tyr Gly Leu Thr Glu Gln Asp Val Lys Lys Ile Pro Ala Ser
Val Ile 115 120 125 Cys Lys Asp Ala Pro Lys Glu Val Thr Asn Gly Leu
Glu Ala Ile Gln 130 135 140 Tyr Leu Arg Asn Thr Tyr Lys Lys Ser Ile
Ser Phe Glu Phe Asp His 145 150 155 160 Val His Ile Phe Glu Glu Arg
Asn Trp Leu Met Lys Lys Ile Glu Ser 165 170 175 Gly Glu Leu Phe Thr
Pro Lys Ser Lys Glu Lys Leu Val Glu Val Leu 180 185 190 Arg Arg Leu
Thr Glu Val Glu Ser Leu Glu Gln Phe Leu His Lys Thr 195 200 205 Phe
Val Gly Gln Lys Arg Phe Ser Ile Glu Gly Leu Asp Ala Leu Val 210 215
220 Pro Met Leu Asp Asp Ile Ile Ala Lys Ser Val Ser Ala Gly Thr Thr
225 230 235 240 Asn Val Asn Ile Gly Met Ala His Arg Gly Arg Leu Asn
Val Leu Ala 245 250 255 His Val Leu Gly Lys Pro Tyr Glu Ile Ile Phe
Ser Glu Phe Gln His 260 265 270 Ala Pro Asn Lys Asp Leu Val Pro Ser
Glu Gly Ser Thr Gly Ile Asn 275 280 285 Tyr Gly Trp Thr Gly Asp Val
Lys Tyr His Leu Gly Ala Asn Arg Gln 290 295 300 Ile Gln Asp Glu His
Thr Lys Thr Ala Arg Ile Ala Leu Ala Asn Asn 305 310 315 320 Pro Ser
His Leu Glu Phe Ile Asp Pro Ile Val Glu Gly Ser Thr Arg 325 330 335
Ala Ala Gln Glu Thr Arg Thr Glu Ser Gly Tyr Pro Val Gln Asp Val 340
345 350 Lys Lys Ser Met Ala Ile Leu Ile His Gly Asp Ala Ala Phe Pro
Gly 355 360 365 Glu Gly Ile Val Ala Glu Thr Leu Asn Leu Ser Gln Leu
Lys Gly Tyr 370 375 380 Gln Val Gly Gly Ala Ile His Ile Ile Ala Asn
Asn Met Ile Gly Phe 385 390 395 400 Thr Thr Glu Ser Asn Glu Ser Arg
Ser Thr Lys Tyr Ala Ser Asp Leu 405 410 415 Ala Lys Gly Phe Glu Ile
Pro Ile Val His Val Asn Ala Asp Asp Pro 420 425 430 Glu Ala Cys Leu
Ser Ala Val Gln Leu Ala Val Glu Tyr Arg Met Thr 435 440 445 Phe Asn
Lys Asp Phe Leu Ile Asp Leu Ile Gly Tyr Arg Arg Phe Gly 450 455 460
His Asn Glu Met Asp Glu Pro Ser Ala Thr Gln Pro Met Leu Tyr Asp 465
470 475 480 Ala Val Arg Lys His Pro Thr Val Lys Asn Ile Phe Ala Glu
Lys Leu 485 490 495 Ile His Lys Gly Ile Val Asp Lys Glu Thr Val Gly
Lys Ile Lys Asp 500 505 510 Ala Val Gln Lys Arg Leu Glu Glu Ala Tyr
Arg Lys Val Pro Ala Lys 515 520 525 Lys Glu Asp Met Thr His Glu Ile
Val Leu Pro Glu Pro Val Ser Asn 530 535 540 Gly Phe Pro Asp Val Asp
Thr Ser Val Asp Phe Glu Thr Leu Arg Lys 545 550 555 560 Ile Asn Gln
Glu Leu Val Ser Trp Pro Glu Asn Phe Asn Val Phe Asp 565 570 575 Lys
Leu Lys Arg Ile Leu Glu Arg Arg Ala Lys Ala Phe Glu Asp Asp 580 585
590 Arg Lys Val Asp Trp Ser Leu Ala Glu Ala Met Ala Phe Ala Ser Ile
595 600 605 Leu Lys Asp Gly Thr Pro Leu Arg Leu Thr Gly Gln Asp Ser
Glu Arg 610 615 620 Gly Thr Phe Ala His Arg Asn Leu Val Leu His Asp
Ser Lys Thr Gly 625 630 635 640 Asp Glu Phe Ile Ala Leu His His Leu
Ala Asp Thr Lys Ala Ser Phe 645 650 655 Ala Val His Asn Ser Pro Leu
Ser Glu Gly Ser Val Leu Gly Phe Glu 660 665 670 Tyr Gly Tyr Asn Val
Ser Ser Pro Glu Thr Met Val Ile Trp Glu Ala 675 680 685 Gln Phe Gly
Asp Phe Ala Asn Ala Ala Gln Val Tyr Phe Asp Gln Phe 690 695 700 Ile
Ser Ala Gly Arg Ala Lys Trp Gly Gln Lys Ser Gly Leu Val Val 705 710
715 720 Leu Leu Pro His Gly Tyr Glu Gly Gln Gly Pro Glu His Ser Ser
Gly 725 730 735 Arg Thr Glu Arg Phe Leu Gln Leu Ala Ala Glu Asn Asn
Trp Thr Val 740 745 750 Ala Asn Leu Thr Ser Ala Ala Gln Tyr Phe His
Ile Leu Arg Arg Gln 755 760 765 Ala Lys Met Leu Leu Arg Glu Glu Ile
Arg Pro Leu Ile Ile Met Thr 770 775 780 Pro Lys Ser Leu Leu Arg Asn
Pro Asn Thr Val Ser Glu Val Gln Glu 785 790 795 800 Leu Ser Asn Ser
Ser Phe Lys Pro Val Tyr Glu Met Ser Gly Leu Ser 805 810 815 His Gln
Tyr Asp Lys Val Thr Arg Leu Val Leu Ser Ser Gly Lys Val 820 825 830
Ser Ile Asp Ile Ser Asp His Phe Asn Lys Met Glu Gly Glu Lys Asp 835
840 845 Trp Leu His Ile Ala Arg Val Glu Glu Leu Tyr Pro Phe Pro Ala
Lys 850 855 860 His Ile Lys Ala Ile Phe Ser Lys Leu Pro Asn Leu Glu
Glu Ile Val 865 870 875 880 Trp Val Gln Glu Glu Pro Gln Asn Met Gly
Ala Trp Asn Tyr Ile Glu 885 890 895 Pro Tyr Leu Arg Glu Val Ala Pro
Lys Asp Val Lys Val Arg Tyr Ile 900 905 910 Gly Arg Arg Arg Arg Ser
Ser Pro Ala Glu Gly Asp Pro Thr Val His 915 920 925 Lys Lys Glu Gln
Glu Arg Ile Val Ser Asp Ser Leu Thr Arg Lys Asn 930 935 940
471545DNABacillus licheniformis DSM 13gene(1)..(1545)probable
acid-CoA ligase (E.C. 6.2.1.-), yhfL 47atg aat
tta gtt tca aaa tta ggg gaa aca gct caa tca aag cct gac 48Met Asn
Leu Val Ser Lys Leu Gly Glu Thr Ala Gln Ser Lys Pro Asp 1 5 10 15
aga acc gct tat att ttt gga gag caa acg gaa aca tac ggg gga ttg
96Arg Thr Ala Tyr Ile Phe Gly Glu Gln Thr Glu Thr Tyr Gly Gly Leu
20 25 30 cag cag aaa att gat tgc ttt gcc gaa ggg ctg cgc gaa atc
ggg gtg 144Gln Gln Lys Ile Asp Cys Phe Ala Glu Gly Leu Arg Glu Ile
Gly Val 35 40 45 gaa aag gga gat cat gtc gct ttg cta ctc ggc aat
aca ccg cat ttt 192Glu Lys Gly Asp His Val Ala Leu Leu Leu Gly Asn
Thr Pro His Phe 50 55 60 gtg att gcg ttt tac ggc gcg ctg aag gcc
ggg gct gtc gtc ata ccg 240Val Ile Ala Phe Tyr Gly Ala Leu Lys Ala
Gly Ala Val Val Ile Pro 65 70 75 80 atc aat ccg gcc tat acg ccg act
gag atc ggc tat atg ctg aca aat 288Ile Asn Pro Ala Tyr Thr Pro Thr
Glu Ile Gly Tyr Met Leu Thr Asn 85 90 95 ggc gat gca aaa gtg atc
gtg gct ctg gga cag ctg ctt ccg ctt tac 336Gly Asp Ala Lys Val Ile
Val Ala Leu Gly Gln Leu Leu Pro Leu Tyr 100 105 110 gaa aag gtg cat
gaa tcc ctt ccg aaa gtc ggc tgc gtc gtg ctt tgc 384Glu Lys Val His
Glu Ser Leu Pro Lys Val Gly Cys Val Val Leu Cys 115 120 125 gaa act
gga gag ccg ctt cag gaa ccg gaa aac aca gag gtt aag atg 432Glu Thr
Gly Glu Pro Leu Gln Glu Pro Glu Asn Thr Glu Val Lys Met 130 135 140
aaa ttg aaa tcg ttt aca agc att atg aaa cct cct gtc cgg ccg ttt
480Lys Leu Lys Ser Phe Thr Ser Ile Met Lys Pro Pro Val Arg Pro Phe
145 150 155 160 cct gaa atc gat gac gaa gat acg gcc gcc atc ctc tat
acg tca ggc 528Pro Glu Ile Asp Asp Glu Asp Thr Ala Ala Ile Leu Tyr
Thr Ser Gly 165 170 175 acc acg gga aga cca aaa ggg gcg atg ctc aca
cat caa aat cta ttt 576Thr Thr Gly Arg Pro Lys Gly Ala Met Leu Thr
His Gln Asn Leu Phe 180 185 190 tcg aat gca aat gat aca gcc cgc tat
ctg aca atg aat gaa tct gac 624Ser Asn Ala Asn Asp Thr Ala Arg Tyr
Leu Thr Met Asn Glu Ser Asp 195 200 205 ctt gtc gtc gcc gcc ctg ccg
atg ttc cac gtt ttt tgt tta acg gtc 672Leu Val Val Ala Ala Leu Pro
Met Phe His Val Phe Cys Leu Thr Val 210 215 220 tgc atg aat gca ccg
ctc atg aac ggc gca gcg att ttg atc gtg ccg 720Cys Met Asn Ala Pro
Leu Met Asn Gly Ala Ala Ile Leu Ile Val Pro 225 230 235 240 aaa ttc
agt ccc gcc gag gtt ttc aag ctg att aaa aag cat cag gcg 768Lys Phe
Ser Pro Ala Glu Val Phe Lys Leu Ile Lys Lys His Gln Ala 245 250 255
acg atc ttc tca ggc gtt ccg aca atg tac aat tac ctg tac cag tat
816Thr Ile Phe Ser Gly Val Pro Thr Met Tyr Asn Tyr Leu Tyr Gln Tyr
260 265 270 gaa gga gcg gat gaa aca ggc ttt cgg tcc atc agg ctt tgc
atc tca 864Glu Gly Ala Asp Glu Thr Gly Phe Arg Ser Ile Arg Leu Cys
Ile Ser 275 280 285 ggc gga gcg gcc atg cct gtc gct ctc ctg aaa agc
ttt gaa gaa agg 912Gly Gly Ala Ala Met Pro Val Ala Leu Leu Lys Ser
Phe Glu Glu Arg 290 295 300 ttc ggc gtc ctc gtt tta gaa ggc tac ggc
ttg tcg gag gct tct cct 960Phe Gly Val Leu Val Leu Glu Gly Tyr Gly
Leu Ser Glu Ala Ser Pro 305 310 315 320 gtt aca tgc ttt aac ccg ttc
agc acc ggc cgc aag cca gga tcg atc 1008Val Thr Cys Phe Asn Pro Phe
Ser Thr Gly Arg Lys Pro Gly Ser Ile 325 330 335 ggc acg aac att ctc
aat gtg aaa aac aaa gtc gtc aat gaa ctc ggg 1056Gly Thr Asn Ile Leu
Asn Val Lys Asn Lys Val Val Asn Glu Leu Gly 340 345 350 gaa gag ctg
cct gcc ggc caa gtc ggc gag ctg atc gtt aaa ggg cct 1104Glu Glu Leu
Pro Ala Gly Gln Val Gly Glu Leu Ile Val Lys Gly Pro 355 360 365 aat
gtc atg aaa ggg tac tac aaa atg ccg gat gag acg gcc cat acg 1152Asn
Val Met Lys Gly Tyr Tyr Lys Met Pro Asp Glu Thr Ala His Thr 370 375
380 ata aaa gac gga tgg ctg tat aca ggt gat ttg gca aaa cgg gat gaa
1200Ile Lys Asp Gly Trp Leu Tyr Thr Gly Asp Leu Ala Lys Arg Asp Glu
385 390 395 400 gac ggt tat ttt tat atc gtc gac aga aaa aaa gat atg
att atc gtc 1248Asp Gly Tyr Phe Tyr Ile Val Asp Arg Lys Lys Asp Met
Ile Ile Val 405 410 415 ggc ggc tat aat gtc tat ccg agg gaa atc gaa
gag gtg ctc tac ctc 1296Gly Gly Tyr Asn Val Tyr Pro Arg Glu Ile Glu
Glu Val Leu Tyr Leu 420 425 430 cat cca aaa atc gca gaa gcg gtt gtg
atc gga gtg ccc gat ccg aat 1344His Pro Lys Ile Ala Glu Ala Val Val
Ile Gly Val Pro Asp Pro Asn 435 440 445 acg gga gaa gcc gtt cat tgc
tat gtc gtt ccg aag gat aaa acg ctg 1392Thr Gly Glu Ala Val His Cys
Tyr Val Val Pro Lys Asp Lys Thr Leu 450 455 460 aca gag gag gat gtc
ttg tcg cac tgc aaa aag cat ctt gcc aaa tac 1440Thr Glu Glu Asp Val
Leu Ser His Cys Lys Lys His Leu Ala Lys Tyr 465 470 475 480 aag cgc
ccg tcg gcg atc gtt ttc atg gat gaa att ccg aaa aac tcg 1488Lys Arg
Pro Ser Ala Ile Val Phe Met Asp Glu Ile Pro Lys Asn Ser 485 490 495
acc ggc aaa att tta agg cgc gct tta aaa gac att ctg aca aac aaa
1536Thr Gly Lys Ile Leu Arg Arg Ala Leu Lys Asp Ile Leu Thr Asn Lys
500 505 510 tct tga ccc 1545Ser 48513PRTBacillus licheniformis DSM
13 48Met Asn Leu Val Ser Lys Leu Gly Glu Thr Ala Gln Ser Lys Pro
Asp 1 5 10 15 Arg Thr Ala Tyr Ile Phe Gly Glu Gln Thr Glu Thr Tyr
Gly Gly Leu 20 25 30 Gln Gln Lys Ile Asp Cys Phe Ala Glu Gly Leu
Arg Glu Ile Gly Val 35 40 45 Glu Lys Gly Asp His Val Ala Leu Leu
Leu Gly Asn Thr Pro His Phe 50 55 60 Val Ile Ala Phe Tyr Gly Ala
Leu Lys Ala Gly Ala Val Val Ile Pro 65 70 75 80 Ile Asn Pro Ala Tyr
Thr Pro Thr Glu Ile Gly Tyr Met Leu Thr Asn 85 90 95 Gly Asp Ala
Lys Val Ile Val Ala Leu Gly Gln Leu Leu Pro Leu Tyr 100 105 110 Glu
Lys Val His Glu Ser Leu Pro Lys Val Gly Cys Val Val Leu Cys 115 120
125 Glu Thr Gly Glu Pro Leu Gln Glu Pro Glu Asn Thr Glu Val Lys Met
130 135 140 Lys Leu Lys Ser Phe Thr Ser Ile Met Lys Pro Pro Val Arg
Pro Phe 145 150 155 160 Pro Glu Ile Asp Asp Glu Asp Thr Ala Ala Ile
Leu Tyr Thr Ser Gly 165 170 175 Thr Thr Gly Arg Pro Lys Gly Ala Met
Leu Thr His Gln Asn Leu Phe 180 185 190 Ser Asn Ala Asn Asp Thr Ala
Arg Tyr Leu Thr Met Asn Glu Ser Asp 195 200 205 Leu Val Val Ala Ala
Leu Pro Met Phe His Val Phe Cys Leu Thr Val 210 215 220 Cys Met Asn
Ala Pro Leu Met Asn Gly Ala Ala Ile Leu Ile Val Pro 225 230 235 240
Lys Phe Ser Pro Ala Glu Val Phe Lys Leu Ile Lys Lys His Gln Ala 245
250 255 Thr Ile Phe Ser Gly Val Pro Thr Met Tyr Asn Tyr Leu Tyr Gln
Tyr 260 265 270 Glu Gly Ala Asp Glu Thr Gly Phe Arg Ser Ile Arg Leu
Cys Ile Ser 275 280 285 Gly Gly Ala Ala Met Pro Val Ala Leu Leu Lys
Ser Phe Glu Glu Arg 290 295 300 Phe Gly Val Leu Val Leu Glu Gly Tyr
Gly Leu Ser Glu Ala Ser Pro 305 310 315 320 Val Thr Cys Phe Asn Pro
Phe Ser Thr Gly Arg Lys Pro Gly Ser Ile 325 330 335 Gly Thr Asn Ile
Leu Asn Val Lys Asn Lys Val Val Asn Glu Leu Gly 340 345 350 Glu Glu
Leu Pro Ala Gly Gln Val Gly Glu Leu Ile Val Lys Gly Pro 355 360 365
Asn Val Met Lys Gly Tyr Tyr Lys Met Pro Asp Glu Thr Ala His Thr 370
375 380 Ile Lys Asp Gly Trp Leu Tyr Thr Gly Asp Leu Ala Lys Arg Asp
Glu 385 390 395 400 Asp Gly Tyr Phe Tyr Ile Val Asp Arg Lys Lys Asp
Met Ile Ile Val 405 410 415 Gly Gly Tyr Asn Val Tyr Pro Arg Glu Ile
Glu Glu Val Leu Tyr Leu 420 425 430 His Pro Lys Ile Ala Glu Ala Val
Val Ile Gly Val Pro Asp Pro Asn 435 440 445 Thr Gly Glu Ala Val His
Cys Tyr Val Val Pro Lys Asp Lys Thr Leu 450 455 460 Thr Glu Glu Asp
Val Leu Ser His Cys Lys Lys His Leu Ala Lys Tyr 465 470 475 480 Lys
Arg Pro Ser Ala Ile Val Phe Met Asp Glu Ile Pro Lys Asn Ser 485 490
495 Thr Gly Lys Ile Leu Arg Arg Ala Leu Lys Asp Ile Leu Thr Asn Lys
500 505 510 Ser 49876DNABacillus licheniformis DSM
13gene(1)..(876)Agmatinase (E.C. 3.5.1.11), ywhG 49atg aga ttt gat
gaa gca tat tcc ggc aag gtg ttt atc gca agc cgc 48Met Arg Phe Asp
Glu Ala Tyr Ser Gly Lys Val Phe Ile Ala Ser Arg 1 5 10 15 ccc gat
tgg gaa gag gcg gac gcc atc ctt tac ggc atg ccg atg gat 96Pro Asp
Trp Glu Glu Ala Asp Ala Ile Leu Tyr Gly Met Pro Met Asp 20 25 30
tgg acg gtc agc tac cgt ccc ggc tcc cgc ttc ggt ccg gcg aga atc
144Trp Thr Val Ser Tyr Arg Pro Gly Ser Arg Phe Gly Pro Ala Arg Ile
35 40 45 cgc gag gtg tcc atc gga ctt gaa gaa tac agc cct tat ttg
gac agg 192Arg Glu Val Ser Ile Gly Leu Glu Glu Tyr Ser Pro Tyr Leu
Asp Arg 50 55 60 gag ctg gaa gag gtt cat ttc ttt gac gcc ggc gac
atc ccg ctg cct 240Glu Leu Glu Glu Val His Phe Phe Asp Ala Gly Asp
Ile Pro Leu Pro 65 70 75 80 ttc ggg aac ccg cag aag agc ctc gac atg
att gaa gaa tat gtc gac 288Phe Gly Asn Pro Gln Lys Ser Leu Asp Met
Ile Glu Glu Tyr Val Asp 85 90 95 agc att tta gat aaa gga aaa ttc
ccg ctc ggt atg ggg gga gag cat 336Ser Ile Leu Asp Lys Gly Lys Phe
Pro Leu Gly Met Gly Gly Glu His 100 105 110 ctc gtt tca tgg ccg gtc
att aaa gcg atg tac aaa aaa tat ccc gat 384Leu Val Ser Trp Pro Val
Ile Lys Ala Met Tyr Lys Lys Tyr Pro Asp 115 120 125 ctt gcc atc atc
cat atg gat gcg cac aca gat ctc cgc gtc gac tat 432Leu Ala Ile Ile
His Met Asp Ala His Thr Asp Leu Arg Val Asp Tyr 130 135 140 gaa gga
gag ccg ctc tcg cat tcg acg ccg atc cgt aaa gcc gcg gaa 480Glu Gly
Glu Pro Leu Ser His Ser Thr Pro Ile Arg Lys Ala Ala Glu 145 150 155
160 ctg atc gga ccc ggc aat gtc tat tca ttc ggc atc cgc tcc ggc atg
528Leu Ile Gly Pro Gly Asn Val Tyr Ser Phe Gly Ile Arg Ser Gly Met
165 170 175 aaa gaa gaa ttt gaa tgg gca aaa gaa aac ggc atg cac att
tct aaa 576Lys Glu Glu Phe Glu Trp Ala Lys Glu Asn Gly Met His Ile
Ser Lys 180 185 190 ttt gaa gtg ctt gag ccg ctt aaa gcc gtc ctg ccg
aaa ctc gcg ggg 624Phe Glu Val Leu Glu Pro Leu Lys Ala Val Leu Pro
Lys Leu Ala Gly 195 200 205 cgt ccg gtt tat gtg acg atc gac atc gat
gtc tta gat ccc gcg cat 672Arg Pro Val Tyr Val Thr Ile Asp Ile Asp
Val Leu Asp Pro Ala His 210 215 220 gcg ccg gga acc ggt acg gtt gac
gcc gga ggc atc aca tcc aaa gag 720Ala Pro Gly Thr Gly Thr Val Asp
Ala Gly Gly Ile Thr Ser Lys Glu 225 230 235 240 ctg ctc gct tcg att
cac gaa atc gcc cgc tca gac gtc aat gtc gtt 768Leu Leu Ala Ser Ile
His Glu Ile Ala Arg Ser Asp Val Asn Val Val 245 250 255 gga gga gac
ctt gtc gaa gtc gct cct gtc tat gac cat tcg gaa caa 816Gly Gly Asp
Leu Val Glu Val Ala Pro Val Tyr Asp His Ser Glu Gln 260 265 270 acc
gca aat acg gca agc aag ctg att cgc gaa atg ctg ctc ggc tgg 864Thr
Ala Asn Thr Ala Ser Lys Leu Ile Arg Glu Met Leu Leu Gly Trp 275 280
285 gtg aaa taa aac 876Val Lys 290 50290PRTBacillus licheniformis
DSM 13 50Met Arg Phe Asp Glu Ala Tyr Ser Gly Lys Val Phe Ile Ala
Ser Arg 1 5 10 15 Pro Asp Trp Glu Glu Ala Asp Ala Ile Leu Tyr Gly
Met Pro Met Asp 20 25 30 Trp Thr Val Ser Tyr Arg Pro Gly Ser Arg
Phe Gly Pro Ala Arg Ile 35 40 45 Arg Glu Val Ser Ile Gly Leu Glu
Glu Tyr Ser Pro Tyr Leu Asp Arg 50 55 60 Glu Leu Glu Glu Val His
Phe Phe Asp Ala Gly Asp Ile Pro Leu Pro 65 70 75 80 Phe Gly Asn Pro
Gln Lys Ser Leu Asp Met Ile Glu Glu Tyr Val Asp 85 90 95 Ser Ile
Leu Asp Lys Gly Lys Phe Pro Leu Gly Met Gly Gly Glu His 100 105 110
Leu Val Ser Trp Pro Val Ile Lys Ala Met Tyr Lys Lys Tyr Pro Asp 115
120 125 Leu Ala Ile Ile His Met Asp Ala His Thr Asp Leu Arg Val Asp
Tyr 130 135 140 Glu Gly Glu Pro Leu Ser His Ser Thr Pro Ile Arg Lys
Ala Ala Glu 145 150 155 160 Leu Ile Gly Pro Gly Asn Val Tyr Ser Phe
Gly Ile Arg Ser Gly Met 165 170 175 Lys Glu Glu Phe Glu Trp Ala Lys
Glu Asn Gly Met His Ile Ser Lys 180 185 190 Phe Glu Val Leu Glu Pro
Leu Lys Ala Val Leu Pro Lys Leu Ala Gly 195 200 205 Arg Pro Val Tyr
Val Thr Ile Asp Ile Asp Val Leu Asp Pro Ala His 210 215 220 Ala Pro
Gly Thr Gly Thr Val Asp Ala Gly Gly Ile Thr Ser Lys Glu 225 230 235
240 Leu Leu Ala Ser Ile His Glu Ile Ala Arg Ser Asp Val Asn Val Val
245 250 255 Gly Gly Asp Leu Val Glu Val Ala Pro Val Tyr Asp His Ser
Glu Gln 260 265 270 Thr Ala Asn Thr Ala Ser Lys Leu Ile Arg Glu Met
Leu Leu Gly Trp 275 280 285 Val Lys 290
* * * * *
References