U.S. patent application number 14/344658 was filed with the patent office on 2015-06-18 for production of odd chain fatty acid derivatives in recombinant microbial cells.
This patent application is currently assigned to LS9, INC.. The applicant listed for this patent is John R. Haliburton, Zhihao Hu, Grace J. Lee, Andreas W. Schirmer. Invention is credited to John R. Haliburton, Zhihao Hu, Grace J. Lee, Andreas W. Schirmer.
Application Number | 20150167033 14/344658 |
Document ID | / |
Family ID | 47884064 |
Filed Date | 2015-06-18 |
United States Patent
Application |
20150167033 |
Kind Code |
A1 |
Lee; Grace J. ; et
al. |
June 18, 2015 |
PRODUCTION OF ODD CHAIN FATTY ACID DERIVATIVES IN RECOMBINANT
MICROBIAL CELLS
Abstract
Recombinant microbial cells are provided which have been
engineered to produce fatty acid derivatives having linear chains
containing an odd number of carbon atoms by the fatty acid
biosynthetic pathway. Also provided are methods of making odd chain
fatty acid derivatives using the recombinant microbial cells, and
compositions comprising odd chain fatty acid derivatives produced
by such methods.
Inventors: |
Lee; Grace J.; (South San
Francisco, CA) ; Haliburton; John R.; (South San
Francisco, CA) ; Hu; Zhihao; (South San Francisco,
CA) ; Schirmer; Andreas W.; (South San Francisco,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Lee; Grace J.
Haliburton; John R.
Hu; Zhihao
Schirmer; Andreas W. |
South San Francisco
South San Francisco
South San Francisco
South San Francisco |
CA
CA
CA
CA |
US
US
US
US |
|
|
Assignee: |
LS9, INC.
South San Franciso
CA
|
Family ID: |
47884064 |
Appl. No.: |
14/344658 |
Filed: |
March 8, 2012 |
PCT Filed: |
March 8, 2012 |
PCT NO: |
PCT/US2012/028256 |
371 Date: |
February 18, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13232297 |
Sep 14, 2011 |
8763570 |
|
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14344658 |
|
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|
|
61383086 |
Sep 15, 2010 |
|
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Current U.S.
Class: |
554/223 ;
435/134; 435/252.33; 435/325; 435/455; 435/471 |
Current CPC
Class: |
C12Y 203/01041 20130101;
C12N 9/90 20130101; C12N 9/1217 20130101; C12Y 301/02001 20130101;
C12Y 402/03001 20130101; C12Y 101/01003 20130101; C07C 57/03
20130101; C12Y 401/01041 20130101; C12N 9/88 20130101; C12Y
207/02004 20130101; C12Y 402/01033 20130101; C12Y 201/03001
20130101; C12N 9/16 20130101; C12P 7/6409 20130101; C12N 9/18
20130101; C12Y 401/03022 20130101; C12N 9/1029 20130101; C12Y
504/99002 20130101; C12N 9/0006 20130101; C12Y 101/01085 20130101;
C07C 53/126 20130101; C12Y 403/01019 20130101; C12N 9/1018
20130101 |
International
Class: |
C12P 7/64 20060101
C12P007/64; C07C 57/03 20060101 C07C057/03; C07C 53/126 20060101
C07C053/126 |
Claims
1. A recombinant microbial cell comprising: (a) a polynucleotide
encoding a polypeptide having enzymatic activity effective to
produce an increased amount of propionyl-CoA in the recombinant
microbial cell relative to the amount of propionyl-CoA produced in
a parental microbial cell lacking or having a reduced amount of
said enzymatic activity, wherein said polypeptide is exogenous to
the recombinant microbial cell or wherein expression of said
polynucleotide is modulated in the recombinant microbial cell as
compared to the expression of the polynucleotide in the parental
microbial cell, (b) a polynucleotide encoding a polypeptide having
.beta.-ketoacyl-ACP synthase activity that utilizes propionyl-CoA
as a substrate, and (c) a polynucleotide encoding a polypeptide
having fatty acid derivative enzyme activity, wherein the
recombinant microbial cell produces a fatty acid derivative
composition comprising odd chain fatty acid derivatives when
cultured in the presence of a carbon source under conditions
effective to express the polynucleotides according to (a), (b), and
(c), and wherein at least 10% of the fatty acid derivatives in the
fatty acid derivative composition are odd chain fatty acid
derivatives.
2. The recombinant microbial cell of claim 1, wherein at least 20%
of the fatty acid derivatives in the fatty acid derivative
composition are odd chain fatty acid derivatives.
3. The recombinant microbial cell of claim 1, wherein the cell
produces at least 100 mg/L of odd chain fatty acid derivatives.
4. The recombinant microbial cell of claim 1, wherein expression of
the at least one polynucleotide according to (a) is modulated by
overexpression of the polynucleotide in the recombinant microbial
cell.
5. The recombinant microbial cell of claim 1, wherein the
polynucleotide according to (a) is selected from the group
consisting of: (i) one or more polynucleotide encoding a
polypeptide having aspartokinase activity, homoserine dehydrogenase
activity, homoserine kinase activity, threonine synthase activity,
and threonine deaminase activity; (ii) one or more polynucleotide
encoding a polypeptide having (R)-citramalate synthase activity,
isopropylmalate isomerase activity, and beta-isopropylmalate
dehydrogenase activity; and (iii) one or more polynucleotide
encoding a polypeptide having methylmalonyl-CoA mutase activity,
methylmalonyl-CoA decarboxylase activity and methylmalonyl-CoA
carboxyltransferase activity.
6. The recombinant microbial cell of claim 5, comprising one or
more polynucleotide according to (i) and one or more polynucleotide
according to (ii).
7. The recombinant microbial cell of claim 1, wherein the
polypeptide having .beta.-ketoacyl-ACP synthase activity that
utilizes propionyl-CoA as a substrate is exogenous to the
recombinant microbial cell, and expression of a polypeptide having
beta-ketoacyl-ACP synthase activity endogenous to the recombinant
microbial cell is attenuated.
8. The recombinant microbial cell of claim 1, wherein the fatty
acid derivative enzyme activity comprises thioesterase activity and
the recombinant microbial cell produces a fatty acid composition
comprising odd chain fatty acids, wherein at least 10% of the fatty
acids in the composition are odd chain fatty acids.
9. The recombinant microbial cell of claim 1, wherein the fatty
acid derivative enzyme activity comprises ester synthase activity
and the recombinant microbial cell produces a fatty ester
composition comprising odd chain fatty esters, wherein at least 10%
of the fatty esters in the composition are odd chain fatty
esters.
10. The recombinant microbial cell of claim 1, wherein the fatty
acid derivative enzyme activity comprises fatty aldehyde
biosynthesis activity and the recombinant microbial cell produces a
fatty aldehyde composition comprising odd chain fatty aldehydes,
wherein at least 10% of the fatty aldehydes in the composition are
odd chain fatty aldehydes.
11. The recombinant microbial cell of claim 1, wherein the fatty
acid derivative enzyme activity comprises fatty alcohol
biosynthesis activity and the recombinant microbial cell produces a
fatty alcohol composition comprising odd chain fatty alcohols
wherein at least 10% of the fatty alcohols in the composition are
odd chain fatty alcohols.
12. The recombinant microbial cell of claim 1, wherein the fatty
acid derivative enzyme activity comprises hydrocarbon biosynthesis
activity and the recombinant microbial cell produces a hydrocarbon
composition comprising even chain hydrocarbons, wherein at least
10% of the hydrocarbons in the composition are even chain
hydrocarbons.
13. A cell culture comprising the recombinant microbial cell of
claim 1.
14. A method of making a fatty acid derivative composition
comprising odd chain fatty acid derivatives, the method comprising:
obtaining the recombinant microbial cell of claim 1, culturing the
recombinant microbial cell in a culture medium containing a carbon
source under conditions effective to express the polynucleotides
according to (a), (b), and (c) and produce a fatty acid derivative
composition comprising odd chain fatty acid derivatives wherein at
least 10% of the fatty acid derivatives in the composition are odd
chain fatty acid derivatives, and optionally recovering the odd
chain fatty acid derivative composition from the culture
medium.
15. The method of claim 14, wherein the recombinant microbial cell
expresses one or more polynucleotide encoding a polypeptide having
a fatty acid derivative enzyme activity selected from the group
consisting of: (1) a polypeptide having thioesterase activity; (2)
a polypeptide having decarboxylase activity; (3) a polypeptide
having carboxylic acid reductase activity; (4) a polypeptide having
alcohol dehydrogenase activity (EC 1.1.1.1); (5) a polypeptide
having aldehyde decarboxylase activity (EC 4.1.99.5); (6) a
polypeptide having acyl-CoA reductase activity (EC 1.2.1.50); (7) a
polypeptide having acyl-ACP reductase activity; (8) a polypeptide
having ester synthase activity (EC 3.1.1.67); (9) a polypeptide
having OleA activity; and (10) a polypeptide having OleCD or OleBCD
activity; wherein the recombinant microbial cell produces a
composition comprising one or more of odd chain fatty acids, odd
chain fatty esters, odd chain fatty aldehydes, odd chain fatty
alcohols, even chain alkanes, even chain alkenes, even chain
terminal olefins, even chain internal olefins, or even chain
ketones.
16. A method of making a recombinant microbial cell which produces
a higher titer or higher proportion of odd chain fatty acid
derivatives than produced by a parental microbial cell, the method
comprising: obtaining a parental microbial cell comprising a
polynucleotide encoding a polypeptide having .beta.-ketoacyl-ACP
synthase activity that utilizes propionyl-CoA as a substrate and a
polynucleotide encoding a polypeptide having fatty acid derivative
enzyme activity, and engineering the parental microbial cell to
obtain a recombinant microbial cell which produces or is capable of
producing a greater amount of propionyl-CoA than the amount of
propionyl-CoA produced by the parental microbial cell when cultured
under the same conditions, wherein the recombinant microbial cell
produces a higher titer or higher proportion of odd chain fatty
acid derivatives when cultured in the presence of a carbon source
under conditions effective to express the polynucleotides, relative
to the titer or proportion of odd chain fatty acid derivatives
produced by the parental microbial cell cultured under the same
conditions.
17. The method of claim 16, wherein the step of engineering the
parental microbial cell comprises: engineering the parental
microbial cell to express polynucleotides encoding polypeptides
selected from the group consisting of: (a) polypeptides having
aspartokinase activity, homoserine dehydrogenase activity,
homoserine kinase activity, threonine synthase activity, and
threonine deaminase activity; (b) polypeptides having
(R)-citramalate synthase activity, isopropylmalate isomerase
activity, and beta-isopropylmalate dehydrogenase activity; and (c)
polypeptides having methylmalonyl-CoA mutase activity, either
methylmalonyl-CoA decarboxylase activity or methylmalonyl-CoA
carboxyltransferase activity, and optionally, methylmalonyl-CoA
epimerase activity; wherein at least one polypeptide according to
(a), (b) or (c) is exogenous to the parental microbial cell, or
wherein expression of at least one polynucleotide according to (a),
(b) or (c) is modulated in the recombinant microbial cell as
compared to the expression of the polynucleotide in the parental
microbial cell.
18. The method of claim 16, wherein the recombinant microbial cell
is engineered to express an exogenous polynucleotide encoding a
polypeptide having .beta.-ketoacyl-ACP synthase activity that
utilizes propionyl-CoA as a substrate, and expression of an
endogenous polynucleotide encoding a polypeptide having
.beta.-ketoacyl-ACP synthase activity is attenuated.
19. A method of increasing the titer or the proportion of odd chain
fatty acid derivatives produced by a microbial cell, the method
comprising: obtaining a parental microbial cell which produces
fatty acid derivatives, and engineering the parental microbial cell
to obtain a recombinant microbial cell which produces or is capable
of producing a greater amount of propionyl-CoA than the amount of
propionyl-CoA produced by the parental microbial cell when cultured
under the same conditions, wherein the recombinant microbial cell
produces a higher titer or higher proportion of odd chain fatty
acid derivatives when cultured in the presence of a carbon source
under conditions effective to produce propionyl-CoA and fatty acid
derivatives in the recombinant microbial cell, relative to the
titer or proportion of odd chain fatty acid derivatives produced by
the parental microbial cell cultured under the same conditions.
20. A fatty acid derivative composition produced by the method of
claim 14.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is the National Phase of International
Application No. /US2012/028256, filed Mar. 8, 2012, which
designated the U.S. and that International Application was
published under PCT Article 21(2) in English, which is a
continuation-in-part and claims priority benefit to U.S.
application Ser. No. 13/232,927, filed Sep. 14, 2011 (now U.S. Pat.
No. 8,372,610), and U.S. Provisional Patent Application No.
61/383,086 filed Sep. 15, 2010, all of which applications are
expressly incorporated by reference herein in their entirety.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Mar. 7, 2012, is named LS0033PC.txt and is 350,776 bytes in
size.
BACKGROUND
[0003] Crude petroleum is a very complex mixture containing a wide
range of hydrocarbons. It is converted into a diversity of fuels
and chemicals through a variety of chemical processes in
refineries. Crude petroleum is a source of transportation fuels as
well as a source of raw materials for producing petrochemicals.
Petrochemicals are used to make specialty chemicals such as
plastics, resins, fibers, elastomers, pharmaceuticals, lubricants,
and gels.
[0004] The most important transportation fuels--gasoline, diesel,
and jet fuel--contain distinctively different mixtures of
hydrocarbons which are tailored toward optimal engine performance.
For example, gasoline comprises straight chain, branched chain, and
aromatic hydrocarbons generally ranging from about 4 to 12 carbon
atoms, while diesel predominantly comprises straight chain
hydrocarbons ranging from about 9 to 23 carbon atoms. Diesel fuel
quality is evaluated by parameters such as cetane number, kinematic
viscosity, oxidative stability, and cloud point (Knothe G., Fuel
Process Technol. 86:1059-1070 (2005)). These parameters, among
others, are impacted by the hydrocarbon chain length as well as by
the degree of branching or saturation of the hydrocarbon.
[0005] Microbially-produced fatty acid derivatives can be tailored
by genetic manipulation. Metabolic engineering enables microbial
strains to produce various mixtures of fatty acid derivatives,
which can be optimized, for example, to meet or exceed fuel
standards or other commercially relevant product specifications.
Microbial strains can be engineered to produce chemicals or
precursor molecules that are typically derived from petroleum. In
some instances, it is desirable to mimic the product profile of an
existing product, for example the product profile of an existing
petroleum-derived fuel or chemical product, for efficient drop-in
compatibility or substitution. Recombinant cells and methods
described herein demonstrate microbial production of fatty acid
derivatives with varied ratios of odd:even length chains as a means
to precisely control the structure and function of, e.g.,
hydrocarbon-based fuels and chemicals.
[0006] There is a need for cost-effective alternatives to petroleum
products that do not require exploration, extraction,
transportation over long distances, or substantial refinement, and
avoid the types of environmental damage associated with processing
of petroleum. For similar reasons, there is a need for alternative
sources of chemicals which are typically derived from petroleum.
There is also a need for efficient and cost-effective methods for
producing high-quality biofuels, fuel alternatives, and chemicals
from renewable energy sources.
[0007] Recombinant microbial cells engineered to produce fatty acid
precursor molecules having desired chain lengths (such as, chains
having odd numbers of carbons), and fatty acid derivatives made
therefrom, methods using these recombinant microbial cells to
produce compositions comprising fatty acid derivatives having
desired acyl chain lengths and desired ratios of odd:even length
chains, and compositions produced by these methods, address these
needs.
SUMMARY
[0008] The present invention provides novel recombinant microbial
cells which produce odd chain length fatty acid derivatives and
cell cultures comprising such novel recombinant microbial cells.
The invention also provides methods of making compositions
comprising odd chain length fatty acid derivatives comprising
culturing recombinant microbial cells of the invention,
compositions made by such methods, and other features apparent upon
further review.
[0009] In a first aspect, the invention provides a recombinant
microbial cell comprising a polynucleotide encoding a polypeptide
having enzymatic activity effective to increase the production of
propionyl-CoA in the cell relative to the production of
propionyl-CoA in a parental microbial cell lacking or having a
reduced amount of said enzymatic activity, wherein the recombinant
microbial cell produces a fatty acid derivative composition
comprising odd chain fatty acid derivatives when the cell is
cultured in the presence of a carbon source under conditions
effective to express the polynucleotide. The recombinant microbial
cell comprises: (a) a polynucleotide encoding a polypeptide having
enzymatic activity effective to produce an increased amount of
propionyl-CoA in the recombinant microbial cell, relative to the
amount of propionyl-CoA produced in a parental microbial cell
lacking or having a reduced amount of said enzymatic activity,
wherein the polypeptide is exogenous to the recombinant microbial
cell, or expression of the polynucleotide is modulated in the
recombinant microbial cell as compared to the expression of the
polynucleotide in the parental microbial cell; (b) a polynucleotide
encoding a polypeptide having .beta.-ketoacyl-ACP synthase ("FabH")
activity that utilizes propionyl-CoA as a substrate, and (c) a
polynucleotide encoding a polypeptide having fatty acid derivative
enzyme activity, wherein the recombinant microbial cell produces a
fatty acid derivative composition comprising odd chain fatty acid
derivatives when the cell is cultured in the presence of a carbon
source under conditions effective to express the polynucleotides
according to (a), (b), and (c). In some embodiments, expression of
at least one polynucleotide according to (a) is modulated by
overexpression of the polynucleotide, such as by operatively
linking the polynucleotide to an exogenous promoter.
[0010] In some embodiments, at least 5%, at least 10%, at least
20%, at least 30%, at least 40%, at least 50%, at least 60%, at
least 70%, at least 80% or at least 90% of the fatty acid
derivatives in the composition produced by the microbial cell of
the first aspect are odd chain fatty acid derivatives. In some
embodiments, the recombinant microbial cell produces at least 50
mg/L, at least 75 mg/L, at least 100 mg/L, at least 200 mg/L, at
least 500 mg/L, at least 1000 mg/L, at least 2000 mg/L, at least
5000 mg/L, or at least 10000 mg/L odd chain fatty acid derivatives
when cultured in a culture medium containing a carbon source under
conditions effective to express the polynucleotides according to
(a), (b), and (c).
[0011] In some embodiments, the polynucleotide encoding a
polypeptide having enzymatic activity effective to produce an
increased amount of propionyl-CoA in the recombinant microbial cell
according to (a) is selected from: (i) one or more polynucleotide
encoding a polypeptide having aspartokinase activity, homoserine
dehydrogenase activity, homoserine kinase activity, threonine
synthase activity, or threonine deaminase activity; (ii) one or
more polynucleotide encoding a polypeptide having (R)-citramalate
synthase activity, isopropylmalate isomerase activity, or
beta-isopropylmalate dehydrogenase activity; and (iii) one or more
polynucleotide encoding a polypeptide having methylmalonyl-CoA
mutase activity, methylmalonyl-CoA decarboxylase activity,
methylmalonyl-CoA carboxyltransferase activity, or
methylmalonyl-CoA epimerase activity. In some embodiments, the
microbial cell comprises one or more polynucleotide according to
(i) and one or more polynucleotide according to (ii). In some
embodiments, the microbial cell comprises one or more
polynucleotide according to (i) and/or (ii), and one or more
polynucleotide according to (iii).
[0012] In some embodiments, the polypeptide having
.beta.-ketoacyl-ACP synthase activity that utilizes propionyl-CoA
as a substrate is exogenous to the recombinant microbial cell. In a
more particular embodiment, expression of a polypeptide having
.beta.-ketoacyl-ACP synthase activity endogenous to the recombinant
microbial cell is attenuated.
[0013] The fatty acid derivative enzyme activity may be endogenous
("native") or exogenous. In some embodiments, the fatty acid
derivative enzyme activity comprises thioesterase activity, and the
fatty acid derivative composition produced by the recombinant
microbial cell comprises odd chain fatty acids and even chain fatty
acids. In some embodiments, at least 5%, at least 10%, at least
20%, at least 30%, at least 40%, at least 50%, at least 60%, at
least 70%, at least 80% or at least 90% of the fatty acids in the
composition are odd chain fatty acids. In some embodiments, the
recombinant microbial cell produces at least 50 mg/L, at least 75
mg/L, at least 100 mg/L, at least 200 mg/L, at least 500 mg/L, at
least 1000 mg/L, at least 2000 mg/L, at least 5000 mg/L, or at
least 10000 mg/L odd chain fatty acids when cultured in a culture
medium containing a carbon source under conditions effective to
express the polynucleotides.
[0014] In some embodiments of the first aspect, the fatty acid
derivative enzyme activity comprises ester synthase activity, and
the fatty acid derivative composition produced by the recombinant
microbial comprises odd chain fatty esters and even chain fatty
esters. In some embodiments, at least 5%, at least 10%, at least
20%, at least 30%, at least 40%, at least 50%, at least 60%, at
least 70%, at least 80% or at least 90% of the fatty esters in the
composition are odd chain fatty esters. In some embodiments, the
recombinant microbial cell produces at least 50 mg/L, at least 75
mg/L, at least 100 mg/L, at least 200 mg/L, at least 500 mg/L, at
least 1000 mg/L, at least 2000 mg/L, at least 5000 mg/L, or at
least 10000 mg/L odd chain fatty esters when cultured in a culture
medium containing a carbon source under conditions effective to
express the polynucleotides.
[0015] In some embodiments of the first aspect, the fatty acid
derivative enzyme activity comprises fatty aldehyde biosynthesis
activity, and the fatty acid derivative composition produced by the
recombinant microbial cell comprises odd chain fatty aldehydes and
even chain fatty aldehydes. In some embodiments, at least 5%, at
least 10%, at least 20%, at least 30%, at least 40%, at least 50%,
at least 60%, at least 70%, at least 80% or at least 90% of the
fatty aldehydes in the composition are odd chain fatty aldehydes.
In some embodiments, the recombinant microbial cell produces at
least 50 mg/L, at least 75 mg/L, at least 100 mg/L, at least 200
mg/L, at least 500 mg/L, at least 1000 mg/L, at least 2000 mg/L, at
least 5000 mg/L, or at least 10000 mg/L odd chain fatty aldehydes
when cultured in a culture medium containing a carbon source under
conditions effective to express the polynucleotides.
[0016] In some embodiments of the first aspect, the fatty acid
derivative enzyme activity comprises fatty alcohol biosynthesis
activity, and the fatty acid derivative composition produced by the
recombinant microbial cell comprises odd chain fatty alcohols and
even chain fatty alcohols. In some embodiments, at least 5%, at
least 10%, at least 20%, at least 30%, at least 40%, at least 50%,
at least 60%, at least 70%, at least 80% or at least 90% of the
fatty alcohols in the composition are odd chain fatty alcohols. In
some embodiments, the recombinant microbial cell produces at least
50 mg/L, at least 75 mg/L, at least 100 mg/L, at least 200 mg/L, at
least 500 mg/L, at least 1000 mg/L, at least 2000 mg/L, at least
5000 mg/L, or at least 10000 mg/L odd chain fatty alcohols when
cultured in a culture medium containing a carbon source under
conditions effective to express the polynucleotides.
[0017] In some embodiments of the first aspect, the fatty acid
derivative enzyme activity comprises hydrocarbon biosynthesis
activity, and the fatty acid derivative composition produced by the
recombinant microbial cell is a hydrocarbon composition, such as an
alkane composition, an alkene composition, a terminal olefin
composition, an internal olefin composition, or a ketone
composition, the hydrocarbon composition comprising odd chain
hydrocarbons and even chain hydrocarbons. In some embodiments, at
least 5%, at least 10%, at least 20%, at least 30%, at least 40%,
at least 50%, at least 60%, at least 70%, at least 80% or at least
90% of the hydrocarbons in the composition are even chain
hydrocarbons. In some embodiments, the recombinant microbial cell
produces at least 50 mg/L, at least 75 mg/L, at least 100 mg/L, at
least 200 mg/L, at least 500 mg/L, at least 1000 mg/L, at least
2000 mg/L, at least 5000 mg/L, or at least 10000 mg/L even chain
hydrocarbons when cultured in a culture medium containing a carbon
source under conditions effective to express the
polynucleotides.
[0018] In various embodiments, the carbon source comprises a
carbohydrate, such as a sugar, e.g., a monosaccharide, a
disaccharide, an oligosaccharide, or a polysaccharide. In some
embodiments, the carbon source is obtained from biomass, such as a
cellulosic hydrolysate.
[0019] In various embodiments, the parental (e.g., host) microbial
cell is a filamentous fungi, an algae, a yeast, or a prokaryote
such as a bacterium. In various preferred embodiments, the host
cell is a bacterial cell. In more preferred embodiments the host
cell is an E. coli cell or a Bacillus cell.
[0020] Exemplary pathways for making even chain fatty acid
derivatives and odd chain fatty acid derivatives are shown in FIGS.
1A and 1B, respectively. FIGS. 2 and 3 provide an overview of
various approaches to direct metabolic flux through propionyl-CoA
to increase odd chain fatty acid derivative production; FIG. 2
showing exemplary pathways through the intermediate
.alpha.-ketobutyrate, and FIG. 3 showing an exemplary pathway
through the intermediate methylmalonyl-CoA.
[0021] In one embodiment, the recombinant microbial cell according
to the first aspect comprises a polynucleotide encoding a
polypeptide having .beta.-ketoacyl-ACP synthase activity that
utilizes propionyl-CoA as a substrate, preferably a
.beta.-ketoacyl-ACP synthase III activity categorized as EC
2.3.1.180. In one embodiment, the polypeptide having
.beta.-ketoacyl-ACP synthase activity is encoded by a fabH gene. In
one embodiment, the polypeptide having .beta.-ketoacyl-ACP synthase
activity is endogenous to the parental microbial cell. In another
embodiment, the polypeptide having .beta.-ketoacyl-ACP synthase
activity is exogenous to the parental microbial cell. In another
embodiment, expression of a polynucleotide encoding a polypeptide
having .beta.-ketoacyl-ACP synthase activity is modulated in the
recombinant microbial cell. In some instances, expression of the
polynucleotide is modulated by operatively linking the
polynucleotide to an exogenous promoter, such that the
polynucleotide is overexpressed in the recombinant microbial cell.
In another embodiment, the polypeptide having .beta.-ketoacyl-ACP
synthase activity comprises a sequence selected from SEQ ID NOs: 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 146, 147, 148, or 149, or a
variant or a fragment thereof having .beta.-ketoacyl-ACP synthase
activity that utilizes propionyl-CoA as a substrate and catalyzes
the condensation of propionyl-CoA with malonyl-ACP to form an odd
chain acyl-ACP in vitro or in vivo, preferably in vivo. In another
embodiment, the polypeptide having .beta.-ketoacyl-ACP synthase
activity that utilizes propionyl-CoA as a substrate comprises one
or more sequence motif selected from SEQ ID NOs:14-19 and catalyzes
the condensation of propionyl-CoA with malonyl-ACP to form an odd
chain acyl-ACP in vitro or in vivo, preferably in vivo.
[0022] In one embodiment, the recombinant microbial cell according
to the first aspect comprises an endogenous polynucleotide sequence
(such as, an endogenous fabH gene) encoding a polypeptide having
.beta.-ketoacyl-ACP synthase activity, and expression of such
endogenous polynucleotide sequence in the recombinant microbial
cell is attenuated. In some embodiments, expression of the
endogenous polynucleotide is attenuated by deletion of all or part
of the sequence of the endogenous polynucleotide in the recombinant
microbial cell. Such a recombinant microbial cell comprising an
attenuated endogenous .beta.-ketoacyl-ACP synthase gene preferably
further comprises a polynucleotide sequence encoding an exogenous
polypeptide having .beta.-ketoacyl-ACP synthase activity that
utilizes propionyl-CoA as a substrate.
[0023] In one embodiment, the recombinant microbial cell according
to the first aspect comprises a polynucleotide encoding a
polypeptide having aspartokinase activity which is categorized as
EC 2.7.2.4 (FIG. 2, pathway (A)). In some embodiments, the
polypeptide having aspartokinase activity is encoded by a thrA, a
dapG or a hom3 gene. In one embodiment, the polypeptide having
aspartokinase activity is endogenous to the parental microbial
cell, or is exogenous to the parental microbial cell. In another
embodiment, expression of the polynucleotide encoding the
polypeptide having aspartokinase activity is modulated in the
recombinant microbial cell. In some instances, expression of the
polynucleotide is modulated by operatively linking the
polynucleotide to an exogenous promoter, such that the
polynucleotide is overexpressed in the recombinant microbial cell.
In another embodiment, the polypeptide having aspartokinase
activity comprises a sequence selected from SEQ ID NOs:20, 21, 22,
23, 24, or a variant or a fragment thereof having aspartokinase
activity and which catalyzes the conversion of aspartate to
aspartyl phosphate in vitro or in vivo, preferably in vivo.
[0024] In one embodiment, the recombinant microbial cell according
to the first aspect comprises a polynucleotide encoding a
polypeptide having homoserine dehydrogenase activity which is
categorized as EC 1.1.1.3. In some embodiments, the polypeptide
having homoserine dehydrogenase activity is encoded by a thrA, a
hom or a hom6 gene. In one embodiment, the polypeptide having
homoserine dehydrogenase activity is endogenous to the parental
microbial cell, or is exogenous to the parental microbial cell. In
another embodiment, expression of the polynucleotide encoding the
polypeptide having homoserine dehydrogenase activity is modulated
in the recombinant microbial cell. In some instances, expression of
the polynucleotide is modulated by operatively linking the
polynucleotide to an exogenous promoter, such that the
polynucleotide is overexpressed in the recombinant microbial cell.
In another embodiment, the polypeptide having homoserine
dehydrogenase activity comprises a sequence selected from SEQ ID
NOs:20, 21, 25, 26, 27, or a variant or a fragment thereof having
homoserine dehydrogenase activity and which catalyzes the
conversion of aspartate semialdehyde to homoserine in vitro or in
vivo, preferably in vivo.
[0025] In a particular embodiment, the recombinant microbial cell
according to the first aspect comprises a polynucleotide encoding a
polypeptide having both aspartokinase and homoserine dehydrogenase
activity. In one embodiment, the polypeptide having aspartokinase
and homoserine dehydrogenase activity is endogenous to the parental
microbial cell, or is exogenous to the parental microbial cell. In
another embodiment, expression of the polynucleotide encoding the
polypeptide having aspartokinase and homoserine dehydrogenase
activity is modulated in the recombinant microbial cell. In some
instances, expression of the polynucleotide is modulated by
operatively linking the polynucleotide to an exogenous promoter,
such that the polynucleotide is overexpressed in the recombinant
microbial cell. In one embodiment the polypeptide having
aspartokinase and homoserine dehydrogenase activity comprises the
sequence SEQ ID NO:20 or a variant or a fragment thereof, such as
SEQ ID NO:21, which catalyzes the conversion of aspartate to
aspartyl phosphate and the conversion of aspartate semialdehyde to
homoserine in vitro or in vivo, preferably in vivo.
[0026] In one embodiment, the recombinant microbial cell according
to the first aspect comprises a polynucleotide encoding a
polypeptide having homoserine kinase activity which is categorized
as EC 2.7.1.39. In some embodiments, the polypeptide having
homoserine kinase activity is encoded by a thrB gene or a thr1
gene. In one embodiment, the polypeptide having homoserine kinase
activity is endogenous to the parental microbial cell, or is
exogenous to the parental microbial cell. In another embodiment,
expression of the polynucleotide encoding the polypeptide having
homoserine kinase activity is modulated in the recombinant
microbial cell. In some instances, expression of the polynucleotide
is modulated by operatively linking the polynucleotide to an
exogenous promoter, such that the polynucleotide is overexpressed
in the recombinant microbial cell. In another embodiment, the
polypeptide having homoserine kinase activity comprises a sequence
selected from SEQ ID NOs:28, 29, 30, 31, or a variant or a fragment
thereof having homoserine kinase activity and which catalyzes the
conversion of homoserine to O-phospho-L-homoserine in vitro or in
vivo, preferably in vivo.
[0027] In one embodiment, the recombinant microbial cell according
to the first aspect comprises a polynucleotide encoding a
polypeptide having threonine synthase activity which is categorized
as EC 4.2.3.1. In one embodiment, the polypeptide having threonine
synthase activity is encoded by a thrC gene. In one embodiment, the
polypeptide having threonine synthase activity is endogenous to the
parental microbial cell, or is exogenous to the parental microbial
cell. In another embodiment, expression of the polynucleotide
encoding the polypeptide having threonine synthase activity is
modulated in the recombinant microbial cell. In some instances,
expression of the polynucleotide is modulated by operatively
linking the polynucleotide to an exogenous promoter, such that the
polynucleotide is overexpressed in the recombinant microbial cell.
In another embodiment, the polypeptide having threonine synthase
activity comprises a sequence selected from SEQ ID NOs:32, 33, 34,
or a variant or a fragment thereof having threonine synthase
activity and which catalyzes the conversion of
O-phospho-L-homoserine to threonine in vitro or in vivo, preferably
in vivo.
[0028] In one embodiment, the recombinant microbial cell according
to the first aspect comprises a polynucleotide encoding a
polypeptide having threonine deaminase activity which is
categorized as EC 4.3.1.19. In some embodiments, the polypeptide
having threonine deaminase activity is encoded by a tdcB gene or an
ilvA gene. In one embodiment, the polypeptide having threonine
deaminase activity is endogenous to the parental microbial cell, or
is exogenous to the parental microbial cell. In another embodiment,
expression of the polynucleotide encoding the polypeptide having
threonine deaminase activity is modulated in the recombinant
microbial cell. In some instances, expression of the polynucleotide
is modulated by operatively linking the polynucleotide to an
exogenous promoter, such that the polynucleotide is overexpressed
in the recombinant microbial cell. In another embodiment, the
polypeptide having threonine deaminase activity comprises a
sequence selected from SEQ ID NOs:35, 36, 37, 38, 39, or a variant
or a fragment thereof having threonine deaminase activity and which
catalyzes the conversion of threonine to 2-ketobutyrate in vitro or
in vivo, preferably in vivo.
[0029] In one embodiment, the recombinant microbial cell according
to the first aspect comprises a polynucleotide encoding a
polypeptide having (R)-citramalate synthase activity which is
categorized as EC 2.3.1.182 (FIG. 2, pathway (B)). In one
embodiment, the polypeptide having (R)-citramalate synthase
activity is encoded by a cimA gene. In one embodiment, the
polypeptide having (R)-citramalate synthase activity is endogenous
to the parental microbial cell, or is exogenous to the parental
microbial cell. In another embodiment, expression of the
polynucleotide encoding the polypeptide having (R)-citramalate
synthase activity is modulated in the recombinant microbial cell.
In some instances, expression of the polynucleotide is modulated by
operatively linking the polynucleotide to an exogenous promoter,
such that the polynucleotide is overexpressed in the recombinant
microbial cell. In another embodiment, the polypeptide having
(R)-citramalate synthase activity comprises a sequence selected
from SEQ ID NOs:40, 41, 42, 43, or a variant or a fragment thereof
having (R)-citramalate synthase activity and which catalyzes the
reaction of acetyl-CoA and pyruvate to (R)-citramalate in vitro or
in vivo, preferably in vivo.
[0030] In one embodiment, the recombinant microbial cell according
to the first aspect comprises a polynucleotide encoding a
polypeptide having isopropylmalate isomerase activity which is
categorized as EC 4.2.1.33. In one embodiment, the polypeptide
having isopropylmalate isomerase activity comprises a large subunit
and a small subunit encoded by leuCD genes. In one embodiment, the
polypeptide having isopropylmalate isomerase activity is endogenous
to the parental microbial cell, or is exogenous to the parental
microbial cell. In another embodiment, expression of the
polynucleotide encoding the polypeptide having isopropylmalate
isomerase activity is modulated in the recombinant microbial cell.
In some instances, expression of the polynucleotide is modulated by
operatively linking the polynucleotide to an exogenous promoter,
such that the polynucleotide is overexpressed in the recombinant
microbial cell. In another embodiment, the polypeptide having
isopropylmalate isomerase activity comprises a large subunit and a
small subunit. In other embodiments, the polypeptide having
isopropylmalate isomerase activity comprises a large subunit
sequence selected from SEQ ID NOs:44 and 46 and a small subunit
sequence selected from SEQ ID NOs:45 and 47, or variants or
fragments thereof having isopropylmalate isomerase activity and
which catalyzes the conversion of (R)-citramalate to citraconate
and citraconate to beta-methyl-D-malate in vitro or in vivo,
preferably in vivo.
[0031] In one embodiment, the recombinant microbial cell according
to the first aspect comprises a polynucleotide encoding a
polypeptide having beta-isopropylmalate dehydrogenase activity
which is categorized as EC 1.1.1.85. In some embodiments, the
polypeptide having beta-isopropyl malate dehydrogenase activity is
encoded by a leuB gene or a leu2 gene. In one embodiment, the
polypeptide having beta-isopropylmalate dehydrogenase activity is
endogenous to the parental microbial cell, or is exogenous to the
parental microbial cell. In another embodiment, expression of the
polynucleotide encoding the polypeptide having beta-isopropylmalate
dehydrogenase activity is modulated in the recombinant microbial
cell. In some instances, expression of the polynucleotide is
modulated by operatively linking the polynucleotide to an exogenous
promoter, such that the polynucleotide is overexpressed in the
recombinant microbial cell. In another embodiment, the polypeptide
having beta-isopropyl malate dehydrogenase activity comprises a
sequence selected from SEQ ID NOs:48, 49, 50, or a variant or a
fragment thereof having beta-isopropylmalate dehydrogenase activity
and which catalyzes conversion of beta-methyl-D-malate to
2-ketobutyrate in vitro or in vivo, preferably in vivo.
[0032] In one embodiment, the recombinant microbial cell according
to the first aspect comprises a polynucleotide encoding a
polypeptide having methylmalonyl-CoA mutase activity which is
categorized as EC 5.4.99.2 (FIG. 3). In some embodiments, the
polypeptide having methylmalonyl-CoA mutase activity is encoded by
an scpA (also known as sbm) gene. In one embodiment, the
polypeptide having methylmalonyl-CoA mutase activity is endogenous
to the parental microbial cell, or is exogenous to the parental
microbial cell. In another embodiment, expression of the
polynucleotide encoding the polypeptide having methylmalonyl-CoA
mutase activity is modulated in the recombinant microbial cell. In
some instances, expression of the polynucleotide is modulated by
operatively linking the polynucleotide to an exogenous promoter,
such that the polynucleotide is overexpressed in the recombinant
microbial cell. In another embodiment, the polypeptide having
methylmalonyl-CoA mutase activity comprises a sequence selected
from SEQ ID NOs:51, 52, 53, 54, 55, 56, 57, 58, or a variant or a
fragment thereof having methylmalonyl-CoA mutase activity and which
catalyzes conversion of succinyl-CoA to methylmalonyl-CoA in vitro
or in vivo, preferably in vivo.
[0033] In one embodiment, the recombinant microbial cell according
to the first aspect comprises a polynucleotide encoding a
polypeptide having methylmalonyl-CoA decarboxylase activity which
is categorized as EC 4.1.1.41. In some embodiments, the polypeptide
having methylmalonyl-CoA decarboxylase activity is encoded by an
scpB (also known as ygfG) gene. In one embodiment, the polypeptide
having methylmalonyl-CoA decarboxylase activity is endogenous to
the parental microbial cell, or is exogenous to the parental
microbial cell. In another embodiment, expression of the
polynucleotide encoding the polypeptide having methylmalonyl-CoA
decarboxylase activity is modulated in the recombinant microbial
cell. In some instances, expression of the polynucleotide is
modulated by operatively linking the polynucleotide to an exogenous
promoter, such that the polynucleotide is overexpressed in the
recombinant microbial cell. In another embodiment, the polypeptide
having methylmalonyl-CoA decarboxylase activity comprises a
sequence selected from SEQ ID NOs:59, 60, 61, or a variant or a
fragment thereof having methylmalonyl-CoA decarboxylase activity
and which catalyzes conversion of methylmalonyl-CoA to
propionyl-CoA in vitro or in vivo, preferably in vivo.
[0034] In one embodiment, the recombinant microbial cell according
to the first aspect comprises a polynucleotide encoding a
polypeptide having methylmalonyl-CoA carboxyltransferase activity
which is categorized as EC 2.1.3.1. In one embodiment, the
polypeptide having methylmalonyl-CoA carboxyltransferase activity
is endogenous to the parental microbial cell, or is exogenous to
the parental microbial cell. In another embodiment, expression of
the polynucleotide encoding the polypeptide having
methylmalonyl-CoA carboxyltransferase activity is modulated in the
recombinant microbial cell. In some instances, expression of the
polynucleotide is modulated by operatively linking the
polynucleotide to an exogenous promoter, such that the
polynucleotide is overexpressed in the recombinant microbial cell.
In another embodiment, the polypeptide having methylmalonyl-CoA
carboxyltransferase activity comprises the sequence SEQ ID NO:62,
or a variant or a fragment thereof having methylmalonyl-CoA
carboxyltransferase activity and which catalyzes conversion of
methylmalonyl-CoA to propionyl-CoA in vitro or in vivo, preferably
in vivo.
[0035] In one embodiment, the recombinant microbial cell according
to the first aspect comprises a polynucleotide encoding a
polypeptide having methylmalonyl-CoA epimerase activity which is
categorized as EC 5.1.99.1. In one embodiment, the polypeptide
having methylmalonyl-CoA epimerase activity is endogenous to the
parental microbial cell, or is exogenous to the parental microbial
cell. In another embodiment, expression of the polynucleotide
encoding the polypeptide having methylmalonyl-CoA epimerase
activity is modulated in the recombinant microbial cell. In some
instances, expression of the polynucleotide is modulated by
operatively linking the polynucleotide to an exogenous promoter,
such that the polynucleotide is overexpressed in the recombinant
microbial cell. In another embodiment, the polypeptide having
methylmalonyl-CoA epimerase activity comprises the sequence SEQ ID
NO:63, or a variant or a fragment thereof having methylmalonyl-CoA
epimerase activity and which catalyzes conversion of
(R)-methylmalonyl-CoA to (S)-methylmalonyl-CoA in vitro or in vivo,
preferably in vivo.
[0036] In one embodiment, the recombinant microbial cell according
to the first aspect comprises an endogenous polynucleotide sequence
(such as, an endogenous scpC gene (also known as ygfH)) encoding a
polypeptide having propionyl-CoA::succinyl-CoA transferase
activity, and expression of the endogenous polynucleotide in the
recombinant microbial cell is attenuated. In some embodiments,
expression of the endogenous polynucleotide is attenuated by
deletion of all or part of the sequence of the endogenous
polynucleotide in the recombinant microbial cell.
[0037] In one embodiment, the recombinant microbial cell according
to the first aspect comprises an endogenous polynucleotide sequence
(such as, an endogenous fadE gene) encoding a polypeptide having
acyl-CoA dehydrogenase activity, and expression of the endogenous
polynucleotide in the recombinant microbial cell may or may not be
attenuated.
[0038] In other embodiments, a recombinant microbial cell according
to the first aspect comprises a polynucleotide encoding a
polypeptide having a fatty acid derivative enzyme activity, wherein
the recombinant microbial cell produces a fatty acid derivative
composition comprising odd chain fatty acid derivatives when
cultured in the presence of a carbon source.
[0039] In various embodiments, the fatty acid derivative enzyme
activity comprises a thioesterase activity, an ester synthase
activity, a fatty aldehyde biosynthesis activity, a fatty alcohol
biosynthesis activity, a ketone biosynthesis activity, and/or a
hydrocarbon biosynthesis activity. In some embodiments, the
recombinant microbial cell comprises polynucleotides encoding two
or more polypeptides, each polypeptide having a fatty acid
derivative enzyme activity. In more particular embodiments, the
recombinant microbial cell expresses or overexpresses one or more
polypeptides having fatty acid derivative enzyme activity selected
from: (1) a polypeptide having thioesterase activity; (2) a
polypeptide having decarboxylase activity; (3) a polypeptide having
carboxylic acid reductase activity; (4) a polypeptide having
alcohol dehydrogenase activity (EC 1.1.1.1); (5) a polypeptide
having aldehyde decarbonylase activity (EC 4.1.99.5); (6) a
polypeptide having acyl-CoA reductase activity (EC 1.2.1.50); (7) a
polypeptide having acyl-ACP reductase activity; (8) a polypeptide
having ester synthase activity (EC 3.1.1.67); (9) a polypeptide
having OleA activity; or (10) a polypeptide having OleCD or OleBCD
activity; wherein the recombinant microbial cell produces a
composition comprising odd chain fatty acids, odd chain fatty
esters, odd chain wax esters, odd chain fatty aldehydes, odd chain
fatty alcohols, even chain alkanes, even chain alkenes, even chain
internal olefins, even chain terminal olefins, or even chain
ketones.
[0040] In one embodiment, the fatty acid derivative enzyme activity
comprises a thioesterase activity, wherein a culture comprising the
recombinant microbial cell produces a fatty acid composition
comprising odd chain fatty acids when cultured in the presence of a
carbon source. In some embodiments, the polypeptide has a
thioesterase activity which is categorized as EC 3.1.1.5, EC
3.1.2.-, or EC 3.1.2.14. In some embodiments, the polypeptide
having a thioesterase activity is encoded by a tesA, a tesB, a
fatA, or a fatB gene. In some embodiments, the polypeptide having
thioesterase activity is endogenous to the parental microbial cell,
or is exogenous to the parental microbial cell. In another
embodiment, expression of the polynucleotide encoding the
polypeptide having thioesterase activity is modulated in the
recombinant microbial cell. In some instances, expression of the
polynucleotide is modulated by operatively linking the
polynucleotide to an exogenous promoter, such that the
polynucleotide is overexpressed in the recombinant microbial cell.
In another embodiment, the polypeptide having thioesterase activity
comprises a sequence selected from SEQ ID NO: 64, 65, 66, 67, 68,
69, 70, 71 and 72, or a variant or a fragment thereof having
thioesterase activity and which catalyzes the hydrolysis of an odd
chain acyl-ACP to an odd chain fatty acid, or catalyzes the
alcoholysis of an odd chain acyl-ACP to an odd chain fatty ester,
in vitro or in vivo, preferably in vivo. In some embodiments, the
recombinant microbial cell according to the first aspect,
comprising a polynucleotide encoding a polypeptide having
thioesterase activity, when cultured in the presence of a carbon
source, produces at least 50 mg/L, at least 75 mg/L, at least 100
mg/L, at least 200 mg/L, at least 500 mg/L, at least 1000 mg/L, or
at least 2000 mg/L odd chain fatty acids when cultured in a culture
medium containing a carbon source under conditions effective to
express the polynucleotides. In some embodiments, the recombinant
microbial cell according to the first aspect, comprising a
polynucleotide encoding a polypeptide having thioesterase activity,
produces a fatty acid composition comprising odd chain fatty acids
and even chain fatty acids. In some embodiments, at least 5%, at
least 10%, at least 20%, at least 30%, at least 40%, at least 50%,
at least 60%, at least 70%, at least 80% or at least 90% of the
fatty acids in the composition are odd chain fatty acids.
[0041] The invention includes a cell culture comprising the
recombinant microbial cell according to the first aspect.
[0042] In a second aspect, the invention includes a method of
producing odd chain fatty acid derivatives (or a fatty acid
derivative composition comprising odd chain fatty acid derivatives)
in a recombinant microbial cell, the method comprising expressing
in the cell a recombinant polypeptide having enzymatic activity
effective to increase the production of propionyl-CoA in the cell,
and culturing the cell in the presence of a carbon source under
conditions effective to express the recombinant polypeptide and
produce the odd chain fatty acid derivatives.
[0043] In one embodiment, the method of making a fatty acid
derivative composition comprising odd chain fatty acid derivatives
comprises obtaining a recombinant microbial cell according to the
first aspect, culturing the cell in a culture medium containing a
carbon source under conditions effective to express the
polynucleotides according to (a), (b), and (c) and produce a fatty
acid derivative composition comprising odd chain fatty acid
derivatives, and optionally recovering the composition from the
culture medium.
[0044] In some embodiments, the fatty acid derivative composition
produced by the method according to the second aspect comprises odd
chain fatty acid derivatives and even chain fatty acid derivatives,
wherein at least 5%, at least 6%, at least 8%, at least 10%, at
least 20%, at least 30%, at least 40%, at least 50%, at least 60%,
at least 70%, at least 80% or at least 90% by weight of the fatty
acid derivatives in the composition are odd chain fatty acid
derivatives. In some embodiments, the fatty acid derivative
composition comprises odd chain fatty acid derivatives in an amount
(e.g., a titer) of at least 50 mg/L, at least 75 mg/L, at least 100
mg/L, at least 200 mg/L, at least 500 mg/L, at least 1000 mg/L, at
least 2000 mg/L, at least 5000 mg/L, at least 10000 mg/L, or at
least 20000 mg/L.
[0045] In various embodiments of the second aspect, the fatty acid
derivative enzyme activity comprises a thioesterase activity, an
ester synthase activity, a fatty aldehyde biosynthesis activity, a
fatty alcohol biosynthesis activity, a ketone biosynthesis
activity, and/or a hydrocarbon biosynthesis activity. In some
embodiments, the recombinant microbial cell comprises
polynucleotides encoding two or more polypeptides, each polypeptide
having a fatty acid derivative enzyme activity. In more particular
embodiments, the recombinant microbial cell expresses or
overexpresses one or more polypeptides having fatty acid derivative
enzyme activity selected from: (1) a polypeptide having
thioesterase activity; (2) a polypeptide having decarboxylase
activity; (3) a polypeptide having carboxylic acid reductase
activity; (4) a polypeptide having alcohol dehydrogenase activity
(EC 1.1.1.1); (5) a polypeptide having aldehyde decarbonylase
activity (EC 4.1.99.5); (6) a polypeptide having acyl-CoA reductase
activity (EC 1.2.1.50); (7) a polypeptide having acyl-ACP reductase
activity; (8) a polypeptide having ester synthase activity (EC
3.1.1.67); (9) a polypeptide having OleA activity; or (10) a
polypeptide having OleCD or OleBCD activity; wherein the
recombinant microbial cell produces a composition comprising one or
more of odd chain fatty acids, odd chain fatty esters, odd chain
wax esters, odd chain fatty aldehydes, odd chain fatty alcohols,
even chain alkanes, even chain alkenes, even chain internal
olefins, even chain terminal olefins, and even chain ketones.
[0046] The invention includes a fatty acid derivative composition
comprising odd chain fatty acid derivatives produced by the method
according to the second aspect.
[0047] In a third aspect, the invention includes a method of making
a recombinant microbial cell which produces a higher titer or
higher proportion of odd chain fatty acid derivatives than a
parental microbial cell, the method comprising obtaining a parental
microbial cell comprising a polynucleotide encoding a polypeptide
having fatty acid derivative enzyme activity, and engineering the
parental microbial cell to obtain a recombinant microbial cell
which produces or is capable of producing a greater amount of
propionyl-CoA than the amount of propionyl-CoA produced by the
parental microbial cell when cultured under the same conditions,
wherein the recombinant microbial cell produces a higher titer or
higher proportion of odd chain fatty acid derivatives when cultured
in the presence of a carbon source under conditions effective to
produce propionyl-CoA and fatty acid derivatives in the recombinant
microbial cell, relative to the titer or proportion of odd chain
fatty acid derivatives produced by the parental microbial cell
cultured under the same conditions.
[0048] In a fourth aspect, the invention includes a method of
increasing the titer or proportion of odd chain fatty acid
derivatives produced by a microbial cell, the method comprising
obtaining a parental microbial cell that is capable of producing a
fatty acid derivative, and engineering the parental microbial cell
to obtain a recombinant microbial cell which produces or is capable
of producing a greater amount of propionyl-CoA than the amount of
propionyl-CoA produced by the parental microbial cell when cultured
under the same conditions, wherein the recombinant microbial cell
produces a higher titer or higher proportion of odd chain fatty
acid derivatives when cultured in the presence of a carbon source
under conditions effective to produce propionyl-CoA and fatty acid
derivatives in the recombinant microbial cell, relative to the
titer or proportion of odd chain fatty acid derivatives produced by
the parental microbial cell cultured under the same conditions.
[0049] In some embodiments according to the third or fourth aspect,
the step of engineering the parental microbial cell comprises
engineering the cell to express polynucleotides encoding
polypeptides selected from (a) one or more polypeptides having
aspartokinase activity, homoserine dehydrogenase activity,
homoserine kinase activity, threonine synthase activity, and
threonine deaminase activity; (b) one or more polypeptides having
(R)-citramalate synthase activity, isopropylmalate isomerase
activity, and beta-isopropylmalate dehydrogenase activity; and (c)
one or more polypeptides having methylmalonyl-CoA mutase activity,
methylmalonyl-CoA decarboxylase activity, methylmalonyl-CoA
carboxyltransferase activity, and methylmalonyl-CoA epimerase
activity; wherein at least one polypeptide according to (a), (b) or
(c) is exogenous to the parental microbial cell, or wherein
expression of at least one polynucleotide according to (a), (b) or
(c) is modulated in the recombinant microbial cell as compared to
the expression of the polynucleotide in the parental microbial
cell. In some embodiments, expression of at least one
polynucleotide is modulated by overexpression of the
polynucleotide, such as by operatively linking the polynucleotide
to an exogenous promoter. In some embodiments, the engineered cell
expresses one or more polypeptide according to (a) and one or more
polypeptide according to (b).
[0050] In some embodiments according to the third or fourth aspect,
the parental microbial cell comprises a polynucleotide encoding a
polypeptide having .beta.-ketoacyl-ACP synthase activity that
utilizes propionyl-CoA as a substrate. In some embodiments, the
recombinant microbial cell is engineered to express an exogenous
polynucleotide or to overexpress an endogenous polynucleotide
encoding a polypeptide having .beta.-ketoacyl-ACP synthase activity
that utilizes propionyl-CoA as a substrate. In some embodiments,
the recombinant microbial cell is engineered to express an
exogenous polynucleotide encoding a polypeptide having
.beta.-ketoacyl-ACP synthase activity that utilizes propionyl-CoA
as a substrate, and expression of an endogenous polynucleotide
encoding a polypeptide having .beta.-ketoacyl-ACP synthase activity
is attenuated. In some embodiments, the polynucleotide encoding a
polypeptide having .beta.-ketoacyl-ACP synthase is a modified,
mutant or variant form of an endogenous polynucleotide, which has
been selected for enhanced affinity or activity for propionyl-CoA
as a substrate relative to the unmodified endogenous
polynucleotide. Numerous methods for generation of modified, mutant
or variant polynucleotides are well known in the art, examples of
which are described herein below.
[0051] These and other objects and features of the invention will
become more fully apparent when the following detailed description
is read in conjunction with the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] FIGS. 1A and 1B compare exemplary intermediates and products
of fatty acid biosynthetic pathways when supplied with different
acyl-CoA "primer" molecules: FIG. 1A shows a reaction pathway
utilizing the two-carbon primer acetyl-CoA, which generates the
even chain length .beta.-ketoacyl-ACP intermediate acetoacetyl-ACP,
leading to even chain (ec)-acyl-ACP intermediates and even chain
fatty acid derivatives produced therefrom; and FIG. 1B shows a
reaction pathway utilizing the three carbon primer propionyl-CoA,
which generates the odd chain length .beta.-ketoacyl-ACP
intermediate 3-oxovaleryl-ACP, leading to odd chain (oc)-acyl-ACP
intermediates and odd chain fatty acid derivatives produced
therefrom.
[0053] FIG. 2 depicts exemplary pathways for increased production
of propionyl-CoA via the intermediate .alpha.-ketobutyrate, by a
threonine biosynthetic pathway (pathway (A)) and by a citramalate
biosynthetic pathway (pathway (B)) as described herein.
[0054] FIG. 3 depicts an exemplary pathway for increased production
of propionyl-CoA via a methylmalonyl-CoA biosynthetic pathway
(pathway (C)) as described herein.
DETAILED DESCRIPTION
[0055] The invention is not limited to the specific compositions
and methodology described herein, as these may, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to limit the scope of the present invention.
[0056] Accession Numbers: Sequence Accession numbers throughout
this description were obtained from databases provided by the NCBI
(National Center for Biotechnology Information) maintained by the
National Institutes of Health, U.S.A. (which are identified herein
as "NCBI Accession Numbers" or alternatively as "GenBank Accession
Numbers"), and from the UniProt Knowledgebase (UniProtKB) and
Swiss-Prot databases provided by the Swiss Institute of
Bioinformatics (which are identified herein as "UniProtKB Accession
Numbers"). Unless otherwise expressly indicated, the sequence
identified by an NCBI/GenBank Accession number is version number 1
(that is, the Version Number of the sequence is
"AccessionNumber.1"). The NCBI and UniProtKB accession numbers
provided herein were current as of Aug. 2, 2011.
[0057] Enzyme Classification (EC) Numbers: EC numbers are
established by the Nomenclature Committee of the International
Union of Biochemistry and Molecular Biology (IUBMB), description of
which is available on the IUBMB Enzyme Nomenclature website on the
World Wide Web. EC numbers classify enzymes according to the
reaction catalyzed. EC numbers referenced herein are derived from
the KEGG Ligand database, maintained by the Kyoto Encyclopedia of
Genes and Genomics, sponsored in part by the University of Tokyo.
Unless otherwise indicated, EC numbers are as provided in the KEGG
database as of Aug. 2, 2011.
[0058] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs. Although
any materials and methods similar or equivalent to those described
herein can be used in the practice or testing of the invention, the
preferred compositions and methods are now described.
DEFINITIONS
[0059] As used herein, the term "fatty acid" refers to a carboxylic
acid having the formula R--(C.dbd.O)--OH, wherein R represents a
carbon chain which can be between about 4 and about 36 carbon atoms
in length, more generally between about 4 and about 22 carbon atoms
in length. Fatty acids can be saturated or unsaturated. If
unsaturated, R can have one or more points of unsaturation, that
is, R can be monounsaturated or polyunsaturated. R can be a
straight chain (also referred to herein as a "linear chain") or a
branched chain. The term "fatty acid" may be used herein to refer
to a "fatty acid derivative" which can include one or more
different fatty acid derivative, or mixtures of fatty acids
derivatives.
[0060] An "odd chain fatty acid" (abbreviated "oc-FA") as used
herein refers to a fatty acid molecule having a linear carbon chain
containing an odd number of carbon atoms, inclusive of the carbonyl
carbon. Non-limiting examples of oc-FAs include tridecanoic acid
(C13:0), pentadecanoic acid (C15:0), and heptadecanoic acid
(C17:0), which are saturated oc-FAs, and heptadecenoic acid
(C17:1), which is an unsaturated (i.e., a monounsaturated)
oc-FA.
[0061] The term ".beta.-ketoacyl-ACP" as used herein refers to the
product of the condensation of an acyl-CoA primer molecule with
malonyl-ACP catalyzed by an enzyme having beta ketoacyl-ACP
synthase activity (e.g., EC 2.3.1.180) as represented by part (D)
of the pathways shown in FIGS. 1A and 1B. The acyl-CoA primer
molecule may have an acyl group containing an even number of carbon
atoms, such as acetyl-CoA as represented in FIG. 1A, in which case
the resulting .beta.-ketoacyl-ACP intermediate is acetoacetyl-ACP,
which is an even chain (ec-).beta.-ketoacyl-ACP. The acyl-CoA
primer molecule may have an acyl group containing an odd number of
carbon atoms, such as propionyl-CoA as represented in FIG. 1B, in
which case the resulting .beta.-ketoacyl-ACP intermediate is
3-oxovaleryl-ACP, which is an odd chain (oc-).beta.-ketoacyl-ACP.
The .beta.-ketoacyl-ACP intermediate enters the fatty acid synthase
(FAS) cycle, represented by part (E) of FIGS. 1A and 1B, where it
is subjected to a round of elongation (i.e., keto reduction,
dehydration, and enoyl reduction), adding two carbon units to the
acyl chain, followed by additional elongation cycles, which each
involve condensation with another malonyl-ACP molecule, keto
reduction, dehydration, and enoyl reduction, such that the acyl
chain of the acyl-ACP is elongated by two carbon units per
elongation cycle.
[0062] An "acyl-ACP" generally refers to the product of one or more
rounds of FAS-catalyzed elongation of a .beta.-ketoacyl-ACP
intermediate. Acyl-ACP is an acyl thioester formed between the
carbonyl carbon of an alkyl chain and the sulfhydryl group of the
4'-phosphopantethionyl moiety of an acyl carrier protein (ACP),
and, in the case of a linear carbon chain, typically has the
formula CH3-(CH.sub.2)n-C(.dbd.O)-s-ACP wherein n may be an even
number (e.g., an "even chain acyl-ACP" or "ec-acyl-ACP", which is
produced, for example, when acetyl-CoA is the primer molecule, see
FIG. 1A) or an odd number (e.g., an "odd chain acyl-ACP" or
"oc-acyl-ACP", which is produced, for example, when propionyl-CoA
is the primer molecule, see FIG. 1B).
[0063] Unless otherwise specified, a "fatty acid derivative"
(abbreviated "FA derivative") is intended to include any product
made at least in part by the fatty acid biosynthetic pathway of the
recombinant microbial cell. A fatty acid derivative also includes
any product made at least in part by a fatty acid pathway
intermediate, such as an acyl-ACP intermediate. The fatty acid
biosynthetic pathways described herein can include fatty acid
derivative enzymes which can be engineered to produce fatty acid
derivatives, and in some instances additional enzymes can be
expressed to produce fatty acid derivatives having desired carbon
chain characteristics, such as, for example, compositions of fatty
acid derivatives having carbon chains containing a desired number
of carbon atoms, or compositions of fatty acid derivatives having a
desired proportion of derivatives containing odd numbered carbon
chains, and the like. Fatty acid derivatives include, but are not
limited to, fatty acids, fatty aldehydes, fatty alcohols, fatty
esters (such as waxes), hydrocarbons (such as alkanes and alkenes
(including terminal olefins and internal olefins)) and ketones.
[0064] The term "odd chain fatty acid derivative" (abbreviated
"oc-FA derivative") refers to a product of the reaction of an
oc-acyl-ACP, as defined above, with one or more fatty acid
derivative enzymes. The resulting fatty acid derivative product
likewise has a linear carbon chain containing an odd number of
carbon atoms, unless the fatty acid derivative is itself the
product of decarbonylation or decarboxylation of an oc-FA
derivative or an oc-acyl-ACP, in which case the resulting oc-FA
derivative has an even number of carbon atoms; for example, when
the fatty acid derivative is an ec-alkane or ec-alkene produced by
decarbonylation of an oc-fatty aldehyde, an ec-terminal olefin
produced by decarboxylation of an oc-fatty acid, an ec-ketone or an
ec-internal olefin produced by decarboxylation of an oc-acyl-ACP,
and so forth. It is to be understood that such even chain length
products of oc-FA derivatives or oc-acyl-ACP precursor molecules,
despite having linear chains containing an even number of carbon
atoms, are nevertheless considered to fall under the definition of
"oc-FA derivatives".
[0065] An "endogenous" polypeptide refers to a polypeptide encoded
by the genome of the parental microbial cell (also termed "host
cell") from which the recombinant cell is engineered (or
"derived").
[0066] An "exogenous" polypeptide refers to a polypeptide which is
not encoded by the genome of the parental microbial cell. A variant
(i.e., mutant) polypeptide is an example of an exogenous
polypeptide.
[0067] In embodiments of the invention wherein a polynucleotide
sequence encodes an endogenous polypeptide, in some instances the
endogenous polypeptide is overexpressed. As used herein,
"overexpress" means to produce or cause to be produced a
polynucleotide or a polypeptide in a cell at a greater
concentration than is normally produced in the corresponding
parental cell (such as, a wild-type cell) under the same
conditions. A polynucleotide or a polypeptide can be
"overexpressed" in a recombinant microbial cell when the
polynucleotide or polypeptide is present in a greater concentration
in the recombinant microbial cell as compared to its concentration
in a non-recombinant microbial cell of the same species (such as,
the parental microbial cell) under the same conditions.
Overexpression can be achieved by any suitable means known in the
art.
[0068] In some embodiments, overexpression of the endogenous
polypeptide in the recombinant microbial cell can be achieved by
the use of an exogenous regulatory element. The term "exogenous
regulatory element" generally refers to a regulatory element (such
as, an expression control sequence or a chemical compound)
originating outside of the host cell. However, in certain
embodiments, the term "exogenous regulatory element" (e.g.,
"exogenous promoter") can refer to a regulatory element derived
from the host cell whose function is replicated or usurped for the
purpose of controlling the expression of the endogenous polypeptide
in the recombinant cell. For example, if the host cell is an E.
coli cell, and the polypeptide is an endogenous polypeptide, then
expression of the endogenous polypeptide the recombinant cell can
be controlled by a promoter derived from another E. coli gene. In
some embodiments, the exogenous regulatory element that causes an
increase in the level of expression and/or activity of an
endogenous polypeptide is a chemical compound, such as a small
molecule.
[0069] In some embodiments, the exogenous regulatory element which
controls the expression of a polynucleotide (e.g., an endogenous
polynucleotide) encoding an endogenous polypeptide is an expression
control sequence which is operably linked to the endogenous
polynucleotide by recombinant integration into the genome of the
host cell. In certain embodiments, the expression control sequence
is integrated into a host cell chromosome by homologous
recombination using methods known in the art (e.g., Datsenko et
al., Proc. Natl. Acad. Sci. U.S.A., 97(12): 6640-6645 (2000)).
[0070] Expression control sequences are known in the art and
include, for example, promoters, enhancers, polyadenylation
signals, transcription terminators, internal ribosome entry sites
(IRES), and the like, that provide for the expression of the
polynucleotide sequence in a host cell. Expression control
sequences interact specifically with cellular proteins involved in
transcription (Maniatis et al., Science, 236: 1237-1245 (1987)).
Exemplary expression control sequences are described in, for
example, Goeddel, Gene Expression Technology: Methods in
Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).
[0071] In the methods of the invention, an expression control
sequence is operably linked to a polynucleotide sequence. By
"operably linked" is meant that a polynucleotide sequence and
expression control sequence(s) are connected in such a way as to
permit gene expression when the appropriate molecules (e.g.,
transcriptional activator proteins) are bound to the expression
control sequence(s). Operably linked promoters are located upstream
of the selected polynucleotide sequence in terms of the direction
of transcription and translation. Operably linked enhancers can be
located upstream, within, or downstream of the selected
polynucleotide. Additional nucleic acid sequences, such as nucleic
acid sequences encoding selection markers, purification moieties,
targeting proteins, and the like, can be operatively linked to the
polynucleotide sequence, such that the additional nucleic acid
sequences are expressed together with the polynucleotide
sequence.
[0072] In some embodiments, the polynucleotide sequence is provided
to the recombinant cell by way of a recombinant vector, which
comprises a promoter operably linked to the polynucleotide
sequence. In certain embodiments, the promoter is a
developmentally-regulated, an organelle-specific, a
tissue-specific, an inducible, a constitutive, or a cell-specific
promoter.
[0073] As used herein, the term "vector" refers to a nucleic acid
molecule capable of transporting another nucleic acid, i.e., a
polynucleotide sequence, to which it has been linked. One type of
useful vector is an episome (i.e., a nucleic acid capable of
extra-chromosomal replication). Useful vectors are those capable of
autonomous replication and/or expression of nucleic acids to which
they are linked. Vectors capable of directing the expression of
genes to which they are operatively linked are referred to herein
as "expression vectors." In general, expression vectors of utility
in recombinant DNA techniques are often in the form of "plasmids,"
which refer generally to circular double stranded DNA loops that,
in their vector form, are not bound to the chromosome. The terms
"plasmid" and "vector" are used interchangeably herein, inasmuch as
a plasmid is the most commonly used form of vector. However, also
included are such other forms of expression vectors that serve
equivalent functions and that become known in the art subsequently
hereto.
[0074] In some embodiments, the recombinant vector comprises at
least one sequence selected from the group consisting of (a) an
expression control sequence operatively linked to the
polynucleotide sequence; (b) a selection marker operatively linked
to the polynucleotide sequence; (c) a marker sequence operatively
linked to the polynucleotide sequence; (d) a purification moiety
operatively linked to the polynucleotide sequence; (e) a secretion
sequence operatively linked to the polynucleotide sequence; and (f)
a targeting sequence operatively linked to the polynucleotide
sequence.
[0075] The expression vectors described herein include a
polynucleotide sequence described herein in a form suitable for
expression of the polynucleotide sequence in a host cell. It will
be appreciated by those skilled in the art that the design of the
expression vector can depend on such factors as the choice of the
host cell to be transformed, the level of expression of polypeptide
desired, etc. The expression vectors described herein can be
introduced into host cells to produce polypeptides, including
fusion polypeptides, encoded by the polynucleotide sequences as
described herein.
[0076] Expression of genes encoding polypeptides in prokaryotes,
for example, E. coli, is often carried out with vectors containing
constitutive or inducible promoters directing the expression of
either fusion or non-fusion polypeptides. Fusion vectors add a
number of amino acids to a polypeptide encoded therein, usually to
the amino- or carboxy-terminus of the recombinant polypeptide. Such
fusion vectors typically serve one or more of the following three
purposes: (1) to increase expression of the recombinant
polypeptide; (2) to increase the solubility of the recombinant
polypeptide; and (3) to aid in the purification of the recombinant
polypeptide by acting as a ligand in affinity purification. Often,
in fusion expression vectors, a proteolytic cleavage site is
introduced at the junction of the fusion moiety and the recombinant
polypeptide. This enables separation of the recombinant polypeptide
from the fusion moiety after purification of the fusion
polypeptide. Examples of such enzymes, and their cognate
recognition sequences, include Factor Xa, thrombin, and
enterokinase. Exemplary fusion expression vectors include pGEX
(Pharmacia Biotech, Inc., Piscataway, N.J.; Smith et al., Gene, 67:
31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.), and
pRITS (Pharmacia Biotech, Inc., Piscataway, N.J.), which fuse
glutathione S-transferase (GST), maltose E binding protein, or
protein A, respectively, to the target recombinant polypeptide.
[0077] Vectors can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" refer
to a variety of art-recognized techniques for introducing foreign
nucleic acid (e.g., DNA) into a host cell, including calcium
phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in, for example, Sambrook et al.
(supra).
[0078] For stable transformation of bacterial cells, it is known
that, depending upon the expression vector and transformation
technique used, only a small fraction of cells will take up and
replicate the expression vector. In order to identify and select
these transformants, a gene that encodes a selectable marker (e.g.,
resistance to an antibiotic) can be introduced into the host cells
along with the gene of interest. Selectable markers include those
that confer resistance to drugs such as, but not limited to,
ampicillin, kanamycin, chloramphenicol, or tetracycline. Nucleic
acids encoding a selectable marker can be introduced into a host
cell on the same vector as that encoding a polypeptide described
herein or can be introduced on a separate vector. Host cells which
are stably transformed with the introduced nucleic acid, resulting
in recombinant cells, can be identified by growth in the presence
of an appropriate selection drug.
[0079] Similarly, for stable transfection of mammalian cells, it is
known that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to an antibiotic) can be introduced into the host cells
along with the gene of interest. Preferred selectable markers
include those which confer resistance to drugs, such as G418,
hygromycin, and methotrexate. Nucleic acids encoding a selectable
marker can be introduced into a host cell on the same vector as
that encoding a polypeptide described herein or can be introduced
on a separate vector. Host cells stably transfected with the
introduced nucleic acid, resulting in recombinant cells, can be
identified by growth in the presence of an appropriate selection
drug.
[0080] "Gene knockout", as used herein, refers to a procedure by
which a gene encoding a target protein is modified or inactivated
so to reduce or eliminate the function of the intact protein.
Inactivation of the gene may be performed by general methods such
as mutagenesis by UV irradiation or treatment with
N-methyl-N'-nitro-N-nitrosoguanidine, site-directed mutagenesis,
homologous recombination, insertion-deletion mutagenesis, or
"Red-driven integration" (Datsenko et al., Proc. Natl. Acad. Sci.
USA, 97:6640-45, 2000). For example, in one embodiment, a construct
is introduced into a parental cell, such that it is possible to
select for homologous recombination events in the resulting
recombinant cell. One of skill in the art can readily design a
knock-out construct including both positive and negative selection
genes for efficiently selecting transfected (i.e., recombinant)
cells that undergo a homologous recombination event with the
construct. The alteration in the parental cell may be obtained, for
example, by replacing through a single or double crossover
recombination a wild type (i.e., endogenous) DNA sequence by a DNA
sequence containing the alteration. For convenient selection of
transformants (i.e., recombinant cells), the alteration may, for
example, be a DNA sequence encoding an antibiotic resistance marker
or a gene complementing a possible auxotrophy of the host cell.
Mutations include, but are not limited to, deletion-insertion
mutations. An example of such an alteration in a recombinant cell
includes a gene disruption, i.e., a perturbation of a gene such
that the product that is normally produced from this gene is not
produced in a functional form. This could be due to a complete
deletion, a deletion and insertion of a selective marker, an
insertion of a selective marker, a frameshift mutation, an in-frame
deletion, or a point mutation that leads to premature termination.
In some instances, the entire mRNA for the gene is absent. In other
situations, the amount of mRNA produced varies.
[0081] The phrase "increasing the level of expression of an
endogenous polypeptide" means to cause the overexpression of a
polynucleotide sequence encoding the endogenous polypeptide, or to
cause the overexpression of an endogenous polypeptide sequence. The
degree of overexpression can be about 1.5-fold or more, about
2-fold or more, about 3-fold or more, about 5-fold or more, about
10-fold or more, about 20-fold or more, about 50-fold or more,
about 100-fold or more, or any range therein.
[0082] The phrase "increasing the level of activity of an
endogenous polypeptide" means to enhance the biochemical or
biological function (e.g., enzymatic activity) of an endogenous
polypeptide. The degree of enhanced activity can be about 10% or
more, about 20% or more, about 50% or more, about 75% or more,
about 100% or more, about 200% or more, about 500% or more, about
1000% or more, or any range therein.
[0083] The phrase, "the expression of said polynucleotide sequence
is modified relative to the wild type polynucleotide sequence", as
used herein means an increase or decrease in the level of
expression and/or activity of an endogenous polynucleotide
sequence. In some embodiments, an exogenous regulatory element
which controls the expression of an endogenous polynucleotide is an
expression control sequence which is operably linked to the
endogenous polynucleotide by recombinant integration into the
genome of the host cell. In some embodiments, the expression
control sequence is integrated into a host cell chromosome by
homologous recombination using methods known in the art.
[0084] As used herein, the phrase "under conditions effective to
express said polynucleotide sequence(s)" means any conditions that
allow a recombinant cell to produce a desired fatty acid
derivative. Suitable conditions include, for example, fermentation
conditions. Fermentation conditions can comprise many parameters,
such as temperature ranges, levels of aeration, and media
composition. Each of these conditions, individually and in
combination, allows the host cell to grow. Exemplary culture media
include broths or gels. Generally, the medium includes a carbon
source that can be metabolized by a recombinant cell directly.
Fermentation denotes the use of a carbon source by a production
host, such as a recombinant microbial cell of the invention.
Fermentation can be aerobic, anaerobic, or variations thereof (such
as micro-aerobic). As will be appreciated by those of skill in the
art, the conditions under which a recombinant microbial cell can
process a carbon source into an oc-acyl-ACP or a desired oc-FA
derivative (e.g., an oc-fatty acid, an oc-fatty ester, an oc-fatty
aldehyde, an oc-fatty alcohol, an ec-alkane, an ec-alkene or an
ec-ketone) will vary in part, based upon the specific
microorganism. In some embodiments, the process occurs in an
aerobic environment. In some embodiments, the process occurs in an
anaerobic environment. In some embodiments, the process occurs in a
micro-aerobic environment.
[0085] As used herein, the term "carbon source" refers to a
substrate or compound suitable to be used as a source of carbon for
prokaryotic or simple eukaryotic cell growth. Carbon sources can be
in various forms, including, but not limited to polymers,
carbohydrates (e.g., sugars, such as monosaccharides,
disaccharides, oligosaccharides, and polysaccharides), acids,
alcohols, aldehydes, ketones, amino acids, peptides, and gases
(e.g., CO and CO.sub.2). Exemplary carbon sources include, but are
not limited to: monosaccharides, such as glucose, fructose,
mannose, galactose, xylose, and arabinose; disaccharides, such as
sucrose, maltose, cellobiose, and turanose; oligosaccharides, such
as fructo-oligosaccharide and galacto-oligosaccharide;
polysaccharides, such as starch, cellulose, pectin, and xylan;
cellulosic material and variants such as hemicelluloses, methyl
cellulose and sodium carboxymethyl cellulose; saturated or
unsaturated fatty acids, succinate, lactate, and acetate; alcohols,
such as ethanol, methanol, and glycerol, or mixtures thereof. The
carbon source can be a product of photosynthesis, such as glucose.
In certain preferred embodiments, the carbon source is derived from
biomass. In another preferred embodiment, the carbon source
comprises sucrose. In another preferred embodiment, the carbon
source comprises glucose.
[0086] As used herein, the term "biomass" refers to any biological
material from which a carbon source is derived. In some
embodiments, a biomass is processed into a carbon source, which is
suitable for bioconversion. In other embodiments, the biomass does
not require further processing into a carbon source. The carbon
source can be converted into a biofuel. An exemplary source of
biomass is plant matter or vegetation, such as corn, sugar cane, or
switchgrass. Another exemplary source of biomass is metabolic waste
products, such as animal matter (e.g., cow manure). Further
exemplary sources of biomass include algae and other marine plants.
Biomass also includes waste products from industry, agriculture,
forestry, and households, including, but not limited to,
fermentation waste, ensilage, straw, lumber, sewage, garbage,
cellulosic urban waste, and food leftovers. The term "biomass" also
can refer to sources of carbon, such as carbohydrates (e.g.,
monosaccharides, disaccharides, or polysaccharides).
[0087] To determine if conditions are sufficient to allow
production of a product or expression of a polypeptide, a
recombinant microbial cell can be cultured, for example, for about
4, 8, 12, 24, 36, 48, 72, or more hours. During and/or after
culturing, samples can be obtained and analyzed to determine if the
conditions allow production or expression. For example, the
recombinant microbial cells in the sample or the medium in which
the recombinant microbial cells were grown can be tested for the
presence of a desired product. When testing for the presence of a
desired product, such as an odd chain fatty acid derivative (e.g.,
an oc-fatty acid, an oc-fatty ester, an oc-fatty aldehyde, an
oc-fatty alcohol, or an ec-hydrocarbon), assays such as, but not
limited to, gas chromatography (GC), mass spectroscopy (MS), thin
layer chromatography (TLC), high-performance liquid chromatography
(HPLC), liquid chromatography (LC), GC coupled with a flame
ionization detector (GC-FID), GC-MS, and LC-MS, can be used. When
testing for the expression of a polypeptide, techniques such as,
but not limited to, Western blotting and dot blotting, may be
used.
[0088] As used herein, the term "microorganism" means prokaryotic
and eukaryotic microbial species from the domains Archaea, Bacteria
and Eucarya, the latter including yeast and filamentous fungi,
protozoa, algae, and higher Protista. The terms "microbes" and
"microbial cells" (i.e., cells from microbes) and are used
interchangeably with "microorganisms" and refer to cells or small
organisms that can only be seen with the aid of a microscope.
[0089] In some embodiments, the host cell (e.g., parental cell) is
a microbial cell. In some embodiments, the host cell is a microbial
cell selected from the genus Escherichia, Bacillus, Lactobacillus,
Pantoea, Zymomonas, Rhodococcus, Pseudomonas, Aspergillus,
Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor,
Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium,
Phanerochaete, Pleurotus, Trametes, Chrysosporium, Saccharomyces,
Stenotrophamonas, Schizosaccharomyces, Yarrowia, Streptomyces,
Synechococcus, Chlorella, or Prototheca.
[0090] In other embodiments, the host cell is a Bacillus lentus
cell, a Bacillus brevis cell, a Bacillus stearothermophilus cell, a
Bacillus lichenoformis cell, a Bacillus alkalophilus cell, a
Bacillus coagulans cell, a Bacillus circulans cell, a Bacillus
pumilis cell, a Bacillus thuringiensis cell, a Bacillus clausii
cell, a Bacillus megaterium cell, a Bacillus subtilis cell, or a
Bacillus amyloliquefaciens cell.
[0091] In other embodiments, the host cell is a Trichoderma
koningii cell, a Trichoderma viride cell, a Trichoderma reesei
cell, a Trichoderma longibrachiatum cell, an Aspergillus awamori
cell, an Aspergillus fumigates cell, an Aspergillus foetidus cell,
an Aspergillus nidulans cell, an Aspergillus niger cell, an
Aspergillus oryzae cell, a Humicola insolens cell, a Humicola
lanuginose cell, a Rhodococcus opacus cell, a Rhizomucor miehei
cell, or a Mucor michei cell.
[0092] In yet other embodiments, the host cell is a Streptomyces
lividans cell or a Streptomyces murinus cell.
[0093] In yet other embodiments, the host cell is an Actinomycetes
cell.
[0094] In some embodiments, the host cell is a Saccharomyces
cerevisiae cell.
[0095] In still other embodiments, the host cell is a CHO cell, a
COS cell, a VERO cell, a BHK cell, a HeLa cell, a Cvl cell, an MDCK
cell, a 293 cell, a 3T3 cell, or a PC12 cell.
[0096] In some embodiments, the host cell is a cell from an
eukaryotic plant, algae, cyanobacterium, green-sulfur bacterium,
green non-sulfur bacterium, purple sulfur bacterium, purple
non-sulfur bacterium, extremophile, yeast, fungus, an engineered
organism thereof, or a synthetic organism. In some embodiments, the
host cell is light-dependent or fixes carbon. In some embodiments,
the host cell has autotrophic activity. In some embodiments, the
host cell has photoautotrophic activity, such as in the presence of
light. In some embodiments, the host cell is heterotrophic or
mixotrophic in the absence of light.
[0097] In certain embodiments, the host cell is a cell from
Avabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, Zea
mays, Botryococcuse braunii, Chlamydomonas reinhardtii, Dunaliela
salina, Synechococcus Sp. PCC 7002, Synechococcus Sp. PCC 7942,
Synechocystis Sp. PCC 6803, Thermosynechococcus elongates BP-1,
Chlorobium tepidum, Chlorojlexus auranticus, Chromatiumm vinosum,
Rhodospirillum rubrum, Rhodobacter capsulatus, Rhodopseudomonas
palusris, Clostridium ljungdahlii, Clostridiuthermocellum,
Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Pseudomonas jluorescens, Pantoea citrea
or Zymomonas mobilis. In certain embodiments, the host cell is a
cell from Chlorella fusca, Chlorella protothecoides, Chlorella
pyrenoidosa, Chlorella kessleri, Chlorella vulgaris, Chlorella
saccharophila, Chlorella sorokiniana, Chlorella ellipsoidea,
Prototheca stagnora, Prototheca portoricensis, Prototheca
moriformis, Prototheca wickerhamii, or Prototheca zopfii.
[0098] In some embodiments, the host cell is a bacterial cell. In
some embodiments, the host cell is a Gram-positive bacterial cell.
In some embodiments, the host cell is a Gram-negative bacterial
cell.
[0099] In certain embodiments, the host cell is an E. coli cell. In
some embodiments, the E. coli cell is a strain B, a strain C, a
strain K, or a strain W E. coli cell.
[0100] In certain embodiments of the invention, the host cell is
engineered to express (or overexpress) a transport protein.
Transport proteins can export polypeptides and organic compounds
(e.g., fatty acids or derivatives thereof) out of a host cell.
[0101] As used herein, the term "metabolically engineered" or
"metabolic engineering" involves rational pathway design and
assembly of polynucleotides corresponding to biosynthetic genes,
genes associated with operons, and control elements of such
polynucleotides, for the production of a desired metabolite such
as, for example, an oc-.beta.-ketoacyl-ACP, an oc-acyl-ACP, or an
oc-fatty acid derivative, in a recombinant cell, such as a
recombinant microbial cell as described herein. "Metabolic
engineering" can further include optimization of metabolic flux by
regulation and optimization of transcription, translation, protein
stability and protein functionality using genetic engineering and
appropriate culture conditions including the reduction of,
disruption, or knocking out of, a competing metabolic pathway that
competes with an intermediate leading to a desired pathway. A
"biosynthetic gene" can be endogenous (native) to the host cell
(i.e., a gene which is not modified from the host cell), or, can be
exogenous (heterologous) to the host cell either by virtue of being
foreign to the host cell, or by being modified by mutagenesis,
recombination, and/or association in the recombinant cell with a
exogenous (heterologous) expression control sequence. A
biosynthetic gene encodes a "biosynthetic polypeptide" or a
"biosynthetic enzyme".
[0102] The term "biosynthetic pathway", also referred to as
"metabolic pathway", refers to a set of biochemical reactions,
catalyzed by biosynthetic enzymes, which convert one chemical
species into another. As used herein, the term "fatty acid
biosynthetic pathway" (or more simply, "fatty acid pathway") refers
to a set of biochemical reactions that produces fatty acid
derivatives (e.g., fatty acids, fatty esters, fatty aldehydes,
fatty alcohols, alkanes, alkenes, ketones, and so forth). The fatty
acid pathway includes fatty acid pathway biosynthetic enzymes
(i.e., "fatty acid pathway enzymes") that can be engineered, as
described herein, to produce fatty acid derivatives, and in some
embodiments can be expressed with additional enzymes to produce
fatty acid derivatives having desired carbon chain characteristics.
For example, an "odd chain fatty acid biosynthetic pathway" (i.e.,
an "oc-FA pathway") as described herein includes enzymes sufficient
to produce oc-fatty acid derivatives.
[0103] The term "recombinant microbial cell" refers to a microbial
cell (i.e., a microorganism) that has been genetically modified
(i.e., "engineered") by the introduction of genetic material into a
"parental microbial cell" (i.e., host cell) of choice, thereby
modifying or altering the cellular physiology and biochemistry of
the parental microbial cell. Through the introduction of genetic
material, the recombinant microbial cell acquires a new or improved
property compared to that of the parental microbial cell, such as,
for example, the ability to produce a new intracellular metabolite,
or greater quantities of an existing intracellular metabolite.
Recombinant microbial cells provided herein express a plurality of
biosynthetic enzymes (e.g., fatty acid pathway enzymes, such as
oc-FA pathway enzymes) involved in pathways for the production of,
for example, an oc-acyl-ACP intermediate or an oc-fatty acid
derivative, from a suitable carbon source. The genetic material
introduced into the parental microbial cell may contain gene(s), or
parts of genes, encoding one or more of the enzymes involved in a
biosynthetic pathway (that is, biosynthetic enzymes) for the
production of an oc-fatty acid derivative, and may alternatively or
in addition include additional elements for the expression and/or
regulation of expression of genes encoding such biosynthetic
enzymes, such as promoter sequences. Accordingly, recombinant
microbial cells described herein have been genetically engineered
to express or overexpress biosynthetic enzymes involved in oc-fatty
acid (oc-FA) biosynthetic pathways as described herein.
[0104] It is understood that the terms "recombinant microbial cell"
and "recombinant microorganism" refer not only to the particular
recombinant microbial cell/microorganism, but to the progeny or
potential progeny of such a cell.
[0105] A recombinant microbial cell can, alternatively or in
addition to comprising genetic material introduced into the
parental microbial cell, include a reduction, disruption, deletion
or a "knocking-out" of a gene or polynucleotide to alter the
cellular physiology and biochemistry of the parental microbial
cell. Through the reduction, disruption, deletion or knocking-out
of a gene or polynucleotide (also known as "attenuation" of the
gene or polynucleotide), the recombinant microbial cell acquires a
new or improved property (such as, for example, the ability to
produce a new or greater quantities of an intracellular metabolite,
the ability to improve the flux of a metabolite through a desired
pathway, and/or the ability to reduce the production of an
undesirable by-product) compared to that of the parental microbial
cell.
Engineering Recombinant Microbial Cells to Produce Odd Chain Fatty
Acid Derivatives
[0106] Many microbial cells normally produce straight chain fatty
acids in which the linear aliphatic chains predominantly contain an
even number of carbon atoms, and generally produce relatively low
amounts of fatty acids having linear aliphatic chains containing an
odd number of carbon atoms. The relatively low amounts of linear
odd chain fatty acids (oc-FAs) and other linear odd chain fatty
acid derivatives (oc-FA derivatives) produced by such microbial
cells, such as E. coli, can in some instances be attributed to low
levels of propionyl-CoA present in such cells. Such cells
predominantly utilize acetyl-CoA as the primer molecule for fatty
acid biosynthesis, leading to the majority of fatty acids and other
fatty acid derivatives produced in such cells being linear even
chain fatty acids (ec-FAs) and other linear even chain fatty acid
derivatives (ec-FA derivatives).
[0107] The invention is based in part on the discovery that by
engineering a microorganism to produce an increased amount of
propionyl-CoA compared to that produced by a parental
microorganism, the engineered microorganism produces a greater
amount (titer) of oc-FA derivatives compared to the amount of oc-FA
derivatives produced by the parental microorganism, and/or produces
a fatty acid derivative composition having a higher proportion of
oc-FA derivatives compared to the proportion of oc-FA derivatives
in the fatty acid derivative composition produced by the parental
microorganism.
[0108] As the ultimate goal is to provide environmentally
responsible and cost-effective methods for the production of fatty
acid derivatives, including oc-FA derivatives, on an industrial
scale starting from a carbon source (such as, for example,
carbohydrate or biomass), improvements in yield of microbially
produced oc-FA derivative molecules and/or optimization of the
composition of microbially produced fatty acid derivative molecules
(such as by increasing the proportion of odd chain product relative
to even chain product) is desirable. Accordingly, strategies for
the overproduction of various pathway intermediates have been
examined to increase metabolic flux through pathways leading to odd
chain fatty acid production. Pathways that direct metabolic flux
from a starting material, such as a sugar, to propionyl-CoA,
through an odd chain acyl-ACP (oc-acyl-ACP) intermediate, to an
oc-FA derivative product, can be engineered in an industrially
useful microorganism.
[0109] In one aspect, the invention includes a recombinant
microbial cell comprising one or more polynucleotides encoding
polypeptides (e.g., enzymes) having enzymatic activities which
participate in the biosynthesis of propionyl-CoA, and/or
participate in the biosynthesis of an oc-acyl-ACP intermediate,
when the recombinant microbial cell is cultured in the presence of
a carbon source under conditions effective to expresses the
polynucleotides. In some embodiments, the recombinant microbial
cell further comprises one or more polynucleotides each encoding a
polypeptide having fatty acid derivative enzyme activity, wherein
the recombinant microbial cell produces an odd chain fatty acid
derivative when cultured in the presence of a carbon source under
conditions sufficient to expresses the polynucleotides. The
invention also includes methods of making compositions comprising
odd chain fatty acid derivatives, comprising culturing a
recombinant microbial cell of the invention. The invention also
includes methods of increasing the amount of propionyl-CoA produced
by a microbial cell, and methods of increasing the amount or
proportion of odd chain fatty acid derivatives produced by a
microbial cell, and other features apparent upon further
review.
[0110] The recombinant microbial cell can be a filamentous fungi,
an algae, a yeast, or a prokaryote such as a bacterium (e.g., an E.
coli or a Bacillus sp).
[0111] In general, odd chain fatty acid derivatives (such as, odd
chain fatty acids, odd chain fatty esters (including odd chain
fatty acid methyl esters (oc-FAMEs), odd chain fatty acid ethyl
esters (oc-FAEEs), and odd chain wax esters), odd chain fatty
aldehydes, odd chain fatty alcohols, and, due to decarbonylation or
decarboxylation of an odd chain precursor, even chain hydrocarbons
such as even chain alkanes, even chain alkenes, even chain terminal
olefins, even chain internal olefins, and even chain ketones) can
be produced in a recombinant microbial cell of the invention via
the odd chain fatty acid biosynthetic pathway ("oc-FA pathway")
depicted in FIG. 1B.
[0112] To produce an odd chain fatty acid derivative, the
recombinant microbial cell utilizes propionyl-CoA as a "primer" for
the initiation of the fatty acyl chain elongation process. As shown
in FIG. 1B, the fatty acyl elongation process initially involves
condensation of the odd chain length primer molecule propionyl-CoA
with a malonyl-ACP molecule, catalyzed by an enzyme having
.beta.-ketoacyl ACP synthase activity (such as, a .beta.-ketoacyl
ACP synthase III enzyme), to form an initial odd chain
.beta.-ketoacyl-ACP intermediate (e.g., 3-oxovaleryl-ACP), as
depicted in step (D) of FIG. 1B. The odd chain .beta.-ketoacyl-ACP
intermediate undergoes keto-reduction, dehydration and
enoyl-reduction at the .beta.-carbon via the fatty acid synthase
(FAS) complex to form an initial odd chain acyl-ACP intermediate,
which undergoes further cycles of condensation with malonyl-ACP,
keto-reduction, dehydration, and enoyl-reduction, adding two carbon
units per cycle to form acyl-ACP intermediates of increasing
odd-numbered carbon chain lengths ("oc-acyl-ACP") as depicted in
step (E) of FIG. 1B. The oc-acyl-ACP intermediate reacts with one
or more fatty acid derivative enzymes, as depicted in step (F) of
FIG. 1B, resulting in an odd chain fatty acid derivative (oc-FA
derivative) product. This is in contrast to the process in a cell
that produces relatively low levels of propionyl-CoA (such as, for
example, a wild-type E. coli cell). Such a cell produces
predominantly straight-chain fatty acids having an even number of
carbon atoms, and low or trace amounts of straight-chain fatty
acids having an odd number of carbon atoms. As depicted in FIG. 1A,
the even chain length primer molecule acetyl-CoA initially
condenses with a malonyl-ACP molecule to form an even chain
.beta.-keto acyl-ACP intermediate (e.g., acetoacetyl-ACP), as
depicted in step (D) of FIG. 1A, which likewise undergoes
FAS-catalyzed cycles of keto-reduction, dehydration,
enoyl-reduction and condensation with additional malonyl-ACP
molecules, likewise adding two carbon units per cycle, this time to
form acyl-ACP intermediates of increasing even-numbered carbon
chain lengths ("ec-acyl-ACP") as depicted in step (E) of FIG. 1A.
The ec-acyl-ACP intermediate reacts with one or more fatty acid
derivative enzymes, as depicted in step (F) of FIG. 1A, resulting
in an even chain fatty acid derivative.
[0113] The propionyl-CoA "primer" molecule can be supplied to the
oc-FA biosynthetic pathway of the recombinant microbial cell of the
invention by a number of methods. Methods to increase the
production of propionyl-CoA in a microbial cell include, but are
not limited to, the following:
[0114] Propionyl-CoA can be generated by the native biosynthetic
machinery of the parental microbial cell (e.g., by enzymes
endogenous to the parental microbial cell). If increasing the
amount of propionyl-CoA produced in the parental microbial cell is
desired, one or more enzymes endogenous to the parental microbial
cell which contribute to the production of propionyl-CoA can be
overexpressed in the recombinant microbial cell.
[0115] Propionyl-CoA can be generated by engineering the cell to
overexpress endogenous enzymes and/or express exogenous enzymes
which divert metabolic flux through the intermediate
.alpha.-ketobutyrate, as shown in FIG. 2. Non-limiting examples of
enzymes for use in engineering such pathways are provided in Tables
1 and 2, below.
[0116] Propionyl-CoA can be generated by engineering the cell to
overexpress endogenous enzymes and/or express exogenous enzymes
which divert metabolic flux from succinyl-CoA through the
intermediate methylmalonyl-CoA, as shown FIG. 3. Non-limiting
examples of enzymes for use in engineering such pathways are
provided in Table 3, below.
[0117] In an exemplary approach, propionyl-CoA can be generated by
engineering the cell to overexpress endogenous enzymes and/or
express exogenous enzymes which divert metabolic flux from
malonyl-CoA through the intermediates malonate semialdehyde and
3-hydroxypropionate. Non-limiting examples of enzymes for use in
engineering such pathways are provided, for example, in United
States Patent Application Publication Number US20110201068A1.
[0118] In another approach, propionyl-CoA can be generated by
engineering the cell to overexpress endogenous enzymes and/or
express exogenous enzymes which divert metabolic flux from
D-lactate through the intermediates lactoyl-CoA and acryloyl-CoA.
Non-limiting examples of enzymes for use in engineering such
pathways are provided, for example, in United States Patent
Application Publication Number U520110201068A1.
[0119] As noted above, initiation of the odd chain elongation
process involves condensation of propionyl-CoA with a malonyl-ACP
molecule to form an oc-.beta.-ketoacyl-ACP intermediate. This step,
as represented by part (D) of FIG. 1B, is catalyzed in the
recombinant microbial cell by an enzyme having .beta.-ketoacyl-ACP
synthase activity, preferably .beta.-ketoacyl-ACP synthase III
activity (e.g., EC 2.3.1.180) which utilizes propionyl-CoA as a
substrate. The enzyme can be endogenous to the recombinant
microbial cell, or can exogenous to the recombinant microbial
cell.
[0120] In one embodiment, a polynucleotide encoding a polypeptide
endogenous to the parental microbial cell having
.beta.-ketoacyl-ACP synthase activity that utilizes propionyl-CoA
as a substrate is expressed or is overexpressed in the recombinant
microbial cell. In another embodiment, a polynucleotide encoding a
polypeptide having .beta.-ketoacyl-ACP synthase activity that
utilizes propionyl-CoA as a substrate which is exogenous to the
parental microbial cell is expressed in the recombinant microbial
cell.
[0121] The oc-.beta.-ketoacyl-ACP intermediate generated in step
(D) of the oc-FA pathway (FIG. 1B) can undergo elongation by
successive cycles of keto-reduction, dehydration and
enoyl-reduction at the beta carbon and further condensation with
malonyl-ACP molecules catalyzed by a fatty acid synthase (FAS)
complex, such as for example a Type II FAS complex, adding 2-carbon
units to the lengthening odd-carbon chain of the oc-acyl-ACP
intermediate as represented by step (E) of FIG. 1B. In one
embodiment, an endogenous FAS complex native to the recombinant
microbial cell catalyzes cycles of condensation with
malonyl-ACP/keto-reduction/dehydration/enoyl-reduction to produce
the oc-acyl-ACP intermediate.
[0122] Odd chain fatty acid derivatives (such as oc-fatty acids,
oc-fatty esters, oc-fatty aldehydes, oc-fatty alcohols, ec-ketones,
and ec-hydrocarbons) can be produced from the oc-acyl-ACP
intermediate, as will be described in more detail below.
Accordingly, in some embodiments, the recombinant microbial cell
further comprises one or more polynucleotide sequences each
encoding a polypeptide having fatty acid derivative enzyme
activity, such as thioesterase (e.g., TesA), decarboxylase,
carboxylic acid reductase (CAR; e.g., CarA, CarB, or FadD9),
alcohol dehydrogenase/aldehyde reductase; aldehyde decarbonylase
(ADC), fatty alcohol forming acyl-CoA reductase (FAR), acyl ACP
reductase (AAR), ester synthase, acyl-CoA reductase (ACR1), OleA,
OleCD, or OleBCD, wherein the microbial cell produces a composition
comprising an oc-fatty acid, an oc-fatty ester (such as an oc-fatty
acid methyl ester, an oc-fatty acid ethyl ester, an oc-wax ester),
an oc-fatty aldehyde, an oc-fatty alcohol, an ec-ketone, or an
ec-hydrocarbon (such as an ec-alkane, an ec-alkene, an ec-terminal
olefin, or an ec-internal olefin), when the recombinant microbial
cell is cultured in the presence of a carbon source under
conditions effective to expresses the polynucleotides. The
invention also includes methods for the production of an oc-fatty
acid derivative comprising culturing a recombinant microbial cell
of the invention.
Engineering Microbial Cells to Produce Increased Amounts of
Propionyl-CoA
[0123] In one aspect, the invention includes a method of increasing
the amount of odd chain fatty acid derivatives produced by a
microbial cell, which comprises engineering a parental microbial
cell to produce an increased amount of propionyl-CoA. Engineering
the parental microbial cell to produce an increased amount of
propionyl-CoA can be accomplished, for example, by engineering the
cell to express polynucleotides encoding: (a) polypeptides having
aspartokinase activity, homoserine dehydrogenase activity,
homoserine kinase activity, threonine synthase activity, and
threonine deaminase activity; (b) polypeptides having
(R)-citramalate synthase activity, isopropylmalate isomerase
activity, and beta-isopropylmalate dehydrogenase activity; or (c) a
polypeptide having methylmalonyl-CoA mutase activity and one or
more polypeptides having methylmalonyl-CoA decarboxylase activity
and methylmalonyl carboxyltransferase activity, and optionally a
polypeptide having methylmalonyl epimerase activity; wherein at
least one polypeptide is exogenous to the recombinant microbial
cell, or expression of at least one polynucleotide is modulated in
the recombinant microbial cell as compared to the expression of the
polynucleotide in the parental microbial cell, and wherein the
recombinant microbial cell produces a greater amount of
propionyl-CoA when cultured in the presence of a carbon source
under conditions effective to express the polynucleotides, relative
to the amount of propionyl-CoA produced by the parental microbial
cell cultured under the same conditions.
[0124] In some embodiments, at least one polypeptide encoded by a
polynucleotide according to (a) is an exogenous polypeptide (for
example, a polypeptide originating from an organism other than the
parental microbial cell, or, a variant of a polypeptide native to
the parental microbial cell). In some instances, at least one
polypeptide encoded by a polynucleotide according to (a) is an
endogenous polypeptide (that is, a polypeptide native to the
parental microbial cell), and the endogenous polypeptide is
overexpressed in the recombinant microbial cell.
[0125] In some embodiments, at least one polypeptide encoded by a
polynucleotide according to (b) is an exogenous polypeptide. In
some instances, at least one polypeptide encoded by a
polynucleotide according to (b) is an endogenous polypeptide, and
the endogenous polypeptide is overexpressed in the recombinant
microbial cell.
[0126] In some embodiments, the recombinant microbial cell
comprises one or more polynucleotide according to (a) and one or
more polynucleotide according to (b). In some instances, at least
one polypeptide encoded by a polynucleotide according to (a) or (b)
is an exogenous polypeptide. In some instances, at least one
polypeptide encoded by a polynucleotide according to (a) or (b) is
an endogenous polypeptide, and the endogenous polypeptide is
overexpressed in the recombinant microbial cell.
[0127] In some embodiments, at least one polypeptide encoded by a
polynucleotide according to (c) is an exogenous polypeptide. In
some instances, at least one polypeptide encoded by a
polynucleotide according to (c) is an endogenous polypeptide, and
the endogenous polypeptide is overexpressed in the recombinant
microbial cell.
[0128] By engineering a parental microbial cell to obtain a
recombinant microbial cell that has increased metabolic flux
through propionyl-CoA compared to the parental (e.g.,
non-engineered) microbial cell, the engineered microbial cell
produces a greater amount (titer) of oc-FA derivative compared to
the amount of oc-FA derivative produced by the parental microbial
cell, and/or produces a fatty acid derivative composition having a
higher proportion of oc-FA derivative compared to the proportion of
oc-FA derivative in the fatty acid derivative composition produced
by the parental microbial cell.
[0129] Accordingly, in another aspect, the invention includes a
method of increasing the amount or proportion of odd chain fatty
acid derivatives produced by a microbial cell, the method
comprising engineering a parental microbial cell to obtain a
recombinant microbial cell which produces a greater amount, or is
capable of producing a greater amount, of propionyl-CoA relative to
the amount of propionyl-CoA produced by the parental microbial cell
cultured under the same conditions, wherein, when the recombinant
microbial cell and the parental microbial cell are each cultured in
the presence of a carbon source under identical conditions
effective to increase the level of propionyl-CoA in the recombinant
microbial cell relative to the parental microbial cell, the culture
of the recombinant microbial cell produces a greater amount or a
greater proportion of odd chain fatty acid derivatives relative to
the amount or proportion of odd chain fatty acid derivatives
produced by the parental microbial cell. In some embodiments, the
recombinant microbial cell comprises polynucleotides encoding
polypeptides according to one or more of pathways (a), (b), and
(c), as described in more detail below, wherein at least one
encoded polypeptide is exogenous to the recombinant microbial cell,
or wherein expression of at least one polynucleotide is modulated
in the recombinant microbial cell as compared to the expression of
the polynucleotide in the parental microbial cell. In some
embodiments, the recombinant microbial cell comprises at least one
polynucleotide encoding a polypeptide having fatty acid derivative
enzyme activity. In some embodiments, the recombinant microbial
cell comprises a polynucleotide encoding a polypeptide having
.beta.-ketoacyl-ACP synthase activity that utilizes propionyl-CoA
as a substrate.
[0130] Exemplary metabolic pathways useful for increasing
propionyl-CoA production in a recombinant microbial cell are
described below. It is to be understood that these exemplary
pathways for increasing propionyl-CoA production in a recombinant
cell are not intended to limit the scope of the invention; any
suitable metabolic pathway that increases propionyl-CoA production
in the cell and/or increases metabolic flux in the cell through the
propionyl-CoA intermediate is suitable for use in recombinant
microbial cells, compositions, and methods of the invention.
Metabolic pathways which increase propionyl-CoA production and/or
increase metabolic flux through the propionyl-CoA intermediate are
therefore suitable for use in recombinant microbial cells,
compositions, and methods of the invention.
Production of Propionyl-CoA Via an .alpha.-Ketobutyrate
Intermediate
[0131] Manipulation of various amino acid biosynthetic pathways has
been shown to increase the production of those various amino acids
in microbial cells (Guillouet S., et al., Appl. Environ. Microbiol.
65:3100-3107 (1999); Lee K. H., et al., Mol. Syst. Biol. 3:149
(2007)). Amino acid biosynthetic pathways have been used in the
production of short chain branched alcohols in E. coli (Atsumi S,
and Liao J. C., Appl. Environ. Microbiol. 74(24): 7802-7808 (2008);
Cann A. F. and Liao J. C., Appl Microbiol Biotechnol. 81(1):89-98
(2008); Zhang K., et al., Proc. Natl. Acad. Sci. USA.
105(52):20653-20658 (2008)).
[0132] Directing the flux of certain amino acid biosynthetic
metabolites to the production of the intermediate
.alpha.-ketobutyrate (also known as alpha-ketobutyrate,
2-ketobutyrate, 2-ketobutanoate, 2-oxobutyrate and 2-oxobutanoate)
results in increased propionyl-CoA production. Accordingly, in one
embodiment, the invention includes a recombinant microbial cell
comprising polynucleotides encoding one or more enzymes (i.e.,
"oc-FA pathway enzymes") which participate in the conversion of a
carbon source (for example, a carbohydrate, such as a sugar) to
.alpha.-ketobutyrate when the recombinant microbial cell is
cultured in the presence of the carbon source under conditions
sufficient to expresses the polynucleotides. The
.alpha.-ketobutyrate molecule is an intermediate in the microbial
production of propionyl-CoA which serves as a primer in the
production of linear odd chain fatty acid derivatives according to
the oc-FA pathway (FIG. 1B).
[0133] Pyruvate dehydrogenase complex (PDC) catalyzes the oxidative
decarboxylation of .alpha.-ketobutyrate to produce propionyl-CoA in
bacteria (Danchin, A. et al., Mol. Gen. Genet. 193: 473-478 (1984);
Bisswanger, H., J. Biol. Chem. 256:815-822 (1981)). The pyruvate
dehydrogenase complex is a multienzyme complex that contains three
activities: a pyruvate decarboxylase (E1), a dihydrolipoyl
transacetylase (E2), and a dihydrolipoyl dehydrogenase (E3). Other
suitable ketoacid dehydrogenase complexes exist that use a similar
catalytic scheme employing .alpha.-ketoacid substrates other than
pyruvate. The TCA cycle .alpha.-ketoglutarate dehydrogenase complex
is an example. In one embodiment, the pyruvate dehydrogenase
complex endogenous to the host cell (i.e., the pyruvate
dehydrogenase complex native to the parental cell) is utilized to
catalyze the conversion of .alpha.-ketobutyrate to propionyl-CoA.
In other embodiments, genes encoding one or more PDC complex
polypeptides having pyruvate decarboxylase, dihydrolipoyl
transacetylase, and/or dihydrolipoyl dehydrogenase activity are
overexpressed in the recombinant microbial cell. Other enzymes or
enzyme complexes which catalyze the conversion of
.alpha.-ketobutyrate to propionyl-CoA can be expressed or
overexpressed in the recombinant microbial cell to further increase
metabolic flux from .alpha.-ketobutyrate to propionyl-CoA.
[0134] Conversion of .alpha.-ketobutyrate to propionyl-CoA can also
be accomplished by conversion of .alpha.-ketobutyrate to propionate
and activation of propionate to propionyl-CoA. Conversion of
.alpha.-ketobutyrate to propionate can be catalyzed by pyruvate
oxidase (E.C. 1.2.3.3), such as E. coli pyruvate oxidase encoded by
the poxB gene (Grabau and Cronan, Nucleic Acids Res. 14(13):
5449-5460 (1986)). The native E. coli PoxB enzyme reacts with
.alpha.-ketobutyrate and with pyruvate, with a preference for
pyruvate; however, Chang and Cronan (Biochem J. 352:717-724 (2000))
described PoxB mutant enzymes which retained full activity towards
.alpha.-ketobutyrate and reduced activity towards pyruvate.
Activation of propionate to propionyl-CoA can be catalyzed by an
acyl-CoA synthase, such as Acetyl-CoA synthetase (Doi et al., J.
Chem. Soc. 23: 1696 (1986)). Yeast acetyl-CoA synthetase has been
shown to catalyze the activation of propionate to propionyl-CoA
(Patel and Walt, J. Biol. Chem. 262: 7132 (1987)). Propionate can
also be activated to propionyl-CoA by the actions of acetate kinase
(ackA) and phosphotransacetylase (pta).
[0135] One or more enzymes endogenous to the parental microbial
cell may compete for substrate with enzymes of the engineered oc-FA
biosynthetic pathway in the recombinant microbial cell, or may
break down or otherwise divert an intermediate (such as,
.alpha.-ketobutyrate) away from the oc-FA biosynthetic pathway;
genes encoding such undesired endogenous enzymes may be attenuated
to increase the production of odd chain fatty acid derivatives by
the recombinant microbial cell. For example, in E. coli, endogenous
acetohydroxyacid synthase (AHAS) complexes, such as AHAS I (e.g.,
encoded by ilvBN genes), AHAS II (e.g., encoded by ilvGM genes) and
AHAS III (e.g., encoded by ilvIH genes), catalyze the conversion of
.alpha.-ketobutyrate to oc-aceto-.alpha.-hydroxybutyrate and may
thus divert metabolic flux away from propionyl-CoA and reduce oc-FA
production. Deleting or otherwise reducing the expression of one or
more endogenous AHAS genes may thus direct biosynthesis in the
recombinant microbial cell more towards propionyl-CoA and
ultimately more towards odd chain fatty acid production. Other
endogenous enzymes which may compete with oc-FA biosynthetic
pathway enzymes include enzymes with acetohydroxyacid
isomeroreductase activity (e.g., encoded by an ilvC gene) which
catalyzes the conversion of oc-aceto-.alpha.-hydroxybutyrate to
2,3-dihydroxy-3-methylvalerate, and dihydroxy acid dehydratase
activity (e.g., encoded by an ilvD gene), which catalyzes the
conversion of 2,3-dihydroxy-3-methylvalerate to
2-keto-3-methylvalerate; deleting or otherwise reducing the
expression of one or more of these genes may direct biosynthesis in
the recombinant microbial cell more towards propionyl-CoA and
ultimately more towards odd chain fatty acid production.
[0136] Either or both of the following exemplary pathways can be
engineered in the recombinant microbial cell to increase metabolic
flux through the common .alpha.-ketobutyrate intermediate resulting
in increased propionyl-CoA production in the cell. These exemplary
pathways are shown in FIG. 2 and are described in more detail
below.
Pathway A (Threonine Intermediate)
[0137] The first pathway leading to the common .alpha.-ketobutyrate
intermediate, as represented by pathway (A) of FIG. 2, involves
production of the intermediate threonine by threonine biosynthetic
enzymes, followed by the deamination of threonine to
.alpha.-ketobutyrate catalyzed by an enzyme with threonine
dehydratase activity.
[0138] In pathway (A), increasing metabolic flux to threonine can
be accomplished by expressing polynucleotides encoding enzymes
involved in threonine biosynthesis, including enzymes with
aspartate kinase activity (e.g., EC 2.7.2.4; also termed
aspartokinase activity), which catalyzes the conversion of
aspartate to aspartyl phosphate; aspartate-semialdehyde
dehydrogenase activity (e.g., EC 1.2.1.11), which catalyzes the
conversion of aspartyl phosphate to aspartate semialdehyde;
homoserine dehydrogenase activity (e.g., EC 1.1.1.3), which
catalyzes the conversion of aspartate semialdehyde to homoserine;
homoserine kinase activity (e.g., EC 2.7.1.39), which catalyzes the
conversion of homoserine to O-phospho-L-homoserine; and threonine
synthase activity (e.g., EC 4.2.3.1), which catalyzes the
conversion of O-phospho-L-homoserine to threonine. Not all of the
activities listed above need be engineered in the recombinant
microbial cell to increase metabolic flux through the threonine
intermediate; in some instances, an activity already present in the
parental microbial cell (for example, a polypeptide having that
activity which is produced by a native gene in the parental
microbial cell) will be sufficient to catalyze a step listed above.
In one embodiment, the recombinant microbial cell is engineered to
recombinantly express one or more polynucleotides selected from: a
polynucleotide encoding a polypeptide having aspartate kinase
activity, wherein the polypeptide catalyzes the conversion of
aspartate to aspartyl phosphate; a polynucleotide encoding a
polypeptide having aspartate-semialdehyde dehydrogenase activity,
wherein the polypeptide catalyzes the conversion of aspartyl
phosphate to aspartate semialdehyde; a polynucleotide encoding a
polypeptide having homoserine dehydrogenase activity, wherein the
polypeptide catalyzes the conversion of aspartate semialdehyde to
homoserine; a polynucleotide encoding a polypeptide having
homoserine kinase activity, wherein the polypeptide catalyzes the
conversion of homoserine to O-phospho-L-homoserine; a
polynucleotide encoding a polypeptide having threonine synthase
activity, wherein the polypeptide catalyzes the conversion of
O-phospho-L-homoserine to threonine; wherein the recombinant
microbial cell has increased metabolic flux through the pathway
intermediate threonine compared to the parental microbial cell. In
some instances, the polypeptide encoded by recombinantly expressed
polynucleotide is present in the recombinant microbial cell at a
greater concentration compared to its concentration in the parent
microbial cell when cultured under the same conditions, i.e., the
polypeptide is "overexpressed" in the recombinant cell. For
example, the recombinantly expressed polynucleotide can be
operatively linked to a promoter which expresses the polynucleotide
in the recombinant microbial cell at a greater concentration than
is normally expressed in the parental microbial cell when cultured
under the same conditions. In one embodiment, an E. coli thrA gene
is used, which encodes a bifunctional ThrA with aspartate kinase
and homoserine dehydrogenase activities. In another embodiment, a
mutant E. coli thrA gene is used, encoding a variant enzyme with
aspartate kinase and homoserine dehydrogenase activities and with
reduced feedback inhibition relative to the parent ThrA enzyme
(designated ThrA*; Ogawa-Miyata, Y., et al., Biosci. Biotechnol.
Biochem. 65:1149-1154 (2001); Lee J.-H., et al., J. Bacteriol. 185:
5442-5451 (2003)).
[0139] Threonine can be deaminated to .alpha.-ketobutyrate by an
enzyme with threonine deaminase activity (e.g., EC 4.3.1.19; also
known as threonine ammonia-lyase activity, and was previously
classified as EC 4.2.1.16, threonine dehydratase). In one
embodiment, threonine deaminase activity which is already present
in (i.e., is endogenous to) the parental microbial cell is
sufficient to catalyze the conversion of threonine to
.alpha.-ketobutyrate. In another embodiment, the recombinant
microbial cell is engineered to recombinantly express a polypeptide
having threonine deaminase activity, wherein the polypeptide
catalyzes the conversion of threonine to .alpha.-ketobutyrate. In
some embodiments, the polypeptide having threonine deaminase
activity is overexpressed in the recombinant microbial cell.
[0140] Non-limiting examples of enzymes and polynucleotides
encoding such enzymes for use in engineering pathway (A) are
provided in Table 1.
TABLE-US-00001 TABLE 1 Non-limiting examples of enzymes and nucleic
acid coding sequences for use in pathway A of the oc-FA
biosynthetic pathway shown in FIG. 2 UniProtKB (SwissProt) NCBI
Protein SEQ Accession Number, or Accession ID EC Number Organism
Gene symbol literature reference Number NO EC 2.7.2.4 aspartate
kinase (aspartokinase) E. coli K-12 thrA P00561 NP_414543 20 MG1655
E. coli (mutant) thrA* Ogawa-Miyata et al, 21 2001; Lee et al, 2003
B. subtilis 168 dapG Q04795 ZP_03591402 22 P. putida F1 Pput1442
A5W0E0 YP_001266784 23 S. cerevisiae hom3 NP_010972 24 EC 1.1.1.3
homoserine dehydrogenase E. coli K12 thrA P00561 NP_414543 20
MG1655 E. coli (mutant) thrA* Ogawa-Miyata et al, 21 2001; Lee et
al, 2003 B. subtilis 168 hom P19582 NP_391106 25 P. putida F1
Pput_4251 A5W8B5 YP_001269559 26 S. cerevisiae hom6 P31116
NP_012673 27 EC 2.7.1.39 homoserine kinase E. coli K12 thrB P00547
NP_414544 28 MG1655 B. subtilis 168 thrB P04948 NP_391104 29 P.
putida F1 Pput_0138 A5VWQ3 YP_001265497 30 S. cerevisiae thr1
P17423 NP_011890 31 EC 4.2.3.1 threonine synthase E. coli K12 thrC
P00934 NP_414545 32 MG1655 B. subtilis 168 thrC P04990 NP_391105 33
C. glutamicum thrC P23669 YP_226461 34 ATCC 13032 EC 4.3.1.19
threonine deaminase (threonine ammonia-lyase; previously termed
threonine dehydratase) E. coli K12 tdcB P0AGF6 NP_417587 35 MG1655
E. coli K12 ilvA P04968 NP_418220 36 MG1655 B. subtilis 168 ilvA
P37946 NP_390060 37 C. glutamicum ilvA Q04513 YP_226365 38 ATCC
13032 C. glutamicum tdcB Q8NRR7 YP_225271 39 ATCC 13032
[0141] Additional polypeptides can be identified, for example, by
searching a relevant database (such as the KEGG database
(University of Tokyo), the PROTEIN or the GENE databases (Entrez
databases; NCBI), the UNIPROTKB or ENZYME databases (ExPASy; Swiss
Institute of Bioinformatics), and the BRENDA database (The
Comprehensive Enzyme Information System; Technical University of
Braunschweig)), all which are available on the World Wide Web, for
polypeptides categorized by the above noted EC numbers. For
example, additional aspartokinase polypeptides can be identified by
searching for polypeptides categorized under EC 2.7.2.4; additional
homoserine dehydrogenase polypeptides can be identified by
searching for polypeptides categorized under EC 1.1.1.3; additional
homoserine kinase polypeptides can be identified by searching for
polypeptides categorized under EC 2.7.1.39; additional threonine
synthase polypeptides can be identified by searching for
polypeptides categorized under EC 4.2.3.1; and additional threonine
deaminase polypeptides can be identified by searching for
polypeptides categorized under EC 4.3.1.19.
[0142] In some embodiments, a polynucleotide encoding a parent
fatty acid pathway polypeptide (such as a polypeptide described in
Table 1 or identified by EC number or by homology to an exemplary
polypeptide) is modified using methods well known in the art to
generate a variant polypeptide having an enzymatic activity noted
above (e.g., aspartokinase activity, homoserine dehydrogenase
activity, homoserine kinase activity, threonine synthase activity,
threonine deaminase activity) and an improved property, compared to
that of the parent polypeptide, which is more suited to the
microbial cell and/or to the pathway being engineered; such as, for
example, increased catalytic activity or improved stability under
conditions in which the recombinant microbial cell is cultured;
reduced inhibition (e.g., reduced feedback inhibition) by a
cellular metabolite or by a culture media component, and the
like.
Pathway B (Citramalate Intermediate)
[0143] The second pathway leading to the common
.alpha.-ketobutyrate intermediate, as represented by pathway (B) of
FIG. 2, involves the production of the intermediate citramalate
(which is also known as 2-methylmalate) via an enzyme with
citramalate synthase activity, and the conversion of citramalate to
.alpha.-ketobutyrate by the action of enzymes with isopropylmalate
isomerase and alcohol dehydrogenase activities.
[0144] Citramalate synthase activity (e.g., EC 2.3.1.182), which
catalyzes the reaction of acetyl-CoA and pyruvate to form
(R)-citramalate, can be supplied by expression of a cimA gene from
a bacterium such as Methanococcus jannaschi or Leptospira
interrogans (Howell, D. M. et al., J. Bacteriol. 181(1):331-3
(1999); Xu, H., et al., J. Bacteriol. 186:5400-5409 (2004)) which
encodes a CimA polypeptide such as CimA from M. jannaschii (SEQ ID
NO: 40) or L. interrogans (SEQ ID NO:42). Alternatively, a modified
cimA nucleic acid sequence encoding a CimA variant with improved
catalytic activity or stability in the recombinant microbial cell
and/or reduced feedback inhibition can be used, such as, for
example, a CimA variant described by Atsumi S, and Liao J. C.
(Appl. Environ. Microbiol. 74(24): 7802-7808 (2008)), preferably
the CimA3.7 variant (SEQ ID NO:41) encoded by the cimA3.7 gene.
Alternatively, a Leptospira interrogans CimA variant (SEQ ID NO:43)
can be used. Isopropylmalate isomerase activity (EC 4.2.1.33; also
termed isopropylmalate dehydratase), which catalyzes the conversion
of (R)-citramalate first to citraconate and then to
beta-methyl-D-malate, can be provided, for example, by expression
of a heterodimeric protein encoded by E. coli or B. subtilis leuCD
genes. Alcohol dehydrogenase activity (EC 1.1.1.85; beta-isopropyl
malate dehydrogenase), which catalyzes the conversion of
beta-methyl-D-malate to 2-ketobutyrate (i.e., .alpha.-ketobutyrate)
can be provided, for example, by expression of an E. coli or B.
subtilis leuB gene or a yeast leu2 gene. Non-limiting examples of
fatty acid pathway enzymes and polynucleotides encoding such
enzymes for use in engineering pathway (B) of the oc-FA pathway are
provided in Table 2.
TABLE-US-00002 TABLE 2 Non-limiting examples of enzymes and nucleic
acid coding sequences for use in pathway (B) of the oc-FA
biosynthetic pathway shown in FIG. 2. UniProtKB (Swiss-Prot)
Protein Accession NCBI Protein SEQ Gene Number, or Accession ID EC
number Organism symbol literature reference Number NO EC 2.3.1.182
(R)-citramalate synthase M. jannaschii cimA Q58787 NP_248395 40
M.jannaschii cimA 3.7 Atsumi and Liao (2008) 41 (mutant) Leptospira
cimA Q8F3Q1 AAN49549 42 interrogans Leptospira cimA* (this
disclosure) 43 interrogans (mutant) EC 4.2.1.33 isopropylmalate
isomerase (3-isopropylmalate dehydratase) E. coli K12 leuCD P0A6A6
(C, Lg (C) NP_414614 44 MG1655 subunit); P30126 (D) NP_414613 45
(D, Sm subunit) B. subtilis 168 leuCD P80858 (C, Lg subunit); (C)
NP_390704 46 P94568 (D, Sm subunit) (D) NP_390703 47 EC 1.1.1.85
beta-isopropylmalate dehydrogenase (3-isopropylmalate
dehydrogenase) E. coli K12 leuB P30125 NP_414615 48 MG1655 B.
subtilis leuB P05645 NP_390705.2 49 S. cerevisiae leu2 P04173
NP_009911.2 50
[0145] Additional polypeptides can be identified, for example, by
searching a relevant database (such as the KEGG database
(University of Tokyo), the PROTEIN or the GENE databases (Entrez
databases; NCBI), the UNIPROTKB or ENZYME databases (ExPASy; Swiss
Institute of Bioinformatics), and the BRENDA database (The
Comprehensive Enzyme Information System; Technical University of
Braunschweig)), all which are available on the World Wide Web, for
polypeptides categorized by the above noted EC numbers. For
example, additional (R)-citramalate synthase polypeptides can be
identified by searching for polypeptides categorized under EC
2.3.1.182; additional isopropyl malate isomerase polypeptides can
be identified by searching for polypeptides categorized under EC
4.2.1.33; and additional beta-isopropyl malate dehydrogenase
polypeptides can be identified by searching for polypeptides
categorized under EC 1.1.1.85.
[0146] In some embodiments, a polynucleotide encoding a parent
fatty acid pathway polypeptide (such as a polypeptide described in
Table 2 or identified by EC number or by homology to an exemplary
polypeptide) is modified using methods well known in the art to
generate a variant polypeptide having an enzymatic activity noted
above (e.g., (R)-citramalate synthase activity, isopropyl malate
isomerase activity, beta-isopropyl malate dehydrogenase activity)
and an improved property, compared to that of the parent
polypeptide, which is more suited to the microbial cell and/or to
the pathway being engineered; such as, for example, increased
catalytic activity or improved stability under conditions in which
the recombinant microbial cell is cultured; reduced inhibition
(e.g., reduced feedback inhibition) by a cellular metabolite or by
a culture media component, and the like.
Production of Propionyl-CoA Via Methylmalonyl-CoA
Pathway C (Methylmalonyl-CoA Intermediate)
[0147] The following exemplary pathway can be engineered in the
recombinant microbial cell to increase metabolic flux through a
methylmalonyl-CoA intermediate resulting in increased propionyl-CoA
production in the cell. This exemplary pathway is shown in FIG. 3
and is described in more detail below.
[0148] Directing metabolic flux through methylmalonyl-CoA can
result in increased propionyl-CoA production. Accordingly, in one
embodiment, the invention includes a recombinant microbial cell
comprising polynucleotides encoding which participate in the
conversion of a carbon source (for example, a carbohydrate, such as
a sugar) to succinyl-CoA and to methylmalonyl-CoA when the
recombinant microbial cell is cultured in the presence of the
carbon source under conditions sufficient to expresses the
polynucleotides. Succinyl-CoA and methylmalonyl-CoA are
intermediates in the microbial production of propionyl-CoA, which
serves as a primer in the production of linear odd chain fatty acid
derivatives according to the oc-FA pathway (FIG. 1B).
[0149] The pathway leading to propionyl-CoA as shown in FIG. 3
(also referred to herein as "pathway (C)") involves the conversion
of succinyl-CoA to methylmalonyl-CoA via an enzyme having
methylmalonyl-CoA mutase activity, and the conversion of
methylmalonyl-CoA to propionyl-CoA by the action of an enzyme
having methylmalonyl-CoA decarboxylase activity, and/or by the
action of an enzyme having methylmalonyl-CoA carboxyltransferase
activity. In some instances, depending on the stereoisomer of
methylmalonyl-CoA utilized by the particular methylmalonyl-CoA
decarboxylase or methylmalonyl-CoA carboxyltransferase employed, an
enzyme having methylmalonyl-CoA epimerase activity may be utilized
to interconvert (R)- and (S)-methylmalonyl-CoA.
[0150] Succinyl-CoA can be provided to this pathway by the cellular
TCA cycle. In some instances, flux from fumarate to succinate can
be increased by, for example, overexpressing endogenous frd
(fumurate reductase) or other gene(s) involved in production of
succinate or succinyl-CoA. The conversion of succinyl-CoA to
methylmalonyl-CoA can be catalyzed by an enzyme having
methylmalonyl-CoA mutase activity (e.g., EC 5.4.99.2). Such
activity can be supplied to the recombinant microbial cell by
expression of an exogenous scpA (also known as sbm) gene or by
overexpression of an endogenous scpA gene. An exemplary sbm gene
includes that from E. coli (Haller, T. et al., Biochemistry
39:4622-4629 (2000)) which encodes an Sbm polypeptide (Accession
NP.sub.--417392, SEQ ID NO: 51) having methylmalonyl-CoA mutase
activity. Alternatively, a methylmalonyl-CoA mutase from, for
example, Propionibacterium freundenreichii subsp. shermanii which
comprises an .alpha.-subunit or "large subunit" (MutB, Accession
YP.sub.--003687736) and a .beta.-subunit or "small subunit" (MutA,
Accession CAA33089) can be used. Non-limiting examples of
polypeptides that catalyze the conversion of succinyl-CoA to
methylmalonyl-CoA are provided in Table 3, below.
[0151] In one embodiment, conversion of methylmalonyl-CoA to
propionyl-CoA can be catalyzed by a polypeptide having
methylmalonyl-CoA decarboxylase activity (e.g., EC 4.1.1.41), which
catalyzes the decarboxylation of methylmalonyl-CoA to
propionyl-CoA. Such activity can be supplied to the recombinant
microbial cell by expression of an exogenous scpB (also known as
ygfG) gene or by overexpression of an endogenous scpB gene.
Exemplary methylmalonyl-CoA decarboxylase polypeptides include, for
example, a methylmalonyl-CoA decarboxylase polypeptide encoded by
the E. coli scpB gene (Haller et al., supra), or a
methylmalonyl-CoA decarboxylase polypeptide encoded by Salmonella
enterica or Yersinia enterocolitica. In another embodiment,
conversion of methylmalonyl-CoA to propionyl-CoA can be catalyzed
by a polypeptide having methylmalonyl-CoA carboxyltransferase
activity (e.g., EC 2.1.3.1), such as, for example, a
methylmalonyl-CoA carboxyltransferase from P. freundenreichii
subsp. shermanii (mmdA, NBCI Accession No. Q8GBW6.3). Depending on
the stereoisomer of methylmalonyl-CoA utilized by the
methylmalonyl-CoA decarboxylase or by the methylmalonyl-CoA
carboxyltransferase, conversion between (R)-methylmalonyl-CoA and
(S)-methylmalonyl-CoA may be desired, which can be catalyzed by a
polypeptide having methylmalonyl-CoA epimerase activity (e.g., EC
5.1.99.1), such as, for example, a methylmalonyl-CoA epimerase from
Bacillus subtilis (yqjC; Haller et al., Biochemistry 39:4622-4629
(2000)) or Propionibacterium freundenreichii subsp. shermanii (NCBI
Accession No. YP.sub.--003688018).
[0152] One or more enzymes endogenous to the parental microbial
cell may compete for substrate with enzymes of the engineered oc-FA
biosynthetic pathway in the recombinant microbial cell, or may
break down or otherwise divert an intermediate away from the oc-FA
biosynthetic pathway; genes encoding such undesired endogenous
enzymes may be attenuated to increase the production of odd chain
fatty acid derivatives by the recombinant microbial cell. For
example, in E. coli, the endogenous propionyl-CoA:succinyl-CoA
transferase (NCBI Accession Number NP.sub.--417395), encoded by the
E. coli scpC (also known as ygfH) gene, catalyzes the conversion of
propionyl-CoA to succinyl-CoA and may thus divert metabolic flux
away from propionyl-CoA and reduce oc-FA production. Deleting or
otherwise reducing the expression of the scpC (ygfH) gene may thus
direct biosynthesis in the recombinant microbial cell more towards
propionyl-CoA and ultimately more towards odd chain fatty acid
production.
[0153] Non-limiting examples of fatty acid pathway enzymes and
polynucleotides encoding such enzymes that catalyze the conversion
of succinyl-CoA to methylmalonyl-CoA and the conversion of
methylmalonyl-CoA to propionyl-CoA for use in engineering pathway
(C) of the oc-FA pathway are provided in Table 3.
TABLE-US-00003 TABLE 3 Non-limiting examples of enzymes and nucleic
acid coding sequences for use in pathway (C) of the oc-FA
biosynthetic pathway shown in FIG. 3. UniProtKB (Swiss- Prot)
Protein Accession Number, SEQ Gene or literature NCBI Protein ID EC
number Organism symbol reference Accession Number NO EC 5.4.99.2
Methylmalonyl-CoA mutase E. coli scpA (sbm) P27253 NP_417392 51
Salmonella SARI_04585 A9MRG0 YP_001573500 52 enterica P.
freundenreichii mutA (sm) P11652 (sm) CAA33089 53 subsp. shermanii
mutB (lg) D7GCN5 (lg) YP_003687736 54 Bacillus mutA (sm) D5DS48
(sm) YP_003564880 55 megaterium mutB (lg) D5DS47 (lg) YP_003564879
56 Corynebacterium mcmA (sm) Q8NQA8 (sm) YP_225814 57 glutamicum
mcmB (lg) Q8NQA9 (lg) YP_225813 58 EC 4.1.1.41 Methylmalonyl-CoA
decarboxylase E. coli scpB (ygfG) C6UT22 YP_001731797 59 Salmonella
SARI_04583 A9MRF8 YP_001573498 60 enterica Yersinia YE1894 A1JMG8
YP_001006155 61 enterocolitica EC 2.1.3.1 Methylmalonyl-CoA
carboxyltransferase P.freudenreichii mmdA Q8GBW6 Q8GBW6.3 62 subsp.
shermanii EC 5.1.99.1 Methylmalonyl-CoA epimerase P. freudenreichii
PFREUD_10590; D7GDH1 YP_003688018 63 subsp. shermanii mmcE
[0154] Additional polypeptides can be identified, for example, by
searching a relevant database (such as the KEGG database
(University of Tokyo), the PROTEIN or the GENE databases (Entrez
databases; NCBI), the UNIPROTKB or ENZYME databases (ExPASy; Swiss
Institute of Bioinformatics), and the BRENDA database (The
Comprehensive Enzyme Information System; Technical University of
Braunschweig)), all which are available on the World Wide Web, for
polypeptides categorized by the above noted EC numbers. For
example, additional methylmalonyl-CoA mutase polypeptides can be
identified by searching for polypeptides categorized under EC
5.4.99.2, additional methylmalonyl-CoA decarboxylase polypeptides
can be identified by searching for polypeptides categorized under
EC 4.1.1.41, additional methylmalonyl-CoA carboxyltransferase
polypeptides can be identified by searching for polypeptides
categorized under EC 2.1.3.1, and additional methylmalonyl-CoA
epimerase polypeptides can be identified by searching for
polypeptides categorized under EC 5.1.99.1.
[0155] In some embodiments, a polynucleotide encoding a parent
fatty acid pathway polypeptide (such as a polypeptide described in
Table 3 or identified by EC number or by homology to an exemplary
polypeptide) is modified using methods well known in the art to
generate a variant polypeptide having an enzymatic activity noted
above (e.g., methylmalonyl-CoA mutase activity, methylmalonyl-CoA
decarboxylase activity, methylmalonyl-CoA epimerase activity,
methylmalonyl-CoA carboxyltransferase activity) and an improved
property, compared to that of the parent polypeptide, which is more
suited to the microbial cell and/or to the pathway being
engineered; such as, for example, increased catalytic activity or
improved stability under conditions in which the recombinant
microbial cell is cultured; reduced inhibition (e.g., reduced
feedback inhibition) by a cellular metabolite or by a culture media
component, and the like.
Engineering Microbial Cells to Produce Increased Amounts of oc-FA
Derivatives Propionyl-CoA to oc-.beta.-Ketoacyl-ACP
[0156] As discussed above, propionyl-CoA serves as a primer for
subsequent FAS-catalyzed elongation steps in the production of
oc-FA derivatives. The initiation of this process involves
condensation of propionyl-CoA with a malonyl-ACP molecule to form
the oc-.beta.-ketoacyl-ACP intermediate 3-oxovaleryl-ACP (FIG. 1B).
This initiation step, as represented by step (D) of FIG. 1B, is
catalyzed in the recombinant microbial cell by an enzyme having
.beta.-ketoacyl-ACP synthase activity (such as, a Type III
.beta.-ketoacyl-ACP synthase (e.g., EC 2.3.1.180)) that utilizes
propionyl-CoA as a substrate.
[0157] The substrate specificity of a .beta.-ketoacyl-ACP synthase
from a particular microorganism often reflects the fatty acid
composition of that microorganism (Han, L., et al., J. Bacteriol.
180:4481-4486 (1998); Qui, X., et al., Protein Sci. 14:2087-2094
(2005)). For example, the E. coli FabH enzyme utilizes
propionyl-CoA and acetyl-CoA with a very strong preference for
acetyl-CoA (Choi, K. H., et al., J. Bacteriology 182:365-370
(2000); Qui, et al., supra) reflecting the high proportion of
linear even chain fatty acids produced, while the enzyme from
Streptococcus pneumoniae utilizes short straight chain acyl-CoA
primers of between two and four carbons in length as well as
various branched-chain acyl-CoA primers (Khandekar S. S., et al.,
J. Biol. Chem. 276:30024-30030 (2001)) reflecting the variety of
linear chain and branched chain fatty acids produced. A
polynucleotide sequence encoding a polypeptide having
.beta.-ketoacyl-ACP synthase activity that utilizes propionyl-CoA
as a substrate can generally be obtained from a microbial cell
containing a .beta.-ketoacyl-ACP synthase with a broad acyl-CoA
substrate specificity. Sources of broad-specificity
.beta.-ketoacyl-ACP synthases may include bacteria that produce a
variety of fatty acid structures including branched chain fatty
acids, such as, for example, Bacillus (e.g., B. subtilis), Listeria
(e.g., L. monocytogenes), Streptomyces (e.g., S. coelicolor), and
Propionibacterium (e.g., P. freudenreichii subsp. shermanii).
Particularly preferred .beta.-ketoacyl-ACP synthase enzymes include
those with a greater preference for propionyl-CoA vs. acetyl-CoA
than that exhibited by the endogenous FabH. For example, when an E.
coli cell is engineered, preferred .beta.-ketoacyl-ACP synthase
enzymes may include, but are not limited to, B. subtilis FabH1
(Choi et al. 2000, supra), Streptomyces glauscens FabH (Han, L., et
al., J. Bacteriol. 180:4481-4486 (1998)), Streptococcus pneumoniae
FabH (Khandekar S. S., et al., J. Biol. Chem. 276:30024-30030
(2001), and Staphylococcus aureus FabH (Qui, X. et al., Protein
Sci. 14:2087-2094 (2005)).
[0158] One or more endogenous enzymes may compete for substrate
with enzymes of the engineered oc-FA biosynthetic pathway in the
recombinant microbial cell, or may break down an oc-FA pathway
intermediate or may otherwise divert metabolic flux away from oc-FA
production; genes encoding such undesired endogenous enzymes may be
attenuated to increase the production of oc-FA derivatives by the
recombinant microbial cell. For example, while the endogenous
fabH-encoded .beta.-ketoacyl-ACP synthase of E. coli utilizes
propionyl-CoA as a substrate, it has a much greater preference for
the two-carbon acetyl-CoA molecule than for the three-carbon
propionyl-CoA molecule (Choi et al. 2000, supra). Cells expressing
the E. coli fabH gene thus preferentially utilize acetyl-CoA as a
primer for fatty acid synthesis and predominantly produce even
chain fatty acid molecules in vivo. Deleting or otherwise reducing
the expression of an endogenous fabH gene and expressing an
exogenous gene encoding a .beta.-ketoacyl-ACP synthase with greater
preference for propionyl-CoA than that exhibited by the endogenous
FabH (for example, when engineering E. coli, replacing the
endogenous E. coli FabH with B. subtilis FabH1 or an alternative
exogenous FabH with a greater preference for propionyl-CoA than
acetyl-CoA relative to that exhibited by E. coli FabH) may direct
metabolic flux in the recombinant microbial cell more towards an
oc-.beta.-ketoacyl-ACP intermediate and ultimately more towards
production of oc-FA derivatives.
[0159] Non-limiting examples of fatty acid pathway enzymes and
polynucleotides encoding such enzymes for use in engineering step D
of the oc-FA pathway are provided in Table 4.
TABLE-US-00004 TABLE 4 Non-limiting examples of enzymes and coding
sequences for use in step D of the oc-FA biosynthetic pathways
shown in FIG. 1B. UniProtKB (Swiss- Prot) Protein NCBI Protein SEQ
Gene Accession Number, or Accession ID EC number Organism symbol
literature reference Number NO EC 2.3.1.180 .beta.-ketoacyl-ACP
synthase III E. coli fabH P0A6R0 AAC74175 1 B. subtilis 168 fabH1
O34746 NP_389015 2 B. subtilis 168 fabH2 O07600 NP_388898 3
Streptomyces fabH Q9K3G9 CAB99151 4 coelicolor Streptomyces fabH
Q54206 AAA99447 5 glaucescens Streptomyces fabH3 Q82KT2 NP_823466 6
avermitilis MA-4680 Listeria fabH B8DFA8 YP_002349314 7
monocytogenes L. monocytogenes fabH2 (this disclosure) 8 (mutant)
Staphylococcus fabH Q8NXE2 NP_645682 9 aureus MW2 Streptococcus
fabH P0A3C5 AAK74580 10 pneumoniae Streptococcus fabH Q8DSN2
NP_722071 11 mutans UA159 Lactococcus lactis fabH Q9CHGO NP_266927
12 subsp. lactis Propionibacterium fabH D7GD58 YP_003687907 13
freundenreichii subsp. shermanii Stenotrophomonas fabH B2FR86
YP_001970902 146 maltophila Alicyclobacillus fabH C8WPY3
YP_003183476 147 acidocaldarius Desulfobulbus fabH1 E8RF72
YP_004195454 148 propionicus Desulfobulbus fabH2 E8RBR5
YP_004196088 149 propionicus
[0160] Additional .beta.-ketoacyl-ACP synthase polypeptides can be
identified, for example, by searching a relevant database (such as
the KEGG database (University of Tokyo), the PROTEIN or the GENE
databases (Entrez databases; NCBI), the UNIPROTKB or ENZYME
databases (ExPASy; Swiss Institute of Bioinformatics), and the
BRENDA database (The Comprehensive Enzyme Information System;
Technical University of Braunschweig)), all which are available on
the World Wide Web, for polypeptides categorized under EC
2.3.1.180.
[0161] Additional .beta.-ketoacyl-ACP synthase polypeptides can
also be identified by searching a sequence pattern database, such
as the Prosite database (ExPASy Proteomics Server, Swiss Institute
of Bioinformatics) for a polypeptide comprising one or more of the
sequence motifs listed below. This is readily accomplished, for
example, by using the ScanProsite tool which is available on the
World Wide Web site of the ExPASy Proteomics Server.
[0162] In one embodiment, the .beta.-ketoacyl-ACP synthase
polypeptide comprises one or more sequence motif selected from:
TABLE-US-00005 (SEQ ID NO: 14)
D-T-[N,S]D-[A,E]-W-I-x(2)-[M,R]-T-G-I-x- [N,E]-R-[R,H] (SEQ ID NO:
15) [S,A]-x-D-x(2)-A-[A,V]-C-[A,S]-G-F-x(3)- [M,L]-x(2)-A (SEQ ID
NO: 16) D-R-x-T-[A,I]-[I,V]-x-F-[A,G]-D-G-A-[A,G]- [G,A]-[A,V] (SEQ
ID NO: 17) H-Q-A-N-x-R-I-[M,L] (SEQ ID NO: 18)
G-N-T-[G,S]-A-A-S-[V,I]-P-x(2)-[I,L]- x(6)-G (SEQ ID NO: 19)
[I,V]-x-L-x(2)-F-G-G-G-[L,F]-[T,S]-W-G
wherein the amino acid residues in each of the brackets indicate
alternative amino acid residues at the particular position, each x
indicates any amino acid residue, and each n in "x(n)" indicates
the number of x residues in a contiguous stretch of amino acid
residues.
[0163] In some embodiments, a polynucleotide encoding a parent
fatty acid pathway polypeptide (such as a polypeptide described in
Table 4 or identified by EC number or by motif or by homology to an
exemplary polypeptide) is modified using methods well known in the
art to generate a variant polypeptide having .beta.-ketoacyl-ACP
synthase activity, and an improved property, compared to that of
the parent polypeptide, which is more suited to the microorganism
and/or to the pathway being engineered; such as, for example,
increased catalytic activity and/or increased specificity for
propionyl-CoA (relative to, e.g., acetyl-CoA); improved catalytic
activity or improved stability under conditions in which the
recombinant microbial cell is cultured; reduced inhibition (e.g.,
reduced feedback inhibition) by a cellular metabolite or by a
culture media component, and the like.
[0164] The invention includes a recombinant microbial cell
comprising a polynucleotide encoding a polypeptide, said
polypeptide comprising a polypeptide sequence having at least 80%,
at least 85%, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, or at least 99% identity to one of SEQ ID NOs:1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 146, 147, 148, and 149, wherein
the polypeptide has .beta.-ketoacyl-ACP synthase activity that
utilizes propionyl-CoA as a substrate. In some instances, the
polypeptide sequence comprises one or more sequence motif selected
from SEQ ID NOs:14-19. The invention also includes an isolated
polypeptide comprising said polypeptide sequence, and an isolated
polynucleotide encoding said polypeptide. In one embodiment, the
polypeptide comprises a substitution at position W310 or at an
equivalent position thereto. In one embodiment, the polypeptide
comprises a W310G substitution. In one embodiment, the polypeptide
comprises a sequence having at least 80%, at least 85%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least
99% identity to SEQ ID NO:7 and comprises the substitution W310G.
In some embodiments, the polypeptide exhibits greater specificity
for propionyl-CoA than for acetyl-CoA.
[0165] As used herein, "a polypeptide having .beta.-ketoacyl-ACP
synthase activity that utilizes propionyl-CoA as a substrate"
includes any polypeptide having a detectable level of
.beta.-ketoacyl-ACP synthase activity when supplied with the
substrate propionyl-CoA.
[0166] Enzymatic activity and specificity of .beta.-ketoacyl-ACP
synthases for substrates, such as propionyl-CoA, can be determined
using known methods. For example, Choi et al. (J. Bacteriology
182(2):365-370 (2000)) described in detail a filtered disc assay
suitable for determining .beta.-ketoacyl-ACP synthase ("FabH")
activity against acetyl-CoA substrate, which can be modified to
assay propionyl-CoA as a substrate. The assay contains 25 .mu.M
ACP, 1 mM .beta.-mercaptoethanol, 65 .mu.M malonyl-CoA, 45 .mu.M
[1-.sup.14C]acetyl-CoA (specificity activity about 45.8 Ci/mol), E.
coli FadD (0.2 .mu.g), and 0.1 M sodium phosphate buffer (pH 7.0)
in a final volume of 40 .mu.L. To assay .beta.-ketoacyl-ACP
synthase activity, [1-.sup.14C]acetyl-CoA can be substituted with
.sup.14C labeled propionyl-CoA. The reaction is initiated by the
addition of FabH, and the mixture is incubated at 37.degree. C. for
12 minutes. A 35 mL aliquot is then removed and deposited on a
Whatman 3 MM filter disc. The discs are then washed with three
changes (20 mL/disc for 20 minutes each) of ice-cold
trichloroacetic acid. The concentration of the trichloroacetic acid
is then reduced from 10 to 5 to 1% in each successive wash. The
filters are dried an counted in 3 mL of scintillation cocktail.
[0167] Alternatively, FabH activity can be determined using a
radioactively labeled malonyl-CoA substrate and gel electrophoresis
to separate and quantitate the products (Choi et al. 2000, supra).
The assay mixture contains 25 .mu.M ACP, 1 mM
.beta.-mercaptoethanol, 70 .mu.M [2-.sup.14C] malonyl-CoA (specific
activity, .about.9 Ci/mol), 45 .mu.M of a CoA-substrate (such as
acetyl-CoA or propionyl-CoA), FadD (0.2 .mu.g), 100 .mu.M NADPH,
FabG (0.2 .mu.g) and 0.1 M sodium phosphate buffer (pH 7.0) in a
final volume of 40 .mu.L. The reaction can be initiated by the
addition of FabH. The mixture is incubated at 37.degree. C. for 12
minutes and then placed in an ice slurry, gel loading buffer is
then added, and the mixture is loaded onto a conformationally
sensitive 13% polyacrylamide gel containing 0.5 to 2.0 M urea.
Electrophoresis can be performed at 25.degree. C. at 32 mA/gel. The
gels are then dried, and the bands quantitated by exposure of the
gel to a Phospholmager screen. Specific activity can be calculated
from the slopes of the plot of product formation vs. FabH protein
concentration in the assay.
oc-.beta.-Ketoacyl-ACP to oc-Acyl-ACP
[0168] The oc-.beta.-ketoacyl-ACP intermediate 3-oxovaleryl-ACP
generated in step (D) can undergo elongation by successive cycles
of condensation with
malonyl-ACP/keto-reduction/dehydration/enoyl-reduction, catalyzed
by a fatty acid synthase (FAS) complex, such as, for example, a
type II fatty acid synthase complex, thereby adding 2-carbon units
to the lengthening fatty acid chain of the resulting oc-acyl-ACP,
as represented by step (E) of FIG. 1B. In one embodiment, a FAS
enzyme complex (such as, for example, a Type II FAS complex)
endogenous to the microbial cell is used to catalyze cycles of
condensation with
malonyl-ACP/keto-reduction/dehydration/enoyl-reduction to produce
the oc-acyl-ACP intermediate.
oc-Acyl-ACP to oc-FA Derivative
[0169] Odd chain fatty acid derivatives can be produced by a
recombinant microbial cell of the invention. The oc-acyl-ACP
intermediate is converted to an oc-FA derivative in a reaction
catalyzed by one or more enzymes each having fatty acid derivative
activity (i.e., fatty acid derivative enzymes), as represented by
step (F) of FIG. 1B. A fatty acid derivative enzyme can, for
example, convert an oc-acyl-ACP to an initial oc-FA derivative, or,
can convert the initial oc-FA derivative to a second oc-FA
derivative. In some instances, the initial oc-FA derivative is
converted to a second oc-FA derivative by an enzyme having a
different fatty acid derivative activity. In some instances, the
second oc-FA derivative is further converted to a third oc-FA
derivative by another fatty acid derivative enzyme, and so on.
[0170] Accordingly, in some embodiments, the recombinant microbial
cell further comprises one or more polynucleotides, each
polynucleotide encoding a polypeptide having a fatty acid
derivative enzyme activity, wherein the recombinant microbial cell
produces an oc-FA derivative when cultured in the presence of a
carbon source under conditions effective to express the
polynucleotides.
[0171] In various embodiments, the fatty acid derivative activity
comprises thioesterase activity, wherein the recombinant microbial
cell produces oc-fatty acids; ester synthase activity, wherein the
recombinant microbial cell produces oc-fatty esters; fatty aldehyde
biosynthesis activity, wherein the recombinant microbial cell
produces oc-fatty aldehydes; fatty alcohol biosynthesis activity,
wherein the recombinant microbial cell produces oc-fatty alcohols;
ketone biosynthesis activity, wherein the recombinant microbial
cell produces ec-ketones; or hydrocarbon biosynthesis activity,
wherein the recombinant microbial cell produces ec-hydrocarbons. In
some embodiments, the recombinant microbial cell comprises
polynucleotides encoding two or more polypeptides, each polypeptide
having fatty acid derivative enzyme activity.
[0172] In more particular embodiments, the recombinant microbial
cell expresses or overexpresses one or more polypeptides having
fatty acid derivative enzyme activity as described hereinabove,
wherein the recombinant microbial cell produces an oc-FA
composition comprising oc-fatty acids, oc-fatty esters, oc-wax
esters, oc-fatty aldehydes, oc-fatty alcohols, ec-ketones,
ec-alkanes, ec-alkanes, ec-internal olefins, or ec-terminal
olefins.
[0173] The following are further examples of fatty acid derivative
enzymes, and oc-FA derivatives produced by reactions catalyzed by
such enzymes, in accordance with various embodiments of the
invention.
oc-Fatty Acid
[0174] In one embodiment, the recombinant microbial cell comprises
a polynucleotide encoding a thioesterase, and the oc-acyl-ACP
intermediate produced by the recombinant microbial cell is
hydrolyzed by the thioesterase (e.g., 3.1.1.5, EC 3.1.2.-; such as,
for example, EC 3.1.2.14) resulting in production of an oc-fatty
acid. In some embodiments, a composition comprising fatty acids
(also referred to herein as a "fatty acid composition") comprising
oc-fatty acids is produced by culturing the recombinant cell in the
presence of a carbon source under conditions effective to express
the polynucleotide. In some embodiments, the fatty acid composition
comprises oc-fatty acids and ec-fatty acids. In some embodiments,
the composition is recovered from the cell culture.
[0175] In some embodiments, the recombinant microbial cell
comprises a polynucleotide encoding a polypeptide having
thioesterase activity, and one or more additional polynucleotides
encoding polypeptides having other fatty acid derivative enzyme
activities. In some such instances, the oc-fatty acid produced by
the action of the thioesterase is converted by one or more enzymes
having different fatty acid derivative enzyme activities to another
oc-fatty acid derivative, such as, for example, an oc-fatty ester,
oc-fatty aldehyde, oc-fatty alcohol, or ec-hydrocarbon.
[0176] In one embodiment, an oc-acyl-ACP intermediate reacts with a
thioesterase to form an oc-fatty acid. The oc-fatty acid can be
recovered from the cell culture, or can be further converted to
another oc-FA derivative, such as an oc-fatty ester, an oc-fatty
aldehyde, an oc-fatty alcohol, or an ec-terminal olefin.
[0177] The chain length of a fatty acid, or a fatty acid derivative
made therefrom, can be selected for by modifying the expression of
certain thioesterases. Thioesterase influences the chain length of
fatty acids produced as well as that of the derivatives made
therefrom. Hence, the recombinant microbial cell can be engineered
to express, overexpress, have attenuated expression, or not to
express one or more selected thioesterases to increase the
production of a preferred fatty acid or fatty acid derivative
substrate. For example, C.sub.10 fatty acids can be produced by
expressing a thioesterase that has a preference for producing
C.sub.10 fatty acids and attenuating thioesterases that have a
preference for producing fatty acids other than C.sub.10 fatty
acids (e.g., a thioesterase which prefers to produce C.sub.14 fatty
acids). This would result in a relatively homogeneous population of
fatty acids that have a carbon chain length of 10. In other
instances, C.sub.14 fatty acids can be produced by attenuating
endogenous thioesterases that produce non-C.sub.14 fatty acids and
expressing thioesterases that use C.sub.14-ACP. In some situations,
C.sub.12 fatty acids can be produced by expressing thioesterases
that use C.sub.12-ACP and attenuating thioesterases that produce
non-C.sub.12 fatty acids. Fatty acid overproduction can be verified
using methods known in the art, for example, by use of radioactive
precursors, HPLC, or GC-MS subsequent to cell lysis.
[0178] Additional non-limiting examples of thioesterases and
polynucleotides encoding them for use in the oc-fatty acid pathway
are provided in Table 5 and in PCT Publication No. WO 2010/075483
incorporated by reference herein.
TABLE-US-00006 TABLE 5 Non-limiting examples of thioesterases and
coding sequences thereof for use in the oc-FA pathway shown in FIG.
1B. UniProtKB (Swiss-Prot) SEQ Gene Protein Accession Number, NCBI
Protein ID EC number Organism symbol or literature reference
Accession Number NO EC 3.1.1.5, Thioesterase EC 3.1.2.- E. coli
K-12 tesA P0ADA1 AAC73596 64 MG1655 E. coli 'tesA Cho et al, J.
Biol. Chem., 65 (without 270:4216-4219 (1995) leader sequence) E.
coli K-12 tesB P0AGG2 AAC73555 66 MG1655 Arabidopsis fatA Q42561
NP_189147 67 thaliana Arabidopsis fatB Q9SJE2 NP_172327 68 thaliana
Umbellularia fatB Q41635 AAA34215 69 california Cuphea fatAl Q9ZTF7
AAC72883 70 hookeriana Cuphea fatB2 Q39514 AAC49269 71 hookeriana
Cuphea fatB3 Q9ZTF9 AAC72881 72 hookeriana
oc-Fatty Ester
[0179] In one embodiment, the recombinant microbial cell produces
an oc-fatty ester, such as, for example, an oc-fatty acid methyl
ester or an oc-fatty acid ethyl ester or an oc-wax ester. In some
embodiments, an oc-fatty acid produced by the recombinant microbial
cell is converted into the oc-fatty ester.
[0180] In some embodiments, the recombinant microbial cell
comprises a polynucleotide encoding a polypeptide (i.e., an enzyme)
having ester synthase activity (also referred to herein as an
"ester synthase polypeptide" or an "ester synthase enzyme"), and
the oc-fatty ester is produced by a reaction catalyzed by the ester
synthase polypeptide expressed or overexpressed in the recombinant
microbial cell. In some embodiments, a composition comprising fatty
esters (also referred to herein as a "fatty ester composition")
comprising oc-fatty esters is produced by culturing the recombinant
cell in the presence of a carbon source under conditions effective
to express the polynucleotide. In some embodiments, the fatty ester
composition comprises oc-fatty esters and ec-fatty esters. In some
embodiments, the composition is recovered from the cell
culture.
[0181] Ester synthase polypeptides include, for example, an ester
synthase polypeptide classified as EC 2.3.1.75, or any other
polypeptide which catalyzes the conversion of an acyl-thioester to
a fatty ester, including, without limitation, a wax-ester synthase,
an acyl-CoA:alcohol transacylase, an acyltransferase, or a fatty
acyl-CoA:fatty alcohol acyltransferase. For example, the
polynucleotide may encode wax/dgat, a bifunctonal ester
synthase/acyl-CoA:diacylglycerol acyltransferase from Simmondsia
chinensis, Acinetobacter sp. Strain ADP1, Alcanivorax borkumensis,
Pseudomonas aeruginosa, Fundibacter jadensis, Arabidopsis thaliana,
or Alkaligenes eutrophus. In a particular embodiment, the ester
synthase polypeptide is an Acinetobacter sp. diacylglycerol
O-acyltransferase (wax-dgaT; UniProtKB Q8GGG1, GenBank AAO17391) or
Simmondsia chinensis wax synthase (UniProtKB Q9XGY6, GenBank
AAD38041). In a particular embodiment, the polynucleotide encoding
the ester synthase polypeptide is overexpressed in the recombinant
microbial cell. In some embodiments the recombinant microbial cell
further comprises a polynucleotide encoding a thioesterase.
[0182] In another embodiment, the recombinant microbial cell
produces an oc-fatty ester, such as, for example, an oc-fatty acid
methyl ester or an oc-fatty acid ethyl ester, wherein the
recombinant microbial cell expresses a polynucleotide encoding an
ester synthase/acyltransferase polypeptide classified as 2.3.1.20,
such as AtfA1 (an acyltransferase derived from Alcanivorax
borkumensis SK2, UniProtKB Q0VKV8, GenBank YP.sub.--694462) or
AtfA2 (another acyltransferase derived from Alcanivorax borkumensis
SK2, UniProtKB Q0VNJ6, GenBank YP.sub.--693524). In a particular
embodiment, the polynucleotide encoding the ester synthase
polypeptide is overexpressed in the recombinant microbial cell. In
some embodiments the recombinant microbial cell further comprises a
polynucleotide encoding a thioesterase.
[0183] In another embodiment, the recombinant microbial cell
produces an oc-fatty ester, such as, for example, an oc-fatty acid
methyl ester or an oc-fatty acid ethyl ester, wherein the
recombinant microbial cell expresses a polynucleotide encoding a
ester synthase polypeptide, such as ES9 (a wax ester synthase from
Marinobacter hydrocarbonoclasticus DSM 8798, UniProtKB A3RE51,
GenBank ABO21021, encoded by the ws2 gene), or ES376 (another wax
ester synthase derived from Marinobacter hydrocarbonoclasticus DSM
8798, UniProtKB A3RE50, GenBank ABO21020, encoded by the ws1 gene).
In a particular embodiment, the polynucleotide encoding the ester
synthase polypeptide is overexpressed in the recombinant microbial
cell. In some embodiments the recombinant microbial cell further
comprises a polynucleotide encoding a thioesterase.
[0184] Additional non-limiting examples of ester synthase
polypeptides and polynucleotides encoding them suitable for use in
these embodiments include those described in PCT Publication Nos.
WO 2007/136762 and WO2008/119082 which are incorporated by
reference herein.
oc-Fatty Aldehyde
[0185] In one embodiment, the recombinant microbial cell produces
an oc-fatty aldehyde. In some embodiments, an oc-fatty acid
produced by the recombinant microbial cell is converted into the an
oc-fatty aldehyde. In some embodiments, the oc-fatty aldehyde
produced by the recombinant microbial cell is then converted into
an oc-fatty alcohol or an ec-hydrocarbon.
[0186] In some embodiments, the recombinant microbial cell
comprises a polynucleotide encoding a polypeptide (i.e., an enzyme)
having fatty aldehyde biosynthesis activity (also referred to
herein as a "fatty aldehyde biosynthesis polypeptide" or a "fatty
aldehyde biosynthesis enzyme"), and the oc-fatty aldehyde is
produced by a reaction catalyzed by the fatty aldehyde biosynthesis
polypeptide expressed or overexpressed in the recombinant microbial
cell. In some embodiments, a composition comprising fatty aldehydes
(also referred to herein as a "fatty aldehyde composition")
comprising oc-fatty aldehydes is produced by culturing the
recombinant cell in the presence of a carbon source under
conditions effective to express the polynucleotide. In some
embodiments, the fatty aldehyde composition comprises oc-fatty
aldehydes and ec-fatty aldehydes. In some embodiments, the
composition is recovered from the cell culture.
[0187] In some embodiments, the oc-fatty aldehyde is produced by
expressing or overexpressing in the recombinant microbial cell a
polynucleotide encoding a polypeptide having a fatty aldehyde
biosynthesis activity such as carboxylic acid reductase (CAR)
activity (encoded, for example, by a car gene). Examples of
carboxylic acid reductase (CAR) polypeptides and polynucleotides
encoding them useful in accordance with this embodiment include,
but are not limited to, FadD9 (EC 6.2.1.-, UniProtKB Q50631,
GenBank NP.sub.--217106), CarA (GenBank ABK75684), CarB (GenBank
YP889972) and related polypeptides described in PCT Publication No.
WO 2010/042664 which is incorporated by reference herein. In some
embodiments the recombinant microbial cell further comprises a
polynucleotide encoding a thioesterase.
[0188] In some embodiments, the oc-fatty aldehyde is produced by
expressing or overexpressing in the recombinant microbial cell a
polynucleotide encoding a fatty aldehyde biosynthesis polypeptide,
such as a polypeptide having acyl-ACP reductase (AAR) activity,
encoded by, for example, an aar gene. Examples of acyl-ACP
reductase polypeptides useful in accordance with this embodiment
include, but are not limited to, acyl-ACP reductase from
Synechococcus elongatus PCC 7942 (GenBank YP.sub.--400611) and
related polypeptides described in PCT Publication No. WO
2010/042664 which is incorporated by reference herein.
[0189] In some embodiments, the oc-fatty aldehyde is produced by
expressing or overexpressing in the recombinant microbial cell a
polynucleotide encoding a fatty aldehyde biosynthesis polypeptide,
such as a polypeptide having acyl-CoA reductase activity (e.g., EC
1.2.1.x), encoded by, for example, an acrl gene. Examples of
acyl-CoA reductase polypeptides useful in accordance with this
embodiment include, but are not limited to, ACR1 from Acinetobacter
sp. strain ADP1 (GenBank YP.sub.--047869) and related polypeptides
described in PCT Publication No. WO 2010/042664 which is
incorporated by reference herein. In some embodiments the
recombinant microbial cell further comprises polynucleotides
encoding a thioesterase and an acyl-CoA synthase.
oc-Fatty Alcohol
[0190] In one embodiment, the recombinant microbial cell produces
an oc-fatty alcohol. In some embodiments, an oc-fatty aldehyde
produced by the recombinant microbial cell is converted to the
oc-fatty alcohol. In other embodiments, an oc-fatty acid produced
by the recombinant microbial cell is converted to the oc-fatty
alcohol
[0191] In some embodiments, the recombinant microbial cell
comprises a polynucleotide encoding a polypeptide (i.e., an enzyme)
having fatty alcohol biosynthesis activity (also referred to herein
as a "fatty alcohol biosynthesis polypeptide" or a "fatty alcohol
biosynthesis enzyme"), and the oc-fatty alcohol is produced by a
reaction catalyzed by the fatty alcohol biosynthesis enzyme
expressed or overexpressed in the recombinant microbial cell. In
some embodiments, a composition comprising fatty alcohols (also
referred to herein as a "fatty alcohol composition") comprising
oc-fatty alcohols is produced by culturing the recombinant cell in
the presence of a carbon source under conditions effective to
express the polynucleotide. In some embodiments, the fatty alcohol
composition comprises oc-fatty alcohols and ec-fatty alcohols. In
some embodiments, the composition is recovered from the cell
culture.
[0192] In some embodiments, the oc-fatty alcohol is produced by
expressing or overexpressing in the recombinant microbial cell a
polynucleotide encoding a polypeptide having fatty alcohol
biosynthesis activity such as alcohol dehydrogenase (aldehyde
reductase) activity, e.g., EC 1.1.1.1. Examples of alcohol
dehydrogenase polypeptides useful in accordance with this
embodiment include, but are not limited to, E. coli alcohol
dehydrogenase YqhD (GenBank AP.sub.--003562) and related
polypeptides described in PCT Publication Nos. WO 2007/136762 and
WO2008/119082 which are incorporated by reference herein. In some
embodiments the recombinant microbial cell further comprises a
polynucleotide encoding a fatty aldehyde biosynthesis polypeptide.
In some embodiments the recombinant microbial cell further
comprises a polynucleotide encoding a thioesterase.
[0193] In some embodiments, the oc-fatty alcohol is produced by
expressing or overexpressing in the recombinant microbial cell a
polynucleotide encoding a fatty alcohol biosynthesis polypeptide,
such as a polypeptide having fatty alcohol forming acyl-CoA
reductase (FAR) activity, e.g., EC 1.1.1.x. Examples of FAR
polypeptides useful in accordance with this embodiment include, but
are not limited to, those described in PCT Publication No. WO
2010/062480 which is incorporated by reference herein. In some
embodiments the recombinant microbial cell further comprises
polynucleotides encoding a thioesterase and an acyl-CoA
synthase.
ec-Hydrocarbon
[0194] In one embodiment, the recombinant microbial cell produces
an ec-hydrocarbon, such as an ec-alkane or an ec-alkene (e.g., an
ec-terminal olefin or an ec-internal olefin) or an ec-ketone. In
some embodiments, an oc-acyl-ACP intermediate is converted by
decarboxylation, removing a carbon atom to form an ec-internal
olefin or an ec-ketone. In some embodiments, an oc-fatty aldehyde
produced by the recombinant microbial cell is converted by
decarbonylation, removing a carbon atom to form an ec-hydrocarbon.
In some embodiments, an oc-fatty acid produced by the recombinant
microbial cell is converted by decarboxylation, removing a carbon
atom to form an ec-terminal olefin.
[0195] In some embodiments, the recombinant microbial cell
comprises a polynucleotide encoding a polypeptide (i.e., an enzyme)
having hydrocarbon biosynthesis activity (also referred to herein
as a "hydrocarbon biosynthesis polypeptide" or a "hydrocarbon
biosynthesis enzyme"), and the ec-hydrocarbon is produced by a
reaction catalyzed by the hydrocarbon biosynthesis enzyme expressed
or overexpressed in the recombinant microbial cell. In some
embodiments, a composition comprising hydrocarbons (also referred
to herein as a "hydrocarbon composition") comprising
ec-hydrocarbons is produced by culturing the recombinant cell in
the presence of a carbon source under conditions effective to
express the polynucleotide. In some embodiments, the hydrocarbon
composition comprises ec-hydrocarbons and oc-hydrocarbons. In some
embodiments, the hydrocarbon composition is recovered from the cell
culture.
[0196] In some embodiments, the ec-hydrocarbon is produced by
expressing or overexpressing in the recombinant microbial cell a
polynucleotide encoding a polypeptide having hydrocarbon
biosynthesis activity such as an aldehyde decarbonylase (ADC)
activity (e.g., EC 4.1.99.5), for example, a polynucleotide
encoding an aldehyde decarbonylase from Prochlorococcus marinus
MIT9313 (GenBank NP.sub.--895059) or Nostoc punctiforme (GenBank
Accession No. YP.sub.--001865325). Additional examples of aldehyde
decarbonylase and related polypeptides useful in accordance with
this embodiment include, but are not limited to, those described in
PCT Publication Nos. WO 2008/119082 and WO 2009/140695 which are
incorporated by reference herein. In some embodiments the
recombinant microbial cell further comprises a polynucleotide
encoding a fatty aldehyde biosynthesis polypeptide. In some
embodiments the recombinant microbial cell further comprises a
polynucleotide encoding an acyl-ACP reductase.
[0197] In some embodiments, an ec-terminal olefin is produced by
expressing or overexpressing in the recombinant microbial cell a
polynucleotide encoding a hydrocarbon biosynthesis polypeptide,
such as a polypeptide having decarboxylase activity as described,
for example, in PCT Publication No. WO 2009/085278 which is
incorporated by reference herein. In some embodiments the
recombinant microbial cell further comprises a polynucleotide
encoding a thioesterase.
[0198] In some embodiments, an ec-internal olefin is produced by
expressing or overexpressing in the recombinant microbial cell a
polynucleotide encoding a hydrocarbon biosynthesis polypeptide,
such as a polypeptide having OleCD or OleBCD activity as described,
for example, in PCT Publication No. WO 2008/147781 which is
incorporated by reference herein.
[0199] In some embodiments, an ec-ketone is produced by expressing
or overexpressing in the recombinant microbial cell a
polynucleotide encoding a hydrocarbon biosynthesis polypeptide,
such as a polypeptide having OleA activity as described, for
example, in PCT Publication No. WO 2008/147781 which is
incorporated by reference herein.
Saturation Levels of oc-FA Derivatives
[0200] The degree of saturation of oc-acyl-ACPs (which can then be
converted into various oc-FA derivatives as described hereinabove)
can be controlled by regulating the degree of saturation of fatty
acid intermediates. For example, the sfa, gns, and fab families of
genes can be expressed, overexpressed, or expressed at reduced
levels (e.g., attenuated), to control the amount of saturation of
an oc-acyl-ACP.
oc-FA Pathway Polypeptides and Polynucleotides
[0201] The disclosure identifies polynucleotides useful in the
recombinant microbial cells, methods, and compositions of the
invention; however it will be recognized that absolute sequence
identity to such polynucleotides is not necessary. For example,
changes in a particular polynucleotide sequence can be made and the
encoded polypeptide screened for activity. Such changes typically
comprise conservative mutations and silent mutations (such as, for
example, codon optimization). Modified or mutated (i.e., mutant)
polynucleotides and encoded variant polypeptides can be screened
for a desired function, such as, an improved function compared to
the parent polypeptide, including but not limited to increased
catalytic activity, increased stability, or decreased inhibition
(e.g., decreased feedback inhibition), using methods known in the
art.
[0202] The disclosure identifies enzymatic activities involved in
various steps (i.e., reactions) of the oc-FA biosynthetic pathways
described herein according to Enzyme Classification (EC) number,
and provides exemplary polypeptides (i.e., enzymes) categorized by
such EC numbers, and exemplary polynucleotides encoding such
polypeptides. Such exemplary polypeptides and polynucleotides,
which are identified herein by Accession Numbers and/or Sequence
Identifier Numbers (SEQ ID NOs), are useful for engineering oc-FA
pathways in parental microbial cells to obtain the recombinant
microbial cells described herein. It is to be understood, however,
that polypeptides and polynucleotides described herein are
exemplary and non-limiting. The sequences of homologues of
exemplary polypeptides described herein are available to those of
skill in the art using databases such as, for example, the Entrez
databases provided by the National Center for Biotechnology
Information (NCBI), the ExPasy databases provided by the Swiss
Institute of Bioinformatics, the BRENDA database provided by the
Technical University of Braunschweig, and the KEGG database
provided by the Bioinformatics Center of Kyoto University and
University of Tokyo, all which are available on the World Wide
Web.
[0203] It is to be further understood that a variety of microbial
cells can be modified to contain an oc-FA pathway described herein,
resulting in recombinant microbial cells suitable for the
production of odd chain fatty acid derivatives. It is also
understood that a variety of cells can provide sources of genetic
material, including sequences of polynucleotides encoding
polypeptides suitable for use in a recombinant microbial cell
provided herein.
[0204] The disclosure provides numerous examples of polypeptides
(i.e., enzymes) having activities suitable for use in the oc-FA
biosynthetic pathways described herein. Such polypeptides are
collectively referred to herein as "oc-FA pathway polypeptides"
(alternatively, "oc-FA pathway enzymes"). Non-limiting examples of
oc-FA pathway polypeptides suitable for use in recombinant
microbial cells of the invention are provided in the Tables and
Description and in the Examples herein.
[0205] In some embodiments, the invention includes a recombinant
microbial cell comprising a polynucleotide sequence (also referred
to herein as an "oc-FA pathway polynucleotide" sequence) which
encodes an oc-FA pathway polypeptide.
[0206] Additional oc-FA pathway polypeptides and polynucleotides
encoding them suitable for use in engineering an oc-FA pathway in a
recombinant microbial cell of the invention can be obtained by a
number of methods. For example, EC numbers classify enzymes
according to the reaction catalyzed. Enzymes that catalyze a
reaction in a biosynthetic pathway described herein can be
identified by searching the EC number corresponding to that
reaction in a database such as, for example: the KEGG database
(Kyoto Encyclopedia of Genes and Genomes; Kyoto University and
University of Tokyo); the UNIPROTKB database or the ENZYME database
(ExPASy Proteomics Server; Swiss Institute of Bioinformatics); the
PROTEIN database or the GENE database (Entrez databases; National
Center for Biotechnology Information (NCBI)); or the BRENDA
database (The Comprehensive Enzyme Information System; Technical
University of Braunschweig); all of which are available on the
World Wide Web. In one embodiment, an oc-FA pathway polynucleotide
encoding an oc-FA pathway polypeptide having an enzymatic activity
categorized by an EC number (such as, an EC number listed in the
Description or in one of Tables herein), or a fragment or a variant
thereof having that activity, is used in engineering the
corresponding step of an oc-FA pathway in a recombinant microbial
cell.
[0207] In some embodiments, an oc-FA pathway polynucleotide
sequence encodes a polypeptide which is endogenous to the parental
cell of the recombinant cell being engineered. Some such endogenous
polypeptides are overexpressed in the recombinant microbial cell.
An "endogenous polypeptide", as used herein, refers to a
polypeptide which is encoded by the genome of the parental (e.g.,
wild-type) cell that is being engineered to produce the recombinant
microbial cell.
[0208] An oc-FA pathway polypeptide, such as for example an
endogenous oc-FA pathway polypeptide, can be overexpressed by any
suitable means. As used herein, "overexpress" means to express or
cause to be expressed a polynucleotide or polypeptide in a cell at
a greater concentration than is normally expressed in a
corresponding parental (for example, wild-type) cell under the same
conditions. For example, a polypeptide is "overexpressed" in a
recombinant microbial cell when it is present in a greater
concentration in the recombinant cell as compared to its
concentration in a non-recombinant host cell of the same species
(e.g., the parental cell) when cultured under the same
conditions.
[0209] In some embodiments, the oc-FA pathway polynucleotide
sequence encodes an exogenous or heterologous polypeptide. In other
words, the polypeptide encoded by the polynucleotide is exogenous
to the parental microbial cell. An "exogenous" (or "heterologous")
polypeptide, as used herein, refers to a polypeptide not encoded by
the genome of the parental (e.g., wild-type) microbial cell that is
being engineered to produce the recombinant microbial cell. Such a
polypeptide can also be referred to as a "non-native"
polypeptide.
[0210] In certain embodiments, an oc-FA pathway polypeptide
comprises an amino acid sequence other than that of one of the
exemplary polypeptides provided herein; for example, an oc-FA
pathway polypeptide can comprise a sequence which is a homologue, a
fragment, or a variant of the sequence of the exemplary
polypeptide.
[0211] The terms "homolog," "homologue," and "homologous" as used
herein refer to a polynucleotide or a polypeptide comprising a
sequence that is at least 50%, preferably at least 60%, more
preferably at least 70% (e.g., at least 75%, at least 80%, at least
85%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
or at least 99%) homologous to the corresponding polynucleotide or
polypeptide sequence. One of ordinary skill in the art is well
aware of methods to determine homology between two or more
sequences. Briefly, calculations of "homology" between two
sequences can be performed as follows. The sequences are aligned
for optimal comparison purposes (e.g., gaps can be introduced in
one or both of a first and a second amino acid or polynucleotide
sequence for optimal alignment and non-homologous sequences can be
disregarded for comparison purposes). In a preferred embodiment,
the length of a first sequence that is aligned for comparison
purposes is at least about 30%, preferably at least about 40%, more
preferably at least about 50%, even more preferably at least about
60%, and even more preferably at least about 70%, at least about
80%, at least about 90%, or about 100% of the length of a second
sequence. The amino acid residues or nucleotides at corresponding
amino acid positions or nucleotide positions of the first and
second sequences are then compared. When a position in the first
sequence is occupied by the same amino acid residue or nucleotide
as the corresponding position in the second sequence, then the
molecules are identical at that position (as used herein, amino
acid or nucleic acid "identity" is equivalent to amino acid or
nucleic acid "homology"). The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences, taking into account the number of gaps and the
length of each gap, which need to be introduced for optimal
alignment of the two sequences.
[0212] The comparison of sequences and determination of percent
homology (i.e., percent identity) between two sequences can be
accomplished using a mathematical algorithm, such as BLAST
(Altschul et al., J. Mol. Biol., 215(3): 403-410 (1990)). The
percent homology between two amino acid sequences also can be
determined using the Needleman and Wunsch algorithm that has been
incorporated into the GAP program in the GCG software package,
using either a Blossum 62 matrix or a PAM250 matrix, and a gap
weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2,
3, 4, 5, or 6 (Needleman and Wunsch, J. Mol. Biol., 48: 444-453
(1970)). The percent homology between two nucleotide sequences also
can be determined using the GAP program in the GCG software
package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50,
60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. One of
ordinary skill in the art can perform initial homology calculations
and adjust the algorithm parameters accordingly. A preferred set of
parameters (and the one that should be used if a practitioner is
uncertain about which parameters should be applied to determine if
a molecule is within a homology limitation of the claims) are a
Blossum 62 scoring matrix with a gap penalty of 12, a gap extend
penalty of 4, and a frameshift gap penalty of 5. Additional methods
of sequence alignment are known in the biotechnology arts (see,
e.g., Rosenberg, BMC Bioinformatics, 6: 278 (2005); Altschul et
al., FEBS J., 272(20): 5101-5109 (2005)).
[0213] An "equivalent position" (for example, an "equivalent amino
acid position" or "equivalent nucleic acid position") is defined
herein as a position (such as, an amino acid position or nucleic
acid position) of a test polypeptide (or test polynucleotide)
sequence which aligns with a corresponding position of a reference
polypeptide (or reference polynucleotide) sequence, when optimally
aligned using an alignment algorithm as described herein. The
equivalent amino acid position of the test polypeptide need not
have the same numerical position number as the corresponding
position of the reference polypeptide; likewise, the equivalent
nucleic acid position of the test polynucleotide need not have the
same numerical position number as the corresponding position of the
reference polynucleotide.
[0214] In some embodiments, the oc-FA pathway polypeptide is a
variant of a reference (e.g., a parent) polypeptide, such as a
variant of an exemplary oc-FA pathway polypeptide described herein.
A "variant" (alternatively, "mutant") polypeptide as used herein
refers to a polypeptide having an amino acid sequence that differs
from that of a parent (e.g., wild-type) polypeptide by at least one
amino acid. The variant can comprise one or more conservative amino
acid substitutions, and/or can comprise one or more
non-conservative substitutions, compared to the parent polypeptide
sequence. In some embodiments, the variant polypeptide has 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or more amino acid
substitutions, additions, insertions, or deletions compared to the
parent polypeptide sequence. In some embodiments, the sequence of
the variant polypeptide is at least 80%, at least 85%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least
99% identical to the sequence of the parent polypeptide.
[0215] In some embodiments, the oc-FA pathway polypeptide is a
fragment of a reference (e.g., a parent) polypeptide, such as a
fragment of an exemplary oc-FA pathway polypeptide described
herein. The term "fragment" refers to a shorter portion of a
full-length polypeptide or protein ranging in size from four amino
acid residues to the entire amino acid sequence minus one amino
acid residue. In certain embodiments of the invention, a fragment
refers to the entire amino acid sequence of a domain of a
polypeptide or protein (e.g., a substrate binding domain or a
catalytic domain).
[0216] In some embodiments, a homologue, a variant, or a fragment
comprises one or more sequence motif as defined herein. In one
embodiment, a homologue, a variant, or a fragment of a
.beta.-ketoacyl-ACP synthase polypeptide comprises one or more
sequence motif selected from SEQ ID NOs:14-19. Determination that a
sequence contains a particular sequence motif can be readily
accomplished, for example, using the ScanProsite tool available on
the World Wide Web site of the ExPASy Proteomics Server.
[0217] It is understood that an oc-FA polypeptide may have
conservative or non-essential amino acid substitutions, relative to
a parent polypeptide, which does not have a substantial effect on a
biological function or property of the oc-FA polypeptide. Whether
or not a particular substitution will be tolerated (i.e., will not
adversely affect a desired biological function, such as enzymatic
activity) can be determined, for example, as described in Bowie et
al. (Science, 247: 1306-1310 (1990)).
[0218] A "conservative amino acid substitution" is one in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine), and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
[0219] Variants can be naturally occurring or created in vitro. In
particular, variants can be created using genetic engineering
techniques, such as site directed mutagenesis, random chemical
mutagenesis, exonuclease III deletion procedures, or standard
cloning techniques. Alternatively, such variants, fragments,
analogs, or derivatives can be created using chemical synthesis or
modification procedures.
[0220] Methods of making variants are well known in the art. These
include procedures in which nucleic acid sequences obtained from
natural isolates are modified to generate nucleic acids that encode
polypeptides having characteristics that enhance their value in
industrial or laboratory applications (including, but not limited
to, increased catalytic activity (turnover number), improved
stability, and reduced feedback inhibition). In such procedures, a
large number of modified nucleic acid sequences having one or more
nucleotide differences with respect to the sequence obtained from
the natural isolate are generated and characterized. Typically,
these nucleotide differences result in amino acid changes with
respect to the polypeptides encoded by the nucleic acids from the
natural isolates. For example, variants can be prepared by using
random or site-directed mutagenesis.
[0221] Variants can also be created by in vivo mutagenesis. In some
embodiments, random mutations in a nucleic acid sequence are
generated by propagating the sequence in a bacterial strain, such
as an E. coli strain, which carries mutations in one or more of the
DNA repair pathways. Such "mutator" strains have a higher random
mutation rate than that of a wild-type strain. Propagating a DNA
sequence in one of these strains will eventually generate random
mutations within the DNA. Mutator strains suitable for use for in
vivo mutagenesis are described in, for example, International
Patent Application Publication No. WO 1991/016427.
[0222] Variants can also be generated using cassette mutagenesis.
In cassette mutagenesis, a small region of a double-stranded DNA
molecule is replaced with a synthetic oligonucleotide "cassette"
that differs from the native sequence. The oligonucleotide often
contains a completely and/or partially randomized native
sequence.
[0223] Recursive ensemble mutagenesis can also be used to generate
variants. Recursive ensemble mutagenesis is an algorithm for
protein engineering (i.e., protein mutagenesis) developed to
produce diverse populations of phenotypically related mutants whose
members differ in amino acid sequence. This method uses a feedback
mechanism to control successive rounds of combinatorial cassette
mutagenesis. Recursive ensemble mutagenesis is described in, for
example, Arkin et al., Proc. Natl. Acad. Sci., U.S.A., 89:
7811-7815 (1992).
[0224] In some embodiments, variants are created using exponential
ensemble mutagenesis. Exponential ensemble mutagenesis is a process
for generating combinatorial libraries with a high percentage of
unique and functional mutants, wherein small groups of residues are
randomized in parallel to identify, at each altered position, amino
acids which lead to functional proteins. Exponential ensemble
mutagenesis is described in, for example, Delegrave et al.,
Biotech. Res, 11: 1548-1552 (1993).
[0225] Preferred fragments or variants of a parent polypeptide
(e.g., fragments or variants of a parent oc-FA pathway polypeptide)
retain some or all of a biological function or property (such as,
enzymatic activity, thermal stability) of the parent polypeptide.
In some embodiments, the fragment or variant retains at least 75%
(e.g., at least 80%, at least 90%, or at least 95%) of a biological
function or property of the parent polypeptide. In other
embodiments, the fragment or variant retains about 100% of a
biological function or property of the parent polypeptide.
[0226] In some embodiments, the fragment or variant of the parent
polypeptide exhibits an increased catalytic activity (as reflected
by, for example, a higher turnover number, an altered pH optimum, a
decreased K.sub.m for a desired substrate, or an increased
k.sub.cat/K.sub.m for a desired substrate), relative to that of the
parent polypeptide, under conditions in which the recombinant
microbial cell is cultured. For example, if the parent polypeptide
is endogenous to (that is, is derived from) a thermophilic cell,
and if the recombinant microbial cell is generally cultured at a
lower temperature than the thermophilic cell, the parent
polypeptide may exhibit significantly reduced activity at the lower
temperature; in which case, the variant polypeptide preferably
exhibits an increased catalytic activity (such as, a higher
turnover number), relative to that of the parent polypeptide, at
that lower temperature.
[0227] In other embodiments, the fragment or variant of the parent
polypeptide exhibits improved stability, relative to that of the
parent polypeptide, under conditions in which the recombinant
microbial cell is cultured. Such stability can include stability
towards changes in temperature, ionic strength, pH, or any other
differences in growth or media conditions between the recombinant
microbial cell and the cell from which the parent polypeptide was
derived. For example, if the parent polypeptide is derived from a
psychrotrophic cell, and if the recombinant microbial cell is
generally cultured at a higher temperature than the psychrotrophic
cell, the parent polypeptide may be relatively unstable at the
higher temperature; in which case, the variant polypeptide
preferably exhibits improved stability relative to that of the
parent polypeptide at that higher temperature.
[0228] In other embodiments, the fragment or variant of the parent
polypeptide exhibits reduced inhibition of catalytic activity (such
as, reduced feedback inhibition) by a cellular metabolite or by a
culture media component, relative to such inhibition exhibited by
the parent polypeptide, under conditions in which the recombinant
microbial cell is cultured.
[0229] In certain embodiments, an oc-FA pathway polypeptide is a
homologue, a fragment, or a variant of a parent polypeptide,
wherein the oc-FA pathway polypeptide is effective in carrying out
an oc-FA pathway reaction in a recombinant microbial cell. Such an
oc-FA pathway polypeptide is suitable for use in a recombinant
microbial cell of the invention.
[0230] The effectiveness of a test polypeptide (such as, for
example, an oc-FA pathway polypeptide described herein, or a
homologue, a fragment, or a variant thereof) in carrying out a
reaction of an oc-FA pathway can be determined by a number of
methods. For example, to determine the effectiveness of a test
polypeptide in catalyzing a specific reaction of a biochemical
pathway, first a cell is engineered (if necessary) to obtain a
parental cell that comprises all the activities needed to catalyze
the reactions of the biochemical pathway in question, except for
the specific pathway reaction being tested (although, in some
instances, the parental cell may express endogenous polypeptide(s)
that catalyze the specific pathway reaction being tested; in such
instances the endogenous activity will preferably be low enough to
readily detect an increase in product owing to the activity of the
test polypeptide). A polynucleotide encoding the test polypeptide,
operatively linked to a suitable promoter (e.g., in an expression
vector), is then introduced into the parental cell, generating a
test cell. The test cell and the parental cell are cultured
separately under identical conditions which are sufficient for
expression of the pathway polypeptides in the parental and test
cell cultures and expression of the test polypeptide in the test
cell culture. At various times during and/or after culturing,
samples are obtained from the test cell culture and the parental
cell culture. The samples are analyzed for the presence of a
particular pathway intermediate or product. Presence of the pathway
intermediate or product can be determined by methods including, but
not limited to, gas chromatography (GC), mass spectroscopy (MS),
thin layer chromatography (TLC), high-performance liquid
chromatography (HPLC), liquid chromatography (LC), GC coupled with
a flame ionization detector (GC-FID), GC-MS, and LC-MS. The
presence of an oc-FA pathway intermediate or product in the test
cell culture sample(s), and the absence (or a reduced amount) of
the oc-FA pathway intermediate or product in the parent cell
culture sample(s), indicates that the test polypeptide is effective
in carrying out an oc-FA pathway reaction and is suitable for use
in a recombinant microbial cell of the invention.
Production of Odd Chain Fatty Acid Derivatives in Recombinant
Microbial Cells
[0231] In one aspect, the invention includes a method of making an
odd chain fatty acid derivative composition, the method comprising
culturing a recombinant microbial cell of the invention in a
culture medium containing a carbon source under conditions
effective to express the recombinant polynucleotide sequences, and
optionally isolating the produced odd chain fatty acid derivative
composition.
[0232] An "odd chain fatty acid derivative composition"
(abbreviated "oc-FA derivative composition") is a composition
comprising an odd chain fatty acid derivative as defined herein,
such as, for example, an odd chain fatty acid, an odd chain fatty
ester (e.g., an odd chain fatty methyl ester, an odd chain fatty
ethyl ester, an odd chain wax ester), an odd chain fatty aldehyde,
an odd chain fatty alcohol, an even chain hydrocarbon (such as an
even chain alkane, an even chain alkene, an even chain terminal
olefin, an even chain internal olefin), or an even chain ketone.
Similarly, an "odd chain fatty acid composition" is a composition
comprising odd chain fatty acids, an "odd chain fatty alcohol
composition" is a composition comprising odd chain fatty alcohols,
an "even chain alkane composition" is a composition comprising even
chain alkanes, and so on. It is to be understood that a composition
comprising odd chain fatty acid derivatives may also comprise even
chain fatty acid derivatives.
[0233] In one aspect, the invention includes a method of making a
composition comprising an odd chain fatty acid derivative, the
method comprising: obtaining a recombinant microbial cell (such as,
a culture comprising a recombinant microbial cell) comprising: (a)
polynucleotides encoding polypeptides having enzymatic activities
effective to produce an increased amount of propionyl-CoA in the
recombinant microbial cell, relative to the amount of propionyl-CoA
produced in a parental microbial cell lacking or having a reduced
amount of said enzymatic activity, wherein at least one polypeptide
is exogenous to the recombinant microbial cell or wherein
expression of at least one polynucleotide is modulated in the
recombinant microbial cell as compared to the expression of the
polynucleotide in the parental microbial cell; (b) a polynucleotide
encoding a polypeptide having .beta.-ketoacyl-ACP synthase activity
that utilizes propionyl-CoA as a substrate; and (c) one or more
polynucleotides encoding a polypeptide having fatty acid derivative
enzyme activity, wherein the recombinant microbial cell produces a
fatty acid derivative composition comprising odd chain fatty acid
derivatives and even chain fatty acid derivatives when cultured in
the presence of a carbon source under conditions effective to
express the polynucleotides according to (a), (b), and (c);
culturing the recombinant microbial cell in a culture medium
containing a carbon source under conditions effective to express
the polynucleotides according to (a), (b), and (c) and produce a
fatty acid derivative composition comprising odd chain fatty acid
derivatives and even chain fatty acid derivatives, and optionally
recovering the composition from the culture medium.
[0234] In some embodiments, at least 5%, at least 10%, at least
20%, at least 30%, at least 40%, at least 50%, at least 60%, at
least 70%, at least 80% or at least 90% by weight of the fatty acid
derivatives in the composition are odd chain fatty acid
derivatives. In some embodiments, the fatty acid derivative
composition comprises odd chain fatty acid derivatives in an amount
(e.g., a titer) of at least 10 mg/L, at least 15 mg/L, at least 20
mg/L, at least 25 mg/L, at least 50 mg/L, at least 75 mg/L, at
least 100 mg/L, at least 125 mg/L, at least 150 mg/L, at least 175
mg/L, at least 200 mg/L, at least 225 mg/L, at least 250 mg/L, at
least 275 mg/L, at least 300 mg/L, at least 325 mg/L, at least 350
mg/L, at least 375 mg/L, at least 400 mg/L, at least 425 mg/L, at
least 450 mg/L, at least 475 mg/L, at least 500 mg/L, at least 525
mg/L, at least 550 mg/L, at least 575 mg/L, at least 600 mg/L, at
least 625 mg/L, at least 650 mg/L, at least 675 mg/L, at least 700
mg/L, at least 725 mg/L, at least 750 mg/L, at least 775 mg/L, at
least 800 mg/L, at least 825 mg/L, at least 850 mg/L, at least 875
mg/L, at least 900 mg/L, at least 925 mg/L, at least 950 mg/L, at
least 975 mg/L, at least 1000 mg/L, at least 1050 mg/L, at least
1075 mg/L, at least 1100 mg/L, at least 1125 mg/L, at least 1150
mg/L, at least 1175 mg/L, at least 1200 mg/L, at least 1225 mg/L,
at least 1250 mg/L, at least 1275 mg/L, at least 1300 mg/L, at
least 1325 mg/L, at least 1350 mg/L, at least 1375 mg/L, at least
1400 mg/L, at least 1425 mg/L, at least 1450 mg/L, at least 1475
mg/L, at least 1500 mg/L, at least 1525 mg/L, at least 1550 mg/L,
at least 1575 mg/L, at least 1600 mg/L, at least 1625 mg/L, at
least 1650 mg/L, at least 1675 mg/L, at least 1700 mg/L, at least
1725 mg/L, at least 1750 mg/L, at least 1775 mg/L, at least 1800
mg/L, at least 1825 mg/L, at least 1850 mg/L, at least 1875 mg/L,
at least 1900 mg/L, at least 1925 mg/L, at least 1950 mg/L, at
least 1975 mg/L, at least 2000 mg/L, at least 3000 mg/L, at least
4000 mg/L, at least 5000 mg/L, at least 6000 mg/L, at least 7000
mg/L, at least 8000 mg/L, at least 9000 mg/L, at least 10000 mg/L,
at least 20000 mg/L, or a range bounded by any two of the foregoing
values.
[0235] In various embodiments, the fatty acid derivative enzyme
activity comprises a thioesterase activity, an ester synthase
activity, a fatty aldehyde biosynthesis activity, a fatty alcohol
biosynthesis activity, a ketone biosynthesis activity, and/or a
hydrocarbon biosynthesis activity. In some embodiments, the
recombinant microbial cell comprises polynucleotides encoding two
or more polypeptides, each polypeptide having a fatty acid
derivative enzyme activity.
[0236] In various embodiments, the recombinant microbial cell
produces a composition comprising odd chain fatty acids, odd chain
fatty esters, odd chain wax esters, odd chain fatty aldehydes, odd
chain fatty alcohols, even chain alkanes, even chain alkenes, even
chain internal olefins, even chain terminal olefins, or even chain
ketones.
[0237] In various embodiments, the recombinant microbial cell
comprises polynucleotides encoding polypeptides having enzymatic
activities effective to produce an increased amount of
propionyl-CoA in the recombinant microbial cell, selected from: (i)
polynucleotides encoding polypeptides having aspartokinase
activity, homoserine dehydrogenase activity, homoserine kinase
activity, threonine synthase activity, and threonine deaminase
activity, or (ii) polynucleotides encoding polypeptides having
(R)-citramalate synthase activity, isopropylmalate isomerase
activity, and beta-isopropyl malate dehydrogenase activity, or
(iii) polypeptides having methylmalonyl-CoA mutase activity,
methylmalonyl-CoA decarboxylase activity and/or methylmalonyl-CoA
carboxyltransferase activity, or (i) and (ii), or (i) and (iii), or
(ii) and (iii), or (i), (ii), and (iii), wherein at least one
polypeptide is exogenous to the recombinant microbial cell, or
wherein expression of at least one polynucleotide is modulated in
the recombinant microbial cell as compared to the expression of the
polynucleotide in the parental microbial cell.
[0238] The fatty acid derivative compositions comprising odd chain
fatty acid derivatives produced by the methods of invention may be
recovered or isolated from the recombinant microbial cell culture.
The term "isolated" as used herein with respect to products, such
as fatty acid derivatives, refers to products that are separated
from cellular components, cell culture media, or chemical or
synthetic precursors. The fatty acid derivatives produced by the
methods described herein can be relatively immiscible in the
fermentation broth, as well as in the cytoplasm. Therefore, the
fatty acid derivatives can collect in an organic phase either
intracellularly or extracellularly. The collection of the products
in the organic phase can lessen the impact of the fatty acid
derivative on cellular function and can allow the recombinant
microbial cell to produce more product.
[0239] In some embodiments, the fatty acid derivative composition
(which comprises odd chain fatty acid derivatives) produced by the
methods of invention are purified. As used herein, the term
"purify," "purified," or "purification" means the removal or
isolation of a molecule from its environment by, for example,
isolation or separation. "Substantially purified" molecules are at
least about 60% free (e.g., at least about 70% free, at least about
75% free, at least about 85% free, at least about 90% free, at
least about 95% free, at least about 97% free, at least about 99%
free) from other components with which they are associated. As used
herein, these terms also refer to the removal of contaminants from
a sample. For example, the removal of contaminants can result in an
increase in the percentage of a fatty acid derivative (such as, a
fatty acid or a fatty alcohol or a fatty ester or a hydrocarbon)
relative to other components in a sample. For example, when a fatty
ester or a fatty alcohol is produced in a recombinant microbial
cell, the fatty ester or fatty alcohol can be purified by the
removal of recombinant microbial cell proteins. After purification,
the percentage of the fatty ester or fatty alcohol in the sample
relative to other components is increased.
[0240] As used herein, the terms "purify," "purified," and
"purification" are relative terms which do not require absolute
purity. Thus, for example, when a fatty acid derivative composition
is produced in recombinant microbial cells, a purified fatty acid
derivative composition is a fatty acid derivative composition that
is substantially separated from other cellular components (e.g.,
nucleic acids, polypeptides, lipids, carbohydrates, or other
hydrocarbons).
[0241] The fatty acid derivative composition (which comprises odd
chain fatty acid derivatives) may be present in the extracellular
environment, or it may be isolated from the extracellular
environment of the recombinant microbial cell. In certain
embodiments, the fatty derivative is secreted from the recombinant
microbial cell. In other embodiments, the fatty acid derivative is
transported into the extracellular environment. In yet other
embodiments, the fatty acid derivative is passively transported
into the extracellular environment. The fatty acid derivative can
be isolated from a recombinant microbial cell using methods known
in the art.
[0242] Fatty acid derivatives (including odd chain fatty acid
derivatives produced according to the methods of the present
invention) can be distinguished from organic compounds derived from
petrochemical carbon on the basis of dual carbon-isotopic
fingerprinting or .sup.14C dating. Additionally, the specific
source of biosourced carbon (e.g., glucose vs. glycerol) can be
determined by dual carbon-isotopic fingerprinting (see, e.g., U.S.
Pat. No. 7,169,588).
[0243] The ability to distinguish fatty acid derivatives produced
by recombinant microbial cells from petroleum-based organic
compounds is beneficial in tracking these materials in commerce.
For example, organic compounds or chemicals comprising both
biologically-based and petroleum-based carbon isotope profiles may
be distinguished from organic compounds and chemicals made only of
petroleum-based materials. Hence, the materials prepared in
accordance with the inventive methods may be followed in commerce
on the basis of their unique carbon isotope profile.
[0244] Fatty acid derivatives produced by recombinant microbial
cells can be distinguished from petroleum-based organic compounds
by comparing the stable carbon isotope ratio (.sup.13C/.sup.12C) in
each fuel. The .sup.13C/.sup.12C ratio in a given fatty acid
derivative thereof produced according to the methods of the
invention is a consequence of the .sup.13C/.sup.12C ratio in
atmospheric carbon dioxide at the time the carbon dioxide is fixed.
It also reflects the precise metabolic pathway. Regional variations
also occur. Petroleum, C.sub.3 plants (the broadleaf), C.sub.4
plants (the grasses), and marine carbonates all show significant
differences in .sup.13C/.sup.12C and the corresponding
.delta..sup.13C values. Furthermore, lipid matter of C.sub.3 and
C.sub.4 plants analyze differently than materials derived from the
carbohydrate components of the same plants as a consequence of the
metabolic pathway.
[0245] The .sup.13C measurement scale was originally defined by a
zero set by Pee Dee Belemnite (PDB) limestone, where values are
given in parts per thousand deviations from this material. The
".delta..sup.13C" values are expressed in parts per thousand (per
mil), abbreviated, % o, and are calculated as follows:
.delta..sup.13C(%
o)=[(.sup.13C/.sup.12C).sub.sample-(.sup.13C/.sup.12C).sub.standard]/(.su-
p.13C/.sup.12C).sub.standard.times.1000
[0246] In some embodiments, a fatty acid derivative produced
according to the methods of the invention has a .delta..sup.13C of
about -30 or greater, about -28 or greater, about -27 or greater,
about -20 or greater, about -18 or greater, about -15 or greater,
about -13 or greater, or about -10 or greater. Alternatively, or in
addition, a fatty acid derivative has a .delta..sup.13C of about -4
or less, about -5 or less, about -8 or less, about -10 or less,
about -13 or less, about -15 or less, about -18 or less, or about
-20 or less. Thus, the fatty acid derivative can have a
.delta..sup.13C bounded by any two of the above endpoints. For
example, a fatty acid derivative can have a .delta..sup.13C of
about -30 to about -15, about -27 to about -19, about -25 to about
-21, about -15 to about -5, about -13 to about -7, or about -13 to
about -10. In some embodiments, a fatty acid derivative can have a
.delta..sup.13C of about -10, -11, -12, or -12.3. In other
embodiments, a fatty acid derivative has a .delta..sup.13C of about
-15.4 or greater. In yet other embodiments, a fatty acid derivative
has a .delta..sup.13C of about -15.4 to about -10.9, or a
.delta..sup.13C of about -13.92 to about -13.84.
[0247] A fatty acid derivative produced by a recombinant microbial
cell can also be distinguished from petroleum-based organic
compounds by comparing the amount of .sup.14C in each compound.
Because .sup.14C has a nuclear half-life of 5730 years, petroleum
based fuels containing "older" carbon can be distinguished from
fatty acids or derivatives thereof which contain "newer" carbon
(see, e.g., Currie, "Source Apportionment of Atmospheric
Particles", Characterization of Environmental Particles, J. Buffle
and H. P. van Leeuwen, Eds., Vol. I of the IUPAC Environmental
Analytical Chemistry Series, Lewis Publishers, Inc., pp. 3-74
(1992)).
[0248] As used herein, "fraction of modern carbon" or f.sub.M has
the same meaning as defined by National Institute of Standards and
Technology (NIST) Standard Reference Materials (SRMs) 4990B and
4990C, known as oxalic acids standards HOxI and HOxII,
respectively. The fundamental definition relates to 0.95 times the
.sup.14C/.sup.12C isotope ratio HOxI (referenced to AD 1950). This
is roughly equivalent to decay-corrected pre-Industrial Revolution
wood. For the current living biosphere (plant material), f.sub.M is
approximately 1.1.
[0249] In some embodiments, a fatty acid derivative produced
according to the methods of the invention has a f.sub.m.sup.14C of
at least about 1, e.g., at least about 1.003, at least about 1.01,
at least about 1.04, at least about 1.111, at least about 1.18, or
at least about 1.124. Alternatively, or in addition, the fatty acid
derivative has an f.sub.m.sup.14C of about 1.130 or less, e.g.,
about 1.124 or less, about 1.18 or less, about 1.111 or less, or
about 1.04 or less. Thus, the fatty acid derivative can have a
f.sub.m.sup.14C bounded by any two of the above endpoints. For
example, the fatty acid derivative can have a f.sub.m.sup.14C of
about 1.003 to about 1.124, a f.sub.m.sup.14C of about 1.04 to
about 1.18, or a f.sub.m.sup.14C of about 1.111 to about 1.124.
[0250] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0251] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language ("e.g.", "such as", "for example") provided
herein, is intended merely to better illuminate the invention and
does not pose a limitation on the scope of the invention unless
otherwise claimed. No language in the specification should be
construed as indicating any non-claimed element as essential to the
practice of the invention.
[0252] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
EXAMPLES
Media Compositions
[0253] Che-9 media: M9 supplemented with extra NH.sub.4Cl (an
additional 1 g/L), Bis-Tris buffer (0.2 M), Triton X-100 (0.1%
v/v), and trace minerals (27 mg/L FeCl.sub.3.6 H.sub.2O, 2 mg/L
ZnC1.4H.sub.2O, 2 mg/L CaCl.sub.2. 6H.sub.2O, 2 mg/L
Na.sub.2MoO.sub.4.2H.sub.2O, 1.9 mg/L CuSO.sub.4.5H.sub.2O, 0.5
mg/L H.sub.3B0.sub.3, 100 mL/L concentrated HCl).
[0254] 2NBT: Che-9 supplemented with 20 g/L (2% w/v) glucose.
[0255] 4NBT: Che-9 supplemented with 40 g/L (4% w/v) glucose.
Example 1
Bacterial Strains and Plasmids
[0256] E. coli MG1655 .DELTA.fadE (Strain "D1")
[0257] This example describes the construction of a recombinant
microbial cell in which the expression of a fatty acid degradation
enzyme is attenuated. The fadE gene of E. coli (also known as
yafH), which encodes an acyl coenzyme A dehydrogenase (GenBank
Accession No. AAC73325) involved in fatty acid degradation, was
deleted from E. coli strain MG1655 using the Red system described
by Datsenko, K. A. et al. (Proc. Natl. Acad. Sci. USA 97: 6640-6645
(2000)), with the following modifications.
[0258] The following two primers were used to create the deletion
of fadE:
TABLE-US-00007 Del-fadE-F (SEQ ID NO: 82) 5'AAAAACAGCA ACAATGTGAG
CTTTGTTGTAATTAT ATTGTAA ACATATT GATTCCGGGGATCCGTCGACC; and
Del-fadE-R (SEQ ID NO: 83) 5'AAACGGAGCCT TTCGGCTCCGTTATT
CATTTACGCGGCTTCAAC TTTCCTG TAGGCTGGAGCTGCTTC
[0259] The Del-fadE-F and Del-fadE-R primers were used to amplify
the kanamycin resistance (Km.sup.R) cassette from plasmid pKD13
(Datsenko et al., supra) by PCR. The PCR product was then used to
transform electrocompetent E. coli MG1655 cells containing plasmid
pKD46, which expresses Red recombinase (Datsenko et al., supra),
which had been previously induced with arabinose for 3-4 hours.
Following a 3-hour outgrowth in SOC medium at 37.degree. C., the
cells were plated on Luria agar plates containing 50 .mu.g/mL of
kanamycin. Resistant colonies were identified and isolated after an
overnight incubation at 37.degree. C. Disruption of the fadE gene
was confirmed in some of the colonies by PCR amplification using
primers fadE-L2 and fadE-R1, which were designed to flank the E.
coli fadE gene.
TABLE-US-00008 fadE-L2 (SEQ ID NO: 84) 5'-CGGGCAGGTGCTATGACCAGGAC;
and fadE-R1 (SEQ ID NO: 85) 5'-CGCGGCGTTGACCGGCAGCCTGG
[0260] After the fadE deletion was confirmed, a single colony was
used to remove the Km.sup.R marker using the pCP20 plasmid
(Datsenko et al., supra). The resulting MG1655 E. coli strain with
the fadE gene deleted and the Km.sup.R marker removed was
designated E. coli MG1655 .DELTA.fadE, or strain "D1".
E. coli MG1655 .DELTA.fadE_.DELTA.tonA (Strain "DV2")
[0261] This example describes the construction of a recombinant
microbial cell in which the expression of a fatty acid degradation
enzyme and the expression of an outer membrane protein receptor are
attenuated. The tonA (also known as fhuA) gene of E. coli MG1655,
which encodes a ferrichrome outer membrane transporter which also
acts as a bacteriophage receptor (GenBank Accession No.
NP.sub.--414692) was deleted from strain D1 (described above) using
the Red system according to Datsenko et al., supra, with the
following modifications:
[0262] The primers used to create the tonA deletion were:
TABLE-US-00009 Del-tonA-F (SEQ ID NO: 86)
5'-ATCATTCTCGTTTACGTTATCATTCACTTTACATCAGAGATATAC
CAATGATTCCGGGGATCCGTCGACC; and Del-tonA-R (SEQ ID NO: 87)
5'-GCACGGAAATCCGTGCCCCAAAAGAGAAATTAGAAACGGAAG GTTGCGG
TTGTAGGCTGGAGCTGCTTC
[0263] The Del-tonA-F and Del-tonA-R primers were used to amplify
the kanamycin resistance (Km.sup.R) cassette from plasmid pKD13 by
PCR. The PCR product obtained in this way was used to transform
electrocompetent E. coli MG1655 D1 cells containing pKD46 (Datsenko
et al., supra), which cells had been previously induced with
arabinose for 3-4 hours. Following a 3-hour outgrowth in SOC medium
at 37.degree. C., cells were plated on Luria agar plates containing
50 .mu.g/mL of kanamycin. Resistant colonies were identified and
isolated after an overnight incubation at 37.degree. C. Disruption
of the tonA gene was confirmed in some of the colonies by PCR
amplification using primers flanking the E. coli tonA gene:
tonA-verF and tonA-verR:.
TABLE-US-00010 tonA-verF (SEQ ID NO: 88) 5'-CAACAGCAACCTGCTCAGCAA;
and tonA-verR (SEQ ID NO: 89) 5'-AAGCTGGAGCAGCAAAGCGTT
[0264] After the tonA deletion was confirmed, a single colony was
used to remove the Km.sup.R marker using the pCP20 plasmid
(Datsenko et al., supra). The resulting MG1655 E. coli strain
having fadE and tonA gene deletions was designated E. coli MG1655
.DELTA.fadE .DELTA.tonA, or strain "DV2".
E. coli MG1655 .DELTA.fadE_.DELTA.tonA lacI:tesA (Strain "DV2
`tesA")
[0265] This example describes the construction of a recombinant
microbial cell comprising a polynucleotide encoding a polypeptide
having a fatty acid derivative enzyme activity. The tesA
polynucleotide sequence encoding E. coli acyl-CoA thioesterase I
(EC 3.1.1.5, 3.1.2.-; e.g., GenBank Accession AAC73596; SEQ ID
NO:64) was modified to remove the leader sequence, such that the
resulting `tesA gene product was truncated by 25 amino acids and
the amino acid at the original position 26, alanine, was replaced
with methionine, which then became the first amino acid of the
`TesA polypeptide sequence (SEQ ID NO:65; Cho et al., J. Biol.
Chem., 270:4216-4219 (1995)).
[0266] An integration cassette containing the `tesA coding sequence
operatively linked to the P.sub.Trc promoter plus a kanamycin
resistance gene was PCR-amplified from plasmid pACYC-P.sub.Trc-tesA
(Example 1, below) using the primers lad-forward:
GGCTGGCTGGCATAAATATCTC (SEQ ID NO:90) and lacZ-reverse:
GCGTTAAAGTTGTTCTGCTTCATCAGCAGGATATCCTGCACCATCGTCTGGATTTTGAACT
TTTGCTTTGCCACGGAAC (SEQ ID NO:91), electroporated into strain DV2
and integrated into the chromosome using Red recombinase expressed
from the pKD46 plasmid (Datsenko et al., supra). The transformants
were selected on LB plates supplemented with kanamycin. Correct
integration was assessed using diagnostic PCR.
pDG2 Expression Vector
[0267] The pDG2 expression vector was the base plasmid for many of
the constructs described below. The pCDFDuet-1 vector (Novagen/EMD
Biosciences) carries the CloDF13 replicon, lad gene and
streptomycin/spectinomycin resistance gene (aadA). To construct the
pDG2 plasmid, the C-terminal portion of the plsX gene, which
contains an internal promoter for the downstream fabH gene
(Podkovyrov and Larson, Nucl. Acids Res. (1996) 24 (9): 1747-1752
(1996)) was amplified from E. coli MG1655 genomic DNA using primers
5'-TGAATTCCATGGCGCAACTCACTCTTCTTTTAGTCG-3' (SEQ ID NO:92) and
5'-CAGTACCTCGAGTCTTCGTATACATATGCGCT CAGTCAC-3' (SEQ ID NO:93) These
primers introduced NcoI and XhoI restriction sites near the ends,
as well as an internal NdeI site.
[0268] Both the plsX insert (containing the EcfabH promoter), and
the pCDFDuet-1 vector, were digested with restriction enzymes NcoI
and XhoI. The cut vector was treated with Antarctic phosphatase.
The insert was ligated into the vector and transformed into
transformation-competent E. coli cells. Clones were screened by DNA
sequencing. The pDG2 plasmid sequence is provided herein as SEQ ID
NO:73.
FabH Expression Plasmids
[0269] The pDG6 plasmid, expressing B. subtilis FabH1, was
constructed using the pDG2 plasmid. The fabH1 coding sequence was
amplified from Bacillus subtilis strain 168 using primers
5'-CCTTGGGGCATATGAAAGCTG-3' (SEQ ID NO:94) and
5'-TTTAGTCATCTCGAGTGCACCTCACCTTT-3' (SEQ ID NO:95). These primers
introduced NdeI and XhoI restriction sites at the ends of the
amplification product.
[0270] Both the fabH1 insert and the pDG2 vector were digested with
restriction enzymes NdeI and XhoI. The cut vector was treated with
Antarctic phosphatase. The insert was ligated into the vector and
transformed into transformation-competent E. coli cells. Clones
were screened by DNA sequencing. The pDG6 plasmid sequence is
provided herein as SEQ ID NO:74, and expresses the B. subtilis
FabH1 polypeptide (SEQ ID NO:2) under the control of the EcfabH
promoter.
[0271] Other plasmids based on pDG2 were prepared using a similar
strategy as employed for the pDG6 plasmid. Plasmid pDG7 comprises a
Bacillus subtilis fabH2 coding sequence which expresses the B.
subtilis FabH2 polypeptide (SEQ ID NO:3). Plasmid pDG8 comprises a
Streptomyces coelicolor fabH coding sequence which expresses the S.
coelicolor FabH polypeptide (SEQ ID NO:4).
pACYC-P.sub.Trc-tesA and pACYC-P.sub.Trc2-tesA Plasmids
[0272] Plasmid pACYC-P.sub.Trc was constructed by PCR amplifying
the ladI.sup.q, P.sub.Trc promoter and terminator region from
pTrcHis2A (Invitrogen, Carlsbad, Calif.) using primers
pTrc_F TTTCGCGAGGCCGGCCCCGCCAACACCCGCTGACG (SEQ ID NO:96) and
pTrc_R AAGGACGTCTTAATTAATCAGGAGAGCGTTCACCGACAA (SEQ ID NO:97)
[0273] The PCR product was then digested with AatII and NruI and
inserted into plasmid pACYC177 (Rose, R. E., Nucleic Acids Res.,
16:356 (1988)) digested with AatII and ScaI. The nucleotide
sequence of the pACYC-P.sub.Trc vector is provided herein as SEQ ID
NO: 75.
[0274] To generate the pACYC-P.sub.Trc2 plasmid, a single point
mutation was introduced in the P.sub.Trc promoter of the
pACYC-P.sub.Trc plasmid to generate the variant promoter P.sub.Trc2
and the pACYC-P.sub.Trc2 plasmid. The wild-type P.sub.Trc promoter
sequence is provided herein as SEQ ID NO:76, and the P.sub.Trc2
variant promoter is provided herein as SEQ ID NO:77.
[0275] The nucleotide sequence encoding E. coli acyl-CoA
thioesterase I (TesA, EC 3.1.1.5, 3.1.2.-; e.g., GenBank Accession
AAC73596; SEQ ID NO:64) was modified to remove the leader sequence,
such that the resulting `tesA gene product was truncated by 25
amino acids and the amino acid at the original position 26,
alanine, was replaced with methionine, which then became the first
amino acid of the `TesA polypeptide (SEQ ID NO:65; Cho et al., J.
Biol. Chem., 270:4216-4219 (1995)). DNA encoding the `TesA
polypeptide was inserted into the NcoI and EcoRI sites of the
pACYC-P.sub.Trc vector and the pACYC-P.sub.Trc2 vector, producing
the pACYC-P.sub.Trc-`tesA and pACYC-P.sub.Trc2-`tesA plasmids,
respectively. Correct insertion of `tesA sequence into the plasmids
was confirmed by restriction digestion.
pOP80 Plasmid
[0276] The pOP80 plasmid was constructed by digesting the cloning
vector pCL1920 (GenBank AB236930; Lerner C. G. and Inouye M.,
Nucleic Acids Res. 18:4631 (1990)) with the restriction enzymes
AflII and SfoI. Three DNA fragments were produced by this
digestion. The 3737 bp fragment was gel-purified using a
gel-purification kit (Qiagen, Inc., Valencia, Calif.). In parallel,
a DNA sequence fragment containing the P.sub.Trc promoter and lacI
region from the commercial plasmid pTrcHis2 (Invitrogen, Carlsbad,
Calif.) was amplified by PCR using primers LF302
(5'-atatgacgtcGGCATCCGCTTACAGACA-3', SEQ ID NO:98) and LF303
(5'-aattcttaagTCAGGAGAGCGTTCACCGACAA-3', SEQ ID NO:99) introducing
the recognition sites for the ZraI and AflII enzymes, respectively.
After amplification, the PCR products were purified using a
PCR-purification kit (Qiagen, Inc. Valencia, Calif.) and digested
with ZraI and AflII following the recommendations of the supplier
(New England BioLabs Inc., Ipswich, Mass.). After digestion, the
PCR product was gel-purified and ligated with the 3737 bp DNA
sequence fragment derived from pCL1920 to generate the expression
vector pOP80 containing the P.sub.Trc promoter.
L. monocytogenes fabH1 and fabH2 Plasmids (pTB.079 and pTB.081)
[0277] The genomic DNA of Listeria monocytogenes L123 (ATCC
19114D-5) was used as template to amplify the fabH gene using the
following primers:
TABLE-US-00011 TREE044 (fabH_forward) (SEQ ID NO: 100)
GAGGAATAAACCATGAACGCAGGAATTTTAGGAGTAG; primer 61 (fabH_reverse)
(SEQ ID NO: 101) CCCAAGCTTCGAATTCTTACTTACCCCAACGAATGATTAGG
[0278] The PCR product was then cloned into the NcoI/EcoRI sites of
pDS80 (a pCL1920-based vector carrying the phage lambda P.sub.L
promoter; SEQ ID NO:78) and transformed into
transformation-competent E. coli cells. Individual colonies were
picked for sequence verification of cloned inserts. The nucleic
acid sequence of wild type L. monocytogenes fabH encodes the wild
type LmFabH1 protein (SEQ ID NO:7), and the plasmid expressing this
sequence was designated pTB.079.
[0279] A mutant L. monocytogenes fabH gene was discovered
containing a T to G change at position 928, resulting in a change
in the expressed protein at amino acid position 310 from Tryptophan
(W) to Glycine (G), i.e., a W310G variant. The mutant L.
monocytogenes fabH gene encoding the FabH W310G variant (SEQ ID NO:
8) was designated LmFabH2, and the plasmid expressing this sequence
pTB.081.
pOP80-Based fabH Expression Plasmids
[0280] Each gene was PCR amplified from the indicated template and
primers (Table 6). The native sequence versions of each gene were
used, except for PffabH in which the E. coli codon-optimized
sequence was used (SEQ ID NO:150). Genes PffabH(opt), DpfabH1, and
DpfabH2 were synthesized by DNA2.0 (Menlo Park, Calif.). The
cloning vector was also PCR amplified with primers PTrc_vector_F
and PTrc_vector_R (Table 7), using plasmid OP80 as a template. The
different fabH genes were then cloned into the PCR-amplified OP80
vector backbone using InFusion cloning (Clontech, Mountain View
Calif.). The standard protocol, as outlined by the manufacturer,
was followed. All constructs were verified by sequencing.
TABLE-US-00012 TABLE 6 FabH genes, primers and templates Forward
PCR Reverse PCR Construct Gene primer primer Template Name BsfabH1
BsfabH1_IFF BsfabH1_IFR B. subtilis pCL-BsH1 genomic DNA BsfabH2
BsfabH2_IFF BsfabH2_IFR B. subtilis pCL-BsH2 genomic DNA LmfabH1
LmfabH1-2_IFF LmfabH1_IFR pTB.079 pCL-LmH LmfabH2 LmfabH1-2_IFF
LmfabH2_IFR pTB.081 pCL-LmH2 SmfabH SmfabH_IFF SmfabH_IFR
S.maltophila pCL-SmH genomic DNA PffabH(opt) PffabHopt_IFF
PffabHopt_IFR synthetic pCL-PfH (opt) AafabH AafabH_IFF AafabH_IFR
pSN-21 pCL-AaH DpfabH1 DpfabH1_IFF DpfabH1_IFR synthetic pCL-DpH1
DpfabH2 DpfabH2_IFF DpfabH2_IFR synthetic pCL-DpH2
TABLE-US-00013 TABLE 7 FabH primer sequences SEQ ID Primer Sequence
(5'.fwdarw.3') NO PTrc_vector_F GAATTCGAAGCTTGGGCCCGAAC 151
PTrc_vector_R CATGGTTTATTCCTCCTTATTTAATCGATAC 152 BsfabH1_IFF
GAGGAATAAACCATGAAAGCTGGAATACTTGGTGTTGGAC 153 BsfabH1_IFR
CCAAGCTTCGAATTCttaTCGGCCCCAGCGGATTGC 154 BsfabH2_IFF
GAGGAATAAACCATGTCAAAAGCAAAAATTACAGCTATCGGC 155 BsfabH2_IFR
CCAAGCTTCGAATTCttaCATCCCCCATTTAATAAGCAATCCTG 156 LmfabH1-2_IFF
GAGGAATAAACCATGAACGCAGGAATTTTAGGAGTAGG 157 LmfabH1_IFR
CCAAGCTTCGAATTCttaCTTACCCCAACGAATGATTAGGGC 158 LmfabH2_IFR
CCAAGCTTCGAATTCttaCTTACCCCCACGAATGATTAGGG 159 DpfabH1_IFF
GAGGAATAAACCATGaatagagcagttatcttgggaacc 160 DpfabH1_IFR
CCAAGCTTCGAATTCttaccaacgcatgagcagcgaacc 161 DpfabH2_IFF
GAGGAATAAACCATGactttgcgttacacccaggtc 162 DpfabH2_IFR
CCAAGCTTCGAATTCttaccagtcgatgcccagcatg 163 AafabH_IFF
GAGGAATAAACCATGTACAAGGCCGTGATTCGCG 164 AafabH_IFR
CCAAGCTTCGAATTCtcaATACTCCACCATCGCGCC 165 PffabHopt_IFF
GAGGAATAAACCATGATTGATAGCACACCGGAATGG 166 PffabHopt_IFR
CCAAGCTTCGAATTCttaCGGCAGAACAACAACACGACC 167 SmfabH_IFF
GAGGAATAAACCATGAGCAAGCGGATCTATTCGAGG 168 SmfabH_IFR
CCAAGCTTCGAATTCtcaATAGCGCAGCAGGGCCG 169
Example 2
Engineering E. coli for Production of Odd Chain Fatty Acids by
Pathway (A)
[0281] The following example describes the construction of
recombinant E. coli strains which express exogenous genes and/or
overexpress endogenous genes encoding enzymes which serve to
increase metabolic flux through the intermediates threonine and
.alpha.-ketobutyrate to propionyl-CoA by pathway (A) of FIG. 2,
leading to the increased production of odd chain acyl-ACPs and odd
chain fatty acid derivatives in these recombinant cells.
[0282] This example also demonstrates the effect on oc-FA
production of attenuating the expression of an endogenous gene and
replacing it with an exogenous gene; in this example, expression of
the endogenous E. coli fabH gene encoding .beta.-ketoacyl-ACP
synthase was attenuated by deletion of the gene, and
.beta.-ketoacyl-ACP synthase activity was supplied by expression of
the exogenous B. subtilis fabH1 gene.
DV2 P.sub.L thrA*BC
[0283] A recombinant E. coli strain was constructed in which
chromosomal genes involved in threonine biosynthesis were placed
under control of a strong chromosomally-integrated lambda P.sub.L
promoter, and one of the genes was mutated.
[0284] To introduce a single mutation in the native aspartokinase I
(thrA) gene, the gene was amplified from E. coli MG1655 DNA in two
parts. The first part was amplified using primers TREE026 and
TREE028 while the second part was amplified using TREE029 and
TREE030 (Table 6). The primers used to amplify the two components
contained overlapping sequences which were then used to "stitch"
the individual pieces together. The two PCR products were combined
in a single PCR reaction and primers TREE026 and TREE030 to amplify
the entire thrA gene. Primers TREE028 and TREE029 were designed to
create a mutation in the native thrA at codon 345, which resulted
in an S345F variant of aspartokinase I (SEQ ID NO: 21). This
mutation has been shown to eliminate feedback inhibition of the
enzyme by threonine in the host strain (Ogawa-Miyata, Y., et al.,
Biosci. Biotechnol. Biochem. 65:1149-1154 (2001); Lee J.-H., et
al., J. Bacteriol. 185: 5442-5451 (2003)). The modified version of
this gene was designated "thrA*".
[0285] The P.sub.L promoter was amplified using primers
Km_trc_overF and TREE027 (Table 8) using plasmid pDS80 (a
pCL1920-based vector carrying the phage lambda P.sub.L promoter;
SEQ ID NO:78) as a template. This fragment was then stitched to a
kanamycin resistance cassette flanked by FRT sites, which was
amplified from plasmid pKD13 using primers TREE025 and Km_trc_overR
(Table 8). The resulting PCR product containing the KmFRT cassette
and P.sub.L promoter was stitched to the thrA* PCR product. Primers
TREE025 and TREE030 were used to amplify the entire
KmFRT-P.sub.L-thrA* mutagenic cassette. These primers also contain
approximately 50 bp of homology to the integration site at the 5'
end and the entire thrA gene as homology on the 3' end, targeting
the cassette to the native thrA site in E. coli, which is part of
an operon comprising the thrA, thrB and thrC genes. This mutagenic
cassette was electroporated into the parental strain, E. coli DV2
(Example 1) containing the helper plasmid pKD46 expressing Red
recombinase (Datsenko et al., supra). Clones containing the
chromosomal integration were selected in the presence of kanamycin,
and verified by diagnostic PCR. The kanamycin marker was then
removed by expression of the pCP20 plasmid (Datsenko et al.,
supra). Proper integration and marker removal were verified by PCR
and sequencing. The resulting strain, in which the mutant thrA*
gene and the endogenous thrB and thrC genes were overexpressed by
the chromosomally-integrated lambda P.sub.L promoter, was
designated DV2 P.sub.L thrA*BC.
TABLE-US-00014 TABLE 8 Primers SEQ ID Primer Sequence
(5'.fwdarw.3') NO TREE025
CCTGACAGTGCGGGCTTTTTTTTTCGACCAAAGGTAACGAGGTAACAACC 102
GTGTAGGCTGGAGCTGCTTCG TREE026
GTATATATTAATGTATCGATTAAATAAGGAGGAATAAACCATGCGAGTGT 103 TGAAGTTCGGCG
TREE027 CTGATGTACCGCCGAACTTCAACACTCGCATGGTTTATTCCTCCTTATTT 104
AATCGATAC TREE028 GCGCCCGTATTTTCGTGGTGCTGATTAC 105 TREE029
GTAATCAGCACCACGTAAATACGGGCGC 106 TREE030 TCAGACTCCTAACTTCCATGAGAGG
107 Km_trc_ AATATTTGCCAGAACCGTTATGATGTCGGCATTCCGGGGATCC 108 overR
GTCGACC Km_trc_ CTTCGAACTGCAGGTCGACGGATCCCCGGAATGCCGACATCATAACGGTT
109 overF CTGGC EG238 GCTGATCATTAACTATCCGCTGGATGACC 110 TREE017
ACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTAAG 111 TREE018
TCACTGCCCGCTTTCC 112 TREE019
ACCGGCAGATCGTATGTAATATGCATGGTTTATTCCTCCTTATTTAATCG 113 ATACA
TREE020 ATGCATATTACATACGATCTGCC 114 TREE021
GGTCGACGGATCCCCGGAATTAAGCGTCAACGAAACCG 115 TREE022
GAAGCAGCTCCAGCCTACACCAGACGATGGTGCAGGAT 116 TREE023
GCAAAGACCAGACCGTTCATA 117 Kan/ ATTCCGGGGATCCGTCGACC 118 Chlor1 Kan/
TGTAGGCTGGAGCTGCTTCG 119 Chlor4
DV2 P.sub.L thrA*BC P.sub.L tdcB
[0286] The native E. coli catabolic threonine deaminase (tdcB) gene
(also known as threonine ammonia-lyase) was overexpressed by
integrating an extra copy of the gene into the lacZ locus and
placing it under the control of a strong chromosomally-integrated
lambda P.sub.L promoter.
[0287] Catabolic threonine deaminase catalyzes the degradation of
threonine to .alpha.-keto-butyrate, the first reaction of the
threonine degradation/isoleucine production pathway. The reaction
catalyzed likely involves initial elimination of water (hence the
earlier classification of this enzyme as a threonine dehydratase),
followed by isomerization and hydrolysis of the product with C--N
bond breakage. Increased expression of this gene has been shown to
dramatically increase levels of isoleucine in heterologous
organisms (Guillouet S. et al., Appl. Environ. Microbiol.
65:3100-3107 (1999)). Furthermore, threonine deaminase is
relatively resistant to isoleucine feedback mechanisms (Guillouet
et al., supra).
[0288] E. coli MG1655 genomic DNA was amplified using primers
TREE020 and TREE021 (Table 8) to obtain the native tdcB gene. At
the same time, primers Kan/Chlor 1 and Kan/Chlor 4 (Table 8) were
used to amplify an FRT-Kanamycin resistance cassette to be used for
integration selection/screening as previously described. Using E.
coli MG1655 genomic DNA as template, primers EG238 and TREE018
(Table 8) were used to amplify a region of homology 3' to the lacZ
integration site, while primers TREE022 and TREE023 (Table 8) were
used to amplify a region of homology 5' to the lacZ site. The
plasmid pDS80 (a pCL1920-based vector carrying the phage lambda
P.sub.L, promoter; SEQ ID NO:78) was used as a template to amplify
a fragment containing the P.sub.L promoter by using primers TREE017
and TREE018 (Table 8). Each of these fragments were designed with
overlaps for corresponding adjacent piece and were stitched
together using SOEing PCR techniques. The resulting P.sub.L tdcB
mutagenic cassette (approx. 4.3 kb) contained approximately 700 bp
of homology to the integration site at the 5' end and 750 bp of
homology to the integration site at the 3' end. The P.sub.L tdcB
mutagenic cassette was electroporated into the host strain, E. coli
DV2 P.sub.L thrA*BC (above) containing the helper plasmid, pKD46
(Datsenko et al., supra). Clones containing the chromosomal
integration were selected for in the presence of kanamycin, and
verified by PCR and sequencing analysis. The kanamycin marker was
then removed using the pCP22 plasmid (Datsenko et al., supra). The
resulting strain was designated DV2 P.sub.L, thrA*BC P.sub.L, tdcB.
The strain was transformed with the plasmid pACYC-p.sub.trc2-`tesA
(Example 1), which expressed a truncated form of E. coli tesA.
[0289] The strain was also transformed with plasmid pDG6 (Example
1) expressing the B. subtilis FabH1 enzyme. Fermentation
experiments were conducted, and the titers of free fatty acids
(FFA), odd chain fatty acids (oc-FA), and the fraction of FFA
produced as oc-FA were determined, as shown in Example 5 and Table
11. Alternatively, the strain can be transformed with a plasmid
expressing a different FabH polypeptide, such as, for example, pDG7
expressing B. subtilis FabH2, pDG8 expressing Streptomyces
coelicolor FabH, pTB.079 expressing Listeria monocytogenes FabH,
pTB.081 expressing a Listeria monocytogenes FabH W310G variant, or
a FabH plasmid described in Example 5 and Tables 12A-12C.
Fermentation experiments are conducted, and the titers of free
fatty acids (FFA), odd chain fatty acids (oc-FA), and the fraction
of FFA produced as oc-FA are determined.
DV2 P.sub.L-thrA*BC P.sub.T5-BsfabH1
[0290] A recombinant E. coli strain was constructed in which the B.
subtilis fabH1 gene was integrated into the chromosome and placed
under transcriptional control of the strong constitutive T5
promoter.
[0291] First, a PCR product was generated for the chromosomal
integration of a loxPcat integration cassette comprising a
chloramphenicol resistance gene, a T5 promoter (P.sub.T5), and
BsfabH1 coding sequence, at the site of the fadE deletion scar of
DV2 P.sub.L thrA*BC. The individual components of the integration
cassette were first PCR-amplified. The loxP-cat-loxP P.sub.T5
component was amplified from plasmid p100.38 (SEQ ID NO:79) using
primers TREE133 and TREE135 (Table 9). The BsfabH1 gene was
amplified from a plasmid carrying the BsfabH1 gene using primers
TREE134 and TREE136. Primers TREE133 and TREE136 contain the 5' and
3' 50 bp of homology sequence for integration. The primers used to
amplify the components contain overlapping sequence which were then
used to "stitch" the individual pieces together. The
loxP-cat-P.sub.T5 and BsfabH1 PCR products were stitched together
by combining both pieces in a single PCR reaction and using primers
TREE133 and TREE136 to amplify the final loxPcat-P.sub.T5-BsfabH1
integration cassette.
TABLE-US-00015 TABLE 9 Primers SEQ Primer ID Name Sequence NO
TREE133 AAAAACAGCAACAATGTGAGCTTTGTTGTAATTATATTGTAAACATA 120
TTGTCCGCTGTTTCTGCATTCTTACgt TREE134
GATGACGACGAACACGCATTaagGAGGTGAATAAGGAGGAATAAcat 121
ATGAAAGCTGGCATTCTTGGTGTTG TREE135
GTAACGTCCAACACCAAGAATGCCAGCTTTCATatgTTATTCCTCCT 122
TATTCACCTCcttAATGCGTGTTCG TREE136
AAACGGAGCCTTTCGGCTCCGTTATTCATTTACGCGGCTTCAACTTT 123
CCGTTATCGGCCCCAGCGGATTG TREE137
CGCAGTTTGCAAGTGACGGTATATAACCGAAAAGTGACTGAGCGTAC 124
atgATTCCGGGGATCCGTCGACC TREE138
GCAAATTGCGTCATGTTTTAATCCTTATCCTAGAAACGAACCAGCGC 125
GGATGTAGGCTGGAGCTGCTTCG TREE139 GCAGCGACAAGTTCCTCAGC 126 TREE140
CCGCAGAAGCTTCAGCAAACG 127 fadE-L2 CGGGCAGGTGCTATGACCAGGAC 128
fadE-R2 GGGCAGGATAAGCTCGGGAGG 129
[0292] The loxP-cat-P.sub.T5-BsfabH1 cassette was integrated using
the Red recombinase system (Datsenko, et al., supra). The
loxP-cat-P.sub.T5-BsfabH1 PCR product was used to transform
electrocompetent DV2 P.sub.L-thrA*BC cells containing plasmid
pKD46, which had been previously induced with arabinose for 3-4
hours at 30.degree. C. Following a 3 hour 37.degree. C. outgrowth
in SOC medium, cells were plated on Luria agar plates containing 17
.mu.g/mL chloramphenicol and incubated overnight at 37.degree. C.
Chloramphenicol-resistant colonies were screened by PCR for proper
integration of loxP-cat-P.sub.T5-BsfabH1. Primers fadE-L2 and
fadE-R2 (Table 9) which flank the chromosomal integration site,
were used to confirm the integration. Upon verification of
integration, the chloramphenicol marker gene was removed by
expressing a Cre recombinase which promotes recombination between
the two loxP sites that flank the chloramphenicol resistance gene.
The plasmid pJW168, which harbors the cre recombinase gene, was
transformed into strain DV2 P.sub.L-thrA*BC
loxP-cat-P.sub.T5-BsfabH1 and the marker was removed according to
the method described by Palmeros et al. (Gene 247:255-264 (2000)).
The resulting strain DV2 P.sub.L-thrA*BC P.sub.T5-BsfabH1 was
verified by sequencing.
DV2 P.sub.L-thrA*BC P.sub.T5-BsfabH1 .DELTA.EcfabH
[0293] A recombinant E. coli strain was constructed in which the
expression of an endogenous gene (in this instance, the fabH gene
of E. coli) was attenuated by deletion of that gene.
[0294] The fabH gene of E. coli was deleted from DV2
P.sub.L-thrA*BC P.sub.T5-BsfabH1 using the Red recombinase system
(Datsenko et al., supra). Primers TREE137 and TREE138 (Table 9),
were used to amplify the kanamycin resistance cassette from plasmid
pKD13 by PCR. The PCR product was then used to transform
electrocompetent DV2 P.sub.L-thrA*BCP.sub.T5-BsfabH1 cells
containing plasmid pKD46. Deletion of EcfabH and removal of the
kanamycin marker were carried out according to the method described
by Wanner and Datsenko, supra. Primers TREE139 and TREE140 were
used to confirm the deletion of EcfabH. The final markerless strain
was designated DV2 P.sub.L-thrA*BC P.sub.T5-BsfabH1
.DELTA.EcfabH.
DV2 P.sub.L-thrA*BC P.sub.L-tdcB P.sub.T5-BsfabH1 .DELTA.EcfabH
[0295] A recombinant E. coli strain was constructed containing
chromosomally-integrated genes overexpressing enzymes of pathway
(A) and step (D) of the oc-FA biosynthetic pathway shown in FIG. 2
and FIG. 1B, respectively. The P.sub.L-tdcB mutagenic cassette
(prepared as described above) was integrated into strain DV2
P.sub.L-thrA*BC P.sub.T5-BsfabH1 .DELTA.EcfabH to generate the
strain DV2 P.sub.L-thrA*BC P.sub.L-tdcB P.sub.T5-BsfabH1
.DELTA.EcfabH. In this strain, the integrated E. coli thrA*BC genes
and the integrated E. coli tdcB gene were both under the control of
strong lambda P.sub.L, promoters, the integrated B. subtilis fabH1
gene was under the control of the strong T5 promoter, and the
endogenous E. coli fabH gene was deleted. Fermentation experiments
were conducted, and the results are provided in Table 11.
Example 3
Engineering E. coli for Production of Odd Chain Fatty Acids by
Pathway (B)
[0296] The following example describes the construction of
recombinant E. coli strains which express exogenous genes and/or
overexpress endogenous genes encoding enzymes which serve to
increase metabolic flux through the intermediates citramalate and
.alpha.-ketobutyrate to propionyl-CoA by pathway (B) of FIG. 2,
leading to the increased production of odd chain acyl-ACPs and odd
chain fatty acid derivatives in these recombinant cells.
DV2 P.sub.Trc-cimA3.7 leuBCD
[0297] To prepare an E. coli strain overexpressing endogenous
leuBCD genes and an exogenous cimA3.7 gene, a PCR product was
generated for the chromosomal integration of a KmFRT cassette, a
P.sub.Trc promoter, and cimA3.7 between the endogenous chromosomal
E. coli leuA and leuB genes. This integration disrupted the native
leuABCD operon, placing cimA3.7 and leuBCD in an operon under
control of the strong IPTG-inducible promoter, P.sub.Trc.
[0298] DNA encoding CimA3.7 was synthesized by Geneart AG
(Regensburg, Germany). The DNA was cloned into the SfiI site of
plasmid pMK-RQ (kanR) (Geneart AG, Regensburg, Germany). Flanking
the coding sequence, a 5' KpnI restriction site and a 3' SacI
restriction site were introduced directly upstream of the ATG start
codon and immediately downstream of the TAA stop codon
respectively. The cimA 3.7 cloning vector was verified by
sequencing.
[0299] The individual components of the integration cassette were
PCR-amplified as follows. The KmFRT component was amplified from
plasmid pKD13 using primers TREE146 and Km_trc_overR (Table 10).
The P.sub.Trc promoter was amplified from pOP80 (Example 1) using
primers Km_trc_overF and TREE033.
[0300] The cimA3.7 coding sequence was amplified from the cimA 3.7
cloning vector described above using primers TREE032 and TREE035.
To provide the 3' homology sequence for integration, E. coli native
leuBC genes were amplified using E. coli genomic DNA and primers
TREE034 and TREE104. The forward primer TREE146, which was used to
amplify the KmFRT cassette, included the 5' 50 bp of homology
sequence for integration. Each of the primers used to amplify the
components contained overlapping sequence which were used to
"stitch" the individual pieces together. First, KmFRT and P.sub.Trc
were stitched together by combining both pieces in a single PCR
reaction and using primers TREE146 and TREE033 to amplify the
KmFRT-P.sub.Trc product. KmFRT P.sub.Trc was then stitched with
cimA3.7 using primers TREE146 and TREE035 to generate
KmFRT-P.sub.Trc-cimA3.7. The final piece, leuBC was stitched to
KmFRT-P.sub.trc-cimA3.7 using primers TREE146 and TREE104 to
generate the final integration cassette: KmFRT-P.sub.Trc-cimA3.7
leuBC.
TABLE-US-00016 TABLE 10 Primers SEQ Primer ID Name Primer Sequence
(5'.fwdarw.3') NO Km_trc_ov
CTTCGAACTGCAGGTCGACGGATCCCCGGAATGCCGACATCAT 130 erF AACGGTTCTGGC
Km_trc_ov AATATTTGCCAGAACCGTTATGATGTCGGCATTCCGGGGATCCG 131 erR
TCGACC TREE032 GTATATATTAATGTATCGATTAAATAAGGAGGAATAAACCatgatg 132
gtaaggatatttgatacaacac TREE033
ctaagtgttgtatcaaatatccttaccatcatGGTTTATTCCTCCTTATTTAATCGAT 133 AC
TREE034 gatttgttggctatagttagagaagttactggaaaattgTAACAAGGAAACCGTGTGA
134 TGTCGAAG TREE035
GTAATTCTTCGACATCACACGGTTTCCTTGTTAcaattttccagtaacttctct 135 aactatag
TREE104 GGTAGCGAAGGTTTTGCCCGGC 136 TREE106 GATTGGTGCCCCAGGTGACCTG
137 TREE146 GAGTTGCAACGCAAAGCTCAACACAACGAAAACAACAAGGAA 138
ACCGTGTGaGTGTAGGCTGGAGCTGCTTCG TREE151 CTTCCACGGCGTCGGCCTG 139
[0301] The KmFRT-P.sub.Trc-cimA3.7 leuBC cassette was integrated
into the E. coli genome using the Red recombinase system (Datsenko
et al., supra). The KmFRT-P.sub.Trc-cimA3.7 leuBC PCR product was
used to transform electrocompetent E. coli MG1655 DV2 cells
containing plasmid pKD46, which had been previously induced with
arabinose for 3-4 hours at 30.degree. C. Following a 3-hour
37.degree. C. outgrowth in SOC medium, cells were plated on Luria
agar plates containing 50 .mu.g/mL kanamycin and incubated
overnight at 37.degree. C. Kanamycin-resistant colonies were
screened by PCR for proper integration of KmFRT-P.sub.Trc-cimA3.7.
Primers TREE151 and TREE106, which flank the chromosomal
integration T.sub.Trc site, were used to confirm the integration.
Upon verification of integration, the kanamycin marker gene was
removed in accordance with the method described by Datsenko et al.,
supra. Successful integration of P.sub.Trc-cimA3.7 and removal of
the kanamycin marker gene in the final strain, DV2 P.sub.Trc
cimA3.7 leuBCD, was verified by sequencing.
[0302] The strain was transformed with the plasmid
pACYC-p.sub.trc2-tesA, which expressed a truncated form of E. coli
tesA, and, in some instances, pDG6, which expressed B. subtilis
fabH1. Fermentation experiments were conducted, and the titers of
free fatty acids (FFA), odd chain fatty acids (oc-FA), and the
fraction of FFA produced as oc-FA, are provided in Table 11.
Example 4
Engineering E. coli for Production of Odd Chain Fatty Acids by
Pathways (A) and (B) Combined
[0303] The following example describes the construction of
recombinant E. coli strains which express exogenous genes and/or
overexpress endogenous genes encoding enzymes which serve to
increase metabolic flux through the common intermediate
.alpha.-ketobutyrate to propionyl-CoA by the combined (A) and (B)
pathways of FIG. 2, leading to even greater production of
oc-acyl-ACPs and odd chain fatty acids in these recombinant
cells.
DV2 P.sub.L-thrA*BC P.sub.Trc-cimA3.7_leuBCD P.sub.T5-BsfabH1
.DELTA.EcfabH (Strain "G1")
[0304] To begin combining pathways (A) and (B) of FIG. 2, the
P.sub.Trc-cimA3.7_leuBCD cassette (Example 5) was integrated into
strain DV2 P.sub.L-thrA*BC P.sub.T5-BsfabH1 .DELTA.EcfabH (Example
4) to generate the strain DV2 P.sub.L-thrA*BC
P.sub.Trc-cimA3.7_leuBCD P.sub.T5-BsfabH1 .DELTA.EcfabH, which was
also called strain G1. This strain overexpressed polypeptides
having (R)-citramalate synthase activity, isopropylmalate isomerase
activity, and beta-isopropyl malate dehydrogenase activity
according to pathway (B) of the oc-FA pathway, and overexpressed
polypeptides having aspartokinase activity, homoserine
dehydrogenase activity, homoserine kinase activity, and threonine
synthase activity according to pathway (A) of the oc-FA pathway
(FIG. 2).
DV2 P.sub.L-thrA*BC P.sub.Trc-cimA3.7_leuBCDP.sub.T5-BsfabH1
.DELTA.EcfabH (Strain "G2")
[0305] To create a strain engineered to overexpress polypeptides
having activities corresponding to the combined pathways (A) and
(B) of the of the oc-FA pathway, the P.sub.L-tdcB cassette (Example
4) was integrated into strain G1, to generate strain DV2
P.sub.L-thrA*BC P.sub.L-tdcB P.sub.Trc-cimA3.7_leuBCD
P.sub.T5-BsfabH1 .DELTA.EcfabH, which was also called strain G2. In
this strain, the integrated E. coli thrA*BC genes and the
integrated E. coli tdcB gene (encoding polypeptides having
aspartokinase activity, homoserine dehydrogenase activity,
homoserine kinase activity, threonine synthase activity, and
threonine deaminase activity, corresponding to pathway (A)) were
placed under the control of strong lambda P.sub.L, promoters, and
were overexpressed. The exogenous cimA3.7 gene and the native E.
coli leuBCD genes (encoding polypeptides having (R)-citramalate
synthase activity, isopropylmalate isomerase activity, and
beta-isopropyl malate dehydrogenase activity corresponding pathway
(B)), were also integrated into the E. coli chromosome under
control of the strong IPTG-inducible promoter P.sub.Trc and
therefore were also overexpressed. The integrated B. subtilis fabH1
gene, encoding a branched chain beta ketoacyl-ACP synthase
corresponding to part (D) of the oc-FA pathway (FIG. 1B), was under
the control of the strong T5 promoter. The endogenous E. coli fabH
gene was deleted from this strain.
Example 5
Evaluation of Odd Chain Fatty Acid Production
[0306] The following example demonstrates the production of linear
odd-chain fatty acids in E. coli strains engineered to express
exogenous genes and/or overexpress endogenous genes encoding
enzymes which increase metabolic flux through the common
.alpha.-ketobutyrate intermediate to produce propionyl-CoA, by way
of either the threonine-dependent pathway (pathway (A) of FIG. 2)
or via the citramalate pathway (pathway (B) of FIG. 2).
Propionyl-CoA, which serves as a "primer" molecule for odd-chain
fatty acid production, then condenses with malonyl-ACP by the
action of .beta.-ketoacyl-ACP synthase III (FabH) to form the
odd-chain .beta.-ketoacyl-ACP intermediate which enters the fatty
acid synthase cycle to produce odd-chain fatty acids and oc-FA
derivatives. Accordingly, this example also demonstrates the effect
of exogenous FabH enzymes on odd-chain fatty acid production.
[0307] In the first set of experiments, strains were evaluated for
free fatty acid (FFA) production by performing a 96 deep-well plate
fermentation using the 4N-BT protocol. Single colonies or a
scraping from a glycerol stock were used to inoculate 300 .mu.L of
LB+antibiotic(s). LB seed cultures were grown for 6-8 hours at
37.degree. C. with shaking at 250 rpm until turbid. 20 .mu.L of the
LB cultures were used to inoculate 400 .mu.L of 2N-BT. These were
allowed to grow overnight at 32.degree. C. with shaking at 250 rpm.
The following morning, 20 .mu.L of 2N-BT culture was transferred to
400 .mu.L of 4N-BT. The 4N-BT cultures were allowed to grow for 6
hours at 32.degree. C. with shaking at 250 rpm at which point,
cells were induced with 1 mM IPTG. Upon induction, cultures were
allowed to grow for an additional 16-18 hours before being
extracted and analyzed for FFA production. 40 .mu.L of 1M HCl was
added to each well, followed by 400 .mu.L of butyl acetate spiked
with 500 mg/L C24 alkane internal standard. Cells were extracted by
vortexing for 15 minutes at 2000 rpm. Extracts were derivatized
with an equal volume of N,O-bis(trimethylsilyl)trifluoroacetamide
(BSTFA) before being analyzed by GC/MS.
TABLE-US-00017 TABLE 11 Production of Odd Chain Fatty Acids in
Recombinant E. coli Strains oc-FA/ Total Total Strain fabH tesA FFA
titer oc-FA titer FFA 1 DV2 Ec p 2054 6 <0.01 2 DV2 Ec p 1364
246 0.18 thrA*BC tdcB 3 DV2 Ec p 1460 545 0.37 thrA*BC tdcB pBsHl 4
DV2 .DELTA.Ec p 1148 832 0.72 thrA*BC tdcB IntBsH1 5 DV2 Ec p 1617
73 0.04 cimA3.7 leuBCD 6 DV2 Ec p 1650 214 0.13 cimA3.7 leuBCD
pBsH1 7 "G1": .DELTA.Ec p 1104 286 0.26 DV2 thrA*BC IntBsH1 cimA3.7
leuBCD 8 G1/Tn7-tesA .DELTA.Ec int 885 267 0.30 IntBsH1 9 "G2":
.DELTA.Ec p 617 551 0.89 DV2 thrA*BC tdcB IntBsH1 cimA3.7 leuBCD 10
G2/Tn7-tesA .DELTA.Ec int 923 840 0.91 IntBsH1 all titers are in
milligrams per liter (mg/L) FFA = free fatty acid (oc-FA + ec-FA)
oc-FA = odd chain fatty acid; ec-FA = even chain fatty acid Ec =
chromosomal (native) E. coli fabH gene .DELTA.Ec = deleted
chromosomal E. coli fabH gene pBsH1 = plasmid-expressed BsfabH1
(pDG6 plasmid) IntBsH1 = chromosomally integrated BsfabH1 p =
plasmid-expressed `tesA gene (pACYC-p.sub.Trc2-tesA) int =
chromosomally integrated `tesA gene
[0308] The odd chain fatty acids produced in these experiments
generally included C13:0, C15:0, C17:0 and C17:1 fatty acids, with
C15:0 being the predominant oc-FA produced.
[0309] Comparison of strains 1 and 2 in Table 11 demonstrates that
microbial cells overexpressing genes involved in the biosynthesis
and degradation of threonine, which increased metabolic flux
through the pathway intermediate .alpha.-ketobutyrate,
significantly increased the proportion of odd chain length fatty
acids produced by the cells. While the parental DV2 strain produced
straight chain fatty acids with only a negligible amount of odd
chain length fatty acids, the DV2 strain overexpressing the thrA*BC
and tdcB genes (encoding polypeptides having aspartokinase
activity, homoserine dehydrogenase activity, homoserine kinase
activity, threonine synthase activity and threonine deaminase
activity) produced a significantly greater amount and significantly
greater proportion of odd chain length fatty acids; about 18% (by
weight) of the straight chain fatty acids produced were odd chain
length fatty acids.
[0310] Strains 2 and 3 demonstrate the effect on oc-FA production
by including an exogenous .beta.-ketoacyl ACP synthase with high
specificity towards propionyl-CoA. Strain 2 contained the native
(endogenous) E. coli fabH gene. By introducing a plasmid expressing
the B. subtilis fabH1 gene, oc-FA production was markedly increased
from about 18% (in Strain 2) to about 37% of the straight chain
fatty acids produced (in Strain 3).
[0311] A striking effect on oc-FA production was observed when the
endogenous E. coli fabH gene was deleted and the B. subtilis fabH1
gene was chromosomally integrated. In Strain 4, the proportion of
oc-FA increased to 72% of the straight chain fatty acids
produced.
[0312] Strains 5 and 6 demonstrate that increasing metabolic flux
through .alpha.-ketobutyrate by another approach, this time by a
pathway involving citramalate biosynthesis and degradation, also
increased the proportion of odd chain length fatty acids produced.
Engineering the DV2 strain to overexpress the cimA3.7 and leuBCD
genes (encoding polypeptides having (R)-citramalate synthase
activity, isopropylmalate isomerase activity, and
.beta.-isopropylmalate dehydrogenase activity) resulted in about 4%
of the straight chain fatty acids produced having odd chain
lengths, which increased to about 13% when plasmid-expressed B.
subtilis fabH1 was included.
[0313] Strains 7 and 9 show the effect of combining the threonine
and citramalate pathways on oc-FA production. In strain G1, in
which the thrA*BC, cimA3.7 and leuBCD genes were overexpressed, the
endogenous E. coli fabH gene was deleted and the B. subtilis fabH1
gene was chromosomally integrated, about 26% of the straight chain
fatty acids produced were odd chain fatty acids. In strain G2, in
which the thrA*BC, tdcB, cimA3.7 and leuBCD genes were
overexpressed, the endogenous E. coli fabH gene was deleted and the
B. subtilis fabH1 gene was chromosomally integrated, nearly 90% of
the straight chain fatty acids produced were odd chain fatty acids.
Strains G1/Tn7-tesA and G/Tn7-tesA (strains 8 and 10,
respectively), in which the `tesA gene was chromosomally integrated
at the Tn7 attachment site, showed amounts and proportions of oc-FA
similar to those in strains G1 and G2 (strains 7 and 9,
respectively) in which the `tesA gene was plasmid-expressed.
[0314] In the second set of experiments, the role of propionyl-CoA
production and the effect of FabH enzymes on oc-FA production was
examined. In these experiments, exogenous fabH coding sequences
were cloned into the pOP80 expression vector (Example 1), where
expression was controlled by the strong P.sub.Trc promoter. The
fabH expression constructs (or, in strains lacking exogenous fabH,
the pOP80 vector alone) were transformed, along with `tesA plasmid
pACYC-PTrc2-tesA, into the following strains:
[0315] DV2
[0316] DV2 cimA3.7_leuBCD (increased propionyl-CoA via the
citramalate pathway (B) of FIG. 2)
[0317] DV2 thrA*BC tdcB (increased propionyl-CoA via the
thr-dependent pathway (A) of FIG. 2)
[0318] Single colonies or a scraping from a frozen glycerol stock
were used to inoculate 300 .mu.L of LB+antibiotic(s). LB seed
cultures were grown for 6-8 hours at 37.degree. C. with shaking at
250 rpm until turbid. 20 .mu.L of the LB cultures were used to
inoculate 400 .mu.L of 2N-BT media. These were allowed to grow
overnight at 32.degree. C. with shaking at 250 rpm, at least 14
hours. The following morning, 20 .mu.L of 2N-BT culture was
transferred to 400 .mu.L of 4N-BT. The 4N-BT cultures were allowed
to grow for 6 hours at 32.degree. C. with shaking at 250 rpm at
which point, cells were induced with 1 mM IPTG. Upon induction,
cultures were allowed to grow for an additional 20-22 hours before
being extracted and analyzed for free fatty acid (FFA) production.
40 .mu.L of 1M HCl was added to each well, followed by 400 .mu.L of
butyl acetate. Cells were extracted by vortexing for 15 minutes at
2000 rpm. Extracts were derivatized with an equal volume of
N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) before being
analyzed by GC coupled with a flame ionization detector
(GC-FID).
[0319] Ratios of odd-chain fatty acids relative to total free fatty
acids produced by strains expressing various fabH genes are
presented in Tables 12A-C below. Odd chain fatty acid ratios
produced in the control DV2 strain are presented in Table 12A,
while odd chain fatty acid ratios in strains engineered for
increased metabolic flux to propionyl-CoA by way of either the
citramalate pathway (pathway (B) of FIG. 2) or the
threonine-dependent pathway (pathway (A) of FIG. 2) are shown in
Tables 12B and 12C, respectively.
TABLE-US-00018 TABLE 12A Production of Odd Chain Fatty Acids in
Recombinant E. coli Strains oc-FA/ Total FFA oc-FA Total fabH
Strain titer titer FFA 1 a Ec DV2 2488 23 <0.01 2 a Ec DV2 193 7
0.04 pCL-BsH1 3 a Ec DV2 314 8 0.03 pCL-BsH2 4 a Ec DV2 571 24 0.04
pCL-LmH 5 a Ec DV2 2501 29 0.01 pCL-LmH2 6 a Ec DV2 2132 47 0.02
pCL-PfH(opt) 7 a Ec DV2 806 31 0.04 pCL-SmH 8 a Ec DV2 569 22 0.04
pCL-AaH 9 a Ec DV2 323 7 0.02 pCL-DpH1 10 a Ec DV2 2381 20 <0.01
pCL-DpH2
TABLE-US-00019 TABLE 12B Production of Odd Chain Fatty Acids in
Recombinant E. coli Strains with Increased Flux to Propionyl-CoA
via the Citramalate Pathway (B) of FIG. 2. oc-FA/ Total FFA oc-FA
Total fabH Strain titer titer FFA 1 b Ec DV2 1994 106 0.05 cimA3.7
leuBCD 2 b Ec DV2 189 17 0.09 pCL-BsH1 cimA3.7 leuBCD 3 b Ec DV2
268 14 0.05 pCL-BsH2 cimA3.7 leuBCD 4 b Ec DV2 826 90 0.04 pCL-LmH
cimA3.7 leuBCD 5 b Ec DV2 641 89 0.14 pCL-LmH2 cimA3.7 leuBCD 6 b
Ec DV2 2135 211 0.10 pCL-PfH(opt) cimA3.7 leuBCD 7 b Ec DV2 2054
240 0.12 pCL-SmH cimA3.7 leuBCD 8 b Ec DV2 618 93 0.15 pCL-AaH
cimA3.7 leuBCD 9 b Ec DV2 222 18 0.08 pCL-DpHJ cimA3.7 leuBCD 10 b
Ec DV2 2033 101 0.05 pCL-DpH2 cimA3.7 leuBCD
TABLE-US-00020 TABLE 12C Production of Odd Chain Fatty Acids in
Recombinant E. coli Strains with Increased Flux to Propionyl-CoA
via the Threonine-Dependent Pathway (A) of FIG. 2. oc-FA/ Total FFA
oc-FA Total fabH Strain titer titer FFA 1 c Ec DV2 1871 376 0.20
thrA*BC tdcB 2 c Ec DV2 500 132 0.26 pCL-BsH1 thrA*BC tdcB 3 c Ec
DV2 236 39 0.17 pCL-BsH2 thrA*BC tdcB 4 c Ec DV2 560 151 0.27
pCL-LmH thrA*BC tdcB 5 c Ec DV2 1968 622 0.32 pCL-LmH2 thrA*BC tdcB
6 c Ec DV2 1708 404 0.23 pCL-PfH(opt) thrA*BC tdcB 7 c Ec DV2 471
131 0.28 pCL-SmH thrA*BC tdcB 8 c Ec DV2 528 137 0.26 pCL-AaH
thrA*BC tdcB 9 c Ec DV2 240 45 0.19 pCL-DpH1 thrA*BC tdcB 10 c Ec
DV2 1614 434 0.27 pCL-DpH2 thrA*BC tdcB all titers are in
milligrams per liter (mg/L) all strains also contained
plasmid-expressed 'tesA (pACYC-p.sub.Trc2-tesA) FFA = free fatty
acid (oc-FA + ec-FA) oc-FA = odd chain fatty acid; ec-FA = even
chain fatty acid Ec = chromosomal (native) E. coli fabH gene
pCL-BsH1 = pOP80-expressed Bacillus subtilis fabH1 pCL-BsH2 =
pOP80-expressed Bacillus subtilis fabH2 pCL-LmH = pOP80-expressed
Listeria monocytogenes fabH pCL-LmH2 = pOP80-expressed Listeria
monocytogenes fabH2 pCL-PfH(opt) = pOP80-expressed
Propionibacterium freudenreichii fabH(codon-optimized) pCL-SmH =
pOP80-expressed Stenotrophomonas maltophila fabH pCL-AaH =
pOP80-expressed Alicyclobacillus acidocaldarius fabH pCL-DpH1 =
pOP80-expressed Desulfobulbus propionicus fabH1 pCL-DpH2 =
pOP80-expressed Desulfobulbus propionicus fabH2
[0320] All of the strains depicted in Tables 12A-12C expressed the
endogenous E. coli fabH gene. Strains 2-10 each contained in
addition a plasmid-expressed exogenous fabH gene. It was shown in
Table 11 (above) that deletion of the endogenous E. coli fabH gene
and chromosomal integration of the exogenous B. subtilis fabH1 gene
produced a larger amount and greater proportion of oc-FA (Table 11,
strain 4) compared to the strain containing endogenous E. coli fabH
plus plasmid-expressed exogenous B. subtilis fabH1 (Table 11,
strain 3). Nevertheless, the results presented in Tables 12A-12C
demonstrate that (a) propionyl-CoA is a necessary precursor for
recombinant linear odd chain fatty acid production in bacteria,
since all of the fabH-expressing strains tested exhibited
significant linear oc-fatty acid production in the strains
engineered for elevated .alpha.-ketobutyrate and propionyl-CoA
levels--DV2 cimA3.7 leuBCD (Table 12B) and DV2 thrA*BC tdcB (Table
12C)--but no significant oc-fatty acid production was observed in
the DV2 control strains (Table 12A), and (b) recombinant linear
oc-fatty acid production occurs in the presence of a variety of
heterologous FabH enzymes isolated from organisms whose membranes
contain branched chain fatty acids and/or odd chain fatty acids.
Such FabH enzymes are capable of utilizing the propionyl-CoA
molecule in the priming reaction for fatty acid biosynthesis and
confer odd-chain fatty acid biosynthetic capablities to the
recombinant microorganism.
[0321] In conclusion, this Example demonstrates that a microoganism
which normally produces even-chain fatty acids can be engineered to
produce odd-chain fatty acids by increasing metabolic flux through
propionyl-CoA and expressing a .beta.-ketoacyl synthase (FabH)
enzyme that utilizes propionyl-CoA. Example 6 (below) demonstrates
an alternative pathway than can be engineered to increase metabolic
flux through propionyl-CoA. Recombinant microorganisms engineered
to produce odd-chain fatty acids can be further modified to produce
odd-chain fatty acid derivatives, such as odd-chain fatty alcohols
(Example 7) and even-chain alkanes (Example 8).
Example 6
Engineering E. coli for Production of Odd Chain Fatty Acids by
Pathway (C)
[0322] The following example describes the construction of
recombinant E. coli strains which express exogenous genes and/or
overexpress endogenous genes encoding enzymes which serve to
increase metabolic flux through the intermediate methylmalonyl-CoA
to produce propionyl-CoA by pathway (C) of FIG. 3, leading to the
increased production of odd chain acyl-ACPs and odd chain fatty
acid derivatives in these recombinant cells. In particular, this
example describes production of odd chain fatty acids in an E. coli
strain which overexpresses endogenous methylmalonyl-CoA mutase
(scpA/sbm) and methylmalonyl-CoA decarboxylase (scpB/ygfG) genes on
a plasmid and the chromosomal propionyl-CoA:succinyl-CoA
transferase (scpC/ygfH) and scpB/ygfG genes are deleted.
[0323] E. coli strain DV2, plasmid pDG6 (expressing B. subtilis
FabH1), and plasmid pACYC-p.sub.Trc2-tesA (expressing the truncated
`TesA polypeptide) were prepared as described in Example 1.
Plasmid pACYC-P.sub.Trc-sbm-ygfG
[0324] Plasmid pACYC-P.sub.Trc-sbm-ygfG is the pACYC-P.sub.Trc
plasmid (Example 1), which overexpresses E. coli sbm encoding
methylmalonyl-CoA mutase and E. coli ygfG encoding
methylmalonyl-CoA decarboxylase. The sequence of
pACYC-P.sub.Trc-sbm-ygfG is provided herein as SEQ ID NO:80
Strain sDF4
[0325] Strain sDF4 is E. coli strain DV2 from which the chromosomal
scpB and scpC genes were deleted, the native frd promoter replaced
with the trc promoter, and the `tesA gene was chromosomally
integrated at the Tn7 attachment site.
[0326] To integrate the tesA gene, a P.sub.Trc-`tesA integration
cassette was first prepared by amplifying the pACYC-P.sub.Trc-`tesA
plasmid (Example 1) using the following primers:
TABLE-US-00021 IFF: (SEQ ID NO: 140)
5'-GGGTCAATAGCGGCCGCCAATTCGCGCGCGAAGGCG IFR: (SEQ ID NO: 141)
5'-TGGCGCGCCTCCTAGGGCATTACGCTGACTTGACGGG
[0327] The integration cassette was inserted into the NotI and
AvrII restriction sites of pGRG25 (GenBank Accession No. DQ460223)
creating the Tn7tes plasmid (SEQ ID NO: 81), in which the lacIq,
P.sub.Trc-`tesA cassette is flanked by the left and right Tn7
ends.
[0328] To prepare strain sDF4, plasmid Tn7tes was first
electroporated into E. coli strain DV2 (Example 1) using a protocol
described by McKenzie et al., BMC Microbiology 6:39 (2006). After
electroporation, ampicillin-resistant cells were selected by growth
in an LB medium containing 0.1% glucose and 100 .mu.g/mL
carbenicilin at 32.degree. C. overnight. This was followed by
selection of plasmids comprising the Tn7-transposition fractions,
using the growth of cells on an LB plus 0.1% arabinose plates
overnight at 32.degree. C. Single colonies were selected and
streaked onto new LB medium plates with and without ampicillin, and
they were grown overnight at 42.degree. C. to cure of Tn7tes
plasmid. Thus, the lacIq, P.sub.Trc-`tesA was integrated into the
attTn7 site on the E. coli chromosome located between the pstS and
glmS genes. Integration of these genes was confirmed by PCR and
sequencing. The resulting strain was designated DV2 Tn7-tesA.
[0329] To delete the scpBC genes from DV2 Tn7-tesA, the following
two primers were used:
TABLE-US-00022 ScpBC-KOfwd (SEQ ID NO: 142)
5'-GCTCAGTGAATTTATCCAGACGCAATATTTTGATTAAAGGA ATTTT TATGATTCCG
GGGATCCGTCGACC; and ScpBC-KOrc (SEQ ID NO: 143)
5'-ATTGCTGAAGATCGTGACGGGACGAGTCATTAACCCAGCATCGAGCCGGTTGT AGGCTG
GAGCTGCTTC
[0330] The ScpBC-KOfwd and ScpBC-KOrc primers were used to amplify
the kanamycin resistance (Km.sup.R) cassette from plasmid pKD13
(Datsenko et al., supra) by PCR. The PCR product was then used to
transform electrocompetent E. coli DV2 Tn7-tesA cells containing
plasmid pKD46, which expresses Red recombinase (Datsenko et al.,
supra) which had been previously induced with arabinose for 3-4
hours. Following a 3-hour outgrowth in SOC medium at 37.degree. C.,
the cells were plated on Luria agar plates containing 50 .mu.g/mL
of kanamycin. Resistant colonies were identified and isolated after
an overnight incubation at 37.degree. C. Disruption of the scpBC
genes was confirmed by PCR amplification using the following
primers designed to flank the chromosomal scpBC genes:
TABLE-US-00023 ScpBC check - 60 fwd (SEQ ID NO: 144)
5'-CGGGTTCTGACTTGTAGCG ScpBC check + 60 rc (SEQ ID NO: 145)
5'-CCAACTTCGAAGCAATGATTGATG
[0331] After the scpBC deletion was confirmed, a single colony was
picked and used to remove the Km.sup.R marker using the pCP20
plasmid (Datsenko et al., supra). The native fumarate reductase
(frd) promoter was replaced with the PTrc promoter using a
modification of the procedure of Datsenko et al. (supra). The
resulting E. coli DV2 .DELTA.scpBC::FRT, .DELTA.Pfrd::FRT-PTrc,
attTn7::PTrc-`tesA strain was designated "sDF4".
[0332] Strains were transformed with plasmids as indicated below
and evaluated for fatty acid production using the 96 deep-well
plate fermentation procedure described in Example 5; since ScpA is
a B-12 dependent enzyme, the 4N-BT culture media was supplemented
with cobalamin.
TABLE-US-00024 TABLE 13 Production of Odd Chain Fatty Acids in
Recombinant E. coli Strains Total oc-FA/ Strain fabH tesA FFA oc-FA
total FFA 1 DV2 pACYC-PTrc2-`tesA Ec p 2054 6 <0.01 2 sDF4
pACYC-PTrc-sbm- Ec int 973 39 0.04 ygfG 3 sDF4 pACYC-PTrc-sbm- Ec
int 863 140 0.16 ygfG pDG6 pBsH1 all titers are in milligrams per
liter (mg/L) FFA = free fatty acid (oc-FA + ec-FA) oc-FA = odd
chain fatty acid; ec-FA = even chain fatty acid Ec = chromosomal E.
coli fabH gene; pBsH1 = plasmid-expressed BsfabH1 (pDG6) p =
plasmid-expressed `tesA gene (pACYC-p.sub.Trc2-tesA); int =
chromosomally integrated `tesA gene
[0333] Microbial cells overexpressing genes involved in the
production of propionyl-CoA via the intermediates succinyl-CoA and
methylmalonyl-CoA increased the proportion of odd chain length
fatty acids produced by the cells. While the DV2 strain (strain 1
of Table 13) produced only a negligible amount of odd chain length
fatty acids, the sDF4 strain overexpressing the endogenous E. coli
sbm and ygfG genes (encoding polypeptides having methylmalonyl-CoA
mutase activity and methylmalonyl-CoA decarboxylase activity)
produced an increased amount of odd chain length fatty acids.
[0334] Strains 2 and 3 of Table 13 demonstrate the effect on oc-FA
production by including an exogenous .beta.-ketoacyl ACP synthase
with high specificity towards propionyl-CoA. Strain 2 contained the
native E. coli fabH gene. By introducing a plasmid expressing the
B. subtilis fabH1 gene, oc-FA production further increased from
about 4% of the fatty acids produced in Strain 2 to about 16% of
the fatty acids produced in Strain 3.
Example 7
Production of Odd Chain Fatty Alcohols in E. coli
[0335] The following demonstrates the production of odd chain fatty
alcohols by previously-described strains, which, in this example,
also expressed a polypeptide having acyl-ACP reductase (AAR)
activity. The AAR activity converted the oc-acyl-ACP intermediate
to oc-fatty aldehyde, which reacted with endogenous aldehyde
reductase to form oc-fatty alcohol.
[0336] Strains DV2, DV2 P.sub.L-thrA*BCP.sub.L-tdcB
P.sub.T5-BsfabH1 AEcfabH, and G1 (prepared as described in Examples
1, 2, and 4, respectively) were transformed either with plasmid
pLS9185 or pDS171s. Plasmid pLS9185 expressed a Synechococcus
elongatus fatty acyl-ACP reductase (AAR; GenBank Accession No.
YP.sub.--400611). Plasmid pDS171s expressed S. elongatus AAR, an
acyl carrier protein (ACP) from the cyanobacterium Nostoc
punctiforme (cACP; GenBank Accession No. YP.sub.--001867863) and a
phosphopantetheinyl transferase from Bacillus subtilis (Sfp;
GenBank Accession No. YP.sub.--004206313). These strains were
evaluated for fatty alcohol production using the 96 deep-well plate
fermentation procedure described in Example 5.
TABLE-US-00025 TABLE 14 Production of Odd Chain Fatty Alcohols in
Recombinant E. coli Strains Total oc- oc-FA1c/ FA1c FA1c Total
Strain pLS9185 pDS171s titer titer FA1c 1 DV2 x 432 23 0.05 4 DV2
thrA*BC tdcB x 398 325 0.82 .DELTA.EcFabH IntBsFabH1 7 "G1": x 420
157 0.37 DV2 thrA*BC cimA3.7 leuBCD .DELTA.EcFabH IntBsFabH1 1 DV2
x 847 37 0.04 4 DV2 thrA*BC tdcB x 906 735 0.81 .DELTA.EcFabH
IntBsFabH1 7 "G1": x 775 344 0.44 DV2 thrA*BC cimA3.7 leuBCD
.DELTA.EcFabH IntBsFabH1 all titers are in milligrams per liter
(mg/L) FAlc = fatty alcohol (oc-FAlc + ec-FAlc) oc-FAlc = odd chain
fatty alcohol; ec-FAlc = even chain fatty alcohol .DELTA.EcFabH =
deleted chromosomal E. coli fabH gene IntBsH1 = chromosomally
integrated BsfabH1 pLS9185 = plasmid-expressed AAR
[0337] pDS171s=plasmid-expressed AAR, cACP, and Sfp
[0338] Compared to the control strain DV2, both strains DV2 thrA*BC
tdcB BsfabH1 .DELTA.EcfabH and G1 produced significantly higher
titers and proportions of odd chain fatty alcohols when transformed
with a plasmid expressing AAR, or a plasmid expressing AAR, cACP,
and Sfp (Table 14). The proportion of fatty alcohols produced as
odd chain fatty alcohols roughly reflects the proportions observed
when these strains were evaluated for fatty acid production (Table
11), suggesting that AAR does not show a preference for odd or even
chain fatty acyl-ACPs of similar overall chain length.
Example 8
Production of Even Chain Alkanes in E. coli
[0339] The following example demonstrates the production of even
chain alkanes by a strain which expressed a polypeptide having
acyl-ACP reductase (AAR) activity and a polypeptide having aldehyde
decarbonylase (ADC) activity. The AAR activity converted the
oc-acyl-ACP intermediate to oc-fatty aldehyde, and the ADC activity
decarbonylated the oc-fatty aldehyde to form even chain
(ec-)alkane.
[0340] Strains DV2, DV2 thrA*BC tdcB BsfabH1 .DELTA.EcfabH, and G1
(prepared as described in Examples 1, 2, and 4, respectively) were
transformed with plasmids pLS9185 and pLS9181. Plasmid pLS9185
expressed a Synechococcus elongatus fatty acyl-ACP reductase (AAR;
GenBank Accession No. YP.sub.--400611). Plasmid pLS9181 expressed a
Nostoc punctiforme aldehyde decarbonylase (ADC; GenBank Accession
No. YP.sub.--001865325). Strains transformed with both plasmids
were analyzed for alkane production using the 96 deep-well plate
fermentation procedure described in Example 5 above, but with the
added supplementation of 25 .mu.M MnSO.sub.4 (final concentration)
at induction.
TABLE-US-00026 TABLE 15 Production of Even Chain Alkanes in
Recombinant E. coli Strains Total Alk ec-Alk ec-Alk/ Strain AAR ADC
titer titer Total Alk 1 DV2 x x 432 23 0.05 4 DV2 thrA*BC tdcB x x
398 325 0.82 .DELTA.EcFabH IntBsFabH1 7 "G1": x x 420 157 0.37 DV2
thrA*BC cimA3.7 leuBCD .DELTA.EcFabH IntBsFabH1 all titers are in
milligrams per liter (mg/L) Alk = alkane (oc-Alk + ec-Alk); oc-Alk
= odd chain alkane; ec-Alk = even chain alkane .DELTA.EcFabH =
deleted chromosomal E. coli fabH gene IntBsFabH1 = chromosomally
integrated BsfabH1 AAR = plasmid-expressed aar gene (pLS9185)
[0341] ADC=plasmid-expressed adc gene (pLS9181)
[0342] Compared to the control strain DV2, both DV2 thrA*BC tdcB
BsfabH1 .DELTA.EcfabH and G1 produced significantly higher titers
and proportions of even chain alkanes when transformed with
plasmids expressing AAR and ADC (Table 15). The proportion of
alkanes produced as even chain alkanes roughly reflects the
proportions of odd chain products produced when these strains were
evaluated for fatty acid production (Table 11) and for fatty
alcohol production (Table 14), suggesting that ADC, like AAR, does
not show a preference between odd or even chain substrates of
comparable overall chain length.
[0343] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
1691317PRTEscherichia colisource/note="Beta ketoacyl-ACP synthase
III" 1Met Tyr Thr Lys Ile Ile Gly Thr Gly Ser Tyr Leu Pro Glu Gln
Val 1 5 10 15 Arg Thr Asn Ala Asp Leu Glu Lys Met Val Asp Thr Ser
Asp Glu Trp 20 25 30 Ile Val Thr Arg Thr Gly Ile Arg Glu Arg His
Ile Ala Ala Pro Asn 35 40 45 Glu Thr Val Ser Thr Met Gly Phe Glu
Ala Ala Thr Arg Ala Ile Glu 50 55 60 Met Ala Gly Ile Glu Lys Asp
Gln Ile Gly Leu Ile Val Val Ala Thr 65 70 75 80 Thr Ser Ala Thr His
Ala Phe Pro Ser Ala Ala Cys Gln Ile Gln Ser 85 90 95 Met Leu Gly
Ile Lys Gly Cys Pro Ala Phe Asp Val Ala Ala Ala Cys 100 105 110 Ala
Gly Phe Thr Tyr Ala Leu Ser Val Ala Asp Gln Tyr Val Lys Ser 115 120
125 Gly Ala Val Lys Tyr Ala Leu Val Val Gly Ser Asp Val Leu Ala Arg
130 135 140 Thr Cys Asp Pro Thr Asp Arg Gly Thr Ile Ile Ile Phe Gly
Asp Gly 145 150 155 160 Ala Gly Ala Ala Val Leu Ala Ala Ser Glu Glu
Pro Gly Ile Ile Ser 165 170 175 Thr His Leu His Ala Asp Gly Ser Tyr
Gly Glu Leu Leu Thr Leu Pro 180 185 190 Asn Ala Asp Arg Val Asn Pro
Glu Asn Ser Ile His Leu Thr Met Ala 195 200 205 Gly Asn Glu Val Phe
Lys Val Ala Val Thr Glu Leu Ala His Ile Val 210 215 220 Asp Glu Thr
Leu Ala Ala Asn Asn Leu Asp Arg Ser Gln Leu Asp Trp 225 230 235 240
Leu Val Pro His Gln Ala Asn Leu Arg Ile Ile Ser Ala Thr Ala Lys 245
250 255 Lys Leu Gly Met Ser Met Asp Asn Val Val Val Thr Leu Asp Arg
His 260 265 270 Gly Asn Thr Ser Ala Ala Ser Val Pro Cys Ala Leu Asp
Glu Ala Val 275 280 285 Arg Asp Gly Arg Ile Lys Pro Gly Gln Leu Val
Leu Leu Glu Ala Phe 290 295 300 Gly Gly Gly Phe Thr Trp Gly Ser Ala
Leu Val Arg Phe 305 310 315 2312PRTBacillus
subtilissource/note="Beta ketoacyl-ACP synthase III (FabH1)" 2Met
Lys Ala Gly Ile Leu Gly Val Gly Arg Tyr Ile Pro Glu Lys Val 1 5 10
15 Leu Thr Asn His Asp Leu Glu Lys Met Val Glu Thr Ser Asp Glu Trp
20 25 30 Ile Arg Thr Arg Thr Gly Ile Glu Glu Arg Arg Ile Ala Ala
Asp Asp 35 40 45 Val Phe Ser Ser His Met Ala Val Ala Ala Ala Lys
Asn Ala Leu Glu 50 55 60 Gln Ala Glu Val Ala Ala Glu Asp Leu Asp
Met Ile Leu Val Ala Thr 65 70 75 80 Val Thr Pro Asp Gln Ser Phe Pro
Thr Val Ser Cys Met Ile Gln Glu 85 90 95 Gln Leu Gly Ala Lys Lys
Ala Cys Ala Met Asp Ile Ser Ala Ala Cys 100 105 110 Ala Gly Phe Met
Tyr Gly Val Val Thr Gly Lys Gln Phe Ile Glu Ser 115 120 125 Gly Thr
Tyr Lys His Val Leu Val Val Gly Val Glu Lys Leu Ser Ser 130 135 140
Ile Thr Asp Trp Glu Asp Arg Asn Thr Ala Val Leu Phe Gly Asp Gly 145
150 155 160 Ala Gly Ala Ala Val Val Gly Pro Val Ser Asp Asp Arg Gly
Ile Leu 165 170 175 Ser Phe Glu Leu Gly Ala Asp Gly Thr Gly Gly Gln
His Leu Tyr Leu 180 185 190 Asn Glu Lys Arg His Thr Ile Met Asn Gly
Arg Glu Val Phe Lys Phe 195 200 205 Ala Val Arg Gln Met Gly Glu Ser
Cys Val Asn Val Ile Glu Lys Ala 210 215 220 Gly Leu Ser Lys Glu Asp
Val Asp Phe Leu Ile Pro His Gln Ala Asn 225 230 235 240 Ile Arg Ile
Met Glu Ala Ala Arg Glu Arg Leu Glu Leu Pro Val Glu 245 250 255 Lys
Met Ser Lys Thr Val His Lys Tyr Gly Asn Thr Ser Ala Ala Ser 260 265
270 Ile Pro Ile Ser Leu Val Glu Glu Leu Glu Ala Gly Lys Ile Lys Asp
275 280 285 Gly Asp Val Val Val Met Val Gly Phe Gly Gly Gly Leu Thr
Trp Gly 290 295 300 Ala Ile Ala Ile Arg Trp Gly Arg 305 310
3325PRTBacillus subtilissource/note="Beta ketoacyl-ACP synthase III
(FabH2)" 3Met Ser Lys Ala Lys Ile Thr Ala Ile Gly Thr Tyr Ala Pro
Ser Arg 1 5 10 15 Arg Leu Thr Asn Ala Asp Leu Glu Lys Ile Val Asp
Thr Ser Asp Glu 20 25 30 Trp Ile Val Gln Arg Thr Gly Met Arg Glu
Arg Arg Ile Ala Asp Glu 35 40 45 His Gln Phe Thr Ser Asp Leu Cys
Ile Glu Ala Val Lys Asn Leu Lys 50 55 60 Ser Arg Tyr Lys Gly Thr
Leu Asp Asp Val Asp Met Ile Leu Val Ala 65 70 75 80 Thr Thr Thr Ser
Asp Tyr Ala Phe Pro Ser Thr Ala Cys Arg Val Gln 85 90 95 Glu Tyr
Phe Gly Trp Glu Ser Thr Gly Ala Leu Asp Ile Asn Ala Thr 100 105 110
Cys Ala Gly Leu Thr Tyr Gly Leu His Leu Ala Asn Gly Leu Ile Thr 115
120 125 Ser Gly Leu His Gln Lys Ile Leu Val Ile Ala Gly Glu Thr Leu
Ser 130 135 140 Lys Val Thr Asp Tyr Thr Asp Arg Thr Thr Cys Val Leu
Phe Gly Asp 145 150 155 160 Ala Ala Gly Ala Leu Leu Val Glu Arg Asp
Glu Glu Thr Pro Gly Phe 165 170 175 Leu Ala Ser Val Gln Gly Thr Ser
Gly Asn Gly Gly Asp Ile Leu Tyr 180 185 190 Arg Ala Gly Leu Arg Asn
Glu Ile Asn Gly Val Gln Leu Val Gly Ser 195 200 205 Gly Lys Met Val
Gln Asn Gly Arg Glu Val Tyr Lys Trp Ala Ala Arg 210 215 220 Thr Val
Pro Gly Glu Phe Glu Arg Leu Leu His Lys Ala Gly Leu Ser 225 230 235
240 Ser Asp Asp Leu Asp Trp Phe Val Pro His Ser Ala Asn Leu Arg Met
245 250 255 Ile Glu Ser Ile Cys Glu Lys Thr Pro Phe Pro Ile Glu Lys
Thr Leu 260 265 270 Thr Ser Val Glu His Tyr Gly Asn Thr Ser Ser Val
Ser Ile Val Leu 275 280 285 Ala Leu Asp Leu Ala Val Lys Ala Gly Lys
Leu Lys Lys Asp Gln Ile 290 295 300 Val Leu Leu Phe Gly Phe Gly Gly
Gly Leu Thr Tyr Thr Gly Leu Leu 305 310 315 320 Ile Lys Trp Gly Met
325 4320PRTStreptomyces coelicolorsource/note="Beta ketoacyl-ACP
synthase III" 4Met Ala Arg Gly Ala Gly Arg Leu Thr Gly Ile Gly Val
Tyr Arg Pro 1 5 10 15 Gly Gly Leu Leu Thr Ser Ala Glu Leu Asp Thr
Arg Phe Gly His Glu 20 25 30 Asp Gly Tyr Ile Glu Gln Ile Thr Gly
Ile Arg Thr Arg Leu Lys Ala 35 40 45 Asp Pro Asp Asp Thr Phe Val
Glu Met Ala Ala Gln Ala Ala Asp Lys 50 55 60 Ala Leu Ala His Ala
Gly Val Leu Ala Glu Asp Leu Asp Cys Val Leu 65 70 75 80 Phe Ser Ser
Ala Ser Ser Val Gly Gln Ala Ser Cys Arg Ala Ala Ser 85 90 95 Leu
Thr His Arg Ile Gly Ala Gly Arg Ala Gly Gly Phe Asp Leu Asn 100 105
110 Gly Gly Cys Ala Gly Phe Gly Tyr Gly Leu Thr Leu Ala Ser Gly Leu
115 120 125 Ile Ala Ala Gln Gln Ala Arg Gln Ile Leu Val Val Ala Ala
Glu Arg 130 135 140 Leu Ser Asp Ile Thr Asp Pro Asp Asp Cys Gly Thr
Val Met Val Phe 145 150 155 160 Gly Asp Ala Ala Gly Ala Ala Val Val
Ser Ala Ala Glu His Pro Gly 165 170 175 Ile Gly Pro Ala Val Trp Gly
Thr His Gly Pro Gly Glu Pro Trp Met 180 185 190 Thr Ser Ala Pro Pro
Lys Pro Gly Ala Ala Arg Pro Tyr Met His Met 195 200 205 Asp Gly Thr
Arg Val Val Arg Trp Phe Gly Ser Gln Met Pro Gln Val 210 215 220 Ala
Arg Asp Ala Leu Glu Ala Ala Gly Leu Thr Trp Asp Asp Ile Gly 225 230
235 240 Ala Phe Val Pro His Gln Cys Asn Gly Arg Leu Ile Asp Ala Met
Val 245 250 255 Arg Arg Leu Arg Pro Pro Glu His Val Ala Ile Ala Arg
Ser Ile Val 260 265 270 Thr Asp Gly Asn Thr Ser Ser Ala Ser Ile Pro
Leu Ala Leu Glu Ser 275 280 285 Leu Leu Ala Ser Ala Thr Val Arg Pro
Gly Asp Lys Ala Leu Leu Leu 290 295 300 Gly Phe Gly Ala Gly Leu Thr
Trp Cys Ala Gln Val Val Glu Leu Pro 305 310 315 320
5333PRTStreptomyces glaucescenssource/note="Beta ketoacyl-ACP
synthase III" 5Met Ser Lys Ile Lys Pro Ala Lys Gly Ala Pro Tyr Ala
Arg Ile Leu 1 5 10 15 Gly Val Gly Gly Tyr Arg Pro Thr Arg Val Val
Pro Asn Glu Val Ile 20 25 30 Leu Glu Thr Ile Asp Ser Ser Asp Glu
Trp Ile Arg Ser Arg Ser Gly 35 40 45 Ile Gln Thr Arg His Trp Ala
Asn Asp Glu Glu Thr Val Ala Ala Met 50 55 60 Ser Ile Glu Ala Ser
Gly Lys Ala Ile Ala Asp Ala Gly Ile Thr Ala 65 70 75 80 Ala Gln Val
Gly Ala Val Ile Val Ser Thr Val Thr His Phe Lys Gln 85 90 95 Thr
Pro Ala Val Ala Thr Glu Ile Ala Asp Lys Leu Gly Thr Asn Lys 100 105
110 Ala Ala Ala Phe Asp Ile Ser Ala Gly Cys Ala Gly Phe Gly Tyr Gly
115 120 125 Leu Thr Leu Ala Lys Gly Met Ile Val Glu Gly Ser Ala Glu
Tyr Val 130 135 140 Leu Val Ile Gly Val Glu Arg Leu Ser Asp Leu Thr
Asp Leu Glu Asp 145 150 155 160 Arg Ala Thr Ala Phe Leu Phe Gly Asp
Gly Ala Gly Ala Val Val Val 165 170 175 Gly Pro Ser Asn Glu Pro Ala
Ile Gly Pro Thr Ile Trp Gly Ser Glu 180 185 190 Gly Asp Lys Ala Glu
Thr Ile Lys Gln Thr Val Pro Trp Thr Asp Tyr 195 200 205 Arg Glu Gly
Gly Val Glu Arg Phe Pro Ala Ile Thr Gln Glu Gly Gln 210 215 220 Ala
Val Phe Arg Trp Ala Val Phe Glu Met Ala Lys Val Ala Gln Gln 225 230
235 240 Ala Leu Asp Ala Ala Gly Val Ala Ala Ala Asp Leu Asp Val Phe
Ile 245 250 255 Pro His Gln Ala Asn Glu Arg Ile Ile Asp Ser Met Val
Lys Thr Leu 260 265 270 Lys Leu Pro Glu Ser Val Thr Val Ala Arg Asp
Val Arg Thr Thr Gly 275 280 285 Asn Thr Ser Ala Ala Ser Ile Pro Leu
Ala Met Glu Arg Leu Leu Ala 290 295 300 Thr Gly Glu Ala Lys Ser Gly
Asp Thr Ala Leu Val Ile Gly Phe Gly 305 310 315 320 Ala Gly Leu Val
Tyr Ala Ala Ser Val Val Thr Leu Pro 325 330 6335PRTStreptomyces
avermitilissource/note="Beta ketoacyl-ACP synthase III" 6Met Ser
Gly Gly Arg Ala Ala Val Ile Thr Gly Ile Gly Gly Tyr Val 1 5 10 15
Pro Pro Asp Leu Val Thr Asn Asp Asp Leu Ala Gln Arg Leu Asp Thr 20
25 30 Ser Asp Ala Trp Ile Arg Ser Arg Thr Gly Ile Ala Glu Arg His
Val 35 40 45 Ile Ala Pro Gly Thr Ala Thr Ser Asp Leu Ala Val Glu
Ala Gly Leu 50 55 60 Arg Ala Leu Lys Ser Ala Gly Asp Glu His Val
Asp Ala Val Val Leu 65 70 75 80 Ala Thr Thr Thr Pro Asp Gln Pro Cys
Pro Ala Thr Ala Pro Gln Val 85 90 95 Ala Ala Arg Leu Gly Leu Gly
Gln Val Pro Ala Phe Asp Val Ala Ala 100 105 110 Val Cys Ser Gly Phe
Leu Phe Gly Leu Ala Thr Ala Ser Gly Leu Ile 115 120 125 Ala Ala Gly
Val Ala Asp Lys Val Leu Leu Val Ala Ala Asp Ala Phe 130 135 140 Thr
Thr Ile Ile Asn Pro Glu Asp Arg Thr Thr Ala Val Ile Phe Ala 145 150
155 160 Asp Gly Ala Gly Ala Val Val Leu Arg Ala Gly Ala Ala Asp Glu
Pro 165 170 175 Gly Ala Val Gly Pro Leu Val Leu Gly Ser Asp Gly Glu
Leu Ser His 180 185 190 Leu Ile Glu Val Pro Ala Gly Gly Ser Arg Gln
Arg Ser Ser Gly Pro 195 200 205 Thr Thr Asp Pro Asp Asp Gln Tyr Phe
Arg Met Leu Gly Arg Asp Thr 210 215 220 Tyr Arg His Ala Val Glu Arg
Met Thr Asp Ala Ser Gln Arg Ala Ala 225 230 235 240 Glu Leu Ala Asp
Trp Arg Ile Asp Asp Val Asp Arg Phe Ala Ala His 245 250 255 Gln Ala
Asn Ala Arg Ile Leu Asp Ser Val Ala Glu Arg Leu Gly Val 260 265 270
Pro Ala Glu Arg Gln Leu Thr Asn Ile Ala Arg Val Gly Asn Thr Gly 275
280 285 Ala Ala Ser Ile Pro Leu Leu Leu Ser Gln Ala Ala Ala Ala Gly
Arg 290 295 300 Leu Gly Ala Gly His Arg Val Leu Leu Thr Ala Phe Gly
Gly Gly Leu 305 310 315 320 Ser Trp Gly Ala Gly Thr Leu Val Trp Pro
Glu Val Gln Pro Val 325 330 335 7312PRTListeria
monocytogenessource/note="Beta ketoacyl-ACP synthase III" 7Met Asn
Ala Gly Ile Leu Gly Val Gly Lys Tyr Val Pro Glu Lys Ile 1 5 10 15
Val Thr Asn Phe Asp Leu Glu Lys Ile Met Asp Thr Ser Asp Glu Trp 20
25 30 Ile Arg Thr Arg Thr Gly Ile Glu Glu Arg Arg Ile Ala Arg Asp
Asp 35 40 45 Glu Tyr Thr His Asp Leu Ala Tyr Glu Ala Ala Lys Val
Ala Ile Glu 50 55 60 Asn Ala Gly Leu Thr Pro Asp Asp Ile Asp Leu
Phe Ile Val Ala Thr 65 70 75 80 Val Thr Gln Glu Ala Thr Phe Pro Ser
Val Ala Asn Ile Ile Gln Asp 85 90 95 Arg Leu Gly Ala Thr Asn Ala
Ala Gly Met Asp Val Glu Ala Ala Cys 100 105 110 Ala Gly Phe Thr Phe
Gly Val Val Thr Ala Ala Gln Phe Ile Lys Thr 115 120 125 Gly Ala Tyr
Lys Asn Ile Val Val Val Gly Ala Asp Lys Leu Ser Lys 130 135 140 Ile
Thr Asn Trp Asp Asp Arg Ala Thr Ala Val Leu Phe Gly Asp Gly 145 150
155 160 Ala Gly Ala Val Val Met Gly Pro Val Ser Asp Asp His Gly Leu
Leu 165 170 175 Ser Phe Asp Leu Gly Ser Asp Gly Ser Gly Gly Lys Tyr
Leu Asn Leu 180 185 190 Asp Glu Asn Lys Lys Ile Tyr Met Asn Gly Arg
Glu Val Phe Arg Phe 195 200 205 Ala Val Arg Gln Met Gly Glu Ala Ser
Leu Arg Val Leu Glu Arg Ala 210 215 220 Gly Leu Glu Lys Glu Glu Leu
Asp Leu Leu Ile Pro His Gln Ala Asn 225 230 235 240 Ile Arg Ile Met
Glu Ala Ser Arg Glu Arg Leu Asn Leu Pro Glu Glu 245 250 255 Lys Leu
Met Lys Thr Val His Lys Tyr Gly Asn Thr Ser Ser Ser Ser 260 265 270
Ile Ala Leu Ala Leu Val Asp Ala Val Glu Glu Gly Arg Ile Lys Asp 275
280 285 Asn Asp Asn Val Leu Leu Val
Gly Phe Gly Gly Gly Leu Thr Trp Gly 290 295 300 Ala Leu Ile Ile Arg
Trp Gly Lys 305 310 8312PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
L. monocytogenes beta ketoacyl-ACP synthase III variant
polypeptide" 8Met Asn Ala Gly Ile Leu Gly Val Gly Lys Tyr Val Pro
Glu Lys Ile 1 5 10 15 Val Thr Asn Phe Asp Leu Glu Lys Ile Met Asp
Thr Ser Asp Glu Trp 20 25 30 Ile Arg Thr Arg Thr Gly Ile Glu Glu
Arg Arg Ile Ala Arg Asp Asp 35 40 45 Glu Tyr Thr His Asp Leu Ala
Tyr Glu Ala Ala Lys Val Ala Ile Glu 50 55 60 Asn Ala Gly Leu Thr
Pro Asp Asp Ile Asp Leu Phe Ile Val Ala Thr 65 70 75 80 Val Thr Gln
Glu Ala Thr Phe Pro Ser Val Ala Asn Ile Ile Gln Asp 85 90 95 Arg
Leu Gly Ala Thr Asn Ala Ala Gly Met Asp Val Glu Ala Ala Cys 100 105
110 Ala Gly Phe Thr Phe Gly Val Val Thr Ala Ala Gln Phe Ile Lys Thr
115 120 125 Gly Ala Tyr Lys Asn Ile Val Val Val Gly Ala Asp Lys Leu
Ser Lys 130 135 140 Ile Thr Asn Trp Asp Asp Arg Ala Thr Ala Val Leu
Phe Gly Asp Gly 145 150 155 160 Ala Gly Ala Val Val Met Gly Pro Val
Ser Asp Asp His Gly Leu Leu 165 170 175 Ser Phe Asp Leu Gly Ser Asp
Gly Ser Gly Gly Lys Tyr Leu Asn Leu 180 185 190 Asp Glu Asn Lys Lys
Ile Tyr Met Asn Gly Arg Glu Val Phe Arg Phe 195 200 205 Ala Val Arg
Gln Met Gly Glu Ala Ser Leu Arg Val Leu Glu Arg Ala 210 215 220 Gly
Leu Glu Lys Glu Glu Leu Asp Leu Leu Ile Pro His Gln Ala Asn 225 230
235 240 Ile Arg Ile Met Glu Ala Ser Arg Glu Arg Leu Asn Leu Pro Glu
Glu 245 250 255 Lys Leu Met Lys Thr Val His Lys Tyr Gly Asn Thr Ser
Ser Ser Ser 260 265 270 Ile Ala Leu Ala Leu Val Asp Ala Val Glu Glu
Gly Arg Ile Lys Asp 275 280 285 Asn Asp Asn Val Leu Leu Val Gly Phe
Gly Gly Gly Leu Thr Trp Gly 290 295 300 Ala Leu Ile Ile Arg Gly Gly
Lys 305 310 9313PRTStaphylococcus aureussource/note="Beta
ketoacyl-ACP synthase III" 9Met Asn Val Gly Ile Lys Gly Phe Gly Ala
Tyr Ala Pro Glu Lys Ile 1 5 10 15 Ile Asp Asn Ala Tyr Phe Glu Gln
Phe Leu Asp Thr Ser Asp Glu Trp 20 25 30 Ile Ser Lys Met Thr Gly
Ile Lys Glu Arg His Trp Ala Asp Asp Asp 35 40 45 Gln Asp Thr Ser
Asp Leu Ala Tyr Glu Ala Ser Leu Lys Ala Ile Ala 50 55 60 Asp Ala
Gly Ile Gln Pro Glu Asp Ile Asp Met Ile Ile Val Ala Thr 65 70 75 80
Ala Thr Gly Asp Met Pro Phe Pro Thr Val Ala Asn Met Leu Gln Glu 85
90 95 Arg Leu Gly Thr Gly Lys Val Ala Ser Met Asp Gln Leu Ala Ala
Cys 100 105 110 Ser Gly Phe Met Tyr Ser Met Ile Thr Ala Lys Gln Tyr
Val Gln Ser 115 120 125 Gly Asp Tyr His Asn Ile Leu Val Val Gly Ala
Asp Lys Leu Ser Lys 130 135 140 Ile Thr Asp Leu Thr Asp Arg Ser Thr
Ala Val Leu Phe Gly Asp Gly 145 150 155 160 Ala Gly Ala Val Ile Ile
Gly Glu Val Ser Asp Gly Arg Gly Ile Ile 165 170 175 Ser Tyr Glu Met
Gly Ser Asp Gly Thr Gly Gly Lys His Leu Tyr Leu 180 185 190 Asp Lys
Asp Thr Gly Lys Leu Lys Met Asn Gly Arg Glu Val Phe Lys 195 200 205
Phe Ala Val Arg Ile Met Gly Asp Ala Ser Thr Arg Val Val Glu Lys 210
215 220 Ala Asn Leu Thr Ser Asp Asp Ile Asp Leu Phe Ile Pro His Gln
Ala 225 230 235 240 Asn Ile Arg Ile Met Glu Ser Ala Arg Glu Arg Leu
Gly Ile Ser Lys 245 250 255 Asp Lys Met Ser Val Ser Val Asn Lys Tyr
Gly Asn Thr Ser Ala Ala 260 265 270 Ser Ile Pro Leu Ser Ile Asp Gln
Glu Leu Lys Asn Gly Lys Ile Lys 275 280 285 Asp Asp Asp Thr Ile Val
Leu Val Gly Phe Gly Gly Gly Leu Thr Trp 290 295 300 Gly Ala Met Thr
Ile Lys Trp Gly Lys 305 310 10324PRTStreptococcus
pneumoniaesource/note="Beta ketoacyl-ACP synthase III" 10Met Ala
Phe Ala Lys Ile Ser Gln Val Ala His Tyr Val Pro Glu Gln 1 5 10 15
Val Val Thr Asn His Asp Leu Ala Gln Ile Met Asp Thr Asn Asp Glu 20
25 30 Trp Ile Ser Ser Arg Thr Gly Ile Arg Gln Arg His Ile Ser Arg
Thr 35 40 45 Glu Ser Thr Ser Asp Leu Ala Thr Glu Val Ala Lys Lys
Leu Met Ala 50 55 60 Lys Ala Gly Ile Thr Gly Glu Glu Leu Asp Phe
Ile Ile Leu Ala Thr 65 70 75 80 Ile Thr Pro Asp Ser Met Met Pro Ser
Thr Ala Ala Arg Val Gln Ala 85 90 95 Asn Ile Gly Ala Asn Lys Ala
Phe Ala Phe Asp Leu Thr Ala Ala Cys 100 105 110 Ser Gly Phe Val Phe
Ala Leu Ser Thr Ala Glu Lys Phe Ile Ala Ser 115 120 125 Gly Arg Phe
Gln Lys Gly Leu Val Ile Gly Ser Glu Thr Leu Ser Lys 130 135 140 Ala
Val Asp Trp Ser Asp Arg Ser Thr Ala Val Leu Phe Gly Asp Gly 145 150
155 160 Ala Gly Gly Val Leu Leu Glu Ala Ser Glu Gln Glu His Phe Leu
Ala 165 170 175 Glu Ser Leu Asn Ser Asp Gly Ser Arg Ser Glu Cys Leu
Thr Tyr Gly 180 185 190 His Ser Gly Leu His Ser Pro Phe Ser Asp Gln
Glu Ser Ala Asp Ser 195 200 205 Phe Leu Lys Met Asp Gly Arg Thr Val
Phe Asp Phe Ala Ile Arg Asp 210 215 220 Val Ala Lys Ser Ile Lys Gln
Thr Ile Asp Glu Ser Pro Ile Glu Val 225 230 235 240 Thr Asp Leu Asp
Tyr Leu Leu Leu His Gln Ala Asn Asp Arg Ile Leu 245 250 255 Asp Lys
Met Ala Arg Lys Ile Gly Val Asp Arg Ala Lys Leu Pro Ala 260 265 270
Asn Met Met Glu Tyr Gly Asn Thr Ser Ala Ala Ser Ile Pro Ile Leu 275
280 285 Leu Ser Glu Cys Val Glu Gln Gly Leu Ile Pro Leu Asp Gly Ser
Gln 290 295 300 Thr Val Leu Leu Ser Gly Phe Gly Gly Gly Leu Thr Trp
Gly Thr Leu 305 310 315 320 Ile Leu Thr Ile 11325PRTStreptococcus
mutanssource/note="Beta ketoacyl-ACP synthase III" 11Met Thr Phe
Ala Lys Ile Ser Gln Ala Ala Tyr Tyr Val Pro Ser Gln 1 5 10 15 Val
Val Thr Asn Asp Asp Leu Ser Lys Ile Met Asp Thr Ser Asp Glu 20 25
30 Trp Ile Thr Ser Arg Thr Gly Ile Arg Glu Arg Arg Ile Ser Gln Ser
35 40 45 Glu Asp Thr Ser Asp Leu Ala Ser Gln Val Ala Lys Glu Leu
Leu Lys 50 55 60 Lys Ala Ser Leu Lys Ala Lys Glu Ile Asp Phe Ile
Ile Val Ala Thr 65 70 75 80 Ile Thr Pro Asp Ala Met Met Pro Ser Thr
Ala Ala Cys Val Gln Ala 85 90 95 Lys Ile Gly Ala Val Asn Ala Phe
Ala Phe Asp Leu Thr Ala Ala Cys 100 105 110 Ser Gly Phe Ile Phe Ala
Leu Ser Ala Ala Glu Lys Met Ile Lys Ser 115 120 125 Gly Gln Tyr Gln
Lys Gly Leu Val Ile Gly Ala Glu Val Leu Ser Lys 130 135 140 Ile Ile
Asp Trp Ser Asp Arg Thr Thr Ala Val Leu Phe Gly Asp Gly 145 150 155
160 Ala Gly Gly Val Leu Leu Glu Ala Asp Ser Ser Glu His Phe Leu Phe
165 170 175 Glu Ser Ile His Ser Asp Gly Ser Arg Gly Glu Ser Leu Thr
Ser Gly 180 185 190 Glu His Ala Val Ser Ser Pro Phe Ser Gln Val Asp
Lys Lys Asp Asn 195 200 205 Cys Phe Leu Lys Met Asp Gly Arg Ala Ile
Phe Asp Phe Ala Ile Arg 210 215 220 Asp Val Ser Lys Ser Ile Ser Met
Leu Ile Arg Lys Ser Asp Met Pro 225 230 235 240 Val Glu Ala Ile Asp
Tyr Phe Leu Leu His Gln Ala Asn Ile Arg Ile 245 250 255 Leu Asp Lys
Met Ala Lys Lys Ile Gly Ala Asp Arg Glu Lys Phe Pro 260 265 270 Ala
Asn Met Met Lys Tyr Gly Asn Thr Ser Ala Ala Ser Ile Pro Ile 275 280
285 Leu Leu Ala Glu Cys Val Glu Asn Gly Thr Ile Glu Leu Asn Gly Ser
290 295 300 His Thr Val Leu Leu Ser Gly Phe Gly Gly Gly Leu Thr Trp
Gly Ser 305 310 315 320 Leu Ile Val Lys Ile 325 12325PRTLactococcus
lactissource/note="Beta ketoacyl-ACP synthase III" 12Met Thr Phe
Ala Lys Ile Thr Gln Val Ala His Tyr Val Pro Glu Asn 1 5 10 15 Val
Val Ser Asn Asp Asp Leu Ser Lys Ile Met Asp Thr Asn Asp Glu 20 25
30 Trp Ile Tyr Ser Arg Thr Gly Ile Lys Asn Arg His Ile Ser Thr Gly
35 40 45 Glu Asn Thr Ser Asp Leu Ala Ala Lys Val Ala Lys Gln Leu
Ile Ser 50 55 60 Asp Ser Asn Leu Ser Pro Glu Thr Ile Asp Phe Ile
Ile Val Ala Thr 65 70 75 80 Val Thr Pro Asp Ser Leu Met Pro Ser Thr
Ala Ala Arg Val Gln Ala 85 90 95 Gln Val Gly Ala Val Asn Ala Phe
Ala Tyr Asp Leu Thr Ala Ala Cys 100 105 110 Ser Gly Phe Val Phe Ala
Leu Ser Thr Ala Glu Lys Leu Ile Ser Ser 115 120 125 Gly Ala Tyr Gln
Arg Gly Leu Val Ile Gly Ala Glu Val Phe Ser Lys 130 135 140 Val Ile
Asp Trp Ser Asp Arg Ser Thr Ala Val Leu Phe Gly Asp Gly 145 150 155
160 Ala Ala Gly Val Leu Ile Glu Ala Gly Ala Ser Gln Pro Leu Ile Ile
165 170 175 Ala Glu Lys Met Gln Thr Asp Gly Ser Arg Gly Asn Ser Leu
Leu Ser 180 185 190 Ser Tyr Ala Asp Ile Gln Thr Pro Phe Ala Ser Val
Ser Tyr Glu Ser 195 200 205 Ser Asn Leu Ser Met Glu Gly Arg Ala Ile
Phe Asp Phe Ala Val Arg 210 215 220 Asp Val Pro Lys Asn Ile Gln Ala
Thr Leu Glu Lys Ala Asn Leu Ser 225 230 235 240 Ala Glu Glu Val Asp
Tyr Tyr Leu Leu His Gln Ala Asn Ser Arg Ile 245 250 255 Leu Asp Lys
Met Ala Lys Lys Leu Gly Val Thr Arg Gln Lys Phe Leu 260 265 270 Gln
Asn Met Gln Glu Tyr Gly Asn Thr Ser Ala Ala Ser Ile Pro Ile 275 280
285 Leu Leu Ser Glu Ser Val Lys Asn Gly Ile Phe Ser Leu Asp Gly Gln
290 295 300 Thr Lys Val Val Leu Thr Gly Phe Gly Gly Gly Leu Thr Trp
Gly Thr 305 310 315 320 Ala Ile Ile Asn Leu 325
13300PRTPropionibacterium freudenreichiisource/note="subsp.
shermanii, Beta ketoacyl-ACP synthase III" 13Met Ile Asp Ser Thr
Pro Glu Trp Ile Glu Gln Arg Thr Gly Ile Arg 1 5 10 15 Glu Arg Arg
Trp Ala Thr Lys Asp Glu Thr Val Leu Ser Met Ala Thr 20 25 30 Asp
Ala Gly Arg Lys Ala Leu Asp Met Ala Gly Val Lys Pro Glu Gln 35 40
45 Val Gly Ala Ile Ile Val Ser Thr Val Ser His His Ile Pro Ser Pro
50 55 60 Gly Leu Ser Asp Tyr Leu Ala Glu Glu Leu Gly Cys Pro Ala
Pro Ala 65 70 75 80 Thr Phe Asp Ile Ser Ala Ala Cys Ala Gly Phe Cys
Tyr Ala Leu Thr 85 90 95 Leu Ala Glu Ser Ile Val Arg Ala Gly His
Ala Gly Lys Asp Gly Phe 100 105 110 Val Leu Ile Val Gly Val Glu Arg
Leu Ser Asp Met Thr Asn Met Asp 115 120 125 Asp Arg Gly Thr Asp Phe
Leu Phe Gly Asp Gly Ala Gly Ala Ala Val 130 135 140 Val Gly Pro Ser
Asp Thr Pro Ala Ile Gly Pro Ala Val Trp Gly Ser 145 150 155 160 Lys
Pro Ala Asn Val Lys Thr Ile Glu Ile Gln Ser Trp Thr Glu Ala 165 170
175 Asp Lys Asn Pro Thr Gly Phe Pro Leu Ile Gln Met Asp Gly His Thr
180 185 190 Val Phe Lys Trp Ala Leu Ser Glu Val Ala Asp His Ala Ala
Glu Ala 195 200 205 Ile Asp Ala Ala Gly Ile Thr Pro Glu Gln Leu Asp
Ile Phe Leu Pro 210 215 220 His Gln Ala Asn Asp Arg Ile Thr Asp Ala
Ile Ile Arg His Leu His 225 230 235 240 Leu Pro Asp Ser Val Ser Val
Cys Arg Asp Ile Ala Glu Met Gly Asn 245 250 255 Thr Ser Ala Ala Ser
Ile Pro Ile Ala Met Asp Ala Met Ile Arg Glu 260 265 270 Gly Arg Ala
Lys Ser Gly Gln Thr Ala Leu Ile Ile Gly Phe Gly Ala 275 280 285 Gly
Leu Val Tyr Ala Gly Arg Val Val Val Leu Pro 290 295 300
1417PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic FabH motif peptide" 14Asp Thr Asn Asp Ala Trp
Ile Xaa Xaa Met Thr Gly Ile Xaa Asn Arg 1 5 10 15 Arg
1518PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic FabH motif peptide" 15Ser Xaa Asp Xaa Xaa Ala
Ala Cys Ala Gly Phe Xaa Xaa Xaa Met Xaa 1 5 10 15 Xaa Ala
1615PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic FabH motif peptide" 16Asp Arg Xaa Thr Ala Ile
Xaa Phe Ala Asp Gly Ala Ala Ala Ala 1 5 10 15 178PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
FabH motif peptide" 17His Gln Ala Asn Xaa Arg Ile Met 1 5
1819PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic FabH motif peptide" 18Gly Asn Thr Gly Ala Ala
Ser Val Pro Xaa Xaa Ile Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Gly
1913PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic FabH motif peptide" 19Ile Xaa Leu Xaa Xaa Phe
Gly Gly Gly Leu Thr Trp Gly 1 5 10 20820PRTEscherichia
colisource/note="Aspartate kinase / Homoserine dehydrogenase
(ThrA)" 20Met Arg Val Leu Lys Phe Gly Gly Thr Ser Val Ala Asn Ala
Glu Arg 1 5 10 15 Phe Leu Arg Val Ala Asp Ile Leu Glu Ser Asn Ala
Arg Gln Gly Gln 20 25 30 Val Ala Thr Val Leu Ser Ala Pro Ala Lys
Ile Thr Asn His Leu Val 35 40 45 Ala Met Ile Glu Lys Thr Ile Ser
Gly Gln Asp Ala Leu Pro Asn Ile 50 55 60 Ser Asp Ala Glu Arg Ile
Phe Ala Glu Leu Leu Thr Gly Leu Ala Ala 65 70 75 80 Ala Gln Pro Gly
Phe Pro Leu Ala Gln Leu Lys Thr Phe Val Asp Gln 85 90 95 Glu Phe
Ala Gln Ile Lys His Val Leu His Gly Ile Ser Leu Leu Gly 100 105 110
Gln Cys Pro Asp Ser Ile Asn Ala Ala Leu Ile Cys Arg
Gly Glu Lys 115 120 125 Met Ser Ile Ala Ile Met Ala Gly Val Leu Glu
Ala Arg Gly His Asn 130 135 140 Val Thr Val Ile Asp Pro Val Glu Lys
Leu Leu Ala Val Gly His Tyr 145 150 155 160 Leu Glu Ser Thr Val Asp
Ile Ala Glu Ser Thr Arg Arg Ile Ala Ala 165 170 175 Ser Arg Ile Pro
Ala Asp His Met Val Leu Met Ala Gly Phe Thr Ala 180 185 190 Gly Asn
Glu Lys Gly Glu Leu Val Val Leu Gly Arg Asn Gly Ser Asp 195 200 205
Tyr Ser Ala Ala Val Leu Ala Ala Cys Leu Arg Ala Asp Cys Cys Glu 210
215 220 Ile Trp Thr Asp Val Asp Gly Val Tyr Thr Cys Asp Pro Arg Gln
Val 225 230 235 240 Pro Asp Ala Arg Leu Leu Lys Ser Met Ser Tyr Gln
Glu Ala Met Glu 245 250 255 Leu Ser Tyr Phe Gly Ala Lys Val Leu His
Pro Arg Thr Ile Thr Pro 260 265 270 Ile Ala Gln Phe Gln Ile Pro Cys
Leu Ile Lys Asn Thr Gly Asn Pro 275 280 285 Gln Ala Pro Gly Thr Leu
Ile Gly Ala Ser Arg Asp Glu Asp Glu Leu 290 295 300 Pro Val Lys Gly
Ile Ser Asn Leu Asn Asn Met Ala Met Phe Ser Val 305 310 315 320 Ser
Gly Pro Gly Met Lys Gly Met Val Gly Met Ala Ala Arg Val Phe 325 330
335 Ala Ala Met Ser Arg Ala Arg Ile Ser Val Val Leu Ile Thr Gln Ser
340 345 350 Ser Ser Glu Tyr Ser Ile Ser Phe Cys Val Pro Gln Ser Asp
Cys Val 355 360 365 Arg Ala Glu Arg Ala Met Gln Glu Glu Phe Tyr Leu
Glu Leu Lys Glu 370 375 380 Gly Leu Leu Glu Pro Leu Ala Val Thr Glu
Arg Leu Ala Ile Ile Ser 385 390 395 400 Val Val Gly Asp Gly Met Arg
Thr Leu Arg Gly Ile Ser Ala Lys Phe 405 410 415 Phe Ala Ala Leu Ala
Arg Ala Asn Ile Asn Ile Val Ala Ile Ala Gln 420 425 430 Gly Ser Ser
Glu Arg Ser Ile Ser Val Val Val Asn Asn Asp Asp Ala 435 440 445 Thr
Thr Gly Val Arg Val Thr His Gln Met Leu Phe Asn Thr Asp Gln 450 455
460 Val Ile Glu Val Phe Val Ile Gly Val Gly Gly Val Gly Gly Ala Leu
465 470 475 480 Leu Glu Gln Leu Lys Arg Gln Gln Ser Trp Leu Lys Asn
Lys His Ile 485 490 495 Asp Leu Arg Val Cys Gly Val Ala Asn Ser Lys
Ala Leu Leu Thr Asn 500 505 510 Val His Gly Leu Asn Leu Glu Asn Trp
Gln Glu Glu Leu Ala Gln Ala 515 520 525 Lys Glu Pro Phe Asn Leu Gly
Arg Leu Ile Arg Leu Val Lys Glu Tyr 530 535 540 His Leu Leu Asn Pro
Val Ile Val Asp Cys Thr Ser Ser Gln Ala Val 545 550 555 560 Ala Asp
Gln Tyr Ala Asp Phe Leu Arg Glu Gly Phe His Val Val Thr 565 570 575
Pro Asn Lys Lys Ala Asn Thr Ser Ser Met Asp Tyr Tyr His Gln Leu 580
585 590 Arg Tyr Ala Ala Glu Lys Ser Arg Arg Lys Phe Leu Tyr Asp Thr
Asn 595 600 605 Val Gly Ala Gly Leu Pro Val Ile Glu Asn Leu Gln Asn
Leu Leu Asn 610 615 620 Ala Gly Asp Glu Leu Met Lys Phe Ser Gly Ile
Leu Ser Gly Ser Leu 625 630 635 640 Ser Tyr Ile Phe Gly Lys Leu Asp
Glu Gly Met Ser Phe Ser Glu Ala 645 650 655 Thr Thr Leu Ala Arg Glu
Met Gly Tyr Thr Glu Pro Asp Pro Arg Asp 660 665 670 Asp Leu Ser Gly
Met Asp Val Ala Arg Lys Leu Leu Ile Leu Ala Arg 675 680 685 Glu Thr
Gly Arg Glu Leu Glu Leu Ala Asp Ile Glu Ile Glu Pro Val 690 695 700
Leu Pro Ala Glu Phe Asn Ala Glu Gly Asp Val Ala Ala Phe Met Ala 705
710 715 720 Asn Leu Ser Gln Leu Asp Asp Leu Phe Ala Ala Arg Val Ala
Lys Ala 725 730 735 Arg Asp Glu Gly Lys Val Leu Arg Tyr Val Gly Asn
Ile Asp Glu Asp 740 745 750 Gly Val Cys Arg Val Lys Ile Ala Glu Val
Asp Gly Asn Asp Pro Leu 755 760 765 Phe Lys Val Lys Asn Gly Glu Asn
Ala Leu Ala Phe Tyr Ser His Tyr 770 775 780 Tyr Gln Pro Leu Pro Leu
Val Leu Arg Gly Tyr Gly Ala Gly Asn Asp 785 790 795 800 Val Thr Ala
Ala Gly Val Phe Ala Asp Leu Leu Arg Thr Leu Ser Trp 805 810 815 Lys
Leu Gly Val 820 21820PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic Escherichia coli Thra S345F
variant polypeptide" 21Met Arg Val Leu Lys Phe Gly Gly Thr Ser Val
Ala Asn Ala Glu Arg 1 5 10 15 Phe Leu Arg Val Ala Asp Ile Leu Glu
Ser Asn Ala Arg Gln Gly Gln 20 25 30 Val Ala Thr Val Leu Ser Ala
Pro Ala Lys Ile Thr Asn His Leu Val 35 40 45 Ala Met Ile Glu Lys
Thr Ile Ser Gly Gln Asp Ala Leu Pro Asn Ile 50 55 60 Ser Asp Ala
Glu Arg Ile Phe Ala Glu Leu Leu Thr Gly Leu Ala Ala 65 70 75 80 Ala
Gln Pro Gly Phe Pro Leu Ala Gln Leu Lys Thr Phe Val Asp Gln 85 90
95 Glu Phe Ala Gln Ile Lys His Val Leu His Gly Ile Ser Leu Leu Gly
100 105 110 Gln Cys Pro Asp Ser Ile Asn Ala Ala Leu Ile Cys Arg Gly
Glu Lys 115 120 125 Met Ser Ile Ala Ile Met Ala Gly Val Leu Glu Ala
Arg Gly His Asn 130 135 140 Val Thr Val Ile Asp Pro Val Glu Lys Leu
Leu Ala Val Gly His Tyr 145 150 155 160 Leu Glu Ser Thr Val Asp Ile
Ala Glu Ser Thr Arg Arg Ile Ala Ala 165 170 175 Ser Arg Ile Pro Ala
Asp His Met Val Leu Met Ala Gly Phe Thr Ala 180 185 190 Gly Asn Glu
Lys Gly Glu Leu Val Val Leu Gly Arg Asn Gly Ser Asp 195 200 205 Tyr
Ser Ala Ala Val Leu Ala Ala Cys Leu Arg Ala Asp Cys Cys Glu 210 215
220 Ile Trp Thr Asp Val Asp Gly Val Tyr Thr Cys Asp Pro Arg Gln Val
225 230 235 240 Pro Asp Ala Arg Leu Leu Lys Ser Met Ser Tyr Gln Glu
Ala Met Glu 245 250 255 Leu Ser Tyr Phe Gly Ala Lys Val Leu His Pro
Arg Thr Ile Thr Pro 260 265 270 Ile Ala Gln Phe Gln Ile Pro Cys Leu
Ile Lys Asn Thr Gly Asn Pro 275 280 285 Gln Ala Pro Gly Thr Leu Ile
Gly Ala Ser Arg Asp Glu Asp Glu Leu 290 295 300 Pro Val Lys Gly Ile
Ser Asn Leu Asn Asn Met Ala Met Phe Ser Val 305 310 315 320 Ser Gly
Pro Gly Met Lys Gly Met Val Gly Met Ala Ala Arg Val Phe 325 330 335
Ala Ala Met Ser Arg Ala Arg Ile Phe Val Val Leu Ile Thr Gln Ser 340
345 350 Ser Ser Glu Tyr Ser Ile Ser Phe Cys Val Pro Gln Ser Asp Cys
Val 355 360 365 Arg Ala Glu Arg Ala Met Gln Glu Glu Phe Tyr Leu Glu
Leu Lys Glu 370 375 380 Gly Leu Leu Glu Pro Leu Ala Val Thr Glu Arg
Leu Ala Ile Ile Ser 385 390 395 400 Val Val Gly Asp Gly Met Arg Thr
Leu Arg Gly Ile Ser Ala Lys Phe 405 410 415 Phe Ala Ala Leu Ala Arg
Ala Asn Ile Asn Ile Val Ala Ile Ala Gln 420 425 430 Gly Ser Ser Glu
Arg Ser Ile Ser Val Val Val Asn Asn Asp Asp Ala 435 440 445 Thr Thr
Gly Val Arg Val Thr His Gln Met Leu Phe Asn Thr Asp Gln 450 455 460
Val Ile Glu Val Phe Val Ile Gly Val Gly Gly Val Gly Gly Ala Leu 465
470 475 480 Leu Glu Gln Leu Lys Arg Gln Gln Ser Trp Leu Lys Asn Lys
His Ile 485 490 495 Asp Leu Arg Val Cys Gly Val Ala Asn Ser Lys Ala
Leu Leu Thr Asn 500 505 510 Val His Gly Leu Asn Leu Glu Asn Trp Gln
Glu Glu Leu Ala Gln Ala 515 520 525 Lys Glu Pro Phe Asn Leu Gly Arg
Leu Ile Arg Leu Val Lys Glu Tyr 530 535 540 His Leu Leu Asn Pro Val
Ile Val Asp Cys Thr Ser Ser Gln Ala Val 545 550 555 560 Ala Asp Gln
Tyr Ala Asp Phe Leu Arg Glu Gly Phe His Val Val Thr 565 570 575 Pro
Asn Lys Lys Ala Asn Thr Ser Ser Met Asp Tyr Tyr His Gln Leu 580 585
590 Arg Tyr Ala Ala Glu Lys Ser Arg Arg Lys Phe Leu Tyr Asp Thr Asn
595 600 605 Val Gly Ala Gly Leu Pro Val Ile Glu Asn Leu Gln Asn Leu
Leu Asn 610 615 620 Ala Gly Asp Glu Leu Met Lys Phe Ser Gly Ile Leu
Ser Gly Ser Leu 625 630 635 640 Ser Tyr Ile Phe Gly Lys Leu Asp Glu
Gly Met Ser Phe Ser Glu Ala 645 650 655 Thr Thr Leu Ala Arg Glu Met
Gly Tyr Thr Glu Pro Asp Pro Arg Asp 660 665 670 Asp Leu Ser Gly Met
Asp Val Ala Arg Lys Leu Leu Ile Leu Ala Arg 675 680 685 Glu Thr Gly
Arg Glu Leu Glu Leu Ala Asp Ile Glu Ile Glu Pro Val 690 695 700 Leu
Pro Ala Glu Phe Asn Ala Glu Gly Asp Val Ala Ala Phe Met Ala 705 710
715 720 Asn Leu Ser Gln Leu Asp Asp Leu Phe Ala Ala Arg Val Ala Lys
Ala 725 730 735 Arg Asp Glu Gly Lys Val Leu Arg Tyr Val Gly Asn Ile
Asp Glu Asp 740 745 750 Gly Val Cys Arg Val Lys Ile Ala Glu Val Asp
Gly Asn Asp Pro Leu 755 760 765 Phe Lys Val Lys Asn Gly Glu Asn Ala
Leu Ala Phe Tyr Ser His Tyr 770 775 780 Tyr Gln Pro Leu Pro Leu Val
Leu Arg Gly Tyr Gly Ala Gly Asn Asp 785 790 795 800 Val Thr Ala Ala
Gly Val Phe Ala Asp Leu Leu Arg Thr Leu Ser Trp 805 810 815 Lys Leu
Gly Val 820 22404PRTBacillus subtilissource/note="Aspartate kinase"
22Met Lys Ile Ile Val Gln Lys Phe Gly Gly Thr Ser Val Lys Asp Asp 1
5 10 15 Lys Gly Arg Lys Leu Ala Leu Gly His Ile Lys Glu Ala Ile Ser
Glu 20 25 30 Gly Tyr Lys Val Val Val Val Val Ser Ala Met Gly Arg
Lys Gly Asp 35 40 45 Pro Tyr Ala Thr Asp Ser Leu Leu Gly Leu Leu
Tyr Gly Asp Gln Ser 50 55 60 Ala Ile Ser Pro Arg Glu Gln Asp Leu
Leu Leu Ser Cys Gly Glu Thr 65 70 75 80 Ile Ser Ser Val Val Phe Thr
Ser Met Leu Leu Asp Asn Gly Val Lys 85 90 95 Ala Ala Ala Leu Thr
Gly Ala Gln Ala Gly Phe Leu Thr Asn Asp Gln 100 105 110 His Thr Asn
Ala Lys Ile Ile Glu Met Lys Pro Glu Arg Leu Phe Ser 115 120 125 Val
Leu Ala Asn His Asp Ala Val Val Val Ala Gly Phe Gln Gly Ala 130 135
140 Thr Glu Lys Gly Asp Thr Thr Thr Ile Gly Arg Gly Gly Ser Asp Thr
145 150 155 160 Ser Ala Ala Ala Leu Gly Ala Ala Val Asp Ala Glu Tyr
Ile Asp Ile 165 170 175 Phe Thr Asp Val Glu Gly Val Met Thr Ala Asp
Pro Arg Val Val Glu 180 185 190 Asn Ala Lys Pro Leu Pro Val Val Thr
Tyr Thr Glu Ile Cys Asn Leu 195 200 205 Ala Tyr Gln Gly Ala Lys Val
Ile Ser Pro Arg Ala Val Glu Ile Ala 210 215 220 Met Gln Ala Lys Val
Pro Ile Arg Val Arg Ser Thr Tyr Ser Asn Asp 225 230 235 240 Lys Gly
Thr Leu Val Thr Ser His His Ser Ser Lys Val Gly Ser Asp 245 250 255
Val Phe Glu Arg Leu Ile Thr Gly Ile Ala His Val Lys Asp Val Thr 260
265 270 Gln Phe Lys Val Pro Ala Lys Ile Gly Gln Tyr Asn Val Gln Thr
Glu 275 280 285 Val Phe Lys Ala Met Ala Asn Ala Gly Ile Ser Val Asp
Phe Phe Asn 290 295 300 Ile Thr Pro Ser Glu Ile Val Tyr Thr Val Ala
Gly Asn Lys Thr Glu 305 310 315 320 Thr Ala Gln Arg Ile Leu Met Asp
Met Gly Tyr Asp Pro Met Val Thr 325 330 335 Arg Asn Cys Ala Lys Val
Ser Ala Val Gly Ala Gly Ile Met Gly Val 340 345 350 Pro Gly Val Thr
Ser Lys Ile Val Ser Ala Leu Ser Glu Lys Glu Ile 355 360 365 Pro Ile
Leu Gln Ser Ala Asp Ser His Thr Thr Ile Trp Val Leu Val 370 375 380
His Glu Ala Asp Met Val Pro Ala Val Asn Ala Leu His Glu Val Phe 385
390 395 400 Glu Leu Ser Lys 23411PRTPseudomonas
putidasource/note="Aspartate kinase" 23Met Ala Leu Ile Val Gln Lys
Phe Gly Gly Thr Ser Val Gly Ser Ile 1 5 10 15 Glu Arg Ile Glu Gln
Val Ala Glu Lys Val Lys Lys His Arg Glu Ala 20 25 30 Gly Asp Asp
Leu Val Val Val Leu Ser Ala Met Ser Gly Glu Thr Asn 35 40 45 Arg
Leu Ile Asp Leu Ala Lys Gln Ile Thr Asp Gln Pro Val Pro Arg 50 55
60 Glu Leu Asp Val Ile Val Ser Thr Gly Glu Gln Val Thr Ile Ala Leu
65 70 75 80 Leu Thr Met Ala Leu Ile Lys Arg Gly Val Pro Ala Val Ser
Tyr Thr 85 90 95 Gly Asn Gln Val Arg Ile Leu Thr Asp Ser Ser His
Asn Lys Ala Arg 100 105 110 Ile Leu Gln Ile Asp Asp Gln Lys Ile Arg
Ala Asp Leu Lys Glu Gly 115 120 125 Arg Val Val Val Val Ala Gly Phe
Gln Gly Val Asp Glu His Gly Ser 130 135 140 Ile Thr Thr Leu Gly Arg
Gly Gly Ser Asp Thr Thr Gly Val Ala Leu 145 150 155 160 Ala Ala Ala
Leu Lys Ala Asp Glu Cys Gln Ile Tyr Thr Asp Val Asp 165 170 175 Gly
Val Tyr Thr Thr Asp Pro Arg Val Val Pro Gln Ala Arg Arg Leu 180 185
190 Glu Lys Ile Thr Phe Glu Glu Met Leu Glu Met Ala Ser Leu Gly Ser
195 200 205 Lys Val Leu Gln Ile Arg Ser Val Glu Phe Ala Gly Lys Tyr
Asn Val 210 215 220 Pro Leu Arg Val Leu His Ser Phe Lys Glu Gly Pro
Gly Thr Leu Ile 225 230 235 240 Thr Ile Asp Glu Glu Glu Ser Met Glu
Gln Pro Ile Ile Ser Gly Ile 245 250 255 Ala Phe Asn Arg Asp Glu Ala
Lys Leu Thr Ile Arg Gly Val Pro Asp 260 265 270 Thr Pro Gly Val Ala
Phe Lys Ile Leu Gly Pro Ile Ser Ala Ser Asn 275 280 285 Ile Glu Val
Asp Met Ile Val Gln Asn Val Ala His Asp Asn Thr Thr 290 295 300 Asp
Phe Thr Phe Thr Val His Arg Asn Glu Tyr Glu Lys Ala Gln Ser 305 310
315 320 Val Leu Glu Asn Thr Ala Arg Glu Ile Gly Ala Arg Glu Val Ile
Gly 325 330 335 Asp Thr Lys Ile Ala Lys Val Ser Ile Val Gly Val Gly
Met Arg Ser
340 345 350 His Ala Gly Val Ala Ser Cys Met Phe Glu Ala Leu Ala Lys
Glu Ser 355 360 365 Ile Asn Ile Gln Met Ile Ser Thr Ser Glu Ile Lys
Val Ser Val Val 370 375 380 Leu Glu Glu Lys Tyr Leu Glu Leu Ala Val
Arg Ala Leu His Thr Ala 385 390 395 400 Phe Asp Leu Asp Ala Pro Ala
Arg Gln Gly Glu 405 410 24527PRTSaccharomyces
cerevisiaesource/note="Aspartate kinase" 24Met Pro Met Asp Phe Gln
Pro Thr Ser Ser His Ser Asn Trp Val Val 1 5 10 15 Gln Lys Phe Gly
Gly Thr Ser Val Gly Lys Phe Pro Val Gln Ile Val 20 25 30 Asp Asp
Ile Val Lys His Tyr Ser Lys Pro Asp Gly Pro Asn Asn Asn 35 40 45
Val Ala Val Val Cys Ser Ala Arg Ser Ser Tyr Thr Lys Ala Glu Gly 50
55 60 Thr Thr Ser Arg Leu Leu Lys Cys Cys Asp Leu Ala Ser Gln Glu
Ser 65 70 75 80 Glu Phe Gln Asp Ile Ile Glu Val Ile Arg Gln Asp His
Ile Asp Asn 85 90 95 Ala Asp Arg Phe Ile Leu Asn Pro Ala Leu Gln
Ala Lys Leu Val Asp 100 105 110 Asp Thr Asn Lys Glu Leu Glu Leu Val
Lys Lys Tyr Leu Asn Ala Ser 115 120 125 Lys Val Leu Gly Glu Val Ser
Ser Arg Thr Val Asp Leu Val Met Ser 130 135 140 Cys Gly Glu Lys Leu
Ser Cys Leu Phe Met Thr Ala Leu Cys Asn Asp 145 150 155 160 Arg Gly
Cys Lys Ala Lys Tyr Val Asp Leu Ser His Ile Val Pro Ser 165 170 175
Asp Phe Ser Ala Ser Ala Leu Asp Asn Ser Phe Tyr Thr Phe Leu Val 180
185 190 Gln Ala Leu Lys Glu Lys Leu Ala Pro Phe Val Ser Ala Lys Glu
Arg 195 200 205 Ile Val Pro Val Phe Thr Gly Phe Phe Gly Leu Val Pro
Thr Gly Leu 210 215 220 Leu Asn Gly Val Gly Arg Gly Tyr Thr Asp Leu
Cys Ala Ala Leu Ile 225 230 235 240 Ala Val Ala Val Asn Ala Asp Glu
Leu Gln Val Trp Lys Glu Val Asp 245 250 255 Gly Ile Phe Thr Ala Asp
Pro Arg Lys Val Pro Glu Ala Arg Leu Leu 260 265 270 Asp Ser Val Thr
Pro Glu Glu Ala Ser Glu Leu Thr Tyr Tyr Gly Ser 275 280 285 Glu Val
Ile His Pro Phe Thr Met Glu Gln Val Ile Arg Ala Lys Ile 290 295 300
Pro Ile Arg Ile Lys Asn Val Gln Asn Pro Leu Gly Asn Gly Thr Ile 305
310 315 320 Ile Tyr Pro Asp Asn Val Ala Lys Lys Gly Glu Ser Thr Pro
Pro His 325 330 335 Pro Pro Glu Asn Leu Ser Ser Ser Phe Tyr Glu Lys
Arg Lys Arg Gly 340 345 350 Ala Thr Ala Ile Thr Thr Lys Asn Asp Ile
Phe Val Ile Asn Ile His 355 360 365 Ser Asn Lys Lys Thr Leu Ser His
Gly Phe Leu Ala Gln Ile Phe Thr 370 375 380 Ile Leu Asp Lys Tyr Lys
Leu Val Val Asp Leu Ile Ser Thr Ser Glu 385 390 395 400 Val His Val
Ser Met Ala Leu Pro Ile Pro Asp Ala Asp Ser Leu Lys 405 410 415 Ser
Leu Arg Gln Ala Glu Glu Lys Leu Arg Ile Leu Gly Ser Val Asp 420 425
430 Ile Thr Lys Lys Leu Ser Ile Val Ser Leu Val Gly Lys His Met Lys
435 440 445 Gln Tyr Ile Gly Ile Ala Gly Thr Met Phe Thr Thr Leu Ala
Glu Glu 450 455 460 Gly Ile Asn Ile Glu Met Ile Ser Gln Gly Ala Asn
Glu Ile Asn Ile 465 470 475 480 Ser Cys Val Ile Asn Glu Ser Asp Ser
Ile Lys Ala Leu Gln Cys Ile 485 490 495 His Ala Lys Leu Leu Ser Glu
Arg Thr Asn Thr Ser Asn Gln Phe Glu 500 505 510 His Ala Ile Asp Glu
Arg Leu Glu Gln Leu Lys Arg Leu Gly Ile 515 520 525
25433PRTBacillus subtilissource/note="Homoserine dehydrogenase" 25
Met Lys Ala Ile Arg Val Gly Leu Leu Gly Leu Gly Thr Val Gly Ser 1 5
10 15 Gly Val Val Lys Ile Ile Gln Asp His Gln Asp Lys Leu Met His
Gln 20 25 30 Val Gly Cys Pro Val Thr Ile Lys Lys Val Leu Val Lys
Asp Leu Glu 35 40 45 Lys Lys Arg Glu Val Asp Leu Pro Lys Glu Val
Leu Thr Thr Glu Val 50 55 60 Tyr Asp Val Ile Asp Asp Pro Asp Val
Asp Val Val Ile Glu Val Ile 65 70 75 80 Gly Gly Val Glu Gln Thr Lys
Gln Tyr Leu Val Asp Ala Leu Arg Ser 85 90 95 Lys Lys His Val Val
Thr Ala Asn Lys Asp Leu Met Ala Val Tyr Gly 100 105 110 Ser Glu Leu
Leu Ala Glu Ala Lys Glu Asn Gly Cys Asp Ile Tyr Phe 115 120 125 Glu
Ala Ser Val Ala Gly Gly Ile Pro Ile Leu Arg Thr Leu Glu Glu 130 135
140 Gly Leu Ser Ser Asp Arg Ile Thr Lys Met Met Gly Ile Val Asn Gly
145 150 155 160 Thr Thr Asn Phe Ile Leu Thr Lys Met Ile Lys Glu Lys
Ser Pro Tyr 165 170 175 Glu Glu Val Leu Lys Glu Ala Gln Asp Leu Gly
Phe Ala Glu Ala Asp 180 185 190 Pro Thr Ser Asp Val Glu Gly Leu Asp
Ala Ala Arg Lys Met Ala Ile 195 200 205 Leu Ala Arg Leu Gly Phe Ser
Met Asn Val Asp Leu Glu Asp Val Lys 210 215 220 Val Lys Gly Ile Ser
Gln Ile Thr Asp Glu Asp Ile Ser Phe Ser Lys 225 230 235 240 Arg Leu
Gly Tyr Thr Met Lys Leu Ile Gly Ile Ala Gln Arg Asp Gly 245 250 255
Ser Lys Ile Glu Val Ser Val Gln Pro Thr Leu Leu Pro Asp His His 260
265 270 Pro Leu Ser Ala Val His Asn Glu Phe Asn Ala Val Tyr Val Tyr
Gly 275 280 285 Glu Ala Val Gly Glu Thr Met Phe Tyr Gly Pro Gly Ala
Gly Ser Met 290 295 300 Pro Thr Ala Thr Ser Val Val Ser Asp Leu Val
Ala Val Met Lys Asn 305 310 315 320 Met Arg Leu Gly Val Thr Gly Asn
Ser Phe Val Gly Pro Gln Tyr Glu 325 330 335 Lys Asn Met Lys Ser Pro
Ser Asp Ile Tyr Ala Gln Gln Phe Leu Arg 340 345 350 Ile His Val Lys
Asp Glu Val Gly Ser Phe Ser Lys Ile Thr Ser Val 355 360 365 Phe Ser
Glu Arg Gly Val Ser Phe Glu Lys Ile Leu Gln Leu Pro Ile 370 375 380
Lys Gly His Asp Glu Leu Ala Glu Ile Val Ile Val Thr His His Thr 385
390 395 400 Ser Glu Ala Asp Phe Ser Asp Ile Leu Gln Asn Leu Asn Asp
Leu Glu 405 410 415 Val Val Gln Glu Val Lys Ser Thr Tyr Arg Val Glu
Gly Asn Gly Trp 420 425 430 Ser 26434PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
Pseudomonas putida homoserine dehydrogenase polypeptide" 26Met Lys
Pro Val Lys Val Gly Ile Cys Gly Leu Gly Thr Val Gly Gly 1 5 10 15
Gly Thr Phe Asn Val Leu Gln Arg Asn Ala Glu Glu Ile Ala Arg Arg 20
25 30 Ala Gly Arg Gly Ile Glu Val Ala Gln Ile Ala Met Arg Ser Gln
Asn 35 40 45 Pro Asn Cys Gln Ile Thr Gly Thr Pro Ile Thr Ala Asp
Val Phe Glu 50 55 60 Val Ala Ser Asn Pro Glu Ile Asp Ile Val Ile
Glu Leu Ile Gly Gly 65 70 75 80 Tyr Thr Ile Ala Arg Asp Leu Val Leu
Lys Ala Ile Glu Asn Gly Lys 85 90 95 His Val Val Thr Ala Asn Lys
Ala Leu Ile Ala Val His Gly Asn Glu 100 105 110 Ile Phe Ala Lys Ala
Arg Glu Lys Gly Val Ile Val Ala Phe Glu Ala 115 120 125 Ala Val Ala
Gly Gly Ile Pro Val Ile Lys Ala Ile Arg Glu Gly Leu 130 135 140 Ser
Ala Asn Arg Ile Asn Trp Leu Ala Gly Ile Ile Asn Gly Thr Gly 145 150
155 160 Asn Phe Ile Leu Thr Glu Met Arg Glu Lys Gly Arg Ala Phe Pro
Asp 165 170 175 Val Leu Ala Glu Ala Gln Ala Leu Gly Tyr Ala Glu Ala
Asp Pro Thr 180 185 190 Phe Asp Val Glu Gly Ile Asp Ala Ala His Lys
Leu Thr Ile Leu Ala 195 200 205 Ser Ile Ala Phe Gly Ile Pro Leu Gln
Phe Asp Lys Ala Tyr Thr Glu 210 215 220 Gly Ile Thr Gln Leu Thr Thr
Ala Asp Val Asn Tyr Ala Glu Ala Leu 225 230 235 240 Gly Tyr Arg Ile
Lys His Leu Gly Val Ala Arg Arg Thr Ala Glu Gly 245 250 255 Ile Glu
Leu Arg Val His Pro Thr Leu Ile Pro Ala Asp Arg Leu Ile 260 265 270
Ala Asn Val Asn Gly Val Met Asn Ala Val Met Val Asn Gly Asp Ala 275
280 285 Ala Gly Ser Thr Leu Tyr Tyr Gly Ala Gly Ala Gly Met Glu Pro
Thr 290 295 300 Ala Ser Ser Val Val Gly Asp Leu Val Asp Val Val Arg
Ala Met Thr 305 310 315 320 Ser Asp Pro Glu Asn Arg Val Pro His Leu
Ala Phe Gln Pro Asp Ser 325 330 335 Leu Ser Ala His Pro Ile Leu Pro
Ile Glu Ala Cys Glu Ser Ala Tyr 340 345 350 Tyr Leu Arg Ile Gln Ala
Lys Asp His Pro Gly Val Leu Ala Gln Val 355 360 365 Ala Ser Ile Leu
Ser Glu Arg Gly Ile Asn Ile Glu Ser Ile Met Gln 370 375 380 Lys Glu
Ala Glu Glu Gln Asp Gly Leu Val Pro Met Ile Leu Val Thr 385 390 395
400 His Gly Val Val Glu Gln Arg Ile Asn Asp Ala Ile Val Ala Leu Glu
405 410 415 Ala Leu Gln Asp Val Val Gly Lys Val Val Arg Ile Arg Val
Glu Gln 420 425 430 Leu Asn 27359PRTSaccharomyces
cerevisiaesource/note="Homoserine dehydrogenase" 27Met Ser Thr Lys
Val Val Asn Val Ala Val Ile Gly Ala Gly Val Val 1 5 10 15 Gly Ser
Ala Phe Leu Asp Gln Leu Leu Ala Met Lys Ser Thr Ile Thr 20 25 30
Tyr Asn Leu Val Leu Leu Ala Glu Ala Glu Arg Ser Leu Ile Ser Lys 35
40 45 Asp Phe Ser Pro Leu Asn Val Gly Ser Asp Trp Lys Ala Ala Leu
Ala 50 55 60 Ala Ser Thr Thr Lys Thr Leu Pro Leu Asp Asp Leu Ile
Ala His Leu 65 70 75 80 Lys Thr Ser Pro Lys Pro Val Ile Leu Val Asp
Asn Thr Ser Ser Ala 85 90 95 Tyr Ile Ala Gly Phe Tyr Thr Lys Phe
Val Glu Asn Gly Ile Ser Ile 100 105 110 Ala Thr Pro Asn Lys Lys Ala
Phe Ser Ser Asp Leu Ala Thr Trp Lys 115 120 125 Ala Leu Phe Ser Asn
Lys Pro Thr Asn Gly Phe Val Tyr His Glu Ala 130 135 140 Thr Val Gly
Ala Gly Leu Pro Ile Ile Ser Phe Leu Arg Glu Ile Ile 145 150 155 160
Gln Thr Gly Asp Glu Val Glu Lys Ile Glu Gly Ile Phe Ser Gly Thr 165
170 175 Leu Ser Tyr Ile Phe Asn Glu Phe Ser Thr Ser Gln Ala Asn Asp
Val 180 185 190 Lys Phe Ser Asp Val Val Lys Val Ala Lys Lys Leu Gly
Tyr Thr Glu 195 200 205 Pro Asp Pro Arg Asp Asp Leu Asn Gly Leu Asp
Val Ala Arg Lys Val 210 215 220 Thr Ile Val Gly Arg Ile Ser Gly Val
Glu Val Glu Ser Pro Thr Ser 225 230 235 240 Phe Pro Val Gln Ser Leu
Ile Pro Lys Pro Leu Glu Ser Val Lys Ser 245 250 255 Ala Asp Glu Phe
Leu Glu Lys Leu Ser Asp Tyr Asp Lys Asp Leu Thr 260 265 270 Gln Leu
Lys Lys Glu Ala Ala Thr Glu Asn Lys Val Leu Arg Phe Ile 275 280 285
Gly Lys Val Asp Val Ala Thr Lys Ser Val Ser Val Gly Ile Glu Lys 290
295 300 Tyr Asp Tyr Ser His Pro Phe Ala Ser Leu Lys Gly Ser Asp Asn
Val 305 310 315 320 Ile Ser Ile Lys Thr Lys Arg Tyr Thr Asn Pro Val
Val Ile Gln Gly 325 330 335 Ala Gly Ala Gly Ala Ala Val Thr Ala Ala
Gly Val Leu Gly Asp Val 340 345 350 Ile Lys Ile Ala Gln Arg Leu 355
28310PRTEscherichia colisource/note="Homoserine kinase" 28Met Val
Lys Val Tyr Ala Pro Ala Ser Ser Ala Asn Met Ser Val Gly 1 5 10 15
Phe Asp Val Leu Gly Ala Ala Val Thr Pro Val Asp Gly Ala Leu Leu 20
25 30 Gly Asp Val Val Thr Val Glu Ala Ala Glu Thr Phe Ser Leu Asn
Asn 35 40 45 Leu Gly Arg Phe Ala Asp Lys Leu Pro Ser Glu Pro Arg
Glu Asn Ile 50 55 60 Val Tyr Gln Cys Trp Glu Arg Phe Cys Gln Glu
Leu Gly Lys Gln Ile 65 70 75 80 Pro Val Ala Met Thr Leu Glu Lys Asn
Met Pro Ile Gly Ser Gly Leu 85 90 95 Gly Ser Ser Ala Cys Ser Val
Val Ala Ala Leu Met Ala Met Asn Glu 100 105 110 His Cys Gly Lys Pro
Leu Asn Asp Thr Arg Leu Leu Ala Leu Met Gly 115 120 125 Glu Leu Glu
Gly Arg Ile Ser Gly Ser Ile His Tyr Asp Asn Val Ala 130 135 140 Pro
Cys Phe Leu Gly Gly Met Gln Leu Met Ile Glu Glu Asn Asp Ile 145 150
155 160 Ile Ser Gln Gln Val Pro Gly Phe Asp Glu Trp Leu Trp Val Leu
Ala 165 170 175 Tyr Pro Gly Ile Lys Val Ser Thr Ala Glu Ala Arg Ala
Ile Leu Pro 180 185 190 Ala Gln Tyr Arg Arg Gln Asp Cys Ile Ala His
Gly Arg His Leu Ala 195 200 205 Gly Phe Ile His Ala Cys Tyr Ser Arg
Gln Pro Glu Leu Ala Ala Lys 210 215 220 Leu Met Lys Asp Val Ile Ala
Glu Pro Tyr Arg Glu Arg Leu Leu Pro 225 230 235 240 Gly Phe Arg Gln
Ala Arg Gln Ala Val Ala Glu Ile Gly Ala Val Ala 245 250 255 Ser Gly
Ile Ser Gly Ser Gly Pro Thr Leu Phe Ala Leu Cys Asp Lys 260 265 270
Pro Glu Thr Ala Gln Arg Val Ala Asp Trp Leu Gly Lys Asn Tyr Leu 275
280 285 Gln Asn Gln Glu Gly Phe Val His Ile Cys Arg Leu Asp Thr Ala
Gly 290 295 300 Ala Arg Val Leu Glu Asn 305 310 29309PRTBacillus
subtilissource/note="Homoserine kinase" 29Met Asn Glu Ala Asp Met
Leu Phe Ser Val Thr Val Pro Gly Ser Thr 1 5 10 15 Ala Asn Leu Gly
Pro Gly Phe Asp Ser Val Gly Met Ala Leu Ser Arg 20 25 30 Tyr Leu
Lys Leu Thr Val Phe Glu Ser Asp Lys Trp Ser Phe Glu Ala 35 40 45
Glu Thr Glu Thr Val Ala Gly Ile Pro Ala Gly Thr Asp Asn Leu Ile 50
55 60 Tyr Gln Val Ala Lys Arg Thr Ala Asp Leu Tyr Gly Lys Glu Met
Pro 65 70 75 80 Pro Val His Val Lys Val Trp Ser Asp Ile Pro Leu Ala
Arg Gly Leu 85 90 95 Gly Ser Ser Ala Ala Ala Ile Val Ala Ala Ile
Glu Leu Ala Asp Glu 100 105
110 Leu Cys Gly Leu Lys Leu Ser Glu Ala Asp Lys Leu His Leu Ala Ser
115 120 125 Leu Glu Glu Gly His Pro Asp Asn Ala Gly Ala Ser Leu Val
Gly Gly 130 135 140 Leu Val Ile Gly Leu His Glu Asp Asp Glu Thr Gln
Met Ile Arg Val 145 150 155 160 Pro Asn Ala Asp Ile Asp Val Val Val
Val Ile Pro Phe Tyr Glu Val 165 170 175 Leu Thr Arg Asp Ala Arg Asp
Val Leu Pro Lys Glu Phe Pro Tyr Ala 180 185 190 Asp Ala Val Lys Ala
Ser Ala Val Ser Asn Ile Leu Ile Ala Ala Ile 195 200 205 Met Ser Lys
Asp Trp Pro Leu Val Gly Lys Ile Met Lys Lys Asp Met 210 215 220 Phe
His Gln Pro Tyr Arg Ala Met Leu Val Pro Glu Leu Ser Lys Val 225 230
235 240 Glu His Val Ala Glu Met Lys Gly Ala Tyr Gly Thr Ala Leu Ser
Gly 245 250 255 Ala Gly Pro Thr Ile Leu Val Met Thr Glu Lys Gly Lys
Gly Glu Glu 260 265 270 Leu Lys Glu Gln Leu Ala Leu His Phe Pro His
Cys Glu Val Asp Ala 275 280 285 Leu Thr Val Pro Lys Glu Gly Ser Ile
Ile Glu Arg Asn Pro Leu Tyr 290 295 300 Gln Val Lys Ser Val 305
30316PRTPseudomonas putidasource/note="Homoserine kinase" 30Met Ser
Val Phe Thr Pro Val Thr Arg Pro Glu Leu Glu Thr Phe Leu 1 5 10 15
Ala Pro Tyr Glu Leu Gly Arg Leu Leu Asp Phe Gln Gly Ile Ala Ala 20
25 30 Gly Thr Glu Asn Ser Asn Phe Phe Val Ser Leu Glu Gln Gly Glu
Phe 35 40 45 Val Leu Thr Leu Ile Glu Arg Gly Pro Ser Glu Asp Met
Pro Phe Phe 50 55 60 Ile Glu Leu Leu Asp Thr Leu His Gly Ala Asp
Met Pro Val Pro Tyr 65 70 75 80 Ala Ile Arg Asp Arg Asp Gly Asn Gly
Leu Arg Glu Leu Cys Gly Lys 85 90 95 Pro Ala Leu Leu Gln Pro Arg
Leu Ser Gly Lys His Ile Lys Ala Pro 100 105 110 Asn Asn Gln His Cys
Ala Gln Val Gly Glu Leu Leu Ala His Ile His 115 120 125 Leu Ala Thr
Arg Glu His Ile Ile Glu Arg Arg Thr Asp Arg Gly Leu 130 135 140 Asp
Trp Met Leu Ala Ser Gly Val Glu Leu Leu Pro Arg Leu Thr Ala 145 150
155 160 Glu Gln Ala Ala Leu Leu Gln Pro Ala Leu Asp Glu Ile Ser Ala
His 165 170 175 Lys Ala Gln Ile Leu Ala Leu Pro Arg Ala Asn Leu His
Ala Asp Leu 180 185 190 Phe Arg Asp Asn Val Met Phe Glu Gly Thr His
Leu Thr Gly Val Ile 195 200 205 Asp Phe Tyr Asn Ala Cys Ser Gly Pro
Met Leu Tyr Asp Ile Ala Ile 210 215 220 Thr Val Asn Asp Trp Cys Leu
Asp Glu Gln Gly Ala Val Asp Val Pro 225 230 235 240 Arg Ala Gln Ala
Leu Leu Ala Ala Tyr Ala Ala Leu Arg Pro Phe Thr 245 250 255 Ala Ala
Glu Ala Glu Leu Trp Pro Glu Met Leu Arg Val Gly Cys Val 260 265 270
Arg Phe Trp Leu Ser Arg Leu Ile Ala Ala Glu Ser Phe Ala Gly Met 275
280 285 Asp Val Met Ile His Asp Pro Ser Glu Phe Glu Val Arg Leu Ala
Gln 290 295 300 Arg Gln Gln Val Ala Leu His Leu Pro Phe Ala Leu 305
310 315 31357PRTSaccharomyces cerevisiaesource/note="Homoserine
kinase" 31Met Val Arg Ala Phe Lys Ile Lys Val Pro Ala Ser Ser Ala
Asn Ile 1 5 10 15 Gly Pro Gly Tyr Asp Val Leu Gly Val Gly Leu Ser
Leu Phe Leu Glu 20 25 30 Leu Asp Val Thr Ile Asp Ser Ser Gln Ala
Gln Glu Thr Asn Asp Asp 35 40 45 Pro Asn Asn Cys Lys Leu Ser Tyr
Thr Lys Glu Ser Glu Gly Tyr Ser 50 55 60 Thr Val Pro Leu Arg Ser
Asp Ala Asn Leu Ile Thr Arg Thr Ala Leu 65 70 75 80 Tyr Val Leu Arg
Cys Asn Asn Ile Arg Asn Phe Pro Ser Gly Thr Lys 85 90 95 Val His
Val Ser Asn Pro Ile Pro Leu Gly Arg Gly Leu Gly Ser Ser 100 105 110
Gly Ala Ala Val Val Ala Gly Val Ile Leu Gly Asn Glu Val Ala Gln 115
120 125 Leu Gly Phe Ser Lys Gln Arg Met Leu Asp Tyr Cys Leu Met Ile
Glu 130 135 140 Arg His Pro Asp Asn Ile Thr Ala Ala Met Met Gly Gly
Phe Cys Gly 145 150 155 160 Ser Phe Leu Arg Asp Leu Thr Pro Gln Glu
Val Glu Arg Arg Glu Ile 165 170 175 Pro Leu Ala Glu Val Leu Pro Glu
Pro Ser Gly Gly Glu Asp Thr Gly 180 185 190 Leu Val Pro Pro Leu Pro
Pro Thr Asp Ile Gly Arg His Val Lys Tyr 195 200 205 Gln Trp Asn Pro
Ala Ile Lys Cys Ile Ala Ile Ile Pro Gln Phe Glu 210 215 220 Leu Ser
Thr Ala Asp Ser Arg Gly Val Leu Pro Lys Ala Tyr Pro Thr 225 230 235
240 Gln Asp Leu Val Phe Asn Leu Gln Arg Leu Ala Val Leu Thr Thr Ala
245 250 255 Leu Thr Met Asp Pro Pro Asn Ala Asp Leu Ile Tyr Pro Ala
Met Gln 260 265 270 Asp Arg Val His Gln Pro Tyr Arg Lys Thr Leu Ile
Pro Gly Leu Thr 275 280 285 Glu Ile Leu Ser Cys Val Thr Pro Ser Thr
Tyr Pro Gly Leu Leu Gly 290 295 300 Ile Cys Leu Ser Gly Ala Gly Pro
Thr Ile Leu Ala Leu Ala Thr Glu 305 310 315 320 Asn Phe Glu Glu Ile
Ser Gln Glu Ile Ile Asn Arg Phe Ala Lys Asn 325 330 335 Gly Ile Lys
Cys Ser Trp Lys Leu Leu Glu Pro Ala Tyr Asp Gly Ala 340 345 350 Ser
Val Glu Gln Gln 355 32428PRTEscherichia colisource/note="Threonine
synthase" 32Met Lys Leu Tyr Asn Leu Lys Asp His Asn Glu Gln Val Ser
Phe Ala 1 5 10 15 Gln Ala Val Thr Gln Gly Leu Gly Lys Asn Gln Gly
Leu Phe Phe Pro 20 25 30 His Asp Leu Pro Glu Phe Ser Leu Thr Glu
Ile Asp Glu Met Leu Lys 35 40 45 Leu Asp Phe Val Thr Arg Ser Ala
Lys Ile Leu Ser Ala Phe Ile Gly 50 55 60 Asp Glu Ile Pro Gln Glu
Ile Leu Glu Glu Arg Val Arg Ala Ala Phe 65 70 75 80 Ala Phe Pro Ala
Pro Val Ala Asn Val Glu Ser Asp Val Gly Cys Leu 85 90 95 Glu Leu
Phe His Gly Pro Thr Leu Ala Phe Lys Asp Phe Gly Gly Arg 100 105 110
Phe Met Ala Gln Met Leu Thr His Ile Ala Gly Asp Lys Pro Val Thr 115
120 125 Ile Leu Thr Ala Thr Ser Gly Asp Thr Gly Ala Ala Val Ala His
Ala 130 135 140 Phe Tyr Gly Leu Pro Asn Val Lys Val Val Ile Leu Tyr
Pro Arg Gly 145 150 155 160 Lys Ile Ser Pro Leu Gln Glu Lys Leu Phe
Cys Thr Leu Gly Gly Asn 165 170 175 Ile Glu Thr Val Ala Ile Asp Gly
Asp Phe Asp Ala Cys Gln Ala Leu 180 185 190 Val Lys Gln Ala Phe Asp
Asp Glu Glu Leu Lys Val Ala Leu Gly Leu 195 200 205 Asn Ser Ala Asn
Ser Ile Asn Ile Ser Arg Leu Leu Ala Gln Ile Cys 210 215 220 Tyr Tyr
Phe Glu Ala Val Ala Gln Leu Pro Gln Glu Thr Arg Asn Gln 225 230 235
240 Leu Val Val Ser Val Pro Ser Gly Asn Phe Gly Asp Leu Thr Ala Gly
245 250 255 Leu Leu Ala Lys Ser Leu Gly Leu Pro Val Lys Arg Phe Ile
Ala Ala 260 265 270 Thr Asn Val Asn Asp Thr Val Pro Arg Phe Leu His
Asp Gly Gln Trp 275 280 285 Ser Pro Lys Ala Thr Gln Ala Thr Leu Ser
Asn Ala Met Asp Val Ser 290 295 300 Gln Pro Asn Asn Trp Pro Arg Val
Glu Glu Leu Phe Arg Arg Lys Ile 305 310 315 320 Trp Gln Leu Lys Glu
Leu Gly Tyr Ala Ala Val Asp Asp Glu Thr Thr 325 330 335 Gln Gln Thr
Met Arg Glu Leu Lys Glu Leu Gly Tyr Thr Ser Glu Pro 340 345 350 His
Ala Ala Val Ala Tyr Arg Ala Leu Arg Asp Gln Leu Asn Pro Gly 355 360
365 Glu Tyr Gly Leu Phe Leu Gly Thr Ala His Pro Ala Lys Phe Lys Glu
370 375 380 Ser Val Glu Ala Ile Leu Gly Glu Thr Leu Asp Leu Pro Lys
Glu Leu 385 390 395 400 Ala Glu Arg Ala Asp Leu Pro Leu Leu Ser His
Asn Leu Pro Ala Asp 405 410 415 Phe Ala Ala Leu Arg Lys Leu Met Met
Asn His Gln 420 425 33352PRTBacillus subtilissource/note="Threonine
synthase" 33Met Trp Lys Gly Leu Ile His Gln Tyr Lys Glu Phe Leu Pro
Val Thr 1 5 10 15 Asp Gln Thr Pro Ala Leu Thr Leu His Glu Gly Asn
Thr Pro Leu Ile 20 25 30 His Leu Pro Lys Leu Ser Glu Gln Leu Gly
Ile Glu Leu His Val Lys 35 40 45 Thr Glu Gly Val Asn Pro Thr Gly
Ser Phe Lys Asp Arg Gly Met Val 50 55 60 Met Ala Val Ala Lys Ala
Lys Glu Glu Gly Asn Asp Thr Ile Met Cys 65 70 75 80 Ala Ser Thr Gly
Asn Thr Ser Ala Ala Ala Ala Ala Tyr Ala Ala Arg 85 90 95 Ala Asn
Met Lys Cys Ile Val Ile Ile Pro Asn Gly Lys Ile Ala Phe 100 105 110
Gly Lys Leu Ala Gln Ala Val Met Tyr Gly Ala Glu Ile Ile Ala Ile 115
120 125 Asp Gly Asn Phe Asp Asp Ala Leu Lys Ile Val Arg Ser Ile Cys
Glu 130 135 140 Lys Ser Pro Ile Ala Leu Val Asn Ser Val Asn Pro Tyr
Arg Ile Glu 145 150 155 160 Gly Gln Lys Thr Ala Ala Phe Glu Val Cys
Glu Gln Leu Gly Glu Ala 165 170 175 Pro Asp Val Leu Ala Ile Pro Val
Gly Asn Ala Gly Asn Ile Thr Ala 180 185 190 Tyr Trp Lys Gly Phe Lys
Glu Tyr His Glu Lys Asn Gly Thr Gly Leu 195 200 205 Pro Lys Met Arg
Gly Phe Glu Ala Glu Gly Ala Ala Ala Ile Val Arg 210 215 220 Asn Glu
Val Ile Glu Asn Pro Glu Thr Ile Ala Thr Ala Ile Arg Ile 225 230 235
240 Gly Asn Pro Ala Ser Trp Asp Lys Ala Val Lys Ala Ala Glu Glu Ser
245 250 255 Asn Gly Lys Ile Asp Glu Val Thr Asp Asp Glu Ile Leu His
Ala Tyr 260 265 270 Gln Leu Ile Ala Arg Val Glu Gly Val Phe Ala Glu
Pro Gly Ser Cys 275 280 285 Ala Ser Ile Ala Gly Val Leu Lys Gln Val
Lys Ser Gly Glu Ile Pro 290 295 300 Lys Gly Ser Lys Val Val Ala Val
Leu Thr Gly Asn Gly Leu Lys Asp 305 310 315 320 Pro Asn Thr Ala Val
Asp Ile Ser Glu Ile Lys Pro Val Thr Leu Pro 325 330 335 Thr Asp Glu
Asp Ser Ile Leu Glu Tyr Val Lys Gly Ala Ala Arg Val 340 345 350
34481PRTCorynebacterium glutamicumsource/note="Threonine synthase"
34Met Asp Tyr Ile Ser Thr Arg Asp Ala Ser Arg Thr Pro Ala Arg Phe 1
5 10 15 Ser Asp Ile Leu Leu Gly Gly Leu Ala Pro Asp Gly Gly Leu Tyr
Leu 20 25 30 Pro Ala Thr Tyr Pro Gln Leu Asp Asp Ala Gln Leu Ser
Lys Trp Arg 35 40 45 Glu Val Leu Ala Asn Glu Gly Tyr Ala Ala Leu
Ala Ala Glu Val Ile 50 55 60 Ser Leu Phe Val Asp Asp Ile Pro Val
Glu Asp Ile Lys Ala Ile Thr 65 70 75 80 Ala Arg Ala Tyr Thr Tyr Pro
Lys Phe Asn Ser Glu Asp Ile Val Pro 85 90 95 Val Thr Glu Leu Glu
Asp Asn Ile Tyr Leu Gly His Leu Ser Glu Gly 100 105 110 Pro Thr Ala
Ala Phe Lys Asp Met Ala Met Gln Leu Leu Gly Glu Leu 115 120 125 Phe
Glu Tyr Glu Leu Arg Arg Arg Asn Glu Thr Ile Asn Ile Leu Gly 130 135
140 Ala Thr Ser Gly Asp Thr Gly Ser Ser Ala Glu Tyr Ala Met Arg Gly
145 150 155 160 Arg Glu Gly Ile Arg Val Phe Met Leu Thr Pro Ala Gly
Arg Met Thr 165 170 175 Pro Phe Gln Gln Ala Gln Met Phe Gly Leu Asp
Asp Pro Asn Ile Phe 180 185 190 Asn Ile Ala Leu Asp Gly Val Phe Asp
Asp Cys Gln Asp Val Val Lys 195 200 205 Ala Val Ser Ala Asp Ala Glu
Phe Lys Lys Asp Asn Arg Ile Gly Ala 210 215 220 Val Asn Ser Ile Asn
Trp Ala Arg Leu Met Ala Gln Val Val Tyr Tyr 225 230 235 240 Val Ser
Ser Trp Ile Arg Thr Thr Thr Ser Asn Asp Gln Lys Val Ser 245 250 255
Phe Ser Val Pro Thr Gly Asn Phe Gly Asp Ile Cys Ala Gly His Ile 260
265 270 Ala Arg Gln Met Gly Leu Pro Ile Asp Arg Leu Ile Val Ala Thr
Asn 275 280 285 Glu Asn Asp Val Leu Asp Glu Phe Phe Arg Thr Gly Asp
Tyr Arg Val 290 295 300 Arg Ser Ser Ala Asp Thr His Glu Thr Ser Ser
Pro Ser Met Asp Ile 305 310 315 320 Ser Arg Ala Ser Asn Phe Glu Arg
Phe Ile Phe Asp Leu Leu Gly Arg 325 330 335 Asp Ala Thr Arg Val Asn
Asp Leu Phe Gly Thr Gln Val Arg Gln Gly 340 345 350 Gly Phe Ser Leu
Ala Asp Asp Ala Asn Phe Glu Lys Ala Ala Ala Glu 355 360 365 Tyr Gly
Phe Ala Ser Gly Arg Ser Thr His Ala Asp Arg Val Ala Thr 370 375 380
Ile Ala Asp Val His Ser Arg Leu Asp Val Leu Ile Asp Pro His Thr 385
390 395 400 Ala Asp Gly Val His Val Ala Arg Gln Trp Arg Asp Glu Val
Asn Thr 405 410 415 Pro Ile Ile Val Leu Glu Thr Ala Leu Pro Val Lys
Phe Ala Asp Thr 420 425 430 Ile Val Glu Ala Ile Gly Glu Ala Pro Gln
Thr Pro Glu Arg Phe Ala 435 440 445 Ala Ile Met Asp Ala Pro Phe Lys
Val Ser Asp Leu Pro Asn Asp Thr 450 455 460 Asp Ala Val Lys Gln Tyr
Ile Val Asp Ala Ile Ala Asn Thr Ser Val 465 470 475 480 Lys
35329PRTEscherichia colisource/note="Threonine deaminase (TdcB)"
35Met His Ile Thr Tyr Asp Leu Pro Val Ala Ile Asp Asp Ile Ile Glu 1
5 10 15 Ala Lys Gln Arg Leu Ala Gly Arg Ile Tyr Lys Thr Gly Met Pro
Arg 20 25 30 Ser Asn Tyr Phe Ser Glu Arg Cys Lys Gly Glu Ile Phe
Leu Lys Phe 35 40 45 Glu Asn Met Gln Arg Thr Gly Ser Phe Lys Ile
Arg Gly Ala Phe Asn 50 55 60 Lys Leu Ser Ser Leu Thr Asp Ala Glu
Lys Arg Lys Gly Val Val Ala 65 70 75 80 Cys Ser Ala Gly Asn His Ala
Gln Gly Val Ser Leu Ser Cys Ala Met 85 90 95 Leu Gly Ile Asp Gly
Lys Val Val Met Pro Lys Gly Ala Pro Lys Ser 100 105
110 Lys Val Ala Ala Thr Cys Asp Tyr Ser Ala Glu Val Val Leu His Gly
115 120 125 Asp Asn Phe Asn Asp Thr Ile Ala Lys Val Ser Glu Ile Val
Glu Met 130 135 140 Glu Gly Arg Ile Phe Ile Pro Pro Tyr Asp Asp Pro
Lys Val Ile Ala 145 150 155 160 Gly Gln Gly Thr Ile Gly Leu Glu Ile
Met Glu Asp Leu Tyr Asp Val 165 170 175 Asp Asn Val Ile Val Pro Ile
Gly Gly Gly Gly Leu Ile Ala Gly Ile 180 185 190 Ala Val Ala Ile Lys
Ser Ile Asn Pro Thr Ile Arg Val Ile Gly Val 195 200 205 Gln Ser Glu
Asn Val His Gly Met Ala Ala Ser Phe His Ser Gly Glu 210 215 220 Ile
Thr Thr His Arg Thr Thr Gly Thr Leu Ala Asp Gly Cys Asp Val 225 230
235 240 Ser Arg Pro Gly Asn Leu Thr Tyr Glu Ile Val Arg Glu Leu Val
Asp 245 250 255 Asp Ile Val Leu Val Ser Glu Asp Glu Ile Arg Asn Ser
Met Ile Ala 260 265 270 Leu Ile Gln Arg Asn Lys Val Val Thr Glu Gly
Ala Gly Ala Leu Ala 275 280 285 Cys Ala Ala Leu Leu Ser Gly Lys Leu
Asp Gln Tyr Ile Gln Asn Arg 290 295 300 Lys Thr Val Ser Ile Ile Ser
Gly Gly Asn Ile Asp Leu Ser Arg Val 305 310 315 320 Ser Gln Ile Thr
Gly Phe Val Asp Ala 325 36514PRTEscherichia
colisource/note="Threonine deaminase (IlvA)" 36Met Ala Asp Ser Gln
Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr 1 5 10 15 Leu Arg Ala
Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr 20 25 30 Pro
Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile 35 40
45 Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60 Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys
Ala His 65 70 75 80 Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln
Gly Val Ala Phe 85 90 95 Ser Ser Ala Arg Leu Gly Val Lys Ala Leu
Ile Val Met Pro Thr Ala 100 105 110 Thr Ala Asp Ile Lys Val Asp Ala
Val Arg Gly Phe Gly Gly Glu Val 115 120 125 Leu Leu His Gly Ala Asn
Phe Asp Glu Ala Lys Ala Lys Ala Ile Glu 130 135 140 Leu Ser Gln Gln
Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro 145 150 155 160 Met
Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln 165 170
175 Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190 Ala Ala Gly Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln
Ile Lys 195 200 205 Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu
Lys Ala Ala Leu 210 215 220 Asp Ala Gly His Pro Val Asp Leu Pro Arg
Val Gly Leu Phe Ala Glu 225 230 235 240 Gly Val Ala Val Lys Arg Ile
Gly Asp Glu Thr Phe Arg Leu Cys Gln 245 250 255 Glu Tyr Leu Asp Asp
Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala 260 265 270 Ala Met Lys
Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser 275 280 285 Gly
Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn 290 295
300 Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320 Phe His Gly Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu
Gly Glu Gln 325 330 335 Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu
Glu Lys Gly Ser Phe 340 345 350 Leu Lys Phe Cys Gln Leu Leu Gly Gly
Arg Ser Val Thr Glu Phe Asn 355 360 365 Tyr Arg Phe Ala Asp Ala Lys
Asn Ala Cys Ile Phe Val Gly Val Arg 370 375 380 Leu Ser Arg Gly Leu
Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn 385 390 395 400 Asp Gly
Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys 405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln 420
425 430 Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu
Leu 435 440 445 Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser
Leu Phe His 450 455 460 Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val
Leu Ala Ala Phe Glu 465 470 475 480 Leu Gly Asp His Glu Pro Asp Phe
Glu Thr Arg Leu Asn Glu Leu Gly 485 490 495 Tyr Asp Cys His Asp Glu
Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu 500 505 510 Ala Gly
37422PRTBacillus subtilissource/note="Threonine deaminase (IlvA)"
37Met Lys Pro Leu Leu Lys Glu Asn Ser Leu Ile Gln Val Lys Asp Ile 1
5 10 15 Leu Lys Ala His Gln Asn Val Lys Asp Val Val Ile His Thr Pro
Leu 20 25 30 Gln Arg Asn Asp Arg Leu Ser Glu Arg Tyr Glu Cys Asn
Ile Tyr Leu 35 40 45 Lys Arg Glu Asp Leu Gln Val Val Arg Ser Phe
Lys Leu Arg Gly Ala 50 55 60 Tyr His Lys Met Lys Gln Leu Ser Ser
Glu Gln Thr Glu Asn Gly Val 65 70 75 80 Val Cys Ala Ser Ala Gly Asn
His Ala Gln Gly Val Ala Phe Ser Cys 85 90 95 Lys His Leu Gly Ile
His Gly Lys Ile Phe Met Pro Ser Thr Thr Pro 100 105 110 Arg Gln Lys
Val Ser Gln Val Glu Leu Phe Gly Lys Gly Phe Ile Asp 115 120 125 Ile
Ile Leu Thr Gly Asp Thr Phe Asp Asp Ala Tyr Lys Ser Ala Ala 130 135
140 Glu Cys Cys Glu Ala Glu Ser Arg Thr Phe Ile His Pro Phe Asp Asp
145 150 155 160 Pro Asp Val Met Ala Gly Gln Gly Thr Leu Ala Val Glu
Ile Leu Asn 165 170 175 Asp Ile Asp Thr Glu Pro His Phe Leu Phe Ala
Ser Val Gly Gly Gly 180 185 190 Gly Leu Leu Ser Gly Val Gly Thr Tyr
Leu Lys Asn Val Ser Pro Asp 195 200 205 Thr Lys Val Ile Ala Val Glu
Pro Ala Gly Ala Ala Ser Tyr Phe Glu 210 215 220 Ser Asn Lys Ala Gly
His Val Val Thr Leu Asp Lys Ile Asp Lys Phe 225 230 235 240 Val Asp
Gly Ala Ala Val Lys Lys Ile Gly Glu Glu Thr Phe Arg Thr 245 250 255
Leu Glu Thr Val Val Asp Asp Ile Leu Leu Val Pro Glu Gly Lys Val 260
265 270 Cys Thr Ser Ile Leu Glu Leu Tyr Asn Glu Cys Ala Val Val Ala
Glu 275 280 285 Pro Ala Gly Ala Leu Ser Val Ala Ala Leu Asp Leu Tyr
Lys Asp Gln 290 295 300 Ile Lys Gly Lys Asn Val Val Cys Val Val Ser
Gly Gly Asn Asn Asp 305 310 315 320 Ile Gly Arg Met Gln Glu Met Lys
Glu Arg Ser Leu Ile Phe Glu Gly 325 330 335 Leu Gln His Tyr Phe Ile
Val Asn Phe Pro Gln Arg Ala Gly Ala Leu 340 345 350 Arg Glu Phe Leu
Asp Glu Val Leu Gly Pro Asn Asp Asp Ile Thr Arg 355 360 365 Phe Glu
Tyr Thr Lys Lys Asn Asn Lys Ser Asn Gly Pro Ala Leu Val 370 375 380
Gly Ile Glu Leu Gln Asn Lys Ala Asp Tyr Gly Pro Leu Ile Glu Arg 385
390 395 400 Met Asn Lys Lys Pro Phe His Tyr Val Glu Val Asn Lys Asp
Glu Asp 405 410 415 Leu Phe His Leu Leu Ile 420
38436PRTCorynebacterium glutamicumsource/note="Threonine deaminase
(IlvA)" 38Met Ser Glu Thr Tyr Val Ser Glu Lys Ser Pro Gly Val Met
Ala Ser 1 5 10 15 Gly Ala Glu Leu Ile Arg Ala Ala Asp Ile Gln Thr
Ala Gln Ala Arg 20 25 30 Ile Ser Ser Val Ile Ala Pro Thr Pro Leu
Gln Tyr Cys Pro Arg Leu 35 40 45 Ser Glu Glu Thr Gly Ala Glu Ile
Tyr Leu Lys Arg Glu Asp Leu Gln 50 55 60 Asp Val Arg Ser Tyr Lys
Ile Arg Gly Ala Leu Asn Ser Gly Ala Gln 65 70 75 80 Leu Thr Gln Glu
Gln Arg Asp Ala Gly Ile Val Ala Ala Ser Ala Gly 85 90 95 Asn His
Ala Gln Gly Val Ala Tyr Val Cys Lys Ser Leu Gly Val Gln 100 105 110
Gly Arg Ile Tyr Val Pro Val Gln Thr Pro Lys Gln Lys Arg Asp Arg 115
120 125 Ile Met Val His Gly Gly Glu Phe Val Ser Leu Val Val Thr Gly
Asn 130 135 140 Asn Phe Asp Glu Ala Ser Ala Ala Ala His Glu Asp Ala
Glu Arg Thr 145 150 155 160 Gly Ala Thr Leu Ile Glu Pro Phe Asp Ala
Arg Asn Thr Val Ile Gly 165 170 175 Gln Gly Thr Val Ala Ala Glu Ile
Leu Ser Gln Leu Thr Ser Met Gly 180 185 190 Lys Ser Ala Asp His Val
Met Val Pro Val Gly Gly Gly Gly Leu Leu 195 200 205 Ala Gly Val Val
Ser Tyr Met Ala Asp Met Ala Pro Arg Thr Ala Ile 210 215 220 Val Gly
Ile Glu Pro Ala Gly Ala Ala Ser Met Gln Ala Ala Leu His 225 230 235
240 Asn Gly Gly Pro Ile Thr Leu Glu Thr Val Asp Pro Phe Val Asp Gly
245 250 255 Ala Ala Val Lys Arg Val Gly Asp Leu Asn Tyr Thr Ile Val
Glu Lys 260 265 270 Asn Gln Gly Arg Val His Met Met Ser Ala Thr Glu
Gly Ala Val Cys 275 280 285 Thr Glu Met Leu Asp Leu Tyr Gln Asn Glu
Gly Ile Ile Ala Glu Pro 290 295 300 Ala Gly Ala Leu Ser Ile Ala Gly
Leu Lys Glu Met Ser Phe Ala Pro 305 310 315 320 Gly Ser Val Val Val
Cys Ile Ile Ser Gly Gly Asn Asn Asp Val Leu 325 330 335 Arg Tyr Ala
Glu Ile Ala Glu Arg Ser Leu Val His Arg Gly Leu Lys 340 345 350 His
Tyr Phe Leu Val Asn Phe Pro Gln Lys Pro Gly Gln Leu Arg His 355 360
365 Phe Leu Glu Asp Ile Leu Gly Pro Asp Asp Asp Ile Thr Leu Phe Glu
370 375 380 Tyr Leu Lys Arg Asn Asn Arg Glu Thr Gly Thr Ala Leu Val
Gly Ile 385 390 395 400 His Leu Ser Glu Ala Ser Gly Leu Asp Ser Leu
Leu Glu Arg Met Glu 405 410 415 Glu Ser Ala Ile Asp Ser Arg Arg Leu
Glu Pro Gly Thr Pro Glu Tyr 420 425 430 Glu Tyr Leu Thr 435
39310PRTCorynebacterium glutamicumsource/note="Threonine deaminase
(TdcB)" 39Met Leu Thr Leu Asn Asp Val Ile Thr Ala Gln Gln Arg Thr
Ala Pro 1 5 10 15 His Val Arg Arg Thr Pro Leu Phe Glu Ala Asp Pro
Ile Asp Gly Thr 20 25 30 Gln Ile Trp Ile Lys Ala Glu Phe Leu Gln
Lys Cys Gly Val Phe Lys 35 40 45 Thr Arg Gly Ala Phe Asn Arg Gln
Leu Ala Ala Ser Glu Asn Gly Leu 50 55 60 Leu Asp Pro Thr Val Gly
Ile Val Ala Ala Ser Gly Gly Asn Ala Gly 65 70 75 80 Leu Ala Asn Ala
Phe Ala Ala Ala Ser Leu Ser Val Pro Ala Thr Val 85 90 95 Leu Val
Pro Glu Thr Ala Pro Gln Val Lys Val Asp Arg Leu Lys Gln 100 105 110
Tyr Gly Ala Thr Val Gln Gln Ile Gly Ser Glu Tyr Ala Glu Ala Phe 115
120 125 Glu Ala Ala Gln Thr Phe Glu Ser Glu Thr Gly Ala Leu Phe Cys
His 130 135 140 Ala Tyr Asp Gln Pro Asp Ile Ala Ala Gly Ala Gly Val
Ile Gly Leu 145 150 155 160 Glu Ile Val Glu Asp Leu Pro Asp Val Asp
Thr Ile Val Val Ala Val 165 170 175 Gly Gly Gly Gly Leu Tyr Ala Gly
Ile Ala Ala Val Val Ala Ala His 180 185 190 Asp Ile Lys Val Val Ala
Val Glu Pro Ser Lys Ile Pro Thr Leu His 195 200 205 Asn Ser Leu Ile
Ala Gly Gln Pro Val Asp Val Asn Val Ser Gly Ile 210 215 220 Ala Ala
Asp Ser Leu Gly Ala Arg Gln Ile Gly Arg Glu Ala Phe Asp 225 230 235
240 Ile Ala Thr Ala His Pro Pro Ile Gly Val Leu Val Asp Asp Glu Ala
245 250 255 Ile Ile Ala Ala Arg Arg His Leu Trp Asp Asn Tyr Arg Ile
Pro Ala 260 265 270 Glu His Gly Ala Ala Ala Ala Leu Ala Ser Leu Thr
Ser Gly Ala Tyr 275 280 285 Lys Pro Ala Ala Asp Glu Lys Val Ala Val
Ile Val Cys Gly Ala Asn 290 295 300 Thr Asp Leu Thr Thr Leu 305 310
40491PRTMethanocaldococcus jannaschiisource/note="Citramalate
synthase" 40Met Met Val Arg Ile Phe Asp Thr Thr Leu Arg Asp Gly Glu
Gln Thr 1 5 10 15 Pro Gly Val Ser Leu Thr Pro Asn Asp Lys Leu Glu
Ile Ala Lys Lys 20 25 30 Leu Asp Glu Leu Gly Val Asp Val Ile Glu
Ala Gly Ser Ala Ile Thr 35 40 45 Ser Lys Gly Glu Arg Glu Gly Ile
Lys Leu Ile Thr Lys Glu Gly Leu 50 55 60 Asn Ala Glu Ile Cys Ser
Phe Val Arg Ala Leu Pro Val Asp Ile Asp 65 70 75 80 Ala Ala Leu Glu
Cys Asp Val Asp Ser Val His Leu Val Val Pro Thr 85 90 95 Ser Pro
Ile His Met Lys Tyr Lys Leu Arg Lys Thr Glu Asp Glu Val 100 105 110
Leu Glu Thr Ala Leu Lys Ala Val Glu Tyr Ala Lys Glu His Gly Leu 115
120 125 Ile Val Glu Leu Ser Ala Glu Asp Ala Thr Arg Ser Asp Val Asn
Phe 130 135 140 Leu Ile Lys Leu Phe Asn Glu Gly Glu Lys Val Gly Ala
Asp Arg Val 145 150 155 160 Cys Val Cys Asp Thr Val Gly Val Leu Thr
Pro Gln Lys Ser Gln Glu 165 170 175 Leu Phe Lys Lys Ile Thr Glu Asn
Val Asn Leu Pro Val Ser Val His 180 185 190 Cys His Asn Asp Phe Gly
Met Ala Thr Ala Asn Thr Cys Ser Ala Val 195 200 205 Leu Gly Gly Ala
Val Gln Cys His Val Thr Val Asn Gly Ile Gly Glu 210 215 220 Arg Ala
Gly Asn Ala Ser Leu Glu Glu Val Val Ala Ala Leu Lys Ile 225 230 235
240 Leu Tyr Gly Tyr Asp Thr Lys Ile Lys Met Glu Lys Leu Tyr Glu Val
245 250 255 Ser Arg Ile Val Ser Arg Leu Met Lys Leu Pro Val Pro Pro
Asn Lys 260 265 270 Ala Ile Val Gly Asp Asn Ala Phe Ala His Glu Ala
Gly Ile His Val 275 280 285 Asp Gly Leu Ile Lys Asn Thr Glu Thr Tyr
Glu Pro Ile Lys Pro Glu 290 295 300 Met Val Gly Asn Arg Arg Arg Ile
Ile Leu Gly Lys His Ser Gly Arg 305 310 315 320 Lys Ala Leu Lys Tyr
Lys Leu Asp Leu Met Gly Ile Asn Val Ser Asp 325 330 335 Glu Gln Leu
Asn Lys Ile Tyr Glu Arg Val Lys Glu Phe Gly Asp Leu 340 345 350 Gly
Lys Tyr Ile Ser Asp Ala Asp Leu Leu Ala Ile Val Arg
Glu Val 355 360 365 Thr Gly Lys Leu Val Glu Glu Lys Ile Lys Leu Asp
Glu Leu Thr Val 370 375 380 Val Ser Gly Asn Lys Ile Thr Pro Ile Ala
Ser Val Lys Leu His Tyr 385 390 395 400 Lys Gly Glu Asp Ile Thr Leu
Ile Glu Thr Ala Tyr Gly Val Gly Pro 405 410 415 Val Asp Ala Ala Ile
Asn Ala Val Arg Lys Ala Ile Ser Gly Val Ala 420 425 430 Asp Ile Lys
Leu Val Glu Tyr Arg Val Glu Ala Ile Gly Gly Gly Thr 435 440 445 Asp
Ala Leu Ile Glu Val Val Val Lys Leu Arg Lys Gly Thr Glu Ile 450 455
460 Val Glu Val Arg Lys Ser Asp Ala Asp Ile Ile Arg Ala Ser Val Asp
465 470 475 480 Ala Val Met Glu Gly Ile Asn Met Leu Leu Asn 485 490
41372PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic M. jannaschii Citramalate synthase variant
polypeptide" 41Met Met Val Arg Ile Phe Asp Thr Thr Leu Arg Asp Gly
Glu Gln Thr 1 5 10 15 Pro Gly Val Ser Leu Thr Pro Asn Asp Lys Leu
Glu Ile Ala Lys Lys 20 25 30 Leu Asp Glu Leu Gly Val Asp Val Ile
Glu Ala Gly Ser Ala Val Thr 35 40 45 Ser Lys Gly Glu Arg Glu Gly
Ile Lys Leu Ile Thr Lys Glu Gly Leu 50 55 60 Asn Ala Glu Ile Cys
Ser Phe Val Arg Ala Leu Pro Val Asp Ile Asp 65 70 75 80 Ala Ala Leu
Glu Cys Asp Val Asp Ser Val His Leu Val Val Pro Thr 85 90 95 Ser
Pro Ile His Met Lys Tyr Lys Leu Arg Lys Thr Glu Asp Glu Val 100 105
110 Leu Val Thr Ala Leu Lys Ala Val Glu Tyr Ala Lys Glu Gln Gly Leu
115 120 125 Ile Val Glu Leu Ser Ala Glu Asp Ala Thr Arg Ser Asp Val
Asn Phe 130 135 140 Leu Ile Lys Leu Phe Asn Glu Gly Glu Lys Val Gly
Ala Asp Arg Val 145 150 155 160 Cys Val Cys Asp Thr Val Gly Val Leu
Thr Pro Gln Lys Ser Gln Glu 165 170 175 Leu Phe Lys Lys Ile Thr Glu
Asn Val Asn Leu Pro Val Ser Val His 180 185 190 Cys His Asn Asp Phe
Gly Met Ala Thr Ala Asn Ala Cys Ser Ala Val 195 200 205 Leu Gly Gly
Ala Val Gln Cys His Val Thr Val Asn Gly Ile Gly Glu 210 215 220 Arg
Ala Gly Asn Ala Ser Leu Glu Glu Val Val Ala Ala Ser Lys Ile 225 230
235 240 Leu Tyr Gly Tyr Asp Thr Lys Ile Lys Met Glu Lys Leu Tyr Glu
Val 245 250 255 Ser Arg Ile Val Ser Arg Leu Met Lys Leu Pro Val Pro
Pro Asn Lys 260 265 270 Ala Ile Val Gly Asp Asn Ala Phe Ala His Glu
Ala Gly Ile His Val 275 280 285 Asp Gly Leu Ile Lys Asn Thr Glu Thr
Tyr Glu Pro Ile Lys Pro Glu 290 295 300 Met Val Gly Asn Arg Arg Arg
Ile Ile Leu Gly Lys His Ser Gly Arg 305 310 315 320 Lys Ala Leu Lys
Tyr Lys Leu Asp Leu Met Gly Ile Asn Val Ser Asp 325 330 335 Glu Gln
Leu Asn Lys Ile Tyr Glu Arg Val Lys Glu Phe Gly Asp Leu 340 345 350
Gly Lys Tyr Ile Ser Asp Ala Asp Leu Leu Ala Ile Val Arg Glu Val 355
360 365 Thr Gly Lys Leu 370 42516PRTLeptospira
interroganssource/note="Citramalate synthase" 42Met Thr Lys Val Glu
Thr Arg Leu Glu Ile Leu Asp Val Thr Leu Arg 1 5 10 15 Asp Gly Glu
Gln Thr Arg Gly Val Ser Phe Ser Thr Ser Glu Lys Leu 20 25 30 Asn
Ile Ala Lys Phe Leu Leu Gln Lys Leu Asn Val Asp Arg Val Glu 35 40
45 Ile Ala Ser Ala Arg Val Ser Lys Gly Glu Leu Glu Thr Val Gln Lys
50 55 60 Ile Met Glu Trp Ala Ala Thr Glu Gln Leu Thr Glu Arg Ile
Glu Ile 65 70 75 80 Leu Gly Phe Val Asp Gly Asn Lys Thr Val Asp Trp
Ile Lys Asp Ser 85 90 95 Gly Ala Lys Val Leu Asn Leu Leu Thr Lys
Gly Ser Leu His His Leu 100 105 110 Glu Lys Gln Leu Gly Lys Thr Pro
Lys Glu Phe Phe Thr Asp Val Ser 115 120 125 Phe Val Ile Glu Tyr Ala
Ile Lys Ser Gly Leu Lys Ile Asn Val Tyr 130 135 140 Leu Glu Asp Trp
Ser Asn Gly Phe Arg Asn Ser Pro Asp Tyr Val Lys 145 150 155 160 Ser
Leu Val Glu His Leu Ser Lys Glu His Ile Glu Arg Ile Phe Leu 165 170
175 Pro Asp Thr Leu Gly Val Leu Ser Pro Glu Glu Thr Phe Gln Gly Val
180 185 190 Asp Ser Leu Ile Gln Lys Tyr Pro Asp Ile His Phe Glu Phe
His Gly 195 200 205 His Asn Asp Tyr Asp Leu Ser Val Ala Asn Ser Leu
Gln Ala Ile Arg 210 215 220 Ala Gly Val Lys Gly Leu His Ala Ser Ile
Asn Gly Leu Gly Glu Arg 225 230 235 240 Ala Gly Asn Thr Pro Leu Glu
Ala Leu Val Thr Thr Ile His Asp Lys 245 250 255 Ser Asn Ser Lys Thr
Asn Ile Asn Glu Ile Ala Ile Thr Glu Ala Ser 260 265 270 Arg Leu Val
Glu Val Phe Ser Gly Lys Arg Ile Ser Ala Asn Arg Pro 275 280 285 Ile
Val Gly Glu Asp Val Phe Thr Gln Thr Ala Gly Val His Ala Asp 290 295
300 Gly Asp Lys Lys Gly Asn Leu Tyr Ala Asn Pro Ile Leu Pro Glu Arg
305 310 315 320 Phe Gly Arg Lys Arg Ser Tyr Ala Leu Gly Lys Leu Ala
Gly Lys Ala 325 330 335 Ser Ile Ser Glu Asn Val Lys Gln Leu Gly Met
Val Leu Ser Glu Val 340 345 350 Val Leu Gln Lys Val Leu Glu Arg Val
Ile Glu Leu Gly Asp Gln Asn 355 360 365 Lys Leu Val Thr Pro Glu Asp
Leu Pro Phe Ile Ile Ala Asp Val Ser 370 375 380 Gly Arg Thr Gly Glu
Lys Val Leu Thr Ile Lys Ser Cys Asn Ile His 385 390 395 400 Ser Gly
Ile Gly Ile Arg Pro His Ala Gln Ile Glu Leu Glu Tyr Gln 405 410 415
Gly Lys Ile His Lys Glu Ile Ser Glu Gly Asp Gly Gly Tyr Asp Ala 420
425 430 Phe Met Asn Ala Leu Thr Lys Ile Thr Asn Arg Leu Gly Ile Ser
Ile 435 440 445 Pro Lys Leu Ile Asp Tyr Glu Val Arg Ile Pro Pro Gly
Gly Lys Thr 450 455 460 Asp Ala Leu Val Glu Thr Arg Ile Thr Trp Asn
Lys Ser Leu Asp Leu 465 470 475 480 Glu Glu Asp Gln Thr Phe Lys Thr
Met Gly Val His Pro Asp Gln Thr 485 490 495 Val Ala Ala Val His Ala
Thr Glu Lys Met Leu Asn Gln Ile Leu Gln 500 505 510 Pro Trp Gln Ile
515 43386PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic Leptospira interrogans citramalate
synthase variant polypeptide" 43Met Thr Lys Val Glu Thr Arg Leu Glu
Ile Leu Asp Val Thr Leu Arg 1 5 10 15 Asp Gly Glu Gln Thr Arg Gly
Val Ser Phe Ser Thr Ser Glu Lys Leu 20 25 30 Asn Ile Ala Lys Phe
Leu Leu Gln Lys Leu Asn Val Asp Arg Val Glu 35 40 45 Ile Ala Ser
Ala Arg Val Ser Lys Gly Glu Leu Glu Thr Val Gln Lys 50 55 60 Ile
Met Glu Trp Ala Ala Thr Glu Gln Leu Thr Glu Arg Ile Glu Ile 65 70
75 80 Leu Gly Phe Val Asp Gly Asn Lys Thr Val Asp Trp Ile Lys Asp
Ser 85 90 95 Gly Ala Lys Val Leu Asn Leu Leu Thr Lys Gly Ser Leu
His His Leu 100 105 110 Glu Lys Gln Leu Gly Lys Thr Pro Lys Glu Phe
Phe Thr Asp Val Ser 115 120 125 Phe Val Ile Glu Tyr Ala Ile Lys Ser
Gly Leu Lys Ile Asn Val Tyr 130 135 140 Leu Glu Asp Trp Ser Asn Gly
Phe Arg Asn Ser Pro Asp Tyr Val Lys 145 150 155 160 Ser Leu Val Glu
His Leu Ser Lys Glu His Ile Glu Arg Ile Phe Leu 165 170 175 Pro Asp
Thr Leu Gly Val Leu Ser Pro Glu Glu Thr Phe Gln Gly Val 180 185 190
Asp Ser Leu Ile Gln Lys Tyr Pro Asp Ile His Phe Glu Phe His Gly 195
200 205 His Asn Asp Tyr Asp Leu Ser Val Ala Asn Ser Leu Gln Ala Ile
Arg 210 215 220 Ala Gly Val Lys Gly Leu His Ala Ser Ile Asn Gly Leu
Gly Glu Arg 225 230 235 240 Ala Gly Asn Thr Pro Leu Glu Ala Leu Val
Thr Thr Ile His Asp Lys 245 250 255 Ser Asn Ser Lys Thr Asn Ile Asn
Glu Ile Ala Ile Thr Glu Ala Ser 260 265 270 Arg Leu Val Glu Val Phe
Ser Gly Lys Arg Ile Ser Ala Asn Arg Pro 275 280 285 Ile Val Gly Glu
Asp Val Phe Thr Gln Thr Ala Gly Val His Ala Asp 290 295 300 Gly Asp
Lys Lys Gly Asn Leu Tyr Ala Asn Pro Ile Leu Pro Glu Arg 305 310 315
320 Phe Gly Arg Lys Arg Ser Tyr Ala Leu Gly Lys Leu Ala Gly Lys Ala
325 330 335 Ser Ile Ser Glu Asn Val Lys Gln Leu Gly Met Val Leu Ser
Glu Val 340 345 350 Val Leu Gln Lys Val Leu Glu Arg Val Ile Glu Leu
Gly Asp Gln Asn 355 360 365 Lys Leu Val Thr Pro Glu Asp Leu Pro Phe
Ile Ile Ala Asp Val Ser 370 375 380 Gly Arg 385 44466PRTEscherichia
colisource/note="Isopropylmalate isomerase large subunit" 44Met Ala
Lys Thr Leu Tyr Glu Lys Leu Phe Asp Ala His Val Val Tyr 1 5 10 15
Glu Ala Glu Asn Glu Thr Pro Leu Leu Tyr Ile Asp Arg His Leu Val 20
25 30 His Glu Val Thr Ser Pro Gln Ala Phe Asp Gly Leu Arg Ala His
Gly 35 40 45 Arg Pro Val Arg Gln Pro Gly Lys Thr Phe Ala Thr Met
Asp His Asn 50 55 60 Val Ser Thr Gln Thr Lys Asp Ile Asn Ala Cys
Gly Glu Met Ala Arg 65 70 75 80 Ile Gln Met Gln Glu Leu Ile Lys Asn
Cys Lys Glu Phe Gly Val Glu 85 90 95 Leu Tyr Asp Leu Asn His Pro
Tyr Gln Gly Ile Val His Val Met Gly 100 105 110 Pro Glu Gln Gly Val
Thr Leu Pro Gly Met Thr Ile Val Cys Gly Asp 115 120 125 Ser His Thr
Ala Thr His Gly Ala Phe Gly Ala Leu Ala Phe Gly Ile 130 135 140 Gly
Thr Ser Glu Val Glu His Val Leu Ala Thr Gln Thr Leu Lys Gln 145 150
155 160 Gly Arg Ala Lys Thr Met Lys Ile Glu Val Gln Gly Lys Ala Ala
Pro 165 170 175 Gly Ile Thr Ala Lys Asp Ile Val Leu Ala Ile Ile Gly
Lys Thr Gly 180 185 190 Ser Ala Gly Gly Thr Gly His Val Val Glu Phe
Cys Gly Glu Ala Ile 195 200 205 Arg Asp Leu Ser Met Glu Gly Arg Met
Thr Leu Cys Asn Met Ala Ile 210 215 220 Glu Met Gly Ala Lys Ala Gly
Leu Val Ala Pro Asp Glu Thr Thr Phe 225 230 235 240 Asn Tyr Val Lys
Gly Arg Leu His Ala Pro Lys Gly Lys Asp Phe Asp 245 250 255 Asp Ala
Val Ala Tyr Trp Lys Thr Leu Gln Thr Asp Glu Gly Ala Thr 260 265 270
Phe Asp Thr Val Val Thr Leu Gln Ala Glu Glu Ile Ser Pro Gln Val 275
280 285 Thr Trp Gly Thr Asn Pro Gly Gln Val Ile Ser Val Asn Asp Asn
Ile 290 295 300 Pro Asp Pro Ala Ser Phe Ala Asp Pro Val Glu Arg Ala
Ser Ala Glu 305 310 315 320 Lys Ala Leu Ala Tyr Met Gly Leu Lys Pro
Gly Ile Pro Leu Thr Glu 325 330 335 Val Ala Ile Asp Lys Val Phe Ile
Gly Ser Cys Thr Asn Ser Arg Ile 340 345 350 Glu Asp Leu Arg Ala Ala
Ala Glu Ile Ala Lys Gly Arg Lys Val Ala 355 360 365 Pro Gly Val Gln
Ala Leu Val Val Pro Gly Ser Gly Pro Val Lys Ala 370 375 380 Gln Ala
Glu Ala Glu Gly Leu Asp Lys Ile Phe Ile Glu Ala Gly Phe 385 390 395
400 Glu Trp Arg Leu Pro Gly Cys Ser Met Cys Leu Ala Met Asn Asn Asp
405 410 415 Arg Leu Asn Pro Gly Glu Arg Cys Ala Ser Thr Ser Asn Arg
Asn Phe 420 425 430 Glu Gly Arg Gln Gly Arg Gly Gly Arg Thr His Leu
Val Ser Pro Ala 435 440 445 Met Ala Ala Ala Ala Ala Val Thr Gly His
Phe Ala Asp Ile Arg Asn 450 455 460 Ile Lys 465 45201PRTEscherichia
colisource/note="Isopropylmalate isomerase small subunit" 45Met Ala
Glu Lys Phe Ile Lys His Thr Gly Leu Val Val Pro Leu Asp 1 5 10 15
Ala Ala Asn Val Asp Thr Asp Ala Ile Ile Pro Lys Gln Phe Leu Gln 20
25 30 Lys Val Thr Arg Thr Gly Phe Gly Ala His Leu Phe Asn Asp Trp
Arg 35 40 45 Phe Leu Asp Glu Lys Gly Gln Gln Pro Asn Pro Asp Phe
Val Leu Asn 50 55 60 Phe Pro Gln Tyr Gln Gly Ala Ser Ile Leu Leu
Ala Arg Glu Asn Phe 65 70 75 80 Gly Cys Gly Ser Ser Arg Glu His Ala
Pro Trp Ala Leu Thr Asp Tyr 85 90 95 Gly Phe Lys Val Val Ile Ala
Pro Ser Phe Ala Asp Ile Phe Tyr Gly 100 105 110 Asn Ser Phe Asn Asn
Gln Leu Leu Pro Val Lys Leu Ser Asp Ala Glu 115 120 125 Val Asp Glu
Leu Phe Ala Leu Val Lys Ala Asn Pro Gly Ile His Phe 130 135 140 Asp
Val Asp Leu Glu Ala Gln Glu Val Lys Ala Gly Glu Lys Thr Tyr 145 150
155 160 Arg Phe Thr Ile Asp Ala Phe Arg Arg His Cys Met Met Asn Gly
Leu 165 170 175 Asp Ser Ile Gly Leu Thr Leu Gln His Asp Asp Ala Ile
Ala Ala Tyr 180 185 190 Glu Ala Lys Gln Pro Ala Phe Met Asn 195 200
46472PRTBacillus subtilissource/note="Isopropylmalate isomerase
large subunit" 46Met Met Pro Arg Thr Ile Ile Glu Lys Ile Trp Asp
Gln His Ile Val 1 5 10 15 Lys His Gly Glu Gly Lys Pro Asp Leu Leu
Tyr Ile Asp Leu His Leu 20 25 30 Ile His Glu Val Thr Ser Pro Gln
Ala Phe Glu Gly Leu Arg Gln Lys 35 40 45 Gly Arg Lys Val Arg Arg
Pro Gln Asn Thr Phe Ala Thr Met Asp His 50 55 60 Asn Ile Pro Thr
Val Asn Arg Phe Glu Ile Lys Asp Glu Val Ala Lys 65 70 75 80 Arg Gln
Val Thr Ala Leu Glu Arg Asn Cys Glu Glu Phe Gly Val Arg 85 90 95
Leu Ala Asp Leu His Ser Val Asp Gln Gly Ile Val His Val Val Gly 100
105 110 Pro Glu Leu Gly Leu Thr Leu Pro Gly Lys Thr Ile Val Cys Gly
Asp 115 120 125 Ser His Thr Ser Thr His Gly Ala Phe Gly Ala Leu Ala
Phe Gly Ile 130 135 140
Gly Thr Ser Glu Val Glu His Val Leu Ser Thr Gln Thr Leu Trp Gln 145
150 155 160 Gln Arg Pro Lys Thr Leu Glu Val Arg Val Asp Gly Thr Leu
Gln Lys 165 170 175 Gly Val Thr Ala Lys Asp Val Ile Leu Ala Val Ile
Gly Lys Tyr Gly 180 185 190 Val Lys Phe Gly Thr Gly Tyr Val Ile Glu
Tyr Thr Gly Glu Val Phe 195 200 205 Arg Asn Met Thr Met Asp Glu Arg
Met Thr Val Cys Asn Met Ser Ile 210 215 220 Glu Ala Gly Ala Arg Ala
Gly Leu Ile Ala Pro Asp Glu Val Thr Phe 225 230 235 240 Glu Tyr Cys
Lys Asn Arg Lys Tyr Thr Pro Lys Gly Glu Glu Phe Asp 245 250 255 Lys
Ala Val Glu Glu Trp Lys Ala Leu Arg Thr Asp Pro Gly Ala Val 260 265
270 Tyr Asp Lys Ser Ile Val Leu Asp Gly Asn Lys Ile Ser Pro Met Val
275 280 285 Thr Trp Gly Ile Asn Pro Gly Met Val Leu Pro Val Asp Ser
Glu Val 290 295 300 Pro Ala Pro Glu Ser Phe Ser Ala Glu Asp Asp Lys
Lys Glu Ala Ile 305 310 315 320 Arg Ala Tyr Glu Tyr Met Gly Leu Thr
Pro His Gln Lys Ile Glu Asp 325 330 335 Ile Lys Val Glu His Val Phe
Ile Gly Ser Cys Thr Asn Ser Arg Met 340 345 350 Thr Asp Leu Arg Gln
Ala Ala Asp Met Ile Lys Gly Lys Lys Val Ala 355 360 365 Asp Ser Val
Arg Ala Ile Val Val Pro Gly Ser Gln Ser Val Lys Leu 370 375 380 Gln
Ala Glu Lys Glu Gly Leu Asp Gln Ile Phe Leu Glu Ala Gly Phe 385 390
395 400 Glu Trp Arg Glu Ser Gly Cys Ser Met Cys Leu Ser Met Asn Asn
Asp 405 410 415 Val Val Pro Glu Gly Glu Arg Cys Ala Ser Thr Ser Asn
Arg Asn Phe 420 425 430 Glu Gly Arg Gln Gly Lys Gly Ala Arg Thr His
Leu Val Ser Pro Ala 435 440 445 Met Ala Ala Met Ala Ala Ile His Gly
His Phe Val Asp Val Arg Lys 450 455 460 Phe Tyr Gln Glu Lys Thr Val
Val 465 470 47199PRTBacillus subtilissource/note="Isopropylmalate
isomerase small subunit" 47Met Glu Pro Leu Lys Ser His Thr Gly Lys
Ala Ala Val Leu Asn Arg 1 5 10 15 Ile Asn Val Asp Thr Asp Gln Ile
Ile Pro Lys Gln Phe Leu Lys Arg 20 25 30 Ile Glu Arg Thr Gly Tyr
Gly Arg Phe Ala Phe Phe Asp Trp Arg Tyr 35 40 45 Asp Ala Asn Gly
Glu Pro Asn Pro Glu Phe Glu Leu Asn Gln Pro Val 50 55 60 Tyr Gln
Gly Ala Ser Ile Leu Ile Ala Gly Glu Asn Phe Gly Cys Gly 65 70 75 80
Ser Ser Arg Glu His Ala Pro Trp Ala Leu Asp Asp Tyr Gly Phe Lys 85
90 95 Ile Ile Ile Ala Pro Ser Phe Ala Asp Ile Phe His Gln Asn Cys
Phe 100 105 110 Lys Asn Gly Met Leu Pro Ile Arg Met Pro Tyr Asp Asn
Trp Lys Gln 115 120 125 Leu Val Gly Gln Tyr Glu Asn Gln Ser Leu Gln
Met Thr Val Asp Leu 130 135 140 Glu Asn Gln Leu Ile His Asp Ser Glu
Gly Asn Gln Ile Ser Phe Glu 145 150 155 160 Val Asp Pro His Trp Lys
Glu Met Leu Ile Asn Gly Tyr Asp Glu Ile 165 170 175 Ser Leu Thr Leu
Leu Leu Glu Asp Glu Ile Lys Gln Phe Glu Ser Gln 180 185 190 Arg Ser
Ser Trp Leu Gln Ala 195 48363PRTEscherichia
colisource/note="Beta-isopropylmalate dehydrogenase" 48 Met Ser Lys
Asn Tyr His Ile Ala Val Leu Pro Gly Asp Gly Ile Gly 1 5 10 15 Pro
Glu Val Met Thr Gln Ala Leu Lys Val Leu Asp Ala Val Arg Asn 20 25
30 Arg Phe Ala Met Arg Ile Thr Thr Ser His Tyr Asp Val Gly Gly Ala
35 40 45 Ala Ile Asp Asn His Gly Gln Pro Leu Pro Pro Ala Thr Val
Glu Gly 50 55 60 Cys Glu Gln Ala Asp Ala Val Leu Phe Gly Ser Val
Gly Gly Pro Lys 65 70 75 80 Trp Glu His Leu Pro Pro Asp Gln Gln Pro
Glu Arg Gly Ala Leu Leu 85 90 95 Pro Leu Arg Lys His Phe Lys Leu
Phe Ser Asn Leu Arg Pro Ala Lys 100 105 110 Leu Tyr Gln Gly Leu Glu
Ala Phe Cys Pro Leu Arg Ala Asp Ile Ala 115 120 125 Ala Asn Gly Phe
Asp Ile Leu Cys Val Arg Glu Leu Thr Gly Gly Ile 130 135 140 Tyr Phe
Gly Gln Pro Lys Gly Arg Glu Gly Ser Gly Gln Tyr Glu Lys 145 150 155
160 Ala Phe Asp Thr Glu Val Tyr His Arg Phe Glu Ile Glu Arg Ile Ala
165 170 175 Arg Ile Ala Phe Glu Ser Ala Arg Lys Arg Arg His Lys Val
Thr Ser 180 185 190 Ile Asp Lys Ala Asn Val Leu Gln Ser Ser Ile Leu
Trp Arg Glu Ile 195 200 205 Val Asn Glu Ile Ala Thr Glu Tyr Pro Asp
Val Glu Leu Ala His Met 210 215 220 Tyr Ile Asp Asn Ala Thr Met Gln
Leu Ile Lys Asp Pro Ser Gln Phe 225 230 235 240 Asp Val Leu Leu Cys
Ser Asn Leu Phe Gly Asp Ile Leu Ser Asp Glu 245 250 255 Cys Ala Met
Ile Thr Gly Ser Met Gly Met Leu Pro Ser Ala Ser Leu 260 265 270 Asn
Glu Gln Gly Phe Gly Leu Tyr Glu Pro Ala Gly Gly Ser Ala Pro 275 280
285 Asp Ile Ala Gly Lys Asn Ile Ala Asn Pro Ile Ala Gln Ile Leu Ser
290 295 300 Leu Ala Leu Leu Leu Arg Tyr Ser Leu Asp Ala Asp Asp Ala
Ala Cys 305 310 315 320 Ala Ile Glu Arg Ala Ile Asn Arg Ala Leu Glu
Glu Gly Ile Arg Thr 325 330 335 Gly Asp Leu Ala Arg Gly Ala Ala Ala
Val Ser Thr Asp Glu Met Gly 340 345 350 Asp Ile Ile Ala Arg Tyr Val
Ala Glu Gly Val 355 360 49365PRTBacillus
subtilissource/note="Beta-isopropylmalate dehydrogenase" 49Met Lys
Lys Arg Ile Ala Leu Leu Pro Gly Asp Gly Ile Gly Pro Glu 1 5 10 15
Val Leu Glu Ser Ala Thr Asp Val Leu Lys Ser Val Ala Glu Arg Phe 20
25 30 Asn His Glu Phe Glu Phe Glu Tyr Gly Leu Ile Gly Gly Ala Ala
Ile 35 40 45 Asp Glu His His Asn Pro Leu Pro Glu Glu Thr Val Ala
Ala Cys Lys 50 55 60 Asn Ala Asp Ala Ile Leu Leu Gly Ala Val Gly
Gly Pro Lys Trp Asp 65 70 75 80 Gln Asn Pro Ser Glu Leu Arg Pro Glu
Lys Gly Leu Leu Ser Ile Arg 85 90 95 Lys Gln Leu Asp Leu Phe Ala
Asn Leu Arg Pro Val Lys Val Phe Glu 100 105 110 Ser Leu Ser Asp Ala
Ser Pro Leu Lys Lys Glu Tyr Ile Asp Asn Val 115 120 125 Asp Phe Val
Ile Val Arg Glu Leu Thr Gly Gly Leu Tyr Phe Gly Gln 130 135 140 Pro
Ser Lys Arg Tyr Val Asn Thr Glu Gly Glu Gln Glu Ala Val Asp 145 150
155 160 Thr Leu Phe Tyr Lys Arg Thr Glu Ile Glu Arg Val Ile Arg Glu
Gly 165 170 175 Phe Lys Met Ala Ala Ala Arg Lys Gly Lys Val Thr Ser
Val Asp Lys 180 185 190 Ala Asn Val Leu Glu Ser Ser Arg Leu Trp Arg
Glu Val Ala Glu Asp 195 200 205 Val Ala Gln Glu Phe Pro Asp Val Lys
Leu Glu His Met Leu Val Asp 210 215 220 Asn Ala Ala Met Gln Leu Ile
Tyr Ala Pro Asn Gln Phe Asp Val Val 225 230 235 240 Val Thr Glu Asn
Met Phe Gly Asp Ile Leu Ser Asp Glu Ala Ser Met 245 250 255 Leu Thr
Gly Ser Leu Gly Met Leu Pro Ser Ala Ser Leu Ser Ser Ser 260 265 270
Gly Leu His Leu Phe Glu Pro Val His Gly Ser Ala Pro Asp Ile Ala 275
280 285 Gly Lys Gly Met Ala Asn Pro Phe Ala Ala Ile Leu Ser Ala Ala
Met 290 295 300 Leu Leu Arg Thr Ser Phe Gly Leu Glu Glu Glu Ala Lys
Ala Val Glu 305 310 315 320 Asp Ala Val Asn Lys Val Leu Ala Ser Gly
Lys Arg Thr Arg Asp Leu 325 330 335 Ala Arg Ser Glu Glu Phe Ser Ser
Thr Gln Ala Ile Thr Glu Glu Val 340 345 350 Lys Ala Ala Ile Met Ser
Glu Asn Thr Ile Ser Asn Val 355 360 365 50364PRTSaccharomyces
cerevisiaesource/note="Beta-isopropylmalate dehydrogenase" 50Met
Ser Ala Pro Lys Lys Ile Val Val Leu Pro Gly Asp His Val Gly 1 5 10
15 Gln Glu Ile Thr Ala Glu Ala Ile Lys Val Leu Lys Ala Ile Ser Asp
20 25 30 Val Arg Ser Asn Val Lys Phe Asp Phe Glu Asn His Leu Ile
Gly Gly 35 40 45 Ala Ala Ile Asp Ala Thr Gly Val Pro Leu Pro Asp
Glu Ala Leu Glu 50 55 60 Ala Ser Lys Lys Ala Asp Ala Val Leu Leu
Gly Ala Val Gly Gly Pro 65 70 75 80 Lys Trp Gly Thr Gly Ser Val Arg
Pro Glu Gln Gly Leu Leu Lys Ile 85 90 95 Arg Lys Glu Leu Gln Leu
Tyr Ala Asn Leu Arg Pro Cys Asn Phe Ala 100 105 110 Ser Asp Ser Leu
Leu Asp Leu Ser Pro Ile Lys Pro Gln Phe Ala Lys 115 120 125 Gly Thr
Asp Phe Val Val Val Arg Glu Leu Val Gly Gly Ile Tyr Phe 130 135 140
Gly Lys Arg Lys Glu Asp Asp Gly Asp Gly Val Ala Trp Asp Ser Glu 145
150 155 160 Gln Tyr Thr Val Pro Glu Val Gln Arg Ile Thr Arg Met Ala
Ala Phe 165 170 175 Met Ala Leu Gln His Glu Pro Pro Leu Pro Ile Trp
Ser Leu Asp Lys 180 185 190 Ala Asn Val Leu Ala Ser Ser Arg Leu Trp
Arg Lys Thr Val Glu Glu 195 200 205 Thr Ile Lys Asn Glu Phe Pro Thr
Leu Lys Val Gln His Gln Leu Ile 210 215 220 Asp Ser Ala Ala Met Ile
Leu Val Lys Asn Pro Thr His Leu Asn Gly 225 230 235 240 Ile Ile Ile
Thr Ser Asn Met Phe Gly Asp Ile Ile Ser Asp Glu Ala 245 250 255 Ser
Val Ile Pro Gly Ser Leu Gly Leu Leu Pro Ser Ala Ser Leu Ala 260 265
270 Ser Leu Pro Asp Lys Asn Thr Ala Phe Gly Leu Tyr Glu Pro Cys His
275 280 285 Gly Ser Ala Pro Asp Leu Pro Lys Asn Lys Val Asn Pro Ile
Ala Thr 290 295 300 Ile Leu Ser Ala Ala Met Met Leu Lys Leu Ser Leu
Asn Leu Pro Glu 305 310 315 320 Glu Gly Lys Ala Ile Glu Asp Ala Val
Lys Lys Val Leu Asp Ala Gly 325 330 335 Ile Arg Thr Gly Asp Leu Gly
Gly Ser Asn Ser Thr Thr Glu Val Gly 340 345 350 Asp Ala Val Ala Glu
Glu Val Lys Lys Ile Leu Ala 355 360 51714PRTEscherichia
colisource/note="Methylmalonyl-CoA mutase" 51Met Ser Asn Val Gln
Glu Trp Gln Gln Leu Ala Asn Lys Glu Leu Ser 1 5 10 15 Arg Arg Glu
Lys Thr Val Asp Ser Leu Val His Gln Thr Ala Glu Gly 20 25 30 Ile
Ala Ile Lys Pro Leu Tyr Thr Glu Ala Asp Leu Asp Asn Leu Glu 35 40
45 Val Thr Gly Thr Leu Pro Gly Leu Pro Pro Tyr Val Arg Gly Pro Arg
50 55 60 Ala Thr Met Tyr Thr Ala Gln Pro Trp Thr Ile Arg Gln Tyr
Ala Gly 65 70 75 80 Phe Ser Thr Ala Lys Glu Ser Asn Ala Phe Tyr Arg
Arg Asn Leu Ala 85 90 95 Ala Gly Gln Lys Gly Leu Ser Val Ala Phe
Asp Leu Ala Thr His Arg 100 105 110 Gly Tyr Asp Ser Asp Asn Pro Arg
Val Ala Gly Asp Val Gly Lys Ala 115 120 125 Gly Val Ala Ile Asp Thr
Val Glu Asp Met Lys Val Leu Phe Asp Gln 130 135 140 Ile Pro Leu Asp
Lys Met Ser Val Ser Met Thr Met Asn Gly Ala Val 145 150 155 160 Leu
Pro Val Leu Ala Phe Tyr Ile Val Ala Ala Glu Glu Gln Gly Val 165 170
175 Thr Pro Asp Lys Leu Thr Gly Thr Ile Gln Asn Asp Ile Leu Lys Glu
180 185 190 Tyr Leu Cys Arg Asn Thr Tyr Ile Tyr Pro Pro Lys Pro Ser
Met Arg 195 200 205 Ile Ile Ala Asp Ile Ile Ala Trp Cys Ser Gly Asn
Met Pro Arg Phe 210 215 220 Asn Thr Ile Ser Ile Ser Gly Tyr His Met
Gly Glu Ala Gly Ala Asn 225 230 235 240 Cys Val Gln Gln Val Ala Phe
Thr Leu Ala Asp Gly Ile Glu Tyr Ile 245 250 255 Lys Ala Ala Ile Ser
Ala Gly Leu Lys Ile Asp Asp Phe Ala Pro Arg 260 265 270 Leu Ser Phe
Phe Phe Gly Ile Gly Met Asp Leu Phe Met Asn Val Ala 275 280 285 Met
Leu Arg Ala Ala Arg Tyr Leu Trp Ser Glu Ala Val Ser Gly Phe 290 295
300 Gly Ala Gln Asp Pro Lys Ser Leu Ala Leu Arg Thr His Cys Gln Thr
305 310 315 320 Ser Gly Trp Ser Leu Thr Glu Gln Asp Pro Tyr Asn Asn
Val Ile Arg 325 330 335 Thr Thr Ile Glu Ala Leu Ala Ala Thr Leu Gly
Gly Thr Gln Ser Leu 340 345 350 His Thr Asn Ala Phe Asp Glu Ala Leu
Gly Leu Pro Thr Asp Phe Ser 355 360 365 Ala Arg Ile Ala Arg Asn Thr
Gln Ile Ile Ile Gln Glu Glu Ser Glu 370 375 380 Leu Cys Arg Thr Val
Asp Pro Leu Ala Gly Ser Tyr Tyr Ile Glu Ser 385 390 395 400 Leu Thr
Asp Gln Ile Val Lys Gln Ala Arg Ala Ile Ile Gln Gln Ile 405 410 415
Asp Glu Ala Gly Gly Met Ala Lys Ala Ile Glu Ala Gly Leu Pro Lys 420
425 430 Arg Met Ile Glu Glu Ala Ser Ala Arg Glu Gln Ser Leu Ile Asp
Gln 435 440 445 Gly Lys Arg Val Ile Val Gly Val Asn Lys Tyr Lys Leu
Asp His Glu 450 455 460 Asp Glu Thr Asp Val Leu Glu Ile Asp Asn Val
Met Val Arg Asn Glu 465 470 475 480 Gln Ile Ala Ser Leu Glu Arg Ile
Arg Ala Thr Arg Asp Asp Ala Ala 485 490 495 Val Thr Ala Ala Leu Asn
Ala Leu Thr His Ala Ala Gln His Asn Glu 500 505 510 Asn Leu Leu Ala
Ala Ala Val Asn Ala Ala Arg Val Arg Ala Thr Leu 515 520 525 Gly Glu
Ile Ser Asp Ala Leu Glu Val Ala Phe Asp Arg Tyr Leu Val 530 535 540
Pro Ser Gln Cys Val Thr Gly Val Ile Ala Gln Ser Tyr His Gln Ser 545
550 555 560 Glu Lys Ser Ala Ser Glu Phe Asp Ala Ile Val Ala Gln Thr
Glu Gln 565 570 575 Phe Leu Ala Asp Asn Gly Arg Arg Pro Arg Ile Leu
Ile Ala Lys Met 580 585 590 Gly Gln Asp Gly His Asp Arg Gly Ala Lys
Val Ile Ala Ser Ala Tyr 595 600 605 Ser Asp Leu Gly Phe Asp Val Asp
Leu Ser Pro Met Phe Ser Thr Pro 610 615 620 Glu Glu Ile Ala Arg Leu
Ala Val
Glu Asn Asp Val His Val Val Gly 625 630 635 640 Ala Ser Ser Leu Ala
Ala Gly His Lys Thr Leu Ile Pro Glu Leu Val 645 650 655 Glu Ala Leu
Lys Lys Trp Gly Arg Glu Asp Ile Cys Val Val Ala Gly 660 665 670 Gly
Val Ile Pro Pro Gln Asp Tyr Ala Phe Leu Gln Glu Arg Gly Val 675 680
685 Ala Ala Ile Tyr Gly Pro Gly Thr Pro Met Leu Asp Ser Val Arg Asp
690 695 700 Val Leu Asn Leu Ile Ser Gln His His Asp 705 710
52714PRTSalmonella entericasource/note="Methylmalonyl-CoA mutase"
52Met Ala Asn Leu Gln Ala Trp Gln Thr Leu Ala Asn Asn Glu Leu Ser 1
5 10 15 Arg Arg Glu Lys Thr Val Glu Ser Leu Ile Arg Gln Thr Ala Glu
Gly 20 25 30 Ile Ala Val Lys Pro Leu Tyr Thr Glu Ala Asp Leu Asn
Asn Leu Glu 35 40 45 Val Thr Gly Thr Leu Pro Gly Leu Pro Pro Tyr
Val Arg Gly Pro Arg 50 55 60 Ala Thr Met Tyr Thr Ala Gln Pro Trp
Thr Ile Arg Gln Tyr Ala Gly 65 70 75 80 Phe Ser Thr Ala Lys Glu Ser
Asn Ala Phe Tyr Arg Arg Asn Leu Ala 85 90 95 Ala Gly Gln Lys Gly
Leu Ser Val Ala Phe Asp Leu Ala Thr His Arg 100 105 110 Gly Tyr Asp
Ser Asp Asn Pro Arg Val Ala Gly Asp Val Gly Lys Ala 115 120 125 Gly
Val Ala Ile Asp Thr Val Glu Asp Met Lys Val Leu Phe Asp Gln 130 135
140 Ile Pro Leu Asp Lys Met Ser Val Ser Met Thr Met Asn Gly Ala Val
145 150 155 160 Leu Pro Val Met Ala Phe Tyr Ile Val Ala Ala Glu Glu
Gln Gly Val 165 170 175 Ser Pro Glu Gln Leu Thr Gly Thr Ile Gln Asn
Asp Ile Leu Lys Glu 180 185 190 Tyr Leu Cys Arg Asn Thr Tyr Ile Tyr
Pro Pro Lys Pro Ser Met Arg 195 200 205 Ile Ile Ala Asp Ile Ile Ala
Trp Cys Ser Gly Asn Met Pro Arg Phe 210 215 220 Asn Thr Ile Ser Ile
Ser Gly Tyr His Met Gly Glu Ala Gly Ala Asn 225 230 235 240 Cys Val
Gln Gln Val Ala Phe Thr Leu Ala Asp Gly Ile Glu Tyr Ile 245 250 255
Lys Ala Ala Leu Ser Ala Gly Leu Lys Ile Asp Asp Phe Ala Pro Arg 260
265 270 Leu Ser Phe Phe Phe Gly Ile Gly Met Asp Leu Phe Met Asn Val
Ala 275 280 285 Met Leu Arg Ala Ala Arg Tyr Leu Trp Ser Glu Ala Val
Ser Gly Phe 290 295 300 Gly Ala Thr Asn Pro Lys Ser Leu Ala Leu Arg
Thr His Cys Gln Thr 305 310 315 320 Ser Gly Trp Ser Leu Thr Glu Gln
Asp Pro Tyr Asn Asn Ile Ile Arg 325 330 335 Thr Thr Ile Glu Ala Leu
Gly Ala Thr Leu Gly Gly Thr Gln Ser Leu 340 345 350 His Thr Asn Ala
Phe Asp Glu Ala Leu Gly Leu Pro Thr Asp Phe Ser 355 360 365 Ala Arg
Ile Ala Arg Asn Thr Gln Ile Ile Ile Gln Glu Glu Ser Ser 370 375 380
Ile Cys Arg Thr Val Asp Pro Leu Ala Gly Ser Tyr Tyr Val Glu Ser 385
390 395 400 Leu Thr Asp Gln Ile Val Lys Gln Ala Arg Ala Ile Ile Lys
Gln Ile 405 410 415 Asp Ala Ala Gly Gly Met Ala Lys Ala Ile Glu Ala
Gly Leu Pro Lys 420 425 430 Arg Met Ile Glu Glu Ala Ser Ala Arg Glu
Gln Ser Leu Ile Asp Gln 435 440 445 Gly Glu Arg Val Ile Val Gly Val
Asn Lys Tyr Lys Leu Glu Lys Glu 450 455 460 Asp Glu Thr Ala Val Leu
Glu Ile Asp Asn Val Lys Val Arg Asn Glu 465 470 475 480 Gln Ile Ala
Ala Leu Glu Arg Ile Arg Ala Thr Arg Asp Asn Arg Ala 485 490 495 Val
Asn Ala Ala Leu Gln Ala Leu Thr His Ala Ala Gln His His Glu 500 505
510 Asn Leu Leu Ala Ala Ala Val Glu Ala Ala Arg Val Arg Ala Thr Leu
515 520 525 Gly Glu Ile Ser Asp Ala Leu Glu Ala Ala Phe Asp Arg Tyr
Leu Val 530 535 540 Pro Ser Gln Cys Val Thr Gly Val Ile Ala Gln Ser
Tyr His Gln Ser 545 550 555 560 Asp Lys Ser Ala Gly Glu Phe Asp Ala
Ile Val Ala Gln Thr Gln Gln 565 570 575 Phe Leu Ala Asp Thr Gly Arg
Arg Pro Arg Ile Leu Ile Ala Lys Met 580 585 590 Gly Gln Asp Gly His
Asp Arg Gly Ala Lys Val Ile Ala Ser Ala Tyr 595 600 605 Ser Asp Leu
Gly Phe Asp Val Asp Leu Ser Pro Met Phe Ser Thr Pro 610 615 620 Asp
Glu Ile Ala Arg Leu Ala Val Glu Asn Asp Val His Val Ile Gly 625 630
635 640 Ala Ser Ser Leu Ala Ala Gly His Lys Thr Leu Ile Pro Glu Leu
Val 645 650 655 Ala Ala Leu Lys Lys Trp Gly Arg Glu Asp Ile Cys Val
Val Ala Gly 660 665 670 Gly Val Ile Pro Pro Gln Asp Tyr Ala Phe Leu
Lys Ala His Gly Val 675 680 685 Ala Ala Ile Tyr Gly Pro Gly Thr Pro
Met Leu Glu Ser Val Arg Asp 690 695 700 Val Leu Ala Arg Ile Ser Gln
His His Asp 705 710 53638PRTPropionibacterium
freudenreichiisource/note="Methylmalonyl-CoA mutase beta (small)
subunit" 53Met Ser Ser Thr Asp Gln Gly Thr Asn Pro Ala Asp Thr Asp
Asp Leu 1 5 10 15 Thr Pro Thr Thr Leu Ser Leu Ala Gly Asp Phe Pro
Lys Ala Thr Glu 20 25 30 Glu Gln Trp Glu Arg Glu Val Glu Lys Val
Leu Asn Arg Gly Arg Pro 35 40 45 Pro Glu Lys Gln Leu Thr Phe Ala
Glu Cys Leu Lys Arg Leu Thr Val 50 55 60 His Thr Val Asp Gly Ile
Asp Ile Val Pro Met Tyr Arg Pro Lys Asp 65 70 75 80 Ala Pro Lys Lys
Leu Gly Tyr Pro Gly Val Ala Pro Phe Thr Arg Gly 85 90 95 Thr Thr
Val Arg Asn Gly Asp Met Asp Ala Trp Asp Val Arg Ala Leu 100 105 110
His Glu Asp Pro Asp Glu Lys Phe Thr Arg Lys Ala Ile Leu Glu Gly 115
120 125 Leu Glu Arg Gly Val Thr Ser Leu Leu Leu Arg Val Asp Pro Asp
Ala 130 135 140 Ile Ala Pro Glu His Leu Asp Glu Val Leu Ser Asp Val
Leu Leu Glu 145 150 155 160 Met Thr Lys Val Glu Val Phe Ser Arg Tyr
Asp Gln Gly Ala Ala Ala 165 170 175 Glu Ala Leu Val Ser Val Tyr Glu
Arg Ser Asp Lys Pro Ala Lys Asp 180 185 190 Leu Ala Leu Asn Leu Gly
Leu Asp Pro Ile Ala Phe Ala Ala Leu Gln 195 200 205 Gly Thr Glu Pro
Asp Leu Thr Val Leu Gly Asp Trp Val Arg Arg Leu 210 215 220 Ala Lys
Phe Ser Pro Asp Ser Arg Ala Val Thr Ile Asp Ala Asn Ile 225 230 235
240 Tyr His Asn Ala Gly Ala Gly Asp Val Ala Glu Leu Ala Trp Ala Leu
245 250 255 Ala Thr Gly Ala Glu Tyr Val Arg Ala Leu Val Glu Gln Gly
Phe Thr 260 265 270 Ala Thr Glu Ala Phe Asp Thr Ile Asn Phe Arg Val
Thr Ala Thr His 275 280 285 Asp Gln Phe Leu Thr Ile Ala Arg Leu Arg
Ala Leu Arg Glu Ala Trp 290 295 300 Ala Arg Ile Gly Glu Val Phe Gly
Val Asp Glu Asp Lys Arg Gly Ala 305 310 315 320 Arg Gln Asn Ala Ile
Thr Ser Trp Arg Asp Val Thr Arg Glu Asp Pro 325 330 335 Tyr Val Asn
Ile Leu Arg Gly Ser Ile Ala Thr Phe Ser Ala Ser Val 340 345 350 Gly
Gly Ala Glu Ser Ile Thr Thr Leu Pro Phe Thr Gln Ala Leu Gly 355 360
365 Leu Pro Glu Asp Asp Phe Pro Leu Arg Ile Ala Arg Asn Thr Gly Ile
370 375 380 Val Leu Ala Glu Glu Val Asn Ile Gly Arg Val Asn Asp Pro
Ala Gly 385 390 395 400 Gly Ser Tyr Tyr Val Glu Ser Leu Thr Arg Ser
Leu Ala Asp Ala Ala 405 410 415 Trp Lys Glu Phe Gln Glu Val Glu Lys
Leu Gly Gly Met Ser Lys Ala 420 425 430 Val Met Thr Glu His Val Thr
Lys Val Leu Asp Ala Cys Asn Ala Glu 435 440 445 Arg Ala Lys Arg Leu
Ala Asn Arg Lys Gln Pro Ile Thr Ala Val Ser 450 455 460 Glu Phe Pro
Met Ile Gly Ala Arg Ser Ile Glu Thr Lys Pro Phe Pro 465 470 475 480
Ala Ala Pro Ala Arg Lys Gly Leu Ala Trp His Arg Asp Ser Glu Val 485
490 495 Phe Glu Gln Leu Met Asp Arg Ser Thr Ser Val Ser Glu Arg Pro
Lys 500 505 510 Val Phe Leu Ala Cys Leu Gly Thr Arg Arg Asp Phe Gly
Gly Arg Glu 515 520 525 Gly Phe Ser Ser Pro Val Trp His Ile Ala Gly
Ile Asp Thr Pro Gln 530 535 540 Val Glu Gly Gly Thr Thr Ala Glu Ile
Val Glu Ala Phe Lys Lys Ser 545 550 555 560 Gly Ala Gln Val Ala Asp
Leu Cys Ser Ser Ala Lys Val Tyr Ala Gln 565 570 575 Gln Gly Leu Glu
Val Ala Lys Ala Leu Lys Ala Ala Gly Ala Lys Ala 580 585 590 Leu Tyr
Leu Ser Gly Ala Phe Lys Glu Phe Gly Asp Asp Ala Ala Glu 595 600 605
Ala Glu Lys Leu Ile Asp Gly Arg Leu Phe Met Gly Met Asp Val Val 610
615 620 Asp Thr Leu Ser Ser Thr Leu Asp Ile Leu Gly Val Ala Lys 625
630 635 54728PRTPropionibacterium
freudenreichiisource/note="Methylmalonyl-CoA mutase alpha (large)
subunit" 54Met Ser Thr Leu Pro Arg Phe Asp Ser Val Asp Leu Gly Asn
Ala Pro 1 5 10 15 Val Pro Ala Asp Ala Ala Gln Arg Phe Glu Glu Leu
Ala Ala Lys Ala 20 25 30 Gly Thr Glu Glu Ala Trp Glu Thr Ala Glu
Gln Ile Pro Val Gly Thr 35 40 45 Leu Phe Asn Glu Asp Val Tyr Lys
Asp Met Asp Trp Leu Asp Thr Tyr 50 55 60 Ala Gly Ile Pro Pro Phe
Val His Gly Pro Tyr Ala Thr Met Tyr Ala 65 70 75 80 Phe Arg Pro Trp
Thr Ile Arg Gln Tyr Ala Gly Phe Ser Thr Ala Lys 85 90 95 Glu Ser
Asn Ala Phe Tyr Arg Arg Asn Leu Ala Ala Gly Gln Lys Gly 100 105 110
Leu Ser Val Ala Phe Asp Leu Pro Thr His Arg Gly Tyr Asp Ser Asp 115
120 125 Asn Pro Arg Val Ala Gly Asp Val Gly Met Ala Gly Val Ala Ile
Asp 130 135 140 Ser Ile Tyr Asp Met Arg Glu Leu Phe Ala Gly Ile Pro
Leu Asp Gln 145 150 155 160 Met Ser Val Ser Met Thr Met Asn Gly Ala
Val Leu Pro Ile Leu Ala 165 170 175 Leu Tyr Val Val Thr Ala Glu Glu
Gln Gly Val Lys Pro Glu Gln Leu 180 185 190 Ala Gly Thr Ile Gln Asn
Asp Ile Leu Lys Glu Phe Met Val Arg Asn 195 200 205 Thr Tyr Ile Tyr
Pro Pro Gln Pro Ser Met Arg Ile Ile Ser Glu Ile 210 215 220 Phe Ala
Tyr Thr Ser Ala Asn Met Pro Lys Trp Asn Ser Ile Ser Ile 225 230 235
240 Ser Gly Tyr His Met Gln Glu Ala Gly Ala Thr Ala Asp Ile Glu Met
245 250 255 Ala Tyr Thr Leu Ala Asp Gly Val Asp Tyr Ile Arg Ala Gly
Glu Ser 260 265 270 Val Gly Leu Asn Val Asp Gln Phe Ala Pro Arg Leu
Ser Phe Phe Trp 275 280 285 Gly Ile Gly Met Asn Phe Phe Met Glu Val
Ala Lys Leu Arg Ala Ala 290 295 300 Arg Met Leu Trp Ala Lys Leu Val
His Gln Phe Gly Pro Lys Asn Pro 305 310 315 320 Lys Ser Met Ser Leu
Arg Thr His Ser Gln Thr Ser Gly Trp Ser Leu 325 330 335 Thr Ala Gln
Asp Val Tyr Asn Asn Val Val Arg Thr Cys Ile Glu Ala 340 345 350 Met
Ala Ala Thr Gln Gly His Thr Gln Ser Leu His Thr Asn Ser Leu 355 360
365 Asp Glu Ala Ile Ala Leu Pro Thr Asp Phe Ser Ala Arg Ile Ala Arg
370 375 380 Asn Thr Gln Leu Phe Leu Gln Gln Glu Ser Gly Thr Thr Arg
Val Ile 385 390 395 400 Asp Pro Trp Ser Gly Ser Ala Tyr Val Glu Glu
Leu Thr Trp Asp Leu 405 410 415 Ala Arg Lys Ala Trp Gly His Ile Gln
Glu Val Glu Lys Val Gly Gly 420 425 430 Met Ala Lys Ala Ile Glu Lys
Gly Ile Pro Lys Met Arg Ile Glu Glu 435 440 445 Ala Ala Ala Arg Thr
Gln Ala Arg Ile Asp Ser Gly Arg Gln Pro Leu 450 455 460 Ile Gly Val
Asn Lys Tyr Arg Leu Glu His Glu Pro Pro Leu Asp Val 465 470 475 480
Leu Lys Val Asp Asn Ser Thr Val Leu Ala Glu Gln Lys Ala Lys Leu 485
490 495 Val Lys Leu Arg Ala Glu Arg Asp Pro Glu Lys Val Lys Ala Ala
Leu 500 505 510 Asp Lys Ile Thr Trp Ala Ala Ala Asn Pro Asp Asp Lys
Asp Pro Asp 515 520 525 Arg Asn Leu Leu Lys Leu Cys Ile Asp Ala Gly
Arg Ala Met Ala Thr 530 535 540 Val Gly Glu Met Ser Asp Ala Leu Glu
Lys Val Phe Gly Arg Tyr Thr 545 550 555 560 Ala Gln Ile Arg Thr Ile
Ser Gly Val Tyr Ser Lys Glu Val Lys Asn 565 570 575 Thr Pro Glu Val
Glu Glu Ala Arg Glu Leu Val Glu Glu Phe Glu Gln 580 585 590 Ala Glu
Gly Arg Arg Pro Arg Ile Leu Leu Ala Lys Met Gly Gln Asp 595 600 605
Gly His Asp Arg Gly Gln Lys Val Ile Ala Thr Ala Tyr Ala Asp Leu 610
615 620 Gly Phe Asp Val Asp Val Gly Pro Leu Phe Gln Thr Pro Glu Glu
Thr 625 630 635 640 Ala Arg Gln Ala Val Glu Ala Asp Val His Val Val
Gly Val Ser Ser 645 650 655 Leu Ala Gly Gly His Leu Thr Leu Val Pro
Ala Leu Arg Lys Glu Leu 660 665 670 Asp Lys Leu Gly Arg Pro Asp Ile
Leu Ile Thr Val Gly Gly Val Ile 675 680 685 Pro Glu Gln Asp Phe Asp
Glu Leu Arg Lys Asp Gly Ala Val Glu Ile 690 695 700 Tyr Thr Pro Gly
Thr Val Ile Pro Glu Ser Ala Ile Ser Leu Val Lys 705 710 715 720 Lys
Leu Arg Ala Ser Leu Asp Ala 725 55678PRTBacillus
megateriumsource/note="Methylmalonyl-CoA mutase beta (small)
subunit" 55Met Lys Thr Asn Thr Leu Ser Phe His Glu Phe Thr Arg Thr
Pro Lys 1 5 10 15 Glu Asp Trp Ala Gln Glu Val Ser Lys Asn Thr Ala
Ile Ser Ser Lys 20 25 30 Glu Thr Leu Glu Asn Ile Phe Leu Lys Pro
Leu Tyr Phe Glu Ser Asp 35 40 45 Thr Ala His Leu Asp Tyr Leu Gln
Gln Ser Pro Ala Gly Ile Asp Tyr 50 55 60 Leu Arg Gly Ala Gly Lys
Glu Ser Tyr Ile Leu Gly Glu Trp Glu Ile 65 70 75
80 Thr Gln Lys Ile Asp Leu Pro Ser Ile Lys Glu Ser Asn Lys Leu Leu
85 90 95 Leu His Ser Leu Arg Asn Gly Gln Asn Thr Ala Ala Phe Thr
Cys Ser 100 105 110 Glu Ala Met Arg Gln Gly Lys Asp Ile Asp Glu Ala
Thr Glu Ala Glu 115 120 125 Val Ala Ser Gly Ala Thr Ile Ser Thr Leu
Glu Asp Val Ala His Leu 130 135 140 Phe Gln His Val Ala Leu Glu Ala
Val Pro Leu Phe Leu Asn Thr Gly 145 150 155 160 Cys Thr Ser Val Pro
Leu Leu Ser Phe Leu Lys Ala Tyr Cys Val Asp 165 170 175 His Asn Phe
Asn Met Arg Gln Leu Lys Gly Thr Val Gly Met Asp Pro 180 185 190 Leu
Gly Thr Leu Ala Glu Tyr Gly Arg Val Pro Leu Ser Thr Arg Asp 195 200
205 Leu Tyr Asp His Leu Ala Tyr Ala Thr Arg Leu Ala His Ser Asn Val
210 215 220 Pro Glu Leu Lys Thr Ile Ile Val Ser Ser Ile Pro Tyr His
Asn Ser 225 230 235 240 Gly Ala Asn Ala Val Gln Glu Leu Ala Tyr Met
Leu Ala Thr Gly Val 245 250 255 Gln Tyr Ile Asp Glu Cys Ile Lys Arg
Gly Leu Ser Leu His Gln Val 260 265 270 Leu Pro His Met Thr Phe Ser
Phe Ser Val Ser Ser His Leu Phe Met 275 280 285 Glu Ile Ser Lys Leu
Arg Ala Phe Arg Met Leu Trp Ala Asn Val Val 290 295 300 Arg Ala Phe
Asp Asp Thr Ala Val Ser Val Pro Phe Ile His Thr Glu 305 310 315 320
Thr Ser His Leu Thr Gln Ser Lys Glu Asp Met Tyr Thr Asn Ala Leu 325
330 335 Arg Ser Thr Val Gln Ala Phe Ala Ser Ile Val Gly Gly Ala Asp
Ser 340 345 350 Leu His Ile Glu Pro Tyr Asp Ser Val Thr Ser Ser Ser
Ser Gln Phe 355 360 365 Ala His Arg Leu Ala Arg Asn Thr His Leu Ile
Leu Gln His Glu Thr 370 375 380 His Ile Ser Lys Val Met Asp Pro Ala
Gly Gly Ser Trp Tyr Val Glu 385 390 395 400 Ala Tyr Thr His Glu Leu
Met Thr Lys Ala Trp Glu Leu Phe Gly Asn 405 410 415 Ile Glu Asp His
Gly Gly Met Glu Glu Ala Leu Lys Gln Gly Arg Ile 420 425 430 Gln Asp
Glu Val Glu Gln Met Lys Val Lys Arg Gln Glu Asp Ile Glu 435 440 445
Cys Arg Ile Glu Arg Leu Ile Gly Val Thr His Tyr Ala Pro Lys Gln 450
455 460 Gln Asp Ala Ser Gln Glu Ile Lys Ser Thr Pro Phe Lys Lys Glu
Glu 465 470 475 480 Ile Lys Met Asp Lys Tyr Ser Asp Gln Asn Ala Ser
Glu Phe Ser Ser 485 490 495 Asn Leu Ser Leu Glu Asp Tyr Thr Lys Leu
Ala Ser Lys Gly Val Thr 500 505 510 Ala Gly Trp Met Leu Lys Gln Met
Ala Lys Gln Thr Gln Pro Asp Ser 515 520 525 Val Val Pro Leu Thr Lys
Trp Arg Ala Ala Glu Lys Phe Glu Lys Ile 530 535 540 Arg Val Tyr Thr
Lys Gly Met Ser Ile Gly Ile Met Glu Leu Thr Asp 545 550 555 560 Pro
Ser Ser Arg Lys Lys Ala Glu Ile Ala Arg Ser Leu Phe Glu Ser 565 570
575 Ala Gly Phe Ala Cys Glu Thr Ile Lys Asn Ile Asp Ser Tyr Val Glu
580 585 590 Ile Ala Asp Trp Met Asn Glu Gln Lys His Glu Ala Tyr Val
Ile Cys 595 600 605 Gly Ser Asp Glu Leu Val Glu Lys Leu Leu Thr Lys
Ala Met Thr Tyr 610 615 620 Phe Glu Glu Asp Ser Val Tyr Val Tyr Val
Val Gly Glu Glu His Val 625 630 635 640 Ser Arg Lys Thr Gln Trp Gln
Gln Lys Gly Val Met Ser Val Ile His 645 650 655 Pro Lys Thr Asn Val
Ile Gln Cys Val Lys Lys Leu Leu Cys Ala Leu 660 665 670 Glu Val Glu
Val His Val 675 56716PRTBacillus
megateriumsource/note="Methylmalonyl-CoA mutase alpha (large)
subunit" 56Met Tyr Lys Lys Pro Ser Phe Ser Asn Ile Pro Leu Ser Phe
Ser Lys 1 5 10 15 Gln Gln Arg Glu Asp Asp Val Thr Gln Ser Ser Tyr
Thr Ala Phe Gln 20 25 30 Thr Asn Glu Gln Ile Glu Leu Lys Ser Val
Tyr Thr Lys Lys Asp Arg 35 40 45 Asp Asn Leu Asp Phe Ile His Phe
Ala Pro Gly Val Pro Pro Phe Val 50 55 60 Arg Gly Pro Tyr Ala Thr
Met Tyr Val Asn Arg Pro Trp Thr Ile Arg 65 70 75 80 Gln Tyr Ala Gly
Tyr Ser Thr Ala Glu Glu Ser Asn Ala Phe Tyr Arg 85 90 95 Arg Asn
Leu Ala Ala Gly Gln Lys Gly Leu Ser Val Ala Phe Asp Leu 100 105 110
Ala Thr His Arg Gly Tyr Asp Ser Asp His Pro Arg Val Val Gly Asp 115
120 125 Val Gly Lys Ala Gly Val Ala Ile Asp Ser Met Met Asp Met Lys
Gln 130 135 140 Leu Phe Glu Gly Ile Pro Leu Asp Gln Met Ser Val Ser
Met Thr Met 145 150 155 160 Asn Gly Ala Val Leu Pro Ile Leu Ala Phe
Tyr Ile Val Thr Ala Glu 165 170 175 Glu Gln Gly Val Lys Lys Glu Lys
Leu Ala Gly Thr Ile Gln Asn Asp 180 185 190 Ile Leu Lys Glu Tyr Met
Val Arg Asn Thr Tyr Ile Tyr Pro Pro Glu 195 200 205 Met Ser Met Arg
Ile Ile Ala Asp Ile Phe Lys Tyr Thr Ala Glu Tyr 210 215 220 Met Pro
Lys Phe Asn Ser Ile Ser Ile Ser Gly Tyr His Met Gln Glu 225 230 235
240 Ala Gly Ala Pro Ala Asp Leu Glu Leu Ala Tyr Thr Leu Ala Asp Gly
245 250 255 Leu Glu Tyr Val Arg Thr Gly Leu Lys Ala Gly Ile Thr Ile
Asp Ala 260 265 270 Phe Ala Pro Arg Leu Ser Phe Phe Trp Ala Ile Gly
Met Asn Tyr Phe 275 280 285 Met Glu Val Ala Lys Met Arg Ala Gly Arg
Leu Leu Trp Ala Lys Leu 290 295 300 Met Lys Gln Phe Glu Pro Asp Asn
Pro Lys Ser Leu Ala Leu Arg Thr 305 310 315 320 His Ser Gln Thr Ser
Gly Trp Ser Leu Thr Glu Gln Asp Pro Phe Asn 325 330 335 Asn Val Ile
Arg Thr Cys Val Glu Ala Leu Ala Ala Val Ser Gly His 340 345 350 Thr
Gln Ser Leu His Thr Asn Ala Leu Asp Glu Ala Ile Ala Leu Pro 355 360
365 Thr Asp Phe Ser Ala Arg Ile Ala Arg Asn Thr Gln Leu Tyr Leu Gln
370 375 380 Asn Glu Thr Glu Ile Cys Ser Val Ile Asp Pro Trp Gly Gly
Ser Tyr 385 390 395 400 Tyr Val Glu Ser Leu Thr Asn Glu Leu Met Ile
Lys Ala Trp Lys His 405 410 415 Leu Glu Glu Ile Glu Gln Leu Gly Gly
Met Thr Lys Ala Ile Glu Ala 420 425 430 Gly Val Pro Lys Met Lys Ile
Glu Glu Ala Ala Ala Arg Arg Gln Ala 435 440 445 Arg Ile Asp Ser Gln
Ala Glu Ile Ile Val Gly Val Asn Gln Phe Gln 450 455 460 Pro Glu Gln
Glu Glu Pro Leu Asp Ile Leu Asp Ile Asp Asn Thr Ala 465 470 475 480
Val Arg Met Lys Gln Leu Glu Lys Leu Lys Lys Ile Arg Ser Glu Arg 485
490 495 Asn Glu Gln Ala Val Ile Glu Ala Leu Asn Arg Leu Thr Asn Cys
Ala 500 505 510 Lys Thr Gly Glu Gly Asn Leu Leu Ala Phe Ala Val Glu
Ala Ala Arg 515 520 525 Ala Arg Ala Thr Leu Gly Glu Ile Ser Glu Ala
Ile Glu Lys Val Ala 530 535 540 Gly Arg His Gln Ala Thr Ser Lys Ser
Val Ser Gly Val Tyr Ser Ala 545 550 555 560 Glu Phe Val His Arg Asp
Gln Ile Glu Glu Val Arg Lys Leu Thr Ala 565 570 575 Glu Phe Leu Glu
Gly Glu Gly Arg Arg Pro Arg Ile Leu Val Ala Lys 580 585 590 Met Gly
Gln Asp Gly His Asp Arg Gly Ser Lys Val Ile Ser Thr Ala 595 600 605
Phe Ala Asp Leu Gly Phe Asp Val Asp Ile Gly Pro Leu Phe Gln Thr 610
615 620 Pro Gln Glu Thr Ala Arg Gln Ala Val Glu Asn Asp Val His Val
Ile 625 630 635 640 Gly Ile Ser Ser Leu Ala Ala Gly His Lys Thr Leu
Leu Pro Gln Leu 645 650 655 Val Asp Glu Leu Lys Lys Leu Glu Arg Asp
Asp Ile Val Val Ile Val 660 665 670 Gly Gly Val Ile Pro Lys Gln Asp
Tyr Ser Phe Leu Leu Glu His Gly 675 680 685 Ala Ser Ala Ile Phe Gly
Pro Gly Thr Val Ile Pro Lys Ala Ala Val 690 695 700 Ser Val Leu His
Glu Ile Lys Lys Arg Leu Glu Glu 705 710 715 57616PRTCorynebacterium
glutamicumsource/note="Methylmalonyl-CoA mutase beta (small)
subunit" 57Met Thr Asp Leu Thr Lys Thr Ala Val Pro Glu Glu Leu Ser
Glu Asn 1 5 10 15 Leu Glu Thr Trp Tyr Lys Ala Val Ala Gly Val Phe
Ala Arg Thr Gln 20 25 30 Lys Lys Asp Ile Gly Asp Ile Ala Val Asp
Val Trp Lys Lys Leu Ile 35 40 45 Val Thr Thr Pro Asp Gly Val Asp
Ile Asn Pro Leu Tyr Thr Arg Ala 50 55 60 Asp Glu Ser Gln Arg Lys
Phe Thr Glu Val Pro Gly Glu Phe Pro Phe 65 70 75 80 Thr Arg Gly Thr
Thr Val Asp Gly Glu Arg Val Gly Trp Gly Val Thr 85 90 95 Glu Thr
Phe Gly His Asp Ser Pro Lys Asn Ile Asn Ala Ala Val Leu 100 105 110
Asn Ala Leu Asn Ser Gly Thr Thr Thr Leu Gly Phe Glu Phe Ser Glu 115
120 125 Glu Phe Thr Ala Ala Asp Leu Lys Val Ala Leu Glu Gly Val Tyr
Leu 130 135 140 Asn Met Ala Pro Leu Leu Ile His Ala Gly Gly Ser Thr
Ser Glu Val 145 150 155 160 Ala Ala Ala Leu Tyr Thr Leu Ala Glu Glu
Ala Gly Thr Phe Phe Ala 165 170 175 Ala Leu Thr Leu Gly Ser Arg Pro
Leu Thr Ala Gln Val Asp Gly Ser 180 185 190 His Ser Asp Thr Ile Glu
Glu Ala Val Gln Leu Ala Val Asn Ala Ser 195 200 205 Lys Arg Ala Asn
Val Arg Ala Ile Leu Val Asp Gly Ser Ser Phe Ser 210 215 220 Asn Gln
Gly Ala Ser Asp Ala Gln Glu Ile Gly Leu Ser Ile Ala Ala 225 230 235
240 Gly Val Asp Tyr Val Arg Arg Leu Val Asp Ala Gly Leu Ser Thr Glu
245 250 255 Ala Ala Leu Lys Gln Val Ala Phe Arg Phe Ala Val Thr Asp
Glu Gln 260 265 270 Phe Ala Gln Ile Ser Lys Leu Arg Val Ala Arg Arg
Leu Trp Ala Arg 275 280 285 Val Cys Glu Val Leu Gly Phe Pro Glu Leu
Ala Val Ala Pro Gln His 290 295 300 Ala Val Thr Ala Arg Ala Met Phe
Ser Gln Arg Asp Pro Trp Val Asn 305 310 315 320 Met Leu Arg Ser Thr
Val Ala Ala Phe Ala Ala Gly Val Gly Gly Ala 325 330 335 Thr Asp Val
Glu Val Arg Thr Phe Asp Asp Ala Ile Pro Asp Gly Val 340 345 350 Pro
Gly Val Ser Arg Asn Phe Ala His Arg Ile Ala Arg Asn Thr Asn 355 360
365 Leu Leu Leu Leu Glu Glu Ser His Leu Gly His Val Val Asp Pro Ala
370 375 380 Gly Gly Ser Tyr Phe Val Glu Ser Phe Thr Asp Asp Leu Ala
Glu Lys 385 390 395 400 Ala Trp Ala Val Phe Ser Gly Ile Glu Ala Glu
Gly Gly Tyr Ser Ala 405 410 415 Ala Cys Ala Ser Gly Thr Val Thr Ala
Met Leu Asp Gln Thr Trp Glu 420 425 430 Gln Thr Arg Ala Asp Val Ala
Ser Arg Lys Lys Lys Leu Thr Gly Ile 435 440 445 Asn Glu Phe Pro Asn
Leu Ala Glu Ser Pro Leu Pro Ala Asp Arg Arg 450 455 460 Val Glu Pro
Ala Gly Val Arg Arg Trp Ala Ala Asp Phe Glu Ala Leu 465 470 475 480
Arg Asn Arg Ser Asp Ala Phe Leu Glu Lys Asn Gly Ala Arg Pro Gln 485
490 495 Ile Thr Met Ile Pro Leu Gly Pro Leu Ser Lys His Asn Ile Arg
Thr 500 505 510 Gly Phe Thr Ser Asn Leu Leu Ala Ser Gly Gly Ile Glu
Ala Ile Asn 515 520 525 Pro Gly Gln Leu Val Pro Gly Thr Asp Ala Phe
Ala Glu Ala Ala Gln 530 535 540 Ala Ala Gly Ile Val Val Val Cys Gly
Thr Asp Gln Glu Tyr Ala Glu 545 550 555 560 Thr Gly Glu Gly Ala Val
Glu Lys Leu Arg Glu Ala Gly Val Glu Arg 565 570 575 Ile Leu Leu Ala
Gly Ala Pro Lys Ser Phe Glu Gly Ser Ala His Ala 580 585 590 Pro Asp
Gly Tyr Leu Asn Met Thr Ile Asp Ala Ala Ala Thr Leu Ala 595 600 605
Asp Leu Leu Asp Ala Leu Gly Ala 610 615 58737PRTCorynebacterium
glutamicumsource/note="Methylmalonyl-CoA mutase alpha (large)
subunit" 58Met Thr Ser Ile Pro Asn Phe Ser Asp Ile Pro Leu Thr Ala
Glu Thr 1 5 10 15 Arg Ala Ser Glu Ser His Asn Val Asp Ala Gly Lys
Val Trp Asn Thr 20 25 30 Pro Glu Gly Ile Asp Val Lys Arg Val Phe
Thr Gln Ala Asp Arg Asp 35 40 45 Glu Ala Gln Ala Ala Gly His Pro
Val Asp Ser Leu Pro Gly Gln Lys 50 55 60 Pro Phe Met Arg Gly Pro
Tyr Pro Thr Met Tyr Thr Asn Gln Pro Trp 65 70 75 80 Thr Ile Arg Gln
Tyr Ala Gly Phe Ser Thr Ala Ala Glu Ser Asn Ala 85 90 95 Phe Tyr
Arg Arg Asn Leu Ala Ala Gly Gln Lys Gly Leu Ser Val Ala 100 105 110
Phe Asp Leu Ala Thr His Arg Gly Tyr Asp Ser Asp Asn Glu Arg Val 115
120 125 Val Gly Asp Val Gly Met Ala Gly Val Ala Ile Asp Ser Ile Leu
Asp 130 135 140 Met Arg Gln Leu Phe Asp Gly Ile Asp Leu Ser Ser Val
Ser Val Ser 145 150 155 160 Met Thr Met Asn Gly Ala Val Leu Pro Ile
Leu Ala Phe Tyr Ile Val 165 170 175 Ala Ala Glu Glu Gln Gly Val Gly
Pro Glu Gln Leu Ala Gly Thr Ile 180 185 190 Gln Asn Asp Ile Leu Lys
Glu Phe Met Val Arg Asn Thr Tyr Ile Tyr 195 200 205 Pro Pro Lys Pro
Ser Met Arg Ile Ile Ser Asn Ile Phe Glu Tyr Thr 210 215 220 Ser Leu
Lys Met Pro Arg Phe Asn Ser Ile Ser Ile Ser Gly Tyr His 225 230 235
240 Ile Gln Glu Ala Gly Ala Thr Ala Asp Leu Glu Leu Ala Tyr Thr Leu
245 250 255 Ala Asp Gly Ile Glu Tyr Ile Arg Ala Gly Lys Glu Val Gly
Leu Asp 260 265 270 Val Asp Lys Phe Ala Pro Arg Leu Ser Phe Phe Trp
Gly Ile Ser Met 275 280 285 Tyr Thr Phe Met Glu Ile Ala Lys Leu Arg
Ala Gly Arg Leu Leu Trp 290 295 300 Ser Glu Leu Val Ala Lys Phe Asp
Pro Lys Asn Ala Lys Ser Gln Ser 305 310 315 320 Leu Arg Thr His Ser
Gln Thr Ser Gly Trp Ser Leu Thr Ala Gln Asp 325 330 335 Val Tyr
Asn
Asn Val Ala Arg Thr Ala Ile Glu Ala Met Ala Ala Thr 340 345 350 Gln
Gly His Thr Gln Ser Leu His Thr Asn Ala Leu Asp Glu Ala Leu 355 360
365 Ala Leu Pro Thr Asp Phe Ser Ala Arg Ile Ala Arg Asn Thr Gln Leu
370 375 380 Leu Leu Gln Gln Glu Ser Gly Thr Val Arg Pro Val Asp Pro
Trp Ala 385 390 395 400 Gly Ser Tyr Tyr Val Glu Trp Leu Thr Asn Glu
Leu Ala Asn Arg Ala 405 410 415 Arg Lys His Ile Asp Glu Val Glu Glu
Ala Gly Gly Met Ala Gln Ala 420 425 430 Thr Ala Gln Gly Ile Pro Lys
Leu Arg Ile Glu Glu Ser Ala Ala Arg 435 440 445 Thr Gln Ala Arg Ile
Asp Ser Gly Arg Gln Ala Leu Ile Gly Val Asn 450 455 460 Arg Tyr Val
Ala Glu Glu Asp Glu Glu Ile Glu Val Leu Lys Val Asp 465 470 475 480
Asn Thr Lys Val Arg Ala Glu Gln Leu Ala Lys Leu Ala Gln Leu Lys 485
490 495 Ala Glu Arg Asn Asp Ala Glu Val Lys Ala Ala Leu Asp Ala Leu
Thr 500 505 510 Ala Ala Ala Arg Asn Glu His Lys Glu Pro Gly Asp Leu
Asp Gln Asn 515 520 525 Leu Leu Lys Leu Ala Val Asp Ala Ala Arg Ala
Lys Ala Thr Ile Gly 530 535 540 Glu Ile Ser Asp Ala Leu Glu Val Val
Phe Gly Arg His Glu Ala Glu 545 550 555 560 Ile Arg Thr Leu Ser Gly
Val Tyr Lys Asp Glu Val Gly Lys Glu Gly 565 570 575 Thr Val Ser Asn
Val Glu Arg Ala Ile Ala Leu Ala Asp Ala Phe Glu 580 585 590 Ala Glu
Glu Gly Arg Arg Pro Arg Ile Phe Ile Ala Lys Met Gly Gln 595 600 605
Asp Gly His Asp Arg Gly Gln Lys Val Val Ala Ser Ala Tyr Ala Asp 610
615 620 Leu Gly Met Asp Val Asp Val Gly Pro Leu Phe Gln Thr Pro Ala
Glu 625 630 635 640 Ala Ala Arg Ala Ala Val Asp Ala Asp Val His Val
Val Gly Met Ser 645 650 655 Ser Leu Ala Ala Gly His Leu Thr Leu Leu
Pro Glu Leu Lys Lys Glu 660 665 670 Leu Ala Ala Leu Gly Arg Asp Asp
Ile Leu Val Thr Val Gly Gly Val 675 680 685 Ile Pro Pro Gly Asp Phe
Gln Asp Leu Tyr Asp Met Gly Ala Ala Ala 690 695 700 Ile Tyr Pro Pro
Gly Thr Val Ile Ala Glu Ser Ala Ile Asp Leu Ile 705 710 715 720 Thr
Arg Leu Ala Ala His Leu Gly Phe Asp Leu Asp Val Asp Val Asn 725 730
735 Glu 59261PRTEscherichia colisource/note="Methylmalonyl-CoA
decarboxylase" 59Met Ser Tyr Gln Tyr Val Asn Val Val Thr Ile Asn
Lys Val Ala Val 1 5 10 15 Ile Glu Phe Asn Tyr Gly Arg Lys Leu Asn
Ala Leu Ser Lys Val Phe 20 25 30 Ile Asp Asp Leu Met Gln Ala Leu
Ser Asp Leu Asn Arg Pro Glu Ile 35 40 45 Arg Cys Ile Ile Leu Arg
Ala Pro Ser Gly Ser Lys Val Phe Ser Ala 50 55 60 Gly His Asp Ile
His Glu Leu Pro Ser Gly Gly Arg Asp Pro Leu Ser 65 70 75 80 Tyr Asp
Asp Pro Leu Arg Gln Ile Thr Arg Met Ile Gln Lys Phe Pro 85 90 95
Lys Pro Ile Ile Ser Met Val Glu Gly Ser Val Trp Gly Gly Ala Phe 100
105 110 Glu Met Ile Met Ser Ser Asp Leu Ile Ile Ala Ala Ser Thr Ser
Thr 115 120 125 Phe Ser Met Thr Pro Val Asn Leu Gly Val Pro Tyr Asn
Leu Val Gly 130 135 140 Ile His Asn Leu Thr Arg Asp Ala Gly Phe His
Ile Val Lys Glu Leu 145 150 155 160 Ile Phe Thr Ala Ser Pro Ile Thr
Ala Gln Arg Ala Leu Ala Val Gly 165 170 175 Ile Leu Asn His Val Val
Glu Val Glu Glu Leu Glu Asp Phe Thr Leu 180 185 190 Gln Met Ala His
His Ile Ser Glu Lys Ala Pro Leu Ala Ile Ala Val 195 200 205 Ile Lys
Glu Glu Leu Arg Val Leu Gly Glu Ala His Thr Met Asn Ser 210 215 220
Asp Glu Phe Glu Arg Ile Gln Gly Met Arg Arg Ala Val Tyr Asp Ser 225
230 235 240 Glu Asp Tyr Gln Glu Gly Met Asn Ala Phe Leu Glu Lys Arg
Lys Pro 245 250 255 Asn Phe Val Gly His 260 60261PRTSalmonella
entericasource/note="Methylmalonyl-CoA decarboxylase" 60Met Ser Tyr
Gln Tyr Val Asn Val Ile Ile Ile Gln Lys Val Ala Val 1 5 10 15 Ile
Glu Phe Asn Tyr Ala Arg Lys Leu Asn Ala Leu Ser Lys Val Phe 20 25
30 Ile Asp Asp Leu Met Gln Ala Leu Ser Asp Leu Ser Arg Pro Glu Ile
35 40 45 Arg Cys Ile Ile Leu Arg Ala Pro Ser Gly Ala Lys Val Phe
Ser Ala 50 55 60 Gly His Asp Ile His Glu Leu Pro Ser Gly Arg Arg
Asp Pro Leu Ser 65 70 75 80 Tyr Asp Asp Pro Leu Arg Gln Ile Thr Arg
Leu Ile Gln Lys Tyr Pro 85 90 95 Lys Pro Val Ile Ser Met Val Glu
Gly Ser Val Trp Gly Gly Ala Phe 100 105 110 Glu Met Ile Met Ser Ser
Asp Leu Ile Ile Ala Ala Ser Thr Ser Thr 115 120 125 Phe Ser Met Thr
Pro Val Asn Leu Gly Val Pro Tyr Asn Leu Val Gly 130 135 140 Ile His
Asn Leu Thr Arg Asp Ala Gly Phe His Ile Val Lys Glu Leu 145 150 155
160 Ile Phe Thr Ala Ser Pro Ile Thr Ala Gln Arg Ala Leu Ala Val Gly
165 170 175 Ile Leu Asn His Val Val Glu Ala Asp Glu Leu Glu Asp Phe
Thr Leu 180 185 190 Gln Met Ala His His Ile Ser Glu Lys Ala Pro Leu
Ala Ile Ala Val 195 200 205 Ile Lys Glu Glu Leu Arg Val Leu Gly Glu
Ala His Thr Met Asn Ser 210 215 220 Asp Glu Phe Glu Arg Ile Gln Gly
Met Arg Arg Ala Val Tyr Asp Ser 225 230 235 240 Glu Asp Tyr Gln Glu
Gly Met Asn Ala Phe Leu Glu Lys Arg Lys Pro 245 250 255 His Phe Val
Gly His 260 61261PRTYersinia
enterocoliticasource/note="Methylmalonyl-CoA decarboxylase" 61Met
Ser Tyr Gln Tyr Val Lys Val Leu Ile Ala Asn Arg Val Gly Ile 1 5 10
15 Ile Glu Phe Asn His Ala Arg Lys Leu Asn Ala Leu Ser Lys Val Phe
20 25 30 Met Asp Asp Leu Met Leu Ala Leu His Asp Leu Asn Asn Thr
Asp Ile 35 40 45 Arg Cys Ile Ile Leu Arg Ala Ala Glu Gly Ser Lys
Val Phe Ser Ala 50 55 60 Gly His Asp Ile His Glu Leu Pro Thr Gly
Arg Arg Asp Pro Leu Ser 65 70 75 80 Tyr Asp Asp Pro Leu Arg Gln Ile
Thr Arg Ala Ile Gln Lys Tyr Pro 85 90 95 Lys Pro Ile Ile Ser Met
Val Glu Gly Ser Val Trp Gly Gly Ala Phe 100 105 110 Glu Met Ile Met
Ser Ser Asp Ile Ile Ile Ala Cys Arg Asn Ser Thr 115 120 125 Phe Ser
Met Thr Pro Val Asn Leu Gly Val Pro Tyr Asn Leu Val Gly 130 135 140
Ile His Asn Leu Ile Arg Asp Ala Gly Phe His Ile Val Lys Glu Leu 145
150 155 160 Ile Phe Thr Ala Ala Pro Ile Thr Ala Glu Arg Ala Leu Ser
Val Gly 165 170 175 Ile Leu Asn His Val Val Glu Pro Ser Glu Leu Glu
Asp Phe Thr Leu 180 185 190 Lys Leu Ala His Val Ile Ser Glu Lys Ala
Pro Leu Ala Ile Ala Val 195 200 205 Ile Lys Glu Glu Leu Arg Val Leu
Gly Glu Ala His Thr Met Asn Ser 210 215 220 Asp Glu Phe Glu Arg Ile
Gln Gly Met Arg Arg Ala Val Tyr Asp Ser 225 230 235 240 Asn Asp Tyr
Gln Glu Gly Met Ser Ala Phe Met Glu Lys Arg Lys Pro 245 250 255 Asn
Phe Leu Gly Arg 260 62611PRTPropionibacterium
freudenreichiisource/note="Methylmalonyl-CoA carboxyl transferase"
62Met Ala Glu Asn Asn Asn Leu Lys Leu Ala Ser Thr Met Glu Gly Arg 1
5 10 15 Val Glu Gln Leu Ala Glu Gln Arg Gln Val Ile Glu Ala Gly Gly
Gly 20 25 30 Glu Arg Arg Val Glu Lys Gln His Ser Gln Gly Lys Gln
Thr Ala Arg 35 40 45 Glu Arg Leu Asn Asn Leu Leu Asp Pro His Ser
Phe Asp Glu Val Gly 50 55 60 Ala Phe Arg Lys His Arg Thr Thr Leu
Phe Gly Met Asp Lys Ala Val 65 70 75 80 Val Pro Ala Asp Gly Val Val
Thr Gly Arg Gly Thr Ile Leu Gly Arg 85 90 95 Pro Val His Ala Ala
Ser Gln Asp Phe Thr Val Met Gly Gly Ser Ala 100 105 110 Gly Glu Thr
Gln Ser Thr Lys Val Val Glu Thr Met Glu Gln Ala Leu 115 120 125 Leu
Thr Gly Thr Pro Phe Leu Phe Phe Tyr Asp Ser Gly Gly Ala Arg 130 135
140 Ile Gln Glu Gly Ile Asp Ser Leu Ser Gly Tyr Gly Lys Met Phe Phe
145 150 155 160 Ala Asn Val Lys Leu Ser Gly Val Val Pro Gln Ile Ala
Ile Ile Ala 165 170 175 Gly Pro Cys Ala Gly Gly Ala Ser Tyr Ser Pro
Ala Leu Thr Asp Phe 180 185 190 Ile Ile Met Thr Lys Lys Ala His Met
Phe Ile Thr Gly Pro Gln Val 195 200 205 Ile Lys Ser Val Thr Gly Glu
Asp Val Thr Ala Asp Glu Leu Gly Gly 210 215 220 Ala Glu Ala His Met
Ala Ile Ser Gly Asn Ile His Phe Val Ala Glu 225 230 235 240 Asp Asp
Asp Ala Ala Glu Leu Ile Ala Lys Lys Leu Leu Ser Phe Leu 245 250 255
Pro Gln Asn Asn Thr Glu Glu Ala Ser Phe Val Asn Pro Asn Asn Asp 260
265 270 Val Ser Pro Asn Thr Glu Leu Arg Asp Ile Val Pro Ile Asp Gly
Lys 275 280 285 Lys Gly Tyr Asp Val Arg Asp Val Ile Ala Lys Ile Val
Asp Trp Gly 290 295 300 Asp Tyr Leu Glu Val Lys Ala Gly Tyr Ala Thr
Asn Leu Val Thr Ala 305 310 315 320 Phe Ala Arg Val Asn Gly Arg Ser
Val Gly Ile Val Ala Asn Gln Pro 325 330 335 Ser Val Met Ser Gly Cys
Leu Asp Ile Asn Ala Ser Asp Lys Ala Ala 340 345 350 Glu Phe Val Asn
Phe Cys Asp Ser Phe Asn Ile Pro Leu Val Gln Leu 355 360 365 Val Asp
Val Pro Gly Phe Leu Pro Gly Val Gln Gln Glu Tyr Gly Gly 370 375 380
Ile Ile Arg His Gly Ala Lys Met Leu Tyr Ala Tyr Ser Glu Ala Thr 385
390 395 400 Val Pro Lys Ile Thr Val Val Leu Arg Lys Ala Tyr Gly Gly
Ser Tyr 405 410 415 Leu Ala Met Cys Asn Arg Asp Leu Gly Ala Asp Ala
Val Tyr Ala Trp 420 425 430 Pro Ser Ala Glu Ile Ala Val Met Gly Ala
Glu Gly Ala Ala Asn Val 435 440 445 Ile Phe Arg Lys Glu Ile Lys Ala
Ala Asp Asp Pro Asp Ala Met Arg 450 455 460 Ala Glu Lys Ile Glu Glu
Tyr Gln Asn Ala Phe Asn Thr Pro Tyr Val 465 470 475 480 Ala Ala Ala
Arg Gly Gln Val Asp Asp Val Ile Asp Pro Ala Asp Thr 485 490 495 Arg
Arg Lys Ile Ala Ser Ala Leu Glu Met Tyr Ala Thr Lys Arg Gln 500 505
510 Thr Arg Pro Ala Lys Lys Pro Trp Lys Leu Pro Leu Leu Ser Glu Glu
515 520 525 Glu Ile Met Ala Asp Glu Glu Glu Lys Asp Leu Met Ile Ala
Thr Leu 530 535 540 Asn Lys Arg Val Ala Ser Leu Glu Ser Glu Leu Gly
Ser Leu Gln Ser 545 550 555 560 Asp Thr Gln Gly Val Thr Glu Asp Val
Leu Thr Ala Ile Ser Ala Val 565 570 575 Ala Ala Tyr Leu Gly Asn Asp
Gly Ser Ala Glu Val Val His Phe Ala 580 585 590 Pro Ser Pro Asn Trp
Val Arg Glu Gly Arg Arg Ala Leu Gln Asn His 595 600 605 Ser Ile Arg
610 63148PRTPropionibacterium
freundenreichiisource/note="Methylmalonyl-CoA epimerase" 63Met Ser
Asn Glu Asp Leu Phe Ile Cys Ile Asp His Val Ala Tyr Ala 1 5 10 15
Cys Pro Asp Ala Asp Glu Ala Ser Lys Tyr Tyr Gln Glu Thr Phe Gly 20
25 30 Trp His Glu Leu His Arg Glu Glu Asn Pro Glu Gln Gly Val Val
Glu 35 40 45 Ile Met Met Ala Pro Ala Ala Lys Leu Thr Glu His Met
Thr Gln Val 50 55 60 Gln Val Met Ala Pro Leu Asn Asp Glu Ser Thr
Val Ala Lys Trp Leu 65 70 75 80 Ala Lys His Asn Gly Arg Ala Gly Leu
His His Met Ala Trp Arg Val 85 90 95 Asp Asp Ile Asp Ala Val Ser
Ala Thr Leu Arg Glu Arg Gly Val Gln 100 105 110 Leu Leu Tyr Asp Glu
Pro Lys Leu Gly Thr Gly Gly Asn Arg Ile Asn 115 120 125 Phe Met His
Pro Lys Ser Gly Lys Gly Val Leu Ile Glu Leu Thr Gln 130 135 140 Tyr
Pro Lys Asn 145 64208PRTEscherichia colisource/note="Thioesterase
(TesA)" 64Met Met Asn Phe Asn Asn Val Phe Arg Trp His Leu Pro Phe
Leu Phe 1 5 10 15 Leu Val Leu Leu Thr Phe Arg Ala Ala Ala Ala Asp
Thr Leu Leu Ile 20 25 30 Leu Gly Asp Ser Leu Ser Ala Gly Tyr Arg
Met Ser Ala Ser Ala Ala 35 40 45 Trp Pro Ala Leu Leu Asn Asp Lys
Trp Gln Ser Lys Thr Ser Val Val 50 55 60 Asn Ala Ser Ile Ser Gly
Asp Thr Ser Gln Gln Gly Leu Ala Arg Leu 65 70 75 80 Pro Ala Leu Leu
Lys Gln His Gln Pro Arg Trp Val Leu Val Glu Leu 85 90 95 Gly Gly
Asn Asp Gly Leu Arg Gly Phe Gln Pro Gln Gln Thr Glu Gln 100 105 110
Thr Leu Arg Gln Ile Leu Gln Asp Val Lys Ala Ala Asn Ala Glu Pro 115
120 125 Leu Leu Met Gln Ile Arg Leu Pro Ala Asn Tyr Gly Arg Arg Tyr
Asn 130 135 140 Glu Ala Phe Ser Ala Ile Tyr Pro Lys Leu Ala Lys Glu
Phe Asp Val 145 150 155 160 Pro Leu Leu Pro Phe Phe Met Glu Glu Val
Tyr Leu Lys Pro Gln Trp 165 170 175 Met Gln Asp Asp Gly Ile His Pro
Asn Arg Asp Ala Gln Pro Phe Ile 180 185 190 Ala Asp Trp Met Ala Lys
Gln Leu Gln Pro Leu Val Asn His Asp Ser 195 200 205
65183PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic Escherichia coli Thioesterase (TesA) mutant
polypeptide" 65Met Ala Asp Thr Leu Leu Ile Leu Gly Asp Ser Leu Ser
Ala Gly Tyr 1 5 10 15 Arg Met Ser Ala Ser Ala Ala Trp Pro Ala Leu
Leu Asn Asp Lys Trp 20 25 30 Gln Ser Lys Thr Ser Val Val Asn Ala
Ser Ile Ser Gly Asp Thr Ser 35 40 45 Gln Gln Gly Leu Ala Arg Leu
Pro Ala Leu Leu Lys Gln His Gln Pro 50 55 60 Arg Trp Val Leu
Val Glu Leu Gly Gly Asn Asp Gly Leu Arg Gly Phe 65 70 75 80 Gln Pro
Gln Gln Thr Glu Gln Thr Leu Arg Gln Ile Leu Gln Asp Val 85 90 95
Lys Ala Ala Asn Ala Glu Pro Leu Leu Met Gln Ile Arg Leu Pro Ala 100
105 110 Asn Tyr Gly Arg Arg Tyr Asn Glu Ala Phe Ser Ala Ile Tyr Pro
Lys 115 120 125 Leu Ala Lys Glu Phe Asp Val Pro Leu Leu Pro Phe Phe
Met Glu Glu 130 135 140 Val Tyr Leu Lys Pro Gln Trp Met Gln Asp Asp
Gly Ile His Pro Asn 145 150 155 160 Arg Asp Ala Gln Pro Phe Ile Ala
Asp Trp Met Ala Lys Gln Leu Gln 165 170 175 Pro Leu Val Asn His Asp
Ser 180 66286PRTEscherichia colisource/note="Thioesterase (TesB)"
66Met Ser Gln Ala Leu Lys Asn Leu Leu Thr Leu Leu Asn Leu Glu Lys 1
5 10 15 Ile Glu Glu Gly Leu Phe Arg Gly Gln Ser Glu Asp Leu Gly Leu
Arg 20 25 30 Gln Val Phe Gly Gly Gln Val Val Gly Gln Ala Leu Tyr
Ala Ala Lys 35 40 45 Glu Thr Val Pro Glu Glu Arg Leu Val His Ser
Phe His Ser Tyr Phe 50 55 60 Leu Arg Pro Gly Asp Ser Lys Lys Pro
Ile Ile Tyr Asp Val Glu Thr 65 70 75 80 Leu Arg Asp Gly Asn Ser Phe
Ser Ala Arg Arg Val Ala Ala Ile Gln 85 90 95 Asn Gly Lys Pro Ile
Phe Tyr Met Thr Ala Ser Phe Gln Ala Pro Glu 100 105 110 Ala Gly Phe
Glu His Gln Lys Thr Met Pro Ser Ala Pro Ala Pro Asp 115 120 125 Gly
Leu Pro Ser Glu Thr Gln Ile Ala Gln Ser Leu Ala His Leu Leu 130 135
140 Pro Pro Val Leu Lys Asp Lys Phe Ile Cys Asp Arg Pro Leu Glu Val
145 150 155 160 Arg Pro Val Glu Phe His Asn Pro Leu Lys Gly His Val
Ala Glu Pro 165 170 175 His Arg Gln Val Trp Ile Arg Ala Asn Gly Ser
Val Pro Asp Asp Leu 180 185 190 Arg Val His Gln Tyr Leu Leu Gly Tyr
Ala Ser Asp Leu Asn Phe Leu 195 200 205 Pro Val Ala Leu Gln Pro His
Gly Ile Gly Phe Leu Glu Pro Gly Ile 210 215 220 Gln Ile Ala Thr Ile
Asp His Ser Met Trp Phe His Arg Pro Phe Asn 225 230 235 240 Leu Asn
Glu Trp Leu Leu Tyr Ser Val Glu Ser Thr Ser Ala Ser Ser 245 250 255
Ala Arg Gly Phe Val Arg Gly Glu Phe Tyr Thr Gln Asp Gly Val Leu 260
265 270 Val Ala Ser Thr Val Gln Glu Gly Val Met Arg Asn His Asn 275
280 285 67362PRTArabidopsis thalianasource/note="Thioesterase
(FatA)" 67Met Leu Lys Leu Ser Cys Asn Val Thr Asp Ser Lys Leu Gln
Arg Ser 1 5 10 15 Leu Leu Phe Phe Ser His Ser Tyr Arg Ser Asp Pro
Val Asn Phe Ile 20 25 30 Arg Arg Arg Ile Val Ser Cys Ser Gln Thr
Lys Lys Thr Gly Leu Val 35 40 45 Pro Leu Arg Ala Val Val Ser Ala
Asp Gln Gly Ser Val Val Gln Gly 50 55 60 Leu Ala Thr Leu Ala Asp
Gln Leu Arg Leu Gly Ser Leu Thr Glu Asp 65 70 75 80 Gly Leu Ser Tyr
Lys Glu Lys Phe Val Val Arg Ser Tyr Glu Val Gly 85 90 95 Ser Asn
Lys Thr Ala Thr Val Glu Thr Ile Ala Asn Leu Leu Gln Glu 100 105 110
Val Gly Cys Asn His Ala Gln Ser Val Gly Phe Ser Thr Asp Gly Phe 115
120 125 Ala Thr Thr Thr Thr Met Arg Lys Leu His Leu Ile Trp Val Thr
Ala 130 135 140 Arg Met His Ile Glu Ile Tyr Lys Tyr Pro Ala Trp Gly
Asp Val Val 145 150 155 160 Glu Ile Glu Thr Trp Cys Gln Ser Glu Gly
Arg Ile Gly Thr Arg Arg 165 170 175 Asp Trp Ile Leu Lys Asp Ser Val
Thr Gly Glu Val Thr Gly Arg Ala 180 185 190 Thr Ser Lys Trp Val Met
Met Asn Gln Asp Thr Arg Arg Leu Gln Lys 195 200 205 Val Ser Asp Asp
Val Arg Asp Glu Tyr Leu Val Phe Cys Pro Gln Glu 210 215 220 Pro Arg
Leu Ala Phe Pro Glu Glu Asn Asn Arg Ser Leu Lys Lys Ile 225 230 235
240 Pro Lys Leu Glu Asp Pro Ala Gln Tyr Ser Met Ile Gly Leu Lys Pro
245 250 255 Arg Arg Ala Asp Leu Asp Met Asn Gln His Val Asn Asn Val
Thr Tyr 260 265 270 Ile Gly Trp Val Leu Glu Ser Ile Pro Gln Glu Ile
Val Asp Thr His 275 280 285 Glu Leu Gln Val Ile Thr Leu Asp Tyr Arg
Arg Glu Cys Gln Gln Asp 290 295 300 Asp Val Val Asp Ser Leu Thr Thr
Thr Thr Ser Glu Ile Gly Gly Thr 305 310 315 320 Asn Gly Ser Ala Thr
Ser Gly Thr Gln Gly His Asn Asp Ser Gln Phe 325 330 335 Leu His Leu
Leu Arg Leu Ser Gly Asp Gly Gln Glu Ile Asn Arg Gly 340 345 350 Thr
Thr Leu Trp Arg Lys Lys Pro Ser Ser 355 360 68412PRTArabidopsis
thalianasource/note="Thioesterase (FatB)" 68Met Val Ala Thr Ser Ala
Thr Ser Ser Phe Phe Pro Val Pro Ser Ser 1 5 10 15 Ser Leu Asp Pro
Asn Gly Lys Gly Asn Lys Ile Gly Ser Thr Asn Leu 20 25 30 Ala Gly
Leu Asn Ser Ala Pro Asn Ser Gly Arg Met Lys Val Lys Pro 35 40 45
Asn Ala Gln Ala Pro Pro Lys Ile Asn Gly Lys Lys Val Gly Leu Pro 50
55 60 Gly Ser Val Asp Ile Val Arg Thr Asp Thr Glu Thr Ser Ser His
Pro 65 70 75 80 Ala Pro Arg Thr Phe Ile Asn Gln Leu Pro Asp Trp Ser
Met Leu Leu 85 90 95 Ala Ala Ile Thr Thr Ile Phe Leu Ala Ala Glu
Lys Gln Trp Met Met 100 105 110 Leu Asp Trp Lys Pro Arg Arg Ser Asp
Met Leu Val Asp Pro Phe Gly 115 120 125 Ile Gly Arg Ile Val Gln Asp
Gly Leu Val Phe Arg Gln Asn Phe Ser 130 135 140 Ile Arg Ser Tyr Glu
Ile Gly Ala Asp Arg Ser Ala Ser Ile Glu Thr 145 150 155 160 Val Met
Asn His Leu Gln Glu Thr Ala Leu Asn His Val Lys Thr Ala 165 170 175
Gly Leu Leu Gly Asp Gly Phe Gly Ser Thr Pro Glu Met Phe Lys Lys 180
185 190 Asn Leu Ile Trp Val Val Thr Arg Met Gln Val Val Val Asp Lys
Tyr 195 200 205 Pro Thr Trp Gly Asp Val Val Glu Val Asp Thr Trp Val
Ser Gln Ser 210 215 220 Gly Lys Asn Gly Met Arg Arg Asp Trp Leu Val
Arg Asp Cys Asn Thr 225 230 235 240 Gly Glu Thr Leu Thr Arg Ala Ser
Ser Val Trp Val Met Met Asn Lys 245 250 255 Leu Thr Arg Arg Leu Ser
Lys Ile Pro Glu Glu Val Arg Gly Glu Ile 260 265 270 Glu Pro Tyr Phe
Val Asn Ser Asp Pro Val Leu Ala Glu Asp Ser Arg 275 280 285 Lys Leu
Thr Lys Ile Asp Asp Lys Thr Ala Asp Tyr Val Arg Ser Gly 290 295 300
Leu Thr Pro Arg Trp Ser Asp Leu Asp Val Asn Gln His Val Asn Asn 305
310 315 320 Val Lys Tyr Ile Gly Trp Ile Leu Glu Ser Ala Pro Val Gly
Ile Met 325 330 335 Glu Arg Gln Lys Leu Lys Ser Met Thr Leu Glu Tyr
Arg Arg Glu Cys 340 345 350 Gly Arg Asp Ser Val Leu Gln Ser Leu Thr
Ala Val Thr Gly Cys Asp 355 360 365 Ile Gly Asn Leu Ala Thr Ala Gly
Asp Val Glu Cys Gln His Leu Leu 370 375 380 Arg Leu Gln Asp Gly Ala
Glu Val Val Arg Gly Arg Thr Glu Trp Ser 385 390 395 400 Ser Lys Thr
Pro Thr Thr Thr Trp Gly Thr Ala Pro 405 410 69382PRTUmbellularia
californicasource/note="Thioesterase (FatB)" 69Met Ala Thr Thr Ser
Leu Ala Ser Ala Phe Cys Ser Met Lys Ala Val 1 5 10 15 Met Leu Ala
Arg Asp Gly Arg Gly Met Lys Pro Arg Ser Ser Asp Leu 20 25 30 Gln
Leu Arg Ala Gly Asn Ala Pro Thr Ser Leu Lys Met Ile Asn Gly 35 40
45 Thr Lys Phe Ser Tyr Thr Glu Ser Leu Lys Arg Leu Pro Asp Trp Ser
50 55 60 Met Leu Phe Ala Val Ile Thr Thr Ile Phe Ser Ala Ala Glu
Lys Gln 65 70 75 80 Trp Thr Asn Leu Glu Trp Lys Pro Lys Pro Lys Leu
Pro Gln Leu Leu 85 90 95 Asp Asp His Phe Gly Leu His Gly Leu Val
Phe Arg Arg Thr Phe Ala 100 105 110 Ile Arg Ser Tyr Glu Val Gly Pro
Asp Arg Ser Thr Ser Ile Leu Ala 115 120 125 Val Met Asn His Met Gln
Glu Ala Thr Leu Asn His Ala Lys Ser Val 130 135 140 Gly Ile Leu Gly
Asp Gly Phe Gly Thr Thr Leu Glu Met Ser Lys Arg 145 150 155 160 Asp
Leu Met Trp Val Val Arg Arg Thr His Val Ala Val Glu Arg Tyr 165 170
175 Pro Thr Trp Gly Asp Thr Val Glu Val Glu Cys Trp Ile Gly Ala Ser
180 185 190 Gly Asn Asn Gly Met Arg Arg Asp Phe Leu Val Arg Asp Cys
Lys Thr 195 200 205 Gly Glu Ile Leu Thr Arg Cys Thr Ser Leu Ser Val
Leu Met Asn Thr 210 215 220 Arg Thr Arg Arg Leu Ser Thr Ile Pro Asp
Glu Val Arg Gly Glu Ile 225 230 235 240 Gly Pro Ala Phe Ile Asp Asn
Val Ala Val Lys Asp Asp Glu Ile Lys 245 250 255 Lys Leu Gln Lys Leu
Asn Asp Ser Thr Ala Asp Tyr Ile Gln Gly Gly 260 265 270 Leu Thr Pro
Arg Trp Asn Asp Leu Asp Val Asn Gln His Val Asn Asn 275 280 285 Leu
Lys Tyr Val Ala Trp Val Phe Glu Thr Val Pro Asp Ser Ile Phe 290 295
300 Glu Ser His His Ile Ser Ser Phe Thr Leu Glu Tyr Arg Arg Glu Cys
305 310 315 320 Thr Arg Asp Ser Val Leu Arg Ser Leu Thr Thr Val Ser
Gly Gly Ser 325 330 335 Ser Glu Ala Gly Leu Val Cys Asp His Leu Leu
Gln Leu Glu Gly Gly 340 345 350 Ser Glu Val Leu Arg Ala Arg Thr Glu
Trp Arg Pro Lys Leu Thr Asp 355 360 365 Ser Phe Arg Gly Ile Ser Val
Ile Pro Ala Glu Pro Arg Val 370 375 380 70376PRTCuphea
hookerianasource/note="Thioesterase (FatA1)" 70Met Leu Lys Leu Ser
Cys Asn Ala Ala Thr Asp Gln Ile Leu Ser Ser 1 5 10 15 Ala Val Ala
Gln Thr Ala Leu Trp Gly Gln Pro Arg Asn Arg Ser Phe 20 25 30 Ser
Met Ser Ala Arg Arg Arg Gly Ala Val Cys Cys Ala Pro Pro Ala 35 40
45 Ala Gly Lys Pro Pro Ala Met Thr Ala Val Ile Pro Lys Asp Gly Val
50 55 60 Ala Ser Ser Gly Ser Gly Ser Leu Ala Asp Gln Leu Arg Leu
Gly Ser 65 70 75 80 Arg Thr Gln Asn Gly Leu Ser Tyr Thr Glu Lys Phe
Ile Val Arg Cys 85 90 95 Tyr Glu Val Gly Ile Asn Lys Thr Ala Thr
Val Glu Thr Met Ala Asn 100 105 110 Leu Leu Gln Glu Val Gly Cys Asn
His Ala Gln Ser Val Gly Phe Ser 115 120 125 Thr Asp Gly Phe Ala Thr
Thr Pro Thr Met Arg Lys Leu Asn Leu Ile 130 135 140 Trp Val Thr Ala
Arg Met His Ile Glu Ile Tyr Lys Tyr Pro Ala Trp 145 150 155 160 Ser
Asp Val Val Glu Ile Glu Thr Trp Cys Gln Ser Glu Gly Arg Ile 165 170
175 Gly Thr Arg Arg Asp Trp Ile Leu Lys Asp Tyr Gly Asn Gly Glu Val
180 185 190 Ile Gly Arg Ala Thr Ser Lys Trp Val Met Met Asn Gln Asn
Thr Arg 195 200 205 Arg Leu Gln Lys Val Asp Asp Ser Val Arg Glu Glu
Tyr Met Val Phe 210 215 220 Cys Pro Arg Glu Pro Arg Leu Ser Phe Pro
Glu Glu Asn Asn Arg Ser 225 230 235 240 Leu Arg Lys Ile Ser Lys Leu
Glu Asp Pro Ala Glu Tyr Ser Arg Leu 245 250 255 Gly Leu Thr Pro Arg
Arg Ala Asp Leu Asp Met Asn Gln His Val Asn 260 265 270 Asn Val Ala
Tyr Ile Gly Trp Ala Leu Glu Ser Val Pro Gln Glu Ile 275 280 285 Ile
Asp Ser Tyr Glu Leu Glu Thr Ile Thr Leu Asp Tyr Arg Arg Glu 290 295
300 Cys Gln Gln Asp Asp Val Val Asp Ser Leu Thr Ser Val Leu Ser Asp
305 310 315 320 Glu Glu Ser Gly Thr Leu Pro Glu Leu Lys Gly Thr Asn
Gly Ser Ala 325 330 335 Ser Thr Pro Leu Lys Arg Asp His Asp Gly Ser
Arg Gln Phe Leu His 340 345 350 Leu Leu Arg Leu Ser Pro Asp Gly Leu
Glu Ile Asn Arg Gly Arg Thr 355 360 365 Glu Trp Arg Lys Lys Ser Thr
Lys 370 375 71415PRTCuphea hookerianasource/note="Thioesterase
(FatB2)" 71Met Val Ala Ala Ala Ala Ser Ser Ala Phe Phe Pro Val Pro
Ala Pro 1 5 10 15 Gly Ala Ser Pro Lys Pro Gly Lys Phe Gly Asn Trp
Pro Ser Ser Leu 20 25 30 Ser Pro Ser Phe Lys Pro Lys Ser Ile Pro
Asn Gly Gly Phe Gln Val 35 40 45 Lys Ala Asn Asp Ser Ala His Pro
Lys Ala Asn Gly Ser Ala Val Ser 50 55 60 Leu Lys Ser Gly Ser Leu
Asn Thr Gln Glu Asp Thr Ser Ser Ser Pro 65 70 75 80 Pro Pro Arg Thr
Phe Leu His Gln Leu Pro Asp Trp Ser Arg Leu Leu 85 90 95 Thr Ala
Ile Thr Thr Val Phe Val Lys Ser Lys Arg Pro Asp Met His 100 105 110
Asp Arg Lys Ser Lys Arg Pro Asp Met Leu Val Asp Ser Phe Gly Leu 115
120 125 Glu Ser Thr Val Gln Asp Gly Leu Val Phe Arg Gln Ser Phe Ser
Ile 130 135 140 Arg Ser Tyr Glu Ile Gly Thr Asp Arg Thr Ala Ser Ile
Glu Thr Leu 145 150 155 160 Met Asn His Leu Gln Glu Thr Ser Leu Asn
His Cys Lys Ser Thr Gly 165 170 175 Ile Leu Leu Asp Gly Phe Gly Arg
Thr Leu Glu Met Cys Lys Arg Asp 180 185 190 Leu Ile Trp Val Val Ile
Lys Met Gln Ile Lys Val Asn Arg Tyr Pro 195 200 205 Ala Trp Gly Asp
Thr Val Glu Ile Asn Thr Arg Phe Ser Arg Leu Gly 210 215 220 Lys Ile
Gly Met Gly Arg Asp Trp Leu Ile Ser Asp Cys Asn Thr Gly 225 230 235
240 Glu Ile Leu Val Arg Ala Thr Ser Ala Tyr Ala Met Met Asn Gln Lys
245 250 255 Thr Arg Arg Leu Ser Lys Leu Pro Tyr Glu Val His Gln Glu
Ile Val 260 265 270 Pro Leu Phe Val Asp Ser Pro Val Ile Glu Asp Ser
Asp Leu Lys Val 275 280 285 His Lys Phe Lys Val Lys Thr Gly Asp Ser
Ile Gln Lys Gly Leu Thr 290 295 300 Pro Gly Trp Asn Asp Leu Asp Val
Asn Gln His Val Ser Asn Val Lys 305
310 315 320 Tyr Ile Gly Trp Ile Leu Glu Ser Met Pro Thr Glu Val Leu
Glu Thr 325 330 335 Gln Glu Leu Cys Ser Leu Ala Leu Glu Tyr Arg Arg
Glu Cys Gly Arg 340 345 350 Asp Ser Val Leu Glu Ser Val Thr Ala Met
Asp Pro Ser Lys Val Gly 355 360 365 Val Arg Ser Gln Tyr Gln His Leu
Leu Arg Leu Glu Asp Gly Thr Ala 370 375 380 Ile Val Asn Gly Ala Thr
Glu Trp Arg Pro Lys Asn Ala Gly Ala Asn 385 390 395 400 Gly Ala Ile
Ser Thr Gly Lys Thr Ser Asn Gly Asn Ser Val Ser 405 410 415
72394PRTCuphea hookerianasource/note="thioesterase (FatB3)" 72Met
Val Ala Ala Ala Ala Ser Ser Ala Phe Phe Ser Val Pro Thr Pro 1 5 10
15 Gly Ile Ser Pro Lys Pro Gly Lys Phe Gly Asn Gly Gly Phe Gln Val
20 25 30 Lys Ala Asn Ala Asn Ala His Pro Ser Leu Lys Ser Gly Ser
Leu Glu 35 40 45 Thr Glu Asp Asp Thr Ser Ser Ser Ser Pro Pro Pro
Arg Thr Phe Ile 50 55 60 Asn Gln Leu Pro Asp Trp Ser Met Leu Leu
Ser Ala Ile Thr Thr Ile 65 70 75 80 Phe Gly Ala Ala Glu Lys Gln Trp
Met Met Leu Asp Arg Lys Ser Lys 85 90 95 Arg Pro Asp Met Leu Met
Glu Pro Phe Gly Val Asp Ser Ile Val Gln 100 105 110 Asp Gly Val Phe
Phe Arg Gln Ser Phe Ser Ile Arg Ser Tyr Glu Ile 115 120 125 Gly Ala
Asp Arg Thr Thr Ser Ile Glu Thr Leu Met Asn Met Phe Gln 130 135 140
Glu Thr Ser Leu Asn His Cys Lys Ser Asn Gly Leu Leu Asn Asp Gly 145
150 155 160 Phe Gly Arg Thr Pro Glu Met Cys Lys Lys Gly Leu Ile Trp
Val Val 165 170 175 Thr Lys Met Gln Val Glu Val Asn Arg Tyr Pro Ile
Trp Gly Asp Ser 180 185 190 Ile Glu Val Asn Thr Trp Val Ser Glu Ser
Gly Lys Asn Gly Met Gly 195 200 205 Arg Asp Trp Leu Ile Ser Asp Cys
Ser Thr Gly Glu Ile Leu Val Arg 210 215 220 Ala Thr Ser Val Trp Ala
Met Met Asn Gln Lys Thr Arg Arg Leu Ser 225 230 235 240 Lys Phe Pro
Phe Glu Val Arg Gln Glu Ile Ala Pro Asn Phe Val Asp 245 250 255 Ser
Val Pro Val Ile Glu Asp Asp Arg Lys Leu His Lys Leu Asp Val 260 265
270 Lys Thr Gly Asp Ser Ile His Asn Gly Leu Thr Pro Arg Trp Asn Asp
275 280 285 Leu Asp Val Asn Gln His Val Asn Asn Val Lys Tyr Ile Gly
Trp Ile 290 295 300 Leu Lys Ser Val Pro Thr Asp Val Phe Glu Ala Gln
Glu Leu Cys Gly 305 310 315 320 Val Thr Leu Glu Tyr Arg Arg Glu Cys
Gly Arg Asp Ser Val Met Glu 325 330 335 Ser Val Thr Ala Met Asp Pro
Ser Lys Glu Gly Asp Arg Ser Val Tyr 340 345 350 Gln His Leu Leu Arg
Leu Glu Asp Gly Ala Asp Ile Ala Ile Gly Arg 355 360 365 Thr Glu Trp
Arg Pro Lys Asn Ala Gly Ala Asn Gly Ala Ile Ser Thr 370 375 380 Gly
Lys Thr Ser Asn Arg Asn Ser Val Ser 385 390 734559DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
pDG2 plasmid polynucleotide" 73ggggaattgt gagcggataa caattcccct
gtagaaataa ttttgtttaa ctttaataag 60gagatatacc atggcgcaac tcactcttct
tttagtcggc aattccgacg ccatcacgcc 120attacttgct aaagctgact
ttgaacaacg ttcgcgtctg cagattattc ctgcgcagtc 180agttatcgcc
agtgatgccc ggccttcgca agctatccgc gccagtcgtg ggagttcaat
240gcgcgtggcc ctggagctgg tgaaagaagg tcgagcgcaa gcctgtgtca
gtgccggtaa 300taccggggcg ctgatggggc tggcaaaatt attactcaag
cccctggagg ggattgagcg 360tccggcgctg gtgacggtat taccacatca
gcaaaagggc aaaacggtgg tccttgactt 420aggggccaac gtcgattgtg
acagcacaat gctggtgcaa tttgccatta tgggctcagt 480tctggctgaa
gaggtggtgg aaattcccaa tcctcgcgtg gcgttgctca atattggtga
540agaagaagta aagggtctcg acagtattcg ggatgcctca gcggtgctta
aaacaatccc 600ttctatcaat tatatcggct atcttgaagc caatgagttg
ttaactggca agacagatgt 660gctggtttgt gacggcttta caggaaatgt
cacattaaag acgatggaag gtgttgtcag 720gatgttcctt tctctgctga
aatctcaggg tgaagggaaa aaacggtcgt ggtggctact 780gttattaaag
cgttggctac aaaagagcct gacgaggcga ttcagtcacc tcaaccccga
840ccagtataac ggcgcctgtc tgttaggatt gcgcggcacg gtgataaaaa
gtcatggtgc 900agccaatcag cgagcttttg cggtcgcgat tgaacaggca
gtgcaggcgg tgcagcgaca 960agttcctcag cgaattgccg ctcgcctgga
atctgtatac ccagctggtt ttgagctgct 1020ggacggtggc aaaagcggaa
ctctgcggta gcaggacgct gccagcgaac tcgcagtttg 1080caagtgacgg
tatataaccg aaaagtgact gagcgcatat gtatacgaag actcgagtct
1140ggtaaagaaa ccgctgctgc gaaatttgaa cgccagcaca tggactcgtc
tactagcgca 1200gcttaattaa cctaggctgc tgccaccgct gagcaataac
tagcataacc ccttggggcc 1260tctaaacggg tcttgagggg ttttttgctg
aaacctcagg catttgagaa gcacacggtc 1320acactgcttc cggtagtcaa
taaaccggta aaccagcaat agacataagc ggctatttaa 1380cgaccctgcc
ctgaaccgac gaccgggtca tcgtggccgg atcttgcggc ccctcggctt
1440gaacgaattg ttagacatta tttgccgact accttggtga tctcgccttt
cacgtagtgg 1500acaaattctt ccaactgatc tgcgcgcgag gccaagcgat
cttcttcttg tccaagataa 1560gcctgtctag cttcaagtat gacgggctga
tactgggccg gcaggcgctc cattgcccag 1620tcggcagcga catccttcgg
cgcgattttg ccggttactg cgctgtacca aatgcgggac 1680aacgtaagca
ctacatttcg ctcatcgcca gcccagtcgg gcggcgagtt ccatagcgtt
1740aaggtttcat ttagcgcctc aaatagatcc tgttcaggaa ccggatcaaa
gagttcctcc 1800gccgctggac ctaccaaggc aacgctatgt tctcttgctt
ttgtcagcaa gatagccaga 1860tcaatgtcga tcgtggctgg ctcgaagata
cctgcaagaa tgtcattgcg ctgccattct 1920ccaaattgca gttcgcgctt
agctggataa cgccacggaa tgatgtcgtc gtgcacaaca 1980atggtgactt
ctacagcgcg gagaatctcg ctctctccag gggaagccga agtttccaaa
2040aggtcgttga tcaaagctcg ccgcgttgtt tcatcaagcc ttacggtcac
cgtaaccagc 2100aaatcaatat cactgtgtgg cttcaggccg ccatccactg
cggagccgta caaatgtacg 2160gccagcaacg tcggttcgag atggcgctcg
atgacgccaa ctacctctga tagttgagtc 2220gatacttcgg cgatcaccgc
ttccctcata ctcttccttt ttcaatatta ttgaagcatt 2280tatcagggtt
attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
2340atagctagct cactcggtcg ctacgctccg ggcgtgagac tgcggcgggc
gctgcggaca 2400catacaaagt tacccacaga ttccgtggat aagcagggga
ctaacatgtg aggcaaaaca 2460gcagggccgc gccggtggcg tttttccata
ggctccgccc tcctgccaga gttcacataa 2520acagacgctt ttccggtgca
tctgtgggag ccgtgaggct caaccatgaa tctgacagta 2580cgggcgaaac
ccgacaggac ttaaagatcc ccaccgtttc cggcgggtcg ctccctcttg
2640cgctctcctg ttccgaccct gccgtttacc ggatacctgt tccgcctttc
tcccttacgg 2700gaagtgtggc gctttctcat agctcacaca ctggtatctc
ggctcggtgt aggtcgttcg 2760ctccaagctg ggctgtaagc aagaactccc
cgttcagccc gactgctgcg ccttatccgg 2820taactgttca cttgagtcca
acccggaaaa gcacggtaaa acgccactgg cagcagccat 2880tggtaactgg
gagttcgcag aggatttgtt tagctaaaca cgcggttgct cttgaagtgt
2940gcgccaaagt ccggctacac tggaaggaca gatttggttg ctgtgctctg
cgaaagccag 3000ttaccacggt taagcagttc cccaactgac ttaaccttcg
atcaaaccac ctccccaggt 3060ggttttttcg tttacagggc aaaagattac
gcgcagaaaa aaaggatctc aagaagatcc 3120tttgatcttt tctactgaac
cgctctagat ttcagtgcaa tttatctctt caaatgtagc 3180acctgaagtc
agccccatac gatataagtt gtaattctca tgttagtcat gccccgcgcc
3240caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
tcccggtgcc 3300taatgagtga gctaacttac attaattgcg ttgcgctcac
tgcccgcttt ccagtcggga 3360aacctgtcgt gccagctgca ttaatgaatc
ggccaacgcg cggggagagg cggtttgcgt 3420attgggcgcc agggtggttt
ttcttttcac cagtgagacg ggcaacagct gattgccctt 3480caccgcctgg
ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
3540aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
cggtatcgtc 3600gtatcccact accgagatgt ccgcaccaac gcgcagcccg
gactcggtaa tggcgcgcat 3660tgcgcccagc gccatctgat cgttggcaac
cagcatcgca gtgggaacga tgccctcatt 3720cagcatttgc atggtttgtt
gaaaaccgga catggcactc cagtcgcctt cccgttccgc 3780tatcggctga
atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
3840cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
atgcgaccag 3900atgctccacg cccagtcgcg taccgtcttc atgggagaaa
ataatactgt tgatgggtgt 3960ctggtcagag acatcaagaa ataacgccgg
aacattagtg caggcagctt ccacagcaat 4020ggcatcctgg tcatccagcg
gatagttaat gatcagccca ctgacgcgtt gcgcgagaag 4080attgtgcacc
gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
4140gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
acggcgcgtg 4200cagggccaga ctggaggtgg caacgccaat cagcaacgac
tgtttgcccg ccagttgttg 4260tgccacgcgg ttgggaatgt aattcagctc
cgccatcgcc gcttccactt tttcccgcgt 4320tttcgcagaa acgtggctgg
cctggttcac cacgcgggaa acggtctgat aagagacacc 4380ggcatactct
gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
4440ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
tggtgtccgg 4500gatctcgacg ctctccctta tgcgactcct gcattaggaa
attaatacga ctcactata 4559745502DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
pDG6 plasmid polynucleotide" 74ggggaattgt gagcggataa caattcccct
gtagaaataa ttttgtttaa ctttaataag 60gagatatacc atggcgcaac tcactcttct
tttagtcggc aattccgacg ccatcacgcc 120attacttgct aaagctgact
ttgaacaacg ttcgcgtctg cagattattc ctgcgcagtc 180agttatcgcc
agtgatgccc ggccttcgca agctatccgc gccagtcgtg ggagttcaat
240gcgcgtggcc ctggagctgg tgaaagaagg tcgagcgcaa gcctgtgtca
gtgccggtaa 300taccggggcg ctgatggggc tggcaaaatt attactcaag
cccctggagg ggattgagcg 360tccggcgctg gtgacggtat taccacatca
gcaaaagggc aaaacggtgg tccttgactt 420aggggccaac gtcgattgtg
acagcacaat gctggtgcaa tttgccatta tgggctcagt 480tctggctgaa
gaggtggtgg aaattcccaa tcctcgcgtg gcgttgctca atattggtga
540agaagaagta aagggtctcg acagtattcg ggatgcctca gcggtgctta
aaacaatccc 600ttctatcaat tatatcggct atcttgaagc caatgagttg
ttaactggca agacagatgt 660gctggtttgt gacggcttta caggaaatgt
cacattaaag acgatggaag gtgttgtcag 720gatgttcctt tctctgctga
aatctcaggg tgaagggaaa aaacggtcgt ggtggctact 780gttattaaag
cgttggctac aaaagagcct gacgaggcga ttcagtcacc tcaaccccga
840ccagtataac ggcgcctgtc tgttaggatt gcgcggcacg gtgataaaaa
gtcatggtgc 900agccaatcag cgagcttttg cggtcgcgat tgaacaggca
gtgcaggcgg tgcagcgaca 960agttcctcag cgaattgccg ctcgcctgga
atctgtatac ccagctggtt ttgagctgct 1020ggacggtggc aaaagcggaa
ctctgcggta gcaggacgct gccagcgaac tcgcagtttg 1080caagtgacgg
tatataaccg aaaagtgact gagcgcatat gaaagctggc attcttggtg
1140ttggacgtta cattcctgag aaggttttaa caaatcatga tcttgaaaaa
atggttgaaa 1200cttctgacga gtggattcgt acaagaacag gaatagaaga
aagaagaatc gcagcagatg 1260atgtgttttc atcacacatg gctgttgcag
cagcgaaaaa tgcgctggaa caagctgaag 1320tggctgctga ggatctggat
atgatcttgg ttgcaactgt tacacctgat cagtcattcc 1380ctacggtgtc
ttgtatgatt caagaacaac tcggcgcgaa gaaagcgtgt gctatggata
1440tcagcgcggc ttgtgcgggc ttcatgtacg gggttgtaac cggtaaacaa
tttattgaat 1500ccggaaccta caagcatgtt ctagttgttg gtgtagagaa
gctctcaagc attaccgact 1560gggaagaccg caatacagcc gttctgtttg
gagacggagc aggcgctgcg gtagtcgggc 1620cagtcagtga tgacagagga
atcctttcat ttgaactagg agccgacggc acaggcggtc 1680agcacttgta
tctgaatgaa aaacgacata caatcatgaa tggacgagaa gttttcaaat
1740ttgcagtccg ccaaatggga gaatcatgcg taaatgtcat tgaaaaagcc
ggactttcaa 1800aagaggatgt ggactttttg attccgcatc aggcgaacat
ccgtatcatg gaagctgctc 1860gcgagcgttt agagcttcct gtcgaaaaga
tgtctaaaac tgttcataaa tatggaaata 1920cttctgccgc atccattccg
atctctcttg tagaagaatt ggaagccggt aaaatcaaag 1980acggcgatgt
ggtcgttatg gtagggttcg gcggaggact aacatggggc gccattgcaa
2040tccgctgggg ccgataaaaa aaaggtgagg tgcactcgag tctggtaaag
aaaccgctgc 2100tgcgaaattt gaacgccagc acatggactc gtctactagc
gcagcttaat taacctaggc 2160tgctgccacc gctgagcaat aactagcata
accccttggg gcctctaaac gggtcttgag 2220gggttttttg ctgaaacctc
aggcatttga gaagcacacg gtcacactgc ttccggtagt 2280caataaaccg
gtaaaccagc aatagacata agcggctatt taacgaccct gccctgaacc
2340gacgaccggg tcatcgtggc cggatcttgc ggcccctcgg cttgaacgaa
ttgttagaca 2400ttatttgccg actaccttgg tgatctcgcc tttcacgtag
tggacaaatt cttccaactg 2460atctgcgcgc gaggccaagc gatcttcttc
ttgtccaaga taagcctgtc tagcttcaag 2520tatgacgggc tgatactggg
ccggcaggcg ctccattgcc cagtcggcag cgacatcctt 2580cggcgcgatt
ttgccggtta ctgcgctgta ccaaatgcgg gacaacgtaa gcactacatt
2640tcgctcatcg ccagcccagt cgggcggcga gttccatagc gttaaggttt
catttagcgc 2700ctcaaataga tcctgttcag gaaccggatc aaagagttcc
tccgccgctg gacctaccaa 2760ggcaacgcta tgttctcttg cttttgtcag
caagatagcc agatcaatgt cgatcgtggc 2820tggctcgaag atacctgcaa
gaatgtcatt gcgctgccat tctccaaatt gcagttcgcg 2880cttagctgga
taacgccacg gaatgatgtc gtcgtgcaca acaatggtga cttctacagc
2940gcggagaatc tcgctctctc caggggaagc cgaagtttcc aaaaggtcgt
tgatcaaagc 3000tcgccgcgtt gtttcatcaa gccttacggt caccgtaacc
agcaaatcaa tatcactgtg 3060tggcttcagg ccgccatcca ctgcggagcc
gtacaaatgt acggccagca acgtcggttc 3120gagatggcgc tcgatgacgc
caactacctc tgatagttga gtcgatactt cggcgatcac 3180cgcttccctc
atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
3240catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagcta
gctcactcgg 3300tcgctacgct ccgggcgtga gactgcggcg ggcgctgcgg
acacatacaa agttacccac 3360agattccgtg gataagcagg ggactaacat
gtgaggcaaa acagcagggc cgcgccggtg 3420gcgtttttcc ataggctccg
ccctcctgcc agagttcaca taaacagacg cttttccggt 3480gcatctgtgg
gagccgtgag gctcaaccat gaatctgaca gtacgggcga aacccgacag
3540gacttaaaga tccccaccgt ttccggcggg tcgctccctc ttgcgctctc
ctgttccgac 3600cctgccgttt accggatacc tgttccgcct ttctccctta
cgggaagtgt ggcgctttct 3660catagctcac acactggtat ctcggctcgg
tgtaggtcgt tcgctccaag ctgggctgta 3720agcaagaact ccccgttcag
cccgactgct gcgccttatc cggtaactgt tcacttgagt 3780ccaacccgga
aaagcacggt aaaacgccac tggcagcagc cattggtaac tgggagttcg
3840cagaggattt gtttagctaa acacgcggtt gctcttgaag tgtgcgccaa
agtccggcta 3900cactggaagg acagatttgg ttgctgtgct ctgcgaaagc
cagttaccac ggttaagcag 3960ttccccaact gacttaacct tcgatcaaac
cacctcccca ggtggttttt tcgtttacag 4020ggcaaaagat tacgcgcaga
aaaaaaggat ctcaagaaga tcctttgatc ttttctactg 4080aaccgctcta
gatttcagtg caatttatct cttcaaatgt agcacctgaa gtcagcccca
4140tacgatataa gttgtaattc tcatgttagt catgccccgc gcccaccgga
aggagctgac 4200tgggttgaag gctctcaagg gcatcggtcg agatcccggt
gcctaatgag tgagctaact 4260tacattaatt gcgttgcgct cactgcccgc
tttccagtcg ggaaacctgt cgtgccagct 4320gcattaatga atcggccaac
gcgcggggag aggcggtttg cgtattgggc gccagggtgg 4380tttttctttt
caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
4440agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
tgtttgatgg 4500tggttaacgg cgggatataa catgagctgt cttcggtatc
gtcgtatccc actaccgaga 4560tgtccgcacc aacgcgcagc ccggactcgg
taatggcgcg cattgcgccc agcgccatct 4620gatcgttggc aaccagcatc
gcagtgggaa cgatgccctc attcagcatt tgcatggttt 4680gttgaaaacc
ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
4740tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
gaacttaatg 4800ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
cagatgctcc acgcccagtc 4860gcgtaccgtc ttcatgggag aaaataatac
tgttgatggg tgtctggtca gagacatcaa 4920gaaataacgc cggaacatta
gtgcaggcag cttccacagc aatggcatcc tggtcatcca 4980gcggatagtt
aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
5040tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
cccagttgat 5100cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
gtgcagggcc agactggagg 5160tggcaacgcc aatcagcaac gactgtttgc
ccgccagttg ttgtgccacg cggttgggaa 5220tgtaattcag ctccgccatc
gccgcttcca ctttttcccg cgttttcgca gaaacgtggc 5280tggcctggtt
caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
5340cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
gggcgctatc 5400atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
cgggatctcg acgctctccc 5460ttatgcgact cctgcattag gaaattaata
cgactcacta ta 5502755733DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
pACYC-PTrc vector polynucleotide" 75actcaccagt cacagaaaag
catcttacgg atggcatgac agtaagagaa ttatgcagtg 60ctgccataac catgagtgat
aacactgcgg ccaacttact tctgacaacg atcggaggac 120cgaaggagct
aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
180gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
atgcctgcag 240caatggcaac aacgttgcgc aaactattaa ctggcgaact
acttactcta gcttcccggc 300aacaattaat agactggatg gaggcggata
aagttgcagg accacttctg cgctcggccc 360ttccggctgg ctggtttatt
gctgataaat ctggagccgg tgagcgtggg tctcgcggta 420tcattgcagc
actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
480ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
gcctcactga 540ttaagcattg gtaactgtca gaccaagttt actcatatat
actttagatt gatttaaaac 600ttcattttta atttaaaagg atctaggtga
agatcctttt tgataatctc atgaccaaaa 660tcccttaacg tgagttttcg
ttccactgag cgtcagaccc cttaataaga tgatcttctt 720gagatcgttt
tggtctgcgc gtaatctctt gctctgaaaa cgaaaaaacc gccttgcagg
780gcggtttttc gaaggttctc tgagctacca actctttgaa ccgaggtaac
tggcttggag 840gagcgcagtc accaaaactt gtcctttcag tttagcctta
accggcgcat gacttcaaga 900ctaactcctc taaatcaatt accagtggct
gctgccagtg gtgcttttgc atgtctttcc 960gggttggact caagacgata
gttaccggat aaggcgcagc ggtcggactg aacggggggt 1020tcgtgcatac
agtccagctt ggagcgaact gcctacccgg aactgagtgt caggcgtgga
1080atgagacaaa cgcggccata acagcggaat gacaccggta aaccgaaagg
caggaacagg 1140agagcgcacg agggagccgc cagggggaaa cgcctggtat
ctttatagtc ctgtcgggtt 1200tcgccaccac tgatttgagc gtcagatttc
gtgatgcttg tcaggggggc ggagcctatg 1260gaaaaacggc tttgccgcgg
ccctctcact tccctgttaa gtatcttcct ggcatcttcc 1320aggaaatctc
cgccccgttc gtaagccatt tccgctcgcc gcagtcgaac gaccgagcgt
1380agcgagtcag tgagcgagga agcggaatat atcctgtatc acatattctg
ctgacgcacc 1440ggtgcagcct tttttctcct gccacatgaa gcacttcact
gacaccctca tcagtgccaa 1500catagtaagc cagtatacac tccgctagcg
ctgaggtctg cctcgtgaag aaggtgttgc 1560tgactcatac caggcctgaa
tcgccccatc atccagccag aaagtgaggg agccacggtt 1620gatgagagct
ttgttgtagg tggaccagtt ggtgattttg aacttttgct ttgccacgga
1680acggtctgcg ttgtcgggaa gatgcgtgat ctgatccttc aactcagcaa
aagttcgatt 1740tattcaacaa agccacgttg tgtctcaaaa tctctgatgt
tacattgcac aagataaaaa 1800tatatcatca tgaacaataa aactgtctgc
ttacataaac agtaatacaa ggggtgttat 1860gagccatatt caacgggaaa
cgtcttgctc gaggccgcga ttaaattcca acatggatgc 1920tgatttatat
gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta
1980tcgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca
aaggtagcgt 2040tgccaatgat gttacagatg agatggtcag actaaactgg
ctgacggaat ttatgcctct 2100tccgaccatc aagcatttta tccgtactcc
tgatgatgca tggttactca ccactgcgat 2160ccccgggaaa acagcattcc
aggtattaga agaatatcct gattcaggtg aaaatattgt 2220tgatgcgctg
gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt
2280taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata
acggtttggt 2340tgatgcgagt gattttgatg acgagcgtaa tggctggcct
gttgaacaag tctggaaaga 2400aatgcataag cttttgccat tctcaccgga
ttcagtcgtc actcatggtg atttctcact 2460tgataacctt atttttgacg
aggggaaatt aataggttgt attgatgttg gacgagtcgg 2520aatcgcagac
cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc
2580ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata
tgaataaatt 2640gcagtttcat ttgatgctcg atgagttttt ctaatcagaa
ttggttaatt ggttgtaaca 2700ctggcagagc attacgctga cttgacggga
cggcggcttt gttgaataaa tcgaactttt 2760gctgagttga aggatcagat
cacgcatctt cccgacaacg cagaccgttc cgtggcaaag 2820caaaagttca
aaatcaccaa ctggtccacc tacaacaaag ctctcatcaa ccgtggctcc
2880ctcactttct ggctggatga tggggcgatt caggcctggt atgagtcagc
aacaccttct 2940tcacgaggca gacctcagcg ctcaaagatg caggggtaaa
agctaaccgc atctttaccg 3000acaaggcatc cggcagttca acagatcggg
aagggctgga tttgctgagg atgaaggtgg 3060aggaaggtga tgtcattctg
gtgaagaagc tcgaccgtct tggccgcgac accgccgaca 3120tgatccaact
gataaaagag tttgatgctc agggtgtagc ggttcggttt attgacgacg
3180ggatcagtac cgacggtgat atggggcaaa tggtggtcac catcctgtcg
gctgtggcac 3240aggctgaacg ccggaggatc ctagagcgca cgaatgaggg
ccgacaggaa gcaaagctga 3300aaggaatcaa atttggccgc aggcgtaccg
tggacaggaa cgtcgtgctg acgcttcatc 3360agaagggcac tggtgcaacg
gaaattgctc atcagctcag tattgcccgc tccacggttt 3420ataaaattct
tgaagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
3480gataataatg gtttcttaga cgtcttaatt aatcaggaga gcgttcaccg
acaaacaaca 3540gataaaacga aaggcccagt ctttcgactg agcctttcgt
tttatttgat gcctggcagt 3600tccctactct cgcatgggga gaccccacac
taccatcggc gctacggcgt ttcacttctg 3660agttcggcat ggggtcaggt
gggaccaccg cgctactgcc gccaggcaaa ttctgtttta 3720tcagaccgct
tctgcgttct gatttaatct gtatcaggct gaaaatcttc tctcatccgc
3780caaaacagcc aagctggaga ccgtttaaac tcaatgatga tgatgatgat
ggtcgacggc 3840gctattcaga tcctcttctg agatgagttt ttgttcgggc
ccaagcttcg aattcccata 3900tggtaccagc tgcagatctc gagctcggat
ccatggttta ttcctcctta tttaatcgat 3960acattaatat atacctcttt
aatttttaat aataaagtta atcgataatt ccggtcgagt 4020gcccacacag
attgtctgat aaattgttaa agagcagtgc cgcttcgctt tttctcagcg
4080gcgctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacattat
acgagccgga 4140tgattaattg tcaacagctc atttcagaat atttgccaga
accgttatga tgtcggcgca 4200aaaaacatta tccagaacgg gagtgcgcct
tgagcgacac gaattatgca gtgatttacg 4260acctgcacag ccataccaca
gcttccgatg gctgcctgac gccagaagca ttggtgcacc 4320gtgcagtcga
tgataagctg tcaaaccaga tcaattcgcg ctaactcaca ttaattgcgt
4380tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
taatgaatcg 4440gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
gggtggtttt tcttttcacc 4500agtgagacgg gcaacagctg attgcccttc
accgcctggc cctgagagag ttgcagcaag 4560cggtccacgc tggtttgccc
cagcaggcga aaatcctgtt tgatggtggt tgacggcggg 4620atataacatg
agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
4680cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
gttggcaacc 4740agcatcgcag tgggaacgat gccctcattc agcatttgca
tggtttgttg aaaaccggac 4800atggcactcc agtcgccttc ccgttccgct
atcggctgaa tttgattgcg agtgagatat 4860ttatgccagc cagccagacg
cagacgcgcc gagacagaac ttaatgggcc cgctaacagc 4920gcgatttgct
ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
4980tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
taacgccgga 5040acattagtgc aggcagcttc cacagcaatg gcatcctggt
catccagcgg atagttaatg 5100atcagcccac tgacgcgttg cgcgagaaga
ttgtgcaccg ccgctttaca ggcttcgacg 5160ccgcttcgtt ctaccatcga
caccaccacg ctggcaccca gttgatcggc gcgagattta 5220atcgccgcga
caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
5280agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
attcagctcc 5340gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
cgtggctggc ctggttcacc 5400acgcgggaaa cggtctgata agagacaccg
gcatactctg cgacatcgta taacgttact 5460ggtttcacat tcaccaccct
gaattgactc tcttccgggc gctatcatgc cataccgcga 5520aaggttttgc
accattcgat ggtgtcaacg taaatgcatg ccgcttcgcc ttcgcgcgcg
5580aattgatctg ctgcctcgcg cgtttcggtg atgacggtga aaacctctga
cacatgcagc 5640tcccggagac ggtcacagct tgtctgtaag cggatgccgg
gagcagacaa gcccgtcagg 5700gcgcgtcagc gggtgttggc ggggccggcc tcg
573376193DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic PTrc promoter polynucleotide"
76ctgttgacaa ttaatcatcc ggctcgtata atgtgtggaa ttgtgagcgg ataacaattt
60cacacaggaa acagcgccgc tgagaaaaag cgaagcggca ctgctcttta acaatttatc
120agacaatctg tgtgggcact cgaccggaat tatcgattaa ctttattatt
aaaaattaaa 180gaggtatata tta 19377193DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
PTrc2 promoter polynucleotide" 77ctgttgacaa ttaatcatcc ggctcgtgta
atgtgtggaa ttgtgagcgg ataacaattt 60cacacaggaa acagcgccgc tgagaaaaag
cgaagcggca ctgctcttta acaatttatc 120agacaatctg tgtgggcact
cgaccggaat tatcgattaa ctttattatt aaaaattaaa 180gaggtatata tta
193785978DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic pDS80 plasmid polynucleotide"
78cactatacca attgagatgg gctagtcaat gataattact agtccttttc ctttgagttg
60tgggtatctg taaattctgc tagacctttg ctggaaaact tgtaaattct gctagaccct
120ctgtaaattc cgctagacct ttgtgtgttt tttttgttta tattcaagtg
gttataattt 180atagaataaa gaaagaataa aaaaagataa aaagaataga
tcccagccct gtgtataact 240cactacttta gtcagttccg cagtattaca
aaaggatgtc gcaaacgctg tttgctcctc 300tacaaaacag accttaaaac
cctaaaggcg tcggcatccg cttacagaca agctgtgacc 360gtctccggga
gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
420cagatcaatt cgcgcgcgaa ggcgaagcgg catgcattta cgttgacacc
atcgaatggt 480gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
gagtcaattc agggtggtga 540atgtgaaacc agtaacgtta tacgatgtcg
cagagtatgc cggtgtctct tatcagaccg 600tttcccgcgt ggtgaaccag
gccagccacg tttctgcgaa aacgcgggaa aaagtggaag 660cggcgatggc
ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
720agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
tcgcaaattg 780tcgcggcgat taaatctcgc gccgatcaac tgggtgccag
cgtggtggtg tcgatggtag 840aacgaagcgg cgtcgaagcc tgtaaagcgg
cggtgcacaa tcttctcgcg caacgcgtca 900gtgggctgat cattaactat
ccgctggatg accaggatgc cattgctgtg gaagctgcct 960gcactaatgt
tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
1020ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
ttgggtcacc 1080agcaaatcgc gctgttagcg ggcccattaa gttctgtctc
ggcgcgtctg cgtctggctg 1140gctggcataa atatctcact cgcaatcaaa
ttcagccgat agcggaacgg gaaggcgact 1200ggagtgccat gtccggtttt
caacaaacca tgcaaatgct gaatgagggc atcgttccca 1260ctgcgatgct
ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
1320ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc
gaagacagct 1380catgttatat cccgccgtta accaccatca aacaggattt
tcgcctgctg gggcaaacca 1440gcgtggaccg cttgctgcaa ctctctcagg
gccaggcggt gaagggcaat cagctgttgc 1500ccgtctcact ggtgaaaaga
aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc 1560gcgcgttggc
cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
1620agtgagcgca acgcaattaa tgtaagttag cgcgaattga tctggtttga
cagcttatca 1680tcgactgcac ggtgcaccaa tgcttctggc gtcaggcagc
catcggaagc tgtggtatgg 1740ctgtgcaggt cgtaaatcac tgcataattc
gtgtcgctca aggcgcactc ccgttctgga 1800taatgttttt tgcgccgaca
tcataacggt tctggcaaat attttcagat ctctcaccta 1860ccaaacaatg
cccccctgca aaaaataaat tcatataaaa aacatacaga taaccatctg
1920cggtgataaa ttatctctgg cggtgttgac ataaatacca ctggcggtga
tactgagcac 1980agaatattca cacaggaaac agcgccgctg agaaaaagcg
aagcggcact gctctttaac 2040aatttatcag acaatctgtg tgggcactcg
accggaatta tcgattaact ttattattaa 2100aaattaaaga ggtatatatt
aatgtatcga ttaaataagg aggaataaac catggatccg 2160agctcgagat
ctgcagctgg taccatatgg gaattcgaag cttgggcccg aacaaaaact
2220catctcagaa gaggatctga atagcgccgt cgaccatcat catcatcatc
attgagttta 2280aacggtctcc agcttggctg ttttggcgga tgagagaaga
ttttcagcct gatacagatt 2340aaatcagaac gcagaagcgg tctgataaaa
cagaatttgc ctggcggcag tagcgcggtg 2400gtcccacctg accccatgcc
gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg 2460gggtctcccc
atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc
2520gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc
tgacgcctga 2580tgcggtattt tctccttacg catctgtgcg gtatttcaca
ccgcatatgg tgcactctca 2640gtacaatctg ctctgatgcc gcatagttaa
gccagccccg acacccgcca acacccgctg 2700acgagcttag taaagccctc
gctagatttt aatgcggatg ttgcgattac ttcgccaact 2760attgcgataa
caagaaaaag ccagcctttc atgatatatc tcccaatttg tgtagggctt
2820attatgcacg cttaaaaata ataaaagcag acttgacctg atagtttggc
tgtgagcaat 2880tatgtgctta gtgcatctaa cgcttgagtt aagccgcgcc
gcgaagcggc gtcggcttga 2940acgaattgtt agacattatt tgccgactac
cttggtgatc tcgcctttca cgtagtggac 3000aaattcttcc aactgatctg
cgcgcgaggc caagcgatct tcttcttgtc caagataagc 3060ctgtctagct
tcaagtatga cgggctgata ctgggccggc aggcgctcca ttgcccagtc
3120ggcagcgaca tccttcggcg cgattttgcc ggttactgcg ctgtaccaaa
tgcgggacaa 3180cgtaagcact acatttcgct catcgccagc ccagtcgggc
ggcgagttcc atagcgttaa 3240ggtttcattt agcgcctcaa atagatcctg
ttcaggaacc ggatcaaaga gttcctccgc 3300cgctggacct accaaggcaa
cgctatgttc tcttgctttt gtcagcaaga tagccagatc 3360aatgtcgatc
gtggctggct cgaagatacc tgcaagaatg tcattgcgct gccattctcc
3420aaattgcagt tcgcgcttag ctggataacg ccacggaatg atgtcgtcgt
gcacaacaat 3480ggtgacttct acagcgcgga gaatctcgct ctctccaggg
gaagccgaag tttccaaaag 3540gtcgttgatc aaagctcgcc gcgttgtttc
atcaagcctt acggtcaccg taaccagcaa 3600atcaatatca ctgtgtggct
tcaggccgcc atccactgcg gagccgtaca aatgtacggc 3660cagcaacgtc
ggttcgagat ggcgctcgat gacgccaact acctctgata gttgagtcga
3720tacttcggcg atcaccgctt ccctcatgat gtttaacttt gttttagggc
gactgccctg 3780ctgcgtaaca tcgttgctgc tccataacat caaacatcga
cccacggcgt aacgcgcttg 3840ctgcttggat gcccgaggca tagactgtac
cccaaaaaaa cagtcataac aagccatgaa 3900aaccgccact gcgccgttac
caccgctgcg ttcggtcaag gttctggacc agttgcgtga 3960gcgcatacgc
tacttgcatt acagcttacg aaccgaacag gcttatgtcc actgggttcg
4020tgccttcatc cgtttccacg gtgtgcgtca cccggcaacc ttgggcagca
gcgaagtcga 4080ggcatttctg tcctggctgg cgaacgagcg caaggtttcg
gtctccacgc atcgtcaggc 4140attggcggcc ttgctgttct tctacggcaa
ggtgctgtgc acggatctgc cctggcttca 4200ggagatcgga agacctcggc
cgtcgcggcg cttgccggtg gtgctgaccc cggatgaagt 4260ggttcgcatc
ctcggttttc tggaaggcga gcatcgtttg ttcgcccagc ttctgtatgg
4320aacgggcatg cggatcagtg agggtttgca actgcgggtc aaggatctgg
atttcgatca 4380cggcacgatc atcgtgcggg agggcaaggg ctccaaggat
cgggccttga tgttacccga 4440gagcttggca cccagcctgc gcgagcaggg
gaattaattc ccacgggttt tgctgcccgc 4500aaacgggctg ttctggtgtt
gctagtttgt tatcagaatc gcagatccgg cttcagccgg 4560tttgccggct
gaaagcgcta tttcttccag aattgccatg attttttccc cacgggaggc
4620gtcactggct cccgtgttgt cggcagcttt gattcgataa gcagcatcgc
ctgtttcagg 4680ctgtctatgt gtgactgttg agctgtaaca agttgtctca
ggtgttcaat ttcatgttct 4740agttgctttg ttttactggt ttcacctgtt
ctattaggtg ttacatgctg ttcatctgtt 4800acattgtcga tctgttcatg
gtgaacagct ttgaatgcac caaaaactcg taaaagctct 4860gatgtatcta
tcttttttac accgttttca tctgtgcata tggacagttt tccctttgat
4920atgtaacggt gaacagttgt tctacttttg tttgttagtc ttgatgcttc
actgatagat 4980acaagagcca taagaacctc agatccttcc gtatttagcc
agtatgttct ctagtgtggt 5040tcgttgtttt tgcgtgagcc atgagaacga
accattgaga tcatacttac tttgcatgtc 5100actcaaaaat tttgcctcaa
aactggtgag ctgaattttt gcagttaaag catcgtgtag 5160tgtttttctt
agtccgttat gtaggtagga atctgatgta atggttgttg gtattttgtc
5220accattcatt tttatctggt tgttctcaag ttcggttacg agatccattt
gtctatctag 5280ttcaacttgg aaaatcaacg tatcagtcgg gcggcctcgc
ttatcaacca ccaatttcat 5340attgctgtaa gtgtttaaat ctttacttat
tggtttcaaa acccattggt taagcctttt 5400aaactcatgg tagttatttt
caagcattaa catgaactta aattcatcaa ggctaatctc 5460tatatttgcc
ttgtgagttt tcttttgtgt tagttctttt aataaccact cataaatcct
5520catagagtat ttgttttcaa aagacttaac atgttccaga ttatatttta
tgaatttttt 5580taactggaaa agataaggca atatctcttc actaaaaact
aattctaatt tttcgcttga 5640gaacttggca tagtttgtcc actggaaaat
ctcaaagcct ttaaccaaag gattcctgat 5700ttccacagtt ctcgtcatca
gctctctggt tgctttagct aatacaccat aagcattttc 5760cctactgatg
ttcatcatct gagcgtattg gttataagtg aacgataccg tccgttcttt
5820ccttgtaggg ttttcaatcg tggggttgag tagtgccaca cagcataaaa
ttagcttggt 5880ttcatgctcc gttaagtcat agcgactaat cgctagttca
tttgctttga aaacaactaa 5940ttcagacata catctcaatt ggtctaggtg attttaat
5978793227DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic p100.38 plasmid polynucleotide"
79gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
60cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
120tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
atgcttcaat 180aatattgaaa aaggaagagt atgagtattc aacatttccg
tgtcgccctt attccctttt 240ttgcggcatt ttgccttcct gtttttgctc
acccagaaac gctggtgaaa gtaaaagatg 300ctgaagatca gttgggtgca
cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360tccttgagag
ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
420tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
cgccgcatac 480actattctca gaatgacttg gttgagtact caccagtcac
agaaaagcat cttacggatg 540gcatgacagt aagagaatta tgcagtgctg
ccataaccat gagtgataac actgcggcca 600acttacttct gacaacgatc
ggaggaccga aggagctaac cgcttttttg cacaacatgg 660gggatcatgt
aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
720acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
ctattaactg 780gcgaactact tactctagct tcccggcaac aattaataga
ctggatggag gcggataaag 840ttgcaggacc acttctgcgc tcggcccttc
cggctggctg gtttattgct gataaatctg 900gagccggtga gcgtgggtct
cgcggtatca ttgcagcact ggggccagat ggtaagccct 960cccgtatcgt
agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
1020agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
caagtttact 1080catatatact ttagattgat ttaaaacttc atttttaatt
tgtgcatccg aagatcagca 1140gttcaacctg ttgatagtac gtactaagct
ctcatgtttc acgtactaag ctctcatgtt 1200taacgtacta agctctcatg
tttaacgaac taaaccctca tggctaacgt actaagctct 1260catggctaac
gtactaagct ctcatgtttg aacaataaaa ttaatataaa tcagcaactt
1320aaatagcctc taaggtttta agttttataa gaaaaaaaag aatatataag
gcttttaaag 1380ctagctttta aggtttcacc atgttctttc ctgcgttatc
ccctgattct gtggataacc 1440gtattaccgc ctttgagtga gctgataccg
ctcgccgcag ccgaacgacc gagcgcagcg 1500agtcagtgag cgaggaagcg
gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt 1560ggccgattca
ttaagacagc tgtctcttat acacatctca accctgaagc tcttgttggc
1620tagtgcgtag tcgttggcaa gctttccgct gtttctgcat tcttacgttt
taggatgcat 1680atggcggccg cataacttcg tatagcatac attatacgaa
gttatctaga gttgcatgcc 1740tgcaggtccg cttattatca cttattcagg
cgtagcaacc aggcgtttaa gggcaccaat 1800aactgcctta aaaaaattac
gccccgccct gccactcatc gcagtactgt tgtaattcat 1860taagcattct
gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg
1920gcatcagcac cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg
ggggcgaaga 1980agttgtccat attggccacg tttaaatcaa aactggtgaa
actcacccag ggattggctg 2040agacgaaaaa catattctca ataaaccctt
tagggaaata ggccaggttt tcaccgtaac 2100acgccacatc ttgcgaatat
atgtgtagaa actgccggaa atcgtcgtgg tattcactcc 2160agagcgatga
aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat
2220cccatatcac cagctcaccg tctttcattg ccatacggaa ttccggatga
gcattcatca 2280ggcgggcaag aatgtgaata aaggccggat aaaacttgtg
cttatttttc tttacggtct 2340ttaaaaaggc cgtaatatcc agctgaacgg
tctggttata ggtacattga gcaactgact 2400gaaatgcctc aaaatgttct
ttacgatgcc attgggatat atcaacggtg gtatatccag 2460tgattttttt
ctccatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata
2520cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg
tgccgatcaa 2580cgtctcattt tcgccaaaag ttggcccagg gcttcccggt
atcaacaggg acaccaggat 2640ttatttattc tgcgaagtga tcttccgtca
caggtattta ttcgactcta gataacttcg 2700tatagcatac attatacgaa
gttatggatc cagcttatcg ataccgtcaa acaaatcata 2760aaaaatttat
ttgctttcag gaaaattttt ctgtataata gattcaattg cgatgacgac
2820gaacacgcat taaggaggtg aagagctcga attcgagcca atatgcgaga
acacccgaga 2880aaattcatcg atgatggttg agatgtgtat aagagacagc
tgtcgtaata gcgaagaggc 2940ccgcaccgat cgcccttccc aacagttgcg
cagcctgaat ggcgaatggc gcctgatgcg 3000gtattttctc cttacgcatc
tgtgcggtat ttcacaccgc atatggtgca ctctcagtac 3060aatctgctct
gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
3120gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
ccgtctccgg 3180gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcga
3227807877DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic pACYC-PTrc-sbm-ygfG plasmid266 -
1348lacI 1577 - 1769PTrc1800 - 3944sbm3967 - 4752ygfG5208 -
6020kanR6347 - 7176p15A ori polynucleotide" 80cgaggccggc cccgccaaca
cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 60ccgcttacag acaagctgtg
accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 120catcaccgaa
acgcgcgagg cagcagatca attcgcgcgc gaaggcgaag cggcatgcat
180ttacgttgac accatcgaat ggtgcaaaac
ctttcgcggt atggcatgat agcgcccgga 240agagagtcaa ttcagggtgg
tgaatgtgaa accagtaacg ttatacgatg tcgcagagta 300tgccggtgtc
tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc
360gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc
ccaaccgcgt 420ggcacaacaa ctggcgggca aacagtcgtt gctgattggc
gttgccacct ccagtctggc 480cctgcacgcg ccgtcgcaaa ttgtcgcggc
gattaaatct cgcgccgatc aactgggtgc 540cagcgtggtg gtgtcgatgg
tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca 600caatcttctc
gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga
660tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg
atgtctctga 720ccagacaccc atcaacagta ttattttctc ccatgaagac
ggtacgcgac tgggcgtgga 780gcatctggtc gcattgggtc accagcaaat
cgcgctgtta gcgggcccat taagttctgt 840ctcggcgcgt ctgcgtctgg
ctggctggca taaatatctc actcgcaatc aaattcagcc 900gatagcggaa
cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat
960gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga
tggcgctggg 1020cgcaatgcgc gccattaccg agtccgggct gcgcgttggt
gcggatatct cggtagtggg 1080atacgacgat accgaagaca gctcatgtta
tatcccgccg tcaaccacca tcaaacagga 1140ttttcgcctg ctggggcaaa
ccagcgtgga ccgcttgctg caactctctc agggccaggc 1200ggtgaagggc
aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc
1260caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
tggcacgaca 1320ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
taatgtgagt tagcgcgaat 1380tgatctggtt tgacagctta tcatcgactg
cacggtgcac caatgcttct ggcgtcaggc 1440agccatcgga agctgtggta
tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc 1500tcaaggcgca
ctcccgttct ggataatgtt ttttgcgccg acatcataac ggttctggca
1560aatattctga aatgagctgt tgacaattaa tcatccggct cgtataatgt
gtggaattgt 1620gagcggataa caatttcaca caggaaacag cgccgctgag
aaaaagcgaa gcggcactgc 1680tctttaacaa tttatcagac aatctgtgtg
ggcactcgac cggaattatc gattaacttt 1740attattaaaa attaaagagg
tatatattaa tgtatcgatt aaataaggag gaataaacca 1800tggctaacgt
gcaggagtgg caacagcttg ccaacaagga attgagccgt cgggagaaaa
1860ctgtcgactc gctggttcat caaaccgcgg aagggatcgc catcaagccg
ctgtataccg 1920aagccgatct cgataatctg gaggtgacag gtacccttcc
tggtttgccg ccctacgttc 1980gtggcccgcg tgccactatg tataccgccc
aaccgtggac catccgtcag tatgctggtt 2040tttcaacagc aaaagagtcc
aacgcttttt atcgccgtaa cctggccgcc gggcaaaaag 2100gtctttccgt
tgcgtttgac cttgccaccc accgtggcta cgactccgat aacccgcgcg
2160tggcgggcga cgtcggcaaa gcgggcgtcg ctatcgacac cgtggaagat
atgaaagtcc 2220tgttcgacca gatcccgctg gataaaatgt cggtttcgat
gaccatgaat ggcgcagtgc 2280taccagtact ggcgttttat atcgtcgccg
cagaagagca aggtgttaca cctgataaac 2340tgaccggcac cattcaaaac
gatattctca aagagtacct ctgccgcaac acctatattt 2400acccaccaaa
accgtcaatg cgcattatcg ccgacatcat cgcctggtgt tccggcaaca
2460tgccgcgatt taataccatc agtatcagcg gttaccacat gggtgaagcg
ggtgccaact 2520gcgtgcagca ggtagcattt acgctcgctg atgggattga
gtacatcaaa gcagcaatct 2580ctgccggact gaaaattgat gacttcgctc
ctcgcctgtc gttcttcttc ggcatcggca 2640tggatctgtt tatgaacgtc
gccatgttgc gtgcggcacg ttatttatgg agcgaagcgg 2700tcagtggatt
tggcgcacag gacccgaaat cactggcgct gcgtacccac tgccagacct
2760caggctggag cctgactgaa caggatccgt ataacaacgt tatccgcacc
accattgaag 2820cgctggctgc gacgctgggc ggtactcagt cactgcatac
caacgccttt gacgaagcgc 2880ttggtttgcc taccgatttc tcagcacgca
ttgcccgcaa cacccagatc atcatccagg 2940aagaatcaga actctgccgc
accgtcgatc cactggccgg atcctattac attgagtcgc 3000tgaccgatca
aatcgtcaaa caagccagag ctattatcca acagatcgac gaagccggtg
3060gcatggcgaa agcgatcgaa gcaggtctgc caaaacgaat gatcgaagag
gcctcagcgc 3120gcgaacagtc gctgatcgac cagggcaagc gtgtcatcgt
tggtgtcaac aagtacaaac 3180tggatcacga agacgaaacc gatgtacttg
agatcgacaa cgtgatggtg cgtaacgagc 3240aaattgcttc gctggaacgc
attcgcgcca cccgtgatga tgccgccgta accgccgcgt 3300tgaacgccct
gactcacgcc gcacagcata acgaaaacct gctggctgcc gctgttaatg
3360ccgctcgcgt tcgcgccacc ctgggtgaaa tttccgatgc gctggaagtc
gctttcgacc 3420gttatctggt gccaagccag tgtgttaccg gcgtgattgc
gcaaagctat catcagtctg 3480agaaatcggc ctccgagttc gatgccattg
ttgcgcaaac ggagcagttc cttgccgaca 3540atggtcgtcg cccgcgcatt
ctgatcgcta agatgggcca ggatggacac gatcgcggcg 3600cgaaagtgat
cgccagcgcc tattccgatc tcggtttcga cgtagattta agcccgatgt
3660tctctacacc tgaagagatc gcccgcctgg ccgtagaaaa cgacgttcac
gtagtgggcg 3720catcctcact ggctgccggt cataaaacgc tgatcccgga
actggtcgaa gcgctgaaaa 3780aatggggacg cgaagatatc tgcgtggtcg
cgggtggcgt cattccgccg caggattacg 3840ccttcctgca agagcgcggc
gtggcggcga tttatggtcc aggtacacct atgctcgaca 3900gtgtgcgcga
cgtactgaat ctgataagcc agcatcatga ttaattctag aaaggaggaa
3960taaaccatgt cttatcagta tgttaacgtt gtcactatca acaaagtggc
ggtcattgag 4020tttaactatg gccgaaaact taatgcctta agtaaagtct
ttattgatga tcttatgcag 4080gcgttaagcg atctcaaccg gccggaaatt
cgctgtatca ttttgcgcgc accgagtgga 4140tccaaagtct tctccgcagg
tcacgatatt cacgaactgc cgtctggcgg tcgcgatccg 4200ctctcctatg
atgatccatt gcgtcaaatc acccgcatga tccaaaaatt cccgaaaccg
4260atcatttcga tggtggaagg tagtgtttgg ggtggcgcat ttgaaatgat
catgagttcc 4320gatctgatca tcgccgccag tacctcaacc ttctcaatga
cgcctgtaaa cctcggcgtc 4380ccgtataacc tggtcggcat tcacaacctg
acccgcgacg cgggcttcca cattgtcaaa 4440gagctgattt ttaccgcttc
gccaatcacc gcccagcgcg cgctggctgt cggcatcctc 4500aaccatgttg
tggaagtgga agaactggaa gatttcacct tacaaatggc gcaccacatc
4560tctgagaaag cgccgttagc cattgccgtt atcaaagaag agctgcgtgt
actgggcgaa 4620gcacacacca tgaactccga tgaatttgaa cgtattcagg
ggatgcgccg cgcggtgtat 4680gacagcgaag attaccagga agggatgaac
gctttcctcg aaaaacgtaa acctaatttc 4740gttggtcatt aagaattcga
agcttgggcc cgaacaaaaa ctcatctcag aagaggatct 4800gaatagcgcc
gtcgaccatc atcatcatca tcattgagtt taaacggtct ccagcttggc
4860tgttttggcg gatgagagaa gattttcagc ctgatacaga ttaaatcaga
acgcagaagc 4920ggtctgataa aacagaattt gcctggcggc agtagcgcgg
tggtcccacc tgaccccatg 4980ccgaactcag aagtgaaacg ccgtagcgcc
gatggtagtg tggggtctcc ccatgcgaga 5040gtagggaact gccaggcatc
aaataaaacg aaaggctcag tcgaaagact gggcctttcg 5100ttttatctgt
tgtttgtcgg tgaacgctct cctgattaat taagacgtcc cgtcaagtca
5160gcgtaatgct ctgccagtgt tacaaccaat taaccaattc tgattagaaa
aactcatcga 5220gcatcaaatg aaactgcaat ttattcatat caggattatc
aataccatat ttttgaaaaa 5280gccgtttctg taatgaagga gaaaactcac
cgaggcagtt ccataggatg gcaagatcct 5340ggtatcggtc tgcgattccg
actcgtccaa catcaataca acctattaat ttcccctcgt 5400caaaaataag
gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
5460gcaaaagctt atgcatttct ttccagactt gttcaacagg ccagccatta
cgctcgtcat 5520caaaatcact cgcatcaacc aaaccgttat tcattcgtga
ttgcgcctga gcgagacgaa 5580atacgcgatc gctgttaaaa ggacaattac
aaacaggaat cgaatgcaac cggcgcagga 5640acactgccag cgcatcaaca
atattttcac ctgaatcagg atattcttct aatacctgga 5700atgctgtttt
cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
5760aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
accatctcat 5820ctgtaacatc attggcaacg ctacctttgc catgtttcag
aaacaactct ggcgcatcgg 5880gcttcccata caatcgatag attgtcgcac
ctgattgccc gacattatcg cgagcccatt 5940tatacccata taaatcagca
tccatgttgg aatttaatcg cggcctcgag caagacgttt 6000cccgttgaat
atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta
6060ttgttcatga tgatatattt ttatcttgtg caatgtaaca tcagagattt
tgagacacaa 6120cgtggctttg ttgaataaat cgaacttttg ctgagttgaa
ggatcagatc acgcatcttc 6180ccgacaacgc agaccgttcc gtggcaaagc
aaaagttcaa aatcaccaac tggtccacct 6240acaacaaagc tctcatcaac
cgtggctccc tcactttctg gctggatgat ggggcgattc 6300aggcctggta
tgagtcagca acaccttctt cacgaggcag acctcagcgc tagcggagtg
6360tatactggct tactatgttg gcactgatga gggtgtcagt gaagtgcttc
atgtggcagg 6420agaaaaaagg ctgcaccggt gcgtcagcag aatatgtgat
acaggatata ttccgcttcc 6480tcgctcactg actcgctacg ctcggtcgtt
cgactgcggc gagcggaaat ggcttacgaa 6540cggggcggag atttcctgga
agatgccagg aagatactta acagggaagt gagagggccg 6600cggcaaagcc
gtttttccat aggctccgcc cccctgacaa gcatcacgaa atctgacgct
6660caaatcagtg gtggcgaaac ccgacaggac tataaagata ccaggcgttt
ccccctggcg 6720gctccctcgt gcgctctcct gttcctgcct ttcggtttac
cggtgtcatt ccgctgttat 6780ggccgcgttt gtctcattcc acgcctgaca
ctcagttccg ggtaggcagt tcgctccaag 6840ctggactgta tgcacgaacc
ccccgttcag tccgaccgct gcgccttatc cggtaactat 6900cgtcttgagt
ccaacccgga aagacatgca aaagcaccac tggcagcagc cactggtaat
6960tgatttagag gagttagtct tgaagtcatg cgccggttaa ggctaaactg
aaaggacaag 7020ttttggtgac tgcgctcctc caagccagtt acctcggttc
aaagagttgg tagctcagag 7080aaccttcgaa aaaccgccct gcaaggcggt
tttttcgttt tcagagcaag agattacgcg 7140cagaccaaaa cgatctcaag
aagatcatct tattaagggg tctgacgctc agtggaacga 7200aaactcacgt
taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
7260tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
cttggtctga 7320cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
atctgtctat ttcgttcatc 7380catagttgcc tgactccccg tcgtgtagat
aactacgata cgggagggct taccatctgg 7440ccccagtgct gcaatgatac
cgcgagaccc acgctcaccg gctccagatt tatcagcaat 7500aaaccagcca
gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
7560ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
atagtttgcg 7620caacgttgtt gccattgctg caggcatcgt ggtgtcacgc
tcgtcgtttg gtatggcttc 7680attcagctcc ggttcccaac gatcaaggcg
agttacatga tcccccatgt tgtgcaaaaa 7740agcggttagc tccttcggtc
ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 7800actcatggtt
atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
7860ttctgtgact ggtgagt 78778115179DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
Tn7tes plasmid polynucleotide" 81ggccacgatg cgtccggcgt agaggatctg
ctcatgtttg acagcttatc atcgatgcat 60aatgtgcctg tcaaatggac gaagcaggga
ttctgcaaac cctatgctac tccgtcaagc 120cgtcaattgt ctgattcgtt
accaattatg acaacttgac ggctacatca ttcacttttt 180cttcacaacc
ggcacggaac tcgctcgggc tggccccggt gcatttttta aatacccgcg
240agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata
ggcatccggg 300tggtgctcaa aagcagcttc gcctggctga tacgttggtc
ctcgcgccag cttaagacgc 360taatccctaa ctgctggcgg aaaagatgtg
acagacgcga cggcgacaag caaacatgct 420gtgcgacgct ggcgatatca
aaattgctgt ctgccaggtg atcgctgatg tactgacaag 480cctcgcgtac
ccgattatcc atcggtggat ggagcgactc gttaatcgct tccatgcgcc
540gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc
ccttcccctt 600gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg
cggctggtgc gcttcatccg 660ggcgaaagaa ccccgtattg gcaaatattg
acggccagtt aagccattca tgccagtagg 720cgcgcggacg aaagtaaacc
cactggtgat accattcgcg agcctccgga tgacgaccgt 780agtgatgaat
ctctcctggc gggaacagca aaatatcacc cggtcggcaa acaaattctc
840gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata
taacctttca 900ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt
ggcctcaatc ggcgttaaac 960ccgccaccag atgggcatta aacgagtatc
ccggcagcag gggatcattt tgcgcttcag 1020ccatactttt catactcccg
ccattcagag aagaaaccaa ttgtccatat tgcatcagac 1080attgccgtca
ctgcgtcttt tactggctct tctcgctaac caaaccggta accccgctta
1140ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt
aacaaaagtg 1200tctataatca cggcagaaaa gtccacattg attatttgca
cggcgtcaca ctttgctatg 1260ccatagcatt tttatccata agattagcgg
atcctacctg acgcttttta tcgcaactct 1320ctactgtttc tccatacccg
tttttttggg ctagcgaatt cgagctcggt acccaagtct 1380taaactagac
agaatagttg taaactgaaa tcagtccagt tatgctgtga aaaagcatac
1440tggacttttg ttatggctaa agcaaactct tcattttctg aagtgcaaat
tgcccgtcgt 1500attaaagagg ggcgtggcca agggcatggt aaagactata
ttccatggct aacagtacaa 1560gaagttcctt cttcaggtcg ttcccaccgt
atttattctc ataagacggg acgagtccat 1620catttgctat ctgacttaga
gcttgctgtt tttctcagtc ttgagtggga gagcagcgtg 1680ctagatatac
gcgagcagtt ccccttatta cctagtgata ccaggcagat tgcaatagat
1740agtggtatta agcatcctgt tattcgtggt gtagatcagg ttatgtctac
tgatttttta 1800gtggactgca aagatggtcc ttttgagcag tttgctattc
aagtcaaacc tgcagcagcc 1860ttacaagacg agcgtacctt agaaaaacta
gaactagagc gtcgctattg gcagcaaaag 1920caaattcctt ggttcatttt
tactgataaa gaaataaatc ccgtagtaaa agaaaatatt 1980gaatggcttt
attcagtgaa aacagaagaa gtttctgcgg agcttttagc acaactatcc
2040ccattggccc atatcctgca agaaaaagga gatgaaaaca ttatcaatgt
ctgtaagcag 2100gttgatattg cttatgattt ggagttaggc aaaacattga
gtgagatacg agccttaacc 2160gcaaatggtt ttattaagtt caatatttat
aagtctttca gggcaaataa gtgtgcagat 2220ctctgtatta gccaagtagt
gaatatggag gagttgcgct atgtggcaaa ttaatgaggt 2280tgtgctattt
gataatgatc cgtatcgcat tttggctata gaggatggcc aagttgtctg
2340gatgcaaata agcgctgata aaggagttcc acaagctagg gctgagttgt
tgctaatgca 2400gtatttagat gaaggccgct tagttagaac tgatgaccct
tatgtacatc ttgatttaga 2460agagccgtct gtagattctg tcagcttcca
gaagcgcgag gaggattatc gaaaaattct 2520tcctattatt aatagtaagg
atcgtttcga ccctaaagtc agaagcgaac tcgttgagca 2580tgtggtccaa
gaacataagg ttactaaggc tacagtttat aagttgttac gccgttactg
2640gcagcgtggt caaacgccta atgcattaat tcctgactac aaaaacagcg
gtgcaccagg 2700ggaaagacgt tcagcgacag gaacagcaaa gattggccga
gccagagaat atggtaaggg 2760tgaaggaacc aaggtaacgc ccgagattga
acgccttttt aggttgacca tagaaaagca 2820cctgttaaat caaaaaggta
caaagaccac cgttgcctat agacgatttg tggacttgtt 2880tgctcagtat
tttcctcgca ttccccaaga ggattaccca acactacgtc agtttcgtta
2940tttttatgat cgagaatacc ctaaagctca gcgcttaaag tctagagtta
aagcaggggt 3000atataaaaaa gacgtacgac ccttaagtag tacagccact
tctcaggcgt taggccctgg 3060gagtcgttat gagattgatg ccacgattgc
tgatatttat ttagtggatc atcatgatcg 3120ccaaaaaatc ataggaagac
caacgcttta cattgtgatt gatgtgttta gtcggatgat 3180cacgggcttt
tatatcggct ttgaaaatcc gtcttatgtg gtggcgatgc aggcttttgt
3240aaatgcttgc tctgacaaaa cggccatttg tgcccagcat gatattgaga
ttagtagctc 3300agactggccg tgtgtaggtt tgccagatgt gttgctagcg
gaccgtggcg aattaatgag 3360tcatcaggtc gaagccttag tttctagttt
taatgtgcga gtggaaagtg ctccacctag 3420acgtggcgat gctaaaggca
tagtggaaag cacttttaga acactacaag ccgagtttaa 3480gtcctttgca
cctggcattg tagagggcag tcggatcaaa agccatggtg aaacagacta
3540taggttagat gcatctctgt cggtatttga gttcacacaa attattttgc
gtacgatctt 3600attcagaaat aaccatctgg tgatggataa atacgatcga
gatgctgatt ttcctacaga 3660tttaccgtct attcctgtcc agctatggca
atggggtatg cagcatcgta caggtagttt 3720aagggctgtg gagcaagagc
agttgcgagt agcgttactg cctcgccgaa aggtctctat 3780ttcttcattt
ggcgttaatt tgtggggttt gtattactcg gggtcagaga ttctgcgtga
3840gggttggttg cagcggagca ctgatatagc tagacctcaa catttagaag
cggcttatga 3900cccagtgctg gttgatacga tttatttgtt tccgcaagtt
ggcagccgtg tattttggcg 3960ctgtaatctg acggaacgta gtcggcagtt
taaaggtctc tcattttggg aggtttggga 4020tatacaagca caagaaaaac
acaataaagc caatgcgaag caggatgagt taactaaacg 4080cagggagctt
gaggcgttta ttcagcaaac cattcagaaa gcgaataagt taacgcccag
4140tactactgag cccaaatcaa cacgcattaa gcagattaaa actaataaaa
aagaagccgt 4200gacctcggag cgtaaaaaac gtgcggagca tttgaagcca
agctcttcag gtgatgaggc 4260taaagttatt cctttcaacg cagtggaagc
ggatgatcaa gaagattaca gcctacccac 4320atacgtgcct gaattatttc
aggatccacc agaaaaggat gagtcatgag tgctacccgg 4380attcaagcag
tttatcgtga tacgggggta gaggcttatc gtgataatcc ttttatcgag
4440gccttaccac cattacaaga gtcagtgaat agtgctgcat cactgaaatc
ctctttacag 4500cttacttcct ctgacttgca aaagtcccgt gttatcagag
ctcataccat ttgtcgtatt 4560ccagatgact attttcagcc attaggtacg
catttgctac taagtgagcg tatttcggtc 4620atgattcgag gtggctacgt
aggcagaaat cctaaaacag gagatttaca aaagcattta 4680caaaatggtt
atgagcgtgt tcaaacggga gagttggaga catttcgctt tgaggaggca
4740cgatctacgg cacaaagctt attgttaatt ggttgttctg gtagtgggaa
gacgacctct 4800cttcatcgta ttctagccac gtatcctcag gtgatttacc
atcgtgaact caatgtagag 4860caggtggtgt atttgaaaat agactgctcg
cataatggtt cgctaaaaga aatctgcttg 4920aattttttca gagcgttgga
tcgagccttg ggctcgaact atgagcgtcg ttatggctta 4980aaacgtcatg
gtatagaaac catgttggct ttgatgtcgc aaatagccaa tgcacatgct
5040ttagggttgt tggttattga tgaaattcag catttaagcc gctctcgttc
gggtggatct 5100caagagatgc tgaacttttt tgtgacgatg gtgaatatta
ttggcgtacc agtgatgttg 5160attggtaccc ctaaagcacg agagattttt
gaggctgatt tgcggtctgc acgtagaggg 5220gcagggtttg gagctatatt
ctgggatcct atacaacaaa cgcaacgtgg aaagcccaat 5280caagagtgga
tcgcttttac ggataatctt tggcaattac agcttttaca acgcaaagat
5340gcgctgttat cggatgaggt ccgtgatgtg tggtatgagc taagccaagg
agtgatggac 5400attgtagtaa aactttttgt actcgctcag ctccgtgcgc
tagctttagg caatgagcgt 5460attaccgctg gtttattgcg gcaagtgtat
caagatgagt taaagcctgt gcaccccatg 5520ctagaggcat tacgctcggg
tatcccagaa cgcattgctc gttattctga tctagtcgtt 5580cccgagattg
ataaacggtt aatccaactt cagctagata tcgcagcgat acaagaacaa
5640acaccagaag aaaaagccct tcaagagtta gataccgaag atcagcgtca
tttatatctg 5700atgctgaaag aggattacga ttcaagcctg ttaattccca
ctattaaaaa agcgtttagc 5760cagaatccaa cgatgacaag acaaaagtta
ctgcctcttg ttttgcagtg gttgatggaa 5820ggcgaaacgg tagtgtcaga
actagaaaag ccctccaaga gtaaaaaggt ttcggctata 5880aaggtagtca
agcccagcga ctgggatagc ttgcctgata cggatttacg ttatatctat
5940tcacaacgcc aacctgaaaa aaccatgcat gaacggttaa aagggaaagg
ggtaatagtg 6000gatatggcga gcttatttaa acaagcaggt tagccatgag
aaactttcct gttccgtact 6060cgaatgagct gatttatagc actattgcac
gggcaggcgt ttatcaaggg attgttagtc 6120ctaagcagct gttggatgag
gtgtatggca accgcaaggt ggtcgctacc ttaggtctgc 6180cctcgcattt
aggtgtgata gcaagacatc tacatcaaac aggacgttac gctgttcagc
6240agcttattta tgagcatacc ttattccctt tatatgctcc gtttgtaggc
aaggagcgcc 6300gagacgaagc tattcggtta atggagtacc aagcgcaagg
tgcggtgcat ttaatgctag 6360gagtcgctgc ttctagagtt aagagcgata
accgctttag atactgccct gattgcgttg 6420ctcttcagct aaataggtat
ggggaagcct tttggcaacg agattggtat ttgcccgctt 6480tgccatattg
tccaaaacac ggtgctttag tcttctttga tagagctgta gatgatcacc
6540gacatcaatt ttgggctttg ggtcatactg agctgctttc agactacccc
aaagactccc 6600tatctcaatt aacagcacta gctgcttata tagcccctct
gttagatgct ccacgagcgc 6660aagagctttc cccaagcctt gagcagtgga
cgctgtttta tcagcgctta gcgcaggatc 6720tagggctaac caaaagcaag
cacattcgtc atgacttggt ggcggagaga gtgaggcaga 6780cttttagtga
tgaggcacta gagaaactgg atttaaagtt ggcagagaac aaggacacgt
6840gttggctgaa aagtatattc cgtaagcata gaaaagcctt tagttattta
cagcatagta 6900ttgtgtggca agccttattg ccaaaactaa cggttataga
agcgctacag caggcaagtg 6960ctcttactga gcactctata acgacaagac
ctgttagcca gtctgtgcaa cctaactctg 7020aagatttatc tgttaagcat
aaagactggc agcaactagt gcataaatac caaggaatta 7080aggcggcaag
acagtcttta gagggtgggg tgctatacgc ttggctttac cgacatgaca
7140gggattggct agttcactgg aatcaacagc atcaacaaga gcgtctggca
cccgccccta 7200gagttgattg gaaccaaaga gatcgaattg
ctgtacgaca actattaaga atcataaagc 7260gtctagatag tagccttgat
cacccaagag cgacatcgag ctggctgtta aagcaaactc 7320ctaacggaac
ctctcttgca aaaaatctac agaaactgcc tttggtagcg ctttgcttaa
7380agcgttactc agagagtgtg gaagattatc aaattagacg gattagccaa
gcttttatta 7440agcttaaaca ggaagatgtt gagcttaggc gctggcgatt
attaagaagt gcaacgttat 7500ctaaagagcg gataactgag gaagcacaaa
gattcttgga aatggtttat ggggaagagt 7560gagtggttag gctagctaca
tttaatgaca atgtgcaggt tgtacatatt ggtcatttat 7620tccgtaactc
gggtcataag gagtggcgta tttttgtttg gtttaatcca atgcaagaac
7680ggaaatggac tcgatttact catttgcctt tattaagtcg agctaaggtg
gttaacagta 7740caacaaagca aataaataag gcggatcgtg tgattgagtt
tgaagcatcg gatcttcaac 7800gagccaaaat aatcgatttt cctaatctct
cgtcctttgc ttccgtacgc aacaaggatg 7860gagcgcagag ttcatttatt
tacgaagctg aaacaccata tagcaagact cgttatcaca 7920tcccacagtt
agagctagct cggtcattat ttttagggga tcctctagag tcgacctgca
7980ggcatgcaag cttggctgtt ttggcggatg agagaagatt ttcagcctga
tacagattaa 8040atcagaacgc agaagcggtc tgataaaaca gaatttgcct
ggcggcagta gcgcggtggt 8100cccacctgac cccatgccga actcagaagt
gaaacgccgt agcgccgatg gtagtgtggg 8160gtctccccat gcgagagtag
ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga 8220aagactgggc
ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa
8280atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg
cgggcaggac 8340gcccgccata aactgccagg catcaaatta agcagaaggc
catcctgacg gatggccttt 8400ttgcgtttct acaaactctt ttgtttattt
ttctaaatac attcaaatat gtatccgctc 8460atgagacaat aaccctgata
aatgcttcaa taatattgaa aaaggaagag tatgagtatt 8520caacatttcc
gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
8580cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
acgagtgggt 8640tacatcgaac tggatctcaa cagcggtaag atccttgaga
gttttcgccc cgaagaacgt 8700tttccaatga tgagcacttt taaagttctg
ctatgtggcg cggtattatc ccgtgttgac 8760gccgggcaag agcaactcgg
tcgccgcata cactattctc agaatgactt ggttgagtac 8820tcaccagtca
cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
8880gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
cggaggaccg 8940aaggagctaa ccgctttttt gcacaacatg ggggatcatg
taactcgcct tgatcgttgg 9000gaaccggagc tgaatgaagc cataccaaac
gacgagcgtg acaccacgat gcctgcagca 9060atggcaacaa cgttgcgcaa
actattaact ggcgaactac ttactctagc ttcccggcaa 9120caattaatag
actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
9180ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
tcgcggtatc 9240attgcagcac tggggccaga tggtaagccc tcccgtatcg
tagttatcta cacgacgggg 9300agtcaggcaa ctatggatga acgaaataga
cagatcgctg agataggtgc ctcactgatt 9360aagcattggt aactgtcaga
ccaagtttac tcatatatac tttagattga tttacgcgcc 9420ctgtagcggc
gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
9480tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
ccacgttcgc 9540cgccggccag cctcgcagag caggattccc gttgagcacc
gccaggtgcg aataagggac 9600agtgaagaag gaacacccgc tcgcgggtgg
gcctacttca cctatcctgc ccggcggcat 9660caccggcgcc acaggtgcgg
ttgctggcgc ctatatcgcc gacatcaccg atggggaaga 9720tcgggctcgc
cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc
9780cgtggccggg ggactgttgg gcgccatctc cttgcatgca ccattccttg
cggcggcggt 9840gctcaacggc ctcaacctac tactgggctg cttcctaatg
caggagtcgc ataagggaga 9900gcgtcgatcc ccgacagtaa gacgggtaag
cctgttgatg ataccgctgc cttactgggt 9960gcattagcca gtctgaatga
cctgtcacgg gataatccga agtggtcaga ctggaaaatc 10020agagggcagg
aactgctgaa cagcaaaaag tcagatagca ccacatagca gacccgccat
10080aaaacgccct gagaagcccg tgacgggctt ttcttgtatt atgggtagtt
tccttgcatg 10140aatccataaa aggcgcctgt agtgccattt acccccattc
actgccagag ccgtgagcgc 10200agcgaactga atgtcacgaa aaagacagcg
actcaggtgc ctgatggtcg gagacaaaag 10260gaatattcag cgatttgccc
gagcttgcga gggtgctact taagccttta gggttttaag 10320gtctgttttg
tagaggagca aacagcgttt gcgacatcct tttgtaatac tgcggaactg
10380actaaagtag tgagttatac acagggctgg gatctattct ttttatcttt
ttttattctt 10440tctttattct ataaattata accacttgaa tataaacaaa
aaaaacacac aaaggtctag 10500cggaatttac agagggtcta gcagaattta
caagttttcc agcaaaggtc tagcagaatt 10560tacagatacc cacaactcaa
aggaaaagga ctagtaatta tcattgacta gcccatctca 10620attggtatag
tgattaaaat cacctagacc aattgagatg tatgtctgaa ttagttgttt
10680tcaaagcaaa tgaactagcg attagtcgct atgacttaac ggagcatgaa
accaagctaa 10740ttttatgctg tgtggcacta ctcaacccca cgattgaaaa
ccctacaagg aaagaacgga 10800cggtatcgtt cacttataac caatacgttc
agatgatgaa catcagtagg gaaaatgctt 10860atggtgtatt agctaaagca
accagagagc tgatgacgag aactgtggaa atcaggaatc 10920ctttggttaa
aggctttgag attttccagt ggacaaacta tgccaagttc tcaagcgaaa
10980aattagaatt agtttttagt gaagagatat tgccttatct tttccagtta
aaaaaattca 11040taaaatataa tctggaacat gttaagtctt ttgaaaacaa
atactctatg aggatttatg 11100agtggttatt aaaagaacta acacaaaaga
aaactcacaa ggcaaatata gagattagcc 11160ttgatgaatt taagttcatg
ttaatgcttg aaaataacta ccatgagttt aaaaggctta 11220accaatgggt
tttgaaacca ataagtaaag atttaaacac ttacagcaat atgaaattgg
11280tggttgataa gcgaggccgc ccgactgata cgttgatttt ccaagttgaa
ctagatagac 11340aaatggatct cgtaaccgaa cttgagaaca accagataaa
aatgaatggt gacaaaatac 11400caacaaccat tacatcagat tcctacctac
ataacggact aagaaaaaca ctacacgatg 11460ctttaactgc aaaaattcag
ctcaccagtt ttgaggcaaa atttttgagt gacatgcaaa 11520gtaagtatga
tctcaatggt tcgttctcat ggctcacgca aaaacaacga accacactag
11580agaacatact ggctaaatac ggaaggatct gaggttctta tggctcttgt
atctatcagt 11640gaagcatcaa gactaacaaa caaaagtaga acaactgttc
accgttacat atcaaaggga 11700aaactgtcca tatgcacaga tgaaaacggt
gtaaaaaaga tagatacatc agagctttta 11760cgagtttttg gtgcatttaa
agctgttcac catgaacaga tcgacaatgt aacagatgaa 11820cagcatgtaa
cacctaatag aacaggtgaa accagtaaaa caaagcaact agaacatgaa
11880attgaacacc tgagacaact tgttacagct caacagtcac acatagacag
cctgaaacag 11940gcgatgctgc ttatcgaatc aaagctgccg acaacacggg
agccagtgac gcctcccgtg 12000gggaaaaaat catggcaatt ctggaagaaa
tagcgctttc agcctgtggg cggacaaaat 12060agttgggaac tgggaggggt
ggaaatggag tttttaagga ttatttaggg aagagtgaca 12120aaatagatgg
gaactgggtg tagcgtcgta agctaatacg aaaattaaaa atgacaaaat
12180agtttggaac tagatttcac ttatctggtt ggtcgacact agtattaccc
tgttatccct 12240agatttaaat gatatcggat cctagtaagc cacgttttaa
ttaatcagat gggtcaatag 12300cggccgccaa ttcgcgcgcg aaggcgaagc
ggcatgcatt tacgttgaca ccatcgaatg 12360gtgcaaaacc tttcgcggta
tggcatgata gcgcccggaa gagagtcaat tcagggtggt 12420gaatgtgaaa
ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac
12480cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
aaaaagtgga 12540agcggcgatg gcggagctga attacattcc caaccgcgtg
gcacaacaac tggcgggcaa 12600acagtcgttg ctgattggcg ttgccacctc
cagtctggcc ctgcacgcgc cgtcgcaaat 12660tgtcgcggcg attaaatctc
gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt 12720agaacgaagc
ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt
12780cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
tggaagctgc 12840ctgcactaat gttccggcgt tatttcttga tgtctctgac
cagacaccca tcaacagtat 12900tattttctcc catgaagacg gtacgcgact
gggcgtggag catctggtcg cattgggtca 12960ccagcaaatc gcgctgttag
cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc 13020tggctggcat
aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga
13080ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
gcatcgttcc 13140cactgcgatg ctggttgcca acgatcagat ggcgctgggc
gcaatgcgcg ccattaccga 13200gtccgggctg cgcgttggtg cggatatctc
ggtagtggga tacgacgata ccgaagacag 13260ctcatgttat atcccgccgt
caaccaccat caaacaggat tttcgcctgc tggggcaaac 13320cagcgtggac
cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt
13380gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa
ccgcctctcc 13440ccgcgcgttg gccgattcat taatgcagct ggcacgacag
gtttcccgac tggaaagcgg 13500gcagtgagcg caacgcaatt aatgtgagtt
agcgcgaatt gatctggttt gacagcttat 13560catcgactgc acggtgcacc
aatgcttctg gcgtcaggca gccatcggaa gctgtggtat 13620ggctgtgcag
gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac tcccgttctg
13680gataatgttt tttgcgccga catcataacg gttctggcaa atattctgaa
atgagctgtt 13740gacaattaat catccggctc gtataatgtg tggaattgtg
agcggataac aatttcacac 13800aggaaacagc gccgctgaga aaaagcgaag
cggcactgct ctttaacaat ttatcagaca 13860atctgtgtgg gcactcgacc
ggaattatcg attaacttta ttattaaaaa ttaaagaggt 13920atatattaat
gtatcgatta aataaggagg aataaaccat ggcggacacg ttattgattc
13980tgggtgatag cctgagcgcc gggtatcgaa tgtctgccag cgcggcctgg
cctgccttgt 14040tgaatgataa gtggcagagt aaaacgtcgg tagttaatgc
cagcatcagc ggcgacacct 14100cgcaacaagg actggcgcgc cttccggctc
tgctgaaaca gcatcagccg cgttgggtgc 14160tggttgaact gggcggcaat
gacggtttgc gtggttttca gccacagcaa accgagcaaa 14220cgctgcgcca
gattttgcag gatgtcaaag ccgccaacgc tgaaccattg ttaatgcaaa
14280tacgtctgcc tgcaaactat ggtcgccgtt ataatgaagc ctttagcgcc
atttacccca 14340aactcgccaa agagtttgat gttccgctgc tgcccttttt
tatggaagag gtctacctca 14400agccacaatg gatgcaggat gacggtattc
atcccaaccg cgacgcccag ccgtttattg 14460ccgactggat ggcgaagcag
ttgcagcctt tagtaaatca tgactcataa tgactctaga 14520aataatttaa
atggaattcg aagcttgggc ccgaacaaaa actcatctca gaagaggatc
14580tgaatagcgc cgtcgaccat catcatcatc atcattgagt ttaaacggtc
tccagcttgg 14640ctgttttggc ggatgagaga agattttcag cctgatacag
attaaatcag aacgcagaag 14700cggtctgata aaacagaatt tgcctggcgg
cagtagcgcg gtggtcccac ctgaccccat 14760gccgaactca gaagtgaaac
gccgtagcgc cgatggtagt gtggggtctc cccatgcgag 14820agtagggaac
tgccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc
14880gttttatctg ttgtttgtcg gtgaacgctc tcctgattaa ttaagacgtc
ccgtcaagtc 14940agcgtaatgc cctaggaggc gcgccacggc cgcgtcgacc
ccacgcccct ctttaatacg 15000acgggcaatt tgcacttcag aaaatgaaga
gtttgcttta gccataacaa aagtccagta 15060tgctttttca cagcataact
ggactgattt cagtttacaa ctattctgtc tagtttaaga 15120ctttattgtc
atagtttaga tctattttgt tcagtttaag actttattgt ccgcccaca
151798270DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic Del-fadE-F primer" 82aaaaacagca
acaatgtgag ctttgttgta attatattgt aaacatattg attccgggga 60tccgtcgacc
708368DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic Del-fadE-R primer" 83aaacggagcc tttcggctcc
gttattcatt tacgcggctt caactttcct gtaggctgga 60gctgcttc
688423DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic fadE-L2 primer" 84cgggcaggtg ctatgaccag gac
238523DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic fadE-R1 primer" 85cgcggcgttg accggcagcc tgg
238670DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic Del-tonA-F primer" 86atcattctcg tttacgttat
cattcacttt acatcagaga tataccaatg attccgggga 60tccgtcgacc
708769DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic Del-tonA-R primer" 87gcacggaaat ccgtgcccca
aaagagaaat tagaaacgga aggttgcggt tgtaggctgg 60agctgcttc
698821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic tonA-verF primer" 88caacagcaac ctgctcagca a
218921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic tonA-verR primer" 89aagctggagc agcaaagcgt t
219022DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic lacI-forward primer" 90ggctggctgg cataaatatc tc
229179DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic lacZ-reverse primer" 91gcgttaaagt tgttctgctt
catcagcagg atatcctgca ccatcgtctg gattttgaac 60ttttgctttg ccacggaac
799236DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic primer" 92tgaattccat ggcgcaactc actcttcttt
tagtcg 369339DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic primer " 93cagtacctcg agtcttcgta
tacatatgcg ctcagtcac 399421DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 94ccttggggca tatgaaagct g 219529DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 95tttagtcatc tcgagtgcac ctcaccttt 299635DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
pTrc_F primer" 96tttcgcgagg ccggccccgc caacacccgc tgacg
359739DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic pTrc_R primer" 97aaggacgtct taattaatca
ggagagcgtt caccgacaa 399828DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
LF302 primer" 98atatgacgtc ggcatccgct tacagaca 289932DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
LF303 primer" 99aattcttaag tcaggagagc gttcaccgac aa
3210037DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic TREE044 primer" 100gaggaataaa ccatgaacgc
aggaatttta ggagtag 3710141DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer61" 101cccaagcttc gaattcttac ttaccccaac gaatgattag g
4110271DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic TREE025 primer" 102cctgacagtg cgggcttttt
ttttcgacca aaggtaacga ggtaacaacc gtgtaggctg 60gagctgcttc g
7110362DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic TREE026 primer" 103gtatatatta atgtatcgat
taaataagga ggaataaacc atgcgagtgt tgaagttcgg 60cg
6210459DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic TREE027 primer" 104ctgatgtacc gccgaacttc
aacactcgca tggtttattc ctccttattt aatcgatac 5910528DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
TREE028 primer" 105gcgcccgtat tttcgtggtg ctgattac
2810628DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic TREE029 primer" 106gtaatcagca ccacgtaaat
acgggcgc 2810725DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic TREE030 primer" 107tcagactcct
aacttccatg agagg 2510850DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
Km_trc_overR primer" 108aatatttgcc agaaccgtta tgatgtcggc attccgggga
tccgtcgacc 5010955DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic Km_trc_overF primer" 109cttcgaactg
caggtcgacg gatccccgga atgccgacat cataacggtt ctggc
5511029DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic EG238 primer" 110gctgatcatt aactatccgc tggatgacc
2911140DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic TREE017 primer" 111actggaaagc gggcagtgag
cgcaacgcaa ttaatgtaag 4011216DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
TREE018 primer" 112tcactgcccg ctttcc 1611355DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
TREE019 primer" 113accggcagat cgtatgtaat atgcatggtt tattcctcct
tatttaatcg ataca 5511423DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
TREE020 primer" 114atgcatatta catacgatct gcc 2311538DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
TREE021 primer" 115ggtcgacgga tccccggaat taagcgtcaa cgaaaccg
3811638DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic TREE022 primer" 116gaagcagctc cagcctacac
cagacgatgg tgcaggat 3811721DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
TREE023 primer" 117gcaaagacca gaccgttcat a 2111820DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
Kan/Chlor1 primer" 118attccgggga tccgtcgacc 2011920DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
Kan/Chlor4 primer" 119tgtaggctgg agctgcttcg 2012074DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
TREE133 primer" 120aaaaacagca acaatgtgag ctttgttgta attatattgt
aaacatattg tccgctgttt 60ctgcattctt acgt 7412172DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
TREE134 primer" 121gatgacgacg aacacgcatt aaggaggtga ataaggagga
ataacatatg aaagctggca 60ttcttggtgt tg 7212272DNAArtificial
Sequencesource/note="Description of Artificial Sequence
Synthetic
TREE135 primer" 122gtaacgtcca acaccaagaa tgccagcttt catatgttat
tcctccttat tcacctcctt 60aatgcgtgtt cg 7212370DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
TREE136 primer" 123aaacggagcc tttcggctcc gttattcatt tacgcggctt
caactttccg ttatcggccc 60cagcggattg 7012470DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
TREE137 primer" 124cgcagtttgc aagtgacggt atataaccga aaagtgactg
agcgtacatg attccgggga 60tccgtcgacc 7012570DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
TREE138 primer" 125gcaaattgcg tcatgtttta atccttatcc tagaaacgaa
ccagcgcgga tgtaggctgg 60agctgcttcg 7012620DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
TREE139 primer" 126gcagcgacaa gttcctcagc 2012721DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
TREE140 primer" 127ccgcagaagc ttcagcaaac g 2112823DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
fadE-L2 primer" 128cgggcaggtg ctatgaccag gac 2312921DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
fadE-R2 primer" 129gggcaggata agctcgggag g 2113055DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
Km_trc_overF primer" 130cttcgaactg caggtcgacg gatccccgga atgccgacat
cataacggtt ctggc 5513150DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
Km_trc_overR primer" 131aatatttgcc agaaccgtta tgatgtcggc attccgggga
tccgtcgacc 5013268DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic TREE032 primer" 132gtatatatta
atgtatcgat taaataagga ggaataaacc atgatggtaa ggatatttga 60tacaacac
6813360DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic TREE033 primer" 133ctaagtgttg tatcaaatat
ccttaccatc atggtttatt cctccttatt taatcgatac 6013466DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
TREE034 primer" 134gatttgttgg ctatagttag agaagttact ggaaaattgt
aacaaggaaa ccgtgtgatg 60tcgaag 6613562DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
TREE035 primer" 135gtaattcttc gacatcacac ggtttccttg ttacaatttt
ccagtaactt ctctaactat 60ag 6213622DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
TREE104 primer" 136ggtagcgaag gttttgcccg gc 2213722DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
TREE106 primer" 137gattggtgcc ccaggtgacc tg 2213872DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
TREE146 primer" 138gagttgcaac gcaaagctca acacaacgaa aacaacaagg
aaaccgtgtg agtgtaggct 60ggagctgctt cg 7213919DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
TREE151 primer" 139cttccacggc gtcggcctg 1914036DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
IFF primer" 140gggtcaatag cggccgccaa ttcgcgcgcg aaggcg
3614137DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic IFR primer" 141tggcgcgcct cctagggcat tacgctgact
tgacggg 3714270DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic ScpBC-KOfwd primer" 142gctcagtgaa
tttatccaga cgcaatattt tgattaaagg aatttttatg attccgggga 60tccgtcgacc
7014369DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic ScpBC-KOrc primer" 143attgctgaag atcgtgacgg
gacgagtcat taacccagca tcgagccggt tgtaggctgg 60agctgcttc
6914419DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic ScpBC check -60 fwd primer" 144cgggttctga
cttgtagcg 1914524DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic ScpBC check +60 rc primer"
145ccaacttcga agcaatgatt gatg 24146325PRTStenotrophomonas
maltophilasource/note="Beta ketoacyl-ACP synthase III" 146Met Ser
Lys Arg Ile Tyr Ser Arg Ile Ala Gly Thr Gly Ser Tyr Leu 1 5 10 15
Pro Glu Lys Val Leu Thr Asn Ala Asp Leu Glu Lys Met Val Glu Thr 20
25 30 Ser Asp Glu Trp Ile Gln Ser Arg Thr Gly Ile Arg Glu Arg His
Ile 35 40 45 Ala Ala Glu Gly Glu Thr Thr Ser Asp Leu Gly Tyr Asn
Ala Ala Leu 50 55 60 Arg Ala Leu Glu Ala Ala Gly Ile Asp Ala Ser
Gln Leu Asp Met Ile 65 70 75 80 Val Val Gly Thr Thr Thr Pro Asp Leu
Ile Phe Pro Ser Thr Ala Cys 85 90 95 Leu Ile Gln Ala Lys Leu Gly
Val Ala Gly Cys Pro Ala Phe Asp Val 100 105 110 Asn Ala Ala Cys Ser
Gly Phe Val Phe Ala Leu Gly Val Ala Asp Lys 115 120 125 Phe Ile Arg
Ser Gly Asp Cys Arg His Val Leu Val Ile Gly Thr Glu 130 135 140 Thr
Leu Thr Arg Met Val Asp Trp Asn Asp Arg Thr Thr Cys Val Leu 145 150
155 160 Phe Gly Asp Gly Ala Gly Ala Val Val Leu Lys Ala Asp Glu Asp
Thr 165 170 175 Gly Ile Leu Ser Thr His Leu His Ala Asp Gly Ser Lys
Lys Glu Leu 180 185 190 Leu Trp Asn Pro Val Gly Val Ser Thr Gly Phe
Lys Asp Gly Ala Asn 195 200 205 Gly Gly Gly Thr Ile Asn Met Lys Gly
Asn Asp Val Phe Lys Tyr Ala 210 215 220 Val Lys Ala Leu Asp Ser Val
Val Asp Glu Thr Leu Ala Ala Asn Gly 225 230 235 240 Leu Asp Lys Ser
Asp Leu Asp Trp Leu Ile Pro His Gln Ala Asn Leu 245 250 255 Arg Ile
Ile Glu Ala Thr Ala Lys Arg Leu Asp Met Ser Met Asp Gln 260 265 270
Val Val Val Thr Val Asp Lys His Gly Asn Thr Ser Ser Gly Ser Val 275
280 285 Pro Leu Ala Leu Asp Ala Ala Val Arg Ser Gly Lys Val Glu Arg
Gly 290 295 300 Gln Leu Leu Leu Leu Glu Ala Phe Gly Gly Gly Phe Thr
Trp Gly Ser 305 310 315 320 Ala Leu Leu Arg Tyr 325
147324PRTAlicyclobacillus acidocaldariussource/note="Beta
ketoacyl-ACP synthase III" 147Met Tyr Lys Ala Val Ile Arg Gly Val
Gly Ser Tyr Leu Pro Glu Thr 1 5 10 15 Arg Leu Thr Asn Val Glu Ile
Glu Gln Met Val Ala Thr Ser Asp Glu 20 25 30 Trp Ile Gln Thr Arg
Thr Gly Ile Ala Glu Arg Arg Ile Ala Arg Pro 35 40 45 Asp Glu Ala
Thr Ser Asp Phe Ala Tyr Leu Ala Ala Gln Ala Ala Leu 50 55 60 Ala
Asp Ala Lys Leu His Pro Thr Asp Ile Asp Leu Leu Ile Val Ala 65 70
75 80 Thr Glu Thr Pro Asp Tyr Leu Leu Pro Pro Val Ala Cys Gln Val
Gln 85 90 95 Ala Arg Leu Gly Cys Arg Asn Ile Gly Ala Phe Asp Leu
His Ala Thr 100 105 110 Cys Ala Gly Phe Leu Ser Ala Leu Gln Val Ala
Glu Gln Phe Val Lys 115 120 125 Ser Gly Val His Glu His Val Leu Ile
Val Gly Ala Asp Thr Leu Ser 130 135 140 Arg Phe Thr Asp Tyr Thr Asp
Arg Gly Thr Cys Ile Leu Phe Ala Asp 145 150 155 160 Gly Ala Gly Ala
Phe Val Val Ser Arg Ser Asp Asp Arg Ala Ala Arg 165 170 175 Gly Val
Ile Ala Thr Thr Ile His Ser Asp Gly Thr Tyr Phe His Asn 180 185 190
Leu Tyr Ile Pro Gly Gly Gly Ser Arg Thr Pro Tyr Gly Asp Gly Ala 195
200 205 Lys Ala Lys Ile Val Met Asp Gly Arg Lys Ile Phe Lys Leu Ala
Val 210 215 220 Asn Val Met Ser Ser Thr Val Glu Glu Leu Leu Gln Lys
Thr Gly Arg 225 230 235 240 Gln Arg Asp Glu Ile Asp Trp Leu Ile Pro
His Gln Ala Asn Gln Arg 245 250 255 Ile Ile Asp Ala Val Ala Glu Ser
Leu Asp Phe Pro Gln Glu Lys Val 260 265 270 Val Ser Thr Ile Gln Asn
Ile Gly Asn Asn Ser Ser Ala Thr Ile Pro 275 280 285 Ile Ala Val Asp
Thr Ala Ile Arg Asp Gly Arg Ile Gln Arg Gly Asp 290 295 300 Leu Leu
Met Leu Val Ala Phe Gly Gly Gly Leu Val Trp Gly Gly Ala 305 310 315
320 Met Val Glu Tyr 148325PRTDesulfobulbus
propionicussource/note="Beta ketoacyl-ACP synthase III (FabH1)"
148Met Asn Arg Ala Val Ile Leu Gly Thr Gly Ser Cys Leu Pro Glu Arg
1 5 10 15 Lys Leu Thr Asn Ala Glu Leu Glu Arg Met Val Asp Thr Ser
Asp Glu 20 25 30 Trp Ile Thr Thr Arg Thr Gly Ile Arg Asn Arg His
Ile Ala Gly Lys 35 40 45 Asn Glu Gln Asn Tyr Gln Leu Ala Ala Lys
Ala Gly Arg Arg Ala Leu 50 55 60 Ala Val Thr Gly Ile Asp Ala Glu
Glu Leu Asp Leu Ile Ile Val Ala 65 70 75 80 Thr Val Ser Pro His Met
Ile Met Pro Ser Thr Ala Cys Phe Val Gln 85 90 95 Ala Glu Leu Gly
Ala Val Asn Ala Phe Ala Tyr Asp Ile Asn Ala Ala 100 105 110 Cys Ala
Gly Phe Thr Tyr Gly Leu Asp Leu Ala Ser Asn Tyr Ile Gln 115 120 125
Asn Arg Pro Glu Met Lys Ile Leu Leu Ile Gly Ala Glu Thr Leu Ser 130
135 140 Ala Arg Val Asp Trp Glu Asp Arg Asn Thr Cys Val Leu Phe Gly
Asp 145 150 155 160 Gly Ala Gly Ala Val Val Leu Ser Gly Ser His Asp
Gly Arg Gly Val 165 170 175 Phe Gly Ser Ser Leu His Ser Asp Gly Lys
Leu Trp Asn Leu Leu Cys 180 185 190 Met Asp Ser Pro Glu Ser Leu Asn
Pro Asp Leu Arg Pro Asp Ile Trp 195 200 205 His Gly Pro His Ile Arg
Met Ser Gly Ser Asp Ile Phe Lys His Ala 210 215 220 Val Arg Met Met
Glu Asp Ala Val Thr Ser Leu Leu Arg Lys His Asp 225 230 235 240 Leu
Thr Ile Asp Asp Val Asn Leu Met Ile Pro His Gln Ala Asn Ile 245 250
255 Arg Ile Leu Thr Asn Leu Arg Asp Arg Leu Gly Ile Ala Glu Glu Lys
260 265 270 Val Phe Ile Asn Leu Ser Lys Tyr Gly Asn Thr Ser Ala Ala
Ser Ile 275 280 285 Pro Ile Ala Leu Asp Glu Ala His Arg Glu Gly Arg
Leu Arg Arg Gly 290 295 300 Asp Ile Val Leu Leu Cys Thr Phe Gly Gly
Gly Leu Thr Trp Gly Ser 305 310 315 320 Leu Leu Met Arg Trp 325
149346PRTDesulfobulbus propionicussource/note="Beta ketoacyl-ACP
synthase III (FabH2)" 149Met Thr Leu Arg Tyr Thr Gln Val Cys Leu
His Asp Phe Gly Tyr Gln 1 5 10 15 Leu Pro Pro Val Glu Leu Ser Ser
Ala Ala Ile Glu Glu Arg Leu Gln 20 25 30 Pro Leu Tyr Glu Arg Leu
Lys Leu Pro Ala Gly Arg Leu Glu Leu Met 35 40 45 Thr Gly Ile Asn
Thr Arg Arg Leu Trp Gln Pro Gly Thr Arg Pro Ser 50 55 60 Ala Gly
Ala Ala Ala Ala Gly Ala Asp Ala Met Ala Lys Ala Gly Val 65 70 75 80
Asp Val Ala Asp Leu Gly Cys Leu Leu Phe Thr Ser Val Ser Arg Asp 85
90 95 Met Met Glu Pro Ala Thr Ala Ala Phe Val His Arg Ser Leu Gly
Leu 100 105 110 Pro Ser Ser Cys Leu Leu Phe Asp Ile Ser Asn Ala Cys
Leu Gly Phe 115 120 125 Leu Asp Gly Met Ile Met Leu Ala Asn Met Leu
Glu Leu Gly Gln Val 130 135 140 Lys Ala Gly Leu Val Val Ala Gly Glu
Thr Ala Glu Gly Leu Val Glu 145 150 155 160 Ser Thr Leu Ala His Leu
Leu Ala Glu Thr Gly Leu Thr Arg Lys Ser 165 170 175 Ile Lys Pro Leu
Phe Ala Ser Leu Thr Ile Gly Ser Gly Ala Val Ala 180 185 190 Leu Val
Met Thr Arg Arg Asp Tyr Arg Asp Thr Gly His Tyr Leu His 195 200 205
Gly Gly Ala Cys Trp Ala Gln Thr Val His Asn Asp Leu Cys Gln Gly 210
215 220 Gly Gln Asn Ala Glu Gln Gly Thr Leu Met Ser Thr Asp Ser Glu
Gln 225 230 235 240 Leu Leu Glu Lys Gly Ile Glu Thr Ala Ala Ala Cys
Trp Gln Gln Phe 245 250 255 His Ala Thr Leu Gly Trp Asp Lys Gly Ser
Ile Asp Arg Phe Phe Cys 260 265 270 His Gln Val Gly Lys Ala His Ala
Gln Leu Leu Phe Glu Thr Leu Glu 275 280 285 Leu Asp Pro Ala Lys Asn
Phe Glu Thr Leu Pro Leu Leu Gly Asn Val 290 295 300 Gly Ser Val Ser
Ala Pro Ile Thr Met Ala Leu Gly Ile Glu Gln Gly 305 310 315 320 Ala
Leu Gly Ala Gly Gln Arg Ala Ala Ile Leu Gly Ile Gly Ser Gly 325 330
335 Ile Asn Ser Leu Met Leu Gly Ile Asp Trp 340 345
150903DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic E. coli codon-optimized Propionibacterium
freundenreichii fabHcoding polynucleotide" 150atgattgata gcacaccgga
atggattgaa cagcgtaccg gtattcgtga acgtcgttgg 60gcaaccaaag atgaaaccgt
tctgagcatg gcaaccgatg caggtcgtaa agcactggat 120atggcaggcg
ttaaaccgga acaggttggg gcaattattg ttagcaccgt tagccatcat
180attccgagtc cgggtctgag cgattatctg gcagaagaac tgggttgtcc
ggcaccggca 240acctttgata ttagcgcagc atgtgcaggt ttttgttatg
cactgaccct ggcagaaagc 300attgttcgtg caggtcatgc aggtaaagat
ggttttgttc tgattgttgg tgttgaacgt 360ctgtccgata tgaccaatat
ggatgatcgt ggcaccgatt ttctgtttgg tgatggtgcc 420ggtgcagcag
ttgttggtcc gagcgataca ccggcaattg gtccggcagt ttggggtagc
480aaaccggcaa atgttaaaac cattgaaatt cagagctgga ccgaagcaga
taaaaatccg 540accggttttc cgctgattca gatggatggt cataccgtgt
ttaaatgggc actgagcgaa 600gttgcagatc acgcagccga agcaattgat
gcagcaggta ttactccgga acagctggat 660atctttctgc cgcatcaggc
aaatgatcgt attaccgatg ccattattcg tcatctgcat 720ctgccggata
gcgttagcgt ttgtcgtgat attgcagaaa tgggtaatac cagcgcagca
780agcattccga ttgcaatgga tgcaatgatt cgcgaaggtc gtgcaaaaag
cggtcagacc 840gcactgatta ttggttttgg tgcaggtctg gtttatgccg
gtcgtgttgt tgttctgccg 900taa 90315123DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
PTrc_vector_F oligonucleotide" 151gaattcgaag cttgggcccg aac
2315231DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic PTrc_vector_R oligonucleotide" 152catggtttat
tcctccttat ttaatcgata c 3115340DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
BsfabH1_IFF oligonucleotide" 153gaggaataaa ccatgaaagc tggaatactt
ggtgttggac 4015436DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
BsfabH1_IFR oligonucleotide" 154ccaagcttcg aattcttatc ggccccagcg
gattgc 3615542DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic BsfabH2_IFF oligonucleotide"
155gaggaataaa ccatgtcaaa agcaaaaatt acagctatcg gc
4215644DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic BsfabH2_IFR oligonucleotide" 156ccaagcttcg
aattcttaca tcccccattt aataagcaat cctg 4415738DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
LmfabH1-2_IFF oligonucleotide" 157gaggaataaa ccatgaacgc aggaatttta
ggagtagg 3815842DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic LmfabH1_IFR oligonucleotide"
158ccaagcttcg aattcttact taccccaacg aatgattagg gc
4215941DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic LmfabH2_IFR oligonucleotide" 159ccaagcttcg
aattcttact tacccccacg aatgattagg g 4116039DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
DpfabH1_IFF oligonucleotide" 160gaggaataaa ccatgaatag agcagttatc
ttgggaacc 3916139DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic DpfabH1_IFR oligonucleotide"
161ccaagcttcg aattcttacc aacgcatgag cagcgaacc 3916236DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
DpfabH2_IFF oligonucleotide" 162gaggaataaa ccatgacttt gcgttacacc
caggtc 3616337DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic DpfabH2_IFR oligonucleotide"
163ccaagcttcg aattcttacc agtcgatgcc cagcatg 3716434DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
AafabH_IFF oligonucleotide" 164gaggaataaa ccatgtacaa ggccgtgatt
cgcg 3416536DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic AafabH_IFR oligonucleotide"
165ccaagcttcg aattctcaat actccaccat cgcgcc 3616636DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
PffabHopt_IFF oligonucleotide" 166gaggaataaa ccatgattga tagcacaccg
gaatgg 3616739DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic PffabHopt_IFR oligonucleotide"
167ccaagcttcg aattcttacg gcagaacaac aacacgacc 3916836DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
SmfabH_IFF oligonucleotide" 168gaggaataaa ccatgagcaa gcggatctat
tcgagg 3616935DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic SmfabH_IFR oligonucleotide"
169ccaagcttcg aattctcaat agcgcagcag ggccg 35
* * * * *